Oncogenic transformation associated with virus infection was for a long time considered to result from a mono-infection (infection with a single virus). However, it is now established that in many cases induction of cancer depends on the simultaneous presence and interactions of multiple viral agents in diverse combinations. Viruses co-infecting human tissues may have synergistic or regulatory effects on carcinogenesis, targeting existing neoplastic cells as well as their microenvironment including reactive T-cells, B cells and macrophages, and non-immune cells such as endothelial cells. HIV-1, in particular, potentiates the effects of EBV, KSHV, HCV, and HPV oncogenes, promoting carcinogenesis in individuals co-infected with HIV-1 and EBV, KSHV, HCV, HBV, and HPV. Here, we will focus on molecular interactions of HIV-1 with HBV, HCV, and HPVs.

Progression to liver cancer/HCC in HIV-1/HBV and HIV-1/HCV co-infected patients is promoted by direct and indirect interactions between these viruses and their antigens within the cells harboring HIV-1 due to infection or sequestration of the virion (viral proteins). HIV-1 infection of hepatic cell lines increases the expression of HBV antigens [27]. HIV-1 gp120 causes intracellular accumulation of HBV DNA as well as HBsAg causing hepatotoxicity [99]. Direct interaction of HIV-1 and HBV in liver cells has been demonstrated, with the HBV X protein interacting with HIV-1 Tat to facilitate HIV-1 replication [99]. Upon co-cultivation of HIV-1 infected Jurkat cells with hepatocytes, up-to 16% of the latter acquire Nef. Sequestered Nef alters the size and numbers of lipid droplets (LD), inducing 1.5 to 2.5 fold up-regulation of replication of HCV subgenomic replicon, a remarkable finding in relation to the initially indolent viral replication. Nef also dramatically enhances the ethanol-mediated up-regulation of HCV replication accelerating progression to HCC [100]. HIV-1 gp 120 also causes TGF-β mediated up-regulation of HCV replication [86]. Taken together, these data indicate that HIV-1 and single HIV-1 proteins are critical elements in accelerating progression of liver pathogenesis by enhancing HBV and HCV replication and coordinating production of key intra- and extra-cellular molecules that orchestrate liver decay [100].

One of the mechanisms of HIV-1 potentiation of liver cancer is the induction of oxidative stress. HCV, HIV-1 (and antiretroviral therapy) act together to activate production of ROS in HSCs and hepatocytes. ROS promote phosphorylation of the major mitogenactivated protein kinases active in human cells, p38 kinase, c-JUN N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) that control cell growth, differentiation and apoptosis. In their turn, the phosphorylated p38 MAPK, JNK, and p42/44 ERK phosphorylate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) protein complex, mastering transcriptional regulation of inflammation and cell death [31]. Following these events, phosphorylated NF-κB translocates to the nucleus, and where it normally modulates the production of both pro- and antifibrogenic/antiapoptotic genes, ensuring that liver cells are protected from apoptosis, but are capable to build the required inflammatory and immune responses [101]. In the presence of LPS, NF-κB can upregulate the expression of profibrogenic genes, such as procollagen α1, transforming growth factor β1 (TGF-β1) and tissue inhibitor of MMPs (TIMP-1) [31,101]. This process is accelerated by HIV-1/HIV-1 proteins: exposure of hepatocytes to HIV-1/HIV-1 proteins results in the elevated production of ROS and increased expression of collagen and TIMP1, further amplified by HCV infection, and even exposure to infectious HCV [35]. Taken together, these data indicate that HIV-1-mediated potentiation of hepatocellular carcinogenesis reflects a concerted action of HIV-1, HBV and HCV as viruses and/or individual viral proteins (Figure 1). Based on compelling data, McGivern & Lemon even suggested that the path to hepatocellular carcinoma in chronic hepatitis C shares important features with the carcinogenesis induced by HPV [102].

The increased risk of PLWH developing HPV-associated cancer can also, at least in part, be due to the interactions between HIV-1 and HPV. In general, epithelial cells of PLWH show loss of E-cadherin, and upregulation of vimentin and TGF-b1 expression with spindle-like morphology indicating induction of TGF-b1-dependent EMT, critical for malignant transformation. As noted above, EMT is induced not only due to HIV-1 infection, but also through exposition of epithelial cells to HIV-1 proteins [69,75,95,97]. EMTinduced keratinocytes can then be infected with pseudoviral HPV16 particles (HPV-16 PsVs) and whole HPV16 virus, with infected cells expressing viral oncogenes E6/E7, whereas unexposed keratinocytes could not be infected with either PsVs, or infectious HPV16. Furthermore, "HIV-1-induced" EMT keratinocytes could be transformed with HPV16 DNA, transformed cells showing active proliferation and migration [103]. This confirms that prolonged exposure to and interaction of HIV-1 with oral and anal epithelial cells induces EMT. EMT-induced loss of cell adhesion and increased proliferation and mobility of epithelial cells play a critical role in HPV infection and HPV-associated transformation. HIV-1-induced EMT in the orogenital mucosa may promote progression of pre-cancerous HPV-associated neoplasia to cancer in HIV-1-infected individuals [103].

"Molecular" cooperation between HIV-1 and HPV has not been sufficiently well characterized, but there are relevant examples in this field. Tat protein was shown to transactivate the HPV long control region and increase expression of oncoprotein E7 of HPV18 in HeLa cells [104,105]. Tat can upregulate the expression of E6 and E7 oncoproteins of HPV type 16 in HPV 16-infected human oral keratinocytes, notably enhancing the in vitro proliferative capacity of these cells [106,107], and increase the transcription of E2 modulating HPV replication [108]. The direct angiogenic effects of Tat [109] or its capacity to up-regulate the expression of E6 and E7 of HR-HPVs [110] allows Tat to favor the angiogenic switch in high-grade CIN. We have shown that gp120 and reverse transcriptases (RT) derived from various HIV-1 strains, can increase the expression of HPV 16 E6 in a cervical cancer cell line containing full-length HPV 16 genome Ca Ski (Figure 2), while HIV-1 p24 exerts no effect. In similar conditions, gp120 increases the expression of HPV16 E6 also in HPV16 immortalized anal epithelial AKC2 cells [104,106,111,112]. Furthermore, Tugizov et al. have shown that in the HPV-immortalized anal and cervical epithelial cells Tat and gp120 proteins induce the EMT phenotype, leading to increased migration of cells via collagen membranes [103]. The data on the interaction(s) between HPV and other HIV-1 proteins is missing.

Overall, these findings indicate that the increased incidence of AIDS-defining and non-AIDS defining forms of cancer in PLWH may reflect the direct or indirect, often concerted, carcinogenic effect(s) of HIV-1 and/or individual HIV-1 proteins on diverse infected as well as uninfected bystander cells. Furthermore, some HIV-1 proteins appear to be directly involved in cell transformation and propagation of malignant cells.

**Figure 2.** Transcription of oncoproteins E6 and E7 of HPV 16 in Ca Ski cells treated with HIV-1 proteins. Ca Ski cells harboring 600 full genomic copies of HPV 16 (ATCC CRL-1550) were cultured in RPMI-1640 medium (PanEco, Moscow, Russia) supplemented with 10% FBS and 100 mg/mL penicillin/streptomycin mix at 37 ◦C in an 5% CO2 and split every 4 days. A panel of recombinant HIV-1 proteins: gp120 [113]; p24 (NIBSC ARP 694.1); RT of HIV-1-1 clade B HXB2 strain [114], drug resistant (dr) RT of HIV-1-1 clade B isolated from patient with multiple drug resistance mutation (RT1.14; [114]) and RT of Moloney murine leukemia virus (MMTV) (CRIE, Moscow, Russia) were added to the culture medium, typically in concentration of 1 ng/mL, and incubated for 48 h, according to the methodology described previously by Lein K. et al. [115] Total RNA was extracted and reverse transcribed as described by Jansons et al. 2020 [116]. Gene-specific PCRs were performed on Rotor-Gene 6000 (Qiagen, Darmstadt, Germany) with SYBR Green kit (Evrogen, Moscow, Russia) with primers specific to HPV 16 E6 and E7 [117]. Expression of mRNA, assessed by the standard ddCt method, was normalized to expression of 18S RNA (18Srna\_rt\_f: GTAACCCGTTGAACCCCATT; 18Srna\_rt\_r: CCATCCAATCGGTAGTAGCG), and presented as fold change compared to cells treated with p24, as was recommended earlier [118]. Values represent mean ± SD from two independent assays run in duplicates. \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05 by the ordinary two-way ANOVA with Sidak's multiple comparisons test.

#### **5. HIV-1 Antigens Involved in Cell Transformation and Tumor Propagation**

#### *5.1. Transactivator of Transcription (Tat)*

Tat has long since been known to influence cell cycle progression. In HeLa cells, Tat induces a significant increase in the levels of proliferation markers together with the reduction in the expression of cell cycle inhibitors of transcription [119]; it inhibits epithelial differentiation, blocks apoptosis in vitro and accelerates tumor formation in vivo [119]. In addition, Tat significantly increases in vitro migration in the absence of fetal calf serum [119]. These results suggest that HIV-1 may enhance carcinogenesis by promoting cell cycle progression [111]. Furthermore, it has been shown that binding of Tat to Tat-interacting promoter 30 (TIP30) enhanced EMT and metastasis of non-small cells lung cancer cells by regulating the nuclear translocation of Snail [120]. One of the possible mechanisms of Tat induced carcinogenesis is blocking at the mRNA level of the expression of a Rb family member pRb2/130 and cyclin-dependent kinase inhibitors p21 and p17 [111]. The transduc-

tion domain of Tat specifically attenuates growth of polyamine-deprived tumor cells [121]. Tat is also known to modulate VEGF and targets VEGFRs which increases angiogenesis and supports tumor growth [122]. Furthermore, Tat alters DNA repair in host cells, potentially leading to genomic instability [123,124]. Specifically, Tat induces expression of the DNA polymerase beta gene, which codes for a central mediator in the DNA base-excision repair pathway [125]. It also interferes with double-strand break DNA repair, as cellular extracts containing Tat possess a reduced capacity to re-join linearized DNA [126], indicating that Tat, as well as cellular co-factors of Tat, interfere with repair of double-stranded DNA breaks [123].

#### *5.2. Envelope Glycoprotein gp120*

Glioma cells were shown to interact with the HIV-1 envelope protein gp120. This interaction promotes proliferation, migration, survival and stimulates glycolysis in glioma cell lines and tumor growth in animal models [127]. Increased glycolysis, also known as the Warburg effect characteristic of malignancy [128], results in increased protein and lipid synthesis, and promotes uncontrolled propagation (both proliferation and invasion) of tumor cells, as it provides them with glycolytic intermediary precursors required for the synthesis of DNA, proteins and lipids [127,129]. As Tat, gp120 induces EMT and cell migration through the TGF-B1 and MAPK signaling pathways [115,130].

#### *5.3. Accessory Protein Negative Factor (Nef)*

Nef is one of the earliest and most abundantly expressed HIV-1 proteins. Nef has the ability to modulate multiple cellular signaling pathways in both CD4+ lymphocytes and macrophages. Nef inhibits the apoptotic function of p53 due to its ability to decrease p53 protein half-life and, consequently, p53 DNA binding activity and transcriptional activation [131]. Both internalized and ectopic expression of Nef in endothelial cells synergizes with Kaposi's sarcoma (KS) KSHV oncoprotein K1 to facilitate vascular tube formation and cell proliferation, and enhance angiogenesis in the chicken chorioallantoic membrane (CAM) model. In vivo experiments further indicate that Nef can accelerate K1-induced angiogenesis and tumorigenesis in athymic nu/nu mice [132]. On non-small lung cancer A549 cells, Nef promotes cell proliferation, migration, anchor independent growth and reduces the levels of expression of p53, increasing the aggressiveness of cancer cells [133].

#### *5.4. Reverse Transcriptase (RT)*

We have shown that constitutive expression of HIV-1 RT in murine mammary gland adenocarcinoma 4T1 cells leads to upregulation, in a concentration-dependent manner, of the expression of the transcription factors Twist and Snail tightly involved in EMT [134]. In vivo, expression of RT by 4T1 cells results in enhanced tumor growth and potentiates formation of metastasis in distal organs of immunocompetent syngenic mice [134]. Interestingly, this is not a common property of the reverse transcriptases, as constitutive expression of enzymatically active reverse transcriptase domain of telomerase reverse transcriptase, on contrary, suppressed both tumor growth and metastatic activity of 4T1 cells [116].

#### *5.5. Matrix Protein p17*

Matrix/p17 protein induces expression of chemokines [135], exerts pro-angiogenic [136] and lymphangiogenic [137] activities, and deregulates the biological activity of diverse cells of the immune system [138].  In a HIV-1 transgenic mouse model of lymphoma, only expression of HIV-1 p17, but not of other HIV-1 proteins, induced spontaneous B-cell lymphomas in HIV-1 transgenic mice, with p17 expressed at high levels in the early stages of the disease [140]. Murine lymphoma tissues exhibited enrichment in expression of the recombination-activating genes (Rag1/2) [140].

The latter suggests that intracellular signaling induced by p17 leads to genomic instability and promotes the transformation [140].

Thus, several HIV-1 proteins are directly or indirectly oncogenic, stimulating transformation of healthy cells and propagation and aggressiveness of already existing cancer cells. These oncogenic properties are linked to two essential characteristics of these proteins: their capacity to induce oxidative stress with production of reactive oxygen species and their ability to exit HIV-1-infected cells (active or passive transport).

#### **6. Oncogenic HIV-1 Proteins Induce Oxidative Stress**

Virally-induced cancer evolves over long periods of time in the context of a strongly oxidative microenvironment, on the background of chronic inflammation. Oxidative stress induced by chronic viral infection is one of the factors driving neoplastic transformation, ultimately leading to oncogenic mutations in many cellular signaling cascades that drive cell growth and proliferation [42,141]. Oxidative damage of chromosomal DNA and chronic immune-mediated inflammation are key features of HBV, HCV, HPV, and HIV-1 infections [42,141]. As we have earlier reviewed, numerous lines of evidence show that HIV-1 infection triggers pronounced oxidative stress in both laboratory models and the context of in vivo infection by deregulation of oxidative stress pathways with escalation of ROS production and by inducing mitochondrial dysfunction [141]. As a result, PLWH exhibit multiple markers of oxidative stress including DNA damage [134,142]. The enhancement of ROS production is mediated by the envelope protein Gp120, Tat, Nef, RT, and p17 [141–146].

#### *6.1. Transactivator of Transcription*

Tat induces oxidative stress both directly and indirectly via several independent mechanisms. The first involves the NADPH oxidases [147], and in the second, an enzyme involved in the catabolism of biogenic polyamines, spermine oxidase (SMO) [148], and the third, a mitochondrial dysfunction [149]. A detailed analysis of the levels of ROS in different subcellular compartments of the HIV-1 infected cells revealed a strong increase in the levels of H2O2 in the endoplasmic reticulum (ER), demonstrating with the help of genetically encoded ratiometric sensor HyPER [150,151]. This indicated the involvement in H2O2 production of NOX4 which primarily resides in ER [152]. The levels of H2O2 in the cytoplasm and mitochondria were not elevated [151]. The above activities of Tat are thought to underlie the onset of HIV-1-associated dementia [109,150].

#### *6.2. Envelope Protein Gp120*

Early findings indicated that gp120 increases free radical production from monocytederived macrophages (MDM) detected by spin-trapping methods, and that the spin trap adduct results from a reaction involving nitrogen oxide NO or its closely related oxidized derivatives [153]. We have earlier summarized a profound role of gp120 in the induction of oxidative stress [141], namely gp120 induces ROS production in cell lines of lymphoid origin, in the endothelial brain cells, astrocytes, neurons and microglia. In astrocytes, it enhances ROS production by several parallel mechanisms: via Fenton–Weiss–Haber reaction, NOX2 and NOX4, and cytochrome P450 2E1 (CYP2E1) [154,155]. The latter is mediated through the upregulation of CYP2E1 expression. In cancer (neuroblastoma) cells, gp120 induces proline oxidase that synthesized pyroline-5-carboxylate with concomitant generation of ROS (reviewed in [141]).

The effect of HIV-1/HIV-1 proteins on the cellular antioxidant defense system is controversial. They can both suppress and enhance antioxidant defense pathways [141]. Gp120 was shown to induce oxidative stress response. It up-regulates functional expression in cultured astrocytes of multidrug resistance protein 1 (Mrp1) which effluxes endogenous substrates glutathione and glutathione disulphide involved in cellular defense against oxidative stress [156]. It also upregulates the expression of nuclear factor erythroid derived 2-related factor 2 (Nrf2), a basic leucine zipper transcription factor which is known to regulate antioxidant defensive mechanisms) in human astrocytes, stimulating expression of

key antioxidant defensive enzymes hemoxygenase (HO-1) and NAD(P)H dehydrogenase quinone1 (Nqo1) [157]. Pre-treatment of astrocytes with antioxidants or a specific calcium chelator BAPTA-AM, significantly blocks the upregulation of Nrf2, HO-1 and Nqo1 [157].

#### *6.3. Accessory Protein Negative Factor*

Nef protein has pro-oxidant activity in microglial cells and in neutrophils. It first induces phosphorylation and then translocation of the cytosolic subunit of NADPH oxidase complex p47(phox) into the plasma membrane which in turn induces superoxide anion release from macrophages [158,159]. As a multifunctional HIV-1 protein, Nef also activates the Vav/Rac/p21-activated kinase (PAK) signaling pathway involved in activation of phagocyte NADPH oxidase (thus, Nef indirectly activates NADPH oxidase) [160]. This leads to the dramatic augmentation of the production of ROS [100], and enhancement of cell responses to a variety of stimuli (Ca(2+) ionophore, formyl peptide, endotoxin) [160]. It also leads to decreased tolerance of the cells to hydrogen peroxide, specifically in astrocytes which normally support neuronal function and protects them against cytotoxic substances including ROS [161]. Rac1-dependent NOX2-mediated reactive oxygen species production was shown to contribute to ongoing HIV-1-related vascular dysfunction [162].

#### *6.4. Reverse Transcriptase*

We have previously demonstrated that expression of RT by human cells induces production of ROS [163]. Later studies demonstrated that this is a property of different RT variants, including drug resistant variants, and variants retargeted for lysosomal processing and secretion [114,163]. Expression of all RT variants led to an increase in the levels of expression of Phase II detoxifying enzymes HO-1 and Nqo-1. Artificial secretion of RT resulted in a decrease of RT capacity to induce oxidative stress with a decrease in the production of ROS compared to the parental enzyme [114].

#### *6.5. Matrix Protein p17*

There is no direct evidence of p17-induced oxidative stress. However, p17 possesses specific structural motifs defined as "*coiled coil*" sequences, and has a high propensity to form multimers, mis-fold and aggregate, forming amyloidogenic assemblies [164,165]. This is typical to amyloidogenic proteins actively involved in the pathogenesis of many human diseases, such as Alzheimer's disease and Parkinson' disease. Amyloidogenic assemblies are toxic, specifically to neural cells. Experiments in the invertebrate nematode *Caenorhabditis elegans* as a "biosensor" demonstrated that p17 significantly inhibits its pharyngeal contractions as do the amyloidogenic proteins [166]. Intrahippocampally injected into mice, p17 induced neurocognitive disorders, comparable in strenght to the effects of other known amyloidogenic proteins [166]. Interestingly, amyloidogenic proteins (typically amyloid-beta peptide Aβ) bound to redox active metal ions, such as copper, catalyse the production of ROS, in particular the most reactive one, hydroxyl radical. This effect may underlie the observed oxidative damage exerted by Aβ peptide on itself and on the surrounding molecules (proteins, lipids, DNA) [167]. One can hypothesize that matrix protein p17 with its amyloidogenic assemblies may trigger the production of ROS through a similar mechanism.

Thus, HIV-1 proteins with known oncogenic/mitogenic potential, Tat, gp120, Nef, RT, and potentially p17, have a potential to directly or indirectly induce oxidative stress, which could be one of the mechanisms by which they induce and potentiate carcinogenesis (Figure 3). Interestingly, HIV-1 proteins with an oncogenic potential involved in the induction of oxidative stress, such as Tat, gp120, Nef, RT, and possibly p17, can be found outside of the cells in which they are expressed.

**Figure 3.** Suggestive mechanism of direct carcinogenic effects of HIV-1 proteins. HIV-1 infected cells express and release gp120, Tat, Nef, p17, RT, each capable of the induction of oxidative stress. (1) p17 may trigger the production of ROS through binding of redox active metal ions by its amyloidogenic assemblies [167]. (2) Nef may indirectly activate NADPH oxidase by activating the Vav/Rac/p21-activated kinase (PAK) signaling pathway involved in phagocytic NADPH oxidase activation and produce peroxynitrite [160]. (3) Tat induces oxidative action through several independent mechanisms via NADPH oxidase, spermine oxidase (SMO) induction and mitochondrial dysfunction [148]. (4) RT induces ROS through unknown mechanisms. There is ROS –dependent activation of the Twist [134], which regulates the expression of Nrf2, which stimulating the expression of antioxidant enzymes (HO1, Nqol1). In addition, the Twist regulates the expression of the Snail. Both transcription factors, Twist and Snail, are involved in epithelial to mesenchymal transduction (EMT). (5) Gp120 increases free radical production from monocyte-derived macrophages (MDM) inducing nitrogen oxide (NO). In astrocytes (AS), it enhances ROS production by several parallel mechanisms: via cytochrome P450 2E1 (CYP2E1), NOX2 and NOX4, and the Fenton-Weiss-Haber reaction. Multidrug resistance proteins (Mrps) involved in cellular defense against oxidative stress. Mrp4 (isoform of Mrp) involved in the regulation of ROS and it acts against ROS [156]. In neuroblastoma cells (NB) gp120 was shown to induce proline oxidase that produces pyroline-5-carboxylate with a concomitant generation of ROS [141]. Production of ROS, which damage of bystander cells inducing oxidative damage of DNA, proteins and lipids, apoptosis and inflammation. DNA damage drives genomic instability and promotes transformation of healthy cells, and propagation and dissemination of malignant cells [168]. Arrows indicate: purple arrows—secretion/entering the intercellular space; black arrows—relationships and interactions; red arrows—production of ROS; blue arrows—oxidative stress response. Text above arrows designates the processes leading to the production of ROS, and text below the arrows, forms of ROS.

#### **7. Oncogenic HIV-1 Proteins Inducing Oxidative Stress Are Found in the Extracellular Space** *7.1. Transactivator of Transcription*

Tat protein can be produced and released into the extracellular space by cells harboring actively replicating HIV-1 as well as by latently infected cells, with further uptake by the neighboring uninfected cells. Uptake of Tat would result in upregulation of inflammatory genes and cytotoxicity; this scenario was observed in a number of HIV-1 associated comorbidities, specifically, in neurocognitive disorders, cardiovascular impairment and accelerated aging [169]. Dangerously, the process may occur on the background of successful ART, in the absence of active HIV-1 replication and viral production. Considering that approximately 2/3 of all Tat expressed by infected T cells is secreted [170], the ac-

tivities of Tat described above make a considerable contribution into HIV-1 associated pathologies [171,172].

Soluble Tat, in the absence of the virus, has been shown to cause induction of apoptosis, release of neurotransmitters, oxidative stress and inflammation [169]. Uptake of Tat has been shown to lead to activation of several transcription factors [173,174] including Sp1, NF-κB, and others, resulting in the modulation of expression of both HIV-1 and host genes, including pro-inflammatory cytokines (like TNF-α, CCL2, IL-2, IL-6, and IL-8), adhesion molecules and sometimes, and pro- and anti-apoptotic factors [175–179], p53 and HPV oncoprotein E6 [107].

#### *7.2. Envelope Protein gp120*

Envelope protein gp120 is known to be secreted by chronically infected cells [180,181], particularly from the intraepithelial immune cells even in presence of ART [98]. A subset of PLWH demonstrate persistent circulation in plasma of gp120 [182] and in saliva [98]. Moreover, gp120 was found in tissues of PLWH [183]. Brain cells can be directly exposed to gp120 secreted by infiltrated and infected microglia and astrocytes [127]. Gp120 is internalized by bystander cells through receptor-independent mechanisms [184]. Internalization of gp120 leads to the release of several proinflammatory, angiogenic, and lymphangiogenic factors from affected cells [185].

#### *7.3. Accessory Protein Negative Factor*

Accessory protein negative factor Nef is found in the serum of PLWH [186,187]. Nef can stimulate its own export via the release of extracellular vesicles (exosomes) from HIV-1 infected cells [188]. Of note, exosomes serve as a marker and confirmation of the systemic oxidative stress [189]. Secreted in exosomes, Nef triggers apoptosis in bystander cells. Extracellular Nef has deleterious effects on CD4+ T cells [188,190]; on bystander B cells by suppressing immunoglobulin class switching [191]; and on astrocytes [192] and endothelial cells [162].

#### *7.4. Reverse Transcriptase (RT)*

In our lab, we have shown secretion of RT into cell culture fluids of cells transiently expressing RT [114]. Recently, RT was also detected in the exosomes detected in the uterine of PLWH [193].

#### *7.5. Matrix Protein p17*

Matrix protein p17 is continuously released into the extracellular space from HIV-1 infected cells, and can be detected in the plasma of PLWH and in different organs and tissue specimens [138]. Cellular aspartyl proteases promote the unconventional secretion of biologically active p17 [194]. HIV-1 secretion of biologically active p17 takes place at the plasma membrane and occurs following its interaction with phosphatidylinositol- (4,5)-bisphosphate and its subsequent cleavage from precursor Gag (Pr55Gag) by cellular aspartyl proteases [194]. Extracellularly, p17 deregulates the function of different cells involved in AIDS pathogenesis. Importantly, p17 accumulates and persists in different organs and tissues of PLWH on ART, even in the absence of any replicative activity [136,195,196]. These findings strongly suggest that p17 may be chronically present in HIV-1-I infected cells and tissues, even under ART-associated suppression of HIV-1 replication.

Thus, gp120 and Tat are actively secreted into the endothelial cell micro-environment, Nef can be neighboring uninfected cells including cells which cannot be infected with HIV-1, modulating their metabolism, cell cycle progression, ability to differentiate, motility, and, importantly, the genomic stability, through induction of ROS. Some HIV-1 proteins such as matrix p17 and gp120 can accumulate and persist in lymphoid tissues for at least 1 year after the on-start of ART on the background of successful suppression of viral replication [196]. These proteins are involved in different processes associated with malignant transformation and tumor growth with significant direct and indirect adverse effects on the epithelial cells. These include a range of responses that contribute to endothelial dysfunction, including enhanced adhesiveness, permeability, cell proliferation, apoptosis, as well as activation of cytokine secretion [86], eventually leading to malignant transformation (Figure 3). In this respect, their effect would resemble oncogenesis mediated by known viral oncoproteins originating from EBV, HTLV-1, KSHV, HCV, HBV, HPV, and identified as causative agents of both AIDS-defining and non-AIDS defining forms of cancer.

#### **8. Conclusions**

People living with human immunodeficiency virus receiving antiretroviral therapy are characterized by high prevalence of different forms of cancer affecting epithelial cells. HIV-1 does not infect epithelial cells, however both HIV virions and proteins were shown to be sequestered into epithelial cells and affect their functions. These proteins have three specific properties:

Sequestered/internalized by innocent bystander cells, these proteins modulate their metabolism, cell cycle progression, ability to differentiate, motility, redox balance (induce ROS) and genomic stability. Through this, they can trigger malignant transformation of normal cells. Another outcome is propagation (proliferation and dissemination) of already existing precancerous and cancer cells, and enhanced growth and metastatic activity of tumors expressing or exposed to HIV-1 proteins.

Altogether, we present a new mechanism of HIV-associated malignant transformation of epithelial cells driven by individual HIV proteins through the induction of reactive oxygen species. In this scenario, HIV-1 proteins act in a manner similar to the known viral oncogenes, and can cooperate with them promoting KSHV, EBV, HBV, HCV, and HPVassociated carcinogenesis. Such pathway of HIV associated carcinogenesis can co-occur together with carcinogenesis driven by persistent immune inflammation, and dysfunction of B cells, T cells and cellular components of the innate immune system.

**Funding:** This study was supported the Russian Fund for Basic Research grants 17\_54\_30002 and 20-04-01034 to M.I., Latvian Science Council grants LZP-2018/2-0308 and LZP-2020/2-0376 to M.I., and NCI R01CA 217715 to J.P. The work of Francesca Chiodi is supported by a grant from the Swedish Medical Research Council (Francesca Chiodi; Vetenskapsrådet 2019-01169).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

### *Review* **The Complement System in Ovarian Cancer: An Underexplored Old Path**

**Yaiza Senent 1,2,3, Daniel Ajona 1,2,3,4, \*, Antonio González-Martín 1,5 , Ruben Pio 1,2,3,4,† and Beatriz Tavira 1,3,6,†**

**Simple Summary:** Ovarian cancer is one of the leading causes of death among women and the most lethal cause of death from gynecological malignancy in developed countries. The immune system plays an essential role in ovarian cancer progression, and its modulation may be used as an effective therapeutic tool. In this review, we examine the relevance of the cellular and humoral components of the adaptive and innate immune responses in ovarian cancer, focusing on the role of an essential component of innate immunity, the complement system. Elements of this system show tumor-promoting activities that impede the efficacy of developing treatment strategies. We discuss evidence that suggests a role of complement components in the progression of ovarian cancer and provide a rationale for evaluating the inhibition of complement components in combination with immunotherapies aimed to reactivate antitumor T-cell responses.

**Abstract:** Ovarian cancer is one of the most lethal gynecological cancers. Current therapeutic strategies allow temporary control of the disease, but most patients develop resistance to treatment. Moreover, although successful in a range of solid tumors, immunotherapy has yielded only modest results in ovarian cancer. Emerging evidence underscores the relevance of the components of innate and adaptive immunity in ovarian cancer progression and response to treatment. Particularly, over the last decade, the complement system, a pillar of innate immunity, has emerged as a major regulator of the tumor microenvironment in cancer immunity. Tumor-associated complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and activate cancer-related signaling pathways. Recent insights suggest an important role of complement effectors, such as C1q or anaphylatoxins C3a and C5a, and their receptors C3aR and C5aR1 in ovarian cancer progression. Nevertheless, the implication of these factors in different clinical contexts is still poorly understood. Detailed knowledge of the interplay between ovarian cancer cells and complement is required to develop new immunotherapy combinations and biomarkers. In this context, we discuss the possibility of targeting complement to overcome some of the hurdles encountered in the treatment of ovarian cancer.

**Keywords:** ovarian cancer; adaptive immunity; innate immunity; complement system; immunotherapy; cancer immunology; tumor microenvironment

**Citation:** Senent, Y.; Ajona, D.; González-Martín, A.; Pio, R.; Tavira, B. The Complement System in Ovarian Cancer: An Underexplored Old Path. *Cancers* **2021**, *13*, 3806. https://doi.org/10.3390/ cancers13153806

Academic Editors: Ion Cristóbal and Marta Rodríguez

Received: 2 July 2021 Accepted: 26 July 2021 Published: 28 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Current Status of Ovarian Cancer: Clinical Perspective and Needs**

Ovarian cancer is the most lethal gynecological cancer in developed countries [1]. According to data from the US National Cancer Institute (NIH), the five-year survival rate for ovarian cancer is 49.1% [2]. This can be attributed to a delay in the diagnosis due to the lack of specific symptoms; 70% of cases are diagnosed in stage III or IV, making it difficult to treat with curative intent [3]. Ovarian cancer is a complex disease that comprises different tumor types, of which epithelial ovarian cancer represents 90–95% of all cases [4]. The current standard treatment includes surgery and platinum-based chemotherapy followed by a maintenance period with the anti-angiogenic therapy bevacizumab [5]. Initial responses to chemotherapy are frequently high, but unfortunately, up to 70% of patients experience recurrence within the first three years, especially patients who are late-diagnosed [5]. Survival rates have recently improved with the introduction of a new generation of poly (ADP-ribose) polymerase inhibitors (PARP inhibitors (PARPi)). These drugs, administered after chemotherapy, prolong the time during which the disease does not progress, mainly in patients carrying BRCA mutations [6]. Despite this great advance, the overall survival of patients with ovarian cancer is still low. There are a variety of factors associated with chemoresistance and relapse, including interactions between ovarian cancer cells and their surrounding immune microenvironment [7]. Ovarian cancers are considered "immunogenic tumors" in which spontaneous antitumor immune responses have been demonstrated [8,9]. The presence of tumors infiltrating CD8+ lymphocytes in the tumor microenvironment (TME) is associated with longer recurrence-free and overall survival [10,11], whereas the recruitment of regulatory T (Treg) cells is correlated with a poor outcome [12]. These associations indicate that ovarian cancers could respond to immunotherapy. However, immune checkpoint inhibitors (anti-CTLA-4 or anti-PD-1/PD-L1) have yielded modest clinical results in ovarian cancer patients [13,14]. A better understanding of the interplay between ovarian tumor cells and the immunological players in innate and adaptive immunity is critical for developing strategies to overcome the resistance of ovarian cancers to immunotherapy [15,16].

A major effector of innate immunity is the complement system, which represents one of the first lines of defense that distinguish "self" from "non-self" [17]. This system is composed of more than 50 soluble or membrane-bound effectors, regulators, and receptors, and it plays a relevant role in numerous physiological and pathological processes, including cancer [18]. Some evidence suggests that the modulation of complement activation may be exploited for the development of successful treatments against cancer [19,20]. In this review, we discuss the role played by components of adaptive and innate immunity on the development and progression of ovarian cancer. We mainly focus on the complement system, its role in the TME, and the rationale behind the use of complement modulators for the treatment of ovarian cancer.

#### **2. Cellular and Humoral Immune Components of the Ovarian Tumor Microenvironment**

The continuous feedback between tumor cells and the immune system is now recognized as a distinguished cancer hallmark [21]. Neoplastic transformation is characterized by the acquisition of tumor-associated molecular patterns that can be detected by the immune system. It is believed that upon recognition, innate and adaptive immunity can eliminate the vast majority of incipient cancer cells, avoiding tumor formation. However, the immune system is unable to eliminate all emerging malignant cells. When transforming cells escape from immune-mediated elimination, a dynamic interplay is established between tumor cells and the immune system, resulting in tumor-associated immune responses that may facilitate the development and progression of cancer [22]. In the case of ovarian tumors, a plethora of immune and non-immune cell types and non-cellular elements are found in the TME, not only in primary tumors but also in ascites and metastases [23]. The co-existence of multiple distinct tumor immune microenvironments within a single individual highlights the high plasticity and adaptability of ovarian cancers [24]. Herein, we summarize the

main roles of the cellular and humoral elements of the immune system in ovarian tumor progression.

#### *2.1. Cellular Immune Components*

Tumor cells co-exist with non-immune and immune cells, and this relationship determines the natural history of the tumor and its resistance or response to therapy. The cellular immune components of the ovarian TME include T and B lymphocytes, natural killer (NK) cells, dendritic cells (DCs), polymorphonuclear cells, and macrophages.

T cells are a prominent component of the ovarian TME. Infiltration by CD8+ T cells is indicative of an ongoing immune response and is associated with a favorable prognosis [25]. Upon activation, tumor-specific CD8+ T cells secrete IFN-γ, tumor necrosis factor (TNF) α, and cytotoxic mediators. However, in the ovarian TME, CD8+ T-cell responses are often dysfunctional. The autologous recognition of ovarian tumor antigens is limited to approximately 10% of the intratumoral CD8+ T receptor (TCR) repertoire [26]. This state can be attributed to the upregulation of T-cell exhaustion molecules by persistent antigen exposure and the existence of a hostile TME characterized by nutrient deprivation, hypoxia, oxidative stress, high concentrations of pro-inflammatory molecules, and the presence of immunosuppressive cell subsets [27]. In fact, ovarian cancers are highly enriched in Treg cells [28], a subset of lymphocytes that hamper tumor immunosurveillance by fostering peripheral tolerance to tumor antigens. Treg cells release and metabolize ATP to adenosine by the action of CD39 and CD73, a process that mediates immunosuppression via the adenosine and A2A pathways [29]. Consequently, depletion of Treg cells in ovarian cancerbearing mice effectively restores antitumor antigen-specific T-cell responses [30]. Other lymphoid subsets are important elements of the ovarian cancer immune infiltrate. In an orthotopic syngeneic mouse model, antitumor immunity was driven by CD4+ T cells [15]. A study identified a novel tumor-infiltrating NK subset characterized by a high expression of PD-1, reduced proliferative capability in response to cytokines, low degranulation, and impaired cytokine production upon interaction with tumor targets [31]. The presence of CD20+ B cells was associated with increased survival in ovarian cancer patients [32]. In human metastases of high-grade serous ovarian cancer, B cells develop memory responses in the TME and promote antitumor immune responses [33].

DCs are a diverse group of innate immune cells that infiltrate tumors and present tumor-derived antigens to naïve T cells. High densities of tumor-infiltrating DC-LAMP+ mature DCs suggest the establishment of an antitumor immune response, which is associated with a favorable prognosis in ovarian cancer patients [34]. However, this immune response is often rendered dysfunctional because of a variety of mechanisms, such as the upregulation of B7-H1 [35], the activation of the endoplasmic reticulum stress response factor X-box binding protein 1 (XBP1) [36], the attenuation of the toll-like receptor-mediated DC activation [37], and the activation of the cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) axis to redirect the development of DCs toward the formation of myeloid-derived suppressor cells (MDSCs) [38].

MDSCs represent a heterogeneous population of immature myeloid cells that fail to differentiate into granulocytes, macrophages, or DCs. Two main subsets of MDSCs have been identified: polymorphonuclear MDSC (PMN-MDSC; CD11b+Ly6G+Ly6Clo in mice and CD11b+CD14−CD15+CD66b+LOX-1+ in humans) and monocytic MDSC (M-MDSC; CD11b+Ly6G−Ly6Chi in mice and CD14+CD15−HLA−DR−/lo in humans). PMN-MDSCs and M-MDSCs are morphologically and phenotypically similar to neutrophils and monocytes, respectively [39]. These cells potently inhibit the anti-tumor immune response and reshape the TME to promote tumor growth and metastatic spread. The differentiation of myeloid precursors toward an MDSC phenotype is mediated by the inflammatory factor PGE2 via DNA methyltransferase 3A (DNMT3A)-dependent hypermethylation and the downregulation of a subset of myeloid genes [40]. The infiltration of MDSCs into ovarian tumors is associated with the Snail-mediated upregulation of CXCL1 and CXCL2 chemokines that attract MDSCs to the tumor via CXCR2 [41]. In the tumor niche, granulocyte–monocyte colony-stimulating factor (GM-CSF), through the signal transducer and activator of transcription 5 (STAT-5) pathway, upregulates AMP-activated protein kinase alpha 1 (AMPKα-1) in MDSCs to suppress antitumor CD8+ T-cell responses [42]. Both the presence of TNF-α and the production of NO by MDSCs sustain Th17 responses in the TME and myeloid cell recruitment in an IL-17-dependent manner [43,44].

Tumor-associated neutrophils, a cell population difficult to distinguish from PMN-MDSCs, are also involved in ovarian cancer-associated immune responses. In a KRASdriven ovarian cancer mouse model, neutrophils reduced the amount of tumor-associated Treg cells and M-MDSCs while increasing the antitumor immune response via the upregulation of CD8+ T-cell function [45]. By contrast, the activation of neutrophils by mitochondrial DNA from ascites obstructs anti-tumor immunity and is associated with worse outcomes in patients with advanced ovarian cancer [46]. This study also reported the formation of neutrophil extracellular traps (NETs), networks of neutrophil decondensed chromatin fibers that are capable of binding tumor cells to support metastatic progression [47]. These contrasting roles of neutrophils in ovarian cancer have been attributed to different polarization states induced by the presence of transforming growth factor (TGF)-β and type-1 interferons in the TME [48].

Tumor-associated macrophages (TAMs) play a major role in the pathogenesis of ovarian cancer [49]. Macrophages constitute over 50% of the cells in peritoneal ovarian tumor nodules and malignant ascites and are involved in ovarian cancer initiation, progression, and metastasis [50]. TAMs are highly plastic cells that can exhibit two main phenotypes: anti-tumorigenic M1-like (F4/80hi and CD86+ or CD80+ or iNOS+ in mice; CD68+HLA-DR+CD11c− and CD86+ or CD80+ or iNOS+ in humans) and pro-tumorigenic M2-like (F4/80hi and CD163+ or CD206+ or arginase+ in mice; CD68+HLA-DR+CD11c− and CD163+ or CD206+ in humans). Analyses of TAM polarization in ovarian cancer show that M2 TAMs are associated with a poor prognosis [51,52]. Malignant cells direct TAM differentiation to facilitate tumor progression. The activation of the ovarian TAM protumor phenotype requires the expression of zinc finger E-box binding homeobox 1 (ZEB1), a driver of the epithelial-mesenchymal transition (EMT), and involves direct crosstalk with tumor cells [53]. Tumor-expressed CD24 interacts with the inhibitory receptor sialicacid-binding Ig-like lectin 10 (Siglec-10) expressed by ovarian cancer-inhibiting TAMs to avoid their antitumor effects [54]. Ovarian cancer cells skew co-cultured macrophages to a phenotype similar to that found in ovarian tumors [55]. Ovarian cancer cells promote membrane-cholesterol efflux and depletion of lipid rafts to polarize TAMs toward a tumor-promoting phenotype characterized by the upregulation of IL-4 signaling [56]. In return, TAMs enhance the malignant potential of ovarian cancer cells. Endothelial growth factor (EGF) secreted from TAMs promoted tumor growth at early stages of transcoelomic metastasis in a mouse model of ovarian cancer [57]. Moreover, TAMs enhance ovarian cancer invasiveness through activation of the nuclear factor kappa B (NF-kB) and Jun N-terminal kinase (JNK) pathways in tumor cells [58].

#### *2.2. Humoral Immune Components*

The crosstalk between the different cellular components of the TME is essential to reprogram tumor-associated immune responses. This process is orchestrated by complex networks interconnected by sets of soluble factors and extracellular structures, such as cytokines, chemokines, small metabolites, and microvesicles, among others [59]. In particular, cytokines mediate key interactions between immune and non-immune cells in the TME [60], and cytokine-based immunotherapy is a promising strategy to modulate the host's immune response toward the induction of apoptosis in tumor cells [61]. To date, there are two FDA-approved treatments for melanoma and metastatic renal cell cancer based on the administration of TNF-α and interleukin (IL)-2 [62]. In the case of ovarian cancer, the proinflammatory cytokine IL-6 has been established as a key immunoregulator [63]. IL-6, along with other cytokines, activates pathways such as STAT and NF-kB, whose modulation could be used as a potential therapeutic tool [63].

Many years ago, Bjørge et al. found elevated levels of complement C1q, C3, C3a, and soluble C5b-9 in ascites from ovarian cancer patients, suggesting that local complement activation may constitute an important soluble component of the ovarian TME [64]. More recently, ovarian cancer has been classified as a cancer type with "upregulated complement" [65]. Interestingly, over the last decade, the complement system has emerged as a major non-cellular regulator of the TME in cancer immunity. Tumor-associated complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis, and activate cancer-related signaling pathways [66]. In the case of ovarian cancer, complement dysregulation may even participate in the onset of tumors since complement molecules are already overexpressed in precursor lesions [67]. In the following section, we summarize the evidence supporting the involvement of the complement system in ovarian cancer progression.

#### **3. The Complement System and Its Dual Role in Ovarian Cancer**

In 1896, the complement system was first described as a heat-labile component in the serum able to "complement" heat-stable factors (antibodies). Now, the complement system is broadly known as a central part of the innate immune response composed of soluble and membrane-bound proteins that can coordinate a nonspecific inflammatory response against microbes and unwanted host elements [18]. Complement-circulating effectors are predominantly synthesized in the liver and are distributed throughout the body in an inactivated state. Complement can be activated by three main distinctive pathways: the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP) (Figure 1). The three pathways converge in the cleavage of the complement component C3 into C3a and C3b. The CP is initiated in foreign, damaged, or dying cells when the C1 complex, which includes C1q, C1r, and C1s, recognizes antibody clusters, pathogenassociated molecular patterns (PAMPs), or danger-associated molecular patterns (DAMPs), among other molecules [68]. The LP is initiated by the recognition of carbohydrate patterns by mannose-binding lectin (MBL) or ficolins, along with the mannan-binding lectin serine proteases MASP1 and MASP2 [68]. The initiation of both the CP and the LP leads to the cleavage of C4 into C4a and C4b and, subsequently, C2 into C2a and C2b. The complex formed by C4b and C2b (C4bC2b, formerly C4b2a) constitutes the classical C3 convertase, which is responsible for the cleavage of C3 into C3a and C3b [68]. The AP is initiated by the spontaneous hydrolysis of C3 into C3(H2O), followed by its binding to factor B. This complex is recognized by factor D, which catalyzes the cleavage of factor B to form the fluidphase alternative C3 convertase C3(H2O)Bb. This convertase can mediate the cleavage of C3 into C3a and C3b to form the membrane-bound alternative C3 convertase C3bBb [69]. Subsequently, C3b is able to bind to C4bC2b (in the CP and LP) or C3bBb (in the AP), leading to the formation of C5 convertase. This complex catalyzes the cleavage of C5 into C5a and C5b. The later fragment sequentially binds to C6, C7, C8, and C9 to form the cytolytic membrane attack complex (MAC) [18,68]. Many complement functions are mediated by the anaphylatoxins C3a and C5a, which act as potent inflammatory modulators [70]. These peptides signal through their respective G-protein-coupled receptors C3aR and C5aR1 [71]. A second, lesser-known C5a receptor, C5aR2, also participates in C5a responses, though its role remains unclear. Finally, an array of membrane and soluble complement regulatory proteins (CRPs) protects normal cells from the overactivation of complement [68] (Figure 1).

**Figure 1.** Schematic representation of the effectors and regulators of the complement cascade. Complement is initiated by three distinctive pathways: the classical (blue arrows), the lectin (green arrows), and the alternative (brown arrows) pathways. All three pathways converge in the formation of C3 and C5 convertases, which in turn generate the inflammation modulators C3a and C5a. The terminal steps, which culminate in the assembly of the membrane attack complex (MAC), are common to the three pathways. Inhibitory proteins of the three pathways are shown in red boxes.

Complement plays an essential role in the control of cellular immunity [18], participating in the regulation, differentiation, and trafficking of several immune cell types [17,72]. C3 and C4 depletion impair humoral immune responses in vivo [73–75]. It has been postulated that antigen–antibody clusters interact with complement and are recognized by DCs, B lymphocytes, and macrophages [76]. Further evidence of the interplay between adaptive and innate immunity is the CD21(CR2)-CD19-CD81 complex on B cells, which enhances B-cell receptor function [77,78], partially by recognizing C3d-tagged surfaces [79]. Anaphylatoxins also play an important role in immune regulation. Most immune cell types express C3aR and/or C5aR1 on their surfaces [65]. On B cells, C3a impairs polyclonal immune responses and TNF-α and IL-6 production [80,81]. C5a has been extensively reported to induce the migration of several cell types [82–86]. Interestingly, C5a fosters antigen cross-presentation and the maturation of DCs [87–89]. Moreover, C3a-C3aR and C5a-C5aR1

signaling promote the activation [90] and expansion [91] of T cells and divert their differentiation from Treg cells [92,93]. Complement inhibitory proteins, such as CD46, have been shown to modulate T-cell fate depending on the isoform expressed and the presence of IL-2 [94,95]. Moreover, negative modulation of the inhibitor CD59 was demonstrated to ameliorate antigen-specific T-cell responses [96]. Overall, the information gathered during the past few decades illustrates the interconnections between the complement system and adaptive and innate immunity and endorses the hypothesis that complement's role extends beyond its traditional non-specific, first-defense function.

Dysregulation of complement can lead to the development of several pathologies. Kidney diseases, such as atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathies, are closely related to complement anomalies. C3 glomerulopathies are characterized by the production of C3 fragments in the fluid phase via the alternative pathway and abnormal complement consumption that leads to the damage of the glomerular basement membrane [97]. Activation of the complement system is also involved in the pathogenesis of systemic autoimmune diseases [98]. Alterations in regulatory proteins can trigger serious conditions as well. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematological disorder caused by a deficiency in glycosylphosphatidylinositol anchor synthesis that negatively affects the expression of the CRPs CD55 and CD59 [99]. More recently, cancer progression has been associated with complement activation [66].

In the next sections, we review studies that have reported the participation of components of the complement system in the biology of ovarian cancer or its potential clinical use. The findings of these studies are summarized in Tables 1 and 2.

**Table 1.** *Cont.*

EMT: epithelial-mesenchymal transition, SCID: severe combined immunodeficient, mAb: monoclonal antibody, CDC: complementdependent cytotoxicity.

**Table 2.** Summary of the studies performed with clinical samples reporting the potential clinical use of the determination of complement components.

**Table 2.** *Cont.*

IHC: immunohistochemistry, mAb: monoclonal antibody, CDC: complement-dependent cytotoxicity.

#### *3.1. Complement Initiation Components in Ovarian Cancer*

C1q, the first component of the classical complement activation pathway, links innate and adaptive immunity [123]. Both promoting and inhibitory roles have been reported for C1q in cancer progression, but most studies associate C1q expression with poor clinical outcomes in cancer, as is the case for gliomas and osteosarcomas [124,125]. C1q may act as a tumor-promoting factor through both complement-dependent and complementindependent mechanisms [126,127]. In ovarian cancer, the role of C1q appears to be context-dependent. In vitro, C1q displays an anti-tumor effect in SKOV3 cells by promoting apoptosis through the upregulation of the TNF-α pathway and the downregulation of the mammalian target of rapamycin (mTOR) survival pathway [100]. Conversely, expression levels of C1q in circulating extracellular vesicles isolated from ovarian cancer patients in stages III–IV are significantly elevated compared with those isolated from healthy individuals [115]. Discrepancies have also been observed in the case of the globular C1q receptor (gC1qR), a cell surface receptor for C1q. This molecule is upregulated in tumor cells [128], and its overexpression induces mitochondrial dysfunction and p53-dependent apoptosis in human cervical squamous carcinoma cells in vitro [101]. Consistently, the induction of gC1qR expression by paclitaxel in ovarian cancer cell lines SKOV3 and CAOV3 results in mitochondrial dysfunction and cell apoptosis [102]. However, this consistency observed in vitro disappears when clinical samples from ovarian cancer patients at different stages of the disease are analyzed. gC1qR downregulation was observed in ovarian cancer patients in the early stages of the disease (stages I–II) [102]. By contrast, gC1qR seems to be overexpressed in tumor tissue from ovarian cancer patients in stages III and IV, and this is associated with a poor prognosis and cisplatin resistance [116]. These data suggest an increase in complement activation during ovarian cancer progression. Consistent with this assumption, C4 was detected in ascitic fluid from late-stage patients, while it was undetectable in ascitic fluid from healthy donors [64]. Moreover, C4 levels were found to be upregulated in plasma samples from chemoresistant compared with chemosensitive ovarian cancer patients [119]. In the same study, complement factor I and C3 were found to be downregulated [119]. Finally, MBL and MASP2 serum levels are altered in ovarian cancer patients, and MBL levels are associated with advanced disease stages [117]. The ovarian tumor antigen cancer antigen 125 (CA-125), a highly glycosylated protein, may be a target for pattern recognition molecules, such as collectins and ficolins, which may mediate the interaction with MBL and the activation of the lectin pathway [129]. Serum ficolins have been reported to be elevated in ovarian cancer patients despite their lower tumor expression [118]. In conclusion, several studies have reported the presence of complement initiation factors in ovarian cancer. However, the contribution of these factors

to ovarian cancer progression and response to treatment is still unclear and requires further investigation.

#### *3.2. C3 and C5 in Ovarian Cancer*

The C3- and C5-derived fragments C3a and C5a participate in the establishment of a chronic inflammatory state that may favor tumorigenesis and cancer progression [70]. In ovarian cancer, the implication of C3a and C5a seems to depend on multiple factors, although most of the evidence suggests a tumor-promoting effect. Nuñez-Cruz et al. assessed the role of complement in ovarian tumor progression using C3 and C5aR1-deficient mice. Complement inhibition impaired both tumor vascularization and growth [103]. Some molecular mechanisms have been associated with the tumor-promoting function of C3 and C5 in ovarian cancer tumor cells. These mechanisms include the activation of the phosphatidylinositol-3-kinase (PI3K) pathway and the induction of EMT [104,105]. C3 and C5 and their effector fragments also influence tumor progression by acting on immune cells. Circulating polymorphonuclear cells from ovarian cancer patients can acquire an immunosuppressive phenotype capable of restraining T-cell proliferation after exposure to ascites in a process dependent on C3 [130]. This T-cell non-responsiveness is associated with the production of C5a and is mediated by mTOR signaling and nuclear factor of activated T-cells (NFAT) translocation [131]. Interestingly, C5a may function in a dosedependent manner. Thus, in a SKOV-3 tumor model, low local doses of C5a reduced tumor growth in association with the recruitment of M1 TAMs and NK cells, while high doses promoted tumor progression [107]. Ovarian cancer cells overexpress ribosomal protein S19 (RPS19), which leads to tumor growth through its interaction with C5aR1 in MDSCs [132]. By contrast, the local production of C3 and the release of C5a disrupt the tumor endothelial barrier, facilitating the homing of T cells and their tumor recruitment [106]. This study further stresses the contrasting effects associated with complement effectors in different models of ovarian cancer. Unfortunately, the results reported in patients do not clarify the matter. High levels of C3 or C5aR1 have been associated with decreased overall survival [104,133]. By contrast, reduced expression of C3 was observed in the blood of ovarian cancer patients [134], and this factor was downregulated in the serum of platinum-resistant patients [119].

#### *3.3. Complement Regulatory Proteins in Ovarian Cancer*

CRPs protect host cells from autologous complement attack, but they can render complement ineffective at eliminating cancer cells. Membrane-bound CRPs (mCRPs), such as CD46, CD55, and CD59, are expressed by ovarian cancer tumors [121,135] and cell lines [108,122,135]. These regulators are linked to worse clinical outcomes and may constitute an obstacle for cancer immunotherapy [121,136–138]. Their presence has also been associated with the development of multi-drug resistance in ovarian cancer cells [139]. Neutralization of mCRPs increases the sensitivity to complement-dependent cytotoxicity [111,113,139], reduces ovarian tumor growth [110], and enhances the anti-tumor efficacy of therapeutic antibodies [108,112]. In line with these findings, CD55 silencing restores cisplatin sensitivity to chemotherapy in resistant ovarian cancer cells [109]. Regarding soluble complement regulators, a range of studies has demonstrated their importance in several tumor types [140–143]. In ovarian cancer, some soluble complement inhibitors, such as factor H and factor H-like 1 (FHL-1), have been found in ascitic fluid and primary tumors [64,114]. However, the role of these regulators in ovarian cancer progression has not been defined yet.

In conclusion, the evidence suggests that complement dysregulation drives ovarian cancer progression. Complement effectors, receptors, and regulators have been implicated in different aspects of ovarian cancer biology (Figure 2). Although there are inconsistencies in the description of the role of complement components in some clinical or experimental contexts, the majority of studies point toward a tumor-promoting activity of complement in

well-established tumors. These findings have paved the way for studies aimed to potentiate cancer therapies through the modulation of the complement system.

**Figure 2.** Complement-related mechanisms associated with ovarian cancer progression. Complement components have been implicated in different biological processes associated with ovarian cancer progression. They include modulation of immunosuppression in the tumor microenvironment; regulation of angiogenesis and endothelial permeabilization; autocrine and paracrine effects in tumor cells mediated by C1q, C3a, or C5a; and inhibition of complement-dependent cytotoxicity (CDC) by membrane-bound complement regulators.

#### **4. Therapeutic Potential of Targeting Complement in Ovarian Cancer**

Complement inhibition may be a useful therapeutic strategy against cancer [19]. Agonists of C5aR1 and C3aR increase ovarian tumor cell proliferation, migration, and invasion, suggesting that receptor antagonists could be used to block cancer growth [104]. Complement targeting may also impair angiogenesis, a highly relevant biological process in ovarian cancer. Elevated levels of serum VEGF after chemotherapy treatment have been associated with lower overall survival in ovarian cancer patients [144], and the anti-VEGF antibody bevacizumab has shown therapeutic activity in both patients and animal models [145–147]. Genetic or pharmacological inhibition of C3 or C5aR1 results in smaller and poorly vascularized ovarian tumors in vivo [103], and C5a is able to promote endothelial cell tube formation and migration [103,148]. Therefore, it can be speculated that inhibition of complement may potentiate the efficacy of anti-angiogenic agents.

Another scenario in which complement modulation may be of special relevance is immunotherapy. We previously described the implication of effectors and regulators of the complement system in the ability of T cells to infiltrate tumors and the response against tumor-associated antigens [149]. Using various models of lung cancer, we proposed that the modulation of complement activation can improve the antitumor efficacy of monoclonal antibodies targeting the PD-1/PD-L1 pathway [150]. This synergistic effect has also been reported in other tumor models targeting C5a/C5aR1 [151,152] or C3a/C3aR [151,153]. To our knowledge, these combinations have not been tested yet in models of ovarian cancer, and we can only hypothesize about the outcome of these studies. The inhibition of C3 or C5aR1 abrogates the suppressor phenotype of MDSCs in the ovarian TME [130,131], suggesting that complement inhibition may have a positive effect on the efficacy of anti-PD-1/PD-L1 therapies. Conversely, antitumor T cells require the production of C3 and the release of C5a in the endothelium in order to infiltrate ovarian tumors [106]. The targeting of mCRPs should also be considered in light of their relevance in the TME [136]. The inhibition of mCRPs may be used to sensitize tumors to other drugs. In ovarian cancer, the neutralization of CD46, CD55, and CD59 in combination with the anti-HER2 monoclonal antibodies trastuzumab and pertuzumab induces tumor cell killing in vitro [113]. Nevertheless, considering the dual role of complement molecules in ovarian tumors, in vivo studies are needed to determine whether complement inhibition has any impact on the response to checkpoint-based or antibody-based immunotherapies, and in which direction.

#### **5. The Need for Preclinical Models to Better Delineate the Role of Complement in Ovarian Cancer**

In this review, we discussed the functions that complement components exert in the biology of ovarian tumors. Many questions remain regarding the conflicting results observed in different experimental settings. To address these questions, in vivo models that faithfully recapitulate the complexity of the disease are needed. Currently, there are a few animal models established for the study of ovarian cancer. These include genetically engineered mouse models, xenograft cell transplants of human cell lines, and patient-derived xenografts [154]. These models have facilitated the study of many mechanisms associated with ovarian cancer progression and have allowed the evaluation of many therapeutic molecules [155]. For the study of complement-related mechanisms or treatments, mouse models that capture the complexity of the TME are required. Models based on syngeneic tumor cells injected intraperitoneally in immunocompetent mice represent a practical option. Some studies have used the syngeneic intraperitoneal injection of ID-8-MOSEC, a mouse epithelial ovarian cancer cell line originating in C57BL/6 mice, to evaluate the roles of C3, C5, and C5aR1 in ovarian cancer development and progression (Table 1) [104,106]. This cell line was developed by Dr. Katherine F. Roby in the Department of Anatomy and Cell Biology of the University of Kansas in the early 2000s, and it is one of the most frequently used ovarian cancer cell lines since it has the capacity to induce tumor peritoneal implants observed in stages III and IV [156]. Because of its slow growth rate, some strategies have been developed to increase the aggressiveness of this cell line, including the overexpression of dendritic cell chemoattractant beta-defensin 29 (Defb29) or VEGF [157], two factors associated with increased invasiveness. Nevertheless, this model does not completely recapitulate the human pathophysiology of the disease and does not exactly reproduce the TME [154]. The development of better ovarian cancer models is needed to unravel the mechanisms by which complement components modulate ovarian cancer progression and to evaluate complement-based therapeutic combinations.

#### **6. Conclusions**

A growing body of literature suggests that the complement system is involved in ovarian cancer progression. Nevertheless, the specific role of the different complement components in different clinical scenarios has just started to be unraveled, and many answers remain elusive. The molecular heterogeneity of ovarian cancers and the complexity of the biological interactions in the ovarian TME pose a challenge to our understanding of the mechanisms underlying the complement-associated immune responses and the identification of adequate therapeutic targets. The situation is aggravated by the lack of preclinical models that reliably recreate ovarian cancer traits. Therefore, further studies are needed to better delineate the complement-related mechanisms associated with ovarian cancer progression as well as to determine how complement activation should be modulated to treat ovarian cancer patients.

**Author Contributions:** Y.S., D.A., R.P., and B.T. conceived and designed the manuscript. All authors (Y.S., D.A., A.G.-M., R.P., and B.T.) wrote the manuscript. Y.S. prepared the figures. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors' work was supported by the Foundation for Applied Medical Research (FIMA), Centro de Investigación Biomédica en Red Cáncer (CIBERONC), Grupo Español de Investigación en Cáncer de Ovario (GEICO), Fundación Ramón Areces, and Instituto de Salud Carlos III-EU FEDER "Una manera de hacer Europa" (PI20/00419 and PI20/00959). YS was supported by a predoctoral fellowship from Ministerio de Ciencia, Innovación y Universidades (FPU18/02638).

**Conflicts of Interest:** D.A. and R.P. are authors of patents and patent applications claiming the determination of complement fragments for lung cancer diagnosis. No potential competing interests are disclosed by the other authors.

#### **References**

### *Review* **Role of Oncogenic Pathways on the Cancer Immunosuppressive Microenvironment and Its Clinical Implications in Hepatocellular Carcinoma**

**Naoshi Nishida**

Department of Gastroenterology and Hepatology, Kindai University Faculty of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama 589-8511, Japan<EMAIL_ADDRESS>Tel.: +81-72-366-0221

**Simple Summary:** Hepatocellular carcinoma is known to become resistant to treatments easily by mutations in genes involved in the key cellular pathways targeted by current molecular targeted agents (MTAs). However, the immune checkpoint inhibitor (ICI) is a promising modality for cancer treatment, in which the cancer cells are made recognizable by the immune system. Blockade of the PD1/PDL1 proteins, which help cancers evade the immune system, is currently being tested in clinical trials in combination with MTAs. In this review, several cellular signaling pathways that can alter the immune processes within the tumor and can subsequently affect the patient's response to ICIs are detailed. This review may help scientists and clinicians to better understand the molecular factors that can influence ICI-based therapy and will help in identifying suitable cases for this type of treatment.

**Abstract:** The tumor immune microenvironment, including hepatocellular carcinoma (HCC), is complex, consisting of crosstalk among tumor components such as the cancer cells, stromal cells and immune cells. It is conceivable that phenotypic changes in cancer cells by genetic and epigenetic alterations affect the cancer–stroma interaction and anti-cancer immunity through the expression of immune checkpoint molecules, growth factors, cytokines, chemokines and metabolites that may act on the immune system in tumors. Therefore, predicting the outcome of ICI therapy requires a thorough understanding of the oncogenic signaling pathways in cancer and how they affect tumor immune evasion. In this review, we have detailed how oncogenic signaling pathways can play a role in altering the condition of the cellular components of the tumor immune microenvironment such as tumor-associated macrophages, regulatory T cells and myeloid-derived suppressor cells. The RAS/MAPK, PI3K/Akt, Wnt/β-catenin and JAK/STAT pathways have all been implicated in anti-tumor immunity. We also found that factors that reflect the immune microenvironment of the tumor, including the status of oncogenic pathways such as the volume of tumor-infiltrating T cells, expression of the immune checkpoint protein PD-1 and its ligand PD-L1, and activation of the Wnt/β-catenin signaling pathway, predict a response to ICI therapy in HCC cases.

**Keywords:** cancer; hepatocellular carcinoma; immune evasion; immunotherapy; immune checkpoint inhibitors; oncogenic signaling pathway; molecular targeted agents; genome; epigenome; tumor immune microenvironment

#### **1. Introduction**

Hepatocellular carcinoma (HCC) is highly refractory and is the third leading cause of cancer-related deaths worldwide [1]. Recent advancements in molecular targeted agents (MTAs) for HCC have dramatically improved the prognosis for patients with this disease. Following the approval of sorafenib as the first MTA for advanced HCC, lenvatinib has also been applied as a first-line systemic chemotherapeutic for HCC, while regorafenib, cabozantinib and ramucirumab have been approved as second-line agents [2]. Because MTAs primarily target molecules involved in oncogenic signaling pathways that play an

**Citation:** Nishida, N. Role of Oncogenic Pathways on the Cancer Immunosuppressive Microenvironment and Its Clinical Implications in Hepatocellular Carcinoma. *Cancers* **2021**, *13*, 3666. https://doi.org/10.3390/ cancers13153666

Academic Editors: Ion Cristóbal and Marta Rodríguez

Received: 8 June 2021 Accepted: 19 July 2021 Published: 21 July 2021

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**Copyright:** © 2021 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

important role in the development of cancer cells, the development of clones resistant to MTAs can happen easily by genetic mutations and modifications in the specific molecular pathways [3,4]. Hence, additional chemotherapeutic agents would be required.

In contrast, immune checkpoint inhibitors (ICIs) play a role in tumor regression by a different mechanism from that of MTAs [5]. They are known to interfere with the immunosuppressive mechanism to enhance the anti-tumor immune response [6]. Because the target molecules of ICIs are primarily expressed in the stromal cells as well as the cancer cells, ICIs can be effective even for patients who fail to respond well to MTAs or acquire resistance to them, potentially enabling ICIs to complement treatment with MTAs [5,7–9]. Although the clinical trial of anti-programmed cell death-1 (PD-1) monotherapy failed to show a significant difference in the survival of patients with advanced HCC compared with conventional MTAs, synergic effects of the combination of different kinds of agents can be expected in several ongoing clinical trials of ICI-based therapy. Based on a successful Phase III clinical trial, the combination of the ICI atezolizumab (an anti-PD-1 antibody) with MTA bevacizumab (an anti-VEGF-A antibody) was approved as a first-line therapy for unresectable HCC [10,11].

Because of the complexity of cancer immunity, where immune cells, tumor cells and other types of stromal cells affect each other, understanding the immune microenvironment of the tumor is difficult [5]. While it has been considered that oncogenic mutations in tumor cells do not directly affect the outcome of ICI therapy, recent reports have suggested that mutation-induced changes in the tumor phenotype can affect the tumor–stroma interactions through alterations in the expression of immunosuppressive cytokines, chemokines, receptors and metabolites, thereby potentially affecting the tumor immune microenvironment [5]. Thus, the anti-tumor effect of MTAs in combination therapy with ICIs can be attributed to the direct action of MTAs on the HCC cells, as well as the reduction in the immunosuppressive nature of the tumor microenvironment through the inhibition of specific oncogenic signals [12].

To understand the significance of oncogenic signaling in the establishment of an immunosuppressive tumor microenvironment, and for the application of this knowledge to the treatment of HCC, this review focused on the role of specific genetic mutations involved in the oncogenic pathways responsible for anti-tumor immunity, and the current status of and perspectives on the combination of ICIs and MTAs for the treatment of HCC.

#### **2. Cellular Components and Molecules Associated with an Inhibitory Tumor Immune Microenvironment**

Oncogenic signals affect the expression of several immune-related molecules, including immune regulatory receptors, ligands, growth factors and other humoral factors, which affect diverse stromal cells as well as cancer cells. The cellular components of tumors and their states are major players in the regulation of the tumor immune microenvironment. Therefore, to better understand the impact of oncogenic signals on anti-cancer immunity, the functions of the stromal cells involved in the immune microenvironment of tumors are briefly discussed here.

#### *2.1. Regulatory T-Cells*

Regulatory T-cells (Tregs) are CD4+ T-cells characterized by the expression of the transcription factor Foxp3. They can be induced in tumor tissues through growth factors and cytokines, such as transforming growth factor β (TGF-β), interleukin 10 (IL-10) and vascular endothelial growth factor (VEGF), and inhibit immune responses through various mechanisms [13]. In particular, Tregs express the inhibitory immune checkpoint molecule cytotoxic T-lymphocyte (associated) antigen 4 (CTLA-4), which plays a critical role in the regulation of T cell-mediated anti-tumor immunity. Generally, T cell activation occurs through binding of the co-stimulatory factor B7 (CD80/CD86) on antigen-presenting cells and CD28 on T-cells, in addition to T-cell receptor (TCR) recognition of major histocompatibility complex (MHC)-presented antigens. Binding of CD80/CD86 on dendritic cells (DCs) with CTLA-4 on Tregs results in the inhibition of DC maturation. In addition, the

membrane molecule CD25 (IL-2 receptor subunit) on Tregs induces the depletion of IL-2 and suppression of cytotoxic T-cells (CTLs) by immunosuppressive cytokines such as TGF-β and IL-10, and cytotoxic secretions such as granzyme B and perforin released by Tregs [13]. A subtype of HCC that showed predominant expression of an mRNA related to Treg response has been reported [14]. Tregs also secrete the epidermal growth factor receptor (EGFR) ligand amphiregulin, which can promote the growth of HCC cells carrying EGFR in an autocrine manner [15]. Tregs also express VEGF receptor 2 on their surface, and the VEGF signal induces the expansion of this type of T cell [16].

#### *2.2.  Via their increased arginase activity, degradation of arginine, and uptake of tryptophan, cysteine and other amino acids required for T-cell activation, MDSCs reduce the concentrations of these amino acids in the tissue microenvironment, thereby inhibiting the propagation and activation of T cells [17]. In addition, MDSCs produce TGF-β and IL-10, inducing Tregs and inhibiting natural killer (NK) cell function [18]. Furthermore, MDSCs induce the immunosuppressive M2 macrophages by secreting IL-10, which, in turn, downregulates IL-12 production by tumor-associated macrophages (TAMs) [19,20].

#### *2.3. Tumor-Associated Macrophages*

Generally, two types of macrophages exist in tumor tissues: M1 macrophages and M2 macrophages. Interferon-γ (IFN-γ) and Type 1 helper cell (Th1) cytokines induce the differentiation of inflammatory monocytes into M1 macrophages. Meanwhile, Type 2 helper cell (Th2) cytokines such as IL-4 and IL-13 promote the differentiation of tissue-resident monocytes into M2 macrophages [20]. In tumor immunity, M1 macrophages produce inflammatory cytokines such as tumor necrosis factor α (TNF-α), IL-6 and IL-12, and exert an anti-tumor effect, whereas M2 macrophages produce immunosuppressive cytokines such as IL-10 and TGF-β, and inhibit anti-tumor immune reactions [20]. The microenvironment in cancer is prone to inducing M2 polarization, which is a characteristic phenotype called tumor-associated macrophages (TAMs). The crosstalk between MDSCs and TAMs induces high IL-10 and low IL-12 levels. In addition, naïve CD4+ T cells differentiate into Th2 cells that can produce IL-4 [21]. These processes result in the development of M2 macrophages, which is a disadvantageous state for tumor immunity. A high level of IL-10 induces the downregulation of human leukocyte antigen (HLA) Class II antigens and reduces the antigen presentation capacity of DCs [21]. It also expands the Treg population and inactivates natural killer (NK) cells. Additionally, the TGF-β secreted by MDSCs induces the expression of the inhibitory receptor T-cell immunoglobulin and mucin domain 3 (TIM-3) on TAMs [20].

#### *2.4. Cancer-Associated Fibroblasts and Vascular Endothelial Cells*

Cancer-associated fibroblasts (CAFs) have proangiogenic activity through the production of extracellular matrix and matrix metalloproteinases; they play a role in tissue remodeling [22]. They also inhibit NK cell function through the production of prostaglandin E2 (PGE2) and indoleamine-2,3-dioxygenase (IDO) [23]. Indoleamine-2,3-dioxygenase is an enzyme involved in tryptophan metabolism, and a reduced level of tryptophan in tumors inhibits local T-cell activation. Hence, crosstalk between CAFs and TAMs also plays a role in immunosuppression. CAFs produce IL-8 and cyclooxygenase-2 (COX2), which lead to the release of TNF and platelet-derived growth factor (PDGF) from TAMs, and further activation of CAFs.

#### *2.5. Other Stromal Cells*

The vascular endothelium is stimulated by angiogenic growth factors such as VEGF and PDGF. It stimulates Tregs and MDSCs in tumor tissues via the production of TGF-β, VEGF and the chemokine C-X-C motif chemokine 12 (CXCL12) [20]. Hepatic satellite cells (HSCs), which generally play a critical role in liver fibrogenesis, also participate in the induction of Tregs and MDSCs by releasing hepatocyte growth factors [24,25]. HSCs also produce amphiregulin and CXCL12, which induce Tregs and MDSCs, respectively [26]. A subset of DCs with high expression of CTLA-4 was also observed in HCC tissues, which may carry immune tolerogenic effects through the production of IL-10 and IDO.

#### *2.6. Immunosuppressive Metabolites*

As shown above, the concentrations of metabolites from cancer cells and stromal cells strongly affect the immune state of the tumor. Cyclic adenosine monophosphate (cAMP), which accumulates in tumor tissues, inhibits CD4+ and CD8+ T-cell responses and macrophage activation, and enhances the Treg response by binding to adenosine A2A receptors [27]. In addition, due to the hypoxic environment in tumor tissues, cAMP upregulates the enzyme COX-2, which synthesizes PGE2 from arachidonic acid. Subsequently, PGE2 binds to prostaglandin E receptor 4 on T-cells and affects T-cell activation and cytokine production [20].

MDSC- and TAM-derived arginase hydrolyzes arginine in the urea cycle and inhibits the function of CTLs via this deficiency in L-arginine. In tumor tissues, a hypoxic environment results in the expression of hypoxia-inducible factor 1α (HIF-1α), which is known to activate arginase [17]. Additionally, IDO is reported to be produced by DCs, macrophages, CAFs, vascular endothelial cells and HCC cells via inflammatory cytokines [28]. As previously stated, IDO inhibits T-cell activation and amplification via depletion of tryptophan and stimulates the differentiation of naïve CD4+ T-cells into Tregs [20,29].

#### *2.7. Immune Checkpoint Molecules*

Immune checkpoint molecules regulate excessive T-cell activation and help to maintain immune homeostasis. In cancer cells, however, these immune checkpoint molecules help tumors evade the immune response. Many immune checkpoint molecules and their ligands have been identified, as summarized in Figure 1. Of these, CTLA-4, programmed cell death-1 (PD-1) and its ligand, programmed cell death-ligand 1 (PD-L1), are believed to play central roles in tumors' immune evasion [5,20]. The induction of immune checkpoint molecules is regulated by environmental factors as well as cell signaling. For example, extracellular stimulation of IFN-γ and hypoxia-induced HIF-1 can enhance the expression of PD-L1 in cancer cells, MDSCs and TAMs [30]. The binding of PD-L1 to PD-1 on TAMs induces the release of the immunosuppressive cytokine IL-10. Additionally, activation of the phosphatidylinositol-3 kinase (PI3K)–Akt pathway also reportedly induces PD-L1, and the loss-of-function mutation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a regulator of the PI3K–Akt pathway, is associated with the expression of PD-L1 in cancer [31,32]. We have also shown that activating mutations in PI3KCA are associated with PD-L1 expression in HCC cells [33]. TIM-3, lymphocyteactivation gene 3 (LAG-3) and the B- and T-lymphocyte attenuator (BTLA, CD272) are also known as co-inhibitory molecules on activated T-cells, based on their association with galectin-9, MHC Class II and herpesvirus entry mediator (HVME), respectively (Figure 1). These suppressive receptors are observed in tumor-infiltrating lymphocytes (TILs) in HCC tissues and are considered to be markers of exhausted T-cells [33–35].

**Figure 1.** Immune checkpoint molecules and their ligands.

#### **3. Unique Aspects of Immunological Characteristics in the Liver and Hepatocarcinogenesis**

Although ICIs are becoming one of the key agents for the treatment of HCC, the response to this type of agent is still unsatisfactory in the majority of HCC cases compared with other types of malignancies [36]. The relatively low response rate to the ICIs can probably be attributed to the low antigenicity of HCC, as tumor mutation is not high in this type of tumor [36,37]. In addition, as the liver needs to be immunotolerant to nonpathological and persistent inflammation, it carries tolerance mechanisms to immune reactions, including cancer immunity.

The liver is continually exposed to the pathogen and microbe components from the gut via the blood supply of the portal vein. In this situation, the liver limits hypersensitivity to food-derived antigens and components of the intestinal flora to prevent excessive tissue damage and maintain systemic tolerance [38]. Chronic infection with hepatitis virus and persistent stimulation by metabolites further induce immune suppression in the liver, which is one of the unique characteristics of the underlying condition of hepatocarcinogenesis [39]. Resident macrophages (Kupffer cells) play a key role in hepatic tolerance through the production of anti-inflammatory cytokines, leading to downregulation of co-stimulatory molecules. This immunological environment of the liver results in the development of fully exhausted T-cells, where suppressive anti-tumor immunity is not susceptible to rescue by ICIs [36,39]. It has also been reported that CD8+ PD-1+ T-cells in NASH livers show a lack of immune surveillance and tissue-damaging function, which contribute to the increase in HCC emergence upon anti-PD-1 treatment in a NASH mouse model [40]. Augmentation of CD8+ PD-1+ T-cells was also observed in human NASH; a worse outcome in HCC patients treated with anti-PD-1 antibodies was observed [40]. Although the details of the difference in the response to ICIs between virally induced and NASH-induced HCCs are still unknown, it is possible that different amounts and quality of antigens and the difference in the liver microenvironment, such as the balance between partially exhausted and fully exhausted T-cells, may be involved in the outcome on ICIs [39,41,42].

In addition, ICI may not be effective or may even exacerbate the disease in some HCC patients. It is reported that blockade of the PD-1 and PD-L1 interaction may induce an expansion of PD-1+ Tregs isolated from the liver of patients with chronic hepatitis C, because PD-1 on Tregs generally plays a role in the regulation of the CD4+CD25+FoxP3+ T-cells [43]. Therefore, a blockade of the binding of the ligand with PD-1 on Tregs may result in further suppression of anti-tumor immunity [44]. More importantly, PD-1+ Tregs may be involved in the hyperprogression of tumors in gastric cancer patients treated with anti-PD-1 antibodies [44]. As hyperprogression on anti-PD-1 antibodies has also been reported in HCC cases, ICI can be even detrimental in such cases [45].

#### **4. Signaling Pathways and the Immune Microenvironment of Tumors**

Alterations in oncogenic signaling in cancer not only trigger abnormal differentiation and cell proliferation, but also play a crucial role in the immune evasion of tumors [12]. Cancerrelated signaling affects the state of the tumors' immune components via cytokine, chemokine and growth factor production. To date, genetic alterations in several signaling pathways observed in cancers have been reported to affect the tumor immune microenvironment.

#### *4.1. RAS/MAPK Signaling Pathway*

In malignant melanomas, activating mutations in BRAF (BRAFV600E) induce constitutive activation of the mitogen-activated protein kinase (MAPK) pathway, which stimulates immune-tolerant DCs and inhibits CD8+ T-cells via the expression of the immunosuppressive cytokines IL-6 and IL-10, as well as via VEGF [46]. This effect has been reported to be inhibited by BRAF inhibitors and VEGF inhibitors. Furthermore, RAS/MAPK signaling inhibits antigen presentation on tumor cells, and inhibition of this pathway is associated with the recovery of MHC expression by IFN-γ in malignant melanomas and breast cancer [47,48]. Meanwhile, in a murine model of pancreatic cancer, activating mutations in KRAS (KRASG12D) induced MDSCs via the production of granulocyte macrophage colonystimulating factor (GM-CSF) and inhibition of CD8+ T-cell infiltration into tumor tissues, which contributed to the establishment of an immunosuppressive tumor microenvironment [49]. In fact, GM-CSF is known to be upregulated in human pancreatic intraepithelial neoplasia and pancreatic cancer cells [49]. An association between activating mutations in KRAS and resistance to ICIs has also been reported in colorectal cancer. KRAS-mediated repression of interferon regulatory factor 2 (IRF) results in the high expression of the chemokine C-X-C motif ligand 3 (CXCL3), which induces MDSCs that express C-X-C motif chemokine receptor 2 (CXCR2) as the receptor of CXCL3 in tumor tissues [50]. In this manner, activation of KRAS induces MDSC-mediated resistance to antitumor immunity in patients with colorectal cancer (Figure 2).

**Figure 2.** Effects of oncogenic signal activation on the tumor immune environment.

#### *4.2. PI3K/Akt Signaling Pathway*

Activation of the P13K/AKT signaling pathway is involved in critical cellular functions, including survival, inhibition of apoptosis and proliferation. Activation of P13K/AKT is one of the common features of cancer. PTEN, which regulates this pathway, demonstrates loss-of-function mutations in various cancers. In melanomas, mutations in PTEN are related to resistance to anti-PD-1 antibody treatment and are correlated with a reduced volume of CD8+ TILs [51]. In a murine model, PI3K-β inhibitors improved sensitivity

to treatment with anti-PD-1 antibodies and anti-CTLA4 antibodies when the resistance was induced by loss-of-function mutations in PTEN [51]. While loss of PTEN function is associated with the induction of various immunosuppressive cytokines, it also induces VEGF, which is presumed to be the mechanism by which immunosuppression is induced. In bladder cancer, activating mutations in PI3KCA are associated with a reduction in TIL volume, while PI3K inhibitors lead to an increase in TILs [52]. PI3K inhibitors have also been reported to inhibit Tregs and induce the differentiation of M2 macrophages into the M1 phenotype [53]. In addition, by inducing PD-L1 expression, both the RAS/MAPK and the PI3K/AKT signaling pathways can be involved in the suppression of anti-tumor immunity. We have previously reported that while activating mutations of the PI3K/AKT signaling pathway are associated with increased expression of PD-L1 in HCC, and the volume of TILs is generally high in PD-L1-positive HCC, TILs are deficient in HCCs with activating mutations of the PI3K/AKT signaling pathway [33]. Therefore, aside from external stimulation such as by IFN-γ, induction of PD-L1 is likely to be attributable to genetic mutations in the PI3K/AKT pathway in this setting (Figure 2).

BRAFV600E induces immune-tolerant dendritic cells (DCs) via the induction of IL-6, IL-10 and VEGF, ultimately inhibiting the action of CD8+ T-cells. In addition, KRASG12D induces MDSCs via GM-CSF production, thereby inhibiting CD8+ T-cells from infiltrating tumor tissues. In colorectal cancer, KRAS activation is known to induce CXCL3 expression and the induction of MDSCs with CXCR2, the receptor of CXCL3. In contrast, PI3K/AKT signaling activation is associated with VEGF expression and a decrease in tumor-infiltrating lymphocyte (TIL) volume, and has been reported to induce Tregs and inhibit the shift of M2 macrophages into M1 macrophages. In addition, PI3K/AKT signaling induces PD-L1 expression. Under the activation of WNT/β-catenin signaling, activating transcription factor 3 (ATF3)-mediated CCL4 downregulation is considered to reduce the migration of CD103+ dendritic cells into the tumor and reduce CD8+ TILs in melanoma. CCL5 was suggested to be involved in HCC. The activation of the WNT/β-catenin signaling pathway has also been reported to be involved in the formation of an immune suppressive tumor microenvironment through the upregulation of IL-10. Activation of the transcription factors YAP/TAZ, which are regulated by Hippo signaling, upregulate PD-L1 and are involved in CXCL5-mediated induction of MDSCs. In addition, YAP is involved in the induction of M2 macrophages by enhancing the transcription of CCL2.

#### *4.3. Wnt/β-Catenin Signaling Pathway*

ICIs are presumed to be insufficiently effective in cases where infiltration of CD8+ T-cells in tumors is lacking, and an analysis of human melanomas has revealed that activation of the Wnt/β-catenin signaling pathway is associated with reduced TILs in tumors. In melanomas with activating mutations in the Wnt/β-catenin signaling pathway, C-C chemokine ligand 4 (CCL4) is downregulated, which reduces the migration of CD103+ DCs and leads to a deficiency in CD8+ TILs. Furthermore, the transcriptional repression of CCL4 was attributed to the activation of activating transcription factor 3 (ATF3) as a result of the activation of the β-catenin signaling pathway [54]. Activating mutations of the Wnt/β-catenin pathway occur frequently in HCC, and immunosuppression in the tumor microenvironment based on activation of this signal in liver cancer is presumed to occur via downregulation of CCL5 [55]. In a murine HCC model, induction of CCL5 increased the number of DCs and CD8+ T-cells in tumors (Figure 2). In melanomas, activation of Wnt/β-catenin signaling also reportedly led to the upregulation of IL-10 through the binding of β-catenin/T-cell factor (TCF) on the IL-10 promoter, thereby contributing to the formation of an immunosuppressive environment [56].

Aside from the altered Wnt/β-catenin signaling in tumor cells, activation of this signaling pathway reportedly disturbs the effector function of CD8+ T-cells and induces the exhausted T-cell phenotype in HCC and colorectal cancers, which contributes to the establishment of immune suppressive tumor microenvironment [41]. Interestingly, neutralization of a canonical Wnt ligand, Wnt 3a, enhances the T-cell response through the rescue of DC activation, resulting in tumor regression in a mouse model [42].

#### *4.4. MYC Gene*

The transcription factor c-myc regulates the expression of genes necessary for cell proliferation and survival. In many cancers, amplification and overexpression of c-myc has been observed; these are involved in inducing the expression of immune checkpoint molecules such as PD-L1 and CD47 [57]. CD47 is a cell surface glycoprotein that regulates phagocytosis by binding to signal regulatory protein alpha (SIRP-α), which is specific to macrophages and DCs. Thus, overexpression of c-myc is involved in the immune evasion of cancer cells through CD47 and PD-L1.

#### *4.5. Chromatin Remodeling Pathway*

Genomic DNA is stored in the nucleus as chromatin. During transcription, replication or repair, alterations of the chromatin structure by chromatin remodeling regulate the access of transcription factors to the DNA. The SWItch/sucrose non-fermentable (SWI/SNF) complex is a chromatin remodeling factor that induces the alteration of nucleosomes via ATP hydrolysis. Genetic abnormalities in SWI/SNF complex subunits are frequently observed in HCC and other human tumors [33], and loss-of-function mutations in the polybromo 1 (PBRM1) gene involved in the SWI/SNF complex are common in renal cancer. Intriguingly, this PBRM1 mutation is associated with the therapeutic effect of ICIs in renal cancer. *PBRM1*-deficient renal cancers show altered transcriptional expression in the JAK/STAT (Janus kinase/signal transducers and activators of transcription) and immune signaling pathways [58].

#### *4.6. JAK/STAT Signaling Pathway*

The JAK/STAT pathway, which transmits signals that are crucial for growth, differentiation, survival and immunity, is altered in many types of malignancy. The downstream transcription factor, STAT3, acts on the PD-L1 promoter, thereby inducing upregulation of PD-L1 in cancer cells. In melanomas, JAK1 and JAK2 mutations inhibit signals from interferon receptors and reduce antigen presentation on tumor cells, which results in resistance to ICI therapy [59]. Meanwhile, β2-microglobulin gene mutations have been reported to induce resistance to ICI treatment via the loss of MHC Class I antigen expression on the cell surface [60].

#### *4.7. Hippo Signaling Pathway*

Hippo signaling, which is involved in the regulation of growth and differentiation as well as in controlling organ size, is dysfunctional in many malignancies. Reduced Hippo signaling is also associated with cancer's immune evasion. Hippo signaling regulates yes-associated protein (YAP) and "transcriptional coactivator with PDZ-binding motif" (TAZ), the activation of which leads to the expression of PD-L1 and stimulates MDSCs carrying CXCR2, by upregulation of its ligand, CXCL5 [61]. In a murine model of HCC, YAP was reported to be associated with tumor immunosuppression via the induction of M2 macrophages resulting from enhanced transcription of CCL2 (Figure 2) [62].

#### *4.8. DNA Repair Pathway*

It is well known that cancers carrying mutations in DNA mismatch genes induce a large number of neoantigens that are attributed to the emergence of a variety of passenger mutations that occur in the microsatellite sequences of the DNA, where anti-tumor immunity is enhanced. Therefore, microsatellite instability is a biomarker for efficacy in the treatment of ICIs [63]. Similarly, cancers with a high mutation burden (TMB) are also markers of tumors with an active immune microenvironment because of their high antigenicity [64]. Recently, it was reported that loss-of-function mutations in the breast cancer susceptibility (*BRCA*) 1 and *BRCA 2* genes, which are involved in the homologous

recombination pathway of DNA repair, are also markers of a high TMB and could be predictors of the outcome of ICI-based treatment [65]. From this perspective, alterations in DNA repair pathways are critical for the establishment of high antigenicity and "immune hot" status in cancer.

#### *4.9. VEGF Signaling*

In tumors, external stimulation can lead to the production of growth factors. In HCC, tissue hypoxia leads to the production of VEGF via the activation of hypoxia-inducible factor 1 (HIF-1), resulting in tumor angiogenesis. The cellular components of tumors that suppress tumor immune responses, such as MDSCs, Tregs and TAMs, express VEGF receptors; therefore, inhibition of VEGF/VEGFR can alter anti-tumor immunity [11]. Anti-PD-1 antibodies and anti-VEGFR-2 antibodies have been reported to have a synergistic effect in murine models of HCC. Anti-VEGFR-2 antibodies induce an increase in CTLs and a decrease in TAMs and Tregs [66]. Atezolizumab + bevacizumab is expected to combine the effects of ICIs with inhibition of VEGF signaling to alter immunity. Pembrolizumab + lenvatinib, a multikinase inhibitor (MKI) with a powerful antiangiogenic effect, and atezolizumab + cabozantinib, which is capable of blocking angiogenesis through the inhibition of VEGFR and AXL, and camrelizumab (an anti-PD-1 antibody) + apatinib (a selective VEGFR2-tyrosine kinase inhibitor) are in Phase III clinical trials, while avelumab (an anti-PD-L1 antibody) + axitinib (which strongly inhibits VEGFR) are undergoing Phase I/II trials (Table 1). ICIs and agents with an anti-VEGF/VEGFR effect are currently the most promising combination therapies for HCC because of their synergistic effect on cancer immunity [6,11].

**Table 1.** Clinical trials for combinations of molecular targeted agents and immune checkpoint inhibitors in hepatocellular carcinoma.

**Table 1.** *Cont.*

1 National Clinical Trial number (ClinicalTrials.gov registry number). 2 MTA: molecular targeted agent; ICI: immune checkpoint inhibitor; TKI: tyrosine kinase inhibitor; Ab: antibody.

#### **5. Signaling Pathway Abnormalities and the Immune Microenvironment in HCC**

In a mouse model of HCC, it has been shown that activation of Wnt/β-catenin signaling induces reduced migration of CD103+ DCs and CD8+ TIL deficiency via downregulation of CCL5. Previous reports have also shown that Wnt/β-catenin activation is associated with the reduced expression of T cell-derived genes in HCC tissues. Therefore, HCC with activated Wnt/β-catenin signaling is unlikely to respond to ICIs because of the "immune cold" phenotype [67]. In fact, post-ICI therapy outcomes are reported to be poor in cases of HCC with Wnt/β-catenin activation [68]. Using a cohort of HCC cases from The Cancer Genome Atlas (TCGA), we determined that the expression of T cell-related genes was low in cases of HCC with activating mutations in Wnt/β-catenin (Figure 3). In addition, in an analysis of HCC tissues, we determined that HCCs with activating mutations in Wnt/β-catenin pathway genes are significantly deficient in CD8+ TILs [33]. However, we did not find CD8+ TILs to be associated with mutations in any other oncogenic signaling pathways (Table 2).

**Figure 3.** Tumor-infiltrating lymphocyte status and abnormal WNT/β-catenin activation. Hierarchical cluster analysis was used to classify HCCs based on the expression of eight T cell-related cytokine mRNAs obtained from the TCGA transcriptome dataset (RNA-seq V2 RSEM) (**a**) "Immune hot" and "immune cold" refer to HCCs with high and low levels of T cell-related gene expression, respectively. Thus, "immune hot" indicates a large volume of tumor-infiltrating lymphocytes (TILs), while "immune cold" suggests that TILs are deficient. (**b**) The presence or absence of activating mutations in WNT/β-catenin pathway genes are determined based on the presence or absence of *CTNNB1, AXIN1* and *APC* mutations in the TCGA provisional dataset obtained. Activating mutations in the WNT/β-catenin pathway are more frequently detected in "immune cold" HCCs than in "immune hot" tumors (*p* = 0.0001). The TCGA dataset used in the present study was downloaded in September 2019.

**Table 2.** Association between alterations in oncogenic signaling pathways and the degree of CD8+ tumor infiltrating lymphocytes.

Degree of tumor infiltrating lymphocytes (TILs) are compared between HCCs with mutations in each oncogenic pathway and those without mutations. In total, 154 HCCs were examined for mutations using the Ion AmpliSeq Comprehensive Cancer Panel, and the degree of CD8+ cells was examined using immunohistochemistry. 1 Median: median number of CD8+ TILs/high power field. 2 *p* value by Wilcoxon's rank-sum test.

In a transcriptome analysis, we reported that Wnt/β-catenin signaling activation was associated with the decreased expression of gene sets related to T-cell priming/activation, IFN-γ response, immunosuppression and Tregs; it was most significantly associated with the downregulation of genes related to the IFN-γ response in multivariate analysis [69]. These data are consistent with the deficiency in CD8+ T-cells in HCC tissues. In addition, we also reported that activating mutations in the Wnt/β-catenin pathway is negatively associated with PD-L1 expression in HCC [33]. As the expression of PD-L1 can be induced by the stimulation of IFN-γ, the lack of PD-L1 expression in HCC with Wnt/β-catenin activation can probably be attributed to the low degree of CD8+ TILs that should secrete IFN-γ [69]. On the other hand, a previous study found that mutations in genes involved in chromatin remodeling, such as AT-Rich Interaction Domain 2 (*ARID2*), were also associated with an immunosuppressive tumor microenvironment through the expression of genes involved in the induction of M2 macrophages [14], although there were no associations between mutations of the genes involved in chromatin remodeling and the degree of CD8+ TILs as well as PD-L1 expression [33]. As mutations of *ARID2* are reportedly associated with the TAM subclass of HCC, the immune suppressive mechanism in HCCs with *an ARID2* mutation should be different from that of CTNNB1 [14]. In contrast, PD-L1-positive HCCs often have high levels of CD8+ TILs [33]. This may be due to the fact that PD-L1 expression in HCC cells can be mainly attributed to stimulation by the IFN-γ from TILs. It is possible that, under continuous immune response to cancer cells, many CD8+ TILs are prone to expressing multiple inhibitory receptors (PD1, TIM-3, LAG-3) that result in the exhausted phenotype of T-cells [33]. In many cases, PD-L1 expression is considered to be a favorable prognostic factor of ICI therapy, suggesting that blockade of the PD-1/PD-L1 response could, at least partially, activate the T-cell immune response, even if the immune cells express additional inhibitory receptors. Indeed, we found that the absence of activating mutations in Wnt/β-catenin pathway genes, a high CD8+ TIL volume and

PD-L1 expression were associated with long progression-free survival of HCC patients on anti-PD-1 antibody therapy, regardless of the expression of other inhibitory receptors, such as TIM-3 and LAG-3 [69]. In this way, assessments of gene alterations in cellular signaling pathways are not only useful for finding suitable MTAs that act on the altered cellular signal, but may also, theoretically, serve to predict the response to ICI therapy, based on the tumor immune microenvironment.

#### **6. Conclusions**

Alterations of cell signaling pathways play a critical role not only in the development of a malignant phenotype in cancer cells but also in the determination of anti-cancer immunity. In a Phase III clinical trial with HCC patients, ICI monotherapy failed to yield a significant anti-cancer response, suggesting that ICIs will be used primarily in combination therapy [6]. It has been speculated that the "immune cold" phenotype of the tumor microenvironment is critical for poor prognosis with ICIs, where activation of the Wnt/β-catenin signaling pathway plays an important role. From this point of view, understanding the response of HCCs carrying Wnt/β-catenin mutations to combination therapy with ICI and MTA is clinically important but has not been clarified yet. Currently, a combination of atezolizumab + bevacizumab is applicable for unresectable HCC; the efficacy of this combination on HCCs showing the "immune cold" phenotype is now under investigation [36]. In addition, aside from atezolizumab + bevacizumab, many ongoing clinical trials have examined combinations of ICIs and MTAs for HCCs that are refractory upon ICI monotherapy, mainly with MTA showing an anti-angiogenic effect (Table 1). However, future trials are likely to examine combinations of ICIs with agents that inhibit other oncogenic pathways that are critical for hepatocarcinogenesis, such as the Wnt/β-catenin pathway, the RAS/MAPK pathway and the PI3K/AKT pathway.

**Funding:** This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI: 21K07184, N. Nishida) and a grant from the Smoking Research Foundation (N. Nishida).

**Conflicts of Interest:** The author has not nothing to declare.

#### **References**

### *Systematic Review* **A Broad Overview of Signaling in** *Ph***-Negative Classic Myeloproliferative Neoplasms**

**Ana Guijarro-Hernández 1 and José Luis Vizmanos 1,2, \***

**Simple Summary:** There is growing evidence that *Ph*-negative myeloproliferative neoplasms are disorders in which multiple signaling pathways are significantly disturbed. The heterogeneous phenotypes observed among patients have highlighted the importance of having a comprehensive knowledge of the molecular mechanisms behind these diseases. This review aims to show a broad overview of the signaling involved in myeloproliferative neoplasms (MPNs) and other processes that can modify them, which could be helpful to better understand these diseases and develop more effective targeted treatments.

**Abstract:** *Ph*-negative myeloproliferative neoplasms (polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF)) are infrequent blood cancers characterized by signaling aberrations. Shortly after the discovery of the somatic mutations in JAK2, MPL, and CALR that cause these diseases, researchers extensively studied the aberrant functions of their mutant products. In all three cases, the main pathogenic mechanism appears to be the constitutive activation of JAK2/STAT signaling and JAK2-related pathways (MAPK/ERK, PI3K/AKT). However, some other non-canonical aberrant mechanisms derived from mutant JAK2 and CALR have also been described. Moreover, additional somatic mutations have been identified in other genes that affect epigenetic regulation, tumor suppression, transcription regulation, splicing and other signaling pathways, leading to the modification of some disease features and adding a layer of complexity to their molecular pathogenesis. All of these factors have highlighted the wide variety of cellular processes and pathways involved in the pathogenesis of MPNs. This review presents an overview of the complex signaling behind these diseases which could explain, at least in part, their phenotypic heterogeneity.

**Keywords:** myeloproliferative neoplasms; signaling pathways; JAK2; CALR; MPL; TPOR

#### **1. Introduction**

Myeloproliferative neoplasms (MPNs) are rare hematological malignancies characterized by the clonal expansion of mature myeloid cells. MPNs arise from certain somatic mutations in hematopoietic stem cells (HSCs) which provide a selective advantage and lead to the expansion of aberrant clones.

Classic MPNs consist of chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In the last few years, the advances in molecular biology have provided key insights into the molecular mechanisms behind these diseases. CML is genetically defined by the *Philadelphia* (*Ph*) chromosome, the result of t(9;22)(q34;q11). This translocation leads to the production of a chimeric BCR-ABL1 protein with constitutive tyrosine kinase activity. The description of the *Ph* chromosome as a disease-initiating event in CML revolutionized the diagnosis and treatment of this disease [1]. The targeted therapy imatinib showed a specific inhibitory capacity against

**Citation:** Guijarro-Hernández, A.; Vizmanos, J.L. A Broad Overview of Signaling in *Ph*-Negative Classic Myeloproliferative Neoplasms. *Cancers* **2021**, *13*, 984. https:// doi.org/10.3390/cancers13050984

Academic Editor: Alain Piché

Received: 8 February 2021 Accepted: 22 February 2021 Published: 26 February 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

the tyrosine kinase activity of BCR-ABL1 [2–4] that, despite not being curative [5], increased the 10-year survival of CML patients in chronic phase to more than 83%–84% [6,7].

This review is focused on PV, ET and PMF, all of them *Ph*-negative MPNs that share similar and mostly mutually exclusive driver mutations affecting *JAK2*, *MPL* and *CALR*. The aberrant functions of the mutant products encoded by these genes have been extensively studied and the main mechanisms that lead to the myeloproliferation described. Currently, it is considered that the major hallmark of *Ph*-negative MPNs is the constitutive activation of JAK2-related signaling pathways. In fact, at this time, the only targeted therapy approved in MPNs is the JAK1/2 inhibitor ruxolitinib, which can reduce splenomegaly and other common symptoms in patients with PMF, post-PV/ET MF [8,9] and PV resistant or intolerant to hydroxyurea [9,10]. Although a reduction in the mutant allele burden is rare [9], it could be achieved in long-term treatment [11]. However, the improvement in the overall survival of ruxolitinib-treated patients has been questioned [12–14]. Actually, malignant cells can still survive in these patients and the clinical response could be mainly due to the downmodulation of proinflammatory cytokines derived from the JAK2 inhibition [15]. These arguments have led researchers to question whether JAK2 is really the best drug target in these diseases or not [16].

In the meantime, some non-canonical mechanisms of mutant JAK2 [17–24] and CALR [25–33] have been described. Chronic inflammation [34–62] and the bone marrow microenvironment [63–72] also seem to contribute to the heterogeneous phenotypes found among MPN patients.

Additionally, mutations in disease-modifying genes that seem to increase the risk of leukemic transformation or progression from ET to myelofibrosis have also been identified [73–75]. The products encoded by these genes are involved in epigenetic modification, tumor suppression, transcription regulation, splicing, and some other signaling pathways [76,77]. Other factors, such as genetic predisposition, age or environment have also been shown to influence the heterogeneity of MPN phenotypes [78].

This review presents an overview of the signaling behind *Ph*-negative MPNs attending not only to the activation of JAK2-related canonical signaling pathways, but also to other non-canonical pathways, disease-modifying signaling, and additional factors that have been found to be involved in the pathogenesis of these diseases.

#### **2. JAK2-Related Canonical Signaling Pathways**

JAK2 signaling is activated through a variety of receptors such as those for erythropoietin (EPOR), thrombopoietin (TPOR), and granulocyte/macrophage colony-stimulating factor (GM-CSFR). They regulate the production of the erythroid, megakaryocytic, and granulocytic lineages, respectively. When stimulated by ligands, receptors dimerize and bring JAK2 kinases into proximity. JAK2 is phosphorylated upon receptor binding and induces the phosphorylation of the cytoplasmic portion of the receptor and downstream factors.

In 2005, several research groups simultaneously published the presence of the somatic mutation p.V617F (JAK2V617F) in the exon 14 of *JAK2* in patients with PV (96%), PMF (65%) and ET (55%) [79–84]. This mutation impairs the physiological inhibitory function of the JH2 pseudokinase domain upon the JH1 kinase domain, which acquires a constitutive activation that promotes JAK2 phosphorylation in the absence of ligand stimulation (Figure 1). In 2007, four additional gain-of-function somatic mutations in the exon 12 of *JAK2* were detected in 3% of patients with PV [84,85]: p.N542-E543del (30%), p.K539L (14%), p.E543- D544del (12%), and p.F537-K539delinsL (10%). All of them are located upstream of the JH2 pseudokinase domain and promote an increased phosphorylation of JAK2 compared to p.V617F [86].

**Figure 1.** JAK2-related canonical signaling pathways active in *Ph*-negative myeloproliferative neoplasms (MPNs). Mutations in *CALR* (CALRMUT), *JAK2* (JAK2V617F), and *MPL* (TPORW515) lead to the constitutive activation of JAK2/STAT, PI3K/AKT, and MAPK/ERK signaling that promotes the transport to the nucleus of several transcription factors such as STATs and FOXO. There, they regulate transcription of their target genes, causing increased proliferation and survival of mutant cells. Mutant proteins are depicted in red.

In 2006, the gain-of-function mutation p.W515L in the exon 10 of *MPL* was identified in a minor proportion of MPN patients [87]. p.W515L and p.W515K are the most commonly reported mutations, identified in approximately 5% of PMF patients and 1% of ET patients [88]. *MPL* encodes the thrombopoietin receptor (TPOR), which depends on JAKs to mediate signal transduction. *MPL* mutations (TPORW515) promote the dimerization and activation of TPOR, leading to transphosphorylation and activation of the previously bound JAK2 proteins (Figure 1) [89].

The molecular alteration that causes the 60–90% of PMF and ET cases in patients not harboring *JAK2*/*MPL* mutations was described in 2013 [90]. During that year, two research groups identified mutations in *CALR* [91,92], a gene that encodes calreticulin, a ubiquitous protein found in the endoplasmic reticulum (ER) of all nucleated cells with multiple functions inside and outside this organelle. CALR is a Ca2+-binding chaperone mainly involved in the regulation of intracellular Ca2+ homeostasis and a regulator of protein folding in the cellular response to ER stress (unfolding protein response (UPR)) [93]. However, this protein has been also found associated with other cytoplasmic, nuclear and extracellular proteins, so it could be involved in a wide variety of signaling pathways [94]. In fact, CALR has been associated with cellular stress responses, adipocyte differentiation, cardiogenesis, proliferation, wound healing, apoptosis and immunogenic cell death [90,94].

The structure of wild-type CALR consists of a signal peptide and three domains: an amino-terminal N-domain, a proline-rich P-domain and a carboxy-terminal C-domain, which contains an ER retention signal (KDEL). The *CALR* mutations described to date are insertions or deletions in exon 9 that shift the reading frame by one base pair (+1), mainly a 52-bp deletion (c.1902\_1143del) or type 1 mutation (CALRdel52), and a 5-bp insertion (c.1154\_1155insTTGTC) or type 2 mutation (CALRins5). As a result, mutant CALRs (CALRMut) show a novel C-terminal end that lacks the ER retention motif (KDEL) [91,92] and some Ca2+-binding sites [95]. In 2016, it was published that CALRMut is transported to the cellular membrane where it activates TPOR in a ligand-independent manner (Figure 1) [96–99]. The characterization of the TPOR binding capacity has revealed that the C-terminal end of CALRMut blocks the P-domain of the protein, which constitutively exerts an inhibitory effect on the N-domain. Consequently, the N-domain can bind to immature N-glycans on TPOR [96]. This mechanism is consistent with the observation that the Nglycan binding motif located in N-domain of CALRMut is required for TPOR activation [97]. In fact, blocking N-glycosylation on asparagine 117 of TPOR diminishes CALR-dependent TPOR activation [97,100]. Both wild-type and mutant CALR recognize immature forms of N-glycans and fold the protein correctly, but CALRMut fails to dissociate from the targeted protein [101]. Thus, the CALRMut-TPOR complex moves from the ER to the plasma membrane through the Golgi apparatus and is secreted out of cells [102]. However, secreted CALRMut is only capable to activate TPOR on the cell surface of cells expressing CALRMut since only these cells have the immature N-glycans on TPOR [96,102,103]. Stimulation of TPOR leads to the activation of JAK2-dependent signaling in a similar way to the rest of the *Ph*-negative MPNs.

In conclusion, the mutations described to date in *JAK2*, *MPL* and *CALR* lead to a constitutive activation of JAK2, which ultimately causes the aberrant proliferation and survival of malignant myeloid clones. The three major downstream signaling pathways that are activated by JAK2 are JAK2/STATs, MAPK/ERK, and PI3K/AKT (Figure 1). The evidence suggests that each of these pathways plays an important role in MPNs, although the JAK2/STAT pathway appears to be the main one. In fact, dysregulation of JAK2/STAT signaling has been identified in all MPNs regardless of mutational status [104].

#### *2.1. JAK2/STAT Pathway*

In MPNs, JAK2 phosphorylates and activates STATs (mainly STAT1, STAT3 and STAT5). It seems that STATs are differentially activated depending on the type of MPN. For example, *MPL* mutations increase STAT3 and STAT5 signaling. In PV patients, JAK2V617F binds to EPOR promoting STAT5 activation. In ET patients, both JAK2V617F and CALRMut bind to TPOR; JAK2V617F enhances the phosphorylation of STAT1 and STAT3 but CALRMut promotes STAT3 and STAT5 activation. However, in PMF, phosphorylation of STAT3 is decreased in both JAK2V617F and mutant CALRs. To date, the precise mechanisms that explain differential activation of the STATs remain unclear [78].

Once the STATs are phosphorylated, they form a dimer that enters the nucleus to activate the transcription of target genes (Figure 1). In this way, JAK2/STAT signaling stimulates cell proliferation, differentiation and survival.

#### *2.2. MAPK/ERK Pathway*

The activated JAK2 can also lead to the phosphorylation of ERK, a serine threonine kinase that activates multiple proteins in both the cytoplasm and the nucleus. ERK is a key regulator of a wide variety of signaling pathways, so its deregulation could disrupt multiple processes. In the cytoplasm, ERK contributes to ion transport, apoptosis, and regulation of metabolism, among others. In the nucleus, it targets regulators of cell cycle and multiple transcription factors (Figure 1) [105].

#### *2.3. PI3K/AKT Pathway*

JAK2 activation also stimulates the PI3K/AKT pathway. AKT is a cell survival kinase which inhibits apoptosis by phosphorylating the proapoptotic protein BAD and the transcription factor FOXO3A. In addition, AKT can activate a wide range of mechanisms such as protein translation through mTOR or the cell cycle machinery (Figure 1) [105].

#### **3. Non-Canonical Signaling Pathways**

#### *3.1. JAK2-Related Non-Canonical Signaling*

In 2009, activated JAK2 was described to be in the nucleus of hematopoietic cells and to phosphorylate Y41 on histone 3 (H3Y41). This event prevents the binding of heterochromatin protein 1 alpha (HP1α) to H3Y41 [17]. HP1α shows a proliferationdependent regulation and is involved in gene silencing, genome stability, and chromosome segregation (Figure 2). It has been found overexpressed in some tumors, and it has been proposed as a potential hallmark of cell proliferation that could be relevant in clinical oncology [18].

**Figure 2.** Main non-canonical signaling pathways activated by JAK2V617F in *Ph*-negative MPNs. In PV patients, JAK2V617F (depicted in red) activates the adhesion receptor Lu/BCAM through the RAP1-AKT signaling pathway, making their erythrocytes more adhesive. JAK2V617F has also been described to promote aberrant signaling in the nucleus, where it prevents the binding of heterochromatin protein 1 alpha (HP1α) and inhibits the methylation of histones via protein arginine methyltransferase 5 (PRMT5) impairment. MPN patients with JAK2V617F also seem to be insensitive to the anti-inflammatory cytokine IL-10, increasing TNF-α production through Toll-Like Receptor (TLR) signaling.

JAK2V617F also acquires the ability to phosphorylate the protein arginine methyltransferase 5 (PRMT5) leading to an impairment in its ability to methylate histones (Figure 2). When PRMT5 is knocked down in CD34+ cells, an increased colony formation and erythroid differentiation can be observed [19].

A recent study also suggests that erythrocytes from PV patients are more adhesive since JAK2V617F activates the erythrocyte adhesion receptor Lu/BCAM through an EPORindependent RAP1/AKT signaling pathway (Figure 2) [20].