Caveolin-1 (Cav-1), a principal structural component of caveolar membrane domains, contributes to cancer development but its precise functional roles and regulation remain unclear. In this study, we determined the oncogenic function of Cav-1 in preclinical models of pancreatic cancer and in human tissue specimens. Cav-1 expression levels correlated with metastatic potential and epithelial-to-mesenchymal transition (EMT) in both mouse and human pancreatic cancer cells. Elevated levels in cells promoted EMT, migration, invasion and metastasis in animal models, whereas RNAi-mediated knockdown inhibited these processes. We determined that levels of Cav-1 and the Forkhead transcription factor FoxM1 correlated directly in pancreatic cancer cells and tumor tissues. Enforced expression of FoxM1 increased Cav-1 levels, whereas RNAi-mediated knockdown of FoxM1 had the opposite effect. FoxM1 directly bound to the promoter region of Cav-1 gene and positively transactivated its activity. Collectively, our findings defined Cav-1 as an important downstream oncogenic target of FoxM1, suggesting that dysregulated signaling of this novel FoxM1-Cav-1 pathway promotes pancreatic cancer development and progression.
Progression; angiogenesis; transcription factor; EMT; biomarkers
Dedifferentiation and loss of hepatocyte polarity during primary culture of hepatocytes are major drawbacks for metabolic analyses. As a prominent profibrotic cytokine and potent inducer of epithelial mesenchymal transition (EMT), TGF-β contributes to these processes in liver epithelial cells. Yet, a distinction between culture dependent and TGF-β driven hepatocyte dedifferentiation has not been shown to date.
Here, we show that in both settings, mesenchymal markers are induced. However, upregulation of Snai1 and downregulation of E-Cadherin are restricted to TGF-β effects, neglecting a full EMT of culture dependent hepatocyte dedifferentiation. Mechanistically, the latter is mediated via FAK/Src/ERK/AKT pathways leading to the induction of the oncogene caveolin-1 (Cav1). Cav1 was recently proposed as a new EMT marker, but our results demonstrate Cav1 is not up-regulated in TGF-β mediated hepatocyte EMT, thus limiting validity of its use for this purpose. Importantly, marking differences on Cav1 expression exist in HCC cell lines. Whereas well differentiated HCC cell lines exhibit low and inducible Cav1 protein levels - by TGF-β in a FAK/Src dependent manner, poorly differentiated cell lines display high Cav1 expression levels which are not further modulated by TGF-β.
This study draws a detailed distinction between intrinsic and TGF-β mediated hepatocyte dedifferentiation and elucidates cellular pathways involved. Additionally, by evaluating the regulation of the oncogene Cav1, we provide evidence to argue against Cav1 as a reliable EMT marker.
Epithelial to mesenchymal transition (EMT) promotes cellular motility, invasiveness and metastasis during embryonic development and tumorigenesis. Transforming growth factor-β (TGF-β) signaling pathway is a key regulator of EMT. A lot of evidences suggest that this process is Smad3-dependent. Herein we showed that exposure of aspc-1 and panc-1 pancreatic cancer cells to TGF-β1 resulted in characteristic morphological alterations of EMT, and enhancement of cell motility and gemcitabine (Gem) resistance along with an up-regulation of EMT markers genes such as vimentin, N-cadherin, MMP2 and MMP9. Naringenin (Nar) down-regulated EMT markers expression in both mRNA and protein levels by inhibiting TGF-β1/Smad3 signal pathway in the pancreatic cancer cells. Consequently, Nar suppressed the cells migration and invasion and reversed their resistance to Gem.
Slug, a member of the Snail family of transcription factors, has a crucial role in the regulation of epithelial-mesenchymal transition (EMT) by suppressing several epithelial markers and adhesion molecules, including E-cadherin. A recent study demonstrated that no relationship exists between Slug and E-cadherin in pancreatic cancer. Another study showed that in malignant mesothelioma effusions Slug was associated with matrix metalloproteinase (MMP) expression, but that there was no association with E-cadherin. F-ascin is an actin-bundling protein involved in filopodia assembly and cancer invasion and metastasis of multiple epithelial cancer types. In this study, we investigated Slug, E-cadherin, and MMP-9 expression using immunohistochemistry in 60 patients with pancreatic cancer and their correlation with carcinoma invasion and metastasis. Additionally, we observed the effects of Slug on invasion and metastasis in the pancreatic cancer cell line PANC-1. Alterations in Slug, MMP-9, and E-cadherin were determined by RT-PCR, western blot, and immunohistochemistry. Alterations in MMP-9 and F-actin cytoskeleton were determined by immunofluorescence staining, flow cytometry (FCM), or gelatin zymography. Slug, E-cadherin, and MMP-9 expression in pancreatic cancer was significantly associated with lymph node metastases and we found a significant correlation between Slug and MMP-9 expression; however, no significant correlation was observed between Slug and E-cadherin expression. Slug transfection significantly increased invasion and metastasis in PANC-1 cells and orthotopic tumor of mouse in vivo, and significantly upregulated and activated MMP-9; however, there was no effect on E-cadherin expression. Slug promoted the formation of lamelliopodia or filopodia in PANC-1 cells. The intracellular F-actin and MMP-9 was increased and relocated to the front of the extending pseudopodia from the perinuclear pool in Slug-transfected PANC-1 cells. These results suggest that Slug promotes migration and invasion of PANC-1 cells, which may correlate with the reorganization of MMP-9 and remodeling of the F-actin cytoskeleton, but not with E-cadherin expression.
E-cadherin; epithelial-mesenchymal transition; F-actin cytoskeleton; matrix metalloproteinase; metastasis; pancreatic carcinoma; Slug
Previously, we reported that caveolin-1 (cav-1) is overexpressed in metastatic prostate cancer and that virulent prostate cancer cells secrete biologically active cav-1. We also showed that cav-1 expression leads to prosurvival activities through maintenance of activated Akt and that cav-1 is taken up by other cav-1–negative tumor cells and/or endothelial cells, leading to stimulation of angiogenic activities through PI-3-K-Akt-eNOS signaling. To analyze the functional consequences of cav-1 overexpression on the development and progression of prostate cancer in vivo, we generated PBcav-1 transgenic mice. Adult male PBcav-1 mice showed significantly increased prostatic wet weight and higher incidence of epithelial hyperplasia compared with nontransgenic littermates. Increased immunostaining for cav-1, proliferative cell nuclear antigen, P-Akt, and reduced nuclear p27Kip1 staining occurred in PBcav-1 hyperplastic prostatic lesions. PBcav-1 mice showed increased resistance to castration-induced prostatic regression and elevated serum cav-1 levels compared with nontransgenic littermates. Intraprostatic injection of androgen-sensitive, cav-1–secreting RM-9 mouse prostate cancer cells resulted in tumors that were larger in PBcav-1 mice than in nontransgenic littermates (P = 0.04). Tail vein inoculation of RM-9 cells produced significantly more experimental lung metastases in PBcav-1 males than in nontransgenic male littermates (P = 0.001), and in cav-1+/+ mice than in cav-1−/− mice (P = 0.041). Combination treatment with surgical castration and systemic cav-1 antibody dramatically reduced the number of experimental metastases. These experimental data suggest a causal association of secreted cav-1 and prostate cancer growth and progression.
Bone morphogenetic proteins (BMPs) have an emerging role in human cancers. Here we demonstrate that the BMP-signaling pathway is intact and functional in human pancreatic cancer cells, with several BMP signaling components and transcriptional targets upregulated in human pancreatic cancer specimens compared with normal pancreatic tissue. Functionally, multiple BMP family members, including BMP-2, BMP-4 and BMP-7, induce an epithelial to mesenchymal transition (EMT) in the human pancreatic cancer cell line Panc-1, as demonstrated by morphological alterations and loss of E-cadherin expression. BMP-mediated EMT results in an increase in invasiveness of Panc-1 cells, in part through increased expression and activity of matrix metalloproteinase (MMP)-2, a known mediator of pancreatic cancer cell invasiveness. Accompanying EMT, BMP reduces expression of the transforming growth factor (TGF)-β superfamily receptor, transforming growth factor-β type III receptor (TβRIII), for which we have previously demonstrated loss of expression during pancreatic cancer progression. Maintaining TβRIII expression inhibits BMP-mediated invasion and suppresses Smad1 activation. Further, Smad1 is required for BMP-induced invasiveness and partially responsible for BMP-mediated increases in MMP-2 activity. These data suggest that BMP signaling, through Smad1 induction and upregulation of MMP-2, is an important mediator of pancreatic cancer invasiveness and a potential therapeutic target for treating this deadly disease.
Epithelial-mesenchymal transition (EMT) is a highly conserved process that has been well characterised in embryogenesis. Studies have shown that the aberrant activation of EMT in adult epithelia can promote tumour metastasis by repressing cell adhesion molecules, including epithelial (E)-cadherin. Reduced intracellular adhesion may allow tumour cells to disseminate and spread throughout the body. A number of transcription proteins of the Snail superfamily have been implicated in EMT. These proteins have been shown to be over-expressed in advanced gastrointestinal (GI) tumours including oesophageal adenocarcinomas, colorectal carcinomas, gastric and pancreatic cancers, with a concomitant reduction in the expression of E-cadherin. Regulators of EMT may provide novel clinical targets to detect GI cancers early, so that cancers previously associated with a poor prognosis such as pancreatic cancer can be diagnosed before they become inoperable. Furthermore, pharmacological therapies designed to inhibit these proteins will aim to prevent local and distant tumour invasion.
Epithelial-mesenchymal transition; Transcription proteins; E-cadherin; Gastrointestinal cancer
In the current study we investigated the role of caveolin-1 (cav-1) in pancreatic adenocarcinoma (PC) cell migration and invasion; initial steps in metastasis. Cav-1 is the major structural protein in caveolae; small Ω-shaped invaginations within the plasma membrane. Caveolae are involved in signal transduction, wherein cav-1 acts as a scaffolding protein to organize multiple molecular complexes regulating a variety of cellular events. Recent evidence suggests a role for cav-1 in promoting cancer cell migration, invasion and metastasis; however, the molecular mechanisms have not been described. The small monomeric GTPases are among several molecules which associate with cav-1. Classically, the Rho GTPases control actin cytoskeletal reorganization during cell migration and invasion. RhoC GTPase is overexpressed in aggressive cancers that metastasize and is the predominant GTPase in PC. Like several GTPases, RhoC contains a putative cav-1 binding motif.
Analysis of 10 PC cell lines revealed high levels of cav-1 expression in lines derived from primary tumors and low expression in those derived from metastases. Comparison of the BxPC-3 (derived from a primary tumor) and HPAF-II (derived from a metastasis) demonstrates a reciprocal relationship between cav-1 expression and p42/p44 Erk activation with PC cell migration, invasion, RhoC GTPase and p38 MAPK activation. Furthermore, inhibition of RhoC or p38 activity in HPAF-II cells leads to partial restoration of cav-1 expression.
Cav-1 expression inhibits RhoC GTPase activation and subsequent activation of the p38 MAPK pathway in primary PC cells thus restricting migration and invasion. In contrast, loss of cav-1 expression leads to RhoC-mediated migration and invasion in metastatic PC cells.
Pancreatic cancer; RhoC GTPase; caveolin-1; cell migration; metastasis; MAPK
Caveolin-1 (cav-1) is reportedly overexpressed in prostate cancer cells and is associated with disease progression. Specific oncogenic activities of cav-1 associated with Akt activation also occur in prostate cancer. A membrane-associated protein, cav-1, is nonetheless secreted by prostate cancer cells; results of recent studies showed that secreted cav-1 can stimulate cell survival and angiogenic activities, defining a role for cav-1 in the prostate cancer microenvironment. Serum cav-1 levels were also higher in prostate cancer patients than in control men without prostate cancer, and the preoperative serum cav-1 concentration had prognostic potential in men undergoing radical prostatectomy. Secreted cav-1 is therefore a potential biomarker and therapeutic target for prostate cancer.
caveolin-1; progression; angiogenesis; biomarkers
While prostate cancer is a common disease in men, it is uncommonly life-threatening. To better understand this phenomenon, tumor biologists have sought to elucidate the mechanisms that contribute to the development of virulent prostate cancer. The recent discovery that caveolin-1 (Cav-1) functions as an important oncogene involved in prostate cancer progression reflects the success of this effort. Cav-1 is a major structural coat protein of caveolae, specialized plasma membrane invaginations involved in multiple cellular functions, including molecular transport, cell adhesion, and signal transduction. Cav-1 is aberrantly overexpressed in human prostate cancer, with higher levels evident in metastatic versus primary sites. Intracellular Cav-1 promotes cell survival through activation of Akt and enhancement of additional growth factor pro-survival pathways. Cav-1 is also secreted as a biologically active molecule that promotes cell survival and angiogenesis within the tumor microenvironment. Secreted Cav-1 can be reproducibly detected in peripheral blood using a sensitive and specific immunoassay. Cav-1 levels distinguish men with prostate cancer from normal controls, and preoperative Cav-1 levels predict which patients are at highest risk for relapse following radical prostatectomy for localized disease. Thus, secreted Cav-1 is a promising biomarker in identifying clinically significant prostate cancer.
caveolin-1; prostate cancer; biomarker
Caveolin-1 (CAV1) and caveolin 2 (CAV2) are the principal structural proteins of caveolae, sphingolipid and cholesterol-rich invaginations of the plasma membrane involved in vesicular trafficking and signal transduction. Over the recent years there has been controversy about their role in breast cancer and their suitability as markers of basal-like phenotype. Caveolin-1 and CAV2 protein expression was assessed on a tissue microarray containing 880 unselected invasive breast cancer cases, by means of immunohistochemistry. Caveolin-1 and CAV2 expression was observed in 13.4 and 5.9% of all breast cancer, respectively. Their expression was strongly associated with high histological grade, lack of steroid hormone receptor positivity (ER and PR), and expression of basal markers (basal cytokeratins, P63, P-cadherin). Furthermore, there was a significant association between CAV1 and CAV2 expression and basal-like phenotype. On univariate analysis only CAV2 had a prognostic impact on breast cancer-specific survival; however, this was not independent from other traditional markers on multivariate analysis. Our results demonstrate that both CAV1 and CAV2 are associated with basal-like phenotype. Further studies are warranted to determine whether they play an oncogenic role in basal-like/triple-negative breast cancer development or are just surrogate markers for this subgroup.
caveolin 1; caveolin 2; immunohistochemistry; breast; basal-like
Pancreatic cancer is one of the deadliest of cancers with a dismal 5-year survival rate. Epidemiological studies have identified chronic pancreatitis as a risk factor for pancreatic cancer. Pancreatic cancer cells also demonstrate increased expression of the transcription factor Snail, a key regulator of epithelial-mesenchymal transition. As ethanol is one of the major causes of pancreatitis, we examined the effect of ethanol on Snail family members in immortalized human pancreatic ductal epithelial (HPDE) cells and in pancreatic cancer cells. Ethanol induced Snail mRNA levels 2.5-fold in HPDE cells, with only 1.5-fold mRNA induction of the Snail-related protein Slug. In contrast, ethanol increased Slug mRNA levels 1.5-2-fold in pancreatic cancer cells, with minimal effect on Snail. Because Snail increases invasion of cancer cells, we examined the effect of ethanol on invasion of HPDE and pancreatic cancer cells. Surprisingly, ethanol decreased invasion of HPDE cells, but had no effect on invasion of pancreatic cancer cells. Mechanistically, ethanol increased adhesion of HPDE cells to collagen and increased expression of the collagen binding α2- and β1-integrins. In contrast, ethanol did not affect collagen adhesion or integrin expression in pancreatic cancer cells. Also in contrast to HPDE cells, ethanol did not attenuate ERK1/2 phosphorylation in pancreatic cancer cells; however, inhibiting ERK1/2 decreased pancreatic cancer cell invasion. Overall, our results identify the differential effects of ethanol on premalignant and malignant pancreatic cells, and demonstrate the pleiotropic effects of ethanol on pancreatic cancer progression.
Ethanol; Snail; Slug; ERK1/2; integrins; collagen; adhesion
Caveolin-1 (CAV1) is the main structural component of Caveolae which are plasma membrane invaginations that participate in vesicular trafficking and signal transduction events. Although evidence has recently accumulated describing the function of CAV1 in several cancer types, its role in melanoma tumor formation and progression remains poorly explored. Here, by employing B16F10 melanoma cells as an experimental system, we directly explore the function of CAV1 in melanoma tumor growth and metastasis. We first show that CAV1 expression promotes proliferation, while it suppresses migration and invasion of B16F10 cells in vitro. When orthotopically implanted in the skin of mice, B16F10 cells expressing CAV1 form tumors that are similar in size to their control counterparts. An experimental metastasis assay demonstrates that CAV1 expression suppresses the ability of B16F10 cells to form lung metastases in C57Bl/6 syngeneic mice. Additionally, CAV1 protein and mRNA levels are found to be significantly reduced in human metastatic melanoma cell lines and human tissue from metastatic lesions. Finally, we demonstrate that following integrin activation, B16F10 cells expressing CAV1 display reduced expression levels and activity of FAK and Src proteins. Furthermore, CAV1 expression markedly reduces the expression of integrin β3 in B16F10 melanoma cells. In summary, our findings provide experimental evidence that CAV1 may function as an antimetastatic gene in malignant melanoma.
Hypoxia activates genetic programs that facilitate cell survival; however, in cancer, it may promote invasion and metastasis. In this study, we show that breast cancer cells cultured in 1.0% O2 demonstrate changes consistent with epithelial–mesenchymal transition (EMT). Snail translocates to the nucleus, and E-cadherin is lost from plasma membranes. Vimentin expression, cell migration, Matrigel invasion, and collagen remodeling are increased. Hypoxia-induced EMT is accompanied by increased expression of the urokinase-type plasminogen activator receptor (uPAR) and activation of cell signaling factors downstream of uPAR, including Akt and Rac1. Glycogen synthase kinase-3β is phosphorylated, and Snail expression is increased. Hypoxia-induced EMT is blocked by uPAR gene silencing and mimicked by uPAR overexpression in normoxia. Antagonizing Rac1 or phosphatidylinositol 3-kinase also inhibits development of cellular properties associated with EMT in hypoxia. Breast cancer cells implanted on chick chorioallantoic membranes and treated with CoCl2, to model hypoxia, demonstrate increased dissemination. We conclude that in hypoxia, uPAR activates diverse cell signaling pathways that cooperatively induce EMT and may promote cancer metastasis.
Clathrin and caveolins are known for their involvement in the internalization of numerous receptors. Here we show that in polarized epithelial Madin-Darby canine kidney cells, both the clathrin machinery and caveolins are involved in the endocytosis and delivery to the plasma membrane (PM) of the M1 muscarinic acetylcholine receptor (mAChR). We initially localized this receptor to the lateral membrane, where it accumulates proximal to the tight junctions. From there it is internalized through the clathrin-mediated pathway. In addition, the receptor may associate on the PM with caveolin (cav) 2 or in intracellular compartments with either cav 2, or monomeric or oligomeric cav 1. Association of the PM M1 mAChR with cav 2 inhibits receptor endocytosis through the clathrin-mediated pathway or retains the receptor in an intracellular compartment. This intracellular association attenuates receptor trafficking. Expression of cav 1 with cav 2 rescues the latter's inhibitory effect. The caveolins stimulate M1 mAChR oligomerization thus maintaining a constant amount of monomeric receptor. These results provide evidence that caveolins play a role in the attenuation of the M1 muscarinic receptor's intracellular trafficking to and from the PM.
The accumulation of LDL-derived cholesterol in the artery wall is the initiating event that causes atherosclerosis. However, the mechanisms that lead to the initiation of atherosclerosis are still poorly understood. Here, by using endothelial cell-specific transgenesis of the caveolin-1 (Cav-1) gene in mice, we show the critical role of Cav-1 in promoting atherogenesis. Mice were generated lacking Cav-1 and apoE but expressing endothelial-specific Cav-1 in the double knockout background. Genetic ablation of Cav-1 on an apoE knockout background inhibits the progression of atherosclerosis while re-expression of Cav-1 in the endothelium promotes lesion expansion. Mechanistically, the loss of Cav-1 reduces LDL infiltration into the artery wall, promotes nitric oxide production and reduces the expression of leukocyte adhesion molecules, effects completely reversed in transgenic mice. In summary, this unique model provides physiological evidence supporting the important role of endothelial Cav-1 expression in regulating the entry of LDL into the vessel wall and the initiation of atherosclerosis.
Epithelial-mesenchymal transition (EMT) is an important step in the invasion and progression of cancer and in the development of chemoresistance by cancer cells.
To address the clinical significance of the EMT pathway in lung adenocarcinoma and the association of the pathway with histological subtype, we examined 193 surgically resected lung adenocarcinoma samples for the expression of representative EMT-related proteins (E-cadherin, β-catenin, and vimentin) by immunohistochemistry. Histological subtypes were classified according to the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification. The results for EMT-related protein expression were analyzed for correlation with clinicopathological features and with survival.
The loss of E-cadherin expression and aberrant β-catenin expression were significantly associated with larger tumor size, pleural invasion, lymphatic/vascular invasion, and advanced pathological stage (p<0.05). The alteration of the E-cadherin/β-catenin complex was least frequently observed in the lepidic-predominant group, but these associations were not statistically significant. In the multivariate analysis, altered E-cadherin/β-catenin complex expression was found to be an independent poor prognostic factor (p=0.017; hazard ratio, 1.926; 95% confidence interval, 1.119 to 3.314).
The alteration of the expression of the E-cadherin/β-catenin complex was associated with aggressive tumor behavior in lung adenocarcinoma.
Epithelial-mesenchymal transition; Cadherin/catenin complex; Immunohistochemistry; Lung neoplasms
The Akt/PKB family of kinases is frequently activated in human cancers, including oral squamous cell carcinoma (OSCC). Akt-induced epithelial-to-mesenchymal transition (EMT) involves downregulation of E-cadherin, which appears to result from upregulation of the transcription repressor Snail. Recently, it was proposed that carcinoma cells, especially in metastatic sites, could acquire the mesenchymal-to-epithelial reverting transition (MErT) in order to adapt the microenvironments and re-expression of E-cadherin be a critical indicator of MErT. However, the precise mechanism and biologic or clinical importance of the MErT in cancers have been little known. This study aimed to investigate whether Akt inhibition would restore the expression of E-cadherin and β-catenin, reduce that of Vimentin, and induce the MErT in OSCC cells with low or negative expression of E-cadherin. We also investigate whether inhibition of Akt activity would affect the E-cadherin repressors and signaling molecules like NF-κB, ERK, and p38.
We screened several OSCC cell lines in order to select suitable cell line models for inducing MErT, using immunoblotting and methylation specific-PCR. We examined whether Akt inhibitor phosphatidylinositol ether lipid analogues (PIA) treatment would restore the expression of E-cadherin and β-catenin, reduce that of Vimentin, and induce the MErT in KB and KOSCC-25B cells using RT-PCR, immunoblotting, immunofluorescence analysis, and in vitro migration assay. We also investigated whether inhibition of Akt activity would affect the E-cadherin repressors, including Snail, Twist, and SIP-1/ZEB-2 and signaling molecules like NF-κB, ERK, JNK, and p38 using RT-PCR, immunoblotting, and immunofluorescence analysis.
Of the 7 OSCC cell lines, KB and KOSCC-25B showed constitutively activated phosphorylated Akt and low or negative expression of E-cadherin. Inhibition of Akt activity by PIA decreased NF-κB signaling, but did not affect phosphorylation of ERK, JNK, and p38 in KB and KOSCC-25B cells. Akt inhibition led to downregulation of Snail and Twist expression. In contrast, inhibition of Akt activity by PIA did not induce any changes in SIP-1/ZEB-2 expression. PIA treatment induced the expression of E-cadherin and β-catenin, reduce that of Vimentin, restored their epithelial morphology of a polygonal shape, and reduced tumor cell migration in KB and KOSCC-25B cells, which was the corresponding feature of MErT.
All of these findings suggest that Akt inhibition could induce the MErT through decreased NF-κB signaling and downregulation of Snail and Twist in OSCC cells. A strategy involving Akt inhibition might be a useful therapeutic tool in controlling cancer dissemination and metastasis in oral cancer patients.
Epithelial-mesenchymal transition (EMT) plays a crucial role in the development of cancer metastasis. The MAP kinases ERK, JNK and p38 have been implicated in promoting EMT, but a role for the MAP kinase BMK1 has not been studied. Here we report that BMK1 signaling suppresses EMT. BMK1 elevation augmented E-cadherin-mediated cell-cell adhesion, downregulated mesenchymal markers and decreased cell motility. Conversely, BMK1 silencing attenuated E-cadherin-mediated cell-cell adhesion, upregulated mesenchymal markers and stimulated cell motility. BMK1 depletion dramatically increased the accumulation of endogenous Snail in the nuclear compartment. Snail accumulation was mediated by Akt/GSK3β signaling, which was activated by a modulation in the expression of the mTOR inhibitor DEPTOR. In support of these observations, BMK1 depletion promoted metastasis in vivo. Together, our findings reveal a novel mechanism of EMT control via mTOR/Akt inhibition that suppresses cancer metastasis.
The ability to selectively detect and target cancer cells that have undergone an epithelial-mesenchymal transition (EMT) may lead to improved methods to treat cancers such as pancreatic cancer. The remodeling of cellular glycosylation previously has been associated with cell differentiation and may represent a valuable class of molecular targets for EMT.
As a first step toward investigating the nature of glycosylation alterations in EMT, we characterized the expression of glycan-related genes in three in-vitro model systems that each represented a complementary aspect of pancreatic cancer EMT. These models included: 1) TGFβ-induced EMT, which provided a look at the active transition between states; 2) a panel of 22 pancreatic cancer cell lines, which represented terminal differentiation states of either epithelial-like or mesenchymal-like; and 3) actively-migrating and stationary cells, which provided a look at the mechanism of migration. We analyzed expression data from a list of 587 genes involved in glycosylation (biosynthesis, sugar transport, glycan-binding, etc.) or EMT. Glycogenes were altered at a higher prevalence than all other genes in the first two models (p<0.05 and <0.005, respectively) but not in the migration model. Several functional themes were shared between the induced-EMT model and the cell line panel, including alterations to matrix components and proteoglycans, the sulfation of glycosaminoglycans; mannose receptor family members; initiation of O-glycosylation; and certain forms of sialylation. Protein-level changes were confirmed by Western blot for the mannose receptor MRC2 and the O-glycosylation enzyme GALNT3, and cell-surface sulfation changes were confirmed using Alcian Blue staining.
Alterations to glycogenes are a major component of cancer EMT and are characterized by changes to matrix components, the sulfation of GAGs, mannose receptors, O-glycosylation, and specific sialylated structures. These results provide leads for targeting aggressive and drug resistant forms of pancreatic cancer cells.
Chronic inflammation-promoted metastasis has been considered as a major challenge in cancer therapy. Pro-inflammatory cytokine TNFα can induce cancer invasion and metastasis associated with epithelial–mesenchymal transition (EMT). However, the underlying mechanisms are not entirely clear. In this study, we showed that TNFα induces EMT in human HCT116 cells and thereby promotes colorectal cancer (CRC) invasion and metastasis. TNFα-induced EMT was characterized by acquiring mesenchymal spindle-like morphology and increasing the expression of N-cadherin and fibronectin with a concomitant decrease of E-cadherin and Zona occludin-1(ZO-1). TNFα treatment also increased the expression of transcription factor Snail, but not Slug, ZEB1 and Twist. Overexpression of Snail induced a switch from E-cadherin to N-cadherin expression in HCT116 cells, which is a characteristic of EMT. Conversely, knockdown of Snail significantly attenuated TNFα-induced EMT in HCT116 cells, suggesting that Snail plays a crucial role in TNFα-induced EMT. Interestingly, exposure to TNFα rapidly increased Snail protein expression and Snail nuclear localization but not mRNA level upregulation. Finally, we demonstrated that TNFα elevated Snail stability by activating AKT pathway and subsequently repressing GSK-3β activity and decreasing the association of Snail with GSK-3β. Knockdown of GSK-3β further verified our finding. Taken together, these results revealed that AKT/GSK-3β-mediated stabilization of Snail is required for TNFα-induced EMT in CRC cells. Our study provides a better understanding of inflammation-induced CRC metastasis.
Response gene to complement-32 (RGC-32) is comprehensively expressed in many kinds of tissues and has been reported to be expressed abnormally in different kinds of human tumors. However, the role of RGC-32 in cancer remains controversial and no reports have described the effect of RGC-32 in pancreatic cancer. The present study investigated the expression of RGC-32 in pancreatic cancer tissues and explored the role of RGC-32 in transforming growth factor-beta (TGF-β)-induced epithelial-mesenchymal transition (EMT) in human pancreatic cancer cell line BxPC-3.
Immunohistochemical staining of RGC-32 and E-cadherin was performed on specimens from 42 patients with pancreatic cancer, 12 with chronic pancreatitis and 8 with normal pancreas. To evaluate the role of RGC-32 in TGF-β-induced EMT in pancreatic cancer cells, BxPC-3 cells were treated with TGF-β1, and RGC-32 siRNA silencing and gene overexpression were performed as well. The mRNA expression and protein expression of RGC-32 and EMT markers such E-cadherin and vimentin were determined by quantitative reverse transcription-PCR (qRT-PCR) and western blot respectively. Finally, migration ability of BxPC-3 cells treated with TGF-β and RGC-32 siRNA transfection was examined by transwell cell migration assay.
We found stronger expression of RGC-32 and higher abnormal expression rate of E-cadherin in pancreatic cancer tissues than those in chronic pancreatitis tissues and normal pancreatic tissues. Immunohistochemical analysis revealed that both RGC-32 positive expression and E-cadherin abnormal expression in pancreatic cancer were correlated with lymph node metastasis and TNM staging. In addition, a significant and positive correlation was found between positive expression of RGC-32 and abnormal expression of E-cadherin. Furthermore, in vitro, we found sustained TGF-β stimuli induced EMT and up-regulated RGC-32 expression in BxPC-3 cells. By means of siRNA silencing and gene overexpression, we further demonstrated that RGC-32 mediated TGF-β-induced EMT and migration in BxPC-3 cells.
The results above indicated that RGC-32 might be a novel metastasis promoting gene in pancreatic cancer and it enhances metastatic phenotype by mediating TGF-β-induced EMT in human pancreatic cancer cell line BxPC-3.
Response gene to complement-32; Pancreatic cancer; Transforming growth factor -β; Epithelial-mesenchymal transition; Migration
Caveolin-1 (CAV1) is highly expressed in Ewing’s sarcoma (EWS). We previously showed that increased cellular CAV1 is associated with the regulation of the tumorigenicity, drug resistance and metastatic ability of EWS cells. Because several studies reported that melanoma and prostate cancer cells, which express relatively high CAV1 levels, secrete CAV1, and that secreted CAV1 is associated with tumor progression, our study explored the possibility that EWS cells also secreted CAV1 and that secreted CAV1 may contribute to EWS pathobiology. Results from experiments involving the ectopic expression of a Myc-tagged CAV1 protein in EWS cells as well as the supplementation of culture media with purified CAV1 protein followed by its intracellular localization using immunofluorescence demonstrated that EWS cells secrete CAV1, that they are able to take up the secreted protein, and that extracellular CAV1 enhances EWS cell proliferation. These findings strongly support the notion that secreted CAV1 may also contribute to the malignant properties of EWS.
caveolin-1; cell proliferation; Ewing’s sarcoma; protein secretion; protein uptake
Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that promotes differentiation and cell survival in the stomach. PPARγ upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPARγ is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPARγ signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPARγ and enhanced nuclear translocation and ligand-independent transcription of PPARγ target genes. In contrast, Cav1 overexpression sequestered PPARγ in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPARγ's ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPARγ and to inhibit cell proliferation. Ligand-activated PPARγ also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPARγ regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPARγ to ligands, limiting proliferation of gastric epithelial cells.
Caveolin-1 (Cav-1), a member of the caveolin family, regulates caveolae-associated signaling proteins, which are involved in many biological processes, including cancer development. Cav-1 was found to exert a complex and ambiguous role as oncogene or tumor suppressor depending on the cellular microenvironment. Here we investigated Cav-1 expression and function in a panel of melanomas, finding its expression in all the cell lines. The exception was the primary vertical melanoma cell line, WM983A, characterized by the lack of Cav-1, and then utilized as a recipient for Cav-1 gene transduction to address a series of functional studies. The alleged yet controversial role of phospho-Cav-1 on cell regulation was also tested by transducing the non phosphorylatable Cav-1Y14A mutant. Wild type Cav-1, but not mutated Cav-1Y14A, increased tumorigenicity as indicated by enhanced proliferation, migration, invasion and capacity of forming foci in semisolid medium. Accordingly, Cav-1 silencing inhibited melanoma cell growth reducing some of the typical traits of malignancy. Finally, we detected a secreted fraction of Cav-1 associated with cell released microvesicular particles able to stimulate in vitro anchorage independence, migration and invasion in a paracrine/autocrine fashion and, more important, competent to convey metastatic asset from the donor melanoma to the less aggressive recipient cell line. A direct correlation between Cav-1 levels, the amount of MV released in the culture medium and MMP-9 expression was also observed.
Melanoma; Caveolin-1; microvesicles; tumorigenicity