Jiankang Jin

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.

IL-24 Inhibits Lung Cancer Cell Migration and Invasion by Disrupting The SDF-1/CXCR4 Signaling Axis

Background The stromal cell derived factor (SDF)-1/chemokine receptor (CXCR)-4 signaling pathway plays a key role in lung cancer metastasis and is molecular target for therapy. In the present study we investigated whether interleukin (IL)-24 can inhibit the SDF-1/CXCR4 axis and suppress lung cancer cell migration and invasion in vitro. Further, the efficacy of IL-24 in combination with CXCR4 antagonists was investigated. Methods Human H1299, A549, H460 and HCC827 lung cancer cell lines were used in the present study. The H1299 lung cancer cell line was stably transfected with doxycycline-inducible plasmid expression vector carrying the human IL-24 cDNA and used in the present study to determine the inhibitory effects of IL-24 on SDF-1/CXCR4 axis. H1299 and A549 cell lines were used in transient transfection studies. The inhibitory effects of IL-24 on SDF1/CXCR4 and its downstream targets were analyzed by quantitative RT-PCR, western blot, luciferase reporter assay, flow cytometry and immunocytochemistry. Functional studies included cell migration and invasion assays. Principal Findings Endogenous CXCR4 protein expression levels varied among the four human lung cancer cell lines. Doxycycline-induced IL-24 expression in the H1299-IL24 cell line resulted in reduced CXCR4 mRNA and protein expression. IL-24 post-transcriptionally regulated CXCR4 mRNA expression by decreasing the half-life of CXCR4 mRNA (>40%). Functional studies showed IL-24 inhibited tumor cell migration and invasion concomitant with reduction in CXCR4 and its downstream targets (pAKTS473, pmTORS2448, pPRAS40T246 and HIF-1α). Additionally, IL-24 inhibited tumor cell migration both in the presence and absence of the CXCR4 agonist, SDF-1. Finally, IL-24 when combined with CXCR4 inhibitors (AMD3100, SJA5) or with CXCR4 siRNA demonstrated enhanced inhibitory activity on tumor cell migration. Conclusions IL-24 disrupts the SDF-1/CXCR4 signaling pathway and inhibits lung tumor cell migration and invasion. Additionally, IL-24, when combined with CXCR4 inhibitors exhibited enhanced anti-metastatic activity and is an attractive therapeutic strategy for lung metastasis.

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.

A novel YAP1 inhibitor targets CSC-enriched radiation-resistant cells and exerts strong antitumor activity in esophageal adenocarcinoma

Mounting evidence suggests that the Hippo coactivator Yes-associated protein 1 (YAP1) is a major mediator of cancer stem cell (CSC) properties, tumor progression, and therapy resistance as well as often a terminal node of many oncogenic pathways. Thus, targeting YAP1 may be a novel therapeutic strategy for many types of tumors with high YAP1 expression, including esophageal adenocarcinoma. However, effective YAP1 inhibitors are currently lacking. Here, we identify a small molecule (CA3) that not only has remarkable inhibitory activity on YAP1/Tead transcriptional activity but also demonstrates strong inhibitory effects on esophageal adenocarcinoma cell growth especially on YAP1 high–expressing esophageal adenocarcinoma cells both in vitro and in vivo. Remarkably, radiation-resistant cells acquire strong cancer stem cell (CSC) properties and aggressive phenotype, while CA3 can effectively suppress these phenotypes by inhibiting proliferation, inducing apoptosis, reducing tumor sphere formation, and reducing the fraction of ALDH1+ cells. Furthermore, CA3, combined with 5-FU, synergistically inhibits esophageal adenocarcinoma cell growth especially in YAP1 high esophageal adenocarcinoma cells. Taken together, these findings demonstrated that CA3 represents a new inhibitor of YAP1 and primarily targets YAP1 high and therapy-resistant esophageal adenocarcinoma cells endowed with CSC properties. Mol Cancer Ther; 17(2); 443–54. ©2017 AACR.

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.

Abstract 1713: IL-24 inhibits lung cancer cell migration and invasion by disrupting the SDF-1/CXCR4 signaling axis

Introduction The stromal cell derived factor (SDF)-1/chemokine receptor (CXCR)-4 signaling pathway plays a key role in lung cancer metastasis. Therefore, disrupting the SDF-1/CXCR4 signaling axis will reduce the incidence of lung metastasis. In the present study we investigated whether interleukin (IL)-24 can inhibit the SDF-1/CXCR4 axis and suppress lung cancer cell migration and invasion in vitro. Further, the efficacy of IL-24 in combination with CXCR4 antagonists was investigated. Methods Human H1299 lung tumor cell line was stably transfected with a tetracycline-inducible plasmid vector carrying the IL-24. Upon addition of doxycycline (Dox; 1μg/ml), cells were induced to express IL-24 protein. The expression levels of CXCR4 and its downstream molecular mechanisms in H1299 cells were analyzed. The inhibitory effect of IL-24 on SDF-1/CXCR4 axis is determined by RT-qPCR, western blot, luciferase reporter assay, flow-cytometry and immuno-cytochemistry and the consequence of its inhibition on cell migration, and invasion. Results Endogenous CXCR4 protein expression levels varied among four human lung cancer cell lines with H1299 cells showing the highest expression. Doxycycline-induced IL-24 expression in the H1299-IL-24 cell line resulted in reduced CXCR4 mRNA and protein expression. IL-24 post-transcriptionally regulated CXCR4 mRNA expression by decreasing the half-life of CXCR4 mRNA (>40%). Associated with CXCR4 inhibition was the reduced protein expression of pAKTS473, pmTORS2448, pPRAS40T246 and HIF-1α. IL-24 inhibited tumor cell migration and invasion both in the presence and absence of the CXCR4 agonist, SDF-1. However, the combinatorial effect of either IL-24 combined with CXCR4 inhibitors (AMD3100, SJA5) or with CXCR4 siRNA demonstrated enhanced inhibitory activity on tumor cell migration. Conclusions Our study results demonstrate that IL-24 inhibits lung tumor cell migration and invasion by disrupting the SDF-1/CXCR4 signaling pathway and exhibits enhanced anti-metastatic activity when combined with CXCR4 inhibitors. Citation Format: Janani Panneerselvam, Jiankang Jin, Manish Shanker, Jason Lauderdale, Jonathan Bates, Qi Wang, Daniel Zhao, Stephen Archibald, Timothy Hubin, Rajagopal Ramesh. IL-24 inhibits lung cancer cell migration and invasion by disrupting the SDF-1/CXCR4 signaling axis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1713. doi:10.1158/1538-7445.AM2015-1713

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