YAP Pathway Research Articles

Integrin β3 Promotes Resistance to EGFR-TKI in Non-Small-Cell Lung Cancer by Upregulating AXL through the YAP Pathway.

Integrin β3 plays a key role in the resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), but the development of integrin β3 inhibitors has been stalled due to the failure of phase III clinical trials for cancer treatment. Therefore, it is imperative to find a potentially effective solution to the problem of acquired resistance to EGFR-TKI for patients with integrin-β3 positive non-small-cell lung cancer (NSCLC) by exploring novel downstream targets and action mechanisms of integrin β3. In the present study, we observed that the expression of integrin β3 and AXL was significantly upregulated in erlotinib-resistant NSCLC cell lines, which was further confirmed clinically in tumor specimens from patients with NSCLC who developed acquired resistance to erlotinib. Through ectopic expression or knockdown, we found that AXL expression was positively regulated by integrin β3. In addition, integrin β3 promoted erlotinib resistance in NSCLC cells by upregulating AXL expression. Furthermore, the YAP pathway, rather than pathways associated with ERK or AKT, was involved in the regulation of AXL by integrin β3. To investigate the clinical significance of this finding, the current well-known AXL inhibitor R428 was tested, demonstrating that R428 significantly inhibited resistance to erlotinib, colony formation, epithelial–mesenchymal transformation and cell migration induced by integrin β3. In conclusion, integrin β3 could promote resistance to EGFR-TKI in NSCLC by upregulating the expression of AXL through the YAP pathway. Patients with advanced NSCLC, who are positive for integrin β3, might benefit from a combination of AXL inhibitors and EGFR-TKI therapy.

Atorvastatin favorably modulates a clinical hepatocellular carcinoma risk gene signature.

Lipophilic but not hydrophilic statins have been shown to be associated with reduced risk for hepatocellular carcinoma (HCC) in patients with chronic viral hepatitis. We investigated differential actions of lipophilic and hydrophilic statins and their ability to modulate a clinical prognostic liver signature (PLS) predicting HCC risk in patients with liver disease. Hepatitis C virus (HCV)–infected Huh7.5.1 cells, recently developed as a model to screen HCC chemopreventive agents, were treated with lipophilic statins (atorvastatin and simvastatin) and hydrophilic statins (rosuvastatin and pravastatin), and then analyzed by RNA sequencing and PLS. Lipophilic statins, particularly atorvastatin, more significantly suppressed the HCV‐induced high‐risk pattern of PLS and genes in YAP and AKT pathway implicated in fibrogenesis and carcinogenesis, compared with the hydrophilic statins. While atorvastatin inhibited YAP activation through the mevalonate pathway, the distinctive AKT inhibition of atorvastatin was mediated by stabilizing truncated retinoid X receptor alpha, which has been known to enhance AKT activation, representing a target for HCC chemoprevention. In addition, atorvastatin modulated the high‐risk PLS in an in vitro model of nonalcoholic fatty liver disease (NAFLD). Conclusion: Atorvastatin distinctively inhibits YAP and AKT activation, which are biologically implicated in HCC development, and attenuates a high‐risk PLS in an in vitro model of HCV infection and NAFLD. These findings suggest that atorvastatin is the most potent statin to reduce HCC risk in patients with viral and metabolic liver diseases.

Type I collagen reduces lipid accumulation during adipogenesis of preadipocytes 3T3-L1 via the YAP-mTOR-autophagy axis

The extracellular matrix (ECM) regulates cell behavior through signal transduction and provides a suitable place for cell survival. As one of the major components of the extracellular matrix, type I collagen is involved in regulating cell migration, proliferation and differentiation. We present a system in which 3T3-L1 preadipocyte cells are induced for adipogenic differentiation on type I collagen coated dishes. Our previous study has found that type I collagen inhibits adipogenic differentiation via YAP activation. Here we further reveal that type I collagen inactivates autophagy by up-regulating mTOR activity via the YAP pathway. Under collagen-coating conditions, co-localization of lysosomes with mTOR was increased and the level of downstream protein p-S6K was elevated, accompanied by a decrease in the level of autophagy. Autophagy is negatively correlated with adipogenesis under type I collagen coating. Through the YAP-autophagy axis, type I collagen improves glycolipid metabolism accompanied by increased mitochondrial content, enhanced glucose uptake, reduced release of free fatty acids (FFAs) and decreased intracellular lipid accumulation. Our findings provide insight into the strategy for dealing with obesity: Type I collagen or the drugs with inhibitory effects on autophagy or YAP, have a potential to accelerate the energy metabolism of adipose tissue, so as to better maintain the homeostasis of glucose and lipids in the body, which can be used for achieving weight loss.

Fenofibrate induced‐hepatomegaly and liver regeneration is PPARα dependent and partially related to the activation of YAP pathway

Background and ObjectiveFenofibrate, an agonist of peroxisome proliferator activated receptor α (PPARα), has been widely used as a lipid‐regulating agent in clinical practice. Recent studies found that fenofibrate can significantly induce hepatomegaly in mice, but the involved mechanisms remain unclear.MethodsThe effect of fenofibrate on hepatomegaly and liver regeneration was investigated in several strains of genetically‐modified mice including Pparafl/fland PparaΔHep mice. Staining assays were performed to investigate the effect of fenofibrate on hepatocyte size and proliferation during hepatomegaly and liver regeneration. AAV‐Yap shRNA mice model was further used to determine the role of YAP in PPARα‐induced hepatomegaly. Western blot analysis was used to detect the protein expressions of YAP signaling pathway as well as other factors potentially involved in fenofibrate‐induced hepatomegaly.ResultsThe study demonstrated that fenofibrate significantly induced liver enlargement in wild‐type and Pparafl/fl mice, while this effect was abolished in hepatocyte‐specific Ppara‐deficient (PparaΔHep) mice. Furthermore, fenofibrate significantly promoted liver regeneration in wild‐type mice following 70% partial hepatectomy (PHx). Staining assays revealed that fenofibrate promoted hepatocyte enlargement around the central vein area and hepatocyte proliferation around the portal vein area in wild‐type, Pparafl/fl and PHx mice. Fenofibrate regulated protein expressions of YAP and its downstream targets (CTGF, CRY61 and ANKRD1) as well as proliferation‐related proteins (CCNA1, CCND1 and CCNE1). Suppression of YAP using AAV‐Yap shRNA repressed fenofibrate‐induced hepatomegaly, as well as hepatocyte enlargement and proliferation. Other factors might also participate in fenofibrate‐induced hepatomegaly, including MYC, KRT23, RHOA and RAS.ConclusionThese studies revealed a novel role of fenofibrate in promoting liver enlargement and regeneration, which is PPARα dependent and partially related to the activation of YAP signaling pathway. These findings provide clinical relevance of fenofibrate as a potential medication for promoting liver regeneration.

YAP/Hippo Pathway and Cancer Immunity: It Takes Two to Tango

Hippo pathway with its main molecule YAP is a crucial pathway for development, tissue homeostasis, wound healing, tissue regeneration, and cancer. In this review, we discuss the multiple effects of the YAP/Hippo pathway in the immune system and cancer. We analyzed a series of effects: extracellular vesicles enhanced immunity through inhibition of LATS1/2, ways of modulation of the tumor microenvironment, YAP- and TAZ-mediated upregulation of PDL1, high expression of YAP and PDL1 in EGFR-TKI-resistant cells, enhanced YAP activity in inflammation, and the effect of the Hippo pathway on T cells, B cells, Tregs, macrophages, and myeloid-derived suppressor cells (MDSCs). These pleiotropic effects render the YAP and Hippo pathway a key pathway for exploitation in the future, in order to enhance our immunotherapy treatment strategies in oncology.

The Multiple Interactions of RUNX with the Hippo-YAP Pathway.

The Hippo–YAP signaling pathway serves roles in cell proliferation, stem cell renewal/maintenance, differentiation and apoptosis. Many of its functions are central to early development, adult tissue repair/regeneration and not surprisingly, tumorigenesis and metastasis. The Hippo pathway represses the activity of YAP and paralog TAZ by modulating cell proliferation and promoting differentiation to maintain tissue homeostasis and proper organ size. Similarly, master regulators of development RUNX transcription factors have been shown to play critical roles in proliferation, differentiation, apoptosis and cell fate determination. In this review, we discuss the multiple interactions of RUNX with the Hippo–YAP pathway, their shared collaborators in Wnt, TGFβ, MYC and RB pathways, and their overlapping functions in development and tumorigenesis.

RhoA enhances osteosarcoma resistance to MPPa-PDT via the Hippo/YAP signaling pathway

BackgroundOsteosarcoma (OS) is the most prevalent primary bone malignancy affecting adolescents, yet the emergence of chemoradiotherapeutic resistance has limited efforts to cure affected patients to date. Pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT) is a recently developed, minimally invasive treatment for OS that is similarly constrained by such therapeutic resistance. This study sought to explore the mechanistic basis for RhoA-activated YAP1 (YAP)-mediated resistance in OS.MethodsThe relationship between YAP expression levels and patient prognosis was analyzed, and YAP levels in OS cell lines were quantified. Immunofluorescent staining was used to assess YAP nuclear translocation. OS cell lines (HOS and MG63) in which RhoA and YAP were knocked down or overexpressed were generated using lentiviral vectors. CCK-8 assays were used to examine OS cell viability, while the apoptotic death of these cells was monitored via Hoechst staining, Western blotting, and flow cytometry. Tumor-bearing nude mice were additionally used to assess the relationship between lentivirus-mediated alterations in RhoA expression and MPPa-PDT treatment outcomes. TUNEL and immunohistochemical staining approaches were leveraged to assess apoptotic cell death in tissue samples.ResultsOS patients exhibited higher levels of YAP expression, and these were correlated with a poor prognosis. MPPa-PDT induced apoptosis in OS cells, and such MPPa-PDT-induced apoptosis was enhanced following YAP knockdown whereas it was suppressed by YAP overexpression. RhoA and YAP expression levels were positively correlated in OS patients, and both active and total RhoA protein levels rose in OS cells following MPPa-PDT treatment. When RhoA was knocked down, levels of unphosphorylated YAP and downstream target genes were significantly reduced, while RhoA/ROCK2/LIMK2 pathway phosphorylation was suppressed, whereas RhoA overexpression resulted in the opposite phenotype. MPPa-PDT treatment was linked to an increase in HMGCR protein levels, and the inhibition of RhoA or HMGCR was sufficient to suppress RhoA activity and to decrease the protein levels of YAP and its downstream targets. Mevalonate administration partially reversed these reductions in the expression of YAP and YAP target genes. RhoA knockdown significantly enhanced the apoptotic death of OS cells in vitro and in vivo following MPPa-PDT treatment, whereas RhoA overexpression had the opposite effect.ConclusionsThese results suggest that the mevalonate pathway activates RhoA, which in turn activates YAP and promotes OS cell resistance to MPPa-PDT therapy. Targeting the RhoA/ROCK2/LIMK2/YAP pathway can significantly improve the efficacy of MPPa-PDT treatment for OS.

Ezrin regulates synovial angiogenesis in rheumatoid arthritis through YAP and Akt signalling.

This study aimed to investigate the role and regulatory mechanisms of Ezrin in synovial vessels in rheumatoid arthritis (RA). Synovial tissues were obtained from people with osteoarthritis people and patients with RA patients. We also used an antigen‐induced arthritis (AIA) mice model by using Freund's adjuvant injections. Ezrin expression was analysed by immunofluorescence and immunohistochemical staining in synovial vessels of patients with RA and AIA mice. We investigated the role of Ezrin on vascular endothelial cells and its regulatory mechanism in vivo and in vitro by adenoviral transfection technology. Our results suggest a role for the Ezrin protein in proliferation, migration and angiogenesis of vascular endothelial cells in RA. We also demonstrate that Ezrin plays an important role in vascular endothelial cell migration and tube formation through regulation of the Hippo‐yes‐associated protein 1 (YAP) pathway. YAP, as a key protein, can further regulate the activity of PI3K/Akt signalling pathway in vascular endothelial cells. In AIA mice experiments, we observed that the inhibition of Ezrin or of its downstream YAP pathway can affect synovial angiogenesis and may lead to progression of RA. In conclusion, Ezrin plays an important role in angiogenesis in the RA synovium by regulating YAP nuclear translocation and interacting with the PI3K/Akt signalling pathway.

Abstract 2073: Co-Targeting PARP and ATR in biliary tract cancer

Abstract Background: Targeting DNA damage response (DDR) has potential anti-tumor effects in diverse cancer types. Homologous-recombination DDR gene mutations are found in 28.9% of biliary tract cancer (BTC). We aimed to evaluate the anti-tumor effects of olaparib (PARP inhibitor) with or without ceralasertib (ATR inhibitor) in BTC.Methods: Ten BTC cell lines (SNU245, SNU308, SNU478, SNU869, SNU1079, SNU1196, HuCCT-1, TFK-1, SNU2670, and SNU2773) were tested to evaluate the anti-tumor effects of olaparib alone and in combination with ceralasertib. MTT assay, colony formation assay, cell cycle analysis, western blot, comet assay and co-culture with human PBMC were used as experimental methods. Tumor xenograft model was established to test olaparib with or without ceralasertib in vivo.Results: Olaparib alone showed moderate anti-proliferative effects in all BTC cells and increased p-ATR and PD-L1 expression. Olaparib plus ceralasertib showed synergistic anti-proliferative effects and led to DNA damage breaks in vitro. Ceralasertib also downregulated PD-L1 level through p-STAT3 and YAP pathway with or without human PBMC conditions. In SNU478-xenograft models, Olaparib plus ceralasertib combination significantly suppressed tumor growth and downregulated PD-L1 and YAP, compared with monotherapy. It also effectively reduced CXCR2 and CXCR4 expression. Conclusion: Combination of olaparib plus ceralasertib showed potent anti-tumor effects in BTC cells. This study could provide the preclinical evidence of development of PARP and ATR dual inhibition in BTC patients. Clinical trial using olaparib and ceralasertib combination in advanced BTC is ongoing (ClinicalTrials.gov Identifier: NCT04298021). Citation Format: Ah Rong Nam, Mei Hua Jin, Kyoung-Seok Oh, Hye Rim Seo, Jae-Min Kim, Ju-Hee Bang, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh. Co-Targeting PARP and ATR in biliary tract cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2073.

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1.

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.

Inhibition of YAP activation attenuates renal injury and fibrosis in angiotensin II hypertensive mice.

The Hippo/YAP (yes-associated protein) pathway is an important signaling pathway to control organ development and tissue homeostasis. YAP is a downstream effector of the Hippo pathway and a critical mediator of mechanic stress. Hypertensive nephropathy is characterized with glomerular sclerosis stiffness and renal fibrosis. The present study investigated the role of YAP pathway in angiotensin (Ang) II hypertensive renal injury by using YAP activation inhibitor verteporfin. Ang II increased the protein expression of YAP in renal nucleus fraction, decreased phospho-YAP, and phospho-LATS1/2 (large tumor suppressors 1 and 2) expressions in renal cytoplasmic fraction, suggesting Ang II activation of renal YAP. Ang II significantly increased systolic blood pressure (SBP), proteinuria, glomerular sclerosis, and fibrosis; treatment with verteporfin attenuated Ang II-induced proteinuria and renal injury with a mild reduction in SBP. Moreover, Ang II increased the protein expressions of inflammatory factors including tumor necrosis factor α, interleukin 1β, and monocyte chemoattractant protein-1, and profibrotic factors including transforming growth factor β, phospho-Smad3 and fibronectin. Verteporfin reversed abovementioned Ang II-induced molecule expressions. Our results for the first time demonstrate that the activation of the YAP pathway promotes hypertensive renal inflammation and fibrosis, which may promote hypertensive renal injury. YAP may be a new target for prevention and treatment of hypertensive renal diseases.

BMSC-derived exosomes ameliorate sulfur mustard-induced acute lung injury by regulating the GPRC5A-YAP axis.

Sulfur mustard (SM) is a highly toxic chemical warfare agent that causes acute lung injury (ALI) and/or acute respiratory distress syndrome (ARDS). There are no effective therapeutic treatments or antidotes available currently to counteract its toxic effects. Our previous study shows that bone marrow-derived mesenchymal stromal cells (BMSCs) could exert therapeutic effects against SM-induced lung injury. In this study, we explored the therapeutic potential of BMSC-derived exosomes (BMSC-Exs) against ALI and the underlying mechanisms. ALI was induced in mice by injection of SM (30 mg/kg, sc) at their medial and dorsal surfaces. BMSC-Exs (20 μg/kg in 200 μL PBS, iv) were injected for a 5-day period after SM exposure. We showed that BMSC-Exs administration caused a protective effect against pulmonary edema. Using a lung epithelial cell barrier model, BMSC-Exs (10, 20, 40 μg) dose-dependently inhibited SM-induced cell apoptosis and promoted the recovery of epithelial barrier function by facilitating the expression and relocalization of junction proteins (E-cadherin, claudin-1, occludin, and ZO-1). We further demonstrated that BMSC-Exs protected against apoptosis and promoted the restoration of barrier function against SM through upregulating G protein-coupled receptor family C group 5 type A (GPRC5A), a retinoic acid target gene predominately expressed in the epithelial cells of the lung. Knockdown of GPRC5A reduced the antiapoptotic and barrier regeneration abilities of BMSC-Exs and diminished their therapeutic effects in vitro and in vivo. BMSC-Exs-caused upregulation of GPRC5A promoted the expression of Bcl-2 and junction proteins via regulating the YAP pathway. In summary, BMSC-Exs treatment exerts protective effects against SM-induced ALI by promoting alveolar epithelialbarrier repair and maybe an alternative approach to stem cell-based therapy.

MicroRNA-670 aggravates cerebral ischemia/reperfusion injury via the Yap pathway.

Apoptosis is an important programmed cell death process involved in ischemia/reperfusion injury. MicroRNAs are considered to play an important role in the molecular mechanism underlying the regulation of cerebral ischemia and reperfusion injury. However, whether miR-670 can regulate cell growth and death in cerebral ischemia/reperfusion and the underlying mechanism are poorly understood. In this study, we established mouse models of transient middle artery occlusion and Neuro 2a cell models of oxygen-glucose deprivation and reoxygenation to investigate the potential molecular mechanism by which miR-670 exhibits its effects during cerebral ischemia/reperfusion injury both in vitro and in vivo. Our results showed that after ischemia/reperfusion injury, miR-670 expression was obviously increased. After miR-670 expression was inhibited with an miR-670 antagomir, cerebral ischemia/reperfusion injury-induced neuronal death was obviously reduced. When miR-670 overexpression was induced by an miR-670 agomir, neuronal apoptosis was increased. In addition, we also found that miR-670 could promote Yap degradation via phosphorylation and worsen neuronal apoptosis and neurological deficits. Inhibition of miR-670 reduced neurological impairments after cerebral ischemia/reperfusion injury. These results suggest that microRNA-670 aggravates cerebral ischemia/reperfusion injury through the Yap pathway, which may be a potential target for treatment of cerebral ischemia/reperfusion injury. The present study was approved by the Institutional Animal Care and Use Committee of China Medical University on February 27, 2017 (IRB No. 2017PS035K).

Retracted] Norcantharidin reverses cisplatin resistance and inhibits the epithelial mesenchymal transition of human non‑small lung cancer cells by regulating the YAP pathway.

An interested reader drew to our attention that the western blots featured in Figs.1B and6C contained strikingly similar protein bands, and repeating patterns of bands, comparing across the lanes of the gels. Furthermore, an image representing the Myc‑YAP colony‑formation assay experiment in Fig.2C was strikingly similar to the data shown for the Control colony‑formation assay experiment in Fig.5B. The Editorial office subsequently investigated this matter further, and noted that the western blots shown in Fig.6A andB likewise contained strikingly similar bands that were purportedly showing the results from different experiments. After having considered the various issues that have been brought to light with this paper, together with an appeal from the authors that a Corrigendum be published, the Editor of Oncology Reports has ruled that the article should be retracted from the publication on account of a lack of overall confidence in the presented data. Note that the authors were not in agreement that the errors reported and identified were sufficient to merit the retraction of the article. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in Oncology Reports 40: 609-620, 2018; DOI: 10.3892/or.2018.6486].

Endothelin-1 axis fosters YAP-induced chemotherapy escape in ovarian cancer.

The majority of ovarian cancer (OC) patients recur with a platinum-resistant disease. OC cells activate adaptive resistance mechanisms that are only partially described. Here we show that OC cells can adapt to chemotherapy through a positive-feedback loop that favors chemoresistance. In platinum-resistant OC cells we document that the endothelin-1 (ET-1)/endothelin A receptor axis intercepts the YAP pathway. This cross-talk occurs through the LATS/RhoA/actin-dependent pathway and contributes to prevent the chemotherapy-induced apoptosis. Mechanistically, β-arrestin1 (β-arr1) and YAP form a complex shaping TEAD-dependent transcriptional activity on the promoters of YAP target genes, including EDN1, which fuels a feed-forward signaling circuit that sustains a platinum-tolerant state. The FDA approved dual ET-1 receptor antagonist macitentan in co-therapy with cisplatin sensitizes resistant cells to the platinum-based therapy, reducing their metastatic potential. Furthermore, high ETAR/YAP gene expression signature is associated with a poor platinum-response in OC patients. Collectively, our findings identify in the networking between ET-1 and YAP pathways an escape strategy from chemotherapy. ET-1 receptor blockade interferes with such adaptive network and enhances platinum-induced apoptosis, representing a promising therapeutic opportunity to restore drug sensitivity in OC patients.

Leukemia inhibitory factor promotes gastric cancer cell proliferation, migration, and invasion via the LIFR-Hippo-YAP pathway.

The long-term outcome of gastric cancer (GC) patients remains unsatisfactory despite some recent improvements. Leukemia inhibitory factor (LIF) is a prognostic biomarker for some solid tumors, however its role in GC remains unknown. In this study, we demonstrated that LIF and LIF receptor (LIFR) are overexpressed in GC tissues and established that a correlation exists between them. LIF and LIFR expression are associated with tumor differentiation, lymphovascular invasion, tumor stage, lymph node metastasis, and pTNM stage, indicating that they may be useful prognostic factors. LIF promoted GC cell proliferation, colony formation, invasion, migration, and tumor growth; it also promoted cell cycle progression and inhibited apoptosis; and knocking out the LIFR gene reversed the effects of LIF. LIF inhibited the activity of the Hippo pathway, resulting in reduced phosphorylation of YAP, increased YAP nuclear translocation, and increased cell proliferation. Finally, silencing YAP mRNA expression suppressed cell proliferation. Overall, the results demonstrate that LIF promotes the malignant biological behavior of GC cells through LIFR-Hippo-YAP signaling. LIF may therefore be a useful biomarker for GC.

Abstract 5943: The role of IL-6 family cytokines in gastrointestinal cancer

Abstract The important effector of the Hippo pathway, YAP, is strongly activated in the majority of gastrointestinal cancer. However, the mechanism by which YAP is activated in gastrointestinal cancer remains unknown. Previously, we found that after intestinal injury, IL-6 family cytokines activate the Src family kinases (SFK)-YAP pathway, which enhances intestinal regeneration. Here, we found that in addition to β-catenin, loss of tumor suppressor adenomatous polyposis coli (APC) activates SFKs, YAP and STAT3, which are simultaneously activated in 60-70% of human colorectal cancer specimens. We also found that up-regulation of IL-6 signal transducer (IL-6ST/gp130), which is induced by YAP activation, causes the activation of SFKs, YAP and STAT3. We also found that leukemia inhibitory factor (LIF), one of the IL-6 family cytokines, plays an important role in human esophageal squamous cell carcinoma by activating the SFK-YAP pathway. The combination treatment of SFK and JAK inhibitors results in suppression of gastrointestinal tumors. These results suggest that these signaling pathways might be new attractive therapeutic targets in human gastrointestinal cancer. Citation Format: Koji Taniguchi, Tetsuro Kawazoe. The role of IL-6 family cytokines in gastrointestinal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5943.

Abstract 124: Recycling endosomes in mature epithelia restrain tumorigenic signaling

Abstract The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, while suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis. Statement of significance: Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism. Citation Format: Sayantani Goswami. Recycling endosomes in mature epithelia restrain tumorigenic signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 124.

Abstract A20: Gαq controls the Hippo pathway through MOB1 tyrosine phosphorylation by FAK

Abstract YAP overactivation is an essential molecular event for cancer initiation and growth of most solid tumors, but pharmacologic targeting of the YAP or Hippo pathway has been proven to be challenging. In this regard, YAP activity is also required for stem and progenitor cell maintenance and function in multiple tissues, and as such, YAP is necessary for normal tissue homeostasis and regeneration. Thus, targeting YAP directly may result in normal cell growth arrest and tissue damage. The elucidation of the signaling networks sustaining YAP pervasive activation in each cancer type may afford the opportunity to identify novel suitable precision therapeutic targets for cancer treatment. GNAQ and GNA11 activate mutations (encoding GTPase deficient and constitutively active Gαq proteins) occurs in ~90% of uveal melanoma (UM) and 4% of skin cutaneous melanoma (SKCM), respectively, firmly established UM, and a subset of SKCM as Gαq-driven human malignancies. Approximately 50% of UM patients develop liver metastasis within 5-10 years after diagnosis, independently of the successful treatment of the primary lesions. To date, there are no effective treatment options for metastatic UM disease. Through the bioinformatics analysis of the TCGA UM dataset (n=80) using skin melanoma samples that do not harbor Gαq genomic alteration (n=209) as control, (1) we first identified the genes that are highly overexpressed in UM. (2) We then filtered the genes for those whose reduced expression leads to better patient survival in UM. (3) We finally used large datasets of gene essentiality and drug response screenings in cancer cells to identify genes that are predicted to reduce cell viability when targeted in Gαq-gain cells. (4) Finally, 7 top predicted drug targets were found from a set of genes that can be targeted by approved and experimental drugs (n=756). The top predicted gene target was PTK2, which encodes the nonreceptor tyrosine kinase FAK (focal adhesion kinase). We found that Gαq activates pY379-FAK (reflecting FAK activation) though a Trio-RhoA signaling pathway, independent of PLC-β regulated second messenger activation. Interestingly, genome-wide transcriptional analysis (RNA-seq) of UM cells treated with a FAK inhibitor (VS-4718) revealed that YAP signatures were highly inhibited by FAK blockade. This suggested that FAK signaling actives YAP downstream of Gαq in UM, in addition to our previously found Trio/Rho GTPase/F-actin polymerization regulation of YAP activation mechanism. Further signaling analysis uncovered that FAK stimulates YAP through direct YAP tyrosine phosphorylation (enhancing YAP protein stability) and, unexpectedly, MOB1 tyrosine phosphorylation on Y26. The latter disrupts MOB1/LATS interaction, causing Hippo signaling inhibition and YAP activation. Interestingly, FAK inhibitors are already under clinical evaluation, and showed strong inhibition of YAP activity and UM growth in vitro and in vivo, thus representing a potential precision therapeutic option for UM treatment. Citation Format: Xiaodong Feng, Nadia Arang, Damiano Cosimo Rigiracciolo, Joo Sang Lee, Huwate Yeerna, Zhiyong Wang, Simone Lubrano, Ayush Kishore, Jonathan A. Pachter, Gabriele M. König, Marcello Maggiolini, Evi Kostenis, David D. Schlaepfer, Pablo Tamayo, Qianming Chen, Eytan Ruppin, J. Silvio Gutkind. Gαq controls the Hippo pathway through MOB1 tyrosine phosphorylation by FAK [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A20.

Abstract B42: YAP1/Hippo pathway and SWI/SNF as critical players in squamous cancers and normal development

Abstract Cancer development is a multistep process involving deregulation of transcription/epigenetic factors and signaling pathways. Accordingly, gene expression lies at the nexus of oncogenic cellular processes. The recent advent of The Cancer Genome Atlas (TCGA) has shed light on multiple genes involved in signaling, transcription, and other cellular processes that are subjected to genomic alterations in multiple cancers. These oncogenic drivers are involved in regenerative proliferation, metastasis, and other cellular processes that contribute to tumorigenesis. Epithelial-stromal interactions and the infiltrating immune response contribute significantly to tumor progression. Our recent studies revealed Hippo pathway activation as a critical component in ACTL6A (subunit of SWI/SNF chromatin remodeling complex) amplified squamous cancers. Our published genetic studies indicate that YAP is a key mediator of the oncogenic function of ACTL6A/p63 in head and neck squamous cancers. P63 is a master transcription factor in epithelial development. We have shown that YAP and p63 biochemically interact to maintain airway architecture and regeneration. YAP and p63 deletion are phenocopies and are required to maintain normal respiratory epithelial stem cells. P63/ACTL6A act as upstream regulators of YAP in squamous cancers. ACTL6A and p63 physically interact and cooperatively control a transcriptional program that promotes proliferation and suppresses differentiation, through activation of the Hippo-YAP pathway via regulators including WWC1 and GPRC5A. Consequently, loss of ACTL6A or p63 in tumor cells induces YAP phosphorylation and inactivation, associated with growth arrest and terminal differentiation, all phenocopied by WWC1 overexpression. YAP activation predicts poor patient survival in squamous cancers. These findings are also in keeping with the emerging role of the YAP pathway as a tumor driver in multiple contexts. Given interest in YAP as a therapeutic target, our studies reveal contribution of p63 and TEAD to YAP function as evident from global transcription profiling. Our most recent studies also indicate contribution of YAP in tumor progression by evading host immune response. All these indicate an important intrinsic role of YAP in development and disease, making it one of the ideal therapeutic targets to control cancer. Citation Format: Leif Ellisen, Purushothama Rao Tata, Rui Zhao, Jayaraj Rajagopal, Srinivas Vinod Saladi. YAP1/Hippo pathway and SWI/SNF as critical players in squamous cancers and normal development [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B42.