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Cell competition was originally described in Drosophila as a physiological process based on the comparison of relative fitness between neighbouring cells. At the end of the process, suboptimal cells, called losers, are committed to die while stimulating proliferation of the most performant cells, called winners, so maintaining tissue homeostasis. In Drosophila and mammalian development, cells showing high MYC activity behave like winners, growing at the expense of the surrounding cells which succumb by apoptosis, so unveiling a leading role for this oncoprotein in eliciting cell competition. Since MYC protein is upregulated in a large fraction of human cancers, cell competition has been speculated to play a role in human tumourigenesis. Consistently with this hypothesis, we found massive apoptotic death of stromal cells in proximity to MYC-upregulating cancer cells in a relevant number of human tumour samples, and showed that modulation of MYC activity in human cancer cell lines is sufficient to subvert their competitive drive. In Drosophila, MYC-overexpressing cells have been demonstrated to be unable to execute cell competition in a p53 loss-of-function background. With the aim to find a role for p53 in MYC-mediated cell competition, we first stained the same cancer samples as above for p53. Interestingly, the tumour regions that did not show significant signs of competitive interactions were also found negative for p53 staining, disclosing a possible function for this protein in promoting cancer-associated cell competition. Further experiments in a¬ Drosophila cancer model helped us define a dose-dependent role of p53 in MYC-overexpressing winner cells, and functional assays in co-cultures of human cancer cells confirmed that p53 function is necessary for the winner cells to execute cell competition. Altogether, our findings reveal a pro-oncogenic role of p53, that appears to cooperate with MYC in driving cell competition in different cancer contexts.

Cell competition is a mechanism conserved from Drosophila to mammals, based on the comparison of relative fitness between neighbouring cells, leading to the apoptotic elimination of the weakest. Several molecules are involved in these competitive interactions: in particular, cells expressing high levels of MYC grow at the expense of surrounding cells. In Drosophila epithelia, MYC-Mediated Cell Competition5 (MMCC) selects cells undergoing clonal expansion, and p53 function is necessary in MYC-overexpressing cells to sustain their competitive advantage. Malignant cells often upregulate MYC and results obtained in our lab suggest that MMCC can shape tumour expansion and evolution. p53 is one of the most frequently mutated genes in human cancers, with both loss and gain of function mutations playing roles in carcinogenesis. Through IHC analysis on several kinds of human carcinomas, in vitro co-culture assays and Drosophila experiments, we observed that loss of p53 in the winner cells is sufficient to make them unable to grow, thus suggesting a functional cooperation between MYC and p53 in cancer-associated MMCC. Our results show an oncogenic side of the p53 wild-type protein that appears to help shape cancer progression through selection of the most competitive cells.

MYC-Mediated Cell Competition (MMCC) is a phenomenon of fitness comparison occurring between adjacent cells showing different levels of MYC activity.. It describes a mechanism, conserved from Drosophila to mammalian development, through which cells characterised by high expression of MYC induce apoptotic death of neighbouring low MYC-expressing cells and acquire an advantage in space occupancy. Though it is widely speculated that this phenomenon is relevant to cancer, its characterisation during tumour progression is still missing. Here we show the presence of markers of MMCC in human carcinomas and demonstrate through experiments in human cancer cell lines that MYC modulation is per se sufficient to induce competitive behaviours in both genetically distant and identical cells. Noteworthy, MYC inhibition in the fittest cell line is sufficient to reverse its competitive status. Moreover, data obtained in a Drosophila,cancer model indicate that MMCC is normally at work during tumour growth and that induction of high or low-MYC expressing cells in the growing masses deeply alters the final tumour size, supporting a role for MMCC in cancer evolution.

The term “field cancerisation” describes a precancerous area in which genetically altered but histologically normal tissues proceed towards the development of multiple malignant foci. In the early stages of tumour progression cells may indeed acquire genetic damages that allow them to proliferate in patches of altered cells gradually replacing normal tissue. This mechanism invokes MYC-mediated cell competition (MMCC), a phenomenon characterised in Drosophila consisting in fitness confrontation between cells sharing the same tissue, with cells expressing high MYC levels ultimately killing and replacing cells showing lower MYC activity. These intrinsic features of MMCC make it a candidate mechanism pioneering field cancerisation. Here we mimic field formation by upregulating MYC in a territory of the larval wing epithelium of Drosophila. Analysis of specific markers usually found in mammalian precancerous areas confirmed that MYC upregulation is sufficient to trigger specific cellular responses. Moreover, MYC-expressing fields were susceptible to the development of multifocal tumours upon induction of different second mutations, a typical trait correlated to mammalian field cancerisation. In summary, our study identified an undescribed early genetic change implicated in field cancerisation and established a genetically amenable model which may help study the molecular basis of the initial tumourigenic events.

Clonal analysis is common practice in Drosophila. In particular, induction of cell clusters carrying both loss-of-function alleles of neoplastic tumour suppressors and activated forms of oncogenes has successfully been exploited to study cooperative tumourigenesis in different organs. This strategy has allowed to collect a number of morphological and molecular details on the phenotypic traits associated with cancer evolution. We previously identified Myc as a target of the Hippo pathway in Drosophila, and found out that its expression is sufficient to rescue the growth deficit of cells mutant for polarity genes, releasing their malignant potential. We also previously characterised cancer-associated tracheogenesis, a process analogue to mammalian neo-angiogenesis, as an intrinsic trait of Drosophila epithelial tumours. Here we expand on previous work, showing that tumour growth and tracheogenesis are separable traits in Drosophila epithelia. While in situ cancer expansion is mainly supported by Myc, this is not the case for migration and tracheogenesis, which mostly depend on Fos activity. These proteins are strategically found at the crossroads of the Hippo, JNK and Ras/MAPK pathways, which current literature recognises as key players in cancer progression.

Despite MYC is one of the most referenced molecules in biology, new unanticipated roles are being found for this key protein, proving we are still far from understanding its full biological potential. During my PhD thesis, I investigated MYC function in a number of cancer traits using Drosophila larval organs as a model. Given that tumours develop in a complex landscape, where the entire cancer community continuously remodels local microenvironment, the conceptual scaffold of my work was built as to take into account both the cell-autonomous and non cell-autonomous MYC functions. The central findings of my study are: - Field cancerisation: I show that MYC, contrariwise to other growth-promoting molecules, is able to form a field where secondary mutations may originate multifocal cancers. This has great implication in human recurrent cancers. - Growth and migration/tracheogenesis: I show that MYC plays opposite but correlated roles in these two central cancer hallmarks: while it exerts a pro-growth effect following stabilisation by different upstream pathways, its presence inhibits cell migration by counteracting the JNK signalling. This has implication in cancer metastasis. - Cell competition/apoptosis: I show that apoptosis induced by MYC-mediated cell competition stimulates tumour growth; its inhibition indeed favours cancer collapse and prolongs animal life under heavy cancer burden. This has great implications in cancer treatment: while proapoptotic therapies may transiently reduce tumour mass, they may create a cytokine-rich environment which helps feed the tumour.

OBJECTIVE While current therapies are conceived to target cancer cells, it is widely acknowledged that several non-cancer populations are involved in tumourigenesis. Dysfunction of epithelial-mesenchymal interactions are indeed known to promote progression of pre-neoplastic lesions to malignancy. What are the major molecular changes occurring in the mesenchymal cells co-opted by the growing cancer? Are these cells more treatable than cancer cells? We believe the success of cancer therapy requires an intimate understanding of the bi-directional feedback between the evolving tumour cells and the associated stromal tissue. The identification of specific molecules at work within the mesenchymal compartment during cancer growth may thus be instrumental for the development of novel therapeutics. Here we present the scheme of a project which starts with the development of a Drosophila cancer model aimed at analysing the expression profile of the mesenchymal cells in an organ whose epithelial cells are undergoing clonal malignant transformation. The resulting findings will be extended to the context of human cancer with the aim to identify a group of mesenchyma-derived molecular factors able to modulate cancer progression. MATERIALS AND METHODS We developed a Drosophila model of Ras-driven carcinogenesis that will be used to capture the expression profile of the mesenchymal cells populating the epithelial organ undergoing cancer growth. Mesenchymal cells will then be isolated from human Ras-driven epithelial cancer samples and a qRT-PCR analysis will be carried out to evaluate the expression of the human orthologues of the transcripts/miRs found deregulated in the fly model. The selected molecules whose expression will result conserved will be screened in vivo in Drosophila to assess their contribution to tumour progression. RESULTS This project aims at identifying a pool of mesenchymal players with cooperative or inhibitory effects on tumour progression, widening our understanding of the role that tumour-associated stroma plays in carcinogenesis. Morover, we expect that our approach will highlight significant molecular conservation between Drosophila and human Ras-driven cancer stroma and will thus represent an invaluabe model for further studies in the field. CONCLUSION It is well recognised that tumour microenvironment plays an essential role in cancer development, and it is thus considered as an additional cancer hallmark. Understanding the molecular basis of the social cell biology of cancer is thus indispensable to develop novel therapeutic approaches targeting the whole cancer-associated cell community.

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials.

Inhibition of respiratory complex I (CI) is becoming a promising anti-cancer strategy, encouraging the design and the use of inhibitors, whose mechanism of action, efficacy and specificity remain elusive. As CI is a central player of cellular bioenergetics, a finely tuned dosing of targeting drugs is required to avoid side effects. We compared the specificity and mode of action of CI inhibitors metformin, BAY 87-2243 and EVP 4593 using cancer cell models devoid of CI. Here we show that both BAY 87-2243 and EVP 4593 were selective, while the antiproliferative effects of metformin were considerably independent from CI inhibition. Molecular docking predictions indicated that the high efficiency of BAY 87-2243 and EVP 4593 may derive from the tight network of bonds in the quinone binding pocket, although in different sites. Most of the amino acids involved in such interactions are conserved across species and only rarely found mutated in human. Our data make a case for caution when referring to metformin as a CI-targeting compound, and highlight the need for dosage optimization and careful evaluation of molecular interactions between inhibitors and the holoenzyme. ispartof: OPEN BIOLOGY vol:12 issue:11 ispartof: location:England status: published

Small cell neuroendocrine carcinoma is most frequently found in the lung (SCLC), but it has been also reported, albeit with a very low incidence, in the ovary. Here, we analyze a case of primary small cell carcinoma of the ovary of pulmonary type (SCCOPT), a rare and aggressive tumor with poor prognosis, whose biology and molecular features have not yet been thoroughly investigated. The patient affected by SCCOPT had a residual tumor following chemotherapy which displayed pronounced similarity with neuroendocrine tumors and lung cancer in terms of its microRNA expression profile and mTOR-downstream activation. By analyzing the metabolic markers of the neoplastic lesion, we established a likely glycolytic signature. In conclusion, this in-depth characterization of SCCOPT could be useful for future diagnoses, possibly aided by microRNA profiling, allowing clinicians to adopt the most appropriate therapeutic strategy.

Anticancer strategies aimed at inhibiting Complex I of the mitochondrial respiratory chain are increasingly being attempted in solid tumors, as functional oxidative phosphorylation is vital for cancer cells. Using ovarian cancer as a model, we show that a compensatory response to an energy crisis induced by Complex I genetic ablation or pharmacological inhibition is an increase in the mitochondrial biogenesis master regulator PGC1α, a pleiotropic coactivator of transcription regulating diverse biological processes within the cell. We associate this compensatory response to the increase in PGC1α target gene expression, setting the basis for the comprehension of the molecular pathways triggered by Complex I inhibition that may need attention as drawbacks before these approaches are implemented in ovarian cancer care. ispartof: SCIENTIFIC REPORTS vol:12 issue:1 ispartof: location:England status: published

Converting carcinomas in benign oncocytomas has been suggested as a potential anti-cancer strategy. One of the oncocytoma hallmarks is the lack of respiratory complex I (CI). Here we use genetic ablation of this enzyme to induce indolence in two cancer types, and show this is reversed by allowing the stabilization of Hypoxia Inducible Factor-1 alpha (HIF-1α). We further show that on the long run CI-deficient tumors re-adapt to their inability to respond to hypoxia, concordantly with the persistence of human oncocytomas. We demonstrate that CI-deficient tumors survive and carry out angiogenesis, despite their inability to stabilize HIF-1α. Such adaptive response is mediated by tumor associated macrophages, whose blockage improves the effect of CI ablation. Additionally, the simultaneous pharmacological inhibition of CI function through metformin and macrophage infiltration through PLX-3397 impairs tumor growth in vivo in a synergistic manner, setting the basis for an efficient combinatorial adjuvant therapy in clinical trials., Lack of respiratory complex I is a hallmark of oncocytomas. Here the authors show that inactivation of this complex via knockout of the NDUFS3 subunit or using metformin, converts tumors from an aggressive phenotype into low-proliferative oncocytomas, which can be further inhibited by targeting pro-tumorigenic macrophages.

MYC-mediated cell competition is a cell-cell interaction mechanism known to play an evolutionary role during development from Drosophila to mammals. Cells expressing low levels of MYC, called losers, are committed to die by nearby cells with high MYC activity, called winners, that overproliferate to compensate for cell loss, so that the fittest cells be selected for organ formation. Given MYC’s consolidated role in oncogenesis, cell competition is supposed to be relevant to cancer, but its significance in human malignant contexts is largely uncharacterised. Here we show stereotypical patterns of MYC-mediated cell competition in human cancers: MYC-upregulating cells and apoptotic cells were indeed repeatedly found at the tumour-stroma interface and within the tumour parenchyma. Cell death amount in the stromal compartment and MYC protein level in the tumour were highly correlated regardless of tumour type and stage. Moreover, we show that MYC modulation in heterotypic co-cultures of human cancer cells is sufficient as to subvert their competitive state, regardless of genetic heterogeneity. Altogether, our findings suggest that the innate role of MYC-mediated cell competition in development is conserved in human cancer, with malignant cells using MYC activity to colonise the organ at the expense of less performant neighbours.

none 4 no Deregulation of MYC family proteins in cancer is associated with a global reprogramming of gene expression, ultimately promoting glycolytic pathways, cell growth, and proliferation. It is well known that MYC upregulation triggers cell-autonomous apoptosis in normal tissues, while frankly malignant cells develop resistance to apoptotic stimuli, partly resulting from MYC addiction. As well as inducing cell-autonomous apoptosis, MYC upregulation is able to trigger non cell-autonomous apoptotic death through an evolutionarily conserved mechanism known as "cell competition". With regard to this intimate and dual relationship between MYC and cell death, recent evidence obtained in Drosophila models of cancer has revealed that, in early tumourigenesis, MYC upregulation guides the clonal expansion of mutant cells, while the surrounding tissue undergoes non-cell autonomous death. Apoptosis inhibition in this context was shown to restrain tumour growth and to restore a wild-type phenotype. This suggests that cell-autonomous and non cell-autonomous apoptosis dependent on MYC upregulation may shape tumour growth in different ways, soliciting the need to reconsider the role of cell death in cancer in the light of this new level of complexity. Here we review recent literature about MYC and cell competition obtained in Drosophila, with a particular emphasis on the relevance of cell death to cell competition and, more generally, to cancer. Possible implications of these findings for the understanding of mammalian cancers are also discussed. open Di Giacomo, Simone; Sollazzo, Manuela; Paglia, Simona; Grifoni, Daniela Di Giacomo, Simone; Sollazzo, Manuela; Paglia, Simona; Grifoni, Daniela

The thriving field that constitutes cancer metabolism has unveiled some groundbreaking facts over the past two decades, at the heart of which is the TCA cycle and its intermediates. As such and besides its metabolic role, α-ketoglutarate was shown to withstand a wide range of physiological reactions from protection against oxidative stress, collagen and bone maintenance to development and immunity. Most importantly, it constitutes the rate-limiting substrate of 2-oxoglutarate-dependent dioxygenases family enzymes, which are involved in hypoxia sensing and in the shaping of cellular epigenetic landscape, two major drivers of oncogenic transformation. Based on literature reports, we hereby review the benefits of this metabolite as a possible novel adjuvant therapeutic opportunity to target tumor progression. This article is part of the special issue "Mitochondrial metabolic alterations in cancer cells and related therapeutic targets".