It is widely accepted that p16 acts as a tumor suppressor by inhibiting Rb phosphorylation and inducing cell cycle arrest in the G1 phase in response to potentially genotoxic stimuli (Alcorta et al., 1996
). This p16-mediated cell cycle arrest allows time for repair of DNA damage prior to replication, thereby reducing the chance of propagating mutations (). However, it is unknown whether other mechanisms exist by which p16 may protect against tumor formation. Here we show that in addition to UV irradiation, exogenous oxidative stress rapidly upregulates expression of p16. We present evidence that p16 deficiency leads to dysregulation of intracellular ROS in multiple cell types and accumulation of oxidative DNA damage, and that p16 is both necessary and sufficient for normal regulation of p38 SAPK signaling and intracellular oxidative status. Our findings further suggest that compromise of p16 may allow cells to progress to S phase bearing oxidative DNA lesions that may result in carcinogenic mutations (). Our results indicate that p16-mediated regulation of intracellular oxidative stress appears to be independent of the Rb pathway, and not secondary to potential cell cycle effects, thus confirming a novel function for p16.
Figure 6 Potential tumor-suppressive functions of p16. In the canonical pathway, p16 mediates cell cycle arrest as part of the DNA damage response pathway, allowing time for DNA repair enzymes to correct potentially oncogenic mutations or avoiding transformation (more ...)
It is unknown why compromise of p16 is more commonly associated with melanoma over other cancers. While we demonstrate here that p16 is necessary for proper oxidative regulation in multiple cell types, our data also indicate that melanocytes maintain higher levels of intracellular ROS and incur greater oxidative DNA damage in response to exogenous oxidative stress than other cell types (). This finding also holds true in the context of p16 depletion when different skin cell types were matched by donor and analyzed for intracellular ROS (). This inherent predisposition to oxidative damage may underlie an increased susceptibility of melanocytes over other cell types to transformation in the setting of p16 deficiency. The basis for higher basal levels of oxidative stress in melanocytes may relate to their synthesis of melanin pigment, which is distributed to adjacent keratinocytes for the purpose of absorbing photons and scavenging free radicals (Riley, 1997
). While UV exposure stimulates melanocytes to proliferate and produce melanin that can absorb UV-generated ROS (Gilchrest et al., 1996
), higher UV doses can oxidize melanin and increase ROS production (Wood et al., 2006
), which may further increase oxidative stress in melanocytes (Urabe et al., 1994
). Moreover, under conditions of increased oxidative stress, there is less efficient repair of 8-OG lesions (Eiberger et al., 2008
). Consistent with this notion, one recent study has shown that melanocytes are deficient (compared to fibroblasts) in repair of oxidative DNA damage (Wang et al.
It has been suggested that loss of p16 in melanocytes may lead to failure of senescence which underlies malignant transformation of melanocytic nevi () (Mooi et al., 2006
). Oncogene activation in melanocytes also generates oxidative stress, and increased ROS and p16 expression have been implicated in oncogene-induced melanocyte senescence (Leikam et al., 2008
). Consistent with this notion, it has been shown that several melanoma-associated p16 mutants lack the capacity to induce senescence (Haferkamp et al., 2008
), although p16 is not consistently expressed in melanocytes of senescent nevi in vivo
(Gray-Schopfer et al., 2006
) and more recent studies indicate that oncogene-induced senescence does not require p16 or p14ARF
(Dhomen et al., 2009
; Haferkamp et al., 2009
Our data showing negative control of ROS by p16 may seem at odds with these (Leikam et al., 2008
) and other reports in the literature, suggesting that the relationship between p16 levels and oxidative stress may be highly dependent on the circumstances and cell system utilized. For example, it was reported that over-expression of p16 increases cellular ROS levels in a human diploid fibroblast line (TIG-3) and p16 knockdown decreases ROS in a conditionally immortalized human fibroblast line (SVts8) (Takahashi et al., 2006
). Another study using EJ human carcinoma cells found that while tet-regulated over-expression of p21 elevated cellular ROS, induction of p16 over-expression had no discernable effect on ROS (Macip et al., 2002
). By contrast, our studies utilized siRNAi to knockdown p16 in primary normal human cells expressing wild-type p16, or lentivirus to express p16 in freshly-isolated mouse fibroblasts that were genetically deficient in p16. These various findings suggest that regulation of ROS by p16 in melanocytes and melanoma cells may be context-specific, and could reflect differences between cell types, or the immortalized or senescent state of the cells. Nevertheless, it seems plausible from our findings that p16 may act to suppress tumorigenesis through two inter-related pathways (): first mediating cell cycle arrest to facilitate repair of DNA damage, and second, to control accumulation of ROS that may cause oxidative DNA damage. Moreover, melanocytes may be more perturbed by oxidative dysregulation induced by compromise of p16 than other cell types, leading to increased susceptibility to melanoma over other cancers.
The precise mechanism by which p16 deficiency increases intracellular ROS in our system remains to be elucidated. Several recent reports also describe possible novel tumor-suppressive roles of p16 that are independent of its role in cell cycle control. For example, several p16 mutants responsible for inherited melanoma susceptibility in humans retain robust CDK4-binding capacity (Becker et al., 2001
). It has been reported that p16 interacts with brahma-related gene 1 (BRG1), a chromatin remodeling factor (Bochar et al., 2000
) whose expression is frequently lost in primary and metastatic melanomas (Becker et al., 2009
). Given the myriad of possible transcriptional targets of the BRG1-p16 complex, it is possible that p16 may regulate cellular oxidative stress through this newly discovered interaction. It has also been reported that p16 can bind to c-Jun N-terminal kinase (JNK) 3, thereby blocking JNK-mediated phosphorylation of c-Jun following UV exposure and activation of the Ras-JNK-Jun-AP-1 signaling cascade (Choi et al., 2005
). Therefore it is possible that loss of p16 removes an important inhibitor of Ras-JNK-Jun-AP-1 signaling (and cellular transformation), resulting in an upregulation of genes responsible for increased intracellular ROS. Interestingly, the residue Arg24 of p16 is thought to play a crucial role in the stabilization of the p16-JNK3 complex, and an Arg24Pro mutation in p16 co-segregates in nine melanoma-prone families (Becker et al., 2001
Taken together, our work presented here suggests that p16 may exert tumor-suppressive effects that extend beyond its known function as a cell cycle regulator. Further study of the mechanistic basis for oxidative regulation by p16 may shed further light on why loss of p16 commonly occurs in tumors, and why inherited p16 mutations predispose to melanoma susceptibility.