Transfection of MCF-7 carcinoma cells with human integrin alpha7 cDNA promotes adhesion to laminin.

The laminin-binding alpha7beta1 integrin receptor is highly expressed by skeletal and cardiac muscles, and has been suggested to be a crucial molecule during myogenic cell migration and differentiation. Absence of integrin alpha7 subunit contributes to a form of muscular dystrophy in integrin alpha7 null mice, whereas specific mutations in the alpha7 gene are associated in humans with congenital myopathy. To examine in more detail the potential role of integrin alpha7 in human-related muscular disorders, we cloned alpha7 cDNA by RT-PCR from human skeletal muscle mRNA and then expressed the full-length human integrin alpha7 cDNA by transfection in several cell lines including MCF-7, COS-7, and NIH3T3 cells. The isolated cDNA corresponds to the human alpha7X2B alternative splice form. Expression of human alpha7 was further confirmed by transfection of chimeric human/mouse alpha7 cDNA constructs. To demonstrate the functionality of expressed human alpha7, adhesion experiments with transfected MCF-7 cells have confirmed the specific binding of human alpha7 to laminin. In addition, mouse polyclonal and monoclonal antibodies were generated against the extracellular domain of human alpha7 and used to analyze by flow cytometry MCF-7 and NIH3T3 cells transfected with the full-length of human alpha7 cDNA. These results show for the first time the exogenous expression of functional full-length human alpha7 cDNA, as well as the development of monoclonal antibodies against the human alpha7 extracellular domain. Antibodies developed will be useful for further analysis of human disorders involving alpha7 dysfunction and facilitate isolation of muscle stem cells (satellite cells) and thereby expand the opportunities for genetically modified transplantation treatment of human disease.

Tumor deposition of laminin-5 and the relationship with perineural invasion.

OBJECTIVES/HYPOTHESIS: Perineural invasion (PNI) is increasingly being recognized as an important indicator of aggressiveness in head and neck squamous cell carcinoma. The mechanisms of PNI are poorly understood. Laminin-5, an important basement membrane constituent, has been shown to be essential in head and neck squamous cell carcinoma invasion and motility. We hypothesized that tumors exhibiting increased expression of laminin-5 are more likely to be neurotropic. STUDY DESIGN: Analysis of archived surgical specimens with and without PNI for presence and intensity of laminin-5 tumor staining. METHODS: Immunohistochemistry of archived head and neck squamous cell carcinoma specimens with known PNI was performed with anti-laminin-5 antibodies and appropriate positive and negative control specimens. The staining patterns were characterized as follows: A, few to no tumor cells positive; B, some peripheral cells positive; C, all peripheral cells positive; and D, almost all tumor cells positive. Statistical analysis was by chi2 analysis. RESULTS: Forty-six PNI-positive and 18 PNI-negative specimens were analyzed. The staining distribution for the PNI-positive specimens was as follows: 2% for A, 41% for B, 46% for C, and 11% for D. For tumors without PNI, the distribution was 28% for A, 50% for B, 22% for C, and 0% for D (P = .005). In PNI-positive tumors, no significant difference in staining was seen between areas with and without PNI. CONCLUSIONS: We found a significant correlation between laminin-5 staining and the presence of PNI in head and neck squamous cell carcinoma. Expression of laminin-5 by tumors is, possibly, an important step in the process of PNI. These preliminary findings support the concept that deposition of basement membrane constituents are required in the multistep process of nerve invasion.

Adhesive mechanisms regulating invasion and metastasis in oral cancer.

It is the relentless invasion and growth into surrounding tissue that characterize oral squamous cell carcinoma. Metastasis is perhaps the most challenging and important aspect of cancer progression, in that it generally signifies limited survival and ineffective therapy. Inherent in metastasis is invasion, the process by which cells infiltrate into adjacent tissues, degrading basement membranes and extracellular matrix and disrupting tissue architecture and sometimes organ function. The factors that regulate these processes are complex and likely involve loss of the controls that are normally in place in physiologic tissue modeling. Adhesion receptors and their ligands are important in modulating not only invasion of oral squamous cell carcinoma cells but also their survival and proliferation. Normal oral mucosal epithelial cells use integrins to maintain their anchorage to the basement membrane, whereas the formation of stratifying cell layers depends on the formation of intercellular adhesions mediated by cadherins. The process of squamous cell carcinoma invasion and dissemination requires active cell migration through the extracellular matrix with the simultaneous remodeling of intercellular adhesions. Integrins are clearly important in the invasive process, whereas intercellular adhesion receptors restrain invasion and promote a more differentiated phenotype.

Type I collagen degradation by invasive oral squamous cell carcinoma.

Expression of extracellular matrix-degrading proteases is required for tumor cell invasion. In the present study we examined the production of type I collagen-degrading matrix metalloproteinases (MMPs) in the invasive oral squamous cell carcinoma-derived cell line HSC-3. In the absence of serum or exogenous growth factors, HSC-3 cells displayed no collagen degradation activity. Addition of serum slightly increased collagen proteolysis. However, addition of epidermal growth factor (EGF) resulted in nearly complete degradation of the collagen matrix. Zymography showed that MMP-2 and -9 are secreted by HSC-3 cells. EGF stimulated secretion of an additional gelatinase with a molecular weight similar to that of MMP-1. Immunoblotting of conditioned medium confirmed that EGF and, to a lesser degree type I collagen, increased production of MMP-1. Finally, in situ hybridization revealed intense expression of MMP-1 in oral squamous cell carcinoma specimens. Together, these results indicate that MMP-1 is expressed, induced by EGF, and required for type I collagen degradation in oral squamous cell carcinoma.

Expression of the alpha7beta1 laminin receptor suppresses melanoma growth and metastatic potential.

The alpha7beta1 integrin is a laminin-binding receptor that was originally identified in melanoma. Here, we show that, in clonally derived mouse K1735 melanoma variant cell lines with high (M-2) and low (C-23) metastatic potential, elevated expression of alpha7 correlates with reduced cell motility, metastasis, and tumor growth. Both cell lines showed similar beta1 integrin-dependent adhesion to laminin-1 and the E8 laminin fragment. However, the highly metastatic M-2 cells rapidly migrated on laminin, whereas the nonmetastatic C-23 cells were minimally motile. Laminin-binding integrin profiles showed that the M-2 cells expressed moderate amounts of alpha1 and abundant alpha6 but low or undetectable levels of alpha2 and alpha7. By contrast, C-23 cells expressed low or undetectable levels of alpha1, alpha2, and alpha6 but had up-regulated levels of alpha7. Consistent with the protein data, Northern blot analysis showed that levels of alpha7 mRNA were highest in the poorly metastatic variant cells, whereas alpha6 message was not detected; in contrast, alpha6 mRNA was elevated in the highly metastatic cells, whereas alpha7 message was not detected. Forced expression of alpha7 in the M-2 cells suppressed cell motility, tumor growth, and metastasis. Collectively, these results indicate that, during melanoma progression, acquisition of a highly tumorigenic and metastatic melanoma phenotype is associated with loss of the alpha7beta1 laminin receptor.

Apoptosis-associated proteins and the development of oral squamous cell carcinoma.

Expression of apoptosis-associated proteins was evaluated in premalignant and malignant oral epithelial lesions, to test the hypothesis that protein regulation of apoptosis may be altered in the development of oral squamous cell carcinoma. Ninety archived paraffin-embedded specimens from 25 patients (two or more sequential biopsies each) and eight control specimens were evaluated in immunohistochemically stained sections for tumor suppressor protein p53, p53 binding protein mdm-2, and apoptosis regulatory proteins Bcl-2, Bcl-X, Bax, and Bak. The initial histologic diagnosis for 17/25 patients was either focal keratosis, mild dysplasia, or moderate dysplasia; the initial diagnosis for the remaining eight patients ranged from severe dysplasia to moderately differentiated squamous cell carcinoma. Thirty of 90 specimens showed positive p53 expression, nine of which were dysplasias. In patients with one or more lesions displaying p53 expression, there was increased intensity of staining with disease progression. Bak was expressed in 57/90 specimens, including 27 dysplasias of various grades. There was also a significantly increased intensity of Bak staining with disease progression, which did not appear to be dependent upon p53 status. Bcl-X was expressed in 73/90 specimens, with staining displayed earlier in premalignant lesions than either p53 or Bak. Ten of 90 specimens were positive for Bcl-2 (all were dysplasias or carcinomas), and only 2/90 specimens were positive for Bax. Eleven of 90 specimens were positive for mdm-2; six of which were also positive for p53. These data show that apoptosis-associated proteins are altered in variable patterns in both premalignant and malignant oral epithelial lesions. p53 and especially Bak and Bcl-X are expressed early; Bax is largely absent; and Bcl-2 and mdm-2 show sporadic expression in the development of oral premalignant and malignant disease.

Cell cycle proteins and the development of oral squamous cell carcinoma.

Expression of cell cycle regulatory proteins was evaluated in premalignant and malignant oral epithelial lesions, to test the hypothesis that protein regulation of the cell cycle may be altered in the development of oral squamous cell carcinoma. Archived paraffin-embedded specimens (n = 90) from 25 patients with recurrent or persistent lesions were evaluated in immunohistochemically stained sections for cell cycle regulatory proteins p53, Rb, Cyclin D1, p27, and p21. The cell cycle was also evaluated by expression of nuclear protein Ki 67. Sections were graded semiquantitatively using a 0-3 + scale to indicate the percentage of positively stained cells. The initial histologic diagnosis for 17/25 patients was either focal keratosis, mild dysplasia, or moderate dysplasia; the initial diagnosis for the remaining eight patients ranged from severe dysplasia to moderately differentiated squamous cell carcinoma. Thirty-three of 90 specimens showed positive p53 expression, 11 of which were dysplasias. Eighty-nine of 90 specimens, from all stages of disease, showed positive Rb expression. Twenty-three of 90 specimens showed positive Cyclin D1 expression, typically in the later stages (carcinoma) of a patient's disease. Eighty-four of 90 specimens showed positive p21 expression; while 55 of 90 specimens were positive for p27. In control mucosa, p27 was highly expressed, while Rb and p21 proteins were expressed at relatively low levels; p53 and Cyclin D1 proteins were largely absent. Generally, staining of p53, Rb, p21, and Ki 67 increased with time in serial biopsies, while p27 showed decreased staining with disease progression. These data show that cell cycle regulatory proteins are altered in both premalignant and malignant disease, and that protein phenotypes are heterogeneous. P53 expression is seen early, and Cyclin D1 expression is seen late in the development of oral premalignant and malignant disease. Expression of p53, Rb, p21 and Ki67 increased, while p27 decreased, with disease progression.

Apoptosis-associated proteins in oral lymphomas from HIV-positive patients.

Extranodal oral lymphomas, seen with increasing frequency in HIV infection, may have dysfunctional apoptotic mechanisms that favor tumor progression. The purpose of this study was to evaluate extranodal lymphomas from HIV-positive patients for expression of apoptosis-associated proteins. Correlations were made with 10 histologically comparable extranodal lymphomas from HIV-negative patients and 6 hyperplastic lymph nodes from otherwise healthy young adults. Formalin-fixed tissue sections were immunohistochemically stained for apoptosis-associated proteins (Bcl-2, Bcl-x, Bax, Bak, p53, MDM2, BHRF). In situ hybridization was also done on deparaffinized sections for Epstein-Barr virus EBER mRNA. Eighteen consecutive oral lymphomas were studied in HIV/AIDS-positive patients. Four of 5 intermediate-grade lymphomas expressed Bcl-2 to a greater degree than did high-grade lymphomas (4 of 13). Most lymphomas were positive for Bcl-x and Bax, and few expressed Bak. The staining patterns for these proteins were similar to those seen in HIV-negative patients. Staining patterns were relatively consistent in the hyperplastic lymph nodes, whereas such patterns were irregular in lymphomas. Positive p53 staining was seen in 11 of 18 HIV-positive cases; 9 of these were also MDM2-positive. Double stains suggested that both p53 and MDM2 proteins were expressed in the same cells in these nine cases. Epstein-Barr virus-EBER mRNA was detected in 14 of 18 cases and in 3 of 10 cases from HIV-negative patients. BHRF staining was evident in only a few cells of three HIV-positive lymphomas. The irregular expression of Bcl-2, Bcl-x, Bax, and Bak in oral lymphomas indicates dysfunctional apoptotic mechanisms in these tumors. Bcl-2 staining differs with tumor grade. Positive staining for p53 and MDM2 proteins is a notable feature of lymphomas in HIV-positive patients and may relate to binding of MDM2 to wild-type p53. Epstein-Barr virus is more commonly associated with oral lymphomas in HIV-positive patients, although the Epstein-Barr virus-produced protein BHRF, which has Bcl-2-like activity, is minimally expressed.

Aberrant regulation of transforming growth factor-alpha during the establishment of growth arrest and quiescence of growth factor independent cells.

Autocrine transforming growth factor alpha (TGFalpha) is an important positive growth effector in malignant cells and plays a significant role in generating the growth factor-independent phenotype associated with malignant progression. However, the molecular mechanisms by which TGFalpha confers a growth advantage in progression is poorly understood. The highly tumorigenic cell line HCT116 up-regulates TGFalpha mRNA expression during growth arrest, whereas the poorly tumorigenic growth factor-dependent FET cell line down-regulates TGFalpha mRNA expression as it becomes quiescent. We have identified a 25-bp sequence at -201 to -225 within the TGFalpha promoter which mediates the differential regulation of TGFalpha expression during quiescence establishment in these two cell lines. This same sequence confers TGFalpha promoter responsiveness to exogenous growth factor or autocrine TGFalpha. The abberant upregulation of TGFalpha mRNA in quiescent HCT116 cells may allow them to return to the dividing state under more stringent conditions (nutrient replenishment alone) then quiescent FET cells (requires nutrients and growth factors). Antisense TGFalpha approaches showed that the dysregulated TGFalpha expression in quiescent HCT116 cells is a function of the strong TGFalpha autocrine loop (not inhibited by blocking antibodies) in these cells.

Mutations in the integrin alpha7 gene cause congenital myopathy.

The basal lamina of muscle fibers plays a crucial role in the development and function of skeletal muscle. An important laminin receptor in muscle is integrin alpha7beta1D. Integrin beta1 is expressed throughout the body, while integrin alpha7 is more muscle-specific. To address the role of integrin alpha7 in human muscle disease, we determined alpha7 protein expression in muscle biopsies from 117 patients with unclassified congenital myopathy and congenital muscular dystrophy by immunocytochemistry. We found three unrelated patients with integrin alpha7 deficiency and normal laminin alpha2 chain expression. To determine if any of these three patients had mutations of the integrin alpha7 gene, ITGA7, we cloned and sequenced the full-length human ITGA7 cDNA, and screened the patients for mutations. One patient had splice mutations on both alleles; one causing a 21-bp insertion in the conserved cysteine-rich region, and the other causing a 98-bp deletion. A second patient was a compound heterozygote for the same 98-bp deletion, and had a 1-bp frame-shift deletion on the other allele. A third showed marked deficiency of ITGA7 mRNA. Clinically, these patients showed congenital myopathy with delayed motor milestones. Our results demonstrate that mutations in ITGA7 are involved in a form of congenital myopathy.

Regulation of transforming growth factor alpha expression in a growth factor-independent cell line.

Aberrant transcriptional regulation of transforming growth factor alpha (TGF alpha) appears to be an important contributor to the malignant phenotype and the growth factor independence with which malignancy is frequently associated. However, little is known about the molecular mechanisms responsible for dysregulation of TGF alpha expression in the malignant phenotype. In this paper, we report on TGF alpha promoter regulation in the highly malignant growth factor-independent cell line HCT116. The HCT116 cell line expresses TGF alpha and the epidermal growth factor receptor (EGFR) but is not growth inhibited by antibodies to EGFR or TGF alpha. However, constitutive expression of TGF alpha antisense RNA in the HCT116 cell line resulted in the isolation of clones with markedly reduced TGF alpha mRNA and which were dependent on exogenous growth factors for proliferation. We hypothesized that if TGF alpha autocrine activation is the major stimulator of TGF alpha expression in this cell line, TGF alpha promoter activity should be reduced in the antisense TGF alpha clones in the absence of exogenous growth factor. This was the case. Moreover, transcriptional activation of the TGF alpha promoter was restored in an antisense-TGF alpha-mRNA-expressing clone which had reverted to a growth factor-independent phenotype. Using this model system, we were able to identify a 25-bp element within the TGF alpha promoter which conferred TGF alpha autoregulation to the TGF alpha promoter in the HCT116 cell line. In the TGF alpha-antisense-RNA-expressing clones, this element was activated by exogenous EGF. This 25-bp sequence contained no consensus sequences of known transcription factors so that the TGF alpha or EGF regulatory element within this 25-bp sequence represents a unique element. Further characterization of this 25-bp DNA sequence by deletion analysis revealed that regulation of TGF alpha promoter activity by this sequence is complex, as both repressors and activators bind in this region, but the overall expression of the activators is pivotal in determining the level of response to EGF or TGF alpha stimulation. The specific nuclear proteins binding to this region are also regulated in an autocrine-TGF alpha-dependent fashion and by exogenous EGF in EGF-deprived TGF alpha antisense clone 33. This regulation is identical to that seen in the growth factor-dependent cell line FET, which requires exogenous EGF for optimal growth. Moreover, the time response of the stimulation of trans-acting factor binding by EGF suggests that the effect is directly due to growth factor and not mediated by changes in growth state. We conclude that this element appears to represent the major positive regulator of TGF alpha expression in the growth factor-independent HCT116 cell line and may represent the major site of transcriptional dysregulation of TGF alpha promoter activity in the growth factor-independent phenotype.

The laminin-binding activity of the alpha 7 integrin receptor is defined by developmentally regulated splicing in the extracellular domain.

The expression pattern of the laminin-binding alpha 7 beta 1 integrin is developmentally regulated in skeletal, cardiac, and smooth muscle. The X1/X2 alternative splicing in the extracellular domain of alpha 7 is found in the variable region between conserved alpha-chain homology repeat domains III and IV, a site implicated in ligand binding. To assess differences in X1/X2 isoform activity, we generated MCF-7 cell lines transfected with alpha 7-X1/X2 cDNAs. Transfectants expressing the alpha 7-X2 variant adhered rapidly to laminin 1, whereas those expressing alpha 7-X1 failed to attach. That alpha 7-X1 exists in an inactive state was established in assays using an activating beta 1 antibody that induced X1-dependent cell adhesion and spreading. Furthermore, the activation of alpha 7-X1 was cell type specific, and when expressed in HT1080 cells, the integrin was converted into a fully functional receptor capable of promoting adhesion. Thus, the expression of the alpha 7-X1/X2 integrin is a novel mechanism that regulates receptor affinity states in a cell-specific context and may modulate integrin-dependent events during muscle development and repair.

Expression of the c-Met/HGF receptor in human breast carcinoma: correlation with tumor progression.

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell motility and tissue remodeling of various epithelial cells through its receptor, the product of the proto-oncogene c-met. High levels of HGF/SF have been correlated with poor prognosis in human breast carcinoma. In this study, we examined the expression of the c-Met receptor in human breast-carcinoma cells in vivo and in cultured cell lines. Immunohistochemical analysis of biopsy samples of human breast carcinoma indicated that, in normal mammary gland, c-Met is localized in the ductal epithelium. The level of expression of c-Met in primary carcinomas was maintained in autologous metastatic lymph-node lesions in some cases, and in other cases was elevated. Frequently there was evidence of heterogeneity in cellular expression of c-Met within individual tumors, suggesting that micro-environmental factors may regulate receptor expression. In an analysis of a panel of human breast-carcinoma cell lines, we found that moderately differentiated cell lines did not express detectable levels of c-Met and were not responsive to HGF. In contrast, poorly differentiated and invasive cell lines did express high levels of the receptor and responded to HGF by increased motility and invasiveness. Sensitivity to HGF/SF also correlated with expression of the c-Met 9-kb mRNA. No correlation was found between gene copy number and the expression level of c-Met protein or mRNA. When the moderately differentiated and c-Met-negative T47D cell line was transfected with c-DNA for c-met, the transfectants showed delayed cell scattering and migratory response to HGF. Thus, over-expression of c-Met in moderately differentiated carcinoma cells may be one of several attributes that contribute to an invasive phenotype during the progression of breast cancer.

Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor.

The alpha 7 beta 1 integrin is specifically expressed by skeletal and cardiac muscles, and its expression and alternative mRNA splicing at the cytoplasmic domain are developmentally regulated. We analyzed the role of alpha 7 integrin in mediating myoblast adhesion and motility on different laminin isoforms. Mouse C2C12 and MM14 myoblast cell lines were found by flow cytometry and immunoprecipitation to express high levels of the alpha 7 integrin. Overall expression of alpha 7 increased as the C2C12 myoblasts differentiated; myoblasts expressed only the alpha 7B cytoplasmic variant whereas in differentiating myotubes alpha 7A increased markedly. Function-perturbing monoclonal antibodies generated to alpha 7 integrin efficiently blocked both adhesion and migration of MM14 and C2C12 mouse myoblasts on laminin 1. Other studies with MM14 myoblasts showed that alpha 7 is also a receptor for laminin 2/4 (human placental merosins) but not for epithelial-cell-specific laminin 5. Blocking antibody to alpha 7 only partially inhibited adhesion to laminin 2/4 but almost completely blocked motility on this substrate. Finally, to assess the potential role of the alpha 7 cytoplasmic domain, CHO cells were stably transfected to expressed chimeric alpha 5 cDNA constructs containing the wild-type alpha 5 or the alpha 7A or alpha 7B cytoplasmic domain; all forms of the integrin showed identical activities for adhesion, migration, proliferation, and matrix assembly on fibronectin substrates. These results established that alpha 7 beta 1 receptor can promote myoblast adhesion and motility on a restricted number of laminin isoforms and may be important in myogenic precursor recruitment during regeneration and differentiation.

Alpha7 integrin mediates cell adhesion and migration on specific laminin isoforms.

The laminin-binding alpha7beta1 integrin receptor is expressed at high levels by skeletal and cardiac muscles and by certain melanocytic cells. We have assessed the potential role of the alpha7A/B integrin isoforms in mediating cell adhesion and motility and determined the laminin isoform specificity of this integrin. When MCF-7 breast carcinoma cells, normally nonadherent to laminin 1, were stably transfected with cDNA for mouse alpha7, they adhered with high efficiency and migrated on laminin 1 substrates. Function-perturbing monoclonal antibodies generated to mouse alpha7 subunit blocked both adhesion and migration of alpha7 transfectants on laminin 1 substrates. Additional studies with MCF-7 transfectants revealed that alpha7beta1 binds well to laminin 1 and to a mixture of laminin 2 and 4 but not to laminin 5. Importantly, alpha7beta1 was capable of promoting motility on both laminin 1 and laminin 2/4 substrates. However, MCF-7 cells transfected with cDNA for either alpha7A or alpha7B showed no significant differences in cell adhesion or motility on laminin 1 substrates. Although the role for the alternatively spliced cytoplasmic variants of alpha7 remains unknown, the results establish that alpha7beta1 mediates cell adhesive activities on a restricted number of laminin isoforms.

Identification and characterization of the cell type-specific and developmentally regulated alpha7 integrin gene promoter.

Expression of alpha7 is mainly confined to skeletal and cardiac muscle in which it appears to be the major laminin-binding integrin. When myoblasts differentiate to myotubes, alpha7 mRNA and protein expression is up-regulated. To explore the mechanisms involved in the tissue-specific and developmentally regulated expression of alpha7, we isolated and characterized a genomic clone containing approximately 2.8 kilobase pairs (kb) of the 5'-flanking region of the murine alpha7 gene. The 5'-flanking region lacks both TATA and CCAAT boxes but contains five putative Sp1 binding sites located in a CpG island. Two transcription start sites, located near an initiator-like sequence, are 176 and 170 base pairs upstream of the translation start site. There are numerous binding sites for developmental and cell type-specific transcription factors, including AP-1, AP-2, GATA, and several AT-rich sites. There are also eight consensus E-boxes that bind the basic helix-loop-helix family of muscle-specific transcription factors. The approximately 2.8-kb 5'-flanking region was an active promoter in C2C12 skeletal myoblasts and exhibited increased expression upon conversion to myotubes but was inactive in HtLM2 cells, a mouse breast carcinoma epithelial cell line that does not express alpha7. Deletion analysis identified both positive and negative regulatory elements within the approximately 2.8-kb fragment. In 10T1/2 fibroblasts the approximately 2.8-kb alpha7 promoter was trans-activated by the myogenic basic helix-loop-helix proteins myogenin and MyoD but not by MRF4 and myf5. These results suggest that muscle-specific transcription factors play a role in regulating the cell-type expression of the alpha7 gene during development.

Laminin-binding integrins in tumor progression and metastasis.

Interactions between tumor cells and extracellular matrix occur at several points during the metastatic cascade. Epithelial tumors, which represent nearly 90% of human neoplasia, must invade their underlying basement membrane to enter the interstitial stroma. For distant metastasis, malignant cells must penetrate basement membranes to gain access to blood vessels and organ parenchyma. Integrin receptors that bind to multiple laminin isoforms appear to mediate tumor cell adhesion to basement membranes before and during invasion. It is notable that changes in several laminin-binding integrins occur during tumor progression. These changes may include increased or decreased expression, or changes in distribution from a polarized to a dispersed pattern. Integrins not only mediate cell adhesion and motility but also transduce important downstream signaling events that regulate cell growth, survival, and gene expression. During tumor progression, the development of variant cells with changes in integrin expression and the associated signaling pathways could result in cells with a highly invasive and metastatic phenotype.

Growth factor regulation of integrin-mediated cell motility.

Cell motility, a primary component of tumor cell invasion, is a continuum of sequential events in which the cell extends pseudopodia, forms nascent attachments, assembles and contracts the cytoskeleton, and finally, as it translocates forward, disengages distal adhesions. What triggers cells to move? Substratum contact mediated by integrin adhesion receptors is important, but other signals such as chemokinetic factors appear to be required for continued crawling. It is now apparent that integrins do not simply bind cells to matrix in a Velcro-like fashion, but also are potent signaling molecules. Initial engagement of integrins induces their condensation into focal contacts, forming anchors to the extracellular matrix and discrete signal-transducing complexes on the cytoplasmic surface. A number of growth factors, through either autocrine or paracrine pathways, can activate the cellular machinery that mobilizes the cell. Thus, these two classes of receptors--the integrin receptors that bind specific extracellular adhesion molecules, and growth factor receptors that bind their respective ligands--can regulate cell locomotion. Not surprisingly, there is 'cross-talk' between integrin and growth factor receptors that occurs through their common intracellular signaling pathways. In this way, each receptor type can either amplify or attenuate the other's signal and downstream response. An example of growth factor-induced motility is the epithelial-mesenchymal transition induced by hepatocyte growth factor/scatter factor (HGF/SF). When bound to its receptor, the c-met proto-oncogene product, HGF/SF induces a phenotypic conversion that appears to be an important aspect of tumor progression in malignant carcinomas. The motogenic response produced by HGF/SF in carcinoma cells occurs in discrete steps in which integrins and focal adhesion kinase (p125FAK) are first recruited to focal contacts. This is rapidly followed by cell spreading, disruption of focal adhesions and cell-cell contacts, and, finally, cell crawling. The precise mechanism by which growth factors such as HGF/SF and its receptor induce this motogenic response and modulate integrin function has not been clearly defined but appears to involve several signaling pathways. Understanding the process by which growth factor and integrin receptors interact and regulate motility may suggest novel targets for therapeutic intervention.

Regulation of autocrine gastrin expression by the TGF alpha autocrine loop.

Gastrin is transcriptionally responsive to EGF stimulation (Merchant et al., 1991, Mol. Cell. Biol., 11:2686-2696). Consequently, we hypothesized that previously recognized gastrin autocrine loops (Hoosein et al., 1990, Exp. Cell. Res., 186:15-21), might be controlled by autocrine TGF alpha in human colon carcinoma cells. Therefore, we examined the interaction between these two autocrine growth factors in two colon carcinoma cell lines which utilize TGF alpha. The FET cell line requires exogenous TGF alpha/EGF for optimal growth and has a classical TGF alpha autocrine loop which is disrupted by TGF alpha or epidermal growth factor receptor (EGFr) antibodies. The HCT 116 cell line is not dependent on exogenous TGF alpha/EGF and exhibits a nonclassical TGF alpha autocrine loop which is not disrupted by neutralizing antibodies to either TGF alpha itself or the EGFr. Basal gastrin mRNA production is significantly higher in HCT 116 than FET as measured by RNase protection assay. In the FET cells, exogenous EGF stimulates gastrin mRNA production but not in HCT 116. When the TGF alpha autocrine loop in HCT 116 is disrupted by constitutive expression of antisense TGF alpha mRNA, the gastrin mRNA level is significantly repressed. In xenografts derived from these antisense clones, TGF alpha reverted to high expression, and the gastrin mRNA level was again increased. This interaction between the strong TGF alpha loop in HCT 116 and the gastrin autocrine loop may confer a growth advantage to these colon cells. Such interactions between growth factors may promote enhanced tumorigenicity to transformed cells with these strong, nonclassical autocrine loops.

Alternative extracellular and cytoplasmic domains of the integrin alpha 7 subunit are differentially expressed during development.

Examination of cDNAs for the laminin-binding alpha 7 integrin subunit identified two different sequences (designated X1 and X2) coding for the variable region between the III and IV homology repeat domains near the putative ligand-binding site. Sequencing of a mouse alpha 7 genomic clone established that the X1 and X2 regions are derived by mutually exclusive alternative mRNA splicing. Reverse transcriptase-polymerase chain reaction analysis of alpha 7 mRNA indicated that the X1 and X2 isoforms were present in equal amounts in mouse skeletal myoblasts and adult heart. However, in adult skeletal muscle, the X2 variant was exclusively expressed. Amino acid sequence homologies in the III/IV segment suggest that alpha 3 and alpha 6 are also alternatively spliced at this site. We identified alternatively spliced exons in a human alpha 6 genomic clone that encode X1- and X2-like segments. Analysis of the alpha 7 cytoplasmic domain indicated that this region was also alternatively spliced and like alpha 3 and alpha 6 could exist as the A or B form. In mouse skeletal and cardiac muscle the B form of alpha 7 was strongly expressed. However, we identified alpha 7A in neonate and adult skeletal muscle but not in cardiac tissue. High levels of alpha 7A were detected in differentiating myotubes, but in proliferating myoblasts only the alpha 7B isoform was present. These results indicate that alternative splicing of alpha 7 mRNA is differentially regulated during development and generates variant integrin chains with structurally and presumably functionally unique ligand-binding and cytoplasmic domains.