Potential dual functional roles of the Y-linked RBMY in hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a highly heterogeneous liver cancer with significant male biases in incidence, disease progression, and outcomes. Previous studies have suggested that genes on the Y chromosome could be expressed and exert various male-specific functions in the oncogenic processes. In particular, the RNA-binding motif on the Y chromosome (RBMY) gene is frequently activated in HCC and postulated to promote hepatic oncogenesis in patients and animal models. In the present study, immunohistochemical analyses of HCC specimens and data mining of The Cancer Genome Atlas (TCGA) database revealed that high-level RBMY expression is associated with poor prognosis and survival of the patients, suggesting that RBMY could possess oncogenic properties in HCC. To examine the immediate effect(s) of the RBMY overexpression in liver cancer cells, cell proliferation was analyzed on HuH-7 and HepG2 cells. The results unexpectedly showed that RBMY overexpression inhibited cell proliferation in both cell lines as its immediate effect, which led to vast cell death in HuH-7 cells. Transcriptome analysis showed that genes involved in various cell proliferative pathways, such as the RAS/RAF/MAP and PIP3/AKT signaling pathways, were downregulated by RBMY overexpression in HuH-7 cells. Furthermore, in vivo analyses in a mouse liver cancer model using hydrodynamic tail vein injection of constitutively active AKT and RAS oncogenes showed that RBMY abolished HCC development. These findings support the notion that Y-linked RBMY could serve dual tumor-suppressing and tumor-promoting functions, depending on the spatiotemporal and magnitude of its expression during oncogenic processes, thereby contributing to sexual dimorphisms in liver cancer.

Role of a novel race-related tumor suppressor microRNA located in frequently deleted chromosomal locus 8p21 in prostate cancer progression.

The prostate cancer (PCa) genome is characterized by deletions of chromosome 8p21-22 region that increase significantly with tumor grade and are associated with poor prognosis. We proposed and validated a novel, paradigm-shifting hypothesis that this region is associated with a set of microRNA genes-miR-3622, miR-3622b, miR-383-that are lost in PCa and play important mechanistic roles in PCa progression and metastasis. Extending our hypothesis, in this study, we evaluated the role of a microRNA gene located in chromosome 8p-miR-4288-by employing clinical samples and cell lines. Our data suggests that (i) miR-4288 is widely downregulated in primary prostate tumors and cell lines; (ii) miR-4288 expression is lost in metastatic castration-resistant PCa; (ii) miR-4288 downregulation is race-related PCa alteration that is prevalent in Caucasian patients and not in African Americans; (iii) in Caucasians, miR-4288 was found to be associated with increasing tumor grade and high serum prostate-specific antigen, suggesting that miR-4288 downregulation/loss may be associated with tumor progression specifically in Caucasians; (iv) miR-4288 possess significant potential as a molecular biomarker to predict aggressiveness/metastasis; and (v) miR-4288 is anti-proliferative, is anti-invasive and inhibits epithelial-to-mesenchymal transition; and (vi) miR-4288 directly represses expression of metastasis/invasion-associated genes MMP16 and ROCK1. Thus, the present study demonstrates a tumor suppressor role for a novel miRNA located with a frequently lost region in PCa, strengthening our hypothesis that this locus is causally related to PCa disease progression via loss of microRNA genes. Our study suggests that miR-4288 may be a novel biomarker and therapeutic target, particularly in Caucasians.

NMR quantification of lactate production and efflux and glutamate fractional enrichment in living human prostate biopsies cultured with [1,6-13 C2 ]glucose.

PURPOSE: Image-guided prostate biopsies are routinely acquired in the diagnosis and treatment monitoring of prostate cancer, yielding useful tissue for identifying metabolic biomarkers and therapeutic targets. We developed an optimized biopsy tissue culture protocol in combination with [1,6-13 C2 ]glucose labeling and quantitative high-resolution NMR to measure glycolysis and tricarboxcylic acid (TCA) cycle activity in freshly acquired living human prostate biopsies. METHODS: We acquired 34 MRI-ultrasound fusion-guided prostate biopsies in vials on ice from 22 previously untreated patients. Within 15 min, biopsies were transferred to rotary tissue culture in 37°C prostate medium containing [1,6-13 C2 ]glucose. Following 24 h of culture, tissue lactate and glutamate pool sizes and fractional enrichments were quantified using quantitative 1 H high resolution magic angle spinning Carr-Purcell-Meiboom-Gill (CPMG) spectroscopy at 1°C with and without 13 C decoupling. Lactate effluxed from the biopsy tissue was quantified in the culture medium using quantitative solution-state high-resolution NMR. RESULTS: Lactate concentration in low-grade cancer (1.15 ± 0.78 nmol/mg) and benign (0.74 ± 0.15 nmol/mg) biopsies agreed with prior published measurements of snap-frozen biopsies. There was substantial fractional enrichment of [3-13 C]lactate (≈70%) and [4-13 C]glutamate (≈24%) in both low-grade cancer and benign biopsies. Although a significant difference in tissue [3-13 C]lactate fractional enrichment was not observed, lactate efflux was significantly higher (P < 0.05) in low-grade cancer biopsies (0.55 ± 0.14 nmol/min/mg) versus benign biopsies (0.31 ± 0.04 nmol/min/mg). CONCLUSION: A protocol was developed for quantification of lactate production-efflux and TCA cycle activity in single living human prostate biopsies, allowing metabolic labeling on a wide spectrum of human tissues (e.g., metastatic, post-non-surgical therapy) from patients not receiving surgery.

Influence of lifestyle choices on risks of CYP1B1 polymorphisms for prostate cancer.

Cytochrome P450 1B1 (CYP1B1) converts xenobiotics to carcinogens and how lifestyle choices may interact with CYP1B1 polymorphisms and affect prostate cancer risk was assessed. Blood genomic DNA from a Caucasian population was analysed at polymorphic sites of the 5' untranslated region of CYP1B1 using TaqMan genotyping assays. Overall, drinker status and minor alleles at rs2551188, rs2567206 and rs10175368 were associated with prostate cancer. Linkage was observed between rs2551188, rs2567206, rs2567207 and rs10175368, and the G-C-T-G haplotype (major allele at respective sites) was decreased in cancer. Interestingly when classified by lifestyle factors, no associations of genotypes were found for non-smokers and non-drinkers, whereas on the contrary, minor type at rs2567206 and rs10175368 increased and major G-C-T-G decreased risk for cancer among smokers and drinkers. Interestingly, rs2551188, rs2567206 and rs10175368 minor genotypes correlated with increased tissue CYP1B1 as determined by immunohistochemistry. Further, rs10175368 enhanced luciferase activity and mobility shift show stronger binding of nuclear factor for the minor allele. These results demonstrate smoking and alcohol consumption to modify the risks of CYP1B1 polymorphisms for prostate cancer which may be through rs10175368, and this is of importance in understanding their role in the pathogenesis and as a biomarker for this disease.

SRY interference of normal regulation of the RET gene suggests a potential role of the Y-chromosome gene in sexual dimorphism in Hirschsprung disease.

The Hirschsprung disease (HSCR) is a complex congenital disorder, arising from abnormalities in enteric nervous system (ENS) development. There is a gender disparity among the patients, with the male to female ratio as high as 5 : 1. Loss-of-function mutations of HSCR genes and haploinsufficiency of their gene products are the primary pathogenic mechanisms for disease development. Recent studies identified over half of the HSCR disease susceptibility genes as targets for the sex-determining factor SRY, suggesting that this Y-encoded transcription factor could be involved in sexual dimorphism in HSCR. Among the SRY targets, the tyrosine kinase receptor RET represents the most important disease gene, whose mutations account for half of the familial and up to one-third of the sporadic forms of HSCR. RET is regulated by a distal and a proximal enhancer at its promoter, in which PAX3 and NKX2-1 are the resident transcription factors respectively. We show that the SRY-box 10 (SOX10) co-activator interacts and forms transcriptional complexes with PAX3 and NKX2-1 in a sequence-independent manner and exacerbates their respective transactivation activities on the RET promoter. SRY competitively displaces SOX10 in such transcription complexes and represses their regulatory functions on RET. Hence SRY could be a Y-located negative modifier of RET expression; and if it is ectopically expressed during ENS development, such SRY repression could result in RET protein haploinsufficiency and promotion of HSCR development, thereby contributing to sexual dimorphism in HSCR.

Cytochrome P450 1B1 polymorphisms and risk of renal cell carcinoma in men.

The cytochrome P450 1B1 (CYP1B1) enzyme activates xenobiotics to reactive forms as well as convert estradiol to 4-hydroxy-estradiol that has been shown to play a role in the carcinogenesis process of the kidney in male but not female animals. Prior reports show polymorphic variants of CYP1B1 to alter catalytic activity, and thus, we hypothesize that polymorphisms of the CYP1B1 gene are involved in the malignant transformation of the renal cell in men. The genetic distributions of five CYP1B1 polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism in 480 normal healthy subjects and 403 sporadic renal cell carcinoma cases. All subjects were Caucasian men. The sites evaluated were codons 48 (C → G, Arg → Gly, rs10012), 119 (G → T, Ala → Ser, rs1056827), 432 (C → G, Leu → Val, rs1056836), 449 (C → T, Asp, rs1056837), and 453 (A → G, Asn → Ser, rs1800440). A trend was demonstrated for the 432 Val/Val (χ2, P = 0.06) and 449 T/T (χ2, P = 0.1) genotypes to play a protective role against renal cancer. Odds ratio (95 % confidence interval) for Val/Val compared to Leu/Leu at codon 432 was 0.65 (0.44-0.95) and T/T compared to C/C at codon 449 was 0.67 (0.45-0.99). Codons 432 and 449 were observed to be linked (D = 0.24), and haplotype involving 432 Val and 449 T was significantly reduced in cancer cases (P = 0.04). No association was found, however, when analyzing polymorphic sites with clinical stage of cancer. These results demonstrate polymorphisms of CYP1B1 to be associated with renal carcinogenesis and are of importance in understanding their role in the pathogenesis of renal cell carcinoma.

Utility and performance of cell blocks in urine cytology: Experience at three teaching hospitals.

BACKGROUND: The use of cell block (CB) preparation is underused in urine cytology (UC) and varies among hospitals. In addition to confirming a diagnosis, CBs can be useful in cases of metastatic disease, diagnoses requiring immunohistochemical (IHC) staining, and for ancillary studies. The role of this study is to examine the performance of CBs for UC at three affiliated teaching hospitals. MATERIALS AND METHODS: A retrospective review of UC cases with a CB was conducted at a county hospital, Veterans Affairs hospital, and tertiary university-based hospital. For each specimen, patient demographics, specimen type, volume, original diagnosis, and IHC stains were recorded. Each case was reviewed for diagnosis based on ThinPrep alone, diagnosis based on ThinPrep and CB, utility of CB for diagnosis, and CB cellularity. RESULTS: A total of 250 UC specimens with CB from 186 patients was identified. Bladder washes were the most common (72.1%). IHC stains were performed on 17.2% of cases. On blinded review, CB preparation was deemed useful in 61.2% of cases, with the highest rate for suspicious for high-grade urothelial carcinoma (SHGUC) cases (87.0%). The diagnosis based on ThinPrep review changed with incorporation of CB in 13.2% of cases, with the highest rate for SHGUC cases (43.5%). CONCLUSIONS: The results demonstrate that use of CB in UC confirms the final diagnosis in more than one-half of cases and changes the diagnosis in a subset of cases. Use of CB was most helpful in the SHGUC category. Further evaluation of the types of cases in which CB are prepared is warranted.

Current State of Cytologic-Histologic Correlation Implementation for North American and International Laboratories: Results of the College of American Pathologists Cytopathology Committee Laboratory Practices in Gynecologic Cytology Survey.

CONTEXT.­: The College of American Pathologists (CAP) updated the Laboratory Accreditation Program Cytopathology Checklist to assist laboratories in meeting and exceeding the Clinical Laboratory Improvement Amendments standards for gynecologic cytologic-histologic correlation (CHC). OBJECTIVE.­: To survey the current CHC practices. DESIGN.­: Data were analyzed from a survey developed by the committee and distributed to participants in the CAP Gynecologic Cytopathology PAP Education Program mailing. RESULTS.­: Worldwide, CHC practice is nearly universally adopted, with an overall rate of 87.0% (568 of 653). CHC material was highly accessible. CHC was commonly performed real time/concurrently at the time the corresponding surgical pathology was reviewed. Investigation of CHC discordances varied with North American laboratories usually having a single pathologist review all discrepant histology and cytology slides to determine the reason for discordance, while international laboratories have a second pathologist review histology slides to determine the reason for discordance. The cause of CHC discordance was primarily sampling issues. The more common statistical metrics for CHC monitoring were the total percentage of cases that correlated with subsequent biopsies, screening error rate by cytotechnologist, and interpretative error rate by cytotechnologist. CONCLUSIONS.­: Many laboratories have adopted and implemented the CHC guidelines with identifiable differences in practices between North American and international laboratories. We identify the commonalities and differences between North American and international institutional practices including where CHC is performed, how CHC cases are identified and their accessibility, when CHC is performed, who investigates discordances, what discordances are identified, and how the findings affect quality improvement.

Cytologic-Histologic Correlation Practices for Nongynecologic Cytology Specimens: A Survey by the College of American Pathologists Cytopathology Committee.

CONTEXT.­: Cytologic-histologic correlation (CHC) is a Clinical Laboratory Improvement Amendments-mandated requirement for gynecologic cytology, but no similar requirement exists for nongynecologic cytology. This study presents the findings from a College of American Pathologists' survey of nongynecologic cytology practice patterns. OBJECTIVE.­: To survey the current CHC practices for nongynecologic cytology. DESIGN.­: Data were analyzed from a survey developed by the committee and distributed to participants in the Nongynecologic Cytopathology Education Program mailing. RESULTS.­: Adoption of CHC for nongynecologic cytology cases is worldwide, with 88.5% of institutions performing CHC on these specimens, a substantial increase from previous years. Performance of CHC varied by institution type, with clinic or regional/local independent laboratories and national/corporate laboratories performing CHC significantly less frequently than hospitals, university hospitals/academic medical centers, and Veterans Administration/Department of Defense hospital institutions. Most CHC was performed concurrently in real time, when the corresponding surgical specimen was reviewed. Selection for real-time concurrent CHC was by the interpreting pathologist, the pathologist diagnosing the surgical biopsy sample or cytopathology case, or both. Sampling was by far the most common reason for discordance. A 2-step difference was the most frequent threshold for discordance between cytology and surgical specimens, but this criterion varied among institutions, with no majority definition. The positive predictive value of a positive cytology finding was calculated rarely in North American institutions but was calculated more frequently in international institutions. CONCLUSIONS.­: CHC practices for nongynecologic cytopathology mirror those found for CHC of gynecologic cytopathology.

MicroRNA-1826 targets VEGFC, beta-catenin (CTNNB1) and MEK1 (MAP2K1) in human bladder cancer.

The Wnt/beta-catenin (CTNNB1) and Ras-Raf-MEK-ERK signaling pathway play an important role in bladder cancer (BC) progression. Tumor-suppressive microRNAs (miRNAs) targeting these cancer pathways may provide a new therapeutic approach for BC. We initially identified miRNA-1826 potentially targeting CTNNB1, VEGFC and MEK1 using several target scan algorithms. Also 3' untranslated region luciferase activity and protein expression of these target genes were significantly downregulated in miR-1826-transfected BC cells (J82 and T24). The expression of miR-1826 was lower in BC tissues and inverse correlation of miR-1826 with several clinical parameters (pT, grade) was observed. Also the expression of miR-1826 was much lower in three BC cell lines (J82, T24 and TCCSUP) compared with a normal bladder cell line (SV-HUC-1). We then performed analyses to look at miR-1826 function and found that miR-1826 inhibited BC cell viability, invasion and migration. We also found increased apoptosis and G(1) cell cycle arrest in miR-1826-transfected BC cells. To examine whether the effect of miR-1826 was through CTNNB1 (beta-catenin) or MEK1 knockdown, we knocked down CTNNB1/MEK1 messenger RNA using a small interfering RNA (siRNA) technique. We observed that CTNNB1 or MEK1 siRNA knockdown resulted in effects similar to those with miR-1826 in BC cells. In conclusion, our data suggest that the miR-1826 plays an important role as tumor suppressor via CTNNB1/MEK1/VEGFC downregulation in BC.

The diagnostic utility of D2-40, calretinin, CK5/6, desmin and MOC-31 in the differentiation of mesothelioma from adenocarcinoma in pleural effusion cytology.

OBJECTIVE: To evaluate the utility of the lymphatic endothelial marker D2-40, along with calretinin, CK5/6, desmin and MOC-31, in differentiating mesothelioma and adenocarcinoma in pleural effusion cytology. STUDY DESIGN: Forty-five pleural effusion cases representing confirmed reactive effusions (13), mesotheliomas (11) and metastatic adenocarcinomas (21) were immunostained with antibodies against D2-40, calretinin, CK5/6, desmin and MOC-31. RESULTS: D2-40 showed membranous staining in 82% of mesotheliomas and 77% of reactive effusions. Calretinin and CK5/6 were positive in 100 and 64% of mesotheliomas, and 92 and 31% of reactive effusions, respectively. All adenocarcinomas showed lack of staining with these markers. Desmin was negative in all malignant cases and positive in 85% of reactive effusions. All adenocarcinomas were positive for MOC-31 and negative for the remaining markers. CONCLUSION: Calretinin was the most sensitive in detecting mesothelial differentiation, followed by D2-40. Although useful, D2-40 necessitated cautious interpretation due to occasional focal/weak positivity, particularly in limited cellularity samples. The muscle marker desmin was useful in differentiating benign from malignant effusions but not in distinguishing mesotheliomas from adenocarcinomas. MOC-31 was both highly sensitive and specific for detecting adenocarcinoma and was useful as part of a panel of stains in differentiating cells of mesothelial origin from adenocarcinoma.

Cytologic features of micropapillary variant urothelial carcinoma in urinary tract cytology: Case series and review of literature.

BACKGROUND: The micropapillary variant of urothelial carcinoma (MPVUC) is rare and aggressive. Surgical specimens often show atypical micro-clusters (AMCs) of cells with hyperchromatic, pyknotic, peripheral, irregular nuclei with variable nuclear to cytoplasmic ratios. We reviewed urinary tract cytology (UTC) from patients with MPVUC and hypothesized that AMCs would be present similar to those in surgical specimens. METHODS: The archives were searched from 2000 to 2020 for patients with surgical cases with either MPVUC or conventional high-grade urothelial carcinoma (HGUC) and with prior abnormal UTC. Two pathologists reviewed UTC cases and controls in a blinded manner for AMCs, with quantitation of none, low, moderate, and high. Interrater reliability was compared by quadratic weighted Cohen's Kappa test. The association between numerical average score and MPVUC status was determined by logistic regression. RESULTS: Five patients with invasive MPVUC, one patient with a noninvasive micropapillary component, and 15 control patients with conventional HGUC were included. All patients had prior or concurrent abnormal UTC samples. Increasing category of quantities of AMCs on cytology was associated with micropapillary status (OR 7.9, 95% CI 2.7-118, p = .045), with moderate agreement between raters (Cohen's Kappa 0.54, 95% CI 0.19-0.89, p = .004). CONCLUSIONS: In patients with MPVUC on surgical specimen, AMCs were frequently observed on cytology. Similar atypical clusters were observed in patients with nonmicropapillary HGUC, albeit at lower frequency. However, given the WHO recommendation to diagnose micropapillary only if an invasive micropapillary component is present, a specific diagnosis of MPVUC on UTC cannot be based solely on the presence of AMCs.

Performance of specific morphologic features in distinguishing low-grade squamous intraepithelial lesions from high-grade squamous intraepithelial lesions in borderline cases: a College of American Pathologists Cytopathology Committee multiobserver study.

INTRODUCTION: Distinguishing between low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL) can be difficult on certain Papanicolaou (Pap) tests, hindering interobserver concordance. We investigated the variables influencing the interpretation of LSIL versus HSIL in Pap test slides rejected from the College of American Pathologists PAP education program. MATERIALS AND METHODS: Eleven cytologists, who were unaware of the reference interpretation, examined 21 Pap slides (11 submitted as LSIL and 10 as HSIL) rejected from the PAP education program and recorded the number of LSIL cells, HSIL cells, keratinized dysplastic cells, LSIL clusters with mixed HSIL cells, atypical squamous metaplasia, atypical glandular cells, the presence of inflammation or infectious organisms, and the overall interpretation (LSIL or HSIL). We evaluated the significance of these 11 variables using a nonlinear mixed model analysis. RESULTS: LSIL had greater concordance (92 of 121 responses; 76.0% concordance) than HSIL (68 of 110 responses; 61.8% concordance; P < 0.001). The only predictors of misclassified cases were the number of atypical squamous metaplastic cells and the number of HSIL cells (P < 0.001). The more of these cells identified, the more likely the reviewers were to classify the slide as HSIL. The reproducibility of the diagnosis was fair (Gwet's agreement coefficient, 0.33). CONCLUSIONS: Interobserver reproducibility is a challenge for a subset of cases with features intermediate between LSIL and HSIL. Atypical squamous metaplasia and dysplastic nuclei with a nuclear/cytoplasmic ratio greater than one half of the cell volume (HSIL) present on a Pap test influenced the likelihood that a reviewer would interpret the case as HSIL rather than LSIL.

MicroRNA-145 is regulated by DNA methylation and p53 gene mutation in prostate cancer.

MiR-145 is downregulated in various cancers including prostate cancer. However, the underlying mechanisms of miR-145 downregulation are not fully understood. Here, we reported that miR-145 was silenced through DNA hypermethylation and p53 mutation status in laser capture microdissected (LCM) prostate cancer and matched adjacent normal tissues. In 22 of 27 (81%) prostate tissues, miR-145 was significantly downregulated in the cancer compared with the normal tissues. Further studies on miR-145 downregulation mechanism showed that miR-145 is methylated at the promoter region in both prostate cancer tissues and 50 different types of cancer cell lines. In seven cancer cell lines with miR-145 hypermethylation, 5-aza-2'-deoxycytidine treatment dramatically induced miR-145 expression. Interestingly, we also found a significant correlation between miR-145 expression and the status of p53 gene in both LCM prostate tissues and 47 cancer cell lines. In 29 cell lines with mutant p53, miR-145 levels were downregulated in 28 lines (97%), whereas in 18 cell lines with wild-type p53 (WT p53), miR-145 levels were downregulated in only 6 lines (33%, P < 0.001). Electrophoretic mobility shift assay showed that p53 binds to the p53 response element upstream of miR-145, but the binding was inhibited by hypermethylation. To further confirm that p53 binding to miR-145 could regulate miR-145 expression, we transfected WT p53 and MUT p53 into PC-3 cells and found that miR-145 is upregulated by WT p53 but not with MUTp53. The apoptotic cells are increased after WT p53 transfection. In summary, this is the first report documenting that downregulation of miR-145 is through DNA methylation and p53 mutation pathways in prostate cancer.

IGFBP-4 activates the Wnt/beta-catenin signaling pathway and induces M-CAM expression in human renal cell carcinoma.

The Wnt/ß-catenin signaling pathway is inactivated by Wnt antagonists in most cancers and IGFBP-4 is an antagonist of the Wnt/ ß-catenin signaling pathway. However, the function of IGFBP-4 is not currently understood in renal cell carcinoma (RCC). We initially found that the expression of IGFBP-4 was significantly lower in primary RCC and higher in metastatic RCC compared to normal human kidney tissues. To assess the function of IGFBP4, we established IGFBP4 transfectants (primary renal cancer cell line) and performed functional analyses including Tcf reporter assays, cell viability, invasive capability, mortality, and in vivo tumor growth. Interestingly IGFBP-4 transfectants promoted cell growth (in vitro and in vivo), invasion, and motility in primary renal cancer. Tcf transcriptional activity was significantly increased in IGFBP-4 transfectants compared to mock cells and ß-catenin expression was increased. Also the ß-catenin downstream effector, MT1-MMP showed increased expression in IGFBP4 transfectants. Additionally IGFBP4 induced the expression of M-CAM, a marker of tumor progression. In order to assess the role of IGFBP4 in metastatic renal cancer, IGFBP-4 mRNA in a metastatic renal cancer cell lines (ACHN) was knocked-down using a siRNA technique. The cell growth and motility was decreased in si-IGFBP4 transfected ACHN cells compared to cells transfected with control siRNA. Tcf activity in ACHN cells was also decreased with si-IGFBP-4 transfection. This is a first report documenting that IGFBP-4 expression in RCC activates cell growth, metastasis, Wnt/beta-catenin signaling and may be involved in RCC metastasis.

Wnt antagonist DICKKOPF-3 (Dkk-3) induces apoptosis in human renal cell carcinoma.

The Wnt signaling pathway is activated in most cancers while Wnt antagonist genes are inactivated. However, the functional significance and mechanisms of inactivation of Wnt antagonist Dkk-3 gene in renal cell carcinoma (RCC) has not been reported. In this study, we examined potential epigenetic mechanisms regulating Dkk-3 expression in RCC cells and whether Dkk-3 expression affects cell growth and apoptosis. The expression of Dkk-3 is regulated by histone modification rather than CpG island DNA methylation in renal cancer cells. Renal cancer cell proliferation was significantly inhibited and apoptosis was promoted in Dkk-3 transfected renal cancer cells. Dkk-3 did not inhibit the Wnt/beta-catenin signaling pathway but induced apoptosis via the noncanonical JNK pathway in renal cancer cells. Expression of p21, MDM-2, and Puma genes were increased after transfecting RCC cell lines with a Dkk-3 expression plasmid. Overexpression of Dkk-3 induced G(0)/G(1) arrest together with an increase in p21 expression. Growth of stable Dkk-3 transfected cells in nude mice was decreased compared to controls. Our data show for the first time that mRNA expression of Dkk-3 is regulated by histone modification and that Dkk-3 inhibits renal cancer growth through modulation of cell cycle and apoptotic pathways.

A lncRNA TCL6-miR-155 Interaction Regulates the Src-Akt-EMT Network to Mediate Kidney Cancer Progression and Metastasis.

Metastasis is the leading cause of mortality from kidney cancer, and understanding the underlying mechanism of this event will provide better strategies for its management. Here we investigated the biological, functional, and clinical significance of lncTCL6 and its interacting miR-155 in clear cell renal cell carcinoma (ccRCC). We employed a comprehensive approach to investigate the lncTCL6-miR-155-Src/Akt-mediated epithelial-to-mesenchymal transition (EMT) pathway as a novel regulatory mechanism in ccRCC progression. Expression analyses revealed that lncTCL6 is downregulated in ccRCC compared with normal tissues. Overexpression of lncTCL6 in ccRCC cell lines impaired their oncogenic functions, such as cell proliferation and migration/invasion, and induced cell-cycle arrest and apoptosis; conversely, depletion of lncTCL6 rescued these phenotypic effects. Furthermore, lncTCL6 directly interacted with miR-155. Unlike lncTCL6, miR-155 was overexpressed in ccRCC. Stable knockdown of miR-155 phenocopied the effects of lncTCL6 overexpression. Conversely, reconstitution of miR-155 and suppression of lncTCL6 in noncancerous renal cell HK2 induced tumorigenic characteristics. Patients with higher expression of lncTCL6 and lower expression of miR-155 had better survival probability. When overexpressed, lncTCL6 recruited STAU1 and mediated decay of Src mRNA, followed by a marked downregulation of an integrated network of Src target genes involved in migration, invasion, and EMT. However, the interaction between miR-155 and lncTCL6 attenuated the regulatory role of lncTCL6 on Src-mediated EMT. In conclusion, this study is the first report documenting the lncTCL6-miR155-Src/Akt/EMT network as a novel regulatory mechanism in aggressive ccRCC and a promising therapeutic target to inhibit renal cancer. SIGNIFICANCE: This study's investigation of noncoding RNA interactions in renal cell carcinoma identify miRNA-155-lncRNA TCL6-mediated regulation of the Src-Akt-EMT network as a novel mechanism of disease progression and metastasis.

Suppressor effect of catechol-O-methyltransferase gene in prostate cancer.

Catechol-estrogens can cause genetic mutations and to counteract their oncogenicity, the catechol-O-methyltransferase (COMT) gene is capable of neutralizing these reactive compounds. In this study, we determined the functional effects and regulation of COMT in prostate cancer. Both the Cancer Genome Atlas (TCGA) and immunohistochemical analysis of clinical specimens demonstrated a reduction of COMT expression in prostate cancer. Also, western analyses of prostate cancer cell lines show COMT levels to be minimal in DuPro and DU145 and thus, these cells were used for further analyses. Re-expression of COMT led to suppressed migration ability (wound healing assay) and enhanced apoptosis (flow cytometric analyses), and when challenged with 4-hydroxyestradiol, a marked reduction of cell proliferation (MTT assay) was observed. Xenograft growth in athymic mice also resulted in inhibition due to COMT. As a mechanism, western analyses show cleaved CASP3 and BID were increased whereas XIAP and cIAP2 were reduced due to COMT. As COMT expression is low in prostate cancer, its regulation was determined. Databases identified several miRNAs capable of binding COMT and of these, miR-195 was observed to be increased in prostate cancer according to TCGA. Real-time PCR validated upregulation of miR-195 in clinical prostate cancer specimens as well as DuPro and DU145 and interestingly, luciferase reporter showed miR-195 capable of binding COMT and overexpressing miR-195 could reduce COMT in cells. These results demonstrate COMT to play a protective role by activating the apoptosis pathway and for miR-195 to regulate its expression. COMT may thus be a potential biomarker and gene of interest for therapeutic development for prostate cancer.

Metabolic, pathologic, and genetic analysis of prostate tissues: quantitative evaluation of histopathologic and mRNA integrity after HR-MAS spectroscopy.

The impact of high-resolution magic angle spinning (HR-MAS) spectroscopy on the histopathologic and mRNA integrity of human prostate tissues was evaluated. Forty prostate tissues were harvested at transrectal ultrasound (TRUS) guided biopsy (n = 20) or radical prostatectomy surgery (n = 20), snap-frozen on dry ice, and stored at -80°C until use. Twenty-one samples (n = 11 biopsy, n = 10 surgical) underwent HR-MAS spectroscopy prior to histopathologic and cDNA microarray analysis, while 19 control samples (n = 9 biopsy, n = 10 surgical) underwent only histopathologic and microarray analysis. Frozen tissues were sectioned at 14-µm intervals and placed on individual histopathology slides. Every 8th slide was stained with hematoxylin and eosin (H&E) and used to target areas of predominantly epithelial tissue on the remaining slides for mRNA integrity and cDNA microarray analysis. Histopathologic integrity was graded from 1 (best) to 5 (worst) by two 'blinded' pathologists. Histopathologic integrity scores were not significantly different for post-surgical tissues (HR-MAS vs controls); however, one pathologist's scores were significantly lower for biopsy tissues following HR-MAS while the other pathologist's scores were not. mRNA integrity assays were performed using an Agilent 2100 Bioanalyzer and the electrophoretic traces were scored with an RNA integrity number (RIN) from 1 (degraded) to 10 (intact). RIN scores were not significantly different for surgical tissues, but were significantly lower for biopsy tissues following HR-MAS spectroscopy. The isolated mRNA then underwent two rounds of amplification, conversion to cDNA, coupling to Cy3 and Cy5 dyes, microarray hybridization, imaging, and analysis. Significance analysis of microarrays (SAM) identified no significantly over- or under-expressed genes, including 14 housekeeping genes, between HR-MAS and control samples of surgical and biopsy tissues (5% false discovery rate). This study demonstrates that histopathologic and genetic microarray analysis can be successfully performed on prostate surgical and biopsy tissues following HR-MAS analysis; however, biopsy tissues are more fragile than surgical tissues.