MPP5 (Membrane Palmitoylated Protein 5), a novel upstream regulator of yes-associated protein (YAP) and its paralogue TAZ

The Hippo pathway plays a central role in the initiation and progression of liver cancer. The overexpression of the pathway effector yes-associated protein YAP is detectable in more than 60% of all hepatocellular carcinomas (HCC) and correlates with higher tumor cell proliferation rate and with poor clinical outcome. However, the mechanistic connection between cell surface and the two pathway effectors YAP and TAZ (WWTR1; WW Domain Containing Transcription Regulator 1) in hepatocarcinogenesis is poorly understood. In this project we aim to identify relevant proteins that link cell polarity with YAP/TAZ localization and activity.

Six different HCC cell line (HepG2, HuH1, HuH6, HLE, HLF, Hep3B) were tested for their ability to translocate YAP/TAZ from the cytoplasm to the nucleus under varying cell density conditions by immunofluorescence (IF). To identify regulators of YAP/TAZ localization, an siRNA screen was performed containing customized siRNAs targeting 26 different proteins involved in hepatocellular polarity. Identified factors were further investigated by IF and IHC (immunohistochemistry), co- immunoprecipitation (Co-IP) experiments, and expression profiling.

The culture of HCC cells using different cell density conditions revealed that three cell lines showed a dynamic shuttling of YAP/TAZ from the cytoplasm to the nucleus with changing confluence (HepG2, HuH1, HuH6). In contrast, HLE, HLF, and Hep3B cells YAP/TAZ localization was uncoupled from cell-density. The results of the siRNA screening in cell density-sensing HepG2 cells showed that silencing of 16/26 candidate proteins resulted in either nuclear YAP or TAZ enrichment. Notably, most positive candidates are part of the apical Crumbs complex. Higher expression levels of MPP5 (Membrane Palmitoylated Protein 5), a Crumbs complex constituent was found to correlate both with better overall and recurrence free survival of HCC patients. In human tissues, the membranous staining of MPP5 gradually decreased from healthy liver tissue to poorly differentiated HCCs and negatively correlated with YAP levels. Co-IP experiments showed that MPP5 physically interact to YAP/TAZ. First expression profiling results after siRNA-mediated inhibition of MPP5 illustrated that many YAP/TAZ target genes were inversely regulated.

Our results illustrate that the apical protein MPP5 is a central negative regulator of YAP/TAZ abundance and activity in hepatocellular cells. Its loss or mislocalization might be one reason for the nuclear enrichment of oncogenic YAP and TAZ in HCC.