solid tumors , such as those in prostate cancer , contribute the majority of all cancers and result in significant distant tumor metastasis to vital organs such as the lungs , brain , and bones [ 1 , 2 ] . prostate cancer contributes significantly to the morbidity and mortality of men in the united states . advanced prostate cancer is associated with significant mortality because the cancer metastasizes and spreads throughout the body , making recovery nearly impossible [ 1 , 4 ] . the high rates of prostate cancer metastasis are , in part , caused by aggressive primary tumor growth in prostate . prostate tumorigenesis is a result of several upregulated signaling pathways , including notch , egf , fgf , and wnt signaling , which promote tumor cell proliferation [ 611 ] . understanding the mechanisms responsible for upregulated signaling during early tumorigenesis is an important step in identifying key regulators and potential therapeutic targets . more importantly , targeting early stages of tumorigenesis will facilitate stabilization of rapid growing tumors , leading to effective surgical removal of primary tumors and inhibition of further tumor metastasis . epsins are endocytic adaptor proteins that regulate clathrin - mediated endocytosis of cell surface receptors by binding ubiquitin moieties on the receptor cytoplasmic tail [ 1220 ] . mammals express three epsins ; epsins 1 and 2 are ubiquitously expressed in all tissues while epsin 3 is exclusively expressed in the stomach [ 14 , 20 , 21 ] . epsins 1 and 2 likely play redundant functions in mediating endocytosis because a single deletion of either epsin 1 or 2 does not present with a significant phenotype [ 22 , 23 ] . however , epsins 1 and 2 are essential during development because deletion of both alleles ( dko ) resulted in embryonic lethality ( e9.5 ) . we recently reported , using inducible epsin depletion , that postnatal loss of epsins 1 and 2 ( idko ) presented a normal phenotype , suggesting epsins play an important role in the development , but not the maintenance , of normal healthy tissues . in contrast , endothelial cell - specific epsin depletion significantly impaired the growth of several tumor types as a result of aberrant tumor angiogenesis . solid tumorigenesis , including tumorigenesis of the prostate , is dependent on both tumor angiogenesis and tumor cell proliferation [ 5 , 24 ] . several signaling pathways involved in tumor cell proliferation involve clathrin - mediated receptor internalization and , therefore , may be mediated by epsins . elevated levels of epsins have been reported in human skin and lung cancers , as well as canine mammary cancers [ 23 , 2528 ] . epsin overexpression promotes cancer cell invasion through the inhibition of cdc42 and rac1 gap activity and by binding to ralbp1 [ 25 , 27 , 2931 ] . intriguingly , in this study , we have also discovered that expression of epsins is upregulated in both human and mouse prostate cancer cells and prostate cancer tissues . we have utilized both the human prostate lncap xenograft model   and the spontaneous tramp mouse model   to determine if epsin depletion affects prostate tumorigenesis . our findings implicate epsins as oncogenic proteins whose expression is upregulated in prostate epithelium and facilitates growth and progression of prostate cancer . furthermore , our data suggest that depletion or inhibition of epsins may provide novel therapeutic targets for solid tumor therapies . we recently reported a strategy for generating a global epsins 1 and 2 double knockout ( dko ) mouse model . c57bl/6 background mice with epsin 1 flanked by loxp sites ( epn1 ) on a global epsin-2-deficient background ( epn1 , epn2 ) were used to create idko mice by crossing epn1 , epn2 mice with ercre deleter mice that express cre recombinase in all cell types upon tamoxifen injection ( idko ) . we crossed idko and wt mice with transgenic adenocarcinoma of mouse prostate ( tramp ) mice on c57bl/6j background   to generate tramp - wt or tramp - idko mice . ten - week - old , sex and genetic background matched tramp - idko or tramp - wt mice were ip - injected with 4-hydroxytamoxifen ( 150  g per 30  g of body weight ) for five to seven consecutive days to induce deletion of epsin 1 . mortality of tramp - wt and tramp - idko mice was recorded and survival rates plotted as a kaplan - meier plot . mice were also dissected at weeks 20 , 24 , 28 , 32 , and 36 and checked for prostate tumors . tumor growth in two groups of mice was monitored by measuring tumor size with digital calipers . we recognized tumors more than 2  mm in diameter as positive and calculated tumor volume based on the formula : 0.5326 ( length [ mm ]  width [ mm ] ) . polyclonal rabbit antibodies for epsins 1 and 2 were obtained as previously described [ 14 , 17 ] ; goat anti - epsin 1 and mouse anti - gapdh were obtained from santa cruz . human lncap cells ( atcc ) were cultured at 37c with 5% co2 in rpmi 1640 medium supplemented with 10% fetal bovine serum ( fbs ) and 1% penicillin / streptomycin . human lncap cells were treated with either control gfp lentivirus or epsins 1 and 2 shrna lentivirus for 2 days then incubated with fresh media for an additional 2 days . cells were lysed with ripa buffer ( 1%triton x-100/0.1%sds/0.5% sodium deoxycholic acid/5  mm tetrasodium pyrophosphate/50  mm sodium fluoride/5  mm edta/150  mm nacl/25  mm tris , ph 7.5/5  mm na3vo4/5  mm n - ethylmaleimide , and protease inhibitor cocktail ) then the deletion of epsins 1 and 2 was analyzed by western blot using goat anti - epsin 1 and rabbit anti - epsin 2 . deletion of epsins 1 and 2 in normal prostate tissues and prostate tissue with tumor was analyzed by western blotting homogenized tissues using rabbit anti - epsin 1 and rabbit anti - epsin 2 antibodies .    to assess tumor growth , we subcutaneously implanted human prostate wt or epsins- 1- and 2-deficient lncap cells ( 3  10  cells / tumor ) in twelve - week - old scid mice . we estimated the time of tumor appearance and monitored the tumor growth in two groups of mice by measuring tumor size with digital calipers . we recognized tumors more than 2  mm in diameter as positive and calculated tumor volume based on the formula : 0.5326 ( length [ mm ]  width [ mm ] ) . human prostate frozen tissue samples with stage 2c tumors , and their corresponding surrounding nonneoplastic ( normal ) tissues , used for epsins 1 and 2 immunofluorescence studies were purchased as accumax array from isu abxis . images were collected and quantified using an olympus spinning disc confocal microscope equipped with 20x objective and slidebook 5.0 software . all animal studies were performed in compliance with institutional guidelines and were approved by institutional animal care and use committee ( iacuc ) , oklahoma medical research foundation , oklahoma city . data was analyzed by the two - tailed student 's t - test or anova , where appropriate . the wilcoxon signed - rank test was used to compare data that did not satisfy the student 's t test or anova . epsins are reportedly upregulated in several types of cancer [ 23 , 25 , 26 ] . to begin investigating a potential correlation between epsins and the development of prostate cancer , we measured the protein levels of epsins 1 and 2 in nonneoplastic ( normal ) human prostate and tumorigenic human prostate tissues via immunofluorescent staining . we found that epsins 1 and 2 were present at low levels in epithelial cells of acini of normal human prostate tissue ( figure 1(a ) ) . furthermore , epithelial epsins 1 and 2 were significantly upregulated in the human prostate tumor tissues ( figures 1(a ) and 1(b ) ) . these data demonstrate a positive correlation between protein levels of epsins and prostate tumorigenesis .   to investigate whether the increased proteins detected in human prostate cancer tissues were a result of increased epsins 1 and 2 gene expression , we employed the spontaneous transgenic adenocarcinoma of mouse prostate ( tramp ) mouse model [ 3336 ] . we first confirmed , via western blotting and immunohistochemistry ( ihc ) , that epsins 1 and/or 2 protein levels were upregulated in prostate tumors extracted from tramp mice ( figures 2(a)-2(b ) ) . then , we used quantitative real - time pcr ( qrt - pcr ) to investigate the gene expression of epsins 1 and 2 . consistent with increased protein detection , we found that the gene expression of epsins 1 and 2 increased by 3.5- or 2.5-fold , respectively , in extracted tramp prostate tumors , compared to normal prostate ( figure 2(c ) ) . these data demonstrate that the expression of epsins 1 and 2 is upregulated in prostate tumors in vivo and further support a correlation between increased epsins 1 and 2 and prostate tumorigenesis . human prostate cancer lncap cells are a common model for prostate cancer progression because they share many characteristics of early prostate cancer progression including low tumorigenesis and androgen sensitivity [ 7 , 32 , 37 , 38 ] . we transfected lncap cells with control gfp lentivirus or epsins 1 and 2 shrna lentivirus and confirmed epsin depletion by western bot ( figure 3(a ) ) . we subcutaneously inoculated control or epsin - depleted lncap cells into scid mice to determine if epsin depletion would alter lncap tumorigenesis . we found that knockdown of epsins significantly impaired lncap tumor growth ( figures 3(b)3(d ) ) and reduced tumor incidence ( figure 3(c ) ) . these data suggest an oncogenic role for epsins in prostate tumorigenesis and cancer progression in a human xenograft model . to further investigate the role of epsins 1 and 2 in the development and progression of prostate cancer in a spontaneous cancer model , we generated an epsin - deficient tramp mouse model ( figure 4 ) . first , we created mice with a conditional epsin 1 allele ( epn1 ) ( figure 4(a ) ) then crossed them with epsin 2 null mice ( epn2 ) , thereby creating epn1 , epn2 mice . to obtain epsins 1 and 2 inducible - double knockout mice ( idko ) , the epn1 , epn2 mice were crossed with mice expressing tamoxifen - inducible cre recombinase ( ercre ) ( figure 4(b ) ) . the epn1 , epn2 , ercre mice were further crossed with tramp mice to generate tramp , epn1 , epn2 , ercre mice . ten - week old tramp , epn1 , epn2 , ercre mice , sex and genetic background matched to tramp - wt mice , were treated every day for two weeks with 5  mg of 4-hydroxytamoxifen per kg body weight via intraperitoneal injection ( i.p . ) to induce epsin 1 deletion ( tramp - idko ) ( figure 4(c ) ) . to examine the effects of epsin deletion on spontaneous prostate tumor progression in the tramp background , we examined tumor development in the male urogenital tract of tramp - wt and tramp - idko mice . we found that similar to the lncap model , depletion of epsins impaired spontaneous prostate tumorigenesis ( figures 4(d)4(g ) ) . furthermore , epsin deletion in tramp mice significantly improved prostate cancer survival rates ( figure 4(h ) ) . in summary , our data demonstrates that epsins are upregulated in , and likely facilitate tumorigenesis of , the prostate . furthermore , interfering with epsins may protect against the growth and progression of prostate cancer . studies investigating the physiological and pathological roles of epsins have revealed their importance in many biological processes [ 16 , 20 , 22 , 23 , 26 ] . however , the full extent with which epsins are involved in the pathological development of diseases , such as cancer , or their tissue and cell type - specific contributions is still largely unknown . in this study , we investigated the potential role of epsins in the development and progression of prostate cancer . our findings suggest that epsins act as oncogenic proteins to promote prostate tumor growth and progression . furthermore , our results suggest that inhibition of epsins may provide a mechanism to impair the onset and progression of prostate cancer . we chose to study the effects of epsin depletion in two common models of prostate cancer , subcutaneous xenograft of the lncap human prostate cancer cells and the genetic tramp mouse model . in both models we found that depletion of epsins impaired the growth of prostate tumors . in addition , using the tramp mouse model , which closely mimics the spontaneous growth and progression of prostate tumors in human prostate cancer [ 35 , 36 , 39 ] , we were able to show that depletion of epsins significantly prolonged prostate cancer survival . increased survival is likely a result of lower tumor incidence , impaired tumor growth , and reduced metastasis . interestingly , both human prostate tumors and tramp mouse prostate tumors exhibited elevated epsin proteins . furthermore , using the tramp model , we determined that increased epsin proteins were due to significant increases in the gene expression of epsins 1 and 2 in tumor tissue . it is not clear from these results whether upregulated epsin expression is a cause or consequence of tumorigenesis . however , the fact that loss of epsins protected against tumor growth suggests the former , rather than the latter , is likely . the transcriptional regulation of epsins is not currently understood but this data suggests it may provide important information about what mediates early prostate cancer progression . lastly , epsins are characterized as endocytic adaptors that regulate the internalization of several ubiquitinated receptors . although not addressed herein , it is tempting to speculate that the overexpression of epsins may play an important role in upregulating signaling pathways dependent on receptor internalization . alternatively , the multidomain nature of epsins may facilitate the stabilization of cell surface receptor complexes , thereby providing a mechanism to prolong signals that promote tumor growth . in summary , epsins are upregulated in human and mouse prostate carcinoma and this upregulation positively correlates with tumor growth . loss of epsins impaired prostate tumor progression and promoted survival in the tramp mouse model . our results suggest targeted inhibition of epsins may provide novel therapeutics to combat prostate cancer .
epsins have an important role in mediating clathrin - mediated endocytosis of ubiquitinated cell surface receptors . the potential role for epsins in tumorigenesis and cancer metastasis by regulating intracellular signaling pathways has largely not been explored . 
 epsins are reportedly upregulated in several types of cancer including human skin , lung , and canine mammary cancers . 
 however , whether their expression is elevated in prostate cancer is unknown . in this study 
 , we investigated the potential role of epsins in prostate tumorigenesis using the wild type or epsin - deficient human prostate cancer cells , lncap , in a human xenograft model , and the spontaneous tramp mouse model in wild type or epsin - deficient background . here , we reported that the expression of epsins 1 and 2 is upregulated in both human and mouse prostate cancer cells and cancerous tissues . 
 consistent with upregulation of epsins in prostate tumors , we discovered that depletion of epsins impaired tumor growth in both the human lncap xenograft and the tramp mouse prostate . 
 furthermore , epsin depletion significantly prolonged survival in the tramp mouse model . in summary , 
 our findings suggest that epsins may act as oncogenic proteins to promote prostate tumorigenesis and that depletion or inhibition of epsins may provide a novel therapeutic target for future prostate cancer therapies .