Poor Prognosis In Triple-negative Breast Cancer Research Articles

High Caveolin-1 mRNA expression in triple-negative breast cancer is associated with an aggressive tumor microenvironment, chemoresistance, and poor clinical outcome.

Currently, there are few treatment-predictive and prognostic biomarkers in triple-negative breast cancer (TNBC). Caveolin-1 (CAV1) is linked to chemoresistance and several important processes involved in tumor progression and metastasis, such as epithelial-mesenchymal transition (EMT). Herein, we report that high CAV1 gene expression is an independent factor of poor prognosis in TNBC. CAV1 gene expression was compared across different molecular features (e.g., PAM50 subtypes). CAV1 expression was assessed in relation to clinical outcomes using Cox regression adjusted for clinicopathological predictors. Differential gene expression and gene set enrichment analyses were applied to compare high- and low-expressing CAV1 tumors. Tumor microenvironment composition of high- and low-expressing CAV1 tumors was estimated using ECOTYPER. Tumor tissue microarrays were used to evaluate CAV1 protein levels in stromal and malignant cells. In the SCAN-B (n = 525) and GSE31519 (n = 327) cohorts, patients with CAV1-high tumors had an increased incidence of early recurrence adjusted HR 1.78 (95% CI 1.12-2.81) and 2.20 (95% CI 1.39-3.47), respectively. In further analysis, high CAV1 gene expression was associated with a molecular profile indicating altered metabolism, neovascularization, chemoresistance, EMT, suppressed immune response, and active tumor microenvironment. Protein levels of CAV1 in malignant and stromal cells were not correlated with CAV1 gene expression. CAV1 gene expression in TNBC is a biomarker that merits further investigation in clinical trials and as a therapeutic target.

Abstract 549: PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation

Abstract Background: Triple-negative breast cancer (TNBC) is lack of effective targeted therapies. Epidermal growth factor receptor (EGFR) is a potential therapeutic target for TNBC. However, TNBC is resistant to EGFR-targeted drugs in clinical trials, and the tolerance mechanism remains unclear. This study aimed to elucidate the possible resistance mechanism for TNBC to EGFR-targeted therapy and how sensitize TNBC cells to erlotinib. Methods: PDZ domain containing 1 (PDZK1) correlated with TNBC development was screened out by bioinformatics analysis and its correlation with resistance to erlotinib was identified by western blotting (WB). Its interaction with EGFR and structural basis were determined by GST-Pull down and coimmunoprecipitation. Its regulation on EGFR signaling activation and expression level were detected by WB in PDZK1 overexpression and knockdown TNBC cells, xenograft tissues and correlation analyses. Correlation was performed by gene set enrichment analysis (GSEA) and immunohistochemistry of tissue microarray followed by Pearson analysis. TNBC-suppressing effects of PDZK1 and its sensitizing effects on erlotinib were investigated using cell viability assay, colony-forming assay, wound healing assay, Matrigel invasion assay and xenograft model in combination with PDZK1 wild type/mutant transfection and rescue experiment. Results: Here we found that PDZK1 was correlated with TNBC development and its level was downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation was related to erlotinib resistance in TNBC. PDZK1 bound with EGFR. Through the interaction, PDZK1 promoted EGFR degradation by enhancing the binding of EGFR with c-Cbl and inhibited EGFR phosphorylation by hindering EGFR dimerization. PDZK1 was specifically downregulated in TNBC tissues and was correlated with poor prognosis of TNBC. Functional assays in vitro and in vivo showed that PDZK1 suppressed TNBC development. Restoring EGFR expression and kinase inhibitor treatment reversed the malignancy caused by PDZK1 overexpression and knockdown, respectively. PDZK1 wild type, but not mutant overexpression enhanced the inhibitory effect of erlotinib on EGFR signaling and TNBC malignancy in vitro and in vivo. Conclusion: PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for TNBC to reverse the tolerance of TNBC cells to erlotinib. Citation Format: Junfang Zheng, Yuanzhen Ma, Zhiyu Fang, Yijun Qi. PDZK1 sensitizes TNBC cells to erlotinib via promoting c-Cbl-mediated EGFR degradation and inhibiting EGFR phosphorylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 549.

PD-L1-mediated immune evasion in triple-negative breast cancer is linked to the loss of ZNF652

The intrinsic regulation of programmed death ligand-1 (PD-L1) expression remains unclear. Here, we report that zinc-finger protein 652 (ZNF652) is a potent transcription repressor of PD-L1. ZNF652 frequently experiences loss of heterozygosity (LOH) in various cancers. Higher LOH rate and lack of estrogen-inducible transcription lead to suppressed expression of ZNF652 in triple-negative breast cancer (TNBC). Mechanistically, ZNF652 is physically associated with the NuRD transcription co-repressor complex to repress a cohort of genes, including PD-L1. Overexpression of ZNF652 inhibits PD-L1 transcription, whereas depletion of ZNF652 upregulates PD-L1. Loss of ZNF652 in TNBC unleashes PD-L1-mediated immune evasion both invitro and invivo. Significantly, ZNF652 expression is progressively lost during breast cancer progression, and a low ZNF652 level is correlated with elevated PD-L1 expression, less infiltrated CD8+ Tcells, and poor prognosis in TNBC. Our study provides insights into PD-L1 regulation and supports the pursuit of ZNF652 as a potential biomarker and drug target for breast cancer immunotherapy.

Identification of TUBB2A as a Cancer-Immunity Cycle-Related Therapeutic Target in Triple-Negative Breast Cancer.

Triple negative breast cancer (TNBC) is a malignant subtype of breast cancer characterized by the absence of ER, PR, and HER2. We aimed to explore target gene from the perspective of cancer-immunity cycle, providing insights into treatment of TNBC. We obtained TNBC samples from METABRIC database and downloaded 4 datasets from GEO database, as well as an IMvigor210 dataset. WGCNA was applied to screen genes associated with cancer-immunity cycle in TNBC. GO, KEGG and GSEA analyses were performed to explore the target gene's potential functions and pathways. The binding motifs with transcription factors were predicted with FIMO. Immune infiltration analysis was conducted by CIBERSORT. TUBB2A was screened out as our target gene which was negatively correlated with T cell recruitment in cancer-immunity cycle. TUBB2A expressed higher in TNBC samples than in normal samples. High expression of TUBB2A was associated with poor prognosis of TNBC. 12 transcription factors and 5 miRNAs might regulate TUBB2A's expression. The infiltration ratios of 7 types of immune cells such as CD8+ T cells, naive CD4+ T cells and activated memory CD4+ T cells were significantly lower in TUBB2A high expression group. TUBB2A was a potential drug target. We screened a cancer-immunity cycle-related gene TUBB2A which was negatively correlated with T cell recruiting in TNBC. TUBB2A expressed higher in TNBC samples than in normal samples, associated with poor prognosis.

FSIP1 enhances the therapeutic sensitivity to CDK4/6 inhibitors in triple-negative breast cancer patients by activating the Nanog pathway.

CDK4/6 inhibitors are routinely recommended agents for the treatment of advanced HR+HER2- breast cancer. However, their therapeutic effectiveness in triple-negative breast cancer (TNBC) remains controversial. Here, we observed that the expression level of fibrous sheath interacting protein 1 (FSIP1) could predict the treatment response of TNBC to CDK4/6 inhibitors. High FSIP1 expression level was related to a poor prognosis in TNBC, which was associated with the ability of FSIP1 to promote tumor cell proliferation. FSIP1 downregulation led to slowed tumor growth and reduced lung metastasis in TNBC. FSIP1 knockout caused cell cycle arrest at the G0/G1 phase and reduced treatment sensitivity to CDK4/6 inhibitors by inactivating the Nanog/CCND1/CDK4/6 pathway. FSIP1 could form a complex with Nanog, protecting it from ubiquitination and degradation, which may facilitate the rapid cell cycle transition from G0/G1 to S phase and exhibit enhanced sensitivity to CDK4/6 inhibitors. Our findings suggest that TNBC patients with high FSIP1 expression levels may be suitable candidates for CDK4/6 inhibitor treatment.

RUNX1 Is Regulated by Androgen Receptor to Promote Cancer Stem Markers and Chemotherapy Resistance in Triple Negative Breast Cancer.

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype for which no effective targeted therapies are available. Growing evidence suggests that chemotherapy-resistant cancer cells with stem-like properties (CSC) may repopulate the tumor. The androgen receptor (AR) is expressed in up to 50% of TNBCs, and AR inhibition decreases CSC and tumor initiation. Runt-related transcription factor 1 (RUNX1) correlates with poor prognosis in TNBC and is regulated by the AR in prostate cancer. Our group has shown that RUNX1 promotes TNBC cell migration and regulates tumor gene expression. We hypothesized that RUNX1 is regulated by the AR and that both may work together in TNBC CSC to promote disease recurrence following chemotherapy. Chromatin immunoprecipitation sequencing (ChIP-seq) experiments in MDA-MB-453 revealed AR binding to RUNX1 regulatory regions. RUNX1 expression is upregulated by dihydrotestosterone (DHT) in MDA-MB-453 and in an AR+-TNBC HCI-009 patient-derived xenograft (PDX) tumors (p < 0.05). RUNX1 is increased in a CSC-like experimental model in MDA-MB-453 and SUM-159PT cells (p < 0.05). Inhibition of RUNX1 transcriptional activity reduced the expression of CSC markers. Interestingly, RUNX1 inhibition reduced cell viability and enhanced paclitaxel and enzalutamide sensitivity. Targeting RUNX1 may be an attractive strategy to potentiate the anti-tumor effects of AR inhibition, specifically in the slow-growing CSC-like populations that resist chemotherapy which lead to metastatic disease.

Inhibition of IκB Kinase Is a Potential Therapeutic Strategy to Circumvent Resistance to Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) remains as an intractable malignancy with limited therapeutic targets. High expression of epidermal growth factor receptor (EGFR) has been associated with a poor prognosis of TNBC; however, EGFR targeting has failed with unfavorable clinical outcomes. Here, we performed a combinatorial screening of fifty-five protein kinase inhibitors with the EGFR inhibitor gefitinib in the TNBC cell line MDA-MB-231 and identified the IκB kinase (IKK) inhibitor IKK16 as a sensitizer of gefitinib. Cell viability and clonogenic survival assays were performed to evaluate the antiproliferative effects of the gefitinib and IKK16 (Gefitinib + IKK16) combination in TNBC cell lines. Western blot analyses were also performed to reveal the potential mode of action of this combination. In addition, next-generation sequencing (NGS) analysis was performed in Gefitinib+IKK16-treated cells. The Gefitinib+IKK16 treatment synergistically reduced cell viability and colony formation of TNBC cell lines such as HS578T, MDA-MB-231, and MDA-MB-468. This combination downregulated p-STAT3, p-AKT, p-mTOR, p-GSK3β, and p-RPS6. In addition, p-NF-κB and the total NF-κB were also regulated by this combination. Furthermore, NGS analysis revealed that NF-κB/RELA targets including CCL2, CXCL8, EDN1, IL-1β, IL-6, and SERPINE1 were further reduced and several potential tumor suppressors, such as FABP3, FADS2, FDFT1, SEMA6A, and PCK2, were synergistically induced by the Gefitinib-+IKK16 treatment. Taken together, we identified the IKK/NF-κB pathway as a potential target in combination of EGFR inhibition for treating TNBC.

Evaluation of CD44+/CD24- and Aldehyde Dehydrogenase Enzyme Markers in Cancer Stem Cells as Prognostic Indicators for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has been extensively studied not just for its aggressive behavior but also to understand its complex molecular nature. This type of heterogeneous tumor shows no expression of estrogen receptor (ER) or progesterone receptor (PR) and does not express the HER2 gene, and often these tumors are high grade with distinct histological groups. The basal-like subtype is most commonly related to the TNBC type of neoplasms; it can be further classified according to Lehmann and Burstein expert’s criteria. TNBC is related to breast stem cell markers such as CD44+/CD24- and high levels of enzyme aldehyde dehydrogenase (ALDH), which have been shown to possess stem cell features that are involved in differentiation, vascular invasion, tumorigenesis, and metastatic potential. CD44+/CD24- and high levels of ALDH have significance as markers as well as indicators of poor prognosis in TNBC. The databases used in this review are PMC, PubMed, and Google Scholar.

Breast-conserving surgery versus mastectomy for older women with triple-negative breast cancer: population-based study.

Purpose: To assess whether the poor prognosis of triple-negative breast cancer (TNBC) necessitates a more aggressive surgical approach. Methods: We examined the association of: breast-conserving surgery (BCS); BCS plus radiotherapy; mastectomy; and mastectomy plus radiotherapy with overall and breast cancer-specific survival of stage I-III TNBC patients aged 66 years and older. We used unweighted and inverse probability of treatment weighted Cox proportional hazards regression and the Fine and Gray sub-distribution model. Results: Among 4333 women, individuals who were selected for BCS, mastectomy or mastectomy plus radiotherapy had lower adjusted overall and breast cancer-specific survival compared with women who had BCS plus radiotherapy. Conclusion: In this population-based study, women with TNBC treated with BCS plus radiotherapy have a better prognosis than those treated with BCS, mastectomy or mastectomy plus radiotherapy. Given the poor prognosis of TNBC and selection bias inherent in observational studies, these findings should be confirmed in further studies such as randomized clinical trials.

Druggable molecular targets of triple-negative breast cancer and prospects of therapeutic application

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, which is characterized by deficient expression of estrogen receptor, progesterone receptor expression and the absence of human epidermal growth factor receptor 2 (HER2) expression/amplification. For the past few decades, due to the lack of druggable targets, conventional chemotherapy has been the mainstay of systemic treatment for TNBC. However, lack of molecular targeted therapies and poor prognosis of TNBC, much research has been devoted to identifying specific molecular targets that can be utilized for targeted cancer therapy to improve the clinical outcomes. Now, poly (ADP-ribose) polymerase (PARP) inhibitors, immune checkpoint inhibitors, and antibody-drug conjugates have been approved by FDA for the treatment of TNBC. Moreover, agents that target signal transduction, angiogenesis, epigenetic modifications, and cell cycle are under active preclinical or clinical investigations. In this review, we summarize current significant achievements applied in treatment of TNBC with molecular targeted therapy, immunotherapy, and antibody-drug conjugates and upcoming drugs for triple negative breast cancer will be summarized. Key words: triple-negative breast cancer (TNBC), breast cancer, estrogen

CircEIF3H-IGF2BP2-HuR scaffold complex promotes TNBC progression via stabilizing HSPD1/RBM8A/G3BP1 mRNA

Triple-negative breast cancer (TNBC) is a molecular subtype with an unfavorable prognosis, and metastasis is the main reason for the failure of clinical treatment. However, the expression profile and regulatory function of circRNAs in TNBC progression are not fully understood. Herein, we performed high-throughput RNA-seq in paired breast cancer tissues and adjacent normal tissues and discovered a novel circRNA, circEIF3H, which was upregulated in breast cancer tissues. Large cohort survival analysis confirmed the association between high circEIF3H expression and poor prognosis of TNBC, indicating the vital function of circEIF3H in TNBC progression. Then we conducted both in vitro and in vivo experiments which illustrated that circEIF3H was essential for TNBC proliferation and metastasis. Further experiments showed that circEIF3H did not function as a microRNA sponge as in the most well-established pathway, but as a scaffold for IGF2BP2 and HuR to regulate the mRNA stability of HSPD1, RBM8A, and G3BP1. Our findings provide insight into a novel circRNA, circEIF3H, with significant cancer-promoting function via serving as a scaffold for IGF2BP2/HuR. These results identified circEIF3H as a potential target for developing individualized therapy of TNBC in the approaching future.

Functional Interaction Between the Oncogenic Kinase NEK2 and Sam68 Promotes a Splicing Program Involved in Migration and Invasion in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) represents the most aggressive breast cancer subtype. Poor prognosis in TNBC is partly due to lack of efficacious targeted therapy and high propensity to metastasize. Dysregulation of alternative splicing has recently emerged as a trait of TNBC, suggesting that unveiling the molecular mechanisms underlying its regulation could uncover new druggable cancer vulnerabilities. The oncogenic kinase NEK2 is significantly upregulated in TNBC and contributes to shaping their unique splicing profile. Herein, we found that NEK2 interacts with the RNA binding protein Sam68 in TNBC cells and that NEK2-mediated phosphorylation of Sam68 enhances its splicing activity. Genome-wide transcriptome analyses identified the splicing targets of Sam68 in TNBC cells and revealed a common set of exons that are co-regulated by NEK2. Functional annotation of splicing-regulated genes highlighted cell migration and spreading as biological processes regulated by Sam68. Accordingly, Sam68 depletion reduces TNBC cell migration and invasion, and these effects are potentiated by the concomitant inhibition of NEK2 activity. Our findings indicate that Sam68 and NEK2 functionally cooperate in the regulation of a splicing program that sustains the pro-metastatic features of TNBC cells.

Propolin G-Suppressed Epithelial-to-Mesenchymal Transition in Triple-Negative Breast Cancer Cells via Glycogen Synthase Kinase 3β-Mediated Snail and HDAC6-Regulated Vimentin Degradation.

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer with a poor prognosis. The incidence and mortality rate of TNBC are frequently found in younger women. Due to the absence of a good therapeutic strategy, effective remedies for inhibiting TNBC have been developed for improving the cure rate. Epithelial-to-mesenchymal transition (EMT) is a critical mechanism to regulate cancer cell motility and invasion. Furthermore, ectopic expression of EMT molecules correlates with the metastasis and poor prognosis of TNBC. Targeting EMT might be a strategy for the therapy and prevention of TNBC. Propolin G, an active c-prenylflavanone in Taiwanese propolis, has been shown to possess anti-cancer activity in many cancers. However, the anti-metastasis activity of propolin G on TNBC is still unclear. The present study showed that the migration and invasion activities of TNBC cells was suppressed by propolin G. Down-regulated expression of Snail and vimentin and up-regulated expression of E-cadherin were dose- and time-dependently observed in propolin G-treated MDA-MB-231 cells. Propolin G inhibited Snail and vimentin expressions via the signaling pathways associated with post-translational modification. The activation of glycogen synthase kinase 3β (GSK-3β) by propolin G resulted in increasing GSK-3β interaction with Snail. Consequently, the nuclear localization and stability of Snail was disrupted resulting in promoting the degradation. Propolin G-inhibited Snail expression and the activities of migration and invasion were reversed by GSK-3β inhibitor pretreatment. Meanwhile, the outcomes also revealed that histone deacetylase 6 (HDAC6) activity was dose-dependently suppressed by propolin G. Correspondently, the amounts of acetyl-α-tubulin, a down-stream substrate of HDAC6, were increased. Dissociation of HDAC6/Hsp90 with vimentin leading to increased vimentin acetylation and degradation was perceived in the cells with the addition of propolin G. Moreover, up-regulated expression of acetyl-α-tubulin by propolin G was attenuated by HDAC6 overexpression. On the contrary, down-regulated expression of vimentin, cell migration and invasion by propolin G were overturned by HDAC6 overexpression. Conclusively, restraint cell migration and invasion of TNBC by propolin G were activated by the expression of GSK-3β-suppressed Snail and the interruption of HDAC6-mediated vimentin protein stability. Aiming at EMT, propolin G might be a potential candidate for TNBC therapy.

Abstract PO-150: Comparison of RUNX1, RUNX2, RUNX3 and CBFβ gene expression in breast tumors Indicate ethnic differences and similarities by receptor status

Abstract The RUNX family of transcription factors RUNX1, RUNX2 and RUNX3 encode nuclear proteins with a Runt DNA-binding domain that functions in a complex with their obligate partner core-binding factor CBFβ to regulate gene expression. RUNX genes are known for paradoxical roles, with oncogenic as well as tumor suppressor functions, dependent on context. Interestingly, RUNX2 has been shown to play a potentially important functional role in Triple-Negative Breast Cancer (TNBC), and RUNX1 expression associates with poor prognosis in TNBC in a cohort of women of European descent. Women of African descent experience more aggressive TNBC with higher mortality rates than their women of European ancestry counterparts. TNBC is the most difficult breast cancer subtype to treat globally and has a poor prognosis and low survival which remains an important clinical challenge. There is a paucity of genomics studies interrogating TNBC of diverse populations. Hence, RNAseq datasets of 782 breast cancer patients were leveraged from a web-based platform -CbioPortal and published literature. 685 patients were queried for the RUNX1, RUNX2, RUNX3, and CBFβ genes, and an additional 97 patient data were harnessed from research publications. The cohort includes 595 (70%) patients of European ancestry (EA), 90 (11%) of African descent (AD), both from The Cancer Genome Atlas (TCGA), and 97 (13%) patients indigenous African from Nigeria (IAN). The queried genes were compared for race/ethnic disparity by estrogen receptor status (er). Mutual exclusivity was analyzed and P-values were determined by Fisher's extract test with the null hypothesis that a pair of genes alteration frequency of occurrence is proportional to uncorrelated occurrence in each gene. The queried genes enrichment in TP53, PIK3CA, CDH1, MAP3K1, and GATA3 between patients of European ancestry and African descent were analyzed and the Kaplan-Meier plots with P-value from the Long rank test were determined. The queried genes were altered in 69 patients (12%) of EA and 9 patients of AD due to unavailability of data on RUNX2 and RUNX 3 in IAN only RUNX1 and CBfβ was altered in 4 (4%) patients of 97 patients. Ethnic disparities and similarities were indicated in the queried altered genes in RUNX1 and RUNX 2 and showed similar expression in ER-negative correlating with the TNBC poor prognosis however RUNX3 genes were silenced in that of AD and was expressed in EA. They were differences in mutual exclusivity and co-occurrence but were not statistically significant, this may be due to the small data size. Interestingly, there was statistically significant equal gene enrichment of RUNX1 and CBfβ in CDH1mutations of both EA and AD. Larger cohorts in particular the AD cohort is needed, to further elucidate some of these borderline findings. In conclusion, our findings are insightful towards deciphering the characteristic aggressive nature of TNBC in ethnic disparity. Citation Format: Uzoamaka A. Okoli. Comparison of RUNX1, RUNX2, RUNX3 and CBFβ gene expression in breast tumors Indicate ethnic differences and similarities by receptor status [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-150.

A panel of miRNAs as prognostic markers for African-American patients with triple negative breast cancer

BackgroundTo investigate the global expression profile of miRNAs, their impact on cellular signaling pathways, and their association with poor prognostic parameters in African-American (AA) patients with triple negative breast cancer (TNBC).MethodsTwenty-five samples of AA TNBC patients were profiled for global miRNA expression and stratified considering three clinical-pathological parameters: tumor size, lymph node (LN), and recurrence (REC) status. Differential miRNA expression analysis was performed for each parameter, and their discriminatory power was determined by Receiver Operating Characteristic (ROC) curve analysis. KMplotter was assessed to determine the association of the miRNAs with survival, and functional enrichment analysis to determine the main affected pathways and miRNA/mRNA target interactions.ResultsA panel of eight, 23 and 27 miRNAs were associated with tumor size, LN, and REC status, respectively. Combined ROC analysis of two (miR-2117, and miR-378c), seven (let-7f-5p, miR-1255b-5p, miR-1268b, miR-200c-3p, miR-520d, miR-527, and miR-518a-5p), and three (miR-1200, miR-1249-3p, and miR-1271-3p) miRNAs showed a robust discriminatory power based on tumor size (AUC = 0.917), LN (AUC = 0.945) and REC (AUC = 0.981) status, respectively. Enrichment pathway analysis revealed their involvement in proteoglycans and glycan and cancer-associated pathways. Eight miRNAs with deregulated expressions in patients with large tumor size, positive LN metastasis, and recurrence were significantly associated with lower survival rates. Finally, the construction of miRNA/mRNA networks based in experimentally validated mRNA targets, revealed nodes of critical cancer genes, such as AKT1, BCL2, CDKN1A, EZR and PTEN.ConclusionsAltogether, our data indicate that miRNA deregulated expression is a relevant biological factor that can be associated with the poor prognosis in TNBC of AA patients, by conferring to their TNBC cells aggressive phenotypes that are reflected in the clinical characteristics evaluated in this study.

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.

Correction: Epigenetic Regulation of NAMPT by NAMPT-AS Drives Metastatic Progression in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is highly heterogeneous and has a poor prognosis. It is therefore important to identify the underlying molecular mechanisms in order to develop novel therapeutic strategies. Although emerging research has revealed long noncoding RNAs (lncRNA) as vital to carcinogenesis and cancer progression, their functional involvement in TNBC has not been well defined. In this study, we utilized the The Cancer Genome Atlas (TCGA) database and analyzed clinical samples to show that the long noncoding antisense transcript of nicotinamide phosphoribosyltransferase (NAMPT), NAMPT-AS, is upregulated in TNBC and is associated with poor prognosis, lymph node involvement, metastasis, and advanced stage. NAMPT-AS was cotranscribed with NAMPT from a bidirectional promoter, where the distributions of H3K4me3 and H3K27Ac chromatin modifications were enriched based on ENCODE and FANTOM5, suggesting the potential enhancer-RNA characteristics of NAMPT-AS. NAMPT-AS epigenetically regulated the expression of NAMPT in two divergent ways: NAMPT-AS recruited POU2F2 to activate the transcription of NAMPT, and NAMPT-AS acted as a competing endogenous RNA to rescue NAMPT degradation from miR-548b-3p. NAMPT-AS/NAMPT promoted tumor progression and regulated autophagy through the mTOR pathway in vitro and in vivo. In a cohort of 480 breast cancer patients, NAMPT was associated with breast cancer-specific survival and overall survival. These results demonstrate that NAMPT-AS is an oncogenic lncRNA in TNBC that epigenetically activates NAMPT to promote tumor progression and metastasis. Furthermore, these data identify NAMPT-AS/NAMPT as promising therapeutic targets in patients with TNBC. SIGNIFICANCE: Upregulation of the long noncoding antisense RNA of NAMPT gene (NAMPT-AS) is associated with metastasis and poor prognosis in TNBC.

Actin-Like Protein 8 Promotes the Progression of Triple-Negative Breast Cancer via Activating PI3K/AKT/mTOR Pathway.

ObjectiveThe purpose of this study was to investigate the function of actin-like protein 8 (ACTL8) on triple-negative breast cancer (TNBC) and its potential mechanisms.MethodsIn our study, ACTL8 expression and the prognostic values of ACTL8 were evaluated via the dataset from the Cancer Genome Atlas (TCGA). At the same time, the expression of ACTL8 in TNBC cells was measured by Western blot and qRT-PCR. Then, the effects of ACTL8 on the growth and metastasis of TNBC were investigated by using 5-ethynyl-20-deoxyuridine (EdU), colony formation, flow cytometry, wound healing and transwell assays. Mechanistically, Western blot was performed to confirm the interaction between ACTL8 and phosphatidylinositol 3′-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway in TNBC.ResultsACTL8 expression was upregulated in TNBC and associated with the poor prognosis of TNBC. Silencing ACTL8 suppressed the proliferation, migration and invasion, also promoted the apoptosis in MDA-MB-231 and BT-549 cells. Moreover, we found that silencing ACTL8 could inhibit the activation of PI3K/AKT/mTOR signaling pathway in MDA-MB-231 and BT-549 cells. Meanwhile, the impact of silencing ACTL8 on the proliferation, apoptosis, migration and invasion was enhanced by PI3K/AKT/mTOR pathway inhibitor (Wortmannin) and reversed by PI3K/AKT/mTOR pathway activator (740Y-P).ConclusionOur data demonstrated that ACTL8 may facilitate the proliferation, migration and invasion, while inhibiting apoptosis through activating PI3K/Akt/mTOR signaling pathway in TNBC.

Identification of potential oncogenes in triple-negative breast cancer based on bioinformatics analyses.

Triple-negative breast cancer (TNBC) is a subtype with high rates of metastasis, poor prognosis and limited therapeutic options. The present study aimed to identify the potential pivotal genes for prognosis and treatment in TNBC. A total of two microarray expression datasets, GSE38959 and GSE65212, were downloaded from the Gene Expression Omnibus database, and RNA-sequencing data of breast cancer from The Cancer Genome Atlas database were analyzed to screen out differentially expressed genes (DEGs) between TNBC tissues and normal tissues. The intersection of DEGs was submitted to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. A protein-protein interaction (PPI) network was constructed and visualized using Cytoscape software. Furthermore, module, centrality and survival analyses were performed to identify the potential hub genes. Reverse transcription-quantitative (RT-q)PCR analysis was performed to detect the expression levels of key genes in TNBC samples, and 377 DEGs were identified. Functional analysis revealed that the DEGs were significantly involved in cell cycle process, nuclear division and the p53 signaling pathway. A PPI network was constructed with these DEGs, and 66 core genes with high centrality features in module 1 were selected. Relapse-free survival analysis confirmed that high expression levels of five genes [cyclin B1 (CCNB1), GINS complex subunit 2, non-SMC condensin I complex subunit G (NCAPG), minichromosome maintenance 4 (MCM4) and ribonucleotide reductase regulatory subunit M2 (RRM2)] were significantly associated with poor prognosis in TNBC. RT-qPCR analysis demonstrated that CCNB1, NCAPG, MCM4 and RRM2 were significantly upregulated in 25 TNBC tissues compared with adjacent normal breast tissues. Furthermore, gene set enrichment analysis revealed that CCNB1, NCAPG, MCM4 and RRM2 were closely associated with tumor proliferation. Taken together, these results suggest that CCNB1, NCAPG, MCM4 and RRM2 are associated with tumorigenesis and TNBC progression, and thus may act as promising prognostic biomarkers and therapeutic targets for TNBC.

A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment.

Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments.Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/-knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays.Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion.Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.