We previously reported that the upregulation of
mortalin, an Hsp70 family chaperone, is important for B-Raf(V600E)
tumor cells to bypass p21(CIP1) expression, which is activated as a
tumor-suppressive mechanism in response to aberrant
MEK/ERK activation (Wu et al., 2013). Interestingly,
mortalin depletion induced p21(CIP1) transcription not only in wild-type TP53 but also in TP53-mutated B-Raf(V600E)
cancer cells, suggesting the presence of an additional mechanism for p21(CIP1) regulation. In the present study, using
luciferase reporter truncation analysis in a TP53-mutated B-Raf(V600E)
cancer cell line, SK-MEL28, we identified a proximal p21(CIP1) promoter region responsive to
mortalin depletion. Interestingly, when Sp1-like cis-elements in this promoter region were mutagenized, the p21(CIP1) promoter
luciferase reporter was no longer responsive to
mortalin depletion. Consistent with this, our ChIP analysis revealed that
mortalin knockdown could induce Sp1 binding to p21(CIP1) promoter in a
MEK/ERK-dependent manner. Moreover, RNA interference of Sp1 substantially attenuated p21(CIP1) expression induced by
mortalin depletion in SK-MEL28 cells. Consistent with this observation in SK-MEL28 cells, Sp1 was necessary for the
tamoxifen-regulated ∆Raf-1:ER to induce p21(CIP1) transcription in U251 cells, in which TP53 is mutated. However, in contrast, Sp1 was not necessary for ∆Raf-1:ER to induce p21(CIP1) transcription in LNCaP cells, in which TP53 is wild type. These data suggest that Sp1 may address TP53-independent p21(CIP1) transcription in Raf/
MEK/ERK-activated
cancer cells and that its requirement in Raf/
MEK/ERK-induced p21(CIP1) transcription is subject to TP53 status.