Abstract P5-05-12: Heparan Sulfatase 2 Regulates Breast Cancer Tumorigenesis and Promotes Invasive Ductal Lesions

Abstract

Abstract Ductal carcinoma in situ (DCIS) of the breast is a heterogenous group of proliferative cellular lesions that have the potential to become invasive. Very little is known about the molecular alterations involved in the progression from DCIS to invasive ductal carcinoma (IDC). Heparan endosulfatases HSulf-1 and -2 catalyze or edit sulfate moieties on heparan sulfate proteoglycans and have been implicated in modulating heparin binding growth factor signaling. Several studies have suggested that while expression of HSulf-1 is down-regulated and is considered a tumor suppressor gene, expression of HSulf-2 is over-expressed in primary breast tumors and cell lines. However, the role of HSulf-2 in breast tumorigenesis is poorly understood. Here we have evaluated the effect of HSulf-2 in an in vivo model of breast cancer progression (MCF10DCIS.com). These cells form comedo type DCIS and progress to IDC when transplanted in immune-deficient mice and therefore are an ideal model to study breast cancer progression. Methods: Stable clones depleted of HSulf-2 in MCF10DCIS.com cells were generated and injected into mouse mammary fat pads with non-targeted control shRNA clones (NTC) as controls. HSulf-1, HSulf-2, smooth muscle actin immunostaining and H&E staining was perfromed at weeks 3, 5 and 7. Matrixmetallo Protease (MMP) expression by real time PCR was evaluated at Weeks 3 and 5 in both groups. Results: A significant reduction in tumor growth was observed in HSulf-2 depleted clones compared to NTC clones (p=<0.01). HSulf-2 immunohistochemical staining showed distinct pattern of staining localized to ductal lesions as well as stromal (myoepithelial and myofibroblasts cells). In contrast, the closely related family member, HSulf-1 expression was restricted largely to stromal and myoepithelial layer in ductal lesions. While HSulf-2 staining remained intense from weeks 3 to 7, HSulf-1 expression was diminished during the same time period. In NTC clones, ductal like lesions were observed by week 3 with disruption of the basement membrane and appearance of invasive phenotype by weeks 5 and 7. In contrast, HSulf2 knockdown xenografts showed retention of ductal like structures even at week 5 and retention of basement membrane (p=<0.05). Additionally marked necrotic areas were also observed in HSulf-2 silenced clones. Previous studies have shown that the malignant epithelium induces the development of the stroma necessary for the development of invasive phenotype. Consistent with this observation, we demonstrated decreased MMP expression in xenografts derived from HSulf-2 depleted clones compared with NTC xenografts (p=<0.05). Conclusions: Our data suggest that HSulf-2 may play an important role in the transition from DCIS to an invasive phenotype (IDC) potentially by up regulating MMP expression and activity. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-12.