The study described here represents the first report on the involvement of the PCPH protein and the mt-PCPH oncoprotein in determining the chemoresistant response of human PCa. Using sh-RNA-mediated gene expression knockdown, ectopic protein expression and re-expression studies, our results, which are consistent among PCa-derived cell lines manipulated to express different levels of PCPH or mtPCPH, show that expression of PCPH and, particularly, of mt-PCPH in PCa cells antagonizes the Cp-induced apoptotic process by enhancing the activating phosphorylation of PKCα at Thr638 and increasing the total expression and phosphorylated levels of Bcl-2. This anti-apoptotic effects ultimately rendered PCa cells resistant to apoptotic cell death, thus favoring tumor cell survival and malignant proliferation. Results reported here identify a new pathway [PCPH/mt-PCPH → PKCα → Bcl-2] that, taking into consideration that PCPH/mt-PCPH expression increases at the PIN stage and is maintained at high levels in malignant PCa, may likely contribute to the acquisition of chemoresistance by PCa cells during tumor progression. Indeed, the same [PCPH/mt-PCPH → PKCα → Bcl-2] pathway was also found to be functional in the Cp response of C4-2 cells, a more metastatic LNCaP-derived line that expresses both PCPH and mt-PCPH (Suppl. Fig. 3
), and PCPH knockdown also sensitized CWR22Rv1 PCa cells, which only express PCPH, to Cp treatment (data not shown). We propose a mechanistic paradigm for the sequence of events that, in the presence of PCPH or, specially, of mt-PCPH, culminates in the acquisition by PCa cells of resistance to Cp-induced apoptosis. The model () takes into account two scenarios: 1) in the absence of PCPH or mt-PCPH, phosphorylation of Bcl-2 at Ser70 by PKCα protects Bcl-2 from proteasome-mediated degradation; thereby creating an anti-apoptotic environment; this setting is subverted when exposure to Cp promotes PKCα inactivation and, consequently, decreases the levels of phosphoSer70 Bcl-2, thus favoring the proteasome-mediated degradation of non-phosphorylated Bcl-2 and ultimately resulting in the apoptotic death of the cells; and 2) the expression of PCPH or, more efficiently, of mt-PCPH prevents the Cp-induced dephosphorylation of PKCα at Thr638, maintaining it in an activated state that phosphorylates Bcl-2 at Ser70 and prevents its degradation, eventually leading to increased phosphorylated Bcl-2 levels that protect PCa cells against apoptosis.
Model proposed for the mechanism of promotion of resistance to cisplatin-induced apoptosis by PCPH and mt-PCPH
The significance of PKC activation in the cellular response to apoptosis-inducing stimuli, including Cp, has long been recognized (31
). Although there are still controversies regarding how PKCs influence apoptosis, the general consensus is that the anti- or pro-apoptotic function of individual PKC isoforms is regulated through a spatiotemporally coordinated cascade of PKC activation (39
) that is differentially triggered by diverse stimuli and is also dependent on the distinct isoform repertoires found in different cellular contexts. This complex interplay among PKC isoforms leads to the establishment of unique and redundant response pathways, which determine the outcome of the cellular exposure to different apoptosis-inducing agents (40
). Most of what is known on the role of individual PKC isoforms in the regulation of apoptosis in human PCa cells derives from mechanistic studies about the cell death-inducing activity of phorbol esters, a group of carcinogenesis promoters and well characterized PKC activators (31
). Results from several laboratories demonstrated that, in response to phorbol esters, PKCε preferentially mediates survival signaling, that PKCα and PKCδ are pro-apoptotic kinases, and that PKCδ is essential for the apoptosis process, whereas PKCα and PKCε are not so stringently required and may have redundant functions under certain conditions (41
). Unfortunately, there is little information available on the expression of different PKC isoforms in tumor specimens from PCa patients (16
), thus making it difficult to translate these observations to the physiology of PCa and their response to treatment.
The mechanism of Cp-induced apoptosis in PCa cells is not so well characterized, and there is little information on the involvement of individual PKC isoforms. Our findings demonstrate that the apoptotic process triggered by Cp in PCa cells is quite different from that elicited by phorbol esters. In contrast to the latter case, PKCδ is dispensable for Cp-induced apoptosis (Suppl. Figs. 1
), and Cp causes the down-regulation, rather than the increase, of the levels of active, Thr638-phosphorylated PKCα (). The fact that, similar to PCPH/mt-PCPH knockdown, PKCα knockdown also sensitizes LNCaP cells to Cp () provides strong evidence in support of the existence of a functional interaction between PCPH and PKCα. Moreover, the fact that PKCα knockdown also efficiently sensitized PC-3 cells, which are androgen independent, to Cp () strongly suggests that such PCPH-PKCα interaction influences the response of PCa cells to Cp irrespective of their androgen responsiveness status. Our results also show a clear role for PCPH and, especially, for mt-PCPH in preventing the down-regulation of Thr638-phosphorylated PKCα () and, consequently, providing resistance to Cp-induced apoptosis (). Whether PCPH and mt-PCPH expression prevent the dephosphorylation of pre-existing Thr638-phosphorylated PKCα or somehow stimulate the Thr638-phosphorylation process remains to be elucidated. The involvement of PKCα rather than PKCδ in the response of PCa to Cp-induced apoptosis is likely a cell type-specific characteristic, as PKCδ has been reported to be the major determinant of the response to Cp in other tumor cells (46
). Our data agree with reports indicating that chemical or ribozyme-mediated inhibition of PKCα sensitized PCa cells to Cp and other anticancer drugs (36
), and are also consistent with the fact that the expression of both PCPH/mt-PCPH (16
) and PKCα (45
) is elevated in PCa. Results from our experiments on the susceptibility to Cp-induced apoptosis of LNCaP and PC-3 cells in which Bcl-2 was knocked down, as well as those from studies on the effect of re-expressing Bcl-2 in PCPH knockdown LNCaP cells conclusively identify Bcl-2 as a mediator of the pro-survival function of PCPH and mt-PCPH. These findings are also consistent with current knowledge on the central role of Bcl-2 in determining the life-or-death outcome after apoptotic stimulation of PCa and other tumor cells (49
) and on the regulation by PKCα-mediated phosphorylation of the susceptibility of Bcl-2 to proteasome-dependent degradation (34
A direct comparison between the relative anti-apoptotic activities of PCPH and mt-PCPH was not possible in LNCaP cells because, although they express both PCPH and mt-PCPH, the single base pair difference between the normal and mutated mRNAs [6
] did not allow the selective knockdown of one or the other. In this regard, ectopic expression of PCPH or mt-PCPH in PC-3 cells, in which they are not endogenously expressed, became more informative. Our results on the response to Cp of PCPH- and mt-PCPH-expressing PC-3 cells agree with previous reports on the pro-survival function of PCPH and mt-PCPH that assigned a greater anti-apoptotic activity to the mt-PCPH oncoprotein than to the normal PCPH protein in the response to ionizing radiation (8
), as well as with our previous results from the characterization of a PCPH/mt-PCPH-inducible system developed to study their involvement in mechanisms of chemo-response of primary mouse embryo fibroblasts (Tirado et al
., unpublished data). Furthermore, although western immunoblotting is not a quantitative technique, it was consistently apparent that the expression levels of PCPH and mt-PCPH achieved in PC-3 cells were quite similar (16
), and this is consistent with previous estimates indicating that a substantially greater expression of PCPH was required to attain levels of pro-survival activity directly comparable to those induced by the expression of mt-PCPH [8
It would be extremely interesting to investigate whether PCPH and/or mt-PCPH may also play a role in the response of PCa to radiation, usually successful in the treatment of localized disease, or to chemotherapeutic agents currently under investigation such as docetaxel, which yields modest benefits to patients with disseminated PCa. The fact that in the same cellular system (cultured mouse embryo fibroblasts) PCPH/mt-PCPH conferred resistance to radiation by interacting with the mTOR pathway (8
) while modulating a different pathway to promote resistance to chemotherapeutic drugs (Tirado et al
., unpublished data) suggests that a similar involvement of PCPH/mt-PCPH with different signaling pathways in the response to different stress stimuli may also be the case in PCa cells. Experiments designed to test these possibilities are currently ongoing in our laboratory.
Finally, it is important to note that PCPH and mt-PCPH appeared to be able to functionally interact with two different PKC isoforms in PCa cells (through PKC-δ, to modulate their invasiveness (16
), and through PKC-α, to regulate their chemo-response), and that invasiveness and chemo-resistance are properties typically acquired during tumor progression to advanced PCa. Therefore, on the basis of the increased levels of PCPH along the process of malignant PCa progression reported previously (16
) and of the increased probability of accumulating mt-PCPH mutations during advanced PCa progression stages, it seems reasonable to propose that PCPH and mt-PCPH may be important contributors to the development of the malignant phenotype of PCa cells. In this context, targeted blockage of PCPH/mt-PCPH expression may be an effective strategy to sensitize PCa to therapeutic treatment and a useful approach to improve the treatment outcome for advanced PCa patients.