Abstract B29: Oncogenic K-ras addiction in NSCLC switches PKCδ from a pro-apoptotic to a pro-survival signal

Abstract

Abstract Oncogenic mutations in K-ras occur in about 25% of lung cancers, however only a subset of lung tumors with KRAS mutations are functionally dependent upon oncogenic K-ras for survival. Our previous studies show that the function of Protein Kinase C-δ (PKCδ), a serine/threonine kinase that regulates apoptosis in non-transformed cells, is altered in non-small cell lung cancers (NSCLC) that are dependent on oncogenic K-ras, such that these cells now require PKCδ for survival and transformed growth (Symonds et al, Cancer Research, 2011). The purpose of the current studies was to determine if this switch in PKCδ function alters the response of lung cancer cells to chemotherapeutic drugs. We analyzed apoptosis and PKCδ function in five NSCLC cells lines previously characterized in our laboratory as dependent on oncogenic K-ras for survival, and five NSCLC cell lines characterized as K-ras independent. Our results show that NSCLC cell lines that are functionally dependent on K-ras are highly resistant to apoptosis induced by DNA damaging agents such as etoposide. In contrast, K-ras independent NSCLC cell lines are highly sensitive to apoptosis. Our previous studies have shown that PKCδ translocates to the nucleus in response to apoptotic agents and that nuclear localization of PKCδ is essential for apoptosis. To determine the mechanism underlying the switch in PKCδ function from pro-apoptotic to pro-survival, we analyzed PKCδ expression and cytoplasmic/nuclear localization in apoptosis sensitive (K-ras independent) and apoptosis resistant (K-ras dependent) NCSCL cells. Relative to insensitive NSCLC cells, apoptosis sensitive cells showed increased expression of PKCδ by qrtPCR, a reduced cytoplasmic:nuclear ratio of PKCδ under basal conditions, and increased nuclear import of PKCδ in response to etoposide. This suggests that PKCδ may be a pro-apoptotic signal in K-ras independent cells, similar to what we have shown for non-transformed cells. In contrast, nuclear import of PKCδ in response to etoposide was suppressed in K-ras dependent/apoptosis resistant NSCLC cells as was phosphorylation of PKCδ on tyrosines Y64 and Y155, which we have previously shown is required for importin -α binding and nuclear import. Exclusion of PKCδ from the nucleus may explain the resistance of K-ras dependent cells to apoptotic agents. To probe the contribution of PKCδ to these two phenotypes more directly, we depleted PKCδ from K-ras independent/apoptosis sensitive A549 and K-ras dependent/apoptosis resistant H2009 cells using an lentivirus delivered shRNA to PKCδ or a scrambled shRNA control. Depletion of PKCδ in A549 cells resulted in suppression of etoposide-induced apoptosis, similar to what we have previously reported in non-transformed cells. Suppression of apoptosis was accompanied by increased activation of the Akt and MEK/ERK cell survival pathways in A549 cells. In contrast, depletion of PKCδ in H2009 cells resulted in increased apoptosis in response to etoposide, and suppression of the Akt and MEK/ERK cell survival pathways. Our studies suggest NSCLC cells functionally dependent on oncogenic K-ras may be resistant to chemotherapeutic agents in part due to loss of the pro-apoptotic function of PKCδ.