Circulating tumor cell-neutrophil clusters in breast cancer metastasis
Xia Liu, PhD

Basic Science Project Mentors: Andrew Lane, PhD and Yadi Wu, PhD
Clinical Project Mentor: Gerhard Hildebrandt, MD
External Mentor: Danny N. Dhanasekaran, PhD

More than 90% of cancer-related death is caused by spreading of tumor cells from the primary sites to distant organs (metastasis). Triple-negative breast cancers (TNBCs) are more aggressive and metastatic than other types of breast cancers.  To metastasize, tumor cells have to enter into the blood vessels (circulation). It has been recognized that these circulating tumor cells (CTCs) contain a rare subset of metastasis-initiating cells with the ability to generate metastatic tumors. However, the properties of these metastasis-initiating CTCs are largely unknown. Both my preliminary data and other studies have shown that neutrophils can interact with CTCs (CTC-neutrophil clusters) to facilitate metastasis, but little is known about how they interact. By single-cell RNA sequencing, I found that ICAM-1 was highly expressed in lung metastatic cells compared to the primary tumor cells in TNBCs. In addition, knockdown of ICAM-1 inhibited self-renewal ability of tumor cells in vitro, uPAR secretion, and TNBC metastasis in vivo. It has been known that neutrophil-endothelial cell interactions are mediated by Mac-1 and ICAM-1, and uPAR participates to the recruitment of neutrophils, and facilitate Mac-1-mediated adhesion. Based on these findings, I hypothesize that ICAM-1+ CTCs contain metastasis-initiating cells which secrete uPAR to recruit neutrophils, and then associate with them through ICAM-1-Mac-1 interaction to promote metastasis. I propose to test this hypothesis in Aim1. Accumulating evidence indicate that neutrophils have both anti-metastatic and pro-metastatic activities (plasticity). Inhibition of Alox5, one of the key enzymes in the arachidonic acid (AA)-Alox5 metabolic pathway, abrogates pro-metastatic activity of neutrophils and consequently reduces metastasis. In addition, AA-cyclooxygenase (COX) metabolic product prostaglandin E2 (PGE2) is also involved in pro-tumor function of neutrophils. Interestingly, both PLA2 (the initial enzyme of the AA metabolism) and Alox5 can increase Mac-1 expression, and I have found that Mac-1 on circulating neutrophils from tumor bearing mice is upregulated. Based on these findings, I hypothesize that AA metabolism regulates pro-metastatic activity of neutrophils, and facilitates CTC-neutrophil interaction by upregulating Mac-1 expression. I will test this hypothesis in Aim2. The results from this proposal will not only reveal new mechanisms of metastasis, but also have significant implication for understanding how AA metabolism regulates pro-metastatic function of neutrophils, which will facilitate the development of the novel cancer immunotherapeutic strategies by targeting pro-metastatic neutrophils.

An intra-vital metabolic microscope to reveal the mechanisms of radiation resistance in head and neck carcinomas
Caigang Zhu, PhD

Basic Science Project Mentors: Teresa Fan, PhD and Ren Xu, PhD
Clinical Project Mentor: Susanne M. Arnold MD, FACP
External Project Mentor: Danny N. Dhanasekaran, PhD

Radio-resistance (RR) leads to poor prognosis in head and neck squamous cell cancer (HNSCC) patients. The failure of radiotherapy (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. 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.

Targeting mitochondrial redox capacity to modulate radiation resistant cancer.
Luksana Chaiswing, PhD

Basic Science Project Mentors: Natasha Kyprianou, MD and Richard M. Higashi, PhD
Clinical Project Mentor: William St. Clair, MD, PhD
External Project Mentor: Douglas R. Spitz, PhD

Radiation therapy (RT) is widely used to treat localized prostate cancer (PCa). However, cancer cells often develop resistance to RT through unknown mechanisms, resulting in cancer recurrence. To improve RT, there is a dire need to uncover cellular events that cause cells to become resistant. We previously demonstrated that PCa heterogeneity, particularly in prostate cancers with an abundant mitochondria subpopulation, often survive and regrow after RT (termed radiation resistant prostate cancer, or RR-PCa). Elevation of mitochondrial mass, number, reactive oxygen species (ROS), and biogenesis markers is acquired in RR-PCa cells. We further demonstrated that knockdown of the mitochondrial biogenesis regulator, transcription factor A, mitochondrial (TFAM), significantly restored the sensitivity of RR-PCa cells to RT. Hence, our overarching hypothesis is that RT-activated mitochondrial biogenesis, via ROS, is an acquisition mechanism that drives PCa survival post-RT, a premise that will undergo stringent examination in the proposed studies. ROS are known to directly and indirectly regulate mitochondrial homeostasis through fusion, fission, mitophagy, and biogenesis. We screened FDA-approved drugs in search of compounds that are nontoxic to normal cells and have the ability to raise the level of mitochondrial hydrogen peroxide (mtH₂O₂) in PCa cells while blocking mitochondrial protein translation. We found azithromycin (AZM), a macrolide antibiotic, to be an effective prototype compound that possesses both properties. We further demonstrated that AZM combined with RT enhances the death of PCa cells with an abundant mitochondrial subpopulation, compared to AZM or RT alone. Thus, we propose to advance our findings and identify the mechanism(s) that effectively inhibit the survival of post-irradiated cancer cells, to improve RT efficacy. The specific aims are: 1) to define the molecular mechanism(s) by which RT-activated mitochondrial biogenesis promotes cell survival and metabolic adaptations of PCa cells with abundant mitochondria, both in vitro and in vivo; 2) to determine if overloading mtH₂O₂ to target pre-existing mitochondria and RT-acquired mitochondria while blocking mitochondrial protein translation in RT-acquired mitochondria enhances radiosensitivity of RR-PCa cells; and 3) to improve RT using a mtH₂O₂ generator and a mitochondrial protein translation inhibitor, AZM as prototype, in an orthotopic mouse xenograft model and a patient-derived xenograft model of PCa with activated mitochondrial biogenesis. This study uses state-of-the-art platforms including reverse phase protein array, stable isotope-resolved metabolomics, a total internal reflection fluorescence microscopy with Imaris software, TEMPOL-enhanced MRI imaging, and a high resolution O₂k-FluoRespirometer. The proposed studies are expected to uncover novel molecular insights by which concurrently targeting mitochondrial redox capacity and mitochondrial biogenesis improve RT efficacy in the treatment of RR-PCa.

Identifying fundamental mechanisms that mediate resistance to anti-cancer therapies
Elizabeth Duncan, PhD

Basic Science Project Mentors: Andrew Lane, PhD and Christine Brainson, PhD
Clinical Mentor: John D'Orazio, MD, PhD
External Mentor: K. Sreekumaran Nair, MD, PhD

Tumor heterogeneity is a complex, challenging and critically important problem in designing effective cancer treatments. Distinct molecular and functional differences are not only found between cancer cells isolated from different patients, tissues and sites, but also among cells of a single tumor. Planarians have a nearly unlimited regenerative capacity and have great potential as a model to study tumor heterogeneity and therapy resistance. The goal of this proposal is to exploit the cancer-like features of planarian stem cells and the experimental tractability of this in vivo model to uncover fundamental new mechanisms that link metabolic reprogramming and changes of histone H3K4me3 to the development of cisplatin resistance in human lung cancer.

This study will yield novel insights into stem cell biology and also open new therapeutic avenues for overcoming cisplatin resistance. Dr. Duncan was a CCM pilot grant awardee in 2019 and became a new project leader of this Phase II application after generating sufficient data toward CCM thematic research.