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Characterizing Determinants of Tamoxifen Resistance in Human Breast Cancer: Role of CtIP (RBBP8)
Characterizing Determinants of Tamoxifen Resistance in Human Breast Cancer: Role of CtIP (RBBP8). Tamoxifen (TAM) is the most commonly used anti-estrogen for the treatment and prevention of breast cancer. However, most of the tumors that initially respond to TAM therapy will develop resistance. This constitutes a major clinical challenge in breast cancer therapy. Unfortunately, our understanding of the mechanisms involved in TAM resistance is very limited. In order to better understand this complex process, we have generated MCF-7 breast cancer cell line variants that are resistant to the inhibitory growth effects of TAM as well as variants that are completely independent of estrogen for growth. These new isogenic breast cancer cell lines represent a unique model that closely resembles the in vivo scenario. We have performed extensive Serial Analysis of Gene Expression (SAGE) on the various isogenic cell lines growth under various conditions (generated 7 libraries, > 500,000 tags, > 35,000 genes). This has allowed us to identify numerous key changes in gene expression that take place during the development of TAM resistance. By mining the SAGE databases using a novel suite of bioinformatic tools (i-Sight Discovery, Silico Insights), we observed that the expression of CtIP (RBBP8) is down-regulated 15-fold in TAM resistant cells when compared to the parental MCF-7 cells. This result has been independently confirmed using quantitative real-time PCR. CtIP was initially identified as a protein that contains an RB-binding domain and an E1A/CtBP-binding motif. Recent studies have shown that the tumor suppressor protein BRCA1 interacts with CtIP through its BRCT domain. It has also been suggested that the function of BRCA1 could be modulated by CtIP, requiring the formation of a BRCA1-CtIP complex. It is well known that BRCA1 plays a critical role in transcriptional regulation, DNA repair and cell growth and differentiation. Moreover, BRCA1 can mediate transactivation of critical cell cycle inhibitors, such as P21WAF1/CIP1 and P27Kip1. Interestingly, a recent report has shown that down-regulation of P21WAF1/CIP1 or P27Kip1 can lead to the abrogation of cell cycle arrest mediated by TAM. Thus, we hypothesize that down-regulation of CtIP constitutes a critical event for the development of TAM resistance in breast cancer cells. We further speculate that this phenomenon is mediated through BRCA1 and its downstream targets. Therefore, the Specific aims of this project are: 1: determine whether silencing the expression of CtIP in TAM sensitive cells can induce the TAM resistant phenotype. We will test the effect of this intervention using both in vitro and in vivo approaches; 2: determine whether restoration of CtIP expression in TAM resistant cells leads to regain sensitivity to the inhibitory growth effects of TAM (also using in vivo/in vitro assays); 3: compare the level of the formation of the BRCA1-CtIP complex in both TAM sensitive and resistant cells and determine whether this complex is able to change the transcriptional activity of BRCA1. We will also test whether disruption of normal complex formation (using dominant negative approaches) leads to a TAM resistant phenotype; 4: investigate the mechanisms causing CtIP down-regulation in TAM resistant cells; 5: analyze the expression of CtIP in human breast cancer samples from patients that are resistant and non-resistant to TAM treatment.
We will also continue mining our SAGE databases to identify other critical genes involved in TAM resistance for future investigation. The ultimate goal of the studies described here is to lead to the discovery of novel therapeutic approaches for the prevention of anti-estrogen resistance development, ultimately precluding breast cancer recurrence.
Characterizing Determinants of Tamoxifen Resistance in Human Breast Cancer: Role of CtIP (RBBP8). Most patients with early, localized breast cancer are treated with surgery and receive adjuvant therapy. Usually these tumors are dependent on the ovarian hormone estrogen for growth. Estrogen can stimulate cancer cell growth by binding to its natural receptor (ER). Tamoxifen (TAM) can compete effectively with estrogen for binding ER. As a result, the growth effect of estrogen on cancer cells is blocked by the action of TAM. Therefore, the anti-estrogen TAM becomes the first line of choice of adjuvant therapy for patients with ER+ tumors. Unfortunately, a significant fraction of the patients that were treated with TAM will return to the clinic with a recurrence of the original cancer. Such recurrence can be either at the local or at a distant site (i.e. metastasis). We still do not understand why and how this happens. In order to help in the understanding of this complex process, we have developed ER+ breast cancer cell lines that became resistant to TAM in contrast to their parental line that is susceptible to the inhibitory effects of TAM. We also have developed derivative lines from the same lineage that are completely independent on estrogen for growth. By using a methodology called SAGE, we have obtained a snapshot of all genes being expressed in these various cancer lines under various conditions. Now we have vast amounts of gene expression data and we need to make sense of all these information to understand the complex gene interactions and the various gene pathways involved. Among the many changes detected, we observed that the expression of a gene called CtIP (RBBP8) is 15-fold lower in the TAM resistant lines than in their parental line. Interestingly, previous studies have shown that CtIP can interact with an important breast cancer tumor suppressor, BRCA1. BRCA1 takes part in and controls many physiological processes, such as growth, differentiation, DNA repair and regulation of the expression of other important genes. Moreover, recent studies suggested that the function of BRCA1 could be regulated by CtIP, requiring the interaction between each other in order to have a functional complex. It has also been shown that BRCA1 can induce the production of various critical cell cycle inhibitors, such as P21WAF1/CIP1 and P27Kip1. Furthermore, it is reported that decreased production of P21WAF1/CIP1 or P27Kip1 can abrogate the inhibitory growth effects of TAM. In this proposal, we will use a combination of molecular and cellular biology techniques to test our hypothesis that decreased expression of CtIP is a critical event for the development of TAM resistance in breast cancer and this phenomenon is mediated via BRCA1 and its downstream targets. We will determine whether decreasing the expression of CtIP can induce TAM resistance in otherwise sensitive breast cancer cells. We will also test the opposite, i.e. whether increasing the expression of CtIP can restore TAM sensitivity in the resistant cells. We will compare the level of the interaction between CtIP and BRCA1 in both TAM sensitive and resistant lines, and determine whether this interaction can affect transcriptional activity of BRCA1. Additionally, we will test whether disruption of this normal interaction leads to TAM resistance. The mechanisms by which the expression of CtIP is decreased in TAM resistant cell lines will also be investigated. More importantly, we will test the expression of CtIP in recurrent and non-recurrent human breast cancer samples obtained from patients.
We will continue mining our SAGE databases to find other promising genes involved in TAM resistance for future study. Our ultimate goal is to help develop novel therapeutic strategies for the prevention of TAM resistance development, ultimately precluding breast cancer recurrence.