Human Triple-negative Breast Cancer Tissues Research Articles

The Prognostic Value of CD39 as a Marker of Tumor-Specific T Cells in Triple-Negative Breast Cancer in Asian Women

Triple-negative breast cancer (TNBC) has a poor prognosis with limited therapeutic options available for affected patients. Efforts are ongoing to identify surrogate markers for tumor-specific CD8+ T cells that can predict the response to immune checkpoint inhibitor (ICI) therapies, such as programmed cell death protein 1 or programmed cell death ligand-1 blockade. We have previously identified tumor-specific CD39+CD8+ T cells in non–small cell lung cancer that might help predict patient responses to programmed cell death protein 1 or programmed cell death ligand-1 blockade. Based on this finding, we conducted a comparative interrogation of TNBC in an Asian cohort to evaluate the potential of CD39 as a surrogate marker of tumor-specific CD8+ T cells. Using ICI-treated TNBC mouse models (n = 24), flow cytometric analyses of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes revealed that >99% of tumor-specific CD8+ T cells also expressed CD39. To investigate the relationship between CD39+CD8+ T-cell density and CD39 expression with disease prognosis, we performed multiplex immunohistochemistry staining on treatment-naive human TNBC tissues (n = 315). We saw that the proportion of CD39+CD8+ T cells in human TNBC tumors correlated with improved overall survival, as did the densities of other CD39+ immune cell infiltrates, such as CD39+CD68+ macrophages. Finally, increased CD39 expression on CD8+ T cells was also found to predict the response to ICI therapy (pembrolizumab) in a separate cohort of 11 TNBC patients. These findings support the potential of CD39+CD8+ T-cell density as a prognostic factor in Asian TNBC patients.

Abstract 5841: Exploring the role of versican in immune exclusion within triple negative breast cancer

Abstract Immunotherapy has found limited success in solid tumors such as triple negative breast cancer (TNBC). The poor response can be attributed to the presence of the extracellular matrix (ECM) that can act biochemically or physically to interfere with cytotoxic T cell migration, leading to a limitation in tumor cell killing. Through previous work on deconstructing this matrix and outlining the spatial arrangement of proteins, we were able to identify versican as a key protein in T cell location. Versican is a chondroitin sulfate proteoglycan and has multiple functions in the ECM due to its ability to bind many ECM components. Using immunohistochemistry in this study, we analyzed the localization of CD8+ T cells within human TNBC tissues to classify them as inflamed (tumorhi, stromahi), desert (tumorlo, stromalo) or immune excluded (tumorlo, stromahi). From the analysis of 26 tissues, nearly half were classified as immune excluded with most of the CD8+ T cells restricted to the stroma, whilst only a quarter were inflamed. When comparing this to versican accumulation we noticed that immune excluded tissues have significantly higher levels of versican in the tumor epithelium compared to inflamed tissues where versican is mostly found within the stroma. To investigate the function of versican’s structure, we carried out transwell migration assays using versican enriched protein treated with or without chondroitinase ABC and showed that the chondroitin sulfates attached to versican’s peptide backbone are an important factor towards T cell trafficking with a reduction in migration following chondroitinase ABC treatment. Comparison of versican levels to chondroitin sulfate via immunohistochemistry showed a positive correlation within inflamed tissues, however no correlation was observed in immune excluded tissues. Versican isoforms expression in TNBC tissues and cell lines was examined with qRT-PCR which showed that within all tissues and cell lines the V0 and V1 isoform are the highest expressed out of the 5 isoforms. Overall, in this work, we have found that versican is differentially expressed in excluded tissues in comparison to inflamed tissues. With chondroitin sulfate shown to play a major role in immune cell trafficking, understanding how it differs between inflamed and excluded tumors can help identify potential targets to improve immunotherapy response. Citation Format: Priyanka Hirani, Kimberly M. Alonge, Daniel J. Pennington, Pedro R. Cutillas, Thomas N. Wight, Oliver M. Pearce. Exploring the role of versican in immune exclusion within triple negative breast cancer. [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 5841.

Cyclin B2 (CCNB2) Stimulates the Proliferation of Triple-Negative Breast Cancer (TNBC) Cells In Vitro and In Vivo.

Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer. Currently, targeting therapy makes great advances for the treatment of TNBC, whereas more effective therapeutic targets are urgently needed. Cyclin B2 (CCNB2), which belongs to B-type cyclins, is known as a cell cycle regulator. CCNB2 is synthesized at G1 phase in cancer cells and downregulated at anaphase. The defects of CCNB2 led to the abnormal cell cycle and tumorigenesis. Though there are wide effects of CCNB2 on multiple types of tumors, the potential role of CCNB2 in TNBC progression is still unclear. Herein, we found that CCNB2 was highly expressed in human TNBC tissues and correlated with the prognosis and clinical pathological features including tumor size (p = 0.022∗) and pTNM stage (p = 0.021∗) of patients with TNBC. CCNB2 could promote the proliferation of TNBC cells in vitro and in mice. Our findings therefore confirmed the involvement of CCNB2 in TNBC progression and provided the evidence that CCNB2 could serve as a promising molecular target of TNBC.

Activation of the eIF2α/ATF4 axis drives triple-negative breast cancer radioresistance by promoting glutathione biosynthesis

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Radiotherapy is an effective option for the treatment of TNBC; however, acquired radioresistance is a major challenge to the modality. In this study, we show that the integrated stress response (ISR) is the most activated signaling pathway in radioresistant TNBC cells. The constitutive phosphorylation of eIF2α in radioresistant TNBC cells promotes the activation of ATF4 and elicits the transcription of genes implicated in glutathione biosynthesis, including GCLC, SLC7A11, and CTH, which increases the intracellular level of reduced glutathione (GSH) and the scavenging of reactive oxygen species (ROS) after irradiation (IR), leading to a radioresistant phenotype. The cascade is significantly up-regulated in human TNBC tissues and is associated with unfavorable survival in patients. Dephosphorylation of eIF2α increases IR-induced ROS accumulation in radioresistant TNBC cells by disrupting ATF4-mediated GSH biosynthesis and sensitizes them to IR in vitro and in vivo. These findings reveal ISR as a vital mechanism underlying TNBC radioresistance and propose the eIF2α/ATF4 axis as a novel therapeutic target for TNBC treatment.

Novel Long Noncoding RNA 005620 Induces Epirubicin Resistance in Triple-Negative Breast Cancer by Regulating ITGB1 Expression.

Triple-negative breast cancer (TNBC) is often treated with anthracyclines (e.g., epirubicin or doxorubicin), but very little is known about anthracycline resistance, especially epirubicin resistance in TNBC. To identify novel long noncoding RNAs (lncRNAs) involved in epirubicin resistance in TNBC, we established a new TNBC MDA-MB-231 cell line that was resistant to epirubicin (Epi-R). A total of 12 differentially expressed lncRNAs were identified using RNA sequencing analysis of Epi-R cells. Among these lncRNAs, we found a novel intronic lncRNA, lnc005620, was highly expressed in Epi-R cells and human TNBC tissues. Further gain- and loss-of-function studies demonstrated that lnc005620 played an oncogenic role and partially abrogated the effects of epirubicin on TNBC cells. Using iTRAQ proteomics analysis, we found that three members of the integrin family, integrin β4, integrin β1 and integrin α6, were all upregulated in Epi-R MDA-MB-231 cells. Integrin β1, encoded by the ITGB1 gene, was validated to be a downstream target of lnc005620 in Epi-R MDA-MB-231 cells. Our study demonstrates that novel lnc005620 promotes TNBC progression and chemoresistance to epirubicin via integrin β1 both in vitro and in vivo and provides a promising therapeutic target for TNBC patients in terms of enhancing the benefits of epirubicin treatment.

Lamin B2 promotes the progression of triple negative breast cancer via mediating cell proliferation and apoptosis.

Triple negative breast cancer (TNBC) is a more common type of breast cancer with high distant metastasis and poor prognosis. The potential role of lamins in cancer progression has been widely revealed. However, the function of lamin B2 (LMNB2) in TNBC progression is still unclear. The present study aimed to investigate the role of LMNB2 in TNBC. The cancer genome atlas (TCGA) database analysis and immunohistochemistry (IHC) were performed to examine LMNB2 expression levels. LMNB2 short hairpin RNA plasmid or lentivirus was used to deplete the expression of LMNB2 in human TNBC cell lines including MDA-MB-468 and MDA-MB-231. Alterations in cell proliferation and apoptosis in vitro and the nude mouse tumorigenicity assay in vivo were subsequently analyzed. The human TNBC tissues shown high expression of LMNB2 according to the bioinformation analysis and IHC assays. LMNB2 expression was correlated with the clinical pathological features of TNBC patients, including pTNM stage and lymph node metastasis. Through in vitro and in vivo assays, we confirmed LMNB2 depletion suppressed the proliferation and induced the apoptosis of TNBC cells, and inhibited tumor growth of TNBC cells in mice, with the decrease in Ki67 expression or the increase in caspase-3 expression. In conclusion, LMNB2 may promote TNBC progression and could serve as a potential therapeutic target for TNBC treatment.

LncRNA FAM83H-AS1 promotes triple-negative breast cancer progression by regulating the miR-136-5p/metadherin axis.

In this study, we evaluated the function and regulation of the long non-coding RNA (lncRNA) FAM83H-AS1 in triple-negative breast cancer (TNBC). Our data show that the FAM83H-AS1 levels are increased in human TNBC cells and tissues. Proliferation, migration, and invasion of TNBC cells are decreased by FAM83H-AS1 suppression, but increased by FAM83H-AS1 overexpression. Bioinformatics analysis revealed that miR-136-5p is a potential target of FAM83H-AS1. MiR-136-5p expression is decreased in TNBC tissues, and its overexpression suppresses TNBC cell proliferation, migration, and invasion. MiR-136-5p suppression reverses the FAM83H-AS1 silencing-mediated inhibition of TNBC cell proliferation, migration, and invasion, suggesting that FAM83H-AS1 exerts its oncogenic effect by inhibiting miR-136-5p. Our data identify metadherin (MTDH) as the target gene of miR-136-5p, and demonstrate that the MTDH expression is increased in human TNBC tissues, which induces proliferation, migration, and invasion of TNBC cells. Importantly, our in vivo data show that FAM83H-AS1 also promotes tumor growth in TNBC mouse xenografts. Together, our results demonstrate that FAM83H-AS1 functions as an oncogenic lncRNA that regulates miR-136-5p and MTDH expression during TNBC progression, and suggest that targeting the FAM83H-AS1/miR-136-5p/MTDH axis may serve as a novel therapeutic target in TNBC.

Effects of HOX transcription antisense RNA on carcinogenesis and development of triple-negative breast cancer

Objective To investigate the significance of HOX transcription antisense RNA (HOTAIR) expression in triple-negative breast cancer (TNBC) and explore the molecular modulation of HOTAIR on the progression of TNBC. Methods Sixty-four human TNBC tissues and their corresponding non-tumor tissues were collected, and the HOTAIR levels were detected by real-time quantitative polymerase chain reaction (Real-time PCR). The different HOTAIR levels in tumor and non-tumor tissues were compared. And then we analyzed its significance in TNBC with different pathological stages. In MDA-MB231 cells, the effects of down-regulation of HOTAIR by small interfering RNA (siRNA) on the proliferation (MTT) and apoptosis (Western blotting) were detected. The effects of down-regulation of HOTAIR on the expression of enhancer of zeste homolog2 (EZH2), the enzymatic subunit of ploycomb repressor complex (PRC2), were detected. SPSS19.0 statistical software was used for analysis. Results Compared with the non-tumor tissues, the HOTAIR expression was significantly increased in TNBC tumors (0.71±0.10 vs. 0.50±0.06, t=20.286, P<0.01), especially in TNBC with advanced stage (0.78±0.05 vs. 0.64±0.07, t=8.625, P<0.01). In TNBC cells, treatment of siEZH2 showed similar effects on the cell proliferation and apoptosis. In TNBC cells, EZH2 expression was down-regulated by siHOTAIR. However, HOTAIR expression had no significant change after siEZH2 treatment. Conclusion HOTAIR is highly expressed in TNBC and associated with poor prognosis. Our results suggest HOTAIR may induce the carcinogenesis in TNBC probably through regulating EZH2. Key words: Triple negative breast cancer; HOX transcription antisense RNA; Enhancer of zeste homolog 2

Assessment of Copper Nanoclusters for Accurate in Vivo Tumor Imaging and Potential for Translation.

Nanoparticles have been widely used for preclinical cancer imaging. However, their successful clinical translation is largely hampered by potential toxicity, unsatisfactory detection of malignancy at early stages, inaccurate diagnosis of tumor biomarkers, and histology for imaging-guided treatment. Herein, a targeted copper nanocluster (CuNC) is reported with high potential to address these challenges for future translation. Its ultrasmall structure enables efficient renal/bowel clearance, minimized off-target effects in nontargeted organs, and low nonspecific tumor retention. The pH-dependent in vivo dissolution of CuNCs affords minimal toxicity and potentially selective drug delivery to tumors. The intrinsic radiolabeling through the direct addition of 64Cu to CuNC (64Cu-CuNCs-FC131) synthesis offers high specific activity for sensitive and accurate detection of CXCR4 via FC131-directed targeting in novel triple negative breast cancer (TNBC) patient-derived xenograft mouse models and human TNBC tissues. In summary, this study not only reveals the potential of CXCR4-targeted 64Cu-CuNCs for TNBC imaging in clinical settings, but also provides a useful strategy to design and assess the translational potential of nanoparticles for cancer theranostics.

ΔNp63 activates EGFR signaling to induce loss of adhesion in triple-negative basal-like breast cancer cells.

The basal-A subtype of triple-negative breast cancer is characterized by high levels of ΔNp63. Various functions have been proposed for p63 in breast cancer initiation and growth, and p63 mediates chemotherapeutic response in a subset of triple-negative breast cancers. We investigated the signaling pathways that are controlled by ΔNp63 in basal-A triple-negative breast cancer. Human basal-A triple-negative breast cancer cell lines with ΔNp63α induction or inhibition were studied, along with primary human triple-negative breast cancer tissues. Proteomic, phospho-kinase array, mRNA measurements, and immunohistochemistry were employed. Global phosphoproteomics identified increased EGFR phosphorylation in MDA-MB-468 cells expressing ΔNp63α. ΔNp63α expression increased EGFR mRNA, total EGFR protein, and phospho-EGFR(Y1086), whereas silencing endogenous ΔNp63 in HCC1806 cells reduced both total and phospho-EGFR levels and inhibited the ability of EGF to activate EGFR. EGFR pathway gene expression analysis indicated that ΔNp63 alters EGFR-regulated genes involved in cell adhesion, migration, and angiogenesis. Addition of EGF or neutralizing EGFR antibodies demonstrated that EGFR activation is responsible for ΔNp63-mediated loss of cellular adhesion. Finally, immunohistochemical staining showed that p63-positive triple-negative breast cancers were more likely to express high levels of EGFR than p63-negative cancers, corroborated by in silico analysis of gene expression profiling data. These data identify EGFR as a major target for ΔNp63 regulation that influences cancer cell adhesion in basal-like triple-negative breast cancer.

HMAGEA2 promotes progression of breast cancer by regulating Akt and Erk1/2 pathways.

Breast cancer is the most abundant cancer worldwide and a severe problem for women. Notably, breast cancer has a high mortality rate, mainly because of tumor progression and metastasis. Triple-negative breast cancer (TNBC) is highly progressive and lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Therefore, there are no established therapeutic targets against TNBC. In this study, we investigated whether the expression of human melanoma-associated antigen A2 (MAGEA2) is associated with TNBC. We found that hMAGEA2 is significantly overexpressed in human TNBC tissues; we also observed oncogenic properties using TNBC cell lines (MDA-MB-231 and MDA-MB-468). The overexpression of hMAGEA2 in MDA-MB-231 cell line showed dramatically increased cellular proliferation, colony formation, invasion, and xenograft tumor formation and growth. Conversely, knockdown of hMAEGA2 in MDA-MB-468 cell line suppressed cellular proliferation, colony formation, and xenograft tumor formation. Additionally, we showed that hMAGEA2 regulated the activation of Akt and Erk1/2 signaling pathways. These data indicate that hMAGEA2 is important for progression of TNBC and may serve as a novel molecular therapeutic target.

Suppressive role exerted by microRNA-29b-1-5p in triple negative breast cancer through SPIN1 regulation.

MiR-29 family dysregulation occurs in various cancers including breast cancers. We investigated miR-29b-1 functional role in human triple negative breast cancer (TNBC) the most aggressive breast cancer subtype. We found that miR-29b-1-5p was downregulated in human TNBC tissues and cell lines. To assess whether miR-29b-1-5p correlated with TNBC regenerative potential, we evaluated cancer stem cell enrichment in our TNBC cell lines, and found that only MDA-MB-231 and BT-20 produced primary, secondary and tertiary mammospheres, which were progressively enriched in OCT4, NANOG and SOX2 stemness genes. MiR-29b-1-5p expression inversely correlated with mammosphere stemness potential, and miR-29b-1 ectopic overexpression decreased TNBC cell growth, self-renewal, migration, invasiveness and paclitaxel resistance repressing WNT/βcatenin and AKT signaling pathways and stemness regulators. We identified SPINDLIN1 (SPIN1) among predicted miR-29b-1-5p targets. Consistently, SPIN1 was overexpressed in most TNBC tissues and cell lines and negatively correlated with miR-29b-1-5p. Target site inhibition showed that SPIN1 seems to be directly controlled by miR-29b-1-5p. Silencing SPIN1 mirrored the effects triggered by miR-29b-1 overexpression, whereas SPIN1 rescue by SPIN1miScript protector, determined the reversal of the molecular effects produced by the mimic-miR-29b-1-5p. Overall, we show that miR-29b-1 deregulation impacts on multiple oncogenic features of TNBC cells and their renewal potential, acting, at least partly, through SPIN1, and suggest that both these factors should be evaluated as new possible therapeutic targets against TNBC.

Up-Regulation of RFC3 Promotes Triple Negative Breast Cancer Metastasis and is Associated With Poor Prognosis Via EMT

Triple-negative breast cancer (TNBC) was regarded as the most aggressive and mortal subtype of breast cancer (BC) since the molecular subtype system has been established. Abundant studies have revealed that epithelial-mesenchymal transition (EMT) played a pivotal role during breast cancer metastasis and progression, especially in TNBC. Herein, we showed that inhibition the expression of replication factor C subunit 3 (RFC3) significantly attenuated TNBC metastasis and progression, which was associated with EMT signal pathway. In TNBC cells, knockdown of RFC3 can down-regulate mesenchymal markers and up-regulate epithelial markers, significantly attenuated cell proliferation, migration and invasion. Additionally, silencing RFC3 expression can decrease nude mice tumor volume, weight and relieve lung metastasis in vivo. Furthermore, we also demonstrated that overexpression of RFC3 in TNBC showed increased metastasis, progression and poor prognosis. We confirmed all of these results by immunohistochemistry analysis in 127 human TNBC tissues and found that RFC3 expression was significantly associated with poor prognosis in TNBC. Taken all these findings into consideration, we can conclude that up-regulation of RFC3 promotes TNBC progression through EMT signal pathway. Therefore, RFC3 could be an independent prognostic factor and therapeutic target for TNBC.

A prognostic risk model for patients with triple negative breast cancer based on stromal natural killer cells, tumor-associated macrophages and growth-arrest specific protein 6.

The aim of this study was to establish a prognostic risk model for patients with triple negative breast cancer (TNBC). A total of 278 specimens of human TNBC tissues were investigated by immunohistochemistry for growth‐arrest specific protein 6 expression, infiltrations of stromal natural killer cells and tumor‐associated macrophages. According to their prognostic risk scores based on the model, patients were divided into three groups (score 0, 1–2, 3). Correlations of prognostic risk scores, clinicopathologic features and overall survival (OS) were analyzed. To study the clinical value of this stratification model in early disease recurrence or metastasis, 177 patients were screened out for further analysis. Based on disease free survival (DFS), 90 patients fell within the DFS ≤3 years group and 87 patients within the DFS ≥5 years group. We analyzed the differences in prognostic risk scores between the two groups. The prognostic risk scores were negatively related to tumor size, lymph node metastasis and P53 status (P < 0.001 for all). Patients with low prognostic risk scores had longer OS (P = 0.001). Using multivariate analysis, it was determined that TNM stage (HR = 0.432, 95% confidence interval [CI] = 0.281–0.665, P = 0.003), FOXP3 positive lymphocytes (HR = 1.712, 95% CI = 1.085–2.702, P = 0.021) and prognostic risk scores (HR = 1.340, 95% CI = 1.192–1.644, P = 0.005) were independent prognostic factors for OS. Compared with the DFS ≥5 years group, the DFS ≤3 years group patients had significantly higher prognostic risk scores (P < 0.001). In conclusion, the prognostic risk score of the model was a significant indicator of prognosis for patients with TNBC.

Abstract 4410: An ICAM-1-targeted, Lcn2 siRNA-encapsulating liposome as a potent anti-angiogenic agent for triple-negative breast cancer

Abstract Introduction: The significant growth and metastasis of triple negative breast cancer (TNBC) tumors, coupled with limited treatment options, have made TNBC a clinically challenging disease. We have previously shown that the intercellular adhesion molecule-1 (ICAM-1) is highly overexpressed in human TNBC tissues and cell lines, suggesting that ICAM-1 could be a potential TNBC molecular target. Lipocalin 2 (Lcn2), a 25-kDa protein belonging to the lipocalin protein superfamily. We have recently reported that Lcn2 is a regulator of angiogenesis. We have also found that down-regulation of TNBC-secreted Lcn2 is associated with reduced tumor angiogenesis in preclinical studies of breast cancer, making it a promising TNBC anti-angiogenic target. Here, we describe a novel liposomal drug delivery system that integrates TNBC targeting via ICAM-1, with Lcn2 siRNA silencing, to synergistically inhibit TNBC angiogenesis in vitro and in vivo, representing a novel anti-angiogenic therapeutic approach for the treatment of TNBC. Materials and Methods: ICAM-1 antibody, or non-targeted IgG conjugated, Lcn2 siRNA encapsulating liposomes (ICAM-Lcn2-LPs or IgG-Lcn2-LPs (non-targeting control)) were prepared by the extrusion method. Lcn2 siRNA silencing in TNBC cells was determined by qRT-PCR, and the anti-angiogenic functions of ICAM-Lcn2-LP were evaluated via a series of in vitro angiogenic assays including: endothelial cell proliferation, endothelial cell migration, and endothelial cell tube formation. These in vitro anti-angiogenic results were further confirmed in vivo using the chick chorioallantoic membrane (CAM) assay. Results: We have engineered a liposomal siRNA delivery system to target triple negative breast cancer (TNBC) via a novel molecular target, ICAM-1, and selectively silenced a TNBC-overexpressing angiogenic mediator, Lcn2 using siRNA. This ICAM-1 targeted, Lcn2 siRNA delivery liposome binds human TNBC MDA-MB-231 cells 3.1-fold greater than non-neoplastic MCF-10A cells. Potent Lcn2 knockdown by ICAM-Lcn2-LP led to a significant reduction in VEGF secretion by MDA-MB-231 cells, which led to reduced TNBC-induced angiogenesis both in vitro and in vivo. Conclusions: We have successfully engineered an immunoliposome that synergistically couples TNBC-targeting with Lcn2 siRNA silencing. Synthesized ICAM-Lcn2-LPs can significantly suppress in vitro and in vivo angiogenic activities of TNBC cells by regulating VEGF secretion. The modular design introduced here provides a basis for future TNBC-targeted therapeutics. Acknowledgements: This research is supported by NIH(NCI 1DP2CA174495) and the Breast Cancer Research Foundation. Citation Format: Peng Guo, Jiang Yang, Marsha Moses, Debra Auguste. An ICAM-1-targeted, Lcn2 siRNA-encapsulating liposome as a potent anti-angiogenic agent for triple-negative 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 4410. doi:10.1158/1538-7445.AM2015-4410

Inhibition of iNOS as a novel effective targeted therapy against triple-negative breast cancer.

IntroductionTriple-negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in patients with breast cancer by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing tumor initiation and metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors.MethodsiNOS protein levels were determined in 83 human TNBC tissues and correlated with clinical outcome. Proliferation, mammosphere-forming efficiency, migration, and EMT transcription factors were assessed in vitro after iNOS inhibition. Endogenous iNOS targeting was evaluated as a potential therapy in TNBC mouse models.ResultsHigh endogenous iNOS expression was associated with worse prognosis in patients with TNBC by gene expression as well as immunohistochemical analysis. Selective iNOS (1400 W) and pan-NOS (L-NMMA and L-NAME) inhibitors diminished cell proliferation, cancer stem cell self-renewal, and cell migration in vitro, together with inhibition of EMT transcription factors (Snail, Slug, Twist1, and Zeb1). Impairment of hypoxia-inducible factor 1α, endoplasmic reticulum stress (IRE1α/XBP1), and the crosstalk between activating transcription factor 3/activating transcription factor 4 and transforming growth factor β was observed. iNOS inhibition significantly reduced tumor growth, the number of lung metastases, tumor initiation, and self-renewal.ConclusionsConsidering the effectiveness of L-NMMA in decreasing tumor growth and enhancing survival rate in TNBC, we propose a targeted therapeutic clinical trial by re-purposing the pan-NOS inhibitor L-NMMA, which has been extensively investigated for cardiogenic shock as an anti-cancer therapeutic.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0527-x) contains supplementary material, which is available to authorized users.

Efficacy of a leptin receptor antagonist peptide in a mouse model of triple-negative breast cancer

Triple-negative breast cancers, which represent 10–20% of all mammary tumours, are characterised by the aggressive phenotype, are often found in younger women and have been associated with poor prognosis. Obesity increases the risk for triple-negative breast cancer development. Because triple-negative breast cancer patients are unresponsive to current targeted therapies and other treatment options are only partially effective, new pharmacological modalities are urgently needed. Here we examined if the leptin (obesity hormone) receptor is a viable target for the treatment of this cancer subtype. In human triple-negative breast cancer tissues, the leptin receptor was expressed in 92% (64/69) and leptin in 86% (59/69) of cases. In a model triple-negative breast cancer cell line MDA-MB-231, the leptin receptor antagonist peptide Allo-aca inhibited leptin-induced proliferation at 50pM concentration. In an MDA-MB-231 orthotopic mouse xenograft model, Allo-aca administered subcutaneously significantly extended the average survival time from 15.4days (untreated controls) to 24 and 28.1days at 0.1 and 1mg/kg/day doses, respectively. In parallel, conventional treatment with 1mg/kg/day intraperitoneal cisplatin prolonged the average survival time to 18.6days, while administration of 20mg/kg/day oral Tamoxifen (negative control) had no significant survival effects relative to controls. In normal CD-1 mice, Allo-aca produced no systemic toxicity up to the highest studied subcutaneous bolus dose of 50mg/kg, while, as expected, it induced a modest 6–10% body weight increase. Our results indicate that leptin receptor antagonists could become attractive options for triple-negative breast cancer treatment, especially in the obese patient population.