Evaluating the Role of the RIN1 Gene in Breast Cancer

Institution: University of California, Los Angeles
Investigator(s): Marc Milstein, B.S. -
Award Cycle: 2005 (Cycle 11) Grant #: 11GB-0038 Award: $71,835
Award Type: Dissertation Award
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2005)
RAS proteins are key regulators of signal transduction pathways that control cell growth, differentiation, and cell death. RAS proteins alternate between inactive (GDP-bound) and active (GTP-bound) states. In about 30% of tumors RAS becomes “activated.” Our interest is in the downstream targets of the RAS signaling pathways. RIN1 is a RAS “effector” that regulates epithelial cell functions. The RIN1 gene is silenced in most breast cancer cell lines and in many breast tumors. Because RIN1 binding to RAS is competitive with other effectors, reduced RIN1 levels could lead to elevated signaling through the mitogenic RAS-RAF-MEK-ERK pathway and perhaps other tumor progression RAS pathways. This hypothesis directly addresses the contrasting observations that RAS signaling is an important component of breast cancer, but that RAS mutations are rarely seen in these tumors. The main objective of the proposed research is to critically evaluate the hypothesis that the silencing of RIN1 contributes to breast cancer progression. As part of this goal we will also determine the mechanism of silencing of RIN1 in breast tumors.

Our specific objectives are to: 1. Test the tumor suppressor properties of RIN1. 2. Characterize the involvement of RIN1 in breast cancer. 3. Determine the mechanism of RIN1 gene silencing in breast tumors.

For these studies we will test the ability of RIN1 to suppress the anchorage independent growth phenotype. Breast cancer cells are able to grow in soft agar while normal mammary epithelial cells cannot. We will use viral expression systems to introduce RIN1. Immunohistochemistry and real-time PCR will measure RIN1 levels. We will determine if there is a correlation between the reduction of RIN1 and the expression of other genes involved in breast cancer. The UCLA Family Cancer Registry will also be screened to determine if there is a hereditary component to the involvement of RIN1 in breast cancer. Furthermore, we will determine if there are overall lower levels of RIN1 in the breast epithelial tissue of those that develop breast tumors and if these lower levels cause a predisposition to developing breast cancer. Methylation-specific PCR will be employed to examine changes at the RIN1 locus. This technique will be performed on DNA from breast tumor cell lines, already characterized with regard to RIN1 expression, and on DNA from breast tissue (normal and tumor).

RIN1 is a previously unexplored factor in breast cancer initiation and malignancy. Our hypothesis may provide a new way to approach breast tumor biology and should directly facilitate the identification of new targets for breast tumor therapeutics.


Final Report (2007)
Our lab at UCLA has previously identified an unknown breast tumor suppressor protein, called RIN1. RIN1 is a Ras “effector” that regulates epithelial cell functions. Proteins in the Ras family act as molecular switches for a wide variety of signal pathways that control such processes as cell proliferation, cell adhesion, apoptosis, and cell migration. Ras and ras-related proteins are often deregulated in cancers, leading to increased invasion and metastasis, and decreased apoptosis.

We determined that RIN1 expression is transcriptionally “silenced” (i.e., RNA transcription blocked) at a high frequency in breast cancer cell lines and human breast tumors. We characterized two mechanisms that silence RIN1. First, the transcriptional repressor, called SNAI1, negatively regulates RIN1 expression. In addition, treatment with TGFß, an inducer of SNAI1 and promoter of tumor metastasis, caused a reduction in RIN1 expression in normal mammary epithelial cells and tumor cells. Second, DNA methylation (epigenetic regulation) was found in the promoter and first exon of the RIN1 gene, suggesting methylation as another silencing mechanism. Furthermore, we found that restoration of RIN1 inhibited the growth of tumor cells in anchorage independent assays (in vitro) and reduced the growth of mammary tumors in nude mice (in vivo), consistent with a tumor suppressor function.

The RIN1 gene lies in a tight cluster with two other tumor suppressor genes, the BRMS1 (Breast Tumor Metastasis Suppressor 1) and B3GNT1 (ß, -1,3-N-acetylglucosaminyltransferase 1). This alignment is highly conserved in mammals. We have shown that this three gene locus (B3GNT1, BRMS1, RIN1) displays coordinated silencing in multiple breast tumor cell lines and a tissue sample. We found that SNAI1 knockdown restored B3GNT1, BRMS1 and RIN1 expression coordinately. Furthermore, treatment with TGFß, caused a reduction in B3GNT1, BRMS1 and RIN1 expression in normal mammary epithelial cells and tumor cells. Finally, B3GNT1, RIN1 (this work) and BRMS1 (published elsewhere) each independently act as negative regulators of cell migration. The discovery of a tumor suppressor gene cluster (B3GNT1-BRMS1-RIN1) represents a unique opportunity to understand how the silencing of this locus may cooperatively lead to tumor formation and metastatic spread.

The identification of RIN1 as a tumor suppressor, and the investigation of its role as part of breast tumor suppressor locus, should facilitate the identification of new targets for breast tumor therapeutics as well as provide insight into epithelial plasticity during mammary tissue development.


Symposium Abstract (2005)
RAS proteins are key regulators of signal transduction pathways that control cell growth, differentiation, and cell death. RAS proteins alternate between inactive (GDP-bound) and active (GTP-bound) states. In about 30% of tumors RAS becomes “activated.” Our interest is in the downstream targets of the RAS signaling pathways. RIN1 is a RAS “effector” that regulates epithelial cell functions. The RIN1 gene is silenced in most breast cancer cell lines and in many breast tumors. Because RIN1 binding to RAS is competitive with other effectors, reduced RIN1 levels could lead to elevated signaling through the mitogenic RAS-RAF-MEK-ERK pathway and perhaps other tumor progression RAS pathways. This hypothesis directly addresses the contrasting observations that RAS signaling is an important component of breast cancer, but that RAS mutations are rarely seen in these tumors. The main objective of the proposed research is to critically evaluate the hypothesis that the silencing of RIN1 contributes to breast cancer progression. As part of this goal we will also determine the mechanism of silencing of RIN1 in breast tumors.

Our specific objectives are to: 1. Test the tumor suppressor properties of RIN1. 2. Characterize the involvement of RIN1 in breast cancer. 3. Determine the mechanism of RIN1 gene silencing in breast tumors.

For these studies we will test the ability of RIN1 to suppress the anchorage independent growth phenotype. Breast cancer cells are able to grow in soft agar while normal mammary epithelial cells cannot. We will use viral expression systems to introduce IN1. Immunohistochemistry and real-time PCR will measure RIN1 levels. We will determine if there is a correlation between the reduction of RIN1 and the expression of other genes involved in breast cancer. The UCLA Family Cancer Registry will also be screened to determine if there is a hereditary component to the involvement of RIN1 in breast cancer. Furthermore, we will determine if there are overall lower levels of RIN1 in the breast epithelial tissue of those that develop breast tumors and if these lower levels cause a predisposition to developing breast cancer. Methylation-specific PCR will be employed to examine changes at the RIN1 locus. This technique will be performed on DNA from breast tumor cell lines, already characterized with regard to RIN1 expression, and on DNA from breast tissue (normal and tumor).

RIN1 is a previously unexplored factor in breast cancer initiation and malignancy. Our hypothesis may provide a new way to approach breast tumor biology and should directly facilitate the identification of new targets for breast tumor therapeutics.

RIN1 is a Breast Tumor Suppressor Gene. Cancer Research. 2007 Dec. 15:67 (24): 11510-6.
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Milstein M, Mooser CK, Hu H, Fejzo M, Slamon D, Goodglick L, Dry S, Colicelli J
Yr: 2007 Vol: 67 Nbr: 24 Abs: Pg:11510-6