Background: Homeodomain-interacting protein kinase-2 (HIPK2), a transcriptional co-repressor with apoptotic function, can affect hypoxia-inducible factor 1 (HIF-1) transcriptional activity, through downmodulation of its HIF-1α subunit, in normoxic condition. Under hypoxia, a condition often found in solid tumors, HIF-1α is activated to induce target genes involved in chemoresistance, inhibition of apoptosis and tumor progression. Here, we investigated whether the HIPK2 overexpression could downregulate HIF-1α expression and activity in tumor cells treated with hypoxia-mimicking condition, and evaluated whether HIPK2-dependent downregulation of HIF-1α could sensitize chemoresistant tumor cells to adriamycin (ADR)-induced apoptosis.

The p53 protein is the most studied tumor suppressor and the p53 pathway has been shown to mediate cellular stress responses that are disrupted when cancer develops. After DNA damage, p53 is activated as transcription factor to directly induce the expression of target genes involved in cell-cycle arrest, DNA repair, senescence and, importantly, apoptosis. Post-translational modifications of p53 are essential for the activation of p53 and for selection of target genes. The tumor suppressor homeodomain-interacting protein kinase-2 (HIPK2) is a crucial regulator of p53 apoptotic function by phosphorylating its N-terminal serine 46 (Ser46) and facilitating Lys382 acetylation at the C-terminus. HIPK2 is activated by numerous genotoxic agents and can be deregulated in tumors by several conditions including hypoxia. Recent findings suggest that HIPK2 active/inactive protein can affect p53 function in multiple and unexpected ways. This makes p53 as well as HIPK2 interesting targets for cancer therapy. Hence, understanding the role of HIPK2 as p53 activator may provide important insights in the process of tumor progression, and may also serve as the crucial point in the diagnostic and therapeutical aspects of cancer.

About half of cancers sustain mutations in the TP53 gene, whereas the other half maintain a wild-type p53 (wtp53) but may compromise the p53 response because of other alterations. Homeodomain-interacting protein kinase-2 (HIPK2) is a positive regulator of p53 oncosuppressor function. Here, we show, by microarray analysis, that wtp53 lost the target gene activation following stable knockdown of HIPK2 (HIPK2i) in colon cancer cell line. Our data show that the stable knockdown of HIPK2 led to wtp53 misfolding, as detected by p53 immunoprecipitation with conformationspecific antibodies, and that p53 protein misfolding impaired p53 DNA binding and transcription of target genes. We present evidence that zinc supplementation to HIPK2i cells increased p53 reactivity to conformation-sensitive PAb1620 (wild-type conformation) antibody and restored p53 sequence-specific DNA binding in vivo and transcription of target genes in response to Adriamycin treatment. Finally, combination of zinc and Adriamycin suppressed tumor growth in vivo and activated misfolded p53 that induced its target genes in nude mice tumor xenografts derived from HIPK2i cells. Bioinformatics analysis of microarray data from colon cancer patients showed significant association of poor survival with low HIPK2 expression only in tumors expressing wtp53. These results show a critical role of HIPK2 in maintaining the transactivation activity of wtp53 and further suggest that low expression of HIPK2 may impair the p53 function in tumors harboring wtp53.

BackgroundHypoxia inducible factor-1α (HIF-1α) is responsible for the majority of HIF-1-induced gene expression changes under hypoxia and for the “angiogenic switch” during tumor progression. HIF-1α is often upregulated in tumors leading to more aggressive tumor growth and chemoresistance, therefore representing an important target for antitumor intervention. We previously reported that zinc downregulated HIF-1α levels. Here, we evaluated the molecular mechanisms of zinc-induced HIF-1α downregulation and whether zinc affected HIF-1α also in vivo.Methodology/Principal FindingsHere we report that zinc downregulated HIF-1α protein levels in human prostate cancer and glioblastoma cells under hypoxia, whether induced or constitutive. Investigations into the molecular mechanisms showed that zinc induced HIF-1α proteasomal degradation that was prevented by treatment with proteasomal inhibitor MG132. HIF-1α downregulation induced by zinc was ineffective in human RCC4 VHL-null renal carcinoma cell line; likewise, the HIF-1αP402/P564A mutant was resistant to zinc treatment. Similarly to HIF-1α, zinc downregulated also hypoxia-induced HIF-2α whereas the HIF-1β subunit remained unchanged. Zinc inhibited HIF-1α recruitment onto VEGF promoter and the zinc-induced suppression of HIF-1-dependent activation of VEGF correlated with reduction of glioblastoma and prostate cancer cell invasiveness in vitro. Finally, zinc administration downregulated HIF-1α levels in vivo, by bioluminescence imaging, and suppressed intratumoral VEGF expression.Conclusions/SignificanceThese findings, by demonstrating that zinc induces HIF-1α proteasomal degradation, indicate that zinc could be useful as an inhibitor of HIF-1α in human tumors to repress important pathways involved in tumor progression, such as those induced by VEGF, MDR1, and Bcl2 target genes, and hopefully potentiate the anticancer therapies.

Interleukin-17 (IL-17Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionNonmelanoma skin cancer (NMSC) represents one of the most common malignancies in humans and keratinocyte-derived basalcell carcinomas (BCCs) and squamous-cell carcinomas (SCCs) are responsible approximately for the 80 and 20% NMSC cases, respectively [1,2]. Both types of NMSCs share important risk factors, such as fair skin and high degree of sun exposure [3]. Nevertheless, BCCs and SCCs have distinct metastatic attitude, being rare in BCC and much more common in SCC [4].Correspondence: Dr. Andrea Cavani e-mail: cavani@idi.itTumor-infiltrating lymphocytes (TILs) are frequently found in malignant tumors, being suggestive of a host antitumoral immune response. Several independent studies have demonstrated that the presence of TILs correlated with increased survival and represents a predictive as well as prognostic biomarker in patients with breast cancer, colorectal cancer, glioma, and gastric cancer [5][6][7]. However, dissimilar data revealed that TILs functionally promoted epithelial carcinogenesis [8], and that TIL-secreted chemokines contributed to tumor angiogenesis and growth [9]. Although few studies have described the inflammatory infiltrate of BCCs and Table 1), but not that of the Th1-cell clone 6.1, was able to induce proliferation of both M77015 ( Fig. 2D and E) and CAL27 cells (Fig. 2F and G). To demonstrate that tumor-cell growth induced by supernatant of activated TIL-derived cell clones was dependent on IL-17 and IL-22, we performed blocking experiments with anti-IL-17 and anti-IL-22 Abs. As shown in Figure 2, results clearly indicate the capacity of both IL-17 and IL-22 blocking antibodies to reduce M77015 and CAL27 proliferation induced by supernatant of TILderived cell clones ( Fig. 2D-G). Furthermore, IL-17 and IL-22 were able to induce CAL27-and M7715-cell migration in an in vitro scratch assay (Fig. 3), indicating that the cytokines promote local invasiveness of tumor cells. IL-6 and IL-8 are induced in SCC cells by IL-17 treatmentThe next step was to investigate whether IL-17 and IL-22 alone or in combination with other Th17-and/or Th22-derived cytokines could induce the secretion of cytokines and/or chemokines, known to influence tumor survival and progression, in tumor cells. Indeed, our previous data showed that in normal human keratinocytes [28,29], IL-17, alone or in combination with TNF-α, induced the secretion of IL-8. To verify whether IL-17 had similar effects on BCC-and SCC-cell lines, we exposed CAL27 and M77015 cells to recombinant human IL-17, IL-22, with or without TNF-α, and cytokine release was investigated by ELISA. In particular, we decided to analyze IL-6 and IL-8 release, as it is known that both cytokines induce proliferation and invasion of many tumor cells [30][31][32]. As shown in Figure 4, IL-17, but not IL-22 alone, augmented both IL-6 ( Fig. 4A) and IL-8 ( Fig. 4B) secretion by CAL27 cells. Moreover, IL-17 showed a s...