Simultaneous knockdown of BRAF and expression of INK4A in melanoma cells leads to potent growth inhibition and apoptosis

Simultaneous Knockdown of Mutant BRAF and Expression of INK4A in Melanoma Cells Leads to Potent Growth Inhibition and Apoptosis

“…Therefore, the enhanced apoptosis and decreased proliferation by simultaneously inhibiting ERK and RB pathways could result from the double hitting of ERK-cyclin D:CDK4-RB that regulate cell cycle progression and cell survival. Alternatively, in support of our previous results that BRAF and INK4A have a non-linear functional interaction (Rotolo et al, 2005;Zhao et al, 2008), additional cellular processes could be affected when cells are exposed to both PD98059 and 219476. ERK pathway has pleiopotent activities that regulate cell proliferation, survival, and differentiation through both cyclin D:CDK4 dependent and independent routes (Fecher et al, 2008;Panka et al, 2006a;Sekulic et al, 2008).…”

“…5E). The expression of wild-type INK4A can be restored in melanoma cells by exogenous expression of INK4A cDNA (Rotolo et al, 2005;Zhao et al, 2008). As shown in Fig.…”

“…The transfection efficiencies in 624Mel cells are approximately 30-50%, and the effects of transfection are assessed on unselected mass cultures. The RAS-RAF-MEK-ERK signaling pathway is viewed as upstream of the cyclin D-CDK4/6-p16-RB cascade; consistently, only melanomas with wild-type NRAS/BRAF have amplified CDK4 and cyclin D1 genes (Curtin et al, 2005 (Zhao et al, 2008) and colony formation (Fig. 9B).…”

“…A straightforward explanation for the observed apoptosis is that changes in the pro-and anti-apoptotic factors offset the balance and lead to apoptosis (Adams & Cory, 2007). Sequencing analysis of TP53 cDNA (Zhao et al, 2008) showed that 624Mel and OM431cells harbor a T1076G (Cys275Trp) and a G1048A (Gly266Glu) mutations, respectively, in the DNA binding domain that is likely to compromise the transcriptional and apoptotic function of p53 (Petitjean et al, 2007). No TP53 mutation has been detected in A101D cells.…”

“…We have demonstrated recently that expression of HERV-K correlates with ERK activation and p16 loss in human melanoma specimens, and that inhibition of MEK and CDK4, especially in combination, suppresses HERV-K expression (Li et al, 2010b), which suggests that HERV-K may mediate BRAF-MEK-ERK and p16-CDK4-RB signaling pathways in melanoma cells. It is interesting to note that the growth characteristics of melanoma cells that can be modified by HERV-K activation (e.g., changes in cell shape, loss of melanin, anchorageindependent growth) (Serafino et al, 2009) overlap with those that can be blocked by suppression of the BRAF-MEK-ERK signaling pathway, especially with simultaneous restoration of p16 or inhibition of CDK4 (Li et al, 2010a;Rotolo et al, 2005;Zhao et al, 2008). This observation, together with the knowledge that aberrations in BRAF-MEK-ERK and p16-CDK4 pathways are early events and often co-exist during melanomagenesis, and the evidence that the RAF-MEK-ERK signaling pathway is required for the completion of HIV-1 reverse transcription (Mettling et al, 2008), prompted us to hypothesize that HERV-K is regulated by BRAF-MEK-ERK and p16-CDK4 pathways.…”