BACKGROUND
while treatment of the primary breast tumor is often well managed with surgery and radiation, metastatic spread to the brain, bones, liver and lungs frequently places women in an incurable state of disease  <cit> .
the basic helix-loop-helix  transcription factor twist <dig> was previously demonstrated to be a potent promoter of cancer cell dissemination into circulation and metastasis  <cit> , providing an ideal target for investigation and a promising therapeutic target for intervention.
based on its role in mesodermal development during mammalian embryogenesis  <cit> , twist <dig> is proposed to induce an embryonic event termed epithelial-mesenchymal transition  in tumor cells to promote the expression of mesenchymal junction proteins in epithelial cells and reduce intercellular junctions in the meantime  <cit> .
induction of emt enables epithelial cells to acquire the properties of mesenchymal lineages, including enhanced mobility and invasiveness that are tightly correlated with cancer metastasis  <cit> .
additionally, twist <dig> was shown to regulate the expression of akt <dig>  <cit>  and mirna-10b  <cit> , which are subsequently involved in the migratory and invasive properties of twist1-overexpressing cells.
eckert et al.
recently reported that twist <dig> up-regulates the expression of platelet derived growth factor receptor, which in turn promotes the formation of invadopodia and matrix degradation  <cit> , presenting the first evidence that twist <dig> causes extracellular matrix  remodeling.
however, the mechanisms by which twist <dig> actively promote cell invasion are still largely unstudied.
twist <dig> is a class ii member of the bhlh super family  <cit> .
it homo- or heterodimerizes with class i hlh family members, such as e proteins , through the hlh domain, and binds dna that contains the e-box sequence  via the basic domain to regulate gene expression  <cit> .
loss of function mutations in the bhlh domain in one allele of the twist <dig> gene cause saethre-chotzen syndrome  in humans, an autosomal dominant craniofacial disease caused by gene haploinsufficiency  <cit> , denoting the functional importance of the twist <dig> bhlh domain.
twist <dig> also contains a highly conserved carboxy-terminus , the wr  domain , which shares 100% sequence homology among jellyfish, xenopus, mice and humans  <cit> .
this region was shown to mediate the association between twist <dig> and runx <dig>  a zinc-finger protein, and prevent runx <dig> from inducing premature osteoblast differentiation during bone development in mice  <cit> .
however, whether the highly conserved wr domain plays any roles in twist1-induced cancer metastasis is an open question.
here we describe a unique property of the twist <dig> wr domain in mediating il <dig> production and breast cancer cell invasion.
using gene set enrichment analysis  of genetic profiles and cytokine array analyses, we found that il <dig> was specifically up-regulated by twist <dig> over-expression in the human breast epithelial cell line mcf10a as well as other breast cancer cell lines.
we also discovered that twist <dig> activates the il <dig> promoter, but, surprisingly, in a manner that is independent of its canonical dna binding bhlh domain.
in contrast, twist1-induced il <dig> promoter activation is dependent on the c-terminal wr domain, through which twist <dig> interacts with the nf-κb subunit rela.
this physical association of twist <dig> and rela activated and synergized the transcriptional activity of nf-κb and increased the binding affinity of rela to dna, which in turn stimulated il <dig> expression.
finally, we demonstrate that the il <dig> autocrine pathway in breast epithelial and cancer cells increases matrix metalloproteinase  production and enables autonomous invasion of the cells.
RESULTS
twist <dig> induces il <dig> cytokine production
to elucidate the molecular function of twist <dig> in human breast epithelial cells, we compared the gene expression profiles of a twist <dig> over-expressing mammary epithelial cell line  and the parental line from which it was derived .
we analyzed these data by gsea  using the c <dig> canonical pathway gene sets  <cit>  on paired independent experiments performed in triplicate using these cell lines.
the returned gene set permutation test showed a significant difference in the biocarta_cytokine_pathway between mcf10a and mcf10atw cells at a false discovery rate of  <dig>  .
the gene set included the pro-inflammatory cytokine interleukin  <dig> alpha  and the leukocyte chemokine il <dig> as two significant contributors to the biocarta_cytokine_pathway enrichment, with respective ranks of  <dig> and  <dig> as determined by signal-to-noise metric among all genes on the gene array, and in concordance with twist <dig> expression .
to verify the up-regulation of these genes, we performed a cytokine array blot with conditioned media collected from mcf10a or mcf10atw cell cultures.
in this experiment, il <dig>  along with growth related oncogene  and angiogenin , showed the most pronounced up-regulation in mcf10atw cells; however, secreted il1α was not detected .
quantification of il <dig> revealed 34-fold greater mrna expression and 3-fold greater secretion of il <dig> protein into culture media of mcf10atw relative to mcf10a cells .
to test if il <dig> was selectively up-regulated in the mcf10atw stable cell line, we transiently transduced mcf10a parental cells with adenoviruses that expressed twist <dig> at serial dilutions and observed that il <dig> transcripts were up-regulated in a dose dependent manner that correlated with the levels of twist <dig> expressed .
since il <dig> was up-regulated at both the mrna and protein levels, indicating that il <dig> is a downstream target of twist <dig>  we, therefore, focused on elucidating the regulatory pathway of il <dig>
to evaluate the induction of il <dig> by twist <dig> under pathological conditions, we expressed twist <dig> in the skbr <dig>  and mcf <dig>  cell lines, which represent two different subtypes of breast cancer, and observed respective 20-fold and 4-fold increases in il <dig> transcript levels .
the difference in il <dig> transcript levels between these two cell lines may be due to the influence of erbb <dig> expression on nf-κb transcriptional activity  <cit> .
in addition, short hairpin rna -mediated knock-down  of endogenous twist <dig> reduced the expression of il <dig> mrna in bt <dig> cells , a triple-negative breast cancer cell line , er- and her2-) that expresses high endogenous levels of both twist <dig> and il <dig>  this reduction in il <dig> expression correlated with the amount of twist <dig> protein knocked down .
taken together, these data show that twist <dig> regulates and maintains il <dig> expression in breast epithelial and cancer cells.
twist1-induced il <dig> transcription is mediated by the twist <dig> wr domain
to investigate whether twist <dig> activates il <dig> transcription, we cloned the il <dig> minimal promoter  <cit> , which contains a putative twist <dig> binding site  and a consensus κb response element, into a luciferase reporter construct .
skbr <dig> and mcf <dig> cells transfected with this il <dig> promoter construct displayed a consistent increase in luciferase activities when co-transfected with twist <dig>  indicating transcriptional activation of il <dig> .
correspondingly, in bt <dig> cells, twist <dig> kd resulted in a decrease in il <dig> promoter activity, and this activity was rescued upon exogenous twist <dig> expression .
to further clarify whether twist <dig> directly binds and activates the il <dig> promoter, we transfected skbr <dig> and mcf <dig> cells with an il <dig> promoter construct that was mutated at the putative twist <dig> binding e-box .
in this experiment, twist <dig> failed to stimulate luciferase transcription , indicating that integrity of the e-box sequence is required for il <dig> promoter activation.
however, we were concerned that these mutations may affect the binding of c/ebp, a known regulator of il <dig> because the e-box sequence heavily overlaps with the c/ebp binding site  <cit> .
therefore, we generated two dna binding deficient twist <dig> mutants by introducing the s144r/k145e and r118c mutations .
these naturally occurring mutations found in scs patients abolish the dna binding ability of twist1/e <dig> heterodimers without affecting its subcellular localization  <cit> .
surprisingly, both the s144r/k145e  and r118c  twist <dig> mutants stimulated the il <dig> promoter at similar levels as wild type  twist <dig> in skbr <dig> and mcf <dig> cells .
to further validate that the dna binding ability of twist <dig> is not required for il <dig> promoter activation, we completely removed the bhlh domain of twist <dig>  and still observed il <dig> promoter activity induced by this mutant comparable to that of wild type twist <dig> in mcf <dig> cells .
more importantly, when we performed chromatin immunoprecipitation  assays with solubilized chromatin collected from mcf10a cells transfected with s144r/k145e dna-binding deficient twist <dig> mutant, we did not detect any statistically significant enrichment of mutant twist <dig> capable of activating il <dig> expression on the il <dig> promoter , and transfected this Δwr mutant protein into skbr <dig> and mcf <dig> cells.
intriguingly, unlike the dna binding deficient twist <dig> mutants, the Δwr truncation mutant protein completely lost its ability to activate the il <dig> promoter.
furthermore, combining the s144r/k145e mutations with the wr deletion showed similar activation of the il <dig> promoter as compared to Δwr alone , illustrating that the wr domain is essential for twist1-induced il <dig> expression.
twist1-induced il <dig> expression is mediated by rela
it was reported by sosic et al.
that twist <dig> and  <dig> regulate the transcriptional activity of nf-κb in mice and specifically twist <dig> does so through its c-terminal region, which contains a wr domain  <cit> .
given that nf-κb is a central regulator of il <dig>  <cit>  and that the wr domain is highly conserved between twist <dig> and twist <dig>  we hypothesized that nf-κb is involved in twist1-mediated il <dig> promoter activation.
to address this hypothesis, we made use of a dominant negative  form of iκbα  that constitutively suppresses the activation of nf-κb  <cit> , and found that twist <dig> could no longer activate the il <dig> promoter when co-transfected with this suppressor in both skbr <dig> and mcf <dig> cells .
to determine if this was due to the inference of iκbsr on the expression or activity of twist <dig>  we examined the expression of twist <dig> in the nuclei of cells co-transfected with iκbsr, and in fact found greater amounts of twist <dig> protein in the nuclear fractions of these cells than cells transfected with twist <dig> alone .
additionally, in bt <dig> cells, which express high endogenous levels of twist <dig>  transfection of iκbsr showed attenuated il <dig> promoter activity, which was completely abolished by mutations in the κb consensus site that disrupts binding of nf-κb to the il <dig> promoter  <cit>  , indicating that nf-κb transcriptional activity is crucial in the expression regulation of il <dig> in these cells.
to compare the role of twist <dig> and tumor necrosis factor alpha , an activator of nf-κb, on il <dig> promoter activation, we treated skbr <dig> and mcf <dig> cells transfected with the il <dig> promoter construct and dna vector or twist <dig> with tnf-α.
although the il <dig> promoter was activated to greater levels by tnf-α alone than by twist <dig> transfection in both cell lines, the expression of twist <dig> synergized the effect of tnf-α on il <dig> promoter activation in both cell lines.
moreover, mutations in the κb consensus binding site eliminated this synergy, indicating that the synergistic effect of twist <dig> and nf-κb is dependent upon nf-κb/dna binding .
the above data also suggest that twist <dig> activates the transcriptional activity of nf-κb, possibly through a mechanism upstream of iκbα, different from the canonical tnf-α pathway.
to further understand the mechanism by which the activation and synergy of nf-κb occurs, we focused on the nf-κb subunit rela, which is a conserved binding partner of twist <dig>  <cit> .
to address whether rela is the nf-κb subunit that mediates twist1-induced il <dig> expression, we performed shrna-mediated kd of rela in skbr <dig> and mcf <dig> cells, and found that il <dig> promoter-driven luciferase activity under twist <dig> over-expression was attenuated by rela kd in both cell lines .
when we performed double kd of twist <dig> and rela in bt <dig> cells, we observed a slight decrease of the il <dig> promoter activity compared to twist <dig> kd , indicating that rela alone is partially responsible for the basal expression of il <dig>  we also noticed that the kd of rela in twist <dig> over-expressing cells was less efficient than in twist1-null or low expressing cells, suggesting that twist <dig> expression may stabilize rela protein when its expression is reduced without affecting its overall basal expression levels .
additionally, forced expression of twist <dig> in hek <dig> cells, which do not express detectable levels of twist <dig> or rela , failed to stimulate il <dig> promoter activity; whereas transfection of these cells with rela activated the il <dig> promoter, which was further synergized by the co-transfection with twist <dig> .
furthermore, mutations in the κb consensus binding site abolished this synergistic effect between twist <dig> and rela , indicating that the association of nf-κb with dna is necessary for il <dig> transcriptional activation and synergism induction by twist <dig>
the twist <dig> wr domain was previously shown to mediate the interaction between twist <dig> and non-bhlh protein partners, such as runx <dig>  <cit> .
an association between recombinant murine twist <dig> and rela was also previously reported  <cit> .
therefore, we speculated that the twist <dig> wr domain bridges an interaction between human twist <dig> and rela.
to address this, we performed rela co-immunoprecipitation  experiments using hek <dig> cells transfected with wt or mutant twist <dig> using α-rela antibodies .
consistent with earlier reports, wt human twist <dig> co-immunoprecipitated with rela; and the twist <dig> bhlh mutant  was also precipitated by α-rela antibodies in hek <dig> cells transfected with the twist <dig> mutant and rela.
however, neither the Δwr nor s144r k145e Δwr twist <dig> mutant could be detected by α-rela antibodies in co-ip experiments when equal amounts of rela were precipitated.
collectively, these results demonstrate that the twist <dig> wr domain is necessary for the association between rela and twist <dig>  and is crucial for transcriptional activation of twist1-induced il <dig> expression.
to determine if this interaction between rela and twist <dig> regulates the activity of nf-κb, we used a luciferase construct driven by an artificial promoter that contains three repeats of the κb consensus sites .
in this experiment, both wt and s144r k145e twist <dig> stimulated the κb consensus site in skbr <dig> cells, but this stimulation was completely blocked by iκbsr expression, indicating that the activation was mediated by the endogenous nf-κb .
in contrast, neither the Δwr nor s144r k145e Δwr twist <dig> mutant could stimulate nf-κb transcriptional activity in a similar manner in luciferase assays .
furthermore, the increase in nf-κb transcriptional activity was dependent upon the amount of twist <dig> being expressed , whereas Δwr twist <dig> in any given amount failed to activate nf-κb transcriptional activity in hek <dig> cells transfected with the 3x κb luciferase construct .
these results demonstrate that the twist <dig> wr domain, which mediates the association between twist <dig> and rela, is essential for the activation of nf-κb transcriptional activity and synergy.
twist <dig> increases the association of rela with the il <dig> promoter
to confirm the physiologic relevance of the association between twist <dig> and rela, we performed co-ip experiments with bt <dig> cells, which express endogenous twist <dig>  using α-twist <dig> antibodies, we found that rela was exclusively pulled down with twist <dig> in the nuclear but not the cytosolic fraction of bt <dig> cells , suggesting that twist <dig> and rela form a protein complex in the nuclear compartment.
next, to understand if the twist1/rela protein complex is associated with the il <dig> promoter sequence and whether this protein/dna association is essential for promoter activation, we performed chip assays using α-twist <dig> antibodies with mcf10a cells transfected with wt twist <dig>  s144r/k145e or Δwr twist <dig> mutants .
we found that although the wt and the Δwr twist <dig> mutant were associated with the il <dig> promoter, the s144r/k145e dna binding mutant is not presented at the same region, confirming that these point mutations indeed disrupt the dna binding ability of the protein.
more importantly, it demonstrates that the activation of rela by twist <dig> can be achieved away from the il <dig> promoter dna and that the direct or indirect association of twist <dig> via rela to the il <dig> promoter is not required for il <dig> transcription activation.
in addition, we also observed that there is no difference in the enrichment of the il <dig> promoter between wt twist <dig> and Δwr-expressing cells indicating that the interaction between twist <dig> and rela does not appear to affect the ability of twist <dig> to become associated with the il <dig> promoter, presumably through the e-box sequence .
we further compared the association of rela with the il <dig> promoter between mcf10a and mcf10atw cells and detected statistically enriched signals of rela on the il <dig> promoter in mcf10atw relative to mcf10a cells , which indicates that the binding affinity of rela to the il <dig> promoter is greatly enhanced in the presence of twist <dig>  reciprocally, the percentage of rela-associated il <dig> promoter was significantly reduced in bt <dig> cells when twist <dig> was kd  compared to those transduced with non-targeting shrna control  .
together, these data demonstrate that twist <dig> increases the recruitment of rela to the il <dig> promoter.
elevated levels of il <dig> enhance mmp activation and increase cellular invasive potential of breast epithelial cells
under physiological conditions, il <dig> activates endothelial cells and neutrophils to produce and release mmps to enhance their migration and invasion in order to facilitate angiogenesis and extravasation, respectively  <cit> .
these observations suggest that il <dig> produced by twist1-expressing breast cells may also alter their migratory and invasive potentials, features seen with many invasive cancer cells.
to examine this possibility, we compared mcf10a and mcf10atw cells and found that expression of twist <dig> significantly increased the migratory and invasive potential of mcf10a cells in transwell assays towards serum-rich media .
when mcf10atw cells were treated with il <dig> neutralizing antibodies or inhibitors against the il <dig> receptors , the number of invasive cells was reduced by 50%.
interestingly, no significant difference was seen in the number of migratory cells in response to il <dig> signaling blockage , indicating that twist <dig> mediates the production of il <dig> to induce ecm degradation but not to enhance the cell's motility.
to test whether blockage of the il <dig> autocrine pathway affected cell division in the migration/invasion assays, we performed cell proliferation assays and found cell growth was not affected by the treatment of neutralizing antibodies or inhibitors , verifying that the decrease in number of invaded cells was not a result of reduced proliferation.
because il <dig> autocrine signaling significantly altered the invasive properties of mcf10atw cells, we analyzed the expression and activation levels of mmps, which play important roles in ecm degradation.
using gelatin zymography, we found that the production and activation of mmp <dig> were greatly enhanced by twist <dig> over-expression in mcf10a cells.
addition of repertaxin or sb <dig> to mcf10atw cell cultures markedly reduced the expression and activation of both mmp <dig> and mmp <dig> to below detectable levels .
these results indicate that twist <dig> over-expression establishes an il <dig> autocrine loop that regulates the production and activation of mmp <dig> and  <dig>
to investigate whether il <dig> also mediates cellular invasiveness in cancer cells, we performed shrna-mediated kd of il <dig> in bt <dig> cells, and found a 40% reduction in the number of invasive cells as compared to the non-targeting shrna control.
this reduction in the number of invasive cells as a result of il <dig> kd was similar to that of the twist <dig> kd of bt <dig> cells in comparison to both non-targeting shrna controls .
in addition, neither il <dig> nor twist <dig> kd of bt <dig> cells displayed significant differences in their proliferation rates during the course of the invasion assay .
collectively, these data demonstrate that twist1-induced il <dig> production regulates the invasive properties of breast epithelial and cancer cells through an autocrine loop.
twist <dig> and il <dig> are selectively co-expressed in the basal subtype of human breast cancers
we compared the expression of twist <dig> and il <dig> in multiple breast cancer cell lines, which revealed that both twist <dig> and il <dig> are expressed in the basal but not in the non-basal breast cancer cell lines .
although the amounts of il <dig> transcript did not appear to be proportional to the detected levels of twist <dig>  which is likely due to the presence of other il <dig> regulators in these cell lines, these results indicate an overall correlation between twist <dig> and il <dig> expression in the aggressive basal subtype of human breast cancers.
to determine the relevance of the in vitro mechanistic studies in relation to patient samples, we analyzed the relative expression levels of twist <dig> and il <dig> in the stockholm  and uppsala  human breast tumor datasets .
these samples were divided into luminal a, luminal b, normal-like, erbb <dig> and basal subtypes based on their molecular intrinsic signatures identified by calza et al.
.
results from these analyses demonstrate significantly higher levels of il <dig> expression in the basal compared to the luminal a subtype in both datasets, which was also seen in the erbb <dig> subtype in the uppsala dataset .
moreover, although the overall differences in relative expression levels of twist <dig> were small across different subtypes, twist <dig> expression was significantly elevated in the basal as well as the erbb <dig> subtypes in both datasets when compared to luminal a subtype .
more importantly, the co-expression of twist <dig> and il <dig> is correlated in the basal and erbb <dig> subtypes, the two most aggressive subtypes of human breast cancers  <cit> , supporting the important roles of the transcription factor and the cytokine in the pathology of advanced breast cancers.
discussion
herein, we show that twist <dig> up-regulates il <dig> expression to induce cell autonomous invasion through the conserved c-terminal wr domain.
the wr domain mediates the association of twist <dig> and rela, which is essential for twist1-induced stimulation and synergism of nf-κb transcriptional activity and il <dig> production.
additionally, twist <dig> forms a protein complex with rela and enhances the association of rela with the il <dig> promoter, thus inducing il <dig> expression.
finally, twist1-mediated secretion of il <dig> establishes an autocrine loop in breast cells to regulate mmp production and cell invasion.
twist <dig> is commonly characterized by its bhlh domain, which is thought to be responsible for the transcriptionally regulated events controlled by this molecule  <cit> .
however, the functional domains of twist <dig> have not been thoroughly studied and many questions are unanswered regarding their potential interacting partners, which in turn can be highly valuable for understanding the mechanism of cancer cell dissemination.
here we demonstrate that instead of direct il <dig> gene activation, twist <dig> interacts with rela, a non-hlh binding partner, and activates/synergizes transcriptional activity of nf-κb to up-regulate the nf-κb downstream gene target, il <dig>  which in turn regulates mmp production and cell invasion.
this example indicates that twist <dig> can recruit non-hlh transcription factors to form protein complexes and modify gene expression downstream of this partner, which is supported by previous developmental and biochemical studies showing that twist <dig> can regulate the activity of its interacting co-factors  <cit> .
rela is a subunit of the nf-κb complex, which is a central mediator of inflammatory responses  <cit>  and causes many pathophysiological conditions upon activation  <cit> , including tumorigenesis and metastasis  <cit> .
previous reports indicated that during normal mesodermal tissue development, twist <dig> and  <dig> inhibited the transcriptional activity of nf-κb and suppressed expression of the pro-inflammatory cytokines tnf-α and il-1β  <cit> .
in contrast, our results show that in breast tumor cell lines, twist <dig> stimulates nf-κb through the twist <dig> wr domain and up-regulates the expression of the nf-κb downstream target gene il <dig>  these findings are in agreement with an earlier report indicating that twist <dig> synergizes the transcriptional activity of nf-κb in a manner that is independent of its bhlh domain  <cit> .
the seemingly contradicting data in normal mesodermal tissues and breast tumor cell lines may indicate there are additional players modifying the functional relationships between twist <dig> and rela during different processes in development.
we hypothesize that the potential regulatory mechanisms may involve the initial availability of additional co-factors in a given cell type as well as the transcription factor binding sites present on specific target promoters.
as an example, during dorsal-ventral development in drosophila embryos, the cell types along the lateral wall of the embryo are determined by the expression of genes  <cit> , which are differentially regulated by gradients of the morphogens dorsal  and twist, as well as the proximity of the binding sites on promoters of these genes  <cit> .
here, the selective induction of il <dig> among many cytokines also argues for the importance of promoter organization as a critical factor that determines gene regulation by twist <dig> and nf-κb as in comparison to a non-cytokine nf-κb target gene, iκbα, whose promoter was not enriched with either twist <dig> or rela in our chip assays .
our data that twist <dig> activates rela to induce il <dig> expression suggest that in breast epithelial and cancer cells, the expression of twist <dig> can translate an extracellular signal, such as hypoxia, and signal intracellularly to modify the activity of endogenous rela and induce il <dig> expression specifically when there is no apparent extracellular signal for nf-κb activation.
furthermore, that twist <dig> can synergize the il <dig> promoter activation induced by tnf-α indicates that twist1- and tnf-α-induced rela/nf-κb activation contain overlapping yet non-identical biological effects.
finally, pham et al.
<cit>  confirm sosic et al.
's findings  <cit>  that twist <dig> is a downstream target of rela, yet they found that twist <dig> acts as a rela effector protein to block programmed cell death mediated by tnf-α treatment, implying that the functional relationships between twist <dig> and tnf-α in nf-κb activation can depend on the genes being regulated and the physiological conditions of the specific cell type.
despite some discrepancies  <cit> , il <dig> expression was elevated in both invasive cancer cells  <cit>  and in the sera of patients with aggressive cancers  <cit> .
it is well known that il <dig> induces potent neutrophil chemotaxis  <cit> , which causes cytoskeletal reorganization, as well as release and activation of mmps from neutrophils to induce directed migration of the granulocytes  <cit> .
here we show that twist1-induced il <dig> expression in breast epithelial cells up-regulates mmp production and enhances cellular invasive property without affecting migratory ability, suggesting that the biological effects of il <dig> on motivating cytoskeleton remodeling are slightly different in cancer cells.
this also implies that secreted il <dig> primarily enhances the cellular ability to degrade basement membrane , a phenomenon common in metastatic cancers.
blockage of il <dig> signaling or knockdown of the cytokine with shrnas against twist <dig> did not cause mcf10atw  or bt <dig>  cells to lose their mesenchymal phenotype, indicating the reduced invasive properties resulted from il <dig> signaling inhibition was not a result of reversion of emt.
this observation may be because of the fact that mesenchymal-epithelial transition is a lengthy process; however, immunoblots of epithelial and mesenchymal markers of bt <dig> cells stably transduced with shil <dig> or shtw did not appear to be morphologically or biochemically different from the bt <dig> shctrl cells , suggesting the increased cellular invasiveness mediated by il <dig> expression is independent of the emt process.
the fact that il <dig> is a chemokine for neutrophils is another important element for tumorigenesis and the development of tumor microenvironment.
neutrophils are short-lived multinuclear white blood cells that have a profound role in ecm degradation and are responsible for several diseases characterized by severe damage of tissue structure  <cit> .
the expression of il <dig> by tumor cells can cause infiltration of neutrophils , which has been evident for many years  <cit> .
it was recently discovered that, like tumor-associated macrophages, tan also displayed two phases of actions, switching from the anti-tumorigenic to the proto-tumorigenic stage  <cit> , providing evidence of the involvement of neutrophils in cancer progression.
moreover, il <dig> is a known effective pro-angiogenic factor  <cit>  that promotes endothelial cell survival and mmp production critical for vascularization  <cit> .
this characteristic of il <dig> may partially be responsible for twist1-associated tumor angiogenesis that was previously noted by our group and collaborators in twist <dig> over-expressing mcf <dig> breast tumor cells  <cit> .
overall, increase of basal expression of il <dig> can bring forth its pleiotropic effects in affecting tumor progression, remodeling of the tumor microenvironment and the formation of cancer metastasis.
CONCLUSIONS
our study demonstrates that the twist <dig> wr domain is functionally critical to twist1-induced cell invasion.
this domain is essential for the interaction between twist <dig> and the nf-κb subunit rela, which is key to il <dig> induction.
twist1-mediated up-regulation of il <dig> leads to mmp production and ecm degradation, resulting in enhanced cell-autonomous invasion.

 while treatment of the primary breast tumor is often well managed with surgery and radiation, metastatic spread to the brain, bones, liver and lungs frequently places women in an incurable state of disease. the basic helix-loop-helix transcription factor twist was previously demonstrated to be a potent promoter of cancer cell dissemination into circulation and metastasis, providing an ideal target for investigation and a promising therapeutic target for intervention. based on its role in mesodermal development during mammalian embryogenesis, twist is proposed to induce an embryonic event termed epithelial-mesenchymal transition in tumor cells to promote the expression of mesenchymal junction proteins in epithelial cells and reduce intercellular junctions in the meantime. induction of emt enables epithelial cells to acquire the properties of mesenchymal lineages, including enhanced mobility and invasiveness that are tightly correlated with cancer metastasis. additionally, twist was shown to regulate the expression of akt and mirna-10b, which are subsequently involved in the migratory and invasive properties of twist1-overexpressing cells. eckert et al. recently reported that twist up-regulates the expression of platelet derived growth factor receptor, which in turn promotes the formation of invadopodia and matrix degradation, presenting the first evidence that twist causes extracellular matrix remodeling. however, the mechanisms by which twist actively promote cell invasion are still largely unstudied. twist is a class ii member of the bhlh super family. it homo- or heterodimerizes with class i hlh family members, such as e proteins, through the hlh domain, and binds dna that contains the e-box sequence via the basic domain to regulate gene expression. loss of function mutations in the bhlh domain in one allele of the twist gene cause saethre-chotzen syndrome in humans, an autosomal dominant craniofacial disease caused by gene haploinsufficiency, denoting the functional importance of the twist bhlh domain. twist also contains a highly conserved carboxy-terminus, the wr domain, which shares 100% sequence homology among jellyfish, xenopus, mice and humans. this region was shown to mediate the association between twist and runx a zinc-finger protein, and prevent runx from inducing premature osteoblast differentiation during bone development in mice. however, whether the highly conserved wr domain plays any roles in twist1-induced cancer metastasis is an open question. here we describe a unique property of the twist wr domain in mediating il production and breast cancer cell invasion. using gene set enrichment analysis of genetic profiles and cytokine array analyses, we found that il was specifically up-regulated by twist over-expression in the human breast epithelial cell line mcf10a as well as other breast cancer cell lines. we also discovered that twist activates the il promoter, but, surprisingly, in a manner that is independent of its canonical dna binding bhlh domain. in contrast, twist1-induced il promoter activation is dependent on the c-terminal wr domain, through which twist interacts with the nf-κb subunit rela. this physical association of twist and rela activated and synergized the transcriptional activity of nf-κb and increased the binding affinity of rela to dna, which in turn stimulated il expression. finally, we demonstrate that the il autocrine pathway in breast epithelial and cancer cells increases matrix metalloproteinase production and enables autonomous invasion of the cells.  twist induces il cytokine production to elucidate the molecular function of twist in human breast epithelial cells, we compared the gene expression profiles of a twist over-expressing mammary epithelial cell line and the parental line from which it was derived. we analyzed these data by gsea using the c canonical pathway gene sets on paired independent experiments performed in triplicate using these cell lines. the returned gene set permutation test showed a significant difference in the biocarta_cytokine_pathway between mcf10a and mcf10atw cells at a false discovery rate of. the gene set included the pro-inflammatory cytokine interleukin alpha and the leukocyte chemokine il as two significant contributors to the biocarta_cytokine_pathway enrichment, with respective ranks of and as determined by signal-to-noise metric among all genes on the gene array, and in concordance with twist expression. to verify the up-regulation of these genes, we performed a cytokine array blot with conditioned media collected from mcf10a or mcf10atw cell cultures. in this experiment, il along with growth related oncogene and angiogenin, showed the most pronounced up-regulation in mcf10atw cells; however, secreted il1α was not detected. quantification of il revealed 34-fold greater mrna expression and 3-fold greater secretion of il protein into culture media of mcf10atw relative to mcf10a cells. to test if il was selectively up-regulated in the mcf10atw stable cell line, we transiently transduced mcf10a parental cells with adenoviruses that expressed twist at serial dilutions and observed that il transcripts were up-regulated in a dose dependent manner that correlated with the levels of twist expressed. since il was up-regulated at both the mrna and protein levels, indicating that il is a downstream target of twist we, therefore, focused on elucidating the regulatory pathway of il to evaluate the induction of il by twist under pathological conditions, we expressed twist in the skbr and mcf cell lines, which represent two different subtypes of breast cancer, and observed respective 20-fold and 4-fold increases in il transcript levels. the difference in il transcript levels between these two cell lines may be due to the influence of erbb expression on nf-κb transcriptional activity. in addition, short hairpin rna -mediated knock-down of endogenous twist reduced the expression of il mrna in bt cells, a triple-negative breast cancer cell line, er- and her2-) that expresses high endogenous levels of both twist and il this reduction in il expression correlated with the amount of twist protein knocked down. taken together, these data show that twist regulates and maintains il expression in breast epithelial and cancer cells. twist1-induced il transcription is mediated by the twist wr domain to investigate whether twist activates il transcription, we cloned the il minimal promoter, which contains a putative twist binding site and a consensus κb response element, into a luciferase reporter construct. skbr and mcf cells transfected with this il promoter construct displayed a consistent increase in luciferase activities when co-transfected with twist indicating transcriptional activation of il. correspondingly, in bt cells, twist kd resulted in a decrease in il promoter activity, and this activity was rescued upon exogenous twist expression. to further clarify whether twist directly binds and activates the il promoter, we transfected skbr and mcf cells with an il promoter construct that was mutated at the putative twist binding e-box. in this experiment, twist failed to stimulate luciferase transcription, indicating that integrity of the e-box sequence is required for il promoter activation. however, we were concerned that these mutations may affect the binding of c/ebp, a known regulator of il because the e-box sequence heavily overlaps with the c/ebp binding site. therefore, we generated two dna binding deficient twist mutants by introducing the s144r/k145e and r118c mutations. these naturally occurring mutations found in scs patients abolish the dna binding ability of twist1/e heterodimers without affecting its subcellular localization. surprisingly, both the s144r/k145e and r118c twist mutants stimulated the il promoter at similar levels as wild type twist in skbr and mcf cells. to further validate that the dna binding ability of twist is not required for il promoter activation, we completely removed the bhlh domain of twist and still observed il promoter activity induced by this mutant comparable to that of wild type twist in mcf cells. more importantly, when we performed chromatin immunoprecipitation assays with solubilized chromatin collected from mcf10a cells transfected with s144r/k145e dna-binding deficient twist mutant, we did not detect any statistically significant enrichment of mutant twist capable of activating il expression on the il promoter, and transfected this Δwr mutant protein into skbr and mcf cells. intriguingly, unlike the dna binding deficient twist mutants, the Δwr truncation mutant protein completely lost its ability to activate the il promoter. furthermore, combining the s144r/k145e mutations with the wr deletion showed similar activation of the il promoter as compared to Δwr alone, illustrating that the wr domain is essential for twist1-induced il expression. twist1-induced il expression is mediated by rela it was reported by sosic et al. that twist and regulate the transcriptional activity of nf-κb in mice and specifically twist does so through its c-terminal region, which contains a wr domain. given that nf-κb is a central regulator of il and that the wr domain is highly conserved between twist and twist we hypothesized that nf-κb is involved in twist1-mediated il promoter activation. to address this hypothesis, we made use of a dominant negative form of iκbα that constitutively suppresses the activation of nf-κb, and found that twist could no longer activate the il promoter when co-transfected with this suppressor in both skbr and mcf cells. to determine if this was due to the inference of iκbsr on the expression or activity of twist we examined the expression of twist in the nuclei of cells co-transfected with iκbsr, and in fact found greater amounts of twist protein in the nuclear fractions of these cells than cells transfected with twist alone. additionally, in bt cells, which express high endogenous levels of twist transfection of iκbsr showed attenuated il promoter activity, which was completely abolished by mutations in the κb consensus site that disrupts binding of nf-κb to the il promoter, indicating that nf-κb transcriptional activity is crucial in the expression regulation of il in these cells. to compare the role of twist and tumor necrosis factor alpha, an activator of nf-κb, on il promoter activation, we treated skbr and mcf cells transfected with the il promoter construct and dna vector or twist with tnf-α. although the il promoter was activated to greater levels by tnf-α alone than by twist transfection in both cell lines, the expression of twist synergized the effect of tnf-α on il promoter activation in both cell lines. moreover, mutations in the κb consensus binding site eliminated this synergy, indicating that the synergistic effect of twist and nf-κb is dependent upon nf-κb/dna binding. the above data also suggest that twist activates the transcriptional activity of nf-κb, possibly through a mechanism upstream of iκbα, different from the canonical tnf-α pathway. to further understand the mechanism by which the activation and synergy of nf-κb occurs, we focused on the nf-κb subunit rela, which is a conserved binding partner of twist. to address whether rela is the nf-κb subunit that mediates twist1-induced il expression, we performed shrna-mediated kd of rela in skbr and mcf cells, and found that il promoter-driven luciferase activity under twist over-expression was attenuated by rela kd in both cell lines. when we performed double kd of twist and rela in bt cells, we observed a slight decrease of the il promoter activity compared to twist kd, indicating that rela alone is partially responsible for the basal expression of il we also noticed that the kd of rela in twist over-expressing cells was less efficient than in twist1-null or low expressing cells, suggesting that twist expression may stabilize rela protein when its expression is reduced without affecting its overall basal expression levels. additionally, forced expression of twist in hek cells, which do not express detectable levels of twist or rela, failed to stimulate il promoter activity; whereas transfection of these cells with rela activated the il promoter, which was further synergized by the co-transfection with twist. furthermore, mutations in the κb consensus binding site abolished this synergistic effect between twist and rela, indicating that the association of nf-κb with dna is necessary for il transcriptional activation and synergism induction by twist the twist wr domain was previously shown to mediate the interaction between twist and non-bhlh protein partners, such as runx. an association between recombinant murine twist and rela was also previously reported. therefore, we speculated that the twist wr domain bridges an interaction between human twist and rela. to address this, we performed rela co-immunoprecipitation experiments using hek cells transfected with wt or mutant twist using α-rela antibodies. consistent with earlier reports, wt human twist co-immunoprecipitated with rela; and the twist bhlh mutant was also precipitated by α-rela antibodies in hek cells transfected with the twist mutant and rela. however, neither the Δwr nor s144r k145e Δwr twist mutant could be detected by α-rela antibodies in co-ip experiments when equal amounts of rela were precipitated. collectively, these results demonstrate that the twist wr domain is necessary for the association between rela and twist and is crucial for transcriptional activation of twist1-induced il expression. to determine if this interaction between rela and twist regulates the activity of nf-κb, we used a luciferase construct driven by an artificial promoter that contains three repeats of the κb consensus sites. in this experiment, both wt and s144r k145e twist stimulated the κb consensus site in skbr cells, but this stimulation was completely blocked by iκbsr expression, indicating that the activation was mediated by the endogenous nf-κb. in contrast, neither the Δwr nor s144r k145e Δwr twist mutant could stimulate nf-κb transcriptional activity in a similar manner in luciferase assays. furthermore, the increase in nf-κb transcriptional activity was dependent upon the amount of twist being expressed, whereas Δwr twist in any given amount failed to activate nf-κb transcriptional activity in hek cells transfected with the 3x κb luciferase construct. these results demonstrate that the twist wr domain, which mediates the association between twist and rela, is essential for the activation of nf-κb transcriptional activity and synergy. twist increases the association of rela with the il promoter to confirm the physiologic relevance of the association between twist and rela, we performed co-ip experiments with bt cells, which express endogenous twist using α-twist antibodies, we found that rela was exclusively pulled down with twist in the nuclear but not the cytosolic fraction of bt cells, suggesting that twist and rela form a protein complex in the nuclear compartment. next, to understand if the twist1/rela protein complex is associated with the il promoter sequence and whether this protein/dna association is essential for promoter activation, we performed chip assays using α-twist antibodies with mcf10a cells transfected with wt twist s144r/k145e or Δwr twist mutants. we found that although the wt and the Δwr twist mutant were associated with the il promoter, the s144r/k145e dna binding mutant is not presented at the same region, confirming that these point mutations indeed disrupt the dna binding ability of the protein. more importantly, it demonstrates that the activation of rela by twist can be achieved away from the il promoter dna and that the direct or indirect association of twist via rela to the il promoter is not required for il transcription activation. in addition, we also observed that there is no difference in the enrichment of the il promoter between wt twist and Δwr-expressing cells indicating that the interaction between twist and rela does not appear to affect the ability of twist to become associated with the il promoter, presumably through the e-box sequence. we further compared the association of rela with the il promoter between mcf10a and mcf10atw cells and detected statistically enriched signals of rela on the il promoter in mcf10atw relative to mcf10a cells, which indicates that the binding affinity of rela to the il promoter is greatly enhanced in the presence of twist reciprocally, the percentage of rela-associated il promoter was significantly reduced in bt cells when twist was kd compared to those transduced with non-targeting shrna control. together, these data demonstrate that twist increases the recruitment of rela to the il promoter. elevated levels of il enhance mmp activation and increase cellular invasive potential of breast epithelial cells under physiological conditions, il activates endothelial cells and neutrophils to produce and release mmps to enhance their migration and invasion in order to facilitate angiogenesis and extravasation, respectively. these observations suggest that il produced by twist1-expressing breast cells may also alter their migratory and invasive potentials, features seen with many invasive cancer cells. to examine this possibility, we compared mcf10a and mcf10atw cells and found that expression of twist significantly increased the migratory and invasive potential of mcf10a cells in transwell assays towards serum-rich media. when mcf10atw cells were treated with il neutralizing antibodies or inhibitors against the il receptors, the number of invasive cells was reduced by 50%. interestingly, no significant difference was seen in the number of migratory cells in response to il signaling blockage, indicating that twist mediates the production of il to induce ecm degradation but not to enhance the cell's motility. to test whether blockage of the il autocrine pathway affected cell division in the migration/invasion assays, we performed cell proliferation assays and found cell growth was not affected by the treatment of neutralizing antibodies or inhibitors, verifying that the decrease in number of invaded cells was not a result of reduced proliferation. because il autocrine signaling significantly altered the invasive properties of mcf10atw cells, we analyzed the expression and activation levels of mmps, which play important roles in ecm degradation. using gelatin zymography, we found that the production and activation of mmp were greatly enhanced by twist over-expression in mcf10a cells. addition of repertaxin or sb to mcf10atw cell cultures markedly reduced the expression and activation of both mmp and mmp to below detectable levels. these results indicate that twist over-expression establishes an il autocrine loop that regulates the production and activation of mmp and to investigate whether il also mediates cellular invasiveness in cancer cells, we performed shrna-mediated kd of il in bt cells, and found a 40% reduction in the number of invasive cells as compared to the non-targeting shrna control. this reduction in the number of invasive cells as a result of il kd was similar to that of the twist kd of bt cells in comparison to both non-targeting shrna controls. in addition, neither il nor twist kd of bt cells displayed significant differences in their proliferation rates during the course of the invasion assay. collectively, these data demonstrate that twist1-induced il production regulates the invasive properties of breast epithelial and cancer cells through an autocrine loop. twist and il are selectively co-expressed in the basal subtype of human breast cancers we compared the expression of twist and il in multiple breast cancer cell lines, which revealed that both twist and il are expressed in the basal but not in the non-basal breast cancer cell lines. although the amounts of il transcript did not appear to be proportional to the detected levels of twist which is likely due to the presence of other il regulators in these cell lines, these results indicate an overall correlation between twist and il expression in the aggressive basal subtype of human breast cancers. to determine the relevance of the in vitro mechanistic studies in relation to patient samples, we analyzed the relative expression levels of twist and il in the stockholm and uppsala human breast tumor datasets. these samples were divided into luminal a, luminal b, normal-like, erbb and basal subtypes based on their molecular intrinsic signatures identified by calza et al.. results from these analyses demonstrate significantly higher levels of il expression in the basal compared to the luminal a subtype in both datasets, which was also seen in the erbb subtype in the uppsala dataset. moreover, although the overall differences in relative expression levels of twist were small across different subtypes, twist expression was significantly elevated in the basal as well as the erbb subtypes in both datasets when compared to luminal a subtype. more importantly, the co-expression of twist and il is correlated in the basal and erbb subtypes, the two most aggressive subtypes of human breast cancers, supporting the important roles of the transcription factor and the cytokine in the pathology of advanced breast cancers. discussion herein, we show that twist up-regulates il expression to induce cell autonomous invasion through the conserved c-terminal wr domain. the wr domain mediates the association of twist and rela, which is essential for twist1-induced stimulation and synergism of nf-κb transcriptional activity and il production. additionally, twist forms a protein complex with rela and enhances the association of rela with the il promoter, thus inducing il expression. finally, twist1-mediated secretion of il establishes an autocrine loop in breast cells to regulate mmp production and cell invasion. twist is commonly characterized by its bhlh domain, which is thought to be responsible for the transcriptionally regulated events controlled by this molecule. however, the functional domains of twist have not been thoroughly studied and many questions are unanswered regarding their potential interacting partners, which in turn can be highly valuable for understanding the mechanism of cancer cell dissemination. here we demonstrate that instead of direct il gene activation, twist interacts with rela, a non-hlh binding partner, and activates/synergizes transcriptional activity of nf-κb to up-regulate the nf-κb downstream gene target, il which in turn regulates mmp production and cell invasion. this example indicates that twist can recruit non-hlh transcription factors to form protein complexes and modify gene expression downstream of this partner, which is supported by previous developmental and biochemical studies showing that twist can regulate the activity of its interacting co-factors. rela is a subunit of the nf-κb complex, which is a central mediator of inflammatory responses and causes many pathophysiological conditions upon activation, including tumorigenesis and metastasis. previous reports indicated that during normal mesodermal tissue development, twist and inhibited the transcriptional activity of nf-κb and suppressed expression of the pro-inflammatory cytokines tnf-α and il-1β. in contrast, our results show that in breast tumor cell lines, twist stimulates nf-κb through the twist wr domain and up-regulates the expression of the nf-κb downstream target gene il these findings are in agreement with an earlier report indicating that twist synergizes the transcriptional activity of nf-κb in a manner that is independent of its bhlh domain. the seemingly contradicting data in normal mesodermal tissues and breast tumor cell lines may indicate there are additional players modifying the functional relationships between twist and rela during different processes in development. we hypothesize that the potential regulatory mechanisms may involve the initial availability of additional co-factors in a given cell type as well as the transcription factor binding sites present on specific target promoters. as an example, during dorsal-ventral development in drosophila embryos, the cell types along the lateral wall of the embryo are determined by the expression of genes, which are differentially regulated by gradients of the morphogens dorsal and twist, as well as the proximity of the binding sites on promoters of these genes. here, the selective induction of il among many cytokines also argues for the importance of promoter organization as a critical factor that determines gene regulation by twist and nf-κb as in comparison to a non-cytokine nf-κb target gene, iκbα, whose promoter was not enriched with either twist or rela in our chip assays. our data that twist activates rela to induce il expression suggest that in breast epithelial and cancer cells, the expression of twist can translate an extracellular signal, such as hypoxia, and signal intracellularly to modify the activity of endogenous rela and induce il expression specifically when there is no apparent extracellular signal for nf-κb activation. furthermore, that twist can synergize the il promoter activation induced by tnf-α indicates that twist1- and tnf-α-induced rela/nf-κb activation contain overlapping yet non-identical biological effects. finally, pham et al. confirm sosic et al.'s findings that twist is a downstream target of rela, yet they found that twist acts as a rela effector protein to block programmed cell death mediated by tnf-α treatment, implying that the functional relationships between twist and tnf-α in nf-κb activation can depend on the genes being regulated and the physiological conditions of the specific cell type. despite some discrepancies, il expression was elevated in both invasive cancer cells and in the sera of patients with aggressive cancers. it is well known that il induces potent neutrophil chemotaxis, which causes cytoskeletal reorganization, as well as release and activation of mmps from neutrophils to induce directed migration of the granulocytes. here we show that twist1-induced il expression in breast epithelial cells up-regulates mmp production and enhances cellular invasive property without affecting migratory ability, suggesting that the biological effects of il on motivating cytoskeleton remodeling are slightly different in cancer cells. this also implies that secreted il primarily enhances the cellular ability to degrade basement membrane, a phenomenon common in metastatic cancers. blockage of il signaling or knockdown of the cytokine with shrnas against twist did not cause mcf10atw or bt cells to lose their mesenchymal phenotype, indicating the reduced invasive properties resulted from il signaling inhibition was not a result of reversion of emt. this observation may be because of the fact that mesenchymal-epithelial transition is a lengthy process; however, immunoblots of epithelial and mesenchymal markers of bt cells stably transduced with shil or shtw did not appear to be morphologically or biochemically different from the bt shctrl cells, suggesting the increased cellular invasiveness mediated by il expression is independent of the emt process. the fact that il is a chemokine for neutrophils is another important element for tumorigenesis and the development of tumor microenvironment. neutrophils are short-lived multinuclear white blood cells that have a profound role in ecm degradation and are responsible for several diseases characterized by severe damage of tissue structure. the expression of il by tumor cells can cause infiltration of neutrophils, which has been evident for many years. it was recently discovered that, like tumor-associated macrophages, tan also displayed two phases of actions, switching from the anti-tumorigenic to the proto-tumorigenic stage, providing evidence of the involvement of neutrophils in cancer progression. moreover, il is a known effective pro-angiogenic factor that promotes endothelial cell survival and mmp production critical for vascularization. this characteristic of il may partially be responsible for twist1-associated tumor angiogenesis that was previously noted by our group and collaborators in twist over-expressing mcf breast tumor cells. overall, increase of basal expression of il can bring forth its pleiotropic effects in affecting tumor progression, remodeling of the tumor microenvironment and the formation of cancer metastasis.  our study demonstrates that the twist wr domain is functionally critical to twist1-induced cell invasion. this domain is essential for the interaction between twist and the nf-κb subunit rela, which is key to il induction. twist1-mediated up-regulation of il leads to mmp production and ecm degradation, resulting in enhanced cell-autonomous invasion. 
however, the molecular events triggered by twist to motivate dissemination of cancer cells are largely unknown. here we show that twist induces the production of interleukin, which activates matrix metalloproteinases and promotes invasion of breast epithelial and cancer cells. blockage of il signaling by il neutralizing antibodies or receptor inhibition reduced the invasiveness of both breast epithelial and cancer cells, indicating that twist induces autonomous cell invasion by establishing an il antocrine loop. co-immunoprecipitation analyses revealed that the wr domain mediates the formation of a protein complex comprised of twist and the nuclear factor-kappab subunit rela, which synergistically activates the transcriptional activity of nf-κb. in this novel mechanism, twist1-mediated il transcription is induced through the twist carboxy-terminal wr domain instead of the classic dna binding bhlh domain. the produced il signals through an autocrine loop and promotes extracellular matrix degradation to enable cell invasion across the basement membrane.
 metastasis is the primary cause of death for cancer patients. twist an evolutionarily conserved basic helix-loop-helix transcription factor, is a strong promoter of metastatic spread and its expression is elevated in many advanced human carcinomas. however, the molecular events triggered by twist to motivate dissemination of cancer cells are largely unknown.  here we show that twist induces the production of interleukin, which activates matrix metalloproteinases and promotes invasion of breast epithelial and cancer cells. in this novel mechanism, twist1-mediated il transcription is induced through the twist carboxy-terminal wr domain instead of the classic dna binding bhlh domain. co-immunoprecipitation analyses revealed that the wr domain mediates the formation of a protein complex comprised of twist and the nuclear factor-kappab subunit rela, which synergistically activates the transcriptional activity of nf-κb. this activation leads to increased dna binding affinity of rela to the il promoter and thus induces the expression of the cytokine. blockage of il signaling by il neutralizing antibodies or receptor inhibition reduced the invasiveness of both breast epithelial and cancer cells, indicating that twist induces autonomous cell invasion by establishing an il antocrine loop.  our data demonstrate that the twist wr domain plays a critical role in twist1-induced il expression through interactions with and activation of nf-κb. the produced il signals through an autocrine loop and promotes extracellular matrix degradation to enable cell invasion across the basement membrane. twist1wr domainrelanf-κbil8 