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Simple Summary: Integrin α6β4 makes triple-negative breast cancer (TNBC) more aggressive by controlling genes that drive tumor invasion and metastasis. PTPRZ1 is linked to cancer relapse, but its role in TNBC is not well understood. We discovered that PTPRZ1 expression is significantly increased by integrin α6β4 in TNBC. We also found that integrin β4 gene expression correlates with PTPRZ1 in breast cancer patient samples. Importantly, we found that integrin α6β4 controls PTPRZ1 through UCHL1, which in turn stabilized Hif-1α. When UCHL1 or PTPRZ1 are blocked, the aggressiveness driven by integrin β4 signaling decreased substantially. Data analysis showed that high levels of these genes (ITGB4, UCHL1, Hif1α, PTPRZ1) are linked to worse survival rates, especially in patients who received chemotherapy treatment. Our findings suggest that integrin α6β4 helps TNBC become invasive through UCHL1-Hif-1α regulation of PTPRZ1, pointing to a new approach to targeting integrin α6β4 signaling using PTPRZ1 and UCHL1 inhibitors. Integrin α6β4 drives triple-negative breast cancer (TNBC) aggressiveness through the transcriptional regulation of key genes. Here, we investigated how integrin α6β4 regulates protein tyrosine phosphatase receptor type Z1 (PTPRZ1). Using stable re-expression of integrin β4 (ITGB4) in cells naturally devoid of integrin α6β4 or knockdown or knockout (KO) of ITGB4, we found that integrin α6β4 regulates PTPRZ1 expression. To gain mechanistic insight, we focused on Hif-1α due to the impact of integrin α6β4 on a hypoxia-associated signature. We found that nuclear localization of Hif-1α, but not Hif-2α, was substantially enhanced with integrin α6β4 signaling. Hif-1α knockdown by shRNA or chemical inhibition decreased PTPRZ1 expression, while chemical activation of Hif-1α increased it. Upstream of Hif-1α, integrin α6β4 upregulates UCHL1 to stabilize Hif-1α and ultimately regulate PTPRZ1. Inhibition of UCHL1 and PTPRZ1 dramatically decreases integrin α6β4-mediated cell migration and three-dimensional invasive growth. Finally, public breast cancer database analyses demonstrated that ITGB4 correlates with PTPRZ1 and that high expression of ITGB4, UCHL1, HIF1A, and PTPRZ1 associated with decreased overall survival, distant metastasis free survival, post progression survival, and relapse-free survival. In summary, these findings provide a novel function of integrin α6β4 in promoting tumor invasive phenotypes through UCHL1-Hif-1α-mediated regulation of PTPRZ1. [ABSTRACT FROM AUTHOR]

(1) Background: Breast cancer is a frequent heterogeneous disorder diagnosed in women and causes a high number of mortality among this population due to rapid metastasis and disease recurrence. Ferroptosis can inhibit breast cancer cell growth, improve the sensitivity of chemotherapy and radiotherapy, and inhibit distant metastases, potentially impacting the tumor microenvironment. (2) Methods: Through data mining, the ferroptosis/extracellular matrix remodeling literature text-mining results were integrated into the breast cancer transcriptome cohort, taking into account patients with distant relapse-free survival (DRFS) under adjuvant therapy (anthracyclin + taxanes) with validation in an independent METABRIC cohort, along with the MDA-MB-231 and HCC338 transcriptome functional experiments with ferroptosis activations (GSE173905). (3) Results: Ferroptosis/extracellular matrix remodeling text-mining identified 910 associated genes. Univariate Cox analyses focused on breast cancer (GSE25066) selected 252 individual significant genes, of which 170 were found to have an adverse expression. Functional enrichment of these 170 adverse genes predicted basal breast cancer signatures. Through text-mining, some ferroptosis-significant adverse-selected genes shared citations in the domain of ECM remodeling, such as TNF, IL6, SET, CDKN2A, EGFR, HMGB1, KRAS, MET, LCN2, HIF1A, and TLR4. A molecular score based on the expression of the eleven genes was found predictive of the worst prognosis breast cancer at the univariate level: basal subtype, short DRFS, high-grade values 3 and 4, and estrogen and progesterone receptor negative and nodal stages 2 and 3. This eleven-gene signature was validated as regulated by ferroptosis inductors (erastin and RSL3) in the triple-negative breast cancer cellular model MDA-MB-231. (4) Conclusions: The crosstalk between ECM remodeling-ferroptosis functionalities allowed for defining a molecular score, which has been characterized as an independent adverse parameter in the prognosis of breast cancer patients. The gene signature of this molecular score has been validated to be regulated by erastin/RSL3 ferroptosis activators. This molecular score could be promising to evaluate the ECM-related impact of ferroptosis target therapies in breast cancer. [ABSTRACT FROM AUTHOR]

Gene co-expression networks are a useful tool in the study of interactions that have allowed the visualization and quantification of diverse phenomena, including the loss of co-expression over long distances in cancerous samples. This characteristic, which could be considered fundamental to cancer, has been widely reported in various types of tumors. Since copy number variations (CNVs) have previously been identified as causing multiple genetic diseases, and gene expression is linked to them, they have often been mentioned as a probable cause of loss of co-expression in cancerous networks. In order to carry out a comparative study of the validity of this statement, we took 477 proteincoding genes from chromosome 8, and the CNVs of 101 genes, also proteincoding, belonging to the 8q24.3 region, a cytoband that is particularly active in the appearance of breast cancer. We created CNVS-conditioned co-expression networks of each of the 101 genes in the 8q24.3 region using conditional mutual information. The study was carried out using the four molecular subtypes of breast cancer (Luminal A, Luminal B, Her2, and Basal), as well as a case corresponding to healthy samples. We observed that in all cancer cases, the measurement of the Kolmogorov-Smirnov statistic shows that there are no significant differences between one and other values of the CNVs for any case. Furthermore, the co-expression interactions are stronger in all cancer subtypes than in the control networks. However, the control network presents a homogeneously distributed set of co-expression interactions, while for cancer networks, the highest interactions are more confined to specific cytobands, in particular 8q24.3 and 8p21.3. With this approach, we demonstrate that despite copy number alterations in the 8q24 region being a common trait in breast cancer, the loss of long-distance co-expression in breast cancer is not determined by CNVs. [ABSTRACT FROM AUTHOR]

(1) Background: Breast cancer is a frequent heterogeneous disorder diagnosed in women and causes a high number of mortality among this population due to rapid metastasis and disease recurrence. Ferroptosis can inhibit breast cancer cell growth, improve the sensitivity of chemotherapy and radiotherapy, and inhibit distant metastases, potentially impacting the tumor microenvironment. (2) Methods: Through data mining, the ferroptosis/extracellular matrix remodeling literature text-mining results were integrated into the breast cancer transcriptome cohort, taking into account patients with distant relapse-free survival (DRFS) under adjuvant therapy (anthracyclin + taxanes) with validation in an independent METABRIC cohort, along with the MDA-MB-231 and HCC338 transcriptome functional experiments with ferroptosis activations (GSE173905). (3) Results: Ferroptosis/extracellular matrix remodeling text-mining identified 910 associated genes. Univariate Cox analyses focused on breast cancer (GSE25066) selected 252 individual significant genes, of which 170 were found to have an adverse expression. Functional enrichment of these 170 adverse genes predicted basal breast cancer signatures. Through text-mining, some ferroptosis-significant adverse-selected genes shared citations in the domain of ECM remodeling, such as TNF, IL6, SET, CDKN2A, EGFR, HMGB1, KRAS, MET, LCN2, HIF1A, and TLR4. A molecular score based on the expression of the eleven genes was found predictive of the worst prognosis breast cancer at the univariate level: basal subtype, short DRFS, high-grade values 3 and 4, and estrogen and progesterone receptor negative and nodal stages 2 and 3. This eleven-gene signature was validated as regulated by ferroptosis inductors (erastin and RSL3) in the triple-negative breast cancer cellular model MDA-MB-231. (4) Conclusions: The crosstalk between ECM remodeling-ferroptosis functionalities allowed for defining a molecular score, which has been characterized as an independent adverse parameter in the prognosis of breast cancer patients. The gene signature of this molecular score has been validated to be regulated by erastin/RSL3 ferroptosis activators. This molecular score could be promising to evaluate the ECM-related impact of ferroptosis target therapies in breast cancer.

Neuronal calcium sensor‐1 (NCS‐1) is a positive modulator of IP3 receptors and was recently associated with poorer survival in breast cancers. However, the association between NCS‐1 and breast cancer molecular subtypes and the effects of NCS‐1 silencing on calcium (Ca2+) signaling in breast cancer cells remain unexplored. Herein, we report for the first time an increased expression of NCS‐1 in breast cancers of the basal molecular subtype, a subtype associated with poor prognosis. Using MDA‐MB‐231 basal breast cancer cells expressing the GCaMP6m Ca2+ indicator, we showed that NCS‐1 silencing did not result in major changes in cytosolic free Ca2+ increases as a result of endoplasmic reticulum Ca2+ store mobilization. However, NCS‐1 silencing suppressed unstimulated basal Ca2+ influx. NCS‐1 silencing in MDA‐MB‐231 cells also promoted necrotic cell death induced by the chemotherapeutic drug doxorubicin (1 µm). The effect of NCS‐1 silencing on cell death was phenocopied by silencing of ORAI1, a Ca2+ store‐operated Ca2+ channel that maintains Ca2+ levels in the endoplasmic reticulum Ca2+ store and whose expression was significantly positively correlated with NCS‐1 in clinical breast cancer samples. This newly identified association between NCS‐1 and basal breast cancers, together with the identification of the role of NCS‐1 in the regulation of the effects of doxorubicin in MDA‐MB‐231 breast cancer cells, suggests that NCS‐1 and/or pathways regulated by NCS‐1 may be important in the treatment of basal breast cancers in women.

The basal breast cancer subtype is enriched for triple-negative breast cancer (TNBC) and displays consistent large chromosomal deletions. Here, we characterize evolution and maintenance of chromosome 4p (chr4p) loss in basal breast cancer. Analysis of The Cancer Genome Atlas data shows recurrent deletion of chr4p in basal breast cancer. Phylogenetic analysis of a panel of 23 primary tumor/patient-derived xenograft basal breast cancers reveals early evolution of chr4p deletion. Mechanistically we show that chr4p loss is associated with enhanced proliferation. Gene function studies identify an unknown gene, C4orf19 , within chr4p, which suppresses proliferation when overexpressed—a member of the PDCD10-GCKIII kinase module we name PGCKA1. Genome-wide pooled overexpression screens using a barcoded library of human open reading frames identify chromosomal regions, including chr4p, that suppress proliferation when overexpressed in a context-dependent manner, implicating network interactions. Together, these results shed light on the early emergence of complex aneuploid karyotypes involving chr4p and adaptive landscapes shaping breast cancer genomes. [Display omitted] • Chr4p loss evolves early in TNBC • Chr4p loss enhances growth • C4orf19 (PGCKA1) tumor suppressor Kuzmin et al. report that chromosome 4p loss evolves early in triple-negative breast cancer (TNBC) and is associated with enhanced proliferation. C4orf19 (PGCKA1) is a tumor suppressor. Certain regions, including chr4p, suppress proliferation when overexpressed, differentially implicating network rewiring. This study illuminates the early emergence of complex aneuploid karyotypes in TNBC. [ABSTRACT FROM AUTHOR]

Summary: 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.

Neuronal calcium sensor‐1 (NCS‐1) is a positive modulator of IP3 receptors and was recently associated with poorer survival in breast cancers. However, the association between NCS‐1 and breast cancer molecular subtypes and the effects of NCS‐1 silencing on calcium (Ca2+) signaling in breast cancer cells remain unexplored. Herein, we report for the first time an increased expression of NCS‐1 in breast cancers of the basal molecular subtype, a subtype associated with poor prognosis. Using MDA‐MB‐231 basal breast cancer cells expressing the GCaMP6m Ca2+ indicator, we showed that NCS‐1 silencing did not result in major changes in cytosolic free Ca2+ increases as a result of endoplasmic reticulum Ca2+ store mobilization. However, NCS‐1 silencing suppressed unstimulated basal Ca2+ influx. NCS‐1 silencing in MDA‐MB‐231 cells also promoted necrotic cell death induced by the chemotherapeutic drug doxorubicin (1 µm). The effect of NCS‐1 silencing on cell death was phenocopied by silencing of ORAI1, a Ca2+ store‐operated Ca2+ channel that maintains Ca2+ levels in the endoplasmic reticulum Ca2+ store and whose expression was significantly positively correlated with NCS‐1 in clinical breast cancer samples. This newly identified association between NCS‐1 and basal breast cancers, together with the identification of the role of NCS‐1 in the regulation of the effects of doxorubicin in MDA‐MB‐231 breast cancer cells, suggests that NCS‐1 and/or pathways regulated by NCS‐1 may be important in the treatment of basal breast cancers in women. [ABSTRACT FROM AUTHOR]

Breast cancer is characterized as being a heterogeneous pathology with a broad phenotype variability. Breast cancer subtypes have been developed in order to capture some of this heterogeneity. Each of these breast cancer subtypes, in turns retains varied characteristic features impacting diagnostic, prognostic and therapeutics. Basal breast tumors, in particular have been challenging in these regards. Basal breast cancer is often more aggressive, of rapid evolution and no tailor-made targeted therapies are available yet to treat it. Arguably, epigenetic variability is behind some of these intricacies. It is possible to further classify basal breast tumor in groups based on their non-coding transcriptome and methylome profiles. It is expected that these groups will have differences in survival as well as in sensitivity to certain classes of drugs. With this in mind, we implemented a computational learning approach to infer different subpopulations of basal breast cancer (from TCGA multi-omic data) based on their epigenetic signatures. Such epigenomic signatures were associated with different survival profiles; we then identified their associated gene co-expression network structure, extracted a signature based on modules within these networks, and use these signatures to find and prioritize drugs (in the LINCS dataset) that may be used to target these types of cancer. In this way we are introducing the analytical workflow for an epigenomic signature-based drug repurposing structure. [Display omitted] [ABSTRACT FROM AUTHOR]

Ionizing radiation and cumulative exposure to steroid hormones are known risk factors for breast cancer. There is increasing evidence that breast tumors are driven by a subpopulation of tumor-initiating cancer stem cells (CSCs). In MCF10A non-cancerous basal-like PR − cells, progesterone treatment and X-rays generated ALDH + and CD44 + /CD24 − CSCs. Here, we report that in irradiated MCF10A cells, progesterone activated the PI3K/Akt pathway via membrane progesterone receptor (mPR). Inhibition of the PI3K/Akt pathway counteracted the generation of CSCs by progesterone and irradiation. The stimulation of PI3K/Akt via mPR resulted in the inactivation of FOXO transcriptional activity, the upregulation of snail and slug expression and a downregulation of miR-29 expression, which led to increased levels of KLF4, a transcription factor required for breast CSC maintenance. Stabilization of miR-29 expression impeded the generation of CSCs, while its inhibition alone was sufficient to generate CSCs. This study provides a new mechanistic basis for progesterone and radiation-induced breast cancer risk in basal cells. In addition, the elucidation of new pathways and miRNA regulations involved in CSC generation and maintenance may open the door to potential novel anti-CSC strategies. [ABSTRACT FROM AUTHOR]

We have compared the proteome, transcriptome, and metabolome of two cell lines: the human breast epithelial line MCF-10A and its mutant descendant MCF-10A-H1047R. These cell lines are derived from the same parental stock and differ by a single amino acid substitution (H1047R) caused by a single nucleotide change in one allele of the PIK3CA gene, which encodes the catalytic subunit p110a of PI3K (phosphatidylinositol 3-kinase). They are considered isogenic. The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF- 10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse, with examples in structural protein levels, the DNA repair machinery, and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer. [ABSTRACT FROM AUTHOR]

Background Young age breast cancers are quite prevalent in our setup, a significant number of which exhibit triple negative phenotype. These cancers behave in an aggressive fashion and unresponsive to targeted adjuvant therapy. We aimed to evaluate clinical and histopathologic features of triple negative cancers in our population. Methods We retrospectively evaluated 1104 cases of primary breast cancers. Immunohistochemical studies for ER, PR and Her2neu followed by Her2neu gene amplification by FISH testing were done to identify 205 (18.6%) cases of triple negative breast cancers. Results Mean age for triple negative breast cancer patients was 48.4 years (±12.3) and 60% of patients were diagnosed at less than 50 years of age. Although ductal carcinoma was the most frequent histologic type, a meaningful number of cases exhibited metaplastic and medullary like features (10.7% and 5.9% respectively). Similarly geographic necrosis involving more than 40% of tumor and extensive lymphocytic infiltration was a considerable finding. Mean Ki67 index was 45.2% (±25.2) and as a reflection of tumor grade, a significantly higher proportion of cases (66.3%) were under high risk Ki67 category (>30%). Conclusion Triple negative breast cancers typify high grade breast cancers with a higher frequency of atypical medullary and metaplastic histologies. Their prevailing occurrence at a younger age raises question of under lying BRCA mutations in our population. Therefore, we suggest that risk factors including BRCA 1 mutations should be uncovered in reproductive age group breast cancers especially those disclosing basal like phenotype. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9042440621102239. [ABSTRACT FROM AUTHOR]

Basal-like and triple-negative breast cancers usually display a high level of genomic instability and often carry TP53 mutations. Mutations in EGFR have been reported in about 10 % triple-negative tumours from Chinese women, and there is some evidence that triple-negative and basal-like tumours might carry additional mutations against which targeted therapies are available. We, therefore, sought to determine the frequency of 238 targetable mutations in 19 oncogenes (including EGFR) in a panel of basal-like and triple-negative breast cancers from Caucasian women. We used the OncoCarta panel to screen for 238 mutations across 19 common oncogenes in 107 basal-like and triple-negative breast cancers from Caucasian women. Mutations were then verified using Sanger sequencing or primer extension by iPLEX. We identified and validated 10 mutations across five genes. Most of the mutations were observed in the PIK3CA gene (18/107, 16.8 %), while mutations in KRAS, NRAS, MET and AKT1 were present in only one tumour each (1/107, 0.9 %). Among the missense substitutions in PIK3CA the point mutation resulting in the amino acid change H1047R was the most frequent (8/18, 44 %). All mutations were mutually exclusive, apart from one basal-like breast tumour which harboured mutations in both MET (p.T992I) and PIK3CA (p.H1047R). We did not identify any mutations in the EGFR gene. In conclusion, we found that with the exception of mutations in PIK3CA, these actionable oncogenic mutations on the Oncocarta panel are rare in basal-like and triple-negative breast cancers from Caucasian women. Custom panels, designed to detect mutations identified by exome sequencing of basal-like and triple-negative breast cancers, are, therefore, needed to identify women who might be eligible for targeted treatment. [ABSTRACT FROM AUTHOR]

The transcription factor FOXA1, which is a member of the forkhead class of DNA-binding proteins, interacts with Estrogen Receptor (ER) to mediate breast cancer progression. However, its role in basal breast cancer cells remains unclear. Although the overall levels of FOXA1 are decreased in the basal subtype of clinical TCGA breast cancer samples, the high levels of FOXA1 improve the survival of the patients from this subtype. This clinical phenomenon is consistent with that of FOXA1 stimulating apoptosis in FOXA1-low expressing basal breast cancer cells, such as MDA-MB-231, and MDA-MB-468 cells. In this study, we have constructed an inducible expression system of FOXA1 and demonstrated the induced expression of FOXA1 resulting in apoptosis and cell cycle arrest in MDA-MB-231 cells, as confirmed by transcriptomic analysis and in vivo tumor-grafted models. Furthermore, the low levels of Estrogen Receptor-1 (ESR1) are critical for FOXA1 in terms of its repressive roles in the cells, as evidenced by clinical data analysis indicating that the high levels of FOXA1 improve the survival of ESR1 Low patients, but worsen the survival of ESR1 High patients of breast cancer. When introduced into MDA-MB-231 cells, ESR1 counteracts the tumor suppressor roles of FOXA1 by altering the FOXA1-regulated gene transcription and the two proteins together maintain the tumor progression in vivo . Our cumulative results suggest that FOXA1 suppresses the basal breast cancer cells with FOXA1-low expressing status independent of ESR1 by inducing apoptosis and inhibiting cell proliferation, thereby implicating its potential therapeutic role in this group of breast cancer., Competing Interests: None., (AJCR Copyright © 2022.)

The purpose of this study is to explore the relationship between tumor hypoxia assessed by CA IX protein expression and loss of BRCA1 function in triple negative breast cancer (TNBC). Protein expression of CA IX and BRCA1 was evaluated by AQUA™ technology on two breast cancer cohorts: an unselected cohort of 637 breast cancer patients and a TNBC cohort of 120 patients. Transcriptional profiling was performed on FFPE samples from the TNBC cohort to evaluate a gene expression signature associated with BRCA1 mutation (van't Veer et al., Nature 415(6871):530-536, ). CA IX is expressed in 7 % of the unselected breast cancer cohort and in 25 % of the TNBCs and is significantly associated with the triple negative phenotype. CA IX protein expression and BRCA1 protein expression are inversely correlated in both cohorts. Patients expressing high levels of CA IX show significantly worse overall survival ( p = 0.02). Importantly, high CA IX protein expression occurs in patients who show the BRCA1 mutant signature and low levels of BRCA1 protein. These data suggest that elevated CA IX protein in TNBC is associated with a BRCA1 mutant signature and loss of BRCA1 function. CA IX may be a useful biomarker to identify triple negative patients with defective homologous recombination, who might benefit from PARP inhibitor therapy. [ABSTRACT FROM AUTHOR]

Summary: Global gene expression profiling studies led to the recent classification of breast cancer into 4 distinct molecular subtypes including luminal, human epidermal growth factor receptor 2 enriched, basal like, and unclassified. Here, we used immunohistochemistry to evaluate expression of the antiapoptotic protein Survivin and its recently described acetylated form, Survivin acetyl129, in normal breast tissue and in 226 primary breast tumors of different molecular subtypes. Correlation of Survivin expression with molecular markers and its impact on patient outcomes were analyzed. Eighty-four percent of basal-like tumors expressed high levels of total Survivin, whereas 52% of luminal tumors expressed high levels of acetylated Survivin (P < .001). Overall survival (91%) for tumors expressing low levels of total Survivin was better than that for tumors expressing high levels of total Survivin (72%, P = .02), whereas the reverse was true for tumors expressing acetylated Survivin. In hierarchical cluster analysis, total Survivin clustered with basal marker expression, whereas acetylated Survivin clustered with luminal marker expression. In multivariate analysis, high total Survivin expression was an independent predictor of worse overall survival in patients with breast cancer (relative risk, 11; P < .01). These data indicate that high levels of total Survivin predict poor outcome in patients with grade 3 invasive ductal carcinoma and correlate directly with a basal-like phenotype. In contrast, high expression of the acetylated form of the protein associates with a favorable outcome and preferentially correlates with luminal-type tumors. Survivin likely has different functions in distinct breast cancer subtypes, and diagnostic strategies that incorporate immunohistochemical markers that detect both Survivin forms may help better strategize patient risk and direct therapy. [Copyright &y& Elsevier]

Prognosis of basal breast cancers is poor but heterogeneous. Medullary breast cancers (MBC) display a basal profile, but a favorable prognosis. We hypothesized that a previously published 368-gene expression signature associated with MBC might serve to define a prognostic classifier in basal cancers. We collected public gene expression and histoclinical data of 2145 invasive early breast adenocarcinomas. We developed a Support Vector Machine (SVM) classifier based on this 368-gene list in a learning set, and tested its predictive performances in an independent validation set. Then, we assessed its prognostic value and that of six prognostic signatures for disease-free survival (DFS) in the remaining 2034 samples. The SVM model accurately classified all MBC samples in the learning and validation sets. A total of 466 cases were basal across other sets. The SVM classifier separated them into two subgroups, subgroup 1 (resembling MBC) and subgroup 2 (not resembling MBC). Subgroup 1 exhibited 71% 5-year DFS, whereas subgroup 2 exhibited 50% ( P = 9.93E-05). The classifier outperformed the classical prognostic variables in multivariate analysis, conferring lesser risk for relapse in subgroup 1 (HR = 0.52, P = 3.9E-04). This prognostic value was specific to the basal subtype, in which none of the other prognostic signatures was informative. Ontology analysis revealed effective immune response (IR), enhanced tumor cell apoptosis, elevated levels of metastasis-inhibiting factors and low levels of metastasis-promoting factors in the good-prognosis subgroup, and a more developed cell migration system in the poor-prognosis subgroup. In conclusion, based on this 368-gene SVM model derived from an MBC signature, basal breast cancers were classified in two prognostic subgroups, suggesting that MBC and basal breast cancers share similar molecular alterations associated with aggressiveness. This signature could help define the prognosis, adapt the systemic treatment, and identify new therapeutic targets. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in some but not all breast cancer cell lines. Breast cancers can be divided into those which express the estrogen (ER) and progesterone (PR) receptors, those with HER-2 amplification, and those without expression of ER, PR, or HER-2 amplification (referred to as basal or triple-negative breast cancer). We tested a panel of 20 breast cancer cell lines representing the different types of breast cancer to evaluate if the molecular phenotype of the breast cancer cells determined their response to TRAIL. The most striking finding was that eight of eleven triple-negative cell lines are sensitive to TRAIL-mediated apoptosis. The eight TRAIL-sensitive triple-negative cell lines have a mesenchymal phenotype while the three TRAIL-resistant triple-negative cell lines have an epithelial phenotype. Two of five cell lines with HER-2 amplification were sensitive to TRAIL and none of the five ER positive cell lines were sensitive. RNAi-mediated knockdown of TRAIL receptor expression demonstrated that TRAIL Receptor 2 (TRAIL-R2) mediates the effects of TRAIL, even when both TRAIL-R1 and TRAIL-R2 are expressed. Finally, inhibition of EGFR, expressed in both TRAIL-sensitive and TRAIL-resistant triple-negative breast cancer cell lines, using a small molecule tyrosine kinase inhibitor (AG1478), enhanced TRAIL-induced apoptosis in TRAIL-sensitive cell lines but did not convert resistant cells into TRAIL-sensitive cells. Together, these findings suggest that a subset of triple-negative breast cancer, those with mesenchymal features, may be the most likely to benefit from TRAIL targeted therapy. These findings could form the basis to select breast cancer patients for clinical trials of TRAIL-R2 ligands. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Drop-seq reveals a strong cancer cell heterogeneity in PyMT mammary tumors • Elf5 redefines cancer cell states toward basal-like with pro-metastatic features • PyMT/Elf5 tumors show cellular and molecular characteristics of involution mimicry • Involution CAFs in PyMT/Elf5 tumors interconnect the TME and cancer cells Using single-cell transcriptomics, Valdés-Mora et al. show that Elf5 drives alveolar lineage differentiation in PyMT tumors, resulting in cancer cell states with basal-like characteristics and a multicellular process of unresolved lactation, with concomitant acquisition of metastasis. This study may partially explain the poor prognosis of breast cancer associated with childbirth. [ABSTRACT FROM AUTHOR]

Recently, we have shown that the CCL5/CCR5 axis is active in patients affected by an aggressive basal subtype of breast cancer. Using preclinical models, we have demonstrated that CCR5 promotes breast cancer invasiveness and metastatic potential, while CCR5 inhibition abrogates them. Thus, CCR5 antagonists may constitute an alternative therapeutic approach for patients affected by metastatic basal breast cancer. [ABSTRACT FROM AUTHOR]

FOXM1 is frequently overexpressed in cancer, but this has not been studied in a comprehensive manner. We utilized genotype-tissue expression (GTEx) normal and The Cancer Genome Atlas (TCGA) tumor data to define FOXM1 expression, including its isoforms, and to determine the genetic alterations that promote FOXM1 expression in cancer. Additionally, we used human fallopian tube epithelial (FTE) cells to dissect the role of Retinoblastoma (Rb)-E2F and Cyclin E1 in FOXM1 regulation, and a novel human embryonic kidney cell (HEK293T) CRISPR FOXM1 knockout model to define isoform-specific transcriptional programs. FOXM1 expression, at the mRNA and protein level, was significantly elevated in tumors with FOXM1 amplification, p53 inactivation, and Rb-E2F deregulation. FOXM1 expression was remarkably high in testicular germ cell tumors (TGCT), high-grade serous ovarian cancer (HGSC), and basal breast cancer (BBC). FOXM1 expression in cancer was associated with genomic instability, as measured using aneuploidy signatures. FTE models confirmed a role for Rb-E2F signaling in FOXM1 regulation and in particular identified Cyclin E1 as a novel inducer of FOXM1 expression. Among the three FOXM1 isoforms, FOXM1c showed the highest expression in normal and tumor tissues and cancer cell lines. The CRISPR knockout model demonstrated that FOXM1b and FOXM1c are transcriptionally active, while FOXM1a is not. Finally, we were unable to confirm the existence of a FOXM1 auto-regulatory loop. This study provides significant and novel information regarding the frequency, causes, and consequences of elevated FOXM1 expression in human cancer. [ABSTRACT FROM AUTHOR]

Background: We investigated the effect of basal protein expression on trastuzamab response in patients with HER2-positive (HER2(+)) breast cancer who received trastuzamab (T) and in HER2(+) breast cancer cell lines., Patients and Methods: Expression of cytokeratin (CK) 5/6, CK14, and epidermal growth factor receptor (EGFR) was evaluated after immunohistochemical staining in paraffin-embedded tissue of 97 patients with stage I to III HER2(+) breast cancer treated with chemotherapy/T. Groups with and without basal protein expression were compared with respect to clinicopathologic parameters and survival. We treated 4 cell lines (2 basal-HER2 [HCC1569, HCC1954] and 2 nonbasal HER2 [BT474, SKBR3]) each with vehicle, T 20 μg/mL, paclitaxel 0.01 μM (P), and T with P (T + P). Cell viability was assessed and HER2 pathway suppression was compared between groups using immunoblot analysis. Mammosphere formation was used to assess breast cancer stem cell properties., Results: EGFR expression was significantly associated with cancer-specific survival (CSS) (P = .05). CK5/6 expression strongly correlated with overall and disease-free survival, and CSS (P = .03, P = .04, and P = .03, respectively). Statistical significance was maintained for EGFR and CK5/6 after adjustment for covariates. CK14 was not associated with survival. All cell lines expressed similar levels of HER2. T and P alone inhibited proliferation of nonbasal cell lines; T + P had an additive cytotoxic effect. Basal cells were resistant to T, P inhibited proliferation, but T + P had no additive cytotoxic effect on cell growth in basal cells. Immunoblot analysis showed a significant decrease in phosphorylated Akt levels after treatment with T or T + P in nonbasal cells but not in basal cells. Akt blockade suppressed growth of basal and nonbasal HER2(+) cells. Furthermore, basal HER2 cell lines had increased mammosphere formation, which suggests increased stem cell properties compared with nonbasal HER2 cell lines., Conclusion: CK5/6 and EGFR expression are predictive of worse prognosis in HER2(+) breast cancer patients treated with T. Basal HER2 breast cancer cell lines are resistant to trastuzamab, which is mediated through the Akt pathway; AKT inhibition abrogates this resistance. Basal HER2 cell lines also have increased stem cell properties, which might play a role in the resistance pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Dysregulation of the epigenome is a common event in malignancy; however, deciphering the earliest cancer-associated epigenetic events remains a challenge. Cancer epigenome studies to date have primarily utilised cancer cell lines or clinical samples, where it is difficult to identify the initial epigenetic lesions from those that occur over time. Here, we analysed the epigenome of human mammary epithelial cells (HMEC) and a matched variant cell population (vHMEC) that have spontaneously escaped senescence and undergone partial carcinogenic transformation. Using this model of basal-like breast carcinogenesis, we provide striking new insights into the very first epigenetic changes that occur during the initial stages of malignancy., Results: The first phase of malignancy is defined by coordinated changes in the epigenome. At the chromatin level, this is embodied in long-range epigenetic deregulation, which involves the concomitant but atypical acquisition or loss of active and repressive histone modifications across large regional blocks. Changes in DNA methylation also occurs in a highly coordinated manner. We identified differentially methylated regions (DMRs) in the very earliest passages of vHMECs. Notably, we find that differential methylation targets loci regulated by key transcription factors including p53, AHR and E2F family members suggesting that epigenetic deregulation of transcription factor binding is a key event in breast carcinogenesis. Interestingly, DMRs identified in vHMEC are extensively methylated in breast cancer, with hypermethylation frequently encroaching into neighbouring regions. A subset of vHMEC DMRs exhibited a strong basal-like cancer specific hypermethylation., Conclusions: Here, we generated epigenome-wide maps of the earliest phase of breast malignancy and show long-range epigenetic deregulation and coordinated DNA hypermethylation targets loci regulated by key transcription factors. These findings support a model where induction of breast cancer occurs through epigenetic disruption of transcription factor binding leading to deregulation of cancer-associated transcriptional networks. With their stability and very early occurrence, vHMECs hypermethylated loci could serve as excellent biomarkers for the initial detection of basal breast cancer.

Cancer stem cells (CSCs) represent a subset of tumor cells with tumor-initiating potential. We recently demonstrated that inhibition of the sumoylation pathway cleared the CSC population and repressed the outgrowth of basal breast cancer xenografts. Targeting the sumoylation pathway offers a novel treatment strategy for basal breast cancer.

Triple-negative breast cancers (TNBC) are defined by their failure to express the estrogen receptor, progesterone receptor, and HER2/neu protein markers. This basic feature is clinically relevant because it indicates that these cancers cannot be managed with endocrine or anti-HER2 systemic therapies. Furthermore, most TNBC cases are also characterized as being of the genetically defined basal subtype, which is an inherently and biologically more aggressive pattern of disease. The two terms, however, are not synonymous, and some TNBC cases are prognostically more favorable. TNBC differs from non-TNBC in risk-factor profile, pattern, and rate of metastatic spread., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The protein 14-3-3σ (stratifin) is frequently described as a tumor suppressor silenced in about 80% of breast tumors. Intriguingly, we show that 14-3-3σ expression, which in normal breast is localized to the myoepithelial cells, tracks with malignant phenotype in two models of basal-like breast cancer progression, and in patients, it is associated with basal-like subtype and poor clinical outcome. We characterized a mechanism by which 14-3-3σ guides breast tumor invasion by integrating cytoskeletal dynamics: it stabilizes a complex of solubilized actin and intermediate filaments to maintain a pool of "bioavailable" complexes for polarized assembly during migration. We show that formation of the actin/cytokeratin/14-3-3σ complex and cellular migration are regulated by PKCζ-dependent phosphorylation, a finding that could form the basis for intervention in aggressive breast carcinomas expressing 14-3-3σ. Our data suggest that the biology of this protein is important in cellular movement and is contingent on breast cancer subtype.