| Pancreatic cancer is highly resistant to current chemotherapies and continues to be the 4th leading cause of cancer deaths in the United States. Identification of the critical components of signaling pathways that mediate pancreatic cancer transformed growth and metastasis is necessary for the development of more effective therapeutic treatments. Recently, we demonstrated that atypical protein kinase C iota promotes pancreatic cancer transformed growth, invasion, tumorigenesis and tumor metastasis and is a critical, upstream regulator of Rac1/ERK signaling. In this study, we first investigate the role of the other atypical protein kinase C, PKCzeta in pancreatic cancer cells. We find that PKCzeta expression is either maintained or elevated in primary human pancreatic tumors, but is never lost, consistent with PKCzeta playing a promotive role in the pancreatic cancer phenotype. Genetic inhibition of PKCzeta reduced adherent growth, cell survival and anchorage-independent growth of human pancreatic cancer cells in vitro. Furthermore, PKCzeta inhibition reduced orthotopic tumor size in vivo by inhibiting tumor cell proliferation and increasing tumor necrosis. In addition, PKCzeta inhibition reduced tumor metastases in vivo, and caused a corresponding reduction in pancreatic cancer cell invasion in vitro. Signal transducer and activator of transcription 3 (STAT3) is often constitutively active in pancreatic cancer, and plays an important role in pancreatic cancer cell survival and metastasis. Interestingly, inhibition of PKCzeta significantly reduced constitutive STAT3 activation in pancreatic cancer cells in vitro and in vivo. Pharmacologic inhibition of STAT3 mimicked the phenotype of PKCzeta inhibition, and expression of a constitutively active STAT3 construct rescued the transformed phenotype in PKCzeta-deficient cells. We conclude that PKCzeta is required for pancreatic cancer cell transformed growth and invasion in vitro and tumorigenesis in vivo, and that STAT3 is an important downstream mediator of the pro-carcinogenic effects of PKCzeta in pancreatic cancer cells.;Both PKCiota and PKCzeta are tumor promotive and regulate distinct oncogenic signaling pathways in pancreatic cancer; therefore, we next investigate the therapeutic potential of targeting the aPKCs. We show that 1) PKCiota and PKCzeta play non-redundant roles in the promotion of pancreatic cancer transformed growth; 2) two gold-containing small molecules known to disrupt aPKC/Par6 interaction, Aurothiomalate and Auranofin, inhibit downstream signaling of PKCiota and PKCzeta and transformed growth of pancreatic cancer cells in vitro; and 3) Aurothiomalate inhibits pancreatic cancer tumor growth and metastasis in vivo. These data provide convincing evidence that small molecule inhibitors of atypical PKC signaling inhibit two oncogenic signaling pathways in pancreatic cancer and significantly reduce tumor growth and metastasis.;Recently a distinct population of cancer cells with 'stem cell', 'initiating', or 'progenitor' properties has been identified. This tumor sub-population is characterized to play a significant role in tumor metastases and therapy resistance, two of the factors that promote the high lethality of pancreatic cancer. Interestingly, we show that PKCzeta regulates STAT3, which has been characterized to promote the cancer stem cell (CSC) phenotype, and PKCiota promotes lung cancer stem cell maintenance. Therefore, lastly in this study, we investigate the effects of genetic and pharmacologic inhibition of the aPKC isozymes on the transformed growth and maintenance of pancreatic CSCs (PCSCs). We generated PCSCs in "low adherent" 3D culture and demonstrated that our PCSC population exhibits enhanced expression of putative cancer stem cell markers, including pSTAT3, the ability to clonally expand, and increased anchorage independent growth compared to their respective adherent, 'bulk-tumor' population. We also show that PKCzeta RNAi-expressing PCSCs exhibit a reduction in various stem cell characteristics, including reduced putative CSC marker expression, reduced ability to form colonies in soft agar, and reduced cell growth and clonal expansion. We next sought to determine if pharmacologic inhibition of PKCzeta would mimic the effects of genetic PKCzeta inhibition on PCSC maintenance. Indeed, ATM treatment drastically reduced PCSC viability, proliferation (measured as cell count over time), anchorage independent growth, and clonal expansion. Taken together, these data suggest that inhibition of atypical PKC signaling is a promising therapeutic strategy to treat pancreatic cancer. |
