Multiple genetic changes contribute to the development of breast cancer. These include amplification and mutation of numerous oncogenes and loss or mutations of recessive oncogenes. Amplification or mutation of growth factor receptors such as neu is considered significant because of the potential for either autocrine growth of the tumor or growth factor independent proliferation. The hypothesis to be examined in this proposal is whether the inappropriate activation of the jak2 tyrosine-specific protein kinase, the signalling component of the prolactin receptor system, contributes to the development of breast cancer. jak2 has recently been described as the signaling component of receptors for cytokines such as growth hormone, erythropoietin and interleukin-3. The receptor for prolactin is a member of this family of receptors and it would be expected that stimulation of cells with prolactin will activate either jak2 or a jak-related tyrosine kinase. The inappropriate activation of tyrosine kinases results in the oncogenic transformation of many different cell types including: fibroblast, epithelial, neuroblastoma and hematopoietic and breast. Thus one might expect that the constitutive activation of jak2 could contribute to breast cancer. The immune complex protein kinase assay will be used to examine the catalytic activity of jak2 in the absence and presence of stimulation by prolactin of human breast cancer cell lines. jak2 catalytic activity will be standardized to the amount of jak2 protein as measured by immunoblotting with an anti-jak2 antibody. Should the constitutive activation of jak2 be observed we will attempt to determine whether this is due to 1) the autocrine production of prolactin (measure prolactin mRNA levels), 2) an activating mutation in jak2 (cDNA cloning and sequencing) or 3) a mutation in the prolactin receptor resulting in ligand-independent activation of the receptor (cDNA cloning and sequencing). Prolactin- independent cells have been isolated from factor-dependent cells by other investigators supporting the possibility of our model. Furthermore, prolactin mRNA has been detected in both normal and neoplastic breast tissue. Genetic evidence for the role of jak2 in stimulating cellular proliferation will be obtained through the use of dominant negative mutants of jak2 or anti sense techniques. This analysis will determine whether the receptor for prolactin and its cognate signaling molecule jak2, can contribute to development of breast carcinoma.
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