A major obstacle to achieving long-term cancer remission is the ability of some cancer cells to resist therapy. Strikingly, the fundamental cell biology underlying therapy resistance in human tumors is remarkably similar to that which confers incredible regenerative capacity in many planarian species. In these flatworm species, even a small piece of excised tissue can recreate an entirely new animal. This remarkable ability relies on the maintenance of a heterogeneous pool of planarian stem cells that bear many similarities to cancer stem cells. For example, a subset of these cells can tolerate high doses of γ-radiation and restore the entire stem cell population. Yet little is known about the mechanisms that confer this tolerance and almost nothing is known about their resistance to chemotherapies. Preliminary research revealed that planarians show resistance to the drug cisplatin. Treatment of planarians with 80-100μM cisplatin induced a phenotype that broadly mimicked the development of chemoresistance in human cancers: animals exhibited an initial loss of tissue followed by regeneration. Based on these data and known effects of cisplatin, the central hypothesis is that cisplatin differentially affects mitochondrial function across the stem and progenitor populations, which differentially alters a conserved chromatin signature that marks tumor suppressor genes i.e., broad H3 lysine 4 trimethylation (H3K4me3), increases cellular heterogeneity, and promotes chemoresistance. The goal of this study is to leverage the remarkable functional similarities between human tumors and the planarian stem cell population to uncover fundamental mechanisms that link the development of chemoresistance to metabolic and epigenetic regulation in vivo through the following specific aims: 1) To identify cisplatin-responsive differences in mitochondrial function in stem and progenitor cell populations using mitochondrial probes, high resolution imaging, and biochemical assays. This study uses state-of the-art approaches to determine the metabolic heterogeneity of stem cells in vivo. 2) To establish mechanistic links between metabolism, chromatin state, and chemoresistance by perturbing metabolite availability and measuring the impact on H3K4me3, transcriptional heterogeneity, and chemoresistance. This study will reveal critical links between metabolic and epigenetic regulation in chemoresistance. 3) To assess the conservation of metabolic H3K4me3 regulatory mechanisms in lung cancer using established airway organoid models. This study will test the hypothesis that resistant lung cancer cells respond to cisplatin-induced ROS by reducing H3K4me3. The proposed research will uncover novel mechanisms and target genes underlying chemoresistance.
Most deaths from head and neck squamous cell cancer (HNSCC) result from tumor recurrence following radiotherapy (RT). More than 75% of HNSCC patients receive RT as part of their care and over 50% of them are at risk for developing recurrence post RT. Radio-resistance (RR) leads to poor prognosis in HNSCC patients. The failure of RT has been attributed to hypoxia. However, new studies found that RT-induced re-oxygenation rates alone cannot distinguish primary from recurring HNSCC tumors, as some recurrent tumors also showed re-oxygenation after RT. Overexpression of RT-induced HIF-1α has been shown to be associated with an increased risk of failure of RT. Hypoxia-Inducible Factor-1 (HIF-1) is known to regulate many growth factors to promote aerobic glycolysis and angiogenesis. We hypothesize that RT-induced HIF-1 expression and subsequent alterations in metabolism/vasculature underlie HNSCC RR. Unraveling metabolic traits of cells that evade RT and recur, and the role of the supporting vasculature, is critical to developing strategies to prevent HNSCC recurrence and improve patient survival. However, there are surprisingly few techniques available to provide a systems-level view of these hallmarks together in vivo. To fill these gaps, I will build a portable multi-parametric microscope to measure the major axes of metabolism and vasculature in small animal models in vivo. I will then use these platforms to study the effect of radiation on HNSCC tumors and test our hypothesis on HNSCC RR development. This technology fills an important gap between in vitro studies on cells and whole body imaging, and is complementary to metabolomics and immunohistochemistry (IHC). I envision that this system will be well suited to study tumor RR and recurrence in patient-derived xenograft (PDX) and organoid models, which can faithfully recapitulate many micro-environmental features of patient tumors. Successful completion of the proposed studies will set the foundation for translating the optical technology to image patient derived tumor lines in PDX models, allowing us identify predictors of recurrence and develop improved radiotherapeutics (HIF-1 inhibitors) for HNSCC.
Radiation therapy is widely used to treat localized prostate tumors. However, cancer cells often develop resistance to radiation through unknown mechanisms and pose an intractable challenge. Radiation resistance is highly unpredictable, rendering the treatment less effective in many patients and frequently resulting in cancer recurrence. There is a dire need to uncover the molecular events that cause cells to become resistant in order to improve radiation therapy. In our in-depth investigations of radiation-resistant prostate cancer (RR-PCa), we found that mitochondrial heat shock protein 90 (mtHsp90) level and mitochondrial metabolism were aberrantly high when compared to radiosensitive PCa. mtHsp90 is a chaperone that maintains the stability of many diverse proteins, including those that are necessary for tumor survival and metabolism. We further demonstrated that decreasing mtHsp90 protein level significantly restored the sensitivity of RR-PCa cells to radiation. Hence, our overarching hypothesis is that mtHsp90 defines resistance of prostate cancer cells to radiation, a premise that will be put under stringent testing in this proposal. Reactive oxygen species (ROS) are known to reduce the level of mtHsp90 by
with its transcriptional and post-translational levels. We screened 768 FDA-approved drugs in search of a potent drug that could raise the level of ROS, but not be toxic to normal cells. We found Azithromycin (AZM), a macrolide antibiotic, to be the most effective drug that selectively increases mitochondrial ROS and reduces mtHsp90 protein level. We further demonstrated that AZM enhances the death of cancer cells with radiation treatment. Encouraged by robust results, we aim to advance our findings in this project, test our hypotheses, and develop a paradigm for adjuvant treatment that will ultimately
radiation therapy as a more effective procedure. The goals are: Aim 1, to determine the functional importance of mtHsp90 in RR-PCa cell survival and adaptive metabolisms, Aim 2, to determine mechanistically how ROS down-regulates mtHsp90 protein level and sensitizes RR-PCa, and Aim 3, to validate in preclinical models if AZM-generated ROS down-regulates mtHsp90 and enhances radiation treatment. The results will establish a novel link between mtHsp90 and RR-PCa. This study using state-of-the art metabolomics, imaging techniques, and model systems and has the potential to be
into a clinical practice because AZM already has a good safety record. In the era of precision medicine, we are confident of the prospects of our closely-focused studies, which will push boundaries and make radiation therapy a better procedure, and our approach will set a precedent for many cancer treatments where radiation therapy is preferred.