PVT1 Expression Research Articles

Salivary HOTAIR and PVT1 as novel biomarkers for early pancreatic cancer.

Sensitive and non-invasive biomarkers for pancreatic cancer (PC) are lacking. We aimed to identify salivary long non-coding RNAs (lncRNAs) as biomarkers in diagnosis of resectable PC. Five well-documented lncRNAs: H19, HOTAIR, HOTTIP, MALAT1, PVT1, which are most closely associated with pancreatic cancer from previous studies were selected as putative lncRNA biomarkers. Their expression in pancreatic tissues and saliva of cancer patients and healthy controls was measured by quantification polymerase chain reaction (qPCR). Compared with benign pancreatic tumour (BPT) and normal pancreatic tissues (NPT), HOTAIR, HOTTIP and PVT1 were significantly up-regulated in pancreatic cancer tissues (PCT). As compared to BPT or healthy groups, the salivary levels of HOTAIR and PVT1 were significantly higher in PC group. They were significantly reduced after the curative pancreatectomy. Both salivary lncRNAs distinguished PC patients from healthy controls and BPT patients with sensitivities and specificities ranging from 60–97%. The expression of salivary HOTAIR and PVT1 did not differ significantly between healthy controls and any one of eight leading cancers worldwide. Collectively, our findings indicate that salivary HOTAIR and PVT1 show potential as novel non-invasive biomarkers for detecting PC.

Abstract B10: Overexpression of the lncRNA PVT1 contributes to the cervical cancer phenotype, possibly via association with nucleolin

Abstract The plasmacytoma variant translocation 1 gene (PVT1) is an lncRNA that has been designated as an oncogene due to its contribution to the phenotype of multiple cancers. Although the mechanism by which PVT1 influences disease processes has been studied in multiple cancer types, its role in cervical tumorigenesis remains unknown. Thus, the present study was designed to investigate the role of PVT1 in cervical cancer in vitro and in vivo. PVT1 expression was measured by quantitative PCR (qPCR) in 121 invasive cervical carcinoma (ICC) samples, 19 normal cervix samples, and a cervical cell line (SiHa). Further, we used targeted knockdown in conjunction with functional assays to examine PVT1's effects on cervical cancer cell proliferation, migration and invasion, apoptosis, and cisplatin resistance. Our results demonstrate that PVT1 expression is significantly increased in ICC tissue versus normal cervix. PVT1 knockdown in cervical cancer cell lines resulted in significantly decreased cell proliferation, migration and invasion. Further, apoptosis and cisplatin cytotoxicity were significantly increased in PVT1 siRNA-transfected cells. Finally, we show that PVT1 associates with Nucleolin protein and may aid in its stabilization of GADD45A mRNA providing a possible mechanism by which PVT1 overexpression drives cervical carcinogenesis. Citation Format: Marissa Iden, Samantha Fye, Keguo Li, Tamjid Chowdhury, Ramani Ramchandran, Janet S. Rader. Overexpression of the lncRNA PVT1 contributes to the cervical cancer phenotype, possibly via association with nucleolin. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B10.

Identification of crucial regulatory relationships between long non-coding RNAs and protein-coding genes in lung squamous cell carcinoma

PurposeThis study aimed to analyze the relationships of long non-coding RNAs (lncRNAs) and protein-coding genes in lung squamous cell carcinoma (LUSC). MethodsRNA-seq data of LUSC deposited in the TCGA database were used to identify differentially expressed protein-coding genes (DECGs) and differentially expressed lncRNA genes (DE-lncRNAs) between LUSC samples and normal samples. Functional enrichment analysis of DECGs was then performed. Subsequently, the target genes and regulators of DE-lncRNAs were predicted from the DECGs. Additionally, expression levels of target genes of DE-lncRNAs were validated by RT-qPCR after the silence of DE-lncRNAs. ResultsIn total, 5162 differentially expressed genes (DEGs) were screened from the LUSC samples, and there were seven upregulated lncRNA genes in the DEGs. The upregulated DECGs were enriched in GO terms like RNA binding and metabolic process. Meanwhile, the downregulated DECGs were enriched in GO terms like cell cycle. Furthermore, the lncRNAs PVT1 and TERC targeted multiple DECGs. PVT1 targeted genes related to cell cycle (e.g. POLA2, POLD1, MCM4, MCM5 and MCM6), and reduced expression of PVT1 decreased expression of the genes. TERC regulated several genes (e.g. NDUFAB1, NDUFA11 and NDUFB5), and reduced expression of TERC increased expression of the genes. Additionally, PVT1 was regulated by multiple transcription factors (TFs) identified from DECGs, such as HSF1; and TERC was modulated by TFs, such as PIR. ConclusionA set of regulatory relationships between PVT1 and its targets and regulators, as well as TERC and its targets and regulators, may play crucial roles in the progress of LUSC.

Aberrant expression of long noncoding RNA PVT1 and its diagnostic and prognostic significance in patients with gastric cancer.

Emerging evidences indicate that dysregulated long noncoding RNAs (lncRNAs) are implicated in cancer tumorigenesis and progression and might be used as diagnosis and prognosis biomarker, or potential therapeutic targets. LncRNA PVT1 has been reported to be upregulated in diverse human cancers; however, its clinical significance in gastric cancer (GC) remains elusive. This study was to evaluate the expression of PVT1 in GC and further explore its clinical significance.Previous microarray datasets were analyzed to conduct apreliminary screening for candidate lncRNAs of gastric cancer biomarkers in human gastric cancer tissues. Expression levels of PVT1 in 111pairs of gastric cancer and adjacent normal tissues, gastric cancer cell lines and gastric cancer juices compared to their corresponding controls were detected by real-time quantitative RT-PCR assay. Areceiver operating characteristic (ROC) curve and Kaplan-Meier analysis were constructed to evaluate the diagnostic and prognostic values. Univariate and multivariate analysis were performed using the Cox proportional hazard analysis.PVT1 expression was remarkably increased in gastric cancer tissues and cell lines compared with that in the normal control, and its up-regulation was significantly correlated to invasion depth (P < 0.001), advanced TNM stage (P = 0.002) and regional lymph nodes metastasis (P < 0.001) in gastric cancer. PVT1 levels were robust in differentiating gastric cancer tissues from controls [area under the curve (AUC) = 0.728; 95 % confidence interval (CI) = 0.665-0.786, p<0.01]. Kaplan-Meier analysis demonstrated that increased PVT1 expression contributed to poor overall survival (P < 0.01) and disease-free survival (P < 0.01) of patients. Amultivariate survival analysis also indicated that PVT1 could be an independent prognostic marker. The levels of PVT1 in gastric juice from gastric patients were significantly higher than those from normal subjects (P = 0.03). PVT1 might serve as apromising biomarker for early detection and prognosis prediction of gastric cancer.

Overexpression of the long non-coding RNA PVT1 is correlated with leukemic cell proliferation in acute promyelocytic leukemia

BackgroundAcute promyelocytic leukemia (APL) is associated with chromosomal translocation t(15;17), which results in the proliferation of morphologically abnormal promyelocytes. Gain of supernumerary copies of the 8q24 chromosomal region, which harbors MYC and PVT1, has been shown to be the most common secondary alteration in human APL. Increased MYC can accelerate the development of myeloid leukemia in APL. However, the role that the expression of the long non-coding RNA (lncRNA) PVT1 plays in the pathogenesis of APL remains largely unknown.FindingsIn this study, we first analyzed the lncRNA PVT1 expression level in peripheral blood cells from 28 patients with de novo APL, and significantly upregulated PVT1 was found in APL patients compared with healthy donors. We then observed significantly lower MYC and PVT1 expression during all-trans retinoic acid (ATRA)-induced differentiation and cell cycle arrest in the APL cell line. MYC knockdown in NB4 cells led to PVT1 downregulation. Moreover, PVT1 knockdown by RNA interference led to suppression of the MYC protein level, and cell proliferation was inhibited.ConclusionOur findings reveal that the lncRNA PVT1 may play an important role in the proliferation of APL cells and may be useful for future therapeutic management.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-015-0223-4) contains supplementary material, which is available to authorized users.

Long non-coding RNA PVT1 as a novel biomarker for diagnosis and prognosis of non-small cell lung cancer.

Accumulating evidence has indicated that long non-coding RNA PVT1 is upregulated in various human cancers. However, it remains unclear whether PVT1 is involved in the development and progression of non-small cell lung cancer (NSCLC). The present study was designed to investigate the expression, biological role, and clinical significance of PVT1 in NSCLC. Our results indicated that PVT1 expression was significantly increased in NSCLC tissues and cell lines, and its upregulation was associated with advanced T-stage and tumor-node-metastasis (TNM) stage and regional lymph node metastasis. PVT1 expression levels were robust in differentiating NSCLC tissues from controls. Kaplan-Meier curve and Cox regression analysis showed that high expression of PVT1 was associated with poor overall survival and disease-free survival in NSCLC patients. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays indicated that knockdown of PVT1 remarkably inhibited NSCLC cell proliferation, whereas overexpression of PVT1 significantly promoted cellular proliferation. In addition, PVT1 knockdown increased the number of cells in the G0/G1 phase and reduced the number of cells in the S phase, while overexpression of PVT1 could promote cell cycle progression. Furthermore, our findings also revealed that the messenger RNA (mRNA) and protein expression of P15 and P21 was significantly upregulated in NSCLC cells transfected with PVT1 small interfering RNA (siRNA) and downregulated in cells transfected with pcDNA3.1-PVT1. In conclusion, our study demonstrated that PVT1 might serve as a promising biomarker for diagnosis and prognosis of NSCLC, and it could promote the proliferation of NSCLC cells by downregulating p15 and p21 expression.

Tetracycline-inducible shRNA targeting long non-coding RNA PVT1 inhibits cell growth and induces apoptosis in bladder cancer cells.

Recent studies show that long non-coding RNAs (lncRNAs) may be significant functional regulators in tumor development, including bladder cancer. Here, we found that PVT1 was upregulated in bladder cancer tissues and cells. Further experiments revealed that PVT1 promoted cell proliferation and suppressed cell apoptosis. Furthermore we also used the emerging technology, synthetic biology, to create tetracycline-inducible small hairpin RNA (shRNA) vectors which silenced PVT1 in a dosage-dependent manner to inhibit the progression of bladder cancer. In conclusion, data suggest that PVT1 could be an oncogene and may be a therapeutic target in bladder cancer. Synthetic "tetracycline-on" switch system can be used to quantitatively control the expression of PVT1 in bladder cancer in response to different concentration of doxycycline to suppress the progression of bladder cancer.

Long noncoding RNA PVT1 modulates thyroid cancer cell proliferation by recruiting EZH2 and regulating thyroid-stimulating hormone receptor (TSHR).

The purposes of this study were to investigate the potential roles of long noncoding RNA (lncRNA) PVT1 in thyroid cancer cell proliferation and to explore their possible mechanisms. A total of 84 patients who were diagnosed as having thyroid cancer (papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC)) in Renji Hospital were enrolled in this study. Expressions of lncRNA PVT1 in thyroid cancer tissues and cell lines (IHH-4, FTC-133, and 8505C) were analyzed using RT-polymerase chain reaction (PCR) and western blotting analysis. The effects of lncRNA PVT1 expression on thyroid cancer cell proliferation and cell cycle were analyzed using flow cytometry. Furthermore, the effects of lncRNA expression on thyroid-stimulating hormone receptor (TSHR) expression and polycomb enhancer of zeste homolog 2 (EZH2) were also analyzed using RNA immunoprecipitation (RIP) assay and chromatin immunoprecipitation (ChIP) assay, respectively. Compared to the controls, lncRNA PVT1 was significantly up-regulated in thyroid tissues, as well as in three kinds of tumor cell lines (P < 0.05). Silenced PVT1 significantly inhibited thyroid cell line IHH-4, FTC-133, and 8505C cell proliferation and arrested cell cycle at G0/G1 stage and significantly decreased cyclin D1 and TSHR expressions (P < 0.05). Moreover, lncRNA PVT1 could be enriched by EZH2, and silencing PVT1 resulted in the decreased recruitment of EZH2. This study suggested that lncRNA PVT1 may contribute to tumorigenesis of thyroid cancer through recruiting EZH2 and regulating TSHR expression.

Abstract 149: Overexpression of the long non-coding RNA PVT1 and its role in cervical carcinogenesis

Abstract Although it is becoming increasingly clear that long non-coding RNAs (lncRNAs) are intricately involved in numerous cancer types, the mechanisms by which they influence carcinogenesis remain poorly understood. The plasmacytoma variant translocation 1 gene (PVT1) is a lncRNA that has been designated as an oncogene due to its contribution to the phenotype of multiple cancers. Further, our lab has recently demonstrated that human papillomavirus (HPV) integration, a hallmark of invasive cervical cancer (ICC), into the PVT1 locus occurs in multiple cervical tumors. The present study was designed to investigate the role of PVT1 in cervical carcinogenesis. PVT1 expression was measured by quantitative PCR (qPCR) in 41 ICC samples, 20 normal cervix samples, and four cervical cell lines (SiHa, HeLa, DoTc2, and E6/E7-transformed ectocervical epithelial cells). Further, we used siRNA-targeted knockdown in conjunction with functional assays to examine PVT1's effects on cervical cancer cell proliferation, migration and invasion, apoptosis, and cisplatin-resistance. Our results demonstrate that PVT1 expression is significantly increased in ICC tissue versus normal cervix. PVT1 knockdown in cervical cancer cell lines resulted in significantly decreased cell proliferation, migration and invasion. Further, apoptosis and cisplatin cytotoxicity were significantly increased in PVT1 siRNA-transfected cells. Collectively, our data suggest that PVT1 may play a crucial role in cervical carcinogenesis. Future work will focus on determining the mechanism(s) by which this lncRNA exerts its multiple effects on cancer-related processes. Citation Format: Marissa Iden, Samantha Fye, Ramani Ramchandran, Janet S. Rader. Overexpression of the long non-coding RNA PVT1 and its role in cervical carcinogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 149. doi:10.1158/1538-7445.AM2015-149

Long noncoding RNA PVT1 indicates a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically regulating p15 and p16

BackgroundMounting evidence indicates that long noncoding RNAs (lncRNAs) could play a pivotal role in cancer biology. However, the overall biological role and clinical significance of PVT1 in gastric carcinogenesis remains largely unknown.MethodsExpression of PVT1 was analyzed in 80 GC tissues and cell lines by qRT-PCR. The effect of PVT1 on proliferation was evaluated by MTT and colony formation assays, and cell apoptosis was evaluated by Flow-cytometric analysis. GC cells transfected with shPVT1 were injected into nude mice to study the effect of PVT1 on tumorigenesis in vivo. RIP was performed to confirm the interaction between PVT1 and EZH2. ChIP was used to study the promoter region of related genes.ResultsThe higher expression of PVT1 was significantly correlated with deeper invasion depth and advanced TNM stage. Multivariate analyses revealed that PVT1 expression served as an independent predictor for overall survival (p = 0.031). Further experiments demonstrated that PVT1 knockdown significantly inhibited the proliferation both in vitro and in vivo. Importantly, we also showed that PVT1 played a key role in G1 arrest. Moreover, we further confirmed that PVT1 was associated with enhancer of zeste homolog 2 (EZH2) and that this association was required for the repression of p15 and p16. To our knowledge, this is the first report showed that the role and the mechanism of PVT1 in the progression of gastric cancer.ConclusionsTogether, these results suggest that lncRNA PVT1 may serve as a candidate prognostic biomarker and target for new therapies in human gastric cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0355-8) contains supplementary material, which is available to authorized users.

MP61-06 INCREASED PVT1 EXPRESSION CORRELATES WITH ADVANCED STAGE AND HORMONE RESISTANCE OF PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2015MP61-06 INCREASED PVT1 EXPRESSION CORRELATES WITH ADVANCED STAGE AND HORMONE RESISTANCE OF PROSTATE CANCER Ayman Soubra, Badrinath Konety, and Anindya Bagchi Ayman SoubraAyman Soubra More articles by this author , Badrinath KonetyBadrinath Konety More articles by this author , and Anindya BagchiAnindya Bagchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2187AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Myelocytomatosis (MYC) oncogene is known to be over expressed in many tumors including prostate cancer, and is associated with poor prognosis. Recently, a long non-coding RNA PVT1 which maps adjacent to MYC at 8q24, has been shown to regulate MYC (Bagchi et al. Nature 2014). In this study we examined the correlation between PVT1 expression and hormone sensitivity of prostate cancer in vitro. METHODS We used three human prostate cancer cell lines for the detection of gain of PVT-1 and one benign prostate cell line as a control: Primary prostate epithelial cells (PrEC), hormone sensitive/Androgen receptor positive cells (LNCaP), castrate resistant/Androgen receptor (AR) positive cells (C4-2) and castrate resistant/Androgen receptor (AR) negative cells (DU145). The 3 cancer cell lines represent the stages of the disease with different aggressiveness and responsiveness to hormonal therapy. RNA isolated from these cell lines was used to prepare complementary DNA (cDNA) and quantitative polymerase chain reaction (qPCR) was used to quantify the copies of PVT-1 present. We also used data available through The Cancer Genome Atlas (TCGA) and published by Grasso et al (nature 2012) to compare the level of PVT1 expression in human prostate tissues obtained from prostatectomies and autopsies from cases of lethal, castrate resistant prostate cancer. RESULTS Non-coding RNA gene PVT1 was not found to be over expressed in the hormone sensitive cell line LNCaP compared to the benign PrEC. However, there was 7.4 and 9.5 fold increase in PVT-1 gene in C4-2 (CRPC AR+)and DU145 (CRPC AR-) cell lines respectively. Data from TCGA analysis demonstrates an insignificant increase in PVT1 expression in prostate tumor tissues compared to normal prostate tissue. CONCLUSIONS PVT1 expression appears to be correlated with hormone sensitivity and androgen receptor status of prostate cancer cell lines in vitro. If similar variation can be demonstrated in human tumor tissue, PVT1 could be a candidate for targeted therapy of advanced prostate cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e748-e749 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ayman Soubra More articles by this author Badrinath Konety More articles by this author Anindya Bagchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Long non-coding RNA PVT1 is associated with tumor progression and predicts recurrence in hepatocellular carcinoma patients.

PVT1, which maps to chromosome 8q24, is a copy number amplification-associated long non-coding RNA. Overexpression of PVT1 is a powerful predictor of tumor progression and patient survival in a diverse range of cancer types. However, the association between PVT1 and hepatocellular carcinoma (HCC) remains unclear. The aim of the present study was to examine the expression pattern of PVT1, and its clinical significance in HCC. Between 2003 and 2012, reverse transcription-quantitative polymerase chain reaction was used to determine the expression levels of PVT1 in two independent cohorts: Cohort one, 58 HCC resection samples; and cohort 2, 214 HCC transplant samples. Additionally, the correlation between PVT1 expression levels and clinical parameters and outcomes was analyzed. The relative expression levels of PVT1 were significantly higher in cancerous tissues compared with the corresponding non-cancerous tissues (cohort one, P=0.0016; cohort two, P=0.0274). Furthermore, overexpression of PVT1 was associated with a higher serum α-fetoprotein expression level (P=0.011) and a higher recurrence rate (P=0.004). Kaplan-Meier analysis indicated that the patients with high PVT1 expression exhibited poor recurrence-free survival (P=0.021), and multivariate analysis demonstrated that high levels of PVT1 expression are an independent predictor for HCC recurrence (P=0.042; hazard ratio, 1.653). Thus, the high expression levels of PVT1 in HCC may serve as a novel biomarker for predicting tumor recurrence in HCC patients, and as a potential therapeutic target.

Amplification of PVT-1 is involved in poor prognosis via apoptosis inhibition in colorectal cancers

Background:We previously conducted gene expression microarray analyses to identify novel indicators for colorectal cancer (CRC) metastasis and prognosis from which we identified PVT-1 as a candidate gene. PVT-1, which encodes a long noncoding RNA, mapped to chromosome 8q24 whose copy-number amplification is one of the most frequent events in a wide variety of malignant diseases. However, PVT-1 molecular mechanism of action remains unclear.Methods:We conducted cell proliferation and invasion assays using colorectal cancer cell lines transfected with PVT-1siRNA or negative control siRNA. Gene expression microarray analyses on these cell lines were also carried out to investigate the molecular function of PVT-1. Further, we investigated the impact of PVT-1 expression on the prognosis of 164 colorectal cancer patients by qRT–PCR.Results:CRC cells transfected with PVT-1 siRNA exhibited significant loss of their proliferation and invasion capabilities. In these cells, the TGF-β signalling pathway and apoptotic signals were significantly activated. In addition, univariate and multivariate analysis revealed that PVT-1 expression level was an independent risk factor for overall survival of colorectal cancer patients.Conclusion:PVT-1, which maps to 8q24, generates antiapoptotic activity in CRC, and abnormal expression of PVT-1 was a prognostic indicator for CRC patients.

Abstract 1834: Noncoding RNAs of the 8q24 locus: Consequences of the over-expression or suppression of miR-1204 and PVT1 in developing B cells.

Abstract Immortalized models of the mouse B lymphocyte lineage have proven to be valuable resources for a wide variety of B cell neoplasms and for the study of normal B cell differentiation. Whether these models have been engineered in mice (in vivo) or in cell culture (in vitro), there are distinct advantages and disadvantages to both. Homogeneity and ease of accessibility can be considered a benefit of working with cultured cells, whereas the contribution from accessory or stromal cells can be an important element not readily available in the in vitro model system. We originally used immortalized Burkitt's Lymphoma (BL) cells to discover a cluster of microRNAs, miR-1204∼1208, that map within the noncoding PVT1 locus in the region of human 8q24 region down-stream of MYC. The BL cell lines allowed us to isolate a large amount of RNA from a homogeneous resource that enhanced discovery of low-level transcripts. Using probes for mouse miR-1204∼1208 to examine expression in a panel of mouse cell lines representing different stages of B cell development, we were able to show that expression of miR-1204∼1208 appeared to arise at the small B cell stage and that these higher levels of expression continued through to the mature plasma cell. This suggested that the pre-B cell or naïve small B cell stages may be most illuminating in assigning targets and function to miR-1204∼1208 or PVT1. To determine if over-expression of one of these miRNAs, miR-1204, influences the latency and/or type of B cell malignancy we used two mouse transgenic (TG) models of B cell malignancy, H2-Ld-hu-IL6 and iMyc, lentiviral expression of miR-1204 reduced the latency of tumor development in both models and in the case of iMyc-TG, there was also a shift in tumor type from late stage plasmacytoma to the earlier stage of large B cell lymphoma. However, further interpretation of these results was confounded by heterogeneity of lentiviral integration and expression among tumors. Thus, we turned to several human and mouse in vitro models of B cell development, representing pro-B, pre-B, small B, mature B and plasma cell stages to address the effects of modulating the expression of miR-1204 and its host noncoding transcript, PVT1. Over-expression of miR-1204 has been achieved through the use of lentiviruses or synthetic mimics and suppressed expression has been achieved through application of synthetic inhibitors. We also over-expressed PVT1 using a lentiviral vector and suppressed PVT1 expression through the use of siRNA corresponding to various exons of PVT1. Resultant changes in growth and morphology of these cell lines hint that microarray expression analyses will reveal functional targets of miR-1204 in normal and malignant lymphoid development. It will also be of interest to examine whether over-expression or inhibition of miR-1204 plays an additional role in maturation of the normal B cell. Citation Format: Konrad Huppi, Oliver L. Ou, Jason J. Pitt, Brady Wahlberg, Tamara L. Jones, Vishala Neppalli, Siegfried Janz, Natasha J. Caplen. Noncoding RNAs of the 8q24 locus: Consequences of the over-expression or suppression of miR-1204 and PVT1 in developing B cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1834. doi:10.1158/1538-7445.AM2013-1834

Abstract 3288: A chromosome engineering mouse model to identify mechanisms by which duplication of 8q24 promotes human breast cancer

Abstract The early latency and poor prognosis of human breast cancer is often associated with the gain of extra copy of 8q24 locus containing the c-Myc oncogene. Evaluation of aberrations at the 8q24 locus by high-resolution SNP array identified a 2.1 Mb region which only includes the c-Myc, Pvt1, and Gsdmc genes. Interestingly, the transgenic c-myc mouse has shown that the overexpression of c-myc alone is not sufficient to develop the mammary adenocarcinoma, suggesting that the overexpression of c-myc may not truly reflect breast cancer patients who were diagnosed with low copy-number gain of 8q24. It also implies that the Pvt1 or Gsdmc may play an important role with the c-Myc during tumor progression. However, functional analysis of this 2.1 Mb interval of 8q24 has been limited by a lack of suitable mouse models. Here, we report a novel mouse model, namely c-myc-gsdmcdp/+ (3N), containing the extra copy-number gain of 2.1 Mb interval as observed in breast cancer patients. To demonstrate the effect of the 3N gain, we have performed in vitro mammary epithelium experiments and in vivo mammary gland analyses in female virgin mice at 6 and 10 weeks of age. Interestingly, the mouse mammary epithelium with the 3N gain displays above 3 fold expression of c-Myc and Pvt-1 and significantly larger acinar structures in three-dimensional (3D) culture compared to the wild type (wt). Moreover, we found that mammary glands from 3N mice show excessive side-branching as early as 6 weeks. In 10 weeks, mammary glands from 3N mice display not only excessive side-branching but also precocious lobulo-alveolar development and contain numerous epithelial cells that are hyperproliferative as demonstrated by BrdU incorporation analysis when compared to wt. Using combined stem cell markers, the mammary stem cells (MaSCs)/progenitor cells-enriched population was expanded in mammary tissue from 3N mice, suggesting that a higher number of MaSCs deregulates a branching network between ducts and lobulo-alveolar structures similarly to other premalignant mouse models. Strikingly, further molecular mechanistic investigation shows that increased expression of c-myc and pvt1 promotes upregulation of epithelial-mesenchymal transition markers in mammary glands from 3N mice. Previous studies have reported that ∼40% of HER2+ breast cancers have a low copy-number gain of 8q24. Therefore, we crossed our 3N mice with MMTVneu transgenic mice. In these double-mutation mice, we observed accelerated malignant properties in 3D culture and migratory analyses. These results suggest that low copy number-gain of 8q24 may collaborate with Her2 oncogenic pathway to accelerate malignant transformation. Thus, our 3N mouse model establishes a new milestone in explaining how the low copy-number gain of 8q24 participates breast carcinogenesis and will be further analyzed to determine how aggressive tumor phenotypes are linked to poor clinical outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3288. doi:1538-7445.AM2012-3288

A Functional Variant at a Prostate Cancer Predisposition Locus at 8q24 Is Associated with PVT1 Expression

Genetic mapping studies have identified multiple cancer susceptibility regions at chromosome 8q24, upstream of the MYC oncogene. MYC has been widely presumed as the regulated target gene, but definitive evidence functionally linking these cancer regions with MYC has been difficult to obtain. Here we examined candidate functional variants of a haplotype block at 8q24 encompassing the two independent risk alleles for prostate and breast cancer, rs620861 and rs13281615. We used the mapping of DNase I hypersensitive sites as a tool to prioritise regions for further functional analysis. This approach identified rs378854, which is in complete linkage disequilibrium (LD) with rs620861, as a novel functional prostate cancer-specific genetic variant. We demonstrate that the risk allele (G) of rs378854 reduces binding of the transcription factor YY1 in vitro. This factor is known to repress global transcription in prostate cancer and is a candidate tumour suppressor. Additional experiments showed that the YY1 binding site is occupied in vivo in prostate cancer, but not breast cancer cells, consistent with the observed cancer-specific effects of this single nucleotide polymorphism (SNP). Using chromatin conformation capture (3C) experiments, we found that the region surrounding rs378854 interacts with the MYC and PVT1 promoters. Moreover, expression of the PVT1 oncogene in normal prostate tissue increased with the presence of the risk allele of rs378854, while expression of MYC was not affected. In conclusion, we identified a new functional prostate cancer risk variant at the 8q24 locus, rs378854 allele G, that reduces binding of the YY1 protein and is associated with increased expression of PVT1 located 0.5 Mb downstream.

Functional characterization of the plasmacytoma variant translocation 1 gene (PVT1) in diabetic nephropathy.

We previously observed association between variants in the plasmacytoma variant translocation 1 gene (PVT1) and end-stage renal disease (ESRD) attributed to both type 1 and type 2 diabetes, and demonstrated PVT1 expression in a variety of renal cell types. While these findings suggest a role for PVT1 in the development of ESRD, potential mechanisms for involvement remain unknown. The goal of this study was to identify possible molecular mechanisms by which PVT1 may contribute to the development and progression of diabetic kidney disease. We knocked-down PVT1 expression in mesangial cells using RNA interference, and analyzed RNA and protein levels of fibronectin 1 (FN1), collagen, type IV, alpha 1 (COL4A1), transforming growth factor beta 1 (TGFB1) and plasminogen activator inhibitor-1 (SERPINE1 or PAI-1) by qPCR and ELISA, respectively. PVT1 expression was significantly upregulated by glucose treatment in human mesangial cells, as were levels of FN1, COL4A1, TGFB1, and PAI-1. Importantly, PVT1 knockdown significantly reduced mRNA and protein levels of the major ECM proteins, FN1 and COL4A1, and two key regulators of ECM proteins, TGFB1 and PAI-1. However, we observed a higher and more rapid reduction in levels of secreted FN1, COL4A1, and PAI-1 compared with TGFB1, suggesting that at least some of the PVT1 effects on ECM proteins may be independent of this cytokine. These results indicate that PVT1 may mediate the development and progression of diabetic nephropathy through mechanisms involving ECM accumulation.

Association of germline or somatic TP53 missense mutation with oncogene amplification in tumors developed in patients with Li‐Fraumeni or Li‐Fraumeni‐like syndrome

Germline TP53 mutations are found in Li-Fraumeni syndrome (LFS) patients, predisposed to soft tissue sarcoma and other malignancies. The mutations and succeeding genetic events are thought to cause LFS-associated cancer, whose genetic alterations have rarely been investigated. Here, we study two LFS or Li-Fraumeni-like syndrome (LFLS) patients whose cancers showed aggressive phenotypes. Patient 1 with LFS and TP53(R273H) developed a rhabdomyosarcoma twice at the ages of 18 months and 21 years. A single-nucleotide polymorphism array-based analysis revealed two amplicons in the second tumor; one at 5q11.2 containing MAP3K1 and the other at 11q22.2 containing BIRC2/3 and YAP1. Increase of kinase signaling of MAP3K1 along with anti-apoptosis function of BIRC2/3 may have facilitated progression of this tumor. Patient 2 with LFLS and wild-typeTP53 suffered from acute myeloid leukemia. The leukemic cells had TP53(I195T) and two amplicons; one at 8q24.1 containing DEPDC6 and the other at 8q24.2 containing TRIB1, MYC, and PVT1. Quantitative PCR confirmed amplification of the genes and FISH revealed co-amplification of DEPDC6 and PVT1 in the same double minutes. Quantitative RT-PCR revealed increased expression levels of TRIB1, but no or little expression of DEPDC6, MYC, and PVT1. The results indicate that TRIB1 may be the target gene in the amplicon in the leukemia cells. Mutant TP53 can be engaged in pathways triggering gene amplification through impairment of DNA double-stranded break repair. The amplified candidate oncogenes identified in this study may have played a part in cancer development and lead to the poor outcome of LFS or LFLS-associated tumors.

C017 Expression of proteins p16INK4a, p53 and Bmi-1 in hematopoietic stem cells of patients with MDS. The role of cellular senescence

Increasing evidences indicated that Long noncoding RNAs (lncRNAs) played pivotal roles during tumorigenesis in multiple types of cancers, including breast cancer. This study aimed to investigate the role and function of long noncoding RNA PVT1 in the progression of breast cancer and explore the transcription factor which contributes to the regulation of PVT1 in breast cancer. Our results indicated that the expression of PVT1 was significantly upregulated in breast cancer tissues, compared with adjacent normal tissues (ANTs). And the increasing expression of PVT1 was associated with clinical stage, lymph nodes metastasis, and overall survival in breast cancer patients. Using computational screening, a transcriptional factor binding site was found between SOX2 and PVT1 promoter and the interaction between each other was further verified by chromatin immunoprecipitation (ChIP) analysis. In addition, ectopically overexpression of SOX2 can enhance breast cancer cell proliferation and invasion, while knockdown of SOX2 or PVT1 can severely attenuate this effect both in epithelial and mesenchymal types of breast cancer cells. Further evidences confirmed that overexpression of SOX2 can promote breast cancer cell EMT process; while silencing SOX2 or PVT1 expression can undermine this effect. These data suggest that elevated expression of SOX2 can activate lncRNA PVT1 expression promoted breast cancer tumorigenesis and progression. PVT1 may be a prognostic predictive biomarker for breast cancer, and the interaction of PVT1-SOX2 could be a therapeutic target in breast cancer.

Amplification of PVT1 Contributes to the Pathophysiology of Ovarian and Breast Cancer

This study was designed to elucidate the role of amplification at 8q24 in the pathophysiology of ovarian and breast cancer because increased copy number at this locus is one of the most frequent genomic abnormalities in these cancers. To accomplish this, we assessed the association of amplification at 8q24 with outcome in ovarian cancers using fluorescence in situ hybridization to tissue microarrays and measured responses of ovarian and breast cancer cell lines to specific small interfering RNAs against the oncogene MYC and a putative noncoding RNA, PVT1, both of which map to 8q24. Amplification of 8q24 was associated with significantly reduced survival duration. In addition, small interfering RNA-mediated reduction in either PVT1 or MYC expression inhibited proliferation in breast and ovarian cancer cell lines in which they were both amplified and overexpressed but not in lines in which they were not amplified/overexpressed. Inhibition of PVT1 expression also induced a strong apoptotic response in cell lines in which it was overexpressed but not in lines in which it was not amplified/overexpressed. Inhibition of MYC, on the other hand, did not induce an apoptotic response in cell lines in which MYC was amplified and overexpressed. These results suggest that MYC and PVT1 contribute independently to ovarian and breast pathogenesis when overexpressed because of genomic abnormalities. They also suggest that PVT1-mediated inhibition of apoptosis may explain why amplification of 8q24 is associated with reduced survival duration in patients treated with agents that act through apoptotic mechanisms.