Alongside its crucial role in fluid volume regulation, the RAS is also important for stem cell maintenance and differentiation in several cell types. ATII expression drives the differentiation of mesenchymal stem cells into adipocytes (19), while other components of the RAS drive differentiation into insulin producing cells (20). ACE is required for hemangioblast expansion, and modulation of ATIIR1 or ATIIR2 signaling can direct the fate of the blasts toward either an endothelial or hematopoietic lineage (21). The RAS also plays a role in hematopoiesis (22, 23), vasculogenesis (24), erythropoiesis (25, 26), and myeloid differentiation (27).

Importantly, the RAS not only acts to promote stem cell differentiation in diverse cell populations, but also appears to act in a feedback loop with Wnt/β-catenin signaling, where pro-renin receptor (PRR) can induce Wnt/β-catenin (28), and components of the RAS themselves are targets of Wnt/βcatenin (29). Wnt/β-catenin signaling is crucial for embryonic development and induces differentiation of pluripotent stem cells into progenitor cells (30). Given that Wnt signaling is also involved in cancer development (31), and downstream Wnt targets include the CSC markers CD44 and c-Myc (32), it may be that CSCs require activation of Wnt signaling (33). This suggests that RAS modulators could be employed in these cells to indirectly inhibit Wnt signaling and its effects.

Having identified these roles in normal stem cell maintenance and differentiation and feedback loops with a canonical developmental signaling pathway, it may be that the expression of the RAS also plays a role in the regulation or function of CSCs.

### Retrospective Studies and Clinical Trials Indicate Potential Benefit of RAS Modulators in Reducing Cancer Risk

The widespread use of RAS modulators as anti-hypertensives and their potential effect on cancer risk have been extensively documented. A seminal study has shown that the use of ACEI and ARBs is associated with a reduced risk of developing some cancer types, particularly cancers affecting women (34). Many other retrospective population studies have reported differing effects on cancer risk depending on the cancer type, cohort characteristics, and the RAS inhibitor used. As a result, several meta-analyses have been undertaken (35), again with differing results, which could be due to the nature of the original

FIGURE 1 | Overview of the renin-angiotensin system with its bypass loops and convergent signaling pathways. The renin-angiotensin system (black) regulates blood pressure, stem cell differentiation, and tumor development. Bypass loops of the RAS involving enzymes such as chymase and cathepsins B, D, and G (green) provide redundancy, while convergent inflammatory and developmental signaling pathways (blue) have multiple roles and effects. Angiotensinogen (AGN) is physiologically synthesized and released by the liver and is cleaved by renin to form angiotensin I (ATI). Renin is formed following binding of pro-renin to the pro-renin receptor. ATI is converted to angiotensin II (ATII) by angiotensin converting enzyme (ACE). ATII interacts with the G-protein coupled receptors ATII receptor 1 (ATIIR1) and ATII receptor 2 (ATIIR2) to restore homeostasis, via vasoconstriction and vasodilation, respectively. ATII can also give rise to angiotensin III via the action of aminopeptidase A, and Angiotensin 1–7 which binds and activates the G-protein coupled receptor MAS. Cathepsins B and D are also renin-activating enzymes that convert pro-renin to renin. Cathepsin D converts AGN to ATI, and cathepsin G converts ATI to ATII or AGN directly to ATII. Chymase converts ATI to ATII. Pro-renin also induces Wnt/β-catenin signaling in a feedback loop. ATIIR1 can also result in inflammatory signaling via the NOX-ROS-NFκB-COX2 signaling axis. ROS, reactive oxygen species.

studies included and inherent publication bias. Aside from the reported effects on cancer risk, many retrospective population studies have also assessed the effect of RAS inhibitors on cancer death. Again, these results have been mixed, though metaanalyses have indicated that β-blocker use is not associated with survival in breast cancer patients (36), and a metaanalysis looking at ACEI use in all cancers showed no effect on cancer survival (37). A more recent meta-analysis looking at the use of different RAS inhibitors in all cancers showed that RAS inhibitor use extended overall, progression-free and disease-free survival (38). This is mainly due to ARBs and not ACEI use, with some site-specific effects. These studies need

to be interpreted critically and with caution as they do not prove causality and the effect on cancer risk and mortality could be due to other factors. It may also be that a defined patient group will derive benefit from these treatments and that a more holistic approach of targeting the RAS in cancer is required to achieve a sustained treatment for patients. Due to the nature of the RAS, with its inherent bypass loops conferring redundancies, and the presence of many other pathways that converge on the RAS, it is likely that a multifaceted approach to target the RAS will be required for effective cancer treatment.

Despite these disparate observations in retrospective population studies, the data around the involvement of the RAS in tumor models is clear, leading to many clinical trials using RAS inhibitors and the development of new targeted agents (39–41). Several of these studies have trialed ARBs in cancer patients, with Losartan being shown to enhance the efficacy of chemotherapy and improve overall survival in ovarian cancer patients (42). Another ARB, Candesartan, has been shown to decrease prostate specific antigen levels in hormone-refractory prostate cancer patients (43), and is tolerated in advanced pancreatic cancer (44, 45). The ACEI Captopril is tolerated in patients with advanced cancer (46), and has been shown to reduce biochemical recurrence in prostate cancer patients (47), while Perindopril reduced the risk of recurrence of hepatocellular carcinoma as a combination therapy with other non-traditional treatments (48, 49). Several trials have targeted the Ang1-7/MAS axis in breast cancer before or after chemotherapy (50), and in metastatic sarcoma, where it is well tolerated (51), and a number of advanced solid tumors where it provides benefit for some patients (52).

#### TABLE 1 | Components of the RAS are expressed in tumors.

TABLE 2 | β-blockers inhibit tumorigenesis in cell and animal models.

β-blockers work by blocking β-adrenergic receptors to prevent neurotransmitter binding. This prevents renin secretion and its actions and subsequently results in lowered blood pressure (**Figure 2**). The non-selective β-blocker Propranolol has been shown in several case reports to be efficacious in treating angiosarcoma (53), and in combination with chemotherapy treatment induced responses in seven patients with advanced angiosarcoma (54). Another study showed that addition of Propranolol or another non-selective βblocker Carvedilol to treatment regimens for metastatic angiosarcoma improved progression-free and overall survival (55). Propranolol has also been used in a proof of concept study in multiple myeloma patients receiving hematopoietic cell transplantations (56), and in a prospective cohort study in melanoma patients where its use was associated with reduced recurrence (57).

While these trials have demonstrated promise for targeting the RAS in cancer treatment, the mechanisms by which this is achieved are yet to be elucidated. Current clinical trials and the development of new RAS targets should help to further define which patient groups may benefit from these treatments.

### In vitro and in vivo Cancer Models Rationalize the RAS as a Therapeutic Target

Given the potential effects on reducing cancer risk observed in retrospective population studies, expression of components of the RAS have been assessed in many different tumor types to clarify the potential role of the RAS in tumorigenesis (**Table 1**).

These studies have helped define the role of the RAS in tumorigenesis, and collectively show that components of the RAS are expressed in many different cancer types (39, 85). The effects on tumor growth, angiogenesis, metastasis and survival indicate that the RAS plays a role in the hallmarks of cancer (39, 86, 87). It is also thought to contribute to an immunosuppressive microenvironment in tumors and reduce infiltration of tumor-associated macrophages (88). The increased

#### TABLE 3 | ACE inhibitors inhibit tumorigenesis in cell and animal models.

#### TABLE 4 | ARBs inhibit tumorigenesis in cell and animal models.

expression of components of the RAS in different cancer types may contribute to tumorigenesis and the poor clinical outcome seen in some cancer types. This suggests that regulation of the RAS may be a general mechanism for cancer prevention and warrants further investigation to understand the precise underlying mechanisms.

Given that the RAS is over-expressed in many cancer types and the use of RAS modulators may affect cancer risk and cancer survival, numerous studies have assessed the effect of RAS inhibitors in vitro and on tumor models in vivo. These have focused on β-blockers (**Table 2**), ACEI (**Table 3**), and ARBs (**Table 4**) to assess the role of the RAS in tumor development.

Studies investigating β-blockers in cancer (**Table 2**) have largely used the β-blocker Propranolol and have shown that across a wide range of cancer types, Propranolol inhibits the growth of tumors and tumor cells. This suggests that Propranolol could be repurposed for cancer treatment (130, 131), as has been the case for the benign vascular tumor infantile hemangioma for which it is an effective treatment (132–134).

Given the effects of β-blockers on cancer and cancer cell growth, other studies have investigated the impact of other classes of drugs that modulate the RAS on neoplastic processes. One of these classes is ACEIs, which block the action of ACE and hence downstream production of ATII (**Figure 2**). Studies looking at ACEIs (**Table 3**) are extensive and demonstrate that this class of drugs (including Captopril, Enalapril, and Perindopril) appear to prevent tumor growth and invasion in many different tumor types and models.

Another broad class of drugs that modulate the RAS are ARBs, which block ATIIR1 (**Figure 2**). Studies using ARBs to assess cancer development in cell and animal models (**Table 4**) have also shown that different drugs within this class (Candesartan, Irbesartan, Losartan, Olmesartan, and Telmisartan) inhibit tumor development across several tumor types.

These studies underscore the complex nature of the RAS and suggests that different RAS modulators may have different effects in different tumor types. Taken together, they suggest that anti-hypertensive drugs which target the RAS have shown promise for repurposing in the cancer setting. Across several classes of drugs (β-blockers, ACEIs, and ARBs) in both in vitro and in vivo models, they have been shown to reduce tumor cell growth, migration, invasion, and metastasis in numerous cancer types. These processes comprise many of the characteristics of CSCs and of the hallmarks of cancer (135), and are consistent with the expression of some components of the RAS in high-grade disease and the associated poor survival (**Table 1**). This suggests that there is merit in repurposing RAS inhibitors for cancer treatment. Many clinical trials using this approach are currently underway, despite limited functional work and mechanistic understanding about how this approach might work in cancer patients. With the development of new agents targeting specific parts of the pathway, including the bypass loops, and the refinement of existing drugs, new opportunities are emerging for modulating the RAS pathway, either in combination with current therapies or by targeting the entire RAS and its bypass loops, and pathways converging on the RAS.

### CANCER STEM CELLS EXPRESS COMPONENTS OF THE RAS

Given the well characterized role of the RAS in both stem cell maintenance and tumorigenesis, it is possible that these functions are directed by RAS signaling in CSCs. In order to demonstrate this, it is important to first establish that CSCs express both CSC markers and components of the RAS. This has been shown to be the case in numerous cancer types, including glioblastoma (136– 138), metastases to the liver from colon adenocarcinoma (139, 140), head and neck cutaneous squamous cell carcinoma (141), and oral cavity squamous cell carcinoma affecting the buccal mucosa (142, 143), oral tongue (144–146), and lip (147, 148). In addition, components of the RAS have also been demonstrated on the tumor stem cells of benign tumors such as meningioma (149, 150), infantile hemangioma (151, 152), and pyogenic granuloma (153). Importantly, the expression of cathepsins B, D, and G in some of these cancer types (74, 138, 140, 146, 150) suggests the presence of bypass loops of the RAS which could circumvent the action of traditional RAS inhibitors and offer a potential explanation for the differing findings of cancer risk and cancer survival with long-term use of traditional RAS inhibitors. Given the presence of components of the RAS in CSCs in these cancers, it is possible that the expression of these components is controlling the differentiation and function of the CSCs within these tumors.

Despite the indirect evidence from retrospective population studies and more substantial direct evidence from in vitro studies and in vivo tumor models, very little is known about the mechanism by which RAS modulators influence tumor development. Although expression of components of the RAS has been demonstrated in CSCs, their function and how they might respond to RAS modulators has yet to be characterized. However, the fact that many clinical trials involving targeting of the RAS in cancer have taken place and are currently underway underscores the role of RAS in tumorigenesis and the need for further investigations into this system. Importantly, the findings in tumor model systems are seen consistently across a broad range of tumor types, suggesting its common role in cancer biology which may be affected through CSCs and their functions.

The expression of both components of the RAS and CSC markers in several cancer types may indicate that the CSCs

targeting Wnt signaling is known strategy for CSC elimination (157, 158). CONCLUSION The involvement of the RAS in both tumor development and stem cell maintenance suggests that these roles may converge on CSC maintenance and function. Given the ability of CSCs to promote cell migration, invasion and metastasis (17), and the

reduction of these processes by RAS inhibitors in vitro and in vivo, it may be that the success of RAS inhibitors in reducing cancer risk and improving cancer survival is due to their effects on CSCs. In support of this, components of the RAS and enzymes that constitute bypass loops of the RAS have been shown to be expressed in CSCs of several different cancer types. This offers an avenue for targeted therapies using RAS inhibitors, modulators of the bypass loops, and agents targeting other signaling pathways that converge on the RAS. Importantly, RAS inhibitors are commonly available, well tolerated and inexpensive and have been shown to be effective in controlling tumor growth in several settings. However, many of these studies have relied on immortalized cancer cell lines and xenograft tumor models, and in order to better understand the mechanisms of these drugs and the discrepancies observed in their effects clinically, models closer to the patient need to be employed. In addition, the nature of the RAS and its crosstalk with other pathways means a systemwide approach simultaneously targeting multiple key steps of the RAS is needed to achieve effective cancer control.

may be a novel therapeutic target through modulation of the RAS. It is possible that a multi-faceted strategy simultaneously targeting multiple critical points of the RAS and related signaling pathways may result in durable cancer treatments by altering CSC function. Indeed, Phase II trials in metastatic renal cell carcinoma using either Perindopril or Candesartan in combination with other agents, including a cyclooxygenase-2 inhibitor have shown potential for stabilizing the disease and reducing recurrence (154). Propranolol treatment in combination with a cyclooxygenase-2 inhibitor is well tolerated in breast cancer patients and transcriptional profiling showed the combination reduced markers of invasion and inflammation (155). Furthermore, targeting other pathways which converge on the RAS may also prove worthwhile, as Metformin selectively kills CSCs in mouse breast cancer models (156), and

## AUTHOR CONTRIBUTIONS

IR drafted the manuscript. AW, SW, PD, and ST commented on the manuscript. All authors approved the manuscript.

#### REFERENCES

Roth et al. Cancer Stem Cells and RAS

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the angiotensin I-converting enzyme gene insertion/deletion polymorphism. Clin Cancer Res. (2005) 11:2526–30. doi: 10.1158/1078-0432.CCR-04-1922

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**Conflict of Interest Statement:** PD and ST are inventors of the of the patent Cancer Stem Cells (US15/503025) and the PCT patent Cancer Therapeutic (PCT/NZ2018/050006), and the provisional patent application Novel Pharmaceutical Compositions for Cancer Therapy (US/62/711709).

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 © 2019 Roth, Wickremesekera, Wickremesekera, Davis and Tan. 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.

## Therapeutic Strategies Targeting Cancer Stem Cells and Their Microenvironment

Hao-Ran Sun1 , Shun Wang1 , Shi-Can Yan1 , Yu Zhang1 , Peter J. Nelson2 , Hu-Liang Jia1 , Lun-Xiu Qin1 \* and Qiong-Zhu Dong1 \*

1 Department of General Surgery, Cancer Metastasis Institute, Institutes of Biomedical Sciences, Huashan Hospital, Fudan University, Shanghai, China, 2 Medizinische Klinik und Poliklinik IV, Ludwig-Maximilian-University (LMU), Munich, Germany

Cancer stem cells (CSCs) have been demonstrated in a variety of tumors and are thought to act as a clonogenic core for the genesis of new tumor growth. This small subpopulation of cancer cells has been proposed to help drive tumorigenesis, metastasis, recurrence and conventional therapy resistance. CSCs show self-renewal and flexible clonogenic properties and help define specific tumor microenvironments (TME). The interaction between CSCs and TME is thought to function as a dynamic support system that fosters the generation and maintenance of CSCs. Investigation of the interaction between CSCs and the TME is shedding light on the biologic mechanisms underlying the process of tumor malignancy, metastasis, and therapy resistance. We summarize recent advances in CSC biology and their environment, and discuss the challenges and future strategies for targeting this biology as a new therapeutic approach.

#### Edited by:

Alexandre Prieur, ECS-Screening SA, Switzerland

#### Reviewed by:

Marco Falasca, Curtin University, Australia Nor Eddine Sounni, University of Liège, Belgium

#### \*Correspondence:

Lun-Xiu Qin<EMAIL_ADDRESS>Qiong-Zhu Dong<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Oncology

> Received: 26 June 2019 Accepted: 07 October 2019 Published: 24 October 2019

#### Citation:

Sun H-R, Wang S, Yan S-C, Zhang Y, Nelson PJ, Jia H-L, Qin L-X and Dong Q-Z (2019) Therapeutic Strategies Targeting Cancer Stem Cells and Their Microenvironment. Front. Oncol. 9:1104. doi: 10.3389/fonc.2019.01104

Keywords: cancer stem cells, tumor microenvironments, therapeutic resistance, mechanism, strategy

#### INTRODUCTION

Cancer remains one of the leading causes of death worldwide (1). Tumor malignancy is linked to tumor heterogeneity, which has been proposed to be driven by a minor subpopulation of cancer cells referred to as cancer stem cells (CSCs) (2, 3). This subpopulation of tumor cells have the capacity to sustain tumorigenesis and drive tumor heterogeneity, processes that underlie tumor progression, metastasis, and resistance to anti-cancer therapies (4). To date, CSCs identification has been largely based on surface markers as well as their ability to self-renew and propagate. However, CSC surface markers alone are not a reliable means of identifying these populations which has led to some confusion and controversy in the field. It is unlikely that these methods can afford a universal specific marker for the identification of these cells. However, some functional markers including the ATP-binding cassette (ABC) transporter and aldehyde dehydrogenase (ALDH) activity (5), the activation of some key signaling pathways (6), live-cell RNA, and single-cell DNA detection (7) have been found to improve CSCs identification in some instances.

The self-renewal potential and extensive clonogenic properties of CSCs are dependent on the tumor microenvironment (TME) (8, 9). The interaction between CSCs and their tumor niche is strongly linked to the characterization of CSCs (10). Through this interaction, CSCs are able to preserve the tumor heterogeneity that underlies the important malignant behaviors of invasion, metastasis, and therapy resistance (11). The influence of TME on CSCs physiology has been shown to act through intrinsic and extrinsic actions. The intrinsic mechanisms include DNA methylation or demethylation, and gene mutation, while the extrinsic

**162**

actions involve the production of diverse growth factors and cytokines by the TME leading to the activation of specific signaling pathways (12). In addition, many studies have shown that CSCs may be responsible for tumor resistance to conventional cancer therapy (4, 13). The resistance that is enhanced through the cross-talk between CSCs and the TME include activation of the DNA repair system (8, 14), increased resistance to hypoxic environments (15), and the phenomenon of epithelial to mesenchymal transition (EMT) (16). These features may help explain the therapeutic failures that are often encountered in different tumor settings.

Despite the enormous challenges seen, a series of promising new therapeutic approaches based on this biology are currently under development. Notably, targeted therapeutic approaches have emerged as important tools in treatment strategies. To this end, both the CSCs and the TME represent important therapeutic targets. Emerging research has shown that CSCtargeted approaches have proven to be effective in prolonging survival time (17). In this review, recent advances in CSC biology are summarized, and the potential challenges and future strategies for targeted therapy and combination therapy to eliminate both cancer and CSC populations are discussed.

### IDENTIFICATION OF CSC

CSCs are a small subpopulation of cells with characteristics that include the capacity to cycle slowly, self-renew, and initiate a novel tumor (18–20). Leukemic stem cells (LSCs) were first described in acute myeloid leukemia (AML), where it was demonstrated that CD34+CD38− AML include a subpopulation of LSCs with a capacity to differentiate and self-renew (20). The early study first demonstrated the existence of a unique tumor subpopulation with the ability to drive tumor progression and recurrence. CSC-like subpopulations have been subsequently isolated from a variety of solid tumors (21). Because some CSCs have been identified via specific surface markers, a door has been opened for potential targeted therapy approaches directed against these cells (22). However, because of the wide diversity underlying this general biology across tumor types, general markers for the global identification of these cells are not available.

The functional relevance of surface markers for CSC identification is still disputed (23, 24). It is suggested that CSCs may arise from normal stem cells, progenitor cells, or even more differentiated cells. In tumor patients, the expression of CSC surface markers in normal organs implies potential metastasis and poor prognosis (25). CD24, CD26, CD44, CD133, CD166, and EpCAM (epithelial cell adhesion molecule, CD326) are surface markers commonly used in CSC characterization (26). CD133 is a special marker that has been widely used for identifying CSCs in different tumor settings (27), especially in solid tumors, such as prostate (28), pancreas (29), brain (30), liver (31), colorectal (32), ovarian (33), osteosarcoma (34), and lung cancer (35). In colorectal cancer, a subpopulation of cells expressing CD133, which comprise 1% of the tumor cells, was shown to efficiently induce xenografts in vivo (36). However, CD133 expression appears to represent only one subset of CSCs and the surface marker can also be found to be ubiquitously expressed on many differentiated cells (37). EpCAM is expessed by most adenocarcinomas and is thought to participatein tumor progression (38). In liver and pancreatic cancer (29, 39), a high expression of EpCAM is associated with the dedifferentiation of tumor cells that have regained stem cell-like features. CD24 is highly expressed in embryonic stem cells (40) and has been widely detected in different tumor settings. The combined surface markers C44/CD24 have been used to identify CSCs in breast tumors (41, 42). CD26 (dipeptidyl peptidase-4, DPP4) is expressed on various cell types, which includes cells with stem traits and is thought to influence progenitor cell migration (43). CD26 is widely detected in leukemic and colorectal cancer (44). Aldehyde dehydrogenase 1A1 (ALDH1A1) has also been identified as a potential CSC marker. ALDH expression is associated with the oxidation of aldehydes to carboxylic acid. ALDH activity has proven useful for the prediction of poor tumor outcome in prostate, breast and lung cancer (45, 46). The ABC transporters are able to pump chemotherapy agents out of the cells that express these proteins. These transporters are widely expressed by CSCs and are thus thought to represent an important component for the failure of cancer chemotherapy. The expression of ABC transporters has been used to identify or isolate CSCs from solid tumors (47). Importantly, CSCs have also been functionally identified in what would represent CSC negative populations based on surface markers (48). Thus, it is generally important to make use of multiple markers to more reliably identify CSCs. To this end, the activation of CSC-related signaling pathways such as the canonical Wnt pathway, has been shown to provide an addition level of information to better identify CSCs from colon and ovarian cancer (49).

Some surface markers used to characterize CSCs are also expressed by normal stem cells. CD29 (integrin β1) is widely expressed on CSCs and also on some normal cells, and is regarded as a marker for breast cancer CSCs. CD29 is important for breast cancer cell adhesion to extracellular matrix, and is thought to promote self-renewal and chemoresistance (50). CD9 (MRP-1) is widely expressed in normal tissues. However, it can also act as an effective marker to diagnose B-acute lymphoblastic leukemia (B-ALL) and is linked to drug resistance. CD44s is frequently used as a CSC marker (51). CD44 is composed of different subtypes (CD44V1-V10) (52, 53) and is expressed by both CSCs and normal cells. CD44 expression is associated with cancer progression and metastasis (51). For example, the CD44V9 is a predictive marker in solid tumors, including head and neck squamous carcinoma and gastric cancer. CD44V3 and V6 have been shown to be linked to invasion, metastasis, and resistance to apoptosis in colorectal cancer (54). The CD44V3-7 varients are highly expressed in non-small cell lung carcinoma (NSCLC) (55, 56). In addition, CD44V6 is associated with lymph node metastasis (6). In examples of breast cancer, high expression of CD44V3, V5, and V6 have been detected and shown to be related to the invasive properties of the tumor (57, 58). ABCB5 (ATP-binding cassette transporter) is a member of the ATP-binding cassette transporter family. ABCB5 expressed by normal cells and contributes to cell proliferation

and differentiation (59). However, the expression of ABCB5 has also been demonstrated in several malignant stem cells, including ocular surface squamous neoplasm (OSSN) (60) and melanoma (61, 62). The ABCB5 subpopulation was shown to have an unlimited self-renewal potential, and is thought to foster tumor progression, metastasis, and therapy resistance (63, 64).

CSCs with unlimited self-renewal potential express potential specific markers that can help dinstinguish them from other cells. By making use of markers in CSCs, it may be possible selectively eradicate CSCs in various tumors (22, 65). While there is a growing list of markers that have been used for identification and isolation of CSCs, very few reliable specific surface markers have been found that clearly identify CSCs because CSCs, for the most part, are heterogeneous. The identification of more universal CSC markers across diverse cancer types would clearly redine the field. Finally, what is emerging is that the application of multiple markers used in combination represents the most reliable means of characterizing these cells absence the functional criteria used to define CSCs.

#### CSC MICROENVIRONMENT

Accumulating evidence suggests that cancer cells acquire a "stemness" feature in part through environment input. Because of this, even differentiated cancer cells can revert to a more dedifferentiated state which has been linked to the ability to form tumors (66). CSCs co-injected into mice with stromal cells extracted from a tumor environment form more aggressive tumors that do CSCs alone suggesting an important role for the stromal matrix surrounding CSCs, also known as the "CSC niche" (67, 68). Cancer cells in a such a niche are capable of maintaining their stemness state (12, 69). The niche can contain various cell types and growth factors providing a tumor promoting microenvironment. This can involve endothelial cells, immune cells, cancer associated fibroblasts (CAFs), various growth factors, and cytokines. In addition to these components, environment changes, such as hypoxia, and pH have been proposed to contribute to the CSC niche (70–72). The perivascular niche, which is best studied in brain tumors, is recognized as a hallmark of glioblastoma (GBM). The perivascular niche enhances GBM stemness and ability for self-renewal and invasion (73).

Low levels of oxygen, referred to as hypoxia, is an important feature of TME. Hypoxia appears to help drive the maintenance of stemness and thus malignancy of CSCs, which promotes tumor survival and metastasis (74). The hypoxia-inducible factors (HIFs) are transcription factors that are increased in response to t hypoxia, and high expression of HIFs (HIF-1α, HIF-2α) is correlated with tumor malignancy (75). The octamer-binding transcription factor 4 (Oct4) is activated by HIF-2α and is linked to control of CSCs self-renewal and an increase in the malignant potential of embryonic stem cellderived tumors (76). Another transcription factor-Sox2 is also linked to stemness through modulation of Oct4 levels in CSCs (77). A reduction in miR-145 was shown to significantly reduce expression Oct4 and Sox2, and thus lead to a decrease in the CSCs population and chemosensitivity in colon cancer (78). In pancreatic ductal adenocarcinoma (PDAC), YAP/HIF-1α signaling is activated by HGF stimulation through its receptor cMET (79). Dysregulation of YAP is related to tumor proliferation, epithelial mesenchymal transition (EMT) and therapy resistance. In the context of a low oxygen environment, CSCs can obtain energy by both OXPHOS and glycolysis activity. During hypoxia, glycolytic enzymes and glucose transporters become induced by HIF-1. Pyruvate dehydrogenase kinase 1(PKD1) plays a role in converting pyruvate to acetyl-coenzyme A (80). As an essential glycolytic enzyme, PDK1 is associated with tumor proliferation, metastasis and poor prognosis (81). In breast cancer, PDK1 stimulates glycolytic activity to stimulate cancer cells to take on stemness traits (82). The use of PDK1 inhibitors can help block glycolysis activity and also limit maintenance of breast cancer stem cells (82). In many instances, CSCs have been shown to be primarily glycolytic, or to preferentially shift from OXPHOS to glycolysis in a tumor typedependent manner. In lung cancer (83), glioblastoma (84), and PDCA (85), CSCs were also shown to utilize OXPHOS as the preferred energy production process, however, the mechanism is still unclear. To target this metabolic biology it is thought that a combined therapy targeting both aerobic glycolysis and OXPHOS dependent cells may be the most effective therapy to block CSCs.

CAFs, as a part of TME, are believed to drive tumor progression and dedifferentiation by their secretion of key growth factors and their interplay with other stromal cells. HGF secreted by CAFs was found to activate the canonical Wnt pathway and promote cancer cells to dedifferentiate to the CSCs state (26). Cytokines secreted by CAFs, such as CCL2, IGF-1, and TGF-β affect the expansion and selfrenewal of CSCs in breast, lung and gastric cancer (86, 87). In a hypoxic environment, CD44 is highly expressed by CAFs that in turns helps mediate cancer cell migration and stemness sustainability (88). The high-mobility group box 1 (HMGB1) released from CAFs was demonstrated to stimulate CSCs through the TLR4 receptor in breast cancer (89). CAFs induced expression of Notch3 is responsible for the activation of lysine demethylase 1 (LSD1) in CSCs, driving self-renewal in HCC (90, 91). In addition, CAFs facilitate tumor cells migration and metastasis indirectly through EMT. In prostate cancer, CAFs secret CXCL12 and promote EMT by inducing the expression of CXCR4 (one of the EMT phenotypes), which enhances metastasis (92). Recent reports also show that CSCs can differentiate into CAF-like cells through TGFβ secretion that promotes self-renewal and proliferation (93). CSCs also secret the Hedgehog ligand SHH that is known to increase the proliferation of CAF in the mammary tumor, and the CAFs secret factors to improve the ability of CSCs malignancy (94).

The biological cross-talk between CSCs and TME is quite complicated, and changes between different tumors and environments. By better understanding these processes, we can develop novel strategies to better target CSCs. **Figures 1**, **2** gives insight into illustration of hypoxia and CAF interactions on CSC.

## THERAPEUTIC RESISTANCE DRIVEN BY CSC AND THEIR MICROENVIRONMENT

Tumors recurrence often means poor prognosis and increased resistance to therapy (95, 96). Increasing evidence suggests that the interplay between CSCs and their TME is important in tumor development. The tumor is a complex tissue composed of different subpopulations of tumor cells and tumor-associated stromal cells. Through interaction with the microenvironment, CSCs can avoid target exposure and this may be a key to therapy resistance (97–99).

The general therapeutic resistance of CSCs allows them to escape from elimination and re-establish tumor. When the treatment cycle comes to an end, CSCs are revived from their quiescence and promote tumorgenesis (100, 101). The mechanisms by which CSCs achieve therapeutic resistance involves heightened DNA damage repair capacity, high expression of multiple drug resistance (MDR) transporters and high expression of anti-apoptosis proteins (102–105).

The DNA damaging repair (DDR) system is important in tumor progression. When under chemotherapy or radiotherapy, damaged DNA triggers the DDR, which enables CSCs to survive and thereby remain resistent to treatment. Several pathways can be activated in cancer cells includiong the double-strand breaks (DSBs) repair (homologous and non-homologous end joining), base excision repair (BER), transcription coupled nucleotide excision repair (NER), and mismatch repair (MMR) systems (106). Previous study have showen that the high expression of apurinic/apirimidinic endonuclease/redox effector factor (Ape1/Ref-1), corresponding to an activation of the BER pathway, has been implicated in the development of CSCs (107). Overexpressed Ape1/Ref-1 was also shown to maintain a low level of reactive oxygen radicals (ROS) that prevented DNA damage and cell death in CSCs (108–110). The Mre11-Rad50- Nbs1 (MRN) complex has the capacity to repair DNA and modulate cells apoptosis, and gene stability and is an important part in the DSBs pathway (111, 112). In nasopharyngeal and gastric cancer, MRN-ATM meditated DNA repair induced resistance to common chemotherapy agents, such as cisplatin and 5-FU (113, 114). The MRN complex also acts as one of the DSB pathway key elements to produce radio-resistance in various cancer types (115). Transcription factors such as forkhead box protein m1 (FOXM1), P53, glioma-associated oncogene (GLI1), and c-MYC, were also shown to be important for the DNA repair response (116, 117). Treating colon cancer with doxorubicin (a chemotherapy agent) was shown to lead to the activation of SMAD, which binds to P53 to produce chemoresistance (118).

The expression of multi-drug resistance (MDR) transporters in CSCs results in drug efflux and decreased intracellular drug concentration (119, 120). The ATP-binding cassette (ABC) transporters encompass 49 members in humans and are organized into seven subfamilies (ABCA-G) (121, 122). Three well-studied members of the family are ABCB1, ABCG2, and ABCC1 (123, 124). Overexpression of ABCG2 is associated with resistance to a large number of chemotherapy agents, such as mitoxantrone, camptothecins and flavopiridol (125).  However, this resistance could be reverted by the MiR-487a target for the expression of BRCP (126). In ovarian cancer, c-MET/PI3K/AKT pathway activation was shown to induce the expression of BRCP/ABCG2, which is important in doxorubicin resistance (125, 128). ABCB1 is also called P-glycoprotein (Pgp) or multidrug resistance gene 1 (MDR1). In AML, P-gp acts as an adverse prognostic factor for drug resistance (129). It was also found that the absence of miR-298 is related to an over expression of P-gp. The upregulation of miR-298 could reduce the expression P-gp, leading to increased concentration and cytotoxicity of doxorubicin in doxorubicin-resistant breast cells (130). In addition, oncogene kinases such as MEK1/2, ERK1/2, c-Raf, EGF, and FGF, can increase the expression of P-gp and effect drug resistance and therefore may also represent potential targets (131). The ACBC1 transporter is encoded by the MRP1gene. This subfamily plays a role in affecting MDR in lung, bladder, and breast cancer (124). In neuroblastoma, the high expression of MRP1 is associated with a poor outcome and sensitivity to chemotherapy should be regained by targeting MRP1 (132).

Hypoxia influences cancer progression, and therapy resistance, and it leads to poor outcomes. The ROS level is affected by oxygen density. In tumors, hypoxia leads to a low ROS level that in turn can be protective for CSCs and lead to therapy failure (133). HIFs are considered as negative factors for effective tumor therapy. It has been suggested that HIFs influence the pathways that contribute to the quiescence of CSCs, such as cell cycle control via cyclin dependent kinase, metabolic control via pyruvate dependent kinase, anti-apoptosis via BCL-XL, and self-renewal via OCT-4 (134–136). In some reports, HIFs are believed to be related to MDR, such as ABCG2, and to affect drug efficacy. VEGF has also been proven to be induced by hypoxia, leading to chemo/radiotherapy resistance (137, 138). In colon cancer, dual specificity phosphatase-2 (DUSP-2) was suppressed by hypoxic culture, which led to the upregulation of COX-2 expression. DUSP-2 has a negative function in cancer malignancy and COX-2 is closely related to cancer stemness, tumor growth and drug resistance (139).

Anti-apoptosis protein expression in CSCs is another component of therapy resistance. BCL2 and BCL-XL are highly expressed in breast and AML CSCs (136, 140). P53 is a tumor suppressor that is frequently mutated in most human cancers. Due to programs such as inactivation of caspase-9 and protease activating factor1 (Apaf-1), and the activation of gain-offunction, the mutant P53 shows acquisition of dedifferentiation and stemness that leads to drug resistance (141, 142). An altered apoptosis pathway has also been demonstrated to be involved in the formation of drug resistance. The high expression of Bcl-2 due to Notch and Hh signaling pathways translates into docetaxel resistance in prostate CSCs (143). Additionally, Hh signaling pathway activation in AML, especially in CD34+ leukemic cell lymphoma, induces the function of anti-apoptosis that lead to chemotherapy (144).

EMT activities in the CSC environment include wound healing, tissue fibrosis, and carcinoma progression. Non-small lung cancer (NSCLC) treated with Erlotinib targeting EGFR mutation shows more drug resistance due to mesenchymal-like expression. With the reversion of EMT, an elevated epigenetic like the expression of E-cadherin, is associated with sensitivity to the EGFR kinase inhibitor (145). It is generally accepted that the Notch pathway is associated with gemcitabine resistance in PDAC and is regulated by EMT (146). The expression of mesenchymal-like regulators such as Zeb2/SIP1 can protect cells from DNA damage-induced apoptosis in bladder cancer, leading to poor prognosis (147). In non-small lung cancer (NSCLC), EMT is thought to act in the process of quiescence. Overexpressed CSC surface markers, such as ALDH1 and CD133 in NSCLC stem cells are proposed to develop resistance to conventional therapy agents 5-FU (148). CSC-mediated therapeutic resistance relies on different mechanisms. **Figure 3** gives insight into illustration of therapeutic resistance driven by CSC and microenvironment.

### THERAPEUTIC STRATEGIES TARGETING CSC AND THEIR MICROENVIREMENTS

#### CSC Targeting Strategies

The selective targeting CSCs is a promising therapeutic strategy to eliminate the development of human cancer and reduce the risk of recurrence (149). Therapeutic strategies discussed include disrupting the central regulating signaling pathways important for the cell type, targeting specific markers, inhibition of the ABC transporters, manipulating miRNA expression, or inducing the differentiation and apoptosis of CSCs.

Signaling pathways that underlie CSC biology and have been identified as potential targets. Key pathways identified include Sonic hedgehog (Shh)/Patched (Ptch)/Smoothened (Smo), Notch/Delta-like ligand (DLL), CXC chemokine receptor 1-2/CXCL8/FAK, and Wnt pathways. Downstream transcription factors such as β-catenin, STAT3, and Nanog have also been identified as potential clinical targets (150). However, the fact that CSCs and normal stem cells share the expression of many genes and signaling pathways, as well as the redundancy of the regulatory pathways, may effectively limit the efficacy and clinical impact of the therapeutic approaches.

Drugs targeting CSC markers may play an adjunctive role together with surgery, chemotherapy, and radiotherapy (151). CD44, the transmembrane protein that is the receptor for matrix components such as hyaluronic acid selectin, collagen, and osteopontin, has been proven to help treat acute myeloid leukemia (AML) by inhibiting tumor proliferation, and increasing apoptosis (152, 153). CD133, the glycoprotein also known as prominin-1, is another well-known marker on the CSCs surface and it has been reported to be a useful target in cancers with a large CD133 subpopulation (154, 155). In addition, other drugs approved by FDA are also used for targeting CSC markers such as rituximab (anti-CD20) (156), cetuximab (anti-EGFR) (157–159).

The aberrant expression of ABC transporters, which are drug efflux pumps, is a major mechanism of chemoresistance in CSCs cells (160). Three generations of inhibitor drugs have been developed and the fourth generation is underway (49, 160), which should be more selective and less toxic. New technology has been applied to improve therapy efficacy, such as the application of miRNAs targeting specific RNAs or the use of nanomedicines for bypassing efflux pumps. However, currently no inhibitors have been approved for clinical practice. Accumulating studies have reported that different tyrosine kinase inhibitors (TKIs) including erlotinib, lapatinib, imatinib, and nilotinib can reverse drug resistance mediated by ABC transporters (161). The ability to inhibit the multiple regulatory targets of ABC transporters synergisticly, combined with other therapy strategies to overcome chemoresistance in CSCs may represent a promising approach.

CSCs have non-coding RNA profiles that are different from those seen in other cancer cells. Non-coding RNAs act as regulators in maintaining and modifying CSCs properties and functions (162). As such, they represent not only potential drugs but also therapeutic targets for the treatment of CSCs. Accumulating evidence suggests that non-coding RNAs, including microRNA (miRNA) and long non-coding RNA (LncRNA), regulate the stemness of CSCs by acting as suppressors or promoters of pathways that modulate the CSC carcinogenesis, differentiation, and EMT. In breast CSC and CD133 positive pancreatic cancer cells, miR-30 was found to be decreased and inhibited anoikis resistance (163). Three novel LncRNAs including Lnc TCF7, Lnc-b-Catm, and Lnc BRM were reported to sustain the self-renewal of CSCs by regulating the classic signaling pathways related to development and progression of liver CSC (164). Non-coding RNAs are regarded as very useful targets for potential therapeutic strategies due to their limited and selective expression in tumor tissues. MiRNAbased therapeutics are also emerging as tumor treatment options and are currently entering clinical trials (165). For example, a phase 1 clinical trial that targomiRs, minicells loaded with miR-16-based mimic miRNA and targeted to EGFR, is being evaluated in patients with malignant pleural mesothelioma and non-small cell lung cancer (166).

The impairment of apoptosis contributes to cancer development and progression, and the reactivation of apoptosis programs might be useful in the treatment of cancer. It has been reported that targeting TRAIL could cause caspase-8 reactivation, ultimately initiating mitochondrial outer membrane permeabilization and triggering the apoptosis (167). In PACA, JNK inhibition attenuated resistance of TRAIL-induced apoptosis and reduced the self-renewal capacity of CSCs (168). Finally, inhibition of NF-κB by the molecule MG-132, has also been reported to induce cell death (169). Another approach that has been evaluated focused on various means to induce CSCs differentiation. Promising agents are under research currently include the use of retinoic acid and its analogs (ATRA) for the treatment of promyelocytic leukemia (170–172). These may also show utilityto induce differentiation of glioma and breast CSCs. Other molecules such as histone deacetylase inhibitors (HDACI), tyrosine kinase have also been proposed in many CSCs studies (151). Most recent antibody targets in CSC were summarized in **Table 1**.

### Targeting the CSC Environment

The tumor environment is comprised of various components including CAFs, immune cells, multipotent stromal cells (MSCs), endothelial and perivascular cells, and their secreted factors including growth factors and cytokines.  The tumor stroma is thought ot help foster the generation and maintenance of CSCs, protect the tumor from the immune system (173), and contribute to the induction of EMT, leading to enhanced tumor progression, invasion, and recolonization as secondary tumors. Furthermore, CSCs can acquire drug resistance by interacting with niche components in TME. Thus, targeting the TME may represent an effective indirect therapeutic strategy for the treatment of CSCs and for the prevention of drug resistance.

#### Stromal Cells in the CSC Niche

It is recognized that tumor stromal cells can not only provide physical shelter for CSC by limiting drug access, but also

TABLE 1 | The antibody target in CSC through different mechanisms in different tumors.

Frontiers in Oncology | www.frontiersin.org

transporters (MDR).

promote CSC growth, migration, and metastasis by producing important growth factors, cytokines and chemokines (174). Since the cross-talk between CSCs and stromal cells can stimulate tumor aggressiveness, directly targeting the stromal cells may serve as an alternative therapeutic strategy to treat CSCs (175).

Vascular endothelial cells (ECs), a type of stromal cell in CSC niche, which are required for angiogenesis, can also secrete growth factors and cytokines that enhance the proliferation of cancer cells, and promote the maintenance of CSCs properties in head and neck squamous cell carcinoma (176, 177). Interfering with tumor EC growth and survival could in theory inhibit not only angiogenesis but also the self-replication of CSCs (178). VEGF is a strong proangiogenic factor secreted by cancer cells, that is a well-recognized therapeutic target. Various angiogenic inhibitors have been developed that can also inhibit the self-renewal of CSCs leading to reduced tumor growth. In GBM, the VEGF tyrosine kinase inhibitor—bevacizumab has proven successful in expanding survival time by targeting the perivascular niche (179). Also in GBM, the VEGF-VEGF2- NRP1 axis is seen as an attractive target in order to decrease CD133+GBM CSCs (180). Bevacizumab combined with antihepatoma-derived growth factor (HDGF) antibody has been shown to suppress CSC populations in NSCLC (181). However, in breast cancer, inhibition of VEGFR may increase CSCs population by inducing hypoxia (182). To address this it may be that use of a VEGFR inhibitor in combination witt HIF inhibition in combination therapy may provide a more effective treatment strategy (183).

CAFs represent the major component of tumor stroma andalso play an important role in cancer therapeutic resistance and radiotherapy resistance. Thus, the direct targeting of CAFs may enhance clinical outcomes. Surface markers of CAFs such as FAP, S1004A, and TEM8 have been directly targeted through administration of sibrotuzumab, 5C3, and ADC, respectively, in various tumor types (184–186). In breast cancer, CAF activation was blocked by inhibition of Hedgehog (Hh) signaling, which also increased the sensitivity of resident CSCs to chemotherapy agents (187). CAFs secret TGF-β, and the inhibition of TGF-β signaling by using LY364947 administered via nanoparticle therapy showed a potent therapeutic effect by disrupting CSC biology (188). PTK7 as a special marker of HNSCC stem cells and is demonstrated to have a close relationship with tumor persistence, metastasis, and recurrence. The use of a PTK7 inhibitor was found to increase the sensitivity of HNSCC to erlotinib (189). In addition to a direct activity on CSCs, the inhibition of important signaling pathways may represent a prospective strategy in tumor treatment. Notch signaling is over-activated in HCC, and is thought to help maintain stemness in liver CSCs by regulating LSD1 deacetylation in CAFs (190). CD10+GPR77+ CAFs were demonstrated to promote cancer stemness and chemoresistance. An antibody against GPR77 was demonstrated to reverse chemoresistance by targeting the CD10+GPR77+ CAF subset in solid tumors, such as in breast, lung cancer (191).

Tumor-associated macrophages (TAMs), feature M2-like characteristics and are important components of TME. TAMs have been demonstrated to promote CSC immunosuppressive traits leading to immune escape (192). It has been suggested that TAMs may represent potential targets for immunotherapy. In inflammatory breast cancer (IBC), tumor cells interact with immune suppressing M2-TAM leading to the production of high levels of IL-8 and CCL18 chemokines that in turn activate STAT3, which induces a CSCs-like phenotype in IBC cells and drives EMT during IBC progression (193). Targeting CXCL8/GRP/STAT3 may represent a therapeutic choice in the treatment of IBC (194). In lung cancer, the Src kinase is associated with metastasis and stemness (195). For example, it has been demonstrated that overexpressing Src in M2-TAMs induces cisplatin resistance in lung cancer. Inhibition of Src using the small molecular agent dasatinib, was found to re-sensitize lung cancer cells to cisplatin (196). CAF derived CXCL12 can help attract monocytes, which display M2 TAM characteristics. Blocking the CXCL12/CXCR4 axis significantly reduced the effect of M2 TME leading to reduced cell proliferation, migration and resistance to vineristine in OSCC chemotherapy (197). A member of the immunoglobulin family, CD47 is found to be overexpressed on the surface of many cancer types. It binds to the signal regulatory protein alpha (SIRPα) to prevent cancer cells from undergoing phagocytosis in the tumor environment (198, 199). Targeting CD47, or interfering with the CD47-SIRPα axis leads to enhanced tumor phagocytosis by macrophages and represents a promising therapeutic strategy to treat CSCs (200– 202). It has been shown in HCC that the miRNA 125 delivered via TAM exosomes may significantly suppress the CSC phenotype and limit drug resistance (203). The same function of miRNA125 or TAM exosomes have also been seen in cervical cancer (204), nasopharyngeal cancer (205), and AML (206).

#### Immunotherapy

Immunotherapy is an emerging field and the exact mechanism by which these therapies may abrogate the ability of CSCs to reinitiate tumors is still under investigation. Over the past decade, therapeutic approaches have utilized the cytotoxic T-lymphocyteassociated antigen 4 (CTLA-4) (207) or programmed death 1 (PD-1)/programmed death receptor ligand-1 (PD-L1) (208) blocking antibodies, which have yielded notable response rates and have shown a remarkable clinical response in patients with advanced cancer. Despite the recent successes, the utilization of single antibodies is often limited and leads to poor treatment outcome. To achieve improved immune responses, the use of combination strategies for checkpoint inhibitors with other therapeutics may offer a stronger response against cancer as well as higher recovery rates (209, 210). PD-1 blockade was shown to enhance a specific antitumor efficacy of streptavidingranulocyte-macrophage-CSF surface-modified bladder cancer stem cells vaccine (211). A recent study showed that targeting CSCs using the CSC-dendritic cell vaccine with CTLA-4 and PD-L1 blockades simultaneously enhanced the eradication of melanoma stem cells in the mouse model (212). In addition, CAR-T cells have produced remarkable antitumor activities in different types of tumors (213, 214). In prostate cancer and NSCLC tumor models, CAR T-cells targeted against EpCAM and EGFR antigens could successfully eradicate CSCs and cancer cells

(215, 216). Most recent target factors and chemokines in CSC were summarized in **Table 2**.

#### CONCLUSIONS AND FUTURE PERSPECTIVES

Based on the central impact of CSCs on tumor progression with accompanying poor patient outcome, CSCs-targeted therapy approaches have emerged as an important new strategy for future tumor treatments. However, the identification of CSCs remains a challenge. Markers expressed on CSCs may also be displayed by normal stem cells, which may limit the accuracy of CSCs identification and compromise the targeted treatment. In addition, CSCs appear to represent by heterogeneous populations within tumor settings. Therefore, CSC surface markers alone are not broadly reliable for their identification. The best results, absence functional criteria, results from the use of multiple markers which provide a better means of identifying CSC in specific tumor types and may provide information regarding potential drug responsiveness and tumor recurrence.

CSCs have been demonstrated to influence tumor metastasis, immune escape, and drug resistance. Targeting CSCs via their unique signaling pathways, by metabolism reprogramming, hitting the ABC transporters, and even the use of noncoding RNA, represent promising strategies to control tumor progression through CSC-based targeting. However, due to the inherent heterogeneity of CSCs the targeting a single molecule or pathway may not be an effective strategy. Combination therapy may represent the most efficient means for the treatment of CSCs.

In cross-talk with CSCs, the tissue environment plays an important role in the development of tumor metastasis and recurrence. In the TME, CSCs are thought to reside in a special "CSC niche," which helps maintain their self–renewal and stemness. Immunotherapy represents an important emerging field in tumor therapy. Recent impressive results have been seen in immune targeting of CSCs through the use of PD-1/PDL-1 inhibitors. However, some studies have reported that CSCs are less immunogenic than non-CSCs, and thereby limiting antitumor response to CSCs. CAR-T cells also hold promise in overcoming cancer resistance in different types of tumors. Thus, targeting both CSCs and TME may represent the best option in the anti-cancer approach. Although the interconnected networks between CSCs and TME are complex, and most mechanisms are still obscure, various CSC targeting agents have been developed and successfully tested in several tumor types. In contrast with the single focus of CSCs-targeted therapy, because TME components include different types of stroma cells, cytokines, and growth factors, many of them have proved to be targeted in the eradication of CSCs. However, although there is growing literature in this promising area, the therapeutics of targeting CSCs and their environment are still in its infancy. Research on CSCs and their related environments will provide new targets for the development of anti-tumor strategies. In addition, clarifying the interconnectiveness of CSCs and TME will be important for the design of effective therapeutic approaches. The focus of future trials may include combination therapies that target multiple mechanisms in the tumor. However, this field is still undeveloped, and considerable research will be required to produce viable products. Many important challenges remain, including how to achieve drug selectivity and efficacy, reduce toxicity to normal cells, reduce adverse side effects, and explore new approaches to deliver and keep an effective drug concentration in place. In conclusion, the combined regimen of CSC-targeted therapy together with conventional treatment methods shows great potential and deserves further research.

#### AUTHOR CONTRIBUTIONS

Q-ZD and L-XQ designed and supervised the entire project. H-RS, SW, and S-CY wrote the manuscript. Q-ZD and PN made significant revisions to the manuscript. All authors read and approved the final manuscript.

### FUNDING

This work was supported by the following: the National Key Research and Development Program of China (2017YFC1308604); the National Key Basic Research Program of China (2014CB542101 and 2013CB910500); National Natural Science Foundation of China (81672820, 81772563 and 81872356).

### REFERENCES

human acute lymphoblastic leukemia. Cancer Res. (2011) 71:1374– 84. doi: 10.1158/0008-5472.CAN-10-2238

**Conflict of Interest:** 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 © 2019 Sun, Wang, Yan, Zhang, Nelson, Jia, Qin and Dong. 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.

# CPT1A/2-Mediated FAO Enhancement—A Metabolic Target in Radioresistant Breast Cancer

Shujun Han1,2†, Ryan Wei 1,3†, Xiaodi Zhang1‡, Nian Jiang1‡, Ming Fan1 , Jie Hunter Huang1 , Bowen Xie1‡, Lu Zhang1‡, Weili Miao4 , Ashley Chen-Ping Butler 1 , Matthew A. Coleman1,5 , Andrew T. Vaughan1,5, Yinsheng Wang4 , Hong-Wu Chen5,6, Jiankang Liu2 \* and Jian Jian Li 1,5 \*

*1 Department of Radiation Oncology, School of Medicine, University of California, Davis, Sacramento, CA, United States, 2 Center for Mitochondrial Biology and Medicine, Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology and Frontier Institute of Science and Technology, Xi'an Jiaotong University, Xi'an, Shaanxi, China, 3 Lewis Katz School of Medicine/St. Luke's University Regional Campus, Temple University, Philadelphia, PA, United States, 4 Department of Chemistry, University of California, Riverside, Riverside, CA, United States, 5 NCI-Designated Compressive Cancer Center, School of Medicine, University of California, Davis, Sacramento, CA, United States, 6 Department of Biochemistry and Molecular Medicine, University of California, Davis, Sacramento, CA, United States*

Tumor cells, including cancer stem cells (CSCs) resistant to radio- and chemotherapy, must enhance metabolism to meet the extra energy demands to repair and survive such genotoxic conditions. However, such stress-induced adaptive metabolic alterations, especially in cancer cells that survive radiotherapy, remain unresolved. In this study, we found that CPT1 (Carnitine palmitoyl transferase I) and CPT2 (Carnitine palmitoyl transferase II), a pair of rate-limiting enzymes for mitochondrial fatty acid transportation, play a critical role in increasing fatty acid oxidation (FAO) required for the cellular fuel demands in radioresistant breast cancer cells (RBCs) and radiation-derived breast cancer stem cells (RD-BCSCs).  Blocking FAO via a FAO inhibitor or by CRISPR-mediated CPT1A/CPT2 gene deficiency inhibited radiation-induced ERK activation and aggressive growth and radioresistance of RBCs and RD-BCSCs. These results revealed that switching to FAO contributes to radiation-induced mitochondrial energy metabolism, and CPT1A/CPT2 is a potential metabolic target in cancer radiotherapy.

Keywords: breast cancer stem cells, CPT1A/CPT2, FAO, metabolism, radioresistance, breast cancer

### INTRODUCTION

Radiation therapy (RT) is the major modality in treatment of solid cancer, including breast cancer (BC), with reported clinical benefits (1, 2). A meta-analysis of 10,801 women with or without RT after breast-conserving surgery in randomized trials demonstrated that RT reduced the 10-year risk of any first recurrence (locoregional or distant tumors) from 35.0 to 19.3% and reduced the 15-year risk of BC mortality rate from 25.2 to 21.4% (1). Cancer stem cells (CSCs; also termed as tumor-initiating cells, TICs) are suggested to be the carcinogenic cell source and responsible for tumor aggressive phenotype and failure of anti-tumor therapy (3, 4). To further improve the BC long-term efficacy by RT, the mechanisms linked with the adaptive radioresistance and recurrent risk in CSCs are to be investigated (5–8).

#### Edited by:

*Alexandre Prieur, ECS-Screening SA, Switzerland*

#### Reviewed by:

*Erina Vlashi, University of California, Los Angeles, United States Patricia Sancho, Aragon Institute for Health Research (IIS Aragon), Spain*

#### \*Correspondence:

*Jiankang Liu<EMAIL_ADDRESS>Jian Jian Li<EMAIL_ADDRESS>
*†These authors have contributed equally to this work*

#### ‡Present address:

*Xiaodi Zhang, Department of Toxicology, Forth Military Medical University, Xian, China Nian Jiang, Bowen Xie and Lu Zhang, Xiangya Hospital, Southcentral University, Changsha, China*

#### Specialty section:

*This article was submitted to Pharmacology of Anti-Cancer Drugs, a section of the journal Frontiers in Oncology*

> Received: *17 April 2019* Accepted: *22 October 2019* Published: *15 November 2019*

#### Citation:

*Han S, Wei R, Zhang X, Jiang N, Fan M, Huang JH, Xie B, Zhang L, Miao W, Butler AC-P, Coleman MA, Vaughan AT, Wang Y, Chen H-W, Liu J and Li JJ (2019) CPT1A/2-Mediated FAO Enhancement—A Metabolic Target in Radioresistant Breast Cancer. Front. Oncol. 9:1201. doi: 10.3389/fonc.2019.01201*

**176**

Elucidation of the metabolic dynamics of resistant breast cancer cells (RBCs) will help to identify metabolic targets to synergize the efficacy of RT. The theory of aerobic glycolysis in malignant cells (Warburg Effect) (9–11) is being updated, with emerging new evidence indicating an adaptive energy reprogramming in tumor cells with mitochondrial reactivation and oxidative phosphorylation (12, 13). We have reported that mitochondrial MnSOD activity is required for radioresistance in BC cells (14, 15) and radiation can enhance mitochondrial OXPHOS in tumor cells (16). Enhanced mitochondrial bioenergetics are found to be the major cellular fuel supplement for cell cycle progression (17–19), tumor aggressive phenotype and metastasis (20). Accumulating new evidence indicates that reprogramming in mitochondrial metabolism is actively involved in tumor proliferation and metastasis (12, 21–23). Mitochondrial energy output is also required for nuclear DNA repair after IR (24), and mitochondrial FAO is linked with BC metastasis (25, 26). With such a flexible adaptive energy metabolism detected in the malignant cells (13, 27), it is reasonable to look into the deep mechanistic insights of reprogramming mitochondrial bioenergetics in aggressive tumors, especially the RBCs.

Under genotoxic stress conditions, such as chemotherapy or radiotherapy, tumor cells must acquire additional cellular fuel resource to meet the increased demands of energy consumption for damage repair and survive (24). In addition to glucose, fatty acids with high ATP yield are a relevant energy-rich source in cancer cells under such genotoxic crisis. The FAO-mediated mitochondrial bioenergetics has been assumed to play a critical role in cell proliferation, cancer stemness and chemoresistance (28–30). Inhibition of mitochondrial FAO by etomoxir impairs NADPH production and increases reactive oxygen species (ROS), resulting in ATP depletion and cell death in human glioblastoma cells (31). In mitochondrial FAO pathways, targeting CPT1A generates clinic benefits in RT for nasopharyngeal carcinoma patients (32). However, the precise network of FAO enhancement in reprogramming mitochondrial energy metabolism, especially in resistant breast cancer cells, remains to be elucidated.

To mimic clinical radioresistance, in this study, RBCs were generated from wild type breast cancer cells via continuous ironizing radiation (IR), and the radioresistant BCSCs cells were sorted from the RBCs (8), termed as radiation-derived BCSCs (RD-BCSCs). Using proteomics of RD-BCSCs and CRISPR-mediated FAO gene editing, here we revealed a novel mitochondrial lipid metabolic reprogramming in RBCs and RD-BCSCs. The mitochondrial fatty acid oxidation (FAO) was enhanced in both RBCs and RD-BCSCs and linked with recurrent BC and a worse prognosis in BC patients. Blocking FAO by CRISPR-mediated CPT1A/CPT2 KO inhibited aggressive phenotype of the radioresistant BC with downregulation of the ERK pathway, indicating a potential metabolic target in breast cancer radiotherapy.

### MATERIALS AND METHODS

#### RBCs, RD-BCSCs, and Other Reagents

MCF7 and MDA-MB-231 human breast cancer cells were purchased from ATCC (Manassas, VA, USA). MCF7/C6 radioresistant clone is a single clone surviving from MCF7/WT cells after fractionated ionizing radiation treatment (2 Gy × 15) (33, 34). MDA-MB-231/C4 are radioresistant breast cancer cells surviving from MDA-MB-231/WT cells after fractionated radiation (2 Gy × 30) (35). MCF7, MCF7/C6, MDA-MB-231, and MDA-MB-231/C4 cells were cultured in DMEM medium with 10% fetal bovine serum (FBS, Life Technologies, NY, USA) and 1% penicillin/streptomycin (Life Technologies). RD-BCSCs were sorted as previously described (8, 36). Cell pellets of MCF7/C6 were rinsed with cold PBS with 2% FBS and then suspended with PBS containing 0.5% FBS and 0.5 mg/mL PI (Sigma, St. Louis, MO, USA). Then, the cell suspension was sorted using Cytopeia influx Cell Sorter (BD Biosciences, San Jose, CA, USA). The antibody against HER2/neu was conjugated to allophycocyanin (APC, BD Biosciences, San Jose, CA), anti-human CD44 was conjugated to FITC, and human CD24 was conjugated to phycoerythrin (PE; Invitrogen, Carlsbad, CA). Cell viability was assessed by 7-AA staining during cell sorting and then determined by trypan blue exclusion after sorting (**Figure S1A**). The isolated RD-BCSCs (CD44+/CD24−/low/HER2+) were maintained in CSC medium containing free serum and supplemented with B27 (Life Technology, Carlsbad, CA, USA), 20 ng/ml EGF (Biovision, Mountain View, CA, USA), 20 ng/ml basic-FGF, and 4µg/ml heparin (VWR, Philadelphia, PA, USA). Cells were cultured in ultralow-attachment Petri dishes with 5% CO2 at 37◦C. All cell lines were tested mycoplasma free before experiments. Etomoxir was purchased from Sigma-Aldrich. Oil Red O was obtained from Millipore Sigma (St. Louis, MO, USA).

#### Western Blot

Western blot was performed as previously described (37). Briefly, the cell lysates were separated on SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% milk for 1 h, and then incubated with primary antibodies with shaking at 4◦C overnight. In the next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein blots were developed using an enhanced chemiluminescence western blot detection system (BioRad, Hercules, CA, USA). Antibodies against HER2/Neu (C-18, #SC284), CPT2 (G-5, #SC-377294), HADHA (E-8, #SC374497), HADHB (E-1, #SC-271495), ERK1/2 (C-9, #SC514302), and c-Fos (E-8, SC#166940) were all diluted at 1:500 and purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-CPT1A (clone D3B3, #12252), Phosphop44/42 MAPK (Thr202/Tyr204, clone 197G2, #4377), Phospho-GSK3 β (Ser9, #9336), GSK3 β (clone 27C10, #9315), Phospho-STAT3 (Ser727, clone D8C2Z, #94994), and Phospho-JNK (Thr183/Tyr185, #9251) were from Cell Signaling Technology (Beverly, MA, USA). Antibody against ACAD9 (#3170340688) was purchased from Novus Biologicals (Littleton, CO, USA). Anti-β-actin at 1:2000 (#A2066) was from Sigma-Aldrich.

### Fatty Acid Oxidation (FAO) Assay

FAO measurement was following the manufacturer's instruction of Fatty Acid Oxidation Assay Kits from Abcam (ab217602). Briefly, 3×104 cells were seeded per well in 96-well plates and cultured overnight. Then the cells were rinsed twice with 100 µl prewarmed FA-Free medium followed by adding 90 µL prewarmed FA Measurement Medium. The wells without cells were used as signal control. A total of 85 µL of FA-Free Measurement medium was added to the wells, and 5 µL of BSA control were included as the FA-free control. All wells except the blank control had 10 µL Extracellular O2 Consumption Reagent added. The FAO activator FCCP (0.625µM) and inhibitor Etomoxir (40µM) were added as the positive and negative controls. Then the wells were sealed with 100 µL pre-warmed mineral oil, and the FAO was measured using the condition as Extracellular Oxygen Consumption. The results were normalized by the protein concentration with the cells in each sample under the BCA assay.

#### Lipid Accumulation Assay

Breast cancer cells were plated and cultured in 96-well plates. In the next day, cells were treated with 250µM free fatty acid (oleic acid: palmitate acid = 2:1) for 48 h. After being washed with PBS twice, cells were fixed with 4% paraformaldehyde under room temperature (RT) for 30 min. Cells were washed with sterilized water once and added into 50 µL Oil Red working solution; they were then incubated for 15 min at RT. Then 50 µL 60% isopropanol was added to the cells for 20 s at RT. Finally, the cells were washed with water twice, and the images were obtained using a Nikon microscope (Eclipse, E1000M, Japan). The red oil dye was eluted with 50 µL DMSO and incubated for 10 min with gentle shaking. The lipid accumulation results were determined by the fluorescence microplate spectrophotometer (Molecular Devices) at 510 nm.

### Oxygen Consumption

Extracellular Oxygen Consumption detection was performed following the instruction of kit from Abcam (ab197243,) with 3 × 104 cells seeded per well in 96-well plates and cultured at 37◦C overnight. The cell medium was replaced with 150 µl fresh culture media followed by adding 10 µl of extracellular oxygen consumption reagent. The wells without oxygen consumption reagent were used as blank control. Wells with Etomoxir (40µM) added were included as the negative control. Wells had 100 µl of pre-warmed mineral oil added to avoid the air bubbles. The plates were read immediately in a fluorescence microplate spectrophotometer (Molecular Devices) at 37◦C. The signals were collected at 1.5 min intervals for 90–120 min at Ex/Em = 380/650 nm. The results were normalized by protein concentration of the cells in each sample under the BCA assay.

#### MTT Assay

Breast cancer cells were seeded in 96-well plates for 24 h. After they proliferated to about 90% confluence, the cells were added into 50∼100 µL MTT solution (M-2128, Sigma) and cultured at 37◦C for 2 h. The results were measured in a microplate spectrophotometer (Molecular Devices) at 540 nm.

#### Cell Apoptosis Assay

Breast cancer cells were rinsed by cold PBS twice, collected and stained using the Annexin-V/PI kit (Biosource, Invitrogen, Carlsbad, CA, USA) according to manufacturer's protocol. Stained cells were analyzed by flow cytometry (Becton Dickinson canto II, BD, NJ, USA). Data were analyzed using Flowjo software (Three Star, Inc., Ashland, OR, USA).

### Colony Formation Assay

For measuring cellular clonogenicity, 1 × 103 cells were seeded into 6-well plates and treated with or without 5 Gy radiation in the next day. Cells were then cultured for 14 days at 37◦C. The colonies were fixed and stained with Coomassie blue, and then colony formation rate was determined by counting colonies in each group. Finally, the colony images were observed and recorded by a Nikon microscope (Eclipse, E1000M, Japan) (8).

### Immunohistochemical Staining (IHC)

The slides of primary biopsy tissues and recurrent tumors from patients with breast cancer were tested by immunohistochemistry (IHC) using Vectastain ABC kit and DAB Peroxidase Substrate kit SK-4100 (Vector Laboratories, Burlingame, CA, USA). The prepared tumor slides were firstly deparaffinized, hydrated, and then covered with blocking buffer for 1 h after heat-induced epitope retrieval. The slides were incubated with anti-CPT1A (Cell Signaling Technology, #12252) and anti-CPT2 (Santa Cruz, sc-20671) at 1:200 at 4◦C overnight, followed by washing with PBST three times, and then incubated with the secondary antibody for 30 min at RT. The slides were then covered with ABC solution for 30 min on the shaker at RT. The slides were incubated with DAB solution about 2 min and then transferred to hematoxylin, HCl solution and Li2CO3 solution quickly several times. Finally, the slides were dehydrated and sealed. The slides were observed and recorded by Nikon microscope (Eclipse, E1000M).

### CRISPR-Mediated Gene Editing

The single guide RNA (sgRNA) was designed according the CRISPR Design in Zhang Lab https://zlab.bio/guide-designresources. We created oligonucleotide to target genes CPT1A and CPT2. The sgRNA sequences are designed as follows: human CPT1A: CTCCGGACGGGATTGACCTG; human CPT2: CGGGGCCCCGCGGTTGGTCC. The LentiCRISPRv2 backbone was used, which contains the hSpCas9 and sgRNA expression cassettes. Plasmids were purchased from the Addgene plasmid repository (Addgene #52961) (https://www. addgene.org/). Backbone LentiCRISPRv2 was annealed to oligonucleotides following the Zhang Lab GeCKO protocol and packaged into lentiviruses. The Lentiviral particles were produced in HEK293T cells following the protocol from Addgene (38), and breast cancer cells were infected with lentiviruses and selected with 0.3µg/ml puromycin. Western blot analysis was performed to identify cell colonies with gene deficiency.

#### Proteomics of RD-BCSCs and MCF7 Cells

The protein mixture from total cell lysates of RD-BCSCs and MCF7 was first treated with dithiothreitol and iodoacetamide for cysteine reduction and alkylation, respectively. The protein samples were then digested using modified sequencing-grade trypsin (Roche, Basel, Switzerland) at an enzyme/substrate ratio of 1:100 in 50 mM NH4HCO3 (pH 8.5) at 37◦C overnight. The peptide mixture was subsequently dried in a Speed-vacuum and desalted by employing OMIX C18 pipet tips (Agilent Technologies, Santa Clara, CA, USA), reconstituted in water and subjected to LC-MS and MS/MS analyses on a Q Exactive Plus mass spectrometer equipped with a nanoelectrospray ionization source. Samples were automatically loaded from a 48 well microplate autosampler using an EASY-nLC 1200 system (Thermo Fisher Scientific, Rockford, IL, USA) at 3 µL/min onto a biphasic precolumn (150µm i.d.) comprised of a 3.5-cm column packed with 5µm C18 120 Å reversed-phase material (ReproSil-Pur 120 C18-AQ, Dr. Maisch). The biphasic trapping column was connected to a 20-cm fused-silica analytical column (PicoTip Emitter, New Objective, 75µm i.d.) with 3µm C18 beads (ReproSil-Pur 120 C18-AQ, Dr. Maisch). The peptides were then separated using a 180-min linear gradient of 2–45% acetonitrile in 0.1% formic acid and at a flow rate on 250 nL/min. The mass spectrometer was operated in a data-dependent scan mode. Full-scan mass spectra were acquired in the range of m/z 350–1,500 using the Orbitrap analyzer with a resolution of 70,000. Up to 25 of most abundant ions found in MS with a charge state of 2 or above were sequentially isolated and collisionally activated in the HCD cell with collision energy of 27 to yield MS/MS.

### Bioinformatics Analysis

Maxquant (Version <IP_ADDRESS>) was used to analyze the LC-MS and MS/MS data for the identification and quantification of proteins in the LFQ mode (39). The maximum number of mis-cleavages for trypsin was two per peptide. Cysteine carbamidomethylation was set as a fixed modification. Methionine oxidation and phosphorylation on serine, threonine, and tyrosine were set as variable modifications. The tolerances in mass accuracy for MS and MS/MS were both 20 ppm. Maximum false discovery rates (FDRs) were set to 0.01 at both peptide and protein levels, and minimum required peptide length was six amino acids. The LC-MS and MS/MS protein data were also analyzed with functional clustering. Of all proteins in our total protein array, only proteins that showed levels of detection were submitted to DAVID Bioinformatics Resources v6.7 (https://david.ncifcrf. gov/). Parameters were established for our functions of interest with a cutoff of p < 0.05.

### Category Selection of Proteomics

DAVID Bioinformatics Resources provide a wealth of information within the Gene Ontology Tool for Biological Function (40, 41). Different broad categories were generated to profile the cluster of proteins related to varied cellular functions including mitochondrial bioenergetics and lipid metabolism as well as FAO. We used the Uniprotein tagging system, UPKeyword due to the high number of hits in the protein list.

#### Tumorsphere Formation

Tumorsphere assay was performed as described (42). Cells were sieved with 40µm cell strainers (Fisher, Failawn, NJ, USA) and single-cell suspensions were seeded into 60 mm Petri dishes. The cells were grown in serum-free mammary epithelial basal medium (MEBM, Lonza, Walkersville, MD, USA), supplemented with B27, 20 ng/ml EGF, 20 ng/ml basic-FGF, and 4µg/ml heparin. Cells were then cultured for 10 days, and tumor spheres were counted under light microscopy.

### Three-Dimensional (3D) Morphogenesis Assay

MCF7 and RD-BCSC cells in 40 µL plug of Matrigel (growth factor reduced and phenol red free, Becton Dickinson, Plymouth, UK) were plated to the well of an 8-well LabTek Chambered coverglass (Nunc, Rochester, USA) at 37◦C for 30 min. On ice, cells were prepared at a concentration of 5,000 cells/ml in KSFM supplemented with 5 ng/ml EGF, 2% (v/v) FCS, 4% (v/v) Matrigel, and 0.2 mL of this cell solution was plated onto the Matrigel plug and incubated for 30 min at 37◦C, after which 0.2 mL of growth medium was added (KSFM supplemented with 5 ng/mL EGF, 2% (v/v) FCS). Culture medium was changed every 2–3 days. At day 10, morphology was assessed by phase microscopy and cells were fixed and processed for immunofluorescence microscopy analysis.

### Statistical Analysis

Statistical significance of differences was evaluated using twotailed student t-test for two groups' comparison or one-way ANOVA test where multiple groups were involved. p < 0.05 was considered statistically significant.

## RESULTS

### Mitochondrial FAO Is Enhanced in Radioresistant Breast Cancer Cells

Lipid metabolism has been linked with cancer therapy response (28–30). Here we address the question of whether reprogramming mitochondrial FAO plays a key role in breast cancer radioresistance. Two radioresistant BC (RBC) cell lines (MCF7/C6 and MDA-MB-231/C4) isolated from surviving MCF7 and MDA-MB-231 residues with HER2 induction and aggressive phenotype after chronical radiation (**Figure S1A**) (8, 35) showed enhanced expressions of mitochondrial FAO genes CPT1A, CPT2, HADHB, and ACAD9 (**Figure 1A**). We then measured the mitochondrial FAO activity by 18C-labeled unsaturated fatty acid oleate as the substrates with CPT1A specific inhibitor Etomoxir (ETX) and the FAO activator FCCP as negative and positive controls, respectively, demonstrating a significant elevation of FAO activity in MCF7/C6 vs. wild type MCF7 cells (**Figure 1B**). Furthermore, enhanced lipid turnover rate was detected in MCF7/C6 cells loaded with free fatty acid (FFA), which was contrasted with the markedly accumulation of FFA in the wild type MCF7 cells (**Figure 1C**), indicating enhanced FAO metabolism in RBC cells.

We next determined whether the FAO is enhanced in the human recurrent breast tumors compared to the primary tumors. CPT1 and CPT2 are the rate-limiting transporters and play a key role in mitochondrial long-chain FAO and lipid metabolism. Remarkably, the enhanced co-expression of CPT1A (an isoform of CPT1) and CPT2 was mostly detected in the pathological sections of recurrent BC tumors compared to the paired original biopsy specimens in a group of 12 BC

FIGURE 1 | FAO is enhanced in radioresistant BC cells and recurrent BC and linked with a poor prognosis in BC patients. (A) Western blot of a cluster of FAO enzymes and HER2 in wild type MCF7, MDA-MB-231, and their counterpart radioresistant MCF7/C6 and MDA-MB-231/C4 cells. (B) FAO activity assay of MCF7 and MCF7/C6 cells with MCF7/C6 treated with FAO inhibitor Etomoxir (40µM) as a negative control and FAO enhancer FCCP (1µM) as a positive control. (C) Fatty acid turnover rate in MCF7 and MCF7/C6 cells treated with or without 250µM Free Fatty Acid (FFA; oleic acid: palmitic acid = 2:1) for 24 h before Oil Red staining *(Continued)* FIGURE 1 | (left: quantitation of lipid accumulation; right: representative images of FFA accumulation). (D) Representative IHC of CPT1A and CPT2 in biopsy and recurrent BC (left). Quantitation of IHC was achieved by scoring staining intensity and positive cells (right). (E) Elevated CPT1A/CPT2 expression correlates a worse overall survival of BC patients in the TCGA database http://bioinformatica.mty.itesm.mx:8080/Biomatec/SurvivaX.jsp. Error bars in (B–D) represent the mean ± standard deviation. Significance was determined by one-way ANOVA test, \**p* < 0.05, \*\**p* < 0.01, \*\*\**p* < 0.001.

patients **(Figure 1D)**. In agreement, the TCGA database revealed a poor prognosis in BC patients with increased expression of CPT1A or CPT2 **(Figure 1E)** (http://bioinformatica.mty. itesm.mx:8080/Biomatec/SurvivaX.jsp). Together, these results indicate that reprograming mitochondrial FAO contributes to BC radioresistance and worse prognosis.

### CPT1A/CPT2 Mediated FAO Is Required for Radioresistant Breast Cancer Stem Cells

Our previous results have indicated that mitochondrial energy enhancement is involved in BC aggressiveness due to HER2 expression (15, 43) that is confirmed in **Figure 1A**. Following the standard biomarkers of breast cancer stem cells (BCSCs) (4) and our established BCSCs from MCF7/C6 with HER2 induction as radiation-derived BCSCs (RD-BCSCs; CD44+/CD24−/low/HER2+ with enhanced ALDH activity (8), we compared the tumorspheres from RD-BCSCs and MCF7 shown in **Figure 2A**. The tumorsphere of RD-BCSCs showed severely disorganized structure with altered distribution of cellular polarity protein (DLG, red) and enhanced HER2 expression (green), indicating an aggressive phenotype of RD-BCSCs.

Consistent with the results of RBC in **Figure 1A**, the key FAO enzymes, CPT1A, CPT2, and HADHB, were also enhanced in RD-BCSCs **(Figure 2B**), and both basal and irradiated FAO levels were elevated in RD-BCSCs whereas no significant FAO elevation was detected in irradiated MCF7 compared to basal level, although the proteomics data showed enhanced mitochondrial proteins in both irradiated both MCF7 and RD-BCSCs (**Figure 2C**, **Figures S2A, S3**), suggesting that reprogramming FAO is an unique feature of RD-BCSCs. This is further evidenced by the specifically enhanced cluster of factors in lipid metabolism rather than other cellular proteins in irradiated RD-BCSCs (47 to 81 in RD-BCSCs vs. 55 to 76 in MCF7); and the protein intensity was also more increased in irradiated RD-BCSCs (5.24 fold) contrasted to 2.31-fold in MCF7 **(Figures S1B,C)**. The FAO inhibitor Etomoxir (ETX) significantly enhanced basal and radiation-induced apoptosis with inhibited tumorsphere formation and ERK activity in RD-BCSCs (**Figures 2D–F**, **Figure S2B**). In agreement, blocking CPT1A/CPT2 by CRISPRmediated gene deficiency **(Figure 2G)**, enhanced apoptosis level and reduced the tumorsphere formation in RD-BCSCs upon IR (**Figures 2H,I**, **Figure S2C**). Additional proteomics evidence suggested that mitochondrial proteins were more enriched in the RD-BCSCs compared to MCF7 cells after radiation. The total mitochondrial protein counts increased from the same basal 117 in MCF7 and RD-BCSCs to 152 and 163, 29.9, and 39.3% respectively, whereas protein intensity had a 2.64-fold increase (4.50E+10 to 1.06E+11) in RD-BCSCs compared to a 1.83 fold increase in MCF7 cells **(Figures S3A,B)**. Additionally, we further analyzed the mitochondrial proteins and found that FAO metabolism was also demonstrated with an increased protein number, from 11 to 17, in irradiated RD-BCSCs including CPT1A and CPT2 (**Figures S4A,B**, **Table S1**), indicating that enhanced mitochondrial FAO contributes to radioresistance of RD-BCSCs.

### ERK Activation Is Linked With Mitochondrial FAO Enhancement

To explore the key factors responsible for FAO-mediated radioresistance, we tested an array of cell proliferating factors in the two RBC cell lines by CRISPR-mediated knockout of CPT1A and CPT2 (**Figure 3A**). Strikingly, the activated form of ERK1/2 was absent in the CPT1A and CPT2 KO cells, although other cell growth factors including phosphor-GSK3β, phospho-STAT3, and phospho-JNK were also reduced to a certain degree (**Figure 3B**). Alternatively, a dose-dependent inhibition of phospho-ERK and its downstream effectors was also determined in MCF7/C6 cells with increasing concentrations of ETX (**Figure 3C**). It turned out that 200µm ETX could dramatically block the phosphorylation of ERK1/2. The dependence of ERK1/2 kinase in the FAO-mediated resistance was again evaluated by a rescue experiment, in which the FAO activity was inhibited by ETX and then activated by L-carnitine. As expected, the induction of phospho-ERK1/2 upon radiation was significantly inhibited by etomoxir while enhanced by L-carnitine. Of note, the combination of etomoxir and Lcarnitine treatment ablated the phospho-ERK1/2 induction by L-carnitine treatment upon radiation (**Figures 3D,E**). Together, our inhibition and rescue experiments consistently demonstrated that FAO mediated radioresistance is linked with ERK1/2 activation for the aggressive phenotype of radioresistant breast cancer cells.

### Deficiency of CPT1A or CPT2 Reduces OCR, Cell Viability, and Slowdown Fatty Acid Turnover Rate in RBC Cells

By comparing the metabolic features of BC cells and RBCs, we found that both the oxygen consumption rate and cell viability were enhanced in MCF7/C6 cells compared to MCF7 cells, but remarkedly reduced by ETX or in CPT1A/CPT2 KO (**Figures 4A,B**). Additionally, ETX treatment and CPT1A/CPT2 KO increased FFA accumulation, indicating a slower fatty acid turnover (**Figure 4C**). The colony formation assay was used to evaluate the cell survival rates when given radiation therapy with FAO inhibition in RBCs. The colonies in the RBC MCF7/C6 and MDA-MB-231/C4 cells were more abundant than the basal clonogenic capacity of the parental MCF7 and MDA-MB-231 cells, indicating an enhanced aggressive phenotype of RBCs. However, the etomoxir treatment and

CPT1A/CPT2 KO markedly reduced the survival colony rate in MCF7/C6 and MDA-MB-231/C4 cells (**Figures 5A–D**). IR induced cellular apoptosis was also evaluated in the RBCs. The FAO inhibition increased radiation-induced apoptosis from 10 to 34% (etomoxir treatment), 31% (CPT1A KO), and 36% (CPT2 KO) in MCF7/C6 cells and from 3.8 to 5% (etomoxir treatment), 9.2% (CPT1A KO), and 7.7% (CPT2 KO) in MDA-MB-231/C4 cells (**Figures 6A,B**). FAO inhibition also reduced tumorsphere formation from 58 to 34.5 (etomoxir treatment), 26.5 (CPT1A KO), and 24.5 (CPT2 KO) with IR in MDA-MB-231/C4 cells (**Figure 6C**). Together, these results indicate that CPT1A/CPT2 mediated FAO enhancement is required for the energy demands and cell survival in RBCs. In summary (**Figure 7**), our results reveal that

(C) Western blot of MCF7/C6 cells treated with increased doses of ETX for 24 h followed by 5 Gy IR. (D) Western blot of pERK in MCF7/C6 cells treated with ETX, L-carnitine (an FAO enhancer) or combined followed by 5 Gy IR. (E) Western blot of pERK in MDA-MB-231 cells were treated ETX, L-carnitine (an FAO enhancer) or combined followed by 5 Gy IR.

reprogramming mitochondrial FAO is the major cellular energy supplement in radioresistant breast cancer cells. Such adaptive mitochondrial energy metabolism is linked with the clinical outcome of BC patients treated with radiotherapy. We also reveal that the ERK-mediated prosurvival pathway is a potential downstream target in FAO-mediated aggressive proliferation in BC with enhanced activation of HER2 leading to promoted cell proliferation.

#### DISCUSSION

It is highly clinically relevant to reveal the major cellular energy driving the growth of therapy-resistant cancer cells. The concept of aerobic glycolysis in cancer metabolism (Warburg Effect), believing the deficient mitochondrial function in cancer cells, has been updated with accumulating results of active tumor metabolic to oncogenic (9) and genotoxic stress including

ionizing radiation (16, 44, 45). A dynamic metabolic feature is linked to the adaptive reprogramming in energy supply, which is required to meet the increased cellular fuel demands for cancer cells to repair the damage and survive anti-cancer therapy. Identification of the metabolic targets required for tumor cell survival under genotoxic stress conditions such as chemoradiotherapy are necessary for improving the therapeutic efficacy. Among other hallmarks of cancer cell progression, cancer cells can adjust energy metabolism to meet the demands of cellular fuel consumption for proliferation and survival to therapeutic stress conditions (11). In this study, we revealed a unique

metabolic feature in RBCs and radioresistant breast cancer stem cells (RD-BCSCs). It showed that FAO enzyme expression and mitochondrial FAO activity were enhanced in response to radiation compared to wild type breast cancer cells. Elevated mitochondrial FAO is required for cell growth and survival in response of radiation therapy. Of note, CPT1A/CPT2 expression was also elevated in human recurrent breast cancer tissues compared to biopsy tumors. CRISPR/Cas9 mediated deficiency of CPT1A/CPT2 efficiently enhanced sensitivity of RBCs and RD-BCSCs to radiation treatment. Finally, we provided evidence that mitochondrial FAO likely functions through the ERK

quantitated (B). (C,D) Same as (A,B) except that MDA231, MDA231/C4 CPT1A KO, MDA231/C4 CPT2 KO, and MDA231/C4 cells were tested. In all experiments,

*n* = 3; Error bars in (B,D) represent mean ± SD; significance was determined by one-way ANOVA test, \*\**p* < 0.01, \*\*\**p* < 0.001.

signaling pathway to confer resistance to radiation therapy in RBCs.

Our proteomics data demonstrate a different adaptive scale of RD-BCSCs vs. wild type MCF7 cells with a higher mitochondrial protein number and density in RD-BCSCs than MCF7 cells. Such differential stress response may apply to the varied response to therapeutic radiation in primary and recurrent/metastatic breast cancers. The plasticity of human cancer cells and the genetic-independent acquisition of therapeutic resistance may be tightly associated with metabolic reprogramming. CSCs are capable of adjusting their unique metabolic plasticity in order to respond in a timely manner and adapt to hostile environments (46). The current study revealed that enhanced FAO could be a critical step for therapy-resistant cancer cells, especially cancer stem cells, to have a survival advantage, thus they could be used for identifying and developing effective metabolic targets. Altered metabolism is served as one of the hallmarks of cancer and has also been observed in CSCs (47, 48). CSCs have been identified in many types of solid tumors and often result in recurrence and both chemo- and radioresistant in tumors because of their self-renewal and tumorigenic properties (49–51). It has been shown that blocking thioredoxinand glutathione-dependent metabolism can enhance radiation response of BCSCs (52), indicating that in addition to FAO enhancement, other metabolites could be critical for the survival advantage of CSCs. Accordingly, metabolic adjustment in the CSC populations under different therapeutic modalities should be further investigated.

The cellular energy shift may require a different signaling network to drive cancer cell proliferation and radioresistance. Our current data also reveal a potential crosstalk between CPT1A/CPT2-mediated lipid metabolism and ERK1/2 controlled cell proliferation (**Figure 7**), implicating a cooperative network of mitochondrial FAO in response to radiation in resistant cancer cells. Although currently there is no direct evidence supporting mitochondrial FAO-mediated ERK1/2 activation, the enhanced mitochondrial products including ATP from TCA cycle may increase the MEK-ERK1/2 cascade. High ATP concentration is indicated in the tumor microenvironment that can activate P2Y2 receptors to enhance BC cell migration through the activation of a MEK-ERK1/2 pathway (53). In addition, mitochondrial OXPHOS can be enhanced by Cyclin B1/CDK1 that can be relocated into mitochondria by radiation leading to phosphorylation of SIRT3, a key keeper for mitochondria homeostasis at the Thr150/Ser159 (54). Furthermore, we have recently observed that mitochondrial homeostasis is enhanced by SIRT3-regulated CTP2 activity in normal mouse liver cells via FAO (unpublished data). Thus, the CPT1A/CPT2-mediated FAO activity may be differently regulated in normal and cancer cells, which warrants further studies.

In summary, this study reveals a previously unknown feature of reprogramming mitochondrial FAO in RBCs due to enhanced CPT1A/CPT2. Thus, targeting CPT1A/CPT2 as well as other mitochondrial FAO elements may serve as a metabolic target to enhance the efficacy of breast cancer radiotherapy.

### DATA AVAILABILITY STATEMENT

All datasets generated for this study are included in the article/**Supplementary Material**.

### AUTHOR CONTRIBUTIONS

JL, JJL, and SH designed the study, wrote the manuscript, and were involved in revising the manuscript. SH, RW, and MF performed experiments with the help of NJ, BX. LZ, XZ, AB, MC, JH, and AV helped to analyze the data, prepare and revise the manuscript. H-WC provided the instruction for CRISPR technology. WM and YW generated and analyzed the proteomics data. All authors have approved the final version.

### FUNDING

This work was partially supported by National Cancer Institute Grants RO1 CA152313 (to JL) and University of California Davis NCI-designated Comprehensive Cancer Center supported by the CCSG Grant awarded by the National Cancer Institute (Dr.

#### REFERENCES

1. Early Breast Cancer Trialists' Collaborative Group (EBCTCG), Darby S, McGale P, Correa C, Taylor C, Arriagada R, et al. Effect of Primo Lara; NCI P30CA093373). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U. S. C. Section 1734 solely to indicate this fact.

## ACKNOWLEDGMENTS

We thank Dr. Irmgard Feldman at the UC Davis Comprehensive Cancer Center Biorepository which was funded by the UC Davis Comprehensive Cancer Center Support Grant (CCSG) awarded by the National Cancer Institute (NCI P30CA093373) in sharing human breast cancer specimens.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc. 2019.01201/full#supplementary-material

Figure S1 | (A) Schematic representation of the process for generating RBCs and RD-BCSCs. (B) Functional clustering of proteomics of MCF7 and RD-BCSCs via DAVID bioinformatics indicating the protein numbers in categories inducing lipid metabolism, oxidation reduction, DNA repair, stress response, glycolysis, and ATP synthesis in MCF7 cells with or without IR. The intensities of proteins in lipid metabolism were shown in right. (C) Repeated proteomics analysis with RD-BCSCs cells, note a group of key FAO enzymes including CPT1A and CPT2 were enhanced by radiation (marked in red in the right).

Figure S2 | (A) FAO measured in MCF7 and RD-BCSCs cells with or without 5 Gy IR treatment for 24 h. RD-BCSCs treated with 40µM Etomoxir (ETX) were used as FAO inhibition control, and RD-BCSCs cells treated with 1µM FCCP as an FAO enhancement control. (B) Radiation-induced apoptotic cell death measured by Annexin-V/PI flow cytometry in RD-BCSCs cells, RD-BCSCs treated with 5 Gy IR, ETX (200µM, 48 h) or combined. (C) Repeated experiment in (B) using RD-BCSCs CPT1A KO and RD-BCSCs CPT2 KO cells.

Figure S3 | (A) Mitochondrial fractions were prepared from MCF7 and RD-BCSCs cells treated with or without 5 Gy IR treatment and analyzed by LC-MS and MS/MS on a Q Exactive Plus mass spectrometer. Numbers of proteins detected comparing MCF7 and RD-BCSCs before and after IR are shown on the left. Percentage of enhanced quantitation of the increased protein numbers by radiation are shown in the pie on the right. (B) The intensities of mitochondrial protein expression of MCF7 and RD-BCSCs treated with –/+ IR are shown.

Figure S4 | (A) Functional clustering of mitochondrial proteins via DAVID bioinformatics show the relatively high enhancement of protein numbers in lipid metabolism, oxidation reduction and ATP synthesis in the mitochondria of irradiated RD-BCSCs. (B) The two key enzymes in mitochondrial FAO metabolism, CPT1A, and CPT2 (in red), were enhanced by IR in RD-BCSCs.

Table S1 | The cluster of proteins involved in fatty acid metabolism enhanced by radiation in RD-BCSCs. The mitochondrial proteomics data were generated with mitochondrial proteins isolated from RD-BCSCs treated with or without IR, followed by digestion with trypsin and analyses by LC-MS and MS/MS on a Q Exactive Plus mass spectrometer. The 17 listed proteins were detected in the proteomic analysis of mitochondrial proteins of RD-BCSCs and classified by UniProtein under the category Lipid Metabolism. The gene symbols, descriptions, and comparison with or without 5 Gy IR are shown in the table, and the CPT1A/CPT2 are marked with yellow.

radiotherapy after breast-conserving surgery on 10-year recurrence and 15-year breast cancer death: meta-analysis of individual patient data for 10 801 women in 17 randomised trials. Lancet. (2011) 378:1707–16. doi: 10.1016/S0140-6736(11)61629-2

**Conflict of Interest:** 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 © 2019 Han, Wei, Zhang, Jiang, Fan, Huang, Xie, Zhang, Miao, Butler, Coleman, Vaughan, Wang, Chen, Liu and Li. 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.