Novel mode of ligand recognition by the Erbin PDZ domain

Erbin contains a class I PDZ domain that binds to the C-terminal region of the receptor tyrosine kinase ErbB2, a class II ligand. The crystal structure of the human Erbin PDZ bound to the peptide EYLGLDVPV corresponding to the C-terminal residues 1247-1255 of human ErbB2 has been determined at 1.25-A resolution. The Erbin PDZ deviates from the canonical PDZ fold in that it contains a single alpha-helix. The isopropyl group of valine at position -2 of the ErbB2 peptide interacts with the Erbin Val(1351) and displaces the peptide backbone away from the alpha-helix, elucidating the molecular basis of class II ligand recognition by a class I PDZ domain. Strikingly, the phenolic ring of tyrosine -7 enters into a pocket formed by the extended beta 2-beta 3 loop of the Erbin PDZ. Phosphorylation of tyrosine -7 abolishes this interaction but does not affect the binding of the four C-terminal peptidic residues to PDZ, as revealed by the crystal structure of the Erbin PDZ complexed with a phosphotyrosine-containing ErbB2 peptide. Since phosphorylation of tyrosine -7 plays a critical role in ErbB2 function, the selective binding and sequestration of this residue in its unphosphorylated state by the Erbin PDZ provides a novel mechanism for regulation of the ErbB2-mediated signaling and oncogenicity.     

Erbin plays a critical role in human umbilical vein endothelial cell migration and tubular structure formation via the Smad1/5 pathway

Angiogenesis is an important process in atherosclerosis. ErbB2 was proved to have an important role in vascular development, but it is still unclear whether Erbin expresses in vessels as well as its location and function in the vessels. In the current study, we investigated the location and function of Erbin in human umbilical veins. The human umbilical veins were prepared, and immunofluorescent analysis was performed to determine the expression of Erbin. Human umbilical vein endothelial cells (HUVECs) were cultured and the lentivirus (LV) containing Erbin RNAi was also prepared. After transfection with the lentivirus, CCK-8 assay and Annexin V-PI assay were used for cell proliferation and apoptosis, respectively. Cell migration was studied using the scratch wound healing assay and the transwell assay. The capillary-like tube formation assay was performed to illustrate the effect of Erbin on HUVEC tube formation. Expression of signaling pathway molecules was assessed with Western blot. The immunofluorescent analysis suggested that Erbin expressed in human umbilical veins and the majority of the Erbin is strongly colocalized in endothelial cells. Although knockdown of Erbin did not affect HUVEC proliferation and apoptosis, it significantly suppressed HUVEC migration and tubular structure formation. Erbin knockdown showed no effect on the ERK1/2 and Smad2/3 signaling pathways but significantly promoted Smad1/5 phosphorylation and nuclear translocation. Ablation of the Smad1/5 pathway decreased the effects of Erbin on endothelial cells. Erbin is mainly localized in endothelial cells in human umbilical veins and plays a critical role in endothelial cell migration and tubular formation via the Smad1/5 pathway.     

A Dominant Role for p53-Dependent Cellular Senescence in Radiosensitization of Human Prostate Cancer Cells

Amplification and overexpression of ErbB2 (HER2/Neu) is one of the most common alterations associated with breast cancer. Activation of ErbB2 via homodimerization in a non-transformed human mammary epithelial cell line, MCF-10A, in basement membrane cultures leads to formation of proliferative structures that share properties with non-invasive early stage lesions. Recently, we have shown that activation of ErbB2 homodimers combined with expression of transforming growth factor (TGF)-beta induces invasive and migratory activity in MCF-10A cells. In this system, migration requires inputs from numerous cellular pathways. We discuss this data and a model for migration induced by ErbB2 and TGF-beta. Concurrent studies by other groups have also shown that ErbB2 and TGF-beta can cooperate to increase metastatic and invasive behavior in murine mammary tumors. Here we discuss these studies and the potential implications of this research on breast cancer therapeutics.     

ErbB2 and TGF-beta: a cooperative role in mammary tumor progression?

Amplification and overexpression of ErbB2 (HER2/Neu) is one of the most common alterations associated with breast cancer. Activation of ErbB2 via homodimerization in a non-transformed human mammary epithelial cell line, MCF-10A, in basement membrane cultures leads to formation of proliferative structures that share properties with non-invasive early stage lesions. Recently, we have shown that activation of ErbB2 homodimers combined with expression of transforming growth factor (TGF)-beta induces invasive and migratory activity in MCF-10A cells. In this system, migration requires inputs from numerous cellular pathways. We discuss this data and a model for migration induced by ErbB2 and TGF-beta. Concurrent studies by other groups have also shown that ErbB2 and TGF-beta can cooperate to increase metastatic and invasive behavior in murine mammary tumors. Here we discuss these studies and the potential implications of this research on breast cancer therapeutics.     

Hyaluronan constitutively regulates ErbB2 phosphorylation and signaling complex formation in carcinoma cells

Hyaluronan is enriched in many types of human cancers, and manipulations of hyaluronan expression or interactions have a major influence on tumor progression in animal models. Increased ErbB2 activity is characteristic of several cancers and is responsible for many aspects of malignant cell behavior in these cancers. In this study we show that constitutively high levels of active, i.e. autophosphorylated, ErbB2 in HCT116 colon carcinoma cells and TA3/St mammary carcinoma cells are dependent on endogenous hyaluronan-CD44 interaction. Dependence on hyaluronan-CD44 interaction was demonstrated by the administration of hyaluronan oligomers, experimentally induced expression of soluble CD44, and small interfering RNA knockdown of CD44 expression. On the other hand, increasing hyaluronan production by overexpression of hyaluronan synthase 2 or emmprin causes elevated ErbB2 phosphorylation in MCF-7 mammary carcinoma cells, which normally exhibit low levels of ErbB2 activity. Furthermore, in HCT116 and TA3/St cells, inhibition of endogenous hyaluronan-CD44 interaction causes disassembly of a constitutive, lipid raft-associated, signaling complex containing phosphorylated ErbB2, CD44, ezrin, phosphoinositide 3-kinase, and the chaperone molecules, Hsp90 and cdc37. Stimulation of hyaluronan production in MCF-7 cells induces assembly of this complex. We conclude that hyaluronan regulates ErbB2 activity and its interactions with other signaling factors in carcinoma cells.     

Estradiol rapidly activates Akt via the ErbB2 signaling pathway

Previously, we have demonstrated that the two mitogenic growth factors epidermal growth factor and IGF-I can activate Akt and estrogen receptor-alpha (ERalpha) in the hormone-dependent breast cancer cell line, MCF-7. In this report we now show that estradiol can also rapidly activate phosphatidylinositol 3-kinase (PI 3-K)/Akt and that this effect is mediated by the ErbB2 signaling pathway. Treatment of cells with estradiol resulted in phosphorylation of Akt and a 9-fold increase in Akt activity in 10 min. Akt activation was blocked by wortmannin and LY 294,002, two inhibitors of PI 3-K; by genistein, a protein tyrosine kinase inhibitor and an ER agonist; by AG825, a selective ErbB2 inhibitor; and by the antiestrogens ICI 182,780 and 4-hydroxy-tamoxifen; but not by rapamycin, an inhibitor of the ribosomal protein kinase p70S6K; nor by AG30, a selective epidermal growth factor receptor inhibitor. Akt activation by estradiol was abrogated by an arginine-to-cysteine mutation in the pleckstrin homology domain of Akt (R25C). Growth factors also activated Akt in the ER-negative variant of MCF-7, MCF-7/ADR, but estradiol did not induce Akt activity in these cells. Transient transfection of ERalpha into these cells restored Akt activation by estradiol, suggesting that estradiol activation of Akt requires the ERalpha. Estradiol did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. In vitro kinase assays using immunoprecipitation and anti-Akt1, -Akt2, and -Akt3-specific antibodies demonstrated that Akt1 is activated by estradiol in MCF-7 cells whereas Akt3 is the activated isoform in ER-negative MDA-MB231 cells, implying that selective activation of Akt subtypes plays a role in the actions of estradiol. Taken together, our data suggest that estradiol, bound to membrane ERalpha, interacts with and activates an ErbB dimer containing ErbB2, inducing activation of PI 3-K/Akt.     

Overexpression of ErbB2 receptor inhibits IGF-I-induced Shc-MAPK signaling pathway in breast cancer cells

Overexpression of the ErbB2 receptor in one-third of human breast cancers contributes to the transformation of epithelial cells and predicts poor prognosis for breast cancer patients. We report that the overexpression of ErbB2 inhibits IGF-I-induced MAPK signaling. IGF-I-induced MAPK phosphorylation and MAPK kinase activity are reduced in ErbB2 overexpressing MCF-7/HER2-18 cells relative to control MCF-7/neo cells. In SKBR3/IGF-IR cells, reduction of ErbB2 by antisense methodology restores the IGF-I-induced MAPK activation. The inhibition of IGF-I-induced MAP kinase activation in ErbB2 overexpressing breast cancer cells is correlated with decreased IGF-I-induced Shc tyrosine-phosphorylation, leading to a decreased association of Grb2 with Shc and decreased Raf phosphorylation. However, IGF-I-induced tyrosine-phosphorylation of IGF-I receptor and IRS-I and AKT phosphorylation were unaffected by ErbB2 overexpression. Consistent with these results, we observed that the proportion of IGF-I-stimulated proliferation blocked by the MAPK inhibitor PD98059 fell from 82.6% in MCF-7/neo cells to 41.2% in MCF-7/HER2-18 cells. These data provide evidence for interplay between the IGF-IR and ErbB2 signaling pathways. They are consistent with the view that the IGF-IR mediated attenuation of trastuzumab-induced growth inhibition we recently described is dependent on IGF-I-induced PI3K signaling rather than IGF-I-induced MAPK signaling.     

The inhibitory effect of ErbB2 on epidermal growth factor-induced formation of clathrin-coated pits correlates with retention of epidermal growth factor receptor-ErbB2 oligomeric complexes at the plasma membrane

By constructing stably transfected cells harboring the same amount of epidermal growth factor (EGF) receptor (EGFR), but with increasing overexpression of ErbB2, we have demonstrated that ErbB2 efficiently inhibits internalization of ligand-bound EGFR. Apparently, ErbB2 inhibits internalization of EGF-bound EGFR by constitutively driving EGFR-ErbB2 hetero/oligomerization. We have demonstrated that ErbB2 does not inhibit phosphorylation or ubiquitination of the EGFR. Our data further indicate that the endocytosis deficiency of ErbB2 and of EGFR-ErbB2 heterodimers/oligomers cannot be explained by anchoring of ErbB2 to PDZ-containing proteins such as Erbin. Instead, we demonstrate that in contrast to EGFR homodimers, which are capable of inducing new clathrin-coated pits in serum-starved cells upon incubation with EGF, clathrin-coated pits are not induced upon activation of EGFR-ErbB2 heterodimers/oligomers.     

5-FdUrd-araC heterodinucleoside re-establishes sensitivity in 5-FdUrd- and AraC-resistant MCF-7 breast cancer cells overexpressing ErbB2

ErbB2 overexpressing breast tumors have a poor prognosis and a high risk to develop chemoresistance to therapeutic treatment. "Chemoresistance" is a response of cells to toxic stress, and, although it is a common phenomenon, it is still poorly defined. However, a detailed understanding is required to target desensitized pathways and mechanisms for successful reactivation as part of a tailored therapy. To gain insight, which malfunctions contribute to chemoresistance, two mechanisms relevant for tissue homeostasis, the regulation of the cell cycle and of apoptosis, were investigated. Maternal MCF-7- and ErbB2-overexpressing MCF-7(erbB2) breast cancer cells were long term pretreated with 2'-deoxy-5-fluorodeoxyuridine (5-FdUrd) or 1-beta-d-arabinofuranosylcytosine (AraC) and the acquisition of drug-insensitivity was analyzed. A phosphate-conjugated heterodinucleoside consisting of one 5-FdUrd- and one AraC-moiety (5-fluoro-2'-desoxyuridylyl-(3'-->5')-Arabinocytidine) was utilized as a tool to assess the type of acquired resistances. ErbB2-overexpression disrupted proper cell cycle regulation and furthermore facilitated the development of an apoptosis-refractory phenotype upon exposure to 5-FdUrd. Experiments with dimer 5-FdUrd-araC in ErbB2-overexpressing MCF-7(erbB2) cells, and also with nucleoside 5-FdUrd in maternal MCF-7 cells, evidenced that the phenotypes of resistance to cell cycle inhibition and to apoptosis induction were differently affected. The expression profile of cyclin D1 (but not that of p53, p21, or p27) correlated with the proliferative phenotypes and nuclear accumulation of apoptosis inducing factor (but not activation of caspase 7) with apoptotic phenotypes. Dimer 5-FdUrd-araC overrode acquired chemoresistances, whereas combined application of 5-FdUrd and AraC exhibited significantly less activity. Dimer 5-FdUrd-araC remained active in MCF-7 clones most likely by circumventing the prerequisite of first-step phosphorylation. The acquisition of chemoresistance encompassed the affection of apoptosis- and cell-cycle regulation to, respectively, different extents. Thus, drug-induced cell cycle arrest and apoptosis induction are independent of each other.     

Enhanced drug resistance in cells coexpressing ErbB2 with EGF receptor or ErbB3

Overexpression of ErbB2 has been found in approximately 25-30% of human breast cancers and has been shown to render the cancer cells more resistant to chemotherapy. However, it is not clear whether ErbB2 overexpression renders the cells more resistant to specific anti-cancer drugs or renders the cells more resistant to a broad range of anti-cancer drugs. It is not clear how the function of ErbB2 in drug resistance is related to expression and activation of the other ErbB receptors. In this communication, we showed that several breast cancer cell lines including BT20, BT474, MCF-7, MDA-MB-453, and SKBR-3 cells had a similar pattern of resistance to a broad range of anti-cancer drugs including 5-Fluorouracil, Cytoxan, Doxorubincin, Taxol, and Vinorelbin, suggesting a mechanism of multidrug resistance. High expression of P-glycoprotein and the ErbB receptors contribute to drug resistance of these breast cancer cells; however, overexpression of ErbB2 alone is not a major factor in determining drug resistance. To further determine the role of the ErbB receptors in drug resistance, we selected various NIH 3T3 cell lines that specifically expressed EGF receptor (EGFR), ErbB2, ErbB3, EGFR/ErbB2, EGFR/ErbB3, or ErbB2/ErbB3. A cytotoxicity assay showed that expression of ErbB2 alone did not significantly enhance drug resistance, whereas coexpression of either EGFR or ErbB3 with ErbB2 significantly enhanced drug resistance. Moreover, ErbB2 was highly phosphorylated in NIH 3T3 cells that coexpress ErbB2 with either EGFR or ErbB3, but not in NIH 3T3 cells that express ErbB2 alone. Together, our results suggest that coexpression of EGFR or ErbB3 with ErbB2 induces high phosphorylation of ErbB2 and renders the cells more resistant to various anti-cancer drugs.     

高表达人核糖核酸酶抑制因子的乳腺癌细胞株的建立及其鉴定

目的建立稳定高表达人核糖核酸酶抑制因子（hRI）的真核细胞系：乳腺癌细胞系,为进一步研究hRI抗肿瘤、抗氧化的作用机制奠定实验基础。方法将hRI通过逆转录病毒载体（pLNCX-hRI）经过病毒包装细胞（PA317）包装后的高滴度病毒上清,感染乳腺癌（MCF-7）细胞系,经G418筛选后,运用RT-PCR、Western blot等方法进行鉴定hRI在MCF-7中的高表达。结果hRI克隆到乳腺癌细胞（MCF-7）基因组,并随着基因组稳定高表达hRI。结论利用逆转录病毒载体感染真核细胞后获得高表达hRI的肿瘤细胞株,从而为进一步研究真核细胞内hRI的抗肿瘤作用机制提供条件。     

Highly altered protein expression profile in the adriamycin resistant MCF-7 cell line

The protein expression pattern in the cytosol fraction of the adriamycin resistant MCF-7 cell line (MCF-7/ADR) was compared to that of the parental MCF-7 cell line using two-dimensional gel electrophoresis and mass spectrometry. Twenty proteins with altered abundances were identified and studied in MCF-7/ADR. Both up regulation and down regulation are characterized. The most striking differences were found for proteins that were uniquely expressed in this cell line and not detectable in the parental MCF-7 cell line. These proteins include annexin I, the neuronal ubiquitin carboxyl hydrolase isoenzyme L-1 (also known as PGP9.5), glutathione-S-transferase pi class, nicotinamide N-methyltransferase, and interleukin-18 precursor. On the other hand, catechol-O-methyltransferase was expressed in the parental cell line, but was not detected in the adriamycin resistant cell line. This protein expression pattern was unique to MCF-7/ADR and not observed in MCF-7 cell lines selected for resistant to etoposide, mitoxantrone or melphalan.     

Expression Profiling during Mammary Epithelial Cell Three-Dimensional Morphogenesis Identifies PTPRO as a Novel Regulator of Morphogenesis and ErbB2-Mediated Transformation

Identification of genes that are upregulated during mammary epithelial cell morphogenesis may reveal novel regulators of tumorigenesis. We have demonstrated that gene expression programs in mammary epithelial cells grown in monolayer cultures differ significantly from those in three-dimensional (3D) cultures. We identify a protein tyrosine phosphate, PTPRO, that was upregulated in mature MCF-10A mammary epithelial 3D structures but had low to undetectable levels in monolayer cultures. Downregulation of PTPRO by RNA interference inhibited proliferation arrest during morphogenesis. Low levels of PTPRO expression correlated with reduced survival for breast cancer patients, suggesting a tumor suppressor function. Furthermore, we showed that the receptor tyrosine kinase ErbB2/HER2 is a direct substrate of PTPRO and that loss of PTPRO increased ErbB2-induced cell proliferation and transformation, together with tyrosine phosphorylation of ErbB2. Moreover, in patients with ErbB2-positive breast tumors, low PTPRO expression correlated with poor clinical prognosis compared to ErbB2-positive patients with high levels of PTPRO. Thus, PTPRO is a novel regulator of ErbB2 signaling, a potential tumor suppressor, and a novel prognostic marker for patients with ErbB2-positive breast cancers. We have identified the protein tyrosine phosphatase PTPRO as a regulator of three-dimensional epithelial morphogenesis of mammary epithelial cells and as a regulator of ErbB2-mediated transformation. In addition, we demonstrated that ErbB2 is a direct substrate of PTPRO and that decreased expression of PTPRO predicts poor prognosis for ErbB2-positive breast cancer patients. Thus, our results identify PTPRO as a novel regulator of mammary epithelial transformation, a potential tumor suppressor, and a predictive biomarker for breast cancer.     

Metabolic Profiling Reveals Sphingosine-1-Phosphate Kinase 2 and Lyase as Key Targets of (Phyto-) Estrogen Action in the Breast Cancer Cell Line MCF-7 and Not in MCF-12A

To search for new targets of anticancer therapies using phytoestrogens we performed comparative metabolic profiling of the breast cancer cell line MCF-7 and the non-tumorigenic breast cell line MCF-12A. Application of gas chromatography-mass spectrometry (GC-MS) revealed significant differences in the metabolic levels after exposure with 17ß-estradiol, genistein or a composition of phytoestrogens within a native root flax extract. We observed the metabolites 3-(4-hydroxyphenyl)-lactic acid, cis-aconitic acid, 11-beta-hydroxy-progesterone, chenodeoxycholic acid and triacontanoic acid with elevated levels due to estrogen action. Particularly highlighted were metabolites of the sphingolipid metabolism. Sphingosine and its dihydro derivate as well as ethanolaminephosphate were significantly altered after exposure with 1 nM 17ß-estradiol in the cell line MCF-7, while MCF-12A was not affected. Treatment with genistein and the flax extract normalized the sphingosine concentrations to the basic levels found in MCF-12A cells. We could further demonstrate that the expression levels of the sphingosine metabolizing enzymes: sphingosine-1-phosphate kinase (Sphk) and lyase (S1P lyase) were significantly influenced by estrogens as well as phytoestrogens. The isoform Sphk2 was overexpressed in the tumorigenic cell line MCF-7, while S1P lyase was predominantly expressed in the non-tumorigenic cell line MCF-12A. Importantly, in MCF-7 the weak S1P lyase expression could be significantly increased after exposure with 10 µM genistein and 1 µg/ml root flax extract. Here, we present, for the first time, an analysis of metabolic response of phytoestrogens to breast cancer cell lines. The contrasting regulation of sphingolipid enzymes in MCF-7 and MCF-12A render them as preferred targets for future anticancer strategies.     

MicroRNA-7 Inhibits Multiple Oncogenic Pathways to Suppress HER2Δ16 Mediated Breast Tumorigenesis and Reverse Trastuzumab Resistance

The oncogenic isoform of HER2, HER2Δ16, is expressed with HER2 in nearly 50% of HER2 positive breast tumors where HER2Δ16 drives metastasis and resistance to multiple therapeutic interventions including tamoxifen and trastuzumab. In recent years microRNAs have been shown to influence multiple aspects of tumorigenesis and tumor cell response to therapy. Accordingly, the HER2Δ16 oncogene alters microRNA expression to promote endocrine resistance. With the goal of identifying microRNA suppressors of HER2Δ16 oncogenic activity we investigated the contribution of altered microRNA expression to HER2Δ16 mediated tumorigenesis and trastuzumab resistance. Using a gene array strategy comparing microRNA expression profiles of MCF-7 to MCF-7/HER2Δ16 cells, we found that expression of HER2Δ16 significantly altered expression of 16 microRNAs by 2-fold or more including a 4.8 fold suppression of the miR-7 tumor suppressor. Reestablished expression of miR-7 in the MCF-7/HER2Δ16 cell line caused a G1 cell cycle arrest and reduced both colony formation and cell migration activity to levels of parental MCF-7 cells. Suppression of miR-7 in the MCF-7 cell line resulted in enhanced colony formation activity but not cell migration, indicating that miR-7 suppression is sufficient to drive tumor cell proliferation but not migration. MiR-7 inhibited MCF-7/HER2Δ16 cell migration through a mechanism involving suppression of the miR-7 target gene EGFR. In contrast, miR-7 inhibition of MCF-7/HER2Δ16 cell proliferation involved a pathway where miR-7 expression resulted in the inactivation of Src kinase independent of suppressed EGFR expression. Also independent of EGFR suppression, reestablished miR-7 expression sensitized refractory MCF-7/HER2Δ16 cells to trastuzumab. Our results demonstrate that reestablished miR-7 expression abolishes HER2Δ16 induced cell proliferation and migration while sensitizing HER2Δ16 expressing cells to trastuzumab therapy. We propose that miR-7 regulated pathways, including EGFR and Src kinase, represent targets for the therapeutic intervention of refractory and metastatic HER2Δ16 driven breast cancer.