Homeodomain-interacting Protein Kinase Research Articles

Regulation of HIPK Proteins by MicroRNAs.

The homeodomain-interacting protein kinase (HIPK) family consists of four evolutionarily conserved and highly related nuclear serine/threonine kinases of recent discovery. They interact with homeobox proteins and other transcription factors, as well as transcriptional coactivators or corepressors depending on the cellular context. HIPK proteins are sensors for various extracellular stimuli, which control key cellular functions such as signal transduction to downstream effectors that regulate apoptosis, embryonic development, DNA-damage response, and cellular proliferation. Thus, HIPKs are involved in proliferative diseases such as cancer and fibrosis. mRNA levels and protein stability tightly regulate expression levels of HIPKs. Here, we review recent works investigating the regulation of HIPKs expression by microRNAs (miRNAs) that are involved in the control of cell proliferation, sensitivity to chemotherapeutic drugs, epithelial-mesenchymal transition, and glucose-stimulated insulin secretion. It appears that HIPK family members, and their related miRNAs, may be considered as novel therapeutic targets for treating cancer, renal fibrosis and type 2 diabetes.

Genetic ablation of homeodomain-interacting protein kinase 2 selectively induces apoptosis of cerebellar Purkinje cells during adulthood and generates an ataxic-like phenotype.

Homeodomain-interacting protein kinase 2 (HIPK2) is a multitalented coregulator of an increasing number of transcription factors and cofactors involved in cell death and proliferation in several organs and systems. As Hipk2−/− mice show behavioral abnormalities consistent with cerebellar dysfunction, we investigated whether Hipk2 is involved in these neurological symptoms. To this aim, we characterized the postnatal developmental expression profile of Hipk2 in the brain cortex, hippocampus, striatum, and cerebellum of mice by real-time PCR, western blot analysis, and immunohistochemistry. Notably, we found that whereas in the brain cortex, hippocampus, and striatum, HIPK2 expression progressively decreased with age, that is, from postnatal day 1 to adulthood, it increased in the cerebellum. Interestingly, mice lacking Hipk2 displayed atrophic lobules and a visibly smaller cerebellum than did wild-type mice. More important, the cerebellum of Hipk2−/− mice showed a strong reduction in cerebellar Purkinje neurons during adulthood. Such reduction is due to the activation of an apoptotic process associated with a compromised proteasomal function followed by an unpredicted accumulation of ubiquitinated proteins. In particular, Purkinje cell dysfunction was characterized by a strong accumulation of ubiquitinated β-catenin. Moreover, our behavioral tests showed that Hipk2−/− mice displayed muscle and balance impairment, indicative of Hipk2 involvement in cerebellar function. Taken together, these results indicate that Hipk2 exerts a relevant role in the survival of cerebellar Purkinje cells and that Hipk2 genetic ablation generates cerebellar dysfunction compatible with an ataxic-like phenotype.

Homeodomain interacting protein kinase 2 is downregulated through the peroxisome proliferator-activated receptor gamma signaling pathway in an insulin-resistant population.

Homeodomain interacting protein kinase 2 (HIPK2), the highly conserved nuclear serine/threonine kinase, is known to act as a co-repressor through interacting with a number of transcription factors. Notably, HIPK2 inhibits cell growth through the activation of p53. However, its in vivo function in insulin resistance is still unknown. Recently, the discovery by Sjolund et al.1 has implicated HIPK2 as a central modulator of adipogenesis. They found that deletion of HIPK2 in mice gave rise to decreased adipogenesis and increased insulin-sensitivity independence of the peroxisome proliferator-activated receptor gamma (PPARγ) signaling pathway. According to their result, we hypothesize that the expression of HIPK2 in the insulin-resistant population might be upregulated. To testify our supposition, we analyzed the publicly available expression data from two human insulin-resistant expression data in GEO datasets that use the same platform. Contrary to our deduction, the HIPK2 gene was downregulated in the insulin-resistant population (Figure ​(Figure11a,b). Figure 1 Decreased expression of homeodomain interacting protein kinase 2 (HIPK2) in an insulin resistant population was associated with the peroxisome proliferator-activated receptor gamma (PPARγ) signaling pathway. (a,b) Expression of HIPK2 detected ... PPARγ is the key transcription factor in adipogenesis, as well as glucose and fatty acid metabolism, and alterations in the pathway lead to insulin resistance and type 2 diabetes mellitus2. According to the report by Sjolund et al.1, the function of HIPK2 was not mediated by a PPARγ-dependent mechanism1. However, we identified that some downstream targets of PPARγ, which Sjolund et al. mentioned, were downregulated in adipocytes in insulin-resistant people, which is synchronously expressed with HIPK2 messenger ribonucleic acid expression (Figure ​(Figure1c).1c). Some transcription cofactors of PPARγ shown by GeneMANIA (http://www.genemania.org/) were also downregulated in the adipocytes of the insulin-resistant population (Figure ​(Figure11d). Rosiglitazone (Rosi), a member of the thiazolidinediones and an agonist of PPARγ, works as an insulin sensitizer in type 2 diabetes mellitus3. The present results showed that HIPK2 messenger ribonucleic acid expression was significantly increased in the presence of both Rosi and overexpressed PPARγ in murine marrow-derived U-33 cells, indicating a tendency of increased HIPK2 expression after administration of insulin sensitizer (Figure ​(Figure1e).1e). In agreement with this, several other articles published the same results that HIPK2 expression was elevated after administration of insulin sensitizer, rosiglitazone and troglitazone (Table S1), suggested by The Comparative Toxicogenomics Database (http://ctdbase.org/). Taken together, despite of Sjolund et al. discovery that HIPK2 deletion in mice induces increased insulin sensitivity and was not mediated by the PPARγ pathway, our data suggest that downexpression of HIPK2 was discovered in a population that suffered from decreased insulin sensitivity (insulin resistance) through the PPARγ-dependent pathway. This discrepancy might be caused by the following reasons: (i) metabolic differences between humans and murine; (ii) the discovery by Sjolund et al. was carried out on skeletal muscle, where the present results were shown in adipocytes; and (iii) our discovery was the result of a subset of humans, accordingly, the discovery by Sjolund et al. was based on a few HIPK2 knockout mice, the two studies all await further verification. Furthermore, we also showed that administration of insulin sensitizer could promote the elevated expression of HIPK2, thus providing a benefit for treatment of insulin resistance.

Targeting ncRNAs in the 3q26.2 amplicon.

Targeting ncRNAs in the 3q26.2 amplicon.

Novel insights about the MDM2/MDM4 heterodimer

ABSTRACTMDM2 (mouse double minute 2 homolog) and MDM4 (double minute 4 human homolog, also known as MDMX) inhibit the activity of tumor protein p53 (TP53, best known as p53) through their heterodimerization. New evidence indicates that under stress conditions the heterodimer is modified, leading to different activities of the single molecules. In particular, following lethal DNA damage, MDM2 and MDM4 dissociate and MDM4 promotes the stabilization of homeodomain-interacting protein kinase 2 (HIPK2) and the phosphorylation of p53, resulting in transcriptional repression of antiapoptotic targets of p53/HIPK2.

Genome-wide gene expression profiling of homeodomain-interacting protein kinase 2 deficient medullary thymic epithelial cells

The establishment of central tolerance essentially depends on the promiscuous gene expression (pGE) of a plethora of tissue restricted antigens by the medullary thymic epithelial cells. The antigens are presented to developing thymocytes in the thymus to select for non-self reactive T-cell receptors in order to prevent autoimmune reactions in the periphery. However the molecular regulation of tissue-restricted antigen expression is still poorly understood. The only regulator known to play a role in the transcriptional regulation so far is the autoimmune regulator (AIRE). AIRE is thought to act in a multi-protein complex, promoting transcription, elongation and splicing of target genes. Yet the full composition of this Aire-associated multi-protein complex and its mode of action remain to be elucidated. Here we describe the experimental details and controls of the gene array analysis on the impact of the homeodomain-interacting protein kinase 2 (Hipk2) on promiscuous gene expression in medullary thymic epithelial cells based on the analysis of newly generated TEC-specific Hipk2 conditional knockout mice. The changes in gene expression are presumably mediated through a regulatory effect of Hipk2 on AIRE as published in the study by Rattay and colleagues in the Journal of Immunology [1]. The gene array data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE63432).

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis.

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser(46) in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser(46), and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.

HIPK2 is a new drug target for anti-fibrosis therapy in kidney disease.

In vitro and animal studies continue to elucidate the mechanisms of fibrosis and have led to advancements in treatment for idiopathic pulmonary fibrosis and cirrhosis, but the search for treatments for renal fibrosis has been more disappointing. Here, we will discuss homeodomain-interacting-protein kinase 2 (HIPK2), a novel regulator of fibrosis that acts upstream of major fibrosis signaling pathways. Its key role in renal fibrosis has been validated in vitro and in several murine models of chronic kidney diseases (CKD).

Programmed cell death 4 protein (Pdcd4) and homeodomain-interacting protein kinase 2 (Hipk2) antagonistically control translation of Hipk2 mRNA

The tumor suppressor protein programmed cell death 4 (Pdcd4) is a highly conserved RNA-binding protein that inhibits the translation of specific mRNAs. Here, we have identified the homeobox-interacting protein kinase-2 (Hipk2) mRNA as a novel translational target of Pdcd4. Unlike most other protein kinases Hipk2 is constitutively active after being synthesized by the ribosome and its expression and activity are thought to be mainly controlled by modulation of the half-life of the kinase. Our work provides the first evidence that Hipk2 expression is also controlled on the level of translation. We show that Hipk2 stimulates the translation of its own mRNA and that Pdcd4 suppresses the translation of Hipk2 mRNA by interfering with this auto-regulatory feedback mechanism. We also show that the translation of the related kinase Hipk1 is controlled by a similar feedback loop and that Hipk2 also stimulates the translation of Hipk1 mRNA. Taken together, our work describes a novel mechanism of translational suppression by Pdcd4 and shows for the first time that Hipk2 controls its own synthesis by an auto-regulatory feedback mechanism. Furthermore, the effect of Hipk2 on the translation of Hipk1 RNA suggests that Hipk2 and Pdcd4 can act in similar manner to control the translation of other mRNAs.

Phosphorylation of Krüppel-like Factor 3 (KLF3/BKLF) and C-terminal Binding Protein 2 (CtBP2) by Homeodomain-interacting Protein Kinase 2 (HIPK2) Modulates KLF3 DNA Binding and Activity

Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3.

HIPK2 expression in progression of cutaneous epithelial neoplasm.

Homeodomain-interacting protein kinase 2 (HIPK2) is responsible for a DNA damage response, centrally regulating p53. The aberrant HIPK2 expression is known to be involved in carcinogenesis in several malignancies. However, the correlation of HIPK2 expression along with progression of cutaneous epithelial neoplasm has not been investigated. Using immunohistochemistry and real-time reverse transcription-polymerase chain reaction, we examined the correlation between HIPK2 and HIPK2-related protein expressions and the progression of some cutaneous epithelial neoplasms (i.e., actinic keratosis, Bowen's disease, keratoacanthoma, squamous cell carcinoma, and basal cell carcinoma). HIPK2 expression was distinct between preinvasive and invasive lesions: the expression decreased in keratoacanthoma (none of eight) and squamous cell carcinoma (five of 35) compared to actinic keratosis (12 of 19) and Bowen's disease (10 of 23) (P<0.001). HIPK2 expression was also negatively correlated with aggressiveness of basal cell carcinoma; high-risk subtypes showed lower HIPK2 expression than did low-risk subtypes (P<0.001). HIPK2 mRNA expression of each tumor group was significantly higher than that of normal skin. HIPK2 mRNA expression of each tumor group was not correlated with the relevant HIPK2 protein expression, which was consistent with previous studies. HIPK2 expression tends to be decreased along tumor progression and may be involved with the invasive potential, suggesting a possible tumor suppressor role for HIPK2.

Homeodomain-interacting protein kinase 2, a novel autoimmune regulator interaction partner, modulates promiscuous gene expression in medullary thymic epithelial cells.

Promiscuous expression of a plethora of tissue-restricted Ags (TRAs) by medullary thymic epithelial cells (mTECs) plays an essential role in T cell tolerance. Although the cellular mechanisms by which promiscuous gene expression (pGE) imposes T cell tolerance have been well characterized, the underlying molecular mechanisms remain poorly understood. The autoimmune regulator (AIRE) is to date the only validated molecule known to regulate pGE. AIRE is part of higher-order multiprotein complexes, which promote transcription, elongation, and splicing of a wide range of target genes. How AIRE and its partners mediate these various effects at the molecular level is still largely unclear. Using a yeast two-hybrid screen, we searched for novel AIRE-interacting proteins and identified the homeodomain-interacting protein kinase 2 (HIPK2) as a novel partner. HIPK2 partially colocalized with AIRE in nuclear bodies upon cotransfection and in human mTECs in situ. Moreover, HIPK2 phosphorylated AIRE in vitro and suppressed the coactivator activity of AIRE in a kinase-dependent manner. To evaluate the role of Hipk2 in modulating the function of AIRE in vivo, we compared whole-genome gene signatures of purified mTEC subsets from TEC-specific Hipk2 knockout mice with control mice and identified a small set of differentially expressed genes. Unexpectedly, most differentially expressed genes were confined to the CD80(lo) mTEC subset and preferentially included AIRE-independent TRAs. Thus, although it modulates gene expression in mTECs and in addition affects the size of the medullary compartment, TEC-specific HIPK2 deletion only mildly affects AIRE-directed pGE in vivo.

Effect of tyrosine autophosphorylation on catalytic activity and subcellular localisation of homeodomain-interacting protein kinases (HIPK).

BackgroundHomeodomain interacting protein kinases (HIPKs) function as modulators of cellular stress responses and regulate cell differentiation, proliferation and apoptosis. The HIPK family includes HIPK1, HIPK2 and HIPK3, which share a similar domain structure, and the more distantly related HIPK4. Although HIPKs phosphorylate their substrates on serine or threonine residues, it was recently reported that HIPK2 depends on the autophosphorylation of a conserved tyrosine in the activation loop to acquire full catalytic activity and correct subcellular localization. In this study we addressed the question whether tyrosine autophosphorylation in the activation loop has a similar function in the other members of the HIPK family.ResultsAll HIPKs contained phosphotyrosine when expressed in HeLa cells. Catalytically inactive point mutants were not tyrosine-phosphorylated, indicating that HIPKs are dual-specificity protein kinases that autophosphorylate on tyrosine residues. HIPK point mutants lacking the conserved tyrosine residue in the activation loop showed reduced catalytic activity towards peptide and protein substrates. Analysis of these mutants revealed that HIPK1, HIPK2 and HIPK3 but not HIPK4 are capable of autophosphorylating on other tyrosines. Inhibition of tyrosine phosphatase activity by treatment with vanadate enhanced global phosphotyrosine content of HIPK1, HIPK2 and HIPK3 but did not affect tyrosine phosphorylation in the activation loop. Mutation of the activation-loop tyrosines resulted in a redistribution of HIPK1 and HIPK2 from a speckle-like subnuclear compartment to the cytoplasm, whereas catalytically inactive point mutants showed the same pattern of cellular distribution as the wild type proteins. In contrast, mutation of the activating tyrosine did not increase the low percentage of cells with extranuclear HIPK3. HIPK4 was excluded from the nucleus with no difference between the wild type kinase and the point mutants.ConclusionsThese results show that HIPKs share the mechanism of activation by tyrosine autophosphorylation with the closely related DYRK family (dual-specificity tyrosine phosphorylation regulated kinase). However, members of the HIPK family differ regarding the subcellular localization and its dependence on tyrosine autophosphorylation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12964-014-0082-6) contains supplementary material, which is available to authorized users.

Role of HIPK2 in kidney fibrosis

Homeodomain interacting protein kinase 2 (HIPK2) functions as either a co-repressor or a co-activator of transcriptional regulators. Dysregulation of HIPK2 is associated with cancer and neurological disease. Recently, we found that HIPK2 is also an important driver of kidney fibrosis in the HIV-1 transgenic murine model, Tg26. HIPK2 protein levels are upregulated in the tubular epithelial cells of Tg26 mice as well as in kidney biopsies of patients with HIV-associated nephropathy, focal segmental glomerulosclerosis, diabetic nephropathy, and IgA nephropathy. We found that HIPK2 regulates pro-apoptotic, pro-fibrotic, and pro-inflammatory pathways including p53, transforming growth factor β (TGF-β)–SMAD family member 3 (Smad3), Notch, Wingless and INT-1 (Wnt)/β-catenin, and nuclear factor kappa-light-chain-enhancer of activated B cells in renal tubular epithelial cells. Our data suggest that HIPK2 may be a potential target for antifibrotic therapy. As mice with germline deletion of HIPK2 do not exhibit any phenotypic change under basal conditions, we do not expect significant side effects with specific HIPK2 inhibitors. However, potential effects of HIPK2 on tumor growth should be considered because of its tumor suppressor effects. Therefore, further understanding of structure–function relationships and post-translational modifications of HIPK2 are necessary to develop more specific drugs targeting the pro-fibrotic effects of HIPK2.

XAF1 directs apoptotic switch of p53 signaling through activation of HIPK2 and ZNF313.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a tumor suppressor that is frequently inactivated in many human cancers. However, the molecular mechanism underlying its growth-inhibitory function remains largely unknown. Here, we report that XAF1 forms a positive feedback loop with p53 and acts as a molecular switch in p53-mediated cell-fate decisions favoring apoptosis over cell-cycle arrest. XAF1 binds directly to the N-terminal proline-rich domain of p53 and thus interferes with E3 ubiquitin ligase MDM2 binding and ubiquitination of p53. XAF1 stimulates homeodomain-interacting protein kinase 2 (HIPK2)-mediated Ser-46 phosphorylation of p53 by blocking E3 ubiquitin ligase Siah2 interaction with and ubiquitination of HIPK2. XAF1 also steps up the termination of p53-mediated cell-cycle arrest by activating zinc finger protein 313 (ZNF313), a p21(WAF1)-targeting ubiquitin E3 ligase. XAF1 interacts with p53, Siah2, and ZNF313 through the zinc finger domains 5, 6, and 7, respectively, and truncated XAF1 isoforms preferentially expressed in cancer cells fail to form a feedback loop with p53. Together, this study uncovers a novel role for XAF1 in p53 stress response, adding a new layer of complexity to the mechanisms by which p53 determines cell-fate decisions.

Abstract 1617: Quantification of pancreatic cancer proteome &amp; phosphorylome: Indicates molecular events likely contributing to cancer &amp; activation status of drug targets

Abstract INTRODUCTION: LC-MS/MS proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed an analytical workflow to quantify the expression and phosphorylation status of thousands of proteins simultaneously in frozen resected human pancreatic tumor (T), relative to matched non-tumor (NT) tissue. These measurements enabled us to determine activity of anti-neoplastic drug targets and other signaling proteins. METHODS: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n=12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using Fe3+ and TiO2, prior to analysis. In-house, commercial and freeware bio-informatics platforms were used to identify relevant biological events from the complex dataset. RESULTS: Of 2,101 proteins identified, 152 demonstrated significant difference in expression between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as homeodomain interacting protein kinase 1 &amp; Myosin light chain kinase. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho GTPase signaling proteins such as Rho guanine nucleotide exchange factors &amp; Serine/threonine-protein kinase MRCKα) as well as proteins involved in disassembly of cell-cell junctions (tight junction, adherens junction) and formation of cell-extracellular matrix (ECM) junctions (focal adhesions). We quantified activator &amp; inhibitory phosphorylation sites on FYN, AKT1, HDAC1&amp;2, GSK3α&amp;β, RAF kinases, MAPKs (p38, ERK1&amp;2), PKCs, Casein Kinases and &amp;gt;20 others, as well as their downstream substrates some of which were often found to be highly modulated (≥ 2 fold) in T versus NT in different cases. CONCLUSION: Application of our LC-MS/MS proteomic workflow to frozen resected human pancreatic T versus NT tissue elucidated molecular events likely contributing to pancreatic cancer in each case, particularly those contributing to cell migration, and in future may help clinicians predict the best targeted anti-cancer therapy bespoke for an individual patient. Citation Format: David Britton, Yoh Zen, Stefan Selzer, Vikram Mitra, Alberto Quaglia, Debashis Sarker, Leandro Castellano, Justin Stebbing, Julia Gee, Rob Nicholson, Nigel Heaton, Ian Pike. Quantification of pancreatic cancer proteome &amp; phosphorylome: Indicates molecular events likely contributing to cancer &amp; activation status of drug targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1617. doi:10.1158/1538-7445.AM2014-1617

204P - Dj-1 Protein Expression in Intrinsic Subtype As a Predictor of Pathological Complete Remission After Neoadjuvant Chemotherapy in Breast Cancer Patients

ABSTRACT Aim: The DJ-1 gene was originally identified as an oncogene that, in conjunction with activated ras, and Parkinson's disease is associated with DJ-1/Parkinson protein 7 dysfunction. Neoplastic transformation is associated with the hyperactivity of DJ-1 . DJ-1 overexpression increases the resistance of neoplastic cells to apoptosis by inhibiting the phosphatase and tensin homolog(PTEN)-mediated protein kinase B/Akt pathway and/or other apoptotic pathways,including death-associated protein 6 and homeodomain-interacting protein kinase1. Recent genetic studies showed that, in addition to apoptosis pathways, DJ-1 is also involved in cellular defense against reactive oxygen species. The activity of apoptotic and cellular defense pathways is key in determining drug sensitivity. DJ-1 overexpression is associated with various cancers. We reported that low DJ-1 protein expression is a significant predictor of pCR after chemotherapy in reast cancer patients.(Kawate, BCRT2013) In this time, we will report that we classified Luminal-A subtype to new Lum-A and Lum-B subtype by ki67 and reanalyzed results. Methods: Expression of DJ-1 in pre-therapeutic needle biopsies and surgical specimens obtained from 198 breast cancer cases that received neoadjuvant chemotherapy was determined using immunohistochemistry and in situ hybridization.Chemotherapy comprised epirubicin / cyclophosphamide taxane-based regimens with or without trastuzumab. Univariate and multivariate analyses were used to evaluate the predictive value of DJ-1 on pCR. Results: Low DJ-1 protein expression was detected in 46.0 % (91/198) of all breast cancer cases and in 80.4 % (37/46) of pCR cases. In multivariate analysis, DJ-1 expression(k2 = 16.05, p Conclusions: Low DJ-1 protein expression is a significant predictor of pCR after neoadjuvant chemotherapy at any Sub Type in breast cancer patients. Disclosure: All authors have declared no conflicts of interest.

Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1γ pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor.

Hipk2 and PP1c Cooperate to Maintain Dvl Protein Levels Required for Wnt Signal Transduction

The phosphoprotein Dishevelled (Dvl) is a common essential component of Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. Here, we show that homeodomain-interacting protein kinase 2 (Hipk2) facilitates protein phosphatase 1 catalytic subunit (PP1c)-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/β-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. We also show that Wnt-3a, a Wnt/β-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/β-catenin signaling.