the human lmo2 gene was first cloned from an acute t lymphocytic leukemia ( t - all ) patient with an ( 11;14)(p13;q11 ) translocation .
it was a pivotal factor for both embryonic and adult hematopoiesis , as well as angiogenesis .
moreover , lmo2 is expressed in all hematopoietic cells except for mature t cells , and its forced ectopic expression caused t - all - like syndrome in mouse models   and several x - scid patients who were treated with lentivirus - mediated gene therapy . in classic studies , lmo2 was recognized as a transcription factor and performed bi - directional regulation functions in its different target genes [ 79 ] .
however , the lmo2 protein , which consists of only 2 tandem lim domains , lacks the direct dna - binding ability and functions as a bridge molecular in a transcriptional complex usually including ldb1 , gata1 , tal1 , and e47 .
notably , recent studies revealed that lmo2 was expressed in a variety of solid tissues and tumors , with either nuclear or cytoplasmic localization , and played dual functions in tumor behaviors in different cancer types .
as an adaptor molecule , many details about lmo2 binding partners , as well as molecular and cytological functions , need to be further investigated .
invasion and metastasis are hallmarks of highly aggressive tumor cells , and result primarily from enhanced cell motility controlled by actin cytoskeleton remodeling .
notably , in actin cytoskeleton regulation , profilin1 is an actin monomer - binding protein that participates in free actin monomer capture and delivery , while arp3 , together with other members of the arp2/3 complex , functions as a core nucleation component for branched microfilament growth during cell migration . in a recent study
, we found that lmo2 had a function of promoting tumor cell invasion and metastasis specifically in basal - type breast cancers . in the present study
, we confirmed that arp3 and profilin1 were 2 binding partners of lmo2 in cytoplasm , and lmo2 facilitated the assembly of the complex including arp3 , profilin1 , and actin monomer , promoting lamellipodia / filopodia formation in basal - type breast cancer cells .
the matchmaker gold yeast two - hybrid system ( clontech , palo alto , ca , usa ) was used for yeast two - hybrid assays .
the lmo2 coding sequence was inserted into the pgbkt7 vector to express the gad4_bd - lmo2 fusion protein as bait .
a pre - transformed library of human universal cdna cloned into pgadt7 vector was purchased from clontech .
positive clones , which presented as blue colonies , were re - seeded onto new selection medium plates and each inserted cdna fragment was pcr - amplified and sequenced .
the human profilin1 and arp3 expression vectors were constructed by amplification of their coding sequences from human peripheral blood cdna and inserting into pcdna6b vector with a myc - tag .
the coding region of lmo2 was inserted into the pmal - c2x vector for the expression of recombinant mbp - lmo2 fusion proteins . the lmo2 expression and lmo2-shrna lentivirus vector , as well as the control lentivirus vector , and the lenti - pac hiv expression packaging kit ,
the breast cancer cell line mda - mb-231 and human embryonic kidney cell line hek-293 t were obtained from atcc ( manassas , va , usa ) and cultured in dmem medium supplemented with 10% fetal bovine serum ( life technologies , austin , tx , usa ) .
transfections were performed using lipofectamine 2000 following the manufacturer s instructions ( life technologies ) .
stable cell strains of mda - mb-231-lmo2 , mda - mb-231-control and mda - mb-231-sh - lmo2 were selected and maintained by cultured in the medium with puromycin ( 2 g / ml ) 3 days after lentivirus infection .
cells transfected with rac1(q61l ) or cdc42(q61l ) expression vector were cultured in the presence of 500 g / ml g418 for 1 week for selection to remove the non - transfected cells before assay .
total proteins were extracted using a protein extraction kit ( cwbio , beijing , china ) .
protein concentrations were assayed with a bca protein assay kit ( pierce biotechnology , rockford , il , usa ) .
immunostaining was detected using an enhanced chemiluminescence system ( amersham pharmacia biotech , buckinghamshire , uk ) .
e. coli cells were transformed with pmal - c2x / pmal - lmo2 , and then activated and cultured in a 1-l flask .
mmol / l ) was added to cultures at a density of od 0.60.8 for 4 h to induce the expression of the recombinant proteins .
the bacterial cells were then harvested by centrifuging at 4000 rpm for 15 min , re - suspended in ste buffer ( 50 mmol / l tris - hcl ph 7.9 , 0.5 mmol / l edta , 50 mmol / l nacl , 5% glycerol ) , and sonicated 5 times ( for 10 s at 250 w for each time ) for protein extraction .
next , 10 mg total bacterial protein from each sample was purified with amylase - resin column ( new england biolabs , ipswich , ma , usa ) following the manufacturer s instructions .
we used 100 l of purified mbp - lmo2 fusion or mbp proteins in an mbp - pulldown assay with 1 mg of total mda - mb-231 cell lysate in a total volume of 1 ml .
each mixture was incubated at 4c overnight with rotation before 100 l of the amylase - resin was added and the samples were incubated for an additional 2 h. then , the resin in each sample was washed 3 times with pbst , mixed with an equal volume of 2sds loading buffer , and heat - denatured for 10 min .
finally , the prepared samples were subjected to sds - page and analyzed by western blotting with the relevant antibodies .
a total of 1/20 of the total protein mixture of each sample was loaded as the input .
after pre - clearing with 5 l of 50% protein g beads ( life technologies ) , the supernatant of each sample was incubated with 5 g anti - lmo2 or -v5 antibodies ( 1 : 100 dilution ) or rabbit igg as the control at 4c overnight with rotation . then , 10 l of 50% protein g beads ( life technologies ) was added to each sample and rotated for an additional 2 h. after washing 3 times with pbst , the beads were mixed with an equal volume of 2sds loading buffer and heat - denatured for 10 min .
the prepared samples were then subjected to sds - page and analyzed using western blotting with relevant antibodies .
a total of 1/20 of the proteins from each sample was used as the input .
mda - mb-231 cells ( 210 ) were seeded onto cell chamber slides ( corning , tewksbury , ma , usa ) in 24-well plates 24 h before assaying .
the cells on the slides were fixed with 4% formaldehyde and then stained by fitc - labeled phalloidin ( sigma - aldrich , st .
louis , mo , usa , 1 : 500 dilution ) , or stained with anti - lmo2 and anti - arp3/anti - profilin1 antibodies ( 1 : 200 dilution ) at 4c overnight , followed by incubating with the appropriate fluorescent secondary antibodies at room temperature for 1 h. images were captured using an fv1000 confocal microscope ( olympus , center valley , pa , usa ) .
the matchmaker gold yeast two - hybrid system ( clontech , palo alto , ca , usa ) was used for yeast two - hybrid assays .
the lmo2 coding sequence was inserted into the pgbkt7 vector to express the gad4_bd - lmo2 fusion protein as bait .
a pre - transformed library of human universal cdna cloned into pgadt7 vector was purchased from clontech .
positive clones , which presented as blue colonies , were re - seeded onto new selection medium plates and each inserted cdna fragment was pcr - amplified and sequenced .
the human profilin1 and arp3 expression vectors were constructed by amplification of their coding sequences from human peripheral blood cdna and inserting into pcdna6b vector with a myc - tag .
the coding region of lmo2 was inserted into the pmal - c2x vector for the expression of recombinant mbp - lmo2 fusion proteins . the lmo2 expression and lmo2-shrna lentivirus vector , as well as the control lentivirus vector , and the lenti - pac hiv expression packaging kit ,
the breast cancer cell line mda - mb-231 and human embryonic kidney cell line hek-293 t were obtained from atcc ( manassas , va , usa ) and cultured in dmem medium supplemented with 10% fetal bovine serum ( life technologies , austin , tx , usa ) .
transfections were performed using lipofectamine 2000 following the manufacturer s instructions ( life technologies ) .
stable cell strains of mda - mb-231-lmo2 , mda - mb-231-control and mda - mb-231-sh - lmo2 were selected and maintained by cultured in the medium with puromycin ( 2 g / ml ) 3 days after lentivirus infection .
cells transfected with rac1(q61l ) or cdc42(q61l ) expression vector were cultured in the presence of 500 g / ml g418 for 1 week for selection to remove the non - transfected cells before assay .
total proteins were extracted using a protein extraction kit ( cwbio , beijing , china ) .
protein concentrations were assayed with a bca protein assay kit ( pierce biotechnology , rockford , il , usa ) .
immunostaining was detected using an enhanced chemiluminescence system ( amersham pharmacia biotech , buckinghamshire , uk ) .
de3 e. coli cells were transformed with pmal - c2x / pmal - lmo2 , and then activated and cultured in a 1-l flask .
mmol / l ) was added to cultures at a density of od 0.60.8 for 4 h to induce the expression of the recombinant proteins .
the bacterial cells were then harvested by centrifuging at 4000 rpm for 15 min , re - suspended in ste buffer ( 50 mmol / l tris - hcl ph 7.9 , 0.5 mmol / l edta , 50 mmol / l nacl , 5% glycerol ) , and sonicated 5 times ( for 10 s at 250 w for each time ) for protein extraction .
next , 10 mg total bacterial protein from each sample was purified with amylase - resin column ( new england biolabs , ipswich , ma , usa ) following the manufacturer s instructions .
we used 100 l of purified mbp - lmo2 fusion or mbp proteins in an mbp - pulldown assay with 1 mg of total mda - mb-231 cell lysate in a total volume of 1 ml .
each mixture was incubated at 4c overnight with rotation before 100 l of the amylase - resin was added and the samples were incubated for an additional 2 h. then , the resin in each sample was washed 3 times with pbst , mixed with an equal volume of 2sds loading buffer , and heat - denatured for 10 min .
finally , the prepared samples were subjected to sds - page and analyzed by western blotting with the relevant antibodies .
a total of 1/20 of the total protein mixture of each sample was loaded as the input .
after pre - clearing with 5 l of 50% protein g beads ( life technologies ) , the supernatant of each sample was incubated with 5 g anti - lmo2 or -v5 antibodies ( 1 : 100 dilution ) or rabbit igg as the control at 4c overnight with rotation . then , 10 l of 50% protein g beads ( life technologies ) was added to each sample and rotated for an additional 2 h. after washing 3 times with pbst , the beads were mixed with an equal volume of 2sds loading buffer and heat - denatured for 10 min .
the prepared samples were then subjected to sds - page and analyzed using western blotting with relevant antibodies .
a total of 1/20 of the proteins from each sample was used as the input .
mda - mb-231 cells ( 210 ) were seeded onto cell chamber slides ( corning , tewksbury , ma , usa ) in 24-well plates 24 h before assaying .
the cells on the slides were fixed with 4% formaldehyde and then stained by fitc - labeled phalloidin ( sigma - aldrich , st .
louis , mo , usa , 1 : 500 dilution ) , or stained with anti - lmo2 and anti - arp3/anti - profilin1 antibodies ( 1 : 200 dilution ) at 4c overnight , followed by incubating with the appropriate fluorescent secondary antibodies at room temperature for 1 h. images were captured using an fv1000 confocal microscope ( olympus , center valley , pa , usa ) .
in a yeast two - hybrid assay using lmo2 as the bait , dozens of candidates that interacted with lmo2 were identified ( presented as blue colonies ; figure 1a ) .
after pcr amplification and sequencing of the inserted cdna fragments for each clone , 2 fragments aligned to the coding sequence of profilin1 and arp3 , respectively , were screened out ( figure 1b ) , suggesting the potential binding between lmo2 and arp3/profilin1 .
further , using mbp - pulldown assay , binding between arp3/profilin1 and recombinant mbp - lmo2 fusion proteins were confirmed ( figure 2a ) . moreover , co - immunoprecipitation assay also confirmed the endogenous lmo2-arp3 and lmo2-profilin1 interaction ( figure 2b ) .
immunofluorescence staining indicated strongly cytoplasmic co - localization of lmo2 and profilin1/arp3 ( figure 2c ) , suggesting that these interactions occurred primarily in cytoplasm . considering that both profilin1 and arp3 can bind with the actin monomer , lmo2 , profilin1 , arp3 , and actin monomer
may form a complex . correspondingly , as shown in figure 3a , co - immunoprecipitation assay revealed that profilin1 and arp3 could indeed be immunoprecipitated by each other , while the amount of arp3 or profilin1 co - immunoprecipitated by each other was increased in lmo2-overexpressing mda - mb-231 cells compared to control cells ( figure 3a , 3b ) .
-actin , an actin monomer , could also be co - immunoprecipitated by arp3 or profilin1 .
the amount of -actin co - precipitated by profilin1 was increased in lmo2-overexpressing mda - mb-231 cells compared to the control , but the change in -actin amount co - precipitated by arp3 was not obvious ( figure 3a , 3b ) . taken together , these data indicated that lmo2 could enhance the interaction between arp3 and profilin1 and facilitated the profilin1-mediated actin monomer delivery to the growing branched microfilaments marked by arp3 , which primarily contributed to cell protrusion formation and cell movement .
notably , in a previous study , we found that basal - type breast cancer cell line mda - mb-231 with lmo2 knocking - down showed more tightly - attached cell morphologies and fewer cell protrusions , while overexpression of lmo2 in mda - mb-231 cells caused the opposite effect .
moreover , the small rho - gtpase family member rac1 and cdc42 are key positive regulators of lamellipodia and filopodia formation , respectively . in general ,
forced expression of the constitutively active form of rac1 ( rac1(q61l ) ) or cdc42 ( cdc42(q61l ) ) in mda - mb-231 cells increased lamellipodia ( figure 4a , 4b ) or filopodia ( figure 4c , 4d ) , respectively . however , knocking - down of lmo2 largely eliminated these effects and maintained the relatively tightly - attached , less - protruding cell morphology ( figure 4a4d ) .
these results indicated the cytological function of lmo2 as a positive regulator of lamellipodia / filopodia formation and cell motility in basal - type breast cancer cells .
in a yeast two - hybrid assay using lmo2 as the bait , dozens of candidates that interacted with lmo2 were identified ( presented as blue colonies ; figure 1a ) .
after pcr amplification and sequencing of the inserted cdna fragments for each clone , 2 fragments aligned to the coding sequence of profilin1 and arp3 , respectively , were screened out ( figure 1b ) , suggesting the potential binding between lmo2 and arp3/profilin1 .
further , using mbp - pulldown assay , binding between arp3/profilin1 and recombinant mbp - lmo2 fusion proteins were confirmed ( figure 2a ) . moreover , co - immunoprecipitation assay also confirmed the endogenous lmo2-arp3 and lmo2-profilin1 interaction ( figure 2b ) .
immunofluorescence staining indicated strongly cytoplasmic co - localization of lmo2 and profilin1/arp3 ( figure 2c ) , suggesting that these interactions occurred primarily in cytoplasm .
considering that both profilin1 and arp3 can bind with the actin monomer , lmo2 , profilin1 , arp3 , and actin monomer may form a complex . correspondingly , as shown in figure 3a , co - immunoprecipitation assay revealed that profilin1 and arp3 could indeed be immunoprecipitated by each other , while the amount of arp3 or profilin1 co - immunoprecipitated by each other was increased in lmo2-overexpressing mda - mb-231 cells compared to control cells ( figure 3a , 3b ) .
-actin , an actin monomer , could also be co - immunoprecipitated by arp3 or profilin1 .
the amount of -actin co - precipitated by profilin1 was increased in lmo2-overexpressing mda - mb-231 cells compared to the control , but the change in -actin amount co - precipitated by arp3 was not obvious ( figure 3a , 3b ) . taken together , these data indicated that lmo2 could enhance the interaction between arp3 and profilin1 and facilitated the profilin1-mediated actin monomer delivery to the growing branched microfilaments marked by arp3 , which primarily contributed to cell protrusion formation and cell movement .
notably , in a previous study , we found that basal - type breast cancer cell line mda - mb-231 with lmo2 knocking - down showed more tightly - attached cell morphologies and fewer cell protrusions , while overexpression of lmo2 in mda - mb-231 cells caused the opposite effect .
moreover , the small rho - gtpase family member rac1 and cdc42 are key positive regulators of lamellipodia and filopodia formation , respectively . in general , forced expression of the constitutively active form of rac1 ( rac1(q61l ) ) or cdc42 ( cdc42(q61l ) ) in mda - mb-231 cells increased lamellipodia ( figure 4a , 4b ) or filopodia ( figure 4c , 4d ) , respectively . however , knocking - down of lmo2 largely eliminated these effects and maintained the relatively tightly - attached , less - protruding cell morphology ( figure 4a4d ) .
these results indicated the cytological function of lmo2 as a positive regulator of lamellipodia / filopodia formation and cell motility in basal - type breast cancer cells .
based on gene expression features , breast cancers can be subdivided into luminal a , luminal b , her2 , and basal subtypes ( the pam50 subtyping system ) . among all these subtypes ,
basal - type breast cancer is a prognostically unfavorable subtype characterized by high aggressiveness and metastasis . in a previous study
, we demonstrated that , specifically in basal - type breast cancer , lmo2 promoted tumor cell migration , invasion , and metastasis , and this function was partially achieved by blocking lim kinase 1 ( limk1)-mediated phosphorylation of cofilin1 .
herein , we found that high lmo2 expression also contributed to the regulation of cell protrusions by interaction with arp3 and profilin1 .
because lmo2 increased the release of actin monomers by targeting cofilin1 , it can be speculated that interactions between lmo2 , arp3/profilin1 , and actin monomers may accelerate free actin monomer delivery by profilin1 as the materials for the growth of branched microfilaments marked by the arp2/3 complex , and these molecular interactions finally promote the branched microfilament growth and lamellipodia / filopodia formation ( figure 5 ) .
traditionally , lmo2 was primarily considered as a transcription factor in cell nuclei in hematopoietic cells and vascular endothelia ; however , some recent studies revealed that lmo2 is expressed in a variety of other tissues and tumors , with either nuclear or cytoplasmic location .
moreover , lmo2 showed predominantly cytoplasmic location in most epithelial - derived tumors   and dual effects on tumor behaviors : some reports indicated that lmo2 played an oncogenic role in glioblastoma   and prostate carcinoma , but was a good prognostic marker for diffuse large b cell lymphoma ( dlbcl ) , acute b lymphocytic leukemia ( b - all ) , and pancreatic carcinoma . in breast cancers ,
lmo2 primarily is located in cytoplasm and plays additionally divergent functions in different breast cancer subtypes .
our data in the current study further support this viewpoint and provided 2 novel cytoplasmic lmo2 binding partners , arp3 and profilin1 , for the annotation of the function of lmo2 in basal - type breast cancers .
the current study identified 2 novel cytoplasmic lmo2-binding partners , arp3 and profilin1 , and revealed a novel functional mechanism of lmo2 in promoting lamellipodia / filopodia formation via lmo2-arp3 and lmo2-profilin1 interactions in basal - type breast cancer cells .
in general , these findings contribute to optimizing the knowledge of lmo2 in biochemistry and cell - level biology .
specifically , lmo2 exhibited complicated effects on tumor behaviors in different cancer types , and our study confirmed a basal - type breast cancer - specific tumor - promoting role of lmo2 .
these findings also suggest the potential of lmo2 for use as a cancer - type - dependent biomarker for precision medicine in clinical practice .
 backgroundthe human lmo2 gene was first cloned from an acute t lymphocytic leukemia patient ; it is primarily expressed in hematopoietic and vascular endothelial systems , and functions as a pivotal transcriptional regulator during embryonic hematopoiesis and angiogenesis . however , some recent reports indicated that lmo2 is widely expressed in many tissues and tumors , predominantly in cytoplasm , and revealed complicated functions on tumor behaviors in a variety of cancer types . 
 as an adaptor molecule , binding partners and function details of lmo2 in these solid tumors need to be further investigated.material/methodsin this study , we used yeast two - hybrid method to screen potential lmo2 interacting partners , mbp - pulldown , and co - immunoprecipitation assay to confirm protein - protein interactions , and confocal microscopy to reveal the subcellular localization of relevant proteins and actin cytoskeleton changes in relevant cells.resultswe found that arp3 and profilin1 were 2 binding partners of lmo2 , primarily in cytoplasm . 
 lmo2 . 
 functionally , lmo2 mediated the assembly of a complex including arp3 , profilin1 , and actin monomer , increased actin monomer binding to profilin1 , and promoted lamellipodia / filopodia formation in basal - type breast cancer cells.conclusionsour data indicate a novel functional mechanism of lmo2 in facilitating the delivery of actin monomers to the branched microfilament and increasing lamellipodia / filopodia formation in basal - type breast cancer cells , suggesting a cancer - promoting role of lmo2 in a subtype - dependent manner and its potential as a subtype - specific biomarker for clinical treatment of breast cancers . 