The role of metabolic ecosystem in cancer progression — metabolic plasticity and mTOR hyperactivity in tumor tissues

Despite advancements in cancer management, tumor relapse and metastasis are associated with poor outcomes in many cancers. Over the past decade, oncogene-driven carcinogenesis, dysregulated cellular signaling networks, dynamic changes in the tissue microenvironment, epithelial-mesenchymal transitions, protein expression within regulatory pathways, and their part in tumor progression are described in several studies. However, the complexity of metabolic enzyme expression is considerably under evaluated. Alterations in cellular metabolism determine the individual phenotype and behavior of cells, which is a well-recognized hallmark of cancer progression, especially in the adaptation mechanisms underlying therapy resistance. In metabolic symbiosis, cells compete, communicate, and even feed each other, supervised by tumor cells. Metabolic reprogramming forms a unique fingerprint for each tumor tissue, depending on the cellular content and genetic, epigenetic, and microenvironmental alterations of the developing cancer. Based on its sensing and effector functions, the mechanistic target of rapamycin (mTOR) kinase is considered the master regulator of metabolic adaptation. Moreover, mTOR kinase hyperactivity is associated with poor prognosis in various tumor types. In situ metabolic phenotyping in recent studies highlights the importance of metabolic plasticity, mTOR hyperactivity, and their role in tumor progression. In this review, we update recent developments in metabolic phenotyping of the cancer ecosystem, metabolic symbiosis, and plasticity which could provide new research directions in tumor biology. In addition, we suggest pathomorphological and analytical studies relating to metabolic alterations, mTOR activity, and their associations which are necessary to improve understanding of tumor heterogeneity and expand the therapeutic management of cancer.

Quantifying the patterns of metabolic plasticity and heterogeneity along the epithelial-hybrid-mesenchymal spectrum in cancer

Cancer metastasis is the leading cause of cancer-related mortality and the process of Epithelial to Mesenchymal Transition (EMT) is crucial for cancer metastasis. Either a partial or complete EMT have been reported to influence the metabolic plasticity of cancer cells in terms of switching among oxidative phosphorylation, fatty acid oxidation and glycolysis pathways. However, a comprehensive analysis of these major metabolic pathways their associations with EMT across different cancers is lacking. Here, we analyse more than 180 cancer cell datasets and show diverse associations of these metabolic pathways with the EMT status of cancer cells. Our bulk data analysis shows that EMT generally positively correlates with glycolysis but negatively with oxidative phosphorylation and fatty acid metabolism. These correlations are also consistent at the level of their molecular master regulators, namely AMPK and HIF1α. Yet, these associations are shown to not be universal. Analysis of single-cell data of EMT induction shows dynamic changes along the different axes of metabolic pathways, consistent with general trends seen in bulk samples. Together, our results reveal underlying patterns of metabolic plasticity and heterogeneity as cancer cells traverse through the epithelial-hybrid-mesenchymal spectrum of states.

Glucocorticoid-Mediated Developmental Programming of Vertebrate Stress Responsivity

Glucocorticoids, vertebrate steroid hormones produced by cells of the adrenal cortex or interrenal tissue, function dynamically to maintain homeostasis under constantly changing and occasionally stressful environmental conditions. They do so by binding and thereby activating nuclear receptor transcription factors, the Glucocorticoid and Mineralocorticoid Receptors (MR and GR, respectively). The GR, by virtue of its lower affinity for endogenous glucocorticoids (cortisol or corticosterone), is primarily responsible for transducing the dynamic signals conveyed by circadian and ultradian glucocorticoid oscillations as well as transient pulses produced in response to acute stress. These dynamics are important determinants of stress responsivity, and at the systemic level are produced by feedforward and feedback signaling along the hypothalamus-pituitary–adrenal/interrenal axis. Within receiving cells, GR signaling dynamics are controlled by the GR target gene and negative feedback regulator fkpb5. Chronic stress can alter signaling dynamics via imperfect physiological adaptation that changes systemic and/or cellular set points, resulting in chronically elevated cortisol levels and increased allostatic load, which undermines health and promotes development of disease. When this occurs during early development it can “program” the responsivity of the stress system, with persistent effects on allostatic load and disease susceptibility. An important question concerns the glucocorticoid-responsive gene regulatory network that contributes to such programming. Recent studies show that klf9, a ubiquitously expressed GR target gene that encodes a Krüppel-like transcription factor important for metabolic plasticity and neuronal differentiation, is a feedforward regulator of GR signaling impacting cellular glucocorticoid responsivity, suggesting that it may be a critical node in that regulatory network.

Microtubule-Based Mitochondrial Dynamics as a Valuable Therapeutic Target in Cancer

Mitochondria constitute an ever-reorganizing dynamic network that plays a key role in several fundamental cellular functions, including the regulation of metabolism, energy production, calcium homeostasis, production of reactive oxygen species, and programmed cell death. Each of these activities can be found to be impaired in cancer cells. It has been reported that mitochondrial dynamics are actively involved in both tumorigenesis and metabolic plasticity, allowing cancer cells to adapt to unfavorable environmental conditions and, thus, contributing to tumor progression. The mitochondrial dynamics include fusion, fragmentation, intracellular trafficking responsible for redistributing the organelle within the cell, biogenesis, and mitophagy. Although the mitochondrial dynamics are driven by the cytoskeleton—particularly by the microtubules and the microtubule-associated motor proteins dynein and kinesin—the molecular mechanisms regulating these complex processes are not yet fully understood. More recently, an exchange of mitochondria between stromal and cancer cells has also been described. The advantage of mitochondrial transfer in tumor cells results in benefits to cell survival, proliferation, and spreading. Therefore, understanding the molecular mechanisms that regulate mitochondrial trafficking can potentially be important for identifying new molecular targets in cancer therapy to interfere specifically with tumor dissemination processes.

Metabolic Plasticity in Melanoma Progression and Response to Oncogene Targeted Therapies

Resistance to therapy continues to be a barrier to curative treatments in melanoma. Recent insights from the clinic and experimental settings have highlighted a range of non-genetic adaptive mechanisms that contribute to therapy resistance and disease relapse, including transcriptional, post-transcriptional and metabolic reprogramming. A growing body of evidence highlights the inherent plasticity of melanoma metabolism, evidenced by reversible metabolome alterations and flexibility in fuel usage that occur during metastasis and response to anti-cancer therapies. Here, we discuss how the inherent metabolic plasticity of melanoma cells facilitates both disease progression and acquisition of anti-cancer therapy resistance. In particular, we discuss in detail the different metabolic changes that occur during the three major phases of the targeted therapy response—the early response, drug tolerance and acquired resistance. We also discuss how non-genetic programs, including transcription and translation, control this process. The prevalence and diverse array of these non-genetic resistance mechanisms poses a new challenge to the field that requires innovative strategies to monitor and counteract these adaptive processes in the quest to prevent therapy resistance.

Combining Arsenic Trioxide and Mitocans Selectively Disrupts Cellular Energetics in Acute Myeloid Leukemia

In our earlier work with arsenic trioxide (ATO) resistance in acute promyelocytic leukemia (APL), we observed that ATO resistant cells reprogrammed their metabolism from glycolysis to oxidative phosphorylation (OXPHOS) as a mechanism of resistance. We further demonstrated that it could be overcome by targeting this metabolic switch using FCCP (mitocan) in combination with ATO (Balasundaram N et al. Biorxiv 2020). There is increasing evidence that acute myeloid leukemia (AML) cells have a greater metabolic plasticity unlike ATO resistant APL cells and most cancers that rely on glycolysis. AML leukemic stem cells preferentially utilize OXPHOS for their survival (Lagadinou ED et al. Cell stem cell 2013). Mitocans like venetoclax used in combination with hypo-methylating agents are already well established in the management of AML (Pollyea D, et al. Nat Med 2021). ATO is also an effective glycolytic inhibitor (Zhang H, et al. PNAS 2015) hence we hypothesized that a combination of ATO and mitocans could potentially target the metabolic plasticity of AML cells. As the combination of ATO and FCCP was found to be non-specific we performed a small-scale screening on an AML cell line (U937) using FDA-approved compounds that are reported to target mitochondria (Gohil V et al. Nature Biotechnology, 2010). Though most of the mitocans showed predicted synergy with ATO. We focused on artesunate (ART) as a candidate for further evaluation due to its specificity for malignant cells, high therapeutic index, bioavailability, route of administration, cost-effectiveness, and global usage as an antimalarial. The combination of ATO+ART significantly reduced the viability of different subtypes of AML cell lines (THP-1, MV4:11, and Kasumi-1) and acute lymphoblastic leukemia cell lines (Jurkat E6.1, SUP B15, and MOLT-4) with minimal effect on the normal cells (CD34 and peripheral blood mononuclear cells; n=10; 48hours) (figure 1a). We noted that the selective specificity of the combination was primarily due to the iron metabolism of the leukemic cells and a requirement of iron for the activity of ART. When an iron chelator deferoxamine (DFO) was used in combination with ATO+ART there was a significant reduction in the activity of the combination on the AML cells (Figure 1b, U937; n=10; 48hours). Seahorse extracellular flux analysis validated that ART (5uM) as a single agent promoted uncoupled mitochondrial respiration and the addition of ATO resulted in a metabolic catastrophe (figure 1c and d). Chemical drug proteomic analysis using biotinylated artesunate and pull down from the leukemic cells revealed that the top interacting partners were localized in the mitochondria. We also noted that ART treatment significantly affected the mitochondrial dynamics of leukemic cells, where ART and ATO+ART treated cells had fragmented mitochondria in comparison to the control and ATO alone treated cells where the mitochondria were more elongated (figure 1e, U937 cells; n=3). We evaluated the effect of ATO+ART and their combination with azacytidine (triplet) in-vitro. Dual and triple combinations showed greater toxicity on AML cell lines and primary AML cells (Figure 1f, n=50) in comparison to the normal peripheral blood mononuclear cells (PBMNCs) and normal CD34+ cells. Taken together, these findings highlight the selective specificity of these combinations and its clinical potential in AML. Figure 1 Figure 1. Disclosures Augustin: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor. Krishna: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; KCM Vellore: Patents & Royalties; SGUL: Patents & Royalties; AML: Other: Co-Inventor. Mathews: Christian Medical College: Patents & Royalties: US 2020/0345770 A1 - Pub.Date Nov.5, 2020; AML: Other: Co-Inventor.

Extracellular Matrix Signals as Drivers of Mitochondrial Bioenergetics and Metabolic Plasticity of Cancer Cells During Metastasis

The role of metabolism in tumor growth and chemoresistance has received considerable attention, however, the contribution of mitochondrial bioenergetics in migration, invasion, and metastasis is recently being understood. Migrating cancer cells adapt their energy needs to fluctuating changes in the microenvironment, exhibiting high metabolic plasticity. This occurs due to dynamic changes in the contributions of metabolic pathways to promote localized ATP production in lamellipodia and control signaling mediated by mitochondrial reactive oxygen species. Recent evidence has shown that metabolic shifts toward a mitochondrial metabolism based on the reductive carboxylation, glutaminolysis, and phosphocreatine-creatine kinase pathways promote resistance to anoikis, migration, and invasion in cancer cells. The PGC1a-driven metabolic adaptations with increased electron transport chain activity and superoxide levels are essential for metastasis in several cancer models. Notably, these metabolic changes can be determined by the composition and density of the extracellular matrix (ECM). ECM stiffness, integrins, and small Rho GTPases promote mitochondrial fragmentation, mitochondrial localization in focal adhesion complexes, and metabolic plasticity, supporting enhanced migration and metastasis. Here, we discuss the role of ECM in regulating mitochondrial metabolism during migration and metastasis, highlighting the therapeutic potential of compounds affecting mitochondrial function and selectively block cancer cell migration.