Tyrosine Phosphorylation Of HER2 Research Articles

Tumor Extrinsic Factors Mediate Primary T-DM1 Resistance in HER2-Positive Breast Cancer Cells

Simple SummaryIn this investigation, we employed an unconventional approach to explore the mechanisms of the primary resistance of human epidermal growth factor 2 (HER2)-positive breast cancer cells to ado-trastuzumab emtansine (also known as T-DM1). Specifically, we used Matrigel matrix as a model of the tumor microenvironment and examined its effect on the sensitivity of HER2-positive breast cancer cells to T-DM1. We found that epidermal growth factor receptor (EGFR) is activated in HER2-positive, T-DM1-sensitive JIMT1 and SKBR-3 cells on the Matrigel matrix. This leads to phosphorylation and degradation of HER2 in these cells, resulting in the loss of or reduced sensitivity to T-DM1. The discovery of extrinsic factors contributing to the primary resistance of HER2-positive breast cancer cells to T-DM1 provides an opportunity to develop a novel therapeutic strategy to overcome T-DM1 resistance.To explore if the tumor microenvironment contributes to the primary resistance of HER2-positive breast cancer cells to T-DM1, we examined whether Matrigel, a basement membrane matrix that provides a three-dimensional (3D) cell culture condition, caused the primary resistance of HER2-positive, T-DM1-sensitive breast cancer cells (JIMT1 and SKBR-3 cells) to T-DM1. This is different from the conventional approach such that the cells are exposed with escalated doses of drug to establish a drug-resistant cell line. We found that these cells were able to grow and form spheroids on the Matrigel in the presence of T-DM1. We further explored the molecular mechanisms that enables these cells to be primarily resistant to T-DM1 and found that EGFR was activated in the spheroids, leading to an increased HER2 tyrosine phosphorylation. This in turn enhances cell growth signaling downstream of EGFR/HER2 in the spheroids. HER2 tyrosine phosphorylation promotes receptor internalization and degradation in the spheroids, which limits T-DM1 access to HER2 on the cell surface of spheroids. Blocking EGFR activity by erlotinib reduces HER2 tyrosine phosphorylation and enhances HER2 cell surface expression. This enables T-DM1 to gain access to HER2 on the cell surface, resumes cell sensitivity to T-DM1, and exhibits synergistic activity with T-DM1 to inhibit the formation of spheroids on Matrigel. The discovery described in this manuscript reveals a novel approach to investigate the primary resistance of HER2-positive breast cancer cells and provides an opportunity to develop a therapeutic strategy to overcome primary resistance to T-DM1 by combing T-DM1 therapy with kinase inhibitors of EGFR.

The G Protein-Coupled Receptor PAC1 Regulates Transactivation of the Receptor Tyrosine Kinase HER3.

Peptide G protein-coupled receptors (GPCRs) for pituitary adenylate cyclase activating polypeptide (PACAP) regulate the growth of non-small cell lung cancer (NSCLC) cells. PACAP binds with high affinity to PAC1, which causes transactivation of receptor tyrosine kinases (RTK) for the EGFR and HER2 but its effect on HER3 is unknown. Using 3 NSCLC cell lines (NCI-H358, NCI-H441, and Calu-3), proteins for EGFR, HER2, HER3, and PAC1 were detected. The increase in EGFR tyrosine phosphorylation caused by PACAP was blocked by the EGFR tyrosine kinase inhibitor (TKI) gefitinib, or PACAP(6-38), a PAC1 antagonist. The increase in HER2 tyrosine phosphorylation caused by PACAP was inhibited by trastuzumab, a monoclonal antibody (mAb) for HER2, or PACAP(6-38). The increase in HER3 tyrosine phosphorylation caused by PACAP was inhibited by HER3 mAb3481 or PACAP(6-38). Immunoprecipitation experiments indicated the PACAP addition to Calu-3 cells resulted in the formation of EGFR/HER3 and HER2/HER3 heterodimers. Addition of the HER3 agonist neuregulin (NRG)-1 increased HER3 tyrosine phosphorylation in non-small-cell lung cancer (NSCLC) cells. PACAP or NRG-1 increased the proliferation of NSCLC cells, whereas PACAP(6-38), gefitinib, trastuzumab, or mAb3481 inhibited proliferation. The results indicate that PAC1 regulates the proliferation of NSCLC cells as a result of transactivation of the EGFR, HER2, and HER3.

Abstract 2075: GPCRs for pituitary adenylate cyclase activating polypeptide regulate transactivation of HER3 in non-small cell lung cancer cells

Abstract HER3, which lacks intrinsic kinase activity, plays an important role in cancer proliferation by formation of heterodimers with the EGFR or HER2 (Kiavue et al., Oncogene, 2019). Peptide GPCRs for pituitary adenylate cyclase activating polypeptide (PACAP) stimulate growth by transactivation of the EGFR and HER2 (Moody et al., JPET, 2012 and Peptides, 2019), but its effect on HER3 is unknown. The ability of PACAP to cause tyrosine phosphorylation of the EGFR, HER2 and HER3 was investigated. Using a panel of 17 non-small cell lung cancer (NSCLC) cell lines, mRNA for EGFR, HER2, HER3 was detected in 94%, 88%, and 100% of the cell lines, respectively. Addition of 100 nM PACAP but not vasoactive intestinal peptide to A549 cells increased EGFR, HER2 and HER3 tyrosine phosphorylation by 420, 240 and 190%, respectively. The increase is EGFR tyrosine phosphorylation was blocked by gefitinib or PACAP(6-38), a PAC1 antagonist. The increase in HER2 tyrosine phosphorylation caused by PACAP was inhibited by Herceptin or PACAP(6-38). The increase in HER3 tyrosine phosphorylation caused by PACAP was inhibited by the HER3 blocking antibody mAb3481 or PACAP(6-38). Immunoprecipitation experiments indicated the PACAP addition to A549 cells resulted in the formation of EGFR/HER2 and HER2/HER3 heterodimers, whereas addition of EGF resulted in EGFR/HER2 heterodimers and addition of the HER3 agonist neuregulin-1 resulted in HER2/HER3 heterodimers. Addition of N-acetyl-cysteine (antioxidant), Tiron (superoxide scavenger) or diphenylene iodonium (Nox/Duox inhibitor) impaired the ability of PACAP to cause EGFR, HER2 or HER3 transactivation. The results indicate that reactive oxygen species are essential for PACAP to cause EGFR, HER2 or HER3 transactivation in non small cell lung cancer cells. Citation Format: Terry W. Moody, Lingaku Lee, Robert T. Jensen. GPCRs for pituitary adenylate cyclase activating polypeptide regulate transactivation of HER3 in non-small cell lung cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2075.

Neurotensin receptors regulate transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner

Neurotensin is a 13 amino acid peptide which is present in many lung cancer cell lines. Neurotensin binds with high affinity to the neurotensin receptor 1, and functions as an autocrine growth factor in lung cancer cells. Neurotensin increases tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and the neurotensin receptor 1 antagonist SR48692 blocks the transactivation of the EGFR. Here the effects of reactive oxygen species on the transactivation of the EGFR and HER2 were investigated. Using non-small cell lung cancer (NSCLC) cell lines, neurotensin receptor 1 mRNA and protein were present. Using NCI–H838 cells, neurotensin or neurotensin8−13 but not neurotensin1−8 increased EGFR, ERK and HER2 tyrosine phosphorylation which was blocked by SR48692. Neurotensin addition to NCI–H838 cells increased significantly reactive oxygen species which was inhibited by SR48692, Tiron (superoxide scavenger) and diphenylene iodonium (DPI inhibits the ability of NADPH oxidase and dual oxidase enzymes to produce reactive oxygen species). Tiron or DPI impaired the ability of neurotensin to increase EGFR, ERK and HER2 tyrosine phosphorylation. Neurotensin stimulated NSCLC cellular proliferation whereas the growth was inhibited by SR48692, DPI or lapatinib (lapatinib is tyrosine kinase inhibitor of the EGFR and HER2). Lapatinib inhibited the ability of the neurotensin receptor 1 to transactivate the EGFR and HER2. The results indicate that neurotensin receptor 1 regulates the transactivation of the EGFR and HER2 in a reactive oxygen species-dependent manner.

A benzimidazole derivative exhibiting antitumor activity blocks EGFR and HER2 activity and upregulates DR5 in breast cancer cells.

Aberrant expression or function of epidermal growth factor receptor (EGFR) or the closely related human epidermal growth factor receptor 2 (HER2) can promote cell proliferation and survival, thereby contributing to tumorigenesis. Specific antibodies and low-molecular-weight tyrosine kinase inhibitors of both proteins are currently in clinical trials for cancer treatment. Benzimidazole derivatives possess diverse biological activities, including antitumor activity. However, the anticancer mechanism of 5a (a 2-aryl benzimidazole compound; 2-chloro-N-(2-p-tolyl-1H-benzo[d]imidazol-5-yl)acetamide, C16H14ClN3O, MW299), a novel 2-aryl benzimidazole derivative, toward breast cancer is largely unknown. Here, we demonstrate that 5a potently inhibited both EGFR and HER2 activity by reducing EGFR and HER2 tyrosine phosphorylation and preventing downstream activation of PI3K/Akt and MEK/Erk pathways in vitro and in vivo. We also show that 5a inhibited the phosphorylation of FOXO and promoted FOXO translocation from the cytoplasm into the nucleus, resulting in the G1-phase cell cycle arrest and apoptosis. Moreover, 5a potently induced apoptosis via the c-Jun N-terminal kinase (JNK)-mediated death receptor 5 upregulation in breast cancer cells. The antitumor activity of 5a was consistent with additional results demonstrating that 5a significantly reduced tumor volume in nude mice in vivo. Analysis of the primary breast cancer cell lines with HER2 overexpression further confirmed that 5a significantly inhibited Akt Ser473 and Bad Ser136 phosphorylation and reduced cyclin D3 expression. On the basis of our findings, further development of this 2-aryl benzimidazole derivative, a new class of multitarget anticancer agents, is warranted and represents a novel strategy for improving breast cancer treatment.

Apigenin induces apoptosis via extrinsic pathway, inducing p53 and inhibiting STAT3 and NFκB signaling in HER2-overexpressing breast cancer cells

Phytoestrogens are known to prevent tumor induction. But their molecular mechanisms of action are still unknown. This study aimed to examine the effect of apigenin on proliferation and apoptosis in HER2-expressing breast cancer cells. In our experiments, apigenin inhibited the proliferation of MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with an increase of sub G(0)/G(1) apoptotic fractions. Overexpression of HER2 did not confer resistance to apigenin in MCF-7 cells. Apigenin-induced extrinsic apoptosis pathway up-regulating the levels of cleaved caspase-8, and inducing the cleavage of poly (ADP-ribose) polymerase, whereas apigenin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease mitochondrial membrane potential maintaining red fluorescence and did not affect the levels of B-cell lymphoma 2 (BCL2) and Bcl-2-associated X protein. Moreover, apigenin reduced the tyrosine phosphorylation of HER2 (phospho-HER2 level) in MCF-7 HER2 cells, and up-regulated the levels of p53, phospho-p53 and p21 in MCF-7 vec and MCF-7 HER2 cells. This suggests that apigenin induces apoptosis through p53-dependent pathway. Apigenin also reduced the expression of phospho-JAK1 and phospho-STAT3 and decreased STAT3-dependent luciferase reporter gene activity in MCF-7 vec and MCF-7 HER2 cells. Apigenin decreased the phosphorylation level of IκBα in the cytosol, and abrogated the nuclear translocation of p65 within the nucleus suggesting that it blocks the activation of NFκB signaling pathway in MCF-7 vec and MCF-7 HER2 cells. Our study indicates that apigenin could be a potential useful compound to prevent or treat HER2-overexpressing breast cancer.

Stromal–epithelial interactions modulate cross-talk between prolactin receptor and HER2/Neu in breast cancer

Prolactin (PRL) promotes the proliferation and survival of breast cancer cells in part via the transactivation of human epidermal growth factor receptor 2 (HER2), also known as Neu in rodents. A PRL receptor (PRLR) antagonist, G129R, has been developed, which indirectly inhibits the tyrosine phosphorylation of HER2 (p-HER2) in human breast cancer cell lines. In this study, we investigate the effects of cancer-associated fibroblasts (CAFs) upon this molecular cross-talk using tumor cells and CAFs derived from spontaneous mammary tumors of female MMTV-neu transgenic mice. Tumors were resected and cultured as small tumor chunks (~3 mm3) or were cultured in monolayer. G129R reduced tyrosine phosphorylation of Neu (p-Neu) in a dose-dependent manner (IC50~10 μg/ml) in tumor chunks, but had no effect on primary tumor epithelial cells grown in monolayer. Direct co-culture of mouse or human tumor epithelial cell lines with CAFs restored the epithelial cells' response to G129R, similar to that observed in mouse tumor chunks. The addition of PRL, as expected, induced p-Neu in both the tumor chunk and co-culture models. The inhibitory effect of G129R was absent when CAFs were physically separated from mouse tumor epithelial cells using a transwell system, or when CAFs were replaced with normal fibroblasts in direct co-culture with human or mouse tumor epithelial cells. In vivo, G129R reduced p-Neu levels in primary mammary tumors of mice in a time- and dose-dependent manner. In conclusion, CAFs play a critical role in bridging the cross-talk between PRL and HER2/Neu in both mouse and human models of breast cancer. The inhibitory effects of G129R on p-Neu and on tumor growth are dependent upon interactions of tumor epithelial cells with CAFs.

Abstract A153: Effect of the multikinase inhibitor TG02 in HER2-positive breast cancer.

Abstract Breast cancer represents a heterogeneous disease in which several distinct genetic and histopathological types have been defined. Depending on the latter, treatment is established based on the presence of estrogen or progesterone receptors, the transmembrane tyrosine kinase HER2, or the absence of these markers. One of the most aggressive subtypes of breast cancer is offered by the HER2 positive tumors. Treatment of these tumors is based on agents, such as the monoclonal antibody trastuzumab, or the small molecule tyrosine kinase inhibitor lapatinib, which target HER2. The incorporation of these therapies to HER2 positive tumors has improved patient outcome. However, in the metastatic setting HER2 positive tumors still represent an important oncological problem. Moreover, some patients are either primary resistant, or acquire resistance to anti-HER2 therapies along time. Therefore, novel therapies are required to fight the disease once disseminated and under instances of anti-HER2 resistance. In addition to HER2, several studies have involved other kinases in the pathophysiology of breast cancer. Because of these facts, we have investigated the antitumoral action of TG02 in HER2 positive breast cancers. TG02 represents a novel molecule with a unique kinase inhibitory sprectrum. TG02 targets several receptor tyrosine kinases, such as FLT3 and various cytosolic serine/threonine kinases, such as ERK5, p38, CDK1, CDK2, CDK5, CDK9. We used HER2 overexpressing cell lines SKBR3 and BT474 to explore the action of TG02 on these models of HER2 positive breast cancer. TG02 caused a dose-dependent decrease in MTT metabolization, an assay used to explore cellular proliferation and viability. IC50 values were in the low nanomolar range. The drug also potentiated the action of trastuzumab or lapatinib, with whom TG02 exerted a synergistic action, as indicated by the Chou and Talalay algorithm. In BT474 cells, which bear constitutively active ERK5, treatment with TG02 caused inhibition of ERK5 phosphorylation, at concentrations of 100 nM and above, without affecting HER2 tyrosine phosphorylation. Cell cycle analyzes indicated that TG02 caused a decrease in G0/G1, and an increase in the subG0 region of the histogram. This result suggested that TG02 could trigger apoptotic cell death in BT474 cells. In fact, treatment with TG02 caused a dose-dependent increase in the processing of PARP, which was accompanied by increased annexin V staining. Therefore TG02 may indice cell cycle effects together with an induction of apoptotic cell death. These data support further development of this compound for the treatment of HER2 positive tumors, especially in combination with other standard of care treatments used in these breast neoplasias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A153.

Trastuzumab and lapatinib modulation of HER2 tyrosine/threonine phosphorylation and cell signaling

Cell surface transmembrane signaling receptors EGFR, HER3, and HER4 are activated by ligand-binding-mediated dimerization and phosphorylation. In contrast, HER2 amplification promotes signaling by increasing homo/heterodimerization and ligand binding. Trastuzumab or lapatinib therapy of HER2 amplicon-positive breast cancer cells induces growth inhibition and intracellular growth pathway signaling modulation. The mechanism(s) by which trastuzumab, an IgG1 humanized antibody, induces modification of cell signaling upon binding to an extracellular determinant on a ligand-less "receptor" membrane protein remains unexplained. Using immune detection methodology comprised of antibodies detecting three distinct domains of HER and five tyrosine/threonine phosphorylation sites, the effects of trastuzumab and lapatinib were defined during steady state growth inhibition. Here, we show that lapatinib markedly reduces HER2 tyrosine phosphorylation, while in contrast, no change in tyrosine phosphate levels is detected during trastuzumab-mediated cell growth inhibition. As trastuzumab treatment does not change either the steady state HER2 protein levels or HER2 mRNA, these findings argue against an antibody-dependent alteration in internalization kinetics. We further show a sequenced relationship between lapatinib-induced blockage of phosphorylation (6-8 h) and induction of delayed cell death (5-6 days), while trastuzumab-treated cells showed no evidence of cell death up to 9 days. Taken together, these results demonstrate that inhibition of HER2 phosphorylation by lapatinib is sufficient to induce apoptosis while trastuzumab binding to the extracellular HER2 domain may function by sterically modulating the detection of phosphate moieties by cytoplasmic signal transducers. This investigation also detected a 20 kD protein, which is down-regulated by lapatinib, further demonstrating the complexity of this signal transduction system.

Sequential Multiplex Analyte Capturing for Phosphoprotein Profiling

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.

Sphingosine 1-Phosphate Receptor 4 Uses HER2 (ERBB2) to Regulate Extracellular Signal Regulated Kinase-1/2 in MDA-MB-453 Breast Cancer Cells

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P(4)) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. This was based on several lines of evidence. First, the S1P stimulation of ERK-1/2 was abolished by JTE013, which we show here is an S1P(2/4) antagonist and reduced by siRNA knockdown of S1P(4). Second, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by a HER2 inhibitor (ErbB2 inhibitor II) and reduced by siRNA knockdown of HER2 expression. Third, phyto-S1P, which is an S1P(4) agonist, stimulated ERK-1/2 activation in an S1P(4)- and HER2-dependent manner. Fourth, FTY720 phosphate, which is an agonist at S1P(1,3,4,5) but not S1P(2) stimulated activation of ERK-1/2. Fifth, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013. HER2 which is an orphan receptor tyrosine kinase is the preferred dimerization partner of the EGF receptor. However, EGF-stimulated activation of ERK-1/2 was not affected by siRNA knockdown of HER2 or by ErbB2 (epidermal growth factor receptor 2 (or HER2)) inhibitor II in MDA-MB-453 cells. Moreover, S1P-stimulated activation of ERK-1/2 does not require an EGF receptor. Thus, S1P and EGF function in a mutually exclusive manner. In conclusion, the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P(4) may have an important role in breast cancer progression.

508 Radiation-induced c-Met expression sensitizes lung cancer cells to c-Met antagonists

Two members of the epidermal growth factor receptor family, EGFR and HER2, have been implicated in radioresistance in breast cancer and other malignancies. To gauge the potential clinical utility of targeting both EGFR and HER2 to control growth and radiosensitize human breast cancers, we examined the effect of a dual EGFR/HER2 inhibitor, GW572016, on the proliferation and radiation response of either EGFR- or HER2-overexpressing human breast cancer cell lines.Primary human breast cancer cell lines that endogenously overexpress EGFR or HER2 and luminal mammary epithelial H16N2 cells stably transfected with HER2 were evaluated for the effect of GW572016 on inhibition of ligand-induced or constitutive receptor phosphorylation, proliferation, radiosensitization, and inhibition of downstream signaling.GW572016 inhibited constitutive and/or ligand-induced EGFR or HER2 tyrosine phosphorylation of all five cell lines, which correlated with the antiproliferative response in all but one cell line. GW572016 radiosensitized EGFR-overexpressing cell lines, but HER2-overexpressing cells were unable to form colonies after brief exposure to GW572016 even in the absence of radiation, and thus could not be evaluated for radiosensitization. One cell line was resistant to the antiproliferative and radiosensitizing effects of GW572016, despite receptor inhibition. Exploration of potential mechanisms of resistance in SUM185 cells revealed failure of GW572016 to inhibit downstream ERK and Akt activation, despite inhibition of HER2 phosphorylation. In contrast, sensitive HER2-overexpressing cell lines demonstrated inhibition of both ERK and Akt phosphorylation.GW572016 potently inhibits receptor phosphorylation in either EGFR- or HER2-overexpressing cell lines and has both antiproliferative and radiosensitizing effects. Resistance to GW572016 was not due to a lack of receptor inhibition, but rather with a lack of inhibition of ERK and Akt, suggesting that measurement of inhibition of crucial signaling pathways may better predict response than inhibition of receptor phosphorylation. The SUM185 cell line provides a valuable model for studying mechanisms of resistance of EGFR/HER2 inhibitor therapy.

Lewis(y) antigen stimulates the growth of ovarian cancer cells via regulation of the epidermal growth factor receptor pathway.

Lewis(y) antigen is an oligosaccharide containing two fucoses, and is expressed variously in 75% of ovarian tumors, where its high expression level predicts poor prognosis. The effect and the possible mechanism of Lewis(y) on the proliferation of human ovarian cancer cells are still largely unkown. We report here that transfecting alpha1,2-FT gene into RMG-I cells increased the expression of Lewis(y) and promoted cell proliferation. In alpha1,2-FT-transfected cells, the Lewis(y) content of EGFR was increased dramatically. Tyrosine phosphorylation of EGFR was elevated. Concomitantly, tyrosine phosphorylation of Akt, ERK1/2 was also upregulated. Moreover, the expression of HER2/neu mRNA and protein, the tyrosine phosphorylation of HER2/ neu were also elveated, while the expression of p27 was significantly reduced. However, the expression of EGFR and the relative content of Lewis(y) on HER2/neu were unchanged. The above-mentioned alterations were correlated with the Lewis(y) content of EGFR and alpha1,2-FT expression in cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of alpha1,2-FT were attenuated significantly by the inhibitor of EGFR tyrosine kinase and by the mono-antibody to Lewis(y). Meanwhile, the reduction in p27 and the difference in its expression among the two cell lines were also blocked by the Lewis(y) antibody. The PI3K signaling pathway was more important than the MAPK pathway in the regulation of p27 expression. These findings provide strong evidence that increased expression of Lewis(y) promotes cell proliferation through regulating the phosphorylation and expression of some molecules involved in the EGFR/PI3K-signaling pathway.

Molecular Mechanism for SHP2 in Promoting HER2-induced Signaling and Transformation

The Src homology phosphotyrosyl phosphatase 2 (SHP2) plays a positive role in HER2-induced signaling and transformation, but its mechanism of action is poorly understood. Given the significance of HER2 in breast cancer, defining a mechanism for SHP2 in the HER2 signaling pathway is of paramount importance. In the current report we show that SHP2 positively modulates the Ras-extracellular signal-regulated kinase 1 and 2 and the phospoinositide-3-kinase-Akt pathways downstream of HER2 by increasing the half-life the activated form of Ras. This is accomplished by dephosphorylating an autophosphorylation site on HER2 that serves as a docking platform for the SH2 domains of the Ras GTPase-activating protein (RasGAP). The net effect is an increase in the intensity and duration of GTP-Ras levels with the overall impact of enhanced HER2 signaling and cell transformation. In conformity to these findings, the HER2 mutant that lacks the SHP2 target site exhibits an enhanced signaling and cell transformation potential. Therefore, SHP2 promotes HER2-induced signaling and transformation at least in part by dephosphorylating a negative regulatory autophosphorylation site. These results suggest that SHP2 might serve as a therapeutic target against breast cancer and other cancers characterized by HER2 overexpression.

Single-Molecule Study of Lateral Mobility of Epidermal Growth Factor Receptor 2/HER2 on Activation

The transmembrane protein HER2, a member of the epidermal growth factor receptor family of tyrosine kinase, plays important roles in many fundamental cellular processes as well as the pathogenesis of many cancers. In this work, we have applied the single-molecule fluorescence microscopic method to study lateral mobility change of HER2 on activation by imaging and tracking individual GFP-tagged HER2 molecules on the membrane of living cells. The single HER2 molecules displayed different diffusion rates and modes. It was interesting to find that the mobility of HER2 increased upon stimulation by heregulin beta1, the specific ligand of HER3. The faster diffusion was related to the tyrosine phosphorylation of HER2 or EGFR. The results provided new information for the understanding of HER2 activation and molecular mechanism of signal transduction through HER2/HER3 heterodimerization.

Leptin Augments Proliferation of Breast Cancer Cells via Transactivation of HER2

Excessive fat mass is a risk factor for postmenopausal breast cancer. Leptin, a fat cell-derived peptide hormone, elicits a growth-stimulating effect in breast cancer cells with leptin receptor expression, although the leptin-induced signal in malignant cells is not fully understood. Here, we found that exogenous leptin induced tyrosine phosphorylation of HER2 in SKBR3 cells, which showed marked overexpression of HER2. Phosphorylation of HER2 was detected at 2 min and continued up to 120 min after the start of stimulation. Leptin-induced HER2 phosphorylation was partially reduced by an epidermal growth factor receptor inhibitor, AG1478, or a Janus-activated kinase inhibitor, AG490. Leptin also induced phosphorylation of extracellular signal-regulated kinase 1/2, which was mostly abrogated by a HER2 tyrosine kinase inhibitor, AG825. In a proliferation assay, addition of 500 ng/mL leptin increased the proliferation of SKBR3, which was totally inhibited by AG825. Collectively, our data suggest that leptin can transactivate HER2 through both epidermal growth factor receptor and Janus-activated kinase 2 activation, which can cause the growth of breast cancer cells with HER2 overexpression.

Transactivation of HER2 by vasoactive intestinal peptide in experimental prostate cancer: Antagonistic action of an analog of growth-hormone-releasing hormone

Receptors for vasoactive intestinal peptide (VIP) and the human epidermal growth factor family of tyrosine kinase receptors (HER) are potent promoters of cell proliferation, survival, migration, adhesion and differentiation in prostate cancer cell lines. In this study, we analyzed the cross-talk between both classes of receptors through the regulation of HER2 transactivation and expression by VIP. Three growth-hormone-releasing hormone analogs endowed with antagonistic activity for VIP receptors (JV-1-51, -52, and -53) abrogated the autocrine/paracrine stimuli of VIP on androgen-independent PC3 cells in the absence or the presence of 10% fetal bovine serum. Semiquantitative and real-time quantitative RT-PCR together with Western blotting showed increased expression levels of both mRNA and proteins for HER2 and HER3 in PC3 and androgen-dependent LNCaP prostate cancer cells as compared to non-neoplastic RWPE-1 cells. VIP (100 nM) stimulated the expression levels of both HER2 and HER3 in PC3 cells in a time-dependent manner. Whereas these effects were relatively slow, VIP rapidly (0.5 min) increased HER2 tyrosine phosphorylation. This pattern of HER transactivation was blocked by H89, a protein kinase A (PKA) inhibitor, as well as by the specific VIP antagonist JV-1-53, indicating the involvement of VIP receptors and PKA activity in phosphorylated HER2 formation. These findings support the merit of further studies on the potential usefulness of VIP receptor antagonists and both HER2 antibodies and tyrosine kinase inhibitors for prostate cancer therapy.

Additive effects of a prolactin receptor antagonist, G129R, and herceptin on inhibition of HER2-overexpressing breast cancer cells

Breast cancers overexpressing human epidermal growth factor receptor 2 (HER2) have been reported to have higher proliferative and metastatic activity in the presence of autocrine prolactin (PRL), indicating potential cooperation between HER2 and the PRL receptor (PRLR) during breast cancer progression. PRL can induce the tyrosine phosphorylation of HER2 which stimulates mitogen-activated protein kinase (MAPK) activity. To determine if this transactivation of HER2 by PRL contributes to anti-HER2 therapy resistance we examined the potential of combining Herceptin with a PRLR antagonist, G129R, which inhibits PRL-induced signaling, as a novel therapeutic strategy. Two PRL-expressing human breast cancer cell lines (T-47D and BT-474) that overexpress PRLR and HER2 to different degrees were chosen for this study. The phosphorylation status of HER2 and activation of MAPK, signal transducers and activators of transcription (STAT), as well as phosphatidylinositol-3 kinase (PI3K) signaling cascades were examined in response to Herceptin, G129R or a combination of the two in either the absence or presence of exogenous PRL. As a single agent, Herceptin was more effective than G129R at inhibiting AKT phosphorylation; whereas, G129R was superior at blocking STAT3 and STAT5 activation. G129R was also able to directly inhibit the HER2 phosphorylation. The combination of Herceptin and G129R had an additive inhibitory effect on HER2 and MAPK phosphorylation, confirming that the MAPK signaling is a converging pathway shared by both HER2 and the PRLR. Combination of Herceptin and G129R also additively inhibited cell proliferation in vitro and in vivo as measured by inhibition of the growth of T-47D and BT-474 xenografts in athymic nude mice. We conclude that an anti-HER2 and anti-PRLR regimen may offer a new approach to treat HER2-overexpressing breast cancers.

GRB-7 facilitates HER-2/Neu-mediated signal transduction and tumor formation

Growth factor receptor-bound protein-7 (GRB-7), an adaptor molecule, can interact with multiple signal transduction molecules. GRB-7 is amplified concurrently with HER-2/Neu in most, if not all, of breast cancer with chromosome 17q11-21 amplification. GRB-7 gene amplification is associated with RNA over-expression. We show GRB-7 protein is over-expressed by immunoblotting in breast cancer cell lines and primary breast tumors with HER-2/Neu protein over-expression. Over-expression of GRB-7 in MCF-7 breast cancer cells that over-express HER-2/Neu leads to activation of tyrosine phosphorylation of HER-2/Neu. Knockdown of GRB-7 expression in SKBR-3 breast cancer cells with naturally occurring HER-2/Neu gene amplification decreases tyrosine phosphorylation of HER-2/Neu. Activation of HER-2/Neu phosphorylation is associated with increase in tyrosine phosphorylation of phosphoinositide-specific lipase C-gamma-1 (PLC-gamma-1) and recruitment of PLC-gamma-1 to HER-2/Neu protein molecule. Activation of downstream protein kinase C (PKC) pathway is evidenced by increase in the phosphorylation of a common PKC substrate-myristoylated alanine-rich protein kinase C substrate (MARCKS). In addition, over-expression of GRB-7 in MCF-7 breast cancer cells that over-express HER-2/Neu leads to activation of AKT phosphorylation. Knockdown of GRB-7 expression in MB-453 and SKBR-3 breast cancer cells results in decrease in AKT phosphorylation. GRB-7 over-expression therefore facilitates activation of phosphorylation of HER-2/Neu and AKT in breast cancer cells with HER-2/Neu over-expression. GRB-7 over-expression in MCF-7 cells over-expressing HER-2/Neu leads to morphologic change of cells and promotes tumor xenograft growth in nude mice. GRB-7 over-expression therefore plays pivotal roles in activating signal transduction and promoting tumor growth in breast cancer cells with chromosome 17q11-21 amplification.

Inactivation of neuregulin-1 by nitration

Nitration is a posttranslational modification that can compromise protein function. We hypothesized that nitration of growth factors secreted in the lung may alter their interaction with their respective receptors and modulate the normal growth and differentiation program induced by ligand-receptor interaction. We tested this hypothesis in vitro by nitration of neuregulin-1's (NRG-1) EGF-like domain and studying the effect on NRG-1's activity. Nitration of NRG-1's (nNRG-1) EGF-like domain resulted in an inability to activate its receptor, the human epidermal growth factor receptors 2 and 3 (HER2/HER3) heterodimer, as defined by loss of HER2 tyrosine phosphorylation induced by nNRG-1 in MCF-7 cells. Receptor activation was not restored with increasing nNRG-1 concentration or exposure times. nNRG-1 did not compete with NRG-1 for HER2/HER3 binding in competition assays. In addition, nNRG-1 no longer induced proliferation of the MCF-7 cell line, as MCF-7 cells exposed to nNRG-1 and NRG-1 concurrently had the same proliferation rate as that induced by NRG-1 alone. Thus nitration of NRG-1's EGF-like domain caused it to lose its ability to bind and activate its receptor with loss of ligand-induced proliferation. Posttranslational nitration of growth factors in states where reactive nitrogen species are increased may be an important means of regulating growth factor receptor effects in the lung.