Functional microRNA high throughput screening reveals miR-9 as a central regulator of liver oncogenesis by affecting the PPARA-CDH1 pathway

Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths, reflecting the aggressiveness of this type of cancer and the absence of effective therapeutic regimens. MicroRNAs have been involved in the pathogenesis of different types of cancers, including liver cancer. Our aim was to identify microRNAs that have both functional and clinical relevance in HCC and examine their downstream signaling effectors. Methods: MicroRNA and gene expression levels were measured by quantitative real-time PCR in HCC tumors and controls. A TargetScan algorithm was used to identify miR-9 downstream direct targets. Results: A high-throughput screen of the human microRNAome revealed 28 microRNAs as regulators of liver cancer cell invasiveness. MiR-9, miR-21 and miR-224 were the top inducers of HCC invasiveness and also their expression was increased in HCC relative to control liver tissues. Integration of the microRNA screen and expression data revealed miR-9 as the top microRNA, having both functional and clinical significance. MiR-9 levels correlated with HCC tumor stage and miR-9 overexpression induced SNU-449 and HepG2 cell growth, invasiveness and their ability to form colonies in soft agar. Bioinformatics and 3’UTR luciferase analyses identified E-cadherin (CDH1) and peroxisome proliferator-activated receptor alpha (PPARA) as direct downstream effectors of miR-9 activity. Inhibition of PPARA suppressed CDH1 mRNA levels, suggesting that miR-9 regulates CDH1 expression directly through binding in its 3’UTR and indirectly through PPARA. On the other hand, miR-9 inhibition of overexpression suppressed HCC tumorigenicity and invasiveness. PPARA and CDH1 mRNA levels were decreased in HCC relative to controls and were inversely correlated with miR-9 levels. Conclusions: Taken together, this study revealed the involvement of the miR-9/PPARA/CDH1 signaling pathway in HCC oncogenesis

Transcriptomic and CRISPR/Cas9 technologies reveal FOXA2 as a tumor suppressor gene in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with low survival rates and limited therapeutic options. Thus elucidation of signaling pathways involved in PDAC pathogenesis is essential for identifying novel potential therapeutic gene targets. Here, we used a systems approach to elucidate those pathways by integrating gene and microRNA profiling analyses together with CRISPR/Cas9 technology to identify novel transcription factors involved in PDAC pathogenesis. FOXA2 transcription factor was found to be significantly downregulated in PDAC relative to control pancreatic tissues. Functional experiments revealed that FOXA2 has a tumor suppressor function through inhibition of pancreatic cancer cell growth, migration, invasion, and colony formation. In situ hybridization analysis revealed miR-199a to be significantly upregulated in pancreatic cancer. Bioinformatics and luciferase analyses showed that miR-199a negatively but directly regulates FOXA2 expression through binding in its 3′-untranslated region (UTR). Evaluation of the functional importance of miR-199a on pancreatic cancer revealed that miR-199a acts as an inhibitor of FOXA2 expression, inducing an increase in pancreatic cancer cell proliferation, migration, and invasion. Additionally, gene ontology and network analyses in PANC-1 cells treated with a small interfering RNA (siRNA) against FOXA2 revealed an enrichment for cell invasion mechanisms through PLAUR and ERK activation. FOXA2 deletion (FOXA2Δ) by using two CRISPR/Cas9 vectors in PANC-1 cells induced tumor growth in vivo resulting in upregulation of PLAUR and ERK pathways in FOXA2Δ xenograft tumors. We have identified FOXA2 as a novel tumor suppressor in pancreatic cancer and it is regulated directly by miR-199a, thereby enhancing our understanding of how microRNAs interplay with the transcription factors to affect pancreatic oncogenesis

Non-coding RNAs in pancreatic ductal adenocarcinoma: New approaches for better diagnosis and therapy

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies with a 5-year survival rate less than 8%, which has remained unchanged over the last 50 years. Early detection is particularly difficult due to the lack of disease-specific symptoms and a reliable biomarker. Multimodality treatment including chemotherapy, radiotherapy (used sparingly) and surgery has become the standard of care for patients with PDAC. Carbohydrate antigen 19–9 (CA 19–9) is the most common diagnostic biomarker; however, it is not specific enough especially for asymptomatic patients. Non-coding RNAs are often deregulated in human malignancies and shown to be involved in cancer-related mechanisms such as cell growth, differentiation, and cell death. Several micro, long non-coding and circular RNAs have been reported to date which are involved in PDAC. Aim of this review is to discuss the roles and functions of non-coding RNAs in diagnosis and treatments of PDAC

Integration of Transcriptomic and CRISPR-Cas9 Technologies Reveal FOXA2 as a Tumor Suppressor Gene in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a median survival of six months and a five-year survival of <5%, making it one of the most lethal human cancers. This poor prognosis is due to the uniformly advanced disease stage at the time of diagnosis and to its profound resistance to existing therapies. Ductal adenocarcinomas makes up between 75 to 92% of pancreatic neoplasms. Premalignant lesions, known as pancreatic intraepithelial neoplasms (PanINs) are of ductal origin and found in close physical proximity with advanced malignant tumors. PanINs are thought to be precursors of ductal adenocarcinoma, as they progress toward increasingly atypical histological stages. Previous studies have implicated the role of different signaling pathways in the pathogenesis of pancreatic cancer, however the role of transcriptomic alterations have not been well characterized. The aim of this dissertation was to characterize the role and function of a new transcription factor gene, called FOXA2, in the pathogenesis of pancreatic cancer. Our preliminary analysis revealed that FOXA2 gene was highly down-regulated in pancreatic cancer tissues relative to control tissue samples. Interestingly, FOXA2 gene has not been implicated previously in pancreatic oncogenesis. FOXA2 is a transcription factor that was initially identified in hepatocytes, where it binds in the promoter areas of important liver-enriched genes transthyretin, alpha 1-antitrypsin and albumin. Our data revealed that FOXA2 is significantly down-regulated in pancreatic cancer, according to qPCR and immunohistochemical analysis in human pancreatic tissues. In order to study the role of FOXA2 deletion in vivo, we utilized the CRISPR/Cas9 system to delete FOXA2 from the PANC-1 cell line (FOXA2 deletion). Subcutaneous injections in immunodeficient mice demonstrated FOXA2 deletion had a significantly higher weight and volume than the control tumors. Moreover, we identified a negative regulator of FOXA2 expression, microRNA-199a, which targets directly and reduces FOXA2 mRNA and protein expression levels in pancreatic cancer. Overexpression of microRNA-199a was observed in human pancreatic cancer tissue by qPCR and in-situ hybridization methods. In an effort to identify downstream targets of FOXA2, we transiently knocked-down FOXA2 with an siRNA and performed a gene expression array in a human pancreatic cell line. The expression data revealed plasminogen urokinase activator receptor (PLAUR) was significantly up-regulated upon FOXA2 inhibition. PLAUR is a marker of invasiveness and is responsible for the invasive phenotype we observed upon FOXA2 knockdown

Jack and Mike : Incredible Drums and Piano Duets

This audio recording contains 13 improvised compositions by Toronto artists/musicians Snow and Vorvis

Integration of Transcriptomic and CRISPR-Cas9 Technologies Reveal FOXA2 as a Tumor Suppressor Gene in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a median survival of six months and a five-year survival of <5%, making it one of the most lethal human cancers. This poor prognosis is due to the uniformly advanced disease stage at the time of diagnosis and to its profound resistance to existing therapies. Ductal adenocarcinomas makes up between 75 to 92% of pancreatic neoplasms. Premalignant lesions, known as pancreatic intraepithelial neoplasms (PanINs) are of ductal origin and found in close physical proximity with advanced malignant tumors. PanINs are thought to be precursors of ductal adenocarcinoma, as they progress toward increasingly atypical histological stages. Previous studies have implicated the role of different signaling pathways in the pathogenesis of pancreatic cancer, however the role of transcriptomic alterations have not been well characterized. The aim of this dissertation was to characterize the role and function of a new transcription factor gene, called FOXA2, in the pathogenesis of pancreatic cancer. Our preliminary analysis revealed that FOXA2 gene was highly down-regulated in pancreatic cancer tissues relative to control tissue samples. Interestingly, FOXA2 gene has not been implicated previously in pancreatic oncogenesis. FOXA2 is a transcription factor that was initially identified in hepatocytes, where it binds in the promoter areas of important liver-enriched genes transthyretin, alpha 1-antitrypsin and albumin. Our data revealed that FOXA2 is significantly down-regulated in pancreatic cancer, according to qPCR and immunohistochemical analysis in human pancreatic tissues. In order to study the role of FOXA2 deletion in vivo, we utilized the CRISPR/Cas9 system to delete FOXA2 from the PANC-1 cell line (FOXA2 deletion). Subcutaneous injections in immunodeficient mice demonstrated FOXA2 deletion had a significantly higher weight and volume than the control tumors. Moreover, we identified a negative regulator of FOXA2 expression, microRNA-199a, which targets directly and reduces FOXA2 mRNA and protein expression levels in pancreatic cancer. Overexpression of microRNA-199a was observed in human pancreatic cancer tissue by qPCR and in-situ hybridization methods. In an effort to identify downstream targets of FOXA2, we transiently knocked-down FOXA2 with an siRNA and performed a gene expression array in a human pancreatic cell line. The expression data revealed plasminogen urokinase activator receptor (PLAUR) was significantly up-regulated upon FOXA2 inhibition. PLAUR is a marker of invasiveness and is responsible for the invasive phenotype we observed upon FOXA2 knockdown