Activation of lysosomal iron triggers ferroptosis in cancer.

Iron catalyses the oxidation of lipids in biological membranes and promotes a form of cell death referred to as ferroptosis 1-3 . Identifying where this chemistry takes place in the cell can inform the design of drugs capable of inducing or inhibiting ferroptosis in various disease-relevant settings. Whereas genetic approaches have revealed underlying mechanisms of lipid peroxide detoxification 1,4,5 , small molecules can provide unparalleled spatiotemporal control of the chemistry at work 6 . Here, we show that the ferroptosis inhibitor liproxstatin-1 (Lip-1) exerts a protective activity by inactivating iron in lysosomes. Based on this, we designed the bifunctional compound fentomycin that targets phospholipids at the plasma membrane and activates iron in lysosomes upon endocytosis, promoting oxidative degradation of phospholipids and ferroptosis. Fentomycin effectively kills primary sarcoma and pancreatic ductal adenocarcinoma cells. It acts as a lipolysis-targeting chimera (LIPTAC), preferentially targeting iron-rich CD44 high cell-subpopulations 7,8 associated with the metastatic disease and drug resistance 9,10 . Furthermore, we demonstrate that fentomycin also depletes CD44 high cells in vivo and reduces intranodal tumour growth in an immunocompetent murine model of breast cancer metastasis. These data demonstrate that lysosomal iron triggers ferroptosis and that lysosomal iron redox chemistry can be exploited for therapeutic benefits.

A novel bioinformatic approach reveals cooperation between Cancer/Testis genes in basal-like breast tumors.

Breast cancer is the most prevalent type of cancer in women worldwide. Within breast tumors, the basal-like subtype has the worst prognosis, prompting the need for new tools to understand, detect, and treat these tumors. Certain germline-restricted genes show aberrant expression in tumors and are known as Cancer/Testis genes; their misexpression has diagnostic and therapeutic applications. Here we designed a new bioinformatic approach to examine Cancer/Testis gene misexpression in breast tumors. We identify several new markers in Luminal and HER-2 positive tumors, some of which predict response to chemotherapy. We then use machine learning to identify the two Cancer/Testis genes most associated with basal-like breast tumors: HORMAD1 and CT83. We show that these genes are expressed by tumor cells and not by the microenvironment, and that they are not expressed by normal breast progenitors; in other words, their activation occurs de novo. We find these genes are epigenetically repressed by DNA methylation, and that their activation upon DNA demethylation is irreversible, providing a memory of past epigenetic disturbances. Simultaneous expression of both genes in breast cells in vitro has a synergistic effect that increases stemness and activates a transcriptional profile also observed in double-positive tumors. Therefore, we reveal a functional cooperation between Cancer/Testis genes in basal breast tumors; these findings have consequences for the understanding, diagnosis, and therapy of the breast tumors with the worst outcomes.

Experimental and spontaneous metastasis assays can result in divergence in clonal architecture.

Intratumoural heterogeneity is associated with poor outcomes in breast cancer. To understand how malignant clones survive and grow in metastatic niches, in vivo models using cell lines and patient-derived xenografts (PDX) have become the gold standard. Injections of cancer cells in orthotopic sites (spontaneous metastasis assays) or into the vasculature (experimental metastasis assays) have been used interchangeably to study the metastatic cascade from early events or post-intravasation, respectively. However, less is known about how these different routes of injection impact heterogeneity. Herein we directly compared the clonality of spontaneous and experimental metastatic assays using the human cell line MDA-MB-231 and a PDX model. Genetic barcoding was used to study the fitness of the subclones in primary and metastatic sites. Using spontaneous assays, we found that intraductal injections resulted in less diverse tumours compared to other routes of injections. Using experimental metastasis assays via tail vein injection of barcoded MDA-MB-231 cells, we also observed an asymmetry in metastatic heterogeneity between lung and liver that was not observed using spontaneous metastasis assays. These results demonstrate that these assays can result in divergent clonal outputs in terms of metastatic heterogeneity and provide a better understanding of the biases inherent to each technique.

MiR-662 is associated with metastatic relapse in early-stage breast cancer and promotes metastasis by stimulating cancer cell stemness.

BACKGROUND: Breast cancer (BC) metastasis, which often occurs in bone, contributes substantially to mortality. MicroRNAs play a fundamental role in BC metastasis, although microRNA-regulated mechanisms driving metastasis progression remain poorly understood. METHODS: MiRome analysis in serum from BC patients was performed by TaqMan™ low-density array. MiR-662 was overexpressed following MIMIC-transfection or lentivirus transduction. Animal models were used to investigate the role of miR-662 in BC (bone) metastasis. The effect of miR-662-overexpressing BC cell conditioned medium on osteoclastogenesis was investigated. ALDEFLUOR assays were performed to study BC stemness. RNA-sequencing transcriptomic analysis of miR-662-overexpressing BC cells was performed to evaluate gene expression changes. RESULTS: High levels of hsa-miR-662 (miR-662) in serum from BC patients, at baseline (time of surgery), were associated with future recurrence in bone. At an early-stage of the metastatic disease, miR-662 could mask the presence of BC metastases in bone by inhibiting the differentiation of bone-resorbing osteoclasts. Nonetheless, metastatic miR-662-overexpressing BC cells then progressed as overt osteolytic metastases thanks to increased stem cell-like traits. CONCLUSIONS: MiR-662 is involved in BC metastasis progression, suggesting it may be used as a prognostic marker to identify BC patients at high risk of metastasis.

Disruption of lineage integrity as a precursor to breast tumor initiation.

Increase in lineage infidelity and/or imbalance is frequently observed around the earliest stage of breast tumor initiation. In response to disruption of homeostasis, differentiated cells can partially lose their identity and gain cellular plasticity, a process involving epigenome landscape remodeling. This increase of cellular plasticity may promote the malignant transformation of breast tumors and fuel their heterogeneity. Here, we review recent studies that have yield insights into important regulators of lineage integrity and mechanisms that trigger mammary epithelial lineage derail, and evaluate their impacts on breast tumor development.

Production of CRISPRi-engineered primary human mammary epithelial cells with baboon envelope pseudotyped lentiviral vectors.

Primary human mammary epithelial cells (pHMECs) are known to be remarkably difficult to engineer genetically. Here, we present a protocol for efficient transduction of pHMECs using a baboon retroviral envelope glycoprotein for pseudotyping of lentiviral vectors (BaEV-LVs). We describe the preparation of the BaEV-LVs, the isolation of pHMECs from breast samples, and the subsequent transduction of pHMECs. We also detail the use of CRISPRi technology to efficiently silence gene expression in pHMECs, which can then be used for functional assays. For complete details on the use and execution of this protocol, please refer to Richart et al. (2022).1.

Genome-wide RNA interference screen in cancer stem cells.

Tumor heterogeneity represents a major hurdle for therapy. This cellular heterogeneity is mainly sustained by different subpopulations of tumorigenic cells, the so-called cancer stem cells (CSCs). CSCs burden is associated with disease progression and patient poor prognosis. In this context, deciphering molecular mechanisms regulating stemness is a key step in the development of new therapeutic strategy. Here, we provide a detailed protocol for high-throughput screening (HTS) strategy to detect modulators of CSC proportion. It is based on a miniaturized ALDEFLUOR-probed CSC assay quantitated by high-content imaging, that allows monitoring the changes in CSC proportions in response to gene silencing. Gene loss-of-function is achieved by transfecting a genome-wide RNA interference library. These genome-wide HTS strategies could lead to the identification of new therapeutic approaches in the treatment of various cancers.

Computational Screening of Anti-Cancer Drugs Identifies a New BRCA Independent Gene Expression Signature to Predict Breast Cancer Sensitivity to Cisplatin.

The development of therapies that target specific disease subtypes has dramatically improved outcomes for patients with breast cancer. However, survival gains have not been uniform across patients, even within a given molecular subtype. Large collections of publicly available drug screening data matched with transcriptomic measurements have facilitated the development of computational models that predict response to therapy. Here, we generated a series of predictive gene signatures to estimate the sensitivity of breast cancer samples to 90 drugs, comprising FDA-approved drugs or compounds in early development. To achieve this, we used a cell line-based drug screen with matched transcriptomic data to derive in silico models that we validated in large independent datasets obtained from cell lines and patient-derived xenograft (PDX) models. Robust computational signatures were obtained for 28 drugs and used to predict drug efficacy in a set of PDX models. We found that our signature for cisplatin can be used to identify tumors that are likely to respond to this drug, even in absence of the BRCA-1 mutation routinely used to select patients for platinum-based therapies. This clinically relevant observation was confirmed in multiple PDXs. Our study foreshadows an effective delivery approach for precision medicine.

XIST loss impairs mammary stem cell differentiation and increases tumorigenicity through Mediator hyperactivation.

X inactivation (XCI) is triggered by upregulation of XIST, which coats the chromosome in cis, promoting formation of a heterochromatic domain (Xi). XIST role beyond initiation of XCI is only beginning to be elucidated. Here, we demonstrate that XIST loss impairs differentiation of human mammary stem cells (MaSCs) and promotes emergence of highly tumorigenic and metastatic carcinomas. On the Xi, XIST deficiency triggers epigenetic changes and reactivation of genes overlapping Polycomb domains, including Mediator subunit MED14. MED14 overdosage results in increased Mediator levels and hyperactivation of the MaSC enhancer landscape and transcriptional program, making differentiation less favorable. We further demonstrate that loss of XIST and Xi transcriptional instability is common among human breast tumors of poor prognosis. We conclude that XIST is a gatekeeper of human mammary epithelium homeostasis, thus unveiling a paradigm in the control of somatic cell identity with potential consequences for our understanding of gender-specific malignancies.

BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells.

Replication stress (RS) has a pivotal role in tumor initiation, progression, or therapeutic resistance. In this study, we depicted the mechanism of breast cancer stem cells' (bCSCs) response to RS and its clinical implication. We demonstrated that bCSCs present a limited level of RS compared with non-bCSCs in patient samples. We described for the first time that the spatial nuclear location of BMI1 protein triggers RS response in breast cancers. Hence, in bCSCs, BMI1 is rapidly located to stalled replication forks to recruit RAD51 and activate homologous-recombination machinery, whereas in non-bCSCs BMI1 is trapped on demethylated 1q12 megasatellites precluding effective RS response. We further demonstrated that BMI1/RAD51 axis activation is necessary to prevent cisplatin-induced DNA damage and that treatment of patient-derived xenografts with a RAD51 inhibitor sensitizes tumor-initiating cells to cisplatin. The comprehensive view of replicative-stress response in bCSC has profound implications for understanding and improving therapeutic resistance.

CD95/Fas suppresses NF-κB activation through recruitment of KPC2 in a CD95L/FasL-independent mechanism.

CD95 expression is preserved in triple-negative breast cancers (TNBCs), and CD95 loss in these cells triggers the induction of a pro-inflammatory program, promoting the recruitment of cytotoxic NK cells impairing tumor growth. Herein, we identify a novel interaction partner of CD95, Kip1 ubiquitination-promoting complex protein 2 (KPC2), using an unbiased proteomic approach. Independently of CD95L, CD95/KPC2 interaction contributes to the partial degradation of p105 (NF-κB1) and the subsequent generation of p50 homodimers, which transcriptionally represses NF-κB-driven gene expression. Mechanistically, KPC2 interacts with the C-terminal region of CD95 and serves as an adaptor to recruit RelA (p65) and KPC1, which acts as E3 ubiquitin-protein ligase promoting the degradation of p105 into p50. Loss of CD95 in TNBC cells releases KPC2, limiting the formation of the NF-κB inhibitory homodimer complex (p50/p50), promoting NF-κB activation and the production of pro-inflammatory cytokines, which might contribute to remodeling the immune landscape in TNBC cells.

CD95/Fas protects triple negative breast cancer from anti-tumor activity of NK cells.

The apoptosis inducing receptor CD95/Fas has multiple tumorigenic activities. In different genetically engineered mouse models tumor-expressed CD95 was shown to be critical for cell growth. Using a combination of immune-deficient and immune-competent mouse models, we now establish that loss of CD95 in metastatic triple negative breast cancer (TNBC) cells prevents tumor growth by modulating the immune landscape. CD95-deficient, but not wild-type, tumors barely grow in an immune-competent environment and show an increase in immune infiltrates into the tumor. This growth reduction is caused by infiltrating NK cells and does not involve T cells or macrophages. In contrast, in immune compromised mice CD95 k.o. cells are not growth inhibited, but they fail to form metastases. In summary, we demonstrate that in addition to its tumor and metastasis promoting activities, CD95 expression by tumor cells can exert immune suppressive activities on NK cells, providing a new target for immune therapy.

A stem cell population at the anorectal junction maintains homeostasis and participates in tissue regeneration.

At numerous locations of the body, transition zones are localized at the crossroad between two types of epithelium and are frequently associated with neoplasia involving both type of tissues. These transition zones contain cells expressing markers of adult stem cells that can be the target of early transformation. The mere fact that transition zone cells can merge different architecture with separate functions implies for a unique plasticity that these cells must display in steady state. However, their roles during tissue regeneration in normal and injured state remain unknown. Here, by using in vivo lineage tracing, single-cell transcriptomics, computational modeling and a three-dimensional organoid culture system of transition zone cells, we identify a population of Krt17+ basal cells with multipotent properties at the squamo-columnar anorectal junction that maintain a squamous epithelium during normal homeostasis and can participate in the repair of a glandular epithelium following tissue injury.

CD44 regulates epigenetic plasticity by mediating iron endocytosis.

CD44 is a transmembrane glycoprotein linked to various biological processes reliant on epigenetic plasticity, which include development, inflammation, immune responses, wound healing and cancer progression. Although it is often referred to as a cell surface marker, the functional regulatory roles of CD44 remain elusive. Here we report the discovery that CD44 mediates the endocytosis of iron-bound hyaluronates in tumorigenic cell lines, primary cancer cells and tumours. This glycan-mediated iron endocytosis mechanism is enhanced during epithelial-mesenchymal transitions, in which iron operates as a metal catalyst to demethylate repressive histone marks that govern the expression of mesenchymal genes. CD44 itself is transcriptionally regulated by nuclear iron through a positive feedback loop, which is in contrast to the negative regulation of the transferrin receptor by excess iron. Finally, we show that epigenetic plasticity can be altered by interfering with iron homeostasis using small molecules. This study reveals an alternative iron-uptake mechanism that prevails in the mesenchymal state of cells, which illuminates a central role of iron as a rate-limiting regulator of epigenetic plasticity.

miRViz: a novel webserver application to visualize and interpret microRNA datasets.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in the regulation of major pathways in eukaryotic cells through their binding to and repression of multiple mRNAs. With high-throughput methodologies, various outcomes can be measured that produce long lists of miRNAs that are often difficult to interpret. A common question is: after differential expression or phenotypic screening of miRNA mimics, which miRNA should be chosen for further investigation? Here, we present miRViz (http://mirviz.prabi.fr/), a webserver application designed to visualize and interpret large miRNA datasets, with no need for programming skills. MiRViz has two main goals: (i) to help biologists to raise data-driven hypotheses and (ii) to share miRNA datasets in a straightforward way through publishable quality data representation, with emphasis on relevant groups of miRNAs. MiRViz can currently handle datasets from 11 eukaryotic species. We present real-case applications of miRViz, and provide both datasets and procedures to reproduce the corresponding figures. MiRViz offers rapid identification of miRNA families, as demonstrated here for the miRNA-320 family, which is significantly exported in exosomes of colon cancer cells. We also visually highlight a group of miRNAs associated with pluripotency that is particularly active in control of a breast cancer stem-cell population in culture.

CD95/Fas and metastatic disease: What does not kill you makes you stronger.

CD95 (also known as Fas) is the prototype of death receptors; however, evidence suggests that this receptor mainly implements non-apoptotic signaling pathways such as NF-κB, MAPK, and PI3K that are involved in cell migration, differentiation, survival, and cytokine secretion. At least two different forms of CD95 L exist. The multi-aggregated transmembrane ligand (m-CD95 L) is cleaved by metalloproteases to release a homotrimeric soluble ligand (s-CD95 L). Unlike m-CD95 L, the interaction between s-CD95 L and its receptor CD95 fails to trigger apoptosis, but instead promotes calcium-dependent cell migration, which contributes to the accumulation of inflammatory Th17 cells in damaged organs of lupus patients and favors cancer cell invasiveness. Novel inhibitors targeting the pro-inflammatory roles of CD95/CD95 L may provide attractive therapeutic options for patients with chronic inflammatory disorders or cancer. This review discusses the roles of the CD95/CD95 L pair in cell migration and metastasis.

A genome-wide RNAi screen reveals essential therapeutic targets of breast cancer stem cells.

Therapeutic resistance is a major clinical challenge in oncology. Evidence identifies cancer stem cells (CSCs) as a driver of tumor evolution. Accordingly, the key stemness property unique to CSCs may represent a reservoir of therapeutic target to improve cancer treatment. Here, we carried out a genome-wide RNA interference screen to identify genes that regulate breast CSCs-fate (bCSC). Using an interactome/regulome analysis, we integrated screen results in a functional mapping of the CSC-related processes. This network analysis uncovered potential therapeutic targets controlling bCSC-fate. We tested a panel of 15 compounds targeting these regulators. We showed that mifepristone, salinomycin, and JQ1 represent the best anti-bCSC activity. A combination assay revealed a synergistic interaction of salinomycin/JQ1 association to deplete the bCSC population. Treatment of primary breast cancer xenografts with this combination reduced the tumor-initiating cell population and limited metastatic development. The clinical relevance of our findings was reinforced by an association between the expression of the bCSC-related networks and patient prognosis. Targeting bCSCs with salinomycin/JQ1 combination provides the basis for a new therapeutic approach in the treatment of breast cancer.

Transcriptomic Analysis of Breast Cancer Stem Cells and Development of a pALDH1A1:mNeptune Reporter System for Live Tracking.

Many solid cancers are hierarchically organized with a small number of cancer stem cells (CSCs) able to regrow a tumor, while their progeny lacks this feature. Breast CSC is known to contribute to therapy resistance. The study of those cells is usually based on their cell-surface markers like CD44high /CD24low/neg or their aldehyde dehydrogenase (ALDH) activity. However, these markers cannot be used to track the dynamics of CSC. Here, a transcriptomic analysis is performed to identify segregating gene expression in CSCs and non-CSCs, sorted by Aldefluor assay. It is observed that among ALDH-associated genes, only ALDH1A1 isoform is increased in CSCs. A CSC reporter system is then developed by using a far red-fluorescent protein (mNeptune) under the control of ALDH1A1 promoter. mNeptune-positive cells exhibit higher sphere-forming capacity, tumor formation, and increased resistance to anticancer therapies. These results indicate that the reporter identifies cells with stemness characteristics. Moreover, live tracking of cells in a microfluidic system reveals a higher extravasation potential of CSCs. Live tracking of non-CSCs under irradiation treatment show, for the first time, live reprogramming of non-CSCs into CSCs. Therefore, the reporter will allow for cell tracking to better understand the implication of CSCs in breast cancer development and recurrence.

Stem Cells Inhibition by Bevacizumab in Combination with Neoadjuvant Chemotherapy for Breast Cancer.

Preclinical works have suggested cytotoxic chemotherapies may increase the number of cancer stem cells (CSC) whereas angiogenesis inhibition may decrease CSC proliferation. We developed a proof of concept clinical trial to explore bevacizumab activity on breast CSC. Breast cancer patients requiring preoperative chemotherapy were included in this open-label, randomized, prospective, multicenter phase II trial. All received FEC-docetaxel combination, and patients randomized in the experimental arm received concomitant bevacizumab. The primary endpoint was to describe ALDH1 (Aldehyde dehydrogenase 1) positive tumor cells rate before treatment and after the fourth cycle. Secondary objectives included safety, pathological complete response (pCR) rate, disease-free survival (DFS), relapse-free survival (RFS), and overall survival (OS). Seventy-five patients were included. ALDH1+ cells rate increase was below the predefined 5% threshold in both arms for the 32 patients with two time points available. Grade 3 or 4 adverse events rates were similar in both arms. A non-significant increase in pCR was observed in the bevacizumab arm (42.6% vs. 18.2%, p = 0.06), but survival was not improved (OS: p = 0.89; DFS: p = 0.45; and RFS: p = 0.68). The increase of ALDH1+ tumor cells rate after bevacizumab-based chemotherapy was less than 5%. However, as similar results were observed with chemotherapy alone, bevacizumab impact on breast CSC cells cannot be confirmed.

Publisher Correction: PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

In the version of this article originally published, several co-authors had incorrect affiliation footnote numbers listed in the author list. Tatiana Cañeque and Angelica Mariani should each have affiliation numbers 3, 4 and 5, and Emmanuelle Charafe-Jauffret should have number 6. Additionally, there was an extra space in the name of co-author Robert P. St.Onge. These errors have been corrected in the HTML and PDF versions of the paper and the Supplementary Information PDF.