The Tyrosine Phosphatase Shp2 Regulates Yapy357 Phosphorylation, And In Vivo Sensitivity To Cytotoxic Chemotherapy In Cholangiocarcinoma

Abstract

Presenter: EeeLN Buckarma MD | Mayo Clinic, Rochester Background: Cancer of the biliary tract, cholangiocarcinoma (CCA), is increasing in incidence and has limited treatment options. In an attempt to further understand signaling pathways driving oncogenesis and impacting response to therapy we, and others, have identified altered activation of the transcriptional co-activator, Yes- associated protein (YAP) in CCA. Canonical regulatory pathways impacting YAP consist of serine kinases; however, more recently we have demonstrated a central role for tyrosine phosphorylation in regulating YAP function in CCA. Herein we explore the role of tyrosine phosphatases in regulating YAP tyrosine phosphorylation and sensitivity to current cytotoxic chemotherapy. Methods: Molecular studies utilized the human CCA cell lines HuCCT-1 and KMCH. Baseline tyrosine phosphatase levels were assessed by RT-PCR and immunoblot. YAP-interacting phosphatases were identified by co-immunoprecipitation. The selective tyrosine phosphatse inhibitors NSC87877 and SHP099 were utilized. Tyrosine phosphorylation was assessed by immunoblot. YAP transcriptional activity was evaluated by RT-PCR for YAP target genes. SHP2 levels were modulated utilizing knockout via a doxycycline-inducible CRISPR/Cas9 system. Enforced expression of SHP2 was explored utilizing catalytically active and dead constructs. YAP sub-cellular localization was assessed by immunofluorescence. Proliferation was evaluated by MTS assay and cell viability was assessed with Cell Titer-Glo. Cytotoxic agents’ gemcitabine and cisplatin were utilized for in vitro and in vivo studies. CCA xenografts (via mouse flank model) were generated with non-targeting small guide-bearing KMCH and sgSHP2-KMCH cells. Mice were treated with q3 day cytotoxic treatment x 3 weeks and tumors were evaluated by growth rate, weight and volume. Results: Profiling of tyrosine phosphatase levels by RT-PCR and immunoblot demonstrated higher levels in KMCH cells compared to HuCCT-1; notably the YAP-interacting phosphatase PTPN11 (SHP2) was elevated. Consistent with the anticipated function of the phosphatases, immunoblot demonstrated lower levels of tyrosine phosphorylated YAP (p-YAPY357) in KMCH cells compared to HuCCT-1. The role of SHP2 was further probed by incubation of KMCH and HuCCT-1 cells with NSC87877 which was associated with an increase in p-YAPY357 levels and YAP co-transcriptional activity. In addition, KMCH cells demonstrated increased YAP nuclear localization following incubation with NSC87877. The effects seen with YAP tyrosine phosphorylation were paralleled in doxycycline-inducible CRISPR/Cas9 sgSHP2-KMCH cells. In HuCCT1 cells overexpressing SHP2 (WT-SHP2), pYAPY357 levels and transcriptional activity were decreased, and the localization of YAP was shifted towards the cytoplasmic compartment. Expression of a phosphatase-dead construct (C459S-SHP2) had no demonstrable effect. A deletion-reconstitution approach utilizing induced sgSHP2-KMCH cells followed by reconstitution with expression of either wild-type or phosphatase-dead constructs was associated with a decrease in p-YAP357 levels, YAP transcriptional activity, and YAP baseline nuclear localization in wild-type alone. Increased cell death was observed in KMCH cells as compared the HuCCT-1 and sgSHP2-KMCH cells incubated with gemcitabine and cisplatin. In keeping with these observations, in vivo studies demonstrated that the sgSHP2-KMCH cell lines when compared to wild type, developed tumors with faster growth rates, and subsequently these tumors were less sensitive to the combination of gemcitabine and cisplatin therapy. Conclusion: The tyrosine phosphatase SHP2 regulates p-YAPY357 levels, YAP co-transcriptional activity, and in vivo sensitivity to current standard chemotherapy in cholangiocarcinoma.