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About 20% of breast cancer patients are positive for HER2. The efficacy of current treatments is limited by primary and secondary resistance to trastuzumab. tRNA-derived fragments (tRFs) have shown crucial regulatory roles in various cancers. This study aimed to evaluate the role of tRF-27 in regulating the resistance of HER2-positive breast cancer against trastuzumab. tRF-27 was highly expressed in trastuzumab-resistant cells, and its expression level could predict the resistance to trastuzumab. High expression of tRF-27 promoted the growth and proliferation of trastuzumab-exposed cells. RNA-pulldown assay and mass spectrometry were performed to identify Ras GTPase-activating protein-binding proteins 1 and 2 (G3BPs) (two proteins targeted by tRF-27 ); RNA-immunoprecipitation (RIP) to confirm their bindings; co-immunoprecipitation (co-IP) and RNA-pulldown assay to determine the binding domains between G3BPs and tRF-27 . tRF-27 bound to the nuclear transport factor 2 like domain(NTF2 domain) of G3BPs through a specific sequence. tRF-27 relied on G3BPs and NTF2 domain to increase trastuzumab tolerance. tRF-27 competed with lysosomal associated membrane protein 1(LAMP1) for NTF2 domain, thereby inhibiting lysosomal localization of G3BPs and tuberous sclerosis complex (TSC). Overexpression of tRF-27 inhibited phosphorylation of TSCs and promoted the activation of mechanistic target of rapamycin complex 1(MTORC1) to enhance cell proliferation and entice the resistance of HER2-positive breast cancer against trastuzumab., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Secondary trastuzumab resistance represents an evolutionary adaptation of HER2-positive breast cancer during anti-HER2 treatment. Most current studies have tended to prioritize HER2 and its associated signaling pathways, often overlooking broader but seemingly less relevant cellular processes, along with their associated genetic and epigenetic mechanisms. Here, transcriptome data is not only characterized but also examined epigenomic and 3D genome architecture information in both trastuzumab-sensitive and secondary-resistant breast cancer cells. The findings reveal that the global metabolic reprogramming associated with trastuzumab resistance may stem from genome-wide alterations in both histone modifications and chromatin structure. Specifically, the transcriptional activities of key genes involved in lipid metabolism appear to be regulated by variant promoter H3K27me3 and H3K4me3 modifications, as well as promoter-enhancer interactions. These discoveries offer valuable insights into how cancer cells adapt to anti-tumor drugs and have the potential to impact future diagnostic and treatment strategies., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Background: NME1 has been exploited as a potential translational target for decades. Substantial efforts have been made to upregulate the expression of NME1 and restore its anti-metastasis function in metastatic cancer., Methods: Cycloheximide (CHX) chase assay was used to measure the steady-state protein stability of NME1 and HSP90α. The NME1-associating proteins were identified by immunoprecipitation combined with mass spectrometric analysis. Gene knockdown and overexpression were employed to examine the impact of HSP90AA1 on intracellular NME1 degradation. The motility and invasiveness of breast cancer cells were examined in vitro using wound healing and transwell invasion assays. The orthotopic spontaneous metastasis and intra-venous experimental metastasis assays were used to test the formation of metastasis in vivo, respectively., Results: HSP90α interacts with NME1 and increases NME1 lifetime by impeding its ubiquitin-proteasome-mediated degradation. HSP90α overexpression significantly inhibits the metastatic potential of breast cancer cells in vitro and in vivo. A novel cell-permeable peptide, OPT22 successfully mimics the HSP90α function and prolongs the life span of endogenous NME1, resulting in reduced metastasis of breast cancer., Conclusion: These results not only reveal a new mechanism of NME1 degradation but also pave the way for the development of new and effective approaches to metastatic cancer therapy., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Tamoxifen is a drug used for treating breast cancer (BC), especially for individuals diagnosed with estrogen receptor-positive (ER+) BC. Its prolonged use could reduce the risk of recurrence and significantly lengthen the survival rate of BC patients. However, an increasing number of patients developed resistance to tamoxifen treatment, which reduced therapeutic efficiency and caused substandard prognosis. Therefore, the exploration of the molecular processes involved in tamoxifen resistance (TR) is urgently required. This investigation aimed to elucidate the relationship of microRNA-330 (miR-330-3p) with the TR of BC. There is little information on miR-330-3p's link with drug-resistant BC, although it is well known to regulate cell proliferation and apoptosis. Primarily, miR-330-3p expression in parental BC (MCF7/T47D), TR (MCF7-TR), and T47D/TR cell lines was detected by qRT-PCR. Then, the impact of miR-330-3p on the TR of BC cells was assessed by a cell proliferation assay. Lastly, dual-luciferase reporter, qRT-PCR, and western blot assessments were carried out to identify histone deacetylase 4 (HDAC4) as the potential miR-330-3p target gene. The data indicated that miRNA-330 was overexpressed in TR ER+ BC cells and its overexpression could induce TR. Furthermore, miRNA-330 could also reduce the expression of HDAC4, which is closely linked to TR, and overexpression of HDAC4 could reverse miRNA-330-induced drug resistance. In summary, miR-330-3p could induce TR of ER+ BC cells by downregulating HDAC4 expression, which might be a novel marker of TR and a possible treatment target against BC patients who are tamoxifen-resistant., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Meng Zhang et al.)

Breast cancer is very heterogeneous and the most frequently diagnosed cancer worldwide, and precise therapy targeting specific subtypes may improve the survival rates of breast cancer patients. In this study, we designed a biomimetic vesicle by camouflaging catalytic DNA machinery with a breast cancer cell membrane, which enabled the molecular classification of circulating exosomes for subtype-based diagnosis through homotypic recognition. In addition, the vesicles specifically targeted and fused with breast cancer exosomes with phenotypic homology and manipulated the DNA machinery to amplify electrochemical signaling using exosomal RNA as an endogenous trigger. The biomimetic vesicles prepared with MCF-7 cancer cell-derived membranes were shown to recognize estrogen receptor-positive breast cancer exosomes and exhibited a low detection limit of 557 particles mL -1 with microRNA-375 used as the endogenous biomarker. Furthermore, the biomimetic vesicles prepared with MDA-MB-231 cancer cell-derived membranes displayed satisfactory performance in a homotypic analysis of triple-negative breast cancer exosomes with a potential therapeutic target, PD-L1 mRNA, used as the endogenous biomarker. Most importantly, cross-validation experiments confirmed the high accuracy and selectivity of this homotypic recognition-driven analysis for molecular subtyping of breast cancer. When applied to clinical samples of breast cancer patients, the vesicles demonstrated feasibility and reliability for evaluating the molecular features of cancer cell-derived exosomes and enabled stage-specific monitoring of breast cancer patients because the electrochemical signals showed a positive correlation with disease progression. Therefore, this work may provide new ideas for the precise diagnosis and personalized treatment of breast cancer patients throughout the whole disease process.

Background: With life span extending, breast cancer (BC) survivors may face the possibility of developing second primary cancer (SPC) and considerably shorten survivorship. However, little is known about multiple primary cancer (MPC) patients with nonmetastatic breast cancer as a first primary malignancy (BCFPM)., Methods: Here, we retrospectively analyzed data on cancer survivors with BCFPM diagnosed between 2010 and 2015 from the Surveillance, Epidemiology, and End Results (SEER) database. The prognostic factors for breast cancer-specific survival (BCSS) were ascertained by the stepwise regression analysis and a competing risk model, and were integrated to the establishment of prognostic nomogram, of which the accuracy was measured by the calibration curve and the concordance index (C-index)., Results: In total, 8616 patients were identified with 4.6% of 3-year breast cancer- specific death (BCSD) and 8.6% of 5-year BCSD. The most common SPC among BCFPM patients were female BC and lung cancer. Besides, the median latency time between BC and SPC was 22 months. At a ratio of 7:3, all patients were randomly categorized into a training cohort (n = 6032) and a validation cohort (n = 2584). By a proportional subdistribution hazards regression analysis, the following factors were considered to own independent prognostic abilities of BCSS: subtypes, grade, T classification, N classification, radiation, and sites of SPC. The nomogram could accurately predict 3-year and 5-year breast cancer-associated survival of BCFPM patients with high internal and external validated C-index, 0.715 (95% CI, 0.691-0.739), and 0.683 (95% CI, 0.642-0.724), respectively., Conclusions: BC survivors remained a high risk of developing SPC and considerably shortened survival time. In this study, a favorable nomogram was constructed to as a prediction model for 3-year and 5-year BCSS of BCFPM patients, largely intending to prolong the life of these patients by assisting clinicians to make individualized follow-up plans., (© 2021 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Breast cancer is a heterogeneous disease with a complex microenvironment consisting of tumor cells, immune cells, fibroblasts and vascular cells. These cancer-associated cells shape the tumor microenvironment (TME) and influence the progression of breast cancer and the therapeutic responses in patients. The exact composition of the intra-tumoral cells is mixed as the highly heterogeneous and dynamic nature of the TME. Recent advances in single-cell technologies such as single-cell DNA sequencing (scDNA-seq), single-cell RNA sequencing (scRNA-seq) and mass cytometry have provided new insights into the phenotypic and functional diversity of tumor-infiltrating cells in breast cancer. In this review, we have outlined the recent progress in single-cell characterization of breast tumor ecosystems, and summarized the phenotypic diversity of intra-tumoral cells and their potential prognostic relevance.

Human epidermal growth factor receptor 2 (HER2)-positive is a particularly aggressive type of the breast cancer. Trastuzumab-based therapy is a standard treatment for HER2-positive breast cancer, but some patients are resistant to the therapy. Serum proteins have been used to predict therapeutic benefit for various cancers, but whether serum proteins can serve as biomarkers for HER2-positive breast cancer remains unclear. Using an isobaric Tandem Mass Tag (TMT) label-based quantitative proteomic, we discovered 18 differentially expressed proteins in the serum of trastuzumab-based therapy resistant patients before therapy. Then, four proteins were selected and validated using an LC-MS/MS-based multiple reaction monitoring quantification method, and it was confirmed that three proteins (SRGN, LDHA and CST3) were correlated with trastuzumab-based therapy resistance. Finally, the trastuzumab-based therapy resistance diagnostic score was calculated and acquired by means of a logistic regression pattern based on the level of these three proteins. In summary, we develop a serum-based protein signature that potentially predicts the therapeutic effects of trastuzumab-based therapy for HER2-positive breast cancer patients., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Breast cancer is the leading cause of cancer death in women worldwide. Long non-coding RNA small nucleolar RNA host gene 1 ( SNHG1 ) has been reported to be involved in human diseases, including cancer. Here, we found that SNHG1 expression was significantly upregulated in human breast cancer tissues and cell lines. We explored the function of SNHG1 in breast cancer cells using in vitro and in vivo experiments and found that SNHG1 promotes breast cancer metastasis and proliferation. The potential molecular mechanism of SNHG1 in breast cancer cells may involve SNHG1 acting as a sponge of miR-193a-5p to activate the expression of the oncogene HOXA1 . In summary, our study reveals a novel SNHG1 / miR-193a-5p/HOXA1 competing endogenous RNA regulatory pathway in breast cancer progression and may provide new strategies for breast cancer therapy.

Background: The efficacy and safety of lapatinib plus capecitabine (LC or LX) versus trastuzumab plus chemotherapy in patients with HER-positive metastatic breast cancer who are resistant to trastuzumab is unknown., Methods: We retrospectively analyzed data from breast cancer patients who began treatment with regimens of lapatinib plus capecitabine (LC or LX) or trastuzumab beyond progression (TBP) at eight hospitals between May 2010 and October 2017., Results: Among 554 patients who had developed resistance to trastuzumab, the median PFS (progression free survival) was 6.77 months in the LX group compared with 5.6 months in the TBP group (hazard ratio 0.804; 95% CI, 0.67 to 0.96; P = 0.019). The central nervous system progression rate during treatment was 5.9% in the LX group and 12.5% in the TBP group (P = 0.018)., Conclusion: The combination of lapatinib and capecitabine showed a prolonged PFS relative to TBP in patients who had progressed on trastuzumab.

Trastuzumab is a successful, rationally designed therapy that provides significant clinical benefit for human epidermal growth factor receptor-2 (HER2)-positive breast cancer patients. However, about half of individuals with HER2-positive breast cancer do not respond to trastuzumab treatment because of various resistance mechanisms, including but not limited to: 1) shedding of the HER2 extracellular domain, 2) steric hindrance ( e.g., MUC4 and MUC1), 3) parallel pathway activation (this is the general mechanism cited in the quote above), 4) perturbation of downstream signaling events ( e.g., PTEN loss or PIK3CA mutation), and 5) immunologic mechanisms (such as FcR polymorphisms). EPHA5, a receptor tyrosine kinase, has been demonstrated to act as an anticancer agent in several cancer cell types. In this study, deletion of EPHA5 can significantly increase the resistance of HER2-positive breast cancer patients to trastuzumab. To investigate how EPHA5 deficiency induces trastuzumab resistance, clustered regularly interspaced short palindromic repeat technology was used to create EPHA5-deficient variants of breast cancer cells. EPHA5 deficiency effectively increases breast cancer stem cell (BCSC)-like properties, including NANOG, CD133+, E-cadherin expression, and the CD44 + /CD24 -/low phenotype, concomitantly enhancing mammosphere-forming ability. EPHA5 deficiency also caused significant aggrandized tumor malignancy in trastuzumab-sensitive xenografts, coinciding with the up-regulation of BCSC-related markers and intracellular Notch1 and PTEN/AKT signaling pathway activation. These findings highlight that EPHA5 is a potential prognostic marker for the activity of Notch1 and better sensitivity to trastuzumab in HER2-positive breast cancer. Moreover, patients with HER2-positive breast cancers expressing high Notch1 activation and low EPHA5 expression could be the best candidates for anti-Notch1 therapy.-Li, Y., Chu, J., Feng, W., Yang, M., Zhang, Y., Zhang, Y., Qin, Y., Xu, J., Li, J., Vasilatos, S. N., Fu, Z., Huang, Y., Yin, Y. EPHA5 mediates trastuzumab resistance in HER2-positive breast cancers through regulating cancer stem cell-like properties.

As a class of endogenous noncoding RNAs, circular RNAs (circRNAs) have been recently identified to regulate tumourigenesis and progression in multiple malignancies. However, the expression profiles and function of circRNAs in breast cancer metastasis are largely unknown. Here, we determined that the expression of a novel circRNA, which we named circIRAK3, was increased in metastatic breast cancer (BC) cells and predictive of BC recurrence. Gain-of-function and loss-of-function studies in BC cells demonstrated that circIRAK3 promoted cell migration, invasion and metastasis in vitro and in vivo but did not affect cell proliferation, colony formation or cell cycle progression. Using circIRAK3 in vivo precipitation and luciferase reporter assays, we identified miR-3607 as a circIRAK3-associated miRNA. Furthermore, RNA sequencing and bioinformatics analysis showed that forkhead box C1 (FOXC1), the target of miR-3607, was downregulated in circIRAK3-silenced cells and mediated circIRAK3-induced BC cell migration. Intriguingly, FOXC1 could, in turn, bind to the IRAK3 promoter, triggering a positive-feedback loop that perpetuated the circIRAK3/miR-3607/FOXC1 signaling axis. Collectively, our findings indicated that circIRAK3 may exert regulatory roles in BC metastasis and may be a potential target for metastatic BC therapy., (Copyright © 2018. Published by Elsevier B.V.)

Placenta-specific protein 1 (Plac1) is a cancer/testis antigen that plays a critical role in promoting cancer initiation and progression. However, the clinical significance and mechanism of Plac1 in cancer progression remain elusive. Here, we report that Plac1 is an important oncogenic and prognostic factor, which physically interacts with Furin to drive breast cancer invasion and metastasis. We have shown that Plac1 expression positively correlates with clinical stage, lymph node metastasis, hormone receptor status, and overall patient survival. Overexpression of Plac1 promoted invasion and metastasis of breast cancer cells in vitro and in vivo. Co-immunoprecipitation and immunofluorescence cell staining assays revealed that interaction of Plac1 and Furin degraded Notch1 and generated Notch1 intracellular domain (NICD) that could inhibit PTEN activity. These findings are consistent with the results of microarray study in MDA-MB-231 cells overexpressing Plac1. A rescue study showed that inhibition of Furin and overexpression of PTEN in Plac1 overexpression cells blocked Plac1-induced tumor cell progression. Taken together, our findings suggest that functional interaction between Plac1 and Furin enhances breast cancer invasion and metastasis and the Furin/NICD/PTEN axis may act as an important therapeutic target for breast cancer treatment., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in females worldwide. Chemoresistance has been a major reason for the drug therapy failure. The present study performed a microarray analysis between MCF‑7 and MCF‑7/adriamycin (ADR) cells, and intended to identify long non‑coding (lnc)RNA expression character in drug resistant breast cancer cells. MCF‑7/ADR cells were induced from MCF‑7 cells via pulse‑selection with doxorubicin for 4 weeks, and the resistance to doxorubicin of ADR cells was confirmed by MTT assay. Microarray analysis was performed between MCF‑7 and MCF‑7/ADR cells. Total RNA was extracted from the two cell lines respectively and was transcribed into cDNA. The results of the microarray were verified by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). Gene Ontology (GO) and pathways analysis were conducted to enrich the dysregulated lncRNAs presented in the microarray results. Compared to the MCF‑7 cells, 8,892 lncRNAs were differentially expressed in MCF/ADR cells (absolute fold‑change >2.0). A total of 32 lncRNAs were selected for RT‑qPCR by fold‑change filtering, standard Student's t‑test, and multiple hypothesis testing. Among the dysregulated lncRNAs, AX747207 was prominent because its associated gene RUNX3 was previously reported to be relative to malignant tumor chemoresistance. GO analysis results also indicated some biological processes and molecular functions linked to chemoresistance. The pathway enrichment results provided some potential pathways associated with chemoresistance. In the present study, the authors intended to identify lncRNA expression character in drug resistant cell line MCF‑7/ADR, corresponding to the parental MCF‑7 cell line. In addition, the study identified the lncRNA AX747207, and its potential targeted gene RUNX3, may be related to chemoresistance in breast cancer. These results may new insights into exploring the mechanisms of chemoresistance in breast cancer.

Breast cancer is a serious health problem worldwide. Acquisition of multi-drug resistance (MDR) during the treatment of breast cancer is still considered a major clinical obstacle. Despite the biological functions of miRNAs becoming increasingly apparent, the function of miRNAs in regulating drug resistance of breast cancer remains under investigation. Quantitative real-time PCR (qRT-PCR) was used to quantify the expression of miR-132/-212 (miR-132 and miR-212) in doxorubicin (DOX)-resistant and -sensitive breast cancer tumors and cells. The function of miR-132/-212 in drug resistance was investigated in vitro (MTT assay, TUNEL assay, fluorescence, immunohistochemistry, luciferase reporter assay, Western blotting). We found that miR-132/-212 were commonly overexpressed in DOX-resistant breast cancer tumors and cells. Silenced miR-132/-212 expression induced DOX accumulation in MCF-7/ADR cells, while overexpression of miR-132/-212 led to breast cancer resistance protein (BCRP)-based DOX efflux in MCF-7 cells. Further study showed that up-regulation of miR-132/-212 in MCF-7/ADR cells suppressed the expression of PTEN, a target gene of miR-132/-212, which activated AKT phosphorylation and the NF-κB pathway and led to increased BCRP expression. Down-regulation of miR-132/-212 sensitized MCF-7/ADR cells to DOX. Mechanistic investigations suggested that miR-132/-212 enhancement was a result of NF-κB-mediated transactivation of the pri-miR-132/-212 gene. Taken together, our findings are among the first to demonstrate a novel aspect of the miR-132/-212-PTEN-AKT/NF-κB-BCRP pathway in the generation of breast cancer resistance and provides a potential method to reverse drug resistance., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Background/aims: Resistance to trastuzumab remains a common challenge to HER-2 positive breast cancer. Up until now, the underlying mechanism of trastuzumab resistance is still unclear. tRNA-derived small non-coding RNAs, a new class of small non-coding RNA (sncRNAs), have been observed to play an important role in cancer progression. However, the relationship between tRNA-derived fragments and trastuzumab resistance is still unknown., Methods: We detected the levels of tRNA-derived fragments expression in normal breast epithelial cell lines, trastuzumab-sensitive and -resistant breast cancer cell lines using high-throughput sequencing. qRT-PCR was conducted to validate the differentially expressed fragments in serums from trastuzumab-sensitive and -resistant patients. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the power of specific tRNA-derived fragments. Progression-free survival (PFS) was analyzed using Cox-regression., Results: Our sequence results showed that tRNA-derived fragments were differentially expressed in the HBL-100, SKBR3, and JIMT-1 cell lines. tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN were found significantly upregulated in trastuzumab-resistant patients compared to sensitive individuals, and the ROC analysis showed that tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN were correlated with trastuzumab resistance. In a multivariate analysis, higher levels of tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN expression were associated with significantly shorter PFS in patients with metastatic HER-2 positive breast cancer., Conclusion: Our results suggest that tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN play important roles in trastuzumab resistance. Patients with high levels of tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN expression benefitted less from trastuzumab-based therapy than those that express lower-levels of these molecules. tRF-30-JZOYJE22RR33 and tRF-27-ZDXPHO53KSN may be potential biomarkers and intervention targets in the clinical treatment of trastuzumab-resistant breast cancer., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Breast cancer is the most common cancer diagnosed and is the second leading cause of cancer death among women in the US. For breast cancer, early diagnosis and efficient therapy remains a significant clinical challenge. Therefore, it is necessary to identify novel tumor associated molecules to target for biomarker development and immunotherapy. In this regard, cancer testis antigens (CTAs) have emerged as a potential clinical biomarker targeting immunotherapy for various malignancies due to the nature of its characteristics. CTAs are a group of tumor associated antigens (TAAs) that display normal expression in immune-privileged organs, but display aberrant expression in several types of cancers, particularly in advanced cancers. Investigation of CTAs for the clinical management of breast malignancies indicates that these TAAs have potential roles as novel biomarkers, with increased specificity and sensitivity compared to those currently used in the clinic. Moreover, TAAs could be therapeutic targets for cancer immunotherapy. This review is an attempt to address the promising CTAs in breast cancer and their possible clinical implications as biomarkers and immunotherapeutic targets with particular focus on challenges and future interventions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The oncogene, mouse double minute 2 (MDM2), has been implicated in the pathogenesis of numerous cancers. In this study, we investigated the role of MDM2 in epithelial-to-mesenchymal transition (EMT) and the underlying mechanisms in breast cancer cells in vitro and in vivo. The results showed that up-regulation of MDM2 in MCF-7 cells altered the cell morphology to a mesenchymal phenotype. Knockdown of MDM2 in MDA-MB-231 cells altered the cell morphology to the epithelial phenotype. In addition, overexpression of MDM2 increased the expression of N-cadherin and Vimentin and decreased the expression of E-cadherin, at both the mRNA and protein levels, in vitro and in vivo. Conversely, down-regulation of MDM2 decreased the expression of N-cadherin and Vimentin, and increased the expression of E-cadherin in vitro. Furthermore, MDM2 up-regulated both the mRNA and protein expression of Snail in vitro and in vivo. Knockdown of Snail almost abolished MDM2 induced EMT in vitro. Finally, we found that MDM2 expression correlated with EMT markers and Snail: Snail expression was inversely associated with E-cadherin in human breast cancer samples. Our findings demonstrated that MDM2 induces EMT by enhancing Snail expression in vitro and in vivo. Thus, MDM2 may be a potential target for therapy against human metastatic breast cancer., Competing Interests: The authors declare that they have no competing interests.

Di(2-ethylhexyl)phthalate (DEHP) is an estrogenic chemical that is widely used in polyvinyl products. We aimed to determine the mechanisms behind the effects of DEHP on ERα-negative breast cancer cells MDA-MB-231 invasion and matrix metalloproteinases-2/-9 (MMP-2/-9) up-regulation in this study. Transwell assay indicated that DEHP exposure (>50 μg/ml) significantly enhanced the invasion ability of MDA-MB-231 cells. Quantitative real-time PCR (qPCR) and western blotting revealed that MMP-2/-9 is overexpressed in mRNA and protein levels after DEHP treatment. Gelatin zymography consistently demonstrated that DEHP exposure also enhances the activity of MMP-2/-9. Immunofluorescence assay showed that DEHP could accelerate NF-kappaB (NF-κB) subunits-p65 translocation into the nucleus, which is confirmed by western blotting assay, suggesting that the ratio of nuclear/cytosolic level of p65 was significantly increased. Furthermore, the invasion and MMP-2/-9 overexpression of MDA-MB-231 cells after DEHP-treated were reversed by the NF-κB chemical inhibitor JSH-23 via drug inhibition assay. This study suggested that DEHP could promote ERα-negative breast cancer cells MDA-MB-231 invasion through activating NF-κB and MMP-2/-9 overexpression.

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer with high heterogeneity. To date, there is no efficient therapy for TNBC patients and the prognosis is poor. It is urgent to find new biomarkers for the diagnosis of TNBC or efficient therapy targets. As an area of focus in the post-genome period, long non-coding RNAs (lncRNAs) have been found to play critical roles in many cancers, including TNBC. However, there is little information on differentially expressed lncRNAs between TNBC and non-TNBC. We detected the expression levels of lncRNAs in TNBC and non-TNBC tissues separately. Then we analyzed the lncRNA expression signature of TNBC relative to non-TNBC, and found dysregulated lncRNAs participated in important biological processes though Gene Ontology and Pathway analysis. Finally, we validated these lncRNA expression levels in breast cancer tissues and cells, and then confirmed that 4 lncRNAs (RP11-434D9.1, LINC00052, BC016831, and IGKV) were correlated with TNBC occurrence through receiver operating characteristic curve analysis. This study offers helpful information to understand the initiation and development mechanisms of TNBC comprehensively and suggests potential biomarkers for diagnosis or therapy targets for clinical treatment.

Heterogeneity is the major obstacle to breast cancer target therapy. Classification of breast cancer with significant biological process may reduce the influence of heterogeneity of intrinsic tumor. We used survival analysis to filter 95 gene sets and classify 638 breast cancer samples into two subtypes based on those gene sets associated with prognosis. Clinical outcome of two subtypes were evaluated with disease-free survival, distant metastasis-free survival, and overall survival levels in three databases and ER+, PR+ HER2+, and TNBC groups. We established a novel classification with 95 prognostic gene sets. In the training and validation cohorts, the subtype 1 was characterized by significant gene sets associated with regulation of metabolic process and enzyme activity and predicted obviously improved clinical outcome than subtype 2, which was enriched by tumor cell division, mitosis, and cell cycle-related gene sets (P < 0.05). When evaluated prognostic impact of subtypes in ER+, PR+ HER2+, and TNBC groups, we found that patients in subtype 1 showed better prognosis in ER+ and PR+ groups (P < 0.05) but had no difference from prognosis of subtype 2 in HER2+ and TNBC groups. These findings may have implications in understanding of breast cancer and filtering effective therapeutic strategies for targeted therapy.

Background/aim: Breast cancer is the most common type of tumor in female and chemoresistance has been a major clinical obstacle to the treatment in clinical patients. miRNA was one of the factors demonstrated to play certain roles in chemoesistance in breast cancer. In this study, we exploited Solexa deep sequencing technology to identify differentially expressed miRNA from samples in vitro, trying to find novel relationship between miRNA and chemoresistance in breast cancer., Methods: The human breast cancer MCF-7 cell line was pulse-selected with doxorubicin (10 pulses, once a week for 4h, with 1μM doxorubicin) to generate MCF-7/Adr cells. Total RNA was extracted from the treated and untreated MCF-7 cells and subsequently subjected to real time PCR. Two small RNA libraries of MCF7NON and MCF7ADR were established to record the Solexa sequencing results of the PCR products above. All the sequencing results were verified by Stem-loop real-time PCR. GO annotation and KEGG analysis program were exploited to enrich the differentially expressed miRNAs., Results: The results showed that 214,822 and 378,597 reads were mapped in the MCF7ADR and MCF7NON libraries when aligned to hairpin structure respectively. Meanwhile, 1323 and 520 reads were mapped when aligned to mature sequences. In addition, 310 known mature miRNAs were coexpressed in both libraries. Comparing the MCF7ADR group to the MCF7NON group, 18 miRNAs were significantly differentially expressed. GO annotation and KEGG analysis showed that the target genes were enriched in regulation of transcription and development as well as Wnt signaling pathway, MAPK signaling pathway and TGF-ß signaling pathway., Conclusion: The results proved that the Solexa deep sequencing was a powerful and reliable platform to analyze small RNAs. And further investigation should be conducted for the biological process and pathways that have been identified and more efforts should be made to research the mechanism of chemoresistance in breast cancer., (Copyright © 2015. Published by Elsevier Masson SAS.)

Background/aims: Chemoresistance of breast cancer is a growing problem and still a major clinical obstacle to successful treatment in clinical patients. miR-760 was significantly downregulated in chemoresistance breast cancer tissues compared to chemo-sensitive tissues in our previous study. However, the role of miR-760 in modulating drug resistance remains largely unexplored. In this study, we sought to determine the expression pattern of miR-760 targeted mRNAs, and explore their potential functions and participated-pathways in breast cancer drug resistance cells., Results: Compared to parental cell line MCF-7, miR-760 was downregulated by 6.15 folds in MCF-7/Adr cells. The qRT-PCR result showed that compared to miR-760 negative control cells group, miR-760 was up-regulated 15.817 folds after miR-760 lentiviral transfection in miR-760 mimics group. The microarray data showed that 270 genes were dysregulated over 2-fold change in MCF-7/Adr cells after miR-760 overexpressed, including 241 up-regulated and 29 downregulated genes. GO analysis result appeared that the predicted target genes of miR-760 mainly regulated DNA binding, protein binding, molecular function, nucleic acid binding, and so on; the pathway analysis data demonstrated that these target genes mainly involved in cell cycle, TGF-beta signaling pathway, mRNA processing reactome, G protein signaling, apoptosis, Wnt signaling pathway, and other signaling pathways. There were 3 predicted target genes (RHOB, ANGOTL4, ABCA1) of miR-760 were selected at a P value<0.05 and the fold enrichment was>40., Conclusion: Our study explored the genes expression pattern after miR-760 overexpresssed, and confirmed 3 dominantly dysregulated genes, which could expand the insights into the miR-760 function and molecular mechanisms in drug resistance of breast cancer. This study might afford a comprehensive understanding of miR-760 as prognostic biomarkers during clinical treatment, and we supposed that the miR-760 expression levels in drug resistance carcinoma tissues could be pursued to develop new strategies for targeted therapies in chemoresistant breast cancer patients., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Mammary fat is the main composition of breast, and is the most probable candidate to affect tumor behavior because the fat produces hormones, growth factors and adipokines, a heterogeneous group of signaling molecules. Gene expression profiling and functional characterization of mammary fat in Chinese women has not been reported. Thus, we collected the mammary fat tissues adjacent to breast tumors from 60 subjects, among which 30 subjects had breast cancer and 30 had benign lesions. We isolated and cultured the stromal vascular cell fraction from mammary fat. The expression of genes related to adipose function (including adipogenesis and secretion) was detected at both the tissue and the cellular level. We also studied mammary fat browning. The results indicated that fat tissue close to malignant and benign lesions exhibited distinctive gene expression profiles and functional characteristics. Although the mammary fat of breast tumors atrophied, it secreted tumor growth stimulatory factors. Browning of mammary fat was observed and browning activity of fat close to malignant breast tumors was greater than that close to benign lesions. Understanding the diversity between these two fat depots may possibly help us improve our understanding of breast cancer pathogenesis and find the key to unlock new anticancer therapies.

Background/aims: Breast cancer chemoresistance is a major obstacle to the successful treatment of patients. miRNAs perform critical roles in biological processes, including tumorigenesis and chemoresistance. However, little clinical data are available regarding the relationship between miRNA expression patterns and breast cancer chemoresistance., Methods: We created a doxorubicin-resistant MCF-7 (MCF-/Adr) cell line using a pulse-selection method; then verified the resistance of the MCF-7/Adr cell line to doxorubicin by using the methyl thiazolyl tetrazolium (MTT) assay, terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, and Intracellular doxorubicin accumulation assay. Then, we performed qRT-PCR to detect the expression patterns of 14 selected miRNAs (which are related to breast cancer resistance) in both cell lines. Subsequently, we performed a bioinformatics analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, to determine the putative functions of 13 differentially expressed miRNA-targeted genes. Finally, we tested the expression levels of these 13 miRNAs in 10 chemotherapy non-responder breast cancer tissues and 29 responder tissues. All statistical analyses were performed by a two-tailed Student's t-test, and a P value less than 0.05 was considered statistically significant., Results: The results of the MTT assay showed that the MCF-7/Adr cell line was significantly more resistant to doxorubicin compared to the MCF-7 cells The results of the TUNEL assay indicated that doxorubicin induced an increase in the number apoptotic cells in the MCF-7 group. Additionally, the accumulation of doxorubicin was higher in MCF-7 cells compared to MCF-7/Adr cells, which was consistent with the MTT and TUNEL results. The qRT-PCR results demonstrated that compared to the parental MCF-7 cell line, miR-200a, miR-141, miR-200c, miR-31, miR-429, and miR-196b were over-expressed, and let-7e, miR-576-3p, miR-125b-1, miR-370, miR-145, miR-765, and miR-760 were significantly down-regulated in MCF-7/Adr cells. The GO analysis results revealed that the predicted target genes of these 14 miRNAs primarily regulated protein binding, zinc ion binding, DNA binding, and transcription factor activity. The KEGG data demonstrated that these target genes are mainly involved in the MAPK signaling pathway, regulation of the actin cytoskeleton, cytokine-cytokine receptor interaction, and other signaling pathways. Compared to the breast cancer tissues from chemotherapy responders, 10 miRNAs were identified to be dysregulated in the chemoresistant breast cancer tissues. Three of these miRNAs were up-regulated (miR-141, miR-200c, and miR-31), and 7 were down-regulated (let-7e, miR-576-3p, miR-125b-1, miR-370, miR-145, miR-765, and miR-760)., Conclusion: In this study, we identified 10 dysregulated miRNAs in both breast cancer cells and chemoresistant tissues, which might be biomarkers for the prognosis of breast cancer chemoresistance. Our study contributes to a comprehensive understanding of prognostic biomarkers during clinical treatment, and we hypothesize that the miRNA signatures of drug-resistant carcinoma tissues could be useful for developing new strategies for targeted therapies in patients with chemoresistant breast cancer., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Perfluorooctanoic acid (PFOA) is widely used because of its stain-resistant and water-repellant properties. This study aimed to explore the molecular mechanisms undergoing the stimulation effects of PFOA on cancer cell invasion and matrix metalloproteinases (MMPs) expression. Trans-well filter assay showed that PFOA exposure (≥5 nM) evidently enhanced the invasion ability of the breast cancer cells MDA-MB-231. Luciferase reporter assay, quantitative real-time PCR, western blotting and gelatin zymography consistently demonstrated that mRNA and protein levels of MMP-2/-9 were increased in the cells after PFOA treatment (P<0.05 each). Western blotting revealed that PFOA could activate nuclear factor kappaB (NF-κB) by accelerating NF-κB translocation into the nucleus. Furthermore, addition of NF-κB inhibitor in culture medium could suppress the breast cancer cells invasiveness enhancement and MMP-2/-9 overexpression. This study indicates that PFOA can stimulate breast cancer cells invasion and up-regulate matrix metalloproteinase-2/-9 expression mediated by activating NF-κB, which deserves more environmental health concerns., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Breast cancer has been considered to be a multifactorial disease with a wide array of well-characterized gene mutations and chromosomal abnormalities. However, it is becoming evident that the onset or development of breast cancer also depends on epigenetic factors, although the mechanisms have not been fully elucidated. We performed a genome-wide analysis of DNA methylation of breast carcinomatous tissues and paired normal tissues to examine the differences in methylation between them. Methylation-specific polymerase chain reaction (MSP) was used to validate the hypermethylated genes screened out by DNA methylation microarray. We found that hypomethylation and hypermethylation occurred in 2753 and 1795 genes, respectively, in breast carcinomatous tissues. Meanwhile, gene ontology analysis and ingenuity pathway analysis revealed the function and pathway of several genes whose methylation status was altered in breast carcinomatous tissues. In addition, we investigated the promoter methylation status of four genes in breast carcinomatous tissue and paired normal tissues (n=30) by MSP. Promoter hypermethylation of CRABP1, HOXB13, IFNGR2, and PIK3C3 was found in 37% (11/30), 23% (7/30), 17% (5/30), and 2% (2/30) of the carcinomas, respectively. Mutation of these four important genes was critical to many types of cancer. Our results suggest that DNA methylation mechanisms may be involved in regulating the occurrence and development of breast cancer., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)