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Disruption of Breast Cancer Chemoresistance by the Translational Antagonist 4E-BP1: A Bitransgenic Mouse Model
DISRUPTION OF BREAST CANCER CHEMORESISTANCE BY THE
TRANSLATIONAL ANTAGONIST 4E-BP1: A BITRANSGENIC MOUSE MODEL
(a) Background: The major challenge for using anticancer therapeutics is to sensitize tumor cells to non-toxic doses of cytostatic agents by disrupting acquired resistance of cancer cells to apoptosis. One striking differencebetween normal and neoplastic cells is the functional activity of the cap-dependent translational complex eIF4F which is constitutively elevated in a broad spectrum of naturally occurring tumors, including breast cancers. In prior studies, we have demonstrated that aberrant activation of eIF4F suppresses drug-induced apoptosis and synergizes in a Ras-like manner with pre-neoplastic alterations in promoting malignant transformation. Consistent with this, the eIF4F antagonist, translational repressor 4E-BP1, displays attributes of a tumor suppressor. Specifically, we have discovered that 4E-BP1 protein is functionally inactivated in aggressive breast carcinomas by its hyperphosphorylation. When overexpressed, 4E-BP1 inhibits tumorigenicity of human breast cancer cells in immunodeficient mice and sensitizes transformed cells to drug-induced apoptosis in a manner dependent on its phosphorylation status. The results of our xenograft and in vitro experiments give us a strong rationale for applying a similar strategy for testing the anti-tumor potency of 4E-BP1 in the physiological context of a whole mammalian organism by using an adequate transgenic mouse model.
(b) Objective/Hypothesis: We hypothesize that disruption of oncogenic activation of eIF4F in mouse mammary glands by ectopic expression of 4E-BP1 can reduce the risk of breast tumorigenesis and sensitize breast cancer cells to safe doses of cytostatic agents. Our objective is to develop a murine model of translationally regulated breast cancer and apply this model for monitoring effects of ectopic 4E-BP1 and its highly active A70 mutant form on breast cancer incidence and tumor cell chemoresistance.
(c) Specific Aims:
Aim 1. Develop a mouse bitransgenic model of translational regulation of breast cancer in which the inducible promoter of the whey acidic protein (WAP) gene will target expression of wild type 4E-BP1 or its highly active A70 mutant to the mammary epithelial cells of transgenic mice that exhibit a high incidence of breast cancer owing to oncogenic alterations in the HER-2/Ras-regulated eIF4F signaling pathway.
Aim 2. Utilize the developed transgenic murine model of breast cancer to examine whether induced overexpression of 4E-BP1 in mammary epithelial cells decreases breast tumorigenesis and/or sensitizes cancer cells to well-tolerated doses of doxorubicin and paclitaxel.
(d) Study Design: To direct 4E-BP1 expression to mammary epithelial cells, GFP- and HA double tagged wild type 4E-BP1 or its most active form, the A70 mutant, will be placed downstream of the mammary specific WAP gene promoter which becomes active in mid-to-late pregnancy. We will use the resulting WAP-4E-BP1wt, WAP-4E-BP1A70 or WAP-GFP constructs to create F1 progeny of FVB/WAP-4E-BP1 transgenic mice. One strategy used by cancer cells for avoiding the anti-translational activity of 4E-BPs is by hyperphosphorylation through a plethora of Ras-dependent signal transduction pathways. We, therefore, will interbreed the resulting WAP/4E-BP1 animals with available FVB/MMTV-Ha-ras and/or FVB/MMT-v-erb/HER-2 mice to generate MMTV-ras or –erb/HER-2 mammary tumors that differ primary in their eIF4F functional status. To induce ectopic 4E-BP1, mono- and bitransgenic WAP/4E-BP1mice will be mated before or after breast tumor onset and continuous pregnancy will be maintained during ras- or erb/HER-2-driven tumorigenesis by continuous housing with male mice. At different time points after beginning of 4E-BP1 induction, we will evaluate effects of ectopic 4E-BP1 alone or in combination with conventional cytostatic therapy on histopathological alterations in mammary glands, frequencies of tumor incidence and dynamics of tumor sizes.
(e) Potential Outcomes and Benefits of Research: By successful completion of this project, we hope to validate the translational repressor 4E-BP1 gene as an element that contributes to reducing risk of breast cancer incidence and decreasing tumor drug resistance. The results will also provide theoretical and methodological support for in vivo preclinical testing of compounds that will be designed to mimic effect of 4E-BP1 in suppressing growth of breast cancer cells.
DISRUPTION OF BREAST CANCER CHEMORESISTANCE BY THE TRANSLATIONAL ANTAGONIST 4E-BP1: A BITRANSGENIC MOUSE MODEL
Breast cancer is a major public health problem, killing more than 40,000 women in the United States each year. While considerable therapeutic progress has been made, long-term survival is limited by toxicity of anti-cancer drugs toward normal tissues and the need to use very high doses of drugs to kill breast cancer cells. One solution to this problem is to use higher doses of chemotherapy and rescue the immune system with bone marrow transplantation. An alternative approach which we propose here is to take advantage of some recent discoveries in our laboratory that permit us to sensitize breast cancer cells to gentler forms and lower doses of chemotherapy.
Breast cancer cells differ from their normal counterparts in many ways. One important difference is abnormal activation of the protein synthesis machinery to support the high requirements of tumor cells for increased synthesis of proteins that are responsible for their growth and viability. In mammalian cells, almost all proteins are synthesized through a mechanism that requires a specific modification of genetic messengers (mRNAs) with a structure called a cap. Environmental and intracellular cues tightly control rates of cap-dependent protein synthesis in normal cells. In contrast, cancer cells commonly display permanently increased activity of the complex eIF4F, which is responsible for initiation of cap-dependent protein synthesis. Moreover, available data suggest that enforced activation of eIF4F in non-malignant cells cooperates with other oncogenic alterations in promoting neoplastic transformation. Correspondingly, targeted disruption of the aberrantly activated eIF4F in cancer cells by expression of its naturally occurring antagonist 4E-BP1 decreases viability of cancer cells and sensitizes cells to anti-cancer drugs. Most importantly from a therapeutic point of view, while overexpressed 4E-BP1 kills cancer cells, normal cells remain viable. Based on these findings, we hypothesized that the gene encoding 4E-BP1 function as a natural tumor suppressor element and that stably overexpressed 4E-BP1 can decrease risk of breast tumor incidence and prevent acquired resistance of tumor cells to anticancer drugs.
The mentioned experimental data were obtained by using cells that were cultivated outside of living organisms in an artificial environment (in vitro). The goal of this proposal is to apply the advanced mouse model of breast tumorigenesis for analysis the 4E-BP1 anti-cancer potency in the context of a whole mammalian organism. To test our hypothesis, we plan to genetically modify mice that are predisposed to a high incidence of breast cancer, to direct expression of a switchable form of 4E-BP1 and its hyperactive mutant version (‘super 4E-BP1’) specifically to their mammary glands. We plan to engineer the introduced 4E-BP1 to turn on its activity in mid-to-late pregnancy by mating. We expect that enforced activation of 4E-BP1 before onset of breast cancer will decrease frequency of tumor formation, while its high activity during breast tumorigenesis will help to kill cancer cells by safe doses of regular anti-cancer therapy.
By successful completion of this project, we hope to confirm in vivo the proof of principle that was earlier established in vitro: expression of 4E-BP1 is an important genetic factor for decreasing risk of breast cancer incidence and evading acquired resistance to chemotherapy. We also believe that our transgenic animal model for the translational control of breast tumorigenesis can be used for testing new anti-eIF4F pharmaceuticals designed for suppressing growth and chemoresistance in breast cancer cells.