Bromoenol Lactone Attenuates Nicotine-Induced Breast Cancer Cell Proliferation and Migration

Objectives

Calcium independent group VIA phospholipase A₂ (iPLA₂β) and Matrix Metalloproteinase-9 (MMP-9) are upregulated in many disease states; their involvement with cancer cell migration has been a recent subject for study. Further, the molecular mechanisms mediating nicotine-induced breast cancer cell progression have not been fully investigated. This study aims to investigate whether iPLA₂β mediates nicotine-induced breast cancer cell proliferation and migration through both in-vitro and in-vivo techniques. Subsequently, the ability of Bromoenol Lactone (BEL) to attenuate the severity of nicotine-induced breast cancer was examined.

Method and Results

We found that BEL significantly attenuated both basal and nicotine-induced 4T1 breast cancer cell proliferation, via an MTT proliferation assay. Breast cancer cell migration was examined by both a scratch and transwell assay, in which, BEL was found to significantly decrease both basal and nicotine-induced migration. Additionally, nicotine-induced MMP-9 expression was found to be mediated in an iPLA₂β dependent manner. These results suggest that iPLA₂β plays a critical role in mediating both basal and nicotine-induced breast cancer cell proliferation and migration in-vitro. In an in-vivo mouse breast cancer model, BEL treatment was found to significantly reduce both basal (p<0.05) and nicotine-induced tumor growth (p<0.01). Immunohistochemical analysis showed BEL decreased nicotine-induced MMP-9, HIF-1alpha, and CD31 tumor tissue expression. Subsequently, BEL was observed to reduce nicotine-induced lung metastasis.

Conclusion

The present study indicates that nicotine-induced migration is mediated by MMP-9 production in an iPLA₂β dependent manner. Our data suggests that BEL is a possible chemotherapeutic agent as it was found to reduce both nicotine-induced breast cancer tumor growth and lung metastasis.     

GRP78在食管癌细胞ECA-109增殖过程中的功能研究

食管癌在中国是高发性肿瘤,并具有较高的致死率。肿瘤细胞的持续增殖与细胞增殖失调密切相关。肿瘤细胞在增殖过程中需要合成大量蛋白质,葡萄糖调节蛋白78(glucose regulatedprotein,GRP78)作为分子伴侣,在蛋白质的折叠、组装、修饰和错误折叠蛋白的降解过程中发挥着重要作用。该研究通过构建pGRP78-EGFP-N1重组质粒,瞬时转染ECA-109细胞,研究GRP78过表达对细胞增殖能力的影响;采用RNA干扰技术,瞬时转染靶向GRP78的siRNA,研究敲低GRP78对细胞增殖能力的影响。该研究发现GRP78过表达后,更多的细胞从G1期进入S期和G2/M期,细胞增殖速率加快,细胞克隆形成率亦明显提高;敲低GRP78后,细胞更多地被阻滞在G1期而无法进入S期和G2/M期,细胞增殖速率减慢,细胞克隆形成率降低。GRP78可能通过调节细胞周期而促进ECA-109细胞的增殖。     

Rho Isoform-specific Interaction with IQGAP1 Promotes Breast Cancer Cell Proliferation and Migration

We performed a proteomics screen for Rho isoform-specific binding proteins to clarify the tumor-promoting effects of RhoA and C that contrast with the tumor-suppressive effects of RhoB. We found that the IQ-motif-containing GTPase-activating protein IQGAP1 interacts directly with GTP-bound, prenylated RhoA and RhoC, but not with RhoB. Co-immunoprecipitation of IQGAP1 with endogenous RhoA/C was enhanced when RhoA/C were activated by epidermal growth factor (EGF) or transfection of a constitutively active guanine nucleotide exchange factor (GEF). Overexpression of IQGAP1 increased GTP-loading of RhoA/C, while siRNA-mediated depletion of IQGAP1 prevented endogenous RhoA/C activation by growth factors. IQGAP1 knockdown also reduced the amount of GTP bound to GTPase-deficient RhoA/C mutants, suggesting that IQGAP enhances Rho activation by GEF(s) or stabilizes Rho-GTP. IQGAP1 depletion in MDA-MB-231 breast cancer cells blocked EGF- and RhoA-induced stimulation of DNA synthesis. Infecting cells with adenovirus encoding constitutively active RhoA^L63 and measuring absolute amounts of RhoA-GTP in infected cells demonstrated that the lack of RhoA^L63-induced DNA synthesis in IQGAP1-depleted cells was not due to reduced GTP-bound RhoA. These data suggested that IQGAP1 functions downstream of RhoA. Overexpression of IQGAP1 in MDA-MB-231 cells increased DNA synthesis irrespective of siRNA-mediated RhoA knockdown. Breast cancer cell motility was increased by expressing a constitutively-active RhoC^V14 mutant or overexpressing IQGAP1. EGF- or RhoC-induced migration required IQGAP1, but IQGAP1-stimulated migration independently of RhoC, placing IQGAP1 downstream of RhoC. We conclude that IQGAP1 acts both upstream of RhoA/C, regulating their activation state, and downstream of RhoA/C, mediating their effects on breast cancer cell proliferation and migration, respectively.     

Novel Etoposide Analogue Modulates Expression of Angiogenesis Associated microRNAs and Regulates Cell Proliferation by Targeting STAT3 in Breast Cancer

Tumor microenvironment play role in angiogenesis and carcinogenesis. Etoposide, a known topoisomerase II inhibitor induces DNA damage resulting in cell cycle arrest. We developed a novel Etoposide analogue, Quinazolino-4β-amidopodophyllotoxin (C-10) that show better efficacy in regulating cell proliferation and angiogenesis. We evaluated its role on expression of microRNAs-15, 16, 17 and 221 and its targets Bcl-2, STAT3 and VEGF that dictate cell proliferation and angiogenesis. Docking studies clearly demonstrated the binding of Etoposide and C-10 to STAT3. We conclude that combination of Etoposide or C-10 with miR-15, 16, 17 and 221 as a new approach to induce apoptosis and control angiogenesis in breast cancer.     

STAT3基因沉默降低小鼠乳腺癌细胞的体外迁移能力

信号转导子与转录活化子3(STAT3)是一个具有信号转导和转录调控双重功能的转录因子,有文献报道STAT3在乳腺癌中的表达显著升高,并能促进乳腺癌的转移。为了深入探索STAT3在肿瘤发生发展中的作用和影响乳腺癌转移的分子机制,采用RNA干扰技术在小鼠乳腺癌细胞株4T1中沉默STAT3的表达。MTT实验结果显示STAT3沉默对4T1细胞的增殖能力没有影响;细胞迁移实验结果表明STAT3表达被沉默后4T1细胞的迁移能力明显被抑制;定量PCR结果显示,STAT3基因沉默后4T1细胞中VEGF和IL-6的mRNA水平下降,E-cadherin表达上升,mosin表达下降;信号通路检测显示STAT3基因表达沉默后MAPK的活化明显降低。研究表明STAT3在小鼠乳腺癌细胞的迁移过程中发挥重要作用,为以STAT3基因为靶向的治疗提供了一定的实验依据。     

Aquaporin-3 Controls Breast Cancer Cell Migration by Regulating Hydrogen Peroxide Transport and Its Downstream Cell Signaling

Most breast cancer mortality is due to clinical relapse associated with metastasis. CXCL12/CXCR4-dependent cell migration is a critical process in breast cancer progression; however, its underlying mechanism remains to be elucidated. Here, we show that the water/glycerol channel protein aquaporin-3 (AQP3) is required for CXCL12/CXCR4-dependent breast cancer cell migration through a mechanism involving its hydrogen peroxide (H₂O₂) transport function. Extracellular H₂O₂, produced by CXCL12-activated membrane NADPH oxidase 2 (Nox2), was transported into breast cancer cells via AQP3. Transient H₂O₂ accumulation was observed around the membrane during CXCL12-induced migration, which may be facilitated by the association of AQP3 with Nox2. Intracellular H₂O₂ then oxidized PTEN and protein tyrosine phosphatase 1B (PTP1B) followed by activation of the Akt pathway. This contributed to directional cell migration. The expression level of AQP3 in breast cancer cells was related to their migration ability both in vitro and in vivo through CXCL12/CXCR4- or H₂O₂-dependent pathways. Coincidentally, spontaneous metastasis of orthotopic xenografts to the lung was reduced upon AQP3 knockdown. These findings underscore the importance of AQP3-transported H₂O₂ in CXCL12/CXCR4-dependent signaling and migration in breast cancer cells and suggest that AQP3 has potential as a therapeutic target for breast cancer.     

乳腺癌细胞迁移的调节与转移

细胞迁移是乳腺癌侵袭和转移中的关键步骤之一.癌细胞在迁移过程中主要受到Rho GTPases的调节,发生肌动蛋白骨架重组,获得定向迁移的能力;高迁移能力的癌细胞通过与胞外基质成分相互作用,为迁移创造合适的微环境;最后迁移的癌细胞在靶器官的趋化作用下在特定部位驻足生长,这些环节共同作用导致乳腺癌转移.研究细胞迁移复杂的分子机制将为控制乳腺癌转移提供新的策略.     

Cancer Cells Resistant to Therapy Promote Cell Surface Relocalization of GRP78 Which Complexes with PI3K and Enhances PI(3,4,5)P3 Production

Traditionally, GRP78 has been regarded as an endoplasmic reticulum (ER) lumenal protein due to its carboxyl KDEL retention motif. Recently, a subfraction of GRP78 is found to localize to the surface of specific cell types, serving as co-receptors and regulating signaling. However, the physiological relevance of cell surface GRP78 (sGRP78) expression in cancer and its functional interactions at the cell surface are just emerging. In this report, we combined biochemical, imaging and mutational approaches to address these issues. For detection of sGRP78, we utilized a mouse monoclonal antibody highly potent and specific for GRP78 or epitope-tagged GRP78, coupled with imaging and biochemical techniques that allowed detection of sGRP78 but not intracellular GRP78. Our studies revealed that breast and prostate cancer cells resistant to hormonal therapy actively promote GRP78 to the cell surface, which can be further elevated by a variety of ER stress-inducing conditions. We showed that sGRP78 forms complex with PI3K, and overexpression of sGRP78 promotes PIP3 formation, indicative of PI3K activation. We further discovered that an insertion mutant of GRP78 at its N-terminus domain, while retaining stable expression and the ability to translocate to the cell surface as the wild-type protein, exhibited reduced complex formation with p85 and production of PIP3. Thus, our studies provide a mechanistic explanation for the regulation of the PI3K/AKT signaling by sGRP78. Our findings suggest that targeting sGRP78 may suppress therapeutic resistance in cancer cells and offer a novel strategy to suppress PI3K activity.     

RNAi沉默STAT3基因对人大细胞肺癌细胞增殖的影响

旨在研究RNAi沉默STAT3基因对人大细胞肺癌NCI-H460细胞增殖的影响。针对STAT3基因mRNA设计合成5条短发夹DNA,构建重组SiRNA-ST3质粒(命名为SiRNA-ST3-1,2,3,4,N)。用重组质粒分别转染NCI-H460细胞,RT-PCR法检测转染24 h后STAT3 mRNA的表达;Western blotting法检测转染24 h和48 h后STAT3、pSTAT3蛋白表达;MTT法检测转染24 h、48 h、72 h后NCI-H460细胞增殖情况。结果显示,SiRNA-ST3载体构建成功。RT-PCR和Western blotting检测结果表明,NCI-H460细胞转染重组质粒SiRNA-ST3-2和SiRNA-ST3-3后STAT3基因mRNA转录和STAT3、pSTAT3蛋白表达都明显下降(P<0.05)。与未转染组比,SiRNA-ST3-2组和SiRNA-ST3-3组NCI-H460增殖能力24 h、48 h降低明显(P<0.05);与SiRNA-ST3-N组比,SiRNA-ST3-2组和SiRNA-ST3-3组NCI-H460增殖能力48 h降低明显(P<0.05)。由此证实,构建的重组质粒SiRNA-ST3-2、SiRNA-ST3-3能有效靶向沉默STAT3基因,并抑制人大细胞肺癌NCI-H460细胞的增殖能力。     

内质网应激分子GRP78 对非小细胞肺癌的诊断价值研究

目的:探讨内质网应激分子葡萄糖调节蛋白78(GRP78)对非小细胞肺癌(NSCLC)的诊断价值。方法:收集45例NSCLC患者术后癌组织标本,及其18例癌旁组织,采用实时定量PCR法检测所有组织中GRP78的表达情况,分析NSCLC癌组织中GRP78的表达与患者临床特征的关系,及其对患者生存期的影响。结果:NSCLC组织中GRP78高表达率明显高于癌旁组织,差异有统计学意义(P0.05)。而Ⅲa期NSCLC患者癌组织中GRP78的表达水平明显高于Ⅰ-Ⅱ期,两者存在显著性的差异(P0.05)。Kaplan-Meier分析显示,GRP78高表达的患者其生存期明显低于GRP78低表达的患者(P0.05)。结论:NSCLC患者GRP78的表达可能与肿瘤细胞的发生、发展及患者的生存期有关,可作为一个预测NSCLC患者诊断及治疗的重要的分子标志物。     

CRISPR/Cas9沉默PAK5抑制乳腺癌细胞增殖并促进细胞衰老

p21活化激酶5(p21-activated kinase 5,PAK5)是一种丝氨酸/苏氨酸激酶,调节多种细胞进程,包括细胞骨架重构、细胞增殖、迁移和侵袭.研究表明,PAK5是调控乳腺癌进程的关键因子,但与衰老关系的研究尚未见报道.本研究利用CRISPR/Cas9慢病毒感染方法,构建敲低PAK5的人乳腺癌MDA-MB...     

Diversin Is Overexpressed in Breast Cancer and Accelerates Cell Proliferation and Invasion

Diversin was recently reported to play roles in Wnt and JNK pathways. However, the expression pattern and biological roles of diversin in human breast cancer have not been reported. In the present study, we found that diversin was overexpressed in breast cancer specimens by immunohistochemistry and western blot. Significant association was observed between diversin overexpression and TNM stage (p = 0.0036), nodal metastasis (p = 0.0033), negative estrogen receptor expression (p = 0.0012) and triple-negative status (p = 0.0017). Furthermore, colony formation assay and matrigel invasion assay showed that knockdown of diversin expression in MDA-MB-231 cell line with high endogenous expression decreased cell proliferation and cell invasion. Transfection of diversin plasmid in MCF-7 cell line increased cell proliferation and invasion. Further analysis showed that diversin depletion downregulated JNK phosphorylation while its overexpression upregulated JNK phosphorylation. In conclusion, our study demonstrated that diversin was overexpressed in human breast cancers. Diversin could contribute to breast cancer cell proliferation and invasion.     

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.     

乳腺癌干细胞和乳腺癌

肿瘤干细胞既包含干细胞的特性也包含肿瘤细胞的特性。乳腺癌起源于乳腺癌干细胞的说法能够合理地解释乳腺癌的不均一性及其治疗后的复发,这些变异的干细胞可能作为肿瘤预防策略的靶标。而且,由于乳腺癌干细胞能够抵抗辐射治疗和化学治疗,所以要想更好地治疗乳腺癌就需要寻找针对这些干细胞的靶标。我们综述了乳腺癌干细胞的发现、富集和分离、相关的信号途径,以及在乳腺癌治疗中的应用。     

MiR-122 Inhibits Cell Proliferation and Tumorigenesis of Breast Cancer by Targeting IGF1R

miRNAs are emerging as critical regulators in carcinogenesis and tumor progression. Recently, microRNA-122 (miR-122) has been proved to play an important role in hepatocellular carcinoma, but its functions in the context of breast cancer (BC) remain unknown. In this study, we report that miR-122 is commonly downregulated in BC specimens and BC cell lines with important functional consequences. Overexpression of miR-122 not only dramatically suppressed cell proliferation, colony formation by inducing G1-phase cell-cycle arrest in vitro, but also reduced tumorigenicity in vivo. We then screened and identified a novel miR-122 target, insulin-like growth factor 1 receptor (IGF1R), and it was further confirmed by luciferase assay. Overexpression of miR-122 would specifically and markedly reduce its expression. Similar to the restoring miR-122 expression, IGF1R downregulation suppressed cell growth and cell-cycle progression, whereas IGF1R overexpression rescued the suppressive effect of miR-122. To identify the mechanisms, we investigated the Akt/mTOR/p70S6K pathway and found that the expression of Akt, mTOR and p70S6K were suppressed, whereas re-expression of IGF1R which did not contain the 3′UTR totally reversed the inhibition of Akt/mTOR/p70S6K signal pathway profile. We also identified a novel, putative miR-122 target gene, PI3CG, a member of PI3K family, which further suggests miR-122 may be a key regulator of the PI3K/Akt pathway. In clinical specimens, IGF1R was widely overexpressed and its mRNA levels were inversely correlated with miR-122 expression. Taken together, our results demonstrate that miR-122 functions as a tumor suppressor and plays an important role in inhibiting the tumorigenesis through targeting IGF1R and regulating PI3K/Akt/mTOR/p70S6K pathway. Given these, miR-122 may serve as a novel therapeutic or diagnostic/prognostic-target for treating BC.     

IGF-1 Regulates Cyr61 Induced Breast Cancer Cell Proliferation and Invasion

Background

Studies from our laboratory and others have shown that cysteine-rich 61 (Cyr61) may be involved in tumor proliferation and invasion. In earlier studies, we demonstrated increased insulin-like growth factor-I (IGF-1) is associated with breast tumor formation and poor clinical outcomes. In our current study we have investigated IGF-1 regulation of Cyr61 and whether targeting IGF-1 could inhibit Cyr61 induced tumor growth and proliferation.

Methods

Several ATCC derived normal and breast cancer cell lines were used in this study: MDA-MB231, BT474, MCF-7, and SKBR3. We also tested cells stably transfected in our laboratory with active Akt1 (pAkt; SKBR3/AA and MCF-7/AA) and dominant negative Akt1 (SKBR3/DN and MCF-7/DN). In addition, we used MCF-7 cells transfected with full length Cyr61 (CYA). Monolayer cultures treated with IGF-1 were analyzed for Cyr61 expression by RT-PCR and immunohistochemical staining. Migration assays and MTT based proliferation assays were used to determine invasive characteristics in response to IGF-1/Cyr61 activation.

Results

Cells with activated Akt have increased levels of Cyr61. Conversely, cells with inactive Akt have decreased levels of Cyr61. IGF-1 treatment increased Cyr61 expression significantly and cells with high level of Cyr61 demonstrate increased invasiveness and proliferation. Cyr61 overexpression and activation led to decrease in E-cadherin and decrease in FOXO1. Inhibition of the PI3K and MAPK pathways resulted in significant decrease in invasiveness and proliferation, most notably in the PI3K pathway inhibited cells.

Conclusion

The findings of this study show that IGF-1 upregulates Cyr61 primarily through activation of the Akt-PI3K pathway. IGF-1 induced MAPK plays a partial role. Increase in Cyr61 leads to increase in breast cancer cell growth and invasion. Hence, targeting Cyr61 and associated pathways may offer an opportunity to inhibit IGF-1 mediated Cyr61 induced breast cancer growth and invasion.