Targeting Hypoxia in Cancer Cells by Restoring Homeodomain Interacting Protein-Kinase 2 and p53 Activity and Suppressing HIF-1α

Lavinia Nardinocchi,Rosa Puca,Ada Sacchi,Gabriella D'Orazi,Gideon Rechavi,David Givol,Mikhail V Blagosklonny

doi:10.1371/journal.pone.0006819

Abstract

BackgroundThe tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) by phosphorylating serine 46 (Ser46) is a crucial regulator of p53 apoptotic function. HIPK2 is also a transcriptional co-repressor of hypoxia-inducible factor-1α (HIF-1α) restraining tumor angiogenesis and chemoresistance. HIPK2 can be deregulated in tumors by several mechanisms including hypoxia. Here, we sought to target hypoxia by restoring HIPK2 function and suppressing HIF-1α, in order to provide evidence for the involvement of both HIPK2 and p53 in counteracting hypoxia-induced chemoresistance.Methodology/Principal FindingsUpon exposure of colon and lung cancer cells to hypoxia, by either low oxygen or cobalt, HIPK2 function was impaired allowing for increased HIF-1α expression and inhibiting the p53-apoptotic response to drug. Cobalt suppressed HIPK2 recruitment onto HIF-1α promoter. Hypoxia induced expression of the p53 target MDM2 that downregulates HIPK2, thus MDM2 inhibition by siRNA restored the HIPK2/p53Ser46 response to drug. Zinc supplementation to hypoxia-treated cells increased HIPK2 protein stability and nuclear accumulation, leading to restoration of HIPK2 binding to HIF-1α promoter, repression of MDR1, Bcl2, and VEGF genes, and activation of the p53 apoptotic response to drug. Combination of zinc and ADR strongly suppressed tumor growth in vivo by inhibiting HIF-1 pathway and upregulating p53 apoptotic target genes.Conclusions/SignificanceWe show here for the first time that hypoxia-induced HIPK2 deregulation was counteracted by zinc that restored HIPK2 suppression of HIF-1 pathway and reactivated p53 apoptotic response to drug, underscoring the potential use of zinc supplementation in combination with chemotherapy to address hypoxia and improve tumor treatment.

Highlights

Solid tumors can survive hypoxic condition by using protective mechanisms including the activation of hypoxiainducible factor-1a (HIF-1a) a transcription factor that induces, among others, antiapoptotic Bcl2, multidrug resistance (MDR), VEGF gene expression, and reprogramming of glucose metabolism that account for cell proliferation, angiogenesis, and chemoresistance [1]
Homeodomain-interacting protein kinase-2 (HIPK2) is an important regulator of p53 apoptotic function, we have previously shown that homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates p53 at serine 46 (Ser46) after severe DNA damage, inducing p53 specific apoptotic transcriptional activity [7,8,9]
The mechanistic explanation of hypoxia-induced chemoresistance involved upregulation of HIF-1 pathway and inhibition of the p53 pathway that were partly interconnected by the hypoxia-induced HIPK2 deregulation

Summary

Introduction

Solid tumors can survive hypoxic condition (the high cell density of a tumor limits the availability of oxygen to cells) by using protective mechanisms including the activation of hypoxiainducible factor-1a (HIF-1a) a transcription factor that induces, among others, antiapoptotic Bcl, multidrug resistance (MDR), VEGF gene expression, and reprogramming of glucose metabolism that account for cell proliferation, angiogenesis, and chemoresistance [1]. Homeodomain-interacting protein kinase-2 (HIPK2) is an important regulator of p53 apoptotic function, we have previously shown that HIPK2 phosphorylates p53 at serine 46 (Ser46) after severe DNA damage, inducing p53 specific apoptotic transcriptional activity [7,8,9]. A major auto-regulatory, negative feed-back loop of p53 involves p53-dependent MDM2 induction that in turn binds and inactivates p53 by driving it to proteasomal degradation [12,13,14] In this regard, we have shown that HIPK2 neutralizes MDM2 inhibition rescuing p53 transcriptional activity and apoptotic function [15]. We sought to target hypoxia by restoring HIPK2 function and suppressing HIF-1a, in order to provide evidence for the involvement of both HIPK2 and p53 in counteracting hypoxia-induced chemoresistance

Methods

Results

Conclusion