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    HoxA9, the Tumor Suppressors BRCA1 and PTEN and Breast Cancer

    Scientific Abstract:
    HoxA9, the tumor suppressors BRCA1 and PTEN and breast cancer The tumor suppressors BRCA1 and PTEN are known to play a critical role in familial and sporadic breast cancers. While genomic deletions and mutations in BRCA1 and PTEN are known to underlie the loss of BRCA1 and PTEN expression in familial breast cancers, less is known about what contributes to down regulation and/or loss of function of these tumor suppressors in sporadic breast tumors. Using affymetrix micro array analysis of micro-dissected breast tumors and their adjacent normal tissue we determined that HoxA9, a homeobox gene, was reduced four-fold in 80 percent of the normal to tumor paired samples we assayed. We then confirmed that HoxA9 was significantly reduced in 90 percent of an expanded subset of primary human breast tumors and a number of breast cancer cell lines when compared to normal controls. Re expression of HoxA9 in a highly malignant, metastatic breast tumor cell line repressed tumor cell growth, motility and invasiveness in culture and inhibited anchorage-independent growth and survival in a soft agar assay. Experiments showed that inhibition of malignant behavior was associated with a significant decrease in expression of ƒÒ1 and ƒÒ4 integrin, reduced adhesion to collagens I and IV and fibronectin, and loss of focal adhesion formation. Studies aimed at clarifying the underlying mechanism(s) for these HoxA9 induced effects on tumor phenotype revealed that HoxA9 significantly up regulated BRCA1 and PTEN gene and protein expression in this model. Upon further investigation we identified previously unrecognized Hox consensus binding sites in the BRCA1 and PTEN promoters. Our studies have since confirmed that the BRCA1 consensus site is functionally relevant using a wild type and mutant BRCA1 promoter reporter assay. These data suggest that HoxA9 may function as a novel tumor suppressor in mammary epithelial cells through regulation of expression of the tumor suppressor genes BRCA1 and PTEN. We propose to test this hypothesis by achieving the following specific aims: 1. Investigate whether HoxA9 also inhibits the malignant behavior of other breast cancer cell lines in culture and in vivo using conventional 2D and 3D culture assays and nude mice and whether this too is associated with up regulation of PTEN and BRCA1, 2. Determine whether HoxA9 mediates its tumor suppressor actions directly through modulation of BRCA1 and PTEN expression and identify additional HoxA9 target genes, 3. Assay a larger subset of primary human breast tissues for changes in HoxA9 expression and determine if there is a correlation between loss of HoxA9 expression and changes in PTEN and BRCA1 expression.

    Lay Abstract:
    HoxA9, the tumor suppressors BRCA1 and PTEN and breast cancer The tumor suppressors BRCA1 and PTEN are known to play a critical role in familial and sporadic breast cancers. Although we know much about why BRCA1 and PTEN expression is lost in hereditary breast tumors we know relatively little about why the expression of these genes is decreased in sporadic breast tumors. Using a global technique to identify novel tumor suppressor genes that could play a role of breast cancer progression we found that the developmental gene HoxA9 was down regulated 80 percent of the time in the samples we examined. We also confirmed that HoxA9 was significantly down regulated in 90 percent of the time when we examined its expression levels in an expanded tumor and normal breast sample set and several human breast tumor cell lines. When we re expressed HoxA9 in an aggressive tumorigenic and metastatic tumor cell line we found that HoxA9 strongly repressed tumor growth, motility and invasion and significantly inhibited anchorage-independent growth. We also observed that HoxA9 altered the ability of the cells to interact with the extracellular matrix, induced changes in ECM receptors called integrins expression and activity. Based upon these results we believe that HoxA9 might constitute a novel tumor suppressor. Studies aimed at clarifying how HoxA9 might repress tumorigenic behavior showed that HoxA9 re expression up regulated the tumor suppressor genes BRCA1 and PTEN. When we looked at the gene promoters of BRCA1 and PTEN we identified a region that could potentially bind to HoxA9 to regulate its expression. We also determined that HoxA9 can up regulate the activity of this gene promoter. This has led us to formulate the hypothesis that HoxA9 might constitute a novel tumor suppressor which represses malignant behavior of mammary epithelial cells through regulation of downstream tumor suppressors such as BRCA1 and PTEN. We would now like to investigate this possibility further by: 1. Investigating whether HoxA9 inhibits the tumor-like behavior of other breast cancer cell lines using various assays ex vivo and in vivo in animals and to determining whether HoxA9s ability to repress tumorigenic behavior in these cancer cells is also associated with regulation of BRCA1 and PTEN, 2. Determining whether HoxA9 mediates its tumor suppressor actions directly through modulation of BRCA1 and PTEN expression and also conducting additional experiments aimed at identifying additional tumor suppressor genes which might be regulated by HoxA9 and, 3. Assaying for changes in HoxA9 expression in a larger subset of primary human breast tissues and determining if there is a correlation between loss of HoxA9 expression and changes in PTEN and BRCA1 expression in these samples.