Honghe Wang

Nuclear Kaiso Indicates Aggressive Prostate Cancers and Promotes Migration and Invasiveness of Prostate Cancer Cells

Kaiso, a p120 catenin-binding protein, is expressed in the cytoplasmic and nuclear compartments of cells; however, the biological consequences and clinical implications of a shift between these compartments have yet to be established. Herein, we report an enrichment of nuclear Kaiso expression in cells of primary and metastatic prostate tumors relative to the normal prostate epithelium. Nuclear expression of Kaiso correlates with Gleason score (P < 0.001) and tumor grade (P < 0.001). There is higher nuclear expression of Kaiso in primary tumor/normal matched samples and in primary tumors from African American men (P < 0.0001). We further found that epidermal growth factor (EGF) receptor up-regulates Kaiso at the RNA and protein levels in prostate cancer cell lines, but more interestingly causes a shift of cytoplasmic Kaiso to the nucleus that is reversed by the EGF receptor-specific kinase inhibitor, PD153035. In both DU-145 and PC-3 prostate cancer cell lines, Kaiso inhibition (short hairpin RNA-Kaiso) decreased cell migration and invasion even in the presence of EGF. Further, Kaiso directly binds to the E-cadherin promoter, and inhibition of Kaiso in PC-3 cells results in increased E-cadherin expression, as well as re-establishment of cell-cell contacts. In addition, Kaiso-depleted cells show more epithelial morphology and a reversal of the mesenchymal markers N-cadherin and fibronectin. Our findings establish a defined oncogenic role of Kaiso in promoting the progression of prostate cancer.

Clinical and Biological Significance of KISS1 Expression in Prostate Cancer

For men in the United States, prostate cancer (PCa) is the most frequent malignancy and the second leading cause of cancer mortality. The metastatic spread of PCa is responsible for most deaths related to PCa. Although KISS1 functions as a metastasis suppressor in various cancers, its expression levels and functions in PCa development and progression remain undetermined. The goals of this study were to correlate the expression levels of KISS1 in PCas with clinicopathologic characteristics and to assess the biological relevance of KISS1 to the viability and motility of PCa cells. Strong KISS1 staining was detected in benign prostate tissues, but the staining was weaker in primary and metastatic PCas (both P < 0.001, t-test). Furthermore, the low expression levels of KISS1 in PCas correlated with clinical stage (P < 0.01) and with KISS1R expression (P < 0.001). Overexpression of full-length KISS1 in low KISS1-expressing PC-3M cells, but not KFMΔSS, which lacks the secretion signal sequence, induced re-sensitization of cells to anoikis, although it had no effect on either cell proliferation or apoptosis. Overexpression of KISS1 also suppressed steps in the metastatic cascade, including motility and invasiveness. Moreover, cells overexpressing KISS1 were found to enhance chemosensitivity to paclitaxel. Collectively, our data suggest that KISS1 functions as a metastasis suppressor in PCas and may serve as a useful biomarker as well as a therapeutic target for aggressive PCas.

Side population rather than CD133+ cells distinguishes enriched tumorigenicity in hTERT-immortalized primary prostate cancer cells

BackgroundSubpopulations of cancer cells with the capacity of generating solid tumors have been characterized. In various cancer types, including prostate cancer cells, a side population (SP) and CD133-expressing cells have been proposed as containing a population cancer cells with stem-like ability. Therefore the aim of this work was to determine, in prostate cancer cell lines, the frequency and tumorigenic potential of SP and CD133+ cells.ResultsIn vitro 2D colony-forming assay and sphere-forming assay, Flow cytometry analysis and magnetic cell sorting were utilized to sort CD133+, CD133- and Side population (SP) cells. Our findings indicate that CD44 and integrin α-6 are uniformly expressed in the hTERT cell lines; however, CD133 is expressed only in a small population (< 0.1%). FACS-sorted CD133+ and CD133- cells exhibited similar tumorigenicity in vitro and in vivo. Additionally, for the hTERT cells, SP rather than CD133 expression showed an 8-fold enhanced tumorigenic potential. The data suggest that SP cells, rather than those with CD133 marker, contain the rare population of CSC capable of producing prostate tumors.ConclusionCollectively, our data suggest that although CD133 is expressed only in a small population of hTERT-immortalized prostate cancer cells, it is not likely to be associated with stem cells, as CD133- and CD133+ cells exhibited similar tumorigenicity. However, SP isolated cells, appear to be enriched with tumorigenic stem-like cells capable of generating palpable tumors.

Detecting gene-gene interactions in prostate disease in African American men

BackgroundThe most common male malignancy in the United States is prostate cancer; however its rate of occurrence varies significantly among ethnic groups. In a previous cDNA microarray study on CaP tumors from African American (AA) and Caucasian (CA) patients, we identified 97 candidate genes that exhibited opposite gene expression polarity with respect to race groups; genes up-regulated in AA were simultaneously down-regulated in CA.PurposeThe purpose of this study was to narrow the 97 member gene list, to a smaller number of genes in order to focus studies on a limited number of genes/SNPs that might explain prostate cancer disparity in African Americans.MethodsWe performed genotype-phenotype, SNP and expression transcript levels correlations using HapMap Yoruba population with 85 of our 97 prostate candidate genes using SCAN database.ResultsFindings revealed an association of SNPs surrounding ABCD3 gene with basal gene expression of RanGAP1 is important in prostate tumors in AA. Hence, to confirm our results in clinical biospecimen, we monitored expression of ABCD3 in a novel panel of African American and Caucasian prostate cancer paired cell lines. The LNCaP, C4-2B showed 2-fold increase; MDA-2PC-2B cell line, derived from AA, showed highest fold-change, 10-fold. The EGFR over expressing DU-145 WT cell line exhibited a 4-fold increase in expression relative to non transfected DU-145 prostate cell lines. Furthermore, Ingenuity Network analysis implicated our AA prostate candidate genes are involved in three network hubs, ERK, MapK and NFkB pathways.ConclusionsTaken together, these findings are intriguing because other members of the ABC gene family, namely, ABCC3, ABCD1, and ABCD2 have been shown to confer chemoresistance in certain cancer types. Equally important, is the fact that activation of the MapK/ERK pathway via EGFR stimulation is vital for increased transcription of numerous cancer related genes. It is especially noteworthy that overexpression of EGFR has been widely observed in AA prostate tumors. Collectively our findings lead us to think that a novel signaling cascade, through which increased aggressiveness and chemoresistance is achieved, may explain prostate cancer health disparity in AA males and the nature of aggressive CaP tumors in general.

Kaiso, a transcriptional repressor, promotes cell migration and invasion of prostate cancer cells through regulation of miR-31 expression

Kaiso, a member of the BTB/POZ zinc finger protein family, functions as a transcriptional repressor by binding to sequence-specific Kaiso binding sites or to methyl-CpG dinucleotides. Previously, we demonstrated that Kaiso overexpression and nuclear localization correlated with the progression of prostate cancer (PCa). Therefore, our objective was to explore the molecular mechanisms underlying Kaiso-mediated PCa progression. Comparative analysis of miRNA arrays revealed that 13 miRNAs were significantly altered (> 1.5 fold, p < 0.05) in sh-Kaiso PC-3 compared to sh-Scr control cells. Real-time PCR validated that three miRNAs (9, 31, 636) were increased in sh-Kaiso cells similar to cells treated with 5-aza-2′-deoxycytidine. miR-31 expression negatively correlated with Kaiso expression and with methylation of the miR-31 promoter in a panel of PCa cell lines. ChIP assays revealed that Kaiso binds directly to the miR-31 promoter in a methylation-dependent manner. Over-expression of miR-31 decreased cell proliferation, migration and invasiveness of PC-3 cells, whereas cells transfected with anti-miR-31 restored proliferation, migration and invasiveness of sh-Kaiso PC-3 cells. In PCa patients, Kaiso high/miR-31 low expression correlated with worse overall survival relative to each marker individually. In conclusion, these results demonstrate that Kaiso promotes cell migration and invasiveness through regulation of miR-31 expression.

MicroRNAs that affect prostate cancer: emphasis on prostate cancer in African Americans.

Abstract Although concerted efforts have been directed toward eradicating health disparities in the United States, the disease and mortality rates for African American men still are among the highest in the world. We focus here on the role of microRNAs (miRNAs) in the signaling pathways of androgen receptors and growth factors that promote the progression of prostate cancer to more aggressive disease. We explore also how differential expression of miRNAs contributes to aggressive prostate cancer including that of African Americans.

Transcriptional repressor Kaiso promotes epithelial to mesenchymal transition and metastasis in prostate cancer through direct regulation of miR-200c

The loss of miR-200 family, through DNA methylation, results in cancer cells undergoing an epithelial to mesenchymal transition (EMT), and metastasis. In this study, we established that the transcriptional repressor Kaiso directly binds methylated regions of the miR-200 family, and this is reversed with 5-aza treatment. sh-Kaiso PC-3 cells display increased miR-200-a/b/c, miR-141, and miR-429 expression, with miR-200c demonstrating the most significant increase. Interestingly, overexpression of EGFR or treatment with EGF decreases miR-200c expression and this is reversed after treatment with EGFR specific kinase inhibitor PD153035. However, EGF did not have a significant effect on miR-200c in sh-Kaiso DU-145 or PC-3 cell lines, suggesting Kaiso silences miR-200c through the activation of EGFR signaling. Overexpression of Kaiso in LNCaP cells results in decreased expression of miR-200-a/b/c, miR-141, and miR-429, along with increased expression of ZEB1, p-EGFR and total EGFR levels. Overexpression of miR200c in PC-3 cells results in decreased expression of EGFR, ZEB1, ERK1/2 and Kaiso. Additionally, sh-Kaiso PC-3 demonstrates reduced in vivo tumor formation and metastasis. Thus, our data suggests that EGFR signaling regulates the silencing of miR-200 family through Kaiso binding to methylated regions in the promoter.

Abstract 5414: Kaiso influences the exosome profile required to induce cell proliferation, migration and metabolism in breast cancer

Purpose: Breast cancer is the most frequent tumor in women, afflicting African American (AA) females to a greater degree than Caucasians (CAs). Recently, we and others have found that Kaiso expression is elevated in AA patients relative to CA patients, and its expression correlates with tumor recurrence and metastasis. Exosomes are considered important modulators of cellular behavior through their cellular communication by transferring mRNA, microRNAs, proteins among cells. Here, we study the role of Kaiso on the biological function of breast cancer exosomes. Experimental procedures: Exosomes were isolated by ultracentrifugation method and characterized by antibody array. We performed exosome internalization assay, cell proliferation assay and migration assay. Cellular metabolic activity was measured by Seahorse Analyzer. Exosome proteomics was performed by LC-MS/MS and analyzed quantitatively by Panther and DAVID bioinformatics resources. Results: Isolated exosomes were effectively internalized by MCF7 cells. MCF7 cells treated with exosomes of Kaiso-knock-downed MDA-MB-231 cells (sh-Kaiso) showed a decreased proliferation as compared to exosomes of Kaiso-scrambled MDA-MB-231 cells (sh-Scr). We further observed that treatment of MCF7 cells with sh-Kaiso exosomes decreased cell migration when compared to sh-Scr. To determine the proteins responsible for this observation, we performed exosome proteomics profiling. Exosomes released from sh-Kaiso compared to sh-Scr cells showed differential enrichment of protein expression. In sh-Kaiso exosomes, 36 proteins were down-regulated and most of these proteins are involved in cell invasion and metastasis; whereas 172 proteins were up-regulated in sh-Kaiso of which most of them are involved in protein folding, protein complex assembly, biogenesis and repair. In proteomics data, lactate dehydrogenases (LDH) AB in sh-SCR exosome treated MCF7 cells as compared to sh-Kaiso exosome treated MCF7 cells. Conclusions: Our findings demonstrate that Kaiso plays an important role in the content of exosome cargo, which in turn has an effect promoting cell growth, migration and metabolism of breast cancer cells. We suggest that Kaiso has defined role in limiting important cellular information in breast cancer exosome cargo. Citation Format: Md Shakir U. Ahmed, Shweta Triphati, William E. Grizzle, Honghe Wang, Clayton C. Yates. Kaiso influences the exosome profile required to induce cell proliferation, migration and metabolism in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5414. doi:10.1158/1538-7445.AM2017-5414

Abstract PL01-02: Functional biomarkers that promote African American prostate cancer through epigenetic regulation

Prostate cancer is the most commonly diagnosed malignancy in men, with African American men experiencing a rate 60% higher than white patients. At the time of diagnosis, approximately 50% of men have clinically advanced disease. African American men have almost twice the incidence and death rates related to prostate cancer compared to Caucasian men. Several studies have suggested that some of these differences may be attributed to the elevated expression of different genes. Many factors have been associated with why prostate tumors in AA patients are more aggressive. However there is a lack of knowledge, whether this is the result of genes that promote aggressiveness or promote tumor development. Methylation profiling of prostate tumors has demonstrated that the loss of a number of key regulatory genes are not via mutation, but rather hypermethylation. This is particularly evident in the AA patients where hypermethylation of a number of genes in normal or pre-malignant areas are thought to predispose a full-blown malignancy. However the underlining mechanism of these acquired methylation patterns is poorly understood. To better understand the epigenetic regulation of genes associated with promoting aggressiveness and specifically to determine if this can explain the more aggressive nature of AA tumors , we conducted 662 microRNA microarray profiling utilizing novel isogenic non-malignant and malignant cell lines derived from both AA and CA tumor patients. Utilizing multiple criteria, included pathological stage and race, we were able to determine a distinct microRNA signature that was unique the AA derived cell lines, independent of tumor status. Validation by q-PCR of the 10 most significantly differentially expressed miRNA9s across our 11 cell line panel demonstrated that loss of miR-152 expression was associated with aggressiveness. Restoring miR-152 expression in miR-152 deficient cell lines, resulted in decreased proliferation, and S phase arrest of the cell cycle. In silico informatics analysis predicted that miR-152 contains a significant number of CpG islands within the promoter region upstream of the start site. We confirmed this analysis with bisulfite conversion and sequencing of the promoter, as well as treatment with demethylation agent 5-Aza-2′-deoxycytidine. Furthermore, we observed an inverse relationship of miR-152 with predicted target DNA methyltransferase-1 (DNMT1), suggesting a reciprocal maintenance of hypermethylation for not only miR-152, but also a number of other methylated genes. This is plausible given the well-established role of DNMT1 in methylation of a large number of genes found to promote aggressiveness in prostate tumors . Lastly, a comparison of normal/tumor ratios of miR-152 expression in 20 CA and 20 AA prostate cancer patients, we observed significantly decreased expression in tumors from CA patients as expected, however this was not observed in AA patients. AA patients also displayed an overall lower expression compared to CA tumors. In summary, these results are the first to identify unique miRNAs that contribute to aggressive prostate cancers in AA patients. Furthermore epigenetic regulation of the miR-152/DNMT1 regulatory loop may play an import ant role in multiple events that contribute to the aggressiveness of PCa tumors. Citation Format: Shaniece C. Theodore, Honghe Wang, Jhong Rhim, Timothy Turner, Melissa Davis, William Grizzle, Clayton C. Yates. Functional biomarkers that promote African American prostate cancer through epigenetic regulation. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr PL01-02. doi:10.1158/1538-7755.DISP13-PL01-02

Abstract C70: ABCD3 expression in prostate and breast tumors

Background: In a previous study, we identified the ABCD3 gene as important in aggressive prostate tumors. The ABCD3 gene is a member of the ABC (ATP-Binding Cassette) family. The ABC gene family give instructions for making transporter proteins that carry many types of molecules, such as fats, sugars, protein building blocks (amino acids) and drugs, across cell membranes and is known to play a role in chemoresistance is some cancers. Purpose: Little is known about role of ABCD3 in prostate cancer, hence in the present study we assessed the expression of ABCD3 in normal and malignant prostate tissue (without regard to racial ethnicity) and determined correlations between ABCD3 expression and clinicopathologic characteristics. Methods: Clinically annotated duplicate core tissue high-density prostate adenocarcinoma tissue microarray of 92 cases of adenocarcinoma, 2 prostate transitional cell carcinoma, 12 prostate adjacent normal tissue and 8 normal prostate tissue were stained with a monoclonal antibody against ABCD3 and scored for percentage of visible staining. Results: Increased ABCD3 expression correlated with increasing Gleason score (p=0.0094), age (p=0.0014) and pathology grade (p=0.0007). Interestingly, we also observed that ABCD3 expression was elevated to varying degrees in other types of cancers, breast (stage II a), brain, cervical, head and neck, kidney and pancreatic. Conclusions: We postulate that ABCD3 may be a putative prognostic biomarker that discriminates between indolent and aggressive cancers, albeit ABCD3 function in cancer progression is still under investigation. Citation Format: R. Renee Reams, Jacqueline D. Jones, Daniel Osborne, Honghe Wang, Clayton C. Yates, III. ABCD3 expression in prostate and breast tumors. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C70. doi:10.1158/1538-7755.DISP13-C70