G9a in Cancer: Mechanisms, Therapeutic Advancements, and Clinical Implications.

G9a, also named EHMT2, is a histone 3 lysine 9 (H3K9) methyltransferase responsible for catalyzing H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). G9a contributes to various aspects of embryonic development and tissue differentiation through epigenetic regulation. Furthermore, the aberrant expression of G9a is frequently observed in various tumors, particularly in prostate cancer, where it contributes to cancer pathogenesis and progression. This review highlights the critical role of G9a in multiple cancer-related processes, such as epigenetic dysregulation, tumor suppressor gene silencing, cancer lineage plasticity, hypoxia adaption, and cancer progression. Despite the increased research on G9a in prostate cancer, there are still significant gaps, particularly in understanding its interactions within the tumor microenvironment and its broader epigenetic effects. Furthermore, this review discusses the recent advancements in G9a inhibitors, including the development of dual-target inhibitors that target G9a along with other epigenetic factors such as EZH2 and HDAC. It aims to bring together the existing knowledge, identify gaps in the current research, and suggest future directions for research and treatment strategies.

Deciphering the Transcription Factor Landscape in Neuroendocrine Prostate Cancer Progression: A Novel Approach to Understand NE Transdifferentiation.

Background and Objective: Prostate cancer (PCa) is a leading cause of cancer mortality in men, with neuroendocrine prostate cancer (NEPC) representing a particularly resistant subtype. The role of transcription factors (TFs) in the progression from prostatic adenocarcinoma (PRAD) to NEPC is poorly understood. This study aims to identify and analyze lineage-specific TF profiles in PRAD and NEPC and illustrate their dynamic shifts during NE transdifferentiation. Methods: A novel algorithmic approach was developed to evaluate the weighted expression of TFs within patient samples, enabling a nuanced understanding of TF landscapes in PCa progression and TF dynamic shifts during NE transdifferentiation. Results: unveiled TF profiles for PRAD and NEPC, identifying 126 shared TFs, 46 adenocarcinoma-TFs, and 56 NEPC-TFs. Enrichment analysis across multiple clinical cohorts confirmed the lineage specificity and clinical relevance of these lineage-TFs signatures. Functional analysis revealed that lineage-TFs are implicated in pathways critical to cell development, differentiation, and lineage determination. Novel lineage-TF candidates were identified, offering potential targets for therapeutic intervention. Furthermore, our longitudinal study on NE transdifferentiation highlighted dynamic TF expression shifts and delineated a three-phase hypothesis for the process comprised of de-differentiation, dormancy, and re-differentiation. and proposing novel insights into the mechanisms of PCa progression. Conclusion: The lineage-specific TF profiles in PRAD and NEPC reveal a dynamic shift in the TF landscape during PCa progression, highlighting three distinct phases of NE transdifferentiation.

Clinically-observed FOXA1 mutations upregulate SEMA3C through transcriptional derepression in prostate cancer.

FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.

Semaphorin 3C promotes de novo steroidogenesis in prostate cancer cells.

Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.

Influence of ADT on B7-H3 expression during CRPC progression from hormone-naïve prostate cancer.

Androgen deprivation therapy (ADT) is the standard care for advanced prostate cancer (PCa) patients. Unfortunately, although tumors respond well initially, they enter dormancy and eventually progress to fatal/incurable castration-resistant prostate cancer (CRPC). B7-H3 is a promising new target for PCa immunotherapy. CD276 (B7-H3) gene has a presumptive androgen receptor (AR) binding site, suggesting potential AR regulation. However, the relationship between B7-H3 and AR is controversial. Meanwhile, the expression pattern of B7-H3 following ADT and during CRPC progression is largely unknown, but critically important for identifying patients and determining the optimal timing of B7-H3 targeting immunotherapy. In this study, we performed a longitudinal study using our unique PCa patient-derived xenograft (PDX) models and assessed B7-H3 expression during post-ADT disease progression. We further validated our findings at the clinical level in PCa patient samples. We found that B7-H3 expression was negatively regulated by AR during the early phase of ADT treatment, but positively associated with PCa proliferation during the remainder of disease progression. Our findings suggest its use as a biomarker for diagnosis, prognosis, and ADT treatment response, and the potential of combining ADT and B7-H3 targeting immunotherapy for hormone-naïve PCa treatment to prevent fatal CRPC relapse.

Dependency of Tamoxifen Sensitive and Resistant ER+ Breast Cancer Cells on Semaphorin 3C (SEMA3C) for Growth.

Estrogen receptor positive (ER+) breast cancer (BCa) accounts for the highest proportion of breast cancer-related deaths. While endocrine therapy is highly effective for this subpopulation, endocrine resistance remains a major challenge and the identification of novel targets is urgently needed. Previously, we have shown that Semaphorin 3C (SEMA3C) is an autocrine growth factor that drives the growth and treatment resistance of various cancers, but its role in breast cancer progression and endocrine resistance is poorly understood. Here, we report that SEMA3C plays a role in maintaining the growth of ER+ BCa cells and is a novel, tractable therapeutic target for the treatment of ER+ BCa patients. Analyses of publicly available clinical datasets indicate that ER+ BCa patients express significantly higher levels of SEMA3C mRNA than other subtypes. Furthermore, SEMA3C mRNA expression was positively correlated with ESR1 mRNA expression. ER+ BCa cell lines (MCF7 and T47D) expressed higher levels of SEMA3C mRNA and protein than a normal mammary epithelial MCF10A cell line. ER siRNA knockdown was suppressed, while dose-dependent beta-estradiol treatment induced SEMA3C expression in both MCF7 and T47D cells, suggesting that SEMA3C is an ER-regulated gene. The stimulation of ER+ BCa cells with recombinant SEMA3C activated MAPK and AKT signaling in a dose-dependent manner. Conversely, SEMA3C silencing inhibited Estrogen Receptor (ER) expression, MAPK and AKT signaling pathways while simultaneously inducing apoptosis, as monitored by flow cytometry and Western blot analyses. SEMA3C silencing significantly inhibited the growth of ER+ BCa cells, implicating a growth dependency of ER+ BCa cells on SEMA3C. Moreover, the analysis of tamoxifen resistant (TamR) cell models (TamC3 and TamR3) showed that SEMA3C levels remain high despite treatment with tamoxifen. Tamoxifen-resistant cells remained dependent on SEMA3C for growth and survival. Treatment with B1SP Fc fusion protein, a SEMA3C pathway inhibitor, attenuated SEMA3C-induced signaling and growth across a panel of tamoxifen sensitive and resistant ER+ breast cancer cells. Furthermore, SEMA3C silencing and B1SP treatment were associated with decreased EGFR signaling in TamR cells. Here, our study implicates SEMA3C in a functional role in ER+ breast cancer signaling and growth that suggests ER+ BCa patients may benefit from SEMA3C-targeted therapy.

SEMA3C induces androgen synthesis in prostatic stromal cells through paracrine signaling.

BACKGROUND: Castration resistant prostate cancer progression is associated with an acquired intratumoral androgen synthesis. Signaling pathways that can upregulate androgen production in prostate tumor microenvironment are not entirely known. In this study, we investigate the potential effect of a secreted signaling protein named semaphorin 3C (SEMA3C) on steroidogenic activities of prostatic stromal cells. METHODS: We treated human primary prostate stromal cells (PrSC) with 1uM recombinant SEMA3C protein and androgen precursor named dehydroepiandrosterone (DHEA) 1.7uM. Also, to test SEMA3C's effect on the conversion of DHEA to androgens, we exposed PrSCs to the conditioned media derived from LNCaP cells that were transduced with a lentiviral vector harboring full length SEMA3C gene or empty vector (CM-LNSEMA3C or CM-LNVector ). Then, liquid chromatography-mass spectrometry was performed on steroids isolated from PrSCs media. The messnger RNA expression of steroidogenic enzymes in PrSCs was quantified by quantitative polymerase chain reaction. RESULTS: Recombinant SEMA3C had no effect on steroidogenic activities in PrSCs. However, key steroidogenic enzymes expression and androgen synthesis were upregulated in PrSCs treated with CM-LNSEMA3C , compared to those treated with CM-LNVector . These results suggest that steroidogenic activities in PrSCs were upregulated in response to a signaling factor in CM-LNSEMA3C , other than SEMA3C. We hypothesized that SEMA3C overexpression in LNCaP cells affected androgen synthesis in PrSCs through sonic hedgehog (Shh) pathway activation in PrSCs. We verified this effect by blocking Shh signaling with smoothened antagonist. CONCLUSION: Based on known ability of Shh signaling pathway to activate steroidogenesis in stromal cells, we suggest that SEMA3C overexpression in LNCaP cells can upregulate Shh which in turn is able to stimulate steroidogenic activities in prostatic stromal cells.

Interleukin-10 and Small Molecule SHIP1 Allosteric Regulators Trigger Anti-inflammatory Effects through SHIP1/STAT3 Complexes.

The anti-inflammatory actions of interleukin-10 (IL10) are thought to be mediated primarily by the STAT3 transcription factor, but pro-inflammatory cytokines such as interleukin-6 (IL6) also act through STAT3. We now report that IL10, but not IL6 signaling, induces formation of a complex between STAT3 and the inositol polyphosphate-5-phosphatase SHIP1 in macrophages. Both SHIP1 and STAT3 translocate to the nucleus in macrophages. Remarkably, sesquiterpenes of the Pelorol family, which we previously described as allosteric activators of SHIP1 phosphatase activity, could induce SHIP1/STAT3 complex formation in cells and mimic the anti-inflammatory action of IL10 in a mouse model of colitis. Using crystallography and docking studies we identified a drug-binding pocket in SHIP1. Our studies reveal new mechanisms of action for both STAT3 and SHIP1 and provide a rationale for use of allosteric SHIP1-activating compounds, which mimic the beneficial anti-inflammatory actions of IL10. VIDEO ABSTRACT.

Interleukin-10 Induces Expression of Neuroendocrine Markers and PDL1 in Prostate Cancer Cells.

Interleukin-10 (IL10) is best studied for its inhibitory action on immune cells and ability to suppress an antitumour immune response. But IL10 also exerts direct effects on nonimmune cells such as prostate cancer epithelial cells. Elevated serum levels of IL10 observed in prostate and other cancer patients are associated with poor prognosis. After first-line androgen-deprivation therapy, prostate cancer patients are treated with androgen receptor antagonists such as enzalutamide to inhibit androgen-dependent prostate cancer cell growth. However, development of resistance inevitably occurs and this is associated with tumour differentiation to more aggressive forms such as a neuroendocrine phenotype characterized by expression of neuron specific enolase and synaptophysin. We found that treatment of prostate cancer cell lines in vitro with IL10 or enzalutamide induced markers of neuroendocrine differentiation and inhibited androgen receptor reporter activity. Both also upregulated the levels of PDL1, which could promote tumour survival in vivo through its interaction with the immune cell inhibitory receptor PD1 to suppress antitumour immunity. These findings suggest that IL10's direct action on prostate cancer cells could contribute to prostate cancer progression independent of IL10's suppression of host immune cells.

Semaphorin 3C as a Therapeutic Target in Prostate and Other Cancers.

The semaphorins represent a large family of signaling molecules with crucial roles in neuronal and cardiac development. While normal semaphorin function pertains largely to development, their involvement in malignancy is becoming increasingly evident. One member, Semaphorin 3C (SEMA3C), has been shown to drive a number of oncogenic programs, correlate inversely with cancer prognosis, and promote the progression of multiple different cancer types. This report surveys the body of knowledge surrounding SEMA3C as a therapeutic target in cancer. In particular, we summarize SEMA3C's role as an autocrine andromedin in prostate cancer growth and survival and provide an overview of other cancer types that SEMA3C has been implicated in including pancreas, brain, breast, and stomach. We also propose molecular strategies that could potentially be deployed against SEMA3C as anticancer agents such as biologics, small molecules, monoclonal antibodies and antisense oligonucleotides. Finally, we discuss important considerations for the inhibition of SEMA3C as a cancer therapeutic agent.

Targeting Semaphorin 3C in Prostate Cancer With Small Molecules.

Despite the amenability of early-stage prostate cancer to surgery and radiation therapy, locally advanced and metastatic prostate cancer is clinically problematic. Chemical castration is often used as a first-line therapy for advanced disease, but progression to the castration-resistant prostate cancer phase occurs with dependable frequency, largely through mutations to the androgen receptor (AR), aberrant AR signaling, and AR-independent mechanisms, among other causes. Semaphorin 3C (SEMA3C) is a secreted signaling protein that is essential for cardiac and neuronal development and has been shown to be regulated by the AR, to drive epithelial-to-mesenchymal transition and stem features in prostate cells, to activate receptor tyrosine kinases, and to promote cancer progression. Given that SEMA3C is linked to several key aspects of prostate cancer progression, we set out to explore SEMA3C inhibition by small molecules as a prospective cancer therapy. A homology-based SEMA3C protein structure was created, and its interaction with the neuropilin (NRP)-1 receptor was modeled to guide the development of the corresponding disrupting compounds. Experimental screening of 146 in silico‒identified molecules from the National Cancer Institute library led to the discovery of four promising candidates that effectively bind to SEMA3C, inhibit its association with NRP1, and attenuate prostate cancer growth. These findings provide proof of concept for the feasibility of inhibiting SEMA3C with small molecules as a therapeutic approach for prostate cancer.

Selectively targeting the dimerization interface of human androgen receptor with small-molecules to treat castration-resistant prostate cancer.

Prostate cancer (PCa) is a leading cause of death for men in North America. The androgen receptor (AR) - a hormone inducible transcription factor - drives expression of tumor promoting genes and represents an important therapeutic target in PCa. The AR is activated by steroid recruitment to its ligand binding domain (LBD), followed by receptor nuclear translocation and dimerization via the DNA binding domain (DBD). Clinically used small molecules interfere with steroid recruitment and prevent AR-driven tumor growth, but are rendered ineffective by emergence of LBD mutations or expression of constitutively active variants, such as ARV7, that lack the LBD. Both drug-resistance mechanisms confound treatment of this 'castration resistant' stage of PCa (CRPC), characterized by return of AR signalling. Here, we employ computer-aided drug-design to develop small molecules that block the AR-DBD dimerization interface, an attractive target given its role in AR activation and independence from the LBD. Virtual screening on the AR-DBD structure led to development of prototypical compounds that block AR dimerization, inhibiting AR-transcriptional activity through a LBD-independent mechanism. Such inhibitors may potentially circumvent AR-dependent resistance mechanisms and directly target CRPC tumor growth.

Heterochromatin Protein 1α Mediates Development and Aggressiveness of Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer arising mostly from adenocarcinoma via neuroendocrine transdifferentiation following androgen deprivation therapy. Mechanisms contributing to both NEPC development and its aggressiveness remain elusive. In light of the fact that hyperchromatic nuclei are a distinguishing histopathologic feature of NEPC, we utilized transcriptomic analyses of our patient-derived xenograft (PDX) models, multiple clinical cohorts, and genetically engineered mouse models to identify 36 heterochromatin-related genes that are significantly enriched in NEPC. Longitudinal analysis using our unique, first-in-field PDX model of adenocarcinoma-to-NEPC transdifferentiation revealed that, among those 36 heterochromatin-related genes, heterochromatin protein 1α (HP1α) expression increased early and steadily during NEPC development and remained elevated in the developed NEPC tumor. Its elevated expression was further confirmed in multiple PDX and clinical NEPC samples. HP1α knockdown in the NCI-H660 NEPC cell line inhibited proliferation, ablated colony formation, and induced apoptotic cell death, ultimately leading to tumor growth arrest. Its ectopic expression significantly promoted NE transdifferentiation in adenocarcinoma cells subjected to androgen deprivation treatment. Mechanistically, HP1α reduced expression of androgen receptor and RE1 silencing transcription factor and enriched the repressive trimethylated histone H3 at Lys9 mark on their respective gene promoters. These observations indicate a novel mechanism underlying NEPC development mediated by abnormally expressed heterochromatin genes, with HP1α as an early functional mediator and a potential therapeutic target for NEPC prevention and management.Significance: Heterochromatin proteins play a fundamental role in NEPC, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(10); 2691-704. ©2018 AACR.

SEMA3C drives cancer growth by transactivating multiple receptor tyrosine kinases via Plexin B1.

Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand-independent manner via Plexin B1. SEMA3C expression levels increase in castration-resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide-resistant progression. Plexin B1 sema domain-containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post-castration in vivo SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer.

Semaphorin 3 C drives epithelial-to-mesenchymal transition, invasiveness, and stem-like characteristics in prostate cells.

Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in cancer-related deaths. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted signalling proteins whose normal roles reside in embryogenesis and neuronal development. In this context, semaphorins help establish chemotactic gradients and direct cell movement. Various semaphorin family members have been found to be up- and down-regulated in a number of cancers. One family member, Semaphorin 3 C (SEMA3C), has been implicated in prostate, breast, ovarian, gastric, lung, and pancreatic cancer as well as glioblastoma. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes epithelial-to-mesenchymal transition (EMT) and stem-like phenotypes in prostate cells. In the present study we show that ectopic expression of SEMA3C in RWPE-1 promotes the upregulation of EMT and stem markers, heightened sphere-formation, and cell plasticity. In addition, we show that SEMA3C promotes migration and invasion in vitro and cell dissemination in vivo.

Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner.

The androgen receptor (AR) is a member of the nuclear receptor superfamily of transcription factors and is central to prostate cancer (PCa) progression. Ligand-activated AR engages androgen response elements (AREs) at androgen-responsive genes to drive the expression of gene batteries involved in cell proliferation and cell fate. Understanding the transcriptional targets of the AR has become critical in apprehending the mechanisms driving treatment-resistant stages of PCa. Although AR transcription regulation has been extensively studied, the signaling networks downstream of AR are incompletely described. Semaphorin 3C (SEMA3C) is a secreted signaling protein with roles in nervous system and cardiac development but can also drive cellular growth and invasive characteristics in multiple cancers including PCa. Despite numerous findings that implicate SEMA3C in cancer progression, regulatory mechanisms governing its expression remain largely unknown. Here we identify and characterize an androgen response element within the SEMA3C locus. Using the AR-positive LNCaP PCa cell line, we show that SEMA3C expression is driven by AR through this element and that AR-mediated expression of SEMA3C is dependent on the transcription factor GATA2. SEMA3C has been shown to promote cellular growth in certain cell types so implicit to our findings is the discovery of direct regulation of a growth factor by AR. We also show that FOXA1 is a negative regulator of SEMA3C. These findings identify SEMA3C as a novel target of AR, GATA2, and FOXA1 and expand our understanding of semaphorin signaling and cancer biology.

Suppression of LIM and SH3 Domain Protein 1 (LASP1) Negatively Regulated by Androgen Receptor Delays Castration Resistant Prostate Cancer Progression.

BACKGROUND: LIM and SH3 domain protein 1 (LASP1) has been implicated in several human malignancies and has been shown to predict PSA recurrence in prostate cancer. However, the anti-tumor effect of LASP1 knockdown and the association between LASP1 and the androgen receptor (AR) remains unclear. The aim of this study is to clarify the significance of LASP1 as a target for prostate cancer, and to test the effect of silencing LASP1 in vivo using antisense oligonucleotides (ASO). METHODS: A tissue microarray (TMA) was performed to characterize the differences in LASP1 expression in prostate cancer treated after hormone deprivation therapy. Flow cytometry was used to analyze cell cycle. We designed LASP1 ASO for knockdown of LASP1 in vivo studies. RESULTS: The expression of LASP1 in TMA was increased after androgen ablation and persisted in castration resistant prostate cancer (CRPC). Also in TMA, compared with LNCaP cell, LASP1 expression is elevated in CRPC cell lines (C4-2 and VehA cells). Interestingly, suppression of AR elevated LASP1 expression conversely, AR activation decreased LASP1 expression. Silencing of LASP1 reduced cell growth through G1 arrest which was accompanied by a decrease of cyclin D1. Forced overexpression of LASP1 promoted cell cycle and induced cell growth which was accompanied by an increase of cyclin D1. Systemic administration of LASP1 ASO with athymic mice significantly inhibited tumor growth in CRPC xenografts. CONCLUSIONS: These results indicate that LASP1 is negatively regulated by AR at the transcriptional level and promotes tumor growth through induction of cell cycle, ultimately suggesting that LASP1 may be a potential target in prostate cancer treatment. Prostate 77:309-320, 2017. © 2016 Wiley Periodicals, Inc.

Prognostic significance of the expression of nuclear eukaryotic translation initiation factor 5A2 in human melanoma.

Eukaryotic translation initiation factor 5A2 (EIF5A2) expression is upregulated in various cancers. The present authors previously demonstrated that cytoplasmic EIF5A2 expression increases with melanoma progression and inversely correlates with patient survival. Other studies have suggested that nuclear EIF5A2 may also play a role in oncogenesis. The present study used immunohistochemistry and tissue microarray with a large number of melanocytic lesions (n=459) and demonstrated that nuclear EIF5A2 expression was significantly upregulated between common acquired nevi, dysplastic nevi and primary melanomas, and between primary melanomas and metastatic melanomas. Nuclear EIF5A2 expression was inversely associated with overall and disease-specific 5-year survival rate for all (P<0.001) and primary (P=0.014 and P=0.015, respectively) melanoma patients. Nuclear EIF5A2 expression was directly associated with melanoma thickness (P=0.036) and American Joint Committee on Cancer staging (P<0.001), which suggests the possible role of nuclear EIF5A2 in melanoma cell invasion. Subsequently, the present study investigated the association between the expression of nuclear EIF5A2 and matrix metalloproteinase-2 (MMP-2), which is an important factor for promoting cancer cell invasion. Nuclear EIF5A2 and a strong MMP-2 expression were directly associated, and their concurrent expression was significantly associated with a poorer overall and disease-specific 5-year survival rate for all and primary melanoma patients. Nuclear and cytoplasmic EIF5A2 expression were also demonstrated to be significantly associated, and simultaneous expression of the two forms of EIF5A2 was significantly associated with poor overall and disease-specific 5-year survival rates for all and primary melanoma patients. Multivariate Cox regression analysis revealed that nuclear EIF5A2 expression alone and in combination with cytoplasmic EIF5A2 expression was an adverse independent prognostic factor for all and primary melanoma patients. In conclusion, the present study for the first time, to the best of our knowledge, demonstrated that nuclear EIF5A2 expression is an independent prognostic marker in melanoma, and revealed its role in melanoma progression and patient survival. Therefore, nuclear EIF5A2 may have the potential to serve as a therapeutic marker for melanoma.

Transcriptome-Based Analysis of Molecular Pathways for Clusterin Functions in Kidney Cells.

Clusterin (CLU) is a chaperone-like protein and plays a protective role against renal ischemia-reperfusion injury (IRI); however, the molecular pathways for its functions in the kidney are not fully understood. This study was designed to investigate CLU-mediating pathways in kidney cells by using bioinformatics analysis. CLU null renal tubular epithelial cells (TECs) expressing human CLU cDNA (TEC-CLU(hCLU) ) or empty vector (TEC-CLU(-/-) ) were exposed to normoxia or hypoxia (1% O2 ). Transcriptome profiling with a significant twofold change was performed using SurePrint G3 Mouse Gene Expression 8 × 60 K microarray, and the signaling pathways was ranked by using Ingenuity pathway analysis. Here, we showed that compared to CLU null controls, ectopic expression of human CLU in CLU null kidney cells promoted cell growth but inhibited migration in normoxia, and enhanced cell survival in hypoxia. CLU expression affected expression of 3864 transcripts (1893 up-regulated) in normoxia and 3670 transcripts (1925 up-regulated) in hypoxia. CLU functions in normoxia were associated mostly with AKT2/PPP2R2B-dependent PI3K/AKT, PTEN, VEGF, and ERK/MAPK signaling and as well with GSK3B-mediated cell cycle progression. In addition to unfolded protein response (UPR) and/or endoplasmic reticulum (ER) stress, CLU-enhanced cell survival in hypoxia was also associated with PIK3CD/MAPK1-dependent PI3K/AKT, HIF-α, PTEN, VEGF, and ERK/MAPK signaling. In conclusion, our data showed that CLU functions in kidney cells were mainly mediated in a cascade manner by PI3K/AKT, PTEN, VEGF, and ERK/MAPK signaling, and specifically by activation of UPR/ER stress in hypoxia, providing new insights into the protective role of CLU in the kidney. J. Cell. Physiol. 231: 2628-2638, 2016. © 2016 Wiley Periodicals, Inc.

The role of the metastasis suppressor gene KAI1 in melanoma angiogenesis.

The tetraspan protein KAI1 (CD82) has been previously shown to have important roles in cell migration, invasion, and melanoma prognosis. In this study, we investigated the role of KAI1 regarding melanoma angiogenesis. KAI1 overexpression strongly suppressed the growth of the human umbilical vein endothelial cells and their tubular structure formation in vitro. Also, KAI1 was able to inhibit both interleukin-6 (IL-6) and VEGF at mRNA and protein levels. Using nude mice in the in vivo study, we showed that KAI1, through the regulation of ING4, inhibited blood vessel formation in matrigel plugs along with the downregulation of IL-6 and VEGF, and the recruitment of CD31-positive cells. Finally, we found that KAI1 was able to suppress the activity of a serine/threonine kinase Akt by suppressing Akt phosphorylation (Ser473). Taken together, our results suggested that KAI1 was able to suppress melanoma angiogenesis by downregulating IL-6 and VEGF expression, and the restoration of KAI1 functionality offered a new approach in human melanoma treatment.