Low BCL-xL expression in triple-negative breast cancer cells favors chemotherapy efficacy, and this effect is limited by cancer-associated fibroblasts.

Triple negative breast cancers (TNBC) present a poor prognosis primarily due to their resistance to chemotherapy. This resistance is known to be associated with elevated expression of certain anti-apoptotic members within the proteins of the BCL-2 family (namely BCL-xL, MCL-1 and BCL-2). These regulate cell death by inhibiting pro-apoptotic protein activation through binding and sequestration and they can be selectively antagonized by BH3 mimetics. Yet the individual influences of BCL-xL, MCL-1, and BCL-2 on the sensitivity of TNBC cells to chemotherapy, and their regulation by cancer-associated fibroblasts (CAFs), major components of the tumor stroma and key contributors to therapy resistance remain to be delineated. Using gene editing or BH3 mimetics to inhibit anti-apoptotic BCL-2 family proteins in TNBC line MDA-MB-231, we show that BCL-xL and MCL-1 promote cancer cell survival through compensatory mechanisms. This cell line shows limited sensitivity to chemotherapy, in line with the clinical resistance observed in TNBC patients. We elucidate that BCL-xL plays a pivotal role in therapy response, as its depletion or pharmacological inhibition heightened chemotherapy effectiveness. Moreover, BCL-xL expression is associated with chemotherapy resistance in patient-derived tumoroids where its pharmacological inhibition enhances ex vivo response to chemotherapy. In a co-culture model of cancer cells and CAFs, we observe that even in a context where BCL-xL reduced expression renders cancer cells more susceptible to chemotherapy, those in contact with CAFs display reduced sensitivity to chemotherapy. Thus CAFs exert a profound pro-survival effect in breast cancer cells, even in a setting highly favoring cell death through combined chemotherapy and absence of the main actor of chemoresistance, BCL-xL.

Mesenchymal-like immune-altered is the fourth robust triple-negative breast cancer molecular subtype.

BACKGROUND: Robust molecular subtyping of triple-negative breast cancer (TNBC) is a prerequisite for the success of precision medicine. Today, there is a clear consensus on three TNBC molecular subtypes: luminal androgen receptor (LAR), basal-like immune-activated (BLIA), and basal-like immune-suppressed (BLIS). However, the debate about the robustness of other subtypes is still open. METHODS: An unprecedented number (n = 1942) of TNBC patient data was collected. Microarray- and RNAseq-based cohorts were independently investigated. Unsupervised analyses were conducted using k-means consensus clustering. Clusters of patients were then functionally annotated using different approaches. Prediction of response to chemotherapy and targeted therapies, immune checkpoint blockade, and radiotherapy were also screened for each TNBC subtype. RESULTS: Four TNBC subtypes were identified in the cohort: LAR (19.36%); mesenchymal stem-like (MSL/MES) (17.35%); BLIA (31.06%); and BLIS (32.23%). Regarding the MSL/MES subtype, we suggest renaming it to mesenchymal-like immune-altered (MLIA) to emphasize its specific histological background and nature of immune response. Treatment response prediction results show, among other things, that despite immune activation, immune checkpoint blockade is probably less or completely ineffective in MLIA, possibly caused by mesenchymal background and/or an enrichment in dysfunctional cytotoxic T lymphocytes. TNBC subtyping results were included in the bc-GenExMiner v5.0 webtool ( http://bcgenex.ico.unicancer.fr ). CONCLUSION: The mesenchymal TNBC subtype is characterized by an exhausted and altered immune response, and resistance to immune checkpoint inhibitors. Consensus for molecular classification of TNBC subtyping and prediction of cancer treatment responses helps usher in the era of precision medicine for TNBC patients.

Stress in the metastatic journey - the role of cell communication and clustering in breast cancer progression and treatment resistance.

Breast cancer stands as the most prevalent malignancy afflicting women. Despite significant advancements in its diagnosis and treatment, breast cancer metastasis continues to be a leading cause of mortality among women. To metastasize, cancer cells face numerous challenges: breaking away from the primary tumor, surviving in the circulation, establishing in a distant location, evading immune detection and, finally, thriving to initiate a new tumor. Each of these sequential steps requires cancer cells to adapt to a myriad of stressors and develop survival mechanisms. In addition, most patients with breast cancer undergo surgical removal of their primary tumor and have various therapeutic interventions designed to eradicate cancer cells. Despite this plethora of attacks and stresses, certain cancer cells not only manage to persist but also proliferate robustly, giving rise to substantial tumors that frequently culminate in the patient's demise. To enhance patient outcomes, there is an imperative need for a deeper understanding of the molecular and cellular mechanisms that empower cancer cells to not only survive but also expand. Herein, we delve into the intrinsic stresses that cancer cells encounter throughout the metastatic journey and the additional stresses induced by therapeutic interventions. We focus on elucidating the remarkable strategies adopted by cancer cells, such as cell-cell clustering and intricate cell-cell communication mechanisms, to ensure their survival.

EMT and primary ciliogenesis: For better or worse in sickness and in health.

Epithelial-mesenchymal transition (EMT) and primary ciliogenesis are two cell-biological programs that are essential for development of multicellular organisms and whose abnormal regulation results in many diseases (i.e., developmental anomalies and cancers). Emerging studies suggest an intricate interplay between these two processes. Here, we discuss physiological and pathological contexts in which their interconnections promote normal development or disease progression. We describe underlying molecular mechanisms of the interplay and EMT/ciliary signaling axes that influence EMT-related processes (i.e., stemness, motility and invasion). Understanding the molecular and cellular mechanisms of the relationship between EMT and primary ciliogenesis may provide new insights in the etiology of diseases related to EMT and cilia dysfunction.

ChemR23 activation reprograms macrophages toward a less inflammatory phenotype and dampens carcinoma progression.

Introduction: Tumor Associated Macrophages (TAM) are a major component of the tumor environment and their accumulation often correlates with poor prognosis by contributing to local inflammation, inhibition of anti-tumor immune response and resistance to anticancer treatments. In this study, we thus investigated the anti-cancer therapeutic interest to target ChemR23, a receptor of the resolution of inflammation expressed by macrophages, using an agonist monoclonal antibody, αChemR23. Methods: Human GM-CSF, M-CSF and Tumor Associated Macrophage (TAM)-like macrophages were obtained by incubation of monocytes from healthy donors with GM-CSF, M-CSF or tumor cell supernatants (Breast cancer (BC) or malignant pleural mesothelioma (MPM) cells). The effects of αChemR23 on macrophages were studied at the transcriptomic, protein and functional level. Datasets from The Cancer Genome Atlas (TCGA) were used to study CMKLR1 expression, coding for ChemR23, in BC and MPM tumors. In vivo, αChemR23 was evaluated on overall survival, metastasis development and transcriptomic modification of the metastatic niche using a model of resected triple negative breast cancer. Results: We show that ChemR23 is expressed at higher levels in M-CSF and tumor cell supernatant differentiated macrophages (TAM-like) than in GM-CSF-differentiated macrophages. ChemR23 activation triggered by αChemR23 deeply modulates M-CSF and TAM-like macrophages including profile of cell surface markers, cytokine secretion, gene mRNA expression and immune functions. The expression of ChemR23 coding gene (CMKLR1) strongly correlates to TAM markers in human BC tumors and MPM and its histological detection in these tumors mainly corresponds to TAM expression. In vivo, treatment with αChemR23 agonist increased mouse survival and decreased metastasis occurrence in a model of triple-negative BC in correlation with modulation of TAM phenotype in the metastatic niche. Conclusion: These results open an attractive opportunity to target TAM and the resolution of inflammation pathways through ChemR23 to circumvent TAM pro-tumoral effects.

Using mammary organoids to study cilia.

Cilia are hair-like projections that assemble at the surface of cells in various tissues of multicellular organisms through a complex cell biological process called ciliogenesis. Cilia can assemble as single structures per cell (i.e. non-motile primary cilia), which act as cell signaling centers that dictate cell fate, or can be assembled in distinct cell types as many copies per cell (i.e. motile cilia) that beat to move fluids at the cell surface. The mechanisms that orchestrate formation and function of cilia, which are dysregulated in pathological settings such as ciliopathies, remain incompletely understood. Stem cell-derived organoids represent valuable models to study the mechanisms of ciliogenesis, ciliary signaling, and ciliary beating that collectively promote tissue development and homeostasis. Here, we present a comprehensive protocol for the growth of mammary organoids derived from mouse mammary stem cells and for immunofluorescence staining of primary cilia in these three-dimensional structures.

Targeting of MCL-1 in breast cancer-associated fibroblasts reverses their myofibroblastic phenotype and pro-invasive properties.

Cancer-associated fibroblasts (CAF) are a major cellular component of epithelial tumors. In breast cancers in particular these stromal cells have numerous tumorigenic effects in part due to their acquisition of a myofibroblastic phenotype. Breast CAFs (bCAFs) typically express MCL-1. We show here that pharmacological inhibition or knock down of this regulator of mitochondrial integrity in primary bCAFs directly derived from human samples mitigates myofibroblastic features. This decreases expression of genes involved in actomyosin organization and contractility (associated with a cytoplasmic retention of the transcriptional regulator, yes-associated protein-YAP) and decreases bCAFs ability to promote cancer cells invasion in 3D coculture assays. Our findings underscore the usefulness of targeting MCL-1 in breast cancer ecosystems, not only to favor death of cancer cells but also to counteract the tumorigenic activation of fibroblasts with which they co-evolve. Mechanistically, pharmacological inhibition of MCL-1 with a specific BH3 mimetic promotes mitochondrial fragmentation in bCAFs. Inhibition of the mitochondrial fission activity of DRP-1, which interacts with MCL-1 upon BH3 mimetic treatment, allows the maintenance of the myofibroblastic phenotype of bCAFs.

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network.

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

An EMT-primary cilium-GLIS2 signaling axis regulates mammogenesis and claudin-low breast tumorigenesis.

The epithelial-mesenchymal transition (EMT) and primary ciliogenesis induce stem cell properties in basal mammary stem cells (MaSCs) to promote mammogenesis, but the underlying mechanisms remain incompletely understood. Here, we show that EMT transcription factors promote ciliogenesis upon entry into intermediate EMT states by activating ciliogenesis inducers, including FGFR1. The resulting primary cilia promote ubiquitination and inactivation of a transcriptional repressor, GLIS2, which localizes to the ciliary base. We show that GLIS2 inactivation promotes MaSC stemness, and GLIS2 is required for normal mammary gland development. Moreover, GLIS2 inactivation is required to induce the proliferative and tumorigenic capacities of the mammary tumor­initiating cells (MaTICs) of claudin-low breast cancers. Claudin-low breast tumors can be segregated from other breast tumor subtypes based on a GLIS2-dependent gene expression signature. Collectively, our findings establish molecular mechanisms by which EMT programs induce ciliogenesis to control MaSC and MaTIC stemness, mammary gland development, and claudin-low breast cancer formation.

Synthesis and biological studies of new piperidino-1,2,3-triazole hybrids with 3-aryl isoxazole side chains.

A small library of new piperidine-triazole hybrids with 3-aryl isoxazole side chains has been designed and synthesized. Their cytotoxicity against a panel of seven cancer cell lines has been established. For the most promising compound, an IC50 value of 3.8 µM on PUMA/Bcl-xL interaction in live cancer cells was established through BRET analysis. A rationale was proposed for these results through complete molecular modelling studies.

[Interest of the bc-GenExMiner web tool in oncology]. / Intérêt de l'outil web bc-GenExMiner en oncologie.

We are taking advantage of the launch of the latest version (v4.6) of our web-based data mining tool "breast cancer gene-expression miner" (bc-GenExMiner) to take stock of its position within the oncology research landscape and to present an activity report ten years after its establishment (http://bcgenex.ico.unicancer.fr). bc-GenExMiner is an open-access, user-friendly tool for statistical mining on breast tumor transcriptomes, annotated with more than 20 clinicopathologic and molecular characteristics. The database comprises more than 16,000 patients from 64 cohorts - including TCGA, METABRIC and SCAN-B - for whom several thousands of genes have been quantified by microarrays or RNA-seq. Correlation, expression and prognostic analyses are available for targeted, exhaustive or customized explorations of queried genes. bc-GenExMiner facilitates the validation, investigation, and prioritization of discoveries and hypotheses on genes of interest. It allows users to analyse large databases, create data visualizations, and obtain robust statistical analysis, thereby accelerating biomarker discovery. Ten years after its launch, judging by the number of visits, analyses, and scientific citations of bc-GenExMiner, we conclude that this web resource serves its purpose in the international scientific community working in breast cancer research, with a never-ending rise in its use.

bc-GenExMiner 4.5: new mining module computes breast cancer differential gene expression analyses.

'Breast cancer gene-expression miner' (bc-GenExMiner) is a breast cancer-associated web portal (http://bcgenex.ico.unicancer.fr). Here, we describe the development of a new statistical mining module, which permits several differential gene expression analyses, i.e. 'Expression' module. Sixty-two breast cancer cohorts and one healthy breast cohort with their corresponding clinicopathological information are included in bc-GenExMiner v4.5 version. Analyses are based on microarray or RNAseq transcriptomic data. Thirty-nine differential gene expression analyses, grouped into 13 categories, according to clinicopathological and molecular characteristics ('Targeted' and 'Exhaustive') and gene expression ('Customized'), have been developed. Output results are visualized in four forms of plots. This new statistical mining module offers, among other things, the possibility to compare gene expression in healthy (cancer-free), tumour-adjacent and tumour tissues at once and in three triple-negative breast cancer subtypes (i.e. C1: molecular apocrine tumours; C2: basal-like tumours infiltrated by immune suppressive cells and C3: basal-like tumours triggering an ineffective immune response). Several validation tests showed that bioinformatics process did not alter the pathobiological information contained in the source data. In this work, we developed and demonstrated that bc-GenExMiner 'Expression' module can be used for exploratory and validation purposes. Database URL: http://bcgenex.ico.unicancer.fr.

Random forest of perfect trees: concept, performance, applications and perspectives.

MOTIVATION: The principle of Breiman's random forest (RF) is to build and assemble complementary classification trees in a way that maximizes their variability. We propose a new type of random forest that disobeys Breiman's principles and involves building trees with no classification errors in very large quantities. We used a new type of decision tree that uses a neuron at each node as well as an in-innovative half Christmas tree structure. With these new RFs, we developed a score, based on a family of ten new statistical information criteria, called Nguyen information criteria (NICs), to evaluate the predictive qualities of features in three dimensions. RESULTS: The first NIC allowed the Akaike information criterion to be minimized more quickly than data obtained with the Gini index when the features were introduced in a logistic regression model. The selected features based on the NICScore showed a slight advantage compared to the support vector machines-recursive feature elimination (SVM-RFE) method. We demonstrate that the inclusion of artificial neurons in tree nodes allows a large number of classifiers in the same node to be taken into account simultaneously and results in perfect trees without classification errors. AVAILABILITY AND IMPLEMENTATION: The methods used to build the perfect trees in this article were implemented in the 'ROP' R package, archived at https://cran.r-project.org/web/packages/ROP/index.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Development of an absolute assignment predictor for triple-negative breast cancer subtyping using machine learning approaches.

Triple-negative breast cancer (TNBC) heterogeneity represents one of the main obstacles to precision medicine for this disease. Recent concordant transcriptomics studies have shown that TNBC could be divided into at least three subtypes with potential therapeutic implications. Although a few studies have been conducted to predict TNBC subtype using transcriptomics data, the subtyping was partially sensitive and limited by batch effect and dependence on a given dataset, which may penalize the switch to routine diagnostic testing. Therefore, we sought to build an absolute predictor (i.e., intra-patient diagnosis) based on machine learning algorithms with a limited number of probes. To that end, we started by introducing probe binary comparison for each patient (indicators). We based the predictive analysis on this transformed data. Probe selection was first involved combining both filter and wrapper methods for variable selection using cross-validation. We tested three prediction models (random forest, gradient boosting [GB], and extreme gradient boosting) using this optimal subset of indicators as inputs. Nested cross-validation consistently allowed us to choose the best model. The results showed that the fifty selected indicators highlighted the biological characteristics associated with each TNBC subtype. The GB based on this subset of indicators performs better than other models.

Mitochondria at Center of Exchanges between Cancer Cells and Cancer-Associated Fibroblasts during Tumor Progression.

Resistance of solid cancer cells to chemotherapies and targeted therapies is not only due to the mutational status of cancer cells but also to the concurring of stromal cells of the tumor ecosystem, such as immune cells, vasculature and cancer-associated fibroblasts (CAFs). The reciprocal education of cancer cells and CAFs favors tumor growth, survival and invasion. Mitochondrial function control, including the regulation of mitochondrial metabolism, oxidative stress and apoptotic stress are crucial for these different tumor progression steps. In this review, we focus on how CAFs participate in cancer progression by modulating cancer cells metabolic functions and mitochondrial apoptosis. We emphasize that mitochondria from CAFs influence their activation status and pro-tumoral effects. We thus advocate that understanding mitochondria-mediated tumor-stroma interactions provides the possibility to consider cancer therapies that improve current treatments by targeting these interactions or mitochondria directly in tumor and/or stromal cells.

Targeting of BCL-2 Family Members during Anticancer Treatment: A Necessary Compromise between Individual Cell and Ecosystemic Responses?

The imbalance between BCL-2 homologues and pro-death counterparts frequently noted in cancer cells endows them with a cell autonomous survival advantage. To eradicate ectopic cells, inhibitors of these homologues (BH3 mimetics) were developed to trigger, during anticancer treatment, full activation of the canonical mitochondrial apoptotic pathway and related caspases. Despite efficiency in some clinical settings, these compounds do not completely fulfill their initial promise. We herein put forth that a growing body of evidence indicates that mitochondrial integrity, controlled by BCL-2 family proteins, and downstream caspases regulate other cell death modes and influence extracellular signaling by committed cells. Moreover, intercellular communications play a key role in spreading therapeutic response across cancer cell populations and in engaging an immune response. We thus advocate that BH3 mimetics administration would be more efficient in the long term if it did not induce apoptosis in all sensitive cells at the same time, but if it could instead allow (or trigger) death signal production by non-terminally committed dying cell populations. The development of such a trade-off strategy requires to unravel the effects of BH3 mimetics not only on each individual cancer cell but also on homotypic and heterotypic cell interactions in dynamic tumor ecosystems.

Mitotic stress-induced secretome primes cancer cells to apoptosis and maximizes paclitaxel response in breast tumors when combined with BCL-xL-targeting BH3 mimetics.

We recently identified a previously unappreciated ability of antimitotics to propagate apoptotic priming across cancer cell populations. The underlying paracrine cytotoxic signal, fueled by undead cells activating the cGAS/STING pathway, is required for in vivo antitumor response and it can be further exploited by delayed, but not synchronous, BCL-xL inhibition.

STING-dependent paracriny shapes apoptotic priming of breast tumors in response to anti-mitotic treatment.

A fascinating but uncharacterized action of antimitotic chemotherapy is to collectively prime cancer cells to apoptotic mitochondrial outer membrane permeabilization (MOMP), while impacting only on cycling cell subsets. Here, we show that a proapoptotic secretory phenotype is induced by activation of cGAS/STING in cancer cells that are hit by antimitotic treatment, accumulate micronuclei and maintain mitochondrial integrity despite intrinsic apoptotic pressure. Organotypic cultures of primary human breast tumors and patient-derived xenografts sensitive to paclitaxel exhibit gene expression signatures typical of type I IFN and TNFα exposure. These cytokines induced by cGAS/STING activation trigger NOXA expression in neighboring cells and render them acutely sensitive to BCL-xL inhibition. cGAS/STING-dependent apoptotic effects are required for paclitaxel response in vivo, and they are amplified by sequential, but not synchronous, administration of BH3 mimetics. Thus anti-mitotic agents propagate apoptotic priming across heterogeneously sensitive cancer cells through cytosolic DNA sensing pathway-dependent extracellular signals, exploitable by delayed MOMP targeting.

Targeting PUMA/Bcl-xL interaction by new specific compounds to unleash apoptotic process in cancer cells.

We describe the first examples of small molecules able to disrupt the nanomolar interaction between the pro-apoptotic protein PUMA and its anti-apoptotic counterpart BcL-xL in malignant cells. Based on molecular modelling studies, we propose a rationale to this result, through a new "bottle-opener"-type strategy which could be of general use in the area of protein-protein interaction studies.