Inhibition of SK4 Potassium Channels Suppresses Cell Proliferation,Migration and the Epithelial-Mesenchymal Transition in Triple-Negative Breast Cancer Cells

Treatments for triple-negative breast cancer (TNBC) are limited; intermediate-conductance calcium-activated potassium (SK4) channels are closely involved in tumor progression, but little is known about these channels in TNBC. We aimed to investigate whether SK4 channels affect TNBC. First, by immunohistochemistry (IHC) and western blotting (WB), increased SK4 protein expression in breast tumor tissues was detected relative to that in non-tumor breast tissues, but there was no apparent expression difference between various subtypes of breast cancer (p>0.05). Next, functional SK4 channels were detected in the TNBC cell line MDA-MB-231 using WB, real-time PCR, immunofluorescence and patch-clamp recording. By employing SK4 specific siRNAs and blockers, including TRAM-34 and clotrimazole, in combination with an MTT assay, a colony-formation assay, flow cytometry and a cell motility assay, we found that the suppression of SK4 channels significantly inhibited cell proliferation and migration and promoted apoptosis in MDA-MB-231 cells (p<0.05). Further investigation revealed that treatment with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) caused MDA-MB-231 cells to undergo the epithelial-mesenchymal transition (EMT) and to show increased SK4 mRNA expression. In addition, the down-regulation of SK4 expression inhibited the EMT markers Vimentin and Snail1. Collectively, our findings suggest that SK4 channels are expressed in TNBC and are involved in the proliferation, apoptosis, migration and EMT processes of TNBC cells.     

Putative tumor suppressor cytoglobin promotes aryl hydrocarbon receptor ligand–mediated triple negative breast cancer cell death

Nearly 40 000 women die annually from breast cancer in the United States. Clinically available targeted breast cancer therapy is largely ineffective in triple negative breast cancer (TNBC), characterized by tumors that lack expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2). TNBC is associated with a poor prognosis. Previous reports show that aryl hydrocarbon receptor (AhR) partial agonist 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) selectively inhibits the growth of breast cancer cells, including those of the TNBC subtype. We previously demonstrated that 5F 203 induced the expression of putative tumor suppressor gene cytoglobin (CYGB) in breast cancer cells. In the current study, we determined that 5F 203 induces apoptosis and caspase-3 activation in MDA-MB-468 TNBC cells and in T47D ER⁺ PR ⁺ Her2 ⁻ breast cancer cells. We also show that caspases and CYGB promote 5F 203–mediated apoptosis in MDA-MB-468 cells. 5F 203 induced lysosomal membrane permeabilization (LMP) and cathepsin B release in MDA-MB-468 and T47D cells. In addition, silencing CYGB attenuated the ability of 5F 203 to induce caspase-3/-7 activation, proapoptotic gene expression, LMP, and cathepsin B release in MDA-MB-468 cells. Moreover, 5F 203 induced CYGB protein expression, proapoptotic protein expression, and caspase-3 cleavage in MDA-MB-468 cells and in MDA-MB-468 xenograft tumors grown orthotopically in athymic mice. These data provide a basis for the development of AhR ligands with the potential to restore CYGB expression as a novel strategy to treat TNBC.     

Suberoylanilide Hydroxamic Acid,an Inhibitor of Histone Deacetylase,Enhances Radiosensitivity and Suppresses Lung Metastasis in Breast Cancer In Vitro and In Vivo

Triple-negative breast cancer (TNBC), defined by the absence of an estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, is associated with an early recurrence of disease and poor outcome. Furthermore, the majority of deaths in breast cancer patients are from metastases instead of from primary tumors. In this study, MCF-7 (an estrogen receptor-positive human breast cancer cell line), MDA-MB-231 (a human TNBC cell line) and 4T1 (a mouse TNBC cell line) were used to investigate the anti-cancer effects of ionizing radiation (IR) combined with suberoylanilide hydroxamic acid (SAHA, an inhibitor of histone deacetylase (HDAC)) and to determine the underlying mechanisms of these effects in vitro and in vivo. We also evaluated the ability of SAHA to inhibit the metastasis of 4T1 cells. We found that IR combined with SAHA showed increased therapeutic efficacy when compared with either treatment alone in MCF-7, MDA-MB-231 and 4T1 cells. Moreover, the combined treatment enhanced DNA damage through the inhibition of DNA repair proteins. The combined treatment was induced primarily through autophagy and ER stress. In an orthotopic breast cancer mouse model, the combination treatment showed a greater inhibition of tumor growth. In addition, SAHA inhibited the migration and invasion abilities of 4T1 cells and inhibited breast cancer cell migration by inhibiting the activity of MMP-9. In an in vivo experimental metastasis mouse model, SAHA significantly inhibited lung metastasis. SAHA not only enhances radiosensitivity but also suppresses lung metastasis in breast cancer. These novel findings suggest that SAHA alone or combined with IR could serve as a potential therapeutic strategy for breast cancer.     

miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究*

目的: 探讨miR-34a-5p在三阴性乳腺癌(triple negative breast cancer,TNBC)中的表达,分析miR-34a-5p对TNBC细胞增殖、凋亡、迁移的作用,对TNBC荷瘤小鼠肿瘤生长的影响以及在TNBC中对B7-H1表达的影响。方法: 利用RT-qPCR、Western blot分析TNBC细胞中miR-34a-5p、B7-H1的表达,并利用Kaplan-Meier分析二者的表达与TNBC患者的生存关系;将miR-34a-5p转染TNBC细胞,通过CCK-8、流式细胞术及划痕实验检测miR-34a-5p对TNBC细胞增殖、凋亡、迁移的影响;利用RT-qPCR、Western blot检测miR-34a-5p、B7-H1表达水平的变化,双荧光素酶基因报告验证miR-34a-5p与B7-H1的相互作用;利用RT-qPCR、Western blot、IHC检测miR-34a-5p对MDA-MB-231荷瘤小鼠miR-34a、B7-H1表达的影响。结果: TNBC细胞中miR-34a-5p呈低表达,B7-H1呈高表达,二者均与TNBC患者的不良预后有关,差距具有统计学意义(P<0.01);miR-34a-5p抑制TNBC细胞增殖、侵袭,促进细胞凋亡,并且在TNBC细胞中靶向抑制B7-H1;miR-34a-5p agomir在体内抑制MDA-MB-231成瘤裸鼠的肿瘤生长和B7-H1表达。结论: miR-34a-5p在TNBC发生、发展中发挥着重要作用,靶向miR-34a-5p/B7-H1可能成为TNBC患者新的分子治疗策略。     

Suppression of TRPM7 enhances TRAIL-induced apoptosis in triple-negative breast cancer cells

Transient receptor potential cation channel subfamily M member 7 (TRPM7) composed of an ion channel and a kinase domain regulates triple-negative breast cancer (TNBC) cell migration, invasion, and metastasis, but it does not modulate TNBC proliferation. However, previous studies have shown that the combination treatment of nonselective TRPM7 channel inhibitors (2-aminoethoxydiphenyl borate and Gd³⁺) with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) increases antiproliferative effects and apoptosis in prostate cancer cells and hepatic stellate cells. We, therefore, investigated the potential role of TRPM7 in proliferation and apoptosis of TNBC cells (MDA-MB-231 and MDA-MB-468 cells) with TRAIL. We demonstrated that suppression of TRPM7 via TRPM7 knockdown or pharmacological inhibition synergistically increases TRAIL-induced antiproliferative effects and apoptosis in TNBC cells. Furthermore, we showed that the synergistic interaction might be associated with TRPM7 channel activities using combination treatments of TRAIL and TRPM7 inhibitors (NS8593 as a TRPM7 channel inhibitor and TG100-115 as a TRPM7 kinase inhibitor). We reveal that downregulation of cellular FLICE-inhibitory protein via inhibition of Ca²⁺ influx might be involved in the synergistic interaction. Our study would provide both a new role of TRPM7 in TNBC cell apoptosis and a potential combinatorial therapeutic strategy using TRPM7 inhibitors with TRAIL in the treatment of TNBC.     

Long noncoding RNA Linc00339 promotes triple-negative breast cancer progression through miR-377-3p/HOXC6 signaling pathway

Recently, long noncoding RNAs (lncRNAs) have become the key gene regulators and prognostic biomarkers in various cancers. Through microarray data, Linc00339 was identified as a candidate oncogenic lncRNA. We compared the expression levels of Linc00339 in several breast cancer cell lines and normal mammary gland epithelial cell line. The effects of Linc00339 on tumor progression were examined both in vitro and in vivo. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were applied to evaluate the functions of Linc00339, miR-377-3p, and HOXC6 on cell proliferation. Flow cytometry analysis was used to detect apoptosis and cell cycle distribution. Overall survival (OS) was analyzed using data from The Cancer Genome Atlas and molecular taxonomy of breast cancer international consortium (METABRIC). Dual luciferase assay and RNA immunoprecipitation were performed to confirm the interaction between Linc003339 and miR-377-3p. Linc00339 was increased in breast cancer cell lines compared with the normal epithelial cell. Through in vitro and in vivo experiments, Linc00339 overexpression promoted triple-negative breast cancer (TNBC) proliferation, inhibited cell cycle arrest, and suppressed apoptosis. Silencing of Linc00339 obtained the opposite effects. Mechanistic investigations demonstrated that Linc00339 could sponge miR-377-3p and regulate its expression. Higher expression of miR-377-3p indicated longer OS in breast cancer patients, especially in TNBC patients. Overexpression of miR-377-3p retarded TNBC cell growth through regulating cell cycle distribution and apoptosis. And miR-377-3p was involved in Linc00339-mediated TNBC proliferation through regulating HOXC6 expression. Knockdown of HOXC6 inhibited TNBC progression. In conclusion, our results illuminated that the novel Linc00339/miR-377-3p/HOXC6 axis played a critical role in TNBC progression and might be a promising therapeutic target for TNBC treatment.     

HIF1A-AS2 predicts poor prognosis and regulates cell migration and invasion in triple-negative breast cancer

The aberrant expression of hypoxia-inducible factor 1 alpha (HIF1A)-antisense RNA 2 (HIF1A-AS2) was found in various human cancers including breast cancer. The aim of this study was to present more evidence about the role HIF1A-AS2 on triple-negative breast cancer (TNBC). In our results, HIF1A-AS2 was also found to be upregulated in TNBC tissues compared with non-TNBC tissues or adjacent normal tissues. Besides, HIF1A-AS2 expression was also elevated in TNBC cell lines compared with the normal breast epithelial cell line. Moreover, high expression of HIF1A-AS2 was associated with lymph node metastasis, distant metastasis and unfavorable histological grade in TNBC patients. Survival analysis showed a TNBC patient with high HIF1A-AS2 expression had shorter overall survival than patients with low HIF1A-AS2 expression, and HIF1A-AS2 high expression acted as an independent poor prognostic factor for overall survival in TNBC patients. The cell migration and invasion assays suggested inhibition of HIF1A-AS2 obviously depressed TNBC cell migration and invasion. In conclusion, HIF1A-AS2 serves as a novel biomarker for predicting clinical progression and prognosis in TNBC.     

MUC4 Overexpression Augments Cell Migration and Metastasis through EGFR Family Proteins in Triple Negative Breast Cancer Cells

Introduction

Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs.

Method

In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining.

Results

MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.

Conclusions

MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.     

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration,Invasion, and Metastasis

Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.     

SARNP,a participant in mRNA splicing and export,negatively regulates E-cadherin expression via interaction with pinin

Triple-negative breast cancer (TNBC) is associated with a high mortality rate, which is related to the insufficient number of appropriate biomarkers and targets. Therefore, there is an urgent need to discover appropriate biomarkers and targets for TNBC. SARNP (Hcc-1 and CIP29) is highly expressed in several cancers. It binds to UAP56, an RNA helicase component of the TREX complex in messenger RNA (mRNA) splicing and export. However, the role of SARNP in mRNA splicing and export and in the progression of breast cancer, especially of TNBC, remains unknown. Therefore, we examined the role of SARNP in mRNA splicing and export and progression of TNBC. We confirmed that SARNP binds to UAP56 and Aly and that SARNP overexpression enhances mRNA splicing, whereas its knockdown suppressed mRNA export. The SARNP overexpression induced the proliferation of MCF7 cells, whereas its knockdown induced E-cadherin expression and downregulated vimentin and N-cadherin expressions in SK-BR-3 and MDA-MB-231 cells. SARNP downregulates E-cadherin expression by interaction with pinin. Mice injected with MDA-MB-231_shSARNP cells exhibited a significant reduction in tumor growth and lung metastasis compared with those injected with MDA-MB-231_shCon cells in vivo. These findings suggested that SARNP is involved in mRNA splicing and export. SARNP maintains mesenchymal phenotype by escaping from inhibitory interaction with pinin leading to the downregulation of E-cadherin expression.     

Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients’ shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases.     

Harmine induces apoptosis and inhibits tumor cell proliferation,migration and invasion through down-regulation of cyclooxygenase-2 expression in gastric cancer

Cyclooxygenase-2 (COX-2) plays an important role in the carcinogenesis and progression of gastric cancer. Harmine is reported as a promising drug candidate for cancer therapy; however, effects and action mechanism of harmine on the human gastric cancer cells remain unclear. This study evaluated the anti-tumor effects of harmine on human gastric cancer both in vitro and in vivo. The cell proliferation was determined using MTT colorimetric assay. Apoptosis was measured by DAPI staining and flow cytometry analysis. The wound healing and transwell invasion assays were performed to evaluate the effects of harmine on the migration and invasion of gastric cancer cells. The expression of COX-2, proliferating cell nuclear antigen (PCNA), Bcl-2, Bax and matrix metalloproteinase-2 (MMP-2) was detected by Western blot analysis. Our results showed that harmine significantly inhibited cellular proliferation, migration, invasion and induced apoptosis in vitro, as well as inhibited tumor growth in vivo. In addition, harmine significantly inhibited the expression of COX-2, PCNA, Bcl-2 and MMP-2 as well as increased Bax expression in gastric cancer cells. These results collectively indicate that harmine induces apoptosis and inhibits proliferation, migration and invasion of human gastric cancer cells, which may be mediated by down-regulation of COX-2 expression.     

Centchroman suppresses breast cancer metastasis by reversing epithelial–mesenchymal transition via downregulation of HER2/ERK1/2/MMP-9 signaling

Metastatic spread during carcinogenesis worsens disease prognosis and accelerates the cancer progression. Therefore, newer therapeutic options with higher specificity toward metastatic cancer are required. Centchroman (CC), a female oral contraceptive, has previously been reported to possess antiproliferative and proapoptotic activities in human breast cancer cells. Here, we investigated the effect of CC-treatment against breast cancer metastasis and associated molecular mechanism using in vitro and in vivo models. CC significantly inhibited the proliferation of human and mouse mammary cancer cells. CC-treatment also inhibited migration and invasion capacities of highly metastatic MDA-MB-231 and 4T1 cells, at sub-IC₅₀ concentrations. Inhibition of cell migration and invasion was found to be associated with the reversal of epithelial-to-mesenchymal transition (EMT) as observed by the upregulation of epithelial markers and downregulation of mesenchymal markers as well as decreased activities of matrix metalloproteinases. Experimental EMT induced by exposure to TGFβ/TNFα in nontumorigenic human mammary epithelial MCF10A cells was also reversed by CC as evidenced by morphological changes and modulation in the expression levels of EMT-markers. CC-mediated inhibition of cellular migration was, at least partially, mediated through inhibition of ERK1/2 signaling, which was further validated by using MEK1/2 inhibitor (PD0325901). Furthermore, CC-treatment resulted in suppression of tumor growth and lung metastasis in 4T1-syngeneic mouse model. Collectively, our findings suggest that CC-treatment at higher doses specifically induces cellular apoptosis and inhibits cellular proliferation; whereas at lower doses, it inhibits cellular migration and invasion. Therefore, CC could further be developed as an effective drug candidate against metastatic breast cancer.     

HOXD8 inhibits the proliferation and migration of triple-negative breast cancer cells and induces apoptosis in them through regulation of AKT/mTOR pathway

HOXD8 (Homeobox D8) functions as an apoptotic inducer to suppress tumor progression. However, the role of HOXD8 in triple-negative breast cancer (TNBC) has not been fully understood. Firstly, HOXD8 was found to be reduced in TNBC tissues based on the TCGA samples through Ualcan (http://ualcan.path.uab.edu/analysis.html) prediction. Moreover, data from qRT-PCR and western blot confirmed the lower expression of HOXD8 in the TNBC tissues or cells than that in paracancerous tissues or human mammary epithelial cell line (MCF10A), respectively. Secondly, pcDNA-mediated over-expression of HOXD8 were conducted in TNBC cells, and the gain-of functional assays showed that over-expression of HOXD8 promoted TNBC cell progression with repressed cell apoptosis and induced proliferation, migration and invasion. Moreover, xenografted mouse model was constructed by injection of tumor cell line with stable over-expression of HOXD8 to assess the in vivo tumor growth, and the results revealed that over-expression of HOXD8 inhibited tumor growth. Lastly, our results showed that AKT and mTOR phosphorylation were repressed by HOXD8 over-expression in TNBC cells. In conclusion, HOXD8 functioned as an apoptotic inducer to suppress TNBC cell growth and progression by inhibition of AKT/mTOR pathway.     

Chetomin induces apoptosis in human triple-negative breast cancer cells by promoting calcium overload and mitochondrial dysfunction

Human triple-negative breast cancer (TNBC) is poorly diagnosed and unresponsive to conventional hormone therapy. Chetomin (CHET), a fungal metabolite synthesized by Chaetomium cochliodes, has been reported as a promising anticancer and antiangiogenic agent but the complete molecular mechanism of its anticancer potential remains to be elucidated. In our study, we explored the anti-neoplastic action of CHET on TNBC cells. Cytotoxicity studies were performed in human TNBC cells viz. MDA-MB-231 and MDA-MB-468 cells by Sulforhodamine B assay. It exhibited antiproliferative response and induced apoptosis in both the cell types. Cell cycle analysis revealed that it increases the sub G0/G1 phase cell population. Modulation of mitochondrial membrane potential, activation of caspase 3/7 and a remarkable increase in the expression of cleaved PARP and increased chromatin condensation was observed after CHET treatment in MDA-MB-231 and MDA-MB-468 cells. Additionally, an elevated level of intracellular Ca²⁺ played an important role in CHET mediated cell death response. Calcium overload in mitochondria led to release of cytochrome c which in turn triggered caspase-3 mediated cell death. Inhibition of calcium signalling using BAPTA-AM reduced apoptosis confirming the involvement of calcium signalling in CHET induced cell death. Chetomin also inhibited PI3K/mTOR cell survival pathway in human TNBC cells. The overall findings suggest that Chetomin inhibited the growth of human TNBC cells by caspase-dependent apoptosis and modulation of PI3K/mTOR signalling and could be used as a novel chemotherapeutic agent for the treatment of human TNBC in future.     

Inhibition of PKM2 sensitizes triple-negative breast cancer cells to doxorubicin

Cancer cells alter regular metabolic pathways in order to sustain rapid proliferation. One example of metabolic remodeling in cancerous tissue is the upregulation of pyruvate kinase isoenzyme M2 (PKM2), which is involved in aerobic glycolysis. Indeed, PKM2 has previously been identified as a tumor biomarker and as a potential target for cancer therapy. Here, we examined the effects of combined treatment with doxorubicin and anti-PKM2 small interfering RNA (siRNA) on triple-negative breast cancer (TNBC). The suppression of PKM2 resulted in changes in glucose metabolism, leading to decreased synthesis of adenosine triphosphate (ATP). Reduced levels of ATP resulted in the intracellular accumulation of doxorubicin, consequently enhancing the therapeutic efficacy of this drug in several triple-negative breast cancer cell lines. Furthermore, the combined effect of PKM2 siRNA and doxorubicin was evaluated in an in vivo MDA-MB-231 orthotopic breast cancer model. The siRNA was systemically administered through a polyethylenimine (PEI)-based delivery system that has been extensively used. We demonstrate that the combination treatment showed superior anticancer efficacy as compared to doxorubicin alone. These findings suggest that targeting PKM2 can increase the efficacy of chemotherapy, potentially providing a new approach for improving the outcome of chemotherapy in patients with TNBC.     

miR-483-3p suppresses the proliferation and progression of human triple negative breast cancer cells by targeting the HDAC8>oncogene

Triple negative breast cancer (TNBC) is a heterogeneous subclass of breast cancer (BC) distinguished by lack of hormone receptor expression. It is highly aggressive and difficult to treat with traditional chemotherapeutic regimens. Targeted-therapy using microRNAs (miR) has recently been proposed to improve the treatment of TNBC in the early stages. Here, we explore the roles of miR-483-3p/HDAC8 HDAC8 premiR-vector on tumorigenicity in TNBC patients. Clinical TNBC specimens and three BC cell lines were prepared. miR-483-3p and expression levels were measured using quantitative real-time polymerase chain reaction. Cell cycle progression was assessed by a flow-cytometry method. We also investigated cell proliferation by 3-2, 5-diphenyl tetrazolium bromide assay and colony formation assay. We used a to overexpress miR-483-3p, and a HDAC8-KO-vector for knocking out the endogenous production of HDAC8. Our data showed significant downregulation of miR-483-3p expression in TNBC clinical and cell line samples. The HDAC8 was also upregulated in both tissue specimens and BC cell lines. We found that increased levels of endogenous miR-483-3p affects tumorigenecity of MDA-MB-231. Downregulation of HDAC8 using the KO-vector showed the same pattern. Our results revealed that the miR-483-3p suppresses cellular proliferation and progression in TNBC cell lines via targeting HDAC8. Overall, our outcomes demonstrated the role of miR-483-3p as a tumor suppressor in TNBC and showed the possible mechanism via HDAC8. In addition, targeted treatment of TNBC with miR-483-3p might be considered in the future.     

Luteolin suppresses androgen receptor-positive triple-negative breast cancer cell proliferation and metastasis by epigenetic regulation of MMP9 expression via the AKT/mTOR signaling pathway

BackgroundTriple-negative breast cancer (TNBC) represents up to 20% of all breast cancers. This cancer lacks the expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The current therapeutic strategy for patients with this subtype is the use of cytotoxic chemotherapy and surgery. Luteolin is a natural herbal flavonoid and a potential therapeutic candidate for multiple diseases. The use of a treatment that combines Chinese herbal medicine and western medicine is rising in Asia.PurposeThe present study evaluates the effects and molecular mechanisms involved with luteolin treatment and evaluates whether this herb affects androgen receptor-positive breast cancer cell proliferation or metastasis.Study DesignIn vitro evaluation of the effect of luteolin on androgen receptor-positive TNBC cell proliferation and metastasisMethodsCell viability analysis was used for the cytotoxicity test. Colony formation and Bromodeoxyuridine (BrdU) staining-based proliferation experiments were used for cell proliferation. Wound healing and transwell assays were used for in vitro migration/invasion. The RT-qPCR analysis was used for gene expression. Furthermore, ChIP-qPCR analysis was used for epigenetic modification of gene promoters.ResultsLuteolin significantly inhibited the proliferation and metastasis of androgen receptor-positive TNBC. Furthermore, luteolin inactivated the AKT/mTOR signaling pathway and reversed the epithelial-mesenchymal transition (EMT). The combination of luteolin and inhibitors of AKT/mTOR synergistically repressed an androgen receptor-positive TNBC cell proliferation and metastasis. Luteolin also downregulated MMP9 expression by decreasing the levels of the AKT/mTOR promoting H3K27Ac and H3K56A on the MMP9 promoter region.ConclusionOur findings indicate that luteolin inhibited the proliferation and metastasis of androgen receptor-positive TNBC by regulating MMP9 expression through a reduction in the levels of AKT/mTOR-inducing H3K27Ac and H3K56Ac.     

CHL1 is involved in human breast tumorigenesis and progression

Neural cell adhesion molecules (CAM) play important roles in the development and regeneration of the nervous system. The L1 family of CAMs is comprised of L1, Close Homolog of L1 (CHL1, L1CAM2), NrCAM, and Neurofascin, which are structurally related trans-membrane proteins in vertebrates. Although the L1CAM has been demonstrated play important role in carcinogenesis and progression, the function of CHL1 in human breast cancer is limited. Here, we found that CHL1 is down-regulated in human breast cancer and related to lower grade. Furthermore, overexpression of CHL1 suppresses proliferation and invasion in MDA-MB-231 cells and knockdown of CHL1 expression results in increased proliferation and invasion in MCF7 cells in vitro. Finally, CHL1 deficiency promotes tumor formation in vivo. Our results may provide a strategy for blocking breast carcinogenesis and progression.