To test this hypothesis, we used the melanocyte-derived radial growth phase melanoma cell line WM-1552, which was transfected to stably express CSPG4 (Yang et al., 2004) and was designated as WM1552-CSPG4. The expression of CSPG4 in WM1552 cells causes enhanced cell adhesion and spreading, increased motility, enhanced epithelial-tomesenchymal transition (EMT), anchorage independent growth, and tumorigenic potential compared with the CSPG4 negative mock transfected counterparts (Yang et al., 2009; Price et al., 2011). In the current study, we compared TNT formation in WM1552c mock transfectants lacking CSPG4 with WM1552c CSPG4-expressing melanoma cell lines. We cultured WM1552 mock and CSPG4 cells separately in low-serum, hyperglycemic RPMI medium (2.5% FCS, 50 mM glucose) and counted TNTs manually at 24-h intervals for 96 h. To assess the rate of cellular proliferation, we also determined the relative absorbance of a surrogate measure of cell growth over time. We detected significantly higher numbers of TNTs at 96 h in WM1552-CSPG4 culture as compared with WM1552-mock cells (**Figure 5**). This increase in TNT formation was independent of two-dimensional cell growth. After accounting for a higher cellular absorbance rate for the former cell line as a measure of cell proliferation, TNT/absorbance remained significantly higher for WM1552-CSPG4 (1.2-fold higher or 21% higher, at 72 h), suggesting an association and potential stimulatory role of CSPG4 in the formation of TNTs. Functionally, CSPG4 is localized on the tips of filopodia and stimulates key oncogenic signaling pathways, including focal adhesion kinase, constitutively activated BRAF/Erk 1,2 and Rho family GTPases, all of which could be important for the stimulation of TNT formation (Price et al., 2011). Furthermore, the function of CSPG4 requires the cytoplasmic tail of the core protein. Thus, studies that interrogate CSPG4 may shed considerable insight into the molecular mechanisms that govern TNT formation (Price et al., 2011). Since CSPG4 is a potential target, studies that interrogate mechanisms of CSPG4 function may also positively identify the importance of TNTs as a clinical target in malignant progression.

#### THE QUESTION OF ACTIVE vs. PASSIVE DIFFUSION: THE MYOSIN CHAPERONE UNC-45A CORRELATES WITH TNT FORMATION FOR A LIMITED TIME, BUT IS INVERSELY ASSOCIATED WITH TNT FORMATION AFTER 48 H IN CULTURE

There remain many questions regarding the active vs. passive nature of intercellular cargo transport that is mediated by TNTs. Discovery of a defining molecular component that is specific to TNTs remains elusive, and it is conceivable that a single one may not exist. The prevailing thought is that these filamentous actinbased cell protrusions employ the myosin motor complex in a

fashion similar to other well-established modes of actin-based machinery.

UNC-45A is a chaperone protein that has been well characterized for its function in the assembly and maintenance of the myosin II motor complex. It has been shown to be overexpressed in serous ovarian carcinomas and is associated with significantly increased cancer cell proliferation as well as motility (Bazzaro et al., 2007). Furthermore, the UNC-45A protein tracks at the leading edge of cells and is known to accumulate at the cell furrow during cytokinesis. We postulated that as TNTs arise at the cell edge, they might utilize UNC-45 for formation and maintenance. Thus, we examined TNT formation in ovarian cancer cells with intact UNC-45A expression as compared with the same cell line (SKOV3) with shRNA knockdown of UNC-45A; a scramble version of the lentivirus offered an additional form of control. We quantified the number of TNTs and cells in 20X fields of view to establish the TNT index (number of TNTs/cell over time), a method we have described in detail in other publications (Ady, 2014). Interestingly, we found that while the average number of TNTs per cell was significantly lower in the UNC-45A-knockdown group at 24 h and 48 h, by 72 h the reverse was seen, as on average there were more TNTs/cell among the cells in which ∼80% of UNC-45A had been suppressed via shRNA knockdown (**Figure 6**). This finding was initially unexpected, but, upon further analysis, it made sense in the overall context based on the known role of UNC-45A in modulating cellular motility and proliferation.

This finding provides stimulus for examining the time course of TNTs and better understanding their niche, not just spatially but also in time. Tunneling nanotubes are most prolific in subconfluent cell cultures. As cultures become increasingly confluent, the physical space between cells is diminished, and there is less need for TNTs to form bridges between these cells. Knockdown of UNC-45A continued to suppress cell proliferation by 72 h; in comparison with scramble and wild type, the ratio of the number of TNTs/cell was naturally higher (**Figure 6**). This observation provided potential insight into the notion that UNC-45A and other mediators of myosin motors might be essential to the early stages of TNT formation among non-crowded cell populations; however, its importance might diminish over time in more confluent or nearly-confluent cell populations and in terms of maintenance, rather than formation, of TNTs.

#### hENT1 EXPRESSION IS INVERSELY CORRELATED WITH TNTs IN PANCREATIC CANCER

The tumor microenvironment comprises a vast and complex set of players that mediate intracellular and intercellular discussion. Important players of intercellular communication include diffusible soluble hormone signals, exosomes and other ECVs, and connexin-based gap junctions for cells in immediate proximity. In certain cancers, there are other forms of transporters. One such example is hENT1. Human equilibrative nucleoside transporter 1 is highly expressed in malignant pancreatic tumors, has a life cycle of 14 h, and has been associated with improved prognosis in pancreatic cancer (Nivillac et al., 2011), although there is no apparent correlation of hENT1 expression to survival (Poplin et al., 2013).

The rationale for studying hENT is that it functions effectively as a transporter of nucleosides. More specifically, hENT1 permits intercellular transfer of chemotherapeutic agents such as the fluoropyrimidine gemcitabine, a standard of care drug that is potentially effective once it has successfully penetrated the dense stroma-rich microenvironment that is characteristic of this type of cancer. We recently reported that despite the especially dense

FIGURE 6 | Knockdown of the UNC-45A myosin motor protein chaperone diminishes TNT formation, but this finding is limited in duration. Upper left: western blot demonstrating effective knockdown of UNC-45A using shRNA. Based on these results, shRNA #2 was used in our study. Upper right: quantitation of effective UNC45-A knockdown by shRNA #1 and #2. Lower left: mean number of TNTs at 24 h, 48 h, and 72 h in wild type SKOV3 ovarian cancer cells compared with cells transfected with the scramble and UNC-45A shRNA #2. Lower right: mean cell count for the same conditions and time points. Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

nature of the pancreatic stromal reaction, TNTs could be detected in human pancreatic tumor tissue (Desir et al., 2018). We further postulated that if TNTs were the mediators of local and regional invasion and metastasis, then their presence would be inversely proportional to the expression of hENT1. As proof of concept, we examined a metastatic pancreatic carcinoma-derived cell line, S2013, for hENT1 expression using a fluorescent antibody. We detected focal expression of this transporter protein along the membrane, as expected. Quantifying its expression as arbitrary units (a.u.), and accounting for the area of each cell and the TNTs, we reported the expression in a fashion identical to our previously published study that examined lipid raft enrichment in TNT-forming cells (Thayanithy et al., 2014a). Consistent with our hypothesis, we found an inverse correlation between hENT1 expression and TNTs in these pancreatic cancer cells (**Figure 7**). In the S2013 cells, hENT1 expression was 1.8-fold higher in cells not forming TNTs than in cells in the same culture forming TNTs (p = 0.0023).

FIGURE 7 | Cancer cells forming more TNTs demonstrate lower expression of hENT1. (A) hENT1 expression in S2013 pancreatic cancer cells, A270 ovarian cancer cells, and LOVO colon cancer cells with and without TNTs. (B) Corresponding images for each cell line are located beneath each graph. Cells were stained with immunofluorescent antibody that marks the expression of hENT1, and the expression was quantified as described. Scale bars = 50µm for each of these panels. (C) The larger image on the bottom demonstrates a particularly long and curved TNT (indicated by white arrows) connecting hENT1-expressing S2013 cells. Materials and Methods section for experiments shown in the figure is available in the Supplementary Material.

Although hENT1 is most often associated with pancreatic carcinomas, it is also present in other forms of cancer, including ovarian cancer (>90% expression) (Farré et al., 2004) and colon cancer (Liu et al., 2017). Thus, for comparison, we performed the same analysis in representative cell lines from these cancers as well. The results in those cell lines were consistent with our findings of inverse correlations of hENT1 to TNTs, indicating that TNTs may be indicative of more chemoresistant and/or more invasive cells. The expression of hENT1 was 2.4-fold higher in A2780 cells not forming TNTs as compared with those forming TNTs (p = 0.0126); this difference was 1.4-fold in LOVO cells, respectively (p = 0.0049).

It will be important not only to substantiate but also reconcile this finding with some of our own recent findings that TNTs are also conduits for mediating intercellular efflux of chemotherapeutic drugs (Desir et al., 2018). In fact, it has been reported that TNT-like tumor microtubes (TMs) seen in gliomas correlate with a higher degree of cellular invasion and drug resistance (Osswald et al., 2015, 2016; Jung et al., 2017; Weil et al., 2017).

# LOCALIZATION OF CONNEXIN PROTEINS IN RELATION TO TNTs

Connexin channels that compose gap junctions mediate intercellular transfer of calcium and other small soluble factors; they are also not inherently separate from TNTs. Tunneling nanotubes and TMs have also been shown to mediate intercellular calcium flux effectively between malignant cells (Osswald et al., 2015; Lock et al., 2016). In fact, in both

of these cited studies, the localization and attachment of connexins was found to be crucial to this TNT/TM-mediated process.

It is well established that gap junctions are downregulated in cancer cells undergoing EMT, a state that is strongly associated with stem cell-like properties and metastasis, which our own group showed was associated with significant upregulation in TNTs in malignant mesothelioma cells (Lou et al., 2012). Gap junctions have been reported to be localized at the tip of TNTs, for example in astrocytes (Wang and Gerdes, 2012), but reports, to date, have been inconsistent with this finding. The finding may be variable based on the timing of TNT formation, detection in its state at the time (e.g., in the midst of forming, transporting cargo, or disconnecting from recipient cells), the state of confluence of the cell culture being examined, and the cell type (malignant vs. non malignant or even heterogeneous between different cancer cell types). Furthermore, it is not yet firmly established whether gap junctions actually mediate the formation of TNTs from the plasma membrane and/or represent structural foundations of TNTs across all cell types. It remains to be determined whether connexins localize more at the base of TNTs where they emerge from the donor cell or at the point of entry into putative recipient cells.

The question remains as to whether connexin expression differs quantitatively between cancer cells with or without TNTs. The answer is likely time-dependent; that is, dependent on the extent of cell confluency, distance between cells, and number of TNTs. We have extensively characterized TNT formation, function, and characterization in several cell lines of malignant mesothelioma (Lou et al., 2012; Ady, 2014, 2016; Thayanithy et al., 2014a). To examine connexin localization in these cells, we stained MSTO-211H and VAMT cells with immunofluorescent markers and performed inverted microscopy. A preliminary analysis was performed to quantify differences in connexin expression relative to expression in non-TNT forming cells. We found a 1.7-fold increase in connexin expression in MSTO cells not forming TNTs, as compared with cells forming TNTs; for the VAMT cell line, the difference was 1.28-fold (**Figure 8**). In the scope of systems biology, no component of the tumor matrix works independently. Rather, all of the components are dynamic and function as a unified process. For example, it is now established that exosomes interact synergistically with TNTs in cancer (Thayanithy et al., 2014a); connexin channels associated with TNTs may play a role in their initial formation and/or maintenance. It will be important to determine the variability between cancer cell types based on tissue of origin, state of EMT, and molecular status and also between cancer and non-cancer cell types.

# CONCLUSIONS

Cancer cells cannot be studied in isolation, as they interact in a complex biological system. With the improved and rapidly expanded understanding of the function and importance of intercellular communication in modulating the tumor microenvironment, it is critical to investigate malignancy as a unique and continuously evolving ecosystem. These interactions can take many forms. Homotypic interactions of malignant cells are mediated via TNTs, exosomes, gap junctions, and a wide ranging list of diffusible and soluble factors. Heterotypic interactions include interactions of the extracellular matrix with malignant cells, vascular endothelium with cancer, immune cells with cancer, and hematologic components with cancer. Altogether, these events comprise a spectrum of intratumoral interactions that reprogram cells for invasion, metastasis, and emergence of resistance to treatment. The interplay and dynamics of the subtopics that we have begun to examine here are illustrated in the accompanying schematic (**Figure 9**). Perturbations to this complex ecosystem, such as those provided by drug- or radiation-induced injuries, can instead induce cellular stress responses that in turn increase the extent of any or all of these inflammatory interactions. Increased characterization of TNTs and their role in cancer cell invasion and chemoresistance over the past decade continues to provide novel mechanistic insights into how the heterogeneous tumor microenvironment adapts and evolves over time. Continued work in this exciting field, including concepts described here and elsewhere, will clarify to what extent TNTs play an important role in mediating the tumor ecosystem.

# AUTHOR CONTRIBUTIONS

EL wrote the initial drafts of the manuscript. EL, EZ, AS, SD, PW, YI, JM, and MB provided initial analysis of the data and prepared the figures. EL, SS, JM, MB, and CS performed further analysis and interpretation of the data and wrote subsequent drafts of the manuscripts. All authors reviewed and approved the final submitted manuscript.

#### FUNDING

This research was supported by the Minnesota Masonic Charities; the Central Society for Clinical and Translational Research Early Career Development Award; the National Pancreas Foundation Research Grant (provided in partnership with the National Pancreas Foundation, several NPF Chapters and the Horvitz/Lebovitz Research Fund); University of Minnesota's Deborah E. Powell Centre for Women's Health Interdisciplinary Seed Grant support (Grant #PCWH-2013-002); the Institutional Research Grant #118198-IRG-58-001-52-IRG94 from the American Cancer Society; the Mezin-Koats Colon Cancer Research Award; the Randy Shaver Cancer Research and Community Fund; the Litman Family Fund for Cancer Research; family and friends of G. Huntington, G. Derfus, and A. Baffa; cancer research fundraisers by the Mu Sigma Chapter of the Phi Gamma Delta Fraternity, the University of Minnesota, and the Courage and a Cure Foundation, Goodyear, Arizona; the Minnesota Medical Foundation/University of Minnesota Foundation; the Masonic Cancer Center and the Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota; and the NIH Clinical and Translational Science KL2 Scholar Award 8UL1TR000114. The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.

#### ACKNOWLEDGMENTS

We would like to thank Guillermo Marques, Ph.D. and Mark Sanders, Ph.D. of the University Imaging Centers (UIC) at the University of Minnesota for providing assistance with confocal microscopy; Zhilian Xia for providing support with the experiments; Nina Lampen, Electron Microscopy Core at the Memorial Sloan-Kettering Cancer Center, for preparation of cells and for providing assistance with imaging for electron microscopic evaluation; Timothy Starr, Ph.D. for helpful discussion and help with the use of equipment for hypoxic conditions; and Michael Franklin, M.S., for helpful critiques and editorial suggestions for this manuscript.

#### SUPPLEMENTARY MATERIAL

The Materials and Methods section for the experiments described in this paper is located in the Supplementary Material of this article, which can be found online at: https://www.frontiersin. org/articles/10.3389/fcell.2018.00095/full#supplementarymaterial

Supplementary Video 1 | Time-lapse video of a coculture of Mg63.2 osteosarcoma cells with hFOB osteoblast cells.

# REFERENCES

models of osteosarcoma. Cancer Lett. 335, 412–420. doi: 10.1016/j.canlet.2013. 02.050

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Lou, Zhai, Sarkari, Desir, Wong, Iizuka, Yang, Subramanian, McCarthy, Bazzaro and Steer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Indoximod: An Immunometabolic Adjuvant That Empowers T Cell Activity in Cancer

#### Eric Fox 1 , Thomas Oliver 1 , Melissa Rowe1 , Sunil Thomas 2 , Yousef Zakharia3 , Paul B. Gilman1,2, Alexander J. Muller 2,4 and George C. Prendergast 2,4 \*

1 Department of Hematology-Oncology, Lankenau Medical Center, Wynnewood, PA, United States, 2 Lankenau Institute for Medical Research, Wynnewood, PA, United States, 3 Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA, United States, 4 Sidney Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, United States

#### Edited by:

Ubaldo Emilio Martinez-Outschoorn, Thomas Jefferson University, United States

#### Reviewed by:

Paolo Puccetti, University of Perugia, Italy Francesca Fallarino, University of Perugia, Italy

\*Correspondence: George C. Prendergast<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology

Received: 11 July 2018 Accepted: 21 August 2018 Published: 11 September 2018

#### Citation:

Fox E, Oliver T, Rowe M, Thomas S, Zakharia Y, Gilman PB, Muller AJ and Prendergast GC (2018) Indoximod: An Immunometabolic Adjuvant That Empowers T Cell Activity in Cancer. Front. Oncol. 8:370. doi: 10.3389/fonc.2018.00370 Exploding interest in immunometabolism as a source of new cancer therapeutics has been driven in large part by studies of tryptophan catabolism mediated by IDO/TDO enzymes. A chief focus in the field is IDO1, a pro-inflammatory modifier that is widely overexpressed in cancers where it blunts immunosurveillance and enables neovascularization and metastasis. The simple racemic compound 1-methyl-D,L-tryptophan (1MT) is an extensively used probe of IDO/TDO pathways that exerts a variety of complex inhibitory effects. The L isomer of 1MT is a weak substrate for IDO1 and is ascribed the weak inhibitory activity of the racemate on the enzyme. In contrast, the D isomer neither binds nor inhibits the purified IDO1 enzyme. However, clinical development focused on D-1MT (now termed indoximod) due to preclinical cues of its greater anticancer activity and its distinct mechanisms of action. In contrast to direct enzymatic inhibitors of IDO1, indoximod acts downstream of IDO1 to stimulate mTORC1, a convergent effector signaling molecule for all IDO/TDO enzymes, thus possibly lowering risks of drug resistance by IDO1 bypass. In this review, we survey the unique biological and mechanistic features of indoximod as an IDO/TDO pathway inhibitor, including recent clinical findings of its ability to safely enhance various types of cancer therapy, including chemotherapy, chemo-radiotherapy, vaccines, and immune checkpoint therapy. We also review the potential advantages indoximod offers compared to selective IDO1-specific blockade, which preclinical studies and the clinical study ECHO-301 suggest may be bypassed readily by tumors. Indoximod lies at a leading edge of broad-spectrum immunometabolic agents that may act to improve responses to many anticancer modalities, in a manner analogous to vaccine adjuvants that act to boost immunity in settings of infectious disease.

Keywords: immunometabolism, immune adjuvant, Immunotherapy, immuno-chemotherapy, immuno-radiotherapy

## INTRODUCTION

Immune therapy has risen to the forefront of cancer therapy in recent years, providing a new approach to cancer therapy, and in some instances has begun to shift the paradigm of cancer care from chemotherapy to immunotherapy. One of the factors crucial to the success of immunotherapy is reversing tumormediated immunosuppression (1). The tryptophan catabolic enzyme indoleamine 2,3-dioxygenase-1 (IDO1) has received a great deal of attention as a driver of tumor-mediated suppression (2–4). IDO1 has been shown to be active in many human cancers and its expression has been associated widely with poor prognosis (5, 6). Accordingly, inhibitors of the enzymatic activity and effector functions of IDO1 have been developed as tools to leverage cancer therapy (7).

Elevated tryptophan catabolism as a characteristic of patients with cancer was initially reported over 60 years ago (8). The basis for this observation and later observations in various types of cancer patients was not clear until IDO1 was discovered in the 1960s. An association of elevated tryptophan cata olism with inflammation was established in the 1970s−1980s with demonstrations that IDO1 is induced strongly in the lungs by LPS, viral infection and interferon (9–12). In a seminal line of work in the late 1990s by Munn and Mellor and colleagues, tryptophan catabolism was implicated in immunosuppression during pregnancy, based on the preferential sensitivity of T cells to tryptophan deprivation leading to an impairment of antigendependent T cell activation (13–15). In these studies, the key probe in defining this mechanism of immune tolerance was the racemic compound 1-methyl-tryptophan (1MT), a tryptophan mimetic with complex IDO inhibitory effects discussed further below. Indeed, much of the huge amount of subsequent work on IDO and disease pathogenesis has relied on this compound, including most importantly cancer studies.

A causal relationship between IDO1 activity and cancer growth was founded by pivotal studies in the 2000s that have been reviewed in detail elsewhere (7). IDO1 was found to be overexpressed widely in human cancers and 1MT could slow the growth of murine tumors (6, 16, 17). IDO1 overexpression in cancer cells was linked genetically to inactivation of BIN1 (18), a tumor suppressor gene widely attenuated in human cancer (19). Loss of BIN1 empowers IFN/STAT and NFkB mediated IDO1 transcription and later studies also implicated the RAS/MAPK, COX2, and PI3K pathways in driving IDO1 expression (18, 20– 22). Interestingly, drugs that target molecules relying on these pathways may act in part by indirectly blocking IDO1 expression, such as the case with imatinib (Gleevec) (23). Pharmacological blockade with 1MT or true catalytic inhibitors of IDO1 enzyme were found to display unimpressive efficacy unless combined with DNA damaging therapies, which led to regression of otherwise unstoppable tumors (18, 24, 25). Preclinical genetic proofs of IDO1 as a valid therapeutic target in cancer were enabled in IDO1-deficient mice, where fundamental connections between IDO1 expression and cancerous inflammatory programs were also established (21, 26, 27).

In the tumor microenvironment or draining lymph nodes, IDO1 activity suppresses the function of T effector cells (Teff) and natural killer (NK) cells and promotes the induction and activation of T regulatory cells (Treg) and the activation, recruitment and expansion of myeloid-derived suppressor cells (MDSC) (**Figure 1**) [Fallarino(21, 29–36)]. IDO1 effector functions are mediated by the tryptophan catabolite kynurenine (Kyn) and by two stress signals generated by locoregional deprivation of tryptophan (7), as discussed further below. Investigations of IDO1 in immune tolerance have focused heavily on antigen-presenting dendritic cells where IDO1 is upregulated by interferons, TLR ligands and other immune signals (37). Beyond its roles in provoking Treg development, IDO1 also acts in certain dendritic cells to directly suppress effector T cell responses (38, 39).

#### BIOLOGICAL ROOTS OF INDOXIMOD AS AN IMMUNOMETABOLIC ADJUVANT FOR CANCER THERAPY

The racemic compound 1-methyl-D,L-tryptophan (1MT) was first described as a competitive inhibitor of the IDO1 enzyme by Cady and Sono in the early 1990s (40). After the seminal demonstration that 1MT could elicit allogeneic conceptus rejection by ablating T cell tolerance to paternal fetal antigens (13), 1MT was shown to weakly retard the growth of cancer cells in mouse tumor graft or spontaneous transgenic models of cancer (16, 17). While the anticancer effects of 1MT were unremarkable as monotherapy, its striking therapeutic power was revealed in combinations with DNA damaging chemotherapy which elicit regressions of otherwise recalcitrant tumors (18). This discovery was an important advance in providing the first indication of how to use an IDO inhibitor to improve cancer therapy. The regressions achieved by 1MT in combination therapy did not appear to reflect drug-drug interactions that raised the cytotoxicity of the chemotherapies tested, as the efficacy was increased without increasing the known side-effects of the chemotherapies tested (18). Further, T cell depletion in subjects nullified the therapeutic benefits of 1MT administration, establishing that its action was based in provoking T cell attacks in the presence of chemotherapy (18). Overall, these observations challenged the paradigm at the time that active immunotherapy and chemotherapy are fundamentally incompatible by offering one of the first demonstrations of a productive immunochemotherapy regimen based exclusively on small molecule drugs (41).

Careful biochemical studies with purified IDO1 enzyme revealed that only the L racemer of 1MT exerted any catalytic inhibitory activity (42), and it became apparent that L-1MT is actually a weak substrate rather than a true catalytic inhibitor of IDO1 as discussed in detail elsewhere (43). Unexpectedly, the D racemer lacking enzyme inhibitory activity was actually more potent in empowering chemotherapy as well as relieving T cell suppression by IDO1-positive dendritic cells from mouse or human sources (42), although there are conflicting data on T cell suppression (44, 45). Mouse genetic studies were consistent with IDO1 pathway targeting in showing that the anticancer efficacy of D-1MT relied genetically on the presence of a functionally intact

IDO1 gene (42), similar to bona fide IDO1 enzyme inhibitors (24, 25). However, subsequent studies of D-1MT make it clear that its antitumor effects in cells and in animals is likely to be complex (7, 43). Indeed, mechanistic studies have made it clear that neither racemer of 1MT is a valid probe of IDO1 enzyme activity, a question ultimately addressed by isolation of several unique structural classes of true enzymatic inhibitors with related antitumor properties, as reviewed elsewhere (7). Cellular mechanisms of action for indoximod have been defined which involve relief of suppression of Teff cells in tumors, limitations on the generation of Tregs, and re-programming of Tregs to Th17 helper cells in draining lymph nodes (**Figure 1**) (2, 46, 47). The robust preclinical efficacy of D-1MT/indoximod in combination with DNA damaging chemotherapy led to its inclusion on a list of 'top ten' agents for clinical evaluation by an NCI immunotherapy workshop (48, 49). In 2008, a decision was made to advance D-1MT/indoximod (NLG-8189) to first-in-man trials as a single molecular species through an FDA investigational new drug application by a collaborative team of investigators from the Medical College of Georgia, Lankenau Institute for Medical Research, National Cancer Institute and NewLink Genetics Corporation as corporate sponsor.

# CLINICAL EVALUATION OF INDOXIMOD

Phase 1 studies of indoximod as a monotherapy or in combination with chemotherapy showed it to be well-tolerated in patients with advanced breast cancers or other solid tumors (50, 51). In a first-in-man dose escalation study conducted in advanced breast cancer patients receiving standard of care taxane therapy, the administration of indoximod was well-tolerated to a maximum delivered dose of 1,200 mg twice daily. Four partial responses were observed in the patients studied (n = 22) in the absence of any apparent drug-drug interactions (50). In a larger dose escalation study of advanced cancer patients with various solid tumors, the maximum tolerated dose was not reached until 2,000 mg twice daily (51). Notably, several patients on the indoximod trial who had been treated previously with ipilimumab developed hypophysitis, an autoimmune reaction to the pituitary gland which had been documented in patients treated with ipilimumab. In these patients, stable disease >6 months was observed, encouraging the notion that indoximod can reactivate latent T cell immunity in cancer patients. In the initial trials of indoximod, its relative apparent safety is notable given comparisons to the acute side-effects of immune

checkpoint therapy, however, a case of Parkinsonism was reported recently in a patient receiving indoximod treatment (52). While safety studies were not able to identify a maximum tolerated dose (MTD) for indoximod, pharmacokinetic analysis indicated that 1,200 mg twice daily (BID) was the maximum exposure that could be achieved in a patient based on a plateau that occurred in plasma AUC and Cmax beyond this dose. Oral dosing generated a Cmax at 2.9 h with a serum halflife of 10.5 h. Interestingly, there was evidence in indoximod-treated patients of increased levels of both C reactive protein (CRP) and autoantibodies to tumor antigens, consistent with an increased inflammatory response to the chemotherapy onboard (51). Based on these initial studies, multiple Phase 2 studies of indoximod in continuous oral cycles have been conducted at a dose of 1,200 mg twice daily.

Phase 2 data from several trials of indoximod in different types of cancer has been provocative but not uniformly positive in all disease settings examined so far (**Table 1**). All trials have been conducted in combination with standard of care treatments, including in metastatic cutaneous, mucosal, or uveal melanoma with immune checkpoint therapy; advanced breast cancer (BRCA), acute myeloid leukemia (AML), and pancreatic ductal adenocarcinoma (PDAC) with chemotherapy; and advanced prostate carcinoma (PC) with sipuleucel-T (Provenge R ), an approved dendritic cell vaccine. In particular, the melanoma and prostate trials have illustrated significant therapeutic activity of indoximod in empowering anti-PD1 treatment (pembrolizumab) and sipuleucel-T vaccine treatment (Provenge R autologous dendritic cells), respectively.

#### Metastatic Melanoma

The initial phase 1b study in melanoma illustrated the safety of indoximod in combination with the anti-CTLA4 antibody ipilimumab, the standard of care treatment for metastatic melanoma at the time of testing. Nine patients with unresectable stage 3 or 4 melanoma patients were treated with escalating doses of indoximod (600 mg BID, then 1,200 mg BID). Unlike an IDO1 enzyme inhibitor (epacadostat) which yields dose-limiting toxicity (DLT) in combination with ipilimumab, no DLT was encountered with indoximod. Thus, the pre-specified highest dose of indoximod (1,200 mg BID) was deemed tolerable and used as the recommended phase 2 dose (RP2D) in combination with checkpoint inhibitors (59).

The phase 2 melanoma study enrolled over 100 patients in a single-arm trial of indoximod plus provider choice of immune checkpoint antibodies (ipilimumab or the anti-PD1 antibodies nivolimumab or pembrolizumab) (NCT03301636). A preclinical treatment rationale was provided by a study showing that indoximod could improve the response of B16 murine melanoma tumors to immune checkpoint therapy (60). In this single-arm trial (53), 85 patients were treated with pembrolizumab plus indoximod with on-treatment imaging to meet a pre-specified definition of evaluable for efficacy. Overall response rate (ORR) was 53% with a rate of complete response (CR) of 18% and disease control rate (DCR) of 73%. Median progression-free survival (PFS) was 12.4 months (95% confidence interval: 7.1, 24.9). Notably, these efficacy data paralleled those achieved by the approved combination of nivolumab and ipilimumab, but without the elevated rate of severe autoimmune side-effects experienced by patients treated with these agents (61). Stratifying the data by PD-L1 expression status, the ORR in PD-L1 positive (+) patients was 77% vs. 37% in PD-L1 negative (-) patients. Some responses seen in uveal melanomas were encouraging given its extremely aggressive nature and complete lack of response to immune checkpoint therapy (62). Overall, these data suggest the ability of indoximod to safely augment anti-PD1 antibody responses, strongly encouraging a randomized Phase 3 trial in this disease setting. These data are striking in light of the failure of epacadastat, a direct IDO1 enzyme inhibitor, to show any benefit to melanoma patients in the phase 3 ECHO-301 study when administered in combination with pembrolizumab. Given the different mechanism of action of indoximod, its independent evaluation must not be dismissed out of hand.

## Metastatic Castrate-Resistant Prostate Cancer

Further significant evidence of the efficacy of indoximod as an immunometabolic adjuvant has been documented in advanced prostate cancer. In a randomized study of metastatic castrateresistant disease (NCT01560923), 46 patients treated with the dendritic cell vaccine sipuleucel-T (Provenge R ) received placebo (n = 24) or indoximod (n = 22) with the latter cohort displaying a >2-fold increase in overall survival (OS) (54). Indoximod was administered for 10 weeks with 3 additional months in cases where an absence of radiographic or clinical progression was documented. Immune monitoring of patients was the same as performed for the IMPACT study which led to approval of sipuleucel-T (63). Indoximod was well tolerated with no significant difference in adverse events between the two study arms. Median OS had not yet been achieved at the time of report, but median radiographic PFS was 10.3 months in the treatment arm vs. 4.1 months in placebo arm (p = 0.011). Notably, the PFS on the placebo arm was identical to that reported in the pivotal IMPACT study for sipuleucel-T. These positive data align with recent evidence that epithelial-mesenchyme transition (EMT) drives IDO1 expression as part of this key step in metastatic progression of prostate cancer to its deadly castrate-resistant form (64). Overall, the findings of this randomized phase 2 trial with a placebo control arm strongly encourages further study of indoximod as an immunometabolic adjuvant for prostate cancer treatment.

#### Acute Myelogenous Leukemia (AML)

In a Phase 1b trial that includes a randomized Phase 2a component to treat AML, patients with newly diagnosed disease received remission-induction chemotherapy (cytarabine plus idarubicin) plus consolidation chemotherapy (high dose cytarabine), a standard of care regimen, with the addition of indoximod or placebo as maintenance therapy (55) (NCT02835729). The dose escalation was a standard 3+3 design for the phase 1 portion aimed at gauging toxicities in combination with the chemotherapy regimen [400, 600, 1,000, 1,200 mg indoximod]. A different schedule was used in this trial, with indoximod provided every 8 h starting on day 8 of induction

therapy, avoiding administration on days that patients received consolidation chemotherapy, and then stopping it 4 weeks prior to hematopoietic stem cell allo-transplanation. At the time of the report, the evidence presented indicated that indoximod did not add significant toxicity to standard of care treatment, and early response data suggested a high occurrence of minimal residual disease after one cycle of induction chemotherapy.

# Brain Cancer

Phase 1b/2 single-arm trials in adult and pediatric brain cancers are being conducted in which indoximod is combined with chemotherapy or chemo-radiotherapy, with some early but intriguing efficacy data being reported. A preclinical treatment rationale was established in a robust orthotopic model of malignant brain cancer (glioblastoma), where the synergistic effects of indoximod were demonstrated in combination with temozolomide (TMZ) and radiation as a cooperative DNA damaging modality (65). In the latest report from the adult trial (NCT02052648) (56), 12 patients who had progressed on standard of care therapy with TMZ were enrolled in a traditional 3+3 dose escalation study of indoximod (600, 1,000, or 1,200 mg twice daily). No dose-limiting toxicity was encountered nor did indoximod cause a delay or reduction in TMZ dosing in any patient. The best responses documented were 1 patient with partial response per Response Assessment in Neuro-Oncology (RANO) criteria at 15 months and 4 patients with stable disease lasting between 4 and 11 months (66). A phase 2 expansion of the study is ongoing at the 1,200 mg twice daily dose in combination with TMZ, bevacizumab and ateriotactic radiation (SRS) (NCT02052648).

In the pediatric brain cancer trial (NCT02502708) (57), the first trial to evaluate indoximod both in children and in the context of radiotherapy, 17 patients from an original cohort of 29 heavily pretreated patients in the dose escalation phase 1b study who were eligible to receive further treatment were administered indoximod and radiotherapy followed by standard of care cycles of TMZ with indoximod as maintenance therapy. The other 12 patients received only indoximod and TMZ. Both treatments were well tolerated with minimal toxicity attributed to indoximod. Overall, at the time of the report, 29 patients in the dose-escalation phase of the study exhibited a median PFS of 6.2 months and median time to regimen failure (TTRF) of 11.7 months, which compares favorably with historical controls. Notably, patients receiving radiotherapy appeared to benefit significantly when indoximod was added, with a median TTRF of 12 months observed vs. 3.2 months without radiotherapy (p = 0.04). These data suggested a dose-sparing effect of indoximod on conventional chemo-radiotherapy, potentially extending efficacious responses. The notion that targeting the IDO pathway may improve chemo-radiotherapy is supported a recent study in lung cancer (67). Encouraged by these response data, the same regimen is now being tested in patients with diffuse intrinsic pontine glioma (DIPG), a dismal disease with no effective treatment option. Thus far, 3/6 patients enrolled are reported to have achieved good symptomatic and radiographic response.

# Pancreatic Ductal Adenocarcinoma (PDAC) and Breast Cancer (BRCA)

In contrast to the trials above, two phase 2 studies of >100 patients in pancreatic or breast cancer have shown little to no evidence of efficacy. In a single-arm study of metastatic PDAC (NCT02077881), 104 of 135 patients enrolled to receive a standard of care regimen of gemcidabine or nab-paclitaxel plus indoximod were judged evaluable for efficacy by a prespecified definition (58). Patients were enrolled with treatmentnaïve disease or first line therapy following earlier resection and adjuvant therapy. Treatment was administered until disease progression or toxicity occurred. Median OS was 10.9 months with an ORR of 46.2%. Notably, responding patients exhibited an increased density of intratumoral CD8+ T cells. This study did not meet its pre-specified goal of a hazard ratio (HR) = 0.70, but the increased ORR that was observed correlated with a positive immunological response. In contrast, a study of metastatic BRCA patients failed to produce any evidence of efficacy. In this study of 169 newly diagnosed patients treated with taxotere and indoximod (NCT01191216), no statistically significant difference in PFS, OS, or ORR was observed. While these two types of aggressive cancer set a high bar for improvements in efficacy, the selection of subjects who were not heavily pre-treated opened a window of opportunity for indoximod. Taken together, clinical findings clearly encourage further study of indoximod as an immunometabolic adjuvant for immunotherapy in treatment of melanoma and prostate cancer, and possibly for DNA damaging modalities in treatment brain cancer and AML, a diverse set of diseases and combinations that illustrate the potentially broad uses indoximod may realize in the clinical setting.

# MECHANISMS OF ACTION OF INDOXIMOD Relieving Suppression of mTORC1 Activity in T Cells Due To Tryptophan Starvation

The molecular mechanisms of action of indoximod as an inhibitor of the IDO pathway are a subject of continued study. However, only one mechanism of action has been described that is consistent with pharmacokinetic analyses of the blood serum levels of indoximod that are actually achieved in human subjects (68). Specifically, in cells subjected to IDO/TDO-mediated tryptophan depletion, indoximod has been shown to relieve suppression of the master metabolic kinase mTORC1 that occurs in tryptophan-depleted cells, with an IC50 (∼70 nM) that is more potent than L-tryptophan itself (68). mTORC1 controls protein synthesis, coordinating nutrient levels to different cellular physiological responses of autophagy vs. growth. In T cells, mTORC1 is pivotal in determining autophagy/tolerance vs. growth/activation. mTORC1 is downregulated by depletion of essential amino acids like tryptophan, to which it responds by activating autophagy as an attempt to access tryptophan from intracellular stores. Accordingly, depletion of tryptophan by IDO/TDO activation downregulates mTORC1 and promotes autophagy which indoximod reverses as a tryptophan mimetic (**Figure 2**). Although the precise connections between IDO/TDO-mediated downregulation of mTORC1 in T cells are not well understood, there is evidence of an intermediate role for the amino acid sensing kinase GLK1 which acts upstream to regulate not only mTORC1 but also PKC-θ, a T cell receptor regulatory kinase (69). Thus, GLK1 may be a linchpin between tryptophan catabolism by IDO/TDO enzymes and mTORC1 downregulation in T cells (7).

By restoring mTORC1 activity, indoximod acts to reverse mTORC1-activated autophagy triggered by tryptophan depletion (68). Since indoximod is a D-tryptophan analog, it cannot support protein translation, but nevertheless it is interpreted by the mTORC1 kinase as a high-potency L-tryptophan mimetic. Why this is the case is unclear, but a mammalian capability to recognize (if not use) D-amino acids might reflect immune crosstalk with the microbiome given their use in bacteria (70). In any case, mTORC1 has a critical role in human Teff cell activity and indoximod acts directly in human T cells where it exerts a direct effect, unlike IDO1 enzyme inhibitors (71).

There are at least three implications of this mechanism of action. First, by targeting a downstream effector molecule, indoximod differs from IDO1 enzyme inhibitors in being agnostic to the IDO/TDO enzyme(s) contributing to cancer pathogenesis. Thus, indoximod is rationalized to treat tumor cells overexpressing IDO1, IDO2 or TDO (or any combination thereof), which is not the case for an enzyme-selective inhibitor. This is a useful feature in heterogenous plastic tumors which represent the norm in advanced cancer patients. Second, by targeting a convergent effector mechanism used by all IDO/TDO enzymes, indoximod may prove less sensitive to inherent or acquired resistance that may arise in patients due to IDO1 mutation, IDO1 overexpression or other target bypass mechanisms that heterogeneous cancers evolve. On this point, preclinical genetic studies illustrate clearly how tumoral bypass of an IDO1-specific blockade is associated with IDO1-independent elevation of regional kynurenine levels (21), suggesting the availability of resistance pathways via TDO2 or IDO2 activation. The critical question of inherent and acquired resistance to IDO1 selective blockade is discussed in greater depth in a separate review of the failed ECHO-301 phase 3 clinical trial in melanoma patients of pembrolizumab with epacadastat, a direct IDO1 enzyme inhibitor that added no benefit to the immune checkpoint therapy under the conditions of study (72). Lastly, mTORC1 is implicated in tumor cell growth and proliferation as well as in T cell activation. Thus, if indoximod also provokes mTORC1 activation in tumor cells, the drug may also empower tumor cell killing when combined with chemotherapeutic drugs, which generally exhibit greater cytotoxicity against growing cells.

Overall, the evidence that indoximod may broaden the efficacy of pembrolizumab (53) suggests that restoring mTORC1 in effector T cells might be sufficient to improve therapeutic responses with reduced risks of resistance due to IDO1 bypass. On this point, it is known that mTORC1 drives expression of ICOS, a positive-acting T cell co-regulatory receptor, and that elevated expression of ICOS in melanoma patients receiving immune checkpoint therapy correlates with the most favorable outcomes (73). In efforts to further leverage its features as an IDO/TDO effector pathway inhibitor, novel salts of indoximod and a pro-drug form of the drug (NLG-802) with superior pharmacokinetic properties have recently been described which have entered clinical testing (71).

#### Other Mechanisms of Action

Indoximod clearly has complex immunomodulatory properties, as illustrated, for example, by its ability to act on B cells to relieve inflammation in a murine model of autoimmune rheumatoid arthritis (74, 75). Thus, other mechanisms of action that have been described for indoximod are likely to illuminate its therapeutic properties.

#### Indirect Blockade of IDO2 Which Is Implicated in IDO1-Mediated Treg Activation

The catalytic activity of IDO2 has been shown to be inhibited indirectly by indoximod in human kidney cells where the IDO2 gene is expressed normally (76). There is conflicting data in dendritic cells, which express IDO2 as well as IDO1, on the ability of indoximod in this setting to block T cell suppression (42, 77, 78). However, mouse genetic studies support a link between indoximod action and IDO2 function, for example, in demonstrating that the therapeutic benefits of indoximod administration in a model of rheumatoid arthritis that relies on the presence of the Ido2 gene (74), which interacts genetically with IDO1 in IDO1-mediated activation of Treg cells in the mouse (28). Here we note that the ability of indoximod to limit rheumatoid arthritis is highly relevant to combination treatments with immune checkpoint antibodies, which often cause autoimmune side-effect in patients. In this sense, indoximod co-administration with immune checkpoint antibodies may widen the therapeutic window at both ends, by extending efficacy and reducing side-effects, unlike IDO1 selective enzyme inhibitors.

#### AHR Modulation

At high concentrations in cell culture (1 mM), evidence has been presented that D-1MT/indoximod can elevate transcription of IDO1 leading to increased production of kynurenine in cancer cells (79), but the concentrations used in this study, which exceed by ∼100-fold the serum levels of indoximod achieved in patients in clinical trials (50), cast doubt on the physiological relevance of this observation. However, a very recent report offers additional support for the related idea that indoximod may somehow affect IDO1 expression in cell-specific ways via AHR (80), a transcription factor that binds and is activated by kynurenine (81) as a convergent effector pathway downstream of all IDO/TDO enzymes (7). Indeed, other evidence has been presented for an autocrine feedback pathway involving IDO1, AHR, and IL-6 that controls IDO1 expression in cancer cells (82).

The AHR connection for indoximod is complex. There are binding sites for AHR in the IDO1 gene and other genes that influence the differentiation of dendritic cells, T helper cells and Tregs and the proliferation of Teffs and Tregs where AHR has influence (83). In a recent study reported at the 2018 AACR conference (80), indoximod was reported to modulate AHRdependent transcriptional activity in human liver and primary T cells, in the latter case altering the transcription of genes

associated with T helper and Treg phenotypes. These effects were reversed by an AHR inhibitor, suggesting that indoximod acts upstream of AHR (80). In plasmacytoid dendritic cells in vitro and in vivo (in tumor-draining lymph nodes), indoximod was found to downregulate IDO1 expression and function, decrease kynurenine production and increase T cell proliferation, while promoting a phenotypic shift in T cells from Treg to Th17 producing T helper cells (80). Thus, in addition to resuscitating Teff cells in tumors, indoximod may also act in draining lymph nodes to reprogram the AHR effector pathway to shift Tregs to Th17 cells.

and activity; and an influence on gut microbial physiology influencing systemic immunity (see the text).

#### Perspectives of Indoximod on IDO/TDO/AHR Signaling to the Gut Microbiome

Immune homeostasis involves a dynamic balance between tolerance of commensals and suitable immune responses to eradicate or otherwise control pathogens (84, 85). Tolerance is important to avoid tissue injury but at the potential costs of chronic infections and inflammation which in the long term become factors in metabolic diseases, autoimmunity, and, in certain settings, cancer (85). Regarding indoximod mechanisms this is an important area to survey given evidence that the therapeutic impact of anti-PD1 therapy is determined by microbiome character, in both preclinical models (86, 87) and clinical settings (88–90).

Cross-regulatory circuitry between IDO1 and AHR is a key factor in mediating disease tolerance (91). For example, exposure to bacterial lipopolysaccharide will program a state of refractoriness to further LPS challenge (endotoxin tolerance), a phenomenon reflecting the engagment of AHR in longterm control of systemic inflammation only when IDO1 is active, which responds late upon initial stimulation but earlier upon subsequent challenge. Mechanistic studies have revealed a feedback control cycle, with SRC kinase as an intermediate between kynurenine-activated AHR and IDO1 expression in regulating tolerance to bacterial endotoxins, a state that protects against immunopathology in Gram-negative and Gram-positive infections. In this fundamental way, IDO1 and especially AHR contribute to immunologic host fitness (91).

IDO1 and AHR are highly expressed in the small and large intestine (92). IDO1 expression increases further during aging, a key factor in the likelihood of a positive therapeutic response to anti-PD1 treatment (93). In the intestine of adult germfree mice, IDO1 levels are reduced suggesting that commensal microorganisms mediate the age-dependent increase in IDO1. Supporting the likelihood that it modulates mucosal immunity to intestinal microbiota, IDO1-deficient mice exhibit resistance to enteric pathogens, for example, to Citrobacter rodentium (94). Tryptophan catabolites produced by microbiota such as gut Lactobacillus can also act as AHR ligands, confounding a clear interpretation of the link between IDO1 and cancer that may involve microbiota-mediated tryptophan catabolism (85).

In melanoma studies of anti-PD-1/PD-L1 it appears that gut commensals of Bifidobacteria can enhance therapeutic efficacy (86, 88–90). Given evidence that indoximod can heighten the benefit of anti-PD1 therapy, it will be important to evaluate Bifidobacteria as a potential mediator in this effects, which raises the possibility of conceptualizing indoximod as a prebiotic substance. In one clue that this may be the case, indoximod was able to reverse the effects of IDO1 activity in models of colitis that are quieted by Bifidobacteria (95). While still in their infancy, studies of the effects on indoximod and the IDO/TDO/AHR pathways on gut microbial physiology and cancer immunity is a rich area for exploration.

### POTENTIAL BENEFITS OF INDOXIMOD TREATMENT TO QUALITY OF LIFE IN CANCER PATIENTS

Recent studies suggest that indoximod may exert a variety of benefits as an immunometabolic adjuvant on the quality of life of cancer patients and survivors. The conditions that are improved are not critical to overall survival, but are of major importance to affected individuals and their oncologists and caregivers. As noted above, one interesting feature of indoximod is its ability to limit autoimmune arthritis in preclinical models, possibly by limiting IDO2 function implicated in this condition (96). Autoimmune joint inflammation is a common short and long term side effect of immune checkpoint therapy in cancer patients which indoximod may limit. This potential may be confirmed through long-term follow up of melanoma patients receiving combinations of indoximod and pembrolizumab in the phase 2 trial discussed above. Other beneficial effects of indoximod that have been described are behavioral, as evaluated in preclinical models of depression, anhedonia, anxiety or pain (97–100), one or more of which occur commonly in cancer patients and survivors. Given its relative safety in trials to date, it may be possible to consider uses in these settings, not only during cancer therapy but as a palliative adjunctive therapy. In summary, indoximod is a unique immunometabolic adjuvant with a wide potential range of uses to improve cancer therapy in adults and

#### REFERENCES

children, not only safely but with possible collateral benefits to quality of life.

#### AUTHOR CONTRIBUTIONS

All authors contributed to composing the text. GP composed the figures. YZ, PG, AM, and GP made final edits to the text and figures.

#### ACKNOWLEDGMENTS

We thank our many colleagues and collaborators and apologize for omitting citations to many primary research reports cited through reviews due to space limitations. GP and AM acknowledge grant support from the NCI (R01 CA191119) and The W.W. Smith Trust, with additional support from the Lankenau Medical Center Foundation and the Main Line Health System. GP holds The Havens Chair for Biomedical Research at the Lankenau Institute for Medical Research.

inhibitor indoximod plus ipilimumab for the treatment of unresectable stage 3 or 4 melanoma. In: European Cancer Congress 2015 (18th ECCO/40th ESMO), Vienna. abstract #514 (2015).

2, 3-dioxygenase expression. Microbiol Immunol. (2018) 62:71–9. doi: 10.1111/1348-0421.12562

**Conflict of Interest Statement:** GP and AM disclose equity ownership in NewLink Genetics reflecting inventorship of licensed IDO intellectual property including indoximod and its uses in cancer treatment from the Lankenau Institute of Medical Research, as described in U.S. Patents Nos. 7705022, 7714139, 8008281, 8058416, 8383613, 8389568, 8436151, 8476454, and 8586636. GP additionally discloses equity ownership in Incyte and Merck; former and present advisory board roles for NewLink Genetics and Kyn Therapeutics, respectively; and a board director role for Meditope Biosciences. AM additionally discloses roles as an advisory board member and grant recipient for I-O Biotech AG, which is developing IDO vaccines for cancer treatment. YZ discloses research and travel support from NewLink Genetics and advisory board roles for Amgen, Roche Diagnostics, Novartis, Eisai, Castle Bioscience and Exelixis.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Fox, Oliver, Rowe, Thomas, Zakharia, Gilman, Muller and Prendergast. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Transforming Growth Factor-β-Induced Cell Plasticity in Liver Fibrosis and Hepatocarcinogenesis

Isabel Fabregat 1,2,3 \* and Daniel Caballero-Díaz 1,3 \*

1 TGF-β and Cancer Group, Oncobell Program, Bellvitge Biomedical Research Institute, Barcelona, Spain, 2 Department of Physiological Sciences, School of Medicine, University of Barcelona, Barcelona, Spain, 3 Oncology Program, CIBEREHD, National Biomedical Research Institute on Liver and Gastrointestinal Diseases, Instituto de Salud Carlos III, Barcelona, Spain

The Transforming Growth Factor-beta (TGF-β) family plays relevant roles in the regulation of different cellular processes that are essential for tissue and organ homeostasis. In the case of the liver, TGF-β signaling participates in different stages of disease progression, from initial liver injury toward fibrosis, cirrhosis and cancer. When a chronic injury takes place, mobilization of lymphocytes and other inflammatory cells occur, thus setting the stage for persistence of an inflammatory response. Macrophages produce profibrotic mediators, among them, TGF-β, which is responsible for activation -transdifferentiationof quiescent hepatic stellate cells (HSC) to a myofibroblast (MFB) phenotype. MFBs are the principal source of extracellular matrix protein (ECM) accumulation and prominent mediators of fibrogenesis. TGF-β also mediates an epithelial-mesenchymal transition (EMT) process in hepatocytes that may contribute, directly or indirectly, to increase the MFB population. In hepatocarcinogenesis, TGF-β plays a dual role, behaving as a suppressor factor at early stages, but contributing to later tumor progression, once cells escape from its cytostatic effects. As part of its potential pro-tumorigenic actions, TGF-β induces EMT in liver tumor cells, which increases its pro-migratory and invasive potential. In parallel, TGF-β also induces changes in tumor cell plasticity, conferring properties of a migratory tumor initiating cell (TIC). The main aim of this review is to shed light about the pleiotropic actions of TGF-β that explain its effects on the different liver cell populations. The cross-talk with other signaling pathways that contribute to TGF-β effects, in particular the Epidermal Growth Factor Receptor (EGFR), will be presented. Finally, we will discuss the rationale for targeting the TGF-β pathway in liver pathologies.

Keywords: TGF-β, plasticity, liver, cancer biology, fibrosis, HCC, EMT, hepatic stellate cell

# INTRODUCTION

The liver shows an unique regenerative response to injuries produced by physical or toxic treatments, which induce tissue damage (1–3). Liver injuries can be classify depending on their persistence or duration and can develop acute and chronic liver diseases. Acute liver injuries can be completely restored, without any evidence of the injury, only withdrawing the damaging agent in a short period of time. In these cases, the liver architecture and function remain stable. However,

#### Edited by:

Ubaldo Emilio Martinez-Outschoorn, Thomas Jefferson University, United States

#### Reviewed by:

Przemyslaw Blyszczuk, Universität Zürich, Switzerland Anna Laurenzana, Università degli Studi di Firenze, Italy

#### \*Correspondence:

Isabel Fabregat<EMAIL_ADDRESS>Daniel Caballero-Díaz<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology

Received: 22 June 2018 Accepted: 13 August 2018 Published: 10 September 2018

#### Citation:

Fabregat I and Caballero-Díaz D (2018) Transforming Growth Factor-β-Induced Cell Plasticity in Liver Fibrosis and Hepatocarcinogenesis. Front. Oncol. 8:357. doi: 10.3389/fonc.2018.00357

**95**

long-time exposure with the damaging agent generates progressive liver damage, parenchyma alterations and vascular architectural distortion, which eventually results in liver fibrosis, cirrhosis, and ultimately, hepatocellular carcinoma (HCC), which is the end-stage of most chronic liver diseases (4, 5).

Chronic liver diseases are characterized by a parenchyma damage with a continued wound healing response, tissue remodeling, inflammatory environment and an altered molecular signaling pathways. Strong evidences point out the relevant role of the Transforming Growth Factor beta (TGF-β) signaling during all phases of the development of liver fibrosis and hepatocarcinogenesis. Perturbation of signaling by TGF-β family members is often seen in different diseases, including malignancies, inflammatory and fibrotic conditions (6). Under physiological conditions, TGF-β has a cytostatic and proapoptotic role in adult hepatocytes, which is critical for the control of liver mass. Loss of these functions may result in hyperproliferative disorders and cancer (7–9). Indeed, in earlystage carcinomas, TGF-β exerts tumor-suppressing activities, inducing cell cycle arrest and apoptosis. However, in latestage carcinomas, once cells acquire resistance to its suppressive effects, TGF-β actions switch to pro-oncogenic, conferring cell survival, inducing cell migration and invasion, mediating immune alterations and microenvironment modifications (10, 11).

Recent evidences suggest that many of the pathological TGF-β effects could be related with its capacity to regulate cell plasticity, contributing to modifications in the phenotype of different liver cell populations. Cell plasticity refers to the interconversion of different stem cell pools, activation of facultative stem cells, and dedifferentiation, transdifferentiation or phenotypic transition of differentiated cells within a tissue (12) and is related with the ability of cells to reversibly change their phenotype and to take on characteristics of other cell types (13). The most studied and classic event related with cell plasticity is the epithelialmesenchymal transition (EMT) and the opposite mesenchymalepithelial transition (MET) (14). After specific stimuli, the cells suffer genetic and epigenetic changes, as well as cytoskeleton remodeling, which alter their phenotype and functions. TGF-β induces EMT in hepatocytes (15) and it is responsible for activation of hepatic stellate cells (HSC) to myofibroblasts (MFB) (16), both effects contributing to liver fibrosis. Moreover, during hepatocarcinogenesis TGF-β could also mediate an EMT process in liver tumor cells. This review will update recent evidences indicating the relevance of TGF-β signaling pathway in the regulation of the cell plasticity during the progression and pathogenesis of liver chronic diseases, as well as the molecular mechanisms involved. Finally, we will discuss the rationale for targeting the TGF-β pathway in liver pathologies.

#### TGF-β SIGNALING PATHWAYS

In humans, the pleiotropic TGF-β cytokine superfamily includes different members, such as bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3). TGF-β signaling pathways regulates different cellular processes playing essential roles in proliferation, migration, differentiation, or cell death. These processes are essential for the homeostasis of tissues and organs and TGF-β signaling deregulation contributes to human disease. TGF-β1 (TGF-β from now on) has essential roles in liver physiology and pathology and contribute to all stages of disease progression: from liver injury through inflammation, fibrosis, cirrhosis and HCC (7, 8).

Most of the functions of the cells involved in the fibrotic tissue and in the tumor microenvironment are under the control of TGF-β: promotes MFB differentiation, the recruitment of immune cells, affects epithelial and endothelial cell differentiation and inhibits the anti-tumor immune responses (17, 18). Besides TGF-β responses could be different depending on the cell type, its receptors are expressed on most of the cells and its signaling pathway is very similar in all of them (6). All TGF-β isoforms are synthesized within the cell as pro-peptide precursors containing a pro-domain, named Latency-Associated peptide (LAP), and the mature domain. This latent form is secreted to the extracellular matrix (ECM) and stored as a fast and available pool of TGF-β, without a novo synthesis (19). By different mechanisms, TGFβ is cleaved and the bioactive form signals via binding to its specific kinase receptor at the cell surface of target cells. Stored TGF-β could be activated by the cell contractile force, which is transmitted by integrins (20, 21). Specific integrins and matrix protein interactions could be required for activation of the latent form of TGF-β. Integrins αv are the major regulators of the local activation of latent TGF-β and in this activation it is required the RGD (Arg-Gly-Asp) sequence (21). Integrin αv deletion in HSC protected mice from CCl4-induced hepatic fibrosis (22). A recent review summarized the crosstalk between TGF-β and tissue extracellular matrix components (23).

TGF-β binds to its receptors triggering the formation of a heterotetrameric complex of type I and type II serine/threonine kinase receptors, in which the constitutively active type II receptor phosphorylates and activates the type I receptor. There are several types of both type I and type II receptors, but TGF-β preferentially signals through activin receptor-like kinase 5 (ALK5) type I receptor (TβRI) and the TGF-β type II receptor (TβRII). In addition, endoglin and betaglican (TβRIII), also called accessory receptors, bind TGF-β with low affinity and present it to the TβRI and TβRII. Activated receptor complexes mediate canonical TGF-β signaling through phosphorylation of the Receptor Associated SMADs (R-SMADs) at their carboxyterminal. Humans express eight SMAD proteins that can be classified into three groups: R-SMADs, Cooperating SMADs (Co-SMADs) and Inhibitory SMADs (I-SMADs: SMAD6 and SMAD7). Among the R-SMADs, SMAD2 and 3 mediate the TGF-β1 branch of signaling (6, 8). After phosphorylation, R-SMADs form a trimeric complex with SMAD4, which translocates to the nucleus and associates with other transcription factors in order to regulate gene expression (7, 8). In addition to the canonical SMAD pathway, TGF-β is able to use non-SMAD effectors to mediate some of its biological responses, including non-receptor tyrosine kinases proteins such as Src and FAK, mediators of cell survival (e.g., NF-kB, PI3K/Akt pathways), MAPK (ERK1/2, p38 MAPK, and JNK among others), and Rho GTPases like Ras, RhoA, Cdc42, and Rac1. Interestingly, these pathways can also regulate the canonical SMAD pathway and are involved in TGF-β-mediated biological responses (**Figure 1**) (8, 24–26).

# LIVER FIBROSIS

Liver fibrosis is a common pathological chronic liver disease, consequence of a continued injury with a huge accumulation of extracellular matrix proteins, mainly enriched in fibrillar collagens, due to a multiple reparative and regenerative processes (5, 27, 28). After liver damage, reparative mechanisms are triggered to replace necrotic and apoptotic hepatocytes, generating wound healing and inflammatory responses that are essential for liver regeneration (5). However, if the damage persists over a long time, the excessive accumulation of extracellular matrix proteins (collagens I, II, and III, undulin, fibronectin, laminin, elastin, proteoglycans and hyaluronan) could replace parenchymal areas leading fibrosis to a cirrhotic state. In advanced stages, it develops an abnormal liver architecture, altered vascularization and fibrotic septa surroundings with regenerative nodules. Liver systemic failure, portal hypertension, high susceptibility to infection and high risk to develop HCC are the main clinical consequences of cirrhosis (28, 29). Interestingly, multiple clinical reports have reported that liver insult eradication can regret liver fibrosis in huge number of patients, mostly during the first stages (29–32). In the development of liver fibrosis, TGF-β plays crucial roles regulating the different stages of the disease, among them, the control of cell plasticity of different liver cell populations, which is summarize in the **Figure 2** and we detail in the next chapters.

# TGF-β REGULATES MACROPHAGE PLASTICITY DURING LIVER FIBROSIS

Inflammation plays a key role in liver fibrosis development. After injury takes place, infiltration of immune system cells -macrophages, lymphocytes, eosinophils, and plasma cellsarises to the damaged place. Lymphocytes produce cytokines

FIGURE 2 | Role of TGF-β in the cell plasticity of hepatic stellate cells and macrophages during liver fibrosis. Different routes followed by TGF-β signals to mediate activation of HSC into MFB (left) or polarization of macrophages to a M2 state (right), which contribute to sustain a fibrotic and immunosuppressive environment, favorable to the initiation of a hepatocarcinogenic process.

and chemokines, which activate macrophages. Activated macrophages stimulate inflammatory cells such as lymphocyte, among others, over-activating and maintaining the inflammatory environment (33). During fibrosis, macrophages produce profibrotic factors such as TGF-β and platelet derived growth factor (PDGF), control ECM turnover by regulating the balance of various matrix metalloproteases and tissue inhibitors of metalloproteinases (TIMPs) (27, 34, 35) and are found very close to collagen-producing MFB (36–38) suggesting the macrophages relevance in the activation of MFB. In this sense, hepatic macrophages have been described as a potential targets against fibrosis (39, 40).

Macrophages represent a heterogeneous cell population with a huge cell plasticity, where diverse microenvironment stimuli polarize them into different phenotypes (41). There are mainly two sources of hepatic macrophages: liver resident macrophages, also called Kupffer cells (42), and circulating monocytes (inflammatory recruited macrophages) (43). Besides the origin, both could play significant roles in the development of fibrosis. In vitro and in vivo studies described that both Kupffer cells and monocyte-derived macrophages can activate HSC and induce their transdifferentiation by paracrine mechanisms, including TGF-β (44–47). Resident hepatic macrophages secrete the chemokine CCL2 (a potent chemoattractant) in order to recruit monocytes which could increase and promote fibrosis. Although, it was described that the pro-fibrotic functions of these resident macrophages remain functional even when recruited macrophages are pharmacologically inhibited using CCL2 antagonists (48). Transgenic rats that express a mutated form of the CCL2 (acting as a negative-mutant), and tail vein injection of adenovirus that overexpress a truncated form of TGF-β receptor II (acting as a negative-receptor mutant) attenuate liver fibrosis in a DEN-induced fibrosis model in rats (49), suggesting the relevance of inflammation and TGF-β pathway during this disease.

In the early stages, activated macrophages secrete proinflammatory cytokines and produce reactive oxygen species (ROS), while in late stages macrophages have been associated with release of anti-inflammatory factors, attenuating inflammation and promoting tissue regeneration (43, 50). Macrophages are classify into M1, also known as classical or pro-inflammatory; and M2, also known as alternative or anti-inflammatory macrophages (51, 52). It is not easy to strictly separate both liver macrophage populations, since they could show common gene expression, and even more M2 macrophages are classify also in different subclasses. For that reason, it has been proposed that could be more adequate to separate them according to their functions: defensive, restorative and regulatory macrophages (53). In the classical classification, M1 macrophages are associated with inflammatory diseases due to microbicidal activity (through their capacity to produce ROS and their phagocytic functions), antigen presentation and antitumor activity. M1 macrophages prevail during the onset of injury (54) and are related with the release of metalloproteinases that degrade ECM and promote EMT/Endothelial-to-mesenchymal transition (EndMT). On the other hand, M2 macrophages secrete anti-inflammatory factors such as IL-10, arginase, TGF-β, and HO-1. Their polarization is promoted by IL-4 and IL-13, and are characterized for the expression of Arg1, Ym1, and Fizz, secretion of angiogenic factors such as IL-8, VEGF, and EGF4 and increased mannose receptor (CD206), with lower ROS production (47, 50). M2 macrophages stimulate an anti-inflammatory environment and promote regeneration and wound healing. However, if injury becomes chronic, M2 macrophages take up a pro-fibrotic role secreting pro-fibrotic factors such as TGF-β, PDGF, among others (47).

Nowadays it is clear that macrophages are essential players in the regulation of liver fibrosis and they are an important source of TGF-β but, could this cytokine regulate the phenotype between M1 and M2 macrophages and their functions? Recent data described that TGF-β could induce M2-like macrophage polarization via SNAIL (55). SNAIL-overexpression in human THP-1 macrophages promotes M2 markers (such as CD206), induces the expression of the anti-inflammatory IL-10 and inhibits pro-inflammatory M1-related cytokines (TNF-α and IL-12). By contrast, SNAIL knockdown by siRNA technology abolishes TGF-β-M2-induced phenotype and partly restores M1 polarization through up-regulation of pro-inflammatory cytokines. The canonical SMAD2/3 and the non-canonical PI3K/AKT signaling pathways are crucial for TGF-β-induced SNAIL overexpression in THP-1 cells. The blockade of TGFβ/SNAIL signaling restores the production of pro-inflammatory cytokines. Likewise, TGF-β also stimulates murine BMDM macrophages to display an M2-like phenotype characterized by high levels of IL-10 and low levels of IL-12p70, and M1 specific markers. Macrophages isolated from fibrotic mouse livers show higher balance of M2/M1 macrophages in comparison to control mice (56). In other fibrotic animal models, such as lung fibrosis, TGF-β could modulate M2 responses (57); and in kidney, TGF-β/Smad3-dependt pathway could transdifferentiate M2-macrophages to myofibroblast favoring kidney fibrosis (58). Moreover, TβRII–/– mice show a defective polarization to M2-macrophages (59). Fibrosis-induced model in rats by thioacetamide show that both M1 and M2-macrophagues polarizations occur during development of the disease (60). Overall results show up that M2-activation/polarization has a relevant role in the development of fibrosis in mice and patients with liver fibrosis (61). However, due to the heterogenicity and higher plasticity of macrophages and the complexity of their study in vivo models in liver, further works are needed in order to clarify the molecular mechanisms whereby TGF-β pathway promote the polarization and the pro-fibrotic functions of macrophages in vivo models. Current data seems to indicate that both hepatic and recruited macrophages play relevant roles in the progression and reversion of liver fibrosis. Targeting both and the reorientation of their phenotypes are arising as attractive therapies (62).

## TGF-β REGULATES LIVER EPITHELIAL CELLS PLASTICITY DURING LIVER FIBROSIS

"Activated" fibroblast or MFB are the main producing cells of fibrogenesis mediators and ECM components, participating actively in their accumulation (63). In the liver, the most fibrogenic MFB are endogenous and their origin is controversial and still unclear, but nowadays there are accepted different sources (63–65), among them, portal and resident fibroblasts (66), activation and differentiation of HSC (more details in the next section) (16, 67), bone marrow-derived fibrocytes (68), liver epithelial cells (hepatocytes and cholangiocytes) that undergo EMT (69–71), endothelial cells that undergo EndMT (66, 72), vascular smooth muscle cells and pericytes (73).

EMT-clear example of cellular plasticity- is a process that drives a de-differentiation of epithelial cells to a mesenchymallike phenotype increasing their migratory and invasive properties (13, 14, 74, 75). The reverse process is called as MET and allows cells to differentiate into different organs and tissues. In a tumorigenic context, mesenchymal migratory tumor cells undergo MET to metastasize (76). The EMT process includes loss of epithelial genes, such as E-cadherin and cytokeratins (8, 18 and 19), and up-regulation of mesenchymal genes, such as N-cadherin, alfa-Smooth Muscle Actin (α-SMA, ACTA2 gene) which correlate with the expression of EMT transcription factors (EMT-TFs) Snail (SNA1 gene), Slug (SNA2 gene), Twist and ZEB (74, 75). Intermediates states are also found between EMT and MET (77). Partial EMT is described for cells that co-express both epithelial and mesenchymal markers. Even more, EMT is classified into different subclasses related with the biological context (78): Type 1 EMT is involved in development stages; type 2 EMT concerns regenerative process and organ fibrosis; and type 3 EMT is related with metastatic process.

During liver fibrosis, type 2 EMT plays a relevant role in the appearance of a pro-fibrotic fibroblast phenotype. Bipotent adult hepatic progenitor cells, which possess the cell plasticity to differentiate into hepatocytes and cholangiocytes after different stimuli (79), are able to undergo EMT in response to liver injury during cholestatic liver fibrosis. Hepatocyte plasticity could play relevant roles during the progression of chronic liver diseases. Mouse hepatocytes that survive to the apoptotic effects of TGF-β, could regulate -in a TGF-β dependent manner- the expression of fibrosis-related genes, such as Connective Tissue Growth Factor (CTGF) or fibronectin, and EMT-related genes, such as Snail, and β-catenin (15, 71, 80), with downregulation of epithelial markers (81). Even more, primary adult hepatocytes could transdifferentiate to a more fibroblastic-like phenotype with loss of cell–cell contacts and polarity, after TGF-β treatment (80). Indeed, hepatocyte-derived fibroblasts are an additional and significant lineage of mesenchymal cells that contribute to progression of liver fibrosis. Zeisberg and collaborators elegantly demonstrate that adult hepatocytes can undergo an EMT process after TGF-β stimuli, contributing to the in vivo pool of fibroblast during liver fibrosis (82). The role of hepatocytes during liver fibrosis in vivo related with TGF-β was also previously described in a transgenic animal model which overexpress SMAD7 (inhibitor of the pathway) specifically in hepatocytes. These transgenic animals have attenuated the TGF-β signaling and EMT, with less ECM depositions and improved CCl4-dependent liver damage and fibrosis (83). Bone morphogenetic protein-7 (BMP-7), a member of the TGF-β family which plays opposite roles to TGF-β, induces MET. Primary rat hepatocytes treated with TGF-β upregulate the expression levels of fibrotic markers, whereas BMP-7 treatment upregulated E-cadherin and decreases SMAD2/3 phosphorylation levels. Even more, in CCl4-treated rats treated with TGF-β, which show advanced fibrosis with higher expression of α-SMA and lower E-cadherin, the fibrotic situation was rescued after BMP-7 treatment (84). Moreover, cholangiocytes -another epithelial cell population- activate, proliferate and change into a more fibroblastic phenotype, increasing the expression of pro-fibrotic cytokines and factors such as TGF-β, PDGF and CTGF (85). These results open new ideas about how epithelial liver cells, through an EMT process, could generate mesenchymal/fibroblastic cells, which could be relevant in the progression of the fibrotic diseases.

## RELEVANCE OF TGF-β AND HSC DIFFERENTIATION DURING LIVER FIBROGENESIS

In the normal liver, HSC (around 5–8% of the cells in the liver) are in a quiescent phenotype hosted in the space of Disse between hepatic epithelial and the sinusoidal endothelial cells (86). HSC are characterized by the store of vitamin A, lipid droplets and the expression of a large number of adipogenic genes and neural markers. After liver insults, different paracrine and autocrine signals are triggered promoting the HSC activation -transdifferentiation- from a quiescent state to an activated myofibroblastic phenotype. MFB are characterized by the expression of α-SMA, loss of retinoids and lipid droplets and de novo expression of receptors for mitogenic, fibrogenic and chemotactic factors, leading an increase in proliferation and survival, enhanced synthesis of matrix proteins (predominantly fibrillar collagens) and inhibitors of matrix degradation TIMPs, and secretion of pro-inflammatory cytokines and chemokines. This provokes the progressive scar formation and the development of liver fibrosis (29, 32).

HSC activation is one of the most important steps during liver fibrosis and is mediated by different signals, such as growth factors (PDGF and CTGF, among others), lipidic mediators, as well as ROS and cytokines produced by hepatocytes, cholangiocytes, endothelial cells, macrophages (Kupffer cells) and immune cells (67, 86). Among these cytokines, TGF-β plays a master role in the activation of the HSC to MFB (16). In fact, some of the previous factors stimulate the expression, production and activation of TGF-β, which at the end is responsible for the activation of HSC (87). Furthermore, MFB demonstrate a growth stimulatory effect in response to TGF-β (88), which also contributes to the maintenance of their myofibroblastic phenotype (89). SMAD3 has been identified as the main mediator of the TGF-βinduced fibrogenic transcriptional program, particularly the up-regulation of collagen expression (7, 8, 30, 31). HSC isolated from SMAD3 knock-out mice showed lower expression of Collagen1A1 mRNA mediated by p38 MAPK (30). Interestingly, it has been proposed that TGF-β activates the p38 MAPK pathway, further leading to SMAD3 phosphorylation at the linker region in the cultured MFBs, which promoted heterocomplex formation and nuclear translocation of SMAD3 and SMAD4 (31). These results would indicate that non-canonical activation of the SMAD3/SMAD4 transcriptional activity accounts for SMAD3-dependent extracellular matrix production in MFBs.

During liver fibrogenesis, activated HSC express CTGF, which acts downstream of TGF-β modulating the ECM production. CTGF mRNA expression is under the control of the canonical TGF-β/SMAD3 and non-canonical ERKs, JNK, p38, and STAT3 pathways (90, 91). Moreover, receptor for activated C-kinase 1 (RACK1), a scaffold protein involved in numerous cellular processes and signaling pathways, is another TGF-β downstream target involved in the HSC activation. RACK1 is able to induce pro-fibrogenic pathways in a TGF-β-dependent manner, contributing to differentiation, proliferation, and migration of HSC (92, 93). Indeed, in this migratory phenotype and in remodeling of the cytoskeleton in TGF-β-activated HSC is also involved the role of Rho guanosine triphosphatase (Rho GTPase) signaling (94). TGF-β also regulates the expression of TRPM7 (transient receptor potential melastatin 7) in a SMAD3-depend manner, which inhibition attenuates TGF-β-induced expression of MFB markers (95).

Mild to moderate liver fibrosis may be reversible. The reversion process is related with the elimination of the damaging stimuli. During liver fibrosis reversion, activated HSC (or myofibroblast) reverted to an inactivated phenotype. In this state, inactivated HSC decrease the expression of fibrogenic genes (including COL1A1 and ACTA2) and up-regulate the expression of some quiescence-associated genes like PPARγ (96, 97). Furthermore, inactivating some of the TGF-β downstream signals, such as ROS production, allows the reversion of the myofibroblast phenotype (89).

## ROLE OF ROS DURING HSC ACTIVATION BY TGF-β

Oxidative stress plays a relevant role in the sequence of events following TGF-β activation of HSC. Actually, antioxidants can inhibits HSC transdifferentiation into MFB (98, 99). In both normal physiological and pathological conditions, ROS are critical intermediates. Oxidative stress plays a role during both initial inflammatory phase and its progression to fibrosis (100). Oxidative stress markers have been detected in experimental liver fibrosis/cirrhosis animals and in the biopsy and serum samples from liver cirrhotic patients (101). It is well known that ROS may act upstream and downstream of the TGF-β pathway. Upstream, ROS, through LAP activation and subsequent TGF-β release, promote fibrosis activating latent TGF-β (102) and/or via matrix metalloproteinases activation (103). Indeed, LAP/TGF-β complex has been suggested to function as an oxidative stress sensor (104). Furthermore, in many cell types such as HSC and hepatocytes, ROS may up-regulate the expression and secretion of TGF-β in a positive feedback loop (105, 106). ROS may be generated in the liver by multiple sources, including the cytochrome p450 family members, peroxisomes, mitochondrial respiratory chain, xanthine oxidase, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Worthy to note, accumulating evidence indicates that NADPH oxidases (NOX) mediated ROS have a critical role in HSC activation and hepatic fibrogenesis (101) mediating TGF-β actions.

NOX enzyme family generate ROS, either hydrogen peroxide or superoxide as the primary species, during oxygen catalytic metabolism for arrange of signaling functions and host defense. There are described seven NOX isoforms in mammalian cells (NOX1-5, DUOX1, and DUOX2). Liver cells (either parenchymal and non-parenchymal) express different members of the NOX family. Hepatocytes and HSC express NOX1, NOX2, NOX4, DUOX1, and DUOX2; endothelial cells express mainly NOX1, NOX2, and NOX4; and Kupffer cells express the phagocytic NOX2 (101, 107). NOXs proteins could be playing relevant roles during liver fibrosis development (101, 108). Both NOX1- and NOX2-deficient HSC had decreased ROS generation and failed to upregulate collagen and TGF-β in response to angiotensin II (109). Of relevance, NOXes mediate TGF-β activation of HSC to MFB process (89, 110). In different organs, such as heart, the main mediator for the activation of MFB is NOX4, downstream from TGF-β (111), lung (112) and kidney (113). In in vivo models of liver fibrosis, the levels of NOX4 are up-regulated, as well as in patients with chronic hepatitis C virus derived infection, increasing along the fibrosis degree. HSC respond to TGF-β inducing NOX4-derived ROS (105), which play a key role in hepatic MFB in both in vivo and in vitro (89, 114). In NOX4 knock-out animals and in NOX4 downregulated cells the TGF-β-transactivation of HSC is attenuated (89, 114), and even more, the myofibroblastic state could also be reversible (89). During liver fibrosis, NOX4 is required for both HSC activation and maintenance of the activated phenotype in MFB in a TGF-β-dependent manner and mediates the TGF-β pro-apoptotic response in hepatocytes, which might be relevant to blunt regeneration and create a pro-fibrogenic microenvironment. In this sense, apoptotic hepatocytes after liver injury generate apoptotic bodies which were described to promote HSC survival (115). HSC can engulf and clear apoptotic hepatocytes bodies inducing their activation in JAK1/STAT3 dependent pathway and a NOX-dependent PI3K/Akt/NF-κB induction pathway, concomitant with a production of ECM components (115). Recent evidences show up the dual role of NOX1/NOX4 pharmacological inhibitors in decreasing both the apparition of fibrogenic markers and hepatocyte apoptosis in vivo (114, 116), highlighting the relevance of NOX1 and NOX4 in liver fibrosis and opening new perspectives for its treatment. Actually, NOX1 and NOX4 signaling mediates hepatic fibrosis through activation of HSC (114, 117). Indeed, it was recently described that NOX4, downstream TGF-β, would play a role in the acquisition and maintenance of the MFB phenotype (89). Deficiency of NOX1 and NOX4 attenuates liver fibrosis in mice after CCl4 treatment. Activated HSC and ROS generation are also attenuated in HSC lacking NOX1 and NOX4, suggesting NOX1 and NOX4 play important roles in liver fibrosis and injury through regulating inflammation, proliferation and fibrogenesis in HSC (117). Therefore, targeting NOX1/4 is emerging as a new and attractive therapy for liver fibrosis in order to impair the pathological effects of TGF-β over this disease.

## HUMAN HEPATOCELLULAR CARCINOMA (HCC)

HCC is a major public health problem worldwide with almost 800,000 new cases each year and its incidence is increasing in Europe and worldwide. In 2015 reports from World Health Organization, liver cancer is the second leading cause of cancerrelated deaths, following lung cancer (118). HCC is the most common primary liver malignancy in adults. Intriguingly, there are significant differences on the incidence when considering the gender, being the male to female ratio estimated to be 2.4. This difference is mainly attributed to the different exposition to risk factors, as well as the influence of androgens and oestrogens on HCC progression (119). Exposition to risk factors also determines the incidence of liver cancer regarding age or ethnicity and the highest incidence of HCC is found in Asia and sub-Saharian Africa (120–122). In most cases, HCC develops within an established background of chronic liver disease. Progressive hepatic fibrosis frequently evolves to cirrhosis, which is the largest risk factor for developing liver cancer. Up to 90% of cases of HCC arise in the setting of advanced fibrosis or cirrhosis regardless of etiology (121, 123–125).

During hepatocarcinogenesis, a complex multistep process, many signaling cascades are altered as a result of genetic and epigenetic changes that contribute to a heterogeneous molecular profile. Furthermore, cellular plasticity increases the complexity of the cellular heterogenicity. Indeed, tumor heterogeneity in HCC is impressive: it can be observed between patients, between nodules in the same patient (i.e., second primary tumors after curative treatment or synchronous multifocal tumors of different clonality) and even within a single tumor nodule (126). Many molecular mechanisms are known to be clearly involved in HCC (127). Signaling pathways are related mainly with cell proliferation, angiogenesis, invasion, and metastasis. IGF-1, Epidermal Growth Factor (EGF), PDGF, Hepatocyte Growth Factor (HGF), VEGF, as well as TGF-β, are the most frequent growth factors and cytokines involved in HCC development. (128). The role of the microenvironment in tumor initiation and progression in HCC is critical and HCC cells could acquire an abnormal phenotype due to tissue remodeling altering their biological behavior (129, 130).

#### ROLE OF TGF-β DURING HEPATOCARCINOGENESIS

As it was mentioned before, TGF-β signaling -in the liverparticipates in all stages of disease progression, from initial liver injury through inflammation and fibrosis, to cirrhosis and cancer (7, 8). During early stages of tumorigenesis TGF-β acts as a tumor suppressor, while in late stages it acts with a protumorigenic role, promoting invasiveness and metastasis once cells become resistant to its suppressor effects (8, 131). In non-transformed hepatocytes and HSC, the cytostatic effects of TGF-β are often dominant over the opposing mitogenic signals; however, carcinoma-derived cells are usually refractory to growth inhibition by this cytokine. Activation of the TGF-β pathway induces antiproliferative responses due to the regulation of the cell cycle at G1-S by inhibiting c-MYC and cyclin-dependent kinase complex (CDK)-1-6 and 7 and regulates cycling inhibitors such as p21 and p15 (132–134). Smad4 –/– mice could develop head and neck cancer demonstrating the role of the TGF-β pathway as cytostatic regulator (135). Other proteins involved in the regulation of the pathway, such as β-II spectrin, which plays a role as scaffold for SMAD3 and SMAD4 and their subsequent activation after TGF-β. Sptbn2 heterozygote mutants develop HCC indicating that TGF-β signaling and β-II spectrin suppress hepatocarcinogenesis, potentially through cyclin D1 deregulation (136). TGF-β pathway also activates the NRF2 transcription factor which is involved in the expression of many cytoprotective genes which are relevant in the protection of the cells against toxic insults, and its depletion increases tumorigenic process (137). These data show up the relevance of the cytoprotective and suppressor role of TGF-β pathway, which could be altered during the carcinogenesis process. Malignant cells surpass the suppressive effects of TGF-β either through inactivation of core components of the pathway (such as TGF-β receptors and/or SMADs) or by downstream alterations repressing the tumorsuppressive arm. In late stages, liver cancer cells take advantages from the TGF-β-dependent pathways to acquire capabilities that contribute to tumor progression, such as production of autocrine mitogens, release of pro-metastatic cytokines and chemokines and up-regulation of receptors that mediate the response to them (10, 138–140). In this sense, different evidences suggest that TGF-β plays a dual role in hepatocarcinogenesis. On one side, as commented above, TGF-β inhibits proliferation and induces apoptosis in hepatocytes and liver tumor cells (141, 142), but simultaneously, it activates survival pathways, such as Akt, and induces an increase in the expression of anti-apoptotic BCL-2-related proteins (143–145), a process that is related to the capacity of TGF-β to transactivate c-Src and EGFR pathways, among others (146). Interestingly, the inhibition of the EGFR increases the apoptotic response to TGF-β (146, 147). In fact, in hepatocytes, TGF-β-induced apoptosis could be counteracted by EGF (an important survival signal) (141, 148) a process that requires activation of the PI3K/Akt axis to counteract TGF-β-induced upregulation of the NOX4, oxidative stress and mitochondrial-dependent apoptosis (149, 150). Another member of the NOX family, NOX1, is involved in this anti-apoptotic role. TGF-β-mediated activation of NOX1 promotes autocrine growth of liver tumor cells through the activation of the EGFR pathway, via upregulation of EGFR ligands expression through a c-Src (151) and NF-κB (152) dependent mechanism. The autocrine loop of EGFR activated by TGF-β in non-transformed hepatocytes and liver cancer cells requires the activity of the metalloprotease TACE/ADAM17 (142, 146) in a Caveolin-1/Src/NOX1 dependent manner (153, 154). This proliferation can be impaired by the addition of the NOX inhibitor VAS2870 (155). Moreover, TGF-β is able to mediate the production of EGFR ligands, which eventually confers resistance to its proapoptotic effects in hepatocytes (149, 152) and HCC cells (156). Importantly, the capacity of hepatocytes to survive to TGF-β is also dependent on their differentiation status (157). Thus, rat hepatoma cells respond to TGF-β inducing survival signals, whereas adult hepatocytes do not (142). In the same way, different features of HCC cell lines, like the activation of the EGFR or MEK/ERK pathways, may provoke different outcomes after TGF-β exposure (156, 158).

Once cells overcome the cytostatic and apoptotic effects of TGF-β, this cytokine regulates cell plasticity, a fact that has been elegantly evidenced in a study by Coulouarn and col., where they proposed different liver TGF-β gene signatures, defining a cohort of genes related to its tumor suppressor capacity and another cohort of genes related with its tumor promoting effects: the early and the late TGF-β-signatures. The "early" TGF-β signature is associated to genes involved in growth inhibition and apoptosis, whereas the termed "late" TGF-β signature is associated to EMT, migration and invasion (159). Of relevance, this study also discriminated HCC cell lines by degree of invasiveness. Interestingly, the early response pattern is associated with longer, and the late response pattern with shorter, survival in human HCC patients. In addition, tumors expressing the late TGF-βresponsive genes displayed invasive phenotype, increased tumor recurrence and accurately predicted liver metastasis. In the development of liver hepatocarcinogenesis, TGF-β plays crucial roles regulating the different stages of the disease, some of these roles are summarize in the **Figure 3** and we detail in the next chapters.

# TGF-β PROMOTES EMT IN HCC

Tumor cells that overcome the suppressor effects of TGF-β become ready to respond to this cytokine inducing other effects, such as EMT, processes that contribute to either fibrosis and/or tumor dissemination (160). Neoplastic transformation of hepatocytes and progenitor cells, which both are epitheliallike, to a mesenchymal-like phenotype boost heterogeneity in HCC (75).

TGF-β is one of the strongest inducers of EMT under both physiological and pathological context (161), regulating the expression and activity of EMT-TFs (14). Different in vitro studies support the idea that TGF-β induces EMT in non-tumorigenic epithelial cells, transforming them into a fibroblast-like phenotype. For example, alveolar epithelia cells via FoxM1/Snail1 can undergo EMT after TGF-β exposure (162, 163); mammary epithelial cells undergo EMT in a TGF-β/PI3K/mTOR pathway (164, 165). After liver insult, non-transformed hepatocytes can undergo EMT as an adaptative response to move and scape from damaged, inflammatory, hypoxic and redox-activated microenvironment allowing them to find better conditions (166). Moreover, TGF-β induces anti-apoptotic signals in transformed hepatocytes, through the activation of the EGFR pathway (143, 146), and liver cells that overcome TGF-β pro-apoptotic effects undergo EMT in a Snail1-dependent manner conferring resistance to apoptosis (15, 71, 142, 156). Besides apoptotic resistance, mesenchymal-like phenotype increases migratory properties in HCC cells through activation of the CXCR4/CXCL12 axis in TGF-β-dependent manner (139), a mechanism that would contribute to tumor progression in HCC patients (167). Interestingly, CXCR4 is localized in the migratory front of the tumors and is coincident with TGF-β signaling overactivation, suggesting this pathway as a future prognostic factor to predict patient response to TGF-β therapies. MicroRNAs (miRNA) are also involved in the regulation of EMT and in the progression and development of HCC. MiR-181, which is regulated by TGF-β, is overexpressed in HCC samples and is associated with and EMT phenotype (168–170). In hepatocytes, miR-181 induces an EMT-like response and mimics TGF-β-effects, upregulating MMP2, α-SMA and vimentin, downregulating E-cadherin and inducing morphological changes.

Upon HCC development, the excessive growth of transformed cells also generates hostile nodules for cancer cells due to oxygen depletion in internal areas (hypoxic environment) (171) as compared to tumor stroma borders, which induces tumor cell necrosis. Malignant hepatocytes or progenitor cells could undergo EMT as an option to escape from these places and to move toward a cytokine/chemokine better and enriched microenvironment, as well as a resistance mechanisms of survival to cell death stimuli (172, 173). In addition, hypoxic factors, such as HIF-1α, could stimulate EMT in hepatocytes in a TGF-β-dependent manner due to hypoxic hepatocytes secrete enzymes that activate latent TGF-β (174).

#### TGF-β REGULATES CANCER STEM CELL PLASTICITY

The variability in the prognosis of HCC patients suggests that it may comprise several distinct biological phenotypes, but individuals with HCC who shared a gene expression pattern with foetal hepatoblasts showed to have a poor prognosis (175). Several evidences provide insight into the role of TGF-β in regulating the cancer stem cell niche (176), much less is known about the potential crosstalk between TGF-βinduced EMT in the HCC cells and the acquisition of stem cell properties (74, 177). It is proved that liver epithelial cells that undergo a TGF-β-dependent EMT process show a less differentiated phenotype. Chronic TGF-β treatment in rat and human fetal hepatocytes, as well as in human HCC cells, promotes a mesenchymal-like phenotype concomitant with decreased expression of specific hepatic genes and the appearance of stem cell features, reminiscent of a progenitorlike phenotype (156, 177, 178). Cancer stem cells (CSC) or tumor-initiating cells (TICs) in the liver could derive from hepatic progenitor cells exposed to chronic TGF-β-exposure during hepatocarcinogenesis (179). TGF-β is involved in the neoplastic transformation of liver progenitor cells, through a miR-216a/PTEN/Akt-dependent pathway (179), concomitant with FOXO3a nuclear exportation. FOXO transcription factors are implicated in a huge cellular events and are also related with the neoplastic phenotypes linked to PI3K/Akt activation (180).

It is suggested that the expression of stem-related genes could also be mediating the acquisition of an EMT phenotype.

In this sense, the stem-related CD44 or CD133 are not only involved in the acquisition of stem properties, but also in the switch to a more mesenchymal, migratory phenotype (181, 182). TGF-β-mediated mesenchymal-like phenotype is regulated by CD44, and its overexpression provokes down-regulation in E-cadherin expression and up-regulation of vimentin, which correlate with higher phospho-SMAD2-positive nuclei and poor prognosis in HCC patients (181, 183). Moreover, intermediate EMT states have recently been identified as crucial drivers of organ fibrosis and tumor progression (14). During partial-EMT stage, both epithelial and mesenchymal stem genes can be expressed. In this sense, it is worthy to mention that in certain HCC cell lines, TGF-β-treatment induces the expression of mesenchymal genes, such as VIM (vimentin), and the mesenchymal-related stem-related genes CD44 and CD90, but simultaneously, they express E-cadherin and the epithelialrelated stem genes EPCAM or CD133 (177). Interestingly, this partial EMT phenotype confers to the HCC cells the highest stemness stage concomitant with an increased migratory/invasive capacity (177).

CSCs could contribute to the failure of therapies to abolish malignant tumors. In pre-clinical assays, cancer stem-like spheres from de-differentiated HCC-derived cell lines show increased expression of stemness markers (CD44), and higher resistance to anticancer drugs (184). Furthermore, the acquisition of some mesenchymal properties and the expression of CD44 impair the HCC cell response to sorafenib-induced apoptosis (183). For this, novel strategies are focused to target CSC development. It is interesting to mention that treatment with inhibitors of the TGF-β pathway, such as Galunisertib (LY2157299–a selective ATP-mimetic inhibitor of TGF-β receptor I) decreased the stemness related genes of mesenchymal HCC cells and their ability to form colonies, liver spheroids and invasive growth (185). Resminostat, a novel orally histone deacetylases inhibitor, has been demonstrated as a good therapy in the SHELTER study (a phase I/II clinical study) in mono and combination therapy with sorafenib (186). The combination therapy revealed an advantage in terms of overall survival and time to progression. In HCC cells with a mesenchymal phenotype, caused by autocrine expression of TGF-β, resminostat sensitizes them to the apoptotic response induced by sorafenib (187). Mesenchymalrelated gene expression was decreased in resminostat-treated HCC cells. This event is concomitant with an epithelial-related gene expression increase, more organized tight junctions and lower invasive growth. Indeed, resminostat down-regulated CD44 expression is coincident with a decrease in the stemness properties. These results reinforce the strong impact of the TGFβ-induced mesenchymal/stemness phenotype on HCC drug resistance.

#### LIVER CANCER STROMA CELL PLASTICITY AND TGF-β

TGF-β display multiple effects on the microenvironment (188), which plays a relevant role in HCC development and progression. In the stroma, TGF-β induces microenvironment changes, including generation of cancer-associated fibroblasts (CAFs) (23) that play a relevant role in facilitating the production of growth factors and cytokines, which contribute to cell proliferation, invasion and neoangiogenesis, being related with poor prognosis (189–192). Different origins are described, but in the liver CAFs could be originated from epithelial cells– hepatocytes, cholangiocytes- and HSC that undergo an EMT process. To promote their tumoral functions, CAFs need the expression of EMT-TFs. Indeed, Snail expression in CAFs is necessary for their response to TGF-β, increased production of fibronectin and stiffness of the ECM (23). CAFs are also a potent source of TGF-β and are described to promote the migration and invasion of HCC cells in vitro and facilitate the HCC metastasis to the bone, brain and lung in NOD/SCID mice (193).

In HCC microenvironment anti-tumor response is impaired due to various immune suppressive elements, such as Tumor Associated Macrophages (TAMs) and regulatory T cells (Treg) (194–196). Similarly to that occurs with macrophages in fibrosis, during HCC progression, TAMs are mainly polarized toward an M2 phenotype, due to the higher levels of TGF-β (among others factors) (197). M2-like macrophages are major players in the connection between inflammation and cancer, involved in functions such as: promotion of tumor cell proliferation, ECM turnover, inhibition of adaptive immunity, among others (198, 199), TAMs are correlated with angiogenesis, metastasis, and poor prognosis (200, 201). Moreover, TAMs can promote cancer stem cell properties in a TGF-β-dependent manner (202). CAFs can also educate Natural Killer (NK) cells (203), dendritic cells (204) and upregulate the production of Treg in a TGF-β-dependent manner (205). CAFs promote Treg cell induction, through upregulation and activation of the human B7 homolog 1(B7-H1)/programmed death 1 (PD-1) signaling, which are involved in immunosuppressive functions in a mTOR/Akt dependent-manner (206). Accumulating evidence indicates that the immune system microenvironment plays key roles in the development of HCC (207, 208). CD4+ naïve T cells show an enormous cell plasticity and under TGF-βtreatment could differentiate into Treg cells (209). Poor prognosis in HCC patients is associated with infiltration liver tissue prognosis in HCC patients. Treg cells -Foxp3-positive cellsare involved in immune homeostasis, peripheral tolerance and prevention of autoimmunity (210, 211). TGF-β induces the expression of the transcriptional factor Foxp3 involved in the conversion of naïve CD4+CD25 T cells to CD4+CD25+ Treg cells with potent immunosuppressive potential (212). Blocking TGF-β signaling with SM-16 (TGF-β inhibitor) significant decreases the percentage of Treg cells in liver tissue concomitant with an attenuation of the hepatocarcinogenesis process in a DEN-model (213). Interestingly, addition of exogenous TGFβ restores the expression level of Foxp3 and the presence of Treg cells. Exogenous addition of TGF-β normalizes Treg cell numbers and promoted their cell differentiation. Moreover, In HCC patient samples, the expression of both genes, TGFB1 and FOXP3, correlate positively and are involved in tumoral progression. (213). On summary, TGF-β promotes tumor immune escape and survival by maintaining natural Treg levels, inducing Treg cell differentiation and TAMs polarization into M2-phenotype.

# NEW THERAPIES TO INHIBIT THE TGF-β PATHWAY

Developing an effective therapy to target the TGF-β pathway in liver pathologies requires a better understanding of its complex role in this organ, considering its pleiotropic effects on cell proliferation, death and differentiation of different liver cell types, its ability to induce EMT in epithelial cells or EndEMT in the endothelial ones, as well as its capacity to act as an immune modulator. In spite of this, targeting TGF-β was proposed a good approach to delay the progression of liver diseases and, in particular, of HCC (9, 214, 215). Indeed, first experiments indicated that inhibiting the TGF-β pathway in HCC cell lines blocked migration and invasion of HCC cells by upregulating E-cadherin and down-regulating CXCR4 (167, 188), as well as inhibiting the upregulated levels of CTGF induced by TGF-β, reducing the stromal component of the microtumoural environment and slowing the HCC growth in vivo (216). These data suggested that there could be a mechanistic use for targeting TGF-β in HCC clinical trials.

Several different strategies have been proposed to inhibit the TGF-β pathway in liver pathologies, including the use of chimeric proteins, monoclonal antibodies, peptide inhibitors, small molecules that inhibit the receptors' kinase activity and antisense oligonucleotides (217). First studies demonstrated the efficiency of potential peptide inhibitors of TGF-β1 (derived from TGF-β1 and from its type III receptor) in vitro and in vivo in reducing liver fibrosis (218, 219). These peptide inhibitors were proved to be also useful in enhancing the efficacy of antitumor immunotherapy (220). To increase the delivery efficiency, in a recent study, one of these peptides (P-17) was loaded separately into folic acid-functionalized nano-carriers made of bovine serum albumin. Cellular studies demonstrated the targeting efficiency of the hybrid carriers (221).

In the last years the interest has been focused on the TβRI kinase inhibitor Galunisertib, developed by Lilly (LY2157299, a selective ATP-mimetic inhibitor of TβRI) that has proved more efficient than neutralizing humanized antibodies, such as D10 against TβRII, in blocking the canonical TGF-β signaling in HCC cells, experiments that supported the use of this drug in preclinical and clinical trials (9, 222). Despite limited antiproliferative effects, Galunisertib yielded potent anti-invasive properties in HCC models and in ex vivo tumor tissue samples from patients (223). Worthy to note, in combination, Galunisertib potentiated the effect of sorafenib efficiently by inhibiting proliferation and increasing apoptosis. Galunisertib also reduced the expression of stemnessrelated genes, such as CD44 and THY1, in vitro and in ex vivo human HCC specimens, overcoming stemness-derived aggressiveness (185). Furthermore, it also showed antitumor activity through the activation of CD8+ T-cell antitumor responses (224). Recent studies have also suggested the potential efficiency of Galunisertib as antifibrotic drug. In ex vivo studies, using both healthy and cirrhotic human precision-cut liver slices, Galunisertib reduced fibrosis-related transcription, which correlated with a significant inhibition in the production and maturation of collagens (225). Furthermore, in an experimental preclinical model (Abcb4ko mice) the treatment with Galunisertib decreased the expression of several fibrogenic genes, such as collagens (Col1a1 and Col1a2), Tgfb1 and Timp1, and reduced the ECM/stromal components, fibronectin and laminin-332, as well as the carcinogenic β-catenin pathway (226).

A phase II clinical trial using Galunisertib in patients with advanced HCC to test safety, time to progression and overall survival (OS) is ongoing (NCT01246986 http://clinicaltrials. gov). Preliminary data show that patients with higher levels of circulating TGF-β1 are more likely to respond to therapy with Galunisertib. TGF-β1 reduction in response to the treatment is related to improvement in OS when compared to patients with non-TGF-β1 reduction. Some efforts are being made in optimizing the delivery of Galunisertib in form of novel polymeric nano-micelles, to avoid acidic pH of gastrointestinal tract, colon alkaline pH and anti-immune recognition (227).

Once it is proven the safety and the benefit of using Galunisertib in HCC, biomarkers will be extremely useful in the proper selection of patients who might benefit from receiving the drug. In this sense, high TGFB1 expression in HCC patients, concomitant with high expression of the genes that mediate its invasive effects, such as PDGF, CXCR4, or CD44, (167, 177, 228) would anticipate a benefit for the use of Galunisertib. Furthermore, a recent study has proposed SKIL and PMEPA1 as strongly downregulated by Galunisertib, correlating with endogenous TGFβ-1 (185, 229). These target genes identified may also serve as biomarkers for the stratification of HCC patients undergoing treatment with Galunisertib. Since biopsy is not frequent in HCC patients, new areas of research must be focused on the improvement of liquid biopsies in these patients to develop the possibility that this kind of analysis may be done in tumor circulating cells.

Finally, new approaches to interfere not only the TGF-β canonical, but also the non-canonical pathways must be developed in the next future, as previously mentioned, the switch from tumor-suppressive to pro-oncogenic TGF-β actions could be directed by its crosstalk with Receptor Tyrosine Kinases, in particular, EGFR. So, interference with EGFR signaling, by employing approved targeted drugs, in TGF-β/SMAD-positive HCC patients might be effective in improving the effectiveness of Galunisertib.

# CONCLUDING REMARKS

TGF-β plays unique actions in modulating cell plasticity, and the liver reveals as a tissue where these actions would be very relevant during the response to injuries that cause chronic diseases. In general terms, TGF-β-induced changes in cell plasticity may converge in transdifferentiation toward a different phenotype, such as the case of activation of HSC to MFB or the dedifferentiation/acquisition of stem cell properties in hepatocytes and liver tumor cells. But it may also proportionate to the cells new capabilities, such as cell survival or increase in migratory/invasive properties that the liver tumor cells acquire when they undergo EMT in response to TGF-β. And, worthy to note the essential role that TGF-β plays inducing Treg cell differentiation and TAMs polarization into M2-phenotype, which promotes tumor immune escape and survival. Despite all current knowledge, there are still many gaps that need to be clarify. However, these evidences point toward the use of tools that target the TGF-β signaling pathway to counteract liver disease progression.

# AUTHORS CONTRIBUTIONS

Both authors equally contribute to the writing and revision of this work. Figures were designed and elaborated by DC-D and supervised by IF.

### FUNDING

Grants: Ministry of Science, Innovation and Universities, Spain (cofounded by FEDER funds/Development Fund—a way to build Europe): SAF2015-64149-R. DC-D was recipient of a fellowship from the FPI program, Ministry of Economy, Industry and Competitiveness, Spain: BES-2016 0077564. The CIBEREHD, National Biomedical Research Institute on Liver and Gastrointestinal Diseases, is funded by the Instituto de Salud Carlos III, Spain.

# ACKNOWLEDGMENTS

We would like to give thanks to Esther Bertran and Judit López-Luque for all their support and advices.

#### REFERENCES

role in idiopathic pulmonary fibrosis. Am J Pathol. (2005) 166:1321–32. doi: 10.1016/S0002-9440(10)62351-6

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Fabregat and Caballero-Díaz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Phenotypic Basis for Matrix Stiffness-Dependent Chemoresistance of Breast Cancer Cells to Doxorubicin

M. Hunter Joyce1 , Carolyne Lu1 , Emily R. James 1 , Rachel Hegab2 , Shane C. Allen1 , Laura J. Suggs 1,3 and Amy Brock 1,3 \*

1 Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, United States, 2 Department of Biomedical Engineering, Louisiana Tech University, Ruston, LA, United States, 3 Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, United States

#### Edited by:

Ubaldo Emilio Martinez-Outschoorn, Thomas Jefferson University, United States

#### Reviewed by:

Hamid Morjani, Université de Reims Champagne Ardenne, France Saraswati Sukumar, Johns Hopkins University, United States

> \*Correspondence: Amy Brock<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology

Received: 07 May 2018 Accepted: 03 August 2018 Published: 05 September 2018

#### Citation:

Joyce MH, Lu C, James ER, Hegab R, Allen SC, Suggs LJ and Brock A (2018) Phenotypic Basis for Matrix Stiffness-Dependent Chemoresistance of Breast Cancer Cells to Doxorubicin. Front. Oncol. 8:337. doi: 10.3389/fonc.2018.00337 The persistence of drug resistant cell populations following chemotherapeutic treatment is a significant challenge in the clinical management of cancer. Resistant subpopulations arise via both cell intrinsic and extrinsic mechanisms. Extrinsic factors in the microenvironment, including neighboring cells, glycosaminoglycans, and fibrous proteins impact therapy response. Elevated levels of extracellular fibrous proteins are associated with tumor progression and cause the surrounding tissue to stiffen through changes in structure and composition of the extracellular matrix (ECM). We sought to determine how this progressively stiffening microenvironment affects the sensitivity of breast cancer cells to chemotherapeutic treatment. MDA-MB-231 triple negative breast carcinoma cells cultured in a 3D alginate-based hydrogel system displayed a stiffness-dependent response to the chemotherapeutic doxorubicin. MCF7 breast carcinoma cells cultured in the same conditions did not exhibit this stiffness-dependent resistance to the drug. This differential therapeutic response was coordinated with nuclear translocation of YAP, a marker of mesenchymal differentiation. The stiffness-dependent response was lost when cells were transferred from 3D to monolayer cultures, suggesting that endpoint ECM conditions largely govern the response to doxorubicin. To further examine this response, we utilized a platform capable of dynamic ECM stiffness modulation to allow for a change in matrix stiffness over time. We found that MDA-MB-231 cells have a stiffness-dependent resistance to doxorubicin and that duration of exposure to ECM stiffness is sufficient to modulate this response. These results indicate the need for additional tools to integrate mechanical stiffness with therapeutic response and inform decisions for more effective use of chemotherapeutics in the clinic.

Keywords: chemotherapy, resistance, extracellular matrix, tumor microenvironment, 3D cell culture

# INTRODUCTION

Cancer is a complex disease capable of affecting multiple properties of tissue organization and is driven by numerous factors. Hanahan and Weinberg (1, 2) have summarized and defined "hallmarks of cancer" which describe biological conditions characteristic of tumorigenesis. Pickup (3) revisited these hallmarks and described roles that the extracellular matrix (ECM) plays in each. The ECM includes the non-cellular components of a cell's microenvironment responsible for providing physical scaffolding and facilitating signaling from the surrounding tissue (4). Studies aimed at characterizing the role that physical cues play in tumor development have demonstrated that ligand type, ligand density, substrate composition, and substrate stiffness are important factors in tumor initiation, progression, and metastasis (5–12). Additional studies investigate how cells manipulate the ECM and vice-versa, suggesting a reciprocal nature whereby each shapes the response of the other (7, 8, 13–15).

With cellular remodeling of the ECM, tumors become progressively more rigid than surrounding tissue, a characteristic that informs diagnosis by physical palpitation and imaging (16). These serve as early detection methods for breast cancer (16), the second leading cause of cancer death among women (17). The ECM influences organization (15) and differentiation (18, 19) of mammary cells into functional mammary structures and tissues. It plays a role in tumor suppression through trafficking of intercellular signals (20) and exerting physical forces sensed by cell surface proteins, such as integrins (10, 13, 15). Changes in the ECM composition (15, 18, 19, 21) and stiffness (6–8, 15, 22, 23) have been shown to promote malignant phenotypes. Previous work in the field has investigated the role of ECM in determining cellular response to chemotherapy treatment (9, 24). Rice et al. (25) and Zustiak et al. (26) both demonstrate that ECM stiffness can alter chemotherapeutic response for some cancer cell lines. Shin and Mooney (9) examined the relationship between ECM ligands and cancer cell resistance to chemotherapeutics. Here we utilize a hydrogel platform to examine how ECM stiffness and the dynamic modulation of that microenvironmental stiffness affect the response of two distinct breast carcinoma cell lines to chemotherapeutic treatment.

Hydrogels have served to isolate and model aspects of the ECM for in vitro studies of cellular organization (15) and differentiation (18, 26, 27), tumor suppression (10, 13, 15), tumorigenesis (6, 13, 28–30), promotion of malignant phenotypes (6–8, 15, 22, 23), and cellular response to chemotherapy (9, 24, 31). Materials native to mammalian cellular ECM (i.e., scaffolding proteins such as collagens and fibrins) as well as biomimetic materials (such as agar and acrylamide) are used to produce 3D scaffolds for in vitro cultures. The mechanical properties of these hydrogel scaffolds are largely determined during the initial preparation; overall stiffness of the hydrogel is determined by the density of protein/polymer or number of cross-links formed. However, with these systems is it not possible to adjust matrix stiffness in a controlled manner after initial formation of the hydrogel. Recent work has shown that the contractile force of MDA-MB-231 cells can cause significant increases in stiffness of their surrounding ECM (32). Microrheology with optical tweezers has been used to measure the stiffness gradient created by cells grown in collagen hydrogels; the linear stiffness of hydrogels adjacent to MDA-MB-231 cells was measured to be up to two orders of magnitude stiffer than areas of unoccupied hydrogel 200µm away. Similar stiffening was seen for MDA-MB-231 cells in Matrigel cultures and human umbilical vein endothelial cells in fibrin hydrogels, indicating that maintaining hydrogel stiffness within a defined range will be challenging as cells rearrange their ECM over time.

Our studies utilized the alginate-based hydrogel system described by Stowers et al. (33) as a means to predictably modulate hydrogel stiffness. Alginate is bio-inert, and alginate polymers can be cross-linked with calcium ions, providing a means for controlling hydrogel stiffness. Additionally, this system incorporates 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes loaded with calcium and gold nanorods. Irradiation with NIR light causes the gold nanorods to undergo surface plasmon resonance and heat the liposomes. As the liposomes approach the phase transition temperature (41◦C), they undergo gel-to-liquid transition causing the encapsulated calcium to leak out and form additional alginate cross-links. This experimental platform allowed us to seed breast cancer cells into hydrogels of an initial stiffness (200 and 2,000 Pa), further stiffen their ECM during the course of the experiment (200 1,600 Pa and 2,000 3,000 Pa), and treat samples with the chemotherapeutic doxorubicin to determine how dynamic changes to the ECM affect chemotherapeutic resistance. Here we show that stiffness of the ECM is sufficient to modulate MDA-MB-231 breast cancer cell resistance to doxorubicin.

## MATERIALS AND METHODS

## Cell Culture

MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies, REF: 10569-010, 94%) supplemented with FBS (Life Technologies, REF: 10437-028 5%) and P/S (Life Technologies, REF: 15070-063, 1%). MCF7 cells were cultured in Minimum Essential Media (MEM, Life Technologies, REF: 11095-080, 89%) supplemented with Fetal Bovine Serum (FBS, Life Technologies, REF: 10437-028, 10%) and Penicillin/Streptomycin (P/S, Life Technologies, REF: 15070- 063, 1%). All cultures were incubated at 37◦C with 5% CO2.

# Hydrogel Preparation