Protein Kinase C Modulates Bcl-2 Family Protein Levels in Cutaneous T Cell Lymphoma.

Abstract 2755

Poster Board II-731

Cutaneous T cell lymphomas (CTCL) represent a spectrum of several distinct extranodal non-Hodgkin's lymphomas and are characterized by an invasion of the skin by malignant, clonal CD4+ lymphocytes. Although current treatments may achieve remission, early chemotherapeutic treatment of CTCL has not demonstrated survival benefits and no treatments have proven to be curative. Our lab has previously demonstrated that treatment with the Protein Kinase C (PKC) inhibitor Enzastaurin increases apoptosis in CTCL which directly led to the clinical trial of Enzastaurin for CTCL. PKCs phosphorylate a number of substrates important in modulating cell survival, including Bcl-2 family members. To investigate the effects of PKC signaling on Bcl-2 family member levels in CTCL we treated cells with Enzastaurin and measured downstream effects. Inhibition of PKC signaling by Enzastaurin led to a decrease of Bcl-x_L protein levels but not Bcl-2 levels. Apoptosis was assessed by flow cytometry and cells cultured in Enzastaurin showed an increase in cell death which correlated with the decrease in anti-apoptotic Bcl-2 family member levels. Enzastaurin treatment also resulted in a decrease of both Bad and Bcl-2 phosphorylation as well as a decrease in Bcl-x_L but not Mcl-1 or Bcl-2 mRNA levels. To determine if PKC modulates Mcl-1 levels through phosphorylation and inactivation of GSK3β in CTCL, cells were exposed to Enzastaurin for 24 hours and immunoblots were performed for Mcl-1, total GSK3β and phosphor-Ser9 GSK3β. Enzastaurin blocked basal phosphorylation as well as TPA-induced phosphorylation of GSK3β. However, Mcl-1 levels were not affected by Enzastaurin. This suggests that PKC modulates GSK3β activity but that this is independent of Mcl-1. To determine if PKC negative regulation of GSK3β inhibits apoptosis we treated cells with Enzastaurin and the GSK3β inhibitor ARA014418. Surprisingly, the addition of the GSK3β inhibitor did not reverse the effects of Enzastaurin but instead increased apoptosis. This suggests that PKC does not deliver survival signals in CTCL by negatively regulating GSK3β. Treatment with Enzastaurin alone as well as treatment with both Enzastaurin and GSK3β inhibitors resulted in an upregulation of β catenin. However, a higher percentage of β catenin was unphosphorylated when both Enzastaurin and the GSK3β inhibitor were added. Interestingly, the combination of both inhibitors resulted in a dramatic increase in β catenin / TCF transcription as measured by TOPGLOW luciferase assay.

The downregulation of Bcl-2 family member levels by PKC inhibition strongly suggests that PKC modulation of Bcl-2 family members is important for CTCL survival. PKC may be affecting Bcl-2 family members by modulating phosphorylation or by indirectly activating or repressing transcription. Additionally, β catenin upregulation may play a role in apoptosis mediated by inhibition of PKC. β catenin TCF/LEF complexes can activate transcription of several genes linked to apoptosis, including c-Jun, fra-1, p53 and Myc. β catenin may also be functioning to repress transcription of Bcl-2 family members such as Bcl-x_L. Elucidating the underlying mechanism will help to identify new drug targets for CTCL which may be applicable to a broad spectrum of malignancies.