Autocrine signaling in carcinoma: VEGF and the alpha6beta4 integrin

This review highlights an emerging function for vascular endothelial growth factor (VEGF) in carcinoma and discusses mechanisms involved in the elaboration of VEGF autocrine loops. Evidence is provided that autocrine VEGF contributes to the two major components of invasive carcinoma: survival and migration. Moreover, the findings discussed support the hypothesis that carcinoma progression selects for cells that depend on VEGF as a survival factor. Furthermore, a related hypothesis, which is developed, is that the function of the alpha6beta4 integrin, which has been implicated in carcinoma progression, is linked to its ability to regulate VEGF translation and, consequently, autocrine VEGF signaling. The findings reviewed challenge the notion that the function of VEGF in cancer is limited to angiogenesis and suggest that VEGF and VEGF receptor-based therapeutics, in addition to targeting angiogenesis, may also impair tumor cell survival and invasion directly.     

Involvement of alpha6beta4 integrin in the mechanisms that regulate breast cancer progression

Integrin alpha6beta4 is mostly expressed in epithelial tissues and endothelial and Schwann cells. Expression of alpha6beta4 is increased in many epithelial tumours, implicating its involvement in tumour malignancy. Moreover, this integrin activates several key signalling molecules in carcinoma cells, but its ability to activate the phosphatidylinositol 3-kinase/Akt pathway is among the mechanisms by which alpha6beta4 integrin regulates tumour behaviour. In this review we discuss the biological and clinical features of alpha6beta4 integrin that allow it to promote tumour survival and progression of mammary tumours.     

The alpha6beta4 integrin can regulate ErbB-3 expression: implications for alpha6beta4 signaling and function

The integrin alpha(6)beta(4) has been shown to facilitate key functions of carcinoma cells, including their ability to migrate, invade, and evade apoptosis. The mechanism involved seems to be a profound effect of alpha(6)beta(4) on specific signaling pathways, especially the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. An intimate relationship between alpha(6)beta(4) and growth factor receptors may explain this effect of alpha(6)beta(4) on signaling. Previously, we showed that alpha(6)beta(4) and ErbB-2 can function synergistically to activate the PI3K/Akt pathway. Given that ErbB-2 can activate PI3K only when it heterodimerizes with other members of the epidermal growth factor receptor family, these data imply that other receptors cooperate in this process. Here, we report that alpha(6)beta(4) can regulate the expression of ErbB-3 using several different models and that the consequent formation of an ErbB-2/ErbB-3 heterodimer promotes the alpha(6)beta(4)-dependent activation of PI3K/Akt and the ability of this integrin to impede apoptosis of carcinoma cells. Our data also support the hypothesis that alpha(6)beta(4) can regulate ErbB-3 expression at the translational level as evidenced by the findings that alpha(6)beta(4) does not increase ErbB-3 mRNA significantly, and that this regulation is both rapamycin sensitive and dependent on eukaryotic translation initiation factor 4E. These findings provide one mechanism to account for the activation of PI3K by alpha(6)beta(4) and they also provide insight into the regulation of ErbB-3 in carcinoma cells.     

Altered surface expression and increased turnover of the alpha6beta4 integrin in an undifferentiated carcinoma

The integrin alpha6beta4, predominantly expressed on tissues of epithelial origin, is known to be variably expressed on carcinomas. The biochemical changes resulting in altered expression during tumor progression are unknown. We have analyzed the expression of alpha6beta4 in a multi-step mouse model of skin carcinogenesis representing normal keratinocyte, benign papilloma and malignant undifferentiated carcinoma. All cell lines expressed the alpha6 integrin exclusively as the alpha6beta4 integrin heterodimer. Analysis of this integrin by flow cytometry and immunoprecipitation of surface labeled proteins revealed that the undifferentiated carcinoma cells have an approximately 75% reduction in surface expression of the integrin as compared with the keratinocyte and papilloma cell lines. The alpha6beta4 integrin which remains expressed on the carcinoma cells is diffusely distributed in the membrane and has an approximately 2.5-fold increased biological turnover as compared with normal keratinocytes. The decreased biological half-life and the loss of polarized expression of alpha6beta4 on the carcinoma cells suggests an altered functional role for the alpha6beta4 integrin on carcinoma cells during tumor progression. These factors may contribute to the known supression of hemidesmosome structures and the increased migration phenotype associated with some epithelial carcinomas.     

整合素α6β1增强人肝癌细胞侵袭性机制的探讨

目的 研究层粘连蛋白受体α６β１整合素在人肝癌细胞侵行为中的作用。方法 采用ｄｏｍｉｎａｎｔ ｎｅｇａｔｉｖｅ策略，以无功能的α６β４－ＴＲ代替细胞表面α６β１受体，从而消除了人肝癌细胞Ｂｅｌ－７０４２ α６β１受体的功能。用划痕法和Ｂｏｒｄｅｎ小室法分别检测细胞的迁移和侵袭，明胶酶谱分析法检测细胞基质金属蛋白ｍａｔｒｉｘ ｍｅｔａｌｌｏｐｒｏｖｔｅｉｎａｓｅｓ，ＭＭＰａ）的分泌。结果 消除α６β     

Invasive skin carcinoma--Ras and alpha6beta4 integrin lead the way

Although the genesis of invasive squamous cell carcinoma (SCC) from stratified epithelia of the skin is considered to be a complex, multistage process, recent work on human epidermis reveals that sustained Ras signaling coupled with suppression of Ras-induced growth arrest is sufficient to drive the entire process and that the alpha6beta4 integrin and its laminin 5 ligand are essential components of this process.     

Changes in expression of alpha 6/beta 4 integrin heterodimer in primary and metastatic breast cancer.

The alpha 6/beta 4 integrin complex has been shown to be expressed in murine tissues at the basolateral aspect of most epithelial cells including the mammary epithelium, thus suggesting that this heterodimer may interact with components of the basement membrane. Because transformation of mammary epithelium frequently results in disappearance of basement membranes and loss of cell polarisation we have analysed in the present study whether expression of the alpha 6/beta 4 complex is altered in human breast tumours. The results of the present study confirm that in human mammary gland alpha 6 and beta 4 subunits colocalise at the basolateral aspect of the epithelium. While in benign breast lesions this distribution pattern remains mostly unchanged, in primary carcinomas the expression of both chains is either redistributed over the cells surface or significantly reduced. This altered pattern of expression is paralleled by the lack of detection of basement membrane laminin and collagen type IV. In metastatic lesions the expression of the heterodimer is maintained in most of the lymphnodal foci, but less frequently detected in metastasis localised in the pleural cavity and in parenchymal tissues. These findings indicate that in breast epithelium expression of the alpha 6/beta 4 heterodimer is modulated by the presence of basement membrane and is possibly influenced by microenvironmental factors as suggested by the different pattern of alpha 6/beta 4 expression in nodal and extranodal metastatic foci.     

The alpha v beta 6 integrin induces gelatinase B secretion in colon cancer cells

In human cancers, the co-operative role between cell-adhesion receptors and proteases capable of degrading matrix barriers remains poorly understood. We have previously reported that the epithelium-restricted integrin alpha(v)beta6 becomes highly expressed in colon cancer compared with normal mucosa and that heterologous expression of alpha(v)beta6 in colon cancer cells is associated with enhanced cell growth. Herein, we report that alpha(v)beta6 expression in colon cancer cells leads to a relative increase in secretion of the matrix metalloproteinase gelatinase B over its respective inhibitor and that this secretion parallels the level of cell-surface beta6 expression. The alpha(v)beta6-mediated gelatinase B secretion is associated with increased proteolysis of denatured collagen at the cell surface, and inactivation of gelatinase B in beta6-expressing tumour cells inhibits cell spreading and proliferation within 3-dimensional collagen matrices. Our findings suggest that alpha(v)beta6-mediated gelatinase B secretion is important in the progression of human colon cancer.     

Expression of the alpha6beta4 integrin provides prognostic information in bladder cancer

Integrins are cell surface receptors for extracellular matrix components that may participate in metastatic processes. Normal urothelial tissues show a polarized expression of alpha6beta4 integrin on basal cells at their junction with the lamina propria. We have previously shown that bladder cancers frequently overexpress one member of the integrin family, the alpha6beta4 integrin. In this study, we evaluated the level of alpha6beta4 integrin expression in bladder cancer specimens from 57 patients and correlated the expression level with patient survival. Expression was evaluated by immunoperoxidase staining. Three patterns of alpha6beta4 expression were observed: negative (13 patients); strong overexpression throughout the tumor cells (21 patients); and weak expression that most closely resembled expression in normal urothelium (23 patients). Individuals with weak staining tumors had a statistically significantly better survival (p=0.041) than patients whose tumors exhibited either no expression or strong overexpression. These data indicate that evaluation of the expression of alpha6beta4 integrin may provide valuable prognostic information on clinical outcome in patients with bladder cancer.     

Abnormal expression of integrin alpha 6 beta 4 in cervical intraepithelial neoplasia.

We have used subunit-specific monoclonal antibodies (MAbs) and immunohistochemistry to examine the distribution of integrin alpha 6 beta 4 in normal ectocervical epithelium and various grades of cervical intraepithelial neoplasia (CIN). Antibodies were first characterised by immunoprecipitation from two surface-labelled tumour cell lines. Monoclonal antibody G71 was found to precipitate integrin beta 4 from BeWo but not T47D cells, while other anti-beta 4 antibodies precipitated beta 4 from both cell lines. Both G71 and an antiserum to the C-terminal peptide of beta 4 precipitated free beta 4 from surface-iodinated BeWo cells. Neither antibody recognised truncated beta 4 chains observed at approximately 160 kDa. These data suggest that different isoforms of beta 4 are expressed in different tumour cell lines, and that there may be a pool of beta 4 at the cell surface that is not complexed to alpha 6. In normal cervix, both the alpha 6 and beta 4 subunits occur at the basal surface of the basal cell layer. In CIN, the distribution is markedly altered, with strong expression of alpha 6 and beta 4 in the upper cell layers of the ectocervical epithelium. All 40 cases of CIN that were studied exhibited this alteration. Furthermore, the extent of extrabasal staining appeared to correspond with the grade of CIN. The form of integrin beta 4 recognised by antibody G71 also appears in the upper cell layers in CIN, but it shows a more restricted distribution than the normal isoform.     

Integrin alpha6beta1 role in metastatic behavior of human pancreatic carcinoma cells

The factors that determine the metastatic behavior of pancreatic tumor cells are incompletely understood. In this study, we first demonstrate differences in adhesion properties, integrin expression and in vivo integrin function in the metastatic tumor cell line PaTu 8988s compared with the non-metastatic cell line PaTu 8988t. Both cell lines were derived from the same original tumor and exhibit identical genetic fingerprints. Using in vitro adhesion assays performed on purified extracellular matrix components, adhesion of PaTu 8988s cells was significantly increased on the basal membrane component laminin and decreased on the interstitial matrix protein fibronectin compared to PaTu 8988t cells. By immunocytochemistry and flow cytometry, and in correspondence with their adhesive properties, the metastatic PaTu 8988s cells did express a distinct pattern of integrin subunits. Laminin-binding integrins alpha6 and beta4 were overexpressed in PaTu 8988s cells. Fibronectin-binding alpha5 integrins were present at higher levels in the non-metastatic PaTu 8988t cells, whereas the beta1 subunit expression did not differ. Adhesion to laminin or fibronectin was specific and was mediated via integrins alpha6beta1 and alpha5beta1, respectively. In addition, metastasis formation in vivo after injection of cells into the tail vein of nude mice was inhibited by preincubation of PaTu 8988s cells with antibodies directed against the integrin alpha6 or beta1. We conclude that alpha6beta1 integrins are overexpressed and functionally active in metastatic human pancreatic carcinoma cells, and participate in metastasis formation probably through binding to the basal membrane component laminin.     

The A9 antigen associated with aggressive human squamous carcinoma is structurally and functionally similar to the newly defined integrin alpha 6 beta 4.

We previously reported that altered expression of the A9 antigen (defined by monoclonal antibody UM-A9) is a predictive marker of early recurrence and progression of squamous cell carcinoma (SCC). In normal squamous cells A9 expression is limited to the site of contact with the basement membrane in vivo and the culture surface in vitro, whereas aggressive SCCs exhibit loss of polarity and increased intensity of A9 expression. The potential relationship of the A9 antigen to structures known to be involved in cell adhesion was analyzed by immunobiochemical and cell adhesion assays. UM-A9 precipitates a complex of protein chains reminiscent of the alpha and beta heterodimer glycoproteins that characterize the integrin family of extracellular matrix receptors. Proteins were isolated from A9-positive cells using UM-A9 and well-defined antibodies specific for integrin alpha and beta chains. UM-A9, anti-alpha 6, and anti-beta 4 monoclonal antibodies (mAbs) all precipitated proteins with comparable electrophoretic mobilities. Furthermore, UM-A9 mAb precleared the SCC alpha 6 beta 4 integrin complex isolated with anti-alpha 6 or anti-beta 4 mAbs but not that isolated by anti-beta 1 mAb. The isoelectric points of the A9 complex chains were consistent with those reported for alpha 6 and beta 4. Three of the polypeptide chains (140, 175, and 205 kDa) precipitated by UM-A9 showed peptide homology to one another and to the beta 4 chain precipitated by mAb 439-9B. The A9/alpha 6 subunit is composed of 125- and 30-kDa chains and was distinguished from beta 4 and beta 1 chains by its peptide map and isoelectric point. UM-A9 binds to an epitope common to the beta 4 subunits since in pulse-chase analysis the beta 4 species are precipitated at an early time point, whereas detection of alpha-subunit synthesis is detected during assembly of the mature complex. Immunoprecipitation and preclearing experiments demonstrated that in SCC the alpha 6 subunit is associated primarily with the beta 4 species and not with the 130-kDa beta 1 subunit. In cell adhesion assays on extracellular matrix proteins, the alpha 6-specific GoH3 mAb inhibited binding of SCC to laminin, suggesting that alpha 6 beta 4 may function as a laminin receptor in SCC. These data and our prior observations showing an association between altered A9 expression and early recurrence in SCC provide the first evidence that altered expression of alpha 6 beta 4 integrin is associated with the clinical behavior of human squamous cell carcinomas.     

Towards a mechanistic understanding of tumor invasion--lessons from the alpha6beta 4 integrin

This review explores the mechanistic basis of carcinoma migration and invasion by focusing on the contribution of integrins. Integrins are essential for invasion not only for their ability to mediate physical interactions with extracellular matrices, but also for their ability to regulate signaling pathways that control actin dynamics and cell movement, as well as for growth and survival. Our comments center on a unique member of the integrin family, the alpha 6 beta 4 integrin, which is a receptor for the laminin family of basement membrane components. Numerous studies have implicated this integrin in the invasion of solid tumors and have provided a rationale for studying the mechanistic basis of its contribution to the invasive process. Such studies have revealed novel insights into the mechanism of carcinoma invasion that involve both the dynamics of cell migration and signaling pathways that regulate this migration.     

Hypoxia stimulates carcinoma invasion by stabilizing microtubules and promoting the Rab11 trafficking of the alpha6beta4 integrin

Hypoxia plays a key role in tumor cell survival, invasion, and metastasis. Here we show that hypoxia increases tumor cell invasion by the modulation of Rab11, an important molecule for vesicular trafficking, especially membrane protein recycling and translocation of proteins from trans-Golgi network to plasma membrane. Dominant-negative Rab11 dramatically decreased hypoxia-induced invasion of MDA-MB-231 breast carcinoma cells without affecting cell apoptosis. Hypoxia-induced Rab11 trafficking is regulated by microtubule stability, as evidenced by the findings that hypoxia increases Glu tubulin and that colchicine blocks Rab11 trafficking and invasion. Inhibition of GSK-3beta activity by hypoxia seems to be central to microtubule stabilization and invasion. In fact, expression of a dominant-negative GSK-3beta was sufficient to stimulate invasion in normoxia. One target of Rab11-mediated trafficking that contributes to invasion is the integrin alpha6beta4. Hypoxia induced a significant increase in alpha6beta4 surface expression but it had no effect on the surface expression of alpha3beta1. This increase is dependent on Rab11 and stable microtubules. In summary, we identify vesicle trafficking as a novel target of hypoxic stimulation that is important for tumor invasion.     

Adhesion-independent alpha6beta4 integrin clustering is mediated by phosphatidylinositol 3-kinase

Clustering of cell-surface integrins is known to augment integrin-mediated signal transduction, but mechanisms of integrin clustering are poorly understood. Here we report that adhesion-independent clustering of alpha6beta4 integrin, known to be important in mediating tumor cell motility, is driven by phosphatidylinositol 3-kinase (PI3K) but does not require activation of the PI3K-Akt pathway. We observed clustering of alpha6beta4 in breast carcinoma cells after adhesion-independent cross-linking of the beta4 integrin subunit. Clustering was significantly blocked when cross-linking was performed in the presence of PI3K inhibitors LY294002 and wortmannin. In contrast, no significant inhibition of clustering was observed with protein kinase C inhibitor GF109203X, rapamycin, or heparin. Although alpha6beta4 clustering was blocked by PI3K inhibitors, clustering was not associated with increased PI3K lipid kinase activity or increased phosphorylation of Akt. A novel role for PI3K in alpha6beta4 integrin clustering is proposed.     

Heparin coating of human breast carcinoma cells in vivo

Mast cells, the only source of heparin in the body, are common in breast carcinoma. Due to metachromatic staining of heparin-proteoglycans released from the mast cells toluidine blue may stain connective tissue a reddish-purple. This study shows that tumour cells in the vicinity may also be coated with metachromatic substance. Such bl vivo coating does not appear to have been reported previously. In experimental studies in vitro coating has been described following suspension of mouse tumour cells in a heparin solution and preincubation of ascitic tumour cells in heparin has given reduction in survival time after transplantation. In human breast carcinoma stromal metachromasia may under certain circumstances be indicative of poor prognosis. The biological implications of in vivo tumour cell coating with heparin are under investigation.