Sp1 regulates Raf/MEK/ERK-induced p21CIP1 transcription in TP53-mutated cancer cells

Abstract

We previously reported that the upregulation of mortalin, an Hsp70 family chaperone, is important for B-RafV600E tumor cells to bypass p21CIP1 expression, which is activated as a tumor-suppressive mechanism in response to aberrant MEK/ERK activation (Wu et al., 2013). Interestingly, mortalin depletion induced p21CIP1 transcription not only in wild-type TP53 but also in TP53-mutated B-RafV600E cancer cells, suggesting the presence of an additional mechanism for p21CIP1 regulation. In the present study, using luciferase reporter truncation analysis in a TP53-mutated B-RafV600E cancer cell line, SK-MEL28, we identified a proximal p21CIP1 promoter region responsive to mortalin depletion. Interestingly, when Sp1-like cis-elements in this promoter region were mutagenized, the p21CIP1 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 p21CIP1 promoter in a MEK/ERK-dependent manner. Moreover, RNA interference of Sp1 substantially attenuated p21CIP1 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 p21CIP1 transcription in U251 cells, in which TP53 is mutated. However, in contrast, Sp1 was not necessary for ∆Raf-1:ER to induce p21CIP1 transcription in LNCaP cells, in which TP53 is wild type. These data suggest that Sp1 may address TP53-independent p21CIP1 transcription in Raf/MEK/ERK-activated cancer cells and that its requirement in Raf/MEK/ERK-induced p21CIP1 transcription is subject to TP53 status.