We report for the first time that CDK5 or its activators are overexpressed in PDACs (, ) compared to normal pancreatic ducts (, ) (p<0.0005). Primary human PDACs exhibit frequent amplification of the genes encoding p35, p39 or CDK5. Each of these genes is individually amplified in about 33% of tumors, and collectively one or more of these genes is amplified in approximately 67% of tumor samples (, n=39). Results presented by Harada et al. (that did not address or discuss CDK5, p35, and p39 directly) are consistent with our results, except p39 was not as frequently amplified (28
). Overexpression of any of these (p35, p39, or CDK5) is predicted to result in enhanced activation of CDK5 kinase activity. This supports the hypothesis that activation of CDK5, in part through gene amplification and commensurate overexpression of CDK5, p35 or p39, contributes to the progression of pancreatic cancer. Our results also shed light on the types of cells within the pancreas that express CDK5, which is widely reported to be "ubiquitously" expressed in all organs. Within the pancreas, expression of CDK5 is highest in islets, but we find that there is little or only sporadic expression of CDK5, p35 or p39 in normal acinar or ductal cells in the pancreas.
The known contribution of CDK5 to neuronal migration and patterning (5
) suggested to us a link between CDK5 expression and perineural invasion (PNI), a prominent feature of PDACs. Sustained activation of phospho Erk 1/2 was achieved through stimulation by nerve growth factor (NGF) in rat pheochromocytoma PC12 cells, which resulted in Egr1 binding and activation of the p35 promoter and a subsequent increase of p35 transcript and protein (29
). NGF induced upregulation of p35 protein levels may explain in part the high p35 levels observed in PNI (, ), and we propose that the microenvironment around the nerves, where physiologically normal CDK5 expression is highest (30
), induces or selects for increased CDK5 and p39 levels in PDACs undergoing PNI.
We sought to determine if CDK5 activation contributed to pancreatic cancer progression in the context of known early transforming events (32
). Analysis of variants of HPNE, a normal pancreatic cell line immortalized with hTert, that was subsequently transduced with retroviruses expressing mutant active K-RasG12D (HPNE.kras) showed higher p35 or p39 mRNA and protein levels in response to activated K-Ras, while CDK5 levels remained constant in HPNE.kras cells (, data not shown). Differences in CDK5 expression between normal pancreas (, ) and immortalized but non-transformed HPNE cells () could be attributed to the expression of Nestin, which HPNE cells express, as Nestin expression has been correlated to CDK5 expression in differentiating myoblasts and in regenerating muscle tissue (33
). Alternatively, CDK5 may be expressed in pancreatic ductal progenitor cells, which would also explain CDK5 expression in HPNE cells, which are derived from progenitors of ductal cells (22
There were increased p25 levels in HPNE.kras cells, a stable cleavage product of p35 that is highly activating for CDK5 (). p25 lacks the N-terminal myristoylation sequence of p35, which causes relocalization from the cell membrane to cytoplasm and potentiates its capacity to bind and activate CDK5 (13
). p25 expression is likely responsible for increased CDK5 kinase activity in HPNE.kras cells compared to HPNE parental cells (). Similar to SH-SY5Y neuroblastoma cells (34
) and in contrast to normal neuronal cells (35
), we found that CDK5 kinase activity in the context of mutant K-Ras (HPNE.kras cells) increased p25 levels (), while inhibition of CDK5 kinase had no effect on p25 levels in the context of the additional oncogenic effects of E6/7 and small t antigen (). This supports the hypothesis that CDK5 hyperactivation contributes to tumor progression especially in the context of early mutant K-Ras expression. We show further that signaling through MEK and PI3K increased p25 expression (). This leads us to propose that signaling from activated mutant K-Ras (G12D) through MEK and PI3K enhance p25 expression, which in turn increases CDK5 kinase activity (, Supplemental Fig. 1
Our results complement and extend recently published findings by Feldman et al (36
), which showed that inhibition of CDK5 in pancreatic cancer cell lines by the dominant negative construct decreased signaling through the RalA and RalB pathways that are downstream of activated Ras, and that commensurate inhibition of MEK and PI3K pathways reinforced these effects. The Ral pathways were shown to contribute to anchorage independent growth and tumorigenicity of these tumor cells lines and their results suggest that these effects are downstream of CDK5. Our results are consistent with these findings and extend them by demonstrating that K-Ras signaling enhances the activity of CDK5 by increasing the steady state levels of p25 upstream of the Ral pathways.
The influence of mutant K-Ras and CDK5 kinase activity on migration was investigated in HPNE cells bearing selected mutations and transforming insults. CDK5 is necessary for proper neuronal cortical layering, a process that requires CDK5-mediated phosphorylation of FAK at S732 and probably PAK1 at T212 (6
). We found that increased CDK5 activity and p25 expression in HPNE cells expressing mutant K-Ras increased phosphorylation of S732 FAK and T212 PAK1 (). We propose that PDACs have appropriated these phosphorylation events to alter cell morphology and increase cellular migration. Inhibition of CDK5 with roscovitine or the CDK5 dominant negative construct enhanced spreading in HPNE.kras cells, as evidenced by a more flattened appearance with shorter cellular processes compared to the respective controls (), whereas there was no change in cellular morphology in parental HPNE cells with CDK5 inhibition (). Our results support the proposal that CDK5 has a role in modulating cell morphology and cytoskeletal reorganization that has been presented by Mao and Hinds and others (37
), even though the precise morphological findings are not entirely congruent because they were obtained in different cell types and experimental systems that were evaluating morphological changes associated with senescence, which were not investigated in our experimental system.
Along with increased p25 expression and CDK5 kinase activity, mutant K-Ras increased migration of HPNE.kras and HPNE.kras.E6/7.St cells as compared to HPNE.E6/7.St cells lacking mutant K-Ras (). The increase of migration in the HPNE.kras.E6/7.St cells relative to HPNE.kras cells suggested that the transforming insults introduced by E6/7 and small t antigen had little effect on migration (). Inhibition of CDK5 kinase activity significantly reduced migration of HPNE cells expressing mutant K-Ras (HPNE.kras and HPNE.kras.E6/7.St cells) while it had little effect on cells lacking mutant K-Ras (HPNE.E6/7.St cells) (). Inhibiting CDK5 kinase activity with a CDK5 dominant negative construct or roscovitine also significantly decreased migration of S2-013 cells ().
CDK5-inhibition with roscovitine reduced invasion by 37–60% in S2-013, tHPNE, FG, HPAF2, and T3M4 PDAC cell lines (). These results are consistent with previous findings in neuroblastoma, prostate cancer and other pancreatic cancer cell lines, where inhibition of CDK5 kinase activity reduced invasion in vitro
In summary, we demonstrate that CDK5 or its activators p35 and p39 are over-expressed in >90% PDACs () compared to normal pancreases (). CDK5, p35, and p39 expression in PDACs can be attributed in part to genomic amplification, as CDK5, p35, or p39 were amplified in 67% (n=39) of tumors analyzed (). Furthermore, CDK5 is hyperactivated downstream of mutant K-Ras signaling, resulting in increased phosphorylation of CDK5 substrates, increased p35 mRNA and protein expression, increased p35 cleavage to p25 (which is dependent on MEK, PI3K, Calpain, and CDK5 signaling), decreased cell spreading, and increased cell migration (–5, S. Fig. 1
, data not shown). We confirm prior reports that CDK5 inhibition can decrease migration and invasion in vitro
CDK5 represents a novel and unexplored therapeutic target for both early and late stage PDACs, and should be investigated in other solid tumors that harbor K-Ras mutations, including those of lung and colon. R-Roscovitine (Selicilib) has been investigated in phase I clinical trials, with minimal toxicity at lower doses, supporting the feasibility of using a CDK5 inhibitor in the clinic (39
). More potent CDK5 kinase inhibitors have been developed as potential therapies for Alzheimer’s Disease, in an attempt to downregulate hyperactive CDK5 kinase activity associated with p25 expression that is observed in that disease (15
). We are currently investigating the use of such inhibitors in xenograft and other animal models of pancreatic cancer. It is also possible that CDK5 inhibitors may reduce pain in pancreatic cancer patients with PNI. CDK5 has previously been demonstrated to reduce thermal afferent nociceptive pain signaling (40
). Type II Diabetes Mellitus (T2DM) is observed in the majority of PDACs (41
), and increased CDK5 kinase activity is known to increase glucotoxicity and independently decrease insulin synthesis and secretion (42
). Thus, inhibiting CDK5 kinase activity with molecular inhibitors may also improve the associated T2DM in PDAC.