ABSTRACT
HOTAIR is a long intervening non-coding RNA (lincRNA) that associates with the Polycomb Repressive Complex 2 (PRC2) and overexpression is correlated with poor survival for breast, colon and liver cancer patients. In this study, we show that HOTAIR expression is increased in pancreatic tumors compared with non-tumor tissue and is associated with more aggressive tumors. Knockdown of HOTAIR (siHOTAIR) by RNA interference shows that HOTAIR has an important role in pancreatic cancer cell invasion, as reported in other cancer cell lines. In contrast, HOTAIR knockdown in Panc1 and L3.6pL pancreatic cancer cells that overexpress this lincRNA decreased cell proliferation, altered cell cycle progression and induced apoptosis, demonstrating an expanded function of HOTAIR in pancreatic cancer cells compared with other cancer cell lines. Results of gene array studies showed that there was minimal overlap between HOTAIR-regulated genes in pancreatic cells and breast cancer cells, and HOTAIR uniquely suppressed several interferon-related genes and gene sets related to cell cycle progression in pancreatic cancer cells and tumors. Analysis of selected genes suppressed by HOTAIR in Panc1 and L3.6pL cells showed by knockdown of EZH2 and chromatin immunoprecipitation assays that HOTAIR-mediated gene repression was both PRC2-dependent and -independent. HOTAIR knockdown in L3.6pL cells inhibited tumor growth in mouse xenograft model, further demonstrating the pro-oncogenic function of HOTAIR in pancreatic cancer.
Subject(s)
Carcinoma, Pancreatic Ductal/diagnosis , Cell Transformation, Neoplastic/genetics , Pancreatic Neoplasms/diagnosis , RNA, Long Noncoding/physiology , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Biomarkers, Tumor/physiology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Transformation, Neoplastic/drug effects , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , Gene Knockdown Techniques , Humans , Mice , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Prognosis , RNA, Long Noncoding/antagonists & inhibitors , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , RNA, Small Interfering/pharmacology , Transplantation, HeterologousABSTRACT
BACKGROUND: Tolfenamic acid (TA) is an NSAID currently under investigation as an anticancer agent in humans. TA induces proteosome-dependent degradation of transcription factors Sp 1, 3, and 4. These proteins are known to be overexpressed in many human cancers. HYPOTHESIS: To evaluate the protein expression of Sps in canine tissue, and efficacy of TA against several canine tumor cell lines. METHODS: Six canine cell lines (2 osteosarcoma, 2 mammary carcinoma, 2 melanoma) were evaluated. Protein levels of Sp 1-4 and their downstream targets were evaluated using Western Blots. Cell survival and TUNEL assays were performed on cell lines, and Sp1 expression was evaluated on histologic samples from archived canine cases. ANIMALS: Six immortalized canine cancer cell lines derived from dogs were used. Archived tissue samples were also used. RESULTS: Sps were highly expressed in all 6 cell lines and variably expressed in histologic tissues. TA decreased expression of Sps 1-4 in all cell lines. All of the downstream targets of Sps were inhibited in the cell lines. Variable Sp1 expression was identified in all histologic samples examined. TA significantly inhibited cell survival in all cell lines in a dose dependant fashion. The number of cells undergoing apoptosis was significantly increased (P < .05) in all cell lines after exposure to TA in a dose-dependent fashion. CONCLUSIONS, AND CLINICAL IMPORTANCE: Tolfenamic acid is a potential anticancer NSAID and further investigation is needed to determine its usefulness in a clinical setting.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Dog Diseases/drug therapy , Neoplasm Proteins/metabolism , Neoplasms/veterinary , Sp Transcription Factors/metabolism , ortho-Aminobenzoates/pharmacology , Animals , Blotting, Western , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/veterinary , Cell Line, Tumor , Cell Proliferation/drug effects , Dog Diseases/metabolism , Dog Diseases/pathology , Dogs , Female , Gene Expression Regulation, Neoplastic , Immunohistochemistry , Melanoma/drug therapy , Melanoma/pathology , Melanoma/veterinary , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Osteosarcoma/drug therapy , Osteosarcoma/pathology , Osteosarcoma/veterinary , RNA, Neoplasm/chemistry , RNA, Neoplasm/genetics , Reverse Transcriptase Polymerase Chain Reaction/veterinary , Sp Transcription Factors/biosynthesis , Sp Transcription Factors/genetics , Up-Regulation/drug effectsABSTRACT
The human POK family members are transcription factors with a POZ domain and zinc-fingers that act primarily as transcriptional repressors. Several members of this family are involved in oncogenesis and this prompted us to assess whether expression levels of individual POK family members are associated with clinical outcomes in cancer. We have observed that ZBTB4 (zinc-finger and BTB domain containing 4) is downregulated in breast cancer patients, and that its expression is significantly correlated with relapse-free survival. Further integrative analysis of mRNA and microRNA (miR) expression data from the NCI-60 cell lines revealed an inverse correlation between ZBTB4 and oncogenic miRs derived from the miR-17-92 cluster and its paralogs. The experimental results using MDA-MB-231 and MCF-7 human breast cancer cells confirm that miRNAs derived from these clusters, containing miR-17-5p, miR-20a, miR-106a, miR-106b and miR-93, negatively regulate ZBTB4 expression. Overexpression of ZBTB4 or restoration of ZBTB4 by using an antagomir inhibit growth and invasion of breast cancer cells, and this effect is due, in part, to ZBTB4-dependent repression of the specificity protein 1 (Sp1), Sp3 and Sp4 genes, and subsequent downregulation of several Sp-dependent oncogenes, in part, through competition between ZBTB4 and Sp transcription factors for GC-rich promoter sequences. These results confirm that ZBTB4 functions as a novel tumor-suppressor gene with prognostic significance for breast cancer survival, and the oncogenic miR-17-92/ZBTB4/Sp axis may be a potential therapeutic target.
