Role of p21-activated kinases in pancreatic cancer

Abstract

Pancreatic cancer remains one of the most lethal of all solid tumours with an overall 5-year survival rate of 7%. Management has not improved significantly over the last thirty years and based on current trends, is expected to become the second leading cause of cancer-related mortality by 2030. Treatment options are limited and gemcitabine-based chemotherapy remains the standard of care as a single agent. Furthermore, the presence of the dense stroma, characteristic of pancreatic cancer, contributes to therapeutic resistance and poor therapeutic response. Thus, a better understanding of the underlying genetic and molecular mechanisms is urgently required to find targeted and effective therapies.

There is growing evidence that p21-activated kinases (PAKs) are involved in pancreatic carcinogenesis. The PAK family consist of six isoforms, two of which, PAK1 and PAK4, are upregulated and/or hyper-activated in pancreatic cancer. PAK1 can mediate many different cellular processes including the regulation of cytoskeletal dynamics and cell adhesion, the evasion of apoptosis, the promotion of cell survival, proliferation, migration and invasion, the fibrosis that constitutes the stroma, and the interplay between cancer cells and the stroma. PAK1’s role has not been fully elucidated in pancreatic cancer and has not been evaluated as a target for therapeutic intervention. The work presented in this thesis investigates the role of PAK1 in pancreatic cancer and the effect of PAK1 inhibitors, alone and in combination with gemcitabine, on pancreatic cancer growth, metastasis, stroma, and survival.

First, we investigated the effect of glaucarubinone, a known inhibitor that reduces the activity of PAK1 and PAK4, on pancreatic cancer growth, migration and murine survival. Using 4 human and 2 murine pancreatic cancer cell lines, PAK1 and PAK4 was expressed in all pancreatic cancer cell lines tested and proliferation and migration/invasion inhibited by treatment of glaucarubinone with reduction in PAK1 and PAK4 activity in vitro. Synergistic inhibition was observed when combined with gemcitabine with decrease in pancreatic cancer proliferation in vitro, decrease in pancreatic cancer growth in human xenograft tumours in vivo, and increase in murine survival in an orthotopic immunocompetent model in vivo. This was one of the first studies that showed clinical benefit of targeting and reducing PAK1 in pancreatic cancer.

Using more direct methods of reducing PAK1 activity, shRNA knockdown systems, and a PAK1 selective inhibitor, FRAX597, were utilised. shRNA knockdown of PAK1 resulted in a reduction in pancreatic cancer cell proliferation and survival and sensitised cells to gemcitabine in vitro. PAK1 was also found to be key regulator of signalling pathways such as PI3K and HIF1α. FRAX597 treatment decreased pancreatic cancer cell proliferation and migration/invasion and synergised with gemcitabine to decrease cell proliferation in vitro. FRAX597, combined with gemcitabine, reduced pancreatic tumour volume and increased murine survival in preclinical orthotopic immunocompetent murine models in vivo. Although, further clinical validation is required, it illustrates the clinical potential of a PAK1 inhibitor, FRAX597, combined with gemcitabine to improve pancreatic cancer patient outcomes.

PAK1’s role was investigated in pancreatic stellate cells (PSCs), which are primarily responsible for the fibrosis that constitutes the pancreatic cancer stroma. This was the first study to show the presence of PAK1 activity in isolated human PSCs. The treatment of the selective PAK1 inhibitor, FRAX597, on PSCs resulted in a reduction in their activation, proliferation, and increase in apoptosis in vitro. PAK1 knockout mice tumours had decreased expression and activity of PAK1, associated with increased murine survival, showing the effect of depleting host PAK1 in an orthotopic immunocompetent murine model in vivo. These results implicate PAK1 as a regulator of PSC activation, proliferation and apoptosis and targeting stromal PAK1 could increase therapeutic response and survival of patients with pancreatic cancer.

Together, these results illustrate the importance of PAK1 signalling in pancreatic cancer and the possible therapeutic benefit of targeting PAK1 with gemcitabine on pancreatic cancer growth and the stroma to increase the survival of pancreatic cancer patients.