glioblastoma multiforme (GBM), an aggressive, incurable form of brain tumor, have a median

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1 1 A. Statement of Problem, Project Objectives, and Significance Introduction Brain tumors are among the deadliest types of cancer. Patients diagnosed with glioblastoma multiforme (GBM), an aggressive, incurable form of brain tumor, have a median survival of only fifteen months 1. Despite tremendous research efforts, few significant improvements in GBM treatment have been made over the last decade. However, recent advances in our understanding of the developmental pathways involved in cancer progression might uncover new treatment targets, providing fresh hope for brain tumor patients and their families. One emerging treatment target is the hedgehog signaling pathway. Normally active during embryonic development, the hedgehog pathway was recently shown to be active in a subpopulation of GBM cells known as brain tumor stem cells (BTSCs). Although BTSCs make up only a small proportion of the cells in a tumor and divide only occasionally, they are considered to be the tumor s driving force due to their importance in tumor formation, recurrence, and metastasis (the spread of the tumor to new locations). Recent studies have shown that chemically inhibiting the hedgehog signaling pathway with a compound called cyclopamine disables BTSCs, essentially deactivating the tumor. Furthermore, inhibiting BTSCs with cyclopamine enhances the anti-tumor effects of temozolomide (TMZ), the standard drug used to treat GBM patients. These findings have exciting implications for GBM treatment. However, cyclopamine is known to cause severe side effects, and therefore is not safe to use on brain tumor patients. Vitamin D is a naturally occurring substance that causes very few side effects, even when administered at high doses. With a mode of action similar to that of cyclopamine, vitamin D is

2 2 able to inhibit the hedgehog signaling pathway and disable BTSCs. Our lab has already begun to compare vitamin D s efficacy to that of cyclopamine. Currently unexplored, however, are vitamin D s interactions with temozolomide (TMZ). This study seeks to investigate vitamin D s compatibility with TMZ, to find out if, like cyclopamine, vitamin D will work synergistically with TMZ. Since TMZ affects the most rapidly dividing cells in the tumor and vitamin D targets BTSCs, which only divide occasionally, a combination treatment of TMZ and vitamin D is very promising. Such a treatment could very effectively limit tumor growth and potentially even prevent recurrence and/or metastasis. Should the proposed study demonstrate vitamin D s compatibility with TMZ, vitamin D could become a powerful, safe, and cost-effective addition to standard brain tumor treatment. Overall Research Questions: Are vitamin D and TMZ more effective when used together than either compound on its own? Will a combination of vitamin D and TMZ effectively reduce tumor proliferation? Hypothesis and Objectives: Combining vitamin D with TMZ will have a synergistic effect and will more cause a greater decrease in GBM cell proliferation than either compound on its own. Vitamin D Vitamin D + Temozolomide Temozolomide Inhibits BTSCs, decreases proliferation Kills tumor cells, decreases proliferation Inhibitory effect one drug inhibits the other No effect results are comparable to either treatment alone Combined effect the two drugs enhance one another Figure 1: Summary of hypotheses and potential outcomes. Dotted lines indicate possible results which have not been previously described in the literature.

3 3 In order to assess the compatibility and effectiveness of TMZ and vitamin D, a timecourse assay will be performed using the CellTiter-Glo reagent to quantify GBM cell line proliferation. The ability of TMZ or vitamin D alone to reduce proliferation will be compared to the combined effect of TMZ plus vitamin D. The combined treatment is expected to most effectively inhibit proliferation, unless some unknown mechanism causes vitamin D to interfere with TMZ activity, or vice versa (figure 1). B. Project Rationale and Literature Review Small subpopulations of cells have been identified within solid brain tumors that exhibit stem cell-like properties, including the ability to differentiate into many cell types and the capacity for self-renewal 2. These cells, known as brain tumor stem cells (BTSCs), are largely responsible for tumorigenesis, or the formation of tumors. While traditional cancer treatment methods like radiation and chemotherapy often target the most rapidly dividing cells in a tumor, BTSCs avoid destruction because of their limited replication and enhanced ability to repair DNA damage 3. These properties make BTSCs very difficult to kill. Designing cancer therapies that effectively target BTSCs could theoretically impede a tumor s ability to recur or metastasize. The question is, how do we find brain tumor stem cells, and how do we stop them? The hedgehog signaling pathway might help answer these questions. Hedgehog signaling acts in normal stem cells to regulate embryonic development. The pathway is activated when the hedgehog signaling molecule binds to a cell surface receptor called Patched, which causes the activation of another protein called Smoothened. When Smoothened is active, it initiates a cascade of signals, resulting in the activation of several members of the Gli family of proteins (figure 2). Gli proteins regulate the expression of genes involved in cell growth and proliferation 4.

4 4 The hedgehog pathway, which is normally only expressed during embryonic development, has been found to be active in multiple cancers, including leukemia, breast cancer, prostate cancer, basal cell carcinoma, medulloblastoma, gliomas, and lung cancer 5-8. Furthermore, it is suspected that hedgehog signaling is important in the establishment and maintenance of cancer stem cell populations. If this is the case, then inhibition of the hedgehog signaling pathway could potentially disable BTSC activity in glioblastoma multiforme. Vitamin D is a safe, naturally occurring substance that inhibits the hedgehog signaling pathway. Vitamin D binds directly to Smoothened, preventing the protein from sending the signals that would lead to cell growth and proliferation. Since BTSCs have been shown to rely on the hedgehog pathway to function, treatment with vitamin D will repress the hedgehog pathway and disable the BTSCs within the tumor 9,10. Importantly, vitamin D naturally circulates throughout the body and crosses the blood-brain-barrier, which is a network of capillaries that prevents most drugs from accessing the brain. Since vitamin D is involved in calcium regulation, one potential side effect of high-dose vitamin D treatment would be hypercalcemia. However, once vitamin D s therapeutic efficacy has been determined, possible ways to reduce the risk of hypercalcemia can be explored, including individual dose determination, dietary restriction, and treatment screening based on preexisting risk factors. Currently, temozolomide (TMZ) is the clinically accepted standard of care for glioblastoma patients. TMZ acts by inducing DNA damage, which triggers the cell s natural death mechanisms. TMZ and vitamin D make an attractive treatment combination: TMZ affects the rapidly dividing cells in a tumor, while hedgehog signaling inhibitors affect BTSCs, which have limited replication and are resistant to chemotherapy. Furthermore, Clement and colleagues recently described a synergistic effect of TMZ plus another hedgehog inhibitor called

5 5 cyclopamine, demonstrating reduced proliferation and increased cell death in both glioma stem cell cultures and U87 MG glioblastoma cells 8. Interestingly, however, interactions between vitamin D and temozolomide have not been previously described. The proposed study seeks to better understand the interactions between vitamin D and TMZ by observing their combined effects on GBM proliferation. C. Project Plan Cell Culture: U87 MG GBM cells will be cultured in DMEM media supplemented with 10% FBS. Alternate GBM cell lines such as LN229 or U138 can be substituted or added to the study if necessary. All cultures will be grown in standard conditions (37 C with 5% CO 2 ). Proliferation assays: Proliferation assays will determine how U87 MG proliferation is affected by vitamin D, TMZ, or a combination of the two. The CellTiter-Glo assay (Promega) will be used to estimate cell viability. The assay works by disrupting the GBM cell membranes and generating a luminescent signal proportionate to the amount of ATP present within the cell. The amount of luminescent ATP (detected by a luminometer) is directly proportionate to the amount of cells present in the sample, so this is a good way to track whether cells numbers are increasing, decreasing, or stabilizing over time. Three treatment groups will be established: cells treated with vitamin D at 100 nm concentrations, cells treated with TMZ at 400 µm concentrations, and cells treated with both 100 nm vitamin D and 400 µm TMZ. Appropriate ethanol controls will be established for each group (both vitamin D and TMZ will be dissolved in ethanol; control treatments will contain ethanol with no dissolved agents). An additional control population will receive no treatment at all. Luminescence will be assessed at 0, 6, 12, 24, 48, 60, and 72 hour timepoints, and growth curves will be constructed. It is expected that cells treated with vitamin D, cyclopamine, or temozolomide will have reduced proliferation compared to the

6 6 appropriate control populations, and that combined treatments will have even more significantly reduced proliferation. Each treatment will be performed in triplicate (at least), and resulting growth curves from the average of each triplicate will be analyzed for significance using a paired t-test. Timetable of Expected Outcomes May 1 st May 15 th Order supplies, prepare cell cultures May 16 th May 29 th Perform time-course assays May 30 th June 5 th Analyze results June 6 th June 24 th Prepare poster presentation and research paper (Poster presentation will be submitted for acceptance at a conference. In addition, this work constitutes a large part of my master s thesis project, which will be submitted for publication upon completion.) D. Statement of Resource Availability *Laboratory space, equipment, general lab supplies, and any materials in excess of $1500 will be secured through Dr. Winn with the support of the Upper Michigan Brain Tumor Center. Budget and Justification Supplies Product number Source Cost DMEM Media Q Lonza $23.70 necessary for GBM cell culture Fetal Bovine Serum A PAA $ necessary for GBM cell culture Vitamin D D UG Sigma $ Therapy in question Temozolomide T MG Sigma $ Standard therapy Cell-Titer Glo G7570 Promega $92.00 To measure GBM proliferation Other Expenses Cost Housing $ $20.75 per night for the six-week data collection period (estimated from 2010 on-campus apartment summer rates) Meals $ $25.00 per week for the six-week data collection period TOTAL $ (*see part D.)