Triple-negative Breast Cancer Tumor Tissues Research Articles

Molecular and Immune Correlates of PDCD1 (PD-1), PD-L1 (CD274), and PD-L2 (PDCD1LG2) DNA Methylation in Triple Negative Breast Cancer.

Immune checkpoints are important targets in oncological therapy. Recent studies have proven efficacy of immune checkpoint inhibition (ICI) in treatment of triple negative breast cancer (TNBC). However, only a proportion of TNBC-patients benefit from ICI. Thus, current scientific efforts in this context are focused on the identification of a robust biomarker that enables patient stratification. In the present study, we investigated the epigenetic regulation of PD-1 (PDCD1), PD-L1 (CD274), and PD-L2 (PDCD1LG2). Methylation data of PD-1, PD-L1, and PD-L2, and complex immunogenomic data were obtained from The Cancer Genome Atlas (TCGA). Methylation were systematically analyzed with regard to the transcriptional activity of the studied immune checkpoint genes and the tumor microenvironment. We found differential methylation of PD-1, PD-L1, and PD-L2 in normal adjacent tissue and TNBC tumor tissue. In the TNBC-TCGA cohort, methylation status of PD-1, PD-L1, and PD-L2 were significantly correlated with mRNA levels indicating a strong epigenetic regulation of the transcriptional activity. Moreover, PD-1, PD-L1, and PD-L2 methylation status was strongly associated with a distinct immune cell infiltration pattern. Our results indicate an epigenetic regulation of immune checkpoint genes through DNA methylation in TNBC. In addition, the methylation status was associated with a distinct composition of the tumor microenvironment. Overall, this provides a strong rationale for assessing the value of PD-1, PD-L1, and PD-L2 DNA methylation to predict response to ICI and immunogenicity in TNBC.

The lncRNA ADAMTS9-AS2 Regulates RPL22 to Modulate TNBC Progression via Controlling the TGF-β Signaling Pathway

BackgroundLong non-coding RNAs (lncRNAs) are key regulators of triple-negative breast cancer (TNBC) progression, but further work is needed to fully understand the functional relevance of these non-coding RNAs in this cancer type. Herein, we explored the functional role of the lncRNA ADAMTS9-AS2 in TNBC.MethodsNext-generation sequencing was conducted to compare the expression of different lncRNAs in TNBC tumor and paracancerous tissues, after which ADAMTS9-AS2differential expression in these tumor tissues was evaluated via qPCR. The functional role of this lncRNA was assessed by overexpressing it in vitro and in vivo. FISH and PCR were used to assess the localization of ADAMTS9-AS2within cells. Downstream targets of ADAMTS9-AS2 signaling were identified via RNA pulldown assays and transcriptomic sequencing.ResultsThe expression ofADAMTS9-AS2 was decreased in TNBC tumor samples (P < 0.05), with such downregulation being correlated with TNM stage, age, and tumor size. Overexpressing ADAMTS9-AS2 promoted the apoptotic death and cell cycle arrest of tumor cells in vitro and inhibited tumor growth in vivo. From a mechanistic perspective, ADAMTS9-AS2 was found to control the expression of RPL22 and to thereby modulate TGF-β signaling to control TNBC progression.ConclusionADAMTS9-AS2 controls the expression of RPL22 and thereby regulates TNBC malignancy via the TGF-β signaling pathway.

Prognostic values of spindle checkpoint protein BUB1B in triple negative breast cancer

Objective: To identify important prognostic molecular markers of triple negative breast cancer (TNBC) using high throughput sequencing technology and to explore the correlation of spindle checkpoint protein BUB1B and clinicopathological features with patients' prognosis. Methods: The clinicopathological data and prognostic information of TNBC diagnosed at the West China Hospital of Sichuan University from 2009 to 2017 were collected. Forty-seven fresh tumor samples and 139 formalin fixed paraffin-embedded samples were selected. The fresh tumor samples were subject to RNA sequencing (RNA-seq). The enrichment analysis and protein-protein interaction (PPI) analysis were performed after intersection of difference analysis between RNAseq and GEO (Gene Expression Omnibus) datasets GSE38959 and GSE65194. Kaplan-Meier plotter database was used to analyze the relationship between expression of BUB1B and prognosis. Immunohistochemical staining was used to verify its expression in TNBC and correlation with clinicopathological features and prognosis. Results: Using edgeR to perform differential expression analysis between 47 TNBC tumor tissues and 12 normal tissues, 1 559 up-regulated genes and 1 376 down-regulated genes were identified, while only 131 differentially expressed genes were overlapping with those in GSE38959 and GSE65194. Enrichment analysis was mainly enriched in cell cycle, JAK-STAT signaling pathway and p53 signaling pathway. The top 10 genes ranked by degree of association were TOP2A, BUB1B, MKI67, PLK1, RRM2, PCNA, KPNA2, SMC4, PBK and IGF1. Kaplan-Meier plotter database analysis showed that the expression of BUB1B was significantly correlated with the prognosis of TNBC [overall survival, hazard ratio (HR)=0.52, 95%CI (0.35-0.77), P=0.001; distant metastasis-free, HR=0.72, 95%CI (0.52-0.98), P=0.038]. The immunohistochemical analyses of 139 formalin fixed paraffin-embedded samples showed that the low expression of BUB1B was correlated with poor prognosis in TNBC [HR=0.41, 95%CI (0.18-0.95), P=0.024]. Conclusions: The low expression of BUB1B protein is associated with poor prognosis in TNBC patients, and the molecular mechanism related with prognosis and potential therapeutic targets need to be further studied.

A triple combination gemcitabine + romidepsin + cisplatin to effectively control triple-negative breast cancer tumor development, recurrence, and metastasis.

Triple-negative breast cancer (TNBC) is an aggressive, lethal, heterogeneous type of breast cancer (BC). TNBC tends to have a lower response rate to chemotherapy and a lower 5-year survival rate than other types of BC due to recurrence and metastasis. Our previous study revealed that a combination of gemcitabine, romidepsin, and cisplatin was efficacious in controlling TNBC tumor development. In this study, we extended our investigation of gemcitabine + romidepsin + cisplatin in controlling TNBC tumor recurrence and metastasis. We investigated the ability of gemcitabine + romidepsin + cisplatin to control cell survival and invasiveness using cell viability, soft agar colony formation, and transwell invasion assays. We determined the efficacy of gemcitabine + romidepsin + cisplatin in controlling tumor recurrence and metastasis using cell-derived xenograft animal models. We used immunoblotting to study signaling modulators regulated by gemcitabine + romidepsin + cisplatin in TNBC cells and tumor tissues. Treatment with gemcitabine + romidepsin + cisplatin reduced the TNBC MDA-MB231 and MDA-MB468 cell survival to ~ 50% and ~ 15%, as well as invasiveness to ~ 31% and ~ 13%, respectively. Gemcitabine + romidepsin + cisplatin suppressed modulators involved in epithelial-mesenchymal transition in an ROS-dependent manner. Controlling tumor recurrence, the Gem plus Rom + Cis regimen (~ 112%) was more efficacious than the Gem plus Cis regimen (~ 21%) in tumor growth inhibition. The Gem plus Rom + Cis regimen efficaciously reduced the development of metastatic nodules to 20% in animals. The gemcitabine plus romidepsin + cisplatin regimen was highly efficacious in controlling TNBC tumor development, recurrence, and metastasis in animals. The combination regimen should be poised for efficient translation into clinical trials for controlling the recurrence and metastasis, ultimately contributing to reducing mortality and improving TNBC patients' quality of life.

Extracellular circular RNAs to promote the growth and metastasis of triple-negative breast cancer through remodeling the tumor immune microenvironment.

e13047 Background: Triple-negative breast cancer (TNBC) has been confirmed to have a high degree of heterogeneity of breast cancer, which is more common in young women, with a high degree of malignancy, lack of effective clinical treatment and poor prognosis. Recently, many studies have also found that immune microenvironment (IME) can remarkablely affect the occurrence and metastasis of TNBC through exosome-mediated molecular transmission. Therefore, there is an urgent need to find new immune-related molecules as therapeutic targets and to predict the progress of TNBC and prognosis of treatment. Methods: Using RNA-sequencing and bioinformatics analysis, we found the differential-expressed circular RNAs (circRNAs) in exosomes from TNBC cell lines (BT549 and MDA-MB-231), non-TNBC cell lines (MCF-7 and MCF-10A) and tissues from breast cancer patients. Then, we used CIBERSORT and ESTIMATE algorithm to explore the tumor-infiltrating immune cells (TICs) or stromal components in IME, and identified the significant differential-expressed mRNAs in the immune cells between TNBC and non-TNBC samples. RT-qPCR was used to confirm the expression level of selected circRNAs and mRNAs in TNBC cell lines. Results: We profiled the circRNAs in the exosomes from TNBC cell lines and breast cancer patients by RNA sequencing and detected 27 significantly differentially-expressed circRNAs. After bioinformatic analysis and RT-qPCR, circRELB and circANXA1 were chose to further study. TICs and IME scores were found to associated with the survival and clinicopathological characteristics of TNBC patients and IME remodeling. Interestingly, 92 differentially-expressed genes were found to be enriched in the NF-kB signaling pathway and mTOR signaling pathway. Moreover, we identified 33 target genes of circRELB and circANXA1 by cross-analysis. Based on the predicted miRNAs and the corresponding mRNAs and circRNAs, we have established an IME-related circRNA-miRNA-mRNA network. Then, Kaplan-Meier plots showed the expression levels of certain targeted genes (PTEN, CCND1, IL6, CDKN1A, AKT1, TNF and WNT1) were connected to prognosis in TNBC metastasis patients. Conclusions: The expression levels of circRELB and circANXA1 was found significantly upregulated in exosomes from TNBC cell lines and tumor tissues from breast cancer patients. In addition, circRELB and circANXA1could affect the expression of downstream target genes by competing for endogenous RNA (ceRNAs), and ultimately promote the metastasis of TNBC and remodeling of IME. In summary, this study analyzes the mechanisms of exosome-contained circRNAs in TNBC and these targets from the IME-related circRNA-miRNA-mRNA network may be new potential prognostic biomarkers and immune treatment strategies to prevent the development and metastasis of TNBC.

Effect of N6-Methyladenosine Regulators on Progression and Prognosis of Triple-Negative Breast Cancer.

Background: The N6-methyladenosine (m6A) modification plays a critical role in cancer development. Little is known about the m6A modification in triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer. Thus, the prognostic value of m6A RNA methylation in TNBC deserves exploration.Methods: The expression levels of the 13 m6A methylation regulators were compared between the 98 TNBC tumor samples and normal tissue samples based on the transcriptome profiles from The Cancer Genome Atlas (TCGA). The association between the m6A regulators and patients' overall survival was assessed by Kaplan-Meier survival analysis and Cox regression analysis. Lasso regression analysis was conducted to construct a prognostic model based on the m6A methylation system. The prognostic performance of the identified model was validated in GSE88847 and GSE135565 datasets. A nomogram combining the TNM stage and the m6A prognostic model was further constructed for the survival prediction of TNBC patients.Results: The m6A regulator genes were remarkably dysregulated in TNBC tumor tissues, with ALKBH5, YTHDF2, HNRNPC, KIAA1429, and RBM15 significantly up-regulated and FTO, YTHDC1, YTHDC2, METTL3, METTL14, and ZC3H13 significantly down-regulated (P < 0.01). The expression level of ALKBH5 was an independent unfavorable prognostic factor (HR = 3.327, P = 0.006), while METTL14 (HR = 0.425, P = 0.009) was an independent favorable prognostic factor for TNBC patients. A prognostic model consisting of ALKBH5 and METTL14 was therefore proposed displaying higher accuracy of risk prediction when combined with TNM stage with an AUC of 0.791. The prognostic value of the identified signature remained consistent within the two external validation datasets.Conclusion: The m6A methylation regulators were significantly dysregulated in TNBC tissues and could constitute a novel prognostic signature for the survival prediction of TNBC patients.

Abstract PO-095: Comparative analysis of breast tumor microbiome in Black non-Hispanic (BNH) and White non-Hispanic (WNH) women

Abstract Studies have demonstrated that environmental, host genetic, and socioeconomic factors influence the breast cancer prevalence landscape with a far-reaching influence on racial disparity to subtypes of breast cancer. To understand whether breast tissue harbors race-specific microbiota, we performed 16S rRNA gene-based sequencing of retrospective tumor and matched normal tissue adjacent to tumor (NAT) samples collected from Black non-Hispanic (BNH) and White non-Hispanic (WNH) women. Analysis of Triple Negative Breast cancer (TNBC) and Triple Positive Breast Cancer (TPBC) tissue for microbiota composition revealed significant differences in relative abundance of specific taxa at both phylum and genus levels between WNH and BNH women cohorts. Our main findings are that microbial diversity as measured by Shannon index was significantly lower in BNH TNBC tumor tissue as compared to matched NAT zone. In contrast, the WNH cohort had an inverse pattern for the Shannon index, when TNBC tumor tissue was compared to the matched NAT. Unweighted Principle Coordinate Analysis (PCoA) revealed a distinct clustering of tumor and NAT microbiota in both BNH and WNH cohorts. Citation Format: Jamboor K. Vishwanatha, Srikantha Thyagarajan, Michael Allen, Yan Zhang, Nicole Phillips, Pankaj Chaudhary. Comparative analysis of breast tumor microbiome in Black non-Hispanic (BNH) and White non-Hispanic (WNH) women [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-095.

Blocking Y-Box Binding Protein-1 through Simultaneous Targeting of PI3K and MAPK in Triple Negative Breast Cancers.

Simple SummaryTriple-negative breast cancer (TNBC) is associated with the high rates of relapse and metastasis and poor survival. YB-1 is overexpressed in TNBC tumor tissues. In the present study, we demonstrated that S102 phosphorylation of YB-1 in TNBC cell lines depend on the mutation status of the components of the MAPK/ERK and PI3K/Akt pathways. Simultaneous targeting of MEK and PI3K was found to be the most effective approach to block YB-1 phosphorylation and to inhibit YB-1 dependent cell proliferation. YBX1 knockout was sufficient to block TNBC tumor growth.The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described cancer hallmarks including cell proliferation and survival. The MAPK/ERK and PI3K/Akt pathways are also the major pathways involved in cell growth, proliferation, and survival, and are the frequently hyperactivated pathways in human cancers. A gain of function mutation in KRAS mainly leads to the constitutive activation of the MAPK pathway, while the activation of the PI3K/Akt pathway occurs either through the loss of PTEN or a gain of function mutation of the catalytic subunit alpha of PI3K (PIK3CA). In this study, we investigated the underlying signaling pathway involved in YB-1 phosphorylation at serine 102 (S102) in KRAS(G13D)-mutated triple-negative breast cancer (TNBC) MDA-MB-231 cells versus PIK3CA(H1047R)/PTEN(E307K) mutated TNBC MDA-MB-453 cells. Our data demonstrate that S102 phosphorylation of YB-1 in KRAS-mutated cells is mainly dependent on the MAPK/ERK pathway, while in PIK3CA/PTEN-mutated cells, YB-1 S102 phosphorylation is entirely dependent on the PI3K/Akt pathway. Independent of the individual dominant pathway regulating YB-1 phosphorylation, dual targeting of MEK and PI3K efficiently inhibited YB-1 phosphorylation and blocked cell proliferation. This represents functional crosstalk between the two pathways. Our data obtained from the experiments, applying pharmacological inhibitors and genetic approaches, shows that YB-1 is a key player in cell proliferation, clonogenic activity, and tumor growth of TNBC cells through the MAPK and PI3K pathways. Therefore, dual inhibition of these two pathways or single targeting of YB-1 may be an effective strategy to treat TNBC.

RETRACTED: TNFAIP8 Promotes Cisplatin Chemoresistance in Triple-Negative Breast Cancer by Repressing p53-Mediated miR-205-5p Expression

Tumor necrosis factor alpha-induced protein 8 (TNFAIP8) is implicated in the tumor progression and prognosis of triple-negative breast cancer (TNBC), but the detailed regulatory mechanism of TNFAIP8 in cisplatin tolerance in TNBC has not yet been investigated. TNFAIP8 was evidently upregulated in TNBC tumor tissues and cell lines. Knocking down TNFAIP8 led to impaired proliferation and elevated apoptosis of TNBC cells upon cisplatin (DDP) treatment. Mechanistic studies revealed that TNFAIP8 repressed the expression of p53 and p53-promoted microRNA (miR)-205-5p; moreover, miR-205-5p targeted multiple genes required for the cell cycle and repressed Akt phosphorylation, which thus inhibited the proliferation of TNBC cells. In addition, silencing of TNFAIP8 led to the upregulation of miR-205-5p and the restraint of the TRAF2-NF-κB pathway, which thus enhanced the suppressive effects of DDP on tumor growth in nude mice. This study revealed that TNFAIP8 was essential in the DDP tolerance formation of TNBC cells by reducing p53-promoted miR-205-5p expression. Thus, targeting TNFAIP8 might become a promising strategy to suppress TNBC progression.

MiR-452 Reverses Abnormal Glycosylation Modification of ERα and Estrogen Resistance in TNBC (Triple-Negative Breast Cancer) Through Targeting UGT1A1.

Background: The breast epithelial cells in patients with triple-negative breast cancer (TNBC) actually have specific estrogen receptor (ER) expression, and the abnormal glycosylation of UGT1A1 in TNBC cells resulted in abnormal expression and function of ERα through regulating the modification of ERα. Therefore, our study targets the role of UGT1A1 expression, then glycosylation modification of ERα (estrogen receptor α) and estrogen resistance in development of TNBC.Methods: The differential expression of mRNA and miRNA in TNBC tissues was tested. Luciferase activity was analyzed in TNBC cells treated with miR-452. Moreover, the human mammary gland and TNBC cell lines were dealt with estrogen and miR-452 or its inhibitors, then proliferation ability was further determined. Moreover, the role of interaction between UGT1A1 and ERα in the glycosylation modification of ERα and UGT activity, and metabolism of estrogen were assessed. The effects of miR-452 on TNBC by improving abnormal glycosylation modification of ERα by targeting UGT1A1 and estrogen resistance were studied in vitro and in vivo.Results: The expression level of UGT1A1 in TNBC tumor tissues was higher than its matched para-tumorous tissues, but the miR-452 expression was opposite. The glycosylation modification site of ERα expressed in TNBC cells was different from that of normal mammary epithelial cells. The estrogen 17β-estradiol (E2) significantly promoted mitotic entry of TNBC cells. The interaction between UGT1A1 and ERα affected the expression level of each other, as well as the UGT enzyme activity and proliferation of TNBC cells. UGT1A1 induced production of intracellular estrogens and TNBC proliferation, but it could be reversed by overexpression of ERα. Upregulation of ERα caused the downregulation of UGT1A1 and marked decrease of intracellular estrogen products, and then suppressed TNBC proliferation. Moreover, UGT1A1 was the target gene of miR-452; miR-452 antagomir restrained TNBC xenograft.Conclusion: Our results demonstrated that estrogen was a positive factor in the proliferation of TNBC cells at onset of mitosis through accentuating the expression and enzyme activity of UGT1A1. However, miR-452 targeted to UGT1A1, then regulated glycosylation modification of ERα, estrogen metabolism, and TNBC development associated with estrogen resistance.

Comparative analysis of racial differences in breast tumor microbiome

Studies have demonstrated that environmental, host genetic, and socioeconomic factors influence the breast cancer prevalence landscape with a far-reaching influence on racial disparity to subtypes of breast cancer. To understand whether breast tissue harbors race-specific microbiota, we performed 16S rRNA gene-based sequencing of retrospective tumor and matched normal tissue adjacent to tumor (NAT) samples collected from Black non-Hispanic (BNH) and White non-Hispanic (WNH) women. Analysis of Triple Negative Breast cancer (TNBC) and Triple Positive Breast Cancer (TPBC) tissues for microbiota composition revealed significant differences in relative abundance of specific taxa at both phylum and genus levels between WNH and BNH women cohorts. Our main findings are that microbial diversity as measured by Shannon index was significantly lower in BNH TNBC tumor tissue as compared to matched NAT zone. In contrast, the WNH cohort had an inverse pattern for the Shannon index, when TNBC tumor tissue was compared to the matched NAT. Unweighted Principle Coordinates Analysis (PCoA) revealed a distinct clustering of tumor and NAT microbiota in both BNH and WNH cohorts.

Long Non-Coding RNA MNX1-AS1 Promotes Progression of Triple Negative Breast Cancer by Enhancing Phosphorylation of Stat3

Triple negative breast cancer (TNBC) accounts for less than a quarter of breast cancer but has the poorest survival outcome and is prone to relapse as well as metastasis due to its aggressiveness and lack of therapeutic target. Herein, we analyzed the TCGA datasets of lncRNA expressional profiles of breast cancer vs. normal tissue and TNBC vs. Non-TNBC subtypes and screened a long non-coding RNA (lncRNA) MNX1-AS1 overexpressing in TNBC. We found that MNX1-AS1 were upregulated in TNBC tumor tissues and correlated with poor survival outcome in TNBC patients. Silencing MNX1-AS1 reduced the aggressiveness of TNBC in vitro and in vivo. By using RNA pulldown assay followed by western blotting and RNA immunoprecipitation (RIP), we identified Stat3 was the MNX1-AS1 binding protein and MNX1-AS1 upregulated the phosphorylation of Stat3 by enhancing the interaction between p-JAK and Stat3. The present study suggested that targeting MNX1-AS1 may represent a promising therapeutic strategy to TNBC.

Circular RNA circ-ZEB1 acts as an oncogene in triple negative breast cancer via sponging miR-448.

Circular RNAs (circRNAs) play an important role in tumor development. The miRNA sponge is a common role played by circRNAs in various tumors, including breast cancer. This study aimed to explore the role of circ-ZEB1 in the proliferation and apoptosis of triple negative breast cancer (TNBC) cells. The expressions of several circRNAs which were predicted to be bound with miR-448 were detected in 30 clinical TNBC tumor tissues and paired paracancer tissues. The cell counting kit-8 assay was performed to detect the TNBC cell proliferation. The TNBC cell apoptosis was detected using the TUNEL assay. The binding between circ-ZEB1 and miR-448, as well as between miR-448 and eukaryotic elongation factor 2 kinase (eEF2 K), was detected using the RNA pull-down assay and/or the luciferase reporter assay. The effect of circ-ZEB1 knockdown on TNBC tumor growth was detected using the mouse xenograft model. Compared with normal tissues and breast epithelial cells, the expression of circ-ZEB1 was markedly higher in TNBC tumor tissues and tumor cell lines. The small hairpin RNA-mediated circ-ZEB1 knockdown inhibited TNBC cell proliferation and induced cell apoptosis. The RNA pull-down assay and the luciferase reporter assay confirmed the binding between circ-ZEB1 and miR-448, as well as between miR-448 and eEF2 K. The knockdown of circ-ZEB1 was proven to inhibit TNBC cell proliferation and tumor growth via releasing miR-448, and subsequently reducing the expression of the miR-448 target, eEF2 K. In conclusion, our findings identified a new functional circ-ZEB1 in TNBC tumorigenesis, and revealed the important regulatory role of circ-ZEB1 via sponging miR-448, providing a novel insight for TNBC pathogenesis.

Exosomal circular RNAs derived from serum: Promising biomarkers for therapeutic targets and prognosis of triple-negative breast cancer (TNBC).

3528 Background: Exosomes are well known by the "exosomal shuttle" that delivers oncogenic microRNAs (miRNAs), mRNAs, circular RNAs (circRNAs) and proteins to the recipient cells and tumor microenvironment, and may be used as promising biomarkers for disease diagnosis. This study aims to provide a theoretical basis to use stable exosomal circRNAs as new biomarkers for predicting the development, metastasis and therapeutic targets of TNBC. Methods: A strategy combining RNA-sequencing technique, bioinformatic analysis and RT-qPCR was used to determine the level of differential expressed circRNAs in serum exosomes samples (n = 43) from TNBC patients compared with non-TNBC patients. The expression of circHSDL2 were also detected in tumor tissues (n = 20) from TNBC patients and breast cancer cell lines by qRT-PCR. Cell cycle analysis, the wound healing assays and transwell assays were used to investigate the function of circHSDL2 in proliferation, invasion and metastasis of TNBC cells. FISH, dual-luciferase reporter and functional assays were performed to confirm the interaction between circHSDL2 and let-7a-2-3p in TNBC cells. Results: We profiled the circRNAs in the serum exosomes samples from TNBC patients and non-TNBC patients by RNA sequencing and detected 803 significantly differentially-expressed circRNAs. After bioinformatic analysis, circHSDL2 was chose to further study. RT-qPCR results showed that higher expression of circHSDL2 in TNBC cell lines and tumor tissues from TNBC patients. Moreover, overexpression of circHSDL2 promoted TNBC cells proliferation and invasion, while knockdown of circHSDL2 inhibited TNBC cells proliferation and invasion. Mechanistically, circHSDL2 acted as a "miRNAs sponge" to absorb let-7a-2-3p; let-7a-2-3p inhibited TNBC cell invasion and metastasis. Kaplan-Meier plots showed lower expression of let-7a-2-3p was connected to poor prognosis in TNBC metastasis patients from TCGA database. Conclusions: The expression of circHSDL2 was found significantly upregulated in serum exosomes and tumor tissues from TNBC patients. Moreover, circHSDL2 could promote cell proliferation, invasion and metastasis in TNBC cells. CircHSDL2 might be function as competing endogenous RNAs (ceRNAs) by targeting let-7a-2-3p in the progression of TNBC. Therefore, this study provides a fresh perspective on novel therapeutic targets and potential biomarkers for TNBC from exosomal circRNAs.

Expression and significance of Nek2B and β-catenin in triple negative breast cancer

Objective: To investigate the expression and significance of Nek2B and β-catenin expression in triple negative breast cancer (TNBC) at molecule levels. Methods: By using the methods of bioinformatics [GEO2R online tool, gene ontology (GO) function analysis, KEGG biological pathway enrichment analysis], the differentially expressed genes were screened from TNBC microarray data.Expression levels of Nek2B and β-catenin TNBC cell lines were detected by Western blot and qRT-PCR.From January 1, 2007 to December 31, 2012, eighty cases of TNBC were collected from the Second Hospital of Shanxi Medical University. The expression of Nek2B in TNBC tumor tissue was detected by immunohistochemistry and tissue microarray, and the relationship between Nek2B and clinical pathological characteristics of TNBC was analyzed. Results: Through bioinformatics analysis of the cDNA chip sets of 2 TNBC tumors(GSE38959,GSE27447), 998 differentially expressed genes were obtained in the initial screening, and 13 differentially expressed genes were revealed after intersection. The results of biological pathway analysis showed that the common differential expression genes were closely related to Wnt/β-catenin pathway, among which Nek2 expression showed the greatest difference and was associated with poor prognosis. Expression intensity of Nek2B and repeated β-catenin in the same TNBC cell line was consistent.The results of immunohistochemistry showed that the high expression of Nek2B was related to the high histological stage (G3;84.3% vs.37.9%, P<0.001), lymph node metastasis group (76.7% vs.54.1%, P=0.032), high Ki-67 positive index group (78.6% vs.52.6%, P=0.007) and β-catenin positive expression group (72.5% vs.27.3%, P=0.018). Conclusions: The high level of Nek2B expression is related to a poor prognosis in TNBC patients. In TNBC tissues and cells, the expression of Nek2B is correlated with β-catenin, suggesting that Nek2B may affect the occurrence and development of TNBC by regulating the Wnt/β-catenin patients signaling pathway.

Abstract B26: Comparative analysis of breast tumor microbiome in Black non-Hispanic (BNH) and White non-Hispanic (WNH) women

Abstract African American women are approximately twice as likely to be diagnosed with highly aggressive estrogen receptor-negative (ER-) and TNBC subtypes of breast cancer than Caucasian women. Among biologic factors, microbiota has been implicated in the etiology of several kinds of cancer including breast cancer. Few recent studies have shown that the breast niche-specific microbiota may have a major influence on breast tumorigenesis as shown by a decreased abundance of bacterial species with known immunomodulatory and probiotic effect in paired tumor tissue as compared to normal tissue derived from healthy patients, including different microbiota profiles in different subtypes of breast cancer. To understand whether the breast tissue harbors microbiota profiles that are uniquely race-specific we have analyzed tumor and matched normal and tumor tissue using 16S rRNA targeted sequencing strategy. Two distinct breast tumor types derived from women were included in this study: triple-negative breast cancer (TNBC) and triple-positive breast cancer (TPBC) derived from BNH and WNH women. The data were analyzed for microbiota composition, abundance, and diversity parameters, alpha diversity and beta diversity. The preliminary microbiome analysis revealed specific differences in abundance both at the phylum and genus levels between WNH and BNH women breast tissues; the alpha diversity matric, Shannon index measuring both richness and evenness of microbiota diversity showed differences in microbial diversity between normal breast tumor tissue and the matched normal adjacent to tumor tissue. The microbiota richness was relatively lower in BNH TNBC tumor tissue as compared to its matched normal adjacent to tumor zone as defined by pathologic analysis. In contrast, the microbiota richness was higher in WNH TNBC tumor tissue as compared to its matched normal breast tissue. The multivariate analysis of beta diversity revealed a distinct clustering in BNH and WNH TNBC microbiota between both tumors as compared to normal tissue and BNH as compared to WNH racial groups. Research reported in this publication was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Numbers S21MD012472 and U54MD006882. Note: This abstract was not presented at the conference. Citation Format: Srikantha Thyagarajan, Yan Zhang, Santosh Thapa, Michael S. Allen, Nicole Phillips, Jamboor K. Vishwanatha. Comparative analysis of breast tumor microbiome in Black non-Hispanic (BNH) and White non-Hispanic (WNH) women [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B26.

Abstract P6-03-11: Interleukin-enhancer binding factor 2 expression associates with chemotherapy response in triple-negative breast cancer

Abstract Background: Triple-Negative Breast Cancer (TNBC) is one of the aggressive breast cancer (BC) subtype and the most difficult to treat once metastasis occurs. The lack of hormone receptors prevents the usage of standard targeted therapy; moreover the heterogeneity of the TNBC subtype and the high risk of metastasis worsens the problem. Systemic chemotherapy is one of the best option to treat metastatic TNBC. However, the response to chemotherapy treatment is variable and tumors develop resistance. Enhancing the expression of specific splicing factors which generate gene variants is a mechanism the TNBC tumor cells to promote resistance. Interleukin-enhancer binding factor 2 (ILF2) is an RNA binding protein (RBP) involved in RNA metabolism and tumorigenesis. We hypothesize that the binding of the splicing factor Y-Box Binding Protein 1 (YBX1) to ILF2 generates alternative splicing of genes involved in DNA-Damage Response (DDR), in particular after chemotherapy treatment. Methods and Results: To test our hypothesis, we utilized KMplot software (http://kmplot.com/analysis/). Of clinical importance, Relapsed-Free Survival (RFS) after 5 years was analyzed for BC patients with basal subtype. High ILF2 expression determined poorer prognosis in patients with basal subtype and high ILF2 expression (upper quartile, n=154) compared to rest of the patients (n=464) (Log-rank test p=0.0032, HR= 1.53 (1.15-2.03)); furthermore when considered only patients receiving chemotherapy with high ILF2 expression (n=75) compared to rest of the patients (n=224) the prognosis was worst (Log-rank test p=0.000077, HR= 2.11 (1.44-3.07)). In assessing the potential relation of ILF2 expression and BC subtypes, we reviewed the BC TCGA and METABRIC dataset to assess ILF2 expression. A significant higher expression of ILF2 in primary tumors (n=1041) and metastatic (n=7) BC than normal tissue (n=113) was observed using TCGA; similar results were obtained using METABRIC dataset. Moreover, BC primary tumors with basal-like (n=98) subtype presented the highest ILF2 expression compare to other BC subtypes. Likewise, we demonstrated that enhanced expression of ILF2 is associated with copy number amplification in TNBC. In order to select models of TNBC, we profiled TNBC cell lines using western blot; TNBC cells presented higher levels of ILF2 expression than a normal-like cells. Then, using lentiviral transduction we generated stable clones overexpressing ILF2 in MDA-MB-231 cell lines. By functional assays, we demonstrated that higher ILF2 expression enhanced cell growth rates. To identify proteins interacting with ILF2 in TNBC, we performed immunoprecipitation assays and showed that ILF2 interacts with YBX1 in TNBC cell lines. Moreover, in TNBC patients ILF2 and YBX1 expression positively correlate. To validate our findings, we performed immunohistochemistry analysis for ILF2 in a TMA (100 BC and 10 normal breast tissues) and showed that patients with TNBC subtype displayed significantly enhanced ILF2 expression. Conclusions: ILF2 is highly expressed in TNBC cell lines and tumor tissues. ILF2 interacts with YBX1 in TNBC cells. In vitro models showed that overexpression of ILF2 promotes tumor growth. Clinically, increased ILF2 is associated with poorer prognosis and increased chance to relapse in patients with basal subtypes receiving chemotherapy treatment. Citation Format: Matias A Bustos, Xiaoqing Zhang, Dave SB Hoon. Interleukin-enhancer binding factor 2 expression associates with chemotherapy response in 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 P6-03-11.

CD155 contributes to the mesenchymal phenotype of triple-negative breast cancer.

Patients with triple‐negative breast cancer (TNBC) lack molecular targets and have an unfavorable outcome. CD155 is overexpressed in human cancers, but whether it plays a role in TNBC is unexplored. Here we found that CD155 was enriched in both TNBC cell lines and tumor tissues. High CD155 expression was related to poor prognosis of breast cancer patients. CD155 was associated with a mesenchymal phenotype. CD155 knockdown induced a mesenchymal‐epithelial transition in TNBC cells, and suppressed TNBC cell migration, invasion and metastasis in vitro and in vivo. Mechanistically, CD155 cross‐talked with oncogenic IL‐6/Stat3 and TGF‐β/Smad3 pathways. Moreover, CD155 knockdown inhibited TNBC cell growth and survival. Taken together, these data indicate that CD155 contributes to the aggressive behavior of TNBC; targeting CD155 may be beneficial to these patients.

Identification of Metastasis-Associated Genes in Triple-Negative Breast Cancer Using Weighted Gene Co-expression Network Analysis.

Triple-negative breast cancer (TNBC) is the most aggressive and fatal sub-type of breast cancer. This study aimed to identify metastasis-associated genes that could serve as biomarkers for TNBC diagnosis and prognosis. RNA-seq data and clinical information on TNBC from the Cancer Genome Atlas were used to conduct analyses. Expression data were used to establish co-expression modules using average linkage hierarchical clustering. We used weighted gene co-expression network analysis to explore the associations between gene sets and clinical features and to identify metastasis-associated candidate biomarkers. The K-M plotter website was used to explore the association between the expression of candidate biomarkers and patient survival. In addition, receiver operating characteristic curve analysis was used to illustrate the diagnostic performance of candidate genes. The pale turquoise module was significantly associated with the occurrence of metastasis. In this module, 64 genes were identified, and its functional enrichment analysis revealed that they were mainly associated with transcriptional misregulation in cancer, microRNAs in cancer, and negative regulation of angiogenesis. Further, 4 genes, IGSF10, RUNX1T1, XIST, and TSHZ2, which were negatively associated with relapse-free survival and have seldom been reported before in TNBC, were selected. In addition, the mRNA expression levels of the 4 candidate genes were significantly lower in TNBC tumor tissues compared with healthy tissues. Based on the K-M plotter, these 4 genes were correlated with poor prognosis of TNBC. The area under the curve of IGSF10, RUNX1T1, TSHZ2, and XIST was 0.918, 0.957, 0.977, and 0.749. These findings provide new insight into TNBC metastasis. IGSF10, RUNX1T1, TSHZ2, and XIST could be used as candidate biomarkers for the diagnosis and prognosis of TNBC metastasis.

Association Between Plasma Diacetylspermine and Tumor Spermine Synthase With Outcome in Triple-Negative Breast Cancer.

MYC is an oncogenic driver of development and progression in triple-negative breast cancer (TNBC). Ornithine decarboxylase, the rate-limiting enzyme in polyamine metabolism, is a transcriptional target of MYC. We therefore hypothesized that a plasma polyamine signature may be predictive of TNBC development and progression. Using liquid chromatography mass spectrometry, polyamine levels were determined in plasma samples from newly diagnosed patients with TNBC (n = 87) and cancer-free controls (n = 115). Findings were validated in plasma samples from an independent prospective cohort of 54 TNBC, 55 estrogen receptor negative (ER-) and progesterone receptor negative (PR-) and HER2 positive (HER2+), and 73 ER+ case patients, and 30 cancer-free control subjects. Gene expression data and clinical data for 921 and 2359 breast cancer tumors were obtained from The Cancer Genome Atlas repository and the Oncomine database, respectively. Relationships between plasma diacetylspermine (DAS) and tumor spermine synthase (SMS) mRNA expression with metastasis-free survival and overall survival were determined using Cox proportional hazard models; Fisher exact tests were used to assess risk of distant metastasis in relation to tumor SMS mRNA expression. An increase in plasma DAS, a catabolic product of spermine mediated through SMS, was observed in the TNBC subtype of breast cancer. Plasma levels of DAS in TNBC associated with increased risk of metastasis (plasma DAS value ≥ 1.16, hazard ratio = 3.06, 95% confidence interval [CI] = 1.15 to 8.13, two-sided P = .03). SMS mRNA expression in TNBC tumor tissue was also found to be predictive of poor overall survival (top 25th percentile hazard ratio = 2.06, 95% CI = 1.04 to 4.08, one-sided P = .04) and increased risk of distant metastasis in TNBC (comparison of lowest SMS quartile [reference] to highest SMS quartile relative risk = 1.90, 95% CI = 0.97 to 4.06, one-sided Fisher exact test P=.03). Metabolomic profiling identified plasma DAS as a predictive marker for TNBC progression and metastasis.