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DNA Methylation of SLIT-2 as a New Marker for the Early Diagnosis and the Use of Soluble
SLIT-2 as a New Therapeutic for Breast Cancer
Minna J PDF0403266
DNA methylation of SLIT-2 as a new marker for the early diagnosis and the use of soluble SLIT-2 as a new therapeutic for breast cancer.
Background: The candidate tumor suppressor gene (TSG) SLIT-2 (4p15.2) encodes the endogenous ligand SLIT-2 of the Robo1 receptor. The role of SLIT/Robo1 signaling has been extensively studied in the development of the nervous system-acting as a repulsion cue for axon guidance. SLIT-2 and Robo1 are also co-expressed in a number of different embryonic and adult tissues, suggesting function in these tissues also. Recently we have found that SLIT-2, undergoes promoter methylation in ~60% or primary breast cancers (Dallol et al.,2002). This methylation correlated with loss of expression of SLIT-2 mRNA while exogenous re-expression led to inhibition of breast cancer growth. Therefore SLIT-2 represents a novel candidate TSG for breast cancer. By contrast, Wang et al. have proposed that SLIT-2 is over expressed in human tumors and acts to stimulate tumor angiogenesis (Wang et al., 2003). It is important to identify the true role of SLIT-2 in breast cancer and either finding would provide a use of SLIT-2 as a diagnostic and potentially therapeutic target. Objective/Hypothesis: SLIT-2 is an important TSG involved in the pathogenesis of breast cancer. Its DNA methylation represents a key feature that could allow the early diagnosis of breast cancer. In addition, SLIT-2, a soluble, secreted protein, potenitally could be used as a new therapeutic. I am aware that an alternative Wang hypothesis exists and I propose to distinguish between these two opposing views and to test the role of SLIT-2 as a new diagnostic and therapeutic target. Specific Aims: 1. Determine the expression of SLIT-2 in primary breast cancers, normal breast epithelium, and breast cancer cell lines using quantitative real-time PCR (Q-PCR) and Western blotting. Develop a monoclonal antibody directed against SLIT-2. 2. Determine the frequency of methylation of SLIT-2 in paired samples of breast cancers, and non-involved breast as well as from women with differing breast cancer risk. 3. Test the effect of exogenous SLIT-2 on the in vitro growth of breast cancer cell lines, and determine if SLIT-2 induces apoptosis. 4. Determine the portion of SLIT-2 (140 kd N-terminus, 60 kd C-terminus or whole protein) required for growth inhibition; 5. Develop breast cancer xenograft SCID mouse models and test the effect of SLIT-2 treatment on breast cancer and immortalized normal mammary epithelial cell growth in vivo. Study design: SLIT-2 expression in primary breast cancers and breast cancer cell lines will be tested using Q-PCR and Western blotting. Primary tumor samples and normal breast cells from ipsi and contralateral breasts of women with defined breast cancer risk will be analyzed for SLIT-2 promoter methylation by methylation specific assays (MSP and COBRA) to determine the relationship of SLIT-2 methylation to breast cancer risk. SLIT-2 expression constructs will be transfected into a panel of breast cancer cell lines, and the effect on cell growth tested by colony formation in liquid and soft-agar media. Similarly the effect on breast cancer cell growth of soluble SLIT-2 will be tested. Tests for induction of apoptosis (TUNEL and caspase 3 induction) of treated breast cancer cells will be done. Expression constructs containing the entire SLIT-2 gene, or the 140 kd N terminus and 60 kd C-terminus (normally processed portions) will be tested for cell growth inhibition in vitro. SCID mice will be innoculated with breast cancer cells orthotopically (mammary fat pad) or intravenously to develop systemic metastatic disease, and the effect of SLIT-2 ascertained by testing breast cancer cells re-expressing SLIT-2 in xenograft growth, or the effect of co-injection of breast cancer with COS7/SLIT-2 transfected cells, or systemically administered semi-purified soluble SLIT-2 on tumor cell in vivo growth. RNAi mediated SLIT-2 “knockdown” experiments in immortalized normal mammary epithelial cells or conversely overexpression of SLIT-2 in these cells will be tested in SCID mice and seeing the effect on in vivo cell growth and angiogenesis. Relevance: The high frequency of SLIT-2 methylation in breast cancers makes its detection an excellent molecular marker for the early detection of breast cancer. Likewise, the dramatic growth inhibitory effect of SLIT-2 re-expression on breast cancer coupled with its soluble nature makes SLIT-2 a potential new systemic therapeutic. Finally, the expression studies and in vitro and in vivo growth studies will help decipher whether SLIT-2 is acting as a TSG (my hypothesis) or as a oncogene (Wang hypothesis).
Minna J PDF0403266
DNA methylation of SLIT-2 as a new marker for the early diagnosis and the use of soluble
SLIT-2 as a new therapeutic for breast cancer.
A clearer understanding of the molecular basis of breast cancer is crucial in the development of new diagnostic tests and eventually therapeutic agents to enable us to more successfully treat this disease. The SLIT-2 gene represents an important new gene in the development of breast cancer. SLIT-2 encodes a secreted protein whose function has been well studied in the nervous system, along with that of its receptor Robo1. However both SLIT-2 and Robo1 are coexpressed in a variety of embryonic and adult tissues, suggesting that SLIT/Robo1 signaling may have a role to play in these tissues also. Recent studies involving my lab have shown that SLIT-2 expression is inactivated in 59% of primary breast cancers by promoter hypermethylation (Dallol et al 2002). This methylation appears to be highly tumor specific. This raises the hope that detection of methylated SLIT-2 DNA sequences in “normal” breast tissues would provide a new molecular marker for the very early diagnosis of breast cancer. Furthermore re-introduction of SLIT-2 into breast cancer cells lacking its expression (either by genetic transfection or application of soluble SLIT-2 protein) leads to inhibition of breast cancer cell growt in vitro. Since SLIT-2 has no effect on tissues already expressing SLIT-2 this protein has the potential to be developed as a therapeutic agent in the treatment of breast cancers lacking SLIT-2 expression. I have done a lot of preliminary work providing support for these two approaches (diagnostic and therapeutic). However, recently a new study has unexpectedly presented data that SLIT-2 in some human cancers could be operating in 180 degree different fashion; namely that tumors make SLIT-2 and this acts to stimulate tumor blood vessel formation (angiogenesis) (Wang et al 2003). While I think this is unlikely for breast cancer I will also study the possibility that SLIT-2 is over expressed in breast cancer. If this turns out to be the case, SLIT-2 protein detection could still be used as an early diagnostic marker, and now for a therapeutic approach one would want to develop ways to block rather than replace SLIT-2 function. In either event, SLIT-2 represents an important new target to study for the early detection and potential treatment of breast cancer. In this proposal, I intend to evaluate SLIT-2 as an early detection and prognostic marker for breast cancer by testing for its methylation in an extended panel of primary breast tumors, uninvolved breast tissues, for which important clinical data is known and tissues (fine needle aspirates) from women with different levels of breast cancer risk but who do not yet have breast cancer. Subsequently we will carry out statistical analysis to see how SLIT-2 promoter methylation correlates with clinical parameters. I also want to do preclinical studies aimed at developing SLIT-2 as a new therapeutic agent, by testing the growth inhibition effects of SLIT-2 in both in vitro (liquid and soft agar colony formation assays) and in vivo (xenograft mouse models of breast cancer and metastatic disease) studies. In addition to further developing SLIT-2 as a therapeutic and diagnostic agent, these studies should help clarify whether SLIT-2 functions as a tumor suppressor (my hypothesis) or an oncogene (competing hypothesis).