Basal-like Breast Cancer Tumors Research Articles

Abstract PO2-06-08: Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like Breast Cancer

Abstract Introduction: Basal-like breast cancer (BLBC) represents an aggressive subtype of triple-negative breast cancer (TNBC) that exhibits a high risk of recurrence and resistance to treatment. Histone deacetylase (HDAC) inhibitors (HDACis), including panobinostat and romidepsin, have obtained approval for the treatment of hematopoietic malignancies and demonstrated effectiveness in TNBC cells. We recently found that panobinostat and romidepsin potently induced TNBC cell growth inhibition and apoptosis via downregulation of HER3 through suppression of forkhead box protein A1 (FOXA1), a pioneering transcription factor. Nonetheless, the underlying mechanism through which HDACis suppress FOXA1, thereby inhibiting HER3 expression and its downstream signaling in FOXA1/HER3 co-expressing TNBC remains elusive. Methods: Colony formation, MTS, and LIVE/DEAD cell staining assays were used to detect cell viability. Apoptosis was detected by flow cytometry assays. QRT-PCR, western blots, and immunohistochemistry were performed to determine the expression and activation of genes and/or proteins. Co-immunoprecipitation was performed to assess the interaction between proteins. Lentivirus vectors containing cDNA or shRNAs were used to overexpress or knockdown gene expression. Chromatin immunoprecipitation-quantitative PCR and dual luciferase reporter assays were performed to elucidate the regulatory role of gene transcription. Results: Elevated expression of FOXA1 was observed in BLBC specimens and cell lines tested. FOXA1 was co-expressed with HER3 and transcriptionally activated the HER3 expression in BLBC cells. HER3 formed heterodimers with epidermal growth factor receptor (EGFR), activating downstream signaling pathways in BLBC cells. Treatment with panobinostat and romidepsin resulted in growth inhibition and apoptosis in BLBC cells by downregulating FOXA1 expression and inhibiting HER3 signaling. Notably, the combination of HDACis with an EGFR inhibitor (gefitinib) or an anti-HER3 antibody synergistically enhanced the anti-survival effects on BLBC cells. Additionally, gene expression profiling datasets revealed a significant positive correlation between FOXA1 expression and the transcription factors GATA-Binding Protein 2/3 (GATA2/3). Patients with high GATA2/3-FOXA1 expression exhibited worse Relapse-Free Survival (RFS) compared to those with low expressions in BLBC via Kaplan-Meier analysis. Further investigations showed that GATA2/3 directly activated FOXA1 transcription by binding to its promoter. Moreover, ectopic expression of GATA2/3 not only restored FOXA1 expression downregulated by HDACis but also attenuated the anti-survival effects of HDACis on BLBC cells. Conclusion: HDACis exhibit potent inhibitory effects on BLBC cells via downregulation of GATA2/3-mediated repression of FOXA1 gene transcription, which in turn suppresses HER3 expression and signaling. Our findings indicate that epigenetic targeting of the GATA2/3-FOXA1-HER3 axis may be an effective therapeutic strategy for the eradication of BLBC tumors. Keywords: FOXA1, GATA2/3, HER3, HDAC inhibitors, Basal-like breast cancer Citation Format: Bolin Liu, Congcong Tan, Hui Lyu. Suppression of GATA2/3-FOXA1-HER3 Axis by Histone Deacetylase (HDAC) Inhibitors shows Antitumor Activity in Basal-like Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-06-08.

Reprogrammed transsulfuration promotes basal-like breast tumor progression via realigning cellular cysteine persulfidation

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast tumors with poor prognosis and limited molecular-targeted therapy options. We show that BLBC cells have a high Cys demand and reprogrammed Cys metabolism. Patient-derived BLBC tumors from four different cohorts exhibited elevated expression of the transsulfuration enzyme cystathione β-synthetase (CBS). CBS silencing (shCBS) made BLBC cells less invasive, proliferate slower, more vulnerable to oxidative stress and cystine (CySSCy) deprivation, prone to ferroptosis, and less responsive to HIF1-α activation under hypoxia. shCBS xenograft tumors grew slower than controls and exhibited impaired angiogenesis and larger necrotic areas. Sulfur metabolite profiling suggested that realigned sulfide/persulfide-inducing functions of CBS are important in BLBC tumor progression. Supporting this, the exclusion of serine, a substrate of CBS for producing Cys but not for producing sulfide/persulfide, did not exacerbate CySSCy deprivation-induced ferroptosis in shCBS BLBC cells. Impaired Tyr phosphorylation was detected in shCBS cells and xenografts, likely due to persulfidation-inhibited phosphatase functions. Overexpression of cystathione γ-lyase (CSE), which can also contribute to cellular sulfide/persulfide production, compensated for the loss of CBS activities, and treatment of shCBS xenografts with a CSE inhibitor further blocked tumor growth. Glutathione and protein-Cys levels were not diminished in shCBS cells or xenografts, but levels of Cys persulfidation and the persulfide-catabolizing enzyme ETHE1 were suppressed. Finally, expression of enzymes of the oxidizing Cys catabolism pathway was diminished, but expression of the persulfide-producing CARS2 was elevated in human BLBC tumors. Hence, the persulfide-producing pathways are major targetable determinants of BLBC pathology that could be therapeutically exploited.

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.

Cancer Cell Intrinsic and Immunologic Phenotypes Determine Clinical Outcomes in Basal-like Breast Cancer.

Basal-like breast cancer (BLBC) is a particularly aggressive intrinsic molecular subtype of breast cancer that lacks targeted therapies. There is also no clinically useful test to risk stratify patients with BLBC. We hypothesized that a transcriptome-based phenotypic characterization of BLBC tumors and their microenvironments may overcome these challenges. We conducted a retrospective correlative genomic sequencing study using a matched pairs design with validation in five independent cohorts. The study was conducted on a large population-based prospective cohort of the major molecular subtypes of breast cancer conducted in the greater Seattle-Puget Sound metropolitan area. Cases consisted of women 20-69 years of age first diagnosed with invasive breast cancer identified through the population-based Surveillance Epidemiology and End Results program. Patients for this analysis (n = 949) were identified from the 1,408 patients with stage I-III triple-negative breast cancer [estrogen receptor-negative (ER-), progesterone receptor-negative (PR-), HER2-]. Of the 949 women, 248 developed a recurrence after their initial diagnosis. A matched set of 67 recurrent and nonrecurrent BLBC tumors was subjected to transcriptome sequencing. Through RNA sequencing of the matched sets of recurrent and nonrecurrent BLBC tumors, we aimed to identify prognostic phenotypes.To identify nonredundant and uncorrelated prognostic genes, we used an ensemble of variable selection algorithms, which resulted in a ranking of genes on the basis of their expected utility in classification. Using leave-one-out cross-validation, we trained a random forest classifier on the basis of the top 21 genes (BRAVO-DX). Validations were performed in five independent triple-negative or BLBC cohorts, and biomarker robustness and transferability were demonstrated by employing real-time PCR. We found that cancer cell intrinsic and immunologic phenotypes are independent predictors of recurrence. By simultaneously interrogating the tumor and its microenvironment, we developed a compound risk model that stratified patients into low-, medium-, and high-risk groups, with a 14%/56%/74% chance of recurrence, respectively. Biologically, the primary tumors of patients who developed a recurrence had increased growth factor signaling and stem-like features, while nonrecurrent tumors showed high lymphocyte infiltration with clonal expansion of T and B cells, as well as antitumor polarization of macrophages. We validated our model in five independent cohorts, including three large cohorts, where BRAVO-DX was highly informative in identifying patients with disease recurrence [HR, 6.79 (95% confidence interval (CI), 1.89-24.37); HR, 3.45 (95% CI, 2.41-4.93); and HR, 1.69 (95% CI, 1.17-2.46)]. A smaller gene set focused on the tumor immunophenotype, BRAVO-IMMUNE, was highly prognostic in all five cohorts. Together, these results indicate that phenotypic characteristics of BLBCs and their microenvironment are associated with recurrence-free survival and demonstrate the utility of intrinsic and extrinsic phenotypes as independent prognostic biomarkers in BLBC. Pending further evaluation and validation, our prognostic model has the potential to inform clinical decision-making for patients with BLBC as it identifies those at high risk of rapidly progressing on standard chemotherapy, as well as those who may benefit from alternative first-line therapies.

Hyperactive CDK2 Activity in Basal-like Breast Cancer Imposes a Genome Integrity Liability that Can Be Exploited by Targeting DNA Polymerase ε

Knowledge of fundamental differences between breast cancer subtypes has driven therapeutic advances; however, basal-like breast cancer (BLBC) remains clinically intractable. Because BLBC exhibits alterations in DNA repair enzymes and cell-cycle checkpoints, elucidation of factors enabling the genomic instability present in this subtype has the potential to reveal novel anti-cancer strategies. Here, we demonstrate that BLBC is especially sensitive to suppression of iron-sulfur cluster (ISC) biosynthesis and identify DNA polymerase epsilon (POLE) as an ISC-containing protein that underlies this phenotype. In BLBC cells, POLE suppression leads to replication fork stalling, DNA damage, and a senescence-like state or cell death. In contrast, luminal breast cancer and non-transformed mammary cells maintain viability upon POLE suppression but become dependent upon an ATR/CHK1/CDC25A/CDK2 DNA damage response axis. We find that CDK1/2 targets exhibit hyperphosphorylation selectively in BLBC tumors, indicating that CDK2 hyperactivity is a genome integrity vulnerability exploitable by targeting POLE.

A systems medicine-based approach for finding metabolic targets applied to primary basal-like breast cancer.

e12590 Background: Although metabolic disfunction is one of the hallmarks of cancer and metabolism-based therapies are a key class of chemotherapeutics, there remains no systematic way to identify metabolism-based targets for therapeutic intervention. Because of this deficiency, metabolism-based therapies offer a largely untapped area for therapeutic discovery, especially for difficult to target tumors, like basal-like breast cancer (BLBC). Adding to the challenge of discovering metabolism-based therapeutics for BLBC, however, is the heterogeneity of tumor behavior across patients, both in terms of aggressiveness and molecular regulation. Methods: We addressed these challenges by introducing a systems medicine-based technique integrating metabolic network modeling with single-patient RNA-sequencing (RNA-seq). We then applied this technique to investigate metabolic weaknesses in primary BLBC. We constructed a genome-scale metabolic model of breast cancer (ecBRCA) along with RNA-seq profiles constructed from The Cancer Genome Atlas (TCGA) to predict metabolic targets whose inhibition is lethal to BLBC tumors. We then evaluated the effectiveness of drugging these targets across patients by simulating the effect of a targeted knock-out on primary tumors with published RNA-seq profiles from 534 BLBC patients in TCGA and the Swedish Cancerome Analysis Network for Breast (SCANB). Results: We found multiple metabolic targets that may prove clinically useful for complementary treatment of primary BLBC tumors. Among these, we found UDP-Glucose 6-Dehydrogenase (UGDH) and Heparan Sulfate 2-O-Sulfotransferase 1 (HS2ST1) to be particularly promising protein targets for chemotherapy in BLBC, with 80-100% of simulated BLBC tumors responding to their inhibition. Currently FDA-approved drugs down-regulating these proteins, such as metformin (for UGDH) and surfen (for HS2ST1), are potentially viable complementary treatments for BLBC patients. Additionally, we found several metabolite targets whose inhibition may inhibit BLBC growth, including leukotriene A4, creatinine, vitamin D3, and the oncometabolites 2- and 3-hydroxy-glutarate. Conclusions: Taking a systems medicine-based approach to understanding BLBC metabolism allows for a systematic way to identify metabolic targets for therapeutic intervention. Additionally, certain metabolic weaknesses appear to be conserved in BLBC tumors across patients, including a dependence on UGDH and HS2ST1, which could be treated with currently available drugs like metformin and surfen.

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.

LncRNA BLAT1 is Upregulated in Basal-like Breast Cancer through Epigenetic Modifications

Long-noncoding RNAs (lncRNAs) have been shown to participate in oncogenesis across a variety of cancers and may represent novel therapeutic targets. However, little is known about the role of lncRNAs in basal-like breast cancer (BLBC), the aggressive form of breast cancer with no molecularly defined therapeutic target. To examine whether altered lncRNA expression contributes to the aggressive phenotype characteristic of BLBC, we performed a comparative analysis of BLBC versus non-BLBC using microarray profiling and RNA sequencing of primary breast cancer. We identified RP11-19E11.1 as a significantly up-regulated lncRNA in BLBC tumors and named it Basal-Like breast cancer Associated Transcript 1 (BLAT1). Analysis of pan-cancer datasets showed the highest expression of BLAT1 in BLBC tumors compared to all other cancers. Depletion of BLAT1 in breast cancer cells led to significantly increased apoptosis, partly because of accumulation of DNA damage. Mechanistically, BLAT1 expression is regulated at the epigenetic level via DNA methylation at CpG islands in the promoter. Concordantly, patients harboring tumors with BLAT1 hypomethylation showed decreased overall survival. Our results suggest that increased expression of BLAT1 via CpG site hypomethylation may contribute to the aggressive phenotype of BLBC, raising a possibility of new biomarkers for prognosis of aggressive BLBC tumors.

High CD90 (THY-1) expression positively correlates with cell transformation and worse prognosis in basal-like breast cancer tumors.

Breast cancer is the most prevalent cancer among women, with the basal-like triple negative (TNBC) being the most agressive one, displaying the poorest prognosis within the ductal carcinoma subtype. Due to the lack of adequate molecular targets, the diagnosis and treatment of patients with the TNBC phenotype has been a great challenge. In a previous work, we identified CD90/Thy-1 as being highly expressed in the aggressive high malignancy grade Hs578T basal-like breast tumor cell line, pointing to this molecule as a promising breast tumor marker, which should be further investigated. Here, CD90 expression was analyzed in human breast cancer samples and its functional role was investigated to better assess the oncogenic nature of CD90 in mammary cells. Quantification of CD90 expression in human breast cancer samples, by tissue microarray, showed that high CD90 positivity correlates with metastasis and poor patient survival in the basal-like subtype. The functional genetic approach, by overexpression in the CD90 cDNA in a basal-like normal mammary cell line (MCF10A) and knockdown in a highly malignant cell line (Hs578T), allowed us to demonstrate that CD90 is involved with several cellular processes that lead to malignant transformation, such as: morphological change, increased cell proliferation, invasiveness, metastasis and activation of the EGFR pathway. Therefore, our results reveal that CD90 is involved with malignant transformation in breast cancer cell lines and is correlated with metastasis and poor patient survival in the basal-like subtype, being considered as a promising new breast cancer target.

Abstract 717: Diagnosis of basal like breast cancer using DNA methylation markers at the promoters of long noncoding RNAs

Abstract Background: Patients with basal-like breast cancer (BLBC) have poor overall survival and suffer a high rate of metastasis to the brain or lung within three to five years of initial presentation. Absence of a cure for advanced BLBC warrants early detection of BLBC, which might save more lives, especially those of women of African ancestry who are disproportionately affected by young onset BLBC. Aberrant DNA methylation is frequently observed in BLBC. DNA methylation is the most robust epigenetic marks and can be analyzed using clinical specimens including FFPE, tumor biopsies and liquid biopsies. Because expression of long non-coding RNAs (lncRNAs) is controlled temporally in response to neoplastic stimuli, we investigated the potential for lncRNA promoter methylation marks to be used for detection and prediction of BLBC development and progression in African Americans. Methods: To identify lncRNAs dysregulated in BLBC, we performed a Ribo-Zero RNA-seq or microarray analysis on breast tissues isolated from African American (n=63) and European American patients (n=14). Differentially methylated and hydroxymethylated regions of lncRNA genes were identified in African American samples (n=30) using our chemical methods to assay 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The Cancer Genome Atlas (TCGA) datasets were utilized to validate our results. To functionally annotate lncRNAs, we knocked down lncRNAs using LNA-antisense oligonucleotides (ASO) and performed Caspase 3/7 assays, flow cytometry, and qRT-PCR. We also utilized the CRISPR-Cas9 editing tools to knock out lncRNAs. Results: LncRNAs displayed distinct expression patterns between tumors and normal breast tissues. Out of hundreds of lncRNAs specifically expressed in BLBC tumors, we selected two lncRNAs, lnc19 and lnc98, which represent, respectively, a significantly increased and decreased lncRNA in BLBC tumors, compared to normal breast tissues and other subtype tumors. Lnc19 is highly up-regulated (a 39-fold increase) whereas lnc98 is dramatically down-regulated (a 59-fold decrease) in BLBC tumors. Methylation analysis showed significantly lower levels of promoter methylation for lnc19 and higher levels for lnc98 in BLBC tumors. A significant inverse correlation between methylation and expression of lnc19 was observed. Depletion of lnc19 resulted in rapid cell death of BLBC cells, with increased sensitivity to chemotherapy drugs. The data suggests that chemo-resistance of BLBC might be partly due to lnc19 overexpression, which is mediated through epigenetic control. Conclusion: We identified two epigenetically dysregulated lncRNAs in BLBC tumors, which contribute to chemo-resistance of these tumors. Our findings on the regulation of lncRNAs by cytosine methylation raise the possibility of new epigenetic biomarkers for diagnosis and prognosis of aggressive BLBC tumors. Citation Format: Yoo J. Han, Sonja M. Boatman, Jing Zhang, Albert C. Yeh, Yonglan Zheng, Olufunmilayo I. Olopade. Diagnosis of basal like breast cancer using DNA methylation markers at the promoters of long noncoding RNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 717. doi:10.1158/1538-7445.AM2017-717

FOXC1 identifies basal-like breast cancer in a hereditary breast cancer cohort.

Breast cancers arising in the setting of the hereditary breast cancer genes BRCA1 and BRCA2 are most commonly classified as basal-like breast cancer (BLBC) or luminal breast cancer, respectively. BLBC is an aggressive subtype of breast cancer associated with liver and lung metastases and poorer prognosis than other subtypes and for which chemotherapy is the only systemic therapy. Multiple immunohistochemical markers are used to identify the basal-like subtype, including the absence of estrogen receptor alpha, progesterone receptor, and human epidermal growth factor receptor 2. Forkhead box C1 (FOXC1) has been identified as a specific marker expressed in BLBC in general breast cancer cohorts. We examined an institutional cohort of breast cancer patients with germline BRCA1 (n=46) and BRCA2 (n=35) mutations and found that FOXC1 expression on immunohistochemical staining is associated with BRCA1 vs BRCA2 mutations [30/46 vs. 6/35]. In BRCA1 mutant tumors, FOXC1 was expressed in 28/31 BLBC tumors and 2/13 non-BLBC tumors, In BRCA2 mutant tumors, FOXC1 was expressed in 5/5 BLBC tumors and 1/30 non-BLBC tumors. In cell culture models of BRCA1-mutant breast cancer, FOXC1 is associated with increased proliferation and may serve as a marker for sensitivity to PARP-inhibitor therapy with olaparib.

Rho GTPase Transcriptome Analysis Reveals Oncogenic Roles for Rho GTPase-Activating Proteins in Basal-like Breast Cancers.

The basal-like breast cancer (BLBC) subtype accounts for a disproportionately high percentage of overall breast cancer mortality. The current therapeutic options for BLBC need improvement; hence, elucidating signaling pathways that drive BLBC growth may identify novel targets for the development of effective therapies. Rho GTPases have previously been implicated in promoting tumor cell proliferation and metastasis. These proteins are inactivated by Rho-selective GTPase-activating proteins (RhoGAP), which have generally been presumed to act as tumor suppressors. Surprisingly, RNA-Seq analysis of the Rho GTPase signaling transcriptome revealed high expression of several RhoGAP genes in BLBC tumors, raising the possibility that these genes may be oncogenic. To evaluate this, we examined the roles of two of these RhoGAPs, ArhGAP11A (also known as MP-GAP) and RacGAP1 (also known as MgcRacGAP), in promoting BLBC. Both proteins were highly expressed in human BLBC cell lines, and knockdown of either gene resulted in significant defects in the proliferation of these cells. Knockdown of ArhGAP11A caused CDKN1B/p27-mediated arrest in the G1 phase of the cell cycle, whereas depletion of RacGAP1 inhibited growth through the combined effects of cytokinesis failure, CDKN1A/p21-mediated RB1 inhibition, and the onset of senescence. Random migration was suppressed or enhanced by the knockdown of ArhGAP11A or RacGAP1, respectively. Cell spreading and levels of GTP-bound RhoA were increased upon depletion of either RhoGAP. We have established that, via the suppression of RhoA, ArhGAP11A and RacGAP1 are both critical drivers of BLBC growth, and propose that RhoGAPs can act as oncogenes in cancer. Cancer Res; 76(13); 3826-37. ©2016 AACR.

Abstract P2-06-11: Wild type N-Ras, overexpressed in basal-like breast cancer, promotes tumor formation by inducing IL8 secretion via JAK2 activation

Abstract "Basal-like" breast cancer (BLBC) is a very aggressive subtype of breast cancer. BLBC has very poor prognosis — median time to distant recurrence is just 2.6 years vs. 5 years overall, and survival time from diagnosis of distant metastatic disease is 9 months vs. 22 months. BLBC tumors usually do not express ER, Her2, or progesterone receptor. As such, they cannot be treated by the current targeted therapies, which target these molecules. What drive the formation and progression of BLBCs is largely unclear. Ras GTPases are best known for mediating growth factor signaling. Oncogenic mutations in the RAS genes, K-RAS in particular, are found in more than 30% of human tumors. Surprisingly, oncogenic RAS mutations are rare in breast cancer. However, we found that wild-type N-RAS is overexpressed in BLBCs, possibly partly via promoter demethylation, but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, while overexpressing it enhances these processes even in preinvasive BLBC cells. In contrast, in breast cancer cells of other subtypes, repressing N-RAS expression does not affect growth and transforming activities. We identified N-Ras-responsive genes, most of which encode chemokines and cytokines, e.g., IL8. High expression levels of these N-Ras-responsive genes as well as of N-RAS itself in tumors correlate with poor patient outcome. N-Ras, but not K-Ras, induces IL8 by binding and activating the cytoplasmic pool of JAK2; IL8 then acts on both the cancer cells and stromal fibroblasts. In conclusion, N-Ras drives BLBC by promoting transformation in epithelial cells, which may in turn remodel the tumor microenvironment to create a proinvasive state. Although oncogenic mutations affecting RAS are common in many other human cancers, tumorigenesis in an important subset of breast cancers is driven instead by increasing activity of wild-type N-Ras. Thus, to fully assess the impact of Ras on tumorigenesis, the role of wild-type as well as mutant Ras proteins must be carefully examined. Citation Format: Zheng Z-Y, Bu W, Tian L, Fan C, Gao X, Zhang X, Yu C, Wang H, Liao Y-H, Li Y, Lewis MT, Edwards D, Zwaka TP, Hilsenbeck SG, Medina D, Perou CM, Creighton CJ, Zhang XH, Chang EC. Wild type N-Ras, overexpressed in basal-like breast cancer, promotes tumor formation by inducing IL8 secretion via JAK2 activation. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-06-11.

Abstract PR01: Understanding the role of BCL11A in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) has poor prognostic outcome compared to other subtypes of breast cancer. The molecular and cellular mechanisms underlying TNBC pathology are not fully understood. We analyzed both METABRIC and TCGA data found the transcription factor BCL11A is overexpressed in TNBC including basal-like breast cancer (BLBC) and that its genomic locus is amplified in up to 38% of BLBC tumors. Moreover, analysis of the TCGA data revealed that the BCL11A locus is hypo-methylated almost exclusively in BLBC tumors but not other subtypes. We show that exogenous BCL11A overexpression promotes tumor formation, whereas its knockdown in TNBC cell lines suppresses their tumorigenic potential in xenograft models. In the mouse DMBA-induced tumor model, Bcl11a deletion substantially decreases tumor formation, even in p53-null cells and inactivation of Bcl11a in established tumors causes their regression. At the cellular level, Bcl11a deletion causes a reduction in the number of mammary epithelial stem and progenitor cells. Mechanistically, proteomics studies have revealed that BCL11A interacts with a specific set of chromatin modifiers in tumor cells but not in normal cells - the significance of which is currently under investigation. This study highlights the potential for targeting BCL11A as an approach for TNBC-targeted therapy. To this end we are currently using the Crispr/Cas9 system to generate endogenous reporter cell lines for high-throughput drug screens to identify potential inhibitors of BCL11A. Citation Format: Kyren Lazarus, Carlos Caldas, Pentao Liu, Walid T. Khaled. Understanding the role of BCL11A in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr PR01.

Abstract 2139: Wild type N-Ras, overexpressed in basal-like breast cancer, promotes tumor formation by inducing IL8 secretion via JAK2 activation

Abstract “Basal-like” breast cancer (BLBC) is the most aggressive breast cancer subtype, with a very poor prognosis _ median time to distant recurrence is just 2.6 years vs. 5 years overall, and survival time from diagnosis of distant metastatic disease is 9 months vs. 22 months. BLBC tumors usually do not express ER, Her2, or progesterone receptor. As such, they cannot be treated by the current targeted therapies, which target these molecules. What drive the formation and progression of BLBCs is largely unclear. Ras GTPases are best known for mediating growth factor signaling, and oncogenic mutations in the RAS genes, and K-RAS in particular, are found in more than 30% of human tumors. Surprisingly, oncogenic RAS mutations are rare in breast cancer. However, we found that wild-type N-RAS is overexpressed in BLBCs, partly via promoter demethylation, but not in other breast cancer subtypes. Repressing N-RAS inhibits transformation and tumor growth, while overexpression enhances these processes even in preinvasive BLBC cells. In contrast, repressing N-RAS expression does not affect growth and transforming activities of breast cancer cells of other subtypes. We identified N-Ras-responsive genes, most of which encode chemokines, e.g., IL8. High expression levels of these N-Ras-responsive genes as well as of N-RAS itself in tumors correlate with poor patient outcome. N-Ras, but not K-Ras, induces IL8 by binding and activating the cytoplasmic pool of JAK2; IL8 then acts on both the cancer cells and stromal fibroblasts. Thus N-Ras drives BLBC by promoting transformation in epithelial cells, which in turn remodels the tumor microenvironment to create a proinvasive state. These results illustrate an N-Ras-dependent convergence between the growth factor and cytokine pathways to enable efficient transformation of epithelial cells, which then orchestrate the remodeling of the tumor microenvironment. Although oncogenic mutations affecting RAS are common in many other human cancers, tumorigenesis in an important subset of breast cancers is driven instead by increasing activity of wild-type N-Ras. Thus, to fully assess the impact of Ras on tumorigenesis, the role of wild-type as well as mutant Ras proteins must be carefully examined. Citation Format: Ze-Yi Zheng, Wen Bu, Lin Tian, Cheng Fan, Xia Gao, Cuijuan Yu, Liu Jun, Xiaomei Zhang, Yi-Hua Liao, Yi Li, Michael T. Lewis, Dean Edwards, Susan G. Hilsenbeck, Daniel Medina, Thomas P. Zwaka, Charles M. Perou, Chad J. Creighton, Xiang Zhang, Eric C. Chang. Wild type N-Ras, overexpressed in basal-like breast cancer, promotes tumor formation by inducing IL8 secretion via JAK2 activation. [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 2139. doi:10.1158/1538-7445.AM2015-2139

Abstract 3906: Annexin A1 overexpression preferentially predicts poor prognosis of basal-like breast cancer patients by activating m TOR-S6 signaling

Abstract Introduction: Annexin A1 is an anti-inflammatory protein reported to play a role in cell proliferation, apoptosis and to be deregulated in breast cancer. The role and regulation of annexin A1 in the biology and prognosis of breast cancer remain unclear because most of the studies did not consider specific differences between the different subtypes of breast cancer. Thus, in this study, we hypothesize that annexin A1 plays subtype-specific role in basal like breast cancer progression and metastasis. Methods: By mining The Cancer Genome Atlas-Breast Cancer Dataset (TCGA-BRCA) and manipulating annexin A1 levels in breast cancer cell lines, we studied the role of annexin A1 in breast cancer and underlying signaling pathways. Results: Our in-silico analysis of TCGA-BRCA dataset demonstrated that annexin A1 mRNA expression is higher in basal subtype compared to luminal and HER2 subtypes. Within the basal subtype, patients show significantly poorer survival associated with higher expression of annexin A1. In both TCGA patient samples and cell lines, annexin A1 levels were significantly higher in basal-like breast cancer tumors than luminal and Her2/neu-positive breast cancer tumors. Stable annexin A1 knockdown in TNBC cell lines suppressed the mTOR-S6 pathway. Specifically, annexin A1 knockdown inhibited phosphorylation of mTOR and S6 (18-73% inhibition in pmTOR and 50-60% inhibition in pS6). But annexin A1 knockdown had no impact on the MAPK, c-Met, and EGFR pathways. In cell migration assays, annexin A1-depleted TNBC cells showed delayed migration as compared to wild-type cells which we found to be through suppression of MMP9. Conclusions: Our data suggest that annexin A1 is prognostic only in patients with basal like breast cancer. This appears to be due in part to the role of annexin A1 in activating mTOR-pS6 pathway. Citation Format: Kazunoshin Tachibana, Anjana Bhardwaj, Nivetha Ganesan, Kimal Rajapakshe, Constance T. Albarracin, Preethi H. Gunaratn, Cristian Coarfa, Isabelle Bedrosian. Annexin A1 overexpression preferentially predicts poor prognosis of basal-like breast cancer patients by activating m TOR-S6 signaling. [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 3906. doi:10.1158/1538-7445.AM2015-3906

Abstract P4-05-07: Functional genomics of TP53 mutations and its impact in breast cancer progression

Abstract Somatic TP53 mutations are prevalent in basal-like breast cancer (BLBC) tumors. Patients with BLBC tumors have fewer treatment options and respond poorly to current therapies. The majority of TP53 point mutations occurs in the DNA binding domain and can be categorized as either DNA contact or structural mutations. TP53 mutation results in a dominant negative phenotype with neomorphic activity. We predicted that different p53 mutations may lead to different phenotypic characteristics. To investigate this, we generated MCF10A stable transduced cell lines over-expressing the ten most frequent TP53 point mutations associated with breast cancer located in the DNA binding domain of TP53. Ectopic expression of TP53 in these stable cells has been confirmed by qRT-PCR and immunoblot. To assess the impact of mutation on carcinogenesis, we developed a series of high-throughput quantitative assays that measure several hallmarks of cancer, including proliferation, escape from apoptosis, epithelial to mesenchymal transition (EMT), cell migration and invasion, anoikis and morphology in 3D. We observed that one DNA contact mutation with the substitution of a positively charged amino acid with hydrophobic side chains such as R248W, and two structural mutants Y234C and H179R are resistant to apoptosis in presence of doxorubicin, are the most invasive displaying a mesenchymal phenotype characterized by the presence of disrupted B-catenin and E-cadherin staining, with reported worst clinical outcome, suggesting that these are the most aggressive phenotypes. Interestingly, the DNA contact mutants (R248Q, R273H, R248W, and R273C) had a growth advantage in absence of growth factors while structural mutants (R175H, H179R, Y220C, Y234C and Y163C) were more resistant to apoptosis after the cells were challenged with doxorubicin. G245S is comparable to the MCF10Ap53wt and is less proliferative, sensitive to apoptosis, and neither migratory nor invasive. In comparison, R248W which is one of the most aggressive mutants, together with R273C, and H179R resist anoikis; but Y234C, requires matrix for attachment in order to be invasive. In conjunction, these results confirmed our hypothesis that different TP53 point mutants have distinct phenotypes and functional effects on hallmarks of cancer due to distinct underlying cellular programs. Citation Format: Anasuya Pal, Laura Gonzalez-Malerva, Seron Eaton, Mayra Guzman, Donald Chow, Hongwei Yin, Jin Park, Karen Anderson, Joshua LaBaer. Functional genomics of TP53 mutations and its impact in breast cancer progression [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-07.

Expression of miR-34c induces G2/M cell cycle arrest in breast cancer cells.

BackgroundMicroRNA-34 is a family of three miRNAs that have been reported to function as tumor suppressor miRNAs and show decreased expression in various cancers. Here, we examine functions of miR-34c in basal-like breast cancer cells.MethodsData from The Cancer Genome Atlas (TCGA) were used for evaluation of expression in primary breast cancers. Cellular processes affected by miR-34c were investigated by thymidine incorporation, Annexin V-assays and cell cycle analysis using breast cancer cell lines. Effects on potential targets were analyzed with qPCR and Western blot.ResultsTCGA data revealed that miR-34c was expressed at lower levels in basal-like breast cancer tumors and low expression was associated with poor prognosis. Ectopic expression of miR-34c in basal-like breast cancer cell lines resulted in suppressed proliferation and increased cell death. Additionally, miR-34c influenced the cell cycle mainly by inducing an arrest in the G2/M phase. We found that expression levels of the known cell cycle-regulating miR-34 targets CCND1, CDK4 and CDK6, were downregulated upon miR-34c expression in breast cancer cell lines. In addition, the levels of CDC23, an important mediator in mitotic progression, were suppressed following miR-34c expression, and siRNAs targeting CDC23 mimicked the effect of miR-34c on G2/M arrest. However, protein levels of PRKCA, a predicted miR-34c target and a known regulator of breast cancer cell proliferation were not influenced by miR-34c.ConclusionsTogether, our results support the role of miR-34c as a tumor suppressor miRNA also in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-538) contains supplementary material, which is available to authorized users.

Elafin drives poor outcome in high-grade serous ovarian cancers and basal-like breast tumors.

High grade serous ovarian carcinoma (HGSOC) and basal-like breast cancer (BLBC) share many features including TP53 mutations, genomic instability and poor prognosis. We recently reported that Elafin is overexpressed by HGSOC and is associated with poor overall survival. Here, we confirmed that Elafin overexpression is associated with shorter survival in 1000 HGSOC patients. Elafin confers a proliferative advantage to tumor cells through activation of the MAP kinase pathway. This mitogenic effect can be neutralized by RNA interference, specific antibodies, and a MEK inhibitor. Elafin expression in patient-derived samples was also associated with chemoresistance and strongly correlates with bcl-xL expression. We extended these findings into examination of 1100 primary breast tumors and six breast cancer cell lines. We observed that Elafin is overexpressed and secreted specifically by BLBC tumors and cell lines, leading to a similar mitogenic effect through activation of the MAP kinase pathway. Here too, Elafin overexpression is associated with poor overall survival, suggesting that it may serve as a biomarker and therapeutic target in this setting.

Abstract B74: Elafin drives poor outcome in high-grade serous ovarian cancers and basal-like breast tumors

Abstract High grade serous ovarian carcinoma (HGSOC) and basal-like breast cancer (BLBC) share many features including TP53 mutations, genomic instability and poor prognosis. We recently reported that Elafin is overexpressed by HGSOC and is associated with poor overall survival. Here, we confirmed that Elafin overexpression is associated with shorter survival in 1000 HGSOC patients. Elafin confers a proliferative advantage to tumor cells through activation of the MAP kinase pathway. This mitogenic effect can be neutralized by RNA interference, specific antibodies, and a MEK inhibitor. Elafin expression in patient-derived samples was also associated with chemoresistance and strongly correlates with bcl-xL expression. We translated these findings into examination of 1100 primary breast tumors and 6 breast cancer cell lines. We observed that Elafin is overexpressed and secreted specifically by BLBC tumors and cell lines, leading to a similar mitogenic effect through activation of the MAP kinase pathway. Here too, Elafin overexpression is associated with shorter overall survival, suggesting that it may serve as a biomarker or therapeutic target in this setting. Citation Format: Intidhar Labidi-Galy, Adam Clauss, Vivian Ng, Sekhar Duraisamy, Kevin M. Elias, Erhan Bilal, Rachel A. Davidowitz, Yiling Lu, Gayane Badalian-Very, Hui-Ying Piao, Un-Beom Kang, Scott Ficarro, Shridar Ganesan, Gordon B. Mills, Jarrod Marto, Ronny Drapkin. Elafin drives poor outcome in high-grade serous ovarian cancers and basal-like breast tumors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B74.