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Preventing Basal Breast Cancer Recurrence
Investigator Initiated Research
Rationale: Among the various types of breast cancer, basal breast cancer (BBC) and Her2 positive patients have the lowest survival rates. Several effective therapeutic agents are currently in clinical use that selectively kill Her2 positive tumors. However, therapeutics that specifically target BBCs are not available. Poor survival of BBC patients is associated with a lack of response to therapies effective against other types of breast cancer, and a high rate of tumor recurrence after surgery. BBCs do not exhibit defined borders and invade extensively into surrounding tissues. Therefore the intrinsic invasiveness of BBCs is likely responsible for their high rate of relapse and poor outcome. Unfortunately, little is known about the mechanisms responsible for the invasive properties of BBC. We have isolated basal breast cancer cell lines from a transgenic model of breast cancer. These cell lines express molecular markers that define them as basal breast cancers, and form highly invasive tumors when injected into the mammary glands of syngeneic wild type female FvB mice. Analysis of the cell lines in vitro and tumors formed from the cells in vivo indicate that the cells lack proper cell-cell adhesion and that this is associated with mislocalization of E-cadherin from the cell membrane to the cytoplasm. The cells also express high levels of the intermediate filament protein Nestin, which has recently been shown to be essential for the invasiveness of prostate cancer cells. Nestin is only expressed in the basal subtype of breast cancers. Significantly, in our preliminary studies cell-cell adhesion of the BBC cell lines was restored by treatment with the Casein Kinase 1 (CK1) inhibitor IC261. This result indicates that it may be possible to restore Cadherin function in vivo using CK1 inhibitors. Such an effect is predicted to suppress BBC invasion, decrease post-surgical recurrence, and improve patient survival.
Hypotheses: We hypothesize that BBCs are inherently invasive due to defective Cadherin cell-cell cohesive function and because of the selective expression of the intermediate filament protein Nestin in this class of breast tumors. We further hypothesize that it is possible to restore Cadherin cell-cell cohesive functions in BBCs using pharmacological CK1 inhibitors.
Research Aims and Design:
(1) Determine the role of cytoplasmic Cadherin localization in basal breast cancer invasion
(a) Determine whether engineering basal breast cancer cells to express mutants of E- or N-cadherin lacking the conserved Casein Kinase 1 (CK1) phosphorylation site converts tumor growth from an invasive to an expansive pattern, and suppresses post-surgical recurrence.
(b) Determine whether the CK1inhibitor IC261 suppresses the invasion of basal breast tumors into surrounding normal tissues, restores Cadherin function in vivo, and suppresses post-surgical recurrence.
(2) Determine the role of Nestin in basal breast cancer invasion
(a) Examine the role of Nestin in basal breast cancer invasion and the influence of Nestin on the localization and function of E- and N-cadherin, and on the integrity of the cytokeratin cytoskeleton.
(b) Examine the correlation between Nestin expression and cytoplasmic cadherin localization in human breast tumors.
(3) Examine the influence of the initiating oncogene on basal breast cancer invasive properties
Impact on Breast Cancer Mortality: The basal subtype of breast cancer exhibits significant differences in the organization of their cytoskeleton and their cell-cell adhesive properties as compared with other tumor subtypes. Improving the survival of BBC patients requires understanding how the unique biology of these tumors allows extensive local invasion into surrounding tissues that translates into an incomplete surgical removal of the tumors and a high rate of recurrence. The proposed studies will elucidate the mechanisms responsible for basal breast cancer invasion and test whether therapeutic inhibition of CK1 suppresses tumor invasion and post-surgical relapse in a novel model system developed in our laboratory to study BBC invasion in vivo.
Hypothesis and Study Design: The basal subtype of breast cancer represents approximately 16% of American breast cancer patients, but 39% of premenopausal African American women. Basal breast cancer (BBC) is associated with a poor prognosis and a lack of response to drugs that are effective against other types of breast cancer. BBC fatalities are associated with the high rate of tumor recurrence after surgical removal. BBCs invade into surrounding tissue and lack a well-defined border. Therefore it is nearly impossible for the surgeon to remove the tumor without leaving cancer cells behind. We hypothesize that the invasiveness of BBCs is due to the decreased function in the cancer cells of proteins called Cadherins and the increased production of a protein called Nestin. Cadherins make cells tightly adhere to each other and prevents them from moving away from the main tumor mass. We hypothesize that Nestin makes the cells less rigid and allows them to squeeze between the cells that border the tumor as the cancer cells move away from the main tumor mass.
We have isolated cell lines from mouse BBCs and developed a new method for studying the invasion of BBC cells into the mouse mammary gland. We propose to examine the role of Cadherins in BBC invasion in two ways. First, we will engineer the cells to produce more Cadherin protein and determine whether this decreases the invasion of the BBCs through the mouse mammary gland. Second, we will treat the animals with a drug that increases the ability of the Cadherins to make the cells tightly adhere to each other and determine whether this suppresses invasion of the BBCs through the mouse mammary gland. The role of Nestin in BBC invasion will be examined by engineering BBCs to produce lower levels of Nestin protein and determine whether this inhibits the ability of the BBCs to invade into a mouse mammary gland. The functions of the Cadherins and Nestin will be examined in clinical samples obtained from a large number of breast cancer patients to determine whether altered function of these proteins correlates with the invasive properties of human breast tumors. The final set of studies involves isolating BBCs from additional mouse tumor models to determine whether the invasiveness of tumors formed by these other BBC cells is also associated with altered Cadherin and Nestin function. The results will help to determine whether the invasiveness of breast cancers is more closely related to the subtype of the cancer, or to the specific genetic defects that caused the cancer.
Understanding the Mechanisms of Breast Cancer Invasion: The results of the proposed studies will elucidate the roles of Cadherins and Nestin in BBC invasion, and recurrence after surgery. A number of existing therapeutic agents increase Cadherin function. The studies proposed here will provide a "proof of principle" and justify clinical trials to test Cadherin-directed anti-invasive agents in patients. The studies involving Nestin will determine the mechanisms by which Nestin increases cancer cell invasiveness. The analyses of human breast cancer patient samples will verify the relevance of altered Cadherin and Nestin function in invasive human breast cancers. If invasiveness is linked more closely with the tumor type than with the genetic changes that initiate tumor formation, this may lead to the development of new therapeutic strategies that involve diverting cancer cells away from the BBC subtype and toward other tumor subtypes. This would result in tumors responsive to existing therapies and exhibiting a low rate of tumor recurrence.
Potential Impact on Breast Cancer Mortality: The high mortality rate of BBC patients results from the fact that these cancers do not respond to drugs that inhibit other classes of breast cancer, and the fact that these cancers invade extensively into surrounding tissues rendering surgical removal essentially impossible. Targeting the mechanisms responsible for the invasiveness of BBCs has the potential to produce a significant decrease in post-surgical cancer relapse and produce a corresponding increase in patient survival. The studies proposed here will determine whether it is possible to develop drugs to suppress BBC invasion into surrounding tissues, to suppress post-surgical recurrence, and to improve survival.