Research Grants Awarded
Thiamine starvation with thiaminase I for breast cancer therapy
Tumor Cell Biology III
Background: We have previously shown that the expression of the thiamine transporter THTR2 is decreased 7-fold in breast cancer (breast tumors compared to normal breast tissue), which may leave breast cancer cells vulnerable to acute thiamine starvation. To explore thiamine deprivation in breast cancer, we have adapted MDA231 breast cancer cells and human mammary epithelial cells (HMECs) to growth in defined medium, and have shown in preliminary experiments that MDA231 cells can adapt to acute, absolute thiamine starvation, but HMECs can not. Adaptation to thiamine starvation is related to changes in Akt and GSK3?, proteins involved in energy metabolism regulation in the breast cancer cells. In addition, we have developed a method of creating acute thiamine starvation that could be applied in vivo, using an enzyme that digests thiamine, the Bacillus thiaminolyticus thiaminase I enzyme, which we have produced and purified by recombinant expression of the gene. Hypothesis: We hypothesize that acute thiamine starvation with thiaminase I will selectively alter energy regulation in breast cancer cells relative to HMECs, and increase sensitivity of breast cancer cells to chemotherapy and radiation therapy. To determine whether the effect of thiaminase I on breast cancer cells is the direct result of thiamine starvation we will compare acute thiamine withdrawal with thiaminase I exposure. Specific Aims: 1) To improve production and purification of the recombinant Bacillus thiaminolyticus thiaminase I enzyme and determine its kinetic properties; 2) Determine the effect of thiaminase I exposure to thiamine starvation on thiamine-dependent enzymes and on proteins involved the regulation of cell energy metabolism in MDA231 breast cancer cells and HMEC; 3) Determine the effectiveness of combining purified recombinant thiaminase I-induced thiamine starvation with chemotherapy, radiation and metabolic toxins in breast cancer cell lines. Study design: For specific aim 1, the expression system for thiaminase I already constructed in our laboratory will be optimized. For specific aim 2, MDA231 breast cancer cells and HMECs already adapted to growth in defined medium and physiologic thiamine concentrations will be used to compare the biological effects of thiaminase I to absolute thiamine starvation. For specific aim 3, a panel of breast cancer cell lines (including MDA231 cells transfected with THTR2) grown in standard media will be exposed to chemotherapy and radiation in the presence of thiaminase I. Potential outcomes and benefits: Abnormalities of cellular energy regulation in cancer represent a potential vulnerability that could be therapeutically exploited. We have established a rationale for exploring a novel approach ? thiamine starvation- and a novel therapeutic agent- thiaminase I ? for breast cancer therapy. Therefore, the potential outcome and benefit is the development of a completely novel therapeutic approach for breast cancer.
Selective nutrient starvation is an important strategy in the treatment of certain cancers. For example, in the treatment of acute lymphoblastic leukemia, the down-regulation of the enzyme that synthesizes the amino acid asparagine in leukemia cells is exploited clinically by using the bacterial enzyme l-asparaginase, which digests asparagine. This proposal investigates the potential of creating acute thiamine starvation for the treatment of breast cancer. The vitamin thiamine is a critical factor in cellular pathways that turn sugar (glucose) into energy. Cancer cells consistently use glucose in an abnormal way (called the Warburg effect). This is the underlying principle behind PET scans that are currently being used to improve breast cancer imaging. We have previously shown that a protein that transports thiamine into cells, THTR2, is decreased 7-fold in breast cancer compared to normal tissue, a finding related to the Warburg effect. This down-regulation of thiamine uptake may make these tumor cells more sensitive to thiamine starvation. In preliminary studies, we have created models of thiamine starvation in a breast cancer cell line and in normal breast cells, and we have shown that the breast cancer cells react differently than normal breast cells to thiamine starvation. Also, we have cloned the bacterial gene for the enzyme thiaminase I, an enzyme that digests thiamine and which could create acute thiamine starvation in the clinic, and have purified this recombinant enzyme. In our model, we would create acute thiamine starvation for breast cancer treatment by administration of the bacterial enzyme thiaminase I. We hypothesize that the down-regulation of thiamine uptake in breast cancer may expose a metabolic vulnerability in these tumors that could be exploited by thiaminase I treatment. Our specific aims are: 1) To produce and purify adequate amounts of thiaminase I enzyme for cell culture experiments; 2) To compare the effect of thiaminase I exposure to thiamine starvation in breast cancer cells; and 3) To determine whether thiaminase I exposure enhances the effectiveness of chemotherapy in a representative panel of breast cancer cells. We hypothesize that acute thiamine starvation will provide a completely new avenue for the therapeutic exploitation of a nutritional vulnerability, altered glucose metabolism, of breast cancer cells. In acute lymphoblastic leukemia, the metabolic vulnerability of the cancer cell is the down-regulation of asparagine synthase, the essential nutrient is asparagine, and the therapeutic agent to exploit the vulnerability is the bacterial enzyme asparaginase, which digests asparagine. In this proposal for breast cancer, the metabolic vulnerability is the down-regulation of the thiamine transporter gene, the essential nutrient is thiamine, and the therapeutic agent to exploit the vulnerability is the bacterial enzyme thiaminase, which digests thiamine.