Rac1 modulates TGF-β1-mediated epithelial cell plasticity and MMP9 production in transformed keratinocytes

Transforming growth factor-β1 (TGF-β1) activates Rac1 GTPase in mouse transformed keratinocytes. Expression of a constitutively active Q61LRac1 mutant induced an epithelial to mesenchymal transition (EMT) linked to stimulation of cell migration and invasion. On the contrary, expression of a dominant-negative N17TRac1 abolished TGF-β1-induced cell scattering, migration and invasion. Moreover, Q61LRac1 enhanced metalloproteinase-9 (MMP9) production to levels comparable to those induced by TGF-β1, while N17TRac1 was inhibitory. TGF-β1-mediated EMT involves the expression of the E-cadherin repressor Snail1, regulated by the Rac1 and mitogen-activated protein kinase (MAPK) pathways. Furthermore, MMP9 production was MAPK-dependent, as the MEK inhibitor PD98059 decreased TGF-β1-induced MMP9 expression and secretion in Q61LRac1 expressing cells. We propose that regulation of TGF-β1-mediated plasticity of transformed keratinocytes requires the cooperation between the Rac1 and MAPK signalling pathways.     

Autocrine galectin-1 promotes collective cell migration of squamous cell carcinoma cells through up-regulation of distinct integrins

We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and β5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.     

Hypoxia Activated EGFR Signaling Induces Epithelial to Mesenchymal Transition (EMT)

Metastasis is a multi-step process which requires the conversion of polarized epithelial cells to mesenchymal cells, Epithelial–Mesenchymal Transition (EMT). EMT is essential during embryonic morphogenesis and has been implicated in the progression of primary tumors towards metastasis. Hypoxia is known to induce EMT; however the molecular mechanism is still poorly understood. Using the A431 epithelial cancer cell line, we show that cells grown under hypoxic conditions migrated faster than cells grown under normal oxygen environment. Cells grown under hypoxia showed reduced adhesion to the extracellular matrix (ECM) probably due to reduced number of Vinculin patches. Growth under hypoxic conditions also led to down regulation of E-cadherin and up regulation of vimentin expression. The increased motility of cells grown under hypoxia could be due to redistribution of Rac1 to the plasma membrane as opposed to increased expression of Rac1. EGF (Epidermal Growth Factor) is a known inducer of EMT and growth of A431 cells in the absence of oxygen led to increased expression of EGFR (EGF Receptor). Treatment of A431 cells with EGF led to reduced cell adhesion to ECM, increased cell motility and other EMT characteristics. Furthermore, this transition was blocked by the monoclonal antibody Cetuximab. Cetuximab also blocked the hypoxia-induced EMT suggesting that cell growth under hypoxic conditions led to activation of EGFR signaling and induction of EMT phenotype.     

Numb is a negative regulator of HGF dependent cell scattering and Rac1 activation

Numb is an endocytic adaptor protein that regulates internalization and post-endocytic trafficking of cell surface proteins. In polarized epithelial cells Numb is localized to the basolateral membrane, and recent work has implicated Numb in regulation of cell adhesion and migration, suggesting a role for Numb in epithelial–mesenchymal transition (EMT). We depleted MDCK cells of Numb and examined the effects downstream of EMT-promoting stimuli. While knockdown of Numb did not affect apicobasal polarity, we show that depletion of Numb destabilizes E-cadherin-based cell–cell adhesion and promotes loss of epithelial cell morphology. In addition, Numb knockdown in MDCK cells potentiates HGF-induced lamellipodia formation and cell dispersal. Examination of Rac1-GTP levels in Numb knockdown cells revealed hyperactivation of Rac1 following extracellular calcium depletion and HGF stimulation, which corresponds with enhanced loss of cell adhesions and lamellipodia formation. Furthermore, inhibition of Rac1 in Numb depleted cells stabilized cell–cell contacts following depletion of extracellular calcium. Together, these data indicate that Numb acts to suppress Rac1-GTP accumulation, and its loss leads to increased sensitivity toward extracellular signals that disrupt cell–cell adhesion to induce epithelial–mesenchymal transition (EMT) and cell dispersal.     

Epithelium and fibroblast-like phenotypes derived from HPV16 E6/E7-immortalized human gingival keratinocytes following chronic ethanol treatment

Epithelial-mesenchymal transition (EMT) may be critical for neoplastic progression and its eventual tumorigenicity of epithelia. In this context, we investigated whether EMT and EMT-associated features occurred after chronic ethanol treatment of human gingival keratinocytes immortalized with the E6/E7 oncogenes of human papillomavirus (HPV) type 16. Following a nine-week treatment of cells with 30 mM ethanol in keratinocyte growth medium, they were cultured in normal DMEM with 10% serum. These cell populations were able to proliferate in this medium gradually exhibiting elongated morphology indicating that these cells underwent EMT. Control cells without ethanol treatment did not survive subcultures in DMEM. Upon long-term subcultures of ethanol-treated cells, two phenotypes were obtained exhibiting epithelium-like and spindle-shape fibroblast-like morphology (respectively, termed as EPI and FIB cells), the latter indicating EMT. In comparison to EPI cells, the phenotypic transition to FIB cells was concomitant with a decrease in the expression of keratins, desmoplakins and a complete loss of K14. Moreover, FIB cell transition strongly correlates with an increase in the expression of vimentin and simple epithelial keratin K18. These alterations in FIB cells were associated with the ability of these cells to exhibit anchorage-independent growth, while EPI cells exhibited anchorage-dependent growth. Concerning the transformation stage, FIB cells represent a progressively more advanced transformed phenotype which may reflect an early step during HPV- and ethanol-dependent multi-step carcinogenesis.     

p62/Sequestosome 1 regulates transforming growth factor beta signaling and epithelial to mesenchymal transition in A549 cells

Transforming growth factor beta (TGFβ) receptor trafficking regulates many TGFβ-dependent cellular outcomes including epithelial to mesenchymal transition (EMT). EMT in A549 non-small cell lung cancer (NSCLC) cells has recently been linked to the regulation of cellular autophagy. Here, we investigated the role of the autophagy cargo receptor, p62/sequestosome 1 (SQSTM1), in regulating TGFβ receptor trafficking, TGFβ1-dependent Smad2 phosphorylation and EMT in A549 NSCLC cells. Using immunofluorescence microscopy, p62/SQSTM1 was observed to co-localize with TGFβ receptors in the late endosome. Small interfering RNA (SiRNA)-mediated silencing of p62/SQSTM1 resulted in an attenuated time-course of Smad2 phosphorylation but did not alter Smad2 nuclear translocation. However, p62/SQSTM1 silencing promoted TGFβ1-dependent EMT marker expression, actin stress fiber formation and A549 cell migration. We further observed that Smad4-independent TGFβ1 signaling decreased p62/SQSTM1 protein levels via a proteasome-dependent mechanism. Although p62/SQSTM1 silencing did not impede TGFβ-dependent autophagy, our results suggest that p62/SQSTM1 may aid in maintaining A549 cells in an epithelial state and TGFβ1 decreases p62/SQSTM1 prior to inducing EMT and autophagy.     

Proline-rich tyrosine kinase 2 (Pyk2) promotes cell motility of hepatocellular carcinoma through induction of epithelial to mesenchymal transition

Aims

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase of the focal adhesion kinase (FAK) family, is up-regulated in more than 60% of the tumors of hepatocellular carcinoma (HCC) patients. Forced overexpression of Pyk2 can promote the proliferation and invasion of HCC cells. In this study, we aimed to explore the underlying molecular mechanism of Pyk2-mediated cell migration of HCC cells.

Methodology/Principal Findings

We demonstrated that Pyk2 transformed the epithelial HCC cell line Hep3B into a mesenchymal phenotype via the induction of epithelial to mesenchymal transition (EMT), signified by the up-regulation of membrane ruffle formation, activation of Rac/Rho GTPases, down-regulation of epithelial genes E-cadherin and cytokeratin as well as promotion of cell motility in presence of lysophosphatidic acid (LPA). Suppression of Pyk2 by overexpression of dominant negative PRNK domain in the metastatic HCC cell line MHCC97L transformed its fibroblastoid phenotype to an epithelial phenotype with up-regulation of epithelial genes, down-regulation of mesenchymal genes N-cadherin and STAT5b, and reduction of LPA-induced membrane ruffle formation and cell motility. Moreover, overexpression of Pyk2 in Hep3B cells promoted the phosphorylation and localization of mesenchymal gene Hic-5 onto cell membrane while suppression of Pyk2 in MHCC97L cells attenuated its phosphorylation and localization.

Conclusion

These data provided new evidence of the underlying mechanism of Pyk2 in controlling cell motility of HCC cells through regulation of genes associated with EMT.     

上皮间质转化与干细胞自我更新和分化

上皮间质转化(epithelial-mesenchymal transition,EMT)是指上皮细胞失去连接和极性转变为间质细胞的过程,这一现象普遍存在于胚胎发育、创伤愈合、器官纤维化以及肿瘤转移.在胚胎早期发育和晚期发育过程,例如着床、原肠运动、心血管发育等事件中有EMT和间质上皮转化(mesenchymal-ep...     

A multiscale computational model of YAP signaling in epithelial fingering behavior

In epithelial-mesenchymal transition (EMT), cells organized into sheets break away and become motile mesenchymal cells. EMT plays a crucial role in wound healing, embryonic development, and cancer metastasis. Intracellular signaling in response to mechanical, topographic, or chemical stimuli can promote EMT. We present a multiscale model for EMT downstream of the protein YAP, which suppresses the cell-cell adhesion protein E-cadherin and activates the GTPase Rac1 that enhances cell migration. We first propose an ordinary differential equation (ODE) model for intracellular YAP/Rac1/E-cadherin interactions. The ODE model dynamics are bistable, accounting for both motile loose cells and adherent slower cells. We incorporate this model into a cellular Potts model simulation of two-dimensional wound healing using the open-source platform Morpheus. We show that, under suitable stimuli representing topographic cues, the sheet exhibits finger-like projections and EMT. Morphological differences and quantitative differences in YAP levels as well as variations in cell speed across the sheet are consistent with previous experimental observations of epithelial sheets grown on topographic features in vitro. The simulation is also consistent with experiments that knock down or overexpress YAP, inhibit Rac1, or block E-cadherin.     

Neuregulin induces HaCaT keratinocyte migration via Rac1-mediated NADPH-oxidase activation

Neuregulin (NRG), a member of the epidermal growth factor family, plays important roles in the development of the nervous system and heart, and in cancer progression. Recent reports have suggested that NRG is involved in wound healing in keratinocytes, although the cellular mechanisms remain unclear. Here, we showed that NRG treatment increased slingshot-1L (SSH-1L)-mediated cofilin dephosphorylation and activation in HaCaT keratinocytes. Additionally, Rac1 activation and NADPH-oxidase (Nox)-dependent reactive oxygen species (ROS) generation, both known to be upstream regulators of the SSH-cofilin pathway, were increased in NRG-stimulated HaCaT cells. Inhibition of Rac1 or Nox activity blocked NRG-induced cofilin activation and cell migration by HaCaT cells. Moreover, the effects of Rac1 on cofilin activation were dependent on Nox activity. These findings indicate that NRG-induced HaCaT cell migration via the ROS-SSH-1L-cofilin pathway is activated as a consequence of Rac1 and Nox activation.     

Epithelium Expressing the E7 Oncoprotein of HPV16 Attracts Immune-Modulatory Dendritic Cells to the Skin and Suppresses Their Antigen-Processing Capacity

Antigen presenting cells (APCs) in skin can promote either antigen-specific effector functions or antigen tolerance, and thus determine clearance or persistence of cutaneous viral infections. Human papillomavirus (HPV) infections can persist in squamous epithelium in immunocompetent individuals, and some persisting HPV infections, particularly with HPV16, promote malignant epithelial transformation. Here, we investigate whether local expression of the HPV16 protein most associated with malignant transformation, HPV16-E7, affects the phenotype and function of APC subsets in the skin. We demonstrate an expanded population of Langerhans cells in HPV16-E7 transgenic skin with distinct cell surface markers which express immune-modulatory enzymes and cytokines not expressed by cells from non transgenic skin. Furthermore, HPV16-E7 transgene expression in keratinocytes attracts new APC subsets to the epidermis. In vivo migration and transport of antigen to the draining lymph node by these APCs is markedly enhanced in HPV16-E7 expressing skin, whereas antigen-processing, as measured by proteolytic cleavage of DQ-OVA and activation of T cells in vivo by APCs, is significantly impaired. These data suggest that local expression of HPV16-E7 in keratinocytes can contribute to persisting infection with this oncogenic virus, by altering the phenotype and function of local APCs.     

Dioxin Receptor Expression Inhibits Basal and Transforming Growth Factor β-induced Epithelial-to-mesenchymal Transition

Recent studies have emphasized the role of the dioxin receptor (AhR) in maintaining cell morphology, adhesion, and migration. These novel AhR functions depend on the cell phenotype, and although AhR expression maintains mesenchymal fibroblasts migration, it inhibits keratinocytes motility. These observations prompted us to investigate whether AhR modulates the epithelial-to-mesenchymal transition (EMT). For this, we have used primary AhR^+/+ and AhR^−/− keratinocytes and NMuMG cells engineered to knock down AhR levels (sh-AhR) or to express a constitutively active receptor (CA-AhR). Both AhR^−/− keratinocytes and sh-AhR NMuMG cells had increased migration, reduced levels of epithelial markers E-cadherin and β-catenin, and increased expression of mesenchymal markers Snail, Slug/Snai2, vimentin, fibronectin, and α-smooth muscle actin. Consistently, AhR^+/+ and CA-AhR NMuMG cells had reduced migration and enhanced expression of epithelial markers. AhR activation by the agonist FICZ (6-formylindolo[3,2-b]carbazole) inhibited NMuMG migration, whereas the antagonist α-naphthoflavone induced migration as did AhR knockdown. Exogenous TGFβ exacerbated the promigratory mesenchymal phenotype in both AhR-expressing and AhR-depleted cells, although the effects on the latter were more pronounced. Rescuing AhR expression in sh-AhR cells reduced Snail and Slug/Snai2 levels and cell migration and restored E-cadherin levels. Interference of AhR in human HaCaT cells further supported its role in EMT. Interestingly, co-immunoprecipitation and immunofluorescence assays showed that AhR associates in common protein complexes with E-cadherin and β-catenin, suggesting the implication of AhR in cell-cell adhesion. Thus, basal or TGFβ-induced AhR down-modulation could be relevant in the acquisition of a motile EMT phenotype in both normal and transformed epithelial cells.     

Zyxin Is a Transforming Growth Factor-β (TGF-β)/Smad3 Target Gene That Regulates Lung Cancer Cell Motility via Integrin α5β1

Although TGF-β acts as a tumor suppressor in normal tissues and in early carcinogenesis, these tumor suppressor effects are lost in advanced malignancies. Single cell migration and epithelial-mesenchymal transition (EMT), both of which are regulated by TGF-β, are critical steps in mediating cancer progression. Here, we sought to identify novel direct targets of TGF-β signaling in lung cancer cells and have indentified the zyxin gene as a target of Smad3-mediated TGF-β1 signaling. Zyxin concentrates at focal adhesions and along the actin cytoskeleton; as such, we hypothesized that cytoskeletal organization, motility, and EMT in response to TGF-β1 might be regulated by zyxin expression. We show that TGF-β1 treatment of lung cancer cells caused rapid phospho-Smad3-dependent expression of zyxin. Zyxin expression was critical for the formation and integrity of cell adherens junctions. Silencing of zyxin decreased expression of the focal adhesion protein vasodilator-activated phospho-protein (VASP), although the formation and morphology of focal adhesions remained unchanged. Zyxin-depleted cells displayed significantly increased integrin α5β1 levels, accompanied by enhanced adhesion to fibronectin and acquisition of a mesenchymal phenotype in response to TGF-β1. Zyxin silencing led to elevated integrin α5β1-dependent single cell motility. Importantly, these features are mirrored in the K-ras-driven mouse model of lung cancer. Here, lung tumors revealed decreased levels of both zyxin and phospho-Smad3 when compared with normal tissues. Our data thus demonstrate that zyxin is a novel functional target and effector of TGF-β signaling in lung cancer. By regulating cell-cell junctions, integrin α5β1 expression, and cell-extracellular matrix adhesion, zyxin may regulate cancer cell motility and EMT during lung cancer development and progression.     

Overexpression of geranylgeranyl diphosphate synthase contributes to tumour metastasis and correlates with poor prognosis of lung adenocarcinoma

This study aimed to evaluate the biological role of geranylgeranyl diphosphate synthase (GGPPS) in the progression of lung adenocarcinoma. GGPPS expression was detected in lung adenocarcinoma tissues by qRT‐PCR, tissue microarray (TMA) and western blotting. The relationships between GGPPS expression and the clinicopathological characteristics and prognosis of lung adenocarcinoma patients were assessed. GGPPS was down‐regulated in SPCA‐1, PC9 and A549 cells using siRNA and up‐regulated in A549 cells using an adenoviral vector. The biological roles of GGPPS in cell proliferation, apoptosis, migration and invasion were determined by MTT and colony formation assays, flow cytometry, and transwell and wound‐healing assays, respectively. In addition, the regulatory roles of GGPPS on the expression of several epithelial‐mesenchymal transition (EMT) markers were determined. Furthermore, the Rac1/Cdc42 prenylation was detected after knockdown of GGPPS in SPCA‐1 and PC9 cells. GGPPS expression was significantly increased in lung adenocarcinoma tissues compared to that in adjacent normal tissues. Overexpression of GGPPS was correlated with large tumours, high TNM stage, lymph node metastasis and poor prognosis in patients. Knockdown of GGPPS inhibited the migration and invasion of lung adenocarcinoma cells, but did not affect cell proliferation and apoptosis. Meanwhile, GGPPS inhibition significantly increased the expression of E‐cadherin and reduced the expression of N‐cadherin and vimentin in lung adenocarcinoma cells. In addition, the Rac1/Cdc42 geranylgeranylation was reduced by GGPPS knockdown. Overexpression of GGPPS correlates with poor prognosis of lung adenocarcinoma and contributes to metastasis through regulating EMT.     

Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.     

Epithelial‐mesenchymal transition softens head and neck cancer cells to facilitate migration in 3D environments

The biological impact and signalling of epithelial‐mesenchymal transition (EMT) during cancer metastasis has been established. However, the changes in biophysical properties of cancer cells undergoing EMT remain elusive. Here, we measured, via video particle tracking microrheology, the intracellular stiffness of head and neck cancer cell lines with distinct EMT phenotypes. We also examined cells migration and invasiveness in different extracellular matrix architectures and EMT‐related signalling in these cell lines. Our results show that when cells were cultivated in three‐dimensional (3D) environments, the differences in cell morphology, migration speed, invasion capability and intracellular stiffness were more pronounced among different head and neck cancer cell lines with distinct EMT phenotypes than those cultivated in traditional plastic dishes and/or seated on top of a thick layer of collagen. An inverse correlation between intracellular stiffness and invasiveness in 3D culture was revealed. Knock‐down of the EMT regulator Twist1 or Snail or inhibition of Rac1 which is a downstream GTPase of Twist1 increased intracellular stiffness. These results indicate that the EMT regulators, Twist1 and Snail and the mediated signals play a critical role in reducing intracellular stiffness and enhancing cell migration in EMT to promote cancer cells invasion.     

RhoA and Rac1 are both required for efficient wound closure of airway epithelial cells

Repair of the airway epithelium after injury is critical for restoring normal lung. The reepithelialization process involves spreading and migration followed later by cell proliferation. Rho-GTPases are key components of the wound healing process in many different types of tissues, but the specific roles for RhoA and Rac1 vary and have not been identified in lung epithelial cells. We investigated whether RhoA and Rac1 regulate wound closure of bronchial epithelial cells. RhoA and Rac1 proteins were efficiently expressed in a cell line of human bronchial epithelial cells (16HBE) by adenovirus-based gene transfer. We found that both constitutively active RhoA and dominant negative RhoA inhibited wound healing, suggesting that both activation and inhibition of RhoA interfere with normal wound healing. Overexpression of wild-type Rac1 induced upregulation of RhoA, disrupted intercellular junctions, and inhibited wound closure. Dominant negative Rac1 also inhibited wound closure. Inhibition of the downstream effector of RhoA, Rho-kinase, with Y-27632 suppressed actin stress fibers and focal adhesion formation, increased Rac1 activity, and stimulated wound closure. The activity of both RhoA and Rac1 are influenced by the polymerization state of microtubules, and cell migration involves coordinated action of actin and microtubules. Microtubule depolymerization upon nocodazole treatment led to an increase in focal adhesions and decreased wound closure. We conclude that coordination of both RhoA and Rac1 activity contributes to bronchial epithelial wound repair mechanisms in vitro, that inhibition of Rho-kinase accelerates wound closure, and that efficient repair involves intact microtubules.