Expression of beta1-integrins and N-cadherin in bladder cancer and melanoma cell lines

Changes in the expression of integrins and cadherins might contribute to the progression, invasion and metastasis of transitional cell cancer of the bladder and of melanomas. The expression of alpha5 (P < 0.001), alpha2 and beta1 (P < 0.05 - P < 0.001) integrin subunits in melanoma cells from noncutaneous metastatic sites (WM9, A375) were significantly increased as compared to cutaneous primary tumor (WM35) and metastatic (WM239) cell lines. These differences might be ascribed to the invasive character of melanoma cells and their metastasis to the noncutaneous locations. The significantly heterogeneous expression of beta1 integrin subunit in two malignant bladder cancer cell lines (T24 and Hu456) and nonsignificant differences in the expression of alpha2, alpha3, and alpha5 subunits between malignant and non-malignant human bladder cell lines do not allow an unanimous conclusion on the role of these intergrin subunits in the progression of transitional cancer of bladder. The adhesion molecule, expressed in all studied melanoma and bladder cell lines, that reacted with anti-Pan cadherin monoclonal antibodies was identified as N-cadherin except in the HCV29 non-malignant ureter cell line. However, neither this nor any other bladder or melanoma cell line expressed E-cadherin. The obtained results imply that the replacement of E-cadherin by N-cadherin accompanied by a simultaneous increase in expression of alpha2, alpha3 and alpha5 integrin subunits clearly indicates an increase of invasiveness of melanoma and, to a lesser extent, of transitional cell cancer of bladder. High expression of N-cadherin and alpha5 integrin subunit seems to be associated with the most invasive melanoma phenotype.     

Colon cancer cells adhesion and spreading on autocrine laminin-10 is mediated by multiple integrin receptors and modulated by EGF receptor stimulation

Epidermal growth factor (EGF) receptor ligands such as EGF and transforming growth factor-alpha (TGF-alpha) play an important role in controlling the proliferation, survival, morphology, and motility of colonic epithelial cells. There is also increasing evidence that growth factors and extracellular matrix (ECM) proteins cooperate to regulate these cellular processes. We have reported previously that autocrine TGF-alpha and an unidentified ECM protein in the serum-free conditioned medium of the human colon carcinoma cell line LIM1215 synergize to induce spreading of these cells in low-density cultures. We have now purified the ECM protein secreted by LIM1215 cells and show that it synergizes with EGF to induce spreading of LIM1215 cells and other human cell lines from the colon and other tissues. The purified ECM migrated as a single protein band with an apparent molecular mass of approximately 800 kDa on SDS-PAGE under nonreducing conditions and, under reducing conditions, as three protein bands of approximately 360, 210, and 200 kDa. Immunoblotting experiments and mass spectrometry analysis of tryptic digests on the purified protein identified the 360-, 210-, and 200-kDa protein bands as laminin alpha5, beta1, and gamma1 chains, respectively, indicating that LIM1215 cells secrete laminin-10 (alpha5 beta1 gamma1). In serum-free medium, LIM1215 cells adhere to laminin-10 primarily via alpha2 beta1 and alpha3 beta1 integrin receptors. EGF-induced spreading of LIM1215 cells on laminin-10 is partially inhibited by pretreatment of the cells with blocking antibodies directed against integrin alpha3 or beta1 but not alpha2, alpha6, or beta4 subunits. Spreading is almost completely inhibited by blocking alpha3 + alpha2, alpha3 + alpha6, or beta1 + beta4 integrin chains and results in cell death. Increased spreading in the presence of EGF correlates with up-regulation of alpha6 beta4 integrins in these cells after exposure to EGF. These results indicate that colon cancer cells attach and spread on laminin-10 via multiple integrin receptors and suggest a critical role for alpha3 beta1 integrins in the spreading response. Together, our results support the concept that the adhesive properties of colon cancer cells are modulated by autocrine production of TGF-alpha and laminin-10 and autocrine induction of appropriate integrins.     

Activated epidermal growth factor receptor induces integrin alpha2 internalization via caveolae/raft-dependent endocytic pathway

Elevated expression or activity of the epidermal growth factor (EGF) receptor is common in ovarian cancer and is associated with poor patient prognosis. Our previous studies demonstrated that expression of the constitutively active mutant form of the EGF receptor (EGFRvIII) in ovarian cancer cells led to reduction in integrin alpha2 surface expression, defects in cell spreading, and disruption of focal adhesions. Inhibition of EGFRvIII catalytic activity reversed the response, suggesting that EGF receptor activation regulates integrin alpha2. In this study we found that EGF treatment resulted in a transient loss of integrin alpha2 from the cell surface. Before EGF stimulation, integrin alpha2 and EGF receptors were associated based on biochemical and immuno-colocalization approaches. After EGF treatment, EGF receptor and integrin alpha2 were internalized and segregated into different compartments. Integrin alpha2, but not EGF receptor, was associated with caveolin-1 and GM1 (Gal_1,3GalNAc_1,4(Neu5Ac-_ 2,3)Gal_1,4Glc_1,1-ceramide) gangliosides, suggesting caveolae-mediated endocytosis. Moreover, integrin alpha2 was subsequently targeted to the Golgi apparatus and the endoplasmic reticulum. Together, these findings demonstrate that activated EGF receptor transiently modulates integrin alpha2 cell surface expression and stimulates integrin alpha2 trafficking via caveolae/raft-mediated endocytosis, representing a novel mechanism by which the EGF receptor may regulate integrin-mediated cell behavior.     

Injury and EGF mediate the expression of alpha6beta4 integrin subunits in corneal epithelium

Our goal was to evaluate the role of epidermal growth factor and injury on the expression of integrin subunits alpha6(alpha6) and beta4(beta4). An in vitro wound model was used to evaluate corneal wound repair and cellular migration. Primary rabbit corneal epithelial cell cultures were serum-starved and injured in the presence or absence of EGF or tyrphostin AG1478, an inhibitor of EGF receptor kinase activity. Repair was monitored morphologically and expression was analyzed using in situ hybridization and immunohistochemistry accompanied by confocal microscopy. The addition of EGF to cell cultures induced a dose-dependent increase in beta4 mRNA expression but the constitutive expression of alpha6 was several fold greater. In the wounded cultures there was a rapid change in expression at the edge of the wound that was enhanced with EGF. In our model there was an increase in beta4 and alpha6 protein in migrating cells. Changes in integrin expression were accompanied by a transient increase in activation of the EGF receptor. The addition of tyrphostin inhibited migration of cells and wound repair, the activation of the EGF receptor and phosphorylation of beta4 in the cytoplasm. These data indicate that the activation of the EGF receptor plays a critical role in the regulation of integrin receptors and the mediation of cellular migration.     

IGFBP-1 inhibits EGF mitogenic activity in cultured endometrial stromal cells

The properties of the insulin-like growth factor-binding proteins (IGFBP-1 to 6) are not limited to modulation of IGF actions. IGFBP-1, which shares an Arg-Gly-Asp (RGD) motif in its C-terminal domain, modulates cell motility by binding to integrin alpha5beta1. The cross-talks between integrins and growth factor receptor signalling pathways are extensively documented, particularly in the case of the epidermal growth factor receptor (EGFR). However, whether IGFBP-1 can modulate growth factor signalling through its interaction with integrin alpha5beta1 has not yet been studied. As EGF is involved in the decidualisation of endometrial stromal cells (ESCs) and as decidualised ESCs are a source of IGFBP-1, we investigated if IGFBP-1 can modulate EGF effects on ESCs. RGD- and IGF-independent inhibition of EGF mitogenic activity and EGFR signalling by IGFBP-1 were demonstrated in ESC primary cultures, A431, cells and in mouse fibroblasts lacking IGF receptors.     

Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.     

Parathyroid hormone-related protein upregulates integrin expression via an intracrine pathway in PC-3 prostate cancer cells

Parathyroid hormone-related protein (PTHrP) is expressed by human prostatic tissue and prostate cancer cell lines, and enhances prostate tumor cell growth both in vivo and in vitro. PTHrP expression also plays a role in the development of bone metastasis, which is a frequent complication in patients with prostate carcinoma. Tumor cell adhesion to extracellular matrix (ECM) components is mediated via integrin subunits, and plays a major role in the invasion and metastasis of tumor cells. We previously showed that PTHrP overexpression increases adhesion of the human prostate cancer cell line PC-3 to the ECM molecules collagen type I, fibronectin, and laminin. Increased adhesion is accompanied by upregulation in the expression of alpha1, alpha5, alpha6, and beta4 integrin subunits. We used the same cell line to study the mechanism via which PTHrP upregulates integrin expression. Clonal PC-3 cells were established overexpressing wild-type PTHrP or PTHrP mutated in the nuclear localization sequence (NLS). Mutation of the NLS negated the effects of PTHrP on alpha1, alpha5, alpha6, and beta4 integrin expression, indicating that these effects are mediated via an intracrine pathway requiring nuclear localization. Expression of the alpha2, alpha3, alphav, and beta1 integrin subunits were comparable in wild-type and NLS-mutated PTHrP transfectants. These findings indicate that PTHrP may play a role in prostate tumor invasion and metastasis by upregulating the expression of specific integrin subunits via an intracrine pathway.     

Urinary-type plasminogen activator (uPA) expression and uPA receptor localization are regulated by alpha 3beta 1 integrin in oral keratinocytes

Expression of urinary-type plasminogen activator (uPA) and its receptor (uPAR) is correlated with matrix proteolysis, cell adhesion, motility, and invasion. To evaluate the functional link between adhesion and proteolysis in gingival keratinocytes (pp126), cells were treated with immobilized integrin antibodies to induce integrin clustering. Clustering of alpha(3) and beta(1) integrin subunits, but not alpha(2), alpha(5), alpha(6), or beta(4), enhanced uPA secretion. Bead-immobilized laminin-5 and collagen I, two major alpha(3)beta(1) ligands, also induced uPA expression. Coordinate regulation of the serpin plasminogen activator inhibitor 1 was also apparent; however, a net increase in uPA activity was predominant. alpha(3)beta(1) integrin clustering induced extracellular signal-regulated kinase 1/2 phosphorylation, and both uPA induction and extracellular signal-regulated kinase activation were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. Integrin aggregation also promoted a dramatic redistribution of uPAR on the cell surface to sites of clustered alpha(3)beta(1) integrins. Co-immunoprecipitation of beta(1) integrin with uPAR provided further evidence that protein-protein interactions between uPAR and beta(1) integrin control uPAR distribution. As a functional consequence of uPA up-regulation and uPA-mediated plasminogen activation, the globular domain of the laminin-5 alpha(3) subunit, a major pp126 matrix protein, was proteolytically processed from a 190-kDa form to a 160-kDa species. Laminin-5 containing the 160-kDa alpha(3) subunit efficiently nucleates hemidesmosome formation and reduces cell motility. Together, these data suggest that multivalent aggregation of the alpha(3)beta(1) integrin regulates proteinase expression, matrix proteolysis, and subsequent cellular behavior.     

Characterization of the oligosaccharide component of alpha3beta1 integrin from human bladder carcinoma cell line T24 and its role in adhesion and migration

Malignant transformation is highly associated with altered expression of cell surface N-linked oligosaccharides. These changes concern integrins, a family of cell surface glycoproteins involved in the attachment and migration of cells on various extracellular matrix proteins. The integrin alpha3beta1 is particularly interesting because of its role in migration and invasion of several types of metastatic tumours. In this study, alpha3beta1 from human bladder T24 carcinoma cells was purified and treated with peptide N-glycosidase F. Then the N-glycans of the alpha3 and beta1 subunits were characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In alpha3beta1 integrin the presence of high-mannose, hybrid and predominantly complex type N-oligosaccharides was shown. Unlike to normal epithelium cells, in both subunits of alpha3beta1 integrin from cancer cells, the sialylated tetraantennary complex type glycan Hex7HexNAc6FucSia4 was present. In a direct ligand binding assay, desialylated alpha3beta1 integrin exhibited significantly higher fibronectin-binding capability than untreated integrin, providing evidence that sialic acids play a direct role in ligand-receptor interaction. Moreover, alpha3beta1 integrin was shown to take part in T24 cell migration on fibronectin: anti-alpha3 antibodies induced ca 30% inhibition of wound closure. Treatment of T24 cells with swainsonine reduced the rate of bladder carcinoma cell migration by 16%, indicating the role of beta1,6 branched complex type glycans in this process. Our data show that alpha3beta1 integrin function may be altered by glycosylation, that both subunits contribute to these changes, and that glycosylation may be considered a newly found mechanism in the regulation of integrin function.     

The androgen receptor associates with the epidermal growth factor receptor in androgen-sensitive prostate cancer cells

Many recent evidences indicate that androgen-sensitive prostate cancer cells have a lower malignant phenotype that is in particular characterized by a reduced migration and invasion. We previously demonstrated that expression of androgen receptor (AR) by transfection of the androgen-independent prostate cancer cell line PC3 decreases invasion and adhesion of these cells (PC3-AR) through modulation of alpha6beta4 integrin expression. The treatment with the synthetic androgen R1881 further reduced invasion of the cells without, however, modifying alpha6beta4 expression on the cell surface, suggesting an interference with the invasion process in response to EGF. We investigated whether the presence of the AR could affect EGF receptor (EGFR)-mediated signaling in response to EGF by evaluating autotransphosphorylation of the receptor as well as activation of downstream signalling pathways. Immunoprecipitation studies demonstrated a reduction of EGF-induced tyrosine phosphorylation of EGFR in PC3-AR cells. In addition, EGF-stimulated PI3K activity, a key signalling pathway for invasion of these cells, was decreased in PC3-AR cells and further reduced by treatment with R1881, indicating decreased functionality of EGFR. An interaction between EGFR and AR has been demonstrated by immunoconfocal and co-immunoprecipitation analysis in PC3-AR cells, suggesting a possible interference of AR on EGFR signalling by interaction of the two proteins. In conclusion, our results suggest that the expression of AR by transfection in PC3 cells confers a less malignant phenotype by interfering with EGFR autophosphorylation and signalling in response to EGF leading to invasion through a mechanism involving an interaction between AR and EGFR.     

Expression of ATRAP in adipocytes and negative regulation by beta-adrenergic stimulation of JAK/STAT.

Sympatho-adrenergic activity and the renin-angiotensin system are considered critical regulators of obesity and hypertension. The novel angiotensin II type 1 receptor-associated protein (ATRAP) has been demonstrated to modulate angiotensin II signalling in smooth muscle cells and cardiomyocytes. Adipose tissue expresses important renin angiotensin system components and contributes to cardiometabolic disease. However, ATRAP expression and regulation in adipocytes are unknown. We investigated expression of this novel modulator of angiotensin signalling and its regulation by beta-adrenergic receptors. We found ATRAP to be expressed in differentiated brown and white adipocytes. Stimulation of beta-adrenoceptors strongly suppressed ATRAP expression. We hypothesised a role for JAK/STAT signalling elements. Indeed, beta3-adrenergic stimulation robustly stimulated both STAT1 and STAT3 phosphorylation in a time- and dose-dependent manner. This effect was abrogated by inhibition of PKA and JAK2 signalling. Moreover, inhibition of JAK/STAT and PKA signalling reversed the beta3-adrenergic suppression of ATRAP expression. This study provides the first evidence for expression and adrenergic regulation of the angiotensin II signalling modulator ATRAP in adipocytes. Further, it indicates a novel regulatory link between beta-adrenergic and JAK/STAT signalling.     

Differential effect of IL-4 and IL-13 on CD44 expression in the Burkitt's lymphoma B cell line BL30/B95-8 and in Epstein-Barr virus (EBV) transformed human B cells: loss of IL-13 receptors on Burkitt's lymphoma B cells

IL-4 and IL-13, cytokines with similar biological effects may influence growth and progression of B-cell tumors through regulation of key cell surface molecules important in intercellular communications. In this study, we demonstrate that IL-4 and IL-13 exhibited differential effects on CD23 and CD44 expression and binding to hyaluronan in BL30/B95-8, a Burkitt's lymphoma (BL), and MK3.31, an Epstein-Barr virus transformed normal human B cell line (B-LCL). Studies conducted to understand the molecular mechanisms underlying this differential effect show that IL-4 induced phosphorylation of JAK1, JAK3, and STAT6 in BL30/B95-8 cells and of JAK3 and STAT6 in MK 3.31 cells. In contrast, IL-13 failed to induce the phosphorylation of JAK kinases or STAT6 proteins in these cell lines. The inability of BL30/B95-8 cells to respond to IL-13 was attributed to the loss of expression of IL-13R subunits alpha1 and alpha2, a finding confirmed for a number of other BL cell lines examined.     

The SHH/Gli axis regulates CD90‐mediated liver cancer stem cell function by activating the IL6/JAK2 pathway

The cell surface antigen CD90 has recently been established as a promising marker for liver cancer stem cells. This study aimed to investigate potential implications of SHH/Gli signalling in CD90+ liver cancer stem cells. Correlation of the expression of SHH signalling components and CD90 in liver cancer cells and clinical tissues, as well as in enriched CD90+ liver cancer stem cells and the TCGA database, were analysed by quantitative RT‐PCR, Western blotting and flow cytometry. Functional analysis was conducted by siRNA‐mediated CD90, Gli1 and Gli3 gene knockdown, SHH treatment and application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody in CD90+ liver cancer stem cells, followed by cell proliferation, migration, sphere formation and tumorigenicity assays. CD90 expression exhibited a high positive correlation with Gli1 and Gli3 in multiple liver cancer cell lines and human cancerous liver tissues, both of which showed a significant increase in liver cancer. Analysis of TCGA data revealed an association of CD90, Gli1 and Gli3 with a short overall survival and positive correlation between CD90 expression and Gli3 expression level. The stem cell potentials of CD90+ 97L liver cancer cells were greatly impaired by Gli1/3 knockdown with siRNA but enhanced by SHH treatment. Application of the JAK2 inhibitor AZD1480 and IL6 neutralizing antibody showed the CD90 and SHH/Gli‐regulated liver cancer stem cell functions were mediated by the IL6/JAK2/STAT3 pathway. The stem cell properties of CD90+ liver cancer cells are regulated by the downstream SHH/Gli and IL6/JAK2/STAT3 signalling pathways.     

Association between alphavbeta6 integrin expression, elevated p42/44 kDa MAPK, and plasminogen-dependent matrix degradation in ovarian cancer

Altered expression of alphav integrins plays a critical role in tumor growth, invasion, and metastasis. In this study, we show that normal human epithelial ovarian cell line, HOSE, and ovarian cancer cell lines, OVCA 429, OVCA 433, and OVHS-1, expressed alphav integrin and associated beta1, beta3, and beta5 subunits, but only ovarian cancer cell lines OVCA 429 and OVCA 433 expressed alphavbeta6 integrin. The expression of alphavbeta6 in OVCA 429 and OVCA 433 was far higher than alphavbeta3 and alphavbeta5 integrin and correlated with high p42/p44 mitogen activated protein kinase (MAPK) activity and high secretion of high molecular weight urokinase plasminogen activator (HMW-uPA), pro-metalloproteinase 2 and 9 (pro-MMP-9 and pro-MMP-2). In contrast to HOSE and OVHS 1, OVCA 433 and OVCA 429 exhibited approximately 2-fold more plasminogen-dependent [3H]-collagen type IV degradation. Plasminogen-dependent [3H]-collagen IV degradation was inhibited by inhibitor of uPA (amiloride) and MMP (phenanthroline) and by antibodies against uPA or MMP-9 or alphavbeta6 integrin, indicating the involvement of alphavbeta6 integrin, uPA and MMP-9 in the process. The alphavbeta6 correlated increase in HMW-uPA and pro-MMP secretion could be inhibited by tyrosine kinase inhibitor genistein or the MEK 1 inhibitor U0126, consistent with a role of active p42/44 MAPK in the elevation of uPA, MMP-9, and MMP-2 secretion. Under similar conditions, genistein and U0126 inhibited plasminogen-dependent [3H]-collagen type IV degradation. These data suggest that sustained elevation of p42/44 MAPK activity may be required for the co-expression of alphavbeta6 integrin, which in turn may regulate the malignant potential of ovarian cancer cells via proteolytic mechanisms.     

Role of Jagged1/STAT3 signalling in platinum‐resistant ovarian cancer

Jagged1, the essential ligand of the Notch signalling pathway, is highly expressed in metastatic prostate cancer, and its high expression in breast cancer is linked to poor survival rates. However, the mechanism of Jagged1′s involvement in platinum‐resistant ovarian cancer has not been thoroughly elucidated to date. The purpose of the present study was to investigate the roles of Jagged1 in the platinum resistance of ovarian cancer and its possible mechanisms. Compared with a platinum responsive group of ovarian epithelial cell carcinomas, we found the positive staining intensity of Notch1, Notch2, Jagged1, STAT3 and Epithelial‐mesenchymal transition (EMT) proteins were lower in a platinum‐resistant group. The DDP‐resistant ovarian cancer cell line (C13K) had a higher IC50 of DDP than its parental cell line (OV2008) (P < 0.05) and acquired an EMT phenotype and invasive characteristics. Inhibiting or knockdown of Jagged1 expression could not only reduce its capacity of migration and invasion but also reverse EMT and down‐regulate the expression of serine 727‐phosphorylated STAT3 (pS727) at the protein level but not total STAT3 or tyrosine 705‐phosphorylated STAT3 (pY705) in C13K cells. Furthermore, it was found that crosstalk between the Jagged1/Notch and JAK/STAT3 signalling pathways were involved in Jagged1‐promoting EMT in C13K cells. Experiments in vivo showed a reduced micrometastatic tumour burden in the lung, liver and spleen of mice implanted with C13K cells with knocked‐down Jagged1 compared with mice implanted with control cells. All of these results demonstrate that Jagged1 can crosstalk with the JAK/STAT3 pathway, and they all cooperate to promote the aberrant occurrence of EMT, further reinforcing the abilities of invasion and migration of platinum‐resistant ovarian cancer in vivo and in vitro.