Research Grants Awarded
Regulation of Breast Cancer Progression by the ADAMs Family of Proteases
The malignant progression of breast cancer involves the acquisition of a number of properties that are normally tightly controlled. Two of these are the ability to degrade extracellular matrix (ECM) proteins, which allows the cancer to metastasize, and the ability to produce growth factors, which may be particularly important for growth at metastatic sites. Both ECM degradation and growth factor shedding are carried out by proteases. To date three distinct classes of proteases, metallo-, cysteine- and serine-proteases, have been implicated in breast cancer progression. Of these, the matrix metalloproteases (MMPs) have been most intensively studied, but limited success have been seen with MMP inhibitors in the clinic. Thus there remains a significant unmet clinical need, and it is important to investigate other proteases that may regulate breast cancer progression. Based on compelling but correlative data, I hypothesize that members of the ADAM family of proteases, particularly ADAM12, promote breast cancer progression by enhancing cancer cell migration, invasion, and/or growth factor production. The proposed study will test the hypothesis by 1) verify the expression of the ADAMs in normal and malignant breast cancer cells, as well as clinical samples of breast carcinomas, by qPCR, immunoblotting and immunohistochemistry methods. 2) the ADAMs whose expression are elevated in invasive cancer cells, termed ?the priority ADAMs?, will be individually targeted by siRNA knockdown and assessed for the effects on tumor cell migration and invasion using ECM degradation and transwell assays. Structural determinant(s) of any potential function will be mapped using mutational analysis. 3) Determine whether the priority ADAMs mediate growth factor shedding from breast cancer cells. Individual ADAM knockdown achieved in either MCF-7 or MDA-MB-231 cells will be assessed for effects on IGFBP-3 protease activity and growth factor production, respectively. The latter will be measured by the ability of the conditioned medium to stimulate DNA synthesis of quiescent 3T3 cells and/or by alkaline phosphatase (AP) activity assay using transfection of AP-tagged versions of candidate growth factor substrates. Combinations of siRNAs and dominant negative forms of ADAMs will be used in these assays if necessary. These experiments will reveal whether these priority ADAMs are regulators of breast cancer progression, and if positive results are observed, the next steps would be to generate selective small molecule inhibitors and test them in pre-clinical models of human breast cancer.
It is well established that several types of enzymes that cut proteins into small fragments, known as proteases, are involved in breast cancer progression. For example, proteases can degrade the extracellular matrix, the material secreted by and surrounding the cells in our organs. Degradation of the extracellular matrix allows cancer cells to escape their tissue of origin and invade into other organs, a process known as metastasis. Additionally, cancer cells promote their own proliferation and survival by secreting growth factors, some of which also requires processing by proteases to become active. The ADAMs, a family of proteases that are involved in both extracellular matrix degradation and growth factor processing, have been found to increase in breast carcinomas, and thus worth studying for their potential regulatory role in breast cancer. I propose to study the expression and function of the ADAMs in a selected panel of breast cells having different degrees of malignancy and invasiveness. I will first identify the ADAMs that are highly expressed in the invasive cells, termed ?the priority ADAMs?. Then I will determine whether these priority ADAMs are required for the invasive behavior and growth factor production of the cancer cells, taking advantage of a new technology called siRNA knockdown by which target genes can be specifically silenced. If these studies are negative, then we will know to look elsewhere for therapeutic targets to halt breast cancer progression. But if positive results are observed, the next steps would be to generate selective small molecule inhibitors and test them in models of human breast cancer.