Research Grants Awarded
Mechanisms Underlying the Dual Action of Cellular Retinoic Acid Binding Protein II in Breast Tumor Suppression
Tumor Cell Biology II
Retinoic acid (RA) is a potent anticarcinogenic agent. It is currently used as a first line therapy in promyelocytic leukemia, and has yielded promising results in treatment of other types of cancers, including breast cancer. The pleiotropic activities of RA emanate from its ability to regulate transcription of multiple target genes, and are regulated by two classes of proteins: the ligand inducible transcription factors RA-receptors (RAR), and cellular retinoic acid binding proteins (CRABP-I and CRABP-II). CRABP-II functions to deliver RA from the cytosol directly to RAR in the nucleus, thereby facilitating the ligation of the receptor and augmenting its transcriptional activity. Consequently, expression of CRABP-II in certain breast cancer cells markedly enhances their sensitivity to RA-induced growth inhibition. Our recent studies indicated that, in addition to cooperating with RAR in mediating the anticarcinogenic activity of RA, CRABP-II also displays pro-apoptotic activities on its own. Specifically, we found that ectopic expression of CRABP-II in MCF-7 breast cancer cells, in the absence of RA, induces characteristic markers of apoptosis, including increased expression of Apaf1 and activation of caspase 7 and caspase 9. These observations suggest that CRABP-II establishes in these cells a “pro-apoptotic state” even in the absence of its ligand. CRABP-II thus appears to suppress tumor cell growth by a dual pathway, encompassing both RA-dependent and RA-independent mechanisms. Available information thus implies that CRABP-II will sensitize mammary carcinomas to growth inhibition triggered not only by RA but also by other apoptosis-inducing agents. Here, we propose to obtain insights into the mechanisms by which CRABP-II exerts its RA-independent activities, and to assess its potential as a target for novel strategies in breast cancer therapy. Using cultured mammary carcinoma cells as a model system, we will: (1) Investigate whether CRABP-II increases Apaf1 mRNA levels by regulating Apaf1 transcription or by modulating Apaf1 mRNA stability, and examine the mechanisms by which the protein exerts these effects. (2) Dissect between activities of CRABP-II through its RA-dependent vs. RA-independent functions, and evaluate its efficacy in sensitizing breast cancer cells to pro-apoptotic agents other than RA. Three mammary carcinoma cell lines displaying varying degrees of RA-responsiveness will be used. Cells will be treated with two apoptosis-inducing agents, TRAIL and doxorubicin. The effect of CRABP-II on the dose responses and time courses for induction of apoptosis by these agents will be examined. (3) Assess possible synergism between the protein’s dual function in suppressing mammary carcinoma growth. To this end, the ability of CRABP-II to enable synergistic responses to a combination treatment using TRAIL or doxorubicin in conjunction with RA will be evaluated.
The vitamin A metabolite retinoic acid (RA) is currently used in treatment of promyelocytic leukemia and is being tested for therapy of several types of cancer, including breast cancer. However, RA therapy of cancer is confounded by toxicity at pharmacological doses, and by the development of resistance to this drug in tumors. Hence, understanding the mechanisms of action of RA and of the cellular machinery involved in mediating its biological activities is critical for optimizing its usage in cancer therapy and for identifying new therapeutic targets. The anticarcinogenic activities of RA emanate from its ability to regulate the expression of multiple genes. These activities are mediated by two proteins: the ligand-activated transcription factor known as RA receptor (RAR), and the small protein termed cellular RA-binding protein II (CRABP-II). Our recent studies revealed that these two proteins cooperate in regulating the ability of RA to turn genes ‘on’ and ‘off’. Specifically, we found that CRABP-II transports RA to the nucleus where it directly delivers it to RAR, thereby facilitating the activation of the receptor. Consequently, the presence of CRABP-II in a cell markedly enhances the efficiency of RA action. Indeed, CRABP-II was found to dramatically lower the effective dose of RA needed for inhibition of breast cancer cell growth, and to overcome the RA-resistance of breast tumors in a transgenic mouse model of cancer. Surprisingly, we found that, in addition to enhancing the therapeutic efficacy of RA, CRABP-II displays additional activities that are RA-independent. Hence, expression of this protein in mammary carcinoma cells, in the absence of RA, increases the levels of several proteins involved in initiation of programmed cell death (also called apoptosis). Apoptosis is a critical process responsible for removal of abnormal cells, and is often defective in cancer cells, resulting in their survival and growth. Indeed, an important chemotherapeutic strategy relies on usage of agents that trigger cancer cell apoptosis. The observations that CRABP-II is involved in the apoptotic program in breast cancer cells suggest that it may improve the efficacy of known apoptosis-inducing drugs. The dual activity of CRABP-II in increasing the anticarcinogenic activities of RA on one hand, and its potential for enhancing the therapeutic activities of pro-apoptotic drugs on the other, point at this protein as a promising novel target for several combination therapies. Here, we propose to elucidate the mechanisms by which CRABP-II establishes a “pro-apoptotic state” in breast cancer cells, and to directly investigate whether targeted expression of CRABP-II in mammary carcinoma cells increases the efficacy and decreases the toxicity of RA and pro-apoptotic drugs. Overall, these studies will evaluate the potential of CRABP-II as a focal point for new strategies in breast cancer therapy.