Basal Breast Cancer Research Articles

Analyzing mRNAsi-Related Genes Identifies Novel Prognostic Markers and Potential Drug Combination for Patients with Basal Breast Cancer.

Basal breast cancer subtype is the worst prognosis subtypes among all breast cancer subtypes. Recently, a new tumor stemness index-mRNAsi is found to be able to measure the degree of oncogenic differentiation of tissues. The mRNAsi involved in a variety of cancer processes is derived from the innovative application of one-class logistic regression (OCLR) machine learning algorithm to the whole genome expression of various stem cells and tumor cells. However, it is largely unknown about mRNAsi in basal breast cancer. Here, we find that basal breast cancer carries the highest mRNAsi among all four subtypes of breast cancer, especially 385 mRNAsi-related genes are positively related to the high mRNAsi value in basal breast cancer. This high mRNAsi is also closely related to active cell cycle, DNA replication, and metabolic reprogramming in basal breast cancer. Intriguingly, in the 385 genes, TRIM59, SEPT3, RAD51AP1, and EXO1 can act as independent protective prognostic factors, but CTSF and ABHD4B can serve as independent bad prognostic factors in patients with basal breast cancer. Remarkably, we establish a robust prognostic model containing the 6 mRNAsi-related genes that can effectively predict the survival rate of patients with the basal breast cancer subtype. Finally, the drug sensitivity analysis reveals that some drug combinations may be effectively against basal breast cancer via targeting the mRNAsi-related genes. Taken together, our study not only identifies novel prognostic biomarkers for basal breast cancers but also provides the drug sensitivity data by establishing an mRNAsi-related prognostic model.

Opposing effects of BRCA1 mRNA expression on patient survival in breast and colorectal cancer and variations among African American, Asian, and younger patients

Breast cancer (BC) and colorectal cancer (CRC) are common and show poor survival in advanced stages. Using The Cancer Genome Atlas (TCGA) computational tool cBioPortal, we evaluated overall patient survival in BRCA1 mRNA-low versus -high cohorts (<−1.29 versus >1.05 SD from mean BRCA1 expression, respectively). Analysis included 1082 BC patients with mRNA data (PanCancer Atlas), 382 CRCs (Firehose Legacy) and 592 CRCs (PanCancer Atlas). As previously reported, BRCA1 mRNA-low tumor expression positively correlated with BC patient survival but was negatively associated in CRC. We observed a correlation between BRCA1 mRNA-high and age <45 years at CRC diagnosis using a Fisher’s exact test [Firehose Legacy database (p-value = 0.0091); CRC PanCancer Atlas (p-value = 0.0778)]. We correlated BRCA1 mRNA-low expression and basal BC (p-value = 0.0016) and BRCA1 mRNA-low tumors and frequency of African American patients (p-value = 0.0448) with BC. Other trends included higher frequency of advanced lymph node stage and mucinous adenocarcinoma among BRCA1 mRNA-low CRC and higher frequency of males in BRCA1 mRNA-high BC and CRC. African Americans more frequently had BRCA1 mRNA-low BC and BRCA1 mRNA-high CRC and the opposite was observed among Asians. Using a gene co-expression tool (cBioPortal), we observed TOP2A and ATAD5 levels correlate (Spearman’s correlation>0.6) with BRCA1 in BC and CRC, whereas LMNB2 correlates with BRCA1 in CRC, suggesting tissue-specific BRCA1 interactions. Our results indicate potential for BRCA1 mRNA expression levels as a prognostic biomarker in BC and CRC, suggest tissue-specificity in BRCA1 molecular interactions, and point to BRCA1 mRNA-high levels as a characteristic of CRC tumors in younger versus older individuals.

ISGylation drives basal breast tumour progression by promoting EGFR recycling and Akt signalling

ISG15 is an ubiquitin-like modifier that is associated with reduced survival rates in breast cancer patients. The mechanism by which ISG15 achieves this however remains elusive. We demonstrate that modification of Rab GDP-Dissociation Inhibitor Beta (GDI2) by ISG15 (ISGylation) alters endocytic recycling of the EGF receptor (EGFR) in non-interferon stimulated cells using CRISPR-knock out models for ISGylation. By regulating EGFR trafficking, ISGylation enhances EGFR recycling and sustains Akt-signalling. We further show that Akt signalling positively correlates with levels of ISG15 and its E2-ligase in basal breast cancer cohorts, confirming the link between ISGylation and Akt signalling in human tumours. Persistent and enhanced Akt activation explains the more aggressive tumour behaviour observed in human breast cancers. We show that ISGylation can act as a driver of tumour progression rather than merely being a bystander.

OTHR-02. Engineered “of the shelf” allogeneic cellular therapies for metastatic brain tumors

Brain metastatic patients have multiple metastatic lesions or diagnostically challenging asymptomatic lesions, making surgery an inadequate therapeutic option. Given the challenges related to systemic delivery of a majority of therapeutic agents across the BBB, engineered cell based therapies offer an excellent platform to target metastatic tumors in the brain. We have established the use tumor cell surface receptor targeted allogeneic “off the shelf” gene engineered cellular therapies and developed two different approaches to treat brain metastases. In one approach, we have armed allogenic stem cells (SC) with oncolytic herpes virus (oHSV) variants and tested them in different mouse models of brain metastatic (BM) tumor derived from brain seeking metastatic melanoma tumor cells from patients. We show that intracarotid artery administration of SC-oHSV effectively tracks metastatic tumor lesions and significantly prolongs the survival of brain tumor bearing mice. We also show that a combination of SC-oHSV and PD-L1 blockade increases IFNγ-producing CD8+ tumor-infiltrating T lymphocytes and results in a profound extension of the median survival in syngeneic brain metastatic melanoma mouse models. In another approach, we have explored the versatility of cell mediated bi-functional EGFR and DR4/5-targeted treatment in basal like breast cancer (BLBC) mouse models featuring different patterns of brain metastasis. Most BLBC lines demonstrated a high sensitivity to EGFR and DR4/5 bi-targeting therapeutic protein, EVDRL [anti-EGFR VHH (EV) fused to DR ligand (DRL)]. Functional analyses using inhibitors and CRISPR-Cas9 knockouts revealed that the EV domain facilitated in augmenting DR4/5-DRL binding and enhancing DRL-induced apoptosis. EVDRL releasing allogeneic SCs alleviated tumor-burden and significantly increased survival in mouse models of residual-tumor after macrometastasis resection, perivascular niche micrometastasis, and leptomeningeal metastasis. These findings provide a clinically applicable therapeutic platform to target disseminated metastatic lesions in the brain and define a new paradigm for treatment of brain metastases.

AGR2 and AGR3 play an important role in the clinical characterization and prognosis of basal like breast cancer

Introduction: Basal-like Breast Cancer (BLBC) represents an important molecular subtype of breast cancer characterized by an aggressive behavior, molecular pathology poorly understood and a limited treatment. Objective: We aim to search for molecular differences between non-BLBC and BLBC tumors in order to propose possible diagnostic and prognostic biomarkers using databases. Metodology: Microarray processed data were downloaded from GEO database considering non-BLBC and BLBC. Enrichment analysis was evaluated using GO consortium and Ingenuity, protein–protein interaction, gene Ontology and co-expression analysis using STRING. Gene expression data was extracted using TCGA, METABRIC and Breast Cancer Gene-Expression Miner v4.2 databases. The Survival was evaluated using The Kaplan–Meier plotter. Results: Were identified 58 upregulated and 58 downregulated genes enriched in signaling pathways like PDGF, Angiogenesis, Integrin and WNT. AGR2 and AGR3 expression were reduced in BLBC in relation to non-BLBC tumors, patients aged ≤51 years, and with negativity of ER, PR and HER-2 and nodal status. Low expression of AGR2 and AGR3 were associated with worse OS and RFS for all breast cancer cases. But according to the molecular stratification, low AGR2 conferred worst OS in luminal A, worst RFS in BLBC and good OS and RFS in luminal B. High AGR3 conferred worse OS and RFS in BLBC, but low AGR3 attributed worse OS in luminal A. Conclusion: AGR2 and AGR3 expression were able to differentiate non-BLBC from BLBC. Downregulation of AGR2 and AGR3 was associated with BLBC clinical phenotype. Furthermore, both genes behave different when considering prognosis and molecular stratification.

A systems-based framework to computationally describe putative transcription factors and signaling pathways regulating glycan biosynthesis.

Glycosylation is a common posttranslational modification, and glycan biosynthesis is regulated by a set of glycogenes. The role of transcription factors (TFs) in regulating the glycogenes and related glycosylation pathways is largely unknown. In this work, we performed data mining of TF–glycogene relationships from the Cistrome Cancer database (DB), which integrates chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-Seq data to constitute regulatory relationships. In total, we observed 22,654 potentially significant TF–glycogene relationships, which include interactions involving 526 unique TFs and 341 glycogenes that span 29 the Cancer Genome Atlas (TCGA) cancer types. Here, TF–glycogene interactions appeared in clusters or so-called communities, suggesting that changes in single TF expression during both health and disease may affect multiple carbohydrate structures. Upon applying the Fisher’s exact test along with glycogene pathway classification, we identified TFs that may specifically regulate the biosynthesis of individual glycan types. Integration with Reactome DB knowledge provided an avenue to relate cell-signaling pathways to TFs and cellular glycosylation state. Whereas analysis results are presented for all 29 cancer types, specific focus is placed on human luminal and basal breast cancer disease progression. Overall, the article presents a computational approach to describe TF–glycogene relationships, the starting point for experimental system-wide validation.

Abstract 2397: Significance of the combination of biguanides and fatty acid β-oxidation inhibitors in triple-negative breast cancer

Abstract Compared to hormone receptor (HR) positive breast cancer (BC), basal or triple-negative BC (TNBC) suffers a poor prognosis, attributed to the limited understanding of its driver signaling pathways. Although TNBC exhibits unique characteristics, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are essential to reduce its severity. We have previously shown that metabolic reprogramming to fatty acid β-oxidation (FAO) is a dominant metabolic pathway in TNBC. We have also reported that FAO regulates c-Src, one of the frequently upregulated oncopathways in TNBC, via autophosphorylation of Src at Y419. However, single drug therapy using FAO inhibitors or Src inhibitors did not show a significant therapeutic advantage in TNBC. Biguanide such as metformin is one of the most widely administered anti-diabetic agents. Recently increasing evidence demonstrates that biguanide therapy can have an anticancer effect in various cancer types, including BC. Phenformin, a biguanide derivative similar to metformin, has greater potency than metformin. Both biguanides can suppress mitochondrial electron transport chain (ETC) function because of its inhibitory effect on ETC complex I activity. However, biguanides also lead to the activation of AMPK, which plays a vital role in insulin signaling and energy-sensing. Notably, AMPK is an upstream regulator of the FAO pathway because it can phosphorylate ACC and activate FAO. Considering the dependency of TNBC on mitochondrial FAO and the role of AMPK in the activation of FAO, our computational modeling predicted that the combined targeting of both ‘arms' of the pathway could provide a significant advantage to the anticancer effect in TNBC. Thus, we have used the combination of biguanides and FAO inhibitors to target both ETC and FAO respectively. Pharmacological and genetic approaches with multiple in vitro and in vivo analysis using TNBC cell lines and PDX models suggest that the combination of biguanides and FAO inhibitors can provide significant advantage in TNBC therapy. Further studies also show that the combination of biguanides with Src inhibitors can provide a better therapeutic option to target metastatic TNBC. We are currently evaluating these findings' translational significance as a cost-effective approach to managing non-targetable TNBCs. Citation Format: Junhyoung Park, Kwang Hwa Jung, Dongya Jia, Sukjin Yang, Dharaniya Sakthivel, Abha Tiwari, José N. Onuchic, Benny Abraham Kaipparettu. Significance of the combination of biguanides and fatty acid β-oxidation inhibitors in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2397.

Abstract 3111: Notch3 functions as a tumor suppressor in ER-positive breast cancer

Abstract The Notch signaling pathway has been implicated in the development as well as tumorigenesis of the mammary gland. Notch3 promotes luminal cell specification and marks a highly clonogenic luminal progenitor population. Aberrant Notch3 activation has been shown to promote the growth of basal breast cancer as well as resistance to hormonal therapy in metastatic luminal breast cancer. Interestingly, recent studies also suggest a tumor-suppressive function of Notch3 in mammary epithelium through regulation of estrogen receptor α (ER). Here we report the expression and functional analysis of Notch3 in the mouse mammary gland. X-gal staining of the Notch3β-Geo/+ mammary gland showed that Notch3 was highly expressed in luminal cells throughout mammary development with exception at the initiation of post-lactational involution, when Notch3 expression was restricted to the basal cells. Deletion of Notch3 caused decreased Notch activation in the CD24HiCD49fLo subpopulation of the pubescent mammary gland, accompanied by a significant decrease in the number of CD24HiCD49fLoCD61+ luminal progenitor cells. Whole-mount preparation of the Notch3 knockout mammary glands showed normal development during puberty, pregnancy, lactation and involution. Surprisingly, parous Notch3 knockout mice developed mammary ductal hyperplasia by 10 months of age, some of which progressed to ductal carcinoma in situ, and ultimately to invasive and metastatic cancer. Parous Notch3 knockout mice exhibited an expansion of the CD24HiCD49fLo subpopulation, and mammary tumors from these mice were composed predominantly of CD24HiCD49fLo cells. The vast majority of these tumors were ER-positive and relatively well differentiated, with a papillary and/or glandular pattern. They expressed the luminal marker cytokeratin 8, and some of them co-expressed cytokeratin 14, a basal marker. All Notch3 knockout mammary tumors showed high level expression of Cyclin D1. These results suggest that Notch3 may prevent ER-positive breast cancer through suppression of CD24HiCD49fLo progenitor cell self-renewal in the postpartum mammary gland. In addition to its impact on mammary epithelium, deletion of Notch3 altered the mammary microenvironment, in particular, brown adipocyte differentiation in the mammary fat pad. Further experiments will be performed to define a potential role for Notch3-regulated brown adipose tissue in breast cancer initiation and progression. Finally, analysis of TCGA and METABRIC human breast cancer data sets revealed a negative correlation between expressions of NOTCH3 and ESR1 (encoding estrogen receptor α), and association of high NOTCH3 expression with enhanced survival of patients with luminal A and luminal B subtype breast tumors. Taken together, Notch3 can act as a tumor suppressor in the mammary epithelium, and possibly in the mammary microenvironment as well, to prevent development and metastasis of ER-positive breast cancer. Citation Format: Wen-Cheng Chung, Sean E. Egan, Keli Xu. Notch3 functions as a tumor suppressor in ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3111.

Syndecan-1 Promotes Angiogenesis in Triple-Negative Breast Cancer through the Prognostically Relevant Tissue Factor Pathway and Additional Angiogenic Routes.

Simple SummaryTriple-negative breast cancer is an aggressive subtype of breast cancer characterized by tumor angiogenesis and poor patient survival. Here, we analyzed the function of the cell surface molecule Syndecan-1 in tumor angiogenesis in a 3D cell culture system. As a novel finding, we demonstrate that downregulation of Syndecan-1 reduces angiogenesis by decreasing the amount of angiogenesis factors of the tissue factor pathway. Furthermore, we show that the components of this pathway are associated with the prognosis of breast cancer patients. Our study identifies Syndecan-1 and the tissue factor pathway as novel potential therapeutic targets in the aggressive triple-negative subtype of breast cancer, for which no targeted therapies are currently available.Triple-negative breast cancer (TNBC) is characterized by increased angiogenesis, metastasis, and poor survival. Dysregulation of the cell surface heparan sulfate proteoglycan and signaling co-receptor Syndecan-1 is linked to poor prognosis. To study its role in angiogenesis, we silenced Syndecan-1 in TNBC cell lines using a 3D human umbilical vein endothelial cell (HUVEC) co-culture system. Syndecan-1 siRNA depletion in SUM-149, MDA-MB-468, and MDA-MB-231 cells decreased HUVEC tubule network formation. Angiogenesis array revealed reduced VEGF-A and tissue factor (TF) in the Syndecan-1-silenced secretome. qPCR independently confirmed altered expression of F3, F7, F2R/PAR1, F2RL1/PAR2, VEGF-A, EDN1, IGFBP1, and IGFBP2 in SUM-149, MDA-MB-231, and MDA-MB-468 cells. ELISA revealed reduced secreted endothelin-1 (SUM-149, MDA-MB-468) and TF (all cell lines) upon Syndecan-1 depletion, while TF pathway inhibitor treatment impaired angiogenesis. Survival analysis of 3951 patients demonstrated that high expression of F3 and F7 are associated with better relapse-free survival, whereas poor survival was observed in TNBC and p53 mutant basal breast cancer (F3) and in ER-negative and HER2-positive breast cancer (F2R, F2RL1). STRING protein network analysis revealed associations of Syndecan-1 with VEGF-A and IGFBP1, further associated with the TF and ET-1 pathways. Our study suggests that TNBC Syndecan-1 regulates angiogenesis via the TF and additional angiogenic pathways and marks its constituents as novel prognostic markers and therapeutic targets.

Transcriptomic insights into the zinc homeostasis of MCF-7 breast cancer cells via next-generation RNA sequencing.

A significant gap in the knowledge of zinc homeostasis exists for breast cancer cells. In this study, we investigated the transcriptomic response of the luminal breast cancer cells (MCF-7) to the exposure of extracellular zinc using next-generation RNA sequencing. The dataset was collected for three time points (T0, T30, and T120) in the time course of zinc treatment, which revealed the dramatic increase, up to 869-fold, of the gene expression for metallothioneins (MT1B, MT1F, MT1X, and MT2A) and the zinc exporter ZnT1 (SLC30A1) at T30, continuingly through to T120. The similar dynamic expression pattern was found for the autophagy-related gene (VMP1) and numerous genes for zinc finger proteins (e.g. RNF165, ZNF365, ZBTB2, SNAI1, ZNF442, ZNF547, ZNF563, and ZNF296). These findings point to the all-hands-on-deck strategy adopted by the cancer cells for maintaining zinc homeostasis. The stress responsive genes encoding heat shock proteins (HSPA1A, HSPA1B, HSPA1L, HSPA4L, HSPA6, HSPA8, HSPH1, HSP90AA1, and HSP90AB1) and the MTF-1 biomarker genes (AKR1C2, CLU, ATF3, GDF15, HMOX1, MAP1A, MAFG, SESN2, and UBC) were also differentially up-regulated at T120, suggesting a role of heat shock proteins and the MTF-1 related stress proteins in dealing with zinc exposure. It is for the first time that the gene encoding Polo-like kinase 2 (PLK2) was found to be involved in zinc-related response. The top differentially expressed genes were validated by qRT-PCR and further extended to the basal type breast cancer cells (MDA-MB-231). It was found that the expression level of SLC30A1 in MDA-MB-231 was higher than MCF-7 in response to zinc exposure. Taken together, the findings contribute to our knowledge and understanding of zinc homeostasis in breast cancer cells.

A novel role of ADGRF1 (GPR110) in promoting cellular quiescence and chemoresistance in human epidermal growth factor receptor 2‐positive breast cancer

Purpose Adhesion G protein coupled receptors (aGPCRs) remains least explored GPCRs family for their role in health and disease. The aGPCR, ADGRF1 (GPR110), is overexpressed and predicts poor survival in various cancer types. However, ADGRF1's coupling to G proteins and downstream pathways remain unknown in cancer. We have reported that ADGRF1 is overexpressed in tumorigenic population and in anti-HER2 therapy-resistant derivatives of various HER2+ breast cancer (BC) cell line models. Further, we have reported that ADGRF1 knockdown reduces tumorigenesis and metastasis in HER2+ BC cells. In this study, we evaluated the effects of ADGRF1 overexpression (OE) on tumorigenesis, signaling pathways, and outcomes in HER2+ BC. Methods Stable clones of 2 HER2+ BC cell lines, BT474 and SKBR3, were generated to overexpress ADGRF1 construct in doxycycline-inducible manner. The effect of ADGRF1 on tumorigenesis was evaluated in vitro using soft agar assays, mammosphere assay, and Aldefluor assay. The effect on the HER pathway was evaluated by assessing expression of phospho- and total-HER1 and -HER2 and potency of anti-HER2 drugs. G protein coupling of ADGRF1 was determined by co-immunoprecipitation of Gαs and Gαq and detecting second messengers, cAMP and IP1, in presence/absence of synaptamide, an ADGRF1 agonist. Downstream pathways of ADGRF1 were explored by RNAseq and Reverse Phase Protein Arrays (RPPA) and functional assays. ADGRF1 expression was assessed in BC patients from The Cancer Genome Atlas. ADGRF1 OE effects on survival was evaluated using the Molecular Taxonomy of Breast Cancer International Consortium dataset. Results ADGRF1 OE enhanced the number of colonies and mammospheres and increased Aldefluor+ tumorigenic cell population, suggesting a role in tumorigenesis. ADGRF1 OE did not alter the expression total- or phospho-HER1/-HER2 proteins or efficacy of anti-HER2 drugs. Overexpressed ADGRF1 co-immunoprecipitated with Gαs and Gαq proteins and increased cAMP and IP1. However, inhibition of only the Gαs pathway by SQ22536 reversed the pro-tumorigenic effects of ADGRF1 OE, suggesting pro-tumorigenic effects of the ADGRF1-Gαs pathway. Synaptamide, did not alter cAMP or IP1 with/without ADGRF1 OE or the number of mammospheres. Integration of RNA-Seq and RPPA data showed that ADGRF1 OE inhibited cell cycle. We detected G0/1 arrest by cell cycle analysis with ADGRF1 OE. The protein level of the proliferation marker Ki67 was reduced upon ADGRF1 OE. Also, ADGRF1 OE led to a 10-fold reduction in potency of docetaxel, a chemotherapy drug used in HER2+ BC. ADGRF1 was overexpressed and amplified in basal and HER2 compared to luminal A and B BCs. High ADGRF1 expression predicted poor survival in HER2 subtype, but not in basal BC. Conclusions: ADGRF1 represents a novel drug target in HER2+ BC, warranting discovery of novel ADGRF1 antagonists.

TNF-\u03b1 differentially modulates subunit levels of respiratory electron transport complexes of ER/PR +ve/\u2212ve breast cancer cells to regulate mitochondrial complex activity and tumorigenic potential

BackgroundTumor necrosis factor-α (TNF-α) is an immunostimulatory cytokine that is consistently high in the breast tumor microenvironment (TME); however, its differential role in mitochondrial functions and cell survival in ER/PR +ve and ER/PR −ve breast cancer cells is not well understood.MethodsIn the current study, we investigated TNF-α modulated mitochondrial proteome using high-resolution mass spectrometry and identified the differentially expressed proteins in two different breast cancer cell lines, ER/PR positive cell line; luminal, MCF-7 and ER/PR negative cell line; basal-like, MDA-MB-231 and explored its implication in regulating the tumorigenic potential of breast cancer cells. We also compared the activity of mitochondrial complexes, ATP, and ROS levels between MCF-7 and MDA-MB-231 in the presence of TNF-α. We used Tumor Immune Estimation Resource (TIMER) webserver to analyze the correlation between TNF-α and mitochondrial proteins in basal and luminal breast cancer patients. Kaplan-Meier method was used to analyze the correlation between mitochondrial protein expression and survival of breast cancer patients.ResultsThe proteome analysis revealed that TNF-α differentially altered the level of critical proteins of mitochondrial respiratory chain complexes both in MCF-7 and MDA-MB-231, which correlated with differential assembly and activity of mitochondrial ETC complexes. The inhibition of the glycolytic pathway in the presence of TNF-α showed that glycolysis is indispensable for the proliferation and clonogenic ability of MDA-MB-231 cells (ER/PR −ve) as compared to MCF-7 cells (ER/PR +ve). The TIMER database showed a negative correlation between the expressions of TNF-α and key regulators of mitochondrial OXPHOS complexes in basal breast vs lobular carcinoma. Conversely, patient survival analysis showed an improved relapse-free survival with increased expression of identified proteins of ETC complexes and survival of the breast cancer patients.ConclusionThe evidence presented in our study convincingly demonstrates that TNF-α regulates the survival and proliferation of aggressive tumor cells by modulating the levels of critical assembly factors and subunits involved in mitochondrial respiratory chain supercomplexes organization and function. This favors the rewiring of mitochondrial metabolism towards anaplerosis to support the survival and proliferation of breast cancer cells. Collectively, the results strongly suggest that TNF-α differentially regulates metabolic adaptation in ER/PR +ve (MCF-7) and ER/PR −ve (MDA-MB-231) cells by modulating the mitochondrial supercomplex assembly and activity.

A cell-to-patient machine learning transfer approach uncovers novel basal-like breast cancer prognostic markers amongst alternative splice variants

BackgroundBreast cancer is amongst the 10 first causes of death in women worldwide. Around 20% of patients are misdiagnosed leading to early metastasis, resistance to treatment and relapse. Many clinical and gene expression profiles have been successfully used to classify breast tumours into 5 major types with different prognosis and sensitivity to specific treatments. Unfortunately, these profiles have failed to subclassify breast tumours into more subtypes to improve diagnostics and survival rate. Alternative splicing is emerging as a new source of highly specific biomarkers to classify tumours in different grades. Taking advantage of extensive public transcriptomics datasets in breast cancer cell lines (CCLE) and breast cancer tumours (TCGA), we have addressed the capacity of alternative splice variants to subclassify highly aggressive breast cancers.ResultsTranscriptomics analysis of alternative splicing events between luminal, basal A and basal B breast cancer cell lines identified a unique splicing signature for a subtype of tumours, the basal B, whose classification is not in use in the clinic yet. Basal B cell lines, in contrast with luminal and basal A, are highly metastatic and express epithelial-to-mesenchymal (EMT) markers, which are hallmarks of cell invasion and resistance to drugs. By developing a semi-supervised machine learning approach, we transferred the molecular knowledge gained from these cell lines into patients to subclassify basal-like triple negative tumours into basal A- and basal B-like categories. Changes in splicing of 25 alternative exons, intimately related to EMT and cell invasion such as ENAH, CD44 and CTNND1, were sufficient to identify the basal-like patients with the worst prognosis. Moreover, patients expressing this basal B-specific splicing signature also expressed newly identified biomarkers of metastasis-initiating cells, like CD36, supporting a more invasive phenotype for this basal B-like breast cancer subtype.ConclusionsUsing a novel machine learning approach, we have identified an EMT-related splicing signature capable of subclassifying the most aggressive type of breast cancer, which are basal-like triple negative tumours. This proof-of-concept demonstrates that the biological knowledge acquired from cell lines can be transferred to patients data for further clinical investigation. More studies, particularly in 3D culture and organoids, will increase the accuracy of this transfer of knowledge, which will open new perspectives into the development of novel therapeutic strategies and the further identification of specific biomarkers for drug resistance and cancer relapse.

Single-cell transcriptomics reveals involution mimicry during the specification of the basal breast cancer subtype.

Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination. This involution mimicry is characterized by a highly interactive multicellular network, with involution cancer-associated fibroblasts playing a pivotal role in extracellular matrix remodeling and immunosuppression. Our results may partially explain the increased risk and poor prognosis of breast cancer associated with childbirth.

Co-dependency for MET and FGFR1 in basal triple-negative breast cancers

Triple-negative breast cancer (TNBC) is a heterogeneous disease that lacks both effective patient stratification strategies and therapeutic targets. Whilst elevated levels of the MET receptor tyrosine kinase are associated with TNBCs and predict poor clinical outcome, the functional role of MET in TNBC is still poorly understood. In this study, we utilise an established Met-dependent transgenic mouse model of TNBC, human cell lines and patient-derived xenografts to investigate the role of MET in TNBC tumorigenesis. We find that in TNBCs with mesenchymal signatures, MET participates in a compensatory interplay with FGFR1 to regulate tumour-initiating cells (TICs). We demonstrate a requirement for the scaffold protein FRS2 downstream from both Met and FGFR1 and find that dual inhibition of MET and FGFR1 signalling results in TIC depletion, hindering tumour progression. Importantly, basal breast cancers that display elevated MET and FGFR1 signatures are associated with poor relapse-free survival. Our results support a role for MET and FGFR1 as potential co-targets for anti-TIC therapies in TNBC.

Triple negative aggressive phenotype controlled by miR-135b and miR-365: new theranostics candidates

Triple negative breast cancer (TNBC) accounts for about a fifth of all breast cancers and includes a diverse group of cancers. The heterogeneity of TNBC and the lack of target receptors on the cell surface make it difficult to develop specific therapeutic treatments. These aspects cause the high negative prognosis of patients with this type of tumor. The analysis of the molecular profiles of TNBC samples has allowed a better characterization of this tumor, supporting the search for new reliable diagnostic markers. To this end, we have developed a bioinformatic approach to integrate networks of genes differentially expressed in basal breast cancer compared to healthy tissues, with miRNAs able to regulate their expression. We studied the role of these miRNAs in TNBC subtype cell lines. We therefore identified two miRNAs, namely miR-135b and miR-365, with a central role in regulating the altered functional pathways in basal breast cancer. These two miRNAs are differentially expressed in human TNBC immunohistochemistry-selected tissues, and their modulation has been shown to play a role in the proliferation of tumor control and its migratory and invasive capacity in TNBC subtype cell lines. From the perspective of personalized medicine, we managed to modulate the expression of the two miRNAs in organotypic cultures, suggesting their possible use as diagnostic and therapeutic molecules. miR-135b and miR-365 have a key role in TNBC, controlling proliferation and invasion. Their detection could be helpful in TNBC diagnosis, while their modulation could become a new therapeutic tool for TNBC.

YAP and β-Catenin Cooperate to Drive Oncogenesis in Basal Breast Cancer.

Targeting cancer stem cells (CSC) can serve as an effective approach toward limiting resistance to therapies. While basal-like (triple-negative) breast cancers encompass cells with CSC features, rational therapies remain poorly established. We show here that the receptor tyrosine kinase Met promotes YAP activity in basal-like breast cancer and find enhanced YAP activity within the CSC population. Interfering with YAP activity delayed basal-like cancer formation, prevented luminal to basal transdifferentiation, and reduced CSC. YAP knockout mammary glands revealed a decrease in β-catenin target genes, suggesting that YAP is required for nuclear β-catenin activity. Mechanistically, nuclear YAP interacted with β-catenin and TEAD4 at gene regulatory elements. Proteomic patient data revealed an upregulation of the YAP signature in basal-like breast cancers. Our findings demonstrate that in basal-like breast cancers, β-catenin activity is dependent on YAP signaling and controls the CSC program. These findings suggest that targeting the YAP/TEAD4/β-catenin complex offers a potential therapeutic strategy for eradicating CSCs in basal-like breast cancers. SIGNIFICANCE: These findings show that YAP cooperates with β-catenin in basal-like breast cancer to regulate CSCs and that targeting this interaction may be a novel CSC therapy for patients with basal-like breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/8/2116/F1.large.jpg.

Patient-Specific Network for Personalized Breast Cancer Therapy with Multi-Omics Data.

The development of new computational approaches that are able to design the correct personalized drugs is the crucial therapeutic issue in cancer research. However, tumor heterogeneity is the main obstacle to developing patient-specific single drugs or combinations of drugs that already exist in clinics. In this study, we developed a computational approach that integrates copy number alteration, gene expression, and a protein interaction network of 73 basal breast cancer samples. 2509 prognostic genes harboring a copy number alteration were identified using survival analysis, and a protein–protein interaction network considering the direct interactions was created. Each patient was described by a specific combination of seven altered hub proteins that fully characterize the 73 basal breast cancer patients. We suggested the optimal combination therapy for each patient considering drug–protein interactions. Our approach is able to confirm well-known cancer related genes and suggest novel potential drug target genes. In conclusion, we presented a new computational approach in breast cancer to deal with the intra-tumor heterogeneity towards personalized cancer therapy.

Comparative stemness and differentiation of luminal and basal breast cancer stem cell type under glutamine-deprivation.

Glutamine (gln) metabolism has emerged as a cancer therapeutic target in past few years, however, the effect of gln-deprivation of bCSCs remains elusive in breast cancer. In this study, effect of glutamine on stemness and differentiation potential of bCSCs isolated from MCF-7 and MDAMB-231 were studied. We have shown that bCSCs differentiate into CD24+ epithelial population under gln-deprivation and demonstrated increased expression of epithelial markers such as e-cadherin, claudin-1 and decreased expression of mesenchymal protein n-cadherin. MCF-7-bCSCs showed a decrease in EpCAMhigh population whereas MDAMB-231-bCSCs increased CD44high population in response to gln-deprivation. The expression of intracellular stem cell markers such sox-2, oct-4 and nanog showed a drastic decrease in gene expression under gln-deprived MDAMB-231-bCSCs. Finally, localization of β-catenin in MCF-7 and MDAMB-231 cells showed its accumulation in cytosol or perinuclear space reducing its efficiency to transcribe downstream genes. Conclusively, our study demonstrated that gln-deprivation induces differentiation of bCSCs into epithelial subtypes and also reduces stemness of bCSCs mediated by reduced nuclear localization of β-catenin. It also suggests that basal and luminal bCSCs respond differentially towards changes in extracellular and intracellular gln. This study could significantly affect the gln targeting regimen of breast cancer therapeutics.

Regulation of cancer stem cells in triple negative breast cancer.

Triple Negative Breast Cancer (TNBC) is the most lethal subtype of breast cancer. Despite the successes of emerging targeted therapies, relapse, recurrence, and therapy failure rates in TNBC significantly outpace other subtypes of breast cancer. Mounting evidence suggests accumulation of therapy resistant Cancer Stem Cell (CSC) populations within TNBCs contributes to poor clinical outcomes. These CSCs are enriched in TNBC compared to non-TNBC breast cancers. The mechanisms underlying CSC accumulation have been well-characterized and discussed in other reviews. In this review, we focus on TNBC-specific mechanisms that allow the expansion and activity of self-renewing CSCs. We highlight cellular signaling pathways and transcription factors, specifically enriched in TNBC over non-TNBC breast cancer, contributing to stemness. We also analyze publicly available single-cell RNA-seq data from basal breast cancer tumors to highlight the potential of emerging bioinformatic approaches in identifying novel drivers of stemness in TNBC and other cancers.