Neu Tyrosine Kinase Research Articles

Participation of FAS- and TNF-Dependent Pathways in Apoptosis Mechanisms in Hypothalamus in Physiological and Pathological Aging

Cellular resistance to apoptosis can be related to the activity of cytokine-dependent signaling. Thus, the goal of the work is to study the mechanisms of cytokine-dependent, FAS/TNF-mediated regulation of apoptosis of neurosecretory cells in physiological and pathological (overexpression of the oncogene HER-2/Neu) aging. Transgenic, accelerated-aged HER-2/Neu mice of different ages and wild-type FVB/N mice were examined. The apoptosis level of neurons in hypothalamic sections (supraoptic and paraventricular nuclei) (TUNEL) and the expression of caspase-8, CD178 (FASL), FAS, FADD, and TRADD (Western blotting) was determined. It is shown that a proinflammatory component participates in the aging process. The expression of FAS, adapter proteins associated with the death domain (FADD and TRADD), and caspase-8 is activated in the hypothalamus in FVB/N (wild-type) mice during aging, and it correlates with an increase in the apoptosis level. HER-2/Neu expression leads to suppression of the extrinsic apoptotic pathway. In this case, the reception of an apoptotic signal (FAS-receptor expression) and its further transmission (expression of FADD and TRADD) is suppressed. However, a sufficiently high TRADD expression in young transgenic mice and an increased expression of TNF-α in old mice can activate one of the survival pathways (NF-κB, ERK or PI3K-AKT). Thus, the HER-2/Neu tyrosine kinase receptor plays a role in the mechanism of cell resistance to age-dependent apoptosis, and the FAS/TNF-signaling pathway is one of the targets of HER-2/Neu.

Raf-1 oncogenic signaling is linked to activation of mesenchymal to epithelial transition pathway in metastatic breast cancer cells

Aberrant activation of the Raf/MEK/MAPK pathway plays a key role in breast cancer development and progression. Dysregulation of Raf/MEK/MAPK oncogenic signaling often results from overexpression of the HER-2/Neu tyrosine kinase receptor leading to chemoendocrine resistance, development of distant metastases and ultimately poor prognosis in breast cancer patients. HER-2/Neu overexpression is also linked to activation of the epithelial to mesenchymal transition (EMT) pathway, loss of adhesion molecules and metastasis. Recently, it has been demonstrated that cancer cells that undergo EMT acquire a CD44+/CD24-/low basal cancer stem cell-like phenotype and are characterized by activation of HER-2/Neu and TGFβ oncogenic signaling pathways with increased capacity of self-renewal, drug resistance, invasion and distant metastases. Following metastatic dissemination, cancer cells re-activate certain epithelial properties through mesenchymal to epithelial transition (MET) to establish neoplastic lesions at secondary sites, although the molecular mechanisms regulating MET remain elusive. In this study we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces HER-2/Neu overexpression leading to the development of distant metastases in ERα+ MCF-7 breast cancer xenografts. Importantly, development of distant metastases in xenograft models was linked to activation of the MET pathway characterized by reduced expression of EMT inducer genes (TGFB2, TWIST1 and FOXC1) and overexpression of BMB7, CXCR7 and EGR family of transcription factors. In summary, our results demonstrate for the first time that amplification of Raf/MEK/MAPK oncogenic signaling during tumor growth promotes the genesis of metastatic lesions from primary tumors by activating the mesenchymal epithelial transition.

Synergistic Inhibition with a Dual Epidermal Growth Factor Receptor/HER-2/neu Tyrosine Kinase Inhibitor and a Disintegrin and Metalloprotease Inhibitor

The ErbB family of receptors is overexpressed in numerous human tumors. Overexpression correlates with poor prognosis and resistance to therapy. Use of ErbB-specific antibodies to the receptors (Herceptin or Erbitux) or ErbB-specific small-molecule inhibitors of the receptor tyrosine kinase activity (Iressa or Tarceva) has shown clinical efficacy in several solid tumors. An alternative method of affecting ErbB-initiated tumor growth and survival is to block sheddase activity. Sheddase activity is responsible for cleavage of multiple ErbB ligands and receptors, a necessary step in availability of the soluble, active form of the ligand and a constitutively activated ligand-independent receptor. This sheddase activity is attributed to the ADAM (a disintegrin and metalloprotease) family of proteins. ADAM 10 is the main sheddase of epidermal growth factor (EGF) and HER-2/neu cleavage, whereas ADAM17 is required for cleavage of additional EGF receptor (EGFR) ligands (transforming growth factor-alpha, amphiregulin, heregulin, heparin binding EGF-like ligand). This study has shown that addition of INCB3619, a potent inhibitor of ADAM10 and ADAM17, reduces in vitro HER-2/neu and amphiregulin shedding, confirming that it interferes with both HER-2/neu and EGFR ligand cleavage. Combining INCB3619 with a lapatinib-like dual inhibitor of EGFR and HER-2/neu kinases resulted in synergistic growth inhibition in MCF-7 and HER-2/neu-transfected MCF-7 human breast cancer cells. Combining the INCB7839 second-generation sheddase inhibitor with lapatinib prevented the growth of HER-2/neu-positive BT474-SC1 human breast cancer xenografts in vivo. These results suggest that there may be an additional clinical benefit of combining agents that target the ErbB pathways at multiple points.

Synthesis and Evaluation of 2-[123I]iodoemodin for a Potential Breast Cancer Imaging Agent

Emodin (3-methyl-1,6,8-trihydroxyanthraquinone) is a natural chemotherapeutic compound with diverse biological properties including an antitumor activity. Emodin, a specific inhibitor of the protein tyrosine kinase, has a number of cellular targets in related to it. Its inhibition activity affects the mammalian cell cycle regulation in specific oncogene. Practically, it has been proven to inhibit HER-2/neu tyrosine kinase expressing breast cancer cells as an anticancer agent. 2-[123I]iodoemodin has been synthesized and evaluated human breast cancer cells (MDA-MB-231, MCF-7, fibroblast as a control) which express basal levels of HER-2/neu tyrosine kinase to investigate its suitability as a breast cancer imaging agent and 2-iodoemodin has been synthesized as a standard compound. The radiochemical yield of the 2-[123I]iodoemodin was about 72% and its radiochemical purity was over 97% after purification. The radioactivity of the 2-[123I]iodoemodin was increased in a time dependent manner in both cell lines and the ratio of MDA-MB-231 and MCF7 to fibroblast was 2.9 and 1.7, respectively.

Regulation of Cripto-1 Signaling and Biological Activity by Caveolin-1 in Mammary Epithelial Cells

Human and mouse Cripto-1 (CR-1/Cr-1) proteins play an important role in mammary gland development and tumorigenesis. In this study, we examined the relationship between Cripto-1 and caveolin-1 (Cav-1), a membrane protein that acts as a tumor suppressor in the mammary gland. Cripto-1 was found to interact with Cav-1 in COS7 cells and mammary epithelial cells. Using EpH4 mouse mammary epithelial cells expressing Cr-1 (EpH4 Cr-1) or Cr-1 and Cav-1 (EpH4 Cr-1/Cav-1), we demonstrate that Cav-1 expression markedly reduced the ability of Cr-1 to enhance migration, invasion, and formation of branching structures in EpH4 Cr-1/Cav-1 cells as compared to EpH4 Cr-1 cells. Furthermore, coexpression of Cav-1 together with Cr-1 in EpH4 Cr-1/Cav-1 cells inhibited Cr-1-mediated activation of c-src and mitogen-activated protein kinase signaling pathways. Conversely, primary mammary epithelial cells isolated from Cav-1 null(-/-)/mouse mammary tumor virus-CR-1 transgenic animals showed enhanced motility and activation of mitogen-activated protein kinase and c-src as compared to Cav-1(+/-)/CR-1 mammary cells. Finally, mammary tumors derived from mouse mammary tumor virus-CR-1 mice showed a dramatic reduction of Cav-1 expression as compared to mammary tissue from normal FVB/N mice, suggesting that in vivo Cav-1 is down-regulated during the process of CR-1-mediated mammary tumorigenesis.

Protein tyrosine phosphatase epsilon and Neu-induced mammary tumorigenesis

Aberrant regulation of the phosphorylation of proteins on tyrosine residues is a well-established cause of cancer. Protein tyrosine phosphatases (PTPs) share in the crucial function of maintaining appropriate levels of phosphorylation of cellular proteins, making them potentially key players in regulating the transformation process. The receptor-type tyrosine phosphatase Epsilon (RPTPepsilon) participates in supporting the transformed phenotype of mammary tumor cells induced in vivo by the Neu tyrosine kinase. The phosphatase is overexpressed in mammary tumors induced in mice by a Neu transgene and expression of RPTPepsilon in mouse mammary glands leads to massive hyperplasia and associated tumorigenesis. Furthermore, cells isolated from mammary tumors induced by Neu in mice genetically lacking RPTPepsilon appear less transformed and proliferate less well than corresponding mammary tumor cells isolated from mice expressing the phosphatase. At the molecular level, RPTPepsilon dephosphorylates and activates Src and the related kinases Yes and Fyn, and the activities of these kinases are significantly reduced in tumor cells lacking RPTPepsilon. Restoring the activities of these kinases reveals that it is only the reduced activity of Src that causes the aberrant morphology and proliferation rate of tumor cells lacking RPTPepsilon. RPTPepsilon is primed to activate Src, and presumably related kinases, following its phosphorylation by Neu at Y695 within its C-terminus. This event is crucial in enabling RPTPepsilon to activate Src, but appears not to affect the activity of RPTPepsilon towards unrelated substrates. We conclude that a Neu-RPTPepsilon-Src pathway exists in mouse mammary tumor cells, in which Neu phosphorylates RPTPepsilon thereby driving the phosphatase to specifically activate Src family kinases and to assist in maintaining the transformed phenotype.

Correlation between the Her-2/neu Status as Determined by Immunohistochemical Analysis and the Serum Her-2/neu Concentration as Determined by the Use of ADVIA Cencaur® Automated Immunoassay in Breast Cancer Patients

domain of the Her-2/neu tyrosine kinase to serum. We evaluated the correlation between the Her-2/neu status as determined by immunohistochemical analysis (IHC) and the serum Her-2/neu concentration in a population of Korean women with breast cancer. Methods: Serum Her-2/neu levels were examined from 254 female patients with primary breast cancer and 38 patients with metastatic breast cancer. Serum Her-2/neu levels were measured by the use of a chemiluminescence immunoassay (ADVIA centaur system) during the preoperative period. The level of Her-2/neu in all of the breast cancer tissue samples was determined by IHC, and samples with an IHC grade +2 were subject to fluorescence in situ (FISH). When tissue samples exhibited IHC grade +3 or showed amplification of Her-2/neu as determined by FISH analysis, Her-2/neu was considered overexpressed. The cut-off value for serum Her2/neu level was 10.2 ng/mL. Results: The mean serum Her-2/neu level was 10.1 ng/mL in primary breast cancer samples. The serum Her-2/neu concentration significantly correlated with expression of Her2/neu as determined by tissue IHC analysis (grade 1/3, 9.33 ±1.7 ng/mL; grade 2/3, 8.89±1.6 ng/mL; grade 3/3, 12.37 ±4.0 ng/mL, p<0.001). Increased serum HER-2/neu levels were associated with the lymph node status (p=0.003) and hormone unresponsiveness (p<0.001), tumor size (p<0.01) and age group (p<0.001). In metastatic breast cancer samples, the mean serum Her-2/neu level was 13.6 ng/mL. Elevated serum Her-2/neu levels were seen in 71.7% of metastatic breast cancer samples. The serum Her-2/neu level correlated with expression of Her-2/neu in metastatic tissue as determined by IHC analysis (p<0.001) rather than with the Her-2/neu status of the primary breast cancer (p=0.16) Conclusion: Serum Her-2/neu appears to be correlate with tissue Her-2/neu expression in primary and metastatic breast cancer where Her-2/neu is overexpressed. Further studies to determine levels of serum Her-2/neu are required to determine cuttoff values and the clinical application of the finding for breast cancer patients in Korea.

Controlled internalization of Her-2/neu receptors by cross-linking for targeted delivery

Receptor mediated internalization is a crucial step for targeted intracellular delivery of therapeutic and imaging agents. It was recently demonstrated that trastuzumab, an FDA approved humanized monoclonal antibody against Her-2/neu tyrosine kinase receptor, did not induce endocytosis of the internalization resistant Her-2/neu receptor. Here we report that accelerated internalization of trastuzumab can be induced by cross-linking the cell membrane bound antibody-receptor complex with an avidin/streptavidin-biotin system. We demonstrated that internalization was achieved both in vitro and in vivo in Her-2/neu expressing human breast cancer cell lines (BT-474, SK-BR-3, and AU-565) and that repetitive labeling cycles further amplified the loading of cargo molecules within the targeted cells. No trastuzumab binding and internalization was observed in Her-2/neu negative MDA-MB-231 cells, whereas weak membrane binding and negligible internalization were detected in MCF-7 cells with low expression level of Her-2/neu receptor. The method was used to noninvasively image Her-2/neu receptors in isolated cells and in a preclinical breast cancer model with MRI. The controlled internalization of Her-2/neu receptors can potentially enhance intracellular delivery of drugs and imaging probes, and improve imaging sensitivity and selectivity as well as therapeutic efficacy, through antibody-directed binding and internalization using a pretargeting approach.

Expression of Her-2/neu in endometrial carcinoma and its relation with clinicopathological variables

Objective and background: Her-2/neu tyrosine kinase has been implicated in the development and progression of several human cancers and is target for therapeutic intervention. Smaller studies suggest that Her-2/neu may be involved in the tumerogenesis of endometrial adenocarcinoma. The aim of this study was to evaluate Her-2/neu expression in endometrial carcinoma (EC) and its correlation with clinicopathological features in order to define the potential prognostic value of Her-2/neu overexpression in EC. Patients and methods: Nineteen patients with stage I-IV EC were included in this study. Demographic, clinical and pathologic information was obtained and recorded. Her-2/neu expression was evaluated by immunohistochemistry (IHC) on paraffin embedded tissue sections with Her-2/neu antibody. Overexpression was defined as complete membrane staining in greater than 10% of cells. Results: The positive rate of Her-2/neu in EC was 42.1%. Her-2/neu was associated with surgical stage (p 0.05). Conclusion: Our study provides evidence of Her-2/neu overexpression in a considerable proportion of the patients with uterine adenocarcinoma, thus suggesting the opportunity for the possible use of anti-Her-2/neu therapy in this malignancy by selective inhibition of Her-2/neu. The use of Herceptin, a monoclonal antibody directed against Her-2/neu, for therapy of patients harboring Her-2/neu positive EC may be beneficial. Her-2/neu overexpression is related to most of the prognostic variables of EC and may be incorporated into the criteria for determination of tumor aggressiveness as a prognostic marker.

Synergistic inhibition of breast cancer cell lines with a dual inhibitor of EGFR-HER-2/neu and a Bcl-2 inhibitor

The epidermal growth factor receptor (EGFR) (ErbB1) and HER-2/neu (ErbB2) are members of the ErbB family of receptor tyrosine kinases. These receptors are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Lapatinib, a reversible inhibitor of both EGFR and HER-2/neu, has shown some success in achieving clinical responses in heavily pretreated advanced cancer patients. GW2974 is a reversible dual inhibitor similar to lapatinib, but GW2974 was not progressed to clinical trials due to pharmacokinetic issues. Bcl-2, an anti-apoptotic protein, is also overexpressed in a number of human tumors. Bcl-2 inhibitors induce apoptosis and sensitize cancer cells to other therapies. The purpose of this study was to assess the effects of combining ErbB and Bcl-2 inhibitors on the growth of human breast cancer cell lines. EGFR/HER-2/neu tyrosine kinase inhibitors (lapatinib and GW2974) were combined with Bcl-2 inhibitors (HA14-1 or GX15-070) and the anti-proliferative effects were determined by the MTT tetrazolium dye assay. Combinations were tested in MCF-7 human breast cancer cells, a HER-2/neu transfected MCF-7 cell line (MCF/18), and a tamoxifen-resistant MCF-7 cell line (MTR-3). A synergistic inhibitory effect was observed with the combination of inhibitors of EGFR-HER-2/neu (lapatinib or GW2974) and Bcl-2 (GX15-070 or HA14-1) on the growth of the MCF-7, MCF/18, and MTR-3 human breast cancer cell lines. This study suggests that simultaneously blocking the ErbB family of receptor tyrosine kinases and Bcl-2 family of proteins may be a benefit to breast cancer patients.

Caveolin-1 Deficiency (−/−) Conveys Premalignant Alterations in Mammary Epithelia, with Abnormal Lumen Formation, Growth Factor Independence, and Cell Invasiveness

During breast cancer development, the luminal space of the mammary acinar unit fills with proliferating epithelial cells that exhibit growth factor-independence, cell attachment defects, and a more invasive fibroblastic phenotype. Here, we used primary cultures of mammary epithelial cells derived from genetically engineered mice to identify caveolin-1 (Cav-1) as a critical factor for maintaining the normal architecture of the mammary acinar unit. Isolated cultures of normal mammary epithelial cells retained the capacity to generate mammary acini within extracellular matrix. However, those from Cav-1 (-/-) mice exhibited defects in three-dimensional acinar architecture, including disrupted lumen formation and epidermal growth factor-independent growth due to hyperactivation of the p42/44 mitogen-activated protein kinase cascade. In addition, Cav-1-null mammary epithelial cells deprived of exogenous extracellular matrix underwent a spontaneous epithelial-mesenchymal transition, with reorganization of the actin cytoskeleton, and E-cadherin redistribution. Mechanistically, these phenotypic changes appear to be caused by increases in matrix metalloproteinase-2/9 secretion and transforming growth factor-beta/Smad-2 hyperactivation. Finally, loss of Cav-1 potentiated the ability of growth factors (hepatocyte growth factor and basic fibroblast growth factor) to induce mammary acini branching, indicative of a more invasive fibroblastic phenotype. Thus, a Cav-1 deficiency profoundly affects mammary epithelia by modulating the activation state of important signaling cascades. Primary cultures of Cav-1-deficient mammary epithelia will provide a valuable new model to study the spatial/temporal progression of mammary cell transformation.

Role of coordinated molecular alterations in the development of androgen‐independent prostate cancer: an in vitro model that corroborates clinical observations

To investigate the role of potential downstream targets of HER-2/neu, including the cell-cycle regulator p27, proliferation-associated protein Ki-67, apoptosis inhibitor Bcl-2, and signal-transduction molecule Akt (which is associated with cell survival), as the development of androgen-independent prostate cancer (AIPC) in patients who are initially responsive to androgen-ablation therapy (AAT) is a significant clinical problem. Earlier studies showed that high levels of HER-2/neu tyrosine kinase receptor expression as assessed by immunohistochemistry were significantly associated with the development of AIPC, and we hypothesised that HER-2/neu overexpression provides an alternative proliferative stimulus upon androgen depletion. We established a unique clinical model system, comprising patients who received no AAT, or who had preoperative AAT, or those with advanced tumours resistant to AAT. To test our hypothesis in vitro, we stably transfected full-length HER-2/neu cDNA in androgen-responsive LNCaP cells and examined the effects of HER-2/neu overexpression on cell proliferation, apoptosis, androgen-receptor activation, and Akt phosphorylation upon androgen deprivation by using immunohistochemistry and Western blot technique. p27 expression was initially induced on exposure to AAT, and significantly decreased in AIPC (P < 0.001). There was also a significant increase in the Ki-67 index in AIPC (P = 0.001). Elevated Bcl-2 expression was closely associated with AAT (P = 0.002), suggesting that Bcl-2 expression is induced on initial exposure to AAT. Further, Bcl-2 expression was highest in hormone-resistant cancers (P < 0.001). Using the HER-2/neu transfected cell-line model, we confirmed the mechanistic basis of the clinical observations which elucidate the pathway leading to HER-2/neu-mediated androgen independence. On androgen deprivation, the HER-2/neu transfected cells had higher proliferation rates, lower G1 arrest, inhibited p27 up-regulation, a lower apoptotic index, and higher Bcl-2, prostate-specific antigen and phosphorylated Akt expression than the mock-transfected LNCaP cells. This study suggests that prostate cancer cells undergo a series of coordinated changes after exposure to AAT, which eventually result in the development of androgen independence. Further, in support of previous results, it appears that a major factor in this process is the induction of HER-2/neu overexpression, which occurs after initial exposure to AAT. HER-2/neu may contribute to the development of androgen independence through: (i) maintaining cell proliferation; (ii) inhibiting apoptosis; and/or (iii) inducing AR activation in a ligand-independent fashion. These effects may be mediated, at least in part, through activation of the PI3K/Akt pathway.

Role of HER‐2/neu signaling in sensitivity to tumor necrosis factor‐related apoptosis‐inducing ligand: Enhancement of TRAIL‐mediated apoptosis by amiloride

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in numerous transformed cell lines but not in most normal cells. Although this selectivity offers a potential therapeutic application in cancer, not all cancers are sensitive to TRAIL-mediated apoptosis. In this study, we observed that amiloride, a current clinically used diuretic drug, which had little or no cytotoxicity, sensitized TRAIL-resistant human prostate adenocarcinoma LNCaP and human ovarian adenocarcinoma SK-OV-3 cells. The TRAIL-mediated activation of caspase, and PARP cleavage, were promoted in the presence of amiloride. Western blot analysis showed that combined treatment with TRAIL and amiloride did not change the levels of TRAIL receptors (DR4, DR5, and DcR2) and anti-apoptotic proteins (FLIP, IAP, and Bcl-2). However, amiloride dephosphorylated HER-2/neu tyrosine kinase as well as Akt, an anti-apoptotic protein. Interestingly, amiloride also dephosphorylated PI3K and PDK-1 kinases along with PP1alpha phosphatase. In vitro kinase assay revealed that amiloride inhibited phosphorylation of kinase as well as phosphatase by competing with ATP. Taken together, the present studies suggest that amiloride enhances TRAIL-induced cytotoxicity by inhibiting phosphorylation of the HER-2/neu-PI3K-Akt pathway-associated kinases and phosphatase.

Inhibition of HER-2/neu Kinase Impairs Androgen Receptor Recruitment to the Androgen Responsive Enhancer

Advanced prostate cancer invariably recurs despite androgen deprivation therapy. The androgen receptor (AR) likely plays a key role in this progression and in the continued survival and proliferation of prostate cancer cells in the low androgen environment. Cross-talk with growth factor receptors, such as epidermal growth factor receptor (EGFR) family, has been postulated as a potential mechanism to activate AR in recurrent prostate cancer. We have investigated the role of HER-2/neu (ErbB-2) tyrosine kinase in AR function by characterizing the effect of inhibiting endogenous HER-2 activity in LNCaP cells. We used two independent methods, expression of intracellular single-chain antibody against HER-2 and treatment with a novel dual EGFR/HER-2 kinase inhibitor GW572016 (lapatinib). Expression of intracellular HER-2 antibody scFv-5R and treatment with GW572016 inhibited HER-2 signaling. This HER-2 inhibition led to impairment of AR-mediated functions, such as androgen-stimulated growth and the induction of endogenous prostate-specific antigen (PSA) mRNA and protein. Androgen-stimulated recruitment of AR and histone acetylation at the androgen responsive enhancer of the PSA gene, detected by chromatin immunoprecipitation analysis, were impaired by HER-2 inhibition. GW572016 was more potent in its ability to inhibit PSA expression and AR recruitment and histone acetylation than the EGFR-selective kinase inhibitor ZD1839 (gefitinib), consistent with the HER-2 kinase playing the major role in AR regulation. These results show that HER-2 signaling is required for optimal transcriptional activity of AR in prostate cancer cells and suggest that HER-2 inhibition may provide a novel strategy to disrupt AR function in prostate cancer.

Cyclooxygenase-2 induces EP1- and HER-2/Neu-dependent vascular endothelial growth factor-C up-regulation: a novel mechanism of lymphangiogenesis in lung adenocarcinoma.

Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin H synthase, has been implicated in the progression of human lung adenocarcinoma. However, the mechanism underlying COX-2's effect on tumor progression remains largely unknown. Lymphangiogenesis, the formation of new lymphatic vessels, has recently received considerable attention and become a new frontier of tumor metastasis research. Here, we study the interaction between COX-2 and the lymphangiogenic factor, vascular endothelial growth factor (VEGF)-C, in human lung cancer cells and their implication in patient outcomes. We developed an isopropyl-beta-D-thiogalactopyranoside-inducible COX-2 gene expression system in human lung adenocarcinoma CL1.0 cells. We found that VEGF-C gene expression but not VEGF-D was significantly elevated in cells overexpressing COX-2. COX-2-mediated VEGF-C up-regulation was commonly observed in a broad array of non-small cell lung cancer cell lines. The use of pharmacological inhibitors or activators and genetic inhibition by EP receptor-antisense oligonucleotides revealed that prostaglandin EP(1) receptor but not other prostaglandin receptors is involved in COX-2-mediated VEGF-C up-regulation. At the mechanistic level, we found that COX-2 expression or prostaglandin E(2) (PGE(2)) treatment could activate the HER-2/Neu tyrosine kinase receptor through the EP(1) receptor-dependent pathway and that this activation was essential for VEGF-C induction. The transactivation of HER-2/Neu by PGE(2) was inhibited by way of blocking the Src kinase signaling using the specific Src family inhibitor, PP1, or transfection with the mutant dominant negative src plasmid. Src kinase was involved in not only the HER-2/Neu transactivation but also the following VEGF-C up-regulation by PGE(2) treatment. In addition, immunohistochemical staining of 59 lung adenocarcinoma specimens showed that COX-2 level was highly correlated with VEGF-C, lymphatic vessels density, and other clinicopathological parameters. Taken together, our results provided evidence that COX-2 up-regulated VEGF-C and promotes lymphangiogenesis in human lung adenocarcinoma via the EP(1)/Src/HER-2/Neu signaling pathway.

Receptor tyrosine kinases: the main targets for new anticancer therapy.

Because conventional chemotherapy is not specific for cancer cells leading to toxic side effects there is a need for novel agents with high grade antitumor specificity. The major prerequisite to develop such drugs is to understand the targets that these agents should attack. In recent years a number of promising new anticancer drugs have been developed which target intracellular pathways or extracellular cell molecules. The clinically most effective compounds function as tyrosine kinase inhibitors. In the past, various tyrosine kinase receptors have been identified as regulators of tumor or tumor vessel growth. Having shown their expression characteristics in different tumor entities, specific inhibitors of the ATP binding sites of these receptors or antibodies were developed and entered clinical trials. The pathognomonic role of the tyrosine kinase defines the way of action of the inhibiting drug, whereas the amount of expression in tumor tissue defines the rationale to use the inhibitor to treat a specific protein. The future will define indications for such drugs by tumor kinase profiles instead of tumor entities. Gleevec, inhibiting the BCR-ABL tyrosine kinase; Iressa, inhibiting the EGF-receptor tyrosine kinase; Herceptin, inhibiting the Her2/neu tyrosine kinase and PTK787/ZK222584, inhibiting the VEGF-receptor tyrosine kinase will be discussed as representatives of selective tyrosine kinase inhibitors whereas ZD6474 and SU6668 will be discussed as representatives of multitarget tyrosine kinase inhibitors.

Targeting HER2: Recent developments and future directions for breast cancer patients

Overexpression of the HER2/ neu oncogene (also known as c-erbB2) is a frequent molecular event in multiple human cancers, including breast and ovarian cancer. Patients with cancer that overexpress HER2/ neu are associated with unfavorable prognosis, shorter relapse time, and low survival rate. Treatments that target HER2/ neu expression in cancer cells have been shown to be useful strategies to significantly reverse the malignancy induced by HER2/ neu overexpression. The humanized anti-HER2/ neu antibody, trastuzumab (Herceptin; Genentech, Inc, South San Francisco, CA) has proven to be effective in clinical trials in patients with metastatic breast cancer. In addition, tyrosine kinase inhibitors such as emodin can also target the HER2/ neu oncogenic activity. Emodin treatment inhibits HER2/ neu tyrosine kinase activity and preferentially suppresses the transformation of HER2/ neu-overexpressing breast cancer cells. Emodin also sensitizes HER2/ neu-over-expressing cancer cells to chemotherapeutic agents, including cisplatin, doxorubicin, etoposide, and paclitaxel. Alternatively, HER2/ neu overexpression can be repressed by attenuating the promoter activity of the HER2/ neu gene. We have identified a number of potent transcriptional regulators, including the ets family member PEA3 and the adenovirus type 5 EIA, which are able to repress HER2/ neu gene expression. Expression of these transcriptional regulators resulted in downregulation of HER2/ neu promoter activity and reversed the transformed phenotype of the cancer cells in vitro. In vivo studies show that these HER2/ neu repressors can act therapeutically as tumor suppressor genes for tumors that overexpress HER2/ neu. These preclinical studies clearly indicate that transcriptional repressors that downregulate HER2/ neu can be effective regimens for cancer treatment in a gene therapy format. More importantly, the tumor-free survival rate of treated animals is dramatically increased under nontoxic doses compared with untreated animals. A phase I clinical trial using EIA-liposome in breast and ovarian patients has recently been completed. Following treatment, we observed downregulation of the HER2/ neu protein accompanied by EIA expression in both cancer and noncancer cells. Numbers of tumor cells in the pleural effusion or ascites were found to be dramatically reduced after treatment. Furthermore, apoptosis was strongly induced in the tumor cells. A phase II study has been started to further evaluate therapeutic efficacy and tumor suppression mechanisms of El A. These studies show the clinical potential of targeting HER2/neu in cancer therapy.

HER2 overexpression and cancer targeting

Overexpression of the HER2/ neu oncogene is a frequent molecular event in multiple human cancers including breast and ovarian cancer. Patients with breast cancer that overexpress HER2/ neu have a poor prognosis, shorter relapse time, and low survival rate. In this report, the biologic signaling pathway mediated by HER2/ neu tyrosine kinase receptor will be discussed. The mechanisms leading to transformation and tumor-igenesis of HER2/ neu-overexpressing cells will also be addressed. Treatments that target HER2/ neu expression in cancer cells have been shown to be useful strategies to significantly reverse the malignancy induced by HER2/ neu overexpression. In this report we will summarize strategies for targeting the HER2/ neu gene, including targeting the gene product p 185 oncoprotein, or transcriptional downregulation through the oncogene promoter. These fundamental studies, performed by different groups, warrant the clinical potential of targeting HER2/ neu in cancer therapy.

Asymmetric Synthesis of the Nakijiquinones—Selective Inhibitors of the Her-2/Neu Protooncogene

A Wieland-Miescher type ketone and a tetramethoxyaryl derivative are the key building blocks for the enantioselective total synthesis of nakijiquinone C (1). The nakijiquinones are the only natural products known that selectively inhibit the Her-2/Neu tyrosine kinase, a protooncogene product that is vastly overexpressed in about 30 % of primary breast, ovary, and gastric carcinomas.

Enhanced adenoviral transduction efficiency in HER-2/neu-overexpressing human breast cancer cells not induced by an integrin pathway.

Adenoviruses are currently widely used as vectors in gene therapy. The steps involved in adenoviral infection have been investigated, but the factors regulating viral entry to the cell have not been clearly identified. We observed a high adenoviral infection rate in HER-2/neu-overexpressing breast cancer cells in vitro (435.eb1 and MCF-7/H18) and in vivo (435.eb1). We used emodin, a tyrosine kinase inhibitor that suppresses autophosphorylation and transphosphorylation activities of the HER-2/neu tyrosine kinase, to test the role of HER-2/neu in adenoviral transduction. Emodin treatment resulted in a marked decrease in the transduction efficiency of HER-2/neu-overexpressing cells but not in the parental cells. Because previous studies have shown that epidermal growth factor and tumor growth factor-alpha increase the expression level of integrin. Because integrin alphav is known as a promotor of viral internalization, penetration, or both, we investigated whether the observed increased transduction rate in HER-2/neu transfectants was mediated through the increased expression of integrin alphav. To test this hypothesis, we examined the level of integrin alphav of in HER-2/neu overexpressing cells. We found that the level of integrin alphav expression detected in HER-2/neu overexpressing cells by immunoblot analysis was similar to the level of integrin alphav found in its parental cells. These results suggest that HER-2/neu expression may have a significant role in the viral transduction efficiency through an integrin alphav independent pathway.