Specificity and prognostic validation of a polyclonal antibody to detect Six1 homeoprotein in ovarian cancer

Abstract

ObjectiveThe presence of Six1 mRNA gene portends a poor prognosis in ovarian cancer. We describe validation of a Six1 specific antibody and evaluate its association with tumorigenicity and prognosis in ovarian cancer. MethodsA Six1 antibody (Six1cTerm) was raised to residues downstream of the Six1 homeodomain, representing its unique C-terminus as compared to other Six family members. Cells were transfected with Six1–Six6 and Western blot was performed to demonstrate Six1 specificity. Ovarian cancer cell lines were analyzed for Six1 mRNA and Six1cTerm and tumorigenicity was evaluated. Ovarian cancer tissue microarrays (OTMA) were analyzed for Six1cTerm by immunohistochemistry and scored by two blinded observers. The metastatic tumors of 15 stage IIIC high grade serous ovarian cancers were analyzed with Six1 mRNA and Six1cTerm and expression was compared to clinical factors and survival. ResultsThe Six1cTerm antibody is specific for Six1. Cell line tumorigenicity in SCID mice correlates with Six1 levels both by mRNA(p=0.001, Mann–Whitney U test) and by protein (presence vs. absence, p=0.05 Fischer's Exact test). Six1 protein was present in up to 54% of OTMA specimens. Six1 protein expression in omental/peritoneal metastases correlated with worsened survival in a sample (n=15) of high grade serous stage IIIC ovarian cancers (p=0.001). ConclusionsThe Six1cTerm antibody is specific and able to detect Six1 in cell lines and tumor tissue. Six1 protein detection is common in ovarian cancer and is associated with tumorigenicity and poor prognosis in this group of patient samples. Six1cTerm antibody should be further validated as prognostic tool.