RNA Sequencing Identifies Transcriptionally Viable Gene Fusions in Esophageal Adenocarcinomas

Esophageal adenocarcinoma (EAC) is a deadly cancer with increasing incidence in the U.S., but mechanisms underlying pathogenesis are still mostly elusive. In addressing this question, we assessed gene-fusion landscapes by comprehensive RNA sequencing (RNAseq) of 55 pre-treatment EAC and 49 non-malignant biopsy tissues from patients undergoing endoscopy for Barrett’s esophagus. In this cohort, we identified 21 novel candidate EAC-associated fusions occurring in 3.33%-11.67% of EACs. Two candidate fusions were selected for validation by PCR and Sanger sequencing in an independent set of pre-treatment EAC (N=115) and non-malignant (N=183) biopsy tissues. In particular, we observed RPS6KB1–VMP1 gene fusion as a recurrent event occurring in ~10% of EAC cases. Notably, EAC cases harboring RPS6KB1–VMP1 fusions exhibited significantly poorer overall survival as compared to fusion-negative cases. Mechanistic investigations established that the RPS6KB1–VMP1 transcript coded for a fusion protein which significantly enhanced the growth rate of non-dysplastic Barrett’s esophagus cells. Compared to the wild-type VMP1 protein, which mediates normal cellular autophagy, RPS6KB1–VMP1 fusion exhibited aberrant subcellular localization and was relatively ineffective in triggering autophagy. Overall, our findings identified RPS6KB1–VMP1 as a genetic fusion that promotes EAC by modulating autophagy-related processes, offering new insights into the molecular pathogenesis of esophageal adenocarcinomas

Chondroitin sulfates play a major role in breast cancer metastasis: a role for CSPG4 and CHST11gene expression in forming surface P-selectin ligands in aggressive breast cancer cells

INTRODUCTION: We have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread. METHODS: Quantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group). RESULTS: The CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002). CONCLUSIONS: Cell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies

RNA Sequencing Identifies Transcriptionally Viable Gene Fusions in Esophageal Adenocarcinomas

Esophageal adenocarcinoma (EAC) is a deadly cancer with increasing incidence in the U.S., but mechanisms underlying pathogenesis are still mostly elusive. In addressing this question, we assessed gene-fusion landscapes by comprehensive RNA sequencing (RNAseq) of 55 pre-treatment EAC and 49 non-malignant biopsy tissues from patients undergoing endoscopy for Barrett’s esophagus. In this cohort, we identified 21 novel candidate EAC-associated fusions occurring in 3.33%-11.67% of EACs. Two candidate fusions were selected for validation by PCR and Sanger sequencing in an independent set of pre-treatment EAC (N=115) and non-malignant (N=183) biopsy tissues. In particular, we observed RPS6KB1–VMP1 gene fusion as a recurrent event occurring in ~10% of EAC cases. Notably, EAC cases harboring RPS6KB1–VMP1 fusions exhibited significantly poorer overall survival as compared to fusion-negative cases. Mechanistic investigations established that the RPS6KB1–VMP1 transcript coded for a fusion protein which significantly enhanced the growth rate of non-dysplastic Barrett’s esophagus cells. Compared to the wild-type VMP1 protein, which mediates normal cellular autophagy, RPS6KB1–VMP1 fusion exhibited aberrant subcellular localization and was relatively ineffective in triggering autophagy. Overall, our findings identified RPS6KB1–VMP1 as a genetic fusion that promotes EAC by modulating autophagy-related processes, offering new insights into the molecular pathogenesis of esophageal adenocarcinomas