Adjacent Normal Tissue Specimens Research Articles

MiR-363 suppresses cell migration, invasion, and epithelial-mesenchymal transition of osteosarcoma by binding to NOB1

BackgroundOsteosarcoma (OS) is a primary malignant bone tumor with a high rate of metastasis and a short 5-year survival rate. MiR-363 was downregulated in a variety of tumors and played a role in suppressing tumors. However, the roles of miR-363 in osteosarcoma remain unknown; thus, the purpose of this study was to explore the functions of miR-363 in osteosarcoma.MethodsCCK-8 and transwell assays were performed to evaluate the proliferation, migration, and invasion abilities of MG63 cells. The epithelial-mesenchymal transition (EMT) and apoptosis-associated proteins were measured by using Western blot assay. Luciferase reporter assay was utilized to verify whether miR-363 directly bound to the 3′-UTR of NOB1 mRNA.ResultsMiR-363 was downregulated while NOB1 was upregulated in osteosarcoma clinical tissue specimens and cell lines as compared with the adjacent normal tissue specimens and normal cell line. The miR-363 is reversely correlated with the expression of NOB1 in osteosarcoma tissues. Overexpression of miR-363 suppressed the ability of cell migration, invasion, and EMT, whereas low expression of miR-363 promoted this ability. In addition, miR-363 inhibited osteosarcoma proliferation both in vitro and in vivo and inhibited the apoptosis in MG63 cells. Interference of NOB1 could inhibit the migration, invasion, and EMT of osteosarcoma cell line MG63. NOB1 was verified to be a direct target of miR-363 and its expression was mediated by miR-363. Re-expression of NOB1 could partially reverse the inhibitory effect of miR-363 on cell migration and invasion. In addition, low expression of miR-363 or overexpression of NOB1 predicted poor prognosis of osteosarcoma patients.ConclusionMiR-363 inhibited osteosarcoma the proliferation, migration, invasion, and EMT and induced the apoptosis by directly targeting NOB1 in MG63 cells. The newly identified miR-363/NOB1 axis provides novel insights into the pathogenesis of osteosarcoma.

Abstract IA11: Bladder cancers affecting transplant recipients harbor diverse viruses that associate with overall survival

Abstract BK polyomavirus (BKPyV) is a ubiquitous virus that establishes a lifelong subclinical infection in the urinary tract. In transplant recipients, BKPyV can reactivate and cause kidney and bladder damage. Recent sequencing studies of tumors from transplant recipients have revealed that BKPyV sequences are present in approximately 25% of bladder tumors. This is dramatically higher than the <1% rate of BKPyV detection observed in muscle-invasive bladder cancers affecting the general population. Like cancer-causing human papillomaviruses (HPVs), BKPyV has been shown to upregulate the human cytosine deaminase APOBEC3B, which has emerged as the second most abundant source of point mutations in human cancers. Furthermore, the APOBEC3-associated mutation signature is the most abundant mutational signature in bladder cancer. To comprehensively assess the role of BKPyV and other viruses in bladder cancer from solid organ transplant recipients, we used a database linking US transplant and cancer registries to identify and collect 44 archived primary bladder tumors, 5 metastases, and 15 adjacent normal tissue specimens. Whole-genome and total RNA sequencing was performed on the samples. From these data, we detected BKPyV in 18% of primary tumors. In five cases, there is clear evidence of BKPyV integration into the host cell genome, and all cases show transcription of the viral tumor antigens. In a separate set of analyses, BKPyV transcription was detected in 3.7% of non-muscle-invasive early-stage bladder cancer cases affecting the general population. Our surveys also detected other viruses, including high- and low-risk HPVs, JC polyomavirus (JCPyV), and anelloviruses, at lower frequencies. Two primary-metastatic pairs maintained clonally integrated HPV or BKPyV and respective viral oncogene expression in the metastatic lesions. Nearly all tumors had dominant APOBEC3 signature mutations, and BKPyV-positive tumors expressed significantly more APOBEC3B compared to virus-negative tumors or normal tissues. Tumors from four kidney transplant patients showed high mutation burden consistent with exposure to aristolochic acid, a nephrotoxic carcinogen found in some herbal supplements. Clinical data indicate that BKPyV-positive tumors are predominantly high grade with invasive behavior. Strikingly, survival was significantly shorter for patients whose tumors harbored BKPyV or JCPyV (9.7 and 8.4 months, respectively) compared to patients with tumors harboring HPV or no detectable viruses (65.8 and 50.2 months, respectively). This study comprehensively characterizes the genomes and transcriptomes of tumors, revealing several distinct tumor etiologies that impact patient outcome. Citation Format: Gabriel J. Starrett, Kelly Yu, David Petersen, Petra Lenz, Lars Dyrskjøt, Paul Meltzer, Eric Engels, Christopher B. Buck. Bladder cancers affecting transplant recipients harbor diverse viruses that associate with overall survival [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA11.

Using high-resolution magic angle spinning magnetic resonance spectroscopy to characterize the metabolomic profile of renal cell carcinoma.

710 Background: Renal cell carcinoma (RCC) is a metabolic disease, with the various subtypes exhibiting aberrations in different metabolic pathways. Metabolomics may offer greater sensitivity for revealing disease biology than evaluation of tissue morphology. In this study, we investigate the metabolomic profile of RCC using high resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS). Methods: Tissue samples were obtained from radical or partial nephrectomy specimens that were fresh frozen & stored at -80ºC. Tissue HRMAS MRS was performed by a Bruker AVANCE spectrometer. Metabolomic profiles of RCC & adjacent benign renal tissue were compared, and false discovery rates (FDR) were used to account for multiple testing. Regions of interest (ROI) with FDR < 0.05 were selected as potential predictors of malignancy. The Wilcoxon rank sum test was used to compare median MRS relative intensities for the candidate predictors. Logistic regression was used to determine odds ratios for risk of malignancy based on abundance of each metabolite. Results: There were 38 RCC (16 clear cell, 11 papillary, 11 chromophobe) & 13 adjacent normal tissue specimens (matched pairs). Metabolic predictors of malignancy based on FDR include histidine, phenylalanine, phosphocholine, serine, phosphocreatine, creatine, glycerophosphocholine, valine, glycine, myo-inositol, scylla-inositol, taurine, glutamine, spermine, acetoacetate & lactate. Higher levels of spermine, histidine & phenylalanine at 3.15-3.13 ppm were associated with a decreased risk of RCC (OR 4x10−5, 95% CI 7.42x10−8, 0.02), while 2.84-2.82 ppm increased the risk of malignant pathology (OR 7158.67, 95% CI 6.3, 8.3x106), and the specific metabolites characterizing this region remain to be identified. Tumor stage did not appear to affect the metabolomics of malignant tumors, suggesting that metabolites are more dependent on histologic subtype. Conclusions: HRMAS-MRS identified many metabolites that may predict RCC. We demonstrated that those in the 3.14-3.13 ppm ROI were present in lower levels in RCC, while higher levels of metabolites in the 2.84-2.82 ppm ROI substantially increased the risk of RCC.

Analysis of circular RNA-associated competing endogenous RNA network in breast cancer.

As the most common type of cancer in female patients, the morbidity and mortality rates of breast cancer (BC) are high, and its incidence is gradually increasing worldwide. However, the underlying molecular and genetic mechanisms involved in the etiopathogenesis of BC remain unclear. Circular RNAs (circRNAs) are a novel type of non-coding RNAs that have been verified to serve a crucial role in tumorigenesis. However, the majority of functions and mechanisms of circRNAs remain unknown. The present study identified 47 differentially expressed circRNAs in a dataset from Gene Expression Omnibus. Using the cancer-specific circRNA database, the potential microRNA (miRNA) response elements, RNA-binding proteins and open reading frames of the candidate circRNAs were predicted. Combing the predictions of miRNAs and target mRNAs, a competing endogenous RNA network was constructed, which may serve as the theoretical basis for further research. Furthermore, the analyses conducted using Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways indicated that candidate circRNAs may serve a role in transcriptional regulation. Moreover, 20 BC tissue specimens and their paired adjacent normal tissue specimens were used to evaluate the expression levels of the screened circRNAs. Thus, the analyses of the raw microarray data conducted in the present study offer perspectives on the exploration of mechanisms associated with BC tumorigenesis with regard to the circRNA-miRNA-mRNA network.

GSTZ1 deficiency promotes hepatocellular carcinoma proliferation via activation of the KEAP1/NRF2 pathway

BackgroundGlutathione S-transferase zeta 1 (GSTZ1) is the penultimate enzyme in phenylalanine/tyrosine catabolism. GSTZ1 is dysregulated in cancers; however, its role in tumorigenesis and progression of hepatocellular carcinoma (HCC) is largely unknown. We aimed to assess the role of GSTZ1 in HCC and to reveal the underlying mechanisms, which may contribute to finding a potential therapeutic strategy against HCC.MethodsWe first analyzed GSTZ1 expression levels in paired human HCC and adjacent normal tissue specimens and the prognostic effect of GSTZ1 on HCC patients. Thereafter, we evaluated the role of GSTZ1 in aerobic glycolysis in HCC cells on the basis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Furthermore, we assessed the effect of GSTZ1 on HCC proliferation, glutathione (GSH) concentration, levels of reactive oxygen species (ROS), and nuclear factor erythroid 2-related factor 2 (NRF2) signaling via gain- and loss- of GSTZ1 function in vitro. Moreover, we investigated the effect of GSTZ1 on diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) induced hepatocarcinogenesis in a mouse model of HCC.ResultsGSTZ1 was downregulated in HCC, thus indicating a poor prognosis. GSTZ1 deficiency significantly promoted hepatoma cell proliferation and aerobic glycolysis in HCC cells. Moreover, loss of GSTZ1 function depleted GSH, increased ROS levels, and enhanced lipid peroxidation, thus activating the NRF2-mediated antioxidant pathway. Furthermore, Gstz1 knockout in mice promoted DEN/CCl4-induced hepatocarcinogenesis via activation of the NRF2 signaling pathway. Furthermore, the antioxidant agent N-acetylcysteine and NRF2 inhibitor brusatol effectively suppressed the growth of Gstz1-knockout HepG2 cells and HCC progression in Gstz1−/− mice.ConclusionsGSTZ1 serves as a tumor suppressor in HCC. GSH depletion caused by GSTZ1 deficiency elevates oxidative stress, thus constitutively activating the NRF2 antioxidant response pathway and accelerating HCC progression. Targeting the NRF2 signaling pathway may be a promising therapeutic approach for this subset of HCC.

RPL34-AS1 functions as tumor suppressive lncRNA in esophageal cancer

Ribosomal protein L34 antisense RNA 1 (RPL34-AS1) is novel long non-coding RNA, and was found to be down-regulated in colorectal cancer and gastric cancer. The role of RPL34-AS1 was still unknown in esophageal cancer. In our study, we found RPL34-AS1 expression levels in esophageal cancer tissue and specimens and cell lines were lower than in adjacent normal tissue specimens and normal esophageal cell line, respectively. In addition, esophageal cancer patients with Ⅲ-Ⅳ, T3-T4 or positive lymph node metastasis had lower RPL34-AS1expression levels than esophageal cancer patients with Ⅰ-Ⅱ, T1-T2 or negative lymph node metastasis, respectively. In survival analysis suggested esophageal cancer patients with low RPL34-AS1 expression had short overall survival than those with high RPL34-AS1 expression. Our in vitro experiments suggested RPL34-AS1 inhibits esophageal cancer cell proliferation, migration and invasion through down-regulating RPL34 expression. In conclusion, RPL34-AS1 functions as tumor suppressor in esophageal cancer.

The expression and clinical significance of cell division cycle associated 5 in bladder cancer tissues

Objective To investigate the expression of cell division cycle associated 5 (CDCA5) in bladder cancer and its correlation with clinicopathological features. Methods Clinical data, bladder cancer tissues and adjacent normal tissue specimens of 56 patients undergoing radical cystectomy were collected. Immunohistochemical technique was used to detect the expression of CDCA5 in bladder cancer tissues and adjacent normal tissues, and the correlation between CDCA5 expression and clinicopathological features was analyzed. Results Immunohistochemistry showed that the high expression rate of CDCA5 in bladder cancer tissues was 60.7% (34/56), and the low expression rate was 39.3% (22/56). The high expression rate in normal tissues adjacent to cancer was 23.2% (13/56), and the low expression rate was 76.8% (43/56). The expression of CDCA5 in bladder cancer tissues and adjacent normal tissues was statistically significant (χ2=16.168, P 0.05), gender (χ2=0.049, P>0.05) The tumor diameter (χ2=0.443, P>0.05) and lymph node metastasis (χ2=0.225, P>0.05) were irrelevant. Conclusion CDCA5 is up-regulated in bladder cancer tissues and associated with increased clinical stage and pathological grade. It suggests that CDCA5 is associated with the occurrence, development, invasion and malignancy of bladder cancer. Key words: Bladder cancer; Cell division cycle associated 5; Clinicopathological features; Immunohistochemistry

Expression of calpain small subunit 1 in breast cancer and its clinical significance

Objective To investigate the expression of calpain small subunit 1 (Capn4) in breast cancer and its clinical significance. Methods The cancer tissues and the paraffin-embeded specimens of the corresponding adjacent normal tissues from 12 patients with breast cancer between May 2015 and July 2015 in Shanghai General Hospital were collected. The expression of Capn4 was detected by using immunohistochemistry. The paraffin-embeded specimens of cancer tissues from 70 breast cancer patients in Shanghai General Hospital between January 2009 and September 2011 were also collected. And the expression of Capn4 was detected. The correlation between Capn4 expression level and TNM stage, pathological grade, molecular markers as well as the prognosis of breast cancer was also analyzed. Results The expression of Capn4 in breast cancer tissues was higher than that in the corresponding adjacent normal tissues. The expression level of Capn4 was correlated with TNM stage (P = 0.002), T stage (P = 0.004), N stage (P = 0.004), M stage (P = 0.025), estrogen receptor (ER) status (χ 2 = 4.787, P = 0.029) and survival status (χ 2 = 5.826, P = 0.016). Kaplan-Meier survival analysis revealed that the median survival time in the higher expression of Capn4 group 90 months was shorter than that in the lower expression of Capn4 group (not reaching the median survival time), and there were statistical differences (χ 2 = 4.351, P = 0.037). Conclusions The expression of Capn4 in breast cancer tissues is upregulated. The expression difference is correlated with TNM stage, pathological grade, ER status and the prognosis of breast cancer, suggesting that it may become a new target and molecular marker for breast cancer treatment. Key words: Breast neoplasms; Calcium-activated neutral protease; Calpain small subunit 1; Prognosis; Immunohistochemistry

FEZF1-AS1 functions as an oncogenic lncRNA in retinoblastoma.

Long non-coding RNA (lncRNA) FEZF1 antisense RNA 1 (FEZF1-AS1) has been shown to be up-regulated in tumor tissues and cells, and exerts oncogenic effects on various types of malignancies. However, the expression and function of FEZF1-AS1 was still fully unclear in retinoblastoma. The purpose of our study was to investigate the expression and clinical value of FEZF1-AS1 in retinoblastoma patients, and explore the effect of FEZF1-AS1 on retinoblastoma cell proliferation, migration and invasion. In our results, levels of FEZF1-AS1 expression were elevated in retinoblastoma tissue specimens and cell lines compared with adjacent normal retina tissue specimens and human retinal pigment epithelial cell line, respectively. The correlation analysis indicated that high FEZF1-AS1 expression was significantly correlated with present choroidal invasion and optic nerve invasion. Survival analysis suggested that retinoblastoma patients in high FEZF1-AS1 expression group had obviously short disease-free survival (DFS) compared with retinoblastoma patients in low FEZF1-AS1 expression group, and high FEZF1-AS1 expression was an independent unfavorable prognostic factor for DFS in retinoblastoma patients. Loss-of-function study indicated silencing FEZF1-AS1 expression inhibited retinoblastoma cell proliferation, invasion and migration. In conclusion, FEZF1-AS1 functions as an oncogenic lncRNA in retinoblastoma.

GHET1 acts as a prognostic indicator and functions as an oncogenic lncRNA in cervical cancer.

Gastric carcinoma proliferation enhancing transcript 1 (GHET1) has been suggested to serve as a promising oncogenic lncRNA in various types of human cancer. However, the role of GHET1 remained unknown in cervical cancer. In our study, we found GHET1 expression was markedly elevated in cervical cancer tissue specimens and cell lines compared with adjacent normal cervical tissue specimens and human normal cervical cell line, respectively. Then, we found high expression of GHET1 is a useful biomarker to discriminate cervical cancer tissues from non-tumorous tissues, and associated with advanced clinical stage, lymph node metastasis, distant metastasis and poor histological grade in cervical cancer patients. The survival analysis showed high GHET1 expression was an independent unfavorable prognostic factor in cervical cancer patients. Knockdown of GHET1 expression markedly inhibits cervical cancer cell proliferation, migration, and invasion. The loss-of-function study indicated knockdown of GHET1 expression markedly inhibits cervical cancer cell proliferation, migration, and invasion. In conclusion, GHET1 acts as an oncogenic lncRNA in cervical cancer.

Expression of long non-coding RNA MAGI2‑AS3 in human gliomas and its prognostic significance.

It has been confirmed that the dysregulation of long noncoding RNAs (lncRNAs) is associated with various diseases, especially cancer. LncRNA MAGI2 antisense RNA 3 (MAGI2-AS3) has been reported to be involved in the progression of bladder cancer and breast cancer. In this study, we aimed to explore its expression and clinical significance in glioma. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) assays were performed to detect the expression levels of MAGI2-AS3 in fresh glioma and matched adjacent normal brain tissue specimens, which were collected from 178 patients. Then the association between MAGI2-AS3 expression and clinical-pathological parameters were further evaluated using the Chi-square test. The overall survival (OS) was analyzed by the log-rank test, and the survival curves were plotted according to Kaplan-Meier. Univariate and multivariate analyses were performed to analyze the prognostic significance of MAGI2-AS3 expression. We found that the relative expression level of MAGI2-AS3 in glioma tissues was significantly lower than those in adjacent normal brain tissues (p<0.01). Lower MAGI2-AS3 expression was observed to be positively correlated with the World Health Organization (WHO) grade (p=0.031) and KPS score (p=0.003) in glioma patients. The Kaplan-Meier analysis indicated that patients with low MAGI2-AS3 expression levels tended to have worse overall survival than those with high levels of MAGI2-AS3 expression (p=0.0042). In the multivariate analysis, we further observed that MAGI2-AS3 expression in glioma tissues was an independent prognostic factor for overall survival (HR=3.098, 95% CI: 1.289-4.118, p=0.014). MAGI2-AS3 expression represents a significant favorable prognostic factor for patients with glioma.

CCEPR is a novel clinical biomarker for prognosis and regulates cell proliferation through PCNA in osteosarcoma.

CCEPR (cervical carcinoma expressed PCNA regulatory lncRNA) has been found to be upregulated and enhance cell proliferation in human cancers. However, the role of CCEPR in osteosarcoma remains to be discovered. In this study, we found CCEPR expression was elevated in osteosarcoma tissue specimens and cell lines compared with adjacent normal tissue specimens and osteoblast cell line, respectively. Meanwhile, osteosarcoma patients with advanced stage or tumor size greater than 8 cm had higher expression of CCEPR than patients with early stage or tumor size less than or equal to 8 cm, respectively. Survival analysis suggested that osteosarcoma patients with high CCEPR expression had a worse overall survival rate than those with low CCEPR expression. The in vitro study indicated that CCEPR positively regulated proliferating cell nuclear antigen (PCNA) expression in osteosarcoma cells and silencing of CCEPR inhibited osteosarcoma cell proliferation through decreasing PCNA expression. In conclusion, CCEPR is a potential prognostic predictor and functions as oncogenic long non-coding RNA (lncRNA) to regulate cell proliferation via PCNA in osteosarcoma.

Expression and significance of tripartite motif 14 in pancreatic cancer tissue

Objective To investigate the expression of tripartite motif 14 (TRIM14) in pancreatic cancer tissue and its clinical significance. Methods The expression of TRIM14 in 32 specimens of pancreatic cancer tissue and 32 specimens of adjacent normal tissues was detected by immunohistochemistry and Western blotting, and their correlation with clinicopathological features of pancreatic cancer was analyzed. Results As compared with adjacent tissues, the expression of TRIM14 protein was up-regulated in pancreatic cancer tissues (t=19.520, P 0.05), but significantly correlated with clinical stage, differentiated degree and tumor size (P<0.05). The survival time in patients with high TRIM14 expression was significantly shorter than that in patients with low TRIM14 expression (χ2=5.180, P<0.05). Conclusion The positive TRIM14 expression rate is increased in pancreatic cancer tissue, and its positive expression may be associated with the occurrence, development and invasion of pancreatic cancer. The TRIM14 expression predicts a poor prognosis in patients with pancreas cancer. Key words: Pancreatic neoplasms; Tripartite motif 14; Prognosis

MNX1-AS1 is a functional oncogene that induces EMT and activates the AKT/mTOR pathway and MNX1 in breast cancer.

PurposelncRNAs have recently been identified as key regulators of basic biological processes as well as the pathogenesis of various diseases. Previous studies have shown that lncRNA MNX1-AS1 promotes cell migration and invasion in ovarian cancer; however, its role in regulating breast cancer-associated biological processes remains unclear.Materials and methodsWe obtained paired specimens of breast cancer tissues and adjacent normal tissues by modified radical mastectomy from 36 patients, in addition to four breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT-549 and MCF-7). RNA was isolated from these tissues and cell lines and subsequently subjected to quantitative real-time polymerase chain reaction. This was followed by bisulfite deep sequencing. The cells were also transfected with siRNA against MNX1-AS1. The cells were then subject to cell proliferation, Transwell migration and invasion assays. Finally, Western blotting analysis was conducted to determine expression levels of MNX1, 5-cadherin, Snail and Slug.ResultsOur results show that MNX1-AS1 expression was significantly higher in breast cancer tissues than adjacent normal tissues. Moreover, knockdown/overexpression of MNX1-AS1 inhibits/promotes proliferation, migration and invasion of breast cancer cells. MNX1-AS1 and its natural sense transcript MNX1 are expressed synergistically in breast tumor tissues. Our results suggest that MNX1-AS1 is a functional oncogene that induces epithelial–mesenchymal transition, in addition to activating AKT/mTOR pathway and its natural sense transcript MNX1 in breast cancer cells.ConclusionOur data indicate that MNX1-AS1 can serve as a novel therapeutic target in breast cancer.

The effect of centromere protein U silencing by lentiviral mediated RNA interference on the proliferation and apoptosis of breast cancer.

Centromere protein U (CENPU) is a novel transcriptional repressor that is associated with different types of cancer. However, its function in breast cancer is poorly understood. In the present study, it was identified that CENPU was highly expressed in breast cancer tissues compared with expression in normal breast tissues (P=0.001). Furthermore, the CENPU mRNA level in tumors was often elevated, compared with the matched adjacent normal breast cancer tissue specimens in the dataset from The Cancer Genome Atlas database (n=106; P<0.001). To understand the function of CENPU in human breast carcinogenesis, its effects on the proliferation, apoptosis and cell cycle progression of MDA-MB-231 cells were examined using the lentiviral-mediated CENPU knockdown approach. The RNA and protein expression levels in the transfected cells were monitored using reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The mRNA and protein expression levels of the CENPU gene were significantly lower in the CENPU-shRNA transfected cells than in the control (P<0.01), indicating successful gene expression knockdown. Post-transfection, cell counting and MTT analysis revealed that the proliferation activity was significantly suppressed in CENPU knockdown cells relative to the control (P<0.01). Additionally, fluorescence activated cell sorting analysis revealed that the (G2+S) phase fraction was significantly declined in CENPU knockdown cells relative to the control; while the G1 phase fraction was significantly increased (P<0.01) and the percentage of the apoptotic cells was significantly increased (P<0.01). In conclusion, downregulation of CENPU gene expression may inhibit cell proliferation and cell cycle progression, and increase the apoptosis of the breast cancer cells. These results suggested a possible function of this protein in breast cancer pathogenesis and prognosis.

Expression and prognosis of MYOZ2 in gastric cancer.

To investigate the expression of human myozenin 2 (MYOZ2) in cancer tissues and its effect on the prognosis of patients with gastric cancer. Gastric cancer tissue and adjacent normal tissue specimens were obtained from a total of 258 patients together with complete clinicopathological data. Those patients were treated in Harbin Medical University Cancer Hospital from March 2007 to March 2012. Quantitative Real Time-Polymerase Chain Reaction (RT-PCR) was used to detect the expression of MYOZ2 messenger RNA (mRNA) in gastric cancer tissues and normal gastric tissues, and correlations between the expression level of MYOZ2 in gastric cancer tissues and the clinicopathological parameters of patients were analyzed. MYOZ2 protein expression levels in different gastric cancer cells were detected by Western blotting; transwell chamber assay was used to detect the effect of MYOZ2 expression on the in-vitro migration and invasion abilities of gastric cancer cells; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to examine the in-vitro proliferation and growth abilities of gastric cancer cells. The expression level of MYOZ2 mRNA in gastric cancer tissues was significantly higher than that in cancer-adjacent tissues in 258 cases (p<0.05). Western blotting results showed that the expression level of MYOZ2 protein in gastric cancer tissues was significantly increased compared with that in the corresponding adjacent healthy tissues (p<0.05). In-vitro growth, migration and invasion abilities of MYOZ2 positive gastric cancer cells were significantly higher than those of normal tissue cells. Univariate analysis showed that the high expression level of MYOZ2 in gastric cancer tissues was closely related to tumor size, lymph node metastasis, and pathological tumor-node-metastasis (pTNM) staging (p<0.05), but not associated with gender, age, differentiation degree, and tumor location (p>0.05). The 1-, 3- and 5-year survival rates of patients with low expression level of MYOZ2 (n=130) were higher than those of patients with high expression level of MYOZ2 (n=128). Multivariate analysis revealed that the expression level of MYOZ2 (p=0.000), lymph node metastasis (p=0.002), and pTNM staging (p=0.015) were independent risk factors influencing the prognosis of gastric cancer. Our results showed that the expression level of MYOZ2 may play an important role in the occurrence and development of gastric cancer, and can also provide references for the clinical diagnosis and treatment of gastric cancer.

Fos-like antigen 2 (FOSL2) promotes metastasis in colon cancer

Among different cancers, incidence and mortality of colorectal cancer (CRC) is one of the highest. KRAS mutation is one of the underlying features in the pathogenesis of CRC with CRC tumors harboring mutant KRAS exhibiting a more aggressive behavior compared to CRC tumors with wild type KRAS. We had earlier shown that the microRNA-143 (miR-143) replenishment not only chemosensitizers CRC cell line with mutant KRAS instead of wild-type KRAS gene, to paclitaxel-mediated cytotoxicity, but also inhibits cell migration and invasion ability. Hence, the study aimed to determine how miR-143 replenishment is inhibiting pre-metastatic behavior in CRC cells with mutant KRAS. Top ten mRNA targets of miR-143 as predicted by TargetScan were evaluated by qRT-PCR in LoVo cells which were performed mock transfection or miR-143 mimic transfection. Evaluation of the changes in cognate mRNA target(s) was done in 30 paired CRC tissue and tumor adjacent normal tissue specimens and in LoVo cells by western blot. Effect of the mRNA target on pro-metastatic behavior was assayed by gain- and loss-of-function studies using a combination of western blotting and in vitro cell proliferation and transwell migration/invasion assay in LoVo cells and in the normal colonic epithelium cell line FHC. In vivo effect of the cognate mRNA target on CRC metastasis was assayed by xenograft assay. Of the 10 predicted mRNA targets, FOSL2 (P < 0.05) and IGFBP5 (P > 0.05) was down regulated in LoVo cells transfected with the miR-143 mimic. FOSL2 mRNA levels were significantly downregulated in CRC tissue specimens compared with adjacent normal tissue (P < 0.05). Immunoblot analysis showed that FOSL2, but not IGFBP5, protein expression is down regulated in LoVo cells after the miR-143 mimic transfection. FOSL2 overexpression in the normal colonic epithelial cell line FHC or siRNA-mediated silencing in LoVo cells induced and repressed, respectively, pro-mesenchymal cell features. Whereas manipulation of FOSL2 expression did not have any effect on cell proliferation rates, silencing its expression inhibited cell migration and invasion ability in vitro. In addition, silencing of FOSL2 expression in the LoVo cells can significantly inhibited invasion of hepatic, while no effect was found for tumorigenic potential. Our results suggest that FOSL2 is a critical regulator of CRC metastasis and might be an important marker for prognostic in CRC patients.

Expression Analysis of ACSL5 and Wnt2B in Human Congenital Pulmonary Airway Malformations

BackgroundThe objective of this study was to determine acyl-CoA synthetase 5 (ACSL5) and Wnt2B expression patterns in human congenital pulmonary airway malformations (CPAMs) and to identify the possible roles of ACSL5 and Wnt2B in the pathogenesis of CPAM. MethodsExpression of ACSL5 and Wnt2B was evaluated by immunohistochemical staining, Western blotting, and quantitative real-time polymerase chain reaction, which were performed on surgical specimens of CPAM and adjacent normal lung tissues as controls. ResultsImmunohistochemistry revealed that ACSL5 and Wnt2B immunopositive cells were predominantly detected in the mesenchymal cell nucleus, and there were lower expressions of ACSL5 and Wnt2B immunopositive cells in CPAM tissues than those in adjacent normal lung tissues. Western blotting and quantitative real-time polymerase chain reaction showed that ACSL5 and Wnt2B protein and mRNA expressions were significantly decreased in CPAM tissues as compared to the adjacent normal lung tissues (P < 0.05). In addition, there was a reduced level of ACSL5 relative to that of Wnt2B. ConclusionsThe decreased ACSL5 and Wnt2B expressions correlated with aberrations in pulmonary development and in the pathogenesis of CPAM, so downregulation of ACSL5 and Wnt2B could play an important role in the development of bronchial-alveolar structures in CPAM.

PD46-03 GENOMIC HALLMARKS OF RENAL MEDULLARY CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research & Pathophysiology I1 Apr 2018PD46-03 GENOMIC HALLMARKS OF RENAL MEDULLARY CARCINOMA Jesse M. Fox, Denise Young, Yingjie Song, Heng Cheng Hu, Anthony Soltis, Matthew Wilkerson, Clifton L. Dalgard, Inger L. Rosner, Shiv Srivastava, Isabell A. Sesterhenn, and Shyh-Han Tan Jesse M. FoxJesse M. Fox More articles by this author , Denise YoungDenise Young More articles by this author , Yingjie SongYingjie Song More articles by this author , Heng Cheng HuHeng Cheng Hu More articles by this author , Anthony SoltisAnthony Soltis More articles by this author , Matthew WilkersonMatthew Wilkerson More articles by this author , Clifton L. DalgardClifton L. Dalgard More articles by this author , Inger L. RosnerInger L. Rosner More articles by this author , Shiv SrivastavaShiv Srivastava More articles by this author , Isabell A. SesterhennIsabell A. Sesterhenn More articles by this author , and Shyh-Han TanShyh-Han Tan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2151AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Renal medullary carcinoma (RMC) is a rare renal neoplasm accounting for less than 1% of all renal cell carcinomas. RMC, which frequently occurs in young African Americans with sickle cell trait, is an aggressive and lethal cancer with a median overall survival of about 13 months. Recent studies identified the biallelic loss of SMARCB1, a subunit of the SWI/SNF chromatin remodeling complex, as a characteristic feature of RMC tumors. Other reports suggest that the presence of vinculin-anaplastic lymphoma kinase (VCL-ALK) fusion without SMARCB1 loss may represent an RMC variant. We hypothesize that in addition to these alterations, novel driver mutations are present and may become potential targets for treatment. To test this hypothesis, we carried out whole exome sequencing of RMC tumor specimens. METHODS Formalin fixed paraffin embedded (FFPE) RMC tumor and adjacent normal tissue specimens of 21 African American patients with sickle cell trait were obtained from the Joint Pathology Center. RNA and DNA were isolated using the Qiagen FFPE All Prep kit. Quantity and integrity of DNA samples were evaluated by Qubit fluorometer and Advanced Analytical Fragment Analyzer, respectively. Whole exome DNA libraries were prepared from sample using the Illumina TruSeq Exome kit. Paired end sequencing (2x75) were performed on the Illumina HiSeq3000 instrument. In parallel with genomic evaluations, known RMC associated molecular alterations, including SMARCB1, VHL, HIF2A, AURKA, and EZH2, were analyzed by immunohistochemistry (IHC). RESULTS Somatic copy number alterations were estimated from the whole exome sequencing of seven tumor normal pairs. Recurrent amplifications of chromosome 7 and 8 and deletions on chromosome 15 were identified. Analysis of somatic sequence mutations detected recurrent non-silent mutations on NF2 and RBM47, and a canonical hotspot mutation (R132C) in IDH1. IHC results show the loss of SMARCB1 expression in all 21 cases assayed. Furthermore, the up-regulation of EZH2 and AURKA, genes normally repressed by SMARCB1, were detected in 3 of 21 and 11 of 21 cases, respectively. Focal HIF2A staining was detected in 7 of 21 tumor cases. CONCLUSIONS The discovery of recurrent somatic sequence mutations, gene amplifications and deletions reveals potential novel oncogenic drivers of RMC. Further validation of these genomic alterations may offer additional insight into the etiology of RMC and new options to manage the disease. The inactivation of SMARCB1 protein expression and upregulation of EZH2 and AURKA supports the evaluation of epigenetic modulating agents for treatment of RMC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e891-e892 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Jesse M. Fox More articles by this author Denise Young More articles by this author Yingjie Song More articles by this author Heng Cheng Hu More articles by this author Anthony Soltis More articles by this author Matthew Wilkerson More articles by this author Clifton L. Dalgard More articles by this author Inger L. Rosner More articles by this author Shiv Srivastava More articles by this author Isabell A. Sesterhenn More articles by this author Shyh-Han Tan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

High expression of CIP2A protein is associated with tumor aggressiveness in stage I-III NSCLC and correlates with poor prognosis.

The aim of this work was to examine the expression of cancerous inhibitor of protein phosphatase 2A (CIP2A) in non-small cell lung cancer (NSCLC) and analyze its correlation with clinical outcomes. CIP2A protein levels were detected by immunohistochemistry (IHC). One hundred and eighty-four of 209 (88.3%) primary stage I–III NSCLC specimens and 4 of 38 (10.5%) adjacent normal lung tissue specimens expressed CIP2A protein. High expression of CIP2A was detected in 38.8% (81/209) of the NSCLC specimens. Patients diagnosed histologically with late-stage NSCLC (p<0.001) and malignant nodes (p=0.001) exhibited high CIP2A expression. Univariate analysis using the log-rank test identified CIP2A expression as a prognostic predictor for overall survival (p=0.005). In multivariate analyses using the Cox regression test, CIP2A expression, T stage, N stage, histological type, and chemotherapy were identified as independent prognostic factors (p=0.007, 0.001, 0.003, <0.001, and <0.001, respectively). Furthermore, Kaplan–Meier survival curves demonstrated that high CIP2A expression indicated poor prognosis in the subgroup of patients with squamous cell carcinoma (p=0.008). Similar results were noted in the subgroup of patients with adenocarcinoma, but the results did not reach statistical significance (p=0.084). We also used univariate analysis and multivariate analysis to assess the prognostic factors for overall survival in the subgroup of patients who received postoperative chemotherapy. CIP2A expression was also an independent prognostic factor in NSCLC patients who received postoperative chemotherapy (p=0.009), along with histological type (p=0.001) and N stage (p=0.034). In conclusion, adding to the accumulating evidence, our research suggested that the CIP2A expression is associated with aggressiveness and correlates with poor prognosis in NSCLC. Our findings also indicated that CIP2A might be a potential therapeutic target against NSCLC.