Esophageal Adenocarcinoma Cells and Xenograft Tumors Exposed to Erb-b2 Receptor Tyrosine Kinase 2 and 3 Inhibitors Activate Transforming Growth Factor Beta Signaling, Which Induces Epithelial to…

Eva A. Ebbing, Anne Steins, Evelyn Fessler, Phylicia Stathi, Willem Joost Lesterhuis, Kausilia K. Krishnadath, Louis Vermeulen, Jan Paul Medema, Maarten F. Bijlsma, Hanneke W.M. van Laarhoven

Drugs that inhibit the erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2) are the standard treatment of patients with different types of cancer, including HER2-overexpressing gastroesophageal tumors. Unfortunately, cancer cells become resistant to these drugs, so overall these drugs provide little benefit to patients with these tumors. We investigated mechanisms that mediate resistance of esophageal adenocarcinoma (EAC) cells and patient-derived xenograft (PDX) tumors to ERBB inhibitors. We cultured primary tumor cells, isolated from EAC patient samples, and OE19 and OE33 EAC cell lines with trastuzumab (an inhibitor of HER2), with or without pertuzumab (inhibits dimerization of HER2 with HER3) or a specific antibody against HER3 (anti-HER3). HER2 was knocked down by expression of small hairpin RNAs. In addition, cells were incubated with NRG1-beta, a mediator of HER2-HER3 signaling, or A83-01, an inhibitor of transforming growth factor beta (TGFB) signaling. Cells were analyzed for markers of the epithelial to mesenchymal transition (EMT) using flow cytometry, immunofluorescence, and quantitative reverse transcription PCR. We performed limiting dilution, transwell, and cell viability assays to study the functional effects of HER2 and HER3 inhibition and reactivation. We analyzed publicly available EAC gene expression datasets to correlate expression of ERBB genes with genes encoding epithelial and mesenchymal proteins. NSG mice were given subcutaneous injections of AMC-EAC-007B cells and also given injections of single or combined inhibitors; growth of these PDX tumors was quantified. EAC cells incubated with trastuzumab decreased expression of epithelial markers (CD24, CD29, and CDH1) and increased expression of mesenchymal markers (CXCR4, VIM, ZEB1, SNAI2, and CDH2), compared to cells not exposed to trastuzumab, indicating induction of EMT. Addition of NRG1-beta to these cells restored their epithelial phenotype. Incubation of EAC cells with trastuzumab and pertuzumab accelerated the expression of EMT markers, compared to cells incubated with trastuzumab alone. EAC cells cultured for 2 months with a combination of trastuzumab and pertuzumab became resistant to chemotherapeutic agents (5-fluoruracil, carboplatin, cisplatin, eribulin, and paclitaxel), based on their continued viability, which was accompanied with an enhanced migratory capacity in transwell assays, and clonogenicity in limiting dilution analyses. In comparisons of EAC gene expression patterns, we associated high expression of ERBB3 with an epithelial gene expression signature; expression of transforming growth factor beta (TGFB) correlated with expression of EMT-related genes, and we found an inverse correlation between expression of TGFB1 and ERBB3. EAC cells incubated with ERBB inhibitors began to secrete ligands for the TGFB receptor and underwent EMT. Incubation of EAC cells with trastuzumab, followed by 10 days incubation with the TGFB receptor inhibitor in the presence of trastuzumab, caused cells to regain an epithelial phenotype. EAC patient-derived xenograft tumors grew more slowly in mice given the combination of trastuzumab, pertuzumab, and the TGFB inhibitor than in mice given single agents or a combination of trastuzumab and pertuzumab. Tumors exposed to trastuzumab and pertuzumab expressed EMT markers o and were poorly differentiated, whereas tumors exposed to the combination of trastuzumab, pertuzumab, and the TGFB inhibitor expressed epithelial markers and were more differentiated. EAC cells become resistant to trastuzumab and pertuzumab by activating TGFB signaling, which induces EMT. Agents that block TGFB signaling can increase the anti-tumor efficacies of trastuzumab and pertuzumab.