A CGA/EGFR/GATA2 positive feedback circuit confers chemoresistance in gastric cancer
Tianyu Cao, Yuanyuan Lu, Qi Wang, Hongqiang Qin, Hongwei Li, Hao Guo, Minghui Ge, Sarah E. Glass, Bhuminder Singh, Wenyao Zhang, Jiaqiang Dong, Feng Du, Airong Qian, Ye Tian, Xin Wang, Cunxi Li, Kaichun Wu, Daiming Fan, Yongzhan Nie, Robert J. Coffey, Xiaodi Zhao
Tianyu Cao, Yuanyuan Lu, Qi Wang, Hongqiang Qin, Hongwei Li, Hao Guo, Minghui Ge, Sarah E. Glass, Bhuminder Singh, Wenyao Zhang, Jiaqiang Dong, Feng Du, Airong Qian, Ye Tian, Xin Wang, Cunxi Li, Kaichun Wu, Daiming Fan, Yongzhan Nie, Robert J. Coffey, Xiaodi Zhao
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Research Article Gastroenterology Oncology

A CGA/EGFR/GATA2 positive feedback circuit confers chemoresistance in gastric cancer

Abstract

De novo and acquired resistance are major impediments to the efficacy of conventional and targeted cancer therapy. In unselected gastric cancer (GC) patients with advanced disease, trials combining chemotherapy and an anti-EGFR monoclonal antibody have been largely unsuccessful. In an effort to identify biomarkers of resistance so as to better select patients for such trials, we screened the secretome of chemotherapy-treated human GC cell lines. We found that levels of CGA, the α-subunit of glycoprotein hormones, were markedly increased in the conditioned media of chemoresistant GC cells, and CGA immunoreactivity was enhanced in GC tissues that progressed on chemotherapy. CGA levels in plasma increased in GC patients who received chemotherapy, and this increase was correlated with reduced responsiveness to chemotherapy and poor survival. Mechanistically, secreted CGA was found to bind to EGFR and activate EGFR signaling, thereby conferring a survival advantage to GC cells. N-glycosylation of CGA at Asn52 and Asn78 is required for its stability, secretion, and interaction with EGFR. GATA2 was found to activate CGA transcription, whose increase, in turn, induced the expression and phosphorylation of GATA2 in an EGFR-dependent manner, forming a positive feedback circuit that was initiated by GATA2 autoregulation upon sublethal exposure to chemotherapy. Based on this circuit, combination strategies involving anti-EGFR therapies or targeting CGA with microRNAs (miR-708-3p and miR-761) restored chemotherapy sensitivity. These findings identify a clinically actionable CGA/EGFR/GATA2 circuit and highlight CGA as a predictive biomarker and therapeutic target in chemoresistant GC.

Authors

Tianyu Cao, Yuanyuan Lu, Qi Wang, Hongqiang Qin, Hongwei Li, Hao Guo, Minghui Ge, Sarah E. Glass, Bhuminder Singh, Wenyao Zhang, Jiaqiang Dong, Feng Du, Airong Qian, Ye Tian, Xin Wang, Cunxi Li, Kaichun Wu, Daiming Fan, Yongzhan Nie, Robert J. Coffey, Xiaodi Zhao

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Figure 3

CGA functions by binding to EGFR and activating EGFR downstream signaling in GC cells.

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CGA functions by binding to EGFR and activating EGFR downstream signalin...
(A) Human phosphorylated RTK antibody array in SGC7901 cells serum starved for 12 hours and then treated with rCGA for 30 minutes. (B) Immunoblotting of CGA, EGFR, and p-EGFR in indicated serum-starved cells. (C) Immunoblotting of p-EGFR in serum-starved SGC7901 cells treated with indicated concentrations of rCGA (top) or treated with rCGA (20 μg/mL) at different time points (bottom). (D) Immunoblotting with indicated antibodies of lysates from CGA–/– SGC7901ADR cells that were pretreated with cetuximab (10 μg/mL) followed by rCGA treatment. (E) Immunoblotting of lysates from SGC7901 cells that were incubated with rCGA and immunoprecipitated with anti-EGFR antibody or normal IgG. (F) Immunoblotting of lysates from SGC7901 cells transfected with Flag-tagged RFP or EGFR containing FL, ECD, or ICD plasmids, treated with purified His-tagged rCGA, and subjected to anti-Flag and anti-His immunoprecipitation. (G) Molecular docking analysis of CGA to the ECD of EGFR. (H) SPR analysis of the interaction between CGA and the ECD of EGFR. Raw response (RU) curves (top) from a representative experiment were fitted to a 1-site-specific kinetic model (bottom) to derive on and off rates and a Kd value for the interaction. (I) IF staining of CGA, EGFR, and early endosome marker EEA1 in SGC7901 cells treated with rCGA at 37°C for a 30-minute time course. Scale bar: 10 μm. (J) Viability of SGC7901 and NCI-N87 cells stably expressing CGA, treated with fluorouracil, cetuximab, erlotinib (20 nM), or their combination. (K and L) SGC7901 cells stably expressing CGA and control SGC7901 cells (K) or SGC7901ADR cells (L) were injected subcutaneously into nude mice (n = 6–8). When the tumor size reached 100 mm3, mice received indicated treatment every 3 days (fluorouracil, 20 mg/kg, i.p. injection; cetuximab, 1 mg/mouse, i.p. injection). Tumor volume and tumor weight were measured. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01 by 1-way ANOVA with Bonferroni’s post hoc test (J–L) or by repeated-measures ANOVA with Bonferroni’s post hoc test (K and L).