Edward J. Richards

Upregulation of miRNA-155 promotes tumour angiogenesis by targeting VHL and is associated with poor prognosis and triple-negative breast cancer

MicroRNA-155 (miR-155) is frequently upregulated in various types of human cancer; however, its role in cancer angiogenesis remains unknown. Here, we demonstrate the role of miR-155 in angiogenesis through targeting von Hippel-Lindau (VHL) tumour suppressor in breast cancer. Ectopic expression of miR-155 induced whereas knockdown of miR-155 inhibited human umbilical vein endothelial cell network formation, proliferation, invasion and migration. Furthermore, mammary fat pad xenotransplantation of ectopically expressed miR-155 resulted in extensive angiogenesis, proliferation, tumour necrosis and recruitment of pro-inflammatory cells such as tumour-associated macrophages. Expression of VHL abrogated these miR-155 effects. Moreover, miR-155 expression inversely correlates with VHL expression level and is associated with late-stage, lymph node metastasis and poor prognosis, as well as triple-negative tumour in breast cancer. These findings indicate that miR-155 has a pivotal role in tumour angiogenesis by downregulation of VHL, and provide a basis for miR-155-expressing tumours to embody an aggressive malignant phenotype, and therefore miR-155 is an important therapeutic target in breast cancer.

Long Non-coding RNAs (LncRNA) Regulated by Transforming Growth Factor (TGF) β: LncRNA-HIT-MEDIATED TGFβ-INDUCED EPITHELIAL TO MESENCHYMAL TRANSITION IN MAMMARY EPITHELIA*

Background: Long noncoding RNAs (LncRNA) are emerging as key regulators in various biological processes. However, their role in epithelial-to-mesenchymal transition (EMT) remains elusive. Results: A subset of lncRNAs are dysregulated upon transforming growth factor (TGF) β-induced EMT, and lncRNA-HIT mediates this process. Conclusion: LncRNAs such as lncRNA-HIT ((HOXA transcript induced by TGFβ) play a pivotal role in EMT and breast cancer progression. Significance: Here we profiled lncRNAs in TGFβ-induced EMT and identified a novel conserved lncRNA-HIT. Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor β (TGFβ) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFβ induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFβ) mediates TGFβ function, i.e. depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

A functional variant in HOXA11-AS, a novel long non-coding RNA, inhibits the oncogenic phenotype of epithelial ovarian cancer

The homeobox A (HOXA) region of protein-coding genes impacts female reproductive system embryogenesis and ovarian carcinogenesis. The 5-prime end of HOXA includes three long non-coding RNAs (lncRNAs) (HOXA10-AS, HOXA11-AS, and HOTTIP) that are underexplored in epithelial ovarian cancer (EOC). We evaluated whether common genetic variants in these lncRNAs are associated with EOC risk and/or have functional roles in EOC development. Using genome-wide association study data from 1,201 serous EOC cases and 2,009 controls, an exonic variant within HOXA11-AS, rs17427875 (A>T), was marginally associated with reduced serous EOC risk (OR = 0.88 (95% CI: 0.78-1.01, p = 0.06). Functional studies of ectopic expression of HOXA11-AS minor allele T in EOC cells showed decreased survival, proliferation, migration, and invasion compared to common allele A expression. Additionally, stable expression of HOXA11-AS minor allele T reduced primary tumor growth in mouse xenograft models to a greater extent than common allele A. Furthermore, HOXA11-AS expression levels were significantly lower in human EOC tumors than normal ovarian tissues (p < 0.05), suggesting that HOXA11-AS has a tumor suppressor function in EOC which may be enhanced by the T allele. These findings demonstrate for the first time a role for HOXA11-AS in EOC with effects that could be modified by germline variants.

Erratum: Long non-coding RNAs (LncRNA) regulated by transforming growth factor (TGF) β. LncRNA-hit-mediated TGF-induced epithelial to mesenchymal transition in mammary epithelia (Journal of Biological Chemistry (2015) 290 (6857-6867) DOI: 10.1074/jbc.A114.610915)

Background: Long noncoding RNAs (LncRNA) are emerging as key regulators in various biological processes. However, their role in epithelial-to-mesenchymal transition (EMT) remains elusive. Results: A subset of lncRNAs are dysregulated upon transforming growth factor (TGF) β-induced EMT, and lncRNA-HIT mediates this process. Conclusion: LncRNAs such as lncRNA-HIT ((HOXA transcript induced by TGFβ) play a pivotal role in EMT and breast cancer progression. Significance: Here we profiled lncRNAs in TGFβ-induced EMT and identified a novel conserved lncRNA-HIT. Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor β (TGFβ) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFβ induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFβ) mediates TGFβ function, i.e. depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

Abstract 998: LncRNA WDFY3-AS2 contributes to the EMT and metastasis in breast cancer

The role of TGFβ in promoting mammary cell EMT and breast cancer metastasis was well established. However, the involvement of long non-coding RNA (lncRNA) in this process remains elusive. Here we demonstrated that mouse lncRNA BB179049 and its human ortholog WDFY3-AS2 were significantly increased upon TGFβ-induced EMT. WDFY3-AS2 was frequently elevated in invasive, metastatic and triple-negative breast cancer. Depletion of WDFY3-AS2 abrogated TGFβ-induced EMT and breast cancer metastasis. WDFY3-AS2 positively regulates STAT3 and WDFY3 through interaction with hnRNP-R. These data indicate that WDFY3-AS2 could play a pivotal role in TGFβ-induced EMT and metastasis and serves as a critical therapeutic target in aggressive breast cancer. Citation Format: Edward Richards, Sridevi Challa, Yajuan Li, Jennifer Permuth-Wey, Marilyn Bui, Domenico Coppola, Thomas Sellers, Jin Cheng. LncRNA WDFY3-AS2 contributes to the EMT and metastasis in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 998.

Abstract 1904: MiR641 regulates EMT, ovarian cancer stem cell and angiogenesis by targeting p63/miR200 axis

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA MiR641 is an uncharacterized miRNA which locates intron 1 of AKT2, a gene residing in the chromosome 19q13.2 amplicon and frequently amplified/overexpressed in refractory ovarian cancer. Here, we demonstrated that frequent upregulation of miR641 was observed in human ovarian cancer tissues and in ovarian cancer cell lines. Knockdown of miR641 inhibited cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) as well as angiogenesis and ovarian cancer stem cell growth. Enforced expression of miR641 had opposite effect. In addition, ectopic expression of miR641 promoted VEGF protein expression and secretion but had no effect on VEGF mRNA level. Mechanistically, we found that miR641 directly targeted the 3’UTR of p63 and reduced p63 expression, which leads to a decrease of miR200a/b. As a result, ZEB1 and VEGF, two validated targets of miR200, were upregulated by miR641 overexpression. Furthermore, expression of miR641 induced anchorage-independent growth. Taken together, these findings suggest that miR641 is an onco-miRNA and plays a pivotal role in ovarian oncogenesis. Citation Format: Yajuan Li, Edward Richards, Mark Coppola, Cheng-Xiong Xu, Jin Q. Cheng. MiR641 regulates EMT, ovarian cancer stem cell and angiogenesis by targeting p63/miR200 axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1904.

Long non-coding RNAs regulated by TGFβ: lncRNA-HIT mediated TGFβ-induced epithelial to mesenchymal transition in mammary epithelia

Background: Long noncoding RNAs (LncRNA) are emerging as key regulators in various biological processes. However, their role in epithelial-to-mesenchymal transition (EMT) remains elusive. Results: A subset of lncRNAs are dysregulated upon transforming growth factor (TGF) β-induced EMT, and lncRNA-HIT mediates this process. Conclusion: LncRNAs such as lncRNA-HIT ((HOXA transcript induced by TGFβ) play a pivotal role in EMT and breast cancer progression. Significance: Here we profiled lncRNAs in TGFβ-induced EMT and identified a novel conserved lncRNA-HIT. Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. Transforming growth factor β (TGFβ) signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Here we report a genome-wide lncRNA profile in mouse mammary epithelial NMuMG cells upon TGFβ induction of EMT. Among 10,802 lncRNAs profiled, over 600 were up-regulated and down-regulated during the EMT, respectively. Furthermore, we identify that lncRNA-HIT (HOXA transcript induced by TGFβ) mediates TGFβ function, i.e. depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth, and metastasis. E-cadherin was identified as a major target of lncRNA-HIT. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Collectively, these data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers.

Abstract 154: Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer metastasis

Background: Long noncoding RNAs (lncRNAs) are emerging as key regulators in various biological processes. Epithelial-to-mesenchymal transition (EMT) is a developmental process hijacked by tumor cells to depart from the primary tumor site, invade surrounding tissue, and establish distant metastases. TGFβ signaling has been shown to be a major inducer of EMT and to facilitate breast cancer metastasis. However, the role of lncRNAs in this process remains largely unknown. Methods: Genome-wide lncRNA profile was performed using NCode™ Mouse Noncoding Microarray chip from Life Technologies, which contains 10,802 lncRNAs and 25,178 protein-coding genes. Total RNA was isolated from mouse mammary epithelial NMuMG cells, which had been treated with TGFβ for 0, 12 and 24 hours, and was used to hybridize the microarray. The role of a top TGFβ-upregulated lncRNA, lncRNA-HIT, in EMT and metastasis was further characterized in cell culture and orthotopic breast cancer models Results: Among 10,802 lnRNAs profiled, 680 were up-regulated and 633 were down-regulated during TGFβ-induced EMT, respectively. Further, we identify that lncRNA-HIT (HOXA antisense transcript induced by TGFβ) mediates TGFβ function, i.e., depletion of lncRNA-HIT inhibits TGFβ-induced migration, invasion, and EMT in NMuMG. LncRNA-HIT is also significantly elevated in the highly metastatic 4T1 cells. Knockdown of lncRNA-HIT in 4T1 results in decrease of cell migration, invasion, tumor growth and metastasis. Moreover, lncRNA-HIT is conserved in humans and elevated expression associates with more invasive human primary breast carcinoma. Conclusions: These data suggest that a subset of lncRNAs such as lncRNA-HIT play a significant role in regulation of EMT and breast cancer invasion and metastasis, and could be potential therapeutic targets in breast cancers. Citation Format: Edward Richards, Gu Zhang, Jennifer Permuth-Wey, Zhu-Peng Li, Sridevi Challa, William Kong, Dan Su, Domenico Coppola, Wei-min Mao, Thomas Sellers, Jin Q. Cheng. Identification of TGFβ-regulated long noncoding RNAs in mammary epithelia: lncRNA-HIT mediated TGFβ-induced EMT and breast cancer metastasis. [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 154. doi:10.1158/1538-7445.AM2015-154

Abstract 4633: Evidence that long non-coding RNA variants associate with epithelial ovarian cancer risk

Background: Genome wide association studies (GWAS) have identified 20 loci associated with epithelial ovarian cancer (EOC) risk. Additional EOC risk loci await identification, and biological approaches may represent a useful strategy for overcoming sample size limitations. The ENCODE project recently concluded that only ∼1.2% of the genome encodes proteins, but at least 20% exhibits biological function and over 80% exhibits biochemical indices of function. Post-GWAS follow-up studies have firmly established that some associations are due to inter-individual differences in non-coding RNAs (ncRNAs). To estimate the magnitude of impact of variants in long non-coding RNAs (lncRNAs), we assessed enrichment of EOC-associated SNPs in lncRNA regions by comparing the density of EOC-associated loci between lncRNA regions, non-lncRNA regions, and the whole genome. Methods: The study population included ∼18,000 invasive EOC cases and 34,000 healthy controls of European ancestry from the Ovarian Cancer Association Consortium with GWAS data imputed to 1000 Genomes Project (1KGP) density. Density was compared with two measures: average chromosome length required for one EOC-associated SNP (kb/locus) and average number of tested SNPs containing one EOC-associated SNP (SNPs/locus). Coordinates and annotation for 13,870 lncRNA genes were downloaded from the publicly available GENCODE (v19) database and were used to annotate SNPs from the 1KGP imputed GWAS data, yielding 13,192 lncRNA genes with biallelic variants imputed at rsquare ≥0.25. A SNP was considered EOC-associated if it reached a significance level of P −5 . Results: Genome-wide, 11.6% of the 15,123,646 tested SNPs fell within lncRNA regions. Of 5,294 EOC-associated SNPs that were identified, 27.7% were located within lncRNAs, a higher frequency than expected ( = 9.35×10 −22 ). Additionally, EOC-associated SNPs were located on average every 237.6 kb (or 1204 tested SNPs) in lncRNA regions versus every 657.7 kb (or 3489 SNPS) in non-lncRNA regions and every 541.5 kb (or 2857 SNPs) in the whole genome, clearly demonstrating an enrichment of EOC-associated loci in lncRNA regions. A total of 1,464 lncRNA SNPs from 53 unique lncRNAs associated with EOC risk (P −5 ); 70 of the identified lncRNA SNPs had P-values between 10 −20 and 10 −32 , and 221 had P-values between 10 −10 and 10 −15 , representing 21 unique regions > 500kb apart. Conclusions: These results justify efforts to integrate functional and population level genetic data on lncRNA regions to explore a new paradigm that addresses the problem of the missing heritability for cancer where restrictions in sample size and statistical power limit the ability to discover additional risk loci. Citation Format: Thomas A. Sellers, Brett M. Reid, Y. Ann Chen, Hui-Yi Lin, Edward Richards, Jamie Teer, Alvaro Monteiro, Zhihua Chen, Andrew Berchuck, Georgia Chenevix-Trench, Jennifer Doherty, Ellen Goode, Edwin Iverson, Leigh Pearce, Paul Pharoah, Catherine Phelan, Susan Ramus, Mary Anne Rossing, Joellen Schildkraut, Jin Cheng, Simon Gayther, Jennifer Permuth-Wey, on behalf of the Ovarian Cancer Association Consortium. Evidence that long non-coding RNA variants associate with epithelial ovarian cancer risk. [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 4633. doi:10.1158/1538-7445.AM2015-4633

Molecular Diagnostics of Pancreatic Cancer

Pancreatic cancer is a malignant disease relating to uncontrollable cell growth in the tissues of the pancreas. The vast majority of pancreatic cancers are ductal adenocarcinomas, originating in the epithelial layer of the exocrine pancreas, however some pancreatic cancers do originate in the endocrine compartment. Ductal adenocarcinomas are very aggressive cancers, associating with poor prognosis and late staging upon discovery. One reason for this is that many patients do not present symptoms until late onset of disease. Numerous research efforts have been put forth to earlier diagnose pancreatic cancer. Understanding of pancreatic cancer etiology and identification of associated tumor markers has progressed, however there is a clear need for more specific and sensitive technologies. This chapter aims to address current status of molecular diagnostics in pancreatic cancer.