Research Grants Awarded
Radioiodide And Pyruvate, A Novel Combined Approach To Breast Cancer Treatment Based On Nis (Na+/I- Symporter) And Smct (Na+/Monocarboxylate Transporter)
The Na+/I- symporter (NIS)-mediated radioiodide treatment of thyroid cancer is the most effective targeted internal radiation anticancer treatment available. This therapy has been successfully used for over 60 years. The prospect of applying this treatment modality to other cancers is extremely attractive, particularly to breast cancer, the only extrathyroidal cancer that expresses functionally NIS molecules endogenously. Our goal is to develop a novel strategy for the destruction of breast cancer cells based on the use of radioiodide therapy combined with an increased intracellular pyruvate concentration. We hypothesize that radioiodide administration to animals bearing NIS- and Na+/monocarboxylate transporter (SMCT)-expressing mammary tumors will be more effective in destroying cancerous cells when pyruvate is also administered simultaneously. The rationale is based on the observations that a) targeting the unique tumor bioenergetics, i.e. increased glycolysis, mitochondrial dysfunction and hypoxia, is an effective approach in inhibiting tumor growth, and b) in vitro pyruvate administration to breast cancer cells via SMCT causes apoptosis through inhibition of HDACs by pyruvate. We hypothesize that increased intracellular concentration of pyruvate via SMCT will rescue mitochondrial dysfunction by replenishing mitochondrial pyruvate catabolism leading to induction of apoptosis. Thus, pyruvate-induction of apoptosis by these two mechanisms -HDAC inhibition and rescued mitochondria function- combined with the successful NIS-mediated radioiodide therapy may result in effective breast cancer treatment. To test this hypothesis, we propose the following aims:
1) To determine the incidence of SMCT expression in human breast cancer. 2a) To generate and characterize MCF7 cells permanently transfected with NIS, SMCT, or both transporters and assess their transport properties for I- and pyruvate, respectively. 2b) To investigate the effect of pyruvate on cell proliferation in SMCT-transfected MCF7 cells as well on the expression of apoptotic markers. 3) To test the efficacy of combined radioiodide and pyruvate treatment in experimental breast cancer in vivo. Four groups of nude mice bearing mammary xenograft tumors derived from MCF-7 cells will be generated. Xenographts will derive from MCF-7 cells permanently transfected with a) NIS only; b) SMCT only; c) both NIS and SMCT; and d) the control xenografts will be derived from intact MCF-7 cells, which, express neither NIS nor SMCT. We will analyze the effect of radioiodide or pyruvate treatment alone or of both therapies combined on tumor reduction in all animal groups.
1) Our preliminary results showed that the SMCT protein is expressed in human breast cancer samples, but not in noncancerous tissue from mammoplasties. We will obtain human breast cancer samples of various histological types and at different stages of evolution, in which we will ascertain SMCT expression by both immunoblot and immunohistochemistry. We expect to confirm SMCT expression human breast specimens but not in adjacent normal breast tissues. 2) MCF-7 cells will be permanently transfected with human NIS or human SMCT or both. NIS and SMCT protein expression will be confirmed by western blot analysis using high-affinity anti-NIS and anti-SMCT antibodies. FACS, cell-surface biotinylation and immunofluorescence analysis will be conducted to confirm the localization of NIS and SMCT at the plasma membrane, a prerequisite for I- and pyruvate transport respectively. NIS and SMCT transport properties will be studied by steady state uptake of their respective substrates and compared to nontransfected MCF-7 cells. Kinetic analyses of I- and pyruvate transport as a function of the external concentration of the respective substrates will be conducted to determine the Km and Vmax values. The effect of pyruvate treatment on cell proliferation in SMCT-transfected MCF-7 cells will be assessed as well as the expression of apoptotic-related genes. We expect to observe induction of apoptosis upon pyruvate treatment and consequently up-regulation of proapoptotic genes p53, Bax and Bak and down-regulation of antiapoptotic genes Bcl-2 and survivin in established MCF-7 cells expressing SMCT and both SMCT and NIS but not in nontransfected MCF-7 cells or in NIS-MCF-7 cells. 3) The efficacy of radioiodide or pyruvate treatment alone or of both therapies combined will be assessed on tumor growth/reduction in experimental breast cancer in vivo. Different groups of nude mice bearing mammary xenograft tumors derived from MCF-7 cells expressing NIS and/or SMCT will be generated. Animals will received multiple doses of 131I and treated with pyruvate-supplemented drinking water. Animals will be imaged by MRI and with 99mTcO4- to visualize and estimate tumor size and confirm functional NIS expression. We expect to observe a synergistic effect of SMCT-mediated apoptosis and NIS-mediated radioiodide accumulation on tumor regression.
Proof of the effectiveness of the proposed combined therapy would significantly contribute to developing novel strategies for managing breast cancer with possible high-impact clinical applications. Like NIS, SMCT is apparently expressed in human breast cancer but not in normal breast tissue. The ease of delivery of pyruvate and its potential use as a therapeutic tool for induction of apoptosis may represent a nontoxic approach to reversing breast tumor growth in vivo. Therefore, the proposed approach based on the use of radioiodide therapy combined with pyruvate-mediated apoptosis induction may lead to a significant advance in breast cancer treatment.
The goal of this study is to test the hypothesis that our proposed combination therapy of radioiodide and pyruvate in experimental breast cancer will prove more effective than either treatment alone. To explain the proposal, we will first provide some background. The thyroid gland produces thyroid hormones that are crucial for the early physical and mental development of the fetus and the newborn and for overall metabolism in all stages of life. The nutrient iodide, normally supplied in the diet, is an essential constituent of the thyroid hormones. Much of the ingested iodide is taken up from the blood by the thyroid via a specialized transport protein present in the gland, the Na+/I- symporter (NIS). Radioiodide (i.e., a radioactive isotope of iodide) has been used for over 60 years to successfully treat thyroid cancer, a therapy made possible by the presence of NIS in thyroid cancer cells. As NIS transports "cold" (i.e., nonradioactive) iodide and radioiodide equally, NIS specifically translocates administered radioiodide into thyroid cancer cells, thus destroying them with minimal side effects to other cells and tissues. Notwithstanding how long the NIS-targeted radioiodide treatment of thyroid cancer has been in use, NIS itself remained elusive for decades. Our group was the first to finally isolate and identify (i.e., ?clone?) NIS in 1996, and we have maintained a leading position in NIS research ever since. Radioiodide therapy targeted via NIS is such an effective treatment for thyroid cancer that it has long been considered desirable to develop ways to apply it to extrathyroidal cancers. However, for a long time, no other cancers were identified to have NIS to make radioiodide therapy a feasible option. Our group showed that, outside the thyroid, NIS is also present in the healthy lactating (but not the nonlactating) breast, where NIS transports iodide from the blood to the milk and thus supplies it to the lactating newborn. Significantly, our laboratory was then the first to report that NIS is present in breast cancer cells, both in the main tumors and their metastases, raising for the first time the possibility of using radioiodide to treat breast cancer, a disease that affects 10 times as many patients as thyroid cancer.
Another transport protein of considerable medical relevance is the Na+/monocarboxylate transporter (SMCT), a molecule present in a wide variety of cells and tissues, including the thyroid and colon, where SMCT transports molecules known as monocarboxylates, such as pyruvate and lactate. Most cancer cells exhibit altered metabolic pathways to generate energy, unlike healthy cells, cancer cells rely mainly on a pathway called anaerobic glycolysis, in which glucose is converted to pyruvate and then pyruvate is quickly converted to lactate. Thus, researchers have been investigating strategies to intervene in the metabolic properties of cancer cells as a way to selectively destroy them, causing minimal harm to other cells. It has recently been reported that when SMCT was experimentally introduced into human breast cancer cells in culture and the cells were treated with pyruvate, cell death occurred. This was because pyruvate at high intracellular levels causes cell death (also called "apoptosis"); high intracelullar levels of pyruvate were reached because SMCT transported the externally added pyruvate into the cells to an extent that overwhelmed the cells' ability to convert pyruvate to lactate. Most importantly, we have observed in preliminary studies that human breast cancer cells have SMCT. All the above considerations led to our hypothesis that, in breast cancers with both NIS and SMCT, a combination treatment of radioiodide and pyruvate will prove more effective than either agent alone. To test this hypothesis, we will: 1) determine what percentage of a large sample of breast cancer cases contain SMCT; 2a) generate in the laboratory breast cancer cells lines with NIS and/or SMCT and analyze their transport properties for iodide and pyruvate; 2b) assess the effect of pyruvate on cell proliferation and cell death in cells with SMCT; 3) test the efficacy of radioiodide and pyruvate alone or in combination in reducing tumor growth in an experimental breast cancer model.
The ease of delivery of pyruvate and its potential use as a therapeutic tool for induction of cell death may represent a nontoxic approach to reversing breast tumor growth in vivo. In addition, the ample experience available with radioiodide as an agent to treat thyroid cancer makes radioiodide an extremely attractive possible treatment modality in breast cancer. Hence, this project will uniquely advance our understanding of breast cancer by elucidating whether the presence of both NIS and SMCT in breast cancer cells provides an opportunity to develop an effective novel therapeutic modality that combines radioiodide and pyruvate. Such a combined strategy, if found to be effective, has the potential to lead to reductions in breast cancer incidence and mortality. The importance of this research to breast cancer patients lies in the possibility that it may result in a therapeutic regime that may be more effective than existing options while causing fewer side effects.