Selective increase of α2‐integrin sub‐unit expression on human carcinoma cells upon EGF‐receptor activation

The effects of chronic EGF exposure on expression of the α₂β₁ collagen and α₅β₁ fibronectin receptor in a pair of human carcinoma cell lines (A431 and A549) with differential responses to EGF in a short‐term ECM‐cell adhesion assay were investigated. Treatment with EGF at 10 ng/ml for 24 hr increased on both cell lines the expression of the α₂‐ but not the β₁‐ or α₅‐integrin sub‐units, and concomitantly cellular adhesion was increased on collagen IV but not on fibronectin. Increased collagen adhesion of A549 cells could be blocked by α₂‐ and β₁‐integrin‐sub‐unit antibodies down to control levels, while it was blocked by α₂‐integrin‐sub‐unit antibody only by 60% and completely by the β₁‐integrin‐sub‐unit antibody on A431 cells. EGF induced disparate shifts in cell morphologies (dome‐like structures, A431, vs. spindle‐like fibroblastoid, A549) with concomitant opposite changes in the expression/localization of E‐cadherin in cell‐cell contacts. This could be taken as an indication for cell‐type‐specific differential changes in the ratio of cell‐ECM vs. cell‐cell contacts. The EGF‐induced up‐regulation of the α₂β₁ integrin was instrumental in increasing collagen adhesion of A549 but only partly in the case of A431 cells, in which cells the α₂β₁ integrin may have additional functions besides serving as cell‐ECM receptor. Int. J. Cancer 80:546–552, 1999. © 1999 Wiley‐Liss, Inc.     

Adhesion molecules and TGF-β1 are involved in the peritoneal dissemination of NUGC-4 human gastric cancer cells

Peritoneal dissemination frequently occurs after surgery in patients with gastric cancer. The presence of peritoneal metastasis after surgery affects prognosis. Very little is known about the biochemical processes involved in the initial attachment of gastric cancer cells to peritoneal mesothelial cells. We conducted in vitro and in vivo studies to assess the role of adhesion molecules and TGF-β1 in this process, using 4 cell lines derived from human gastric cancers. NUGC-4 cells, which disseminate early after inoculation into the abdominal cavity of nude mice, predominantly express CD44H and β₁ integrin. We found that NUGC-4 cells adhered to monolayers of mesothelial cells more firmly than to other cell lines. Adhesion of NUGC-4 cells to mesothelial cells was partially inhibited by antibodies against CD44H or the β₁ subunit of integrin and was completely blocked by a combination of these 2 antibodies. Treatment with ligands for CD44H and β₁ integrin also inhibited adhesion. In the NUGC-4 cell culture medium, larger amounts of TGF-β₁ were detected in relation to the increase in cancer cells than in the other cell lines. TGF-β1 increased the expression of CD44H in NUGC-4 cells and in mesothelial cells and augmented adhesion and implantation of NUGC-4 cells to mesothelial cells accompanied by accumulation of extracellular matrix (ECM) components. Treatment with antibodies against both CD44H and β₁ integrin inhibited the dissemination of NUGC-4 cells in the peritoneal cavity of nude mice and prolonged their survival time. Our findings suggest that CD44H and integrins mediate the initial attachment of gastric cancer cells to mesothelial cells and that TGF-β1 participates in the promotion of the disease. Increased expression of CD44H and of the amount of ligands for CD44H and integrins induced by TGF-β1 promotes early development of peritoneal dissemination. Int. J. Cancer 70:612–618. © 1997 Wiley-Liss Inc     

Regulation of fibronectin and laminin receptor expression,fibronectin and laminin secretion in human colon cancer cells by transforming growth factor-β1

Transforming growth factor (TGF)-β₁, modulates the expression of extracellular matrix (ECM) glycoproteins, fibronectin and laminin and the adhesion of Moser colon cancer cells to these glycoproteins. Since adhesion can be altered through expression of cell-surface receptors, binding affinities of adhesion molecules for receptors, or both, we investigated the effect of TGF-β₁, on the binding properties of fibronectin and laminin to their cell-surface receptors by saturation binding and Scatchard analyses using radiolabeled fibronectin and laminin. Fibronectin bound to its cell-surface receptor with high affinity (K_d = 1.25 × 10^?9 M), Moser cells had approximately 7.1 × 10⁴ fibronectin-binding sites per cell. TGF-β₁ treatment rapidly up-modulated the number of cell-surface fibronectin-binding sites by 1.9-fold. The binding affinity of fibronectin for the receptor, however, was not altered. Laminin was found to bind to a higher-affinity and a lower-affinity receptor. Moser cells expressed approximately 1.1 × 10³ higher-affinity laminin-binding sites and approximately 3.1 × 10 lower-affinity-binding sites per cell. TGF-β₁ rapidly increased the expression of the higher-affinity sites 3-fold and the lower-affinity sites 5-fold. The binding affinity of both the higher-affinity and lower-affinity laminin receptors increased 3-fold after 2 and 6 hr of TGF-β₁ treatment respectively. Concurrent with receptor modulation, TGF-β₁ induced the secretion of fibronectin and laminin from Moser cells. Northern hybridization analyses showed a concurrent stimulation of the expression of the mRNAs for ligands (fibronectin and laminin) and the mRNAs for the integrin species of the fibronectin and laminin receptors (α5 and α6 subunits). Thus the production of fibronectin and laminin and the expression of their receptors were tightly co-regulated by TGF-β₁.     

Transformation and tumor progression are frequently associated with expression of the α3/β1 heterodimer in solid tumors

The ability of tumor cells to interact with the extracellular matrix (ECM) is functionally mediated by a variety of receptor molecules among which the integrins are a well-characterized family of mediators. In this study we have investigated immuno-histochemically the in vivo expression of the α/β₁ promiscuous receptor for ECM constituents in a variety of human solid malignancies. Although the receptor appears to undergo changes in distribution patterns, its expression is maintained in a high percentage of primary (76%) and metastatic (82%) tumors. Furthermore, the comparative immunohistochemical evaluation of α/β₁ and its ligands, in a selected number of tumors of different histotypes, demonstrated that the expression of this integrin correlates with the presence of at least one ligand, either around nests of neoplastic cells or at the epithelial-stromal interface. The highly conserved expression of α/β₁ shown in this study suggests that this receptor may play a role in tumor growth at the primary as well as at the metastatic site.     

Integrin-receptor-mediated differentiation and growth inhibition are enhanced by transforming growth factor-beta in colorectal tumour cells grown in collagen gel

We have studied the role of cell-matrix interactions and the modulating effect on these of transforming growth factor-beta s (TGF-beta s) in controlling differentiation and proliferation in a series of human colorectal carcinoma cell lines. Two (SW1222 and SW480) out of 7 cell lines specifically bound type-I collagen, via integrin-like (SW1222) and non-integrin-like (SW480) collagen receptors. Binding of these receptors may be responsible for regulating the degree of epithelial differentiation of the cells when grown in a 3-dimensional (3D) collagen gel. We have also shown that TGF-beta s enhance the binding of SW1222 cells to collagen and that this is accompanied by greatly increased crypt-like glandular differentiation and inhibition of cell proliferation. Inhibition of cell proliferation was only seen when cells were grown in 3D collagen gel and were thus expressing a fully differentiated phenotype. The enhanced collagen binding induced by TGF-beta s was partially inhibited by an Arg-Gly-Asp (RGD)-containing peptide which is a cell recognition signal for collagen binding. This suggests that TGF-beta s mediate their effects on differentiation of SW1222 cells specifically by modulating the expression of the integrin-like collagen receptor. The other colorectal carcinoma cell lines which lack this integrin-like receptor either failed to bind collagen or, in the case of SW480 binding, exhibited differentiation and proliferation which were not affected by TGF-beta s. This suggests that cell responsiveness to TGF-beta s may depend, at least in part, upon the cell-matrix interaction.     

Altered in vitro spreading and cytoskeletal organization in human glioma cells by downregulation of urokinase receptor

The interaction of urokinase-type plasminogen activator (uPA) with its cell-surface receptor (uPAR) is implicated in diverse biological processes such as cell migration, tissue remodeling, and tumor cell invasion. Recent studies indicated that uPAR can act as an extracellular matrix receptor during cell adhesion. Recently, we showed that transfection of the humanglioma cell line SNB19 with antisense uPAR resulted in downregulation of uPAR at both the mRNA and protein levels. In this study, we used SNB19 to determine how the presence or absence of uPAR promotes cellspreading and associated changes in cell morphology. Microscopic analysis of cell spreading revealed that antisense uPAR–transfected cells were larger, remained round, and did not spread efficiently over extracellular matrix substrate type IV collagen and fibronectin, unlike parental SNB19 cells, which were smaller and spindle shaped. Biochemical studies showed that antisense uPAR–transfected cells, in addition to not spreading, exhibited increased expression of α3β1 integrin but not α5β1 integrin. However, we could not find a change in the expression of extracellular matrix components or altered growth rate in these cells. Furthermore, despite the increased α3β1 integrin expression, antisenseuPAR–transfected cells failed to form an organized actin cytoskeleton when plated on type IV collagen or fibronectin, unlike parental SNB19 cells, which displayed an organized cytoskeleton. These findings show that the absence of uPAR in human glioma cells leads to morphological changes associated with decreased spreading and a disorganized cytoskeleton resulting in altered cell morphology, suggesting that coordinated expression of uPAR and integrin may be involved in spreading of antisense uPAR–transfected glioma cells. Mol. Carcinog. 20:355–365, 1997. © 1997 Wiley-Liss, Inc.     

Colon-cancer cell variants producing regressive tumors in syngeneic rats,unlike variants yielding progressive tumors,attach to interstitial collagens through integrin α2β1

Nine clones of tumor cells, derived from a single rat colon carcinoma, were analyzed for their adhesive properties and in vivo growth patterns. Four clones (denoted REG) gave rise to regressively growing tumors. Cells from the 4 REG clones attached significantly better to collagen types I and III than did cells from the 5 clones (denoted PRO) which grew progressively in vivo. In contrast, REG and PRO clones did not differ in their attachment to collagen type IV, laminin or fibronectin. The attachment of REG cells to collagen was dependent on Mg²⁺, but not Ca²⁺. Monospecific rabbit IgG to rat integrin β₁-chain inhibited REG cell attachment to collagen, demonstrating involvement of a β₁ integrin in this process. PRO and REG cells expressed an underglycosylated β₁ chain (M_r ∼ 105,000) that was somewhat smaller than β₁-chains described previously on rat fibroblasts and hepatocytes (M_r ∼ 115,000). Monoclonal IgG to rat integrin α₂β₁, but not to α₁β₁, readily inhibited REG cell attachment to collagen, demonstrating the involvement of integrin α₂β₁. However, β₁ and α₂ integrin subunits were found in purified glycoproteins from both PRO and REG cells. This suggests that α₂β₁ integrin is expressed by both cell variants, but is functional as a collagen receptor on REG cells only. In this system of tumor-cell variants, the clear-cut differences in attachment to interstitial collagens of the 9 clones suggest a possible relationship between this attachment and the capacity to induce progressive or regressive tumors. © 1996 Wiley-Liss, Inc.     

Differential localization of transforming growth factor-β isoforms in human gastric mucosa and overexpression in gastric carcinoma

Transforming growth factor β (TGF-β) isoforms comprise a family of multifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. We examined TGF-β expression in normal human gastric mucosa and carcinoma. The distribution and expression of TGF-β isoforms in 4 normal mucosa samples from organ donors, in 12 normal mucosa samples adjacent to gastric cancer and in 12 gastric carcinomas were examined using immunohistochemistry and Northern blot analysis. Because TGF-βs regulate collagen expression, collagen type 1 α₁ mRNA amounts were also examined. Immunohistochemical analysis of normal human gastric tissue samples indicated that TGF-β₁ localized principally in parietal cells but also in some surface mucus cells, TGF-β₂ was present exclusively in chief cells and TGF-β₃ was present in parietal, chief and mucus cells. In the gastric cancers, strong colocalization of TGF-β₁, -β₂ and -β₃ was evident in the cancer cells. Northern blot analysis indicated that, compared to normal gastric tissue, gastric cancers showed a 4.8- and 6-fold increase in mRNA amounts encoding TGF-β₁, and TGF-β₃, respectively. In contrast, TGF-β₂ mRNA amounts were comparable in both groups. Northern blot analysis showed a 10-fold increase in human collagen type 1 α₁ mRNA amounts compared to normal gastric tissue. These findings imply a role for TGF-βs in normal human gastric mucosa function, and raise the possibility that the aberrant colocalization and overexpression of all 3 TGF-β isoforms in human gastric cancer cells in vivo may bcontribute to the pathobiology of gastric carcinoma. Int. J. Cancer 71:131–137, 1997. © 1997 Wiley-Liss, Inc.     

Endothelial expression of TNF receptor-1 generates a proapoptotic signal inhibited by integrin α6β1 in glioblastoma

Activation of TNF receptor 1 (TNF-R1) can generate signals that promote either apoptosis or survival. In this study, we show that these signals can be determined by the character of the extracellular matrix in the tumor microenvironment. Specifically, through studies of glioblastoma, we showed that TNFα stimulation induced apoptosis of primary brain endothelial cells (EC) attached to collagen or fibronectin (which engage integrins α2β1/α3β1 and α5β1, respectively), but did not induce apoptosis of ECs attached to laminin (which engages integrins α6β1 and α3β1). TNF-R1 expression was significantly higher in ECs in glioblastoma (GBM) tumors compared with ECs in normal brain specimens. TNFα was also expressed in GBM tumor-associated ECs, which was associated with longer patient survival. ECs plated on anti-integrin α2 or α3 antibody were susceptible to TNFα-induced apoptosis, whereas those plated on anti-integrin α6 antibody were not. Moreover, the ECs plated on laminin, but not collagen, expressed cellular FLICE inhibitory protein (cFLIP) and TNFα stimulation of laminin-attached cells in which cFLIP had been downregulated resulted in the induction of apoptosis. In contrast, attachment to laminin did not induce cFLIP expression in GBM tumor stem cells. Together, our findings indicate that the laminin receptor integrin α6β1 promotes the survival of brain ECs by inhibiting prodeath signaling by TNF-R1, in part by inducing cFLIP expression.     

Cilengitide downmodulates invasiveness and vasculogenic mimicry of neuropilin 1 expressing melanoma cells through the inhibition of αvβ5 integrin

During melanoma progression, tumour cells show increased adhesiveness to the vascular wall, invade the extracellular matrix (ECM) and frequently form functional channels similar to vascular vessels (vasculogenic mimicry). These properties are mainly mediated by the interaction of integrins with ECM components. Since we had previously identified neuropilin 1 (NRP‐1), a coreceptor of vascular endothelial growth factor A (VEGF‐A), as an important determinant of melanoma aggressiveness, aims of this study were to identify the specific integrins involved in the highly invasive phenotype of NRP‐1 expressing cells and to investigate their role as targets to counteract melanoma progression. Melanoma aggressiveness was evaluated in vitro as cell ability to migrate through an ECM layer and to form tubule‐like structures using transfected cells. Integrins relevant to these processes were identified using specific blocking antibodies. The αvβ5 integrin was found to be responsible for about 80% of the capability of NRP‐1 expressing cells to adhere on vitronectin. In these cells αvβ5 expression level was twice higher than in low‐invasive control cells and contributed to the ability of melanoma cells to form tubule‐like structures on matrigel. Cilengitide, a potent inhibitor of αν integrins activation, reduced ECM invasion, vasculogenic mimicry and secretion of VEGF‐A and metalloproteinase 9 by melanoma cells. In conclusion, we demonstrated that ανβ5 integrin is involved in the highly aggressive phenotype of melanoma cells expressing NRP‐1. Moreover, we identified a novel mechanism that contributes to the antimelanoma activity of the αv integrin inhibitor cilengitide based on the inhibition of vasculogenic mimicry.     

Response of Hepatic Stellate Cells to TGFB1 Differs from the Response of Myofibroblasts. Decorin Protects against the Action of Growth Factor

Regardless to the exact nature of damage, hepatic stellate cells (HSCs) and other non-parenchymal liver cells transform to activated myofibroblasts, synthesizing the accumulating extracellular matrix (ECM) proteins, and transforming growth factor-β1 (TGF-β1) plays a crucial role in this process. Later it was discovered that decorin, member of the small leucin rich proteoglycan family is able to inhibit this action of TGF-β1. The aim of our present study was to clarify whether HSCs and activated myofibroblasts of portal region exert identical or different response to TGF-β1 exposure, and the inhibitory action of decorin against the growth factor is a generalized phenomenon on myofibroblast of different origin? To this end we measured mRNA expression and production of major collagen components (collagen type I, III and IV) of the liver after stimulation and co-stimulation with TGF-β1 and decorin in primary cell cultures of HSCs and myofibroblasts (MFs). Production of matrix proteins, decorin and members of the TGF-β1 signaling pathways were assessed on Western blots. Messenger RNA expression of collagens and TIEG was quantified by real-time RT-PCR. HSCs and MFs responded differently to TGF-β1 exposure. In contrast to HSCs in which TGF-β1 stimulated the synthesis of collagen type I, type III, and type IV, only the increase of collagen type IV was detected in portal MFs. However, in a combined treatment, decorin seemed to interfere with TGF-β1 and its stimulatory effect was abolished. The different mode of TGF-β1 action is mirrored by the different activation of signaling pathways in activated HSCs and portal fibroblasts. In HSCs the activation of pSMAD2 whereas in myofibroblasts the activation of MAPK pathway was detected. The inhibitory effect of decorin was neither related to the Smad-dependent nor to the Smad-independent signaling pathways.     

Organisation and gel contraction by human colonic carcinoma (HCA-7) sublines grown in 3-dimensional collagen gel

The role of cell-matrix interactions in controlling phenotypic heterogeneity in human colonic carcinoma sublines has been investigated. Four cell lines (colony 1, colony 3, colony 6 and colony 30) previously isolated from a single human colonic carcinoma cell line, HCA-7, were grown in 3-dimensional collagen gels. In collagen, the growth of the 4 sublines ranged from well-organised glandular structures (colony 30) to elongated branching structures (colony 3). The capability of cells to organise into glandular structures in collagen correlated with the degree of differentiation observed in their xenografts. Certain sublines, most notably colony 3, were able to contract the collagen gel. Gel contraction could be partially inhibited by a function-blocking antibody directed to the α₂ integrin chain but not by an antibody directed to the α₃ integrin chain demonstrating a role for α₂ integrin in the contraction process. In addition, colony 3 cultures treated with the function-blocking α₂ antibody formed more compact structures with limited outgrowth, suggesting a role for α₂ integrin in cell migration. Gel contraction and cell migration in collagen gel was largely restricted to 1 subline, colony 3. The subsequent demonstration that α₂ integrin is involved in both of these processes suggests that integrin expression and function has a role in generating the phenotypic heterogeneity exhibited by these cell lines.     

Stimulation of glioma-cell migration by laminin and inhibition by anti-α3 and anti-β1 integrin antibodies

An induction of laminin in the confrontation zone between tumor cells and normal brain tissue has been observed in our model systems in vivo and in vitro. In order to study the effects of ECM components on glioma-cell migration and invasion, we have used 2 lacZ-transfected glioma cell lines, ANI/lacZ and U-251/lacZ. Cell migration from multicellular spheroids was studied using different types of media: DMEM with 10% serum, Ultra Culture medium, and filtrated DMEM with serum in which the protein fraction > 100 kDa had been removed by ultrafiltration. Laminin, fibronectin and collagen type-IV were individually added to the different media, and cell migration from the spheroids was studied. The results show that cell migration in both cell lines, was stimulated by laminin and fibronectin. Collagen type-IV stimulated only cell migration of U-251/lacZ cells. Scanning electron microscopy revealed an extensive change in cell shape as a result of laminin stimulation. Flow-cytometric studies showed that both ANI/lacZ and U-251/lacZ strongly express the α3β1 integrin receptor, which can bind to several ECM components (laminin, fibronectin, collagen). Immunofluorescence microscopy demonstrated that the same integrin sub-units were expressed in multicellular spheroids. When monoclonal antibodies to α3 and β1 were added to the laminin-stimulated cultures, cell migration was significantly reduced. This indicates that the α3β1 integrin receptor plays an important role during glioma-cell migration. © 1996 Wiley-Liss, Inc.     

Migration properties of the human ovarian adenocarcinoma cell line IGROV1: Importance of αvβ3 integrins and vitronectin

Cell migration of ovarian tumoral cells is essential for cell dissemination and for invasion of the submesothelial extracellular matrix (ECM). We have conducted a study of the migratory properties of an ovarian adenocarcinoma cell line (IGROV1) by using 2 distinct methods for the evaluation of cell migration. We found that in a short‐term transfilter migration assay, IGROV1 cells migrated toward vitronectin, fibronectin, type IV collagen and laminin in an integrin‐dependent manner. When migration was evaluated in a wound healing assay, the restitution of the wounded area was stimulated solely by added, exogenous vitronectin and was almost totally dependent on αvβ3 integrin function. Moreover, we demonstrated that αvβ3 was localized in focal contacts restricted to the leading edge of migrating cells, whereas vitronectin notably localized with actin stress fibers and cortical actin. On the other hand, several kinase inhibitors were found to impede migration of IGROV1 induced by vitronectin. It thus appears that αvβ3–vitronectin interactions lead to the activation of multiple signaling pathway including activation of protein kinase C, phosphatidyl‐inositol‐3‐phosphate kinase and protein tyrosine kinase. The “αvβ3–vitronectin system” is therefore essential to the migration of human ovarian carcinoma cells.Int. J. Cancer 80:285–294, 1999. © 1999 Wiley‐Liss, Inc.     

Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in fibroblasts: Implications for melanoma invasion

Fibroblast activation protein-α (FAP-α) promotes tumor growth and cell invasiveness through extracellular matrix degradation. How ultraviolet radiation (UVR), the major risk factor for malignant melanoma, influences the expression of FAP-α is unknown. We examined the effect of UVR on FAP-α expression in melanocytes, keratinocytes and fibroblasts from the skin and in melanoma cells. UVR induces upregulation of FAP-α in fibroblasts, melanocytes and primary melanoma cells (PM) whereas keratinocytes and metastatic melanoma cells remained FAP-α negative. UVA and UVB stimulated FAP-α-driven migration and invasion in fibroblasts, melanocytes and PM. In co-culture systems UVR of melanocytes, PM and cells from regional metastases upregulated FAP-α in fibroblasts but only supernatants from non-irradiated PM were able to induce FAP-α in fibroblasts. Further, UV-radiated melanocytes and PM significantly increased FAP-α expression in fibroblasts through secretory crosstalk via Wnt5a, PDGF-BB and TGF-β1. Moreover, UV radiated melanocytes and PM increased collagen I invasion and migration of fibroblasts. The FAP-α/DPPIV inhibitor Gly-ProP(OPh)2 significantly decreased this response implicating FAP-α/DPPIV as an important protein complex in cell migration and invasion. These experiments suggest a functional association between UVR and FAP-α expression in fibroblasts, melanocytes and melanoma cells implicating that UVR of malignant melanoma converts fibroblasts into FAP-α expressing and ECM degrading fibroblasts thus facilitating invasion and migration. The secretory crosstalk between melanoma and tumor surrounding fibroblasts is mediated via PDGF-BB, TGF-β1 and Wnt5a and these factors should be evaluated as targets to reduce FAP-α activity and prevent early melanoma dissemination.     

β1 integrin is required for anchorage-independent growth and invasion of tumor cells in a context dependent manner

Recent studies suggest that extracellular matrix (ECM) components within the tumor microenvironment can influence malignant progression, thus we investigated the influence of the ECM binding receptor β1 integrin, on the hallmark properties of tumorigenesis. Small interfering (si) or short hairpin (sh) RNA approaches were used to deplete β1 integrin in cancer cell lines. β1 integrin-depleted cells were then assessed for their growth and invasive capabilities using 2-dimensional (2D) or 3D culture conditions. Depletion of β1 integrin expression did not impact cell growth in 2D assay systems; however, β1 integrin and its ligand fibronectin were required for growth in 3D. β1 integrin-depleted cells also had reduced invasive capabilities, in part due to increased tissue inhibitor of metalloprotease (TIMP)-2 expression in conjunction with down-regulation of matrix metalloprotease (MMP)-9 levels in β1 integrin-depleted cells. Our results suggest that despite no apparent effect on 2D cell growth, fibronectin-β1 integrin signaling is a critical mediator of the 3D growth and invasive properties of tumor cells. These observations highlight the importance of investigating the role of adhesion molecules in the appropriate context and furthermore identify β1 integrin as a possible therapeutic target to inhibit the aggressive growth and invasion of tumor cells.     

Expression of β1 integrin receptors in transformed mouse epidermal keratinocytes: Upregulation of α5β1 in spindle carcinoma cells

The adhesive properties and the expression of extracellular matrix receptors of the β1-integrin subfamily were analyzed in transformed epidermal keratinocyte cell lines of different stages of mouse skin carcinogenesis. One- and two-dimensional analyses of the immunoprecipitates obtained with anti-β1- and specific anti-α-integrin subunits showed qualitative and quantitative changes in the expression of β1 integrins by the different cell lines. The polyvalent α3β1 integrin was expressed by all analyzed cell lines, although the levels detected in undifferentiated spindle CarC cells were lower than those present in the rest of keratinocyte cell lines. In contrast, spindle cells expressed high levels of the specific fibronectin receptor α5β1, whereas this integrin was absent or expressed at very reduced levels in the other epithelial cell lines. Expression of α5β1 integrin in spindle cells appeared organized in cell-substratum contact areas on spread cells. In addition, high and homogenous expression of α5β1 was detected in fully undifferentiated tumors induced in nude mice by three independent spindle cell lines. These results suggest that the expression of α5β1 integrin is upregulated during the development of spindle cell carcinomas that occur in the last stages of mouse skin carcinogenesis and can be associated with the acquisition of the fibroblastoid phenotype of spindle cells. On the other hand, expression of the collagen receptor α2β1 was demonstrated in a transformed cell line (PDV), and it was apparently also expressed in two other malignant keratinocyte cell lines (PDVC57 and HaCa4). The expression of α2β1 was correlated with the increased adhesion to collagen type I and Collagen type IV exhibited by the tumorigenic cell lines. © 1995 Wiley-Liss Inc.     

Small Molecule Hormone or Hormone-Like Ligands of Integrin αVβ3: Implications for Cancer Cell Behavior

Integrins are heterodimeric structural components of the plasma membrane whose ligands include a large number of extracellular matrix (ECM) proteins. The ligands contain Arg–Gly–Asp (RGD) sequences that enable recognition of ECM proteins by as many as eight integrins, but other distinguishing features of the proteins permit the integrins to generate intracellular signals specific to the ECM molecules. Recently, integrin αvβ3 has been shown to have a panel of previously unappreciated small molecule receptor sites for thyroid hormone and hormone analogues, for dihydrotestosterone, and for resveratrol, a polyphenol that has certain estrogen-like features. These binding sites are close to the RGD recognition site of αvβ3. The thyroid hormone receptor site on the extracellular domain of αvβ3 contains two domains with discrete functions in terms of intracellular protein trafficking and gene expression. Occupancy of the receptor by a deaminated thyroid hormone analogue, tetraiodothyroacetic acid (tetrac), prevents cell responses to agonist thyroid hormones (l-thyroxine; 3, 5, 3′-triiodo-l-thyronine) and modulates expression of a number of cancer cell survival pathway genes in an up- or downregulation pattern coherent to induction of cell death. The small molecule thyroid hormone receptor on the integrin also regulates activity of five vascular growth factor receptors and/or their ligands, providing control of angiogenesis via specific pharmacologic regulation of this thyroid hormone receptor. The resveratrol receptor induces programmed cancer cell death via p53, even when the latter has undergone specific mutations. There is also evidence for the presence of several receptors on integrin αvβ3 for authentic steroids, including a dihydrotestosterone site that supports proliferation of breast cancer cells that lack nuclear androgen and estrogen receptors. The existence of these small molecule hormone receptors on an integrin with a remarkably complex functional profile defines novel pharmacologic options via individual small molecule receptor manipulation for control of cancer cell behavior. This refinement of up-down control at the level of discrete receptors is not a function of the use of αvβ3 antibody or RGD peptides that occlude regions of the integrin.