Kinome-wide RNAi screen identifies key role of dual specificity kinase Mps1 in proliferation and survival of pancreatic cancer cells

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the western world. Currently it is incurable with existing therapies. The median survival rate (˜6 months) and the overall 5-year survival rate (< 5%) has not improved despite significant research and diagnostic efforts, warranting the urgent need for novel therapeutic targets and approaches.

Protein kinases are known to be effective and druggable targets in a variety of disease states. We employed a kinome-wide RNAi screen to identify hitherto unknown protein kinases which when silenced would sensitize the cancer cells but not control cells to apoptosis.

Aim: This project aims to elucidate the pathophysiological role of one of the identified kinases, Mps1 (a.k.a. TTK), in pancreatic cancer. The Mps1 gene has been implicated in regulation of mitosis in certain cancer types, but its functional relevance has not been investigated in PDAC.

Methods: qPCR, RNAi, cell proliferation and viability assays, FACS analysis, Western blot, shRNA inducible clones.

Results: The qRT-PCR data showed that Mps1 kinase is overexpressed in pancreatic cancer tissues and this strong expression is also retained in the majority of pancreatic tumor cell lines in vitro. Functional effects of Mps1 were investigated after transient knockdown in a variety of transformed and non-transformed cell lines. Proliferation (BrdU incorporation assay), viability (MTT assay) and clonogenic ability (Anchorage independent growth assay) of the cancer cells were significantly impaired in the absence of functional Mps1 gene-product. FACS analyses did not show any cell-cyle impairment but Western blot analyses showed activation of classical apoptosis pathway (Caspase-3 and PARP cleavage). Further experiments involving generation of stable inducible knockdown clones for further in vivo analyses as well as experiments for deciphering the pathways involved are currently in progress.

Conclusion: Our experimental data shows that loss of Mps1 function severely compromises the proliferation, viability as well as clonogenic ability of the pancreatic cancer cell and leads them into apoptosis.