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    Research Grants Awarded

    Constitutively Active Erythropoietin Receptor Variants As Novel Therapeutic Targets In Breast Cancer

    Grant Mechanism:
    Investigator Initiated Research

    Scientific Abstract:
    BACKGROUND: Anemia not only impairs the quality of life of patients, but it leads to tumor hypoxia, resistance to therapy and reduces survival. Human recombinant erythropoietin (rHuEpo) is extensively used to treat anemia in breast cancer patients and was shown to be effective in reducing transfusion requirements and improve quality of life. However, its effects on patient survival are controversial. Recent clinical trials suggested an adverse effect of rHuEpo on outcome, prompting the FDA to issue a ?black box warning? for Epo products. Various cancer cells, including breast cancers, were shown to express Epo and Epo receptor (EpoR). Several studies suggested that Epo may enhance cancer cell proliferation, survival and invasiveness, leading to the hypothesis that the adverse effects of Epo in cancer patients are attributable to stimulation of EpoR present on cancer cells by rHuEpo treatment. Others argue, based on in vitro and in vivo data showing lack of a proliferative effect of exogenous Epo in EpoR bearing cancer cells, that while EpoR is present in these cells is not biologically active. Our recent observations question both of these hypotheses: We have found that specific inhibition of EpoR expression by RNA interference in A2780 cancer cells resulted in markedly suppressed proliferation and invasiveness in vitro and in vivo, although these cells fail to respond to Epo. Our findings suggest that EpoR may be constitutively active in some cancer cells and provide the first evidence for a potential role of an Epo independent EpoR mediated pathway in tumor growth. We identified altered EpoR mRNA transcripts in ovarian and breast cancer cells lacking exon 1-3 sequences, strongly suggesting that EpoR variants lacking the ligand binding domain are expressed in these cells. HYPOTHESIS AND OBJECTIVES: Our hypothesis is that different EpoR variants are expressed in human breast cancer and the variant forms have modified functional characteristics. Our objective is to characterize the structure, function and signaling mechanisms of the altered EpoR variant forms, which will not only allow for the categorization of breast carcinomas according to their sensitivity to Epo and thus safe, individualized targeting of treatment of breast cancer patients by rHuEpo, but will also provide a novel therapeutic target for the specific inhibition of tumor growth without affecting hematopoiesis. SPECIFIC AIMS: 1) To identify variant EpoR forms expressed in human breast cancers and characterize their function and signaling mechanisms in vitro and in vivo. 2) To determine the significance of the expression of variant EpoR forms in human breast cancer by analyzing the correlation of their expression levels with clinicopathologic features and outcome in a retrospective cohort of archived breast cancers. 3) To determine the effect of specific inhibition of the expression of variant EpoR forms on the growth and sensitivity to therapeutic agents of human breast cancer cells in vitro and in vivo using RNA interference. STUDY DESIGN: 1) The SMART RACE PCR system will be used to specifically identify complete sequences of EpoR mRNA variants in 20 primary human breast cancers and corresponding benign breast tissue. Amplified fragments of EpoR variants will be sequenced, cloned into expression vectors and the variant EpoR forms will be expressed in benign mammary epithelial and breast cancer cells. The gene expression profile of the modified cells will be compared to those of controls by microarray analysis. Key components of the proposed signaling networks will be subjected to Western blot analysis, along with members of the conventional EpoR regulatory network (JAK2, STAT5, PI3K, Akt, MAPK, NF-?B) in control and EpoR variant transfected cells, with or without Epo treatement. The effect of the expression of variant EpoR forms on the proliferation, apoptotic sensitivity and invasiveness of the cells will be determined in vitro, and in vivo using a tumor xenograft system. 2) The expression levels of full length EpoR and EpoR variant forms will be measured by quantitative RT-PCR in microdissected samples of 250 cases of primary human breast carcinoma and correlated with clinicopathologic tumor features, established prognostic/predictive factors and patient outcome. 3) Short hairpin RNAs will be designed for the selective knock down of full length EpoR and variant EpoR forms in three breast cancer cell lines expressing high levels of the variant EpoR forms. The effect of EpoR variant form knock down on gene expression profile, signaling mechanisms, proliferation, apoptotic sensitivity and invasiveness of the cells in vitro will be assessed as described in Aim 1. The effect of the specific knock down of variant EpoR forms on the proliferation, apoptotic sensitivity and invasiveness of the cells will be determined in vivo using a tumor xenograft system. CLINICAL IMPACT AND INNOVATION: Identifying and understanding the functions of variant EpoR forms in breast cancers will enable us to identify patients can be safely treated with rHuEpo to manage anemia. Specific knock-down of variant EpoR forms necessary for tumor growth will provide a novel form of cancer therapy whereby we can specifically inhibit the growth of breast cancer cells without having an effect on erythropoiesis. Besides their clinical significance, by highlighting the importance of altered mRNA processing in breast cancer biology, our studies will also provide a basis for further studies into similar alterations in potentially hundreds of genes playing important roles in the development and progression of human breast cancer.

    Lay Abstract:
    Low levels of red blood cells, termed anemia, are frequently present in breast cancer patients and it is a side effect experienced by more than 60% of patients undergoing chemotherapy. Because it is the hemoglobin in the red blood cells that carries oxygen throughout the body, a shortage of these oxygen-rich cells can cause low levels of oxygen concentrations in the tissues and results in feelings of weakness, fatigue, dizziness, irritability, shortness of breath and chills. The effects of anemia, such as the lack of energy, affects the lives of cancer patients more than any other side effect of treatment, including nausea, pain and depression, and has a negative impact on their day-to-day activities. In addition to the impairment of quality of life, anemia and low oxygen concentrations also have a negative effect on our ability to fight cancer. In people with cancer, tumors can grow so rapidly that they outgrow their blood supply. Such tumors experience low oxygen levels, which can cause genetic changes that make the tumor cells more aggressive and resistant to chemo- and radiation therapy, technics commonly used today to treat breast cancer. In order to improve quality of life and increase the effect of therapy, doctors have tried various methods, such as blood transfusions, to increase the number of red blood cells in the blood and oxygen levels in the tissues. One of the best known adaptive responses of the human body is that provoked by low levels of oxygen concentration. Low oxygen levels cause increased production of the hormone erythropoietin (Epo) by the kidneys, which results in increased production of red blood cells and higher oxygen carrying capacity of the blood. Recently Epo manufactured by drug companies (Procrit?, Epogen?) became available and revolutionized the treatment of anemia in cancer patients. Although Epo treatment is very effective in improving the quality of life, its effects on the cancers themselves are not well understood. Several recent clinical trials examining the use of Epo in cancer patients, including breast cancer, reported that Epo may have an adverse effect on patient survival due to earlier recurrence of tumors. These recent results raised serious concerns and prompted the Food and Drug Administration (FDA) to issue a warning regarding the use of Epo for treatment of anemia in cancer patients. Although for a long time it was thought that Epo only stimulated the growth of red blood cells, it was recently shown that Epo can also rescue brain cells (neurons) and heart muscle cells from death caused by low oxygen concentrations, such as that occurs in stroke and heart attack. We have recently discovered that breast cancer cells in the culture dish also have the receptor for Epo (called EpoR). Subsequently many kinds of other cancers were found to have EpoR as well, and some studies found that cancer cells in the culture dish can be stimulated to grow by Epo. These findings lead to the suggestion that the adverse effects of Epo in cancer patients may be due to the stimulation of the cancer cells to grow by Epo given to patients to treat anemia. However, the situation appears to be more complicated. Other researchers found that many cancer cells are not stimulated to grow by Epo, even though they have the receptor (EpoR). These investigators argue that EpoR present in cancer cells are not active and are not necessary for the tumors to grow. Our recent findings in breast and ovarian cancer cells, however, question both of these hypotheses. We have recently found that inhibiting the production of EpoR in cancer cells in a specific manner results in a significant reduction in tumor growth, although Epo treatment did not stimulate the cells to grow. Based on our recent data we believe that breast cancer cells make altered forms of EpoR that are active and able to stimulate the cancers to grow even if Epo is not present. In the proposed research we will test this hypothesis in clinical samples of breast cancer in addition to breast cancer cell lines. We will identify the altered, active forms of EpoR and analyze the molecular mechanisms whereby they affect the growth of tumor cells. We will develop a test to identify these altered EpoR forms in archived clinical samples of human breast cancer and correlate their presence with the clinical and pathologic features of the tumors and patient outcome. Our studies can have a great impact on the prevention and treatment of human breast cancer and they will provide evidence for a novel way that production of altered EpoR can promote the growth of breast cancer. An immediate clinical impact of our studies is that they will provide a sensitive and specific method for identifying breast cancer patients whose tumors respond to Epo and may be stimulated by Epo to grow and separate them from those who can be safely treated with Epo to manage their cancer and therapy related anemia. In addition, the identification of altered EpoR forms in breast cancer cells will provide a new approach to block these proteins without affecting red blood cell production and lead to the development a novel, specific way to treat human breast cancers.