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An Investigation of p21CIP1 Function During Breast Cancer Metastasis
The p21Cip1 and p27Kip1 tumor-suppressor proteins are members of the CIP/KIP family of cyclin-dependent kinase inhibitors, important in cell growth and breast cancer. In contrast to p27Kip1 where reduced levels correlate to poor clinical outcome, low expression of p21Cip1 correlates with good prognosis in some studies. While low levels of p21Cip1 could reduce cell growth, its presence might assist metastasis. Aberrant expression of the cadherin adhesion molecules are important events in the progression of breast cancer to metastasis. Loss of E-cadherin and gain of N-cadherin is associated with epithelial to mesenchymal transition (EMT) and tumor invasiveness. Using an EMT model of breast cancer metastasis, our preliminary data shows that FGF-2 treatment of N-cadherin-transfected MCF-7 cells produces a highly invasive phenotype and elevated levels of p21Cip1, effects not seen in FGF-2 treated MCF-7 control cells. Increases in p21Cip1 expression result in a G1-S phase block suggesting invasion and withdrawal from the cell cycle are two interrelated events in metastasis. We hypothesize p21Cip1 expression in tumor cells is important for metastatic progression of breast tumor cells. The p21Cip1 blockade of the cell cycle at the G1-S phase allows for transiently-induced metastatic events to occur, which endow tumor cells with a gain in motility, tissue invasiveness and metastasis. Aim1: Use stable p21Cip1 anti-sense (AS) versus p27Kip1 AS expression to test the loss of p21Cip1 on tumor cell migration and Matrigel invasion using highly versus weakly invasive breast cancer cell lines. Aim2: To test in vivo, if loss of p21Cip1 expression reduces metastasis of breast cancer cell lines using p21Cip1 AS-expressing breast cancer cell lines and a metastasis model where we can follow tumor cell spread from nude mice fat pads into distant organs. Aim3: To test in vivo the effect of p21Cip1 loss on tumor metastasis in onco-mouse models of breast cancer by crossing the p21Cip1 knockout mice with transgenic MMTV-c-neu and/or Polyomavirus middle T antigen mice. Both models cause the growth of metastasis in the lungs. Animals will be analyzed for tumor onset and growth. Lungs and other organs will be analyzed for the presence of metastases. This proposal will elucidate if and why actively metastasizing tumor cells shut down their cell cycle by increasing levels of the p21Cip1 during this process. The outcome of these studies will aid our understanding of tumor metastasis and give us clues for the prevention or treatment of metastatic breast disease.
Cell division in breast epithelium is a complex process which is highly orchestrated with many built-in regulatory controls. Loss of this regulation can lead to un-controlled cell growth and cancer. The Cip1 and Kip1 proteins are members of a family of proteins, who function is to help regulate growth by slowing or stopping cell division. In many breast cancers Kip1 protein levels are kept lower than required, which results in an increase in cell division. Many studies show low Kip1 protein levels correlate well with poor long term survival. This has allowed clinicians to use Kip1 levels as a prognostic indicator of disease outcome. Cip1 levels would also be predicted to be a useful prognostic indicator since both proteins perform similar functions, however this is not the case. In several reported clinical breast cancer studies, the absence or low expression of Cip1 protein is associated with long term, disease-free survival. This suggests Cip1 protein might assist in the metastatic process, which is what dictates long term survival. Work in our laboratory focuses on proteins named cadherins that allow neighboring cells to attach to each other and maintain normal tissue organization. These proteins appear to play important roles in cancer progression and metastasis. The presence of the wrong cadherins in breast cancer cells can lead to a cancer with greater invasive and metastatic abilities. N-cadherin is often found in breast cancer cells and is associated with a more severe form of the disease. Importantly we have found that N-cadherin causes an increase in Cip1 levels in a metastatic breast cancer cell line. We believe that Cip1 might be required by some cancer cells to temporarily halt cell division to allow metastatic-associated changes to take place. Once the cancer cell has disseminated to distant organs, this process can reverse itself, again allowing cell division to occur. Using existing breast cancer cell lines and mouse models of human breast cancer we believe we can show that Cip1 is important in the metastatic process. This result would not only be valuable in prevention and treatment of metastatic breast disease, but also as a useful guide for the therapeutic regimen of breast cancer patients.