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Introduction: Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils and constitutes a major component of the prominent desmoplastic reaction in pancreatic ductal adenocarcinoma (PDA). We have demonstrated recently that a subunit of COL6, COL6A3, is expressed in high levels in PDA tissue. We also showed that COL6A3 gene undergoes tumor-specific alternative splicing to produce 3 isoforms E3, E4 and E6 that are tumor tissue-specific. The aim of this study is to investigate the diagnostic value and clinical significance of circulating COL6A3 isoforms mRNA in PDA.
Methods: Serum samples were obtained from patients that underwent pancreatic resection at a single institution between 2006 and 2009. COL6A3 levels in the sera from patients with pathologically confirmed PDA (n=40), intraductal papillary mucinous neoplasms (IPMN) (n=20), and chronic pancreatitis (n=10) were analyzed by real time PCR using isoform-specific primers for E3, E4 and E6 . In addition, sera from age-matched healthy volunteers were analyzed (n=30). The prediction levels for malignancy were determined by the area under the receiver operating characteristic curve (AUC). In vitro, wound healing, cell proliferation and soft-agar colony formation assays evaluated the functional impact of each isoform in PDA cells (MIAPACA-2 and ASP-C-1) transfected with isoform-specific siRNA. A panel of inflammation- and invasion/angiogenesis-related genes was also evaluated.
Results: Circulating E6 mRNA levels were significantly (p=0.006) elevated in PDA patients when compared to all benign lesions. E3 and E4 were expressed at extremely low levels in all patients. Compared to IPMN alone, E6 levels were significantly higher in PDA (p=0.0036). There were no significant differences between E6 levels in IPMN and Normal sera (p= 0.59). Using a logistic regression model, we found that for each increasing unit of log E6 COL6A3, patients are 9.5 times more likely to harbor a cancer rather than a benign lesion, 95% CI (2.4, 38.1), p=0.002. The area under the ROC curve, AUC, was 0.72. Knocking down E3 or E4 or E6 with isoform-specific siRNA resulted in reduced PDA cell migration and invasion and concomitant reduction of the expression of several inflammation and angiogenesis-related genes, such as MMP-9, OPN, MCP-1 and VEGF. Interestingly, knocking down any of the 3 isoforms resulted in increased expression of TNF-alpha.
Conclusions: Our data show for the first time the potential clinical significance of circulating E6 COL6A3 levels in the diagnosis of pancreatic malignancy. Our in vitro data suggests a role for COL6A3 isoforms in PDA progression and metastatic potential.