Corticotropin Releasing Factor promotes breast cancer cell motility and invasiveness

Introduction  

Cancer cells secrete bioactive peptides that act in an autocrine or paracrine fashion affecting tumor growth and metastasis. Corticotropin-releasing factor (CRF), a hypothalamic neuropeptide that controls the response to stress, has been detected in breast cancer tissues and cell lines. CRF can affect breast cancer cells in an autocrine or paracrine manner via its production from innervating sympathetic neurons or immune cells.     

COX-2 inhibitors suppress lung cancer cell growth by inducing p21 via COX-2 independent signals

COX-2 has been implicated in the control of human non-small cell lung carcinoma (NSCLC) cell growth. The mechanisms by which COX-2 exerts its mitogenic effects have not been entirely elucidated, but stimulation of prostaglandin E2 production and alterations in the expression of the cyclin-dependent kinase inhibitor p21(WAF-1/CIP1/MDA-6)(p2i) have been suggested. Here, we demonstrate that two COX-2 inhibitors (NS398 and Nimesulide) inhibit proliferation and induce apoptosis in NSCLC cells, and these effects were associated with induction of p21 mRNA and protein expression. However, the anti-growth effect of the COX-2 inhibitors and their ability to induce p21 were not affected by COX-2 siRNA suggesting that their actions were COX-2 independent. Instead, activation of the MEK-1/Erk pathway was necessary since COX-2 inhibitors stimulated the phosphorylation of ERKs, and their effects were blocked by PD98095, an inhibitor of this pathway. Furthermore, we show that both NS398 and Nimesulide induced p21 gene promoter activity and this was prevented by PD98095. COX-2 inhibitors increased nuclear protein binding to the Spl site in the promoter region of the p21 gene. Consistent with a role for p21, we found that p21 antisense oligonucleotides prevented the effects of COX-2 inhibitors on cell growth. In summary, our results suggest that COX-2 inhibitors suppress NSCLC cell growth by inducing the expression of the p21 gene through MEK-1/ERK signaling and DNA-protein interactions involving Spl. These observations unveil a mechanism for p21 gene regulation by COX-2 inhibitors in lung carcinoma cell growth and this pathway represents a potential target for therapy.     

S6 kinase 2 promotes breast cancer cell survival via Akt

The 40S ribosomal protein S6 kinase (S6K) acts downstream of mTOR, which plays important roles in cell proliferation, protein translation, and cell survival and is a target for cancer therapy. mTOR inhibitors are, however, of limited success. Although Akt is believed to act upstream of mTOR, persistent inhibition of p70 S6 kinase or S6K1 can activate Akt via a negative feedback loop. S6K exists as two homologues, S6K1 and S6K2, but little is known about the function of S6K2. In the present study, we have examined the effects of S6K2 on Akt activation and cell survival. Silencing of S6K1 caused a modest decrease, whereas knockdown of S6K2 caused a substantial increase in TNF-α and TRAIL (TNF-related apoptosis-inducing ligand)-mediated apoptosis. In contrast to S6K1, depletion of S6K2 by siRNA decreased basal and TNF-induced Akt phosphorylation. Ectopic expression of constitutively active Akt in MCF-7 cells restored cell survival in S6K2-depleted cells. We have previously shown that activation of Akt induces downregulation of Bid via p53. Knockdown of S6K2 caused an increase in p53, and downregulation of p53 by siRNA decreased Bid level. Silencing of Bid blunted the ability of S6K2 deficiency to enhance TNF-induced apoptosis. Taken together, our study shows that the two homologues of S6K have distinct effects on Akt activation and cell survival. Thus, targeting S6K2 may be an effective therapeutic strategy to treat cancers.     

Doxorubicin and vinorelbine act independently via p53 expression and p38 activation respectively in breast cancer cell lines

In the treatment of breast cancer, combination chemotherapy is used to overcome drug resistance. Combining doxorubicin and vinorelbine in the treatment of patients with metastatic breast cancer has shown high response rates; even single-agent vinorelbine in patients previously exposed to anthracyclines results in significant remission. Alterations in protein kinase-mediated signal transduction and p53 mutations may play a role in drug resistance with cross-talk between signal transduction and p53 pathways. The aim of this study was to establish the effects of doxorubicin and vinorelbine, as single agents, in combination, and as sequential treatments, on signal transduction and p53 in the breast cancer cell lines MCF-7 and MDA-MB-468. In both cell lines, increased p38 activity was demonstrated following vinorelbine but not doxorubicin treatment, whether vinorelbine was given prior to or simultaneously with doxorubicin. Mitogen-activated protein kinase (MAPK) activity and p53 expression remained unchanged following vinorelbine treatment. Doxorubicin treatment resulted in increased p53 expression, without changes in MAPK or p38 activity. These findings suggest that the effect of doxorubicin and vinorelbine used in combination may be achieved at least in part through distinct mechanisms. This additivism, where doxorubicin acts via p53 expression and vinorelbine through p38 activation, may contribute to the high clinical response rate when the two drugs are used together in the treatment of breast cancer.     

Deoxycholate induces COX-2 expression via Erk1/2-, p38-MAPK and AP-1-dependent mechanisms in esophageal cancer cells

Background  

The progression from Barrett's metaplasia to adenocarcinoma is associated with the acquirement of an apoptosis-resistant phenotype. The bile acid deoxycholate (DCA) has been proposed to play an important role in the development of esophageal adenocarcinoma, but the precise molecular mechanisms remain undefined. The aim of this study was to investigate DCA-stimulated COX-2 signaling pathways and their possible contribution to deregulated cell survival and apoptosis in esophageal adenocarcinoma cells.     

hsa_circ_0000520通过调控miR-556-5p/SLC38A2促进乳腺癌的发生和转移

目的:探究hsa_circ_0000520促进乳腺癌发生和转移的作用机制。方法:双荧光素酶报告基因实验、RNA pull-down实验验证miR-556-5p与hsa_circ_0000520、溶质载体家族38成员2（SLC38A2）的靶向关系。MCF7细胞分为sh-NC组、sh-hsa_circ_0000520组、sh-hsa_circ_0000520+anti-NC组、sh-hsa_circ_0000520+anti-miR-556-5p组,qRT-PCR或Western blot检测细胞中hsa_circ_0000520、miR-556-5p、SLC38A2表达水平;MTT检测、平板克隆形成实验评估细胞增殖能力;划痕愈合实验、Transwell实验评估细胞迁移、侵袭能力。通过裸鼠成瘤实验评估hsa_circ_0000520对miR-556-5p/SLC38A2的调控作用及对移植瘤生长的影响。结果:经验证,MCF7细胞中miR-556-5p与hsa_circ_0000520、SLC38A2均存在靶向关系。与sh-NC组比较,sh-hsa_circ_0000520组可降低MCF7细胞中hsa_circ_0000520、SLC38A2 mRNA和蛋白表达水平、细胞活力、克隆形成数目、划痕愈合率及迁移、侵袭细胞数（P＜0.05）,升高miR-556-5p表达水平（P＜0.05）;与sh-hsa_circ_0000520+anti-NC组比较,sh-hsa_circ_0000520+anti-miR-556-5p组可降低MCF7细胞中miR-556-5p表达水平（P＜0.05）,升高SLC38A2 mRNA和蛋白表达水平、细胞活力、克隆形成数目、划痕愈合率及迁移、侵袭细胞数（P＜0.05）,而对hsa_circ_0000520表达无显著影响（P＞0.05）。裸鼠成瘤实验结果表明,敲低移植瘤中hsa_circ_0000520的表达可升高miR-556-5p表达水平并降低SLC38A2 mRNA和蛋白表达水平,同时降低肿瘤体积和肿瘤重量（P＜0.05）。结论:hsa_circ_0000520可能通过靶向调控miR-556-5p/SLC38A2促进乳腺癌的发生和转移。     

D4-GDI, a Rho GTPase regulator, promotes breast cancer cell invasiveness

D4-GDI is a Rho GDP dissociation inhibitor that is widely expressed in hematopoietic cells. Its possible expression and function in breast cancer cells has not been described. Here, we found that D4-GDI is expressed in a panel of breast cancer cell lines, but not in benign-derived mammary epithelial cells. Knockdown of D4-GDI expression in MDA-MB-231 cells by RNA interference blocks cell motility and invasion. The cells lacking D4-GDI grown on Matrigel revert to a normal breast epithelial phenotype characterized by the formation of cavitary structures. Silencing D4-GDI expression inhibits beta1-integrin expression and cell-matrix adhesion. Reintroduction of D4-GDI fully restored both beta1-integrin expression and cellular invasion. Knockdown of D4-GDI in BT549 cells results in a similar effect. These results show that D4-GDI modulates breast cancer cell invasive activities.     

Human cytomegalovirus induces cellular tyrosine kinase signaling and promotes glioma cell invasiveness

Given our previous findings that human cytomegalovirus (HCMV) nucleic acids and proteins are expressed in human malignant glioma in vivo, we investigated cellular signaling events associated with HCMV infection of human glioma and astroglial cells. HCMV infection caused rapid activation of the phosphatidylinositol-3 kinase (PI-3K) effector AKT kinase in human astro-glial and fibroblast cells, and induced tyrosine phosphorylation of phospholipase Cγ (PLCγ). Co-immunoprecipitation experiments revealed association of the p85 regulatory subunit of PI-3K with a high-molecular weight protein phosphorylated on tyrosine, following short-term exposure to HCMV. In contrast to a previous report, we were unable to confirm the identity of this high-molecular weight protein as being the epidermal growth factor receptor (EGFR). Stimulation of glioma and fibroblast cell lines over-expressing EGFR with HCMV (whole virus) or soluble glycoprotein B did not induce tyrosine phosphorylation of the receptor, as did the genuine ligand, EGF. Furthermore, we found that expression levels of the human ErbB1-4 receptors were not rate-limiting for HCMV infection. Dispensability of EGFR function during early HCMV infection was substantiated by demonstration of viral immediate early gene expression in cells lacking the EGFR gene, indicating that HCMV may promote oncogenic signaling pathways independently of EGFR activation. Among non-receptor cellular kinases, HCMV infection induced phosphorylation of focal adhesion kinase (FAK) Tyr397, which is indispensable for integrin-mediated cell migration and invasion. HCMV-induced FAK activation was paralleled by increased extracellular matrix-dependent migration of human malignant glioma but not normal astro-glial cells, suggesting that HCMV can selectively augment glioma cell invasiveness. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

CPNE1 predicts poor prognosis and promotes tumorigenesis and radioresistance via the AKT singling pathway in triple-negative breast cancer

Elevated expression of Copine 1 (CPNE1) has been observed in multiple cancers; however, the underlying mechanisms by which it affects cancer cells are unclear. We aimed to study the effect of CPNE1 on the tumorigenesis and radioresistance of triple-negative breast cancer (TNBC). Quantitative real-time polymerase chain reaction was used to detect the expression of CPNE1 in TNBC tissues and cell lines. Western blot, immunohistochemistry, and immunofluorescence were used to investigate the levels of CPNE1, p-AKT, AKT, cleaved caspase-3, cleaved PARP1, and γ-H2AX. Cell viability and apoptosis were measured by CCK-8 and flow cytometry, respectively. CPNE1 was overexpressed in TNBC tissues and cell lines and was associated with tumor size, distant metastases, and survival rates of patients with TNBC. Moreover, function study shows that CPNE1 promoted cell viability and inhibited cell apoptosis in vitro and inhibited the radiosensitivity of TNBC. Importantly, inactivation of AKT signaling inhibited the tumorigenesis and radioresistance mediated by CPNE1 in TNBC cells. In vivo xenograft study also shows that CPNE1 knockdown inhibited tumor growth and promoted cell apoptosis. Overall, our findings suggest that CPNE1 promotes tumorigenesis and radioresistance in TNBC by regulating AKT activation and targeted CPNE1 expression may be a strategy to sensitize TNBC cells toward radiation therapy.     

COX-2和p53在乳腺癌组织中的表达及其相关性

目的:研究乳腺癌中环氧化酶-2(cy-clooxygenase-2,COX-2)和p53的表达及其相关性。方法:采用免疫组织化学法检测48例乳腺癌组织中COX-2与p53的表达情况,分析其相互间关系及与乳腺癌临床病理学特征间的联系。结果:乳腺癌中COX-2表达阳性率60·4%(29/48)。COX-2高表达与肿块>2cm、淋巴结转移阳性、ER阴性、PR阴性显著相关,而与患者年龄、肿瘤病理类型及肿瘤分期无明显关系。在p53阳性的乳腺癌患者中COX-2表达阳性率82·6%(19/23),而p53阴性的乳腺癌患者中,COX-2表达阳性率只有40·0%(10/25),两者相比有统计学意义,P=0·006。结论:COX-2在乳腺癌中高水平表达且与p53密切相关,提示乳腺癌中COX-2与p53存在互相调控机制,共同促进肿瘤的发生和发展。     

Therapeutic IMC-C225 antibody inhibits breast cancer cell invasiveness via Vav2-dependent activation of RhoA GTPase

PURPOSE: Abnormalities in the expression and signaling pathways downstream of epidermal growth factor receptor (EGFR) contribute to progression, invasion, and maintenance of the malignant phenotype in human cancers. Accordingly, biological agents, such as the EGFR-blocking antibody IMC-C225 have promising anticancer potential and are currently in various stages of clinical development. Because use of IMC-C225 is limited, at present, only for treatment of cancer with high EGFR expression, the goal of the present study was to determine the effect of IMC-C225 on the invasiveness of breast cancer cells with high and low levels of EGFR expression. EXPERIMENTAL DESIGN: The effect of IMC-C225 on invasion was studied using breast cancer cell lines with high and low levels of EGFR expression. RESULTS: The addition of EGF led to progressive stress fiber dissolution. In contrast, cells treated with IMC-C225 showed reduced invasiveness and increased stress-fiber formation. Interestingly, IMC-C225 pretreatment was accompanied by EGFR phosphorylation, as detected using an anti-phosphorylated tyrosine antibody (PY99), which correlated with phosphorylation of Vav2 guanine nucleotide exchange factor and activation of RhoA GTPase irrespective of EGFR level, and Vav2 interacted with EGFR only in IMC-C225-treated cells. The underlying mechanism involved an enhanced interaction between beta1 integrins and EGFR upon IMC-C225 treatment. CONCLUSION: Here, we defined a new mechanism for IMC-C225 that cross-links integrins with EGFR, leading to activation of RhoA and inhibition of breast cancer cell invasion irrespective of the level of EGFR in the cells, thus providing a rationale for using IMC-C225 in the metastatic setting independent of the levels of EGFR.     

NFATC1 promotes cell growth and tumorigenesis in ovarian cancer up-regulating c-Myc through ERK1/2/p38 MAPK signal pathway

Tumor Biology - It has been reported that nuclear factor of activated T cells (NFATC1) was up-regulated in cancers mediating malignant behaviors. However, the role of NFATC1 in ovarian cancer has...     

环氧合酶-2与乳腺癌的关系

研究发现乳腺癌组织内环氧合酶(COX)-2的表达与乳腺癌的发生、发展及预后有关。COX-2的表达可能增加乳腺癌细胞的活性和侵袭力,增加前列腺素产物的生成,抑制乳腺癌细胞的凋亡等。因此,COX-2抑制剂可能成为防治乳腺癌的新物质。     

环氧合酶-2与乳腺癌的关系

研究发现乳腺癌组织内环氧合酶（COX）-2的表达与乳腺癌的发生、发展及预后有关。COX-2的表达可能增加乳腺癌细胞的活性和侵袭力,增加前列腺素产物的生成,抑制乳腺癌细胞的凋亡等。因此,COX-2抑制剂可能成为防治乳腺癌的新物质。     

IL-17 expression by breast-cancer-associated macrophages: IL-17 promotes invasiveness of breast cancer cell lines

Introduction

IL-17 plays an important role in autoimmunity, promoting autoimmunity, inflammation and invasion in multiple sclerosis, rheumatoid arthritis and type I diabetes. The role of IL-17 in cancer is unclear, however, as there are few studies examining IL-17 protein expression in cancer. We therefore examined IL-17 protein expression in human breast cancer and modelled its potential biological significance in vitro.

Methods

Immunohistochemistry was used to determine IL-17 expression in breast cancers. Matrigel invasion assays were employed to examine the effect of IL-17 on cancer cell invasion by a panel of breast cancer cell lines. The role of matrix metalloproteinases (MMPs) was investigated with selective antagonists and immunoassays for MMP-2, MMP-3, MMP-9 and tissue inhibitor of MMP.

Results

IL-17-expressing cells with macrophage morphology were identified in the peritumoural area of a proportion of patients (8/19 patients). Macrophages were confirmed by CD68 staining on serial sections. With the exception of occasional lymphocytes, one patient with rare multinucleate giant cells and one patient with occasional expression of IL-17 in tumour cells, no other IL-17-positive cells were detected. Addition of IL-17 to cell lines in vitro stimulated marked invasion of Matrigel. In contrast, IL-17 did not promote the invasion of MCF7 or T47D cell lines. Invasion was initially thought to be dependent on MMPs, as evidenced by the broad-spectrum MMP inhibitor GM6001 and selective antagonists of MMP-2/MMP-9 and MMP-3. Measurement of MMP-2, MMP-3 and MMP-9, and tissue inhibitor of MMP 1 secretion, failed to reveal any changes in expression following IL-17 exposure. In contrast, TNF promoted secretion of MMPs but IL-17 did not augment TNF, indicating that IL-17 acts via an independent mechanism.

Conclusions

The present study is the first to describe in situ expression of IL-17 protein in human breast tumours and to propose a direct association between IL-17 and breast cancer invasion. The precise effectors of IL-17-dependent invasion remain to be characterised but could include a range of proteases such as a disintegrin and metalloproteinase protein or astacins. Nevertheless, this work identifies a novel potential mechanism for breast cancer invasion and tumour progression, the prognostic implication of which is currently under investigation.     

p85β alters response to EGFR inhibitor in ovarian cancer through p38 MAPK-mediated regulation of DNA repair

EGFR signaling promotes ovarian cancer tumorigenesis, and high EGFR expression correlates with poor prognosis. However, EGFR inhibitors alone have demonstrated limited clinical benefit for ovarian cancer patients, owing partly to tumor resistance and the lack of predictive biomarkers. Cotargeting EGFR and the PI3K pathway has been previously shown to yield synergistic antitumor effects in ovarian cancer. Therefore, we reasoned that PI3K may affect cellular response to EGFR inhibition. In this study, we revealed PI3K isoform-specific effects on the sensitivity of ovarian cancer cells to the EGFR inhibitor erlotinib. Gene silencing of PIK3CA (p110α) and PIK3CB (p110β) rendered cells more susceptible to erlotinib. In contrast, low expression of PIK3R2 (p85β) was associated with erlotinib resistance. Depletion of PIK3R2, but not PIK3CA or PIK3CB, led to increased DNA damage and reduced level of the nonhomologous end joining DNA repair protein BRD4. Intriguingly, these defects in DNA repair were reversed upon erlotinib treatment, which caused activation and nuclear import of p38 MAPK to promote DNA repair with increased protein levels of 53BP1 and BRD4 and foci formation of 53BP1. Remarkably, inhibition of p38 MAPK or BRD4 re-sensitized PIK3R2-depleted cells to erlotinib. Collectively, these data suggest that p38 MAPK activation and the subsequent DNA repair serve as a resistance mechanism to EGFR inhibitor. Combined inhibition of EGFR and p38 MAPK or DNA repair may maximize the therapeutic potential of EGFR inhibitor in ovarian cancer.     

SHP2 介导IL-6 促进乳腺癌细胞侵袭作用机制的研究*

目的：探讨非受体型酪氨酸磷酸酶SHP 2 介导白细胞介素- 6（interleukin- 6 ,IL- 6）促进人乳腺癌细胞侵袭的作用,以及相应的分子机制。方法：利用外源性重组IL- 6 处理人乳腺癌细胞T 47D ,采用表达IL- 6 的慢病毒感染T 47D 细胞使其内源性表达IL- 6,观察细胞的形态学改变情况,分析细胞迁移和侵袭能力的变化。采用小RNA 干扰的方法下调IL- 6 信号通路中关键分子SHP 2 的表达,观察其表达下降对IL- 6 促进乳腺癌细胞侵袭能力的影响,同时采用Westernblot方法检测Erk1/ 2 磷酸化变化。结果：上调IL- 6 在乳腺癌细胞中的表达显著促进乳腺癌细胞的迁移和侵袭能力,且细胞发生由上皮形态向类成纤维细胞形态的变化,同时伴随着上皮标志性蛋白E-cadherin 表达下调和间质标志Vimentin 表达上调。下调SHP 2 的表达明显抑制IL- 6 诱导乳腺癌细胞的上皮间质转化（epithelialmesenchymaltransition,EMT ）和侵袭能力,同时伴随着细胞内Erk1/ 2 磷酸化水平的下降。结论：SHP 2 通过介导IL- 6 诱导的EMT 促进乳腺癌细胞的迁移和侵袭能力。