IGFBP-3 Can Either Inhibit or Enhance EGF-mediated Growth of Breast Epithelial Cells Dependent upon the Presence of Fibronectin

Progression of breast cancer is associated with remodeling of the extracellular matrix, often involving a switch from estrogen dependence to a dependence on EGF receptor (EGFR)/HER-2 and is accompanied by increased expression of the main binding protein for insulin-like growth factors (IGFBP-3). We have examined the effects of IGFBP-3 on EGF responses of breast epithelial cells in the context of changes in the extracellular matrix. On plastic and laminin with MCF-10A normal breast epithelial cells, EGF and IGFBP-3 each increased cell growth and together produced a synergistic response, whereas with T47D breast cancer cells IGFBP-3 alone had no effect, but the ability of EGF to increase cell proliferation was markedly inhibited in the presence of IGFBP-3. In contrast on fibronectin with MCF-10A cells, IGFBP-3 alone inhibited cell growth and blocked EGF-induced proliferation. With the cancer cells, IGFBP-3 alone had no effect but enhanced the EGF-induced increase in cell growth. The insulin-like growth factor-independent effects of IGFBP-3 alone on cell proliferation were completely abrogated in the presence of an EGFR, tyrosine kinase inhibitor, Iressa. Although IGFBP-3 did not affect EGFR phosphorylation [Tyr¹⁰⁶⁸], it was found to modulate receptor internalization and was associated with activation of Rho and subsequent changes in MAPK phosphorylation. The levels of fibronectin and IGFBP-3 within breast tumors may determine their dependence on EGFR and their response to therapies targeting this receptor.     

Effect of the tyrosine kinase inhibitor lapatinib on CUB-domain containing protein (CDCP1)-mediated breast cancer cell survival and migration

The surface receptor CUB domain-containing protein 1 (CDCP1) is highly expressed in several adenocarcinomas and speculated to participate in anchorage-independent cell survival and cell motility. Tyrosine kinase phosphorylation seems to be crucial for intracellular signaling of CDCP1. Lapatinib, a tyrosine kinase inhibitor (TKI), is approved for treatment of HER-2/neu overexpressing metastatic breast cancer and functions by preventing autophosphorylation following HER-2/neu receptor activation. This study aimed to investigate the effect of CDCP1 expression on anchorage-independent growth and cell motility of breast cancer cells. Moreover, studies were performed to examine if lapatinib provided any beneficial effect on HER-2/neu^(+)/−/CDCP1⁺ breast cancer cell lines. In our studies, we affirmed that CDCP1 prevents cells from undergoing apoptosis when cultured in the absence of cell–substratum anchorage and that migratory and invasive properties of these cells were decreased when CDCP1 was down-regulated. However, only HER-2/neu⁺, but not HER-2/neu^(+)/− cells showed decreased proliferation and invasion and an enhanced level of apoptosis towards loss of anchorage when treated with lapatinib. Therefore, we conclude that CDCP1 might be involved in regulating adhesion and motility of breast cancer cells but that lapatinib has no effect on tyrosine kinases regulating CDCP1. Nonetheless, other TKIs might offer therapeutic approaches for CDCP1-targeted breast cancer therapy and should be studied considering this aspect.     

Association of ErbB2 Ser1113 phosphorylation with epidermal growth factor receptor co-expression and poor prognosis in human breast cancer

The carboxyterminal domain of the epidermal growth factor receptor (EGFR) – a putative binding site for the ubiquitin ligase Cbl – is the site of serine phosphorylation events which are essential for ligand-dependent EGFR desensitization and degradation. Using a monoclonal antibody (aPS¹¹¹³) which selectively recognizes the homologous phosphorylated domain in the ErbB2 oncoprotein, we show here that wild-type ErbB2 becomes Ser¹¹¹³-phosphorylated following treatment of 3T3 cells with growth factors or tyrosine phosphatase inhibitors. In EGFR-overexpressing A431 cells, ligand-inducible aPS¹¹¹³ immunoreactivity declines more rapidly than other detectable phosphorylation events and is followed by EGFR downregulation. Analysis of 65 ErbB2-expressing primary breast cancers reveals a highly significant relationship between Ser¹¹¹³ phosphorylation and EGFR overexpression (p < 0.0001) as well as an association with poor prognosis (p = 0.005). We submit that ErbB2 Ser¹¹¹³ phosphorylation status represents a novel and informative biomarker of cancer cell biology and tumor behavior.     

Blocking HER-2/HER-3 function with a dominant negative form of HER-3 in cells stimulated by heregulin and in breast cancer cells with HER-2 gene amplification.

Amplification and overexpression of the HER-2 (neu/ erbB-2) gene in human breast cancer are clearly important events that lead to the transformation of mammary epithelial cells in approximately one-third of breast cancer patients. Heterodimer interactions between HER-2 and HER-3 (erbB-3) are activated by neu differentiation factor/heregulin (HRG), and HER-2/HER-3 heterodimers are constitutively activated in breast cancer cells with HER-2 gene amplification. This indicates that inhibition of HER-2/HER-3 heterodimer function may be an especially effective and unique strategy for blocking the HER-2-mediated transformation of breast cancer cells. Therefore, we constructed a bicistronic retroviral expression vector (pCMV-dn3) containing a dominant negative form of HER-3 in which most of the cytoplasmic domain was removed for introduction into cells. By using a bicistronic retroviral vector in which the antibiotic resistance gene and the gene of interest are driven by a single promoter, we attained 100% coordinate coexpression of antibiotic resistance with the gene of interest in target cell populations. Breast carcinoma cells with HER-2 gene amplification (21 MT-1 cells) and normal mammary epithelial cells without HER-2 gene amplification from the same patient (H16N-2 cells) were infected with pCMV-dn3 and assessed for HER-2/ HER-3 receptor tyrosine phosphorylation, p85PI 3-kinase and SHC protein activation, growth factor-dependent and -independent proliferation, and transformed growth in culture. Dominant negative HER-3 inhibited the HRG-induced activation of HER-2/HER-3 and signaling in H16N-2 and 21 MT-1 cells as well as the constitutive activation of HER-2/HER-3 and signaling in 21 MT-1 cells. Responses to exogenous HRG were strongly inhibited by dominant negative HER-3. In contrast, the proliferation of cells stimulated by epidermal growth factor was not apparently affected by dominant negative HER-3. The growth factor-independent proliferation and transformed growth of 21 MT-1 cells were also strongly inhibited by dominant negative HER-3 in anchorage-dependent and independent growth assays in culture. Furthermore, the HRG-induced or growth factor-independent proliferation of 21 MT-1 cells was inhibited by dominant negative HER-3, whereas the epidermal growth factor-induced proliferation of these cells was not: this indicates that dominant negative HER-3 preferentially inhibits proliferation induced by HER-2/HER-3.     

Mammaglobin,GATA-binding protein 3 (GATA3), and epithelial growth factor receptor (EGFR) expression in different breast cancer subtypes and their clinical significance

Increasing evidence has shown that mammaglobin, GATA-binding protein 3 (GATA3), and epithelial growth factor receptor (EGFR) have unique clinical implications for breast cancer subtyping and classification, as well as for breast cancer targeted therapy. It is particularly important to clarify the correlation between their expression and different molecular breast carcinoma subtypes to better understand the molecular basis of the subtypes and to identify effective therapeutic targets for the disease. This study aimed to evaluate mammaglobin, GATA3, and EGFR expression in different breast cancer subtypes, as well as their clinical significance. Subjects of the study included 228 patients with breast cancer at The First Affiliated Hospital of University of Science and Technology of China. They were divided into triple negative (TN), Luminal A, Luminal B, and HER-2 positive (HER-2.P) breast cancer groups based on molecular classification. Immunohistochemical methods were used to detect mammaglobin, GATA3, and EGFR expression in cases of different molecular subtypes before determining the correlation between protein expression and subtype. Mammaglobin and GATA3 expression levels were found to significantly vary with respect to histopathological grade, lymph node status, and molecular subtype; EGFR expression was significantly correlated with breast cancer histopathological grade and molecular subtype. For breast cancer, the expression levels of mammaglobin and GATA3, as well as mammaglobin and EGFR, were significantly correlated. In addition, there was a significantly negative correlation between the expression levels of GATA3 and EGFR in breast cancer tissue samples, especially in HER-2.P samples. These findings provide a theoretical basis for assessing breast cancer clinical prognosis based on the cancer subtype, and hence, have significant practical value.Key words: breast cancer, mammaglobin, GATA-binding protein 3, epithelial growth factor receptor, molecular subtype     

EGFR,HER-2 and KRAS in Canine Gastric Epithelial Tumors: A Potential Human Model?

Epidermal growth factor receptor (EGFR or HER-1) and its analog c-erbB-2 (HER-2) are protein tyrosine kinases correlated with prognosis and response to therapy in a variety of human cancers. KRAS mediates the transduction of signals between EGFR and the nucleus, and its mutation has been identified as a predictor of resistance to anti-EGFR drugs. In human oncology, the importance of the EGFR/HER-2/KRAS signalling pathway in gastric cancer is well established, and HER-2 testing is required before initiating therapy. Conversely, this pathway has never been investigated in canine gastric tumours. A total of 19 canine gastric epithelial neoplasms (5 adenomas and 14 carcinomas) were retrospectively evaluated for EGFR/HER-2 immunohistochemical expression and KRAS mutational status. Five (35.7%) carcinomas were classified as intestinal-type and 9 (64.3%) as diffuse-type. EGFR was overexpressed (≥1+) in 8 (42.1%) cases and HER-2 (3+) in 11 (57.9%) cases, regardless of tumour location or biological behaviour. The percentage of EGFR-positive tumours was significantly higher in the intestinal-type (80%) than in the diffuse-type (11.1%, p = 0.023). KRAS gene was wild type in 18 cases, whereas one mucinous carcinoma harboured a point mutation at codon 12 (G12R). EGFR and HER-2 may be promising prognostic and therapeutic targets in canine gastric epithelial neoplasms. The potential presence of KRAS mutation should be taken into account as a possible mechanism of drug resistance. Further studies are necessary to evaluate the role of dog as a model for human gastric cancer.     

The epidermal growth factor receptor (EGFR) is proteolytically modified by the Matriptase-Prostasin serine protease cascade in cultured epithelial cells

Prostasin is expressed at the apical surface of normal epithelial cells and suppresses in vitro invasion of cancer cells. Prostasin re-expression in the PC-3 prostate carcinoma cells down-regulated the epidermal growth factor receptor (EGFR) protein expression and EGF-induced phosphorylation of the extracellular signal-regulated kinases (Erk1/2). We report here that prostasin and its activating enzyme matriptase are capable of inducing proteolytic cleavages in the EGFR extracellular domain (ECD) when co-expressed in the FT-293 cells, generating two amino-terminally truncated fragments EGFR135 and EGFR110, at 135 and 110 kDa. Prostasin's role in EGFR cleavage is dependent on the serine active-site but not the GPI-anchor. The modifications of EGFR were confirmed to be on the primary structure by deglycosylation. EGFR135 and EGFR110 are not responsive to EGF stimulation, indicating loss of the ligand-binding domains. EGFR110 is constitutively phosphorylated and in its presence Erk1/2 phosphorylation is increased in the absence of EGF. The protease-induced EGFR cleavages are not dependent on EGFR phosphorylation. The EGFR ECD proteolytic modification by matriptase-prostasin is also observed in the BEAS-2B normal lung epithelial cells, the BPH-1 benign prostate hyperplasia and the MDA-MB-231 breast cancer cell lines; and represents a novel mechanism for epithelial cells to modulate EGF-EGFR signaling.     

Targeting the EGFR/PCNA Signaling Suppresses Tumor Growth of Triple-Negative Breast Cancer Cells with Cell-Penetrating PCNA Peptides

Tyrosine 211 (Y211) phosphorylation of proliferation cell nuclear antigen (PCNA) coincides with pronounced cancer cell proliferation and correlates with poor survival of breast cancer patients. In epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)-resistant cells, both nuclear EGFR (nEGFR) expression and PCNA Y211 phosphorylation are increased. Moreover, the resistance to EGFR TKI is a major clinical problem in treating EGFR-overexpressing triple-negative breast cancer (TNBC). Thus, effective treatment to combat resistance is urgently needed. Here, we show that treatment of cell-penetrating PCNA peptide (CPPP) inhibits growth and induces apoptosis of human TNBC cells. The Y211F CPPP specifically targets EGFR and competes directly for PCNA tyrosine Y211 phosphorylation and prevents nEGFR from binding PCNA in vivo; it also suppresses tumor growth by sensitizing EGFR TKI resistant cells, which have enhanced nEGFR function and abrogated classical EGFR membrane signaling. Furthermore, we identify an active motif of CPPP, RFLNFF (RF6 CPPP), which is necessary and sufficient to inhibit TKI-resistant TNBC cell growth of orthotopic implanted tumor in mice. Finally, the activity of its synthetic retro-inverted derivative, D-RF6 CPPP, on an equimolar basis, is more potent than RF6 CPPP. Our study reveals a drug candidate with translational potential for the future development of safe and effective therapeutic for EGFR TKI resistance in TNBC.     

Insulin Induces Swelling-dependent Activation of the Epidermal Growth Factor Receptor in Rat Liver

The aim of the study was to analyze whether the proliferative effects of insulin in rat liver involve cross-signaling toward the epidermal growth factor receptor (EGFR) and whether this is mediated by insulin-induced hepatocyte swelling. Studies were performed in the perfused rat liver and in primary rat hepatocytes. Insulin (35 nmol/liter) induced phosphorylation of the EGFR at position Tyr⁸⁴⁵ and Tyr¹¹⁷³, but not at Tyr¹⁰⁴⁵, suggesting that EGF is not involved in insulin-induced EGFR activation. Insulin-induced EGFR phosphorylation and subsequent ERK1/2 phosphorylation were sensitive to bumetanide, indicating an involvement of insulin-induced hepatocyte swelling. In line with this, hypoosmotic (225 mosmol/liter) hepatocyte swelling also induced EGFR and ERK1/2 activation. Insulin- and hypoosmolarity-induced EGFR activation were sensitive to inhibition by an integrin-antagonistic RGD peptide, an integrin β1 subtype-blocking antibody, and the c-Src inhibitor PP-2, indicating the involvement of the recently described integrin-dependent osmosensing/signaling pathway (Schliess, F., Reissmann, R., Reinehr, R., vom Dahl, S., and Häussinger, D. (2004) J. Biol. Chem. 279, 21294–21301). As shown by immunoprecipitation studies, insulin and hypoosmolarity induced a rapid, RGD peptide-, integrin β1-blocking antibody and PP-2-sensitive association of c-Src with the EGFR. As for control, insulin-induced insulin receptor substrate-1 phosphorylation remained unaffected by the RGD peptide, PP-2, or inhibition of the EGFR tyrosine kinase activity by AG1478. Both insulin and hypoosmolarity induced a significant increase in BrdU uptake in primary rat hepatocytes, which was sensitive to RGD peptide-, integrin β1-blocking antibody, PP-2, AG1478, and PD098059. It is concluded that insulin- or hypoosmolarity-induced hepatocyte swelling triggers an integrin- and c-Src kinase-dependent EGFR activation, which may explain the proliferative effects of insulin.     

ERK/RSK-mediated phosphorylation of Y-box binding protein-1 aggravates diabetic cardiomyopathy by suppressing its interaction with deubiquitinase OTUB1

Diabetic cardiomyopathy (DCM) is a major complication of diabetes, but its underlying mechanisms still remain unclear. The multifunctional protein Y-box binding protein-1 (YB-1) plays an important role in cardiac pathogenesis by regulating cardiac apoptosis, cardiac fibrosis, and pathological remodeling, whereas its role in chronic DCM requires further investigation. Here, we report that the phosphorylation of YB-1 at serine102 (S102) was markedly elevated in streptozotocin-induced diabetic mouse hearts and in high glucose-treated cardiomyocytes, whereas total YB-1 protein levels were significantly reduced. Coimmunoprecipitation experiments showed that YB-1 interacts with the deubiquitinase otubain-1, but hyperglycemia-induced phosphorylation of YB-1 at S102 diminished this homeostatic interaction, resulting in ubiquitination and degradation of YB-1. Mechanistically, the high glucose-induced phosphorylation of YB-1 at S102 is dependent on the upstream extracellular signal-regulated kinase (ERK)/Ras/mitogen-activated protein kinase (p90 ribosomal S6 kinase [RSK]) signaling pathway. Accordingly, pharmacological inhibition of the ERK pathway using the upstream kinase inhibitor U0126 ameliorated features of DCM compared with vehicle-treated diabetic mice. We demonstrate that ERK inhibition with U0126 also suppressed the phosphorylation of the downstream RSK and YB-1 (S102), which stabilized the interaction between YB-1 and otubain-1 and thereby preserved YB-1 protein expression in diabetic hearts. Taken together, we propose that targeting the ERK/RSK/YB-1 pathway could be a potential therapeutic approach for treating DCM.     

De novo and cell line models of human mammary cell transformation reveal an essential role for Yb-1 in multiple stages of human breast cancer

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRAS^G12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.Subject terms: Cancer models, Metastasis, Oncogenes     

Progesterone Receptor A Stability Is Mediated by Glycogen Synthase Kinase-3β in the Brca1-deficient Mammary Gland

Germ line mutations of the BRCA1 gene increase the risk of breast and ovarian cancer, but the basis of this tissue-specific tumor predisposition is not fully understood. Previously, we reported that the progesterone receptors are stabilized in Brca1-deficient mammary epithelial cells, and treating with anti-progesterone delays mammary tumorigenesis in Brca1/p53 conditional knock-out mice, suggesting that the progesterone has a critical role in breast carcinogenesis. To further explore how the stability of progesterone receptor is modulated, here, we have found that glycogen synthase kinase (GSK)-3β phosphorylation of progesterone receptor-A (PR-A) facilitates its ubiquitination. GSK-3β-mediated phosphorylation of serine 390 in PR-A regulates its subsequent ubiquitination and protein stability. Expression of PR-A^S390A mutant in the human breast epithelial cells, MCF-10A, results in enhanced proliferation and formation of aberrant acini structure in the three-dimensional culture. Consistently, reduction of phosphorylation of serine 390 of PR-A and GSK-3β activity is observed in the Brca1-deficient mammary gland. Taken together, these results provide important aspects of tissue specificity of BRCA1-mediated suppression of breast carcinogenesis.     

Alterations in both Heparan Sulfate Proteoglycans and Mitogenic Activity of Fibroblast Growth Factor-2 Are Triggered by Inhibitors of Proliferation in Normal and Breast Cancer Epithelial Cells

Heparan sulfate proteoglycans (HSPG) are involved in the regulation of cellular proliferation, differentiation, and migration. We have studied the effect of three inhibitors of proliferation on³⁵S incorporation into HSPG of the breast cancer cell lines MCF-7 and MDA-MB-231 and the normal breast epithelial cells (NBEC). Transforming growth factor β-1 (TGFβ-1), which inhibits the proliferation of NBEC, but not of MCF-7 and MDA-MB-231, cells induced an increase in³⁵S incorporation of HSPG in NBEC, but had no effect on cancer cells. Sodium butyrate (NaB), which inhibits NBEC as well as cancer cell proliferation, induced an increase in³⁵S incorporation into HSPG in all cell types studied. In contrast, retinoic acid had no effect on HSPG of breast epithelial cells. Modification of HSPG induced by TGFβ-1 or NaB treatments in normal and breast cancer epithelial cells resulted in an increase in¹²⁵I-fibroblast growth factor-2 (FGF-2) binding on HSPG. More importantly, NaB pretreatment resulted in an inhibition of the MCF-7 cell responsiveness to FGF-2, even though these cells remained sensitive to growth stimulation induced by serum or epidermal growth factor. These results indicate that changes in HSPG production are a key process involved in the mechanism of breast epithelial cell growth regulation.     

UVC radiation induces downregulation of EGF receptor via phosphorylation at serine 1046/1047 in human pancreatic cancer cells

Epidermal growth factor receptor (EGFR) is overexpressed in human pancreatic cancer and is one of the clinical targets in its treatment. In the present study we investigated the mechanism underlying ultraviolet C (UVC)-radiation-induced cell growth inhibition and downregulation of EGFR in human pancreatic cancer cells (Panc1 and KP3). The cell proliferation assay indicated that Panc1 and KP3 cells were more sensitive to UVC radiation, and their growth was significantly inhibited compared to cells of the normal human pancreatic epithelial cell line, PE. Although EGFR levels was extremely low in PE cells, EGFR were highly overexpressed in Panc1 and KP3 cells, and UVC radiation downregulated the expression of EGFR in a time-dependent manner and induced phosphorylation of EGFR at Ser1046/1047 (S1046/7) in Panc1 and KP3 cells. UVC radiation induced activation of p38 mitogen-activated protein kinase (MAPK), and EGFR phosphorylation at S1046/7 induced by UVC radiation was markedly attenuated by the inhibition of p38 MAPK. Moreover, fluorescence microscopy revealed that p38 MAPK activated by UVC radiation triggered EGFR internalization and that this was not correlated with c-Cbl, an ubiquitin ligase, which plays an important role in EGF-induced EGFR downregulation. Taken together, our results suggest that in pancreatic cancer cells UVC radiation induced desensitization of the cells to EGFR stimuli via phosphorylation of EGFR at S1046/7 by activation of p38 MAPK, independent of c-Cbl.     

Resveratrol inhibits heregulin-beta1-mediated matrix metalloproteinase-9 expression and cell invasion in human breast cancer cells

The growth factor heregulin-β1 (HRG-β1), which is expressed in breast cancer, activates the HER-2 signaling pathway through induction of heterodimeric complexes of HER-2 with HER-3 or HER-4. It has been shown in many studies that HRG-β1 induces the tumorigenicity and metastasis of breast cancer cells. Matrix metalloproteinase (MMP) 9 is a key enzyme in the degradation of extracellular matrices, and its expression may be dysregulated in breast cancer invasion and metastasis. Resveratrol, a major component in grape, exhibited potential anticarcinogenic activities in both in vitro and in vivo studies. However, the inhibitory effect of resveratrol on HER-2-mediated expression of MMP-9 has not been demonstrated yet.

In the present study, we investigated the anti-invasive mechanism of resveratrol in human breast cancer cells. Human breast cancer MCF-7 cells were exposed to resveratrol (2, 5 and 10 μM). The expression activity of MMP-9 was measured by zymogram analysis. Phosphorylated levels of HER-2 and mitogen-activated protein kinase (MAPK)/ERK were measured by Western blot analysis. Total actin was used as internal control for protein expression. HRG-β1 induced the phosphorylation of HER-2/neu receptor and MMP-9 expression in human breast cancer MCF-7 cells. Resveratrol significantly inhibited HRG-β1-mediated MMP-9 expression in human breast cancer cells. MEK inhibitor induced a marked reduction in MMP-9 expression, and it suggested that ERK1/2 cascade could play an important role in HRG-β1-mediated MMP-9 expression. Furthermore, resveratrol significantly suppressed HRG-β1-mediated phosphorylation of ERK1/2 and invasion of breast cancer cells. However, resveratrol had negligible effects on either HRG-β1-mediated phosphorylation of HER-2 receptor or expression of the tissue inhibitor of MMP, tissue inhibitor metalloproteinase protein 1.

Taken together, our results suggest that resveratrol inhibited MMP-9 expression in human breast cancer cells. The inhibitory effects of resveratrol on MMP-9 expression and invasion of breast cancer cells are, in part, associated with the down-regulation of the MAPK/ERK signaling pathway.     

CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777–788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.     

A CD80-transfected human breast cancer cell variant induces HER-2/neu–specific T cells in HLA-A*02–matched situations in vitro as well as in vivo

Adjuvant treatment is still only working in a small percentage of breast cancer patients. Therefore, new strategies need to be developed. Immunotherapies are a very promising approach because they could successfully attack tumor cells in the stage of dormancy. To assess the feasibility of using an allogeneic approach for vaccination of breast cancer patients, we selected a CD80-transfected breast cancer cell line based on its immunogenic properties. Using CD80⁺ KS breast cancer cells and human leukocyte antigen (HLA)-A*02–matched peripheral blood mononuclear cells (PBMCs) of breast cancer patients in allogeneic mixed lymphocyte–tumor cell cultures (MLTCs), it was possible to isolate HLA-A*02–restricted cytotoxic T cells (CTLs). Furthermore, a genetically modified KS variant expressing influenza A matrix protein serving as a surrogate tumor-associated antigen (TAA) was able to stimulate flu peptide-specific T cells alongside the induction of alloresponses in MLTCs. KS breast cancer cells were demonstrated to express already known TAAs such as CEA, MUC-1, MAGE-1, MAGE-2, and MAGE-3. To further improve antigenicity, HER-2/neu was added to this panel as a marker antigen known to elicit HLA-A*02–restricted CTLs in patients with breast cancer. Thus, the antigen-processing and antigen-presentation capacity of KS cells was further demonstrated by the stimulation of HER-2/neu–specific CD8⁺ T cells in PBMCs of breast cancer patients in vitro. These results gave a good rationale for a phase I/II trial, where the CD80⁺ HER-2/neu–overexpressing KS variant is actually used as a cellular vaccine in patients with metastatic breast cancer. As a proof of principle, we present data from two patients where a significant increase of interferon- (IFN-) release was detected when postvaccination PBMCs were stimulated by allogeneic vaccine cells as well as by HLA-A*02–restricted HER-2/neu epitopes. In whole cell vaccine trials, monitoring is particularly challenging because of strong alloresponses and limited knowledge of TAAs. In this study, a panel of HER-2/neu epitopes, together with the quantitative real time (qRT)-PCR method to analyze vaccine-induced cytokines secreted by T cells, proved to be highly sensitive and feasible to perform an immunological staging following vaccination.