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Ceramide Metabolism As Prognostic Marker Of Chemotherapy Responsiveness And Metastatic Potential In Breast Cancer
BACKGROUND: Knowledge of metastatic potential and drug responsiveness in breast cancer would greatly enhance treatment. Prognostic and/or diagnostic markers to detect these conditions would allow for use of alternate drugs and drug resistance modulators, and spare patients needless exposure to toxic anticancer agents that bring debilitating side effects. This application is a pilot project incorporating basic, translational, and clinical research to determine the feasibility of using a lipid metabolism panel as a marker of chemotherapy responsiveness and metastatic potential in breast cancer. Recent discoveries in lipid metabolism, chemotherapy, and apoptosis have brought us to this point.
OBJECTIVE/HYPOTHESIS: The objective of this research proposal is to assess expression levels of the genes and enzymes of ceramide metabolism to determine whether obtaining a “ceramide panel” would be of utility as a marker of responsiveness to chemotherapy and an indicator of metastatic potential in breast cancer. The hypothesis is that enzymes responsible for clearance of ceramide will be upregulated and/or overexpressed in human breast cancer cells and tumors that are poorly responsive to natural product chemotherapy and in cells and tumors with high metastatic potential (aggressive disease).
SPECIFIC AIMS: (1). Characterize ceramide metabolism in normal mammary epithelial cells, in wild-type and in drug-resistant human breast cancer cell lines (Adriamycin-, etoposide-, cisplatin-, tamoxifen-, Taxol- resistant) and in breast cancer cell lines established from the primary site tumor and from metastases. From this we can determine whether modified ceramide metabolism is associated with resistance to chemotherapy and predictive of metastatic potential. (2). Assess expression levels of the genes/enzymes of ceramide metabolism and evaluate chemotherapy responsiveness in tumor specimens from patients. The following will be used: i) primary tumor sample obtained at surgery, ii) biopsy sample before neoadjuvant chemotherapy (with an Adriamycin-containing regimen) and tumor specimen at time of definitive surgery. These pilot clinical studies will determine the feasibility of using ceramide metabolism as a diagnostic and/or prognostic marker of chemotherapy responsiveness and metastatic potential, and test whether neoadjuvant administration of Adriamycin influences expression of ceramide metabolism genes in breast tumors. (3). Determine whether anthracyclines and taxanes modify the expression of ceramide metabolizing genes/enzymes, using a breast cancer cell line as a model.
STUDY DESIGN: Characterizing the breast cancer cell lines will provide data on applicability of the ceramide panel as a marker of chemotherapy responsiveness and metastatic potential. Ceramide metabolism will be characterized by gene expression (gene array; RT-PCR), protein (Western blot), in vitro enzyme incubations, and metabolic activity measured in intact cells. For tumor specimens from patients (Stage I-IV), a ceramide panel will be obtained by gene array, and drug responsiveness will be evaluated using an in vitro soft agar drug resistance assay (testing Adriamycin, Taxol, 4-HPR). To determine whether chemotherapy influences gene expression, MCF-7 cells, transiently transfected with the full length sequence of the GCS promoter (with luciferase reporter) will be exposed to anthracyclines and taxanes and GCS promoter activity measured. Gene array profiles will be used to determine the influence of neoadjuvant chemotherapy (with Adriamycin) on expression of the genes regulating ceramide metabolism. Profiles before and after neoadjuvant therapy will be compared.
POTENTIAL OUTCOMES AND BENEFITS OF THE RESEARCH: Having patients fail chemotherapy is not an acceptable mode of determining drug responsiveness. This pilot study will set the stage for future, indepth research on the utility of lipid markers for predicting drug responsiveness (drug resistance) and metastatic potential in breast cancer. The potential outcome is to benefit the survivor by providing diagnostic and prognostic indicators allowing for enhanced treatment.
Chemotherapy resistance and its diagnosis in breast cancer remains a major clinical problem, and patients and oncologists would welcome its possible correction. Unfortunately, having patients fail chemotherapy is not an acceptable mode of diagnosing drug resistance. Knowing more about the tumor at diagnosis would greatly enhance treatment options/outcomes and spare patients needless exposure to toxic anticancer agents that bring debilitating side-effects. Markers to detect chemotherapy resistance early-on and to gauge metastatic potential would enhance survival. To this end, this project, which incorporates basic, translational, and clinical research, will evaluate the feasibility of using a lipid metabolism panel as an indicator of chemotherapy responsiveness and metastatic potential in breast cancer. Recent discoveries on the metabolism of ceramide (a lipid) and in chemotherapy mechanisms of action have brought us to this important juncture in cancer biology.
The objective of this proposal is to measure expression levels of the genes that regulate ceramide metabolism (about 9 genes) and determine whether expression levels correlate with breast tumor behavior as regards responsiveness to chemotherapy, Stage of disease, and metastatic potential. The hypothesis is that the genes (and enzymes) responsible for ceramide clearance will be overexpressed and the levels positively correlated with increases in chemoresistance, metastatic potential, and Stage. New findings have shown that Adriamycin efficacy is linked to the formation of ceramide in breast cancer cells and that rapid metabolic removal of ceramide renders cells insensitive to treatment. The research in this proposal will show whether “ceramide metabolism gene panel” will be of utility in predicting chemotherapy responsiveness.
Gene arrays will be used to quantitate expression levels of the ceramide-related genes in a series of human breast cancer cell lines (chemotherapy-sensitive, chemotherapy-insensitive), in cell lines derived from primary and metastatic tumors, and in tumor samples (Stage I-IV) obtained at surgery. Tumor samples will also be tested for chemotherapy sensitivity using in vitro drug resistance assays. The gene arrays will provide a fingerprint that will show whether high expression levels of the ceramide-related genes correlate with drug resistance and tumor aggressiveness. Clinical studies will be conducted (before and after neoadjuvant chemotherapy with Adriamycin) to determine if Adriamycin enhances the expression of these genes, and a laboratory study will be carried out with cultured human breast cancer cells (exposed to several types of anthracyclines, including Adriamycin), to determine if chemotherapy alters the expression of genes that regulate ceramide clearance.
This multicomponent pilot study combines basic and translational research in gene expression with clinical research to give a complete picture as to the feasibility of exploring ceramide gene expression panels to predict chemotherapy responsiveness and metastatic potential in patients with breast cancer. The study is intended to set the stage for future indepth research on the use of lipid markers in breast cancer. The outcome will benefit breast cancer survivors by providing diagnostic and prognostic markers that provide for enhanced treatment and care.