Qiongrong Chen

Hippo Coactivator YAP1 Upregulates SOX9 and Endows Esophageal Cancer Cells with Stem-like Properties

Cancer stem cells (CSC) are purported to initiate and maintain tumor growth. Deregulation of normal stem cell signaling may lead to the generation of CSCs; however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional coactivator YAP1 is a major determinant of CSC properties in nontransformed cells and in esophageal cancer cells by direct upregulation of SOX9. YAP1 regulates the transcription of SOX9 through a conserved TEAD binding site in the SOX9 promoter. Expression of exogenous YAP1 in vitro or inhibition of its upstream negative regulators in vivo results in elevated SOX9 expression accompanied by the acquisition of CSC properties. Conversely, shRNA-mediated knockdown of YAP1 or SOX9 in transformed cells attenuates CSC phenotypes in vitro and tumorigenicity in vivo. The small-molecule inhibitor of YAP1, verteporfin, significantly blocks CSC properties in cells with high YAP1 and a high proportion of ALDH1(+). Our findings identify YAP1-driven SOX9 expression as a critical event in the acquisition of CSC properties, suggesting that YAP1 inhibition may offer an effective means of therapeutically targeting the CSC population.

The Hippo Coactivator YAP1 Mediates EGFR Overexpression and Confers Chemoresistance in Esophageal Cancer

Purpose: Esophageal cancer is an aggressive malignancy and often resistant to therapy. Overexpression of EGFR has been associated with poor prognosis of patients with esophageal cancer. However, clinical trials using EGFR inhibitors have not provided benefit for patients with esophageal cancer. Failure of EGFR inhibition may be due to crosstalk with other oncogenic pathways. Experimental Design: In this study, expression of YAP1 and EGFR were examined in EAC-resistant tumor tissues versus sensitive tissues by IHC. Western blot analysis, immunofluorescence, real-time PCR, promoter analysis, site-directed mutagenesis, and in vitro and in vivo functional assays were performed to elucidate the YAP1-mediated EGFR expression and transcription and the relationship with chemoresistance in esophageal cancer. Results: We demonstrate that Hippo pathway coactivator YAP1 can induce EGFR expression and transcription in multiple cell systems. Both YAP1 and EGFR are overexpressed in resistant esophageal cancer tissues compared with sensitive esophageal cancer tissues. Furthermore, we found that YAP1 increases EGFR expression at the level of transcription requiring an intact TEAD-binding site in the EGFR promoter. Most importantly, exogenous induction of YAP1 induces resistance to 5-fluorouracil and docetaxcel, whereas knockdown of YAP1 sensitizes esophageal cancer cells to these cytotoxics. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and EGFR expression and sensitizes cells to cytotoxics. Conclusions: Our data provide evidence that YAP1 upregulation of EGFR plays an important role in conferring therapy resistance in esophageal cancer cells. Targeting YAP1-EGFR axis may be more efficacious than targeting EGFR alone in esophageal cancer. Clin Cancer Res; 21(11); 2580–90. ©2015 AACR.

Metformin sensitizes chemotherapy by targeting cancer stem cells and the mTOR pathway in esophageal cancer

Our clinical study indicates esophageal adenocarcinoma patients on metformin had a better treatment response than those without metformin. However, the effects of metformin and the mechanisms of its action in esophageal cancer (EC) are unclear. EC cell lines were used to assess the effects of metformin alone or in combination with 5-fluorouracil on survival and apoptosis. RPPA proteomic array and immunoblots were used to identify signaling affected by metformin. Standard descriptive statistical methods were used. Reduction in cell survival and induction of apoptosis by metformin were observed in several EC cell lines. The use of metformin in combination with 5-FU significantly sensitized EC cells to the cytotoxic effect of 5-FU. RPPA array demonstrated that metformin decreased various oncogenes including PI3K/mTORsignaling and survival/cancer stem cell-related genes in cells treated with metformin compared with its control. Immunoblots and transcriptional analyses further confirm that metformin downregulated these CSC-related genes and the components of the mTOR pathway in a dose-dependent manner. Sorted ALDH-1+ cell tumor sphere forming capacity was preferentially reduced by metformin. Finally, metformin reduced tumor growth in vivo and when combined with FU, there was synergistic reduction in tumor growth. Metformin inhibits EC cell growth and sensitizes EC cells to 5-FU cytotoxic effects by targeting CSCs and the components of mTOR. The present study supports our previous clinical observations that the use of metformin is beneficial to EC patients. Metformin can complement other therapeutic combinations to effectively treat EC patients.

Medical management of gastric cancer: A 2014 update

Gastric cancer represents a serious health problem on a global scale. It is the second leading cause of cancer-related death worldwide. Novel therapeutic targets are desperately needed because the meager improvement in the cure rate of about 10% realized by adjunctive treatments to surgery is unacceptable as > 50% patients with localized gastric cancer succumb to their disease. Either postoperative chemoradiotherapy (United States), pre-and post-operative chemotherapy (Europe), and adjuvant chemotherapy after a D2 resection (Asia) can all be regarded as standards of care in the localized gastric cancer management. In metastatic disease the addition of trastuzumab to chemotherapy is standard of care in Her2 positive disease. In the HER2 negative population, the treatments remain limited. In the first line setting, the standard of care is a combination of fluoropyrimidine and platinum containing chemotherapy, with or without epirubicin or docetaxel. The results of targeted therapy trials have by and large been disappointing, but none of these trials looked at an appropriately enriched population. Finally there is a meager overall survival benefit in treating patients with metastatic disease in the second line setting, with either irinotecan, docetaxel or ramucirumab however none of these drugs have been compared head to head in a well-powered randomized controlled trial.

ABT-263 induces apoptosis and synergizes with chemotherapy by targeting stemness pathways in esophageal cancer.

Activation of cancer stem cell signaling is central to acquired resistance to therapy in esophageal cancer (EC). ABT-263, a potent Bcl-2 family inhibitor, is active against many tumor types. However, effect of ABT-263 on EC cells and their resistant counterparts are unknown. Here we report that ABT-263 inhibited cell proliferation and induced apoptosis in human EC cells and their chemo-resistant counterparts. The combination of ABT-263 with 5-FU had synergistic lethal effects and amplified apoptosis that does not depend fully on its inhibition of BCL-2 family proteins in EC cells. To further explore the novel mechanisms of ABT-263, proteomic array (RPPAs) were performed and gene set enriched analysis demonstrated that ABT-263 suppresses the expression of many oncogenes including genes that govern stemness pathways. Immunoblotting and immunofluorescence further confirmed reduction in protein expression and transcription in Wnt/β-catenin and YAP/SOX9 axes. Furthermore, ABT263 strongly suppresses cancer stem cell properties in EC cells and the combination of ABT-263 and 5-FU significantly reduced tumor growth in vivo and suppresses the expression of stemness genes. Thus, our findings demonstrated a novel mechanism of ABT-263 antitumor effect in EC and indicating that combination of ABT-263 with cytotoxic drugs is worthy of pursuit in patients with EC.

Anti-angiogenic agent ramucirumab: meaningful or marginal?

Ramucirumab (IMC-1121B) targets VEGFR-2. Ramucirumab is being investigated in many malignancies including gastric cancer. The Phase III trial in patients with advanced breast cancer failed to improve the primary end point The REGARD trial, a Phase III study, in patients with advanced gastric cancer in the second line setting, had a marginal improvement in overall survival but did not achieve the expected hazard ratio target (of 0.69) and the median duration of therapy with ramucirumab was meager 8 weeks (only 2 weeks longer than the placebo’s). Other notable agents in the second line setting are docetaxel and irinotecan. Preliminary results of the RAINBOW trial suggest that ramucirumab may be providing more than marginal advantage. In this review, we briefly summarize the process of angiogenesis and address the emerging cost-benefit issues that surround all newly developed agents including ramucirumab.

Galectin-3 expression is prognostic in diffuse type gastric adenocarcinoma, confers aggressive phenotype, and can be targeted by YAP1/BET inhibitors

Background:Overexpression of Galectin-3 (Gal-3), a β-galactoside binding protein, has been noted in many tumour types but its functional significance and clinical utility in gastric adenocarcinoma (GAC) are not well known.Methods:We studied 184 GAC patients characterised by histologic grade, sub-phenotypes (diffuse vs intestinal), and ethnicity (Asians vs North Americans). Immunohistochemistry was performed to assess the expression of Gal-3 in human GACs and we correlated it to the clinical outcomes. Cell proliferation, invasion, co-immunoprecipitation and kinase activity assays were done in genetically stable Gal-3 overexpressing GC cell lines and the parental counterparts to delineate the mechanisms of action and activity of inhibitors.Results:Most patients were men, Asian, and had a poorly differentiated GAC. Gal-3 was over-expressed in poorly differentiated (P=0.002) tumours and also in diffuse sub-phenotype (P=0.02). Gal-3 overexpression was associated with shorter overall survival (OS; P=0.026) in all patients. Although, Gal-3 over-expression was not prognostic in the Asian cohort (P=0.337), it was highly prognostic in the North American cohort (P=0.001). In a multivariate analysis, Gal-3 (P=0.001) and N-stage (P=<0.001) were independently prognostic for shorter OS. Mechanistically, Gal-3 induced c-MYC expression through increasing RalA activity and an enhanced YAP1/RalA/RalBP complex to confer an aggressive phenotype. YAP1/BET bromodomain inhibitors reduced Gal-3-mediated aggressive phenotypes in GAC cells.Conclusions:Gal-3 is an independent prognostic marker of shorter OS and a novel therapeutic target particularly in diffuse type GAC in North American patients.

Anal canal cancer: biology and therapy

Introduction: Chemoradiation represents the standard of care for most patients with localized squamous cell carcinoma of the anal canal. In this article, randomized trials and studies on chemoradiation for localized anal cancer are reviewed. Areas covered: Herein the major Phase III randomized controlled trials published on the treatment of anal carcinoma are discussed. A recent randomized trial (Anal Cancer Trial II), after a total follow-up of 5.1 years, demonstrated that there was no difference in the complete response rate or 3-year progression-free survival when 5-fluorouracil (5-FU) with cisplatin chemoradiation was compared with 5-FU plus mitomycin C chemoradiation. Maintenance chemotherapy using 5-FU and cisplatin did not improve 3-year progression-free survival – 74 versus 73%. Recent studies have continued to evaluate intensity-modulated radiation therapy for anal cancer in an effort to reduce acute and long-term toxicity from radiotherapy, but no clear answers have emerged. Expert opinion: Studies on tumor biology, patient genetics and predictive marker are warranted to identify patients that are most likely to benefit from newer locoregional and systemic therapies. Intensity-modulated radiation therapy appears to be a promising approach for reducing treatment-related toxicity in anal cancer patients, but its role still remains to be completely defined.

Abstract 3896: The Hippo coactivator YAP1 upregulates SOX9 and endows cancer stem cell properties in non-transformed cells and esophageal cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cancer stem cells are proposed to initiate and maintain tumor growth. Dysregulation of normal stem cell signaling may lead to the generation of cancer stem cells (CSCs), however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional co-activator YAP1 through direct regulation of SOX9 promotes the generation of CSCs and that the inhibition of YAP1 and SOX9 attenuates CSC formation. SOX9 transcripts and expression are upregulated upon YAP1 activation and several lines of evidence indicate that SOX9 is a direct target of YAP1. The Chromatin Immunoprecipitation analysis and luciferase assays demonstrate direct binding of YAP1 to the SOX9 promoter through a conserved TEAD binding site. Mutation of this site abrogates transcriptional regulation of SOX9 by YAP1 and Tead2. Functional studies demonstrate that YAP1 regulation of SOX9 is necessary and sufficient to confer CSC properties and tumorigenesis in vitro and in vivo. The small molecule inhibitor of YAP1, Verteporfin (VP) significantly blocks CSC self-renewal properties in cells with high YAP1 and a high proportion of the CSC marker aldehyde dehydrogenase 1 (ALDH1) indicating that VP targets the CSC population. These data identify YAP1 as a driver of esophageal cancer (EC) stem cells, in part, by regulation of SOX9 and suggest that pharmacological inhibition of YAP1 may be an effective means of specifically targeting EC stem cells. Citation Format: Shumei Song, Jaffer A. Ajani, Soichiro Honjo, Dipen M. Maru, Qiongrong Chen, Jiankang Jin, Ailing W. Scott, Todd R. Heallen, Lianchun Xiao, Wayne L. Hofstetter, Brian Weston, Jeffrey H. Lee, Roopma Wadhwa, Kazuki Sudo, James F. Martin, John R. Stroehlein, Mien-Chie Hung, Randy L. Johnson. The Hippo coactivator YAP1 upregulates SOX9 and endows cancer stem cell properties in non-transformed cells and esophageal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3896. doi:10.1158/1538-7445.AM2014-3896