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Sp1 regulates Raf/MEK/ERK-induced p21(CIP1) transcription in TP53-mutated cancer cells

Mansi Karkhanis1, Jong-In Park1

  • 1Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.

Cellular Signalling
|January 18, 2015
PubMed

Insights

Mortalin depletion induces p21(CIP1) transcription via Sp1 in cancer cells with mutated TP53. This Sp1-dependent pathway bypasses wild-type TP53, highlighting a novel mechanism in cancer therapy.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Cellular Signaling

Background:

  • Mortalin (Hsp70 chaperone) upregulation aids B-Raf(V600E) tumor cells in overcoming p21(CIP1) tumor suppression.
  • p21(CIP1) transcription is activated by aberrant MEK/ERK signaling, a common event in B-Raf(V600E) cancers.
  • Mortalin depletion unexpectedly induced p21(CIP1) in both wild-type and mutated TP53 cancer cells, suggesting alternative regulatory pathways.

Purpose of the Study:

  • To investigate the mechanism by which mortalin depletion influences p21(CIP1) transcription.
  • To identify the specific promoter regions and transcription factors involved in mortalin-mediated p21(CIP1) regulation.
  • To determine the role of Sp1 and TP53 status in p21(CIP1) transcriptional control under aberrant Raf/MEK/ERK signaling.

Main Methods:

  • Luciferase reporter assays with promoter truncation and cis-element mutagenesis in SK-MEL28 (TP53-mutated) cells.
  • Chromatin immunoprecipitation (ChIP) assays to assess Sp1 binding to the p21(CIP1) promoter.
  • RNA interference (RNAi) to deplete Sp1 and evaluate its effect on p21(CIP1) expression.
  • Experiments in U251 (TP53-mutated) and LNCaP (wild-type TP53) cells to assess Sp1 requirement.

Main Results:

  • A proximal p21(CIP1) promoter region responsive to mortalin depletion was identified.
  • Mutagenesis of Sp1-like cis-elements abolished the responsiveness of the p21(CIP1) promoter to mortalin depletion.
  • Mortalin knockdown increased Sp1 binding to the p21(CIP1) promoter in a MEK/ERK-dependent manner.
  • Sp1 depletion attenuated mortalin depletion-induced p21(CIP1) expression in TP53-mutated cells.
  • Sp1 was essential for p21(CIP1) induction in TP53-mutated cells (U251) but not in wild-type TP53 cells (LNCaP).

Conclusions:

  • Sp1 mediates TP53-independent p21(CIP1) transcription in Raf/MEK/ERK-activated cancer cells.
  • The requirement of Sp1 for p21(CIP1) induction is dependent on the TP53 mutational status.
  • These findings reveal a novel mortalin-Sp1-p21(CIP1) axis that bypasses TP53 in cancer, offering potential therapeutic targets.
Keywords:
MortalinRaf/MEK/ERKSp1TP53p21(CIP1)

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