Clinical Treatment Of Breast Cancer Research Articles

Possible involvement of TGF‑β‑SMAD‑mediated epithelial‑mesenchymal transition in pro‑metastatic property of PAX6.

Paired box 6 (PAX6) is a transcription factor that has oncogenic features. In breast cancer, PAX6 facilitates tumor progression; however, the underlying mechanism is largely unknown. The majority of breast cancer-related mortalities are associated with metastasis of cancer cells. Therefore, the present study aimed to investigate the role of PAX6 in breast tumor metastasis. PAX6 was stably overexpressed in breast cancer cells to perform tumor migration and metastasis assays in vitro and in vivo. In addition, the expression of PAX6 and transforming growth factor β (TGF-β)-SMAD signaling associated proteins on human breast cancer tissue array, as well as key factors involved in epithelial-mesenchymal transition (EMT) were assayed to explore the mechanism underlying metastasis of breast cancer cells. The expression levels of PAX6 were demonstrated to be increased in human breast cancer tissues and associated with poor clinical outcomes. Overexpression of PAX6 markedly promoted metastasis. Further investigation revealed that PAX6 overexpression increased TGF-β-SMAD signaling pathway and induced EMT. These results suggested that highly expressed PAX6 led to EMT through TGF-β-SMAD signaling pathway, thereby promoting cell metastasis and ultimately affecting survival in patients with breast cancer. Taken together, findings indicated that PAX6 may serve as a therapeutic target for the clinical treatment of breast cancer and the underlying mechanism could be used to overcome metastasis of cancer cells.

Revealing the selective mechanisms of inhibitors to PARP-1 and PARP-2 via multiple computational methods.

BackgroundResearch has shown that Poly-ADP-ribose polymerases 1 (PARP-1) is a potential therapeutic target in the clinical treatment of breast cancer. An increasing number of studies have focused on the development of highly selective inhibitors that target PARP-1 over PARP-2, its closest isoform, to mitigate potential side effects. However, due to the highly conserved and similar binding sites of PARP-1 and PARP-2, there is a huge challenge for the discovery and design of PARP-1 inhibitors. Recently, it was reported that a potent PARP-1 inhibitor named NMS-P118 exhibited greater selectivity to PARP-1 over PARP-2 compared with a previously reported drug (Niraparib). However, the mechanisms underlying the effect of this inhibitor remains unclear.MethodsIn the present study, classical molecular dynamics (MD) simulations and accelerated molecular dynamics (aMD) simulations combined with structural and energetic analysis were used to investigate the structural dynamics and selective mechanisms of PARP-1 and PARP-2 that are bound to NMS-P118 and Niraparib with distinct selectivity.ResultsThe results from classical MD simulations indicated that the selectivity of inhibitors may be controlled by electrostatic interactions, which were mainly due to the residues of Gln-322, Ser-328, Glu-335, and Tyr-455 in helix αF. The energetic differences were corroborated by the results from aMD simulations.ConclusionThis study provides new insights about how inhibitors specifically bind to PARP-1 over PARP-2, which may help facilitate the design of highly selective PARP-1 inhibitors in the future.

17‑AAG synergizes with Belinostat to exhibit a negative effect on the proliferation and invasion of MDA‑MB‑231 breast cancer cells.

Breast cancer is one of the most common malignancies that threaten the health of women. Although there are a few chemotherapies for the clinical treatment of breast cancer, these therapies are faced with the problems of drug-resistance and metastasis. Drug combination can help to reduce the adverse side effects of chemotherapies using single drugs, and also help to overcome common drug-resistance during clinical treatment of breast cancer. The present study reported the synergistic effect of the heat shock protein 90 inhibitor 17-AAG and the histone deacetylase 6 inhibitor Belinostat in triple-negative breast cancer (TNBC) MDA-MB-231 cells, by detection of proliferation, apoptosis and cell cycle arrest following treatment with this combination. Subsequently, RNA sequencing (RNA-seq) data was collected and analyzed to investigate the synergistic mechanism of this combination. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways revealed by RNA-seq data analysis, a wound-healing assay was used to investigate the effect of this combination on the migration of MDA-MB-231 cells. Compared with treatment with 17-AAG or Belinostat alone, both the viability inhibition and apoptosis rate of MDA-MB-231 cells were significantly enhanced in the combination group. The combination index values were <1 in three concentration groups. Revealed by the RNA-seq data analysis, the most significantly enriched KEGG pathways in the combination group were closely associated with cell migration. Based on these findings, the anti-migration effect of this combination was investigated. It was revealed that the migration of MDA-MB-231 cells was significantly suppressed in the combination group compared with in the groups treated with 17-AAG or Belinostat alone. In terms of specific genes, the mRNA expression levels of TEA domain family proteins were significantly decreased in the combination group, whereas the phosphorylation of YY1 associated protein 1 and modulator of VRAC current 1 was significantly enhanced in the combination group. These alterations may help to explain the anti-migration effect of this combination. Belinostat has already been approved as a treatment for T-cell lymphoma and 17-AAG is undergoing clinical trials. These findings could provide a beneficial reference for the clinical treatment of patients with TNBC.

Abstract P5-13-04: Gene expression profiling of breast cancers between high and low Ki-67 after short-term preoperative hormone therapy among hormone receptor-positive / human epidermal growth factor receptor 2-negative breast cancers with high Ki-67

Abstract Background In hormone receptor (HR)-positive / human epidermal growth factor receptor 2 (HER2)-negative breast cancers, high Ki-67 predicts poor prognosis. Recently, it has been reported that among breast cancers with high Ki-67, low Ki-67 after short-term preoperative hormone therapy (post Ki-67) might predict favorable prognosis compared to high post Ki-67 (SABCS 2017). However, the differences in gene expression profiling of breast cancers between high and low post Ki-67 among high Ki-67 before treatment are unclear. Therefore, this study aimed to clarify genetic differences in two groups and to explore novel therapeutic targets for the poor prognostic group after the treatment. Methods Seventy-seven patients with primary HR-positive / HER2-negative breast cancer who received an aromatase inhibitor for 2 weeks [NCBI Gene Expression Omnibus repository GSE80077:19 cases, GSE20181:58 cases] were enrolled in this study. Forty-five patients with high pre Ki-67 among them were stratified into two groups, high (H→H) and low (H→L) post Ki-67 after short-term preoperative hormone therapy. We compared gene expression profiling in two groups about the followings. 1) 3 genes (ESR1, PGR and ERBB2) related to classical clinical treatment strategy of breast cancer, immune- and inflammatory-related genes 2) 178 pathways (gene set analysis) 3) 41 genes that are targeted by FDA-approved drugs or have been investigated with clinical trials as molecular target agents for various malignant tumors including breast cancer Results 1) TNF that is inflammatory-related gene were significantly overexpressed in H→L group (P=0.021). However, 3 genes related to clinical treatment of breast cancer and immune-related genes expression had no significant difference between two groups. 2) 5 gene sets (Tryptophan metabolism, Propanoate metabolism, beta-Alanine metabolism, SNARE interactions in vesicular transport and Nucleotide excision repair) were significantly upregulated in H→L group. (P ≤ 0.005) 3) 5 targeted genes (PARP, BRCA2, FLT4, CDK6 and PDCD1LG2) were significantly overexpressed in H→H group (P ≤ 0.05). Conclusions There were different gene expression and biological processes in two groups stratified by post-Ki67. In the future, it is necessary to seek new therapeutic strategies for poor prognostic group with high post-Ki67 in considerations of these differences. Citation Format: Yukiko Kajiwara, Takayuki Iwamoto, Yusuke Otani, Miwa Fujihara, Yoko Suzuki, Minami Hatono, Takahiro Tsukioki, Kengo Kawada, Mariko Kochi, Hirokuni Ikeda, Tadahiko Shien, Naruto Taira, Hiroyoshi Doihara. Gene expression profiling of breast cancers between high and low Ki-67 after short-term preoperative hormone therapy among hormone receptor-positive / human epidermal growth factor receptor 2-negative breast cancers with high Ki-67 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-13-04.

Resibufogenin suppresses tumor growth and Warburg effect through regulating miR-143-3p/HK2 axis in breast cancer.

Increasing evidence confirmed that the Warburg effect plays an important role involved in the progression of malignant tumors. Resibufogenin (RES) has been proved to have a therapeutic effect in multiple malignant tumors. However, the mechanism of whether RES exerted an antitumor effect on breast cancer through regulating the Warburg effect is largely unknown. The effect of RES on glycolysis was determined by glucose consumption, lactate production, ATP generation, extracellular acidification rate and oxygen consumption rate in breast cancer cells. The total RNA and protein levels were respectively measured by RT-qPCR and western blot. Cell proliferation and apoptosis were examined using the CCK-8 assay, colony formation assay, and flow cytometry, respectively. The interaction between miR-143-3p and HK2 was verified by dual-luciferase reporter gene assay. We also evaluated the influence of RES on the tumor growth and Warburg effect in vivo. RES treatment significantly decreased glycolysis, cell proliferation and induced apoptosis of both MDA-MB-453 and MCF-7 cells. Simultaneously, the expression of HK2 was decreased in breast cancer cells treated with RES, which was positively associated with tumor size and glycolysis. Moreover, HK2 was a direct target gene of miR-143-3p. Mechanistically, upregulation of miR-143-3p by RES treatment inhibited tumor growth by downregulating HK2-mediated Warburg effect in breast cancer. Our findings suggested that RES exerted anti-tumorigenesis and anti-glycolysis activities in breast cancer through upregulating the inhibitory effect of miR-143-3p on HK2 expression, which provided a new potential strategy for breast cancer clinical treatment.

Knockdown of SF3B1 inhibits cell proliferation, invasion and migration triggering apoptosis in breast cancer via aberrant splicing.

Splicing factor 3b subunit 1 (SF3B1) was frequently reported to be significantly mutated in breast cancer. However, the status of SF3B1 expression, its function and molecular consequence in breast cancer remained unreported. Immunohistochemistry was used to assess SF3B1expression in 110 breast cancer samples. SF3B1 knock‑down in ZR-75-30 and MDA-MB-231 cells was performed by shRNA transfection. The expression of SF3B1 in cells was detected by quantitative real‑time PCR and western blot. Cell proliferation ability was determined by MTT and colony formation assay. Migration and invasion were determined by transwell assay. Flow cytometry was performed to investigate cell cycle and apoptosis. RNA-sequencing was performed to examine differentially expressed genes and affected alternative splicing events. SF3B1 is overexpressed in breast cancer tissues compared with normal tissues. Overexpression of SF3B1 is associated with lymph node metastasis. SF3B1 knockdown in MDA-MB-231 and ZR-75-30 breast cancer cells significantly induced the suppression of proliferation, migration, invasion and also enhancement of apoptosis. RNA-sequencing data revealed that 860 genes were significantly up-regulated and 776 genes were significantly down-regulated upon SF3B1 knockdown. Differentially expressed genes enriched in the signaling pathways including Ras signaling pathway; cytokine receptor interaction; tight junction; MAPK signaling pathway, Glycine, serine and threonine metabolism. Alternative splicing analysis revealed that exon skipping (SKIP) and cassette exons (MSKIP) were the most common molecular effect upon SF3B1 knockdown. Our study suggests that SF3B1 may be an important molecular target for breast cancer treatment and provides a new clue for clinical treatment of breast cancer.

Inhibition of α-enolase affects the biological activity of breast cancer cells by attenuating PI3K/Akt signaling pathway.

This study aimed at exploring the role of α-enolase (ENO1) in proliferation, invasion, and cell apoptosis in MDA-MB-231 and MCF-7 breast cancer human cells, to provide a theoretical basis for the clinical treatment of breast cancer. MDA-MB-231 and MCF-7 cells were randomly divided into five groups: normal control group (Control group), negative control group (negative control virus, NC group), and shENO1 (sh1, sh2, and sh3) group, respectively. The expressions of ENO1 mRNA and protein were measured by Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Cell proliferation, cell invasion ability, and cell apoptosis rate were detected by methyl thiazolyl tetrazolium (MTT) assay, transwell invasion assay, and flow cytometer, respectively. The expressions of the proteins correlated with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway were analyzed by Western blot. In MDA-MB-231 and MCF-7 cells, the gene and protein expressions of ENO1 in the three sh groups in MDA-MB-231 and MCF-7 cells were significantly lower than those in control group and NC group. In MDA-MB-231 and MCF-7 cells, the gene and protein expressions of ENO1 in the three sh groups were significantly lower than those in control group and NC group. Compared with NC group, the proliferation activity, invasion ability, and apoptosis rate of shENO1 group were significantly decreased (p < 0.01). PI3K and Akt protein levels in shENO1 group were significantly downregulated (p < 0.01). Bcl-2 protein expression was markedly upregulated (p < 0.01), meanwhile Bax protein revealed a significant reduction (p < 0.01). The results revealed that silencing ENO1 reduced proliferation activity, invasion ability, and apoptosis rate of breast cancer cells by decreasing the phosphorylation of PI3K and Akt pathway. Our results suggested that ENO1 may be a potential therapeutic target in breast cancer.

LINC00365-SCGB2A1 axis inhibits the viability of breast cancer through targeting NF-κB signaling.

Breast cancer is the most common high-grade malignancy in women. The lack of therapeutic targets has limited the treatment of breast cancer. Recently, long noncoding RNAs (lncRNAs) have been demonstrated to be dysregulated in various types of cancer. However, the specific mechanisms by which lncRNAs influence breast cancer have remained largely unclear. To bridge this research gap, the present study focused on the lncRNA LINC00365, which is expressed at a low level in breast cancer. Secretoglobin family 2A member 1 (SCGB2A1) was identified as a potential target protein regulated by LINC00365. The results of the present study demonstrated that the overexpression of LINC00365 and SCGB2A1 inhibited cell viability and induced cell apoptosis through the inhibition of the NF-κB signaling pathway in breast cancer cells. These findings indicated that LINC00365 may serve a crucial role in breast cancer and may be considered as a novel target for the clinical treatment of breast cancer.

Stimuli‐Responsive Scaffold for Breast Cancer Treatment Combining Accurate Photothermal Therapy and Adipose Tissue Regeneration

AbstractDevelopment of new therapeutic scaffolds to selectively destruct tumors under gentle conditions meanwhile promoting adipose tissue formation would be a promising strategy for clinical treatment of breast cancer. Herein, a stimuli‐responsive scaffold composed of polyacrylic acid‐g‐polylactic acid (PAA‐g‐PLLA) modified graphene oxide (GO) with a cleavable bond in between (GO‐PAA‐g‐PLLA), gambogic acid (GA), and polycaprolactone (PCL) is fabricated and then preseeded on adipose‐derived stem cells (ADSCs) for breast cancer treatment. This GO–GA‐polymer scaffold is able to simultaneously perform pH‐triggered low temperature (45 °C) photothermal therapy to selectively induce the apoptosis of tumor cells and significantly improve ADSCs growth without any photothermal damage. The low‐temperature photothermal therapy of the scaffolds can induce more than 95% of cell death for human breast cancer (MCF‐7) in vitro, which further completely inhibits tumor growth and finally eliminates tumor tissue in mice. Meanwhile, the prepared GO–GA‐polymer scaffold possesses the improved capability to stimulate the differentiation of ADSCs into adipocytes by upregulating adipo‐related gene expression, and significantly promotes new adipose tissue formation whether with or without NIR irradiation. These results successfully demonstrate that the prepared GO–GA‐polymer scaffolds with bifunctional properties will be a promising candidate for clinical cases involving both tumor treatment and tissue engineering.

MicroRNA-222 promotes drug resistance to doxorubicin in breast cancer via regulation of miR-222/bim pathway.

Resistance to doxorubicin (DOX) is the most common clinical problem in breast cancer therapy, and the underlying molecular mechanism remains to be investigated. MicroRNAs (miRNAs) exhibit important regulatory functions in various malignant tumors including breast cancer. The aim of the present study was to find the relationship between miR-222 and DOX resistance. We found that miR-222 was highly expressed in patients’ serum and DOX-resistant cell line MCF-7-R and that miR-222 could promote proliferation and migration of breast cancer cells. Our results also showed that inhibition of miR-222 in MCF-7-R significantly increased Bcl-2 interacting mediator (Bim) expression both in mRNA and protein levels by using quantitative real-time PCR (qRT-PCR) and Western blot. MTT and flow cytometry suggested that lower expressed miR-222 enhanced apoptosis and decreased IC50 of MCF-7-R cells. Conversely, in MCF-7 cells transfected with miR-222 mimics, up-regulation of miR-222 was associated with decreased Bim level accompanied by less apoptosis and higher IC50. Moreover, miR-222 inhibitors reversed DOX resistance via miR-222-Bim-caspase pathway. Collectively, these data first elucidated that miR-222 could function as an oncogene and was able to reduce the sensitivity of breast cancer cells to DOX through miR-222-Bim-caspase pathway, which provided a potential target to increase DOX sensitivity in clinical breast cancer treatment.

NAMPT regulates PKM2 nuclear location through 14-3-3ζ: Conferring resistance to tamoxifen in breast cancer.

The resistance against tamoxifen therapy has become one of the major obstacles in the clinical treatment of breast cancer. Nicotinamide phosphoribosyltransferase (NAMPT) is an essential enzyme catalyzing nicotinamide adenine dinucleotide biosynthesis and is important for tumor metabolism. The study here sought to explore the effect of NAMPT on breast cancer survival with tamoxifen conditioning. We found that NAMPT was highly expressed in breast cancer cells compared with normal mammary epithelial cells. Inhibition of NAMPT by FK866 inhibited cell viability and aggravated apoptosis in cancer cells treated with 4-hydroxytamoxifen. NAMPT overexpression upregulated 14-3-3ζ expression. Knockdown of 14-3-3ζ reduced cell survival and promoted apoptosis. Activation of Akt signaling, rather than ERK1/2 pathway, is responsible for 14-3-3ζ regulation by NAMPT overexpression. Furthermore, NAMPT overexpression led to PKM2 accumulation in the cell nucleus and could be dampened by 14-3-3ζ inhibition. In addition, NAMPT overexpression promoted xenografted tumor growth and apoptosis in nude mice, while 14-3-3ζ inhibition attenuated its effect. Collectively, our data demonstrate that NAMPT contributes to tamoxifen resistance through regulation of 14-3-3ζ expression and PKM2 translocation.

Ablative Treatment of Breast Cancer; Are We There Yet?

Breast cancer treatment has evolved through continuous integration of technological advances and changes in our understanding of tumor biology. Radical mastectomy was the best operation for the technology and beliefs of its time. Advances in technology, the acknowledgement that care is best delivered through a multi-modal approach, and continued investigation have resulted in less disfiguring procedures. Ablative therapy for the treatment of breast cancer is the natural continuation of this trend. Data from breast cancer ablation trials utilizing various energy forms have been favorable and have led to the development of ongoing multi-center treat and observe studies. This paper examines the current status of cryoablation, laser ablation, and focused ultrasound ablation for the treatment of breast cancers. The advantages and shortcomings of each technique are considered and the challenges to be met in order for ablative therapy to become a mainstream clinical treatment of breast cancer are discussed.

An In silico Approach to Identify High Affinity Small Molecule Targeting m-TOR Inhibitors for the Clinical Treatment of Breast Cancer

Breast cancer is the most frequent malignancy among women. It is a heterogeneous disease with different subtypes defined by its hormone receptor. A hormone receptor is mainly concerned with the progression of the PI3K/AKT/mTOR pathway which is often dysregulated in breast cancer. This is a major signaling pathway that controls the activities such as cell growth, cell division, and cell proliferation. The present study aims to suppress mTOR protein by its various inhibitors and to select one with the highest binding affinity to the receptor protein. Out of 40 inhibitors of mTOR against breast cancer, SF1126 was identified to have the best docking score of -8.705, using Schrodinger Suite which was further subjected for high throughput screening to obtain best similar compound using Lipinski’s filters. The compound obtained after virtual screening, ID: ZINC85569445 is seen to have the highest affinity with the target protein mTOR. The same result based on the binding free energy analysis using MM-GBSA showed that the compound ZINC85569445 to have the the highest binding free energy. The next study of interaction between the ligand and receptor protein with the pharmacophore mapping showed the best conjugates, and the ZINC85569445 can be further studied for future benefits of treatment of breast cancer.

Beclin1 inhibition enhances paclitaxel‑mediated cytotoxicity in breast cancer invitro and invivo.

Beclin1, a key regulator of autophagy, has been demonstrated to be associated with cancer cell resistance to chemotherapy. Paclitaxel is a conventional chemotherapeutic drug used in the clinical treatment of breast cancer. However, the function and mechanism of Beclin1 in paclitaxel‑mediated cytotoxicity in breast cancer are not well defined. The present study demonstrated that paclitaxel suppressed cell viability and Beclin1 expression levels in BT‑474 breast cancer cells in a dose‑ and time‑dependent fashion. Compared with the control, the knockdown of Beclin1 significantly enhanced breast cancer cell death via the induction of caspase‑dependent apoptosis following paclitaxel treatment in vitro (P<0.05). In a BT‑474 xenograft model, paclitaxel achieved substantial inhibition of tumor growth in the Beclin1 knockdown group compared with the control group. Furthermore, analysis of the publicly available Gene Expression Omnibus datasets revealed a clinical correlation between Beclin1 levels and the response to paclitaxel therapy in patients with breast cancer. Collectively, the present results suggest that Beclin1 protects breast cancer cells from apoptotic death. Thus, the inhibition of Beclin1 may be a novel way to improve the effect of paclitaxel. Additionally, Beclin1 may function as a favorable prognostic biomarker for paclitaxel treatment in patients with breast cancer.

Caveolin-1: a multifaceted driver of breast cancer progression and its application in clinical treatment.

Human breast cancer is one of the most frequent cancer diseases and causes of death among female population worldwide. It appears at a high incidence and has a high malignancy, mortality, recurrence rate and poor prognosis. Caveolin-1 (Cav1) is the main component of caveolae and participates in various biological events. More and more experimental studies have shown that Cav1 plays a critical role in the progression of breast cancer including cell proliferation, apoptosis, autophagy, invasion, migration and breast cancer metastasis. Besides, Cav1 has been found to be involved in chemotherapeutics and radiotherapy resistance, which are still the principal problems encountered in clinical breast cancer treatment. In addition, stromal Cav1 may be a potential indicator for breast cancer patients’ prognosis. In the current review, we cover the state-of-the-art study, development and progress on Cav1 and breast cancer, altogether describing the role of Cav1 in breast cancer progression and application in clinical treatment, in the hope of providing a basis for further research and promoting CAV1 gene as a potential target to diagnose and treat aggressive breast cancers.

Prevalence and Reclassification of &lt;i&gt;BRCA1&lt;/i&gt; and &lt;i&gt;BRCA2&lt;/i&gt; Variants in a Large Unselected Chinese Han Breast Cancer Cohort

Background: Accurate interpretation of BRCA1/2 variants is critical for risk assessment, enhanced cancer surveillance, prevention strategies, and precise clinical treatment of breast cancer. However, current worldwide standards depend primarily on sequencing information from Caucasian populations. Methods: Panel-based sequencing served to detect BRCA1/2 variants in a Chinese multicenter cohort of 21,216 breast cancer patients (BCs) and 6,434 healthy controls (HCs). Findings: 1,245 pathogenic variants of BRCA1/2, 262 of which were novel (21.04%), and 2,517 variants of uncertain significance (VUS) were identified. Unlike in the Caucasian population, BRCA2 variants were more frequent than BRCA1 variants (56.34% versus 43.66%). The 48 most common variants accounted for 39.86% of all pathogenic variants, and were not reported as common variants in Caucasians. Furthermore, we unambiguously reclassified VUS in 508 subjects, lowering the VUS ratio from 9.05% to 7.21%. Pathogenic BRCA1/2 variants correlated with younger onset age, higher frequencies of bilateral and triple-negative BC, invasive carcinomas, high histological grades, and family history of BC and other cancers. Interpretation: Strong ethnicity-specific occurrences of pathogenic BRCA1/2 variants were identified in the Chinese population. Hence, the findings provide rationale and sequencing information for the implementation of BRCA1/2 variants tailored to the Chinese population into clinical risk assessment. Funding Statement: National Natural Science Foundation of China 81772802, Shanghai science and technology innovation action plan of China 17JC1404400 and 16DZ19102, Special Funds for Technological Innovation of Shanghai Jiao Tong University YG2016MS22, Chinese Academy of Science XDA12020223 (W.H.X), Natural Science Foundation of Zhejiang Province LZ16H160002 (C.Y.D), Zhejiang Provincial Program for the Cultivation of High-level Innovative Health Talents (C.Y.D). Declaration of Interests: Authors declare no potential conflicts of interest. Ethics Approval Statement: The study was approved by the Ethics Committee of all the hospitals involved and was performed according to the Declaration of Helsinki Principles. Written consent to notify blood samples for future research was obtained for each patient under the Institutional Review Board (IRB)-approved protocol.

Abnormally elevated USP37 expression in breast cancer stem cells regulates stemness, epithelial-mesenchymal transition and cisplatin sensitivity

BackgroundRecent studies have indicated that deubiquitinating enzymes (DUBs) are related to the stem-cell pathway network and chemo-resistance in cancer. Ubiquitin-specific peptidase 37 (USP37), a novel DUB, was identified to be a potential factor associated with tumor progression. However, the biological functions of USP37 in breast cancer remain unclear.MethodsThe distribution of USP37 expression in breast cancer and the correlation between USP37 expression and the overall survival rate were detected by The Cancer Genome Atlas (TCGA) database. Gene set enrichment analysis (GSEA) was utilized to evaluate potential mechanism of USP37 in breast cancer. The USP37 expression in breast cancer tissues and breast cancer cell lines were detected by immunohistochemistry and western blotting. Sorting of breast cancer stem cells (BCSCs) were by using MACS assay. In vitro and in vivo assays were performed to examine the biological functions of USP37 in breast cancer cells. MG132, CHX chase, immunofluorescence staining and co-immunoprecipitation assays were used to test the interaction between USP37 and Gli-1.ResultsBioinformatics analysis demonstrated that USP37 gene was elevated in breast cancer tissues and its overexpression was strongly correlated with the increased mortality rate. GSEA analysis showed that USP37 expression was positively associated with cell growth and metastasis while negatively related to cell apoptosis in the TCGA breast cancer samples. USP37 expression was elevated in breast cancer tissues and breast cancer cell lines. Moreover, we also detected that USP37 was overexpressed in BCSCs. USP37 regulated the ability of cell invasion, epithelial-mesenchymal transition (EMT), stemness and cisplatin sensitivity in breast cancer cell lines. Additionally, USP37 knockdown inhibited tumorigenicity and increased anticancer effect of cisplatin in vivo. Knockdown of USP37 significantly decreased hedgehog (Hh) pathway components Smo and Gli-1. Gli-1 was stabilized by USP37 and they interacted with each other. Further studies indicated that USP37 knockdown could inhibit the stemness, cell invasion and EMT in breast cancer via downregulation of Hh pathway.ConclusionsThese findings reveal that USP37 is highly expressed in BCSCs and is correlated with poor prognosis in breast cancer patients. USP37 can regulate the stemness, cell invasion and EMT via Hh pathway, and decreased USP37 confers sensitivity to cisplatin in breast cancer cells. USP37 is required for the regulation of breast cancer progression, as well as a critical target for clinical treatment of breast cancer.

Tumor suppressor let-7a inhibits breast cancer cell proliferation, migration and invasion by targeting MAGE-A1.

Let-7 was one of the earliest discovered miRNAs and while it reportedly acts as a tumor suppressor in various solid tumors, its function in breast cancer has not been fully studied. Therefore, we examined let-7a and MAGE-A1 expression in breast tissues by qRT-PCR and found that let-7a expression significantly correlates with larger tumor size, higher histological grade (p<0.05) and is significantly lower in patients with Her-2-positive cancers and Ki-67 >14% (p=0.028 and p=0.023). MAGE-A1 expression incidence is 50.8% (33/65) and it inversely correlates with let-7a expression (p=0.008). let-7a inhibition of breast cancer cell proliferation, migration and invasion was also observed in in vitro cell culture experiments, and dual-luciferase reporter assays showed that melanoma-associated antigen A1 (MAGE-A1) was its target gene; the target comprised bases 451-457 of the 3'UTR region of the MAGE-A1 mRNA. RT-qPCR and Western blot analyses showed that let-7a inhibited MAGE-A1 expression at both the nucleic acid and protein levels. In our final co-transfection experiment, we targeted MAGE-A1 in a breast cancer cell line and observed that let-7a inhibited cell proliferation, migration and invasion. These combined results confirm that let-7a functions as a tumor suppressor by targeting MAGE-A1 in breast cancer and it therefore provides a novel target in breast cancer clinical treatment.

Reversal of epigenetic aberrations associated with the acquisition of doxorubicin resistance restores drug sensitivity in breast cancer cells

Acquired resistance against doxorubicin is a major limitation in clinical treatment of breast cancer. The molecular mechanism behind the aberrant expression of genes leading to doxorubicin resistance is not clear. Epigenetic changes play an important role in the regulation of gene expression. Therefore, the objective of this study was to identify the epigenetic mechanism underlying acquired doxorubicin resistance in breast cancer cells. Doxorubicin-resistant cells were selected by repeated exposure of MCF-7 and MDA-MB-231 breast cancer cell lines to clinically relevant doses of doxorubicin for 18 months. MTT assay, cell cycle analysis, colony formation, qRT-PCR, and Western blot analyses were used to characterize the epigenetic and molecular mechanism. Pyrosequencing was used to detect MSH2 promoter hypermethylation. Aberrant expression of epigenetic regulatory genes, a significant increase in H3 acetylation and methylation, as well as promoter hypermethylation-mediated inactivation of MSH2 gene were associated with the acquired resistant phenotype. Demethylating agent 5-Aza-deoxycytidine and HDAC inhibitor Trichostatin A significantly re-sensitized resistant cells to doxorubicin. Findings of this study revealed that epigenetic aberrations including promoter hypermethylation-mediated inactivation MSH2 contribute to the acquisition of doxorubicin resistance in breast cancer cells. Additionally, our data suggest that some of these epigenetic aberrations are progressive during resistance development and therefore can potentially be used as biomarkers for early detection of resistance. These epigenetic aberrations, being reversible, can also serve as targets for epigenetic therapy to re-sensitize doxorubicin-resistant breast cancer cells. Epigenetic inactivation of mismatch repair gene MSH2 further suggests that loss of MMR-dependent apoptotic potential could be a novel mechanistic basis for the acquisition of doxorubicin resistance in breast cancer cells.

PO-432 New combined nanoparticle therapy inhibits metastatic breast tumour growth with superior efficacy and lower side effect profile to docetaxel

IntroductionCombining molecular therapies with chemotherapy may offer an improved clinical outcome for chemoresistant tumours. Sphingosine kinase 1 (SK1) is a proto-oncogene that is highly expressed in breast cancer, especially in oestrogen receptor (ER) negative tumours. SK1 inhibitor FTY720 has promising anticancer properties as monothrerapy. In this study, we have developed and tested polymer and silicon nanoparticles combining docetaxel and FTY720 for enhanced anticancer effect, targeted tumour delivery and reduced systemic toxicity.Material and methodsDocetaxel, FTY720 and glucosamine were embedded or covalently conjugated to poly(lactic-co-glycolic acid) or silicon nanopores. Nanoparticles were characterised by dynamic light scattering and electron microscopy. The cellular uptake, cytotoxicity and in vivo antitumor efficacy of nanoparticles were evaluated.Results and discussionsOur data indicate that in ER negative breast cancer cells FTY720 provides chemosensitisation to docetaxel, allowing a four-fold reduction in the effective dose. We have encapsulated both drugs in nanoparticles, with narrow size distribution of ~100 nm and excellent cancer cell uptake providing sequential, sustained release of both drugs. In mouse models of human ER negative breast cancer nanoparticles had superior efficacy to systemic free docetaxel. Both polymer and silicon nanoparticles had significantly lower side effect profile including reduction of chemotherapy-induced weight loss, liver toxicity and neutropenia.ConclusionHere we have shown for the first time that FTY720 can sensitise ER negative breast cancer to docetaxel. We further demonstrate that encapsulation of free drugs in nanoparticles can improve targeting, provide low off-target toxicity and enhance antitumour efficacy offering potential therapeutic use of FTY720 in clinical breast cancer treatment.