Levels In Breast Cancer Tissues Research Articles

Abstract 3826: The zinc transporters, ZnT5 and ZnT6, in breast cancer

Abstract The zinc levels in breast cancer tissue were reported to be higher than those in normal tissue. In addition, the expression levels of zinc transporters, including ZnT5 and ZnT6, in breast cancer were reported to be higher than those in normal breast tissue. ZnT5 and ZnT6 have been also reported to contribute to the formation of heterodimers and be involved in several different biological functions. Among those, autotaxin, matrix metalloproteinase 9, and carbonic anhydrase IX, which have crucial roles in tumor formation, invasion, and metastasis, were reported to require ZnT5–ZnT6 heterodimers for their activation and secretion in the chicken DT40 cells. However, the functions of ZnT5 and ZnT6 heterodimers has remained virtually unknown in breast cancer. Therefore, we first immunolocalized ZnT5 and ZnT6 in pathology specimens of breast cancer. We examined 88 breast cancer specimens in this study. We then analyzed the interaction between ZnT5 and ZnT6 using proximity ligation assay (PLA) in breast cancer tissues and further evaluated cell proliferation and migration in ZnT5 knockdown MCF-7 and T-47D cells using siRNA. Immunohistochemical analysis revealed that the number of ZnT5-positive breast cancer cells was significantly higher in HER2-positive patients and the number of ZnT6-positive breast cancer cells was significantly higher in patients with low histological grade and high ER and PgR expressions. The number of ZnT5 and ZnT6 double-positive breast cancer cells tended to be associated with low stage (p = 0.0577). In addition, ZnT5 and ZnT6 interaction was detected using PLA through high expression of both ZnT5 and ZnT6 in breast cancer tissues. ZnT5 knockdown exerted no effect on cell proliferation in the presence of 100 µM ZnCl2 in MCF-7 and T-47D cells. The 100 µM ZnCl2 treatment inhibited cell migration in MCF-7 and T-47D cells but ZnT5 knockdown promoted cell migration. To the best of our knowledge, this is the first study demonstrating the expressions of ZnT5 and ZnT6 in breast cancer. The results of this study also indicated that both ZnT5 and ZnT6 could be involved in less aggressive subtypes of breast cancer possibly through inhibition of cell migration. Further studies are required to clarify the clinical significance of their interactions in patients with breast cancer. Citation Format: Erina Iwabuchi, Yasuhiro Miki, Takanori Ishida, Hironobu Sasano. The zinc transporters, ZnT5 and ZnT6, in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3826.

Circular RNA PVT1 promotes the invasion and epithelial-mesenchymal transition of breast cancer cells through serving as a competing endogenous RNA for miR-204-5p.

PurposeCircular RNAs (circRNAs) are a class of covalently closed circular RNA transcripts and have been found to regulate the progression of human malignancies. The objective of this study was to identify the functions of circRNAs in breast cancer (BCa).Patients and methodsBCa and adjacent non-cancerous tissues were collected from 99 patients. Kaplan–Meier analysis was used to analyze the relationship between circPVT1 and prognosis. CircPVT1 expression levels in BCa tissues and cell lines were detected via PCR. Transfection technology was used to silence circPVT1 and overexpress miR-204-5p. Cell biological behavior was analyzed, and epithelial-mesenchymal transition (EMT) related proteins were detected by Western blot. In vivo experiments were performed with the subcutaneous xenograft tumor model.ResultsCircPVT1 was markedly overexpressed in BCa tissues and cell lines. Higher expression of circPVT1 was correlated with poor prognosis of BCa patients. Knockdown circPVT1 significantly suppressed the proliferation, migration and invasion of BCa cells invitro, and suppressed BCa tumor growth invivo. CircPVT1 knockdown upregulated E-cadherin and downregulated N-cadherin, Vimentin, Slug and Twist in BCa cells. Moreover, circPVT1 could serve as a competing endogenous RNA (ceRNA) for miR-204-5p, and restoration of miR-204-5p abrogated the oncogenic role of circPVT1 in BCa cells.ConclusionCircPVT1 as a potentially valuable biomarker for BCa diagnosis and therapeutic target for BCa treatment. CircPVT1 might promote the invasion and EMT of BCa cells by serving as aceRNA for miR-204-5p.

The low expression of miR-1976 in plasma samples indicating its biological functions in the progression of breast cancer.

The incidence of breast cancer (BC) is the highest among women. Identification of miRNAs as biomarkers may help to improve the diagnosis of BC. The purpose of this study was to assess the expression levels of miR-1976 in plasma samples and the biological functions in the progression of BC. The expression levels of miR-1976 in plasma samples and tissues were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The associations between the expression levels and clinicopathological features were studied. Cell supernatants were used to simulate circulation. The biological functions of miR-1976 were assessed in vitro and in vivo. The expression levels of miR-1976 in plasma samples were found significantly lower in patients with BC than those in healthy controls, and were associated with Ki-67. The expression levels in BC tissues were lower than those in adjacent normal tissues, and were correlated with the number of lymph nodes and Ki-67. The expression levels in BC cell supernatants and cell lines were lower than that in normal human breast epithelial cell line HBL-100. miR-1976 knockdown promoted proliferation in vitro and in vivo. miR-1976 may serve as a promising non-invasive biomarker for the diagnosis of BC in the future.

SIRT7 Is a Prognostic Biomarker Associated With Immune Infiltration in Luminal Breast Cancer.

Background: Sirtuin 7 (SIRT7), a protein-coding gene whose abnormal expression and function are associated with carcinogenesis. However, the prognosis of SIRT7 in different breast cancer subtypes and its correlation with tumor-infiltrating lymphocytes remain unclear.Methods: The expression and survival data of SIRT7 in patients with breast cancer were analyzed using Tumor Immune Estimation Resource (TIMER), Gene Expression Profiling Interaction Analysis (GEPIA), The Human Protein Atlas (HPA), UALCAN, Breast Cancer Gene-Expression Miner (BC-GenExMiner), and Kaplan-Meier plotter databases. Also, the expression correlations between SIRT7 and immune infiltration gene markers were analyzed using TIMER and further verified the results using immunohistochemistry.Results: SIRT7 exhibited higher expression levels in breast cancer tissues than the adjacent normal tissues. SIRT7 expression was significantly correlated with sample type, subclass, cancer stage, menopause status, age, nodal status, estrogen receptor (ER), progesterone receptor (PR), and triple-negative status. High SIRT7 expression was associated with poor prognosis in breast cancer-luminal A [overall survival (OS): hazard ratio (HR) = 1.54, p = 1.70e-02; distant metastasis-free survival (DMFS): HR = 1.56, p = 2.60e-03]. Moreover, the expression of SIRT7 was positively correlated with the expression of IRF5 (M1 macrophages marker, r = 0.165, p = 1.13e-04) and PD1 (T cell exhaustion marker, r = 0.134, p = 1.74e-03). These results suggested that the expression of SIRT7 was related to M1 macrophages and T cell exhaustion infiltration in breast cancer-luminal.Conclusions: These findings demonstrate that the high expression of SIRT7 indicates poor prognosis in breast cancer as well as increased immune infiltration levels of M1 macrophages and T cell exhaustion in breast cancer-luminal. Thus, SIRT7 may serve as a candidate prognostic biomarker for determining prognosis associated with immune infiltration in breast cancer-luminal.

SAPCD2 promotes invasiveness and migration ability of breast cancer cells via YAP/TAZ.

The aim of this study was to explore whether SAPCD2 can affect the proliferation of breast cancer cells through YAP/TAZ, thus promoting the development of breast cancer (BCa). Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine SAPCD2 expression level in BCa tissues collected from patients in different tumor TNM-stage. The correlation between SAPCD2 expression and clinicopathological features of patients were analyzed, and the Kaplan-Meier test was used for survival analysis. In addition, after knocking down SAPCD2 in cells, qRT-PCR and Western blot were applied to analyze the expression of the related genes and proteins, respectively. Moreover, Cell Counting Kit-8 (CCK-8) and transwell experiments were conducted to detect cell viability, migration, and invasion abilities. Furthermore, the changes in cell viability and migration and invasion abilities were examined after the simultaneous overexpression of YAP. It was found that SAPCD2 expression levels in BCa tissues were remarkably higher than those in the normal control samples; meanwhile, patients with tumor size >3 cm or in the T3+T4 stage had a relatively higher expression of SAPCD2 than those with tumor size <3 cm or in T1+T2 stage. At the same time, the overall survival rate of BCa patients with highly expressed SAPCD2 was remarkably lower than that of patients in the low expression group. Moreover, it was found that the SAPCD2 level was correlated to the tumor size, TNM stage, and lymph node metastasis. After knocking down SAPCD2 in cells, cell viability, migration, and invasion abilities, as well as the YAP/TAZ protein expression levels were all found to be remarkably attenuated, which, however, were reversed after simultaneous overexpression of YAP in cells. SAPCD2 may be able to enhance the proliferation ability of BCa cells via modulating the expression of YAP/TAZ, thereby prompting the progression of BCa.

MiR-7 reduces the BCSC subset by inhibiting XIST to modulate the miR-92b/Slug/ESA axis and inhibit tumor growth

BackgroundBreast cancer stem cells (BCSCs) are typically seed cells of breast tumor that initiate and maintain tumor growth. MiR-7, as a cancer inhibitor, decreases the BCSC subset and inhibits tumor progression through mechanisms that remain unknown.MethodsWe examined miR-7 expression in breast cancer and developed a BCSC-driven xenograft mouse model, to evaluate the effects of miR-7 overexpression on the decrease of the BCSC subset in vitro and in vivo. In addition, we determined how miR-7 decreased the BCSC subset by using the ALDEFLUOR, lentivirus infection, dual-luciferase reporter, and chromatin immunoprecipitation-PCR assays.ResultsMiR-7 was expressed at low levels in breast cancer tissues compared with normal tissues, and overexpression of miR-7 directly inhibited lncRNA XIST, which mediates the transcriptional silencing of genes on the X chromosome, and reduced epithelium-specific antigen (ESA) expression by increasing miR-92b and inhibiting slug. Moreover, miR-7 suppressed CD44 and ESA by directly inhibiting the NF-κB subunit RELA and slug in breast cancer cell lines and in BCSC-driven xenografts, which confirmed the antitumor activity in mice injected with miR-7 agomir or stably infected with lenti-miR-7.ConclusionsThe findings from this study uncover the molecular mechanisms by which miR-7 inhibits XIST, modulates the miR-92b/Slug/ESA axis, and decreases the RELA and CD44 expression, resulting in a reduced BCSC subset and breast cancer growth inhibition. These findings suggest a potentially targeted treatment approach to breast cancer.

EGFR Promotes the Development of Triple Negative Breast Cancer Through JAK/STAT3 Signaling.

PurposeTo investigate the role of EGFR and STAT3 in breast cancer development and progression.MethodsThrough bioinformatics analysis differently expressed genes (DEGs) including EGFR and STAT3 were identified in breast cancer tissue. QRT-PCR and Western blot analysis were used to investigate EGFR and STAT3 levels in breast cancer tissues and cells. The influence of EGFR and STAT3 on the breast cancer cell proliferation (CCK-8 assay, clone formation assays), migration (wound healing assays) and invasion (transwell assays) were investigated. The influence of EGFR on breast cancer in vivo was examined by Nude mouse transplantation tumor experiments and immunohistochemistry (IHC) staining. The effects of EGFR on breast cancer signaling were assessed via Western blot.ResultsBoth EGFR and p-STAT3 were up-regulated in breast cancer tissues and cell lines. EGFR expression was positively associated with p-STAT3. Moreover, EGFR and p-STAT3 activity enhanced the proliferation and invasion of tumor cells. Breast cancer cell growth was dramatically inhibited by EGFR silencing in vivo.ConclusionEGFR promotes breast cancer progression via STAT3 phosphorylation and JAK/STAT3 signaling.

PARPBP is a prognostic marker and confers anthracycline resistance to breast cancer.

Background:PARPBP (PARP1 binding protein) is an important suppressor of homologous recombination during DNA repair, but the expression and function of PARPBP in breast cancer remain unclear.Methods:PARPBP expression was analyzed in breast cancer patient samples and public datasets for its correlation with clinical outcome. The function of PARPBP in breast cancer cell proliferation and anthracycline treatment response were studied both in vitro and in vivo.Results:PARPBP was upregulated significantly at both mRNA and protein levels in breast cancer tissues compared with normal breast tissues. PARPBP high expression group had poorer overall survival (OS) than the PARPBP low expression group. Knockdown of PARPBP suppressed breast cancer cell proliferation and colony formation while overexpression of PARPBP did the opposite. We found that transcription factor forkhead box M1 (FOXM1) could activate PARPBP expression by directly binding to the promoter of PARPBP. In addition, high expression of PARPBP related with anthracycline resistance in breast cancer. Depletion of PARPBP increased breast cancer cell apoptosis and DNA damage caused by epirubicin. Moreover, tumor xenograft experiments further demonstrated that PARPBP was involved in breast cancer anthracycline resistance.Conclusion:Taken together, our results highlight that PARPBP is a prognostic marker and confers anthracycline resistance on breast cancer.

Expression of pendrin and NIS iodide transporters in human breast tumor and peri-tumoral tissue.

IntroductionThyroid iodide transporters, Na+/I– symporter (NIS) and pendrin (PDS), are responsible for supplying this vital micronutrient for thyroid hormone synthesis by thyroid peroxidase (TPO). Both proteins were shown to be expressed, apart from the thyroid, also in other human tissues, including lactating mammary gland. NIS expression in human breast cancers has been widely studied. On the other hand, until now PDS mRNA levels in breast tumor tissue have been estimated only in high throughput analyses. Previously, we have observed that TPO is expressed in normal and cancerous human breast tissues and shows enzymatic activity. However, biochemical activity of TPO in human breast cancer cells requires iodide transport by NIS and PDS. Therefore, to extend our previous study on TPO expression and function in human breast tumors we performed analysis of NIS and PDS levels in the same group of patients.Material and methodsThe study involved detection of NIS and PDS protein levels by immunohistochemistry and Western blotting, as well as mRNA levels by real-time quantitative polymerase chain reaction.ResultsHere we provide direct evidence that NIS and PDS are expressed in human breast cancer tissue, with NIS levels being increased and PDS levels decreased in tumor tissue. Interestingly, PDS mRNA levels in breast cancer tissue seem to be influenced by the estrogen receptor status and age of the patients, while NIS mRNA levels were dependent on histological type of the tumor.ConclusionsThis study provides valuable information important for consideration in diagnostic or therapeutic application of radioiodine in breast cancer management.

&lt;p&gt;Down-regulation of CCL17 in cancer-associated fibroblasts inhibits cell migration and invasion of breast cancer through ERK1/2 pathway&lt;/p&gt;

ObjectiveCancer-associated fibroblasts (CAFs) in the tumor microenvironment are involved in cancer development and progression, including breast cancer (BC). Up-regulation of CCL17 was observed in BC and predicted a decrease in overall survival, suggesting an important role of CCL17 in BC development. Nonetheless, little is known about the role of CCL17 in the interaction between CAFs and BC.Materials and methodsReal-time quantitative PCR, Western blot, and enzyme-linked immunosorbent assay were performed to examine C-C motif chemokine ligand 17 (CCL17) and C-C motif chemokine receptor 4 (CCR4) levels in BC tissues and CAFs. Cell proliferation, migration, and invasion of CAFs co-cultured with or without BC cell lines were measured by Cell Counting Kit-8 and Transwell analysis. Expression of CCL17, CCR4, dual specificity phosphatase 6 (DUSP6), matrix metallopeptidase 13 (MMP13), extracellular signal-regulated kinase (ERK) 1/2, and phosphor-ERK1/2 (p-ERK1/2) in BC cell lines co-cultured with or without CAFs was measured by Western blotting.ResultsWe found that BC tissues and CAFs demonstrated higher levels of CCL17 compared with adjacent-normal breast tissues and adjacent-normal fibroblasts (NFs), respectively. CCL17 expression is correlated with lymph nodes, TNM stage and tumor size of BC patients. CCL17 knockdown significantly inhibited CCL17 release, CCR4 expression, and the cell proliferation of CAFs, while CCL17 overexpression demonstrated an inverse effect in NFs. Co-culture with CAFs induced the increases in cell proliferation, migration, invasion, and the expression of CCL17, CCR4, MMP13, and p-ERK1/2 in MCF-7 and MDA-MB-231 cells were markedly reversed by CCL17 knockdown in CAFs. Meanwhile, co-culture with NFs induced the malignant phenotype of MCF-7 cells was markedly enhanced by CCL17 overexpression in NFs. Moreover, DUSP6, a negative regulator of ERK1/2, was dose-dependent decrease in response to recombinant CCL17 and inhibited cell migration, invasion, MMP13 expression, and ERK1/2 activation in MCF-7 cells.ConclusionThe findings of this study suggest that CCL17 may function as a novel biomarker as well as potential therapeutic target against BC and CAF-secreted CCL17 promotes BC cell migration and invasion through the DUSP6-dependent ERK1/2 pathway.

CXCL1 stimulates migration and invasion in ER‑negative breast cancer cells via activation of the ERK/MMP2/9 signaling axis.

Chemokine (C-X-C motif) ligand 1 (CXCL1), a member of the CXC chemokine family, has been reported to be a critical factor in inflammatory diseases and tumor progression; however, its functions and molecular mechanisms in estrogen receptor α (ER)-negative breast cancer (BC) remain largely unknown. The present study demonstrated that CXCL1 was upregulated in ER-negative BC tissues and cell lines compared with ER-positive tissues and cell lines. Treatment with recombinant human CXCL1 protein promoted ER-negative BC cell migration and invasion in a dose-dependent manner, and stimulated the activation of phosphorylated (p)- extracellular signal-regulated kinase (ERK)1/2, but not p-STAT3 or p-AKT. Conversely, knockdown of CXCL1 in BC cells attenuated these effects. Additionally, CXCL1 increased the expression of matrix metalloproteinase (MMP)2/9 via the ERK1/2 pathway. Inhibition of MEK1/2 by its antagonist U0126 reversed the effects of CXCL1 on MMP2/9 expression. Furthermore, immunohistochemical analysis revealed a strong positive association between CXCL1 and p-ERK1/2 expression levels in BC tissues. In conclusion, the present study demonstrated that CXCL1 is highly expressed in ER-negative BC, and stimulates BC cell migration and invasion via the ERK/MMP2/9 pathway. Therefore, CXCL1 may serve as a potential therapeutic target in ER-negative BC.

NEAT1/miR‑124/STAT3 feedback loop promotes breast cancer progression.

The long non‑coding RNA nuclear enriched abundant transcript1 (NEAT1) has important roles in the regulation of multiple cell functions, such as proliferation, apoptosis and migration. However, the mechanism by which NEAT1 regulates breast cancer progression is not well elucidated. In the present study, NEAT1 and microRNA‑124 (miR‑124) levels were detected by reverse transcription‑quantitative PCR in breast cancer tissues and cell lines. STAT3 protein levels were detected by western blot analysis. Cell proliferation and cell cycle distribution were determined using MTT and colony formation assays, and flow cytometry, respectively. The results demonstrated that NEAT1 and STAT3 expression levels were increased in breast cancer tissues compared with normal breast tissues, whereas miR‑124 expression was significantly decreased. Functional analyses revealed that NEAT1 promoted cell proliferation and cell cycle progression in breast cancer cells. Additionally, NEAT1 and STAT3 expression levels were negatively correlated with miR‑124 levels in breast cancer tissues. A direct interaction between miR‑124, and NEAT1 and STAT3, was predicted by bioinformatics analysis and confirmed using a luciferase activity assay. NEAT1 overexpression markedly increased STAT3 protein expression levels, and this effect was reversed by miR‑124 overexpression in breast cancer cells. Furthermore, miR‑124 overexpression partially attenuated the effects of NEAT1 on breast cancer cell proliferation and cell cycle progression. The inhibitory effects of miR‑124 overexpression on the proliferation rate and cell cycle progression were abolished by STAT3 overexpression. In turn, STAT3 silencing inhibited NEAT1 transcription in breast cancer cells. In summary, the present findings revealed that NEAT1 and STAT3 formed a feedback loop via sponging miR‑124 to promote breast cancer progression.

SNHG1 contributes to proliferation and invasion by regulating miR-382 in breast cancer.

Introduction: Long non-coding RNAs (lncRNAs) are key regulators in multiple cancers. lncRNA, SNHG1, was shown to be associated with tumorigenesis. However, little is known about the role SNHG1 plays in breast cancer. The aim of the study was to study the role and underlying mechanism of SNHG1 regulation in breast cancer.Methods: Quantitative real-time PCR was used to measure the levels of SNHG1, miR-382 and ZEB1 levels in breast cancer tissue or cells. The proliferation, colony formation, migration and invasion of breast cancer cells, under SNHG1 knockdown achieved by transfection of SNHG1-specific siRNAs, were assessed by Cell Counting Kit-8, colony forming, scratch wound and transwell assays. Bioinformatical analysis and luciferase assay were used to explore the interaction between SNHG1 and its potential miRNA target. Western blot was used to evaluate the expression of epithelial-to-mesenchymal transition (EMT) markers. MDA-MB-231 cells with or without SNHG1 knockdown were used to initiate tumor xenografts in vivo. Tumor growth and expression of SNHG1, miR-382-5p and EMT markers were evaluated.Results: SNHG1 upregulation was observed in breast cancer tissues and cells. Knockdown of SNHG1 attenuated breast cancer proliferation, colony formation, migration and invasion. A miRNA, miR-382-5p, was identified as the target of SNHG1. A reciprocal negative regulation was found between SNHG1 and miR-382-5p. SNHG1 knockdown attenuated EMT both in vitro and in vivo. miR-382-5p transfection reversed the tumor-promoting role by SNHG1. In vivo, SNHG1 knockdown decreased breast tumor growth.Conclusion: SNHG1 promotes breast cancer through the regulation of miR-382-5p and EMT markers. Our results report SNHG1 as a novel miRNA that govern the progression of breast cancer, providing a potential new therapeutic target in breast cancer.

MiR-145-5p Suppresses Breast Cancer Progression by Inhibiting SOX2

BackgroundIn this study, we aimed to investigate the expression and clinical significance of miR-145-5p and its tumor-suppressive effect in breast cancer patients. MethodsWe used luciferase reporter assay, real-time quantitative reverse transcription polymerase chain reaction and Western blot to identify sex-determining region Y-box2 (SOX2) as the target gene of miR-145-5p. Their expression levels in breast cancer tissues (n = 122) were detected by real-time quantitative polymerase chain reaction. We also applied 3-(4,5-dimethyl- 2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry to reveal the effect of miR-145-5p on proliferation in breast cancer. ResultsmiR-145-5p expression is downregulated in breast cancer tissues and negatively correlated with SOX2 expression. Decreased miR-145-5p expression was significantly associated with larger tumor size, distal metastasis, higher Ki67 expression level, and shorter overall survival. miR-145-5p inhibits breast cancer cell proliferation via targeting SOX2, and multivariate regression showed that both miR-145-5p and SOX2 were unfavorable prognostic factors. ConclusionsmiR-145-5p played a suppressive role in the proliferation of breast cancer cells by targeting SOX2, and miR-145-5p is a putative biomarker for risk assessment in patients with breast cancer.

Bromodomain‑containing protein7 sensitizes breast cancer cells to paclitaxel by activating Bcl2‑antagonist/killer protein.

Our previous study demonstrated that bromodomain-containing protein 7 (BRD7) inhibits cell proliferation and tumor growth, restoring the expression of B-cell lymphoma 2 antagonist/killer (Bak) sensitized breast cancer cells to paclitaxel. However, the association between BRD7 and paclitaxel sensitization, as well as BRD7 and Bak in breast cancer remains unknown. In the present study, immunochemical staining was performed to measure the expression of BRD7 and Bak in breast cancer tissues. Cell Counting Kit-8 assay, flow cytometry and tumor xenograft procedures were performed to evaluate the biological role of BRD7 and Bak in breast cancer cells. Western blotting, reverse transcription-quantitative polymerase chain reaction, chromatin immunoprecipitation and luciferase reporter assays were also performed. BRD7 was positively correlated with Bak levels in breast cancer tissues, and the survival rate of patients with low Bak and BRD7 expression was significantly lower than that of patients with high Bak and BRD7 expression. In addition, BRD7 activated Bak promoter activity and induced Bak expression in an indirect manner. Furthermore, ectopic expression of BRD7 inhibited cell proliferation, tumor growth and sensitized cancer cells to paclitaxel, while knockdown of Bak abolished BRD7-mediated inhibitory effects on cell proliferation and paclitaxel sensitization in breast cancer cells whether in vitro and in vivo. The results demonstrated that BRD7 inhibits cell proliferation and sensitizes breast cancer cells to paclitaxel by activating Bak; they also provide promising targets for the diagnosis and treatment of breast cancer.

Establishment of 2D Cell Cultures Derived From 3D MCF-7 Spheroids Displaying a Doxorubicin Resistant Profile.

In vitro 3D cancer spheroids generally exhibit a drug resistance profile similar to that found in solid tumors. Due to this property, these models are an appealing for anticancer compounds screening. Nevertheless, the techniques and methods aimed for drug discovery are mostly standardized for cells cultured in 2D. The development of 2D cell culture models displaying a drug resistant profile is required to mimic the in vivo tumors, while the equipment, techniques, and methodologies established for conventional 2D cell cultures can continue to be employed in compound screening. In this work, the response of 3D-derived MCF-7 cells subsequently cultured in 2D in medium supplemented with glutathione (GSH) (antioxidant agent found in high levels in breast cancer tissues and a promoter of cancer cells resistance) to Doxorubicin (DOX) is evaluated. These cells demonstrated a resistance toward DOX closer to that displayed by 3D spheroids, which is higher than that exhibited by standard 2D cell cultures. In fact, the 50% inhibitory concentration (IC50 ) of DOX in 3D-derived MCF-7 cell cultures supplemented with GSH is about eight-times higher than that obtained for conventional 2D cell cultures (cultured without GSH), and is only about two-times lower than that attained for 3D MCF-7 spheroids (cultured without GSH). Further investigation revealed that this improved resistance of 3D-derived MCF-7 cells may result from their increased P-glycoprotein (P-gp) activity and reduced production of intracellular reactive oxygen species (ROS).

Membrane associated collagen XIII promotes cancer metastasis and enhances anoikis resistance

BackgroundIncreased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. However, function and regulation of membrane-associated collagen in breast cancer have not been determined. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse models show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. In the present study, we determined roles of collagen XIII in breast cancer progression and metastasis.MethodsWe analyzed the association of collagen XIII expression with breast cancer development and metastasis using published gene expression profiles generated from human breast cancer tissues. Utilizing gain- and loss- of function approaches and 3D culture assays, we investigated roles of collagen XIII in regulating invasive tumor growth. Using the tumorsphere/mammosphere formation assay and the detachment cell culture assay, we determined whether collagen XIII enhances cancer cell stemness and induces anoikis resistance. We also inhibited collagen XIII signaling with β1 integrin function-blocking antibody. Finally, using the lung colonization assay and the orthotopic mammary tumor model, we investigated roles of collagen XIII in regulating breast cancer colonization and metastasis. Cox proportional hazard (log-rank) test, two-sided Student’s t-test (two groups) and one-way ANOVA (three or more groups) analyses were used in this study.ResultsCollagen XIII expression is significantly higher in human breast cancer tissue compared with normal mammary gland. Increased collagen XIII mRNA levels in breast cancer tissue correlated with short distant recurrence free survival. We showed that collagen XIII expression promoted invasive tumor growth in 3D culture, enhanced cancer cell stemness, and induced anoikis resistance. Collagen XIII expression induced β1 integrin activation. Blocking β1 integrin activation significantly reduced collagen XIII-induced invasion and mammosphere formation. Importantly, silencing collagen XIII in MDA-MB-231 cells reduced lung colonization and metastasis.ConclusionsOur results demonstrate a novel function of collagen XIII in promoting cancer metastasis, cell invasion, and anoikis resistance.

Effect of postoperative ischemia on steroid hormone receptors and c-erbB-2 levels in breast cancer tissue.

Postoperative ischemia could affect the evaluation of breast cancer tissue for steroid hormone receptors and c-erbB2 levels until fixation using formalin. The misevaluation of steroid hormone receptors and c-erbB2 levels, which are important prognostic factors in the treatment of breast cancer, could change treatment options. The aim of this study was to investigate the effects of postoperative ischemia on a breast cancer tissue sample, particularly on steroid hormone receptors and c-erbB2 expression level. Twenty patients who underwent modified radical mastectomy were included in this study. Two histopathological methods, namely frozen and regular follow-up methods, were performed postoperatively on all specimens. Steroid hormone receptors, estrogen receptors, progesterone receptors, and c-erbB2 expression of breast cancer tissue samples were evaluated using both techniques. Two groups were created based on the results of steroid hormone receptors and c-erbB2 expression levels using the two histopathological techniques. We determined that ischemia has an adverse effect on the evaluation of steroid hormone receptors and c-erbB2, especially its effect on c-erbB2 expression level was significant (p<0.002). A mastectomy specimen should be examined at once to ensure accurate detection of steroid hormone receptors and c-erbB2 levels for the proper treatment of breast cancer patients.

RBBP6 expressional effects on cell proliferation and apoptosis in breast cancer cell lines with distinct p53 statuses.

IntroductionBreast cancer is the most common malignancy amongst women and has a higher incidence rate than lung cancer. Its tumor progression partially results from inactivation of p53 which is caused by overexpression of ubiquitous regulatory proteins possessing p53-binding domain. RBBP6 is regarded as one of the ubiquitous proteins because of its RING finger-like domain which enables it to possess E3 ligase activity. Thus, it has become a potential target in cancer treatment as it is highly expressed in various malignancies including cancer. However, it is not clearly defined whether the effect of RBBP6 on cell growth and apoptosis is cell line-dependent, more especially in breast cancer cell lines that have distinct p53 expression profiles. This study aims at evaluating the effects of RBBP6 on cell growth and apoptosis in breast cancer cell lines with different p53 expressions.MethodsFollowing the analysis at mRNA and protein levels in breast cancer tissue, RBBP6 expression was successfully manipulated using gene silencing and protein overexpression techniques in MCF-7 and MDA-MB-231 cell lines. The cells were co-treated with siRBBP6 and anticancer agents following apoptosis detection, which was confirmed by caspase 3/7 activity and quantification of apoptotic genes.ResultsRBBP6 was overexpressed in breast cancer tissues that were classified as stages 3 and 4, while in stage 1, its expression was much lower. The MCF-7 cell line which expresses wild-type p53 was more sensitive to apoptosis induction than MDA-MB-231 which is a mutant p53-expressing cell line. These data suggest that RBBP6 silencing triggers significant levels of intrinsic apoptosis, and its overexpression appears to promote cell proliferation in wild-type p53-expressing MCF-7 cell line as opposed to MDA-MB-231 cells.ConclusionThe effect of RBBP6 on cell proliferation and apoptosis induction in breast cancer seems to be cell line-dependent based on p53 status.

MAC30 knockdown involved in the activation of the Hippo signaling pathway in breast cancer cells.

Down-regulation of the meningioma-associated protein (MAC30) gene has been found in many solid cancers. This study was carried out to determine the roles and the mechanisms of MAC30 in breast cancer. We used our own data and a public database to analyze the MAC30 mRNA and protein levels in breast cancer tissues. In addition, we established MAC30 knockdown breast cancer cells using MAC30 siRNA. The roles of MAC30 were detected by using the Soft agar assay, Annexin-V-FITC/PI double staining and the Transwell assay. Western blotting was used to analyze the potential mechanism(s) of MAC30 in these cells. We found that MAC30 mRNA and protein were higher in the cancer tissues compared to the matched normal tissues. MAC30 expression was associated with tumor size, tumor differentiation and estrogen receptor (ER) status. Overall survival rate of the patients with low MAC30 expression was obviously higher than the ones with high expression. The apoptotic ratio was lower in MDA-MB-231 and MDA-MB-157 cells with MAC30 expression. By Western blot analysis, we found that increased levels of phosphorylated YAP1, MST1 and LATS1 after MAC30 siRNA transfection in these two cells. In summary, we demonstrate that MAC30 knockdown is involved in the activation of the Hippo signaling pathway.