Abstract A17: The tyrosine phosphatase SHP2 regulates YAPY357 phosphorylation, subcellular localization, and transcriptional coactivity in cholangiocarcinoma
Authors: Buckarma EeeLN , Werneburg Nathan , Kabashima Ayano , Gores Gregory , Smoot Rory 
Published: September 2020

Introduction: The effector protein of the Hippo pathway, Yes-associated protein (YAP), functions as a transcriptional coactivator and has been implicated in the biology of cholangiocarcinoma (CCA). Although the Hippo cascade consists of serine kinases, which act to sequester YAP in the cytosol, we have recently demonstrated the importance of tyrosine phosphorylation in regulating YAP localization and function in CCA. Furthermore, previous evaluation of clinical samples of CCA demonstrated a high frequency of mutations in tyrosine phosphatases. Evaluation of YAP-interacting proteins identified several tyrosine phosphatases, including SHP2. Herein, we describe the ability to modulate YAPY357 phosphorylation status, localization, and activity through manipulation of SHP2 levels and activity.

Methods: The cholangiocarcinoma cell lines HuCCT-1 and KMCH were utilized. SHP2 activity was modulated utilizing the inhibitors NSC87877 and SHP099 (Selleckchem). 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. Tyrosine phosphorylation was assessed via immunoblot. Transcriptional activity was assessed by qRT-PCR for YAP target genes. YAP sub-cellular localization was assessed by immunofluorescence. Proliferation was evaluated by MTS assay utilizing SHP2 inhibitors and the Src-family kinase inhibitor dasatinib (Selleckchem).

Results: The HuCCT-1 and KMCH cell lines were noted to have unique p-YAPY357 profiles (HuCCT-1 [p-YAPY357 high] KMCH [p-YAPY357 low]), and these profiles correlated with SHP2 levels and YAP subcellular localization with HuCCT1 cells demonstrating low levels of SHP2 and prominent nuclear YAP localization, while KMCH cells demonstrated high levels of SHP2 and predominately cytoplasmic YAP. In both cell lines, pharmacologic inhibition of SHP2 was accompanied by an increase in p-YAPY357 levels and YAP cotranscriptional activity (as noted by increased expression of cognate YAP target genes). In KMCH cells SHP2 inhibition shifted YAP from the cytoplasm to the nucleus, and these findings were confirmed by CRISPR/Cas9-mediated deletion of SHP2. In contrast, enforced expression of catalytically active SHP2 in HuCCT1 cells was associated with a decrease in p-YAPY357 levels and a shift of YAP from the nucleus to the cytoplasm. As predicted, based on an increase in YAP activity and nuclear localization, SHP2 inhibition was associated with an increase in cellular proliferation in both cell lines. Incubation of the cell lines with dasatinib was associated with decreased proliferation, which was partially rescued by pretreatment with an SHP2 inhibitor.

Conclusions: The tyrosine phosphatase SHP2 can regulate YAPY357 phosphorylation, transcriptional coactivity, and subcellular localization in CCA. Inhibition of SHP2 activity protects cells from the effects of Src kinase inhibition on cellular proliferation and caspase activation.

Citation Format: EeeLN Buckarma, Nathan Werneburg, Ayano Kabashima, Gregory Gores, Rory Smoot. The tyrosine phosphatase SHP2 regulates YAPY357 phosphorylation, subcellular localization, and transcriptional coactivity in cholangiocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A17.