Understanding The Effect Of Mitochondrial Complex I Deficiency In Cancer Cells And Their Microenvironment

Targeting metabolism has become a valid anticancer strategy to pursue. Among others, respiratory complex-I (CI) has been identified as a valid target. In this context, a genetic ablation of mitochondrial CI enzyme in colorectals (HCT) and osteosarcomas (143B) was associated with the lack of HIF-1α stabilization and subsequent inability to adapt to hypoxic environment, suggesting this may contribute to the anti-tumorigenic effect of CI deficiency. However, most recent data imply that, despite the lack of HIF-1α, the progression of CI-deficient tumors seems to be supported by components of tumor-microenvironment (TME), in particular tumor associated macrophages (TAMs). Thus, the aim of this thesis was (i) to prove that the lack of HIF-1α stabilization and subsequent inability to adapt to hypoxic environment is a generalized phenomenon in murine, human cancers and in an orthotopic system and may contribute to the anti-tumorigenic effect of CI deficiency (ii) to prove that the CI-deficient tumors activate macrophages to progress despite HIF-1α destabilization (iii) to identify common targetable factors responsible for macrophage recruitment among the CI deficient 143B and HCT models. By introducing a non-degradable form of HIF-1α (HIF-TM) in CI deficient models, we demonstrated that HIF-TM rescued their tumorigenic potential, mature vasculature and vessel size. Moreover, in 143B CI deficient model (143B-/-), lack of HIF-1 activity correlated with macrophage migration inhibitory factor (MIF) downregulation and macrophage abundance. This indicated that the disruption of HIF-1-MIF axis leading to vasculature remodeling and TAM recruitment is one of the adaptive mechanisms activated by the 143B-/- tumors. However, this mechanism may not be generalized to the epithelial HCT model. Large-scale omics approach on CI deficient and CI competent 143B and HCT xenografts derived supernatants identified nine common metabolites secreted specifically from both the CI deficient models, allowing to hypothesize a potential cytokine-like function of these metabolites in attracting TAMs

A Humanized Bone Niche Model Reveals Bone Tissue Preservation Upon Targeting Mitochondrial Complex I in Pseudo-Orthotopic Osteosarcoma

A cogent issue in cancer research is how to account for the effects of tumor microenvironment (TME) on the response to therapy, warranting the need to adopt adequate in vitro and in vivo models. This is particularly relevant in the development of strategies targeting cancer metabolism, as they will inevitably have systemic effects. For example, inhibition of mitochondrial complex I (CI), despite showing promising results as an anticancer approach, triggers TME-mediated survival mechanisms in subcutaneous osteosarcoma xenografts, a response that may vary according to whether the tumors are induced via subcutaneous injection or by intrabone orthotopic transplantation. Thus, with the aim to characterize the TME of CI-deficient tumors in a model that more faithfully represents osteosarcoma development, we set up a humanized bone niche ectopic graft. A prominent involvement of TME was revealed in CI-deficient tumors, characterized by the abundance of cancer associated fibroblasts, tumor associated macrophages and preservation of osteocytes and osteoblasts in the mineralized bone matrix. The pseudo-orthotopic approach allowed investigation of osteosarcoma progression in a bone-like microenvironment setting, without being invasive as the intrabone cell transplantation. Additionally, establishing osteosarcomas in a humanized bone niche model identified a peculiar association between targeting CI and bone tissue preservation

Ref-1 redox activity alters cancer cell metabolism in pancreatic cancer: exploiting this novel finding as a potential target

Background: Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1's role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. Results: Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. Conclusion: Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo

The multifaceted effects of metformin on tumor microenvironment

The efficacy of metformin in treating cancer has been extensively investigated since epidemiologic studies associated this anti-diabetic drug with a lower risk of cancer incidence. Since tumors are complex systems, in which cancer cells coexist and interact with several different types of non-malignant cells, it is not surprising that anti-cancer drugs affect not only cancer cells, but also the abundance and functions of cells of the tumor microenvironment. Recent years have seen a wide collection of reports showing how metformin, as well as other complex I inhibitors, may influence cancer progression by modulating the phenotype of non-transformed cells in a tumor. In this review, we particularly focus on the effect of metformin on angiogenesis, cancer-associated fibroblasts, tumor-associated macrophages and cancer immunosuppression

The Neglected Liaison: Targeting Cancer Cell Metabolic Reprogramming Modifies the Composition of Non-Malignant Populations of the Tumor Microenvironment

Metabolic reprogramming is a well-known hallmark of cancer, whereby the development of drugs that target cancer cell metabolism is gaining momentum. However, when establishing preclinical studies and clinical trials, it is often neglected that a tumor mass is a complex system in which cancer cells coexist and interact with several types of microenvironment populations, including endothelial cells, fibroblasts and immune cells. We are just starting to understand how such populations are affected by the metabolic changes occurring in a transformed cell and little is known about the impact of metabolism-targeting drugs on the non-malignant tumor components. Here we provide a general overview of the links between cancer cell metabolism and tumor microenvironment (TME), particularly focusing on the emerging literature reporting TME-specific effects of metabolic therapies

NDUFS3 depletion permits complex I maturation and reveals TMEM126A/OPA7 as an assembly factor binding the ND4-module intermediate

Complex I (CI) is the largest enzyme of the mitochondrial respiratory chain, and its defects are the main cause of mitochondrial disease. To understand the mechanisms regulating the extremely intricate biogenesis of this fundamental bioenergeticmachine, we analyze the structural and functional consequences of the ablation of NDUFS3, a non-catalytic core subunit. We show that, in diverse mammalian cell types, a small amount of functional CI can still be detected in the complete absence of NDUFS3. In addition, we determine the dynamics of CI disassembly when the amount of NDUFS3 is gradually decreased. The process of degradation of the complex occurs in a hierarchical and modular fashion in which the ND4 module remains stable and bound to TMEM126A. We, thus, uncover the function of TMEM126A, the product of a disease gene causing recessive optic atrophy as a factor necessary for the correct assembly and function of CI.

BAFF Attenuates Immunosuppressive Monocytes in the Melanoma Tumor Microenvironment

Emerging evidence indicates B-cell activating factor (BAFF, Tnfsf13b) to he an important cytokine for antitumor immunity. In this study, we generated a BAFF-overexpressing B16.F10 melanoma cell model and found that BAFF-expressing tumors grow more slowly in vivo than control tumors. The tumor microenvironment (TME) of BAFF-overexpressing tumors had decreased myeloid infiltrates with lower PD-L1 expression. Monocyte depletion and anti-PD-L1 antibody treatment confirmed the functional importance of monocytes for the phenotype of BAFF-mediated tumor growth delay. RNA sequencing analysis confirmed that monocytes isolated from BAFF-overexpressing tumors were characterized by a less exhaustive phenotype and were enriched for in genes involved in activating adaptive immune responses and NF-kappa B signaling. Evaluation of patients with late-stage metastatic melanoma treated with inhibitors of the PD-1/PD-L1 axis demonstrated a stratification of patients with high and low BAFF plasma levels. Patients with high BAFF levels experienced lower responses to anti-PD-1 immunotherapies. In summary, these results show that BAFF, through its effect on tumor-infiltrating monocytes, not only impacts primary tumor growth but can serve as a biomarker to predict response to anti-PD-1 immunotherapy in advanced disease. Significance: The BAFF cytokine regulates monocytes in the melanoma microenvironment to suppress tumor growth, highlighting the importance of BAFF in antitumor immunity