we explored the role of myc in metastasis by manipulating expression of the gene in four established cell lines of human breast cancer , two of which express little myc ( mda - mb-231 and bd549 ) , and two of which express myc at high levels ( mcf-7 and t47d ) ( fig . 1a ) , and in human retinal pigment epithelium ( rpe ) cells , a cell line that has been used previously for investigating the effects of myc on induction of proliferation and apoptosis . exogenous overexpression of human myc in mda - mb-231 , bt549 , and rpe cells significantly increased proliferation assessed by brdu labeling ( fig . overexpression of myc in mda - mb-231 and bt549 cells also inhibited the invasive growth pattern of these cells when cultured in three - dimensional , laminin - rich gels ( lrecm ) such as matrigel ( fig . 1f ) . when injected subcutaneously , myc - expressing mda - mb-231 cells produced tumors that showed substantially reduced local invasion ( fig . 1 g ) , even though the expression of myc led to significantly larger tumors ( fig . these results showed that overexpression of myc simultaneously promotes cell proliferation while inhibiting invasion , both in culture and in vivo . we found that mda - mb-231 cells overexpressing myc showed significantly reduced incidence of lung metastases following injection into the tail vein ( fig . 2a , b ) , although occasional myc - expressing cells that did metastasize resulted in larger tumors ( fig . 2c ) . to assess tumor growth and metastasis from the orthotopic site , mda - mb-231-vector and mda - mb-231-myc cells were transduced with luciferase - expressing virus ( fig . we found that the myc - expressing tumors , as expected , grew more rapidly at the orthotopic site ( fig . 2e - g ) . however , isolation and quantification of luminescence of lungs ( fig . 2m , n ) revealed decreased metastatic burden at 6 weeks following injection , though the difference was not statistically significant . metastatic burden is a combination of both number of metastases and the size of the metastatic tumors ; we found that expression of myc significantly reduced the number of metastases but led to increased size of individual metastases in both lung ( fig . we conclude that overexpression of myc can stimulate cell proliferation producing larger tumors that have reduced invasiveness and metastatic potential . these findings are consistent with recent indications that dissemination of tumor cells may occur early during tumor progression and is not necessarily linked to primary tumor size . motility is a dynamic process associated with major changes in cellular phenotype including spreading , actin polymerization , formation of actin - rich protrusions at the leading edge of migrating cells , and creation and dissolution of focal adhesions during cell translocation . we found that exogenous overexpression of myc in both mda - mb-231 and rpe cells ( fig . 3a , b ) reduced cell spreading , decreased stress fiber and focal adhesion formation , and increased cortical actin ( fig . 3c , d ) . since formation of focal adhesions requires the interaction of cell surface integrins with their ligands in the extracellular matrix ( ecm ) , we hypothesized that the effect of overexpression of myc on focal adhesion might reflect a defect in the interaction of cells with specific ecm ligands . to test this hypothesis , we assessed cell attachment to purified human ecm ligands and found that overexpression of myc in mda - mb-231 or rpe cells reduced their attachment to vitronectin ( fig . such defects could in turn impair cellular motility . to explore this possibility , we measured chemotaxis towards purified vitronectin in boyden chamber assays , and found that overexpression of myc in mda - mb-231 and rpe cells decreased migration of both cell types ( fig . 3g , h ) . to explore the mechanism by which myc impedes invasion and motility , we measured the expression of subunits of v3 integrin , which is a well - known cell surface receptor for vitronectin . overexpression of myc in rpe , mda - mb-231 and bt549 cells was accompanied by substantial reductions in both integrin subunits in all three cell lines , and less consistent reductions of 5 , 1 and 5 subunits ( fig . conversely , when the high levels of endogenous myc in mcf7 and t47d cells were knocked down by sirna , the levels of v , 5 , 1 and 5 integrin subunits rose ( fig . irrespective of the level of myc expression , 3 integrin could not be detected by either rt - pcr or western blots in mcf7 cells ( fig . 4a , and data not shown ) , nor in t47d cells ( see below , fig . overexpression of myc in mda - mb-231 cells caused a reduction of rna for both v and 3 ( fig . a canonical e - box binding site for myc is located upstream of the transcription initiation sites for both v ( cacatg ) and 3 ( cacgtg ) integrins in human dna ( fig . quantitative chromatin immunoprecipitation assays ( chip ) showed that myc bound directly to the e - box region of both these genes in mda - mb-231 cells ( fig . 4e , f ) and in rpe cells ( not shown ) , but not to non - specific sequences in the same domain . although myc was originally viewed as a transcriptional activator , it can serve also as a transcriptional repressor . our results show that overexpression of myc represses transcription of integrin genes involved in migration and metastasis . we found that the invasive and migratory phenotype of mda - mb-231 and rpe cells was dependent on v3 integrin ( fig . 5a , b , and data not shown ) as was the invasive growth of mda - mb-231 cells in lrecm ( fig . 5c ) , and that defects in cell invasion and migration caused by overexpression of myc could be partially or completely rescued by expressing v and 3 integrins in mda - mb-231 ( fig . invasive growth of mda - mb-231 in 3d lrecm and cell spreading , focal adhesion and stress fiber formation in rpe cells were rescued as well ( fig . 5 g ) . to evaluate whether exogenous overexpression of v and 3 integrins was sufficient to reconstitute metastatic capability in cells also overexpressing myc , we generated myc - expressing mda - mb-231 cells that also expressed integrin v , and integrin 3 using exogenous promoters unaffected by myc ( fig . 5h ) . when these cells were implanted orthotopically , ectopic expression of integrins v and 3 substantially increased lung metastasis ( fig . these results indicate that suppression of integrin v3 expression is the key mechanism by which myc inhibits breast cancer cell metastasis . we noticed that depletion of either v or 3 integrin resulted in a parallel reduction of the other ( figure 5a , b ) ; conversely , exogenous expression of 3 integrin increased the amount of v integrin ( see below , fig . moreover , exogenous expression of either integrin at least partially rescued the phenotype suppressed by myc ( fig . these findings could be explained by mass action , wherein an increase in either integrin alone can augment the formation of heterodimers and stabilize both components . we found that transfection of v and 3 , either individually or together , substantially increased the amount of heterodimers in extracts of mda - mb-231 cells ( supplementary fig . the relative amounts of heterodimer correlated with the extent to which exogenous expression of the integrins rescued a defect in invasion caused by myc ( compare fig . we conclude that the defects in motility and invasion elicited by overexpression of myc are due to reduced expression of the v and 3 integrin subunits , which in turn reduces formation of the heterodimer . we also explored the effect of depleting the high level of myc in mcf-7 cells with rnai ( fig . depletion of myc significantly reduced cellular proliferation as expected ( data not shown ) , whereas it increased cell adhesion to vitronectin and fibronectin ( fig . 6b ) , as well as cell spreading , focal adhesion and stress fiber formation ( fig . however , knockdown of myc in these cells neither increased cell motility ( data not shown ) nor invasiveness ( fig . 6j ) , implicating the absence of 3 integrin in mcf7 cells and the consequent inability to form v3 heterodimers . accordingly , 6h ) ; similar effects were seen with exogenous expression of 3 integrin in t47d cells ( fig . mcf7 cells grow in a cuboidal morphology with tight cell - cell junctions that restrict individual cell motility . in order to isolate the inhibitory effect of myc on cell motility , we simultaneously knocked down e - cadherin and myc , and expressed exogenous 3 integrin in mcf7 cells ( fig . we conclude that when cell - cell interactions are abrogated , inhibition of v and 3-integrin expression is the primary barrier to cell invasion in mcf7 and possibly other human breast cancer cells that overexpress myc . we explored the role of myc in metastasis by manipulating expression of the gene in four established cell lines of human breast cancer , two of which express little myc ( mda - mb-231 and bd549 ) , and two of which express myc at high levels ( mcf-7 and t47d ) ( fig . 1a ) , and in human retinal pigment epithelium ( rpe ) cells , a cell line that has been used previously for investigating the effects of myc on induction of proliferation and apoptosis . exogenous overexpression of human myc in mda - mb-231 , bt549 , and rpe cells significantly increased proliferation assessed by brdu labeling ( fig . overexpression of myc in mda - mb-231 and bt549 cells also inhibited the invasive growth pattern of these cells when cultured in three - dimensional , laminin - rich gels ( lrecm ) such as matrigel ( fig . 1f ) . when injected subcutaneously , myc - expressing mda - mb-231 cells produced tumors that showed substantially reduced local invasion ( fig . 1 g ) , even though the expression of myc led to significantly larger tumors ( fig . these results showed that overexpression of myc simultaneously promotes cell proliferation while inhibiting invasion , both in culture and in vivo . we found that mda - mb-231 cells overexpressing myc showed significantly reduced incidence of lung metastases following injection into the tail vein ( fig . 2a , b ) , although occasional myc - expressing cells that did metastasize resulted in larger tumors ( fig . 2c ) . to assess tumor growth and metastasis from the orthotopic site , mda - mb-231-vector and mda - mb-231-myc cells were transduced with luciferase - expressing virus ( fig . we found that the myc - expressing tumors , as expected , grew more rapidly at the orthotopic site ( fig . 2e - g ) . however , isolation and quantification of luminescence of lungs ( fig . 2m , n ) revealed decreased metastatic burden at 6 weeks following injection , though the difference was not statistically significant . metastatic burden is a combination of both number of metastases and the size of the metastatic tumors ; we found that expression of myc significantly reduced the number of metastases but led to increased size of individual metastases in both lung ( fig . we conclude that overexpression of myc can stimulate cell proliferation producing larger tumors that have reduced invasiveness and metastatic potential . these findings are consistent with recent indications that dissemination of tumor cells may occur early during tumor progression and is not necessarily linked to primary tumor size . motility is a dynamic process associated with major changes in cellular phenotype including spreading , actin polymerization , formation of actin - rich protrusions at the leading edge of migrating cells , and creation and dissolution of focal adhesions during cell translocation . we found that exogenous overexpression of myc in both mda - mb-231 and rpe cells ( fig . 3a , b ) reduced cell spreading , decreased stress fiber and focal adhesion formation , and increased cortical actin ( fig . 3c , d ) . since formation of focal adhesions requires the interaction of cell surface integrins with their ligands in the extracellular matrix ( ecm ) , we hypothesized that the effect of overexpression of myc on focal adhesion might reflect a defect in the interaction of cells with specific ecm ligands . to test this hypothesis , we assessed cell attachment to purified human ecm ligands and found that overexpression of myc in mda - mb-231 or rpe cells reduced their attachment to vitronectin ( fig . such defects could in turn impair cellular motility . to explore this possibility , we measured chemotaxis towards purified vitronectin in boyden chamber assays , and found that overexpression of myc in mda - mb-231 and rpe cells decreased migration of both cell types ( fig . to explore the mechanism by which myc impedes invasion and motility , we measured the expression of subunits of v3 integrin , which is a well - known cell surface receptor for vitronectin . overexpression of myc in rpe , mda - mb-231 and bt549 cells was accompanied by substantial reductions in both integrin subunits in all three cell lines , and less consistent reductions of 5 , 1 and 5 subunits ( fig . conversely , when the high levels of endogenous myc in mcf7 and t47d cells were knocked down by sirna , the levels of v , 5 , 1 and 5 integrin subunits rose ( fig . irrespective of the level of myc expression , 3 integrin could not be detected by either rt - pcr or western blots in mcf7 cells ( fig . 4a , and data not shown ) , nor in t47d cells ( see below , fig . overexpression of myc in mda - mb-231 cells caused a reduction of rna for both v and 3 ( fig . a canonical e - box binding site for myc is located upstream of the transcription initiation sites for both v ( cacatg ) and 3 ( cacgtg ) integrins in human dna ( fig . quantitative chromatin immunoprecipitation assays ( chip ) showed that myc bound directly to the e - box region of both these genes in mda - mb-231 cells ( fig . 4e , f ) and in rpe cells ( not shown ) , but not to non - specific sequences in the same domain . although myc was originally viewed as a transcriptional activator , it can serve also as a transcriptional repressor . our results show that overexpression of myc represses transcription of integrin genes involved in migration and metastasis . we found that the invasive and migratory phenotype of mda - mb-231 and rpe cells was dependent on v3 integrin ( fig . 5a , b , and data not shown ) as was the invasive growth of mda - mb-231 cells in lrecm ( fig . 5c ) , and that defects in cell invasion and migration caused by overexpression of myc could be partially or completely rescued by expressing v and 3 integrins in mda - mb-231 ( fig . 5f ) . invasive growth of mda - mb-231 in 3d lrecm and cell spreading , focal adhesion and stress fiber formation in rpe cells were rescued as well ( fig . 5 g ) . to evaluate whether exogenous overexpression of v and 3 integrins was sufficient to reconstitute metastatic capability in cells also overexpressing myc , we generated myc - expressing mda - mb-231 cells that also expressed integrin v , and integrin 3 using exogenous promoters unaffected by myc ( fig . 5h ) . when these cells were implanted orthotopically , ectopic expression of integrins v and 3 substantially increased lung metastasis ( fig . 5i , j ) . these results indicate that suppression of integrin v3 expression is the key mechanism by which myc inhibits breast cancer cell metastasis . we noticed that depletion of either v or 3 integrin resulted in a parallel reduction of the other ( figure 5a , b ) ; conversely , exogenous expression of 3 integrin increased the amount of v integrin ( see below , fig . moreover , exogenous expression of either integrin at least partially rescued the phenotype suppressed by myc ( fig . these findings could be explained by mass action , wherein an increase in either integrin alone can augment the formation of heterodimers and stabilize both components . we found that transfection of v and 3 , either individually or together , substantially increased the amount of heterodimers in extracts of mda - mb-231 cells ( supplementary fig . the relative amounts of heterodimer correlated with the extent to which exogenous expression of the integrins rescued a defect in invasion caused by myc ( compare fig . we conclude that the defects in motility and invasion elicited by overexpression of myc are due to reduced expression of the v and 3 integrin subunits , which in turn reduces formation of the heterodimer . we also explored the effect of depleting the high level of myc in mcf-7 cells with rnai ( fig . depletion of myc significantly reduced cellular proliferation as expected ( data not shown ) , whereas it increased cell adhesion to vitronectin and fibronectin ( fig . 6b ) , as well as cell spreading , focal adhesion and stress fiber formation ( fig . however , knockdown of myc in these cells neither increased cell motility ( data not shown ) nor invasiveness ( fig . 6j ) , implicating the absence of 3 integrin in mcf7 cells and the consequent inability to form v3 heterodimers . accordingly , 6h ) ; similar effects were seen with exogenous expression of 3 integrin in t47d cells ( fig . mcf7 cells grow in a cuboidal morphology with tight cell - cell junctions that restrict individual cell motility . in order to isolate the inhibitory effect of myc on cell motility , we simultaneously knocked down e - cadherin and myc , and expressed exogenous 3 integrin in mcf7 cells ( fig . we conclude that when cell - cell interactions are abrogated , inhibition of v and 3-integrin expression is the primary barrier to cell invasion in mcf7 and possibly other human breast cancer cells that overexpress myc . myc has been implicated extensively in tumor growth and cell transformation through studies in cultured cells , targeted expression in mice and retrospective analyses of myc expression in human tumors . examples include the ability to initiate tumorigenesis in mice , destabilization of the genome and a requirement for maintenance of established tumors . to this list we add here a previously unrecognized effect of myc : ability to inhibit metastasis through inhibition of transcription of an integrin involved in metastasis . the possibility that myc could act to inhibit metastasis could have been surmised by the observations that overexpression of the gene is sometimes dissociated from the tendency of tumors to metastasize . why this may be the case , however , had not been explored . in the case of human breast cancer , distal metastases may express myc at the level of cognate normal cells , even though the gene is over - expressed in the primary tumor . similarly , mammary carcinoma and other tumors induced by myc in mice frequently fail to metastasize . examples include murine mammary cancer elicited by myc under the control of the mouse mammary tumor virus ( mmtv ) promoter and aggressive breast tumors elicited by retroviral transduction of both met and myc into normal breast epithelial cells . here we explored the impact of myc on metastasis by using cell lines derived from carcinomas of the human breast . two of these lines express low levels of myc and display a metastatic phenotype , whereas the other two overexpress the gene but do not metastasize . irrespective of the reason for this variation , the differences in myc expression allowed reciprocal studies on induction or repression of the metastatic phenotype by manipulation of myc expression and its downstream targets . clearly human breast cancers that overexpress myc may still metastasize if other factors override its function . in fact our in vivo assays demonstrated that expression of myc will support increased growth of those few metastatic cells that escape the inhibitory function of myc by means of other mutations or by changes in gene expression ( fig . , we sought direct experimental demonstration that myc can indeed inhibit metastasis . using mda - mb-231 , the human cell line that expresses only low levels of myc and is used extensively for metastatic studies , we confirmed their highly metastatic potential , by both tail vein injection ( fig . 2c , however , when exogenous myc was over - expressed in these cells , their ability to metastasize was greatly reduced . further , the behavior of breast cancer cell lines was remarkably malleable when the level of myc was modulated : it was possible to reduce the metastatic properties by over - expressing myc , or to augment those properties by reducing myc levels . at the same time , myc could increase proliferation even in overtly malignant cells ( fig . we reported previously an apparent dichotomy between stimulation of proliferation and inhibition of other malignant properties of breast cancer cells also for the oncogene , akt1 ( protein kinase b1 ) . our finding that myc overexpression leads to inhibition of cell motility and invasiveness through direct downmodulation of v and 3 integrin subunits supports reports that integrins with these subunits are involved in cellular motility , invasiveness , adhesion , and transmigration through endothelium , and are associated with metastasis . there are reports also that myc may be involved in controlling the expression of integrin genes using cells from different tissues . however , none of these reports has linked the effect of myc on integrin expression to cellular components of metastasis , and none has implicated the v3 heterodimer directly in the cellular changes elicited by myc . our results demonstrate that overexpression of myc represses transcription from the promoters of the v and 3 integrin genes , which in turn reduces properties that are essential for metastasis . these data , however , do not mean that myc affects invasion and metastasis only through regulation of integrins v and 3 , nor that myc is the sole and only regulator of integrin v3 function in invasion and metastasis . the fact that myc sirna increases invasion when mcf7 cells are forced to have high levels of integrins v and 3 ( fig . 6j ) suggests that there may be mechanism(s ) other than integrin v3 by which myc can inhibit invasion and metastasis . nevertheless , since exogenous expression of these integrins is sufficient to bypass the repressive effect of myc in invasion ( fig . 6d , f , j ) and metastasis ( fig . 5h , j ) , it is apparent that a primary effect of myc on invasion in breast cancer cells is mediated through its inhibition of integrins v and 3 . tumor progression often culminates in metastatic disease indicating that there must be selection for antidotes to the myc inhibition of the metastatic phenotype . one obvious possibility would be selection against overexpression of myc during the course of tumor progression . indeed , the two breast cancer cell lines used in the present study that express relatively low levels of myc both originated from metastatic tumors : mda - mb-231 from pleural effusion and bt-549 from an invasive ductal tumor in regional lymph nodes . other possibilities include loss of 3 integrin functions , as in the case of mcf-7 and t47d cell lines . constitutive signaling from active ras or overexpression of bcl - xl can also override the inhibition of metastasis by myc ( unpublished results of h.l and d.y ) . these complexities might account for the fact that in some experimental circumstances , myc may appear to favor metastasis . the frequency with which myc is over - expressed , and the variety of tumors in which that over - expression occurs , have made myc a seemingly advantageous therapeutic target . our findings raise the possibility that inhibition of myc in human tumors might at times be contraindicated since its suppression may indeed promote metastasis . mda - mb-231 cells overexpressing myc were transiently transfected with constructs expressing either v or 3 integrin , or both . the lysates of transfectants were immunoprecipitated by antibodies against v integrin or v3 integrin heterodimers , separated by sds - page electrophoresis and immunoblotted with the indicated antibodies . exogenous expression of integrin 3 in mcf7 cells leads to increased generation of v3 heterodimers even if myc is not knocked down . mcf-7 cells with control vector or stably over - expressing 3 integrin were transfected with negative control sirna or 2 different oligoes of myc sirna . the lysates of transfectants were immunoprecipitated by antibodies against v integrin or v3 integrin heterodimers , separated by sds - page electrophoresis and immunoblotted with the indicated antibodies .
over - expression of myc transforms cells in culture , elicits malignant tumors in experimental animals and is found in many human tumors . 
 we now report the paradoxical finding that this powerful oncogene can also act as a suppressor of cell motility , invasiveness and metastasis . 
 overexpression of myc stimulated proliferation of breast cancer cells both in culture and in vivo as expected , but inhibited motility and invasiveness in culture , and lung and liver metastases in xenografted tumors . 
 we show further that myc represses transcription of both subunits of v3 integrin , and that exogenous expression of 3 integrin in human breast cancer cells that do not express this integrin rescues invasiveness and migration when myc is downregulated . 
 these data uncover an unexpected function of myc , provide an explanation for the hitherto puzzling literature on the relationship between myc and metastasis and reveal a variable that should influence the development of therapeutics that target myc .