A time for YAP1: Tumorigenesis, immunosuppression and targeted therapy

Abstract

YAP1 is one of the most important effectors of the Hippo pathway and has crosstalk with other cancer promoting pathways. YAP1 contributes to cancer development in various ways that include promoting malignant phenotypes, expansion of cancer stem cells and drug resistance of cancer cells. Because pharmacologic or genetic inhibition of YAP1 suppresses tumor progression and increases the drug sensitivity, targeting YAP1 may open a fertile avenue for a novel therapeutic approach in relevant cancers. Recent enormous studies have established the efficacy of immunotherapy, and several immune checkpoint blockades are in clinical use or in the phase of development to treat various cancer types. Immunosuppression in the tumor microenvironment (TME) induced by cancer cells, immune cells and associated stromal cells promotes tumor progression and causes drug resistance. Accumulated evidences of scientific efforts from the last few years suggest that YAP1 influences macrophages, myeloid-derived suppressor cells and regulatory T-cells to facilitate immunosuppressive TME. Although the underlying mechanisms is not clearly discerned, it is evident that YAP1 activating pathways in different cellular components induce immunosuppressive TME. In this review, we summarize the evidences involved in the dual roles of YAP1 in cancer development and immunosuppression in the TME. We also discuss the possibility of YAP1 as a novel therapeutic target.

Introduction

The Hippo signaling pathway was originally discovered in Dorsophilia, which is an essential pathway that regulates cell proliferation, apoptosis and organ growth. This pathway is highly conserved in mammals.^1,2 In this pathway, YAP1 and its paralog TAZ act as transcriptional coactivators of mainly TEADs. Because TEADs are not capable of inducing targeted genes expression by themselves,³ YAP1 and TAZ act together with TEADs to mediate this pathway.^4-6 The Hippo pathway is considered as a tumor suppressor pathway, since the component of this pathway plays a key role in regulating organ size by inhibiting cell proliferation, promoting apoptosis and regulating stem/progenitor cell expansion,^7-9 and mutations or downregulations of this pathway components lead to organ overgrowth.¹⁰ Dephosphorylated YAP1 due to inactivated Hippo signaling accumulates in the nucleus, and YAP1 in the nucleus upregulates the transcription factors that regulate the expression of genes associated with cell proliferation, reprogramming, stemness, epithelial-mesenchymal transition (EMT) and anti-apoptosis.^6,8,11,12 In addition, YAP1 also contributes to acquiring resistance of cancer cells to certain drugs treatment.^13-20 Of note, in addition to the Hippo pathway, YAP1/TAZ activities are also regulated by the Hippo-independent pathways²¹ that include: (i) mechanotransduction through the actin cytoskeleton and Rho GTPase;²² (ii) metabolic routes such as mevalonate pathway;^23,24 (iii) stem cell related signals like Wnt/β-catenin pathway.²⁵ These pathways regulate nuclear translocation of YAP1/TAZ. In many cancers, phenotypic changes occur due to dynamics of YAP1 expression,¹² and overexpression of YAP1 is recognized as a poor prognostic marker in various cancer types such as hepatocellular carcinoma (HCC), gastric cancer (GC), colorectal cancer (CRC), non-small-cell lung cancer (NSCLC) and small-cell lung cancer.^17,26-30 Because YAP1 inhibition suppresses tumor progression and recovers drug-sensitivities in pre-clinical setting, YAP1 could be an attractive therapeutic target.^5,15,31-35

Recently, the immunosuppressive tumor microenvironment (TME) is considered as one of the important elements for cancer progression and therapeutic resistance. The TME consists of various cells including tumor cells, myeloid cells, lymphocytes, macrophages, natural killer (NK) cells and stromal cells.³⁶ The main purpose of activated host immune cells in the TME should be attacking tumor cells. However, mainly influenced by tumor cells, myeloid-derived suppressor cells (MDSCs) and regulatory T-cells (Tregs) infiltration in the tumor bed inhibit the immune attacks against tumor cells.³⁷ Cancer immunotherapy facilitates the development of immune responsive TME. Several immune checkpoint blockades such as anti-programmed cell death 1 (PD-1) drugs (nivolumab and pembrolizumab) and anti-cytotoxic T lymphocyte associated antigen 4 (CTLA-4) drug (ipilimumab) are used in clinical practice,³⁸ while many other immune agents are in the process of development for clinical use. Most recently, YAP1 has been revealed to contribute to inducing immunosuppressive TME by upregulating programmed cell death ligand 1 (PD-L1) or stimulating cytokines of tumor cells to recruit tumor-infiltrating macrophages, MDSCs and Tregs.^39-42

In this review, we summarize the most recent advances in the dual roles of YAP1 in cancer development and immunosuppression. We also discuss whether YAP1 can be a promising therapeutic target for cancer treatment.

YAP1 in Cancer Development

Cancer progressive roles of YAP1

YAP1/TAZ, the important effectors in the Hippo pathway, act as oncogenes in various malignant tumors. They are transcriptional coactivators that mainly interact with transcription factors TEADs to exert diverse effects on tumorigenesis and cancer progression. We summarized cancer progressive roles of YAP1 in Table 1, and a brief outline about YAP1 roles in various cancer types is provided below.

Table 1.

Roles of YAP1 in cancer initiation and progression

Cancer type	Observation and/or mechanistic evidence	Reference
Bladder cancer	YAP1 was highly expressed in the majority of bladder cancer patients, and positive expression of YAP1 determined by immunohistochemistry was associated with advanced stage and poor prognosis.	82
	YAP1 promoted cancer cell proliferation, tumor growth and enhanced cancer stem cell generation and expansion by coordinating with other associated molecules.	84-86
Breast cancer	YAP1 promoted cell proliferation, tumorigenesis, and EMT. Nuclear accumulation of YAP1 was associated with poor prognosis.	76-78
	YAP1 knockdown suppressed malignant phenotypes and enhanced tumor growth in mouse model.	80
	Although YAP1 expression was positively associated with proliferation in ER negative patients, low YAP1 expression was associated with poor prognosis in luminal A subtype of patients.	81
	YAP1 enhanced the mutant p53 to promote cell growth by upregulating cyclin A, cyclin B, and CDK1.	132
CRC	Activated YAP1 was associated with poor prognosis and induced tumor cell proliferation and progression.	17,57
	YAP1 had a crosstalk with Wnt/β-catenin pathway. YAP1 directly regulated β-catenin, and β-catenin also enhanced YAP1 expression.	25,58-60,133
GC	YAP1 was overexpressed in nucleus and cytoplasm in advanced GC patients, and high YAP1 expression level was associated with poor prognosis.	27,52,53
	YAP1 promoted RAF/MEK/ERK signaling pathway to enhance malignant phenotypes.	52
	YAP1 and RUNX2 cooperatively played roles for oncogenic transformation. On the other hand,RUNX3 inhibited YAP1-TEAD activity.	54,55
	MiR-15a, miR-16-1, and miR-506 suppressed YAP1 activity.	34,56
HCC	Overexpression of YAP1/TAZ was associated with more advanced pathological stage and poor prognosis.	30,43
	Yap1 expression mediated liver tumorigenesis and progression in animal models.	44-46
	Verteporfin (YAP1 inhibitor) treatment reduced the cancer progression.	46
	YAP1 had a crosstalk with Notch, PI3K-mTOR and MEK pathways to promote carcinogenesis.	47-49,134
	YAP1 was suppressed by miR-132 and miR-375, resulting in tumor regression.	50,51
NSCLC	Elevated YAP1/TAZ expressions were associated with advanced clinicopathological factors and poor prognosis, and YAP1/TAZ activations led to lung tumorigenesis and tumor progression through their transcriptional targets.	28,65-70
	RASSF1A and VGLL4 suppressed YAP1 activity to inhibit tumor progression.	71,72,135
	Germline R331W missense mutation of YAP1 raised the risk of lung adenocarcinoma.	74
	MiR-138, miR-497 and miR-375 downregulated YAP1 to prevent tumor growth.	33,35,73
Pancreatic cancer	YAP1 acted as an oncogene independently of KRAS. When KRAS was suppressed, YAP1 substituted for KRAS and rescued cancer cells from cell death in KRAS dependent cancer cells.	60-62
	Yap1/Taz were associated with cancer development through the activation of Jak-Stat3 pathway in mouse model.	64
	MiR-141 and miR-375 downregulated YAP1 to inhibit cancer cell growth.	136,137

In HCC, several studies have demonstrated that overexpressed YAP1/TAZ are associated with advanced pathological grades, poor cellular differentiation and poor prognosis.^30,43 In transgenic mice, Yap1 overexpression mediated by miR-130a resulted in hepatomegaly and liver tumorigenesis.⁴⁴ Conversely, in a genetically engineered HCC mouse model, Yap1 inhibition by siRNA-lipid nanoparticles restored hepatocyte differentiation and caused tumor regression.⁴⁵ The experiments in resistant-hepatocyte rat model revealed that Yap1 overexpression was an early event in liver tumorigenesis and that verteporfin, a small molecule drug that inhibits YAP1-TEADs complex, significantly reduced cancer cell proliferation.⁴⁶ In HCC, YAP1 influences several oncogenic pathways such as: (i) YAP1 targets Jagged-1 to induce Notch signaling for tumorigenic activities;⁴⁷ (ii) YAP1 influences PI3K-mTOR pathways by suppressing PTEN;⁴⁸ (iii) Concomitant activation of Yap1 and PI3K promoted activation of AKT/mTOR, ERK/MAPK and Notch pathways for liver tumorigenesis in mice.⁴⁹ Several microRNAs have been reported to affect YAP1 expression. In HCC, miR-132 and miR-375 suppress the expression of YAP1 and lead to tumor regression.^50,51

As noted above, YAP1 was overexpressed in GC and associated with poor prognosis.^27,52 Furthermore, high-grade or metastatic GC patients overexpressed YAP1 in the nucleus and cytoplasm.⁵³ Inhibition of YAP1 decreased cancer cell proliferation, colony formation, invasiveness and motility. The signaling pathway analysis revealed that YAP1 promoted RAF/MEK/ERK pathway activities and thus enhanced the expression of FOS in GC.⁵² Furthermore, RUNX2, which plays a key role in gastric development, cooperated with YAP1 to inhibit p21 expression, resulting in oncogenic transformation.⁵⁴ On the other hand, RUNX3, a potential tumor suppressor, reduced the DNA-binding activity of TEAD and decreased the YAP1-TEAD activity.⁵⁵ In regards to micro-RNAs, miR-15a, miR-16–1, and miR-506 suppress YAP1 in GC.^34,56

Activated YAP1 signature is associated with poor prognosis in CRC.¹⁷ Immunohistochemistry of 144 CRC patients’ specimens revealed that active nuclear YAP1 had a positive correlation with Ki-67 and nuclear phosphorylated ERK expression, which induced the tumor cell proliferation and progression.⁵⁷ Previous studies have shown the association between YAP1 and Wnt/β-catenin signaling pathway that is commonly deregulated in CRC.⁵⁸ Among 36 CRC specimens, 86% scored positive for nuclear localization of both YAP1 and β-catenin.⁵⁹ Interestingly, YAP1 and β-catenin regulate each other. Phosphorylated YAP1 (inactive form) binds directly to β-catenin in the cytoplasm and prevents its nuclear translocation, resulting in suppression of Wnt/β-catenin pathway.⁶⁰ Conversely, β-catenin upregulates YAP1 by binding to DNA enhancer element of Yap1, and knockdown of β-catenin led to decreased YAP1 expression.⁵⁹ Recently, Azzolin et al. showed that YAP1/TAZ are restricted in the β-catenin complex in Wnt/β-catenin signaling off cells, while activated Wnt/β-catenin signaling releases YAP1/TAZ from the complex and accumulates them in the nucleus to activate oncogenic pathways for cancer progression.²⁵

In pancreatic cancer patients, KRAS mutation is widely observed. YAP1 expression substitutes for oncogenic KRAS in KRAS mutant colon and pancreatic cancer cell lines.⁶¹ KRAS and YAP1 converge on the transcription factor FOS to regulate EMT, and YAP1 prevents cell death in KRAS dependent cancer cells upon suppression of KRAS.⁶¹ In pancreatic cancer, Yap1 activation enables the bypass of oncogenic KRAS addiction.⁶² These insights reinforce that YAP1 plays its oncogenic roles by an independent pathway from KRAS. In addition, YAP1 acts as a critical transcriptional switch downstream of the KRAS-MAPK pathway and amplifies the expression of genes encoding secretory factors to promote neoplastic proliferation and tumorigenic stromal response in the TME.⁶³ In a recent study, pancreatic tumor initiation was studied in Yap1/Taz disrupted KRas mutant mice.⁶⁴ In this study, Yap1 and Taz directly and independently upregulate transcriptional activation of several genes in the Jak-Stat3 signaling pathway, which induces acinar-to-ductal metaplasia, a precursor of pancreatic ductal adenocarcinoma.

In NSCLC, significant correlations have been demonstrated between elevated YAP1/TAZ expressions and malignant features including high histological grade, positive lymph node metastasis, advanced TNM stage and poor prognosis.^28,65 Knockdown of YAP1 or TAZ decreased in vitro cellular migration and transplantation of metastatic disease, while activated Yap1 enhanced lung tumor progression in mice.⁶⁶ Numerous groups have studied the mechanism of YAP1 in lung oncogenesis and found that YAP1/TAZ are associated with NSCLC through their transcriptional targets such as AXL, CYR61, AREG, and EREG.^67-70 RASSF1A, an upstream regulator of YAP1, inhibits YAP1 via the Hippo pathway and decreases invasion and metastasis of NSCLC cells.⁷¹ Another potential tumor suppressor gene, VGLL4, also negatively regulates YAP1-TEAD transcriptional complex by inhibiting YAP1 binding to TEADs in NSCLC.⁷² Several microRNAs also influence YAP1 expression in NSCLC.^33,35 MiR-138 and miR-497 suppress YAP1 directly to decrease cancer cell proliferation, invasion, migration and tumor growth respectively.^33,35 In neuroendocrine lung cancers, activated miR-375 inhibits YAP1 directly to prevent tumor growth.⁷³ Although several known oncogenes are activated by oncogenic mutation in the coding region, YAP1 mutation is rare in NSCLC. Whole-genome sequencing data of 1312 patients revealed 1.1% germline R331W missense mutation of YAP1 in lung adenocarcinoma.⁷⁴

In breast cancer, YAP1/TAZ influence multiple cancer-associated features such as cell proliferation, survival, migration, metastasis and resistance to chemotherapy.⁷⁵ Different investigators reported inconsistent findings of YAP1 in breast cancer. On one hand, YAP1 has been reported to be an oncogene based on: (i) YAP1 promoted breast cancer cell proliferation and tumorigenesis in mice;⁷⁶ (ii) Nuclear accumulation of YAP1 was associated with shorter disease-free survival;⁷⁷ (iii) YAP1 overexpression was associated with upregulation of mesenchymal markers such as fibronectin, vimentin and N-cadherin and downregulation of the epithelial markers such as E-cadherin and OCLN in the human mammalian cell line;⁷⁸ (iv) High expression of YAP1 was observed in metaplastic carcinoma (characterized by higher stemness and EMT features) compared to triple-negative subtype.⁷⁹ In contrast, some studies reported anti-tumorigenic roles of YAP1: (i) Knockdown of YAP1 suppressed anoikis, increased migration and invasiveness of breast cancer cells and enhanced tumor growth in mice;⁸⁰ (ii) In clinical samples, YAP1 expression was positively correlated with estrogen receptor (ER) negative patients, while it was inversely correlated with ER positive patients;⁸¹ and (iii) Low YAP1 mRNA expression was an independent poor prognostic factor for the luminal A subtype.⁸¹ This study reported that downregulation of YAP1 leads to increased ER and progesterone receptor levels in the luminal A subtype patients and associates with increased response to tamoxifen adjuvant therapy, which resulted in better prognosis. From these conflicting results, YAP1 is considered to play diverse roles in different breast cancer subtypes.²¹

In bladder cancer, YAP1 expression is significantly associated with advanced clinicopathological stage and poor prognosis.⁸² YAP1 promotes bladder cancer cell growth and migration by cooperating with other molecules such as ANKRD17, KLF5, and COX2.^83-86

In summary, although the roles and mechanisms are different among cancer types, accumulated evidences suggest that YAP1 is predominantly an oncogene, and YAP1 interplays with other molecules for the initiation and progression of various cancer types.

YAP1 and cancer stem cells (CSCs)

CSCs are considered to derive from normal counterparts harboring oncogenic mutations or transformed epithelial cells that undergo EMT and acquire a stem-like phenotype such as self-renewal abilities.^87,88 In a recent study, we demonstrated that YAP1 is overexpressed in the basal type of bladder cancer that is considered as stem cell phenotype.⁸⁶ In our study, high YAP1 expression was associated with increased mesenchymal markers including vimentin and Snail. YAP1 overexpression induced CSC properties such as sphere-forming, self-renewal abilities, invasiveness and drug resistance. These YAP1 associated biological features related to CSCs are implemented through regulating SOX2 activity. The role of YAP1 in regulating cellular stemness has been reported in several studies. In NSCLC, YAP1 directly interacts with OCT4 followed by SOX2 upregulation to facilitate self-renewal and vascular mimicry of CSCs, while depletion of YAP1 lowered the expression of core embryonic stem cell factors such as SOX2, OCT4 and NANOG.⁸⁹ YAP1/TEAD regulate cellular pluripotency and chemoresistance in human ovarian cancer.⁹⁰ In HCC, YAP1 concurrently expressed with two potential stemness markers, EPCAM and keratin 19.⁹¹ Recently, the analysis of microarray based transcriptome data revealed that YAP1 signaling influences long noncoding RNA to drive self-renewal of liver CSCs.⁹² All the later evidences suggest that YAP1 regulates CSCs, albeit, incomplete information is available to conclude the exact mechanisms of this regulation. As noted above, substantial evidences suggest that the Hippo pathway is one of the important pathways for CSCs regulation. However, other pathways such as Wnt/β-catenin pathway is also involved in regulating YAP1 for maintenance and expansion of CSCs.^21,58 In a recent study, downregulation of Wnt/β-catenin pathway led to sequestration of YAP1/TAZ in the β-catenin destruction complex, while upregulation of Wnt/β-catenin pathway released YAP1/TAZ from the β-catenin destruction complex, and they accumulated in the nucleus.²⁵ Further investigation about the molecular mechanisms of YAP1-driven CSCs possibly leads to the novel therapeutic strategy for eradication of CSCs that potentially harbor in residual disease.

YAP1 signaling and drug resistance

Recently, various improved formulated cytotoxic chemotherapies and molecular-targeting drugs have been developed to treat malignant tumors, especially for metastatic disease. Drug resistance is one of the fundamental challenges in treating cancer. Inhibiting molecules that are responsible for the drug resistance can enhance the drug efficiency.⁹³ As YAP1 signaling pathway has been consistently associated with the occurrence of intrinsic or acquired resistance to chemotherapeutic agents in several malignancies, inhibition of YAP1 signaling may synergistically enhance the efficacy of chemotherapeutic agents.⁹³ Such a notion solidified by our recent findings that genetically and pharmacologically silencing of YAP1 suffices to render resistant cancer cells more sensitive to chemotherapeutic agents such as gemcitabine and cisplatin.⁸⁶ We summarized the articles associated with YAP1 impacts on drug resistance in Table 2.

Table 2.

Drug response associated with YAP1 expression status

Cancer type	Drug	Result	In vivo	Reference
Bladder cancer	Cisplatin	YAP1 knockdown enhanced cisplatin sensitivity, and verteporfin treatment inhibited tumor cell proliferation and restored the sensitivity to cisplatin in mice.	Yes	86,138
Breast cancer	Lapatinib	Expressions of YAP1/TAZ decreased the response to lapatinib in HER2 positive breast cancer. The combination of YAP1/TAZ inhibition and labatinib had synergistic benefit in mice xenograft.	Yes	139
CRC	5-fluorouracil (5-FU)	YAP1 was overexpressed in 5-FU resistant cells.	No	140
	Doxorubicin	YAP1 was overexpressed in doxorubicin-resistant cells.YAP1 promoted cell survival by increasing survivin expression.	No	18
	Cetuximab	From five patient cohorts, patients with high YAP1 expression in tumor tissue were more likely to be resistant to cetuximab.	No	17
Esophageal cancer	5-FU Docetaxel	YAP1 directly induced EGFR in tumor cells, and inhibition of YAP1 increased the sensitivities to 5-FU and docetaxel through downregulation of YAP1 and EGFR.	Yes	141
HCC	Irinotecan	Hypoxia-induced nuclear translocation and accumulation of YAP1 promoted resistance to irinotecan. YAP1 inhibition improved the anti-cancerous effects.	Yes	14
Head and neck cancer	Cetuximab	Patients with overexpressed YAP1 in tumor tissue tended to be more resistant to cetuximab, and YAP1 silencing restored cetuximab sensitivity.	No	95
Melanoma	Vemurafenib	Overexpression of YAP1 induced in resistance of BRAFV600E mutant melanoma cells to vemurafenib, BRAF inhibitor. Actin remodeling was associated with the resistant mechanism.	No	142
NSCLC	Paclitaxel	MiR-424-3p restored the chemosensitivity of NSCLC cell lines through suppressing YAP1.	No	20
	Erlotinib	Overexpression of YAP1 promoted resistance to erlotinib in the NSCLC cell line. Combinational treatment of verteporfin and erlotinib suppressed the tumorigenic properties of erlotinib-resistant cells.	No	15
	Gefitinib	YAP1 was overexpressed in gefitinib-resistant NSCLC cell lines than parental cells. The combination of verteporfin and gefitinib decreased the viability of gefitinib-resistant cells.	No	16
Oral squamous cell carcinoma	Cisplatin	Increased nuclear translocation of YAP1 was observed in cisplatin-resistant cells, and YAP1 knockdown decreased cisplatin resistance.	No	143
Ovarian cancer	Cisplatin	YAP1 inhibition recovered the sensitivity to cisplatin through the impairment of autophagy.	No	19
	Cisplatin Erlotinib	Knockdown of YAP1 sensitized cancer cells to cisplatin and erlotinib. PTPN14 negatively regulated YAP1.	No	144
Various cancers	RAF/MEK inhibitors	Combination of YAP1 and RAF/MEK inhibitors synergistically suppressed BRAF or RAS mutant tumors.	Yes	145

A very few studies described the mechanisms of drug resistance governed by YAP1. In a recent ovarian cancer study, YAP1 induced EGFRs and EGF-like ligands, and EGF-like ligands also activated YAP1 through EGFR, resulting in cancer cell growth.⁹⁴ It is noted that the combined targeting of YAP1 and EGFR pathways may be more effective than either pathway inhibition. In human NSCLC cells, YAP1 was shown to promote erlotinib (EGFR tyrosine kinase inhibitor) resistance.¹⁵ CRC patients who expressed higher levels of YAP1 were more likely to be resistant to cetuximab (EGFR monoclonal antibody) compared to patients who expressed lower levels of YAP1.¹⁷ Furthermore, in head and neck cancer, YAP1 mRNA expression level was identified as a predicting marker of cetuximab resistance.⁹⁵

In summary, YAP1 expression may decrease the therapeutic efficacy of numerous anti-cancer drugs, and the combination of conventional drugs with YAP1 inhibitor may suppress malignant tumors more effectively. Verteporfin, a member of porphyrin family, is identified as the most effective inhibitory drug against Yap1-TEADs complex, and it is approved by Food and Drug Administration (FDA) for the treatment of macular degeneration.^5,96 Inhibition of YAP1 by genetic silencing or verteporfin can increase the sensitivity of cisplatin, erlotinib and gefitinib in different cancer types.^13,16,18,86 In addition to verteporfin, other drugs such as dasatinib and statins are capable of blocking YAP1/TAZ activities through the suppression of YAP1/TAZ associated components.^21,97

Accumulated evidences implicate that the combination of anti-cancerous drugs and the YAP1-inhibitory agents is a promising strategy to bring cancer expansion under control. However, verteporfin possibly causes adverse events to patients derived from its high phototoxicity, inhibition of autophagy and induction of oligomerization.⁹⁶ Before clinical application, further investigations to clarify the mechanism of YAP1 inhibitory drugs are warranted.

YAP1 and Immunoregulation

Recent development of immune checkpoint blockades and immunosuppression in the TME

Immune activity in the TME is deeply associated with cancer progression and drug resistance. In recent years, immunotherapy has been recognized as one of the standard treatments for several cancer types such as melanoma, NSCLC, bladder cancer and renal cell carcinoma, and anti-PD-1, anti-PD-L1 and anti-CTLA-4 drugs have been approved by FDA.⁹⁸ These immune-oncologic (IO) agents are different from conventional anti-cancerous drugs, as IO agents target the TME rather than tumor cells. However, although comparatively less toxic than traditionally used chemotherapy, the response rate to immunotherapy remains 15–30% depending on cancer types.⁹⁹ The mechanisms defining the sensitivity of IO agents among patients are still elusive, and limited numbers of comprehensive and extensive mechanism based studies have been reported. Further understanding about these mechanisms of IO agents response will contribute to more efficient therapeutic strategies including combinational treatment of several IO agents or combination of IO agents and conventional cytotoxic or molecular-targeting drugs.

Recent studies revealed that the activation of oncogenes or the inhibition of tumor suppressor genes induces immunosuppressive TME. For example, the transcription factors NF-κB and STAT3 promote the production of cytokines, chemokines, growth factors and several enzymes to stimulate inflammation and establish immunosuppressive TME.¹⁰⁰ Conversely, p53 activation stimulates both innate and adaptive immunity, while p53 inactivation alters the immune landscape of the TME towards pro-tumor inflammation, and p53 reactivation changes the TME to promote antitumor immunity.¹⁰¹ Since various molecules induce immunosuppression in the TME, it is understandable that manipulation of those molecules may facilitate immune-responsive TME and will suppress cancer progression and enhance the efficacy of IO agents.

The role of YAP1 in the TME is gradually emerging. In non-cancerous tissues, YAP1 negatively regulates an innate antiviral immune response through blocking interferon regulatory factor 3 in the interferon-β signaling pathway.¹⁰² We summarized the previous reports that described the roles of YAP1 in the TME (Fig. 1a-1c and Table 3).

Figure 1.

(a–c) YAP1 in tumor and immune cellular components of TME. (a) YAP1 in cancer cells upregulates various cytokines, chemokines and molecules associated with recruitment of M2 macrophages, Tregs and MDSCs and inhibition of NK cells. These impacts caused by YAP1 contribute to the suppression of effector T-cells infiltration and activities. (b) YAP1 in naïve T-cells induces the differentiation to Tregs by upregulating TGFBR2. (c) YAP1 in stromal cells promotes the transcription of myofibroblast marker genes like α-SMA, CYR61, and CTGF for the activation of cancer-associated fibroblasts in breast cancer. Then, activated cancer-associated fibroblasts recruit MDSCs and Tregs and lead impairment of CD8+ T-cells. (d) The combinational therapeutic strategy of immune checkpoint blockades and YAP1 and/or COX2 inhibitory agent seems to be fascinating to enhance the efficacy of immunotherapy.

Table 3.

Roles of YAP1 associated with immunosuppression in the TME

Target cell	Target molecule	Effect	Cancer or cell type	In vivo	Reference
Cancer cell	PD-L1	YAP1/TEAD upregulated PD-L1 by directly binding to the PD-L1 promoter region.	NSCLC	No	41,104
CD8+ T-cell	TME	YAP1 and c-MET over-expression and LEF1 down-expression were associated with decreased CD8+ T-cells numbers/function in the tumors.	Melanoma resistant to MAPK inhibitor	No	105
M2 macrophage	CCL2, CSF1	YAP1 in cancer cells directly recruited M2 macrophages by stimulating CCL2 and CSF1 for liver carcinogenesis.	HCC	Yes	40
M1/M2 macrophage	CCL2	YAP1 upregulated CCL2 for the recruitment of M1/M2 macrophages to induce inflammation and tumor formation.	HCC	Yes	108
MDSC	CXCL5	YAP1/TEAD directly upregulated CXCL5 in cancer cells to recruit CXCR2 expressing MDSCs, resulting in decreased infiltration of CD8+ T-cells.	Prostate cancer	Yes	42
MDSC	CSF1–3, IL-6	IL-6 and CSF1–3 induced by YAP1 stimulated the accumulation of MDSCs.	Pancreatic cancer	Yes	110
MDSC, NK cell, cytotoxic T-cell	TNF-α	YAP1 promoted by PRKCI directly upregulated TNF-α to recruit MDSCs, which inhibited NK cells and cytotoxic T-cells infiltration.	Ovarian cancer	Yes	111
MDSC	COX2	YAP1 upregulated COX2. COX2 and PGE2 promoted the recruitment of MDSCs.	Granulosa cells Neurofibromatosis type 2	No Yes	112 113
Treg	FOXP3	YAP1 expression in cancer cells was associated with the infiltrating Treg count.	GC	No	129
Treg	TGFBR2	YAP1 in peripheral T-cells directly upregulated TGFBR2 to stimulate the polarization to Tregs.	HCC	No	39

Programmed cell death ligand 1 (PD-L1)

PD-1 is expressed on the cell membrane in various immune cells including T-cells, and PD-L1 is one of the ligands of PD-1 that is expressed on several types of cancer cells as well as immune cells.¹⁰³ Most recent studies demonstrated the positive correlation of YAP1 and PD-L1 expressions in NSCLC patients.^41,104 In addition, these studies have shown that YAP1 directly upregulates PD-L1 transcription.

In melanoma, an integrated transcriptomic and methylomic analysis was conducted using the tissues from acquired resistant patients to MAPK inhibitor and pre-treatment patients.¹⁰⁵ In this study, overexpression of YAP1 and c-MET and under-expression of LEF1 were found to be associated with reduced sensitivity to MAPK inhibitor. Importantly, resistant tumor tissues with highly expressed YAP1 and c-MET exhibited decreased intra-tumoral CD8⁺ T-cells numbers/function and antigen presentation genes such as B2M, HLA-A, HLA-B, and TAP1. The authors noted that these immunosuppressive changes induced by MAPK inhibitor treatment may eventually cause resistance to anti-PD-1/PD-L1 immunotherapy.

Tumor-infiltrating macrophages

There are two groups of tumor-infiltrating macrophages. While immune activated type (M1) inhibits cell proliferation and causes tissue damages, M2 type promotes cell proliferation and tissue repair.¹⁰⁶ In the TME, M2 type macrophages play important roles in tumorigenesis and tumor progression by suppressing immune clearance, promoting cancer cell proliferation and stimulating angiogenesis.¹⁰⁷ In a recent study, it was found that YAP1 directly recruits M2 macrophages for liver carcinogenesis, and YAP1 activates tumor-initiating cells for the recruitment of M2 macrophages from the very beginning of tumor formation.⁴⁰ In this process, YAP1-TEAD stimulates releasing chemokines of CCL2 and CSF1 to recruit macrophages. A more recent study showed that YAP1 directly upregulates CCL2 in hepatocytes to enhance the infiltration of macrophages for the cancer formation, and macrophage ablation of Ccl2 in mice significantly reduced hepatic inflammation and HCC development.¹⁰⁸

Myeloid-derived suppressor cells (MDSCs)

Recent data revealed that immunosuppression in the TME is mainly facilitated by MDSCs and Tregs. These cells inhibit host effector T-cell activity against tumor-associated antigens, and they inhibit the efficacy of anti-cancer immunotherapy.³⁷ MDSCs represent a phenotypically heterogeneous cell population of immature myeloid cells, and they facilitate tumor progression by suppressing effector T-cell activities, especially CD8⁺ cytotoxic T-cells. YAP1 contributes to stimulating cytokines/chemokines that induce MDSCs recruitment in different cancer types.¹⁰⁹ For example, in prostate cancer, YAP1-TEAD complex upregulates the cancer cell-secreted chemokine CXCL5 to recruit CXCR2-expressing MDSCs, resulting in the decrease of CD8⁺ T-cells.⁴² In this study, more MDSCs were observed in the tumor bed of Cxcr2 overexpressed mice, and CXCR2 inhibitory agent suppressed the tumorigenesis and prolonged mice survival. In pancreatic cancer patients, YAP1 expression levels were correlated with MDSC gene signatures, and high expression of YAP1 or MDSC-related genes predicted poor survival.¹¹⁰ In this study, the authors demonstrated that YAP1 induces the secretion of multiple cytokines/chemokines such as IL-6 and CSF 1–3 and promotes the accumulation of MDSCs, which results in the impairment of T-cell activities. In ovarian cancer, YAP1 nuclear localization is promoted by PRKCI, and high PRKCI expression was associated with YAP1 and TNF-α overexpression and low infiltration of cytotoxic T-cells in the primary tissue.¹¹¹ In this study, TNF-α was found to be a direct transcriptional target of YAP1. Because TNF-α induces MDSCs recruitment and inhibits NK cells and cytotoxic T-cells infiltration, YAP1 can be considered to facilitate immunosuppressive and tumor-promoting microenvironment through TNF-α releasing from cancer cells. In KRAS mutant pancreatic cancer, YAP1 upregulates cytokines (e.g., IL-6 and IL-1α) and COX2 which mediates prostaglandin E2 (PGE2) synthesis to promote inflammation.⁶³ Similar to pancreatic cancer, YAP1 upregulates COX2 in granulosa cells, neurofibromatosis Type 2 and bladder cancer.^86,112,113 Importantly, COX2-PGE2 pathway in cancer cells induces MDSCs recruitment in the TME.^114-119 In addition, COX2 and PGE2 expressions in myeloid cells mediate their differentiation to MDSCs.^120,121 Furthermore, COX2 inhibitory drug decreases the number and function of MDSCs and improves the efficacy of immunotherapy.^114,122-124 Notably, pre-clinical data suggests that adding COX2 inhibitor to PD-1 blockade agent boosts anti-cancerous effects.^125,126 Recently, we reported that YAP1 and COX2 expressions are mutually compensated through the negative feedback of SOX2 expression in bladder cancer.⁸⁶ Considering these evidences, COX2 is possibly associated with YAP1 induced recruitment of MDSCs. Therefore, it is worthwhile to evaluate whether the combined inhibition of YAP1 and COX2 suppresses MDSCs recruitment more efficiently than either inhibition alone and increases the sensitivity to IO agents (Fig. 1d).

Regulatory T-cells (Tregs)

Tregs (CD4⁺CD25⁺ T-cells) were identified in peripheral T-cells.¹²⁷ Increased number of Tregs in the TME indicates poor prognosis, and the TME with high infiltrated Tregs usually indicates immunotherapy resistance.¹²⁸ A small number of studies have shown the relationship between YAP1 and Treg infiltration in the TME.

Immunohistochemical analysis of gastric cancerous tissues showed that the infiltrating Treg count in the TME was positively correlated with YAP1 expression in cancer cells.¹²⁹ In HCC patients, YAP1 overexpression in peripheral T-cells was associated with increased Treg percentage in the peripheral blood mononuclear cells and indicated poor prognosis.³⁹ This study also showed that YAP1 promotes naïve T-cell polarization to Treg through inducing TGFBR2 (Fig. 1b). These findings suggest that YAP1 in T-cells induces Treg differentiation to enhance immunosuppressive TME. Notably, this study focused on YAP1 activities of immune cells. Because the TME is composed of not only cancer cells but also immune cells, endothelial cells and mesenchymal cells, YAP1 in these non-cancerous components of TME possibly plays important roles on immune evasion. For example, in breast cancer stromal cells, downregulation of SPIN90 causes microtubule acetylation, which promotes nuclear localization of YAP1.¹³⁰ Then, YAP1 upregulates the transcription of myofibroblast marker genes such as α-SMA, CYR61 and CTGF for activating cancer-associated fibroblasts that is required for the remodeling of cancer stroma (Fig. 1c). Cancer-associated fibroblasts are involved in immunosuppression as well as tumor growth, angiogenesis, cancer stemness and extracellular matrix remodeling.¹³¹ Chemokines, pro-inflammatory factors and chemical mediators (e.g., IL-6, CCL2, TGF-β, and PGE2) are secreted from the cancer-associated fibroblasts for the impairment of T-cell, modulation of MDSCs and recruitment of Tregs. Although a limited number of studies about YAP1 regulation in immune and stromal cells have been reported, YAP1 seems to fulfill an important part in the TME.

Conclusions

The roles of YAP1 on tumorigenesis and the TME are now beginning to become apparent. However, the substantial evidences discussed above support the idea that YAP1 is associated with numerous cancer promoting molecules for the recruitment of M2 macrophages, MDSCs and Tregs to suppress host effector T-cells in the TME, resulting in cancer progression and drug resistance. Because YAP1 and COX2 inhibitory drugs block the recruitment of these immunosuppressive cells, the combinational therapy of these inhibitors and IO agents may be fascinating treatment strategies (Fig. 1d).

In pre-clinical data, verteporfin treatment suppresses cancer cell proliferation, reduces cellular stemness properties, restores the drug sensitivity and prolongs survival of mice.⁸⁶ In addition to these anti-cancerous effects, YAP1 inhibition may modulate immunosuppressive to immunoresponsive TME by inhibiting recruitment of MDSCs, activating effector T-cells activities and enhancing the sensitivity to IO agents. Although further mechanistic and preclinical studies are needed, YAP1 inhibitory therapy in combination with anti-cancerous drugs has a potential to be a novel therapeutic strategy.

Abbreviations:

CRC

colorectal cancer

CSCs

cancer stem cells

CTLA-4

cytotoxic T lymphocyte associated antigen 4

EMT

epithelial-mesenchymal transition

ER

estrogen receptor

FDA

Food and Drug Administration

GC

gastric cancer

HCC

hepatocellular carcinoma

IO agents

immune-oncologic agents

MDSCs

myeloid-derived suppressor cells

NK cells

natural killer cells

NSCLC

non-small-cell lung cancer

PD-1

programmed cell death 1

PD-L1

programmed cell death ligand 1

PGE2

prostaglandin E2

TME

tumor microenvironment

Tregs

regulatory T-cells