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Genes Providing Resistance to Tamoxifen: Development and Validation of Clinical Biomarkers
To find genes involved in resistance to selective estrogen receptor modulator (SERM) Tamoxifen (TAM) we delivered a full-length cDNA expression library into a population of MCF-7 cells and selected cells that survived TAM treatment. Several delivered cDNAs were recovered from surviving clones and individually reinserted into naïve populations of MCF-7; these populations became resistant to cytotoxic concentrations of TAM. The nature of recovered cDNAs suggests that they reduce sensitivity to TAM by modulation of intracellular levels of reactive oxygen species (ROS), stabilization of mitochondrial transmembrane potential, and modulation of proteasome function. This hypothesis was confirmed when mitochondrial potential were measured in parental and resistant cells after TAM treatment. Since many chemotherapeutic drugs induce similar processes in breast cancer cells, the identified genes can induce resistance against other drugs as well. One of identified genes has been overexpressed in cells resistant to melphalan, while its product has oxidoreductase activity and is elevated in sera of some breast cancer patients. We hypothesize that (1) discovered genes can induce resistance to other therapeutic agents besides TAM and (2) elevated level of corresponding proteins in patients' sera correlates with resistance to TAM and other drugs. We propose to characterize protective effects of discovered cDNAs against other therapeutic agents and in cells with different genetic background, and to test whether serum concentration of proteins produced by identified genes can predict tumor response to TAM and/or other drugs. Design and methodology: we will test sensitivity of cellular clones containing individual cDNA inserts against a number of chemotherapeutic drugs used for treatment of breast cancer, and will evaluate whether response depends on estrogen receptor and p53 status, production of ROS, Ca2+ release, inhibition of proteasome and changes of mitochondria. We will determine concentrations of proteins produced by identified genes in sera of patients resistant and responsive to TAM. Using these data we will build a biomarker assay and will test blinded sera samples to determine if the assay can predict patients' response to TAM and/or other therapy. Data obtained in Aim 1 will expand our knowledge on the mechanism of protection afforded by identified genes, indicate potential cross-resistance with other drugs, and identify potential drug targets. Data obtained in Aim 2 will define and validate a clinically relevant predictive biomarker of drug response.
We have identified four genes that make human breast cancer cells resistant to selective estrogen receptor modulator Tamoxifen (TAM). Literature search revealed that one of the genes is found in cells resistant to another chemotherapeutic agent, melphalan, while high concentration of the protein encoded by this gene is detected in the blood of breast cancer patients. Protein product of one of the identified genes is involved in regulation of reactive oxygen spieces (ROS), which might explain its protective activity against TAM. While the data on other proteins is limited, all of them might be implicated in drug resistance. Our preliminary data indicate that all identified genes can be acting in a similar or even identical way by reducing ROS effects to make cells resistant to TAM. Since many chemotherapeutic drugs cause production of ROS and may thus kill tumor cells through this mechanism, we hypothesize that the same genes that cause resistance to TAM can induce resistance to other drugs as well. To test this hypothesis we will determine whether cells expressing these genes are as sensitive to other chemotherapeutic drugs as parental cells. Next we will determine whether protective effects of these genes are different in tumors with different genetic background: we will test if the level of resistance depends on expression of estrogen receptor and/or p53. Since at least one of these genes is secreted into the bloodstream we will develop immunoassay to test the level of corresponding proteins in patients’ sera; in this case we expect to see different levels of at least one of these proteins depending on the response to TAM and/or other types of chemotherapy. As a result of this project we expect to gain better understanding of mechanisms of resistance to chemotherapy in tumors with different genetic background, and to evaluate potential use of discovered genes as targets for rational drug design. Importantly, we will also determine if the levels of identified proteins in sera of breast cancer patients can predict their response to therapy with TAM and other drugs.