A novel mechanism for integrin-mediated ras activation in breast carcinoma cells: the alpha6beta4 integrin regulates ErbB2 translation and transactivates epidermal growth factor receptor/ErbB2 signaling

ErbB2 (HER2, Neu) and Ras play key roles in tumor invasion and metastasis. We identified a novel mechanism by which integrin alpha(6)beta(4) regulates ErbB2 expression, Ras activation, and the invasion of breast carcinoma cells. Here we show that integrin alpha(6)beta(4) regulates Ras activity especially in serum-depleted condition. Down-regulation of beta(4) integrin by beta(4) short hairpin RNA (shRNA) decreased Ras activity and carcinoma invasion whereas reexpression of this integrin restored Ras activity. ErbB2, a binding partner of epidermal growth factor receptor (EGFR), and EGFR modulated Ras activity, and integrin alpha(6)beta(4) regulated phospho-EGFR level without affecting EGFR expression. We also found that integrin alpha(6)beta(4) is involved in ErbB2 expression. Depletion of beta(4) by shRNA reduced ErbB2 protein level without affecting ErbB2 mRNA level and reexpression of beta(4) increased ErbB2 protein level. Reduction of eukaryotic initiation factor 4E, a rate-limiting factor for cap-dependent translation, decreased ErbB2 protein level, and beta(4) shRNA cells exhibited a shift in ErbB2 mRNA to light polysomes compared with control cells. These results show that integrin alpha(6)beta(4) regulates ErbB2 through translational control. In summary, we propose a novel mechanism for ErbB2 up-regulation and Ras activation in serum-depleted breast cancer cells; integrin alpha(6)beta(4) regulates the expression of ErbB2 and the subsequent phosphorylation of EGFR and activation of Ras. These findings provide a mechanism that substantiates the reported role of alpha(6)beta(4) in carcinoma invasion.     

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.     

Ethanol enhances erbB-mediated migration of human breast cancer cells in culture

Growth factor systems (ligands and their receptors) are targets of ethanol toxicity. Inasmuch as alcohol consumption may increase the risk and development of breast cancer, we hypothesize that ethanol enhances cell migration by up-regulating the activities of erbB receptors. Of the three tested breast cancer cell lines that exhibit low invasion capacity (BT-20, MCF-7, and T47D cells), erbB receptors were specifically affected by ethanol only in the T47D cells. Ethanol increased erbB2, erbB3, and erbB4 expression in T47D human breast cancer cells in a concentration-dependent manner. ErbB1 (epidermal growth factor receptor) was unaffected. Heregulin 1 (ligand for erbB3 and erbB4) induced a modest increase in the invasion potential of the T47D cells. Ethanol alone also promoted modest invasion by the T47D cells, however, ethanol dramatically increased their heregulin-mediated invasion. Knocking-out erbB2 with an anti-sense oligonucleotide eliminated heregulin 1-promoted migration and blocked ethanol-induced chemo-migration. Thus, these data suggest that alcohol may enhance metastasis by altering an erbB system, and pivotally, erbB2.     

Five carboxyl-terminal residues of neuregulin2 are critical for stimulation of signaling by the ErbB4 receptor tyrosine kinase

The neuregulins (NRGs) are members of the epidermal growth factor (EGF) family of peptide growth factors. These hormones are agonists for the ErbB family of receptor tyrosine kinases, a family that includes the epidermal growth factor receptor (EGFR/ErbB1), ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4. We recently observed that the EGF family hormone NRG2beta is a potent agonist for ErbB4. In contrast, NRG2alpha, a splicing isoform of the same gene that encodes NRG2beta, is a poor ErbB4 agonist. We hypothesized that carboxyl-terminal residues of NRG2beta are critical for stimulation of ErbB4 tyrosine phosphorylation and coupling to downstream signaling events. Here, we demonstrate that the substitution of a lysine residue for Phe45 in NRG2beta results in reduced ligand potency. We also demonstrate that substitution of a phenylalanine for Lys45 in NRG2alpha results in increased ligand potency. Finally, analyses of the gain-of-function NRG2alpha Chg5 mutant demonstrate that Gln43, Met47, Asn49, and Phe50 regulate ligand efficacy. Thus, these data indicate that carboxyl-terminal residues of NRG2beta are critical for activation of ErbB4 signaling. Moreover, these NRG2alpha and NRG2beta mutants reveal new insights into models for ligand-induced ErbB family receptor tyrosine phosphorylation and coupling to downstream signaling events.     

Heregulin regulates the actin cytoskeleton and promotes invasive properties in breast cancer cell lines

The metastatic process requires changes in tumor cell adhesion properties, cell motility and remodeling of the extracellular matrix. The erbB2 proto-oncogene is overexpressed in approximately 30% of breast cancers and is a major prognostic parameter when present in invasive disease. A ligand for the erbB2 receptor has not yet been identified but it can be activated by heterodimerization with heregulin (HRG)-stimulated erbB3 and erbB4 receptors. The HRGs are a family of polypeptide growth factors that have been shown to play a role in embryogenesis, tumor formation, growth and differentiation of breast cancer cells. The erbB3 and erbB4 receptors are involved in transregulation of erbB2 signaling. The work presented here suggests biological roles for HRG including regulation of the actin cytoskeleton and induction of motility and invasion in breast cancer cells. HRG-expressing breast cancer cell lines are characterized by low erbB receptor levels and a high invasive and metastatic index, while those which overexpress erbB2 demonstrate minimal invasive potential in vitro and are non-tumorigenic in vivo. Treatment of the highly tumorigenic and metastatic HRG-expressing breast cancer cell line MDA-MB-231 with an HRG-neutralizing antibody significantly inhibited proliferation in culture and motility in the Boyden chamber assay. Addition of exogenous HRG to non-invasive erbB2 overexpressing cells (SKBr-3) at low concentrations induced formation of pseudopodia, enhanced phagocytic activity and increased chemomigration and invasion in the Boyden chamber assay. The specificity of the chemomigration response to HRG is demonstrated by inhibition with the anti-HRG neutralizing antibody. These results suggest that either HRG can act as an autocrine or paracrine ligand to promote the invasive behavior of breast cancer cells in vitro or thus may enhance the metastatic process in vivo.     

Heregulin-induced activation of ErbB3 by EGFR tyrosine kinase activity promotes tumor growth and metastasis in melanoma cells

ErbB3 receptor tyrosine kinase has been shown to induce tumor progression in several types of cancer through heterodimerization with ErbB2. However, the role of ErbB3 and its ligand heregulin (HRG) in tumor metastasis remains poorly understood. In the present study, we tried to clarify their contributions to the metastasis of ErbB3-overexpressing B16-BL6 melanoma cells. Stimulation with HRG induced phosphorylation of ErbB3 and metastatic properties including MMP-9 expression, invasion, adhesion and experimental lung metastasis in vivo. These cellular responses were blocked by inhibiting the tyrosine kinase activity of EGFR with PD153035. In addition, phosphorylation of EGFR was rapidly induced by HRG, suggesting that EGFR is a possible heterodimeric counterpart of ErbB3. RNA interference demonstrated that subcutaneous tumor growth and angiogenesis was attenuated by inactivation of ErbB3 in cancer cells. Although experimental pulmonary metastasis was not affected by the knockdown of ErbB3, spontaneous metastasis was, even when primary tumors in the foot pad were amputated at a similar size. These results indicate that HRG-induced activation of ErbB3 via EGFR promotes tumor growth and metastasis of melanoma cells.     

Truncated ErbB2 receptor (p95ErbB2) is regulated by heregulin through heterodimer formation with ErbB3 yet remains sensitive to the dual EGFR/ErbB2 kinase inhibitor GW572016

The expression of the NH2 terminally truncated ErbB2 receptor (p95ErbB2) in breast cancer correlates with metastatic disease progression compared with the expression of full-length p185ErbB2. We now show that heregulin (HRG), but not EGF, stimulates p95ErbB2 phosphorylation in BT474 breast cancer cells. Furthermore, phospho-p95ErbB2 forms heterodimers with ErbB3, but not EGFR, while p185ErbB2 heterodimerizes with both EGFR and ErbB3. The predilection of p95ErbB2 to heterodimerize with ErbB3 provides an explanation for its regulation by HRG, an ErbB3 ligand. GW572016, a reversible small molecule inhibitor of EGFR and ErbB2 tyrosine kinases, inhibits baseline p95ErbB2 phosphorylation in BT474 cells and tumor xenografts. Inhibition of p95ErbB2, p185ErbB2, and EGFR phosphorylation by GW572016 resulted in the inhibition of downstream phospho-Erk1/2, phospho-AKT, and cyclin D steady-state protein levels. Increased phosphorylation of p95ErbB2 and AKT in response to HRG was abrogated to varying degrees by GW572016. In contrast, trastuzumab did not inhibit p95ErbB2 phosphorylation or the expression of downstream phospho-Erk1/2, phospho-AKT, or cyclin D. It is tempting to speculate that trastuzumab resistance may be mediated in part by the selection of p95ErbB2-expressing breast cancer cells capable of exerting potent growth and prosurvival signals through p95ErbB2-ErbB3 heterodimers. Thus, p95ErbB2 represents a target for therapeutic intervention, and one that is sensitive to GW572016 therapy.     

Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects

Due to heterodimerization and a variety of stimulating ligands, the ErbB receptor system is both diverse and flexible, which proves particularly advantageous to the aberrant signaling of cancer cells. However, specific mechanisms of how a particular receptor contributes to generating the flexibility that leads to aberrant growth regulation have not been well described. We compared the utilization of ErbB2 in response to epidermal growth factor (EGF) and heregulin stimulation in colon carcinoma cells. Anti-ErbB2 monoclonal antibody 2C4 blocked heregulin-stimulated phosphorylation of ErbB2 and ErbB3; activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3'-kinase (PI3K), and Akt; proliferation; and anchorage-independent growth. 2C4 blocked EGF-mediated phosphorylation of ErbB2 and inhibited PI3K/Akt and anchorage-independent growth but did not affect ErbB1 or MAPK. Immunoprecipitations showed that ErbB3 and Grb2-associated binder (Gab) 1 were phosphorylated and associated with PI3K activity after heregulin treatment and that Gab1 and Gab2, but not ErbB3, were phosphorylated and associated with PI3K activity after EGF treatment. These data show that monoclonal antibody 2C4 inhibited all aspects of heregulin signaling as well as anchorage-independent and monolayer growth. Furthermore, we identify ErbB2 as a critical component of EGF signaling to the Gab1/Gab2-PI3K-Akt pathway and anchorage-independent growth, but EGF stimulation of MAPK and monolayer growth can occur efficiently without the contribution of ErbB2.     

Tumor cell invasiveness correlates with changes in integrin expression and localization

In nontumorigenic mammary epithelial cells (EpH4), transforming growth factor-beta (TGFbeta1) causes cell cycle arrest/apoptosis, but induces epitheliomesenchymal transition (EMT) in Ha-Ras-transformed EpH4 cells (EpRas). EMT is closely correlated with late-stage tumor progression and results in fibroblastic, migratory cells displaying a mesenchymal gene expression program (FibRas). EpRas and FibRas cells showed strongly increased cell substrate adhesion to fibronectin, collagens I/IV and laminin 1. Furthermore, Ras transformation caused enhanced or de-novo expression of the integrin subunits beta1, alpha2 and alpha3, or alpha5 and alpha6, respectively, the latter subunits being even more strongly expressed in FibRas cells. Importantly, polarized EpRas cells expressed integrin subunits beta1 and alpha6 at distinct (apical and lateral) membrane domains, while FibRas cells coexpressed these integrins and alpha5 at the entire plasma membrane. During EMT, EpRas cells formed an alpha5beta1 complex and deposited its ligand fibronectin into the extracellular matrix. Function-blocking alpha5 antibodies attenuated migration, and caused massive apoptosis in EpRas cells undergoing TGFbeta1-induced EMT in collagen gels, but failed to affect EpRas- or FibRas-derived structures. We conclude that functional alpha5beta1 integrin is centrally implicated in EMT induction. Importantly, FibRas cells also failed to deposit the alpha6beta4 ligand laminin 5, suggesting that alpha6beta4 is no longer functional after EMT and replaced by mesenchymal integrins such as alpha5beta1.     

Steroid hormone receptor status of mouse mammary stem cells

The estrogen receptor alpha (ERalpha), progesterone receptor (PR), and erbB2 (Her2 in humans) are important prognostic markers of human breast cancer, and they are variably expressed in different subtypes of breast cancer. The basal subtype, for example, is negative for ERalpha, PR, and Her2 by immunohistochemistry. We investigated the expression of these signaling molecules in enriched populations of mouse mammary stem cells and luminal cells that were isolated according to their differential expression of CD24 and the alpha6beta1-integrin complex. We found that the basal population, which is enriched in mouse mammary stem cells, did not express ERalpha, PR, or ErbB2/Her2 but did express epidermal growth factor receptor (EGFR)/ErbB1, whereas the subset of cells enriched for luminal cells expressed ERalpha (37% of cells) and PR (40% of cells) but not ErbB2/Her2 or EGFR/ErbB1. Ovariectomy confirmed the importance of estrogen signaling to luminal cell proliferation but had no effect on the size of the mouse mammary stem cell-enriched population. Thus, mouse mammary stem cells were negative for ERalpha, PR, and ErbB2 and appeared to share common properties with poor-prognosis basal breast cancer.     

Identification of signal transduction pathways involved in constitutive NF-kappaB activation in breast cancer cells

Nuclear factor-kappaB (NF-kappaB) is usually maintained in an inactive form in the cytoplasm through its association with inhibitor of kappaB (IkappaB) proteins, and is activated upon stimulation of cells with a variety of signals. However, constitutive activation of NF-kappaB is observed in a number of cancers including breast cancer. The signaling pathways that are involved in constitutive NF-kappaB activation remain largely unknown. Using breast cancer cell lines derived from transgenic mice that overexpress specific oncogene/growth factors in the mammary gland, we show that heregulin but not her2/neu, c-Myc or v-Ha-ras plays a major role in constitutive NF-kappaB activation. Her2/neu potentiated tumor necrosis factor alpha (TNFalpha)-inducible NF-kappaB activation whereas c-Myc potentiated 12-o-tetracecanyolphorbol-13-acetate (TPA)-induced NF-kappaB activation. Heregulin-mediated NF-kappaB activation correlated with phosphorylation of epidermal growth factor receptor (EGFR) and ErbB3 but not her2/neu. Tryphostin AG1517, which inhibits heregulin-mediated phosphorylation of EGFR, her2/neu and ErbB3 reduced NF-kappaB activation. In contrast, emodin, which blocks phosphorylation of her2/neu by heregulin, failed to reduce NF-kappaB activation. These results suggest that heregulin induces NF-kappaB independent of her2/neu. PI3 kinase/AKT, protein kinase A (PKA) and IkappaB kinase appear to be downstream signaling molecules involved in NF-kappaB activation as specific inhibitors of these kinases but not inhibitors of ERK/MAP kinase or protein kinase C reduced heregulin-mediated NF-kappaB activation. Based on these results, we propose that heregulin increases the expression of pro-invasive, pro-metastatic and anti-apoptotic genes in cancer cells through autocrine activation of NF-kappaB, which leads to invasive and drug-resistant growth of breast cancer.     

Ras oncogene directs expression of a differentially sialylated, functionally altered beta1 integrin

Intense investigation has centered on understanding the regulation of integrin cell adhesion receptors. In the present study, we propose that variant N-glycosylation represents an important mechanism for regulation of beta1, but not beta3 or beta5 integrins. We find that expression of oncogenic ras in HD3 colonocytes causes increased alpha2-6 sialylation of beta1 integrins, whereas expression of dominant-negative ras induces decreased alpha2-6 sialylation, relative to cells with wild-type ras. In contrast, neither beta3 nor beta5 integrins are alpha2-6 sialylated, regardless of the state of ras activation. Results from RT-PCR analyses suggest that differential integrin sialylation is due to a ras-dependent alteration in the expression of ST6Gal I, the enzyme that adds alpha2-6-linked sialic acids. Cells that express differentially sialylated beta1 integrins exhibit altered adhesion to collagen I (a beta1 ligand), but not to vitronectin (a beta3 or beta5 ligand). Similarly, the enzymatic removal of cell surface sialic acids from control cells alters binding to collagen, but not to vitronectin. Finally, using a cell-free receptor/ligand-binding assay, we show that purified, desialylated alpha1beta1 integrins have diminished collagen-binding capability, providing strong evidence that sialic acids play a causal role in regulating beta1 integrin function.     

Involvement of hepatocyte growth factor in increased integrin expression on HepG2 cells triggered by adhesion to endothelial cells.

Adhesion of cancer cells to vascular endothelium is an important step in haematogenous metastasis of cancer. A human hepatocellular carcinoma cell line, HepG2, strongly adheres to human umbilical vein endothelial cells (HUVECs) through the interaction of E-selectin and its carbohydrate ligand sialyl Lewis X. In this study, we investigated alteration in integrin expression on HepG2 cells, which follows the selectin-mediated initial adhesion of HepG2 cells to HUVECs. Expression of alpha2beta1 integrin was markedly increased when the HepG2 cells adhered to HUVECs. Among the tested cytokines that are known to be produced by endothelial cells, recombinant hepatocyte growth factor (rHGF) could replace the effect of HUVECs, and a similar increase in integrin expression was observed by the addition of 20 ng ml-1 rHGF to HepG2. The increment of alpha2beta1 integrin expression was significantly inhibited by anti-HGF neutralizing antibody treatment. HepG2 cells expressed alpha2, alpha6, beta1, and beta4 integrin subunits, but expression of integrins other than alpha2beta1 was not affected by the rHGF treatment. The rHGF treatment of HepG2 cells resulted in augmented adhesion to immobilized collagen. This augmentation in adhesion to collagen was completely blocked by the addition of anti-alpha2- or anti-beta1-integrin antibody. In double-chamber chemoinvasion experiments, transmigration of the HepG2 cells through extracellular matrix (ECM) gel was significantly accelerated by co-cultivation with HUVECs. A similar level of enhancement in transmigration activity of the cancer cells was observed by the addition of rHGF. Our interpretation of the results described above is that the cancer cells received stimulation from cytokines, such as HGF, presented by vascular endothelial cells, following the initial adhesion of cancer cells via selectins. This resulted in the secondary increment in the expression of cell adhesion molecules, such as the alpha2beta1 integrin, and led to the augmented adhesive activities of cancer cells towards extracellular matrices at vascular walls. We suggest that this sequence of events is involved in the facilitated migration of some cancer cells to extravascular tissues.     

Beta 1 integrin expression on human small cell lung cancer cells.

The integrins are a supergene family of cell surface glycoproteins that promote cellular adhesion. Each member of the family is an alpha/beta heterodimer composed of a distinct alpha subunit noncovalently linked to one of at least six common beta subunits. These include the six beta 1 integrins (alpha 1-6/beta 1) which represent receptors for extracellular matrix proteins and the three beta 2 integrins (alpha L, alpha M, alpha X/beta 2) that are expressed by leukocytes and which bind to C3bi and/or endothelial ligands. Recently, it was reported that certain human tumor cells express the beta 1 integrins and that small cell lung cancer (SCLC) cell lines express the beta 2 integrin Mo1 (alpha M/beta 2). To extend these initial observations, we examined SCLC cell lines for integrin expression at the glycoprotein and mRNA levels and assessed the potential function of these integrins in promoting SCLC adhesion. An indirect immunofluorescence analysis of five SCLC cell lines (NCI-H187, H345, H146, H209, and N417) using alpha and beta subunit-specific monoclonal antibodies demonstrated the uniform expression of beta 1 (beta 1 much greater than beta 2 greater than or equal to beta 3 congruent to beta 4). Among the beta 1-associated alpha subunits, alpha 3 was uniformly expressed at high surface density by all five cell lines (as confirmed in H345 cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of anti-beta 1 and anti-alpha 3 immunoprecipitates), while alpha 5 was not detected. The leukocyte (beta 2-associated) alpha M and alpha L subunits were also variably expressed by the five lines. Consistent with the surface expression of beta 1 integrin gene products, beta 1 (but not beta 2) mRNA was detected in SCLC cells by Northern blot analysis. That beta 1 integrin expression was involved in SCLC adhesion was suggested by the adherence of H345 cells to laminin, a known ligand for the alpha 3 beta 1 integrin. Moreover, an antibody specific for the beta 1 subunit inhibited this adhesion, indicating that the beta 1 subunit promotes adhesion to laminin. We conclude that beta 1 integrin molecules are expressed by human SCLC cells (with uniform expression of alpha 3/beta 1) and promote their adhesion to laminin.     

Increased Constitutive Activity of PKB/Akt in Tamoxifen Resistant Breast Cancer MCF-7 Cells

The tamoxifen-resistant (TAM-R) MCF-7 breast cancer cell line has been used as a model to identify the signalling pathways that enable resistant cancer cells to grow independently of steroid hormones. In TAM-R cells, peptide growth factor signalling pathways appear to be important in modified cell behaviour, growth and survival. The PI3 kinase signalling components Akt1 and Akt2 are expressed at similar levels by both parental wild-type MCF-7 and TAM-R cells, but Akt1 phosphorylation is significantly increased in TAM-R cells grown under basal conditions. High levels of basal Akt, GSK3 alpha / beta and p70S6 kinase phosphorylation are all inhibited by the PI3 kinase inhibitor, LY 294002. The ligands for the EGFR/erbB1 receptor, EGF (epidermal growth factor) and TGF alpha (transforming growth factor- alpha ) demonstrate an increased ability to activate Akt in TAM-R compared with parental MCF-7 cells and it is proposed that the preferred autocrine or paracrine activation of Akt occurs via the erbB heterodimer EGFR/erbB2 in TAM-R cells. Akt phosphorylation is reduced by gefitinib ("Iressa"/ZD1839). The results suggest that the PI3 kinase pathway plays a role in proliferation of TAM-R cells and is important in the increased EGF induced membrane ruffling detected in the resistant cells. Increased Akt1 activation may contribute to the aggressive phenotype of tamoxifen resistant ER (oestrogen receptor) positive breast cancers.     

Osteoblasts-derived TGF-beta1 enhance motility and integrin upregulation through Akt, ERK, and NF-kappaB-dependent pathway in human breast cancer cells

Bone metastases are common complications of breast cancer. Integrins are the major adhesive molecules in mammalian cells. Here we found that osteoblast conditioned medium (OBCM) increased the migration and cell surface expression of beta1 or beta3 integrin in human breast cancer cells (MDA-MB-231 cells). beta1 or beta3 integrin monoclonal antibodies (mAbs) or small interference RNA (siRNA) against beta1 or beta3 integrin inhibited the OBCM-induced increase in the migration of breast cancer cells. Transforming growth factor-beta1 (TGF-beta1) siRNA could remarkably blocked OBCM-induced chemomigration and beta1 and beta3 integrin expression in breast cancer cells. Stimulation of cells with OBCM caused an increase in Akt and extracellular signal-regulated kinase (ERK) phosphorylation in a time-dependent manner. In addition, treatment of MDA-MB-231 cells with phosphatidylinositol 3-kinase inhibitor (LY294002), ERK inhibitor (PD98059), NF-kappaB inhibitor (PDTC), or IkappaB protease inhibitor (TPCK) inhibited OBCM-induced cells migration and integrins expression. Treatment of MDA-MB-231 cells with OBCM induced IkappaB kinase alpha/beta (IKK alpha/beta) phosphorylation, IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. The OBCM-mediated increases in IKK alpha/beta phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity were inhibited by LY294002 and PD98059. In addition, TGF-beta1 siRNA also reduced the OBCM-induced ERK, Akt, IKKalpha/beta, IkappaBalpha, and p65 phosphorylation. Taken together, these results suggest that the osteoblast-derived TGF-beta1 acts through Akt and ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of beta1 and beta3 integrins and contributing the migration of breast cancer cell.     

The Asn418-linked N-glycan of ErbB3 plays a crucial role in preventing spontaneous heterodimerization and tumor promotion

ErbB2 and ErbB3, two members of the ErbB family, form a high-affinity heregulin coreceptor that elicits potent mitogenic and transforming signals, and clinical studies indicate that these receptors play an important role in tumor incidence and progression. To determine whether N-glycosylation is involved in the function of ErbB3, a series of human ErbB3 molecules devoid of N-glycans were prepared and transfected to Flp-In-CHO cells for stable expression. A cross-linking study showed that the Asn(418) to Gln mutant (N418Q) of ErbB3 underwent autodimerization without its ligand, heregulin. The wild-type or N418Q mutant of ErbB3 was next coexpressed with ErbB2 in Flp-In-CHO cells, and the effect of N-glycan on heterodimerization was examined. The N418Q mutant of ErbB3 was autodimerized with ErbB2 without ligand stimulation, and receptor tyrosine phosphorylation and subsequent extracellular signal-regulated kinase (ERK) and Akt phosphorylation were promoted in the absence of heregulin. A cell proliferation assay and a soft agar colony formation assay showed that the N418Q mutant of ErbB3 coexpressed with ErbB2 promoted cell proliferation and colony formation in soft agar in an ERK- and Akt-dependent manner. The mutation also promoted the growth of tumors in athymic mice when injected s.c. These findings suggest that the Asn(418)-linked N-glycan in ErbB3 plays an essential role in regulating receptor heterodimerization with ErbB2 and might have an effect on transforming activity.     

Expression and function of CYR61, an angiogenic factor, in breast cancer cell lines and tumor biopsies

We have previously shown that expression of heregulin (HRG) is closely correlated with breast cancer progression. We have subsequently isolated Cyr61, a ligand for the alpha(v)beta3 integrin that is differentially expressed in HRG-positive cells, and have shown that it is expressed in all of the invasive and metastatic breast cancer cell lines tested. Preliminary evaluation of Cyr61 expression in breast tumor biopsies revealed expression of Cyr61 in about 30% of invasive breast carcinomas. Significantly, we demonstrated that Cyr61 is a downstream effector of HRG action, because a Cyr61-neutralizing antibody abolished the ability of HRG-expressing cells to migrate in vitro. Furthermore, we have shown that HRG-expressing cells denote higher levels of alpha(v)beta3 expression, and we have established that Cyr61 action is mediated, at least in part, through its receptor alpha(v)beta3, because a functional blocking antibody of the alpha(v)beta3 blocked the Matrigel outgrowth of HRG-expressing cells. These results strongly suggest that Cyr61 is necessary for HRG-mediated chemomigration and that Cyr61 plays a functional role in breast cancer progression, possibly through its interactions with the alpha(v)beta3 receptor.