Basal-like Phenotype In Breast Cancer Research Articles

Abstract 6544: Identification of epigenetic regulatory networks associated with the basal-like breast cancer subtype

Abstract Purpose: Basal-like breast cancer (BLBC) is a biologically aggressive and molecular heterogeneous breast cancer subtype that confers to the patient´s poor prognosis. The crosstalk of epigenetic regulators, such as methylation events and microRNA (miRNA) deregulation, drives breast cancer progression and modulates aggressive tumor phenotypes. However, few studies have specifically characterized the epigenetic regulatory networks of genome-wide DNA methylation-based modifications, miRNAs and transcription factors (TF) expression in BLBC. Therefore, the main objective of this study was to identify and correlate the DNA methylation profiles and the transcriptional expression of miRNAs and TFs of the BLBC subtype. Methods: To achieve our goal, a comprehensive computational pipeline using the R language was used. First, the transcriptional expression profiles and DNA methylation data of BLBC and non-tumor samples were downloaded from The Cancer Genome Atlas (TCGA). Then, the ELMER package was used to determine the differentially methylated distal probes between these two groups, identifying gene-probes pairs that can be regulated by un/methylated regions that affect the function of TFs. The different expressed (DE) miRNAs were identified using the DESeq2 package. To determine the regulatory pairs (gene, miRNA and TF interactions) the multiMiR, miRWalk, ENCODE, and hTFtarget were used and the regulatory networks were identified by the FANMOD algorithm. Finally, pathway enrichment analysis (PEA) was conducted using the Reactome database and microT-CDS (Diana Tools mirPath v.3). Results: A total of 147 and 199 pairs of gene-probes were identified in hypermethylation and hypomethylation analyses, respectively. Fifty genes and 59 regulatory TFs whose expression was associated with DNA methylation were selected, including members of the E2F (E2Fs) and Krüppel-like factor (KLFs) family. Eleven DE miRNAs, out of the 51 identified, were selected based on their interaction with genes and TFs. These pairs of miRNAs, TF, and genes generated three types of feed-forward loops (FFLs): coherent FFLs, mediated by miRNAs; incoherent FFLs, mediated by TFs; and compost FFLs, mediated by TF and miRNAs. Finally, the PEA analysis showed 51 pathways in which miRNAs, genes, or TFs may interact, including pathways related to DNA repair, cell cycle, pathways in cancer, as well as the defective binding of RB1 mutants to E2F1, and the ErbB signaling pathway. Conclusion: Our findings demonstrated the epigenetic regulation of methylation patterns and miRNA expression and their impact on the expression of TFs that may exert critical roles in the BLBC. The functional analysis of these regulatory networks in well-established BLBC models, targeting the signaling pathways identified, can determine whether they modulate the BLBC phenotype and can be used as potential clinical targets. Citation Format: Larissa Miyuki Okano, Alexandre Luiz Azevedo, Tamyres Mingorance Carvalho, Fernanda Brandão Berti, Luciane Regina Cavalli. Identification of epigenetic regulatory networks associated with the basal-like breast cancer subtype. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6544.

Programmed cell death 1 and programmed cell death ligand 1 expression in invasive breast carcinoma using CAL10 and NAT105 immunostaining

ABSTRACT Increased incidence of breast cancer has stimulated development of new diagnostic and therapeutic methods. The programmed cell death 1 (PD1) pathway and its inhibitors are promising avenues for investigation. PD1 includes PD ligands 1 (PDL1) and 2 (PDL2). We investigated the expression of PD1 and PDL1 in invasive breast carcinomas using immunohistochemical staining. We used 171 invasive breast carcinoma specimens from which tissue microarray blocks were created. Immunohistochemical staining of PD1 using NAT105, and PDL1 using CAL10 was performed on tissue microarray sections. NAT105 and CAL10 are useful clones for detecting expression of PD1 and PDL1. PD1 and PDL1 immunostaining was significantly stronger in carcinomas with basal-like phenotype compared to other molecular breast cancer types. PD1 and PDL1 expression also was associated with a high histologic grade and a high Ki-67 index. PD1 expression also was associated with lymphovascular invasion and axillary metastasis. PD1 and PDL1 expression is associated with aggressive tumor behavior and a basal-like phenotype in breast cancer. We suggest that inhibition of the PD1/PDL1 pathway, particularly in triple negative breast carcinomas with basal-like phenotype, might be useful for targeted immunotherapy.

Modulatory Role of microRNAs in Triple Negative Breast Cancer with Basal-Like Phenotype.

Simple SummaryTriple Negative breast cancer (TNBC) is an aggressive tumor showing high histological grade, high recurrence, and frequent metastasis, accounting for about 25% of breast cancer-related deaths. Emerging roles and molecular mechanisms by which miRNAs impact pathogenesis and prognosis of basal-phenotype TNBC and their potential clinical utility are analyzed in the present review. Progress achieved in TNBC molecular taxonomy had minimal clinical impact, while miRNAs could act as prognostic/predictive biomarkers for TNBC subtypes. As there are currently no other therapeutic options to treat TNBC apart from chemotherapy, various studies were reviewed to draw the conclusion that ncRNAs might be candidates for drug development and drug resistance. Targeted approaches to epigenetic mechanisms and clarification of the molecular mechanisms of specific miRNAs in TNBC subtypes are fully justified. This review might provide a collection of biomarkers potentially useful in clinical settings and shows that the combination of miRNA-based therapeutic strategies with conventional therapies might synergize anticancer effects improving patient outcome.Development of new research, classification, and therapeutic options are urgently required due to the fact that TNBC is a heterogeneous malignancy. The expression of high molecular weight cytokeratins identifies a biologically and clinically distinct subgroup of TNBCs with a basal-like phenotype, representing about 75% of TNBCs, while the remaining 25% includes all other intrinsic subtypes. The triple negative phenotype in basal-like breast cancer (BLBC) makes it unresponsive to endocrine therapy, i.e., tamoxifen, aromatase inhibitors, and/or anti-HER2-targeted therapies; for this reason, only chemotherapy can be considered an approach available for systemic treatment even if it shows poor prognosis. Therefore, treatment for these subgroups of patients is a strong challenge for oncologists due to disease heterogeneity and the absence of unambiguous molecular targets. Dysregulation of the cellular miRNAome has been related to huge cellular process deregulations underlying human malignancy. Consequently, epigenetics is a field of great promise in cancer research. Increasing evidence suggests that specific miRNA clusters/signatures might be of clinical utility in TNBCs with basal-like phenotype. The epigenetic mechanisms behind tumorigenesis enable progress in the treatment, diagnosis, and prevention of cancer. This review intends to summarize the epigenetic findings related to miRNAome in TNBCs with basal-like phenotype.

Abstract 3148: FOXC1 expression is associated with a triple-negative basal-like phenotype in breast cancer

Abstract Introduction: Triple-negative breast carcinoma (TNBC) is a group of aggressive tumors that are resistant to hormonal therapy. Forkhead box C1 (FOXC1) has been shown to play a crucial role in cancer pathogenesis by cancer stem cell regulation, epithelial to mesenchymal transition and tumor progression in basal-like breast cancer (BLBC). This study examined the expression of FOXC1 in basal-like TNBC, which may potential be exploited as a treatment target for this group of tumors. Materials and Methods: Immunohistochemical Peroxidase-DAB system and Opal immunofluorescence staining system were employed to detect FOXC1 and cytokeratin 14 (CK14) expressions in 44 formalin-fixed paraffin-embedded (FFPE) invasive ductal breast carcinoma (IDC) and 150 IDC samples on tissue microarray. CK14 was used to identify the BLBC subtype. The staining was scored semi-quantitatively according to the intensity with a mean score from 0-4. Results: CK14 identified 15 (34%) BLBC in the FFPE tissues and 24 (16%) BLBC in the samples on tissue microarray. Of the 44 FFPE tissues, FOXC1 expression was observed in 100% of the BLBC and 83% of TNBC, while it was only found in 15% of non-TNBC. The mean staining intensity of FOXC1 was lower in non-TNBC (0.30) compared to TNBC (2.16) and basal-like TNBC (2.60). These findings were confirmed in the tissue microarray, which also revealed a strong positive correlation of FOXC1 with basal-like TNBC. In addition, our results showed that FOXC1 was more frequently expressed in ER-negative (22%), PR-negative (21%) and HER2-negative (19%) breast cancers than in ER-positive (3%), PR-positive (3%) and HER2-positive (5%) breast cancers. Moreover, FOXC1 expression was significantly associated with high tumor grade (p = 0.000041) and high tumor-infiltrating lymphocytes (TILs) (p = 0.000001). Conclusion: FOXC1 is highly expressed in TNBC, particularly among the basal-like subgroup, as opposed to other breast cancers. It accurately classifies the BLBC subtype which may be used as a surrogate marker for this molecular subtype. We find that FOXC1 has an inverse correlation with ER, PR and HER2, as well as a positive correlation with tumor grade and TILs. Our finding suggests that FOXC1 may serve as a potential treatment target for basal-like TNBC. Citation Format: William C. Cho, Victor W. Ma, Wah Cheuk, Grace Y. So, Rebecca Y. Chin. FOXC1 expression is associated with a triple-negative basal-like phenotype in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3148.

WNT5B governs the phenotype of basal-like breast cancer by activating WNT signaling

BackgroundBreast cancer is the leading cause of cancer-related death in women worldwide. Metastatic disease remains the primary cause of death in patients with breast cancer. Basal-like breast cancer (BLBC) is associated with aggressive behavior, stem-like phenotype, high histological grade, poor clinical features, and high rates of recurrences and/or metastasis. However, the mechanism of BLBC phenotype shaping remains obscure.MethodsSeventeen normal breast/breast cancer cell lines were used for evaluating the breast cancer subtype-markers, WNT targets and constitutive components, and epithelial mesenchymal transition (EMT) markers analysis by western blot. One hundred and twenty formalin-fixed breast cancer tissues were used for immunohistochemistry (IHC) staining. Nine online platforms (cBioPortal, CCLE, GEPIA, etc.) were used for related analyses.ResultsWe identified Wnt5b as a key regulatory factor that governs the phenotype of BLBC by activating canonical and non-canonical WNT signaling. Wnt5b exhibited basal-like specificity in cells and clinical samples both at the mRNA and protein levels and also showed good correlation with basal-like phenotype at the mRNA level. Besides, Wnt5b was also a promising therapeutic target for LGK-974 treatment. In addition, we identified that CK1α was expressed at low levels in BLBC and that the activation of CK1α by pyrvinium was an alternative strategy for BLBC treatment.ConclusionsWnt5b is not only a diagnostic biomarker but also a potential therapeutic target of BLBC.Graphical abstract

Correction to: Schlafen-11 expression is associated with immune signatures and basal-like phenotype in breast cancer.

In the original publication of the article, the funding information was incorrectly published. The corrected funding statement is given in this correction article.

Schlafen-11 expression is associated with immune signatures and basal-like phenotype in breast cancer.

Breast cancer (BC) is a heterogeneous disorder, with variable response to systemic chemotherapy. Likewise, BC shows highly complex immune activation patterns, only in part reflecting classical histopathological subtyping. Schlafen-11 (SLFN11) is a nuclear protein we independently described as causal factor of sensitivity to DNA damaging agents (DDA) in cancer cell line models. SLFN11 has been reported as a predictive biomarker for DDA and PARP inhibitors in human neoplasms. SLFN11 has been implicated in several immune processes such as thymocyte maturation and antiviral response through the activation of interferon signaling pathway, suggesting its potential relevance as a link between immunity and cancer. In the present work, we investigated the transcriptional landscape of SLFN11, its potential prognostic value, and the clinico-pathological associations with its variability in BC. We assessed SLFN11 determinants in a gene expression meta-set of 5061 breast cancer patients annotated with clinical data and multigene signatures. We found that 537 transcripts are highly correlated with SLFN11, identifying "immune response", "lymphocyte activation", and "T cell activation" as top Gene Ontology processes. We established a strong association of SLFN11 with stromal signatures of basal-like phenotype and response to chemotherapy in estrogen receptor negative (ER-) BC. We identified a distinct subgroup of patients, characterized by high SLFN11 levels, ER- status, basal-like phenotype, immune activation, and younger age. Finally, we observed an independent positive predictive role for SLFN11 in BC. Our findings are suggestive of a relevant role for SLFN11 in BC and its immune and molecular variability.

Clinicopathological characteristics of basal-like breast cancer: a comparative study between Egyptian and British patients

Background Clinicopathological features of basal-like breast cancer (BLBC) in African-American women have been extensively studied. Comparatively, less is known about these tumors in patients from countries in the North African region. The aim of this study was to assess the frequency and clinicopathological characteristics of BLBC in Egyptian patients in comparison with British patients. Patients and methods Tissue microarray blocks were constructed from primary invasive breast cancers from 321 Egyptian and 527 British patients with BC. Sections were stained immunohistochemically with estrogen receptor, progesterone receptor, epidermal growth factor receptor 2, CK19, CK14, EGFR, CK5/6, P53, and Ki-67. BLBC phenotype was identified by the lack of staining of estrogen receptor, progesterone receptor, and epidermal growth factor receptor 2, and positive staining for any of the CK14, CK5/6, and/or EGFR. Results The rate of BLBC phenotype was higher in Egyptian cohort (21%) than the British cohort (13%). BLBC tumors from both Egyptian and British patients were significantly associated with tumors of higher histopathological grade (P Conclusion BLBCs occurred more frequently in Egyptian patients compared with British women and are characterized by unfavorable biological features, akin to BLBC in African-American women. These findings warrant further studies to unravel the genetic background of BLBCs and whether their aggressive features are related to ethnic origin or other multifactorial and environmental variables.

Expression of human endogenous retrovirus-K is strongly associated with the basal-like breast cancer phenotype

Human endogenous retroviruses (HERVs), which make up approximately 8% of the human genome, are overexpressed in some breast cancer cells and tissues but without regard to cancer subtype. We, therefore, analyzed TCGA RNA-Seq data to evaluate differences in expression of the HERV-K family in breast cancers of the various subtypes. Four HERV-K loci on different chromosomes were analyzed in basal, Her2E, LumA, and LumB breast cancer subtypes of 512 breast cancer patients with invasive ductal carcinoma (IDC). The results for all four loci showed higher HERV-K expression in the basal subtype, suggesting similar mechanisms of regulation regardless of locus. Expression of the HERV-K envelope gene (env) was highly significantly increased in basal tumors in comparison with the also-upregulated expression of other HERV-K genes. Analysis of reverse-phase protein array data indicated that increased expression of HERV-K is associated with decreased mutation of H-Ras (wild-type). Our results show elevation of HERV-K expression exclusively in the basal subtype of IDC breast cancer (as opposed to the other subtypes) and suggest HERV-K as a possible target for cancer vaccines or immunotherapy against this highly aggressive form of breast cancer.

Abstract 2429: Nestin expression in tumor cells is associated with BRCA1 positive genotype and a basal-like phenotype in breast cancer

Abstract Background: The basal-like subtype of breast cancer is one of at least five molecular categories. These poor prognosis tumors are most often triple negative, i.e. negative for hormone receptors and HER2, and therefore lack targeted treatment options. There is at present no consensus on how to best define this subtype, and different surrogate immunohistochemistry based markers have been suggested. We here present data supporting nestin as a marker of the basal-like subtype. Originally reported in neuronal progenitor cells, nestin was later observed in the stem cell/progenitor compartment in a variety of tissues as well as in different cancer types, and suggested to be a cancer stem cell marker. In breast cancer, nestin has been reported in the basal-like and triple negative phenotypes. Here, we examined nestin expression in different breast cancer cohorts, including BRCA familial cancer, and breast cancer metastasis. Materials and methods: Immunohistochemical staining of nestin was done in four series of breast cancer, and a semi-quantitative measure of nestin positivity (staining index) was used for evaluation of expression. Series I include 190 breast cancer patients diagnosed during 1996-2001 in Hordaland, Norway. Series II comprise 192 breast cancer cases identified in Kampala, Uganda in 1990-2002. Series III includes 151 patients with breast cancer, 53 BRCA1 and 45 BRCA2 mutation carriers, identified during 1980-1995 in Montreal, Canada, and 51 BRCA negative patients from Norway. Series IV consists of 35 patients with tissue from the primary breast cancer and matched metastasis collected in 1996-2007. Finally, level 3 mRNA microarray data from TCGA (n = 520) and four open access breast cancer gene expression data sets (n = 1148) with information on molecular subtype were included. Results: The proportion of nestin positive breast tumors was 11%, 24% and 28% in Series I-III. Nestin expression was significantly associated with basal cell markers Cytokeratin 5/6 and P-cadherin (p-values <0.0005), five immunohistochemical profiles for the basal-like phenotype (Odds ratio, OR, range 10.9-27.5) and the triple negative phenotype (OR range 11.8-28.2). Further, patients with BRCA1 mutation more often had nestin positive tumors (OR 8.7, Series III). Higher nestin mRNA levels were seen in basal-like tumors, compared with the luminal-like and HER2-enriched subtypes. In Series IV, nestin expression in the primary tumor correlated with expression in the corresponding metastasis, with no statistically significant difference between the two groups (McNemar test, p = 1.0). Conclusion: We found nestin positivity in breast cancer cells to be significantly and strongly associated with the BRCA1 genotype, individual basal cell markers, and the basal-like and triple negative phenotypes. Citation Format: Kristi Krüger, Elisabeth Wik, Hawa Nalwoga, Monica Mannelqvist, Ingunn M. Stefansson, Jarle B. Arnes, Gøril Knutsvik, Turid Aas, William D. Foulkes, Lars A. Akslen. Nestin expression in tumor cells is associated with BRCA1 positive genotype and a basal-like phenotype in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2429. doi:10.1158/1538-7445.AM2015-2429

VGLL1 expression is associated with a triple-negative basal-like phenotype in breast cancer.

Vestigial-like 1 (VGLL1) is a poorly characterized gene encoding a transcriptional co-activator structurally homologous to TAZ and YAP that modulates the Hippo pathway in Drosophila. In this study, we examined the expression of VGLL1 and its intronic miRNA, miR-934, in breast cancer. VGLL1 and miR-934 expression miRNA profiling was carried out on frozen samples of grade 3 invasive ductal carcinomas. VGLL1 protein was also examined in 433 sporadic and BRCA1-associated breast carcinomas on tissue microarrays. RNA-seq data from The Cancer Genome Atlas (TCGA) was used to confirm differences in VGLL1 and miR-934 expression in different breast cancer subtypes, and to correlate their expression with that of other genes and miRNAs. Of 28 miRNAs differentially expressed in estrogen receptor (ER)-positive and ER-negative grade 3 breast carcinomas, miR-934 was most strongly upregulated in ER-negative carcinomas, and its expression was correlated with that of VGLL1. Nuclear VGLL1 expression was observed in 13% of sporadic breast carcinomas, and while VGLL1 was only occasionally found in luminal A (0.70%) and B (5.60%) carcinomas, it was often expressed in HER2-positive (17%), triple-negative (TN) breast carcinomas (>40%) and BRCA1-associated TN carcinomas (>50%). These findings were confirmed in the TCGA dataset, which revealed positive associations with luminal progenitor genes (GABRP, SLC6A14, FOXC1, PROM1, and BBOX1) and strong negative correlations with ER-associated genes (ESR1, C6ORF211, GATA3, and FOXA1). Moreover, VGLL1 expression was associated with reduced overall survival. In conclusion, VGLL1 and miR-934 are mainly expressed in sporadic and BRCA1-associated TN basal-like breast carcinomas, and their coordinated expression, at least partially mediated by the direct modulation of ESR1, might be involved in the maintenance of a luminal progenitor phenotype.

Abstract 4199: Regulation of microRNA-29c expression in different subtypes of breast cancer.

Abstract Introduction: The basal-like subtype of breast cancer is associated with an aggressive phenotype and worse prognosis compared to other subtypes. Cancers of this subtype often do not express the ER, PR, or HER-2 receptor, and lack targets for molecular therapy. Research to identify pathways responsible for driving the malignant phenotype in basal-like breast cancer is critical to help develop more targeted therapies. Our lab has previously identified miR-29c as a significantly down-regulated miRNA in basal-like breast tumors and demonstrated an association with cell invasion and drug sensitivity. However, little is known about the genetic and regulatory factors contributing to the down-regulation of miR-29c in the basal-like subtype. Materials and Methods: To characterize the expression of miR-29c, qRT-PCR was used to measure levels in four basal-like, four luminal, and five claudin-low breast cancer cell lines. We used CAGE-tag analysis and ChIP assays to determine transcription start sites of miR-29c and RNA polymerase II binding sites, respectively. Flouresecent In-Situ Hybridization (FISH) and qPCR were used to assess copy number variation (CNV) of the gene. Luciferase assays were used to measure the promoter activity in the cell lines. Results: MiR-29c has lower expression in basal-like cell lines and higher expression in claudin-low cell lines compared to luminal cell lines. To determine if the changes in expression are driven by the changes in promoter activity, we first identified the promoter of miR-29c at the region 20kb upstream of the gene on Chromosome 1q32. Initial results from promoter activity assays support a trend of promoter activity that correlates with miR-29c expression in cell lines of the different subtypes. Sequencing of the promoter region revealed no significant mutations or SNPs to explain the expression pattern representing the basal, luminal, and claudin-low subtypes in tumors and cell lines. CNV assays using qPCR suggested lower copy numbers of miR-29c in basal-like cell lines with a large variation of copy numbers in tumors. FISH analysis yielded several abnormalities in the number and structure of 1q32 in cell lines. We are currently determining the correlation of CNV with expression pattern of mir-29c seen in the different subtypes as well as identifying a transcriptional factor contributing to differential expression of miR-29c in breast cancer subtypes. Conclusions: Our results suggest that miR-29c has lower expression and promoter activity in basal-like breast cancer, which is most likely due to regulation of promoter activity rather than mutations or CNV in the promoter. More work is required to discover the regulatory factors or epigenetic changes that may be involved in the aberrant expression of mir-29c in breast cancer. Citation Format: Elizabeth Poli, Yoo-Jeong Han, Maria Gomez, Jing Zhang, Olufunmilayo Olopade. Regulation of microRNA-29c expression in different subtypes of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4199. doi:10.1158/1538-7445.AM2013-4199

Elevated expression of podocalyxin is associated with lymphatic invasion, basal-like phenotype, and clinical outcome in axillary lymph node-negative breast cancer

Lymphatic invasion (LVI) is associated with disease recurrence in axillary node-negative (ANN) breast cancer. Using gene expression profiling of 105 ANN tumors, we found that podocalyxin (PODXL) was more highly expressed in tumors with LVI (LVI+) than in those without LVI (LVI-). Differences in PODXL expression were validated using real-time polymerase chain reaction as well as by immunohistochemistry in an independent set of 652 tumors on tissue microarrays. Disease-free survival (DFS) analyses were conducted for association of high PODXL protein expression with risk of distant recurrence overall and within breast cancer subtypes using both Cox and cure-rate models. High PODXL expression was associated with poor prognosis features including large tumor size, high histological grade, estrogen and progesterone receptor negativity, and with clinical alterations characteristic of the basal-like breast cancer phenotype. Surprisingly, despite having other poor prognosis characteristics, women with high PODXL expressing tumors had better long-term DFS in multivariate analysis with traditional clinicopathologic factors including LVI and HER2 status (P=0.001). PODXL has the potential to be a useful biomarker for identifying good prognosis patients in characteristically poor prognosis breast cancer groups and may impact treatment of women with this disease.

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.

Ets-1 is a transcriptional mediator of oncogenic nitric oxide signaling in estrogen receptor-negative breast cancer

IntroductionThe Ets-1 transcription factor is a candidate breast cancer oncogene that regulates the expression of genes involved in tumor progression and metastasis. Ets-1 signaling has also been linked to the development of a basal-like breast cancer phenotype. We recently described a nitric oxide (NO)-induced gene signature that is associated with poor disease outcome in estrogen receptor-negative (ER-) breast cancer and contains both stem cell-like and basal-like components. Thus, we examined the role of Ets-1 in NO signaling and NO-induced phenotypes in ER- human breast cancer cells.MethodsPromoter region analyses were performed on genes upregulated in inducible nitric oxide synthase (NOS2) high expressing tumors for Ets-binding sites. In vitro mechanisms were examined in human basal-like breast cancer cells lines. NO signaling effects were studied using either forced NOS2 expression or the use of a chemical NO-donor, diethlylenetriamine NONOate (DETANO).ResultsPromoter region analysis of genes that are up-regulated in human ER-negative breast tumors with high NOS2 expression revealed that the Ets-binding sequence is the only common promoter element present in all of these genes, indicating that Ets-1 is the key transcriptional factor down-stream of oncogenic NOS2-signaling. Accordingly, both forced NOS2 over-expression and exposure to NO-donors resulted in significant Ets-1 transcriptional activation in ER- breast cancer cells. Functional studies showed that NO activated Ets-1 transcriptional activity via a Ras/MEK/ERK signaling pathway by a mechanism that involved Ras S-nitrosylation. RNA knock-down of Ets-1 suppressed NO-induced expression of selected basal-like breast cancer markers such as P-cadherin, S100A8, IL-8 and αβ-crystallin. Additionally, Ets-1 knock-down reduced NO-mediated cellular proliferation, matrix metalloproteinase and cathepsin B activities, as well as matrigel invasion.ConclusionsThese data show that Ets-1 is a key transcriptional mediator of oncogenic NO signaling that promotes the development of an aggressive disease phenotype in ER- breast cancer in an Ets-1 and Ras-dependent manner, providing novel clues of how NOS2 expression in human breast tumors is functionally linked to poor patient survival.

Nuclear localization of GLI1 and elevated expression of FOXC2 in breast cancer is associated with the basal-like phenotype.

Aberrant sonic hedgehog (SHH)/glioma-associated oncogene (GLI) signaling has been shown in the development of many tumors. The aims of the present study are to determine the expression of two SHH signaling molecules, the glioma-associated oncogene homolog 1 (GLI1) and forkhead box C2 (FOXC2), in invasive breast cancers (IBC), to evaluate their association with clinicopathological parameters, and to determine their prognostic significance in breast cancer patients. Expression of GLI1 and FOXC2 were assessed by immunohistochemical analysis of a tissue microarray containing 262 unselected IBC cases. A statistical analysis was performed to assess the correlation of GLI1 and FOXC2 expression with the patients' clinicopathological parameters, postoperative survival rate, and molecular subtypes. Immunoreactivity of GLI1 and FOXC2 was observed in 84% and 75% of all breast cancer tissues, respectively. There was a significant correlation between nuclear FOXC2 and GLI1 expressions in these breast cancers, which was associated with estrogen receptor (ER) negativity. Furthermore, there was a significant association between nuclear expression of GLI1 and FOXC2 and a basal-like breast cancer phenotype. Patients with nuclear GLI1 or FOXC2-expressing tumors had a significantly shorter survival time than those without nuclear FOXC2 or GLI1 expression. Multivariate analysis showed that nuclear GLI1 or FOXC2 expression was an independent factor for predicting the prognosis of basal-like breast cancer. In conclusion, there was a significant correlation between expression of nuclear GLI1 or FOXC2 and human breast cancer. More specifically, elevated levels of these proteins were associated with the basal-like breast cancer phenotype and with a poor rate of disease-free survival. These data suggest that GLI1 and FOXC2 are involved in tumorigenesis and that they may be useful as diagnostic and therapeutic targets for human basal-like breast cancers. Additional studies are warranted to better understand the biological significance of GLI1 and FOXC2, to further refine statistics related to patient prognosis, and to optimize treatment of patients with basal-like breast cancer.

S-Nitrosation Mediates Multiple Pathways That Lead to Tumor Progression in Estrogen Receptor−Negative Breast Cancer

Chronic inflammation within the tumor microenvironment is a major driver of tumor progression and poor prognosis. Inducible nitric oxide synthase (NOS2) is present in numerous solid tumors. Estrogen receptor-negative (ER-) patients with high expression of tumor NOS2 have a poorer outcome than patients with low expression of NOS2. Furthermore, expression of NOS2 is associated with the basal-like breast cancer phenotype. Using an in vitro model, we have found that nitrosation of critical thiols and nitration of tyrosines lead to the activation of membrane receptors such as epithelial growth factor receptor, Src, Ras, and CD63. These nitric oxide-mediated events in itiate oncogenic signaling pathways such as PI3K/Akt, Ras/ERK, β-catenin, nuclear factor-κB, and AP-1. These data suggest that NOS2 can serve as a major "nonmutatational driver" of ER- breast cancer.

Oxidative stress and counteracting mechanisms in hormone receptor positive, triple-negative and basal-like breast carcinomas

BackgroundTriple-negative breast cancer (TNBC) and basal-like breast cancer (BLBC) are breast cancer subtypes with an especially poor prognosis. 8-Hydroxydeoxyguanosine (8-OHdG) is a widely used marker of oxidative stress and the redox-state-regulating enzymes peroxiredoxins (PRDXs) are efficient at depressing excessive reactive oxygen species. NF-E2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) are redox-sensitive transcription factors that regulate PRDX expression. This is the first study to assess oxidative stress and or cell redox state-regulating enzymes in TNBC and BLBC.MethodsWe assessed immunohistochemical expression of 8-OHdG, Nrf2, Keap1, PRDX III and PRDX IV in 79 women with invasive ductal breast carcinomas. Of these tumors, 37 represented TNBC (grade II-III tumors with total lack of ER, PR and human epidermal growth factor receptor 2 [HER2] expression). Control cases (n = 42) were ER-positive, PR-positive and HER2-negative. Of the 37 TNBCs, 31 had BLBC phenotype (TNBC with expression of cytokeratin 5/6 or epidermal growth factor receptor 1).ResultsPatients with TNBC had worse breast cancer-specific survival (BCSS) than the control group (p = 0.015). Expression of 8-OHdG was significantly lower in TNBC than in the non-TNBC group (p < 0.005). 8-OHdG immunostaining was associated with better BCSS (p = 0.01), small tumor size (p < 0.0001) and low grade (p < 0.0005). Keap1 overexpression was observed in the TNBC cohort (p = 0.001) and Keap1-positive patients had worse BCSS than Keap1-negative women (p = 0.014). PRDX IV was overexpressed in the TNBC vs. the non-TNBC group (p = 0.022).ConclusionsCellular redox state markers may be promising targets when elucidating the pathogenesis of TNBC.

Use of gene expression patterns post neoadjuvant chemotherapy to identify a role for the MAPK phosphatase DUSP4 in therapeutic resistance and a stem-like phenotype in basal-like breast cancer (BLBC).

10509 Background: Neoadjuvant chemotherapy (NAC) leads to a pathological complete response in ~20% of patients with breast cancer. The remaining patients have residual disease and are more likely to develop metastasis. In post-NAC tumors, Ki67 levels, a marker of cell proliferation, have been shown to inform odds of recurrence and death. We hypothesized that post-NAC residual cancers with a high Ki67 harbor molecular alterations that are associated with drug resistance. Methods: We utilized Nanostring digital RNA quantification to measure the expression levels of 350 cancer-related genes in 49 post-NAC breast cancers. Gene expression data were tested for associations with post-NAC Ki67. The cohort was enriched for triple-negative breast cancer (24/49 tumors). The candidate gene DUSP4 was explored in in vitro models of breast cancer. Results: The post-NAC Ki67 (range 2.4 - 99%; median: 31.6%) was statistically different among the molecular subtypes of breast cancers enrolled, being highest in BLBC (p<0.0001). Gene expression signatures of KRAS activation were enriched in BLBC. Low levels of the MAPK phosphatase DUSP4 correlated with a high Ki67 in BLBCs (r=-0.54, p=8x10-4) and with Ras/MAPK pathway activation in human breast tumors and cell lines. Genome-wide studies identified preferential methylation of the DUSP4 promoter in BLBC. Among BLBC cell lines, low DUSP4 expression predicted for sensitivity to MEK inhibitors and resistance to docetaxel. Inhibition of MEK with AZD6244 or overexpression of DUSP4 in DUSP4-low BLBC cell lines improved sensitivity to docetaxel and inhibited mammosphere formation. RNAi-mediated knockdown of DUSP4 in BLBC cells enhanced ETS-1 phosphorylation, mammosphere formation, and MAPK-dependent secretion of the cancer stem cell-promoting cytokines IL6 and IL8. Conclusions: Low levels of DUSP4 correlate with high tumor cell proliferation after NAC. Downregulation of DUSP4 may underlie activation of the MAPK pathway in the absence of KRAS mutations in BLBC while conferring resistance to chemotherapy and a stem cell-like phenotype that is therapeutically targetable with MAPK inhibitors.

MicroRNAs miR-221 and miR-222: a new level of regulation in aggressive breast cancer

MicroRNAs (miRNAs) are crucial in the initiation and progression of tumors. A recent study has reported that the miRNAs miR-221 and miR-222 are involved in the promotion of an aggressive basal-like phenotype in breast cancer, functioning downstream of the RAS pathway and triggering epithelial-to-mesenchymal transition. These new insights into the roles of miR-221/222 in breast cancer metastasis, drug resistance and RAS pathways could potentially have applications in medical practice.