E-Cadherin and EpCAM expression by NSCLC tumour cells associate with normal fibroblast activation through a pathway initiated by integrin αvß6 and maintained through TGFß signalling.

Fibroblasts in the tumour stroma (cancer-associated fibroblasts) influence tumour progression and response to therapeutics; little is known about the mechanisms through which the tumour cell co-opts a normal fibroblast. To study the activation of fibroblasts by tumour cells, a panel of non-small cell lung cancer (NSCLC) cell lines and normal human dermal fibroblasts were co-cultured. A subset of the NSCLC cells induced an activated cancer-associated fibroblast-like fibroblast phenotype defined by induction of fibroblast α-smooth muscle actin expression. Tumour cells that activated fibroblasts were associated with E-Cadherin and EpCAM expression and expression of integrin αvß6. Co-culture of activating tumour cells with fibroblasts resulted in induction of transcripts associated with tumour cell invasion and growth, TGFß1 and TGFBR1, SERPINE-1, BMP6, SPHK1 and MMP9. Fibroblast activation was inhibited by an αvß6/8 integrin blocking antibody (264RAD) and a small molecule inhibitor of the TGF-beta type I receptor activin-like kinase (ALK5) (SB431542), demonstrating that transactivation of the TGFß pathway initiates fibroblast activation. Both integrin and ALK5 antagonists inhibited initiation. Only ALK5 was effective when added after 3 days of co-culture. This suggests that although activation is αvß6-dependent, once fibroblasts are activated alternative TGFß pathway regulators maintain an activation loop. In co-culture activating cells had reduced sensitivity to selumetinib, AZD8931 and afatinib compared with mono-culture. In contrast, non-activating cells were insensitive to selumetinib and AZD8931 in both mono-culture and co-culture. In conclusion NSCLC cell lines, positive for E-Cadherin, EpCAM and αvß6 expression, activate normal fibroblasts through avß6/TGFß signalling in vitro, and influence both gene expression and response to therapeutic agents.

Serum protein profiling reveals baseline and pharmacodynamic biomarker signatures associated with clinical outcome in mCRC patients treated with chemotherapy ± cediranib.

BACKGROUND: This study evaluated soluble serum proteins as biomarkers to subset patients with metastatic colorectal cancer (mCRC) treated with chemotherapy±cediranib, a vascular endothelial growth factor (VEGF) signalling inhibitor (VEGFi). Exploring biomarkers at pre- and on-treatment may identify patient subgroups showing clinical benefit on cediranib combination. METHODS: Two hundred and seven serum proteins were analysed in 588 mCRC patients at pre- and on-treatment with chemotherapy (FOLFOX/CAPOX)±cediranib 20 mg. Patients were enrolled in the phase III trial HORIZON II. We correlated baseline biomarker signatures and pharmacodynamic (PD) biomarkers with PFS and OS. RESULTS: We identified a baseline signature (BS) of 47 biomarkers that included VEGFA, VEGFD, VEGFR2, VEGFR3 and TIE-2, which defined two distinct subgroups of patients. Patients treated with chemotherapy plus cediranib who had 'high' BS had shorter PFS (HR=1.82, P=0.003) than patients with 'low' BS. This BS did not correlate with PFS of the patients treated with chemotherapy plus placebo. In addition, we identified a profile of 16 PD proteins on treatment associated with PFS (HR=0.58, P<0.001) and OS (HR=0.52, P<0.001) in patients treated with chemotherapy plus cediranib. This PD profile did not correlate with PFS and OS in patients treated with chemotherapy plus placebo. CONCLUSIONS: Serum proteins may represent relevant biomarkers to predict the outcome of patients treated with VEGFi-based therapies. We report a BS and PD biomarkers that may identify mCRC patients showing increased benefit of combining cediranib with chemotherapy. These exploratory findings need to be validated in future prospective studies.

Prognostic/predictive value of 207 serum factors in colorectal cancer treated with cediranib and/or chemotherapy.

BACKGROUND: The prognostic and predictive value of multiple serum biomarkers was evaluated using samples from a randomised phase III study (HORIZON II) investigating chemotherapy with or without cediranib in metastatic colorectal cancer (mCRC). METHODS: Baseline levels of 207 protein markers were measured in serum samples from 582 HORIZON II (FOLFOX/XELOX plus cediranib 20 mg (n=330) or placebo (n=252)) patients. Median baseline values of each biomarker were used to categorise patients as high or low. Markers were then assessed for their association with efficacy, defined by progression-free survival (PFS) and overall survival (OS). A generalised boosted regression model identified markers of particular interest. RESULTS: Correlation of protein levels with PFS and OS suggested that multiple factors had a prognostic value, independent of treatment arm, including IL-6, IL-8, C-reactive protein (CRP), ICAM-1 and carcinoembryonic antigen (CEA). Among the angiogenesis regulators, low levels of vascular endothelial growth factor (VEGF), VEGF-D, VEGFR-1, VEGFR-3, NRP1 and Tie-2 correlated with better outcome. CONCLUSION: This large data set generated using serum samples from mCRC patients treated with chemotherapy and VEGF inhibitors, defines baseline characteristics for 207 serum proteins. Multiple prognostic factors were identified that could be disease related or predict which patients derive most benefit from 5-fluorouracil (5-FU)-based chemotherapy, meriting further exploration in prospective studies.

A human monoclonal antibody 264RAD targeting αvß6 integrin reduces tumour growth and metastasis, and modulates key biomarkers in vivo.

αvß6 integrin expression is upregulated on a wide range of epithelial tumours, and is thought to play a role in modulating tumour growth. Here we describe a human therapeutic antibody 264RAD, which binds and inhibits αvß6 integrin function. 264RAD cross-reacts with human, mouse and cynomolgus monkey αvß6, and inhibits binding to all ligands including the latency-associated peptide of TGF-ß. Screening across a range of integrins revealed that 264RAD also binds and inhibits the related integrin αvß8, but not the integrins α5ß1, αvß3, αvß5 and α4ß1. In vitro 264RAD inhibited invasion of VB6 and Detroit 562 cells in a Matrigel invasion assay and αvß6 mediated production of matrix metalloproteinase-9 in Calu-3 cells. It inhibited TGF-ß-mediated activation of dermal skin fibroblasts by preventing local activation of TGF-ß by NCI-H358 tumour cells in a tumour cell-fibroblast co-culture assay. In vivo 264RAD showed dose-dependent inhibition of Detroit 562 tumour growth, regressing established tumours when dosed at 20 mg/kg once weekly. The reduction in growth associated with 264RAD was related to a dose-dependent inhibition of Ki67 and phospho-ERK and a reduction of αvß6 expression in the tumour cells, coupled to a reduction in fibronectin and alpha smooth muscle actin expression in stromal fibroblasts. 264RAD also reduced the growth and metastasis of orthotopic 4T1 tumours. At 20 mg/kg growth of both the primary tumour and the number of metastatic deposits in lung were reduced. The data support the conclusion that 264RAD is a potent inhibitor of αvß6 integrin, with some activity against αvß8 integrin, that reduces both tumour growth and metastasis.

Structural elements of the osteopontin SVVYGLR motif important for the interaction with alpha(4) integrins.

The osteopontin SVVYGLR motif binds the integrins alpha(4)beta(1) and alpha(9)beta(1). We show that alpha(4)beta(7) also interacts with this motif and that an SVVYGLR-OH peptide antagonises the alpha(4)beta(7) MAdCAM interaction. The important elements of this motif required to bind alpha(4)beta(1) and alpha(4)beta(7) were probed using a series of mutated peptides based around SVVYGLR. Leu167 is important for the interaction with alpha(4) integrins, as is the C-terminal carboxylic acid of Arg168 exposed by thrombin cleavage. The importance of the acidic group means that SVVYGLR has structural elements in common with other alpha(4) integrin-binding motifs and suggests why thrombin cleavage activates this motif.

Analysis of the alpha4beta1 integrin-osteopontin interaction.

The integrin alpha4beta1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via alpha4beta1 using GST fusion proteins. We show that alpha4beta1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the alpha4beta1 binding region in OPN lies between amino acid residues 125 and 168 (aa125-168). This region contains the central RGD motif of OPN, which also interacts with integrins alphavbeta3, alphavbeta5, alphavbeta1, alpha8beta1, and alpha5beta1. Mutating the RGD motif to RAD had no effect on the interaction with alpha4beta1. To define the binding site the region incorporating aa125-168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via alpha4beta1, aa132-146, and aa153-168; of these only a synthetic peptide, SVVYGLR (aa162-168), derived from aa153-168 was able to inhibit alpha4beta1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of alpha4beta1, and the primary alpha4beta1 binding site within OPN.

A regulated interaction between alpha5beta1 integrin and osteopontin.

The extracellular matrix protein osteopontin (OPN) interacts with a number of integrins, namely alphavbeta1, alphavbeta3, alphavbeta5, alpha9beta1, alpha8beta1, and alpha4beta1. We have investigated the interaction of alpha5beta1 integrin with OPN using K562 cells, which only express alpha5beta1. alpha5beta1 is in a low activation state in this cell line, but can be stimulated to a higher activation state by the phorbol ester TPA. Treating K562 wild-type cells (K562-WT) with TPA stimulated an interaction between alpha5beta1 and OPN. No interaction was seen in the absence of TPA. alpha5beta1 selectively interacted with a GST fusion protein of the N-terminal fragment of OPN (aa17-168), which is generated in vivo by thrombin cleavage of OPN. Expression of the alpha4 integrin in K562 cells (K562-alpha4beta1) stimulated alpha5beta1-dependent binding to aa17-168 in the absence of TPA, suggesting that alpha4beta1 activates alpha5beta1 in K562 cells. Adhesion via alpha5beta1 is mediated by the Arg-Gly-Asp (RGD) motif of OPN, as mutating this sequence to Arg-Ala-Asp (RAD) blocked binding of both cell types. These data demonstrate that thrombin cleavage regulates the adhesive properties of OPN and that alpha5beta1 integrin can interact with thrombin-cleaved osteopontin when in a high activation state.

Integrin-mediated cell adhesion: the cytoskeletal connection.

Members of the integrin family of cell adhesion molecules play a pivotal role in the interaction between animal cells and the extracellular matrix. This article reviews the evidence (i) that the integrin beta-subunit cytoplasmic domain is important in the localization of integrins to focal adhesions, and for integrin-mediated cell adhesion/spreading; and (ii) that the integrin beta-subunit can be linked to F-actin via the actin-binding proteins talin, alpha-actinin and filamin. Talin has two or more actin-binding sites, and three binding sites for the cytoskeletal protein vinculin. Because vinculin can also bind F-actin, it may cross-link talin and actin, thereby stabilizing the interaction. In addition, vinculin contains a binding site for VASP (vasodilator-stimulated phospho-protein), a protein which may serve to recruit a profilin/G-actin complex to talin, which has actin-nucleating activity. Evidence that talin, vinculin and alpha-actinin are important in the assembly of focal adhesions, obtained using antisense technology and protein microinjection, is reviewed. To analyse the role of talin in focal adhesions, we have disrupted both copies of the talin gene in mouse embryonic stem (ES) cells. Undifferentiated talin (-/-) ES cell mutants are unable to assemble focal adhesions when plated on fibronectin, whereas vinculin (-/-) ES cells are able to do so. Finally, the role of small GTP-binding proteins in the assembly of focal adhesions is discussed, along with our recent studies using streptolysin-O-permeabilized Swiss 3T3 cells which suggest that the GTP-binding protein ADP-ribosylation factor-1 (ARF-1) is important in targeting the protein paxillin to focal adhesions.

ARF1 mediates paxillin recruitment to focal adhesions and potentiates Rho-stimulated stress fiber formation in intact and permeabilized Swiss 3T3 fibroblasts.

Focal adhesion assembly and actin stress fiber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Permeabilization in the presence of GTPgammaS stimulated rho-dependent formation of stress fibers, and the redistribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTPgammaS at 8 min after permeabilization still induced paxillin recruitment to focal adhesion-like structures at the ends of stress fibers, but vinculin remained in the perinuclear region, indicating that the distributions of these two proteins are regulated by different mechanisms. Paxillin recruitment was largely rho-independent, but could be evoked using constitutively active Q71L ADP-ribosylation factor (ARF1), and blocked by NH2-terminally truncated Delta17ARF1. Moreover, leakage of endogenous ARF from cells was coincident with loss of GTPgammaS- induced redistribution of paxillin to focal adhesions, and the response was recovered by addition of ARF1. The ability of ARF1 to regulate paxillin recruitment to focal adhesions was confirmed by microinjection of Q71LARF1 and Delta17ARF1 into intact cells. Interestingly, these experiments showed that V14RhoA- induced assembly of actin stress fibers was potentiated by Q71LARF1. We conclude that rho and ARF1 activate complimentary pathways that together lead to the formation of paxillin-rich focal adhesions at the ends of prominent actin stress fibers.

Requirement for Rho in integrin signalling.

Overnight culture of Swiss 3T3 cells in serum-free medium leads to loss of focal adhesions and associated actin stress fibres, although the cells remain well spread. The small GTP-binding protein Rho is required for the formation of stress fibres and focal adhesions induced by growth factors such as lysophosphatidic acid (LPA) in serum-starved Swiss 3T3 cells, and for the LPA-induced tyrosine phosphorylation of several focal adhesion proteins. Plating of cells on extracellular matrix proteins also stimulates protein tyrosine phosphorylation and the formation of stress fibres and focal adhesions in the absence of added growth factors. These responses were inhibited in cells scrape-loaded with the Rho inhibitor C3 transferase. Focal adhesion and stress fibre formation was also triggered by addition of a peptide GRGDS, which is recognised by a number of integrins and is contained within the cell binding domain of a variety of extracellular matrix proteins. The activity of the GRGDS peptide was blocked by microinjecting cells with C3 transferase, suggesting that peptide binding to integrins stimulates a Rho-dependent assembly of focal adhesions. These experiments indicate that Rho is involved in signalling downstream of integrins.

Monoclonal antibodies recognizing the N- and C-terminal regions of talin disrupt actin stress fibers when microinjected into human fibroblasts.

We have characterized a panel of 6 monoclonal antibodies raised against human platelet talin by Western blotting, immune precipitation, and immunofluorescence, and shown that antibodies TA205 and TD77 disrupt actin stress fibers and focal adhesions, and inhibit cell motility when microinjected into human fibroblasts. Using a series of chick talin fusion proteins spanning the entire length of the molecule, we have mapped the epitopes recognized by these antibodies to the conserved N- and C-terminal regions of the protein. TA205 bound to an epitope contained within residues 139-433, a region which overlaps an F-actin binding site, and which shows homology with the ezrin/radixin/moesin family of cytoskeletal proteins. The epitope recognized by TD77 was located within the C-terminal region of the protein (residues 2269-2541) which also contains an F-actin binding site homologous to that in the yeast actin-binding protein SIa2p. To investigate the possibility that TD77 disrupts actin stress fibers by binding directly to the C-terminal actin binding site, additional talin fusion proteins were generated and analyzed for TD77 and actin binding. Fusion proteins containing residues 2269-2541, 2304-2541, and 2304-2463 all cosedimented with F-actin, whereas TD77 did not recognize the latter fusion protein. These results show that the C-terminal actin-binding site is distinct from the region recognized by the anti-functional antibody TD77, raising the possibility that it binds to a novel functionally important ligand-binding site in the talin molecule.

Focal adhesion and stress fiber formation is regulated by tyrosine phosphatase activity.

Tyrosine phosphorylation of cytoskeletal proteins plays an important role in the regulation of focal adhesions and stress fiber organization. In the present study we examined the role of tyrosine phosphatases in this process using p125FAK and paxillin as substrates. We show that tyrosine phosphatase activity in Swiss 3T3 cells was markedly increased when actin stress fibers were disassembled by cell detachment from the substratum, by serum starvation, or by cytochalasin D treatment. This activity was blocked by phenylarsine oxide, an inhibitor of a specific class of tyrosine phosphatases characterized by two vicinal thiol groups in the active site. Phenylarsine oxide treatment of serum-starved cells induced increased tyrosine phosphorylation of p125FAK and paxillin in a dose-dependent manner and induced assembly of focal adhesions and actin stress fibers, showing that inhibition of one or more phenylarsine oxide-sensitive tyrosine phosphatases is a sufficient stimulus for triggering focal adhesion and actin stress fiber formation in adherent cells.

Acidic phospholipids inhibit the intramolecular association between the N- and C-terminal regions of vinculin, exposing actin-binding and protein kinase C phosphorylation sites.

Chick vinculin polypeptides expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins have been used to identify the sites involved in the intramolecular association between the 90 kDa N-terminal head and the 30 kDa C-terminal tail region of the vinculin molecule. Fusion proteins spanning vinculin residues 1-258 and 1-398, immobilized on glutathione-agarose beads, were shown to bind a C-terminal vinculin polypeptide spanning residues 881-1066 (liberated from GST by thrombin cleavage). However, the C-terminal polypeptide did not bind to a fusion protein spanning residues 399-881 or to itself. Binding was dependent on residues 167-207 within the N-terminal polypeptide, a sequence also essential for talin binding. Conversely, the 90 kDa head polypeptide was shown to bind to residues 1029-1036 in the tail region of vinculin. The association of the head and tail was inhibited by acidic, but not neutral, phospholipids. Pre-incubation of vinculin with acidic phospholipids exposed the binding site for F-actin and a phosphorylation site for protein kinase C. The phosphorylation site was located in the tail region of the vinculin molecule. These results raise the possibility that acidic phospholipids play a role in regulating the activity of vinculin and therefore the assembly of both cell-cell and cell-matrix adherens-type junctions.

The RhoA-dependent assembly of focal adhesions in Swiss 3T3 cells is associated with increased tyrosine phosphorylation and the recruitment of both pp125FAK and protein kinase C-delta to focal adhesions.

Mouse Swiss 3T3 fibroblasts cultured in serum-free medium lose their actin stress fibres and vinculin-containing focal adhesions, a process that can be reversed by the addition of serum, lysophosphatidic acid (LPA) or bombesin, and is mediated by rhoA (A. J. Ridley and A. Hall (1992) Cell 70, 389-399). We have shown that the addition of serum to these cells induces the recruitment of the cytoskeletal proteins talin, vinculin and paxillin, and the protein kinases pp125FAK and PKC-delta, to newly formed focal adhesions, and that alpha-actinin is distributed along the actin stress fibres associated with these structures. The newly formed focal adhesions stained heavily with an antibody to phosphotyrosine. A similar response was elicited by 100 ng/ml LPA. The effect of serum was rapid, with focal staining for paxillin largely restricted to cell margins seen within 2 minutes of serum addition, and preceding the assembly of actin filaments. Phosphotyrosine staining differed in that it was predominantly punctate and was widely distributed throughout the cell. By 5 minutes, the paxillin and phosphotyrosine staining was concentrated at the ends of actin filaments largely at the cell margins. The structures stained ranged from circular to oval, but by 10 minutes they more closely resembled the elongated focal adhesions found in cultured fibroblasts. Within 10 minutes, the addition of serum or LPA induced a marked increase in the levels of pp125FAK and paxillin immune-precipitated by an anti-phosphotyrosine antibody. The results suggest that both pp125FAK and paxillin undergo changes in tyrosine phosphorylation upon activation of rhoA, and that these changes are associated with the assembly of focal adhesions and actin stress fibres. The observation that formation of focal adhesions can be induced by the tyrosine phosphatase inhibitor vanadyl hydroperoxide is consistent with the direct involvement of tyrosine phosphorylation in the assembly process. The localisation of PKC-delta to newly formed focal adhesions suggests that serine/threonine phosphorylation may also be important in this regard.

Rising to the challenge.

An overview of health care in America today. An estimated 37 million men, women, and children lack access to affordable health care. As the cost of medical care continues to rise, millions more face the possibility of joining the ranks of the uninsured. These problems must be addressed. But we also need to consider the many benefits that we derive from our health care system.