Abstract P4-06-17: MRE11/RAD50/NBS1 (MRN) Complex Functions in Invasion in Breast Cells and Tumor Progression

Abstract

Abstract Background: The MRN complex is essential in conserving genetic integrity. It recruits ATM kinase to damage sites, and distinguishes and activates homologous recombinational repair of DNA double-strand breaks. Individuals with mutations in the MRE11 and NBS1 genes experience radiation sensitivity and are associated with a higher cancer rate. Previously, we showed that some genes involved in DNA repair are involved in regulating cell invasiveness. Since the MRN complex is downregulated in invasive breast cancer, we hypothesize that change in MRN complex expression may contribute to the pre-invasive to invasive progression of breast cancer. Materials and Methods: The HMT-3522 series of breast epithelial cell lines containing non-invasive S1, pre-invasive S3-C, and invasive T4-2 counterparts were used in 2D or 3D culture. Western blot and immunohistochemistry (IHC) were used to determine MRE11, RAD50, and NBS1 expression levels. Breast tumor tissues each containing benign, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) from 48 patients were used after IRB approval. Two pathologists and Ariol, an automated image acquisition and analysis system, scored IHC signal. Tissues were scored 0 to 3 (0=negative, 3=strong positive). ANOVA p-value of .01 or lower was considered significant for expression comparisons. Regression analysis was used for correlating Estrogen Receptor ER/Progesterone Receptor PR status with MRN expression patterns. MRE11, RAD50, and NBS1 were downregulated using siRNA. Protein expression levels were quantified by western blot. Boyden Chamber assay was used to measure cellular invasion through laminin-rich ECM, lrECM (Matrigel). Results: In non-invasive S1 cells, 3DlrECM culturing resulted in higher levels of MRN than in 2D. In pre-invasive S3-C and invasive T4-2 cells, 3DlrECM no longer upregulated MRN. Consequently, in 3DlrECM cultures non-invasive S1 cells had a higher expression of MRE11, RAD50 and NBS1 compared to invasive T4-2 cells. IHC on tissue sections showed that, on average, normal tissues have higher levels of MRN expression than DCIS and DCIS have higher levels of expression than IDC. Multiple MRN expression patterns were observed in subsets of patients when benign, DCIS, and IDC transitions were compared. Decreases in MRN expression correlated with ER/PR negativity: “single decrease” in benign to DCIS to IDC progression was associated significantly with ER negative status for MRE11 and RAD50, not NBS1. MRE11, RAD50, and NBS1 siRNAs downregulated their protein levels in invasive T4-2 cells, resulting in a significant increase in cell invasiveness through ECM. Discussion: First, we described a new role for ECM signaling in up-regulating the DNA repair protein complex MRN in non-tumorigenic cells, which gets misregulated in pre-invasive and invasive cells resulting in a progressive decrease in MRN levels in culture and in tissues. Reciprocally, we showed that downregulation of MRN upregulates invasion through ECM, suggesting it is a negative regulator of invasion in addition to its role as a gatekeeper of genome stability. Finally, different patterns of MRN expression changes in progression associated with ER/PR status suggest MRN levels as a candidate biomarker of patient-specific clinical outcomes. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-17.