Temporal and quantitative regulation of mitogen-activated protein kinase (MAPK) modulates cell motility and invasion.

We have shown that ER-negative and invasive human breast cancer cell lines MDA-MB-468 and MDA-MB-231 have constitutively higher mitogen activated protein kinase (ERK1&2/MAPK) when compared to the ER-positive and non-invasive MCF-7 human breast cancer cells. In MCF-7 cells, TGFalpha stimulation induced only transient MAPK activation, leading to a transient increase in cell migration. However, MDA 231 and MDA 468 cells, TGFalpha stimulation induced sustained MAPK activation, which correlated with enhanced cell motility and in vitro invasion. Serum stimulation activates ERK/MAPK activity persistently in both ER-positive and ER-negative breast cancer cells, leading to enhanced and sustained cell migration. Inhibition of MAPK activation by anti-sense MEK expression in MDA-MB-468 cells significantly inhibits cell migration and in vitro invasion. In contrast, MCF-7 cells expressing constitutively activated MEK show a significant increase in MAPK activity and cell migration, but this failed to enhance in vitro invasion. The kinetic profiles of MAPK activation and inhibition show a relationship between the duration and magnitude of MAPK activation and cell migration in both ER-positive and ER-negative human breast cancer cells. These studies show that cell motility is modulated by the magnitude and the duration of MAPK activation; but increased activation of MAPK may not be sufficient to allow in vitro invasion in non-invasive MCF-7 breast cancer cells.     

Caveolin-1 inhibits anchorage-independent growth, anoikis and invasiveness in MCF-7 human breast cancer cells

Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling and oncogenesis. Caveolin-1 is down-regulated in oncogene-transformed and tumor-derived cells. Antisense suppression of caveolin-1 or expression of a dominant negative form are sufficient for inducing cellular transformation. Expression of recombinant caveolin-1 inhibits anchorage-independent growth in cancer cells. The present study was designed to determine whether this is caused by inhibition of cancer cell survival or cell proliferation, and to test if another important property of cancer cells, i.e. matrix invasion, is modulated by expression of caveolin. Utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/Cav1), we demonstrate that caveolin-1 expression decreases MCF-7 cell proliferation rate and markedly reduces their capacity to form colonies in soft agar. The loss of anchorage-independent growth is not associated with stimulation of anoikis; in fact, MCF-7/Cav1 cells exhibit increased survival after detachment as compared with MCF-7 cells, indicating that in these cells caveolin-1 inhibits anoikis. Analysis of matrix metalloprotease release and matrix invasion revealed that expression of caveolin-1 inhibits also these important metastasis-related phenomena. Plating MCF-7 cells on a laminin matrix resulted in activation of ERK1/2, which was dramatically inhibited in MCF-7/Cav1 cells. We conclude that high expression level of caveolin-1 in human breast cancer cells exerts a negative modulatory effect on anchorage-independent growth by inhibiting cell proliferation even though matrix-independent cell survival is enhanced. Caveolin-1 expression inhibits also matrix invasion and blocks laminin-dependent activation of ERK1/2. The inhibitory effect of caveolin-1 on these transformation-dependent processes supports the hypothesis that caveolin-1 acts as a tumor suppressor protein which may impose major phenotypic changes when expressed in human cancer cells.     

Role of TGF-beta in cancer and the potential for therapy and prevention

Transforming growth factor (TGF)-beta is a naturally occurring potent inhibitor of cell growth. TGF-beta binds first to a Type II (TGFBR2), then a Type I receptor (TGFBR1). TGFBR1 activation results in the phosphorylation of intracellular messengers, the SMADs. Unrestricted cell growth due to decreased growth inhibitory activity is a paramount feature of a defect in TGF-beta function. There is growing evidence that common variants of the TGF-beta pathway ligand and receptors that alter TGF-beta signaling modify cancer risk. Approximately 14% of the general population carry TGFBR1*6A, a variant of the TGFBR1 gene that results in decreased TGF-beta-mediated growth inhibition. Recent studies show that overall cancer risk is increased by 70 and 19% among TGFBR1*6A homozygotes and heterozygotes, respectively. This suggests that TGFBR1*6A may contribute to the development of a large proportion of common forms of cancer and may become a target for cancer chemoprevention. While decreased TGF-beta signaling increases cancer risk, TGF-beta secretion and activated TGF-beta signaling enhances the aggressiveness of several types of tumors. The activated TGF-beta signaling pathway is emerging as an attractive target in cancer and the authors predict that assessment of functionally relevant variants of this pathway will lead to the identification of individuals with a higher cancer risk and account for some forms of familial cancer susceptibility. In addition, it is predicted that inhibitors of the TGF-beta signaling pathway will find their way into cancer clinical trials, leading to delays in tumor progression and improvements in overall survival.     

WNT-5A triggers Cdc42 activation leading to an ERK1/2 dependent decrease in MMP9 activity and invasive migration of breast cancer cells

An important role for WNT-5A is implicated in a variety of tumors, including breast carcinoma. We previously showed that WNT-5A signaling inhibits migration and metastasis of breast cancer cells, and that patients with primary breast cancer in which WNT-5A was expressed have a better prognosis. Despite the fact that RhoGTPase Cdc42 is commonly associated with increased cell migration, we here show that recombinant WNT-5A activates the Cdc42 in breast cancer cells (lines MDA-MB468 and MDA-MB231) in a time-dependent manner. Activation of Cdc42 was also observed in MDA-MB468 cells that were stably transfected with a WNT-5A plasmid (MDA-MB468-5A). In all situations, increased Cdc42 activity was accompanied by decreased migration and invasion of the breast cancer cells. To explore these findings further we also investigated the effect of WNT-5A signaling on ERK1/2 activity. Apart from an initial Ca²⁺-dependent rWNT-5A-induced activation of ERK1/2, Cdc42 activity was inversely correlated with ERK1/2 activity in both rWNT-5A-stimulated parental MDA-MB468 and MDA-MB468-5A cells. We also demonstrated increased ERK1/2 activity in MDA-MB468-5A cells following siRNA knockdown of Cdc42. Consistent with these results, breast cancer cells transfected with constitutively active Cdc42 exhibited reduced ERK1/2 activity, migration and invasion, whereas cells transfected with dominant negative Cdc42 had increased ERK1/2 activity in response to rWNT-5A. To gain information on how ERK1/2 can mediate its effect on breast cancer cell migration and invasion, we next investigated and demonstrated that WNT-5A signaling and constitutively active Cdc42 both decreased matrix metalloproteinase 9 (MMP9) activity. These data indicate an essential role of Cdc42 and ERK1/2 signaling and MMP9 activity in WNT-5A-impaired breast cancer cells.     

PAX-6在人乳腺癌中的表达及促进肿瘤转移的机制

目的:研究配对盒基因-6(paired box,Pax-6)在人乳腺癌组织中的表达和临床预后,及PAX-6对乳腺癌细胞MB-231和MCF-7迁移和侵袭的影响及机制。方法:以人正常乳腺上皮、乳腺癌及配对癌旁组织为研究对象;通过实时定量PCR和免疫蛋白印迹实验分析PAX-6在乳腺癌组织、癌旁组织及正常上皮组织中的相对表达;在线数据库分析PAX-6在乳腺癌中表达与预后的相关性;免疫蛋白印迹实验分析PAX-6在乳腺癌细胞系中的相对表达,选取高表达PAX-6基因的乳腺癌MB-231和MCF7细胞进行基因敲除,设定敲除效率高的si-PAX-6(#1)为实验组(P<0.05),si-NC细胞为对照组;Transwell实验分析PAX-6敲除对细胞迁移和侵袭的影响;实时定量PCR和免疫蛋白印迹实验验证PAX-6基因对细胞迁移和侵袭影响的机制。结果:与正常乳腺上皮、配对癌旁组织相比,PAX-6在人乳腺癌组织中表达上调(P<0.001);在线数据发现PAX-6的表达程度与乳腺癌的预后呈负相关,PAX-6基因表达程度越高,患者预后越差(P=0.038);与正常乳腺上皮细胞相比,PAX-6基因在乳腺癌细胞MB231、MCF-7和BT-549中高表达;与对照组相比,实验组si-PAX-6(#1)细胞迁移和侵袭明显受到抑制（P<0.001）;si-PAX-6(#1)敲除后能下调Vimentin、N-cadherin、MMP2、MMP7和MMP9 mRNA和蛋白表达水平,上调E-cadherin mRNA和蛋白表达水平。结论:PAX-6在乳腺癌的发生发展中起着重要的作用,可能作为潜在的癌基因参与乳腺癌的进程。     

Overexpression of CEACAM6 promotes migration and invasion of oestrogen-deprived breast cancer cells

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is an intercellular adhesion molecule that is overexpressed in a wide variety of human cancers, including colon, breast and lung and is associated with tumourigenesis, tumour cell adhesion, invasion and metastasis. In this study, we showed that CEACAM6 was overexpressed in a panel of oestrogen receptor (ERalpha)-positive human breast cancer cell lines (MCF-7:5C and MCF-7:2A) that have acquired resistance to oestrogen deprivation, and this overexpression was associated with a more aggressive invasive phenotype in vitro. Expression array analysis revealed that MCF-7:5C and MCF-7:2A cells overexpressed CEACAM6 mRNA by 27-fold and 12-fold, respectively, and were 6-15-times more invasive compared to non-invasive wild-type MCF-7 cells which expressed low levels of CEACAM6. Suppression of CEACAM6 expression using small interfering RNA (siRNA) completely reversed migration and invasion of MCF-7:5C and MCF-7:2A cells and it significantly reduced phosphorylated Akt and c-Src expression in these cells. In conclusion, our findings establish CEACAM6 as a unique mediator of migration and invasion of drug resistant oestrogen-deprived breast cancer cells and suggest that this protein could be an important biomarker of metastasis.     

The melanoma-associated antigen A3 mediates fibronectin-controlled cancer progression and metastasis

Tumor cells frequently exhibit decreased adhesiveness due to failure to deposit stromal fibronectin (FN), permitting more rapid proliferation, migration, invasion, and metastasis. Although up-regulation of FN has been noted in gene profiles of carcinomas compared with normal tissue, reduced FN expression has been described at the peripheral margins of invading tumors. In this study, we investigate the role of FN in cancer behavior. Using human thyroid carcinoma cells with stably down-regulated FN, we performed gene profiling and created an orthotopic mouse model. We stably overexpressed the FN target, MAGE A3, which has also been identified as a target of the breast cancer risk factor fibroblast growth factor receptor 2, and examined the functional effects in vitro and in vivo in a flank model and an orthotopic model of thyroid cancer. Mouse xenografts showed significantly enhanced tumor growth as well as larger and more numerous lung metastases in response to FN silencing. Gene profiling identified the melanoma-associated antigen (MAGE A3) as significantly up-regulated in response to FN silencing. Forced expression of MAGE A3 resulted in p21 down-regulation, accelerated cell cycle progression, increased cell migration rate, and invasion in vitro and in vivo in an orthotopic mouse model where microcomputed tomography confirmed lung metastases that recapitulate the progression of human thyroid cancer. We conclude that MAGE A3 is a functional integrator of diverse signals, including FGFR2 and FN, to modulate cancer progression.     

Combined genetic assessment of transforming growth factor-beta signaling pathway variants may predict breast cancer risk

There is growing evidence that common variants of the transforming growth factor-beta (TGF-beta) signaling pathway may modify breast cancer risk. In vitro studies have shown that some variants increase TGF-beta signaling, whereas others have an opposite effect. We tested the hypothesis that a combined genetic assessment of two well-characterized variants may predict breast cancer risk. Consecutive patients (n = 660) with breast cancer from the Memorial Sloan-Kettering Cancer Center (New York, NY) and healthy females (n = 880) from New York City were genotyped for the hypomorphic TGFBR1*6A allele and for the TGFB1 T29C variant that results in increased TGF-beta circulating levels. Cases and controls were of similar ethnicity and geographic location. Thirty percent of cases were identified as high or low TGF-beta signalers based on TGFB1 and TGFBR1 genotypes. There was a significantly higher proportion of high signalers (TGFBR1/TGFBR1 and TGFB1*CC) among controls (21.6%) than cases (15.7%; P = 0.003). The odds ratio [OR; 95% confidence interval (95% CI)] for individuals with the lowest expected TGF-beta signaling level (TGFB1*TT or TGFB1*TC and TGFBR1*6A) was 1.69 (1.08-2.66) when compared with individuals with the highest expected TGF-signaling levels. Breast cancer risk incurred by low signalers was most pronounced among women after age 50 years (OR, 2.05; 95% CI, 1.01-4.16). TGFBR1*6A was associated with a significantly increased risk for breast cancer (OR, 1.46; 95% CI, 1.04-2.06), but the TGFB1*CC genotype was not associated with any appreciable risk (OR, 0.89; 95% CI, 0.63-1.21). TGFBR1*6A effect was most pronounced among women diagnosed after age 50 years (OR, 2.20; 95% CI, 1.25-3.87). This is the first study assessing the TGF-beta signaling pathway through two common and functionally relevant TGFBR1 and TGFB1 variants. This approach may predict breast cancer risk in a large subset of the population.     

The Sprouty-related protein, Spred, inhibits cell motility, metastasis, and Rho-mediated actin reorganization

Sprouty and the Sprouty-related protein, Spred (Sprouty-related Ena/vasodilator-stimulated phosphoprotein homology-1 (EVH1) domain-containing protein), inhibit Ras-dependent extracellular signal-regulated kinase (ERK) signaling induced by a variety of growth factors. Since Sprouty proteins have been shown to inhibit not only ERK activation but also cell migration, we postulated that Spreds also inhibit cellular migration. Using stably highly metastatic LM8 cells infected with the Spred1-Sendai virus vector, we demonstrated that Spred1 inhibits the metastasis of LM8 cells in nude mice. Spred1 overexpression also inhibited migration of cells in vitro in response to chemokines, CCL19 and CCL21. We also found that Spred1 overexpression dissolved actin-stress fibers. Both EVH1 domain and C-terminal Sprouty-related domain were required for actin reassembly. Spred1 and Spred2 suppressed constitutively activated RhoA (V14RhoA)-induced stress fiber formation and serum response factor activation. Spred1 bound to activated RhoA, but not cdc42 and Rac. Spred1 also inhibited chemokine-induced RhoA activation and active RhoA-induced Rho-kinase activation. These data suggest that Spreds are key regulators of RhoA-mediated cell motility and signal transduction. Furthermore, our study suggests that the induction of Spreds could be a novel strategy for preventing cancer cell metastasis.     

Cooperative effects of Akt-1 and Raf-1 on the induction of cellular senescence in doxorubicin or tamoxifen treated breast cancer cells

Escape from cellular senescence induction is a potent mechanism for chemoresistance. Cellular senescence can be induced in breast cancer cell lines by the removal of estrogen signaling with tamoxifen or by the accumulation of DNA damage induced by the chemotherapeutic drug doxorubicin. Long term culturing of the hormone-sensitive breast cancer cell line MCF-7 in doxorubicin (MCF-7/DoxR) reduced the ability of doxorubicin, but not tamoxifen, to induce senescence. Two pathways that are often upregulated in chemo- and hormonal-resistance are the PI3K/PTEN/Akt/mTOR and Ras/Raf/MEK/ERK pathways. To determine if active Akt-1 and Raf-1 can influence drug-induced senescence, we stably introduced activated ΔAkt-1(CA) and ΔRaf-1(CA) into drug-sensitive and doxorubicin-resistant cells. Expression of a constitutively-active Raf-1 construct resulted in higher baseline senescence, indicating these cells possessed the ability to undergo oncogene-induced-senescence. Constitutive activation of the Akt pathway significantly decreased drug-induced senescence in response to doxorubicin but not tamoxifen in MCF-7 cells. However, constitutive Akt-1 activation in drug-resistant cells containing high levels of active ERK completely escaped cellular senescence induced by doxorubicin and tamoxifen. These results indicate that up regulation of the Ras/PI3K/PTEN/Akt/mTOR pathway in the presence of elevated Ras/Raf/MEK/ERK signaling together can contribute to drug-resistance by diminishing cell senescence in response to chemotherapy. Understanding how breast cancers containing certain oncogenic mutations escape cell senescence in response to chemotherapy and hormonal based therapies may provide insights into the design of more effective drug combinations for the treatment of breast cancer.     

AQP1通过Wnt通路促进乳腺癌MCF-7细胞增殖、迁移与侵袭

目的:探讨过表达水通道蛋白1（aquaporin 1,AQP1）基因对人乳腺癌细胞MCF-7增殖、迁移、侵袭能力的影响,分析其对Wnt通路的调控作用。方法:构建和包装pBabe-puro-AQP1逆转录病毒载体,建立稳定过表达AQP1基因的MCF-7细胞。细胞免疫荧光染色法检测外源性AQP1在MCF-7细胞内的定位,实时荧光定量PCR（qRT-PCR）和Western blot检测AQP1 mRNA及蛋白表达变化;采用CCK-8法检测细胞的增殖活性;流式细胞法检测细胞周期;划痕实验检测细胞迁移能力;Transwell法检测细胞的侵袭能力;基因表达谱芯片、Western blot检测转染后Wnt细胞通路相关基因表达改变。结果:稳定过表达AQP1后MCF-7细胞增殖、迁移、侵袭能力显著增加,差异有统计学意义（P＜0.001）。基因表达谱芯片结果显示,过表达AQP1后22个差异表达基因富集于Wnt细胞信号通路,mRNAs表达明显增强（P＜0.000 1）,Wnt细胞通路关键基因β-catenin及下游靶基因细胞周期蛋白D1(Cyclin D1)、C-Myc蛋白表达也显著增加。结论:AQP1可能通过激活Wnt信号通路促进MCF-7细胞增殖、迁移与侵袭。     

PPAPDC1A在体内外对乳腺癌细胞增殖、侵袭和转移能力的影响

目的:通过构建稳定过表达和干扰PPAPDC1A的乳腺癌细胞株,探讨PPAPDC1A对乳腺癌细胞增殖、侵袭和转移能力的影响。方法:利用CCK-8和Transwell实验检测PPAPDC1A稳定过表达和干扰后对乳腺癌细胞体外增殖和侵袭能力的影响。采用裸鼠皮下成瘤实验检测PPAPDC1A对乳腺癌细胞体内增殖和裸鼠致瘤性的作用。利用免疫组织化学染色法检测各组肿瘤组织中Ki-67的表达。通过裸鼠尾静脉注射实验检测PPAPDC1A对乳腺癌MCF-7细胞和MDA-MB-231细胞体内转移能力的影响。结果:成功建立稳定过表达PPAPDC1A的乳腺癌MCF-7细胞株和稳定干扰PPAPDC1A的乳腺癌MDA-MB-231细胞株;CCK-8和Transwell实验结果显示,与MCF-7和MCF-7-Vector细胞株相比,MCF-7-PPAPDC1A细胞株的生长速度显著增快,穿膜细胞数量多（P＜0.05）;与此相反,MDA-MB-231-shPPAPDC1A组细胞的生长速度和穿膜细胞数明显少于MDA-MB-231-shNC和MDA-MB-231 细胞株（P＜0.05）。动物实验结果显示,与MCF-7-Vector组相比,MCF-7-PPAPDC1A组的肿瘤生长速度较快,肿瘤的体积较大,Ki-67的阳性率高,肺转移灶的数目增多（P＜0.05）;与此相反,与MDA-MB-231-shNC组相比MDA-MB-231-shPPAPDC1A组的肿瘤生长速度较慢,肿瘤的体积较小,Ki-67的阳性率低,肺转移灶的数目减少（P＜0.05）。结论:PPAPDC1A对乳腺癌细胞的增殖、侵袭和转移有促进作用。     

Cyclic phosphatidic acid inhibits RhoA-mediated autophosphorylation of FAK at Tyr-397 and subsequent tumor-cell invasion

We demonstrated previously that rat ascites hepatoma MM1 cells require both lysophosphatidic acid (LPA) and fibronectin (FN) for phagokinetic motility and transcellular migration and that these events are regulated through the RhoA-ROCK pathway and tyrosine phosphorylation of proteins including focal adhesion kinase (FAK). Moreover, we reported that palmitoyl-cyclic phosphatidic acid (Pal-cPA), a structural analogue of LPA, inhibits LPA-induced migration of MM1 cells and experimental metastasis of B16 murine melanoma cells. However, the molecular mechanisms of action of Pal-cPA remains to be clarified. To examine this, total cellular lysates after stimulation with LPA or FN were subjected to time-course immunoblot analysis with anti-phophotyrosine and anti-pY397-FAK antibodies. Tyrosine-phosphorylation of FAK especially at Tyr-397 was obviously persistent after stimulation with LPA + FN compared to after stimulation with LPA alone. This persistent phosphorylation was necessary for MM1 cell migration and inhibited by Pal-cPA as by C3 exoenzyme Rho inhibitor. RhoA activity (GTP-bound RhoA) was also measured by the pull down assay using the Rho binding domain of Rhotekin. LPA-induced RhoA-activation of MM1 cells was completely inhibited by Pal-cPA. Moreover, we demonstrated that autophosphorylation of FAK at Tyr-397, downstream of RhoA, contributed to formation of focal adhesions and was critical in LPA-induced MM1 cell migration by developing autophosphorylation-deficient (Y397F) FAK-transfectants. Collectively, Pal-cPA hampered LPA-induced morphological changes and transcellular migration of MM1 cells through downregulating active RhoA and inhibiting its downstream events including autophosphorylation of FAK. Pal-cPA also inhibited endogenous (LPA-independent) activation of RhoA in human fibrosarcoma HT-1080 cells. Pal-cPA may potentially provide a new therapy for the treatment of cancer invasion and metastasis.     

Long non-coding RNA ARHGAP5-AS1 inhibits migration of breast cancer cell via stabilizing SMAD7 protein

Purpose

Tumor metastasis is the main cause of death from breast cancer patients and cell migration plays a critical role in cancer metastasis. Recent studies have shown long non-coding RNAs (lncRNAs) play an essential role in the initiation and progression of cancer. In the present study, the role of an LncRNA, Rho GTPase Activating Protein 5- Antisense 1 (ARHGAP5-AS1) in breast cancer was investigated.

Methods

RNA sequencing was performed to find out dysregulated LncRNAs in MDA-MB-231-LM2 cells. Transwell migration assays and F-actin staining were utilized to estimate cell migration ability. RNA pulldown assays and RNA immunoprecipitation were used to prove the interaction between ARHGAP5-AS1 and SMAD7. Western blot and immunofluorescence imaging were used to examine the protein levels. Dual luciferase reporter assays were performed to evaluate the activation of TGF-β signaling.

Results

We analyzed the RNA-seq data of MDA-MB-231 and its highly metastatic derivative MDA-MB-231-LM2 cell lines (referred to as LM2) and identified a novel lncRNA (NR_027263) named as ARHGAP5-AS1, which expression was significantly downregulated in LM2 cells. Further functional investigation showed ARHGAP5-AS1 could inhibit cell migration via suppression of stress fibers in breast cancer cell lines. Afterwards, SMAD7 was further identified to interact with ARHGAP5-AS1 by its PY motif and thus its ubiquitination and degradation was blocked due to reduced interaction with E3 ligase SMURF1 and SMURF2. Moreover, ARHGAP5-AS1 could inhibit TGF-β signaling pathway due to its inhibitory role on SMAD7.

Conclusion

ARHGAP5-AS1 inhibits breast cancer cell migration via stabilization of SMAD7 protein and could serve as a novel biomarker and a potential target for breast cancer in the future.

     

Transforming growth factor beta1 treatment leads to an epithelial-mesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase 2 activation

The aim of this study was to examine the effects of transforming growth factor (TGF) beta1 on the phenotype and the biological behavior of pancreatic cancer cell lines with and without mutations in the TGF-beta signaling pathway and to elucidate whether the Ras signaling cascade participates in mediating these effects of TGF-beta1. TGF-beta-responsive (PANC-1, COLO-357, and IMIM-PC1) and nonresponsive (CAPAN1 and IMIM-PC2) pancreatic cancer cell lines with activating mutations of the Ki-Ras oncogene were treated with 10 ng/ml TGF-beta1 over time. Phenotypic alterations were studied by electron and phase contrast microscopy and by immunohistochemistry and expression analyses of differentiation markers. The influence of TGF-beta on tumor cell scattering, migration, and invasion was determined. The role of the Ras-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade in mediating TGF-beta-induced morphological and functional effects were studied by pretreatment with the MEK1 inhibitor PD 98059 and by measuring ERK2 activation using immune complex kinase assays. TGF-beta1 led to a reversible and time-dependent epithelial-mesenchymal transdifferentiation (EMT) in TGF-beta-responsive pancreatic cancer cell lines, characterized by a fibroblastoid morphology and an up-regulation of mesenchymal markers and a down-regulation of epithelial markers. EMT was associated with an increase in tumor cell migration, invasion, and scattering. In the responsive cell lines, TGF-beta1 induced a moderate but sustained activation of ERK2. EMT, the concomitant changes in gene expression, and the invasive and migratory potential were reduced or abolished by pretreatment with the selective MEK1 inhibitor. Thus, in TGF-beta-responsive pancreatic cancer cells with activating Ki-Ras mutations, TGF-beta1 treatment caused an EMT associated with a more invasive phenotype. Cross-talk with the Ras-MEK-ERK-signaling cascade appears to be essential for mediating these effects of TGF-beta1.     

Rho GTP酶活化蛋白30对宫颈癌C41细胞增殖、迁移的影响及其机制

目的:探究Rho GTP酶活化蛋白30(ARHGAP30)在宫颈癌细胞中的作用，并分析其影响功能的可能机制。方法:收集临床组织样本，验证ARHGAP30的蛋白水平表达量，在公共数据库中分析ARHGAP30与预后之间的关系。构建ARHGAP30过表达质粒，对人宫颈癌细胞C41进行转染，建立C41-NC组及C41-ARHGAP30组，通过RT-PCR、Western Blot验证其转染效率，CCK8、Transwell实验检测其对C41细胞功能影响，并用RT-PCR、Western Blot验证其下游迁移相关通路表达量变化。结果:在肿瘤组织中，ARHGAP30的表达量更低，ARHGAP30表达量较低的患者预后更差。与NC组相比，ARHGAP30过表达组的细胞增殖能力显著下降（72 h，0.41 vs 0.86，P＜0.01）,细胞迁移能力显著下降（62.67 vs 352.67,P＜0.001），并下调整合素ITGA1、ITGB6的转录水平及蛋白水平。结论:ARHGAP30过表达抑制宫颈癌细胞的增殖、迁移功能，并且可以下调整合素蛋白ITGA1、ITGB6的转录及蛋白水平，在宫颈癌的发生发展中具有重要意义。     

Leptin promotes motility and invasiveness in human colon cancer cells by activating multiple signal-transduction pathways

Leptin serum levels are about 5 times higher in obese people than in normal individuals. We aimed at investigating the signaling pathways induced by leptin in the human colonic cell lines LS174T and HM7. Both cells expressed the leptin transmembrane Ob-receptor. Leptin activated the mitogen-activated protein kinase pathway, induced invasion of colonic cells and concomitantly increased the formation of lamellipodial structures. A direct and novel dose- and time-dependent activation of RhoA, Cdc42 and Rac1 by leptin is demonstrated in these aggressive colon cancer cells. The activation of the Rho family of GTPases was amenable to specific inhibition: Wortmannin inhibited leptin-induced Rac1 and Cdc42 activation but did not affect RhoA activation, and inhibited the formation of leptin-induced lamellipodia and cell invasion. The Rac1 inhibitor NSC23766 inhibited only leptin-induced Rac1 activation and concomitantly, lamellipodium formation and cell invasion. The Src kinase inhibitor II (SrcKI-II) exerted a positive effect on RhoA activation, inhibited tyrosine phosphorylation of p190RhoGAP and inhibited leptin-induced Cdc42 activation and leptin-induced lamellopodium formation and cell invasion. The specific JAK2 inhibitor AG490 exerted a positive effect on Rac1 and Cdc42 activation by leptin and concomitantly inhibited RhoA activation. AG490 did not inhibit leptin-induced lamellopodium formation or cell invasion. Our findings clearly indicate that leptin activates PI3K and Src kinase pathways in the metastatic colon cancer cells LS174T and HM7. These signaling pathways induce the activation of Rac1 and Cdc42, lamellopodium formation and concomitantly enhanced cell invasion, but leptin activation of RhoA is not associated with enhanced cell locomotion and invasion. Understanding in-depth the pathways involved in leptin-associated enhanced cell locomotion and invasion may contribute with the design of novel therapeutics to treat obesity-associated advanced colorectal cancer.     

miR-130a-3p通过HGF/MET信号通路抑制乳腺癌细胞MCF-7 侵袭

目的：探究miR-130a-3p 通过HGF/MET信号通路调控上皮间质转化（epithelial-mesenchymal transition,EMT）影响乳腺癌细胞侵袭转移的分子机制。方法：收集承德医学院附属医院2018 年1 月至10 月收治的22 例乳腺癌患者癌组织和配对癌旁组织标本,乳腺癌细胞系（MCF-7、MDA-MB-231 和MDA-MB-453）和正常乳腺上皮细胞MCF10A来自承德医学院基础研究所,然后采用qPCR检测组织和细胞系中miR-130a-3p 的表达情况;将实验分为对照组、miR-130a-3p mimics 组、miR-130a-3p inhibitor组、PHA665752（MET小分子抑制剂）转染组及共转PHA665752+miR-130a-3p inhibitor 组,然后采用CCK-8 法和Transwell 实验分别检测MCF-7 细胞增殖活力、侵袭和迁移能力;WB实验检测MCF-7 细胞EMT和HGF/MET信号通路相关蛋白的表达情况;此外,采用双荧光素酶报告基因检测miR-130a-3p 与MET之间的靶向关系。结果：miR-130a-3p 在乳腺癌组织和细胞系中呈低表达;过表达miR-130a-3p 可抑制MCF-7 细胞增殖、侵袭、迁移和EMT;而抑制miR-130a-3p 出现相反的结果。双荧光素酶报告基因结果证实miR-130a-3p 靶向下调MET的表达水平,且miR-130a-3p 负调控HGF/MET信号通路的表达;进一步实验证明,miR-130a-3p 通过阻断HGF/MET信号通路抑制MCF-7 细胞增殖、侵袭、迁移和EMT。结论：miR-130a-3p 通过阻断HGF/MET信号通路抑制MCF-7 细胞EMT过程,进而抑制MCF-7 细胞侵袭转移。     

Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion

Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However, an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study, we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1, side population and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore, knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised non-obese diabetic/severe combined immunodeficiency mice, supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion, but not for CSC maintenance. Taken together, our study provides evidence that KLF4 has a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell-like features and by promoting cell migration and invasion. Thus, targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer.