Abstract P3-03-01: The role of a newly discovered N-terminal methyltransferase in breast cancer

Abstract

Abstract The recently discovered N-terminal methyltransferase, NRMT, is the first eukaryotic N-terminal methyltransferase to be identified. NRMT is a highly conserved, ubiquitously expressed, nuclear MT. After methionine cleavage, it methylates the X-P-K amino-terminal consensus sequence of its substrates. Loss of N-terminal methylation has been associated with decreased ability to bind to DNA and decreased protein stability. Many NRMT targets are known tumor suppressors or oncogenes, including DDB2, RB, SET, and NEMO. Knockdown of NRMT in HEK cells results in increased cell proliferation and multi-spindle formation, which are characteristics of malignant cells. In addition, microarray analysis has shown that NRMT expression can be either up or down regulated in a variety of human cancers. Our preliminary data in human breast cancer samples verifies the microarray data and shows that NRMT mRNA levels are both significantly increased or decreased in a patient specific manner. NRMT expression levels do not seem dependent on the type of tumor, therefore, we hypothesize that NRMT expression levels may be dependent on the endocrine sensitivity of the tumor. To test if NRMT activity differentially affects endocrine sensitive and endocrine resistant cell types in vitro, we knocked down NRMT expression in a panel of human breast cancer cell lines with decreasing endocrine sensitivity using shRNA. Cells expressing the shRNA against human NRMT (or mouse NRMT as a control) were then assayed for proliferation and migration potential. Knockdown of NRMT significantly increased proliferation of the endocrine sensitive MCF-7 and T47D cancer cell lines over a five-day time course. In contrast, NRMT knockdown only slightly inhibited LCC9 (an antiestrogen-resistant MCF-7 variant line) proliferation and had no effect on endocrine resistant MDA-MB-231 cells. NRMT knockdown also significantly inhibited MCF-7 cell migration at 72 hours but only slightly affected LCC9 cells and had no affect on the MDA-MD-231 cells. These data indicate that NRMT loss early in tumorigenesis has the potential to increase the growth rate of endocrine sensitive primary tumors but has little affect on endocrine resistant cells. We are now interested in determining if we can increase endocrine resistant cell proliferation and migration through NRMT overexpression, thus explaining the microarray profiles. Taken together these studies indicate that N-terminal methylation by NRMT is a biologically important post-translational modification with regard to cell division and tumorigenesis in breast cancer. Next, we will test if NRMT loss can confer endocrine resistance to endocrine sensitive cells and use a murine mammary gland transplantation and regeneration model to study the effect of NRMT knockdown on mammary tumorigenesis and metastasis in vivo. Understanding the general biological function of N-terminal methylation and its relationship to endocrine sensitivity could lead to the use of NRMT expression levels as a biomarker for tumors likely to develop resistance to endocrine therapies. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-01.