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.

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.

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.

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.

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.

The SCRIB Paralog LANO/LRRC1 Regulates Breast Cancer Stem Cell Fate through WNT/ß-Catenin Signaling.

Tumor initiation, progression, and therapeutic resistance have been proposed to originate from a subset of tumor cells, cancer stem cells (CSCs). However, the current understanding of the mechanisms involved in their self-renewal and tumor initiation capacity remains limited. Here, we report that expression of LANO/LRRC1, the vertebrate paralog of SCRIB tumor suppressor, is associated with a stem cell signature in normal and tumoral mammary epithelia. Through in vitro and in vivo experiments including a Lano/Lrrc1 knockout mouse model, we demonstrate its involvement in the regulation of breast CSC (bCSC) fate. Mechanistically, we demonstrate that Lano/LRRC1-depleted cells secrete increased levels of WNT ligands, which act in a paracrine manner to positively deregulate the WNT/ß-catenin pathway in bCSCs. In addition to describing the first function of LANO/LRRC1, our results suggest that its expression level could be used as a biomarker to stratify breast cancer patients who could benefit from WNT/ß-catenin signaling inhibitors.

Salinomycin kills cancer stem cells by sequestering iron in lysosomes.

Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.

A stemness-related ZEB1-MSRB3 axis governs cellular pliancy and breast cancer genome stability.

Chromosomal instability (CIN), a feature of most adult neoplasms from their early stages onward, is a driver of tumorigenesis. However, several malignancy subtypes, including some triple-negative breast cancers, display a paucity of genomic aberrations, thus suggesting that tumor development may occur in the absence of CIN. Here we show that the differentiation status of normal human mammary epithelial cells dictates cell behavior after an oncogenic event and predetermines the genetic routes toward malignancy. Whereas oncogene induction in differentiated cells induces massive DNA damage, mammary stem cells are resistant, owing to a preemptive program driven by the transcription factor ZEB1 and the methionine sulfoxide reductase MSRB3. The prevention of oncogene-induced DNA damage precludes induction of the oncosuppressive p53-dependent DNA-damage response, thereby increasing stem cells' intrinsic susceptibility to malignant transformation. In accord with this model, a subclass of breast neoplasms exhibit unique pathological features, including high ZEB1 expression, a low frequency of TP53 mutations and low CIN.

miR-600 Acts as a Bimodal Switch that Regulates Breast Cancer Stem Cell Fate through WNT Signaling.

Breast cancer stem cells (bCSCs) have been implicated in tumor progression and therapeutic resistance; however, the molecular mechanisms that define this state are unclear. We have performed two microRNA (miRNA) gain- and loss-of-function screens to identify miRNAs that regulate the choice between bCSC self-renewal and differentiation. We find that micro-RNA (miR)-600 silencing results in bCSC expansion, while its overexpression reduces bCSC self-renewal, leading to decreased in vivo tumorigenicity. miR-600 targets stearoyl desaturase 1 (SCD1), an enzyme required to produce active, lipid-modified WNT proteins. In the absence of miR-600, WNT signaling is active and promotes self-renewal, whereas overexpression of miR-600 inhibits the production of active WNT and promotes bCSC differentiation. In a series of 120 breast tumors, we found that a low level of miR-600 is correlated with active WNT signaling and a poor prognosis. These findings highlight a miR-600-centered signaling network that governs bCSC-fate decisions and influences tumor progression.

Brief reports: A distinct DNA methylation signature defines breast cancer stem cells and predicts cancer outcome.

Self-renewal and differentiation are two epigenetic programs that regulate stem cells fate. Dysregulation of these two programs leads to the development of cancer stem cells (CSCs). Recent evidence suggests that CSCs are relatively resistant to conventional therapies and responsible for metastasis formation. Deciphering these processes will help understand oncogenesis and allow the development of new targeted therapies. Here, we have used a whole genome promoter microarray to establish the DNA methylation portraits of breast cancer stem cells (bCSCs) and non-bCSCs. A total of 68 differentially methylated regions (DMRs) were more hypomethylated in bCSCs than in non-bCSCs. Using a differentiation assay we demonstrated that DMRs are rapidly hypermethylated within the first 6 hours following induction of CSC differentiation whereas the cells reached the steady-state within 6 days, suggesting that these DMRs are linked to early CSC epigenetic regulation. These DMRs were significantly enriched in genes coding for TGF-ß signaling-related proteins. Interestingly, DMRs hypomethylation was correlated to an overexpression of TGF-ß signaling genes in a series of 109 breast tumors. Moreover, patients with tumors harboring the bCSC DMRs signature had a worse prognosis than those with non-bCSC DMRs signature. Our results show that bCSCs have a distinct DNA methylation landscape with TGF-ß signaling as a key epigenetic regulator of bCSCs differentiation.

The histone deacetylase inhibitor abexinostat induces cancer stem cells differentiation in breast cancer with low Xist expression.

PURPOSE: Cancer stem cells (CSC) are the tumorigenic cell population that has been shown to sustain tumor growth and to resist conventional therapies. The purpose of this study was to evaluate the potential of histone deacetylase inhibitors (HDACi) as anti-CSC therapies. EXPERIMENTAL DESIGN: We evaluated the effect of the HDACi compound abexinostat on CSCs from 16 breast cancer cell lines (BCL) using ALDEFLUOR assay and tumorsphere formation. We performed gene expression profiling to identify biomarkers predicting drug response to abexinostat. Then, we used patient-derived xenograft (PDX) to confirm, in vivo, abexinostat treatment effect on breast CSCs according to the identified biomarkers. RESULTS: We identified two drug-response profiles to abexinostat in BCLs. Abexinostat induced CSC differentiation in low-dose sensitive BCLs, whereas it did not have any effect on the CSC population from high-dose sensitive BCLs. Using gene expression profiling, we identified the long noncoding RNA Xist (X-inactive specific transcript) as a biomarker predicting BCL response to HDACi. We validated that low Xist expression predicts drug response in PDXs associated with a significant reduction of the breast CSC population. CONCLUSIONS: Our study opens promising perspectives for the use of HDACi as a differentiation therapy targeting the breast CSCs and identified a biomarker to select patients with breast cancer susceptible to responding to this treatment.

ALDH1-positive cancer stem cells predict engraftment of primary breast tumors and are governed by a common stem cell program.

Cancer stem-like cells (CSC) have been widely studied, but their clinical relevance has yet to be established in breast cancer. Here, we report the establishment of primary breast tumor-derived xenografts (PDX) that encompass the main diversity of human breast cancer and retain the major clinicopathologic features of primary tumors. Successful engraftment was correlated with the presence of ALDH1-positive CSCs, which predicted prognosis in patients. The xenografts we developed showed a hierarchical cell organization of breast cancer with the ALDH1-positive CSCs constituting the tumorigenic cell population. Analysis of gene expression from functionally validated CSCs yielded a breast CSC signature and identified a core transcriptional program of 19 genes shared with murine embryonic, hematopoietic, and neural stem cells. This generalized stem cell program allowed the identification of potential CSC regulators, which were related mainly to metabolic processes. Using an siRNA genetic screen designed to target the 19 genes, we validated the functional role of this stem cell program in the regulation of breast CSC biology. Our work offers a proof of the functional importance of CSCs in breast cancer, and it establishes the reliability of PDXs for use in developing personalized CSC therapies for patients with breast cancer.

Mevalonate metabolism regulates Basal breast cancer stem cells and is a potential therapeutic target.

There is increasing evidence that breast tumors are organized in a hierarchy, with a subpopulation of tumorigenic cancer cells, the cancer stem cells (CSCs), which sustain tumor growth. The characterization of protein networks that govern CSC behavior is paramount to design new therapeutic strategies targeting this subpopulation of cells. We have sought to identify specific molecular pathways of CSCs isolated from 13 different breast cancer cell lines of luminal or basal/mesenchymal subtypes. We compared the gene expression profiling of cancer cells grown in adherent conditions to those of matched tumorsphere cultures. No specific pathway was identified to be commonly regulated in luminal tumorspheres, resulting from a minor CSC enrichment in tumorsphere passages from luminal cell lines. However, in basal/mesenchymal tumorspheres, the enzymes of the mevalonate metabolic pathway were overexpressed compared to those in cognate adherent cells. Inhibition of this pathway with hydroxy-3-methylglutaryl CoA reductase blockers resulted in a reduction of breast CSC independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Further modulation of this metabolic pathway demonstrated that protein geranylgeranylation (GG) is critical to breast CSC maintenance. A small molecule inhibitor of the geranylgeranyl transferase I (GGTI) enzyme reduced the breast CSC subpopulation both in vitro and in primary breast cancer xenografts. We found that the GGTI effect on the CSC subpopulation is mediated by inactivation of Ras homolog family member A (RHOA) and increased accumulation of P27(kip1) in the nucleus. The identification of protein GG as a major contributor to CSC maintenance opens promising perspectives for CSC targeted therapy in basal breast cancer.

ZNF703 gene amplification at 8p12 specifies luminal B breast cancer.

Luminal B breast cancers represent a fraction of oestrogen receptor (ER)-positive tumours associated with poor recurrence-free and disease-specific survival in all adjuvant systemic treatment categories including hormone therapy alone. Identification of specific signalling pathways driving luminal B biology is paramount to improve treatment. We have studied 100 luminal breast tumours by combined analysis of genome copy number aberrations and gene expression. We show that amplification of the ZNF703 gene, located in chromosomal region 8p12, preferentially occurs in luminal B tumours. We explored the functional role of ZNF703 in luminal B tumours by overexpressing ZNF703 in the MCF7 luminal cell line. Using mass spectrometry, we identified ZNF703 as a co-factor of a nuclear complex comprising DCAF7, PHB2 and NCOR2. ZNF703 expression results in the activation of stem cell-related gene expression leading to an increase in cancer stem cells. Moreover, we show that ZNF703 is implicated in the regulation of ER and E2F1 transcription factor. These findings point out the prominent role of ZNF703 in transcription modulation, stem cell regulation and luminal B oncogenesis.

CXCR1 blockade selectively targets human breast cancer stem cells in vitro and in xenografts.

Recent evidence suggests that breast cancer and other solid tumors possess a rare population of cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. We report here the development of a strategy to target these breast cancer stem cells (CSCs) through blockade of the IL-8 receptor CXCR1. CXCR1 blockade using either a CXCR1-specific blocking antibody or repertaxin, a small-molecule CXCR1 inhibitor, selectively depleted the CSC population in 2 human breast cancer cell lines in vitro. Furthermore, this was followed by the induction of massive apoptosis in the bulk tumor population via FASL/FAS signaling. The effects of CXCR1 blockade on CSC viability and on FASL production were mediated by the FAK/AKT/FOXO3A pathway. In addition, repertaxin was able to specifically target the CSC population in human breast cancer xenografts, retarding tumor growth and reducing metastasis. Our data therefore suggest that CXCR1 blockade may provide a novel means of targeting and eliminating breast CSCs.

Retinoid signaling regulates breast cancer stem cell differentiation.

The cancer stem cell (CSC) hypothesis implicates the development of new therapeutic approaches to target the CSC population. Characterization of the pathways that regulate CSCs activity will facilitate the development of targeted therapies. We recently reported that the enzymatic activity of ALDH1, as measured by the ALDELFUOR assay, can be utilized to isolate normal and malignant breast stem cells in both primary tumors and cell lines. In this study, utilizing a tumorsphere assay, we have demonstrated the role of retinoid signaling in the regulation of breast CSCs self-renewal and differentiation. Utilizing the gene set enrichment analysis (GSEA) algorithm we identified gene sets and pathways associated with retinoid signaling. These pathways regulate breast CSCs biology and their inhibition may provide novel therapeutic approaches to target breast CSCs.

Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature.

Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have used the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize cancer stem cell (CSC) populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene CSC profile that included genes known to play a role in stem cell function, as well as genes such as CXCR1/IL-8RA not previously known to play such a role. Recombinant interleukin-8 (IL-8) increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally, we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines, establish techniques that can facilitate the characterization of regulatory pathways of CSCs, and identify potential stem cell markers and therapeutic targets.