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Activated p53 is necessary for tumor suppression. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of functional p53. HIPK2 modulates wild-type p53 activity toward proapoptotic transcription and tumor suppression by the phosphorylation of serine 46. Knock-down of HIPK2 interferes with tumor suppression and sensitivity to chemotherapy. Combined administration of adriamycin and zinc restores activity of misfolded p53 and enables the induction of its proapoptotic and tumor suppressor functions in vitro and in vivo. We therefore looked for a cancer model where HIPK2 expression is low. MMTV- neu transgenic mice overexpressing HER2/ neu, develop mammary tumors at puberty with a long latency, showing very low expression of HIPK2. Here we show that whereas these tumors are resistant to adriamycin treatment, a combination of adriamycin and zinc suppresses tumor growth in vivo in these mice, an effect evidenced by the histological features of the mammary tumors. The combined treatment of adriamycin and zinc also restores wild-type p53 conformation and induces proapoptotic transcription activity. These findings may open up new possibilities for the treatment of human cancers via the combination of zinc with chemotherapeutic agents, for a selected group of patients expressing low levels of HIPK2, with an intact p53. In addition, HIPK2 may serve as a new biomarker for tumor aggressiveness. [ABSTRACT FROM AUTHOR]

Abstract: Homeodomain-interacting protein kinase-2 (HIPK2) is a crucial regulator of p53 apoptotic function by phosphorylating serine 46 (Ser46) in response to DNA damage. In tumors with wild-type p53, its tumor suppressor function is often impaired by MDM2 overexpression that targets p53 for proteasomal degradation. Likewise, MDM2 targets HIPK2 for protein degradation impairing p53-apoptotic function. Here we report that zinc antagonised MDM2-induced HIPK2 degradation as well as p53 ubiquitination. The zinc inhibitory effect on MDM2 activity leads to HIPK2-induced p53Ser46 phosphorylation and p53 pro-apoptotic transcriptional activity. These results suggest that zinc derivatives are potential molecules to target the MDM2-induced HIPK2/p53 inhibition. [ABSTRACT FROM AUTHOR]

Abstract: HIPK2 is a stress-induced kinase and a transcriptional corepressor that functionally cooperates with p53 to suppress cancer. Activation of the p53 proapoptotic function requires a cascade of phosphorylations and acetylations, and HIPK2 takes part in both modifications in that it phosphorylates p53 Ser46 and induces p53 Lys382 acetylation. Here, to further investigate the role of HIPK2 in p53 activation, we started with the finding that HIPK2 inhibition upregulated Nox1, a homolog of the catalytic subunit of the superoxide-generating NADPH oxidase, involved in tumor progression and ROS production. We found that Nox1 inhibited p53 Lys382 acetylation, which is a target of SIRT1 deacetylase, and impaired p53 proapoptotic transcriptional activity. By the use of either small interfering RNAs to target SIRT1 or the SIRT1 inhibitor nicotinamide we found that Nox1-dependent inhibition of p53 transcriptional activity was SIRT1-dependent. Thus, Nox1 was unable to inhibit p53 when coexpressed with a SIRT1 deacetylase-defective mutant (SIRT1HY), suggesting a link between Nox1 and SIRT1 activity. Finally, recovery of HIPK2 function downregulated Nox1 expression with rescue of p53 Lys382 acetylation and p53 activity. Together, our findings indicate that Nox1 upregulation may activate SIRT1 and inhibit p53 and that Lys382 is important for p53 proapoptotic function. [Copyright &y& Elsevier]

Abstract: HIPK2 has been implicated in restraining tumor progression by more than one mechanism, involving both its catalytic and transcriptional co-repressor functions. Starting from the finding that HIPK2 knockdown by RNA-interference (HIPK2i) induced significant up-regulation of HIF-1α mRNA and of its target VEGF in tumor cells, we evaluated the role of HIPK2 in transcriptional regulation of HIF-1α. We found that HIPK2 overexpression downmodulated both HIF-1α reporter activity and mRNA levels and showed that HIPK2 was bound in vivo to the HIF-1α promoter likely in a multiprotein co-repressor complex with histone deacetylase 1 (HDAC1). Thus, the HIF-1α promoter was strongly acetylated following HIPK2 knockdown. The HIF-1α-dependent VEGF transcription was evaluated by co-transfection of a dominant negative (DN) construct of HIF-1α that inhibited VEGF reporter activity induced by HIPK2 knockdown. HIF-1α and VEGF up-regulation in HIPK2i cells correlated with increased vascularity of tumor xenografts in vivo and tube formation in HUVEC in vitro. These findings provide the first evidence of HIPK2-mediated transcriptional regulation of HIF-1α that might play a critical role in VEGF expression. [Copyright &y& Elsevier]

Abstract: The maintenance of p53 transactivation activity is important for p53 apoptotic function. We have shown that stable knockdown of HIPK2 induces p53 misfolding with inhibition of p53 target gene transcription. In this study we established a lentiviral-based system for doxycyclin (Dox)-induced conditional interference of HIPK2 expression to evaluate the molecular mechanisms involved in p53 deregulation. We found that HIPK2 knockdown induced metallothionein 2A (MT2A) upregulation as assessed by RT-PCR analysis, increased promoter acetylation, and increased promoter luciferase activity. The MT2A upregulation correlated with resistance to Adriamycin (ADR)-driven apoptosis and with p53 inhibition. Thus, acute knockdown of HIPK2 (HIPK2i) induced misfolded p53 protein in MCF7 breast cancer cells and inhibited p53 DNA-binding and transcription activities in response to ADR treatment. Previous works show that MT may modulate p53 activity through zinc exchange. Here, we found that inhibition of MT2A expression by siRNA in the HIPK2i cells restored p53 transcription activity. Similarly zinc supplementation to HIPK2i cells restored p53 transcription activity and drug-induced apoptosis. These data support the notion that MT2A is involved in p53 deregulation and strengthen the possibility that combination of chemotherapy and zinc might be useful to treat tumors with inactive wtp53. [Copyright &y& Elsevier]

Abstract: Objective: Chemoresistance, due to inhibition of apoptotic response, is the major reason for the failure of anticancer therapies. HIPK2 regulates p53-apoptotic function via serine-46 (Ser46) phosphorylation and activation of p53 is a key determinant in ovarian cancer cell death. In this study we determined whether HIPK2 overexpression restored apoptotic response in chemoresistant cancer cells. Methods: Using cisplatin chemosensitive (2008) and chemoresistant (2008C13) ovarian cancer cell lines we compared drug-induced activation of the HIPK2/p53Ser46 apoptotic pathway. The levels of HIPK2, Ser46 phosphorylation, and PARP cleavage were detected by Western blotting. The p53Ser46 apoptotic commitment was evaluated by luciferase assay using the Ser46 specific AIP1 target gene promoter. The apoptotic pathway was detected by caspase-3, -8, and -9 activities. Results: HIPK2 was expressed differently in sensitive versus chemoresistant cells in response to different chemotherapeutic drugs (i.e., cisplatin and adriamycin), though the p53Ser46 apoptotic pathway was not defective in chemoresistant 2008C13 cells. Thus, 2008C13 cells were resistant to cisplatin but sensitive to adriamycin-induced apoptosis through activation of the HIPK2/p53Ser46 pathway. HIPK2 knock-down inhibited the adriamycin-induced apoptosis in 2008C13 cells. Exogenous HIPK2 triggered apoptosis in chemoresistant cells, associated with induction of p53Ser46-target gene AIP1. Conclusions: HIPK2 is an important regulator of p53 activity in response to a chemotherapeutic drug. These results suggest that different drug-activated pathways may regulate HIPK2 and that HIPK2/p53Ser46 deregulation is involved in chemoresistance. Exogenous HIPK2 might represent a novel therapeutic approach to circumvent inhibition of apoptosis in treatment of chemoresistant ovarian cancers with wtp53. [Copyright &y& Elsevier]