Integrin signaling links protein kinase Cepsilon to the protein kinase B/Akt survival pathway in recurrent prostate cancer cells.

Failure of hormone therapy often involves an outgrowth of protein kinase Cepsilon (PKCepsilon)-positive cells in recurrent prostate cancer. Our previous investigations have uncovered evidence of a complex signaling network operating downstream of this oncogenic protein kinase to actively advance the survival and proliferation of prostate cancer cells. In this study, we present evidence of a functional interplay among integrin receptors, PKCepsilon, and protein kinase B (PKB/Akt) in recurrent CWR-R1 prostate cancer cells. Flow cytometry and confocal microscopy provided evidence that PKCepsilon signaling promoted the assembly of matrix adhesions containing an abundance of colocalized actin filaments and beta1 integrins that exhibited an exposed activation epitope on the surface of live CWR-R1 cells. Reciprocal coimmunoprecipitations provided evidence of signaling complexes containing PKCepsilon, beta1 integrins, Src, and PKB/Akt in CWR-R1 cell cultures. An investigation into the functional significance of these interactions, and of their positive influence on beta1 integrins, demonstrated that PKCepsilon and several key components of the PKB/Akt signaling pathway remain constitutively phosphorylated/activated in adherent but not suspension cultures of PTEN-positive CWR-R1 cells. Gene transfer, antisense and pharmacological experiments provided additional support for the hypothesis that a mutually reinforcing signaling loop sustains the activation of beta1 integrins, PKCepsilon, and PKB/Akt in adherent prostate cancer cells.

Protein kinase Cepsilon interacts with Bax and promotes survival of human prostate cancer cells.

Prostatic glandular epithelial cells express protein kinase Cepsilon (PKCepsilon ), an oncoprotein that coordinately disrupts the reactivation of the tumor suppressor Rb, derepressess transcriptional elongation of the c-myc oncogene, and propagates survival signals in LNCaP cells. Since the activation of such a program may contribute to the progression of human prostate cancer, a proteomic analysis was performed to gain a more global perspective on the signaling network that PKCepsilon might be capable of engaging in prostate cancer cells. Using CWR22 xenografts, we identified at least 18 different structural, signaling, and stress-related proteins that associated with PKCepsilon, including an interaction with the proapoptotic protein Bax that was novel to recurrent CWR22 tumors. An investigation into the biological significance of the PKCepsilon association with Bax provided the first evidence of an inverse relationship between endogenous levels of PKCepsilon and susceptibility of prostate cancer cells to the apoptotic effects of phorbol esters. Western blot and antisense experiments demonstrated that CWR-R1 cells expressed moderate levels of PKCepsilon and relied on this protein to survive in the presence of phorbol esters, while the apoptosis normally induced by phorbol esters in PKCepsilon -deficient LNCaP cells was dependent on the presence of Bax. Forced expression of PKCepsilon in LNCaP cells was sufficient to confer a significant resistance to phorbol esters and this resistance was associated with an inhibition of phorbol ester-induced Bax conformational rearrangements that are important for Bax oligomerization, mitochondrial integration, and cytochrome c release. Considered in their entirety, our data suggest that an association of PKCepsilon with Bax may neutralize apoptotic signals propagated through a mitochondrial death-signaling pathway.

Manipulating expression of endogenous oncogenic proteins using an antisense oligonucleotide approach in prostate cancer cells.

It has been shown that antisense oligodeoxynucleotide (ODN) treatments provide an effective, specific approach to inhibiting the function of target proteins. Using this method, we have acquired additional evidence that protein kinase C-epsilon functions as an oncogenic protein in the progression of recurrent human prostate cancer. This chapter describes the use of antisense ODN to directly target cellular protein kinase C-epsilon as a potential chemotherapeutic agent for blocking the advance of prostatic adenocarcinoma to androgen-independence. Using Lipofectin as the carrier, phosphorothioate-modified antisense ODNs were transferred into prostate cancer cells with high efficiency, effectively inhibiting the expression of endogenous protein kinase C-epsilon and the androgen-independent (AI) proliferation of several independent human prostate cancer cell lines.

Regulation of tumor invasion and metastasis in protein kinase C epsilon-transformed NIH3T3 fibroblasts.

Protein kinase C epsilon is an oncogenic, actin nucleating protein that coordinately regulates changes in cell growth and shape. Cells constitutively expressing PKCepsilon spontaneously acquire a polarized morphology and extend long cellular membrane protrusions. Here we report that the regulatory C1 domain of PKCepsilon contains an actin binding site that is essential for the formation of elongate invadopodial-like structures, increased pericellular metalloproteinase activity, in vitro invasion of a Matrigel barrier, and the invasion and metastasis of tumors grown in vivo by PKCepsilon-transformed NIH3T3 fibroblasts in nude mice. While removing this small actin binding motif caused a dramatic reversion of tumor invasion, the deletion mutant of PKCepsilon remained oncogenic and tumorigenic in this experimental system. We propose that PKCepsilon directly interacts with actin to stimulate polymerization and the extension of membrane protrusions that transformed NIH3T3 cells use in vivo to penetrate and degrade surrounding tissue boundaries.

Protein kinase cepsilon has the potential to advance the recurrence of human prostate cancer.

Prostatic epithelial cells that are capable of surviving in the absence of androgenic steroids were found to express protein kinase Cepsilon (PKCepsilon), an oncogenic protein capable of promoting autocrine cell-signaling events. Gene transfer experiments demonstrated that PKCepsilon overexpression was sufficient to transform androgen-dependent LNCaP cells into an androgen-independent variant that rapidly initiated tumor growth in vivo in both intact and castrated male nude mice. This transformation was associated with an accelerated rate of androgen-independent LNCaP cell proliferation, resistance to apoptosis, hyperphosphorylation of the mitogen-activated protein kinase extracellular signal-regulated kinase and transcriptional repressor protein retinoblastoma, and increased expression of E2F-1 and other 5'-cap-dependent mRNAs, including the G(1) cyclins, c-myc, and caveolin-1. Coimmunoprecipitation experiments indicated that PKCepsilon was associated with members of the extracellular signal-regulated kinase signaling cascade and the scaffolding protein caveolin-1. Caveolin-1, produced by LNCaP cells overexpressing PKCepsilon, was released into the medium, possibly through a Golgi-independent route, and significant growth inhibition was observed when these cells were cultured in the presence of an anti-caveolin-1 antiserum. Finally, antisense experiments established that endogenous PKCepsilon plays an important role in regulating the growth and survival of androgen-independent prostate cancer cells. This study provides several independent lines of evidence supporting the hypothesis that PKCepsilon expression may be sufficient to maintain prostate cancer growth and survival after androgen ablation.