Androgen receptor contributes to repairing DNA damage induced by inflammation and oxidative stress in prostate cancer.

Background: Androgen deprivation therapy remains the first-line therapy option for prostate cancer, mostly resulting in the transition of the disease to a castration-resistant state. The lack of androgen signaling during therapy affects various cellular processes, which sometimes paradoxically contributes to cancer progression. As androgen receptor (AR) signaling is known to contribute to oxidative stress regulation, loss of AR may also affect DNA damage level and the response mechanism in oxidant and inflammatory conditions of the prostate tumor microenvironment. Therefore, this study aimed to investigate the role of AR and AR-regulated tumor suppressor NKX3.1 upon oxidative stress-induced DNA damage response (DDR) in the inflammatory tumor microenvironment of the prostate. Materials and methods: Intracellular reactive oxygen species (ROS) level was induced by either inflammatory conditioned media obtained from lipopolysaccharide-induced macrophages or oxidants and measured by dichlorodihydrofluorescein diacetate. In addition to this, DNA damage was subsequently quantified by counting gH2AX foci using an immunofluorescence-based Aklides platform. Altered expression of proteins function in DDR detected by western blotting. Results: Cellular levels of ROS and ROS-induced DNA double-strand break damage were analyzed in the absence and presence of AR signaling upon treatment of prostate cancer cells by either oxidants or inflammatory microenvironment exposure. The results showed that AR suppresses intracellular ROS and contributes to DNA damage recognition under oxidant conditions. Besides, increased DNA damage due to loss of NKX3.1 under inflammatory conditions was alleviated by its overexpression. Moreover, the activation of the DDR mediators caused by AR and NKX3.1 activation in androgen-responsive and castration-resistant prostate cancer cells indicated that the androgen receptor function is essential both in controlling oxidative stress and in activating the ROS-induced DDR. Conclusion: Taken together, it is concluded that the regulatory function of androgen receptor signaling has a vital function in the balance between antioxidant response and DDR activation.

NKX3.1 Expression Contributes to Epithelial-Mesenchymal Transition of Prostate Cancer Cells.

Studies demonstrate that inflammation synergizes with high-grade aggressive prostate tumor development and ultimately metastatic spread, in which a lot of work has been done in recent years. However, the clear mechanism of inflammation inciting prostate cancer remains largely uncharacterized. Our previous study has shown that the conditioned media (CM)-mediated LNCaP cell migration is partially correlated with the loss of expression of the tumor suppressor NKX3.1. Here, we continue to investigate the inflammation-mediated migration of prostate cancer cells, and the role of NKX3.1 in this process to gain insights into cell migration-related changes comprehensively. Earlier, the model of inflammation in the tumor microenvironment have been optimized by our research group; here, we continue to investigate the time-dependent effect of CM exposure together with NKX3.1 changes, in which we observed that these changes play important roles in gaining heterogeneous epithelial-to-mesenchymal transition (EMT) phenotype. Hence, this is an important parameter of tumor progression; we depleted NKX3.1 expression using the CRISPR/Cas9 system and examined the migrating cell clusters after exposure to inflammatory cytokines. We found that the migrated cells clearly demonstrate reversible loss of E-cadherin expression, which is consistent with subsequent vimentin expression alterations in comparison to control cells. Moreover, the data suggest that the AR-mediated transcriptional program also contributes to mesenchymal-to-epithelial transition (MET) in prostate cancer progression. Furthermore, the quantitative proteomic analysis showed that migrated subpopulations from the same cell line presented different phenotypes in which the proteins overexpressed are involved in cell metabolism and RNA processing. According to KEGG pathway analysis, the ABC transporters were found to be the most significant. Thus, the dynamic process of cellular migration favors diverse genetic compositions under changing tumor microenvironments. The different levels of invasiveness are supported by shifting the cells in between these EMT and MET phenotypes.

The Regulation of Cyclins and Cyclin-Dependent Kinases in the Development of Gastric Cancer.

Gastric cancer predominantly occurs in adenocarcinoma form and is characterized by uncontrolled growth and metastases of gastric epithelial cells. The growth of gastric cells is regulated by the action of several major cell cycle regulators including Cyclins and Cyclin-dependent kinases (CDKs), which act sequentially to modulate the life cycle of a living cell. It has been reported that inadequate or over-activity of these molecules leads to disturbances in cell cycle dynamics, which consequently results in gastric cancer development. Manny studies have reported the key roles of Cyclins and CDKs in the development and progression of the disease in either in vitro cell culture studies or in vivo models. We aimed to compile the evidence of molecules acting as regulators of both Cyclins and CDKs, i.e., upstream regulators either activating or inhibiting Cyclins and CDKs. The review entails an introduction to gastric cancer, along with an overview of the involvement of cell cycle regulation and focused on the regulation of various Cyclins and CDKs in gastric cancer. It can act as an extensive resource for developing new hypotheses for future studies.

HN1 Is Enriched in the S-Phase, Phosphorylated in Mitosis, and Contributes to Cyclin B1 Degradation in Prostate Cancer Cells.

HN1 has previously been shown as overexpressed in various cancers. In Prostate cancer, it regulates AR signaling and centrosome-related functions. Previously, in two different studies, HN1 expression has been observed as inversely correlated with Cyclin B1. However, HN1 interacting partners and the role of HN1 interactions in cell cycle pathways have not been completely elucidated. Therefore, we used Prostate cancer cell lines again and utilized both transient and stable inducible overexpression systems to delineate the role of HN1 in the cell cycle. HN1 characterization was performed using treatments of kinase inhibitors, western blotting, flow cytometry, immunofluorescence, cellular fractionation, and immunoprecipitation approaches. Our findings suggest that HN1 overexpression before mitosis (post-G2), using both transient and stable expression systems, leads to S-phase accumulation and causes early mitotic exit after post-G2 overexpression. Mechanistically, HN1 interacted with Cyclin B1 and increased its degradation via ubiquitination through stabilized Cdh1, which is a co-factor of the APC/C complex. Stably HN1-expressing cells exhibited a reduced Cdt1 loading onto chromatin, demonstrating an exit from a G1 to S phenotype. We found HN1 and Cdh1 interaction as a new regulator of the Cyclin B1/CDK1 axis in mitotic regulation which can be explored further to dissect the roles of HN1 in the cell cycle.

HN1 interacts with γ-tubulin to regulate centrosomes in advanced prostate cancer cells.

Prostate cancer is one of the most common cancer for men worldwide with advanced forms showing supernumerary or clustered centrosomes. Hematological and neurological expressed 1 (HN1) also known as Jupiter Microtubule Associated Homolog 1 (JPT1) belongs to a small poorly understood family of genes that are evolutionarily conserved across vertebrate species. The co-expression network of HN1 from the TCGA PRAD dataset indicates the putative role of HN1 in centrosome-related processes in the context of prostate cancer. HN1 expression is low in normal RWPE-1 cells as compared to cancerous androgen-responsive LNCaP and androgen insensitive PC-3 cells. HN1 overexpression resulted in differential response for cell proliferation and cell cycle changes in RWPE-1, LNCaP, and PC-3 cells. Since HN1 overexpression increased the proliferation rate in PC-3 cells, these cells were used for functional characterization of HN1 in advanced prostate carcinogenesis. Furthermore, alterations in HN expression led to an increase in abnormal to normal nuclei ratio and increased chromosomal aberrations in PC-3 cells. We observed the co-localization of HN1 with γ-tubulin foci in prostate cancer cells, further validated by immunoprecipitation. HN1 was observed as physically associated with γ-tubulin and its depletion led to increased γ-tubulin foci and disruption in microtubule spindle assembly. Higher HN1 expression was correlated with prostate cancer as compared to normal tissues. The restoration of HN1 expression after silencing suggested that it has a role in centrosome clustering, implicating a potential role of HN1 in cell division as well as in prostate carcinogenesis warranting further studies.

Oxidative DNA Damage-Mediated Genomic Heterogeneity Is Regulated by NKX3.1 in Prostate Cancer.

The 8-hydroxy-2'-deoxyguanosine (8-OHdG) damages are base damages induced by reactive oxygen species. We aimed to investigate the role of Androgen Receptor and NKX3.1 in 8-OHdG formation and repair activation by quantitating the DNA damage using Aklides.NUK system. The data demonstrated that the loss of NKX3.1 resulted in increased oxidative DNA damage and its overexpression contributes to the removal of menadione-induced 8-OHdG damage even under oxidative stress conditions. Moreover, 8-oxoguanine DNA glycosylase-1 (OGG1) expression level positively correlates to NKX3.1 expression. Also in this study, first time a reliable cell-based quantitation method for 8-OHdG damages is reported and used for data collection.

Cycloartane-type sapogenol derivatives inhibit NFκB activation as chemopreventive strategy for inflammation-induced prostate carcinogenesis.

Chronic inflammation is associated to 25% of cancer cases according to epidemiological data. Therefore, inhibition of inflammation-induced carcinogenesis can be an efficient therapeutic approach for cancer chemoprevention in drug development studies. It is also determined that anti-inflammatory drugs reduce cancer incidence. Cell culture-based in vitro screening methods are used as a fast and efficient method to investigate the biological activities of the biomolecules. In addition, saponins are molecules that are isolated from natural sources and are known to have potential for tumor inhibition. Studies on the preparation of analogues of cycloartane-type sapogenols (9,19-cyclolanostanes) have so far been limited. Therefore we have decided to direct our efforts toward the exploration of new anti-tumor agents prepared from cycloastragenol and its production artifact astragenol. The semi-synthetic derivatives were prepared mainly by oxidation, condensation, alkylation, acylation, and elimination reactions. After preliminary studies, five sapogenol analogues, two of which were new compounds (2 and 3), were selected and screened for their inhibitory activity on cell viability and NFκB signaling pathway activity in LNCaP prostate cancer cells. We found that the astragenol derivatives 1 and 2 as well as cycloastragenol derivatives 3, 4, and 5 exhibited strong inhibitory activity on NFκB signaling leading the repression of NFκB transcriptional activation and suppressed cell proliferation. The results suggested that these molecules might have significant potential for chemoprevention of prostate carcinogenesis induced by inflammatory NFκB signaling pathway.

Automated Cell-Based Quantitation of 8-OHdG Damage.

Detection of 8-OHdG-base damage has been a big challenge for decades, though different analytical methods are developed. The recent approaches that are used for quantitating either the total amount of base damage or the amount of base damage per cell from different sources of samples are not automated. We have developed a method for automated damage detection from a single cell and applied it to 8-OHdG quantitation.

The redox biology network in cancer pathophysiology and therapeutics.

The review pinpoints operational concepts related to the redox biology network applied to the pathophysiology and therapeutics of solid tumors. A sophisticated network of intrinsic and extrinsic cues, integrated in the tumor niche, drives tumorigenesis and tumor progression. Critical mutations and distorted redox signaling pathways orchestrate pathologic events inside cancer cells, resulting in resistance to stress and death signals, aberrant proliferation and efficient repair mechanisms. Additionally, the complex inter-cellular crosstalk within the tumor niche, mediated by cytokines, redox-sensitive danger signals (HMGB1) and exosomes, under the pressure of multiple stresses (oxidative, inflammatory, metabolic), greatly contributes to the malignant phenotype. The tumor-associated inflammatory stress and its suppressive action on the anti-tumor immune response are highlighted. We further emphasize that ROS may act either as supporter or enemy of cancer cells, depending on the context. Oxidative stress-based therapies, such as radiotherapy and photodynamic therapy, take advantage of the cytotoxic face of ROS for killing tumor cells by a non-physiologically sudden, localized and intense oxidative burst. The type of tumor cell death elicited by these therapies is discussed. Therapy outcome depends on the differential sensitivity to oxidative stress of particular tumor cells, such as cancer stem cells, and therefore co-therapies that transiently down-regulate their intrinsic antioxidant system hold great promise. We draw attention on the consequences of the damage signals delivered by oxidative stress-injured cells to neighboring and distant cells, and emphasize the benefits of therapeutically triggered immunologic cell death in metastatic cancer. An integrative approach should be applied when designing therapeutic strategies in cancer, taking into consideration the mutational, metabolic, inflammatory and oxidative status of tumor cells, cellular heterogeneity and the hypoxia map in the tumor niche, along with the adjoining and systemic effects of oxidative stress-based therapies.

Inflammation contributes to NKX3.1 loss and augments DNA damage but does not alter the DNA damage response via increased SIRT1 expression.

The oxidative stress response is a cellular defense mechanism that protects cells from oxidative damage and cancer development. The exact molecular mechanism by which reactive oxygen species (ROS) contribute to DNA damage and increase genome instability in prostate cancer merits further investigation. Here, we aimed to determine the effects of NKX3.1 loss on antioxidant defense in response to acute and chronic inflammation in an in vitro model. Oxidative stress-induced DNA damage resulted in increased H2AX((S139)) phosphorylation (a hallmark of DNA damage), along with the degradation of the androgen receptor (AR), p53 and NKX3.1, upon treatment with conditioned medium (CM) obtained from activated macrophages or H2O2. Furthermore, the expression and stability of SIRT1 were increased by CM treatment but not by H2O2 treatment, although the level of ATM((S1981)) phosphorylation was not changed compared with controls. Moreover, the deregulated antioxidant response resulted in upregulation of the pro-oxidant QSCN6 and the antioxidant GPX2 and downregulation of the antioxidant GPX3 after CM treatment. Consistently, the intracellular ROS level increased after chronic treatment, leading to a dose-dependent increase in the ability of LNCaP cells to tolerate oxidative damage. These data suggest that the inflammatory microenvironment is a major factor contributing to DNA damage and the deregulation of the oxidative stress response, which may be the underlying cause of the increased genetic heterogeneity during prostate tumor progression.

TNFα-mediated loss of ß-catenin/E-cadherin association and subsequent increase in cell migration is partially restored by NKX3.1 expression in prostate cells.

Inflammation-induced carcinogenesis is associated with increased proliferation and migration/invasion of various types of tumor cells. In this study, altered ß-catenin signaling upon TNFα exposure, and relation to loss of function of the tumor suppressor NKX3.1 was examined in prostate cancer cells. We used an in vitro prostate inflammation model to demonstrate altered sub-cellular localization of ß-catenin following increased phosphorylation of Akt(S473) and GSK3ß(S9). Consistently, we observed that subsequent increase in ß-catenin transactivation enhanced c-myc, cyclin D1 and MMP2 expressions. Consequently, it was also observed that the ß-catenin-E-cadherin association at the plasma membrane was disrupted during acute cytokine exposure. Additionally, it was demonstrated that disrupting cell-cell interactions led to increased migration of LNCaP cells in real-time migration assay. Nevertheless, ectopic expression of NKX3.1, which is degraded upon proinflammatory cytokine exposure in inflammation, was found to induce the degradation of ß-catenin by inhibiting Akt(S473) phosphorylation, therefore, partially rescued the disrupted ß-catenin-E-cadherin interaction as well as the cell migration in LNCaP cells upon cytokine exposure. As, the disrupted localization of ß-catenin at the cell membrane as well as increased Akt(S308) priming phosphorylation was observed in human prostate tissues with prostatic inflammatory atrophy (PIA), high-grade prostatic intraepithelial neoplasia (H-PIN) and carcinoma lesions correlated with loss of NKX3.1 expression. Thus, the data indicate that the ß-catenin signaling; consequently sub-cellular localization is deregulated in inflammation, associates with prostatic atrophy and PIN pathology.

HOXB13 contributes to G1/S and G2/M checkpoint controls in prostate.

HOXB13 is a homeobox protein that is expressed in normal adult prostate and colon tissues; however, its deregulated expression was evidenced in various malignancies. To characterize the putative role of HOXB13 in cell cycle progression, we performed overexpression and siRNA-mediated knockdown studies in PC-3 and LNCaP cells. Immunohistochemistry (IHC) analyses were also performed using formalin-fixed, paraffin-embedded tissues containing normal, H-PIN and PCa sections from 20 radical prostatectomy specimens. Furthermore, when the role of HOXB13 during cell cycle progression, association with cyclins, cell growth and colony formation using real-time cell proliferation were assessed, we observed that ectopic expression of HOXB13 accumulated cells at G1 through decreasing the cyclin D1 level by promoting its ubiquitination and degradation. This loss slowed S phase entry in both cell lines examined, with an associated decrease in pRb((S780) and (S795)) phosphorylations. Contrary, siRNA-mediated depletion of HOXB13 expression noticeably increased cyclin levels, stabilized E2F1 and CDC25C, subsequent to increased pRb phosphorylations. This increase in Cyclin B1 and CDC25C both together facilitated activation of cyclin B complex via dephosphorylating CDK1((T14Y15)), and resumed the G2/M transition after nocodazole synchronization. Despite an increase in the total expression level and cytoplasmic retention of HOXB13 in H-PIN and PCa samples that were observed via IHC evaluation of prostate tissues, HOXB13 depletion facilitated to an increase in PC-3 and LNCaP cell proliferation. Thus, we suggest that HOXB13 expression is required for cell cycle regulation, and increases by an unknown mechanism consequent to its functional loss in cancer.

Inflammation-mediated abrogation of androgen signaling: an in vitro model of prostate cell inflammation.

As a link between inflammation and cancer has been reported in many studies, we established an in vitro model of prostatic inflammation to investigate the loss of androgen receptor (AR)-mediated signaling in androgen responsive prostate cell lines. First, the U937 monocyte cell line was differentiated into macrophages using phorbol acetate (PMA), and cells were induced with lipopolysaccharide (LPS) for cytokine secretion. Next, the cytokine levels (TNFα, IL-6, and IL1ß) in conditioned media (CM) were analyzed. Prostate cells were then fed with CM containing varying concentrations of TNFα, and IkB degradation, nuclear factor kappa B (NFκB) translocation and transactivation, and the expression of matrix metalloproteinase-8 (MMP8) and matrix metalloproteinase-9 (MMP9) were then assessed. As a result of CM treatment, ubiquitin-mediated AR degradation, which was restored using anti-TNFα antibody neutralization, led to both a decrease in KLK4, PSA, and NKX3.1 expression levels and the upregulation of GPX2. In addition to the loss of AR, acute and chronic CM exposure resulted in p53 degradation and consequent p21 downregulation, which was also restored by either androgen administration or ectopic NKX3.1 expression via the stabilization of MDM2 levels in LNCaP cells. Additionally, CM treatment enhanced H2AX((S139)) phosphorylation (a hallmark of DNA damage) and genetic heterogeneity in the absence of androgens in prostate cells without activating mitochondrial apoptosis. Thus, the data suggest that inflammatory cytokine exposure results in the loss of AR and p53 signaling in prostate cells and facilitates genetic heterogeneity via ROS accumulation to promote prostate carcinogenesis.

Early life stress-induced histone acetylations correlate with activation of the synaptic plasticity genes Arc and Egr1 in the mouse hippocampus.

Early life stress (ELS) programs the developing organism and influences the development of brain and behavior. We tested the hypothesis that ELS-induced histone acetylations might alter the expression of synaptic plasticity genes that are critically involved in the establishment of limbic brain circuits. Maternal separation (MS) from postnatal day 14-16 was applied as ELS and two immediate early genes underlying experience-induced synaptic plasticity, Arc and early growth response 1 (Egr1) were analyzed. We show here that repeated ELS induces a rapid increase of Arc and Egr1 in the mouse hippocampus. Furthermore, immunoblotting revealed that these changes are paralleled by histone modifications, reflected by increased acetylation levels of H3 and H4. Most importantly, using native Chromatin immunoprecipitation quantitative PCR (nChIP-qPCR), we show for the first time a correlation between elevated histone acetylation and increased Arc and Egr1 expression in response to ELS. These rapid epigenetic changes are paralleled by increases of dendritic complexity and spine number of hippocampal CA3 pyramidal neurons in ELS animals at weaning age. Our results are in line with our working hypothesis that ELS induces activation of synaptic plasticity genes, mediated by epigenetic mechanisms. These events are assumed to represent early steps in the adaption of neuronal networks to a stressful environment.

Androgen regulated HN1 leads proteosomal degradation of androgen receptor (AR) and negatively influences AR mediated transactivation in prostate cells.

We recently reported that hematological and neurological expressed 1 (HN1) is a ubiquitously expressed, EGF-regulated gene. Expression of HN1 in prostate cell lines down-regulates PI3K-dependent Akt activation. Here, we investigate whether HN1 is regulated by androgens through the putative androgen response elements (AREs) found in its promoter. Knockdown of HN1 expression by siRNA silencing leads to an increase in Akt((S473)) phosphorylation, resulting in the translocation of androgen receptor (AR) to the nucleus; these effects can be abrogated by the non-specific Akt inhibitor LY294002 but not by the ERK inhibitor PD98059. Furthermore, HN1 overexpression correlates with an increase in ubiquitination-mediated degradation (a consequence of the decrease in S213/210 phosphorylation of AR), ultimately resulting in the down-regulation of AR-mediated expression of the KLK3, KLK4, NKX3.1 and STAMP2 genes. We also found that HN1 overexpression suppresses colony formation as well as R1881-mediated growth in LNCaP cells, while it has the opposite effect (increasing colony formation but not proliferation) in PC-3 and DU145 cells. Therefore, we suggest that HN1 maintains a balance between the androgen-regulated nuclear translocation of AR and steady-state Akt phosphorylation, predominantly in the absence of androgens. If so, the balance between cell growth and EGF- and AR-signaling must be tightly regulated by HN1. This work has important implications for prostate cancer research, as AR, EGFR and HN1 are known to be highly expressed in prostate adenocarcinomas.

NKX3.1 contributes to S phase entry and regulates DNA damage response (DDR) in prostate cancer cell lines.

NKX3.1 is an androgen-regulated homeobox gene that encodes a tissue-restricted transcription factor, which plays an important role in the differentiation of the prostate epithelium. Thus, the role of NKX3.1 as a functional topoisomerase I activity enhancer in cell cycle regulation and the DNA damage response (DDR) was explored in prostate cancer cell lines. As an early response to DNA damage following CPT-11 treatment, we found that there was an increase in the γH2AX(S139) foci number and that total phosphorylation levels were reduced in PC-3 cells following ectopic NKX3.1 expression as well as in LNCaP cells following androgen administration. Furthermore, upon drug treatment, the increase in ATM(S1981) phosphorylation was reduced in the presence of NKX3.1 expression, whereas DNA-PKcs expression was increased. Additionally, phosphorylation of CHK2(T68) and NBS1(S343) was abrogated by ectopic NKX3.1 expression, compared with the increasing levels in control PC-3 cells in a time-course experiment. Finally, NKX3.1 expression maintained a high cyclin D1 expression level regardless of drug treatment, while total γH2AX(S139) phosphorylation remained depleted in PC-3, as well as in LNCaP, cells. Thus, we suggest that androgen regulated NKX3.1 maintains an active DDR at the intra S progression and contributes to the chemotherapeutic resistance of prostate cancer cells to DNA damaging compounds.

ALCAPs induce mitochondrial apoptosis and activate DNA damage response by generating ROS and inhibiting topoisomerase I enzyme activity in K562 leukemia cell line.

Endemic Alkanna cappadocica was used to isolate novel antitumor molecules from Turkish landscapes in our previous studies. In this study, deoxyalkannin (ALCAP1), ß,ß-dimethylacrylalkannin (ALCAP2), acetylalkannin (ALCAP3), and alkannin (ALCAP4) as well as the novel isolated compounds 5-methoxydeoxyalkannin (ALCAP5), 8-methoxydeoxyalkannin (ALCAP6), 5-methoxyacetylalkannin (ALCAP7), 5-methoxy-ß,ß-dimethylacrylalkannin (ALCAP8) were characterized. The topoisomerase I (topo I) inhibitory activity of ALCAPs was investigated using in vitro plasmid relaxation assay and found that ALCAP2, 3, 4 and 7 were potent inhibitors at 2-6µM concentrations. Further, DNA damage response to ALCAP treatments was also studied by measuring the H2AX((S139)) and ATM((S1981)) phosphorylations. ALCAP2, 7 and 8 induced the DNA damage and apoptosis, consistently resulted in PARP cleavage at nanomolar concentrations in K562 leukemia cells. Moreover, when the free radical (ROS) generating capacity of the compounds was studied by 2',7'-dichlorofluorescein-diacetate assay using flow cytometry, we found that a known antioxidant N-acetyl-cysteine almost completely abrogated the H2AX((S139)) phosphorylations and the caspase 3 cleavage and activation. Thus, γH2AX((S139)) foci formation remained higher than the control, and an increase in CHK2((T68)) phosphorylation was observed by ALCAP2 and 7 treatments suggested that, these compounds can be potential therapeutics against tumor cell growth because of their unique DNA damaging abilities additional to enzyme inhibition similar to those of doxorubicin.

Ubiquitously expressed hematological and neurological expressed 1 downregulates Akt-mediated GSK3ß signaling, and its knockdown results in deregulated G2/M transition in prostate cells.

As the molecular mechanism of ß-catenin deregulation is not well understood, and stabilized ß-catenin is known to translocate into the nucleus and activate genes for proliferation, a novel regulatory factor, hematological and neurological expressed 1 (HN1), for Akt-GSK3ß-ß-catenin axis is reported here. In our studies, HN1 gene structure was characterized. HN1 expression was found to be epidermal growth factor-responsive in PC-3 cells, and protein expression was also upregulated in PC-3 and LNCaP but not in DU145 cells. Additionally, HN1 was found to be downregulated by the specific AKT inhibitor wortmannin but not with PI3K or MAPK inhibitors, LY294002 and PD98059, respectively, in PC-3 and MCF-7 cells. Further, siRNA-mediated knockdown of HN1 resulted in considerable increase in Akt((S473)) and GSK3ß((S9),(Y216)) phosphorylations; moreover, subsequent accumulation of ß-catenin, increase in c-myc expression, and nuclear accumulation of cyclin D1 were observed in PC-3 cells. Knockdown of HN1 also resulted in prolongation of G(1) phase in cell cycle, increasing tetraploidy, presumably because of cells escaping from abnormal mitosis in PC-3 cells. Consistently, overexpression of HN1 reversed the cell-cycle-specific observations, resulted in accumulation of cells in G(2)/M, and reduced the proliferation rate, which were investigated using flow cytometry and methylthiazol tetrazolium assays. As activating mutations of ß-catenin have been demonstrated in late-stage tumors, and ß-catenin stabilization was correlated with poor prognosis in previous reports, epidermal growth factor-upregulated HN1 expression might have a role in deregulating the AKT-GSK3ß((S9))-mediated signaling as a novel compensating mechanism.