Barriers in radiobiology: Hypoxic radiosensitization by modifying the metabolism of the tumor

Research output: ThesisPhD Thesis

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Abstract

Radiotherapy (RT) represents an essential treatment modality in cancer management, either alone or combined with other therapies. Up to 40% of patients who are cured of cancer will receive RT as part of their treatment, and around 50% of cancer patients will require RT at some point. Over the last decades, significant developments in the use of radiation to treat cancer have been focused on the physically accurate delivery of radiation therapy. RT has dramatically improved both technological point of view and clinical outcomes. About two decades ago, RT was mainly given as simple opposing anteroposterior 'fields', based on bony anatomy and skin marks, with minimal imaging guidance. Nowadays, Intensity-modulated RT can conform radiation dose very precisely to the target of interest thus sparing, as much as possible, surrounding adjacent normal tissues to reduce attendant toxicities. These steep dose gradients demand a precise positioning, based on daily conebeam computed tomography (CT) or magnetic resonance imaging (MRI). Although the personalization of RT already occurs based on anatomical and clinical information, it lacks biological precision related to what is known, or yet to be discovered, with respect to specific individual tumors and normal tissue response to radiation. Personalized medicine with RT may in the future also means rethinking treatment plans to match the tumors' changing biology.
Resistance to RT is polymodal and associated with several biological alterations both within the tumor and the surrounding microenvironment. The first chapter of this thesis summarizes the available literature on how cells become radioresistant and elaborates on the available clinical interventions to counteract this radioresistance. These mechanisms and interventions are described based on the 6R framework of radiobiology. Classically, there were only 4Rs, which described the rationale behind fractionation of radiation namely Repair, Redistribution, Repopulation and Reoxygenation. Repair stands for the capacity of cells to repair radiation-induced DNA damage. Redistribution explains that cancer cells are more sensitive to radiation in certain cell-cycle phases. Cells progress to these sensitive phases with consecutive RT doses, leading to an optimal therapeutic effect. Repopulation takes the presumption that cells proliferate between fractions of radiotherapy into account. Reoxygenation is based upon the fact that sensitivity to radiation increases when solid tumors are remodeled into well-oxygenated tissues. The fifth R, Radiosensitivity, and sixth R, immune Rejection, were added over time. Radiosensitivity explains that the response to radiation varies by tumor intrinsic and individual radiosensitivity. Immune rejection takes into account the connection between the anti-tumor immune response and RT. Each of the Rs is a double-edged sword with changes affecting radiotherapy's net therapeutic effect.
As a first research step, we investigated which possible pathways or biological processes could be targeted to enhance RT. We extracted data from The Cancer Genome Atlas (TCGA) of head-and-neck, cervical and breast cancer patients that underwent RT and demonstrated several genetic prognostic indicators that could potentially influence radioresistance within these cancer types. We analysed the mRNA expression patterns of 50 hallmark gene sets of the MSigDB collection. These hallmark gene sets were divided into eight categories based on a shared biological or functional process (radiobiological, metabolic, proliferation, development, signalling, cellular component, pathway and immune). We observed that gene signatures linked to radiobiological 2 pathways, metabolism and proliferation of the cells had prognostic associations with overall survival of the patients. Based on these results and the available literature, we decided to focus on targeting the metabolism of cells to radiosensitize tumors.
We investigated the potential radiosensitizing effect of a drug that shifts the glycolysis of cancer cells. Dichloroacetate (DCA) is a specific inhibitor of the pyruvate dehydrogenase kinase (PDK), which leads to enhanced ROS production. ROS are the primary effector molecules of radiation and an increase hereof will enhance the radioresponse. We evaluated the effects of DCA and radiotherapy on two triple-negative breast cancer (TNBC) cell lines, namely EMT6 and 4T1, under aerobic and hypoxic conditions. As expected, DCA treatment decreased phosphorylated pyruvate dehydrogenase (PDH) and lowered both extracellular acidification rate (ECAR) and lactate production. Remarkably, DCA treatment led to a significant increase in ROS production (up to 15-fold) in hypoxic cancer cells but not in aerobic cells. Consistently, DCA radiosensitized hypoxic tumor cells and 3D spheroids while leaving the intrinsic radiosensitivity of the tumor cells unchanged. Our results suggest that although described as OXPHOS-promoting drug, DCA can also increase hypoxic radioresponses.
Next, we decided to look at another essential metabolic process, namely OXPHOS. We investigated the anti-diabetic drugs metformin and phenformin as potential radiosensitizers. Metformin and phenformin are two biguanide drugs that target the complex I of the mitochondria. Although metformin and phenformin exhibit anti-tumor activity in various models, their radiomodulatory effect under hypoxic conditions, particularly for phenformin, is largely unknown. We showed that metformin and phenformin inhibited mitochondrial complex I activity and subsequently reduced the cells' oxygen consumption rate (OCR) in a dose-dependent manner. As a result, the hypoxic radioresistance of tumor cells was counteracted by metformin and phenformin. Regarding intrinsic radioresistance, both did not exhibit any effect although there was an increase of phosphorylation of AMPK and ROS production. Metformin or phenformin alone did not show any anti-tumor effect in tumor-bearing mice. While in combination with radiation, both substantially delayed tumor growth and enhanced radioresponse. Our results demonstrated that metformin and phenformin overcome hypoxic radioresistance by inhibiting mitochondrial respiration. In the last step, we decided to look into the literature to investigate how complex 1 inhibitors (focus on metformin) can be used as an immunomodulatory setting for colorectal cancer. Therefore, we discussed the immunological landscape of colorectal cancer and the potential immunocorrective effect of metformin together with modified IL-2 in chapter VI. Taken together, we have demonstrated that multiple possible metabolic paths can be taken to radiosensitize hypoxic cancer cells. We have first demonstrated that DCA can shift glycolysis to OXPHOS of the cancer cells even under hypoxic conditions, which leads to enhanced radioresponse of both aerobic and hypoxic tumor cells through increased ROS. Secondly, we showed that the anti-diabetic drugs metformin and phenformin could block the mitochondrial complex I. By blocking the OXPHOS of the cells, we reduced their OCR. The reduced oxygen consumption of the cells counteracted the hypoxic radioresistance both in cells and tumor-bearing mice. Lastly, we discussed the possibility of using metformin and modified IL-2 as a potential 3 immunocorrective drug in colorectal cancer. DCA, metformin and phenformin are all under clinical investigation. The data from our study will provide new insight into their development as radiosensitizes and accelerate the design of clinical trials in combination with radiotherapy
Original languageEnglish
QualificationDoctor in Medical Sciences
Awarding Institution
  • Vrije Universiteit Brussel
Supervisors/Advisors
  • De Ridder, Mark, Supervisor
  • Dufait, Inès, Co-Supervisor
Award date28 Jun 2022
Publication statusPublished - 2022

Keywords

  • radiobiology
  • Radiotherapy
  • Hypoxic radiosensitization
  • tumor

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