Engaged urokinase receptors enhance tumor breast cell migration and invasion by upregulating alpha(v)beta5 vitronectin receptor cell surface expression

We have previously shown that urokinase receptor physically and functionally interacts with alpha(v)beta5 vitronectin receptor, leading to tumor breast cell migration and invasion. Here, the link between these 2 receptors was further investigated by analyzing the expression levels of urokinase receptor and alpha(v)beta5 integrin in 35 human breast carcinomas and 5 benign breast lesions. The occurrence of a positive correlation between urokinase receptor and alpha(v)beta5 protein levels in benign and malignant tumor specimens prompted us to investigate whether engaged urokinase receptors might modulate alpha(v)beta5 expression. Here, we report the receptor-dependent ability of catalytically inactive urokinase to upregulate the alpha(v) and beta5 chains in MDA-MB-231 and MCF-7 breast carcinoma cell lines in a time- and concentration-dependent manner. This effect is dependent on protein kinase C activity and requires new protein synthesis. Accordingly, the availability of assembled alpha(v)beta5 receptors on the cell surface increases upon urokinase treatment, as shown by immunoprecipitation and immunocytochemical analyses. Exposure to urokinase leads to enhanced tumor cell migration and invasion, which is prevented by the "phosphorylation-like" urokinase receptor antagonist His-uPA(138E/303E), the DNA-binding drug mithramycin, the protein kinase C inhibitor calphostin C and anti-alpha(v)beta5 antibodies. Finally, urokinase enables benign breast MCF-10A cells to cross Matrigel in a alpha(v)beta5- and urokinase receptor-dependent manner, indicating that urokinase controls a regulatory circuitry crucial to breast tumor progression.     

Expression of laminin-5 enhances tumorigenicity of human fibrosarcoma cells in nude mice.

Laminin-5 (LN5), which consists of laminin alpha3, beta3 and gamma2 chains, is a laminin isoform produced by various kinds of normal epithelial cells and tumor cells. Strong activity of LN5 in adhesion, migration and scattering of cells in vitro and its frequent detection in human tumor tissues have suggested a possible role of LN5 in the malignant growth of tumor cells. To examine whether LN5 affects the malignant potential of tumor cells, we prepared human fibrosarcoma HT1080 cell lines producing LN5 by transfecting a cDNA of laminin alpha3 chain into the parent cell line, which constitutively expressed the laminin beta3 and gamma2 chains. The exogenous alpha3 chain associated with the endogenous beta3 and gamma2 chains to secrete the LN5 heterotrimer that has strong cell-scattering and cell adhesion activities. The HT1080 transfectants expressing LN5 efficiently adhered to culture dishes in a serum-free condition as compared with control HT1080 cells, which secreted the monomers and heterodimer of the beta3 and gamma2 chains. When injected into nude mice subcutaneously, the HT1080 transfectants expressing LN5 grew faster and formed much larger tumors than the control cells. This suggests that LN5 promotes tumor growth in vivo.     

尿激酶氨基末端基因抗乳腺癌细胞转移的实验研究

目的：探讨尿激酶氨基末端（ATF）基因转移对肿瘤转移的抑制作用。方法：构建重组ATF基因真核表达载体pcDNA3-ATF,用脂质体Lipofectin介导,将其导入其证明尿激酶（uPA)和尿激酶受体（uPAR）均有表达的人乳腺癌细胞系MCF－7,用Western blot检测转染细胞ATF基因的表达,体外观察并比较了野生型和转基因MCF－7细胞侵袭人工基底膜能力;裸鼠皮下接种癌细胞,复制自发性肿瘤转移模型,观察成瘤性和转移性。结果uPA／uPAR在MCF－7中有较高水平的表达;转ATF基因后,可检测到ATF的明显表达,体外侵袭穿透人工基底膜的能力明显降低,体内原位成瘤性不受影响,但自主性体肺转移能力有一定程度的下降,结论ATF基因转移后,ATF的表达可能竞争 抑制内源性uPA与uPAR的结合,在一定程度上抑制癌细胞的侵袭和转移。     

Aberrant expression of integrin and erbB subunits in breast cancer cell lines

Integrin and growth factor receptors play an important role in cell functions and their aberrant expressions are implicated in breast cancer malignancy. Recent studies have shown that integrins physically and functionally associate with growth factor receptors suggesting the cooperative regulation of these two signals. We studied the expression of integrin and erbB subunits by flow cytometer in human normal mammary epithelial (HME) cell, non-metastatic (MCF-7, ZR-75-1, MDA-MB453) and metastatic tumor cell lines (MDA-MB231, MDA-MB435). Compared with HME cells, all of non-metastatic and metastatic cell lines showed decreased expressions of alpha2 and beta4 integrin subunits. Two metastatic cell lines, but not three non-metastatic tumor cell lines, expressed alpha5 and alpha6 comparable to HME cells. There was no correlation of erbB2 expression with integrin expressions. We isolated MDA-MB435 subpopulations expressing lower amount of alpha6 integrin and found that alpha5, but not alpha2 and alphav integrins, was concomitantly decreased while erbB family was not affected. Then we transfected erbB2 gene into MDA-MB435 and found the induction of erbB3 expression but not erbB1 and erbB4. However, erbB2 transfection had no effect on the expression of alpha6 and beta4 integrin subunits. These data suggest that the expression of alpha5 and alpha6 integrins may contribute to metastasis, and that the regulation of erbB2 and alpha6 integrin expressions is independent in breast cancer cells.     

Ascites induces modulation of alpha6beta1 integrin and urokinase plasminogen activator receptor expression and associated functions in ovarian carcinoma

Interactions between cancer cells and the surrounding medium are not fully understood. In this study, we demonstrate that ascites induces selective changes in the expression of integrins and urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) in ovarian cancer cells. We hypothesise that this change of integrin and uPA/uPAR expression triggers signalling pathways responsible for modulating phenotype-dependent functional changes in ovarian cancer cells. Human ovarian surface epithelial (HOSE) cell lines and epithelial ovarian cancer cell lines were treated with ascites for 48 h. Ascites induced upregulation of alpha6 integrin, without any change in the expression of alphav, beta1 and beta4 integrin subunits. Out of the four ovarian cancer cell lines studied, ascites induced enhancement in the expression of uPA/uPAR in the more invasive OVCA 433 and HEY cell lines without any change in the noninvasive OVHS1 and moderately invasive PEO.36 cell lines. On the other hand, no change in the expression of alpha6 integrin or uPAR, in response to ascites, was observed in HOSE cells. In response to ascites, enhancement in proliferation and in adhesion was observed in all four ovarian cancer cell lines studied. In contrast, no significant increase in proliferation or adhesion by ascites was observed in HOSE cells. Ascites-induced expression of uPA/uPAR correlated with the increased invasiveness of HEY and OVCA 433 cell lines but was not seen in OVHS1, PEO.36 and HOSE cell lines. Upregulation of alpha6 integrin and uPA/uPAR correlated with the activation of Ras and downstream Erk pathways. Ascites-induced activation of Ras and downstream Erk can be inhibited by using inhibitory antibodies against alpha6 and beta1 integrin and uPAR, consistent with the inhibition of proliferation, adhesion and invasive functions of ovarian cancer cell lines. Based on these findings, we conclude that ascites can induce selective upregulation of integrin and uPA/uPAR in ovarian cancer cells and these changes may modulate the functions of ovarian carcinomas.     

Soluble urokinase receptor released from human carcinoma cells: a plasma parameter for xenograft tumour studies.

The urokinase plasminogen activator receptor (uPAR) plays a critical role in urokinase-mediated plasminogen activation and thereby in the process leading to invasion and metastasis. Soluble urokinase receptor (suPAR) is released from tumours, and in cancer patients the blood level of soluble receptor is increased. Using an enzyme-linked, immunosorbent assay (ELISA)-specific for the human urokinase receptor, release of soluble receptor was measured in cultures of human breast carcinoma cells, in tumour extracts and in plasma from mice with xenografted human tumours. Soluble human urokinase receptor (shuPAR) was released into culture supernatant during the growth of the human breast cancer cell line MDA-MB-231 BAG, and the level of shuPAR in conditioned medium determined by ELISA was a linear function of both viable cell number and time of incubation. Western blotting showed that the form of shuPAR measured by ELISA in conditioned medium consisted virtually exclusively of the three-domain full-length protein, while uPAR in cell lysates consisted of full-length uPAR as well as the domains (2+3) cleavage product. shuPAR was also released into the plasma of nude mice during growth of MDA-MB-231 BAG, MDA-MB-435 BAG and HCT 116 cells as subcutaneously xenografted tumours. Western blotting demonstrated that the shuPAR released from the xenografted human tumours into plasma consisted of the three-domain full-length protein, despite the finding of some cleaved uPAR in detergent extracts of tumour tissue. The levels of shuPAR determined by ELISA in the plasma of host mice during the growth of xenografted cell lines were highly correlated with tumour volume.     

Calponin h1 induced a flattened morphology and suppressed the growth of human fibrosarcoma HT1080 cells

Calponin h1 (CNh1) is an actin-binding protein that is expressed mainly in smooth muscle cells and is known to regulate smooth muscle contraction. Recently, re-expression of CNh1 in leiomyosarcoma cell lines is reported to suppress cell proliferation and tumorigenicity. However, little is known about the associated cellular structural and functional changes. Since CNh1 is also detected in normal fibroblasts, we hypothesised that CNh1 would also inhibit cell proliferation of the fibrosarcoma cells, HT1080, in which CNh1 is suppressed. An expression vector of human CNh1 complementary DNA was transfected into human HT1080 cells by a calcium-phosphate precipitation method. CNh1-transfected cells exhibited a flattened morphology with organised actin filaments, a significant decrease in cell motility and enhancement in adhesion to fibronectin in association with an increase in integrin alpha5beta1 expression. Anchorage-independent growth and tumorigenicity in nude mice were suppressed in the CNh1-transfected cells. Our results suggest that CNh1 may have a role as a tumour suppressor in human fibrosarcoma by influencing cytoskeletal activities.     

Protein and mRNA expression of uPAR and PAI-1 in myoepithelial cells of early breast cancer lesions and normal breast tissue

Myoepithelial cells (MEs), which surround ducts and acini of the breast glands, exhibit an anti-invasive phenotype and form a natural border separating proliferating tumour cells of ductal carcinoma in situ (DCIS) from basement membrane (bm) and underlying stroma. Invasion requires penetration of these host cellular and extracellular matrix barriers. This destruction is caused by proteolytic activity of tumour cells and host bystander cells. There is substantial evidence that high concentrations of the urokinase plasminogen-activating system are conducive to tumour cell spread and metastasis. Prompted by the conspicuous absence of studies examining the role of the ME in breast cancer progression, we studied the expression of the urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1) in MEs of 60 DCIS samples. Our results show that nearly all MEs of DCIS and normal breast glands exhibit the uPAR antigen, whereas the PAI-1 antigen was mainly expressed in MEs of high-grade DCIS. In one intermediate DCIS numerous ducts showed an incomplete myoepithelial layer expressing uPAR and PAI-1. We conclude that uPAR in MEs may be necessary to attach them to the bm by uPAR/vitronectin (Vn) interaction. The strong expression of PAI-1, which is known to resolve the uPAR/Vn binding, may be involved in the detachment of MEs of DCIS. Although the role of PAI-1 acting as cell detachment factor could not be demonstrated in our study, we speculate that the loss of the anti-invasive ME layer in DCIS may be triggered by PAI-1 and could be an early sign of subsequent tumour cell infiltration.     

Overexpression of alpha(v)beta6 integrin in serous epithelial ovarian cancer regulates extracellular matrix degradation via the plasminogen activation cascade

Recent evidence suggests that integrins are involved in the multi-step process of tumour metastasis. The biological relevance of alpha(v) integrins and associated beta-subunits in ovarian cancer metastasis was examined by analysing the expression of these cell surface receptors in nine ovarian cancer cell lines and also in the primary human ovarian surface epithelial cell line (HOSE). beta1, beta3 and beta5 subunits were present in all ten ovarian cell lines. beta6 subunit was present at varying levels in eight out of nine cancer cell lines but was absent in the HOSE cell line. Immunohistochemical staining showed that beta6 was present in both non-invasive (borderline) and high-grade ovarian cancer tissues but was absent in benign and normal ovarian tissue. High alpha(v)beta6 integrin expressing ovarian cancer cell lines had high cell surface expression of uPA and uPAR. Ovarian cancer cell lines expressing high to moderate level of alpha(v)beta6 integrin demonstrated ligand-independent enhanced levels of high molecular weight (HMW)-uPA and pro-matrix metalloproteinase 2 and 9 (pro-MMP-2 and pro-MMP-9) expression in the tumour-conditioned medium. High and moderate expression of alpha(v)beta6 integrin correlated with increased plasminogen-dependent degradation of extracellular matrix which could be inhibited by inhibitors of plasmin, uPA and MMPs or by monoclonal antibody against uPA, MMP-9 or alpha(v)beta6 integrin. These results suggest that endogenous de novo expression of alpha(v)beta6 integrin in ovarian cancer cells may contribute to their invasive potential, and that alpha(v)beta6 expression may play a role in ovarian cancer progression and metastasis.     

Bimolecular interaction of insulin-like growth factor (IGF) binding protein-2 with alphavbeta3 negatively modulates IGF-I-mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the alpha v beta 3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and alpha v beta 3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7 beta 3, which overexpressed the alpha v beta 3 integrin. Collectively, these results indicate that alpha v beta 3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.     

Urokinase/urokinase receptor and vitronectin/alpha(v)beta(3) integrin induce chemotaxis and cytoskeleton reorganization through different signaling pathways

Vitronectin (VN) and pro-urokinase (pro-uPA) stimulated migration of rat smooth muscle cells in a dose-dependent and additive way, and induced motile-type changes in cell morphology together with a complete reorganization of the actin filaments and of the microtubules. All these effects were inhibited by pertussis toxin, or by antibodies directed against the urokinase receptor (uPAR) or against the VN receptor alpha(v)beta(3) suggesting that an association between the two receptors is required to mediate both signals. Investigation of the signaling pathways showed that increasing the intracellular cAMP resulted in a selective inhibition of VN-induced cell migration. On the other hand, PD 98059, an inhibitor of MEK, differentially inhibited the pro-uPA- but not the VN-induced cell migration. Phosphorylation and nuclear translocation of Erk by pro-uPA was directly observed. We conclude that the signaling pathways of pro-uPA and VN must be at least in part different.     

Cell signaling by urokinase-type plasminogen activator receptor induces stem cell-like properties in breast cancer cells

Signaling by urokinase-type plasminogen activator receptor (uPAR) can cause epithelial-mesenchymal transition (EMT) in cultured breast cancer cells. In this report, we show that uPAR signaling can also induce cancer stem cell (CSC)-like properties. Ectopic overexpression of uPAR in human MDA-MB-468 breast cancer cells promoted the emergence of a CD24(-)/CD44(+) phenotype, characteristic of CSCs, while increasing the cell surface abundance of integrin subunits β1/CD29 and α6/CD49f that represent putative mammary gland stem cell biomarkers. uPAR overexpression increased mammosphere formation in vitro and tumor formation in an immunocompromized severe combined immunodeficient (SCID) mouse model of orthotopic breast cancer. Hypoxic conditions that are known to induce EMT in MDA-MB-468 cells also increased cell surface β1/CD29, mimicking the effects of uPAR overexpression. Antagonizing uPAR effector signaling pathways reversed the increase in cell surface integrin expression. Whereas uPAR overexpression did not induce EMT in MCF-7 breast cancer cells, CSC-like properties were nevertheless still induced along with an increase in tumor initiation and growth in the orthotopic setting in SCID mice. Notably, in MCF-7 cell mammospheres, which display a well-defined acinus-like structure with polarized expression of E-cadherin and β1-integrin, cell collapse into the central cavity was decreased by uPAR overexpression, suggesting that uPAR signaling may stabilize epithelial morphology. In summary, our findings show that uPAR signaling can induce CSC-like properties in breast cancer cells, either concomitantly with or separately from EMT.     

Inhibition of migration and invasion of carcinoma cells by urokinase-derived antagonists of alphavbeta5 integrin activation

We previously showed that, while binding to urokinase receptor (uPAR) through its growth factor domain (GFD, residues 1-49), urokinase (uPA) can engage alphavbeta5 integrin through an internal domain (CP, residues 132-158). This novel uPA/alphavbeta5 interaction promotes cytoskeletal rearrangements and directional cell migration (Franco et al., J Cell Sci 2006;119:3424-34). We now show that treatment of cells with phosphomimic uPA (uPA138E/303E, serine 138 and 303 substituted with glutamic acid) strongly inhibits matrix-induced cell migration. Unlike uPA, binding of uPA138E/303E to cell surface did not induce F-actin enriched protruding structures and caused a 5-fold reduction in cell translocation speed, as determined by video tracking of living cells. Inhibition of migration was found to be independent of uPAR, since uPA variants lacking the GFD domain, but carrying the relevant Ser to Glu substitutions were as effective inhibitor as uPA138E/303E. Through several independent approaches, we established that the phosphomimics specifically bind to alphavbeta5 integrin through the CP region carrying the S138E mutation. This interaction blocks integrin activation, as determined by a decreased affinity of alphavbeta5 to vitronectin and a reduced association of the beta5 cytoplasmic tail with talin. Finally, stable expression of uPA138E/303E in human squamous carcinoma cells prevented tumor cell invasion in vivo. Thus, when expressed in cancer cells, the inhibitory phosphomimic effect was dominant over the effect of endogenously produced uPA. These results shed light on the regulation of cell migration by uPA phosphorylation and provide a realistic opportunity for a novel antiinvasive/metastatic therapeutic intervention.     

Inhibition of angiogenesis and tumor growth by SCH221153, a dual alpha(v)beta3 and alpha(v)beta5 integrin receptor antagonist

New blood vessel formation is essential for tumor growth and metastatic spread. Integrins alpha(v)beta3 and alpha(v)beta5 are arginine-glycine-aspartic acid-dependent adhesion receptors that play a critical role in angiogenesis. Hence, selective dual alpha(v)beta3 and alpha(v)beta5 antagonists may represent a novel class of angiogenesis and tumor-growth inhibitors. Here, an arginine-glycine-aspartic acid-based peptidomimetic library was screened to identify alpha(v)beta3 antagonists. Selected compounds were then modified to generate potent and selective dual inhibitors of alpha(v)beta3 and alpha(v)beta5 receptors. One of these compounds, SCH 221153, inhibited the binding of echistatin to alpha(v)beta3 (IC50 = 3.2 nM) and alpha(v)beta5 (IC50 = 1.7 nM) with similar potency. Its IC50 values for related alpha(IIb)beta3 and alpha5beta1 receptors were 1294 nM and 421 nM, respectively, indicating that SCH 221153 is highly selective for alpha(v)beta3 and alpha(v)beta5 receptors. In cell-based assays, SCH 221153 inhibited the binding of echistatin to alpha(v)beta3- and alpha(v)beta5-expressing 293 cells and blocked the adhesion of endothelial cells to immobilized vitronectin and fibroblast growth factor 2 (FGF2). SCH 221153, but not the inactive analogue SCH 216687, was effective in inhibiting FGF2 and vascular endothelial growth factor-induced endothelial cell proliferation in vitro with an IC50 equal to 3-10 microM. Angiogenesis induced by FGF2 in the chick chorioallantoic membrane assay was also inhibited by SCH 221153. Finally, SCH 221153 exerted a significant inhibition on tumor growth induced by intradermal or s.c. injection of human melanoma LOX cells in severe combined immunodeficient mice.     

ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK)

After dissemination from a primary tumor, cancer cells may resume growth, leading to overt metastasis, or enter a state of protracted dormancy. However, mechanisms that determine their fate, or markers that predict it, are mostly unavailable. We previously showed that in HEp3 human head and neck carcinoma, the extracellular signal-regulated kinase (ERK)(MAPK)/p38(SAPK) activity ratio predicts whether the cells will proliferate or enter a state of dormancy in vivo. The proliferative balance of high ERK/p38 ratio was induced by high urokinase (uPA) receptor (uPAR) expression, which activated alpha5beta1-integrin and epidermal growth factor receptor. This signaling pathway was additionally enhanced by uPA binding to uPAR and fibronectin binding to alpha5beta1-integrin. We tested whether the ERK/p38 balance is predictive of in vivo behavior in other cancer cell types and whether altering the balance will shift their phenotype between proliferation and dormancy. ERK and p38 activities were determined using either phospho-specific monoclonal antibodies or a trans-reporting system where GAL4-Elk and GAL4-CHOP trans-activation of luciferase gene served as reporters for ERK and p38 activities, respectively. We show that in breast, prostate, melanoma, and fibrosarcoma cell lines, the level of active phospho-ERK and the ERK/p38 activity ratio predict for the in vivo behavior in approximately 90% of the cell lines tested. Modulation of ERK/p38 activity ratio by multiple pharmacological and genetic interventions confirms that high ERK/p38 ratio favors tumor growth, whereas high p38/ERK ratio induces tumor growth arrest (dormancy) in vivo and that ERK is negatively regulated by p38. A melanoma cell line appeared to have developed an escape mechanism to avoid the growth inhibitory effect of high p38 activity. Mechanistic analysis implicated high uPAR expression and its interaction with and activation of alpha5beta1-integrin as determinants of the in vivo growth promoting high ERK/p38 ratio in several cell lines. The small GTPase, Cdc42, was implicated in activation of p38 and growth arrest. These results suggest that even cells that originate in advanced cancers retain a degree of dependence on surface receptors and matrix for their proliferative signals in vivo and provide a therapeutic opportunity to change their phenotype from tumorigenic to dormant.     

Effects of type I and II interferons on cultured human breast cells: interaction with estrogen receptors and with tamoxifen

The combined effects of tamoxifen, a competitive inhibitor of estrogen, and type I or II interferons on the proliferation of several human breast cancer cell lines in vitro were examined. Additive antiproliferative effects were observed with interferons and tamoxifen, in two estrogen receptor positive cell lines, MCF-7 and T-47D. In MCF-7 cells neither beta ser interferon, gamma interferon, nor alpha 6 interferon were able to significantly alter estrogen receptor levels. Antigrowth activities of beta ser interferon and gamma interferon in an estrogen receptor negative cell line, HS578T, were equivalent to those in estrogen receptor-positive cell lines. Consistent with the antiproliferative effects of interferons, high affinity beta ser interferon receptors and interferon inducible 2'5'-oligoadenylate synthetase were present in both MCF-7 and HS578T cell lines. Thus steroid hormone receptor status did not influence the antiproliferative effects of interferons on breast carcinoma cells in vitro.