In the current manuscript, we present data arguing that protein stability is a major factor contributing to the elevated levels of C-MYC in melanoma cells. Furthermore, we demonstrate that among all known regulators of C-MYC stability, the expression of two proteins that regulate PP2A activity against C-MYC (CIP2A and PP2A-B56α) were markedly different between NHM and melanoma cells. Therefore, it is quite remarkable that despite high genetic variability of melanoma cells (Smalley et al., 2005
), C-MYC was similarly up-regulated in cells from all studied melanoma lines and that the expression of two proteins regulating a single post-translational modification of C-MYC (phosphorylation of Ser62
) was also respectively uniformed among these lines. In principal, this uniformity could be due to another feature common for all studied melanoma cells: the activated state of MEK-ERK pathway. Because Ser62
is a target of ERK (Sears et al, 1999
), phosphorylation of Ser62
can be a rheostat for the opposing activities of ERK and PP2A.
Activation of BRAF, NRAS or HRAS plays a controversial role in melanomagenesis. On the one hand, constitutive activity of these oncoproteins is essential for sustained proliferation of melanoma cells which requires high levels of C-MYC (Zhuang et al., 2008
; Welcker et al., 2004
). On the other hand, these oncogenes cause OIS in NHM accompanied by down-regulation of C-MYC (Zhuang et al., 2008
). Intriguingly, BRAFV600E
stimulates proliferation shortly after delivery into NHM and suppresses it at later time points (Denoyelle et al., 2006
; Zhuang et al., 2008
). Our previous and current data support the model that suppression of C-MYC is responsible for the transition from proliferation-promoting to proliferation-inhibitory activities of BRAFV600E
and possibly activated RAS proteins.
We report that overexpression of PP2A-B56α suppressed C-MYC protein rather than mRNA levels in cells from two out of four examined melanoma lines. In the other two lines (SK-Mel-19 and SK-Mel-103), PP2A-B56α overexpression led to 50% and 77% reduction in C-MYC
mRNA, respectively. This phenomenon may be due to the reported ability of PP2A-B56α to facilitate degradation of β-catenin (Arnold et al., 2009
; Hart et al., 1998
), a mediator of Wnt-pathway-dependent up-regulation of C-MYC
transcription (He et al., 1998
). Because the Wnt pathway is often activated in melanoma cells (Larue and Delmas, 2006
) including SK-Mel-103 (D.Z. and M.A.N. unpublished observations), the presumed degradation of β-catenin via PP2A-B56α in these cells may affect C-MYC
PP2A-B56α-dependent depletion of C-MYC in studied melanoma cells resulted in the emergence of senescence phenotypes similar to those caused by depletion of C-MYC via shRNA. However, the timing of senescence varied substantially: 5–6 days post-infection with PP2A-B56α in SK-Mel-19, -29 cells versus 14–20 days post-infection in SK-Mel-103, -147 cells. In the latter group, the amounts of C-MYC did not change until prior to the emergence of senescence phenotypes (data not shown), indicating that ectopic PP2A-B56α in these cells did not suppress C-MYC amounts during a long period of time. One possible explanation for this observation is that unlike BRAFV600E-expressing SK-Mel-19 and SK-Mel-29 cells that contain activated ERK (), SK-Mel-103 and SK-Mel-147 cells bear NRASQ61R and, therefore possess both activated ERK and suppressed GSK3β. This leads to stabilization of C-MYC by increased phosporylation of its Ser62 and hypo-phosphorylation of its Thr58 Therefore, it is conceivable that destabilization of C-MYC in SK-Mel-103 and SK-Mel-147 cells via PP2A-mediated de-phosphorylation of Ser62 is hampered or delayed compared to that in SK-Mel-19 and -29 cells.
Depletion of PP2A-B56α led to the up-regulation of endogenous C-MYC in NHM expressing BRAFV600E
. Yet, C-MYC increase was sufficient to effectively suppress senescence phenotypes caused only by BRAFV600E
. OIS induced by NRASQ61R
was inhibited only partially, whereas HRASG12V
-dependent senescence phenotypes were unaffected by upregulation of C-MYC. These data are concordant with our previous observations that ectopic expression of C-MYC much more efficiently overcomes OIS caused by BRAFV600E
than by NRASQ61R
(Zhuang et al., 2008
). Unlike BRAFV600E
and, to much lesser extent, NRASQ61R
induce the unfolded protein response (UPR) in NHM (Denoyelle et al., 2006
). In our experience, C-MYC overexpression in NHM does not suppress the UPR pathway (Zhuang et al., 2008
), which could account for the inability of PP2A-B56α depletion and subsequent C-MYC up-regulation to overcome HRASG12V
-induced senescence in these cells.
In summary, we demonstrated that increased protein stability contributes substantially to the elevated levels of C-MYC in melanoma cells. We showed that PP2A-B56α can account for differential C-MYC expression between NHM and melanoma cells. Lastly, we demonstrated the functional importance of PP2A-B56α downregulation for both the maintenance of high levels of C-MYC and the suppression of oncogene-induced senescence in NHM and melanoma cells.