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[Cancer Research 65, 10674-10679, December 1, 2005]
© 2005 American Association for Cancer Research

Priority Reports

ß4 Integrin Is a Transforming Molecule that Unleashes Met Tyrosine Kinase Tumorigenesis

Andrea Bertotti, Paolo M. Comoglio and Livio Trusolino

Division of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), University of Torino School of Medicine, Candiolo (Torino), Italy

Requests for reprints: Livio Trusolino, Division of Molecular Oncology, Institute for Cancer Research and Treatment (IRCC), University of Torino School of Medicine, Strada Provinciale 142, km 3.95, 10060 Candiolo, Turin, Italy. Phone: 39-11-993-3202; Fax: 39-11-993-3225; E-mail: livio.trusolino{at}ircc.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 References
 
Cell multiplication in the absence of integrin-derived adhesive signals (anchorage-independent growth) is the phenotypic hallmark of neoplastic transformation. Therefore, the frequently observed up-regulation of some integrins in tumors has been interpreted as an epiphenomenon and not as a causative factor of oncogenic conversion. ß4 integrin stimulates proliferation and survival of epithelial cells and is overexpressed in human carcinomas, often in concomitance with up-regulation of the Met tyrosine kinase receptor for hepatocyte growth factor. Met is not endowed with transforming ability but can exploit the ß4 cytoplasmic tail as a substrate/adaptor for amplification of mitogenic and antiapoptotic responses, independently of cell adhesion. Here, we show that overexpression of ß4 is sufficient to transform rodent fibroblasts, enhances anchorage-independent growth of breast carcinoma cells, and induces tumorigenesis in nude mice; conversely, RNA interference–mediated depletion abrogates the transformed phenotype of neoplastic cells. These autonomous oncogenic properties are dramatically exacerbated upon Met coexpression, suggesting that the integrin can instigate the latent tumorigenic potential of the kinase. A ß4 nonadhesive variant still cooperates with Met for cellular transformation, confirming the adhesion-independent function of ß4 in magnification of Met biological effects. Conversely, a ß4 signaling-incompetent mutant that cannot be efficiently tyrosine phosphorylated by Met and displays reduced ability to activate phosphatidylinositol 3-kinase–dependent and Ras-dependent pathways aborts transformation. Our findings define ß4 as a signaling accomplice (a "servo-oncogene") of tyrosine kinase proto-oncogenes in primary carcinogenesis, evoke an unorthodox function for a prototypic adhesion molecule in the positive regulation of anchorage-independent growth, and suggest the use of ß4 as a target for anticancer therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Conclusions
 References
 
Regulated proliferation of normal cells obeys a bimodal control by integrins and tyrosine kinase receptors, which together prime mitogenic signals in response to cell adhesion and growth factors, respectively (1). In contrast, deranged proliferation of transformed cells circumvents anchorage dependence and relies on constitutive activation of tyrosine kinase–based pathways (2). This tenet has obscured a potential causal role for integrins in oncogenic conversion, despite their frequent up-regulation in tumors (3). A number of anecdotic reports suggest that the ß4 subunit of the {alpha}6ß4 integrin complex might be etiologically involved in cancer onset. ß4 was originally identified as a tumor-associated antigen (4), and ß4 neo or overexpression has been repeatedly documented in human premalignant lesions and incipient neoplasms (5). Moreover, in accordance with the notion that anchorage independence is the major phenotypic hallmark of neoplastic transformation, the tumor-associated functions of ß4 may not require the integrin adhesive activity. In human carcinomas, the topographical localization of ß4 is not restricted to cell membrane domains that are in contact with adhesive ligands but undergoes pericellular redistribution. Similarly, in animal models of experimental skin carcinogenesis, aberrant expression of unligated ß4 in keratinocytes of the upper epidermal layers coincides with suprabasal expansion of the proliferating compartment and with development of squamous cell carcinomas (6). Finally, in cancer cells, ß4 can act as an adhesion-independent signaling substrate for the hepatocyte growth factor (HGF) receptor Met and as a docking platform for additional recruitment of signal transducers, with consequent optimization of HGF-dependent mitogenic and antiapoptotic responses (7). The functional collaboration between ß4 and Met is corroborated by the observation that in human carcinomas, the two molecules are concomitantly up-regulated in the same tumor types (5, 8).

The in vivo expression patterns of ß4 in human tumors and its ability to foster cell accretion and limit cell attrition even when cells are not attached to physiologic substrates prompted us to investigate whether expression of ß4, alone and in combination with Met, can induce cellular transformation and enhance tumorigenicity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Conclusions
 References
 
Antibodies and vectors. We used the following antibodies: anti-human Met and anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-ß4 integrin (Chemicon, Temecula, CA), anti-phosphotyrosine (Upstate Biotechnology, Charlottesville, VA). The ß4cyto-T construct was generated by PCR amplification of the BssHII-NotI fragment of a ß4 template already containing the Y1257F and Y1494F substitutions (from L.M. Shaw). To create the ß4 siRNA expression vector, oligonucleotides used by Chung et al. (9) were annealed and ligated into pSUPER between the BglII and HindIII sites. BamHI- and XhoI-digested inserts were then subcloned into the pRLL5 lentiviral vector. The constructs encoding for wild-type Met, kinase-inactive Met, and ß4{Delta}extra have already been described (7).

Cell culture, transfection, and viral infection. COS, ZR75, MDA-MB-231, and mouse embryonic fibroblasts (MEF; spontaneously immortalized clones that survived senescence crisis; from L. Lanzetti) were cultured in DMEM with 10% fetal bovine serum (FBS; Invitrogen/Life Technologies, Carlsbad, CA); NIH3T3 were cultured in DMEM containing 10% heat-inactivated calf serum (Invitrogen/Life Technologies); T47D cells were cultured in RPMI with 10% FBS and 5 µg/mL insulin (Sigma, St. Louis, MO). Expression of exogenous proteins was obtained with LipofectAMINE (Invitrogen, Carlsbad, CA)–mediated transfection, (for NIH3T3), or with retroviral or lentiviral infection (for ZR75, T47D, MDA-MB-231, and MEFs). Viral hybrid vectors were produced by transient transfection of 293T cells. Transient transfections in COS cells were carried out using DEAE-dextran.

Biochemical methods. For immunoprecipitations, cells were lysed in a buffer containing 50 mmol/L HEPES (pH 7.4), 5 mmol/L EDTA, 2 mmol/L EGTA, 150 mmol/L NaCl, 10% glycerol, and 1% Triton X-100, in the presence of protease and phosphatase inhibitors. Extracts were clarified at 12,000 rpm for 15 minutes, normalized with the Bicinchoninic Acid Protein Assay Reagent kit (Pierce, Rockford, IL), and incubated with anti-ß4 monoclonal antibodies for 2 hours at 4°C. Immune complexes were collected with protein G-Sepharose, washed in lysis buffer, and eluted. Total cellular proteins were extracted with boiling SDS buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% SDS], electrophoresed on SDS-polyacrylamide gels, and transferred onto nitrocellulose membranes (Hybond, Amersham, Piscataway, NJ). Nitrocellulose-bound antibodies were detected by the enhanced chemiluminescence system (Amersham).

Focus-forming and soft agar assays. For focus-forming assays, 3 days after transfection, 5 x 105 cells were split 1:5 and cultured under standard conditions in a nonselective medium to avoid clonal variations. Foci were fixed with glutaraldehyde and stained with Giemsa 2 weeks (NIH3T3) or 3 weeks (MEFs) after transfection. For colony formation in soft agar, 50 x 103 NIH3T3 and 20 x 103 MDA-MB-231, T47D, and ZR75 cells were resuspended in complete medium containing 0.5% Seaplaque agar and then seeded in six-well plates containing a 1% agar underlay. Colonies were stained by incorporation of tetrazolium salts 3 weeks after seeding.

Tumorigenicity assay. Cells derived from the focus-forming assays (2 x 106 for NIH3T3, 2.5 x 106 for MEFs, and 107 for ZR75) were suspended in 200 µL of PBS and inoculated s.c. into the right posterior flank of 6-week-old immunodeficient nu/nu female mice on Swiss CD1 background (Charles River Laboratories, Wilmington, MA); tumors were measured every 2 or 3 days. Tumor volume was calculated with the formula 4/3{pi} x (d/D)2 x D/2, where d is the minor tumor axis and D is the major tumor axis. All animal procedures were approved by the Ethical Commission of the University of Turin, Italy and by the Italian Ministry of Health.

Statistics. Results are means ± SEs. Comparisons were made using the two-tailed Student's t test. Ps < 0.05 were considered statistically significant.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Conclusions
 References
 
Oncogenic properties of ß4 integrin in vitro. Rodent fibroblasts represent prototypic recipients for analysis of the transforming properties of candidate oncogene products and have the advantage of expressing negligible levels of endogenous ß4 and Met (Fig. 1A). NIH3T3 cells with ectopic expression of ß4 were able to form foci on plastic and colonies in soft agar (P < 0.01; Fig. 1B and C; for statistics, see Supplementary Tables 1 and 2), whereas cells expressing only Met did not display any obvious sign of transformation (Fig. 1B and C), in accordance with previous reports (10). Coexpression of ß4 and Met produced a significantly higher number of foci and colonies (P < 0.01; Fig. 1B and C), almost comparable with that produced by the oncogenic variant of Met TPR-MET (data not shown).



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  		Figure 1. Autonomous and Met-dependent oncogenic properties of ß4 integrin in vitro. A, expression of ß4 and Met in NIH3T3 total cell lysates. Cells were transfected with the indicated constructs. In the case of single protein expression, cells were cotransfected with an empty vector. B, focus-forming assay in NIH3T3. Columns, means of two independent experiments done in quadruplicate; bars, SE. C, soft agar assay in NIH3T3. Columns, means of two independent experiments done in quadruplicate; bars, SE. D, expression of Met and ß4 in MEF total cell lysates. Cells were infected with the indicated vectors (plus empty vector when a single protein was expressed). E, focus-forming assay in MEFs. Columns, means of two independent experiments done in quadruplicate; bars, SE.

 
Similar results were obtained upon ß4 and/or Met expression in MEFs (Fig. 1D); again, single Met transfection was not accompanied by visible transformation (Fig. 1E); transfection of ß4 alone resulted in the development of a limited but clearly detectable number of sizeable foci, and cotransfection of Met and ß4 gave rise to a significant amount of foci with considerable dimensions (P < 0.01; Fig. 1E; Supplementary Table 3). Together, these results indicate that ß4 is able to induce cell transformation in vitro, and that this ability is exploited to unleash the silent transforming potential of the Met proto-oncogene.

Integrity of ß4-dependent signals but not of ß4 extracellular portion is required for ß4-mediated transformation. The ß4 cytoplasmic tail can be tyrosine phosphorylated by Met, which is important for amplification of Met-dependent proliferation and survival (7). To verify whether this substrate/adaptor function of the integrin is also involved in Met oncogenic activity, we decided to test the transforming ability of a mutant of ß4 with reduced substrate capacity for Met. To generate this mutant, we did a progressive phenylalanine mutagenesis of some critical tyrosines located in the ß4 intracellular domain and evaluated the Met-dependent tyrosine phosphorylation status of the integrin.

Coexpression of Met and ß4 in COS cells resulted in elevated tyrosine phosphorylation of ß4 (Fig. 2A), whereas expression of ß4 and a kinase-inactive Met isoform (MetK–) did not produce significant tyrosine phosphorylation of the integrin, confirming our previous observation that ß4 is an optimal substrate for Met kinase activity (Fig. 2A; ref. 7). ß4 tyrosine phosphorylation seemed progressively reduced following cotransfection of wild-type Met and ß4 mutants bearing single (ß4cyto-S; Tyr1257Phe) or double (ß4cyto-D; Tyr1257Phe and Tyr1494Phe) phenylalanine substitutions and decreased by almost 90% in the presence of a ß4 triple mutant (ß4cyto-T) with phenylalanine permutations of Tyr1257, Tyr1440, and Tyr1494 (Fig. 2A). All these tyrosines represent crucial residues for ß4 signaling activity: Tyr1257 is one of the major tyrosine phosphorylation sites of the integrin in response to antibody-mediated ligation (11), Tyr1494 is critical for stimulation of phosphatidylinositol 3-kinase (PI3K; ref. 11), and Tyr1440 is primarily responsible for SH2-mediated interaction with the growth factor receptor binding protein 2 (Grb2) upstream effector Src homology and collagen (7, 12). In summary, ß4cyto-T is a very poor substrate for Met catalytic activity and thus an inefficient downstream effector of Met-dependent signals; in addition, ß4cyto-T is endowed with reduced ability to activate oncogenic signaling cascades, such as PI3K-dependent and Grb2/Sos/Ras-dependent pathways. Accordingly, when ß4cyto-T and Met were transfected in NIH3T3 cells, no foci could be observed (P < 0.01 compared with cells expressing Met and wild-type ß4; Fig. 2B; Supplementary Table 1), indicating that the signaling integrity of the ß4 intracellular domain is in fact necessary for inducing Met-dependent oncogenic conversion.



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  		Figure 2. Integrity of ß4-dependent signals but not of ß4 extracellular portion is required for ß4-mediated transformation in vitro and in vivo. A, tyrosine phosphorylation status of wild-type ß4 compared with mutant isoforms of ß4 bearing single (ß4cyto-S; Tyr1257Phe), double (ß4cyto-D; Tyr1257Phe and Tyr1494Phe), and triple (ß4cyto-T; Tyr1257Phe, Tyr1440Phe, and Tyr1494Phe) phenylalanine substitutions of critical signaling tyrosines. B, focus-forming assay in NIH3T3 expressing the indicated cDNAs. Columns, means of two independent experiments done in quadruplicate; bars, SE. C, growth curves of NIH3T3 xenografts in nude mice, monitored during the first 3 weeks after injection (n = 6 for each experimental group). Green, Met/wild-type ß4; red, Met/ß4{Delta}extra; blue, ß4 alone.

 
In vitro transformation assays are experimentally meant to evaluate the ability of cells to grow and survive in the absence of anchorage, suggesting that ß4 can act as a transforming protein irrespective of its default function as an adhesion molecule. To further confirm this notion, we exploited a ß4 truncated variant (ß4{Delta}extra) lacking most of the extracellular region and thus unable to bind matrix ligands. NIH3T3 cells transfected with Met and ß4{Delta}extra produced a number of foci superimposable to that of cells expressing Met and wild-type ß4 (P = 0.7; Fig. 2B; Supplementary Table 1). Together, these results indicate that abolition of the cytoplasmic signaling activity of ß4 curtails its transforming properties, whereas suppression of the integrin adhesive function leaves them intact.

Oncogenic properties of ß4 integrin in vivo. We extended the data obtained in the in vitro transformation assays to tumorigenesis in vivo by implanting s.c. xenografts of NIH3T3 cells in immunocompromised mice. In the first 3 weeks after injection, cells expressing ß4 alone or Met and ß4 gave rise to actively expanding tumors, although growth of lesions produced by fibroblasts expressing only ß4 was more indolent (P < 0.01; Fig. 2C; Supplementary Tables 4-7), in accordance with the in vitro results. Again, mice injected with cells expressing Met and ß4cyto-T did not manifest s.c. masses, whereas the group injected with cells expressing Met and ß4{Delta}extra developed visible tumors (Fig. 2C). In this latter cohort, xenografts grew with slower kinetics compared with cells expressing Met and wild-type ß4, suggesting that the matrix-binding activity of the integrin may provide an additional oncogenic stimulus in vivo.

Prolonged mice monitoring showed that almost all animals, including controls injected with vector cells, started developing tumors after 3 weeks, confirming the observation that NIH3T3 cells are occasionally prone to spontaneous tumorigenesis (Table 1). We thus decided to perform a similar experiment employing MEFs, which proved to be less vulnerable to oncogenic conversion in preliminary experiments. In this setting, only cells expressing Met and ß4 were able to form tumors, whereas all other transfectants (Met alone, ß4 alone, and Met/ß4cyto-T) were nontumorigenic even over an extended period of time (120 days; Table 1). Hence, ß4 displays significant albeit not dramatic oncogenic properties in vivo, which are magnified in the presence of Met.


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  		Table 1. Tumors in nude mice

 
Oncogenic properties of ß4 in human epithelial cells. To further validate a causal role for ß4 in tumorigenesis in a setting that is more relevant for human cancer, we expanded the panel of cell types tested to include human epithelial cells. As cellular models for this analysis, we chose various cell lines derived from the mammary epithelium, in which progression towards the neoplastic phenotype is accompanied by up-regulation of both ß4 (13) and Met (14).

In MDA-MB-231 breast carcinoma cells, which physiologically express both molecules, we either enhanced integrin levels by infection with a ß4-encoding retrovirus, or abated them by lentiviral delivery of small interfering RNAs (siRNA; Fig. 3A). When subjected to a soft agar assay, mock cells infected with a control (scrambled) siRNA were able to form nonadherent colonies at high efficiency. Strikingly, integrin overexpression strongly increased the colony-forming ability of these cells (not only in absolute numbers but especially in size; P = 0.01; Supplementary Table 8), whereas ß4 knockdown resulted in almost complete abolition of anchorage-independent growth (P < 0.01; Fig. 3B).



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  		Figure 3. In vitro and in vivo transforming activity of ß4, ß4 mutants, and Met in human epithelial cells. A, expression levels of ß4 in MDA-MB-231 cells infected with a control siRNA vector (scrambled ß4 sequence; siRNActr), with a ß4-specific siRNA (siRNAß4), or with wild-type ß4 (ß4 ovr). B, soft agar assay in MDA-MB-231. Columns, means of two independent experiments done in quadruplicate; bars, SE. C, expression of ß4, ß4 mutants, and Met in ZR75 cells. ß4{Delta}extra appears as a 140-kDa band, whereas full-length ß4 yields a band of 200 kDa. Met is expressed as both the precursor (top band) and mature form (bottom band). D, soft agar assay in ZR75. Columns, means of two independent experiments done in quadruplicate; bars, SE. E, expression of ß4, ß4 mutants, and Met in T47D cells. F, soft agar assay in T47D. Columns, means of two independent experiments done in quadruplicate; bars, SE. G, growth curves of ZR75 xenografts in nude mice (n = 6 for each experimental group). Green, Met/wild-type ß4; red, Met/ß4{Delta}extra; blue, ß4 alone; gray, Met/ß4cyto-T; yellow, mock; pale blue, Met alone.

 
In MCF-10A and B5-589 cells, derived from the normal mammary epithelium, overexpression of ß4, Met, or both was not sufficient to induce a transformed phenotype (data not shown), confirming the established notion that normal human epithelial cells are more difficult to transform than rodent fibroblasts. We thus analyzed ZR75 and T47D mammary carcinoma cells, which do not express ß4 and Met and, despite being of tumor origin, do not display an overt transformed phenotype: indeed, both lines are well differentiated, synthesize high amounts of E-cadherin, grow poorly in soft agar, and are not tumorigenic in nude mice unless an external estrogen supply is provided. In these cells, expression of ß4 substantially enhanced the number of colonies (P < 0.01; Fig. 3C-F), which was further increased upon coexpression of Met (P < 0.01; Fig. 3C-F; Supplementary Tables 9 and 10), confirming the results obtained in NIH3T3 and MEFs. Cells expressing Met and the ß4{Delta}extra nonadhesive mutant retained the ability to grow in soft agar at higher efficiency compared with cells expressing Met alone (P < 0.01; Supplementary Tables 9 and 10), but they formed less colonies compared with cells expressing Met and wild-type ß4 (Fig. 3C-F). This could be due to the fact that ß4{Delta}extra, although competent for transduction of Met-dependent signals, is unable to convey adhesive and survival signals provided by laminins endogenously produced by epithelial cells (15). In line with that observed in fibroblasts, coexpression of Met and the ß4cyto-T-signaling dead mutant did not significantly affect basal anchorage-independent growth in both cell lines (Fig. 3C-F).

Finally, consistent with the transformed phenotype displayed in vitro, ZR75 cells expressing only ß4 exhibited increased tumorigenic potential in nude mice compared with mock cells, in the absence of any exogenous estradiol treatment (P < 0.05; Fig. 3G; Supplementary Table 11). Cells expressing Met and ß4 developed tumors with faster kinetics, whereas cells expressing Met and ß4{Delta}extra grew more rapidly than cells expressing Met alone (P < 0.05; Fig. 3G; Supplementary Table 11) but slower than cells expressing Met and wild-type ß4. In contrast, the growth curves of cells expressing Met and ß4cyto-T were superimposable to those of mock and Met-expressing cells (P = 0.72; Fig. 3G).


   Conclusions
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
References
 
Our data show that ß4 integrin is endowed with transforming ability and conspires with the tyrosine kinase Met for patent tumorigenesis functioning as a Met substrate. This activity, which significantly induces oncogenic conversion of rodent fibroblasts, is not sufficient for de novo transformation of human normal epithelial cells but is necessary to maintain the tumorigenic phenotype of carcinoma cells and strongly exacerbates their cancerous properties.

A number of phenomenological studies substantiate the observed collaboration between ß4 and Met in cancer formation. Although both molecules are expressed in a limited subset of normal adult tissues, they seem concomitantly overrepresented and similarly localized in a variety of human malignancies, including skin, thyroid, breast, pancreas, lung, nasopharyngeal, and bladder carcinomas (5, 8, 16). In this respect, analysis of the promoter regions of ß4 (17) and Met (18) reveals that both sequences share response elements for the same nuclear factors, implying common regulatory mechanisms for their transcriptional induction. Together with protein coexpression, the cooperation between ß4 and Met in oncogenic conversion might also have a genetic basis. Several germ line and somatic mutations of the Met gene have been found in human renal papillary carcinomas (19), but most them do not confer transforming ability to Met either in fibroblasts (10) or epithelial cell lines (20). Intriguingly, all these tumors bearing Met mutations display trisomy of chromosome 17, where the ß4 genomic locus resides (21). This suggests a potential gene-dosage effect, whereby enhanced production of ß4 could awake the dormant oncogenic activity of Met.

In conclusion, our findings disclose an unanticipated role for ß4 as a servo-oncogene of tyrosine kinase proto-oncogenes, which elevates this integrin from candidate status to culprit status in tumor development. Implicit in these findings is the issue that targeting the signaling function of ß4 in neoplastic contexts could add therapeutic value to experimental approaches aimed at interfering with cancer growth and progression.


   Acknowledgments
 
Grant support: Associazione Italiana per la Ricerca sul Cancro, Compagnia di San Paolo, and Fondazione Cassa di Risparmio di Torino.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank L.M. Shaw (Department of Pathology, Harvard Medical School, Boston, MA) for the Y1257F and Y1494F ß4 mutants; L. Lanzetti (Department of Oncological Sciences, Institute for Cancer Research and Treatment, Candiolo, Italy) for MEFs; F. Girolami and M. Mazzone for help with animal experiments; A. Crivellari for cell cultures; R. Albano, F. Grasso, and L. Palmas for technical assistance; and A. Cignetto for secretarial assistance.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 8/ 9/05. Revised 10/ 7/05. Accepted 10/14/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Conclusions
 References
 

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  12. Dans M, Gagnoux-Palacios L, Blaikie P, Klein S, Mariotti A, Giancotti FG. Tyrosine phosphorylation of the ß4 integrin cytoplasmic domain mediates Shc signaling to extracellular signal-regulated kinase and antagonizes formation of hemidesmosomes. J Biol Chem 2001;276:1494–502.[Abstract/Free Full Text]
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K. Wilhelmsen, S. H.M. Litjens, and A. Sonnenberg
Multiple Functions of the Integrin {alpha}6{beta}4 in Epidermal Homeostasis and Tumorigenesis
Mol. Cell. Biol., April 15, 2006; 26(8): 2877 - 2886.
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