Research Grants Awarded
Role Of The Spdef Transcription Factor In Breast Cancer And Its Putative Implication In The Estrogen-Mediated Transcriptional Network
(1) SCIENTIFIC RATIONALE:
Over two-third of human breast cancers express Estrogen Receptor alpha (ER), which is positively correlated with the expression of a signature set of transcription factors, including FOXA1, GATA3 and XBP1. Our laboratory is interested in the understanding of the transcriptional activity of ER and its cofactors, with a particular emphasis on the whole genome analysis of their direct binding regions through ChIP-on-chip technique. A study performed in our laboratory thus demonstrated that FoxA1 acts as an essential pioneer factor prior to ER recruitment to chromatin. SPDEF (SAM Pointed Domain Ets Factor) is a member of the ?Ets? family. These transcription factors regulate a number of biological processes, including cell proliferation, differentiation and invasion and are thought to play an important role in oncogenesis. SPDEF is expressed exclusively in tissues with a high epithelial content, especially hormone-regulated epithelium, such as prostate, mammary gland, endometrium and ovary. Furthermore, several studies showed SPDEF to be one of the most highly over expressed mRNA in human and mouse mammary tumors. In normal and breast cancer cells, it has been shown that SPDEF could cooperate with ERBB2 to promote motility and invasion.
(2) HYPOTHESIS / EXPECTED RESULTS:
The connection between SPDEF and Estrogen signaling has never been studied. Preliminary results obtained in our laboratory suggest a functional link. Oncomine coexpression analysis revealed that SPDEF is significantly co expressed with ER in ER positive breast carcinomas, together with other ER cofactors such as FOXA1, XBP1 and GATA3. A list of SPDEF targets (established by gene expression chip analysis in immortalized non-tumorigenic breast cells) has been obtained from a collaboration with Dr. Joan Brugge?s laboratory. We studied the overlap of SPDEF targets with our whole genome ER binding regions dataset. We found that there is a bias toward ER regulated genes among SPDEF targets. Directed ChIP experiments show that SPDEF is itself an ER target through 3 binding regions located within the first intron and at the 3? end of the gene. Our working hypothesis is that SPDEF is component of estrogen signaling transcriptional network in breast cancer, and our proposed research project deals with the understanding of this connection.
(3) RESEARCH AIMS AND DESIGN:
We first would like to understand the mechanism of ER and SPDEF co expression. ER and SPDEF could indeed be involved in a positive cross-regulatory loop, which might explain their co expression in breast cancer. Using gene expression chips, we will then compare the transcriptomes of breast cancer cells in presence/absence of SPDEF (siRNA downregulation), in order to establish the full list of its targets. We will also determine all the SPDEF binding sites in the genome by using ChIP-on-chip. This whole genome data will be compared to existing data generated in our laboratory (estrogen induced genes, ER/FOXA1 binding regions). This will allow us to evaluate the extent of the implication of SPDEF in ER transcriptional response. We will then focus on specific subsets of ER/SPDEF common target genes and analyze the mechanisms regulation by these two factors (direct interaction on a common binding region, separate binding on different regions, etc).
The data obtained by the ChIP-on-chip analysis will provide an interesting template to better understand physiological functions of SPDEF. A recent study described a role of SPDEF in epithelial cell migration and invasion. We believe that determination of SPDEF direct targets using ChIP-on-chip will provide information necessary to better understand these functions.
The over expression of SPDEF in mammary carcinomas and its role in migration/invasion suggests a role in tumor progression. SPDEF knockout (KO) mice have been generated in Dr. Towia Libermann?s laboratory. The mammary gland of these animals lacks an obvious functional defect; however, we would like to evaluate the effect of SPDEF in mammary carcinoma progression. Our laboratory developed transgenic animals over expressing ER cofactor AIB1. AIB1 transgenic (Tg) mice display a high incidence of ER positive mammary tumors. We plan on crossing SPDEF KO with AIB1-Tg and examine the effect of SPDEF loss on tumor incidence and progression.
The over expression of SPDEF in breast carcinoma and its positive role in migration and invasion strongly suggest an oncogenic role. Its mechanism of transcriptional regulation and its targets in breast are poorly known. We believe that SPDEF is part of the Estrogen driven transcription factors network and thus a direct or indirect cofactor of ER, a key protein in breast carcinoma treatment. Our study will therefore establish SPDEF as a new therapeutic target.
Breast cancer and cancers in general are tightly linked to abnormal gene expression programs that lead to uncontrolled cell proliferation. About 80% of breast cancers, once established, rely on the supply of the hormone estrogen to grow; these are known as hormone-sensitive or hormone receptor-positive cancers. In cells, estrogen binds to a protein known as the Estrogen Receptor (ER), which, upon interaction, switches specific genes on or off. ER is thus the target of drugs that counteract its action (antihormonal therapy) in breast cancer.
Our laboratory has begun to prove that estrogen induces cell proliferation in breast cancer cells through ER and the network of cofactors, with which it collaborates. We are establishing the list of genes that are directly regulated by ER and its cofactors, through a new powerful technique called ChIP-on-chip. We decided to focus on a potential new cofactor of ER. This cofactor, termed SPDEF, is highly expressed in breast cancer and is already a known marker for detection of metastatic breast cancer in patients. We want to prove that SPDEF collaborates with ER and contributes to ER dependent tumor progression. To test this hypothesis, a key experiment will be to establish the list of SPDEF direct target genes and compare it to ER?s targets. We will then explore this list of target genes and try to connect it with novel or already known roles of SPDEF, especially those linked with tumor progression. We finally are going to determine whether SPDEF plays a role in tumor development in a mouse model, which develops mammary tumors.
The discovery of SPDEF direct target genes and its connection with ER will contribute to better understand the network of proteins involved in breast cancer development, providing new insights for better treatments.