TNBC Progression Research Articles

SLC6A8-mediated intracellular creatine accumulation enhances hypoxic breast cancer cell survival via ameliorating oxidative stress

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with poor prognosis and limited treatment options. Hypoxia is a key hallmark of TNBC. Metabolic adaptation promotes progression of TNBC cells that are located within the hypoxic tumor regions. However, it is not well understood regarding the precise molecular mechanisms underlying the regulation of metabolic adaptions by hypoxia.MethodsRNA sequencing was performed to analyze the gene expression profiles in MDA-MB-231 cell line (20% O2 and 1% O2). Expressions of Slc6a8, which encodes the creatine transporter protein, were detected in breast cancer cells and tissues by quantitative real-time PCR. Immunohistochemistry was performed to detect SLC6A8 protein abundances in tumor tissues. Clinicopathologic correlation and overall survival were evaluated by chi-square test and Kaplan-Meier analysis, respectively. Cell viability assay and flow cytometry analysis with Annexin V/PI double staining were performed to investigate the impact of SLC6A8-mediated uptake of creatine on viability of hypoxic TNBC cells. TNBC orthotopic mouse model was used to evaluate the effects of creatine in vivo.ResultsSLC6A8 was aberrantly upregulated in TNBC cells in hypoxia. SLC6A8 was drastically overexpressed in TNBC tissues and its level was tightly associated with advanced TNM stage, higher histological grade and worse overall survival of TNBC patients. We found that SLC6A8 was transcriptionally upregulated by p65/NF-κB and mediated accumulation of intracellular creatine in hypoxia. SLC6A8-mediated accumulation of creatine promoted survival and suppressed apoptosis via maintaining redox homeostasis in hypoxic TNBC cells. Furthermore, creatine was required to facilitate tumor growth in xenograft mouse models. Mechanistically, intracellular creatine bolstered cell antioxidant defense by reducing mitochondrial activity and oxygen consumption rates to reduce accumulation of intracellular reactive oxygen species, ultimately activating AKT-ERK signaling, the activation of which protected the viability of hypoxic TNBC cells via mediating the upregulation of Ki-67 and Bcl-2, and the downregulation of Bax and cleaved Caspase-3.ConclusionsOur study indicates that SLC6A8-mediated creatine accumulation plays an important role in promoting TNBC progression, and may provide a potential therapeutic strategy option for treatment of SLC6A8 high expressed TNBC.

Alteration of MDM2 by the Small Molecule YF438 Exerts Antitumor Effects in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) exhibits a high mortality rate and is the most aggressive subtype of breast cancer. As previous studies have shown that histone deacetylases (HDAC) may represent molecular targets for TNBC treatment, we screened a small library of synthetic molecules and identified a potent HDAC inhibitor (HDACi), YF438, which exerts effective anti-TNBC activity both in vitro and in vivo. Proteomic and biochemical studies revealed that YF438 significantly downregulated mouse double minute 2 homolog (MDM2) expression. In parallel, loss of MDM2 expression or blocking MDM2 E3 ligase activity rendered TNBC cells less sensitive to YF438 treatment, revealing an essential role of MDM2 E3 ligase activity in YF438-induced inhibition of TNBC. Mechanistically, YF438 disturbed the interaction between HDAC1 and MDM2, induced the dissociation of MDM2-MDMX, and subsequently increased MDM2 self-ubiquitination to accelerate its degradation, which ultimately inhibited growth and metastasis of TNBC cells. In addition, analysis of clinical tissue samples demonstrated high expression levels of MDM2 in TNBC, and MDM2 protein levels closely correlated with TNBC progression and metastasis. Collectively, these findings show that MDM2 plays an essential role in TNBC progression and targeting the HDAC1-MDM2-MDMX signaling axis with YF438 may provide a promising therapeutic option for TNBC. Furthermore, this novel underlying mechanism of a hydroxamate-based HDACi in altering MDM2 highlights the need for further development of HDACi for TNBC treatment. SIGNIFICANCE: This study uncovers the essential role of MDM2 in TNBC progression and suggests that targeting the HDAC1-MDM2-MDMX axis with a hydroxamate-based HDACi could be a promising therapeutic strategy for TNBC.

The Calcium-Sensing Receptor (CaSR) Variants at rs1801725 Increase the Risk of Developing Secondary Malignant Cancers

The dysregulation of systemic calcium homeostasis during malignancy is common in most patients with high-grade tumors. The associated comorbidity is known as cancer-induced hypercalcemia (CIH) which affects up to 30% of cases, in the absence of metastasis. In the course of breast cancer progression, the secretion of parathyroid hormone-related protein (PTHrP) by tumor cells and the associated destruction of bone tissues, leads to a progressive increase in systemic calcium or CIH. The increase in circulating Ca2+ is sensed by the calcium-sensing receptor (CaSR), which plays a significant role in maintaining Ca2+ homeostasis. More than 200 mutations and single nucleotide polymorphisms (SNPs) in the CaSR gene have been described, including the A986S CaSR at rs1801725 and Q1011E CaSR at rs1801726 SNPs with reduced sensitivity to Ca2+. Interestingly, high circulating Ca2+ is associated with aggressive breast tumors in premenopausal women and larger tumors in postmenopausal women; however, the contribution of the CaSR in breast cancer progression remains poorly understood. Unlike SNPs at rs1801726, up to 20% of breast cancer patients with SNPs at rs1801725 may be predisposed to higher circulating Ca2+ in the course of their disease. Since breast cancer frequently metastasizes to Ca2+ rich skeletal tissues, we hypothesize that the development of CIH and subsequent desensitization of the CaSR by sustained high Ca2+ is critical for both the adaptation of TNBC cells to CIH in Ca2+ rich microenvironments and TNBC progression. Our preliminary data reveal that the expression level and mutational status of the CaSR is cell type-specific, and that sustained high Ca2+ desensitizes the receptor, but promotes tumor cell growth and motility. Sustained high Ca2+ also triggers the expression of metastasis promoting genes, including the cancer/testis antigen, MAGEC2, and Plasminogen Activator Inhibitor, PAI-2, potentially via the early response genes FOS/FOSB. In addition, our preliminary data show that the A986S SNP is associated with hypercalcemia, secondary malignancy of bone and respiratory organs, and deficiency of humoral immunity. This study provides novel insights into not only the adaptation of TNBC cells to high circulating Ca2+, but also suggests that mutations of the CaSR at rs1801725 are predictive of the likelihood for metastasis to lungs and bone.

Overexpression of CCNE1 confers a poorer prognosis in triple-negative breast cancer identified by bioinformatic analysis

BackgroundTriple-negative breast cancer (TNBC) is a major subtype of breast cancer. Due to the lack of effective therapeutic targets, the prognosis is poor. In order to find an effective target, despite many efforts, the molecular mechanisms of TNBC are still not well understood which remain to be a profound clinical challenge.MethodsTo identify the candidate genes in the carcinogenesis and progression of TNBC, microarray datasets GSE36693 and GSE65216 were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified, and functional and pathway enrichment analyses were performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases via DAVID. We constructed the protein-protein interaction network (PPI) and performed the module analysis using STRING and Cytoscape. Then, we reanalyzed the selected DEG genes, and the survival analysis was performed using cBioportal.ResultsA total of 140 DEGs were identified, consisting of 69 upregulated genes and 71 downregulated genes. Three hub genes were upregulated among the selected genes from PPI, and biological process analysis uncovered the fact that these genes were mainly enriched in p53 pathway and the pathways in cancer. Survival analysis showed that only CCNE1 may be involved in the carcinogenesis, invasion, or recurrence of TNBC. The expression levels of CCNE1 were significantly higher in TNBC cells than non-TNBC cells that were detected by qRT-PCR (P < 0.05).ConclusionCCNE1 could confer a poorer prognosis in TNBC identified by bioinformatic analysis and plays key roles in the progression of TNBC which may contribute potential targets for the diagnosis, treatment, and prognosis assessment of TNBC.

ADAM10 is involved in the oncogenic process and chemo-resistance of triple-negative breast cancer via regulating Notch1 signaling pathway, CD44 and PrPc

BackgroundTriple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat, because it is so aggressive with shorter survival. Chemotherapy remains the standard treatment due to the lack of specific and effective molecular targets. The aim of the present study is to investigate the potential roles of A Disintegrin and Metalloproteinase 10 (ADAM10) on TNBC cells and the effects of combining ADAM10 expression and neoadjuvant chemotherapy treatment (NACT) to improve the overall survival in breast cancer patients.MethodsUsing a series of breast cancer cell lines, we measured the expression of ADAM10 and its substrates by quantitative real-time PCR assay (qRT-PCR) and western blot analysis. Cell migration and invasion, cell proliferation, drug sensitivity assay, cell cycle and apoptosis were conducted in MDA-MB-231 cells cultured with ADAM10 siRNA. The effect of ADAM10 down-regulation by siRNA on its substrates was assessed by western blot analysis. We performed immunohistochemical staining for ADAM10 in clinical breast cancer tissues in 94 patients receiving NACT.ResultsThe active form of ADAM10 was highly expressed in TNBC cell lines. Knockdown of ADAM10 in MDA-MB-231 cells led to a significant decrease in cell proliferation, migration, invasion and the IC50 value of paclitaxel and adriamycin, while induced cell cycle arrest and apoptosis. And these changes were correlated with down-regulation of Notch signaling, CD44 and cellular prion protein (PrPc). In clinical breast cancer cases, a high ADAM10 expression in pre-NACT samples was strongly associated with poorer response to NACT and shorter overall survival.ConclusionsThese data suggest the previously unrecognized roles of ADAM10 in contributing to the progression and chemo-resistance of TNBC.

Pembrolizumab can delay progression of TNBC.

Pembrolizumab can delay progression of TNBC.

Comprehensive genomic and immunophenotypic analysis of CD4 T cell infiltrating human triple-negative breast cancer

The aim of this study is to investigate the gene expression module of tumor-infiltrating CD4+T cells and its potential roles in modulating immune cell functions in triple-negative breast cancer. Differentially expressed genes were identified by comparison of the expression profile in CD4+T cells isolated from tumor tissues and peripheral blood of TNBC patients respectively. The differential expression analysis was conducted using R, and then the functional and pathway enrichment of the DEGs were analyzed using GSEA, followed by integrated regulatory network construction and genetic analysis of tumor-infiltrating immune cells based on a scientific deconvolution algorithm. As a result, abundant Treg and exhausted lymphocytes were detected, accompanied by largely decreased of effector/memory and cytotoxic T cells. Immune-related gene correlation analysis showed that the extent of follicular helper T cells gene expression signatures were inversely associated with those of CD4+ naive T cells and CD4+ memory resting T cells, but positively correlated with that of CD4+ memory activated T cells. In addition, we found five core genes including IFNG, CTLA4, FAS, CXCR6, and JUN were significantly over expressed in CD4+ TILs which may contribute to exhaustion of lymphocytes and participate in biological processes associated with regulation of chemotaxis. Study provides a comprehensive understanding of the roles of DEGs associated with the chemotactic and exhausted immunophenotypes of CD4+ TILs that are a valuable resource from which future investigation may be carried out to better understand the mechanisms that promote TNBC progression.

CircUBE2D2 (hsa_circ_0005728) promotes cell proliferation, metastasis and chemoresistance in triple-negative breast cancer by regulating miR-512-3p/CDCA3 axis

BackgroundTriple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer with a bad prognosis. Chemotherapy is still the standard of care for TNBC treatment. Circular RNAs (CircRNAs) have been recently discovered to be closely involved in the initiation and development of human cancers. Herein, we focus our attention on the functions and underlying mechanisms of circUBE2D2 in TNBC progression and chemoresistance.MethodsThe expression of circUBE2D2, miR-512-3p, and cell division cycle associated 3 (CDCA3) mRNA were determined by qRT-PCR. CCK-8, colony formation, transwell and flow cytometry assays were performed to detect cell proliferation, migration, invasion and apoptosis. Western blot assay was utilized to measure the protein level of CDCA3. RNA pull-down, luciferase reporter and RIP experiments were employed to examine the possible regulatory mechanism of circUBE2D2.ResultsCircUBE2D2 expression was elevated in TNBC tissues and cells. TNBC patients with high circUBE2D2 expression are inclined to present advanced TNM stage, lymph node metastasis and adverse prognosis. Knockdown of circUBE2D2 repressed cell proliferation, migration and invasion in vitro, and impeded tumor growth in vivo. Moreover, silencing of circUBE2D2 reduced doxorubicin resistance of TNBC cells. In-depth mechanism analysis revealed that circUBE2D2 served as a miRNA sponge to protect CDCA3 from the attack of miR-512-3p. Additionally, the tumor-suppressive effect induced by circUBE2D2 depletion was greatly impaired upon miR512-3p down-regulation or CDCA3 overexpression. Also, depletion of circUBE2D2 decreased the resistance to doxorubicin through regulating miR-512-3p/CDCA3 axis.ConclusionCircUBE2D2 promoted TNBC progression and doxorubicin resistance through acting as a sponge of miR-512-3p to up-regulate CDCA3 expression. Targeting circUBE2D2 combine with doxorubicin might be exploited as a novel therapy for TNBC.

ANGPTL4 overexpression inhibits tumor cell adhesion and migration and predicts favorable prognosis of triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) patients have relatively poor clinical outcomes. A marker predicting the prognosis of patients with TNBC could help guide treatment. Extensive evidence demonstrates that angiopoietin-like 4 (ANGPTL4) is involved in the regulation of cancer growth, metastasis and angiogenesis. Therefore, its role in TNBC is of interest.Methods: We tested the ANGPTL4 expression level in tumor tissues by immunohistochemistry (IHC) and detected its association with the clinical features of TNBC patients. Next, the effects and mechanisms of ANGPTL4 on TNBC cell migration and adhesion were investigated.ResultsWe found that ANGPTL4 overexpression was associated with favorable outcomes in TNBC patients. ANGPTL4 upregulation inhibited cell adhesion, migration and invasion in vitro. Further analyses demonstrated that the possible mechanism might involve suppression of TNBC progression by interacting with extracellular matrix-related genes.ConclusionsThe present findings demonstrated that enhancement of ANGPTL4 expression might inversely correlate with TNBC progression. ANGPTL4 is a promising marker of TNBC and should be evaluated in further studies.Trial registrationRetrospectively registered.

Abstract 3704: MicroRNA-3977 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting FoxM1

Abstract Triple negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). TNBC is associated with poor prognosis, high rates of relapse and distant metastasis despite surgery and adjuvant chemotherapy. The lack of effective targeted therapies for TNBC requires new therapeutic approaches. The Forkhead box protein M1 (FoxM1) is a member of forkhead superfamily transcription factors that has been associated with normal cell growth and proliferation through cell cycle transition and mitotic spindle control. FoxM1 is also identified as a master regulator of tumor metastasis. Based on analysis of pooled clinical datasets derived from online accessible databases as Breast Cancer Gene-Expression Miner v4.3 and Kaplan-Meier plotter, the expression and prognostic values of FoxM1 increase in TNBC compare with other breast cancer subtypes. Directly binding to the 3'-untranslated region (3' UTR) of FoxM1 mRNAs suppress its high expression in TNBC that leads to inhibition of cell proliferation, motility, and invasion. These results implied that FoxM1 is an essential poor prognostic factor and promising candidate target in the treatment of TNBC. We identified miR-3977 as a potential regulator of FoxM1 by using in silico prediction algorithms, and we investigated its role in TNBC. In this study, we found miR-3977 expression is significantly suppressed in TNBC cell lines and inversely correlated with FoxM1 levels. We demonstrate that miR-3977 directly bound to the 3′ UTR of FoxM1 mRNA and repressed its expression. We also find reduced miR-3977 expression is associated with shorter patient survival in TNBC based on Kaplan-Meier plotter database. Moreover, in vivo delivery of miR-3977 nanoparticles inhibited FoxM1 expression and tumor growth in TNBC xenograft mouse models. In conclusion, we provide the first evidence that miR-3977 functions as a tumor suppressor and gain of miR-3977 expression leads to decrease in FoxM1 expression and inhibits to cell growth, colony formation, invasion, and progression of TNBC. Taken together, our data suggest that miR-3977-based gene therapy is a potential therapeutic strategy in TNBC. Citation Format: Selda Karamil, Nuray Altintas, Bulent Ozpolat. MicroRNA-3977 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting FoxM1 [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 3704.

Abstract 492: A Novel QUATRAMER sustained injectable suspension for the intracellular delivery of Salinomycin, a stem cell inhibitor (HSB-1216), for the treatment of triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for approximately 15-20 % of all breast cancers, is an aggressive disease with a poor prognosis, and lacks targeted therapy. Recent studies suggest that cancer stem cells (CSCs) play an important role in TNBC progression and tumorigenesis and that targeting CSCs may be a promising, novel strategy for the treatment of this disease. Both CD44+/CD24- and CD44+/CD24 low CSCs are enriched in TNBCs and may contribute to chemotherapy resistance, emphasizing the need to target this population. In this regard, recent studies have shown that salinomycin (SAL) preferentially targets CSCs; however, the preclinical toxicity associated with this agent has limited its clinical development.In the current studies, we have generated novel biodegradable QUATRAMER polymeric nanoparticles comprised of a polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG modified polylactic acid (PLA)-tetra-block copolymer (SAL-NPs; HSB-1216) for the intracellular delivery of SAL. Treatment of human TNBC MDA-MB-231 cells with HSB-1216 was associated with significant cell death with an IC50 of around 2 uM. Similar results were obtained when other TNBC cell line SUM149 was treated with HSB-1216 (IC50 ~ 0.5 uM). Based on the characteristic formation of TNBC mammospheres in in vitro 3D culture conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity. Cultured MDA-MB-231 mammospheres were therefore exposed to different concentrations and durations of HSB-1216. Our results demonstrate dose-dependent inhibition of mammosphere formation in response to HSB-1216. Additionally, CD44+/CD24- enriched CSCs were isolated from a TNBC patient tumor biopsy, grown as 3D culture/mammospheres and then treated with HSB-1216. Interestingly, in contrast to Paclitaxel, treatment with HSB-1216 was associated with nearly complete inhibition of mammosphere formation. Taken together, these results demonstrate an effective method for sustained delivery of a potent stem cell inhibitor, SAL, and its potential for the treatment of TNBC. Citation Format: Surender M. Kharbanda, Anees Mohammad, Bhawana Gupta, Sireesh Appajosyula, Sandeep Laumas, Jugnu Jain, Donald Kufe, Harpal Singh. A Novel QUATRAMER sustained injectable suspension for the intracellular delivery of Salinomycin, a stem cell inhibitor (HSB-1216), for the treatment of triple negative 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 492.

Abstract 3866: Investigating tumor infiltrating immune cells signature in obese triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive breast cancers and TNBC patients are pathologically negative for estrogen receptor (ER−), progesterone receptor (PR−), and human epidermal growth factor receptor 2 (HER2−) amplification, which impedes the use of targeted therapies used in other breast cancer subtypes. Obesity is a chronic state of inflammation and is associated with increased secretion of pro-inflammatory cytokines, increased infiltration of immune cells and the development of a microenvironment that supports tumor growth. Several epidemiological studies support the association of obesity with TNBC; however, the underlining molecular mechanisms remain unknown. We developed an immunocompetent obese FVB (female) mouse model by feeding ‘Western diet' or control diet for four months and then injecting with syngeneic C0321 (mouse TNBC) cells to investigate the immune signatures and to study the role of obesity related factors in TNBC progression. We found significant body weight increases in ‘Western diet' fed mice group as expected. The percentage of CD8+ and CD4+ T cells, and macrophages in spleen, liver and peripheral blood in obese mice did not change significantly compared to lean mice. However, the percentages of immunosuppressive Myeloid Derived Suppressor cells (MDSCs), particularly Monocytic-MDSCs were elevated in liver and visceral fat tissue in obese mice. Importantly, tumors in ‘Western Diet' fed mice achieved significantly larger volumes. We found no noticeable difference in the percentage of CD8+ and CD4+ T cells, MDSC or macrophages in the spleen of tumor bearing obese mice. Interestingly, there was a decreasing trend in tumor infiltrating CD8+ and CD4+ T cells in tumor bearing obese mice. Further, tumor infiltrating myeloid cells, MDSCs and macrophages were significantly increased in obese mice, suggesting the formation of an immunosuppressive environment. More importantly, tumor infiltrating MDSCs from obese mice were more immunosuppressive than those from lean mice. In summary, our results reveal alterations in the number and function of tumor infiltrating immune cells in obese mice. RNA-Sequencing of lean vs obese tumors revealed significant changes in pathways related to Hypertrophic and Dilated Cardiomyopathy, Adrenergic signaling in Cardiomyocytes, as well as signaling pathways of Insulin, cGMP-PKG, Glucagon, Calcium and Adipocytokine and others. Our data warrants further investigation on the immunosuppressive tumor microenvironment in obese TNBC patients. Citation Format: Fokhrul Hossain, Deniz A Ucar, Maria Sanchez-Pino, Matthew Dean, Samarpan Majumder, Dorota Wyczechowska, Giulia Monticone, Rachel Sabol, Keli Xu, Luis D Valle, Jovanny Zabaleta, Bruce Bunnell, Lucio Miele. Investigating tumor infiltrating immune cells signature in obese triple negative 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 3866.

Extracts of Cordyceps sinensis inhibit breast cancer growth through promoting M1 macrophage polarization via NF-κB pathway activation

Ethnopharmacological relevanceCordyceps sinensis is a traditional Chinese medicine. It is widely reported that Cordyceps sinensis has inhibitory effect on tumor growth and immunoregulation effect on macrophages. However, the exact mechanism of Cordyceps sinensis on macrophage polarization in tumor progression is not known. Aim of studyWe aimed to investigate the role of extracts of Cordyceps sinensis on macrophage polarization and its underlying mechanism in antitumor activity. Materials and methodsThe 4T1 orthotopic xenograft mouse model and immunohistochemical staining were used to investigate the effect of Cordyceps sinensis on breast tumor and the change of the macrophages phenotype in the tumor, respectively. A 3D co-culture assay was used to confirm the activity in vitro. Measurement of cytokines and NO, quantitative real-time PCR and flow cytometry assays were used to investigate the effect of Cordyceps sinensis on the macrophage polarization in vitro. The mechanism of the effect of Cordyceps sinensis on macrophages was investigated by using western blot assays. ResultsIn the orthotopic mouse tumor model, Cordyceps sinensis inhibited the 4T1 tumor growth in a dose dependent manner, and the immunohistochemical staining analysis showed that there is a positive correlation between tumor growth inhibition and macrophage M1-like polarized phenotype. The cytokines and NO measurement, quantitative real-time PCR assay and flow cytometry assays confirmed that Cordyceps sinensis could promote macrophage differentiation toward the M1 phenotype. The 3D co-culture assay and western blot assay showed that Cordyceps sinensis could inhibit tumor growth by promoting macrophage polarization and enhance its activity by activating the NF-κB signaling pathway. ConclusionThese findings suggest that Cordyceps sinensis could potently suppress TNBC progression by promoting M1 phenotypic differentiation of macrophages via activation NF-κB signaling pathway in tumor microenvironment.

Transcriptome network analysis identifies co-expressed CXCL13, CXCR5, and associated genes contributes to the aggressive phenotype of TNBC

Abstract Triple negative breast cancer (TNBC) is an aggressive disease associated with poor prognosis. Currently, there are no targeted therapies for this disease. Standard therapy for TNBC consists of a combination of chemotherapeutic drugs. However, patients often become resistant to this intervention and ultimately succumb to this disease. Hence, understanding the cellular and molecular mechanisms associated with TNBC progression is essential to develop more effective therapies. We hypothesize co-expressed CXCL13, CXCR5, and associated genes are significantly correlated with TNBC and poor overall survival. A bioinformatic approach was used to characterize this new potential drug target for TNBC. Our patient cohort was obtained from The Cancer Genome Atlas. Weighted gene network co-expression analysis identified gene networks associated with factors influencing BrCa prognosis. An ANOVA test identified genes differentially expressed among Luminal A, Luminal B, HER-2, and TNBC tumor groups. Finally, gene ontology analysis was performed via GO-ELITE and EPIC was used to estimate the proportion of various cell types found in bulk samples. We identified seven unique gene networks that were significantly and positively correlated with TNBC. The turquoise module (M1), contained CXCL13, CXCR5, and associated genes that drive tertiary lymphoid structure formation, regulation of immune response, and immune system processes. The M1 module was also significantly and positively correlated with TNBC, but not overall survival. The genes signatures contained in the M1 module were significantly correlated to CD8+ T cell, macrophage, B cell, CD4+ T cell, cancer-associated fibroblast, and NK cell, as determined by EPIC analysis.

Kynurenine 3-monooxygenase upregulates pluripotent genes through β-catenin and promotes triple-negative breast cancer progression.

BackgroundTriple-negative breast cancer (TNBC) is aggressive and has a poor prognosis. Kynurenine 3-monooxygenase (KMO), a crucial kynurenine metabolic enzyme, is involved in inflammation, immune response and tumorigenesis. We aimed to study the role of KMO in TNBC.MethodsKMO alteration and expression data from public databases were analyzed. KMO expression levels in TNBC samples were analyzed using immunohistochemistry. Knockdown of KMO in TNBC cells was achieved by RNAi and CRISPR/Cas9. KMO functions were examined by MTT, colony-forming, transwell migration/invasion, and mammosphere assays. The molecular events were analyzed by cDNA microarrays, Western blot, quantitative real-time PCR and luciferase reporter assays. Tumor growth and metastasis were detected by orthotopic xenograft and tail vein metastasis mouse models, respectively.FindingsKMO was amplified and associated with worse survival in breast cancer patients. KMO expression levels were higher in TNBC tumors compared to adjacent normal mammary tissues. In vitro ectopic KMO expression increased cell growth, colony and mammosphere formation, migration, invasion as well as mesenchymal marker expression levels in TNBC cells. In addition, KMO increased pluripotent gene expression levels and promoter activities in vitro. Mechanistically, KMO was associated with β-catenin and prevented β-catenin degradation, thereby enhancing the transcription of pluripotent genes. KMO knockdown suppressed tumor growth and the expression levels of β-catenin, CD44 and Nanog. Furthermore, mutant KMO (known with suppressed enzymatic activity) could still promote TNBC cell migration/invasion. Importantly, mice bearing CRISPR KMO-knockdown TNBC tumors showed decreased lung metastasis and prolonged survival.InterpretationKMO regulates pluripotent genes via β-catenin and plays an oncogenic role in TNBC progression.

Long non-coding RNA HUMT hypomethylation promotes lymphangiogenesis and metastasis via activating FOXK1 transcription in triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer with highly invasive ability and metastatic nature to the lymph nodes. Long non-coding RNAs (lncRNAs) have been widely explored in cancer tumorigenesis and progression. However, their roles in TNBC lymph node metastasis remains rarely studied.MethodsThe expression of lncRNA highly upregulated in metastatic TNBC (HUMT) in cell lines and tissues was detected by quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). RNA immunoprecipitation (RIP) and RNA pulldown were used to verify the interaction between lncRNA and protein. Chromatin immunoprecipitation (CHIP) and dCas9-gRNA-guided chromatin immunoprecipitation (dCas9-CHIP) were conducted to identify the specific binding site of HUMT-YBX1 complex. Western blot was used to detect the downstream of HUMT.ResultsHUMT was significantly upregulated in lymph node invasive cells and predicted poorer clinical prognosis. Functional study indicated that HUMT promoted lymphangiogenesis and lymph node metastasis. Bioinformatic analysis and qRT-PCR showed that the high expression of HUMT was correlated with the hypomethylation status of its promoter region. Further, HUMT recruited Y-box binding protein 1 (YBX1) to form a novel transcription complex and activated the expression of forkhead box k1 (FOXK1), thus enhancing the expression of vascular endothelial growth factor C (VEGFC). The therapeutic value was further validated in patient-derived xenograft (PDX) models, and a combined marker panel exhibited a better prognostic value for TNBC in receiver operating characteristic (ROC) analysis.ConclusionsOur study identified a novel TNBC lymph node metastasis-associated lncRNA, which promoted TNBC progression and indicated a novel biomarker and potential therapeutic target for TNBC lymph node metastasis.

Abstract P5-04-19: Sulindac sulfide as a non-immune suppressive gamma secretase modifier to target triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is defined as pathologically negative for estrogen receptor (ER-), progesterone receptor (PR-), and human epidermal growth factor receptor 2 amplification (HER2-). TNBCs are a heterogeneous group of clinically aggressive cancers with high risk of recurrence and metastasis, and current treatment options remain limited. Immunotherapy with checkpoint inhibitors shows promise. However, recent data show that crosstalk between cancer stem cells (CSC) and the immune microenvironment leads to immunotherapy resistance, while myeloid-derived suppressor cells (MDSC) promote CSC survival via Notch signaling. Strong evidence supports the involvement of Notch, a prominent CSC pathway, in TNBC progression. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Notch inhibitors, Including Gamma Secretase Inhibitors (GSIs) are quite effective in preclinical models of TNBC. However the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects. We identified Sulindac Sulfide (SS), the active metabolite of FDA-approved NSAID Sulindac, as a potential candidate to replace GSIs. We confirmed that SS has Gamma Secretase Modifier (GSM) activity, in addition to cyclo-oxygenase (COX) inhibition. SS inhibits Notch1 cleavage in TNBC cells, but not in murine T-cells. SS significantly inhibited mammospheres growth in all human and murine TNBC models we tested: 1) human MDA-MB-231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe (LFng-/-); and 3) Two TNBC patient-derived xenograft models, 2K1 and 4IC. In C0321 tumors in mice, we found that SS had remarkable single-agent anti-tumor activity and virtually eliminated Notch1 expression in tumors without intestinal toxicity. SS caused an increase in intra-tumoral CD11c+ dendritic cells and CD8 cells. SS did not affect the numbers of tumor infiltrating macrophages or myeloid-derived suppressor cells (MDSC). However, SS blocked the immunosuppressive function of bone marrow-derived MDSC. RNA-Sequencing of SS-treated tumors revealed significant reduction of CXCL14, EGR1, HOXC6, MAGI2, NCAM1, APOE, CLU (a Wnt target), DTX4 (an E3-ligase positive regulator of Notch activation), and TGFB3 genes and upregulation of CCL17, EPCAM, FABP4, C4A, LTF, ZBTB16, INADL, and FGFR2 genes. Importantly, SS enhanced the antitumor effect of a-PDL1 immunotherapy in our 0321 TNBC mouse model. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy. Repurposing an FDA-approved, safe agent for the treatment of TNBC may be significantly easier and more cost-effective than developing unproven investigational agents. Citation Format: Fokhrul Hossain, Deniz A Ucar, Samarpan Majumder, Margarite Matossian, Giulia Monticone, Keli Xu, Yong Ran, Lisa Minter, Yaguang Xi, Matthew Burow, Todd Golde, Barbara Osborne, Lucio Miele. Sulindac sulfide as a non-immune suppressive gamma secretase modifier to target triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-19.

Abstract P3-06-16: Synergistic effect of biguanides and fatty acid β-oxidation inhibitor in triple-negative breast cancers

Abstract Triple negative breast cancer (TNBC) is one of non-targetable cancers which are still poorly understood for the driver pathways and therapeutic targets. Over the past decade, cancer metabolism researches have a huge advance. Thus, it has enhanced our understanding on metabolic reprogramming in cancer therapy. We have previously shown that metabolic reprogramming to fatty acid β-oxidation (FAO) is a key energy metabolic pathway in TNBC. Moreover, we reported that FAO regulates c-Src, one of the frequently upregulated oncopathways in TNBC via autophosphorylation of Src at Y419. However, single treatment by FAO inhibitor alone cannot effectively control the tumor progression in TNBC because of metabolic reprogramming and flexibility of tumors. Therefore, proper combination therapies with other metabolic targets are necessary. Recently increasing studies demonstrate that anti-diabetic biguanides have attractive anti-cancer effect in various cancer types including breast cancer (BC). However, its significance as an anti-cancer drug is not well established due to parallel metabolic pathways that support tumor growth. Biguanide like metformin is the most widely administered anti-diabetic agent worldwide. Phenformin, a biguanide derivative similar to metformin, has a greater potency than metformin. Like metformin, phenformin also inhibits mitochondrial electron transport chain (ETC) through complex I inhibition. In addition, biguanides lead to the activation of AMPK, which plays a important role in insulin signaling and energy sensing. Importantly, AMPK is an upstream regulator of FAO pathway because it can phosphorylate ACC to activate FAO. Considering the dependency of TNBC to FAO, we evaluated the therapeutic significance of the combination of biguanides (ETC inhibitors) and FAO inhibitors in TNBC progression and metastasis. And we investigated that targeting both ‘arms’ of the pathway can provide more effective and durable responses in TNBC treatments. Our various in vitro and in vivo studies using TNBC cell lines and PDX models suggest that the combination of both inhibitors can provide better therapeutic benefits in TNBCs. This is a rationale and cost-effective metabolic approach to overcome the currently non-targetable TNBCs. Further study toward the clinical effectiveness of this combination may offer better treatment opportunities for TNBC patients. Citation Format: Jun Hyoung Park, Kwang Hwa Jung, Dongya Jia, Benny A Kaipparettu. Synergistic effect of biguanides and fatty acid β-oxidation inhibitor in triple-negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-16.

Abstract C120: The tumor suppressor role of STXBP6 in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is a subtype of breast cancer (BC) that accounts for approximately 15–20% of all BCs. Additionally, TNBC presents with a poor prognosis mainly due to the lack of effective therapeutic options. For this reason, numerous studies have focused to find the potential molecular targets. Syntaxin binding protein 6, encoded by STXBP6, was initially identified in regulating the formation of the SNARE complex and cytogenesis. It has been reported to be associated with many diseases, such as diabetes, autism and systemic lupus erythematosus. However, there are no studies revealing its biological role and its epigenetic regulation in TNBC. Thus, herein we aim to demonstrate antitumor implications of STXBP6 in TNBC. In this study first, we explored the clinical significance of STXBP6 in cancer. Pan-cancer profiling using TCGA data sets identified aberrant expression of STXBP6 in different cancer types. Importantly, we identified significant down regulation of STXBP6 in TNBC samples. Moreover, significantly improved survival rates were observed in patients when STXBP6 expression levels high. The concurrent analysis between gene expression and methylation of STXBP6 highlighted a clear negative correlation. When the luminal subtype and TNBC subtype of BC cells were treated with demethylating agent 5-aza-2’-deoxycytidine, we observed an increase in the expression of STXBP6 in TNBC cells only which further support the regulatory role of DNA methylation in the control of STXBP6 expression in TNBC. Next, we elucidated the function of STXBP6 in TNBC cells. Overexpression of STXBP6 in TNBC cells significantly decreased cell proliferation, colony formation and increased apoptosis. In summary, these findings highlight the suppression effect of STXBP6 on TNBC progression and could be a novel prognostic and therapeutic target for TNBC in the future. Key Words: STXBP6, Gene Expression, Methylation, Tumor Suppressor, Triple Negative Breast Cancer. Citation Format: Govinda Lenka, Jingxuan Shan, Sirin Abuaqel, Lotfi Chouchane. The tumor suppressor role of STXBP6 in triple negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C120. doi:10.1158/1535-7163.TARG-19-C120

Integrated Bioinformatics Data Analysis Reveals Prognostic Significance Of SIDT1 In Triple-Negative Breast Cancer.

BackgroundTriple-negative breast cancer (TNBC) is a heterogeneous disease with a worse prognosis. However, current therapies have rarely improved the outcome of patients with TNBC. Here we sought to identify novel biomarkers or targets for TNBC.Materials and methodsPatients GSE76275 clinic traits and their corresponding mRNA profiles for 198 TNBC and 67 non-TNBC were obtained from the GEO database. Weighted gene co-expression network analysis (WGCNA) of the GSE76275 keyed out hub genes, and the differentially expressed genes (DEGs) were identified with the cut-off of adjusted P (adj. P) <0.01 and |log2 fold-change (FC)| > 1.5. The hub - DEGs overlapping genes, as key genes, were considered for further study using Kaplan-Meier plotter online analysis. Subsequently, Breast Cancer Gene-Expression Miner v4.0 and tissue microarray analysis were applied to determine the transcriptional and translational levels of every key gene. Following plasmid transfection for overexpression, the proliferation of TNBC cells was determined by CCK8 and colony formation assay. Moreover, xenograft tumor models were canvassed to investigate their effect upon in vivo tumor growth.ResultsFour genes (SIDT1, ANKRD30A, GPR160, and CA12) were found to be associated with relapse-free survival (RFS) in TNBC through WGCNA and DEGs integrated analysis. Patients with a higher level of SIDT1 had significantly better RFS compared to those with lower levels. The transcriptional and translational levels of SIDT1 were validated as downregulated in patients with triple-negative status, negative estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Furthermore, SIDT1 inhibited proliferation of breast cancer cells (MDA-MB-231 and MDA-MB-468) and xenograft studies demonstrated that SIDT1 can suppress tumor growth in vivo.ConclusionThis study suggests that SIDT1 may play a crucial role in TNBC progression and has the potential as a prognostic biomarker of TNBC.