Zn(II)-curc targets p53 in thyroid cancer cells

P53 mutation is a common event in many cancers, including thyroid carcinoma. Defective p53 activity promotes cancer resistance to therapies and a more malignant phenotype, acquiring oncogenic functions. Rescuing the function of mutant p53 (mutp53) protein is an attractive anticancer therapeutic strategy. Zn(II)-curc is a novel small molecule that has been shown to target mutp53 protein in several cancer cells, but its effect in thyroid cancer cells remains unclear. Here, we investigated whether Zn(II)-curc could affect p53 in thyroid cancer cells with both p53 mutation (R273H) and wild-type p53. Zn(II)-curc induced mutp53H273 downregulation and reactivation of wild-type functions, such as binding to canonical target promoters and target gene transactivation. This latter effect was similar to that induced by PRIMA-1. In addition, Zn(II)-curc triggered p53 target gene expression in wild-type p53-carrying cells. In combination treatments, Zn(II)-curc enhanced the antitumor activity of chemotherapeutic drugs, in both mutant and wild-type-carrying cancer cells. Taken together, our data indicate that Zn(II)-curc promotes the reactivation of p53 in thyroid cancer cells, providing in vitro evidence for a potential therapeutic approach in thyroid cancers

Reduced chemotherapeutic sensitivity in high glucose condition: implication of antioxidant response

Resistance to chemotherapy represents a major obstacle to successful treatment. The generation of reactive oxygen species (ROS) has been directly linked to the cytotoxic effects of several antitumor agents, including Adriamycin (ADR), and modulation of the oxidative balance has been implicated in the development and/or regulation of resistance to chemotherapeutic drugs. We recently showed that high glucose (HG) markedly diminished the cancer cell death induced by anticancer agents such as ADR. In the present study we attempted to evaluate the mechanism that impaired the cytotoxic effect of ADR in HG. We found that, in colon cancer cells, HG attenuated ADR-induced ROS production that consequently diminished ADR-induced H2AX phosphorylation and micronuclei (MN) formation. Mechanistically, HG attenuation of ADR-induced ROS production correlated with increased antioxidant response promoted by NRF2 activity. Thus, pharmacologic inhibition of NRF2 pathway by brusatol re-established the ADR cytotoxic effect impaired by HG. Together, the data provide new insights into chemotherapeutic-resistance mechanisms in HG condition dictated by increased NRF2-induced antioxidant response and how they may be overcome in order to restore chemosensitivity and ADR-induced cell death

Regulation of vascular endothelial growth factor expression by homeodomain-interacting protein kinase-2

<p>Abstract</p> <p>Background</p> <p>Homeodomain-interacting protein kinase-2 (HIPK2) plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors) involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the β-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF) levels (a β-catenin target gene) and the role of β-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches.</p> <p>Methods</p> <p>The regulation of VEGF expression by HIPK2 was evaluated by using luciferase assay with VEGF reporter construct, after overexpression of the β-catenin transcription factor. Relative quantification of VEGF and β-catenin mRNAs were assessed by reverse-transcriptase-PCR (RT-PCR) analyses, following HIPK2 overexpression, while β-catenin protein levels were evaluated by western immunoblotting.</p> <p>Results</p> <p>HIPK2 overexpression in tumor cells downregulated VEGF mRNA levels and VEGF promoter activity. The VEGF downregulation was partly depending on HIPK2-mediated β-catenin regulation. Thus, HIPK2 could induce β-catenin protein degradation that was prevented by cell treatment with proteasome inhibitor MG132. The β-catenin degradation was dependent on HIPK2 catalytic activity and independent of p53 and glycogen synthase kinase 3β (GSK-3β) activities.</p> <p>Conclusion</p> <p>These results suggest that VEGF might be a target of HIPK2, at least in part, through regulation of β-catenin activity. These findings support the function of HIPK2 as tumor suppressor and hypothesise a role for HIPK2 as antiangiogenic tool in tumor therapy approaches.</p

Interplay between endoplasmic reticulum (ER) stress and autophagy induces mutant p53H273 degradation

The unfolded protein response (UPR) is an adaptive response to intrinsic and external stressors, and it is mainly activated by the accumulation of misfolded proteins at the endoplasmic reticulum (ER) lumen producing ER stress. The UPR signaling network is interconnected with autophagy, the proteolytic machinery specifically devoted to clearing misfolded proteins in order to survive bioenergetic stress and/or induce cell death. Oncosuppressor TP53 may undergo inactivation following missense mutations within the DNA-binding domain (DBD), and mutant p53 (mutp53) proteins may acquire a misfolded conformation, often due to the loss of the DBD-bound zinc ion, leading to accumulation of hyperstable mutp53 proteins that correlates with more aggressive tumors, resistance to therapies, and poorer outcomes. We previously showed that zinc supplementation induces mutp53 protein degradation by autophagy. Here, we show that mutp53 (i.e., Arg273) degradation following zinc supplementation is correlated with activation of ER stress and of the IRE1α/XBPI arm of the UPR. ER stress inhibition with chemical chaperone 4-phenyl butyrate (PBA) impaired mutp53 downregulation, which is similar to IRE1α/XBPI specific inhibition, reducing cancer cell death. Knockdown of mutp53 failed to induce UPR/autophagy activation indicating that the effect of zinc on mutp53 folding was likely the key event occurring in ER stress activation. Recently discovered small molecules targeting components of the UPR show promise as a novel anticancer therapeutic intervention. However, our findings showing UPR activation during mutp53 degradation indicate that caution is necessary in the design of therapies that inhibit UPR components

The beneficial effect of Zinc(II) on low-dose chemotherapeutic sensitivity involves p53 activation in wild-type p53-carrying colorectal cancer cells

BACKGROUND: Activation of wild-type p53 in response to genotoxic stress occurs through different mechanisms including protein conformation, posttranslational modifications, and nuclear localization, leading to DNA binding to sequence-specific promoters. Zinc ion plays a crucial role in stabilizing p53/DNA binding to induce canonical target genes. Mutant p53 proteins undergo protein misfolding that can be counteracted by zinc. However, whether zinc supplementation might have a beneficial antitumor effect in wild-type p53-carrying cells in combination with drugs, has not been addressed so far. METHODS: In this study we compared the effect of two antitumor treatments: on the one hand wild-type p53-carrying colon cancer cells were treated with low and high doses of chemotherapeutic agent Adriamycin and, on the other hand, Adriamycin was used in combination with ZnCl2. Biochemical and molecular analyses were applied to evaluate p53 activity and biological outcomes in this setting. Finally, the effect of the different combination treatments were applied to assess tumor growth in vivo in tumor xenografts. RESULTS: We found that low-dose Adriamycin did not induce p53 activation in wtp53-carrying colon cancer cells, unless in combination with ZnCl2. Mechanistically, ZnCl2 was a key determinant in inducing wtp53/DNA binding and transactivation of target genes in response to low-dose Adriamycin that used alone did not achieve such effects. Finally, in vivo studies, in a model of wtp53 colon cancer xenograft, show that low-dose Adriamycin did not induce tumor regression unless in combination with ZnCl2 that activated endogenous wtp53. CONCLUSIONS: These results provide evidence that ZnCl2 might be a valuable adjuvant in chemotherapeutic regimens of colorectal cancer harboring wild-type p53, able to both activate p53 and reduce the amount of drugs for antitumor purposes

Hyperglycemia triggers HIPK2 protein degradation

Homeodomain interacting protein kinase-2 (HIPK2) is an evolutionary conserved kinase that modulates several key molecular pathways to restrain tumor growth and induce p53-depending apoptotic cell-death in response to anticancer therapies. HIPK2 silencing in cancer cells leads to chemoresistance and cancer progression, in part due to p53 inhibition. Recently, hyperglycemia has been shown to reduce p53 phosphorylation at serine 46 (Ser46), the target residue of HIPK2, thus impairing p53 apoptotic function. Here we asked whether hyperglycemia could, upstream of p53, target HIPK2. We focused on the effect of high glucose (HG) on HIPK2 protein stability and the underlying mechanisms. We found that HG reduced HIPK2 protein levels, therefore impairing HIPK2-induced p53 apoptotic activity. HG-triggered HIPK2 protein downregulation was rescued by both proteasome inhibitor MG132 and by protein phosphatase inhibitors Calyculin A (CL-A) and Okadaic Acid (OA). Looking for the phosphatase involved, we found that protein phosphatase 2A (PP2A) induced HIPK2 degradation, as evidenced by directly activating PP2A with FTY720 or by silencing PP2A with siRNA in HG condition. The effect of PP2A on HIPK2 protein degradation could be in part due to hypoxia-inducible factor-1 (HIF-1) activity which has been previously shown to induce HIPK2 proteasomal degradation through several ubiquitin ligases. Validation analysed performed with HIF-1α dominant negative or with silencing of Siah2 ubiquitin ligase clearly showed rescue of HG-induced HIPK2 degradation. These findings demonstrate how hyperglycemia, through a complex protein cascade, induced HIPK2 downregulation and consequently impaired p53 apoptotic activity, revealing a novel link between diabetes/obesity and tumor resistance to therapies

HIPK2 modulates p53 activity towards pro-apoptotic transcription

<p>Abstract</p> <p>Background</p> <p>Activation of p53-mediated gene transcription is a critical cellular response to DNA damage and involves a phosphorylation-acetylation cascade of p53. The discovery of differences in the response to different agents raises the question whether some of the p53 oncosuppressor functions might be exerted by different posttranslational modifications. Stress-induced homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates p53 at serine-46 (Ser46) for p53 apoptotic activity; p53 acetylation at different C-terminus lysines including p300-mediated lysine-382 (Lys382) is also required for full activation of p53 transcriptional activity. The purpose of the current study was to evaluate the interplay among HIPK2, p300, and p53 in p53 acetylation and apoptotic transcriptional activity in response to drug by using siRNA interference, p300 overexpression or deacetylase inhibitors, in cancer cells.</p> <p>Results</p> <p>Knockdown of HIPK2 inhibited both adriamycin-induced Ser46 phosphorylation and Lys382 acetylation in p53 protein; however, while combination of ADR and zinc restored Ser46 phosphorylation it did not recover Lys382 acetylation. Chromatin immunoprecipitation studies showed that HIPK2 was required <it>in vivo </it>for efficient p300/p53 co-recruitment onto apoptotic promoters and that both p53 modifications at Ser46 and Lys382 were necessary for p53 apoptotic transcription. Thus, p53Lys382 acetylation in HIPK2 knockdown as well as p53 apoptotic activity in response to drug could be rescued by p300 overexpression. Similar effect was obtained with the Sirt1-inhibitor nicotinamide. Interestingly trichostatin A (TSA), the inhibitor of histone deacetylase complexes (HDAC) did not have effect, suggesting that Sirt1 was the deacetylase involved in p53 deacetylation in HIPK2 knockdown.</p> <p>Conclusion</p> <p>These results reveal a novel role for HIPK2 in activating p53 apoptotic transcription. Our results indicate that HIPK2 may regulate the balance between p53 acetylation and deacetylation, by stimulating on one hand co-recruitment of p300 and p53Lys382 on apoptotic promoters and on the other hand by inhibiting Sirt1 deacetylase activity. We attempted to reactivate p53 apoptotic transcriptional activity by rescuing both Ser46 and Lys382 modification in response to drug. Our data propose combination strategies for the treatment of tumors with dysfunctional p53 and/or HIPK2 that include classical chemotherapy with pharmacological or natural agents such as Sirt1-deacetylase inhibitors or zinc, respectively.</p

Unfolded p53 in the pathogenesis of Alzheimer's disease: is HIPK2 the link?

p53 transcriptional activity depends mainly on posttranslational modifications and protein/protein interaction. Another important mechanism that controls p53 function is its conformational stability since p53 is an intrinsically unstable protein. An altered conformational state of p53, independent from point mutations, has been reported in tissues from patients with Alzheimer's disease (AD), leading to an impaired and dysfunctional response to stressors. Recent evidence shows that one of the activators that induces p53 posttranslational modification and wild-type conformational stability is homeodomain interacting protein kinase 2 (HIPK2). Hence, conditions that induce HIPK2 deregulation would result in a dysfunctional response to stressors by affecting p53 activity. Discovering the mechanisms of HIPK2 activation/inhibition and the ways to manipulate HIPK2 activity are an interesting option to affect several biological pathways, including those underlying AD. Soluble beta-amyloid peptides have recently been involved in HIPK2 degradation, in turn regulating the p53 conformational state and vulnerability to a noxious stimulus, before triggering the amyloidogenic cascade. Here we discuss about these findings and the potential relevance of HIPK2 as a target for AD and highlight the existence of a novel amyloid-based mechanism in AD potentially leading to the survival of injured dysfunctional cells

ATF6 prevents DNA damage and cell death in colon cancer cells undergoing ER stress

Colon cancer represents one of the most common and aggressive cancers in its advanced state. Among the most innovative anti-cancer approaches, the manipulation of UPR is a promising one, effective also against cancers carrying dysfunctional p53. Interestingly, it is emerging that UPR cross-talks with DDR and that targeting the interplay between these two adaptive responses may be exploited to overcome the resistance to the single DDR- and UPR-targeting treatments. Previous studies have highlighted the role of IRE1 alpha and PERK UPR sensors on DDR, while the impact of ATF6 on this process remains under-investigated. This study shows for the first time that ATF6 sustains the expression level of BRCA-1 and protects colon cancer cells from the cytotoxic effect of ER stressors DPE and Thapsigargin. At molecular level, ATF6 activates mTOR to sustain the expression of HSP90, of which BRCA-1 is a client protein. Therefore, pharmacological or genetic inhibition of ATF6 promoted BRCA-1 degradation and increased DNA damage and cell death, particularly in combination with Adriamycin. All together this study suggests that targeting ATF6 may not only potentiate the cytotoxic effect of drugs triggering ER stress but may render colon cancer cells more sensitive to Adriamycin and possibly to other DNA damaging agents used to treat colon cancer