These results implicate PKCα as a key mediator of SCCHN proliferation through activation of MAPK and negative regulation of miR-15-a
, an inhibitor of cyclin E expression, leading to increased synthesis of cell cycle proteins and enhanced DNA synthesis. Our findings fit a coherent type 4 feed-forward loop (FFL) network (28
) (). The FFL is initiated by PKCα with one arm comprising a series of activating reactions and the second arm consisting in part of two successive inhibitory reactions that together promote cyclin E expression and DNA synthesis. In the present case, PKCα activates a “driver”, MAPK, as well as releasing a “brake”, miR-15a
(). The key characteristics of FFLs are a delay in the activation of the system upon stimulation as well as a rapid shut-off upon loss of the stimulus. Both of these features represent key elements that ensure the integrity of DNA synthesis by enabling initiation only when two input conditions are satisfied, and causing rapid cessation when either input is lost. However, in cancer cells, the stimulus is constitutively activated so that there is no initial delay in the activation of the cell cycle. It is likely that this type of coherent type 4 FFL network, consisting of a stimulus that activates a positive signal and removes a negative brake, are general characteristics of cell cycle progression in both normal and tumor cells.
Figure 6 Schema illustrating a feed-forward loop initiated by PKCα that regulates DNA synthesis. A) Illustration of a typical feed-forward loop with X as the initiation signal that stimulates Y activation rapidly and Z activation slowly. Y also stimulates (more ...)
Since both PKCα and cyclin E have been implicated in diverse cancers, these findings could have broader relevance. The mechanism described for upregulating cyclin E involves inhibition of miR-15-a
by activated PKCα. miR-15-a
are located in the 13q14 locus, deleted in the majority of chronic lymphocytic leukemias, consistent with tumor suppressor function (29
). Further evidence demonstrated that miR-15a
negatively regulates Bcl-2 and thus induces apoptosis in a leukemic cell line (30
). In pituitary adenomas, suppression of miR-15a
has been associated with tumor growth (31
). However, in endocrine pancreatic tumors the same miRs appear to be overexpressed underscoring the likelihood that their role is tissue specific (32
). A functional screen of the miR16
family that includes miR-15a
in HCT116, HeLa, and TOV21G cell lines demonstrated that these miRs negatively regulate cell cycle progression (11
). The miR16
family in these cells functioned primarily in feedback regulation, and its inhibition of DNA synthesis was mediated by its cumulative effect on multiple cell cycle targets. By contrast, we demonstrate here that, in SCCHN, miR-15a
regulation of cyclin E expression is sufficient to account for the negative regulation of DNA synthesis by PKCα. Our results suggest that miR-15a
is a positive prognostic marker based on its relationship with PKCα expression and may function as a tumor suppressor that negatively regulates cell proliferation.
It is noteworthy that inhibition of PKCα with siRNA did not mimic all the effects of Gö6976 in vitro
suggesting that other PKCs, such as PKCε, could also play a role in proliferation. Thus, the in vivo
effects observed in response to Gö6976 could result in part from inhibition of other PKC isoforms in tumor cells, mouse stromal tissue or immune cells. PKCβ has been implicated as a mediator of angiogenesis through inhibition of GSK3β (33
) but a recent study using Gö6976 and other PKC inhibitors concluded that PKCα rather than PKCβ promotes angiogenesis (34
). PKCs also regulate immune cell function (35
); thus, the contribution of specific isoforms with respect to tumor microenvironment and immune system needs to be further elucidated.
PKC has been shown to regulate cyclins or E2Fs in other cell types but the outcomes can differ depending upon the specific system. For example, overexpression or TPA activation of PKCα in late G1 phase was shown to inhibit rather than activate E2F activity and DNA synthesis in rat 3Y1 fibroblasts (38
). Conversely, constitutively activated PKCα enhances both cyclins D and E promoter activity in NIH3T3 cells (39
). In human keratinocytes, activation of PKCα has been implicated in cell cycle arrest or terminal differentiation (40
) while in some cancers PKCα expression is decreased or possesses tumor suppressor function (3
). This is not necessarily contradictory to our findings since similar mechanisms may be utilized in other tissues but other pathways may also be present that counteract the effect of PKCα or function as feedback regulators. In SQ20B cells, cyclin E gene expression and proliferation was significantly altered by PKCα. It is also important to note that the cell lines we characterized were derived from rapidly proliferating, radiation-resistant, highly EGFR expressing tumors, and the signaling cascade elucidated may not be characteristic of less aggressive SCCHN cancers.
Previous work from our laboratory showed that EGF-stimulated PKCζ regulates SCCHN DNA synthesis by contributing to Raf-1/MAPK activation (2
). Numerous studies have shown that other PKC isoforms such as PKCα also stimulate Raf-1 activation. Here we demonstrate a complementary mechanism for the oncogenic effects of PKCα, namely, stimulation of MAPK together with inhibition of miR-15a
, leading to upregulation of cyclin E synthesis. However, unlike PKCζ PKCα is activated independently of the EGF receptor (data not shown). The mechanism leading to constitutive
activation and overexpression in tumors remains to be determined.
Our results highlighted cyclin E as the key target of PKCα regulation. Cyclin E was able to substantially rescue the inhibition of DNA synthesis upon PKCα kinase inactivation in our cells. In addition to its role in complex with cdks, cyclin E was recently found to be required for loading the MCM replicative helicase onto replicative origins (replicative licensing) and for transformation by Ras in a manner that is independent of cdks (43
). Thus, cyclin E performs dual functions that are both cdk-dependent and cdk-independent, and loss of cyclin E should rapidly prevent DNA synthesis.
Cyclin E overexpression or deregulation has been associated with a number of highly aggressive tumors with poor prognosis but the mechanisms that have been described differ from the one elucidated here (reviewed in (44
)). In laryngeal squamous cell carcinomas (LSCC), cyclin E overexpression alone was a prognostic marker for early stage LSCC, and tumors with a combination of high cyclin E and PCNA expression yielded the poorest prognoses (45
). Cyclin E overexpression in tumors has been attributed to a number of mechanisms including loss or mutation of ubiquitin ligases, decreasing the rate of degradation, cyclin E gene amplification, and mutation of oncogenes upstream of the cyclin D-Rb-E2F pathway that normally regulate cell cycle progression (44
). Our results contribute an additional mechanism for cyclin E overexpression involving regulation of synthesis by PKCα via mir-15a
This study is the first demonstration that PKCα is a mediator of SCCHN proliferation and a marker of progression and prognosis. Our results indicate that PKCα is a primary driver of the cancer phenotype that promotes SCCHN development and thus represents a novel therapeutic target. These results carry important clinical implications by providing a rationale for the advancement of PKC inhibitors that are currently being developed or investigated through clinical trials. Taken together, this evidence suggests that PKCα inhibition will yield efficacy in a variety of cancers.