Growth inhibition of breast cancer cells by Grb2 downregulation is correlated with inactivation of mitogen-activated protein kinase in EGFR, but not in ErbB2, cells

Increased breast cancer growth has been associated with increased expression of epidermal growth factor receptor (EGFR) and ErbB2 receptor tyrosine kinases (RTKs). Upon activation, RTKs may transmit their oncogenic signals by binding to the growth factor receptor bound protein-2 (Grb2), which in turn binds to SOS and activates the Ras/Raf/MEK/mitogen-activated protein (MAP) kinase pathway. Grb2 is important for the transformation of fibroblasts by EGFR and ErbB2; however, whether Grb2 is also important for the proliferation of breast cancer cells expressing these RTKs is unclear. We have used liposomes to deliver nuclease-resistant antisense oligodeoxynucleotides (oligos) specific for the GRB2 mRNA to breast cancer cells. Grb2 protein downregulation could inhibit breast cancer cell growth; the degree of growth inhibition was dependent upon the activation and/or endogenous levels of the RTKs. Grb2 inhibition led to MAP kinase inactivation in EGFR, but not in ErbB2, breast cancer cells, suggesting that different pathways might be used by EGFR and ErbB2 to regulate breast cancer growth.     

Dynamin2 downregulation delays EGFR endocytic trafficking and promotes EGFR signaling and invasion in hepatocellular carcinoma

Dysregulation of endocytosis was viewed as an emerging feature of cancer development and progression. A large GTPase dynamin2 plays a significant role in receptor tyrosine kinases (RTKs) endocytosis. The study was designed to investigate its roles in hepatocellular carcinoma (HCC) metastasis and its underlying mechanism. Dynamin2 expression in cancer tissues from HCC patients was assessed by immunohistochemistry and its prognostic significance for the patients was conducted using univariate and multivariate analysis. Its role in tumor invasion and metastasis was evaluated in vitro by gene silence using siRNA-mediated approach and the small molecule inhibitor of Dynasore. EGFR expression in HCC cell lines and EGFR downstream signaling ERK1/2 was evaluated by Western-blot and immunofluorescence analyses after Dynamin2 inhibition. Our data indicated that low expression of dynamin2 was well correlated with invasion characteristics and shorter overall survival. HCC cell migration, colony formation and invasion were significantly increased after the inhibition of dynamin2 in HCC cells. Internalization of EGFR was markedly reduced when dynamin2 was knock down or inhibition. In addition, we observed that dynamin2 regulated EGF mediated EGFR downstream Ras/ERK1/2 signaling and p-ERK1/2 accumulation in nucleus. The results demonstrate a possible mechanism of dynamin involved EGFR endocytosis and modulation of metastasis in HCC. Dynamin2 inhibits the invasion and metastasis of HCC cells by the promotion of EGFR endocytosis and downregulation of ERK1/2 phosphorylation.     

Csk-binding protein (Cbp) negatively regulates epidermal growth factor-induced cell transformation by controlling Src activation

Epidermal growth factor receptor (EGFR) and Src tyrosine kinase cooperate in regulating EGFR-mediated cell signaling and promoting cell transformation and tumorigenesis in pathological conditions. Activation of Src is tightly regulated by the C-terminal Src kinase (Csk). The Csk-binding protein (Cbp) is a ubiquitously expressed transmembrane protein. Its functions include suppression of T-cell receptor activation through recruiting Csk and inhibiting Src family kinase (SFK). However, a potential role of Cbp in EGF-induced cell activities has not been investigated. Here, we report that EGF-stimulation-induced Cbp tyrosine phosphorylation followed by Cbp-Csk association, in a SFK-dependent manner. Expression of wild-type (wt) Cbp remarkably suppressed EGF-induced activation of Src, ERK1/2, and Akt-1 enzymes, and NIH3T3 cell transformation, as well as colony formation of a breast cancer cell line (MDA-MB-468) in soft agar. In contrast, expression of CbpY317F or knockdown endogenous Cbp in NIH3T3 cells by RNA interference significantly enhanced EGF-induced activation of these enzymes and cell transformation. In addition, overexpression of multiple receptor tyrosine kinases (RTKs)-induced Cbp tyrosine phosphorylation. These results demonstrate that Cbp functions as a negative regulator of cell transformation and tumor cell growth through downregulation of Src activation, suggesting that Cbp might be broadly involved in RTKs-activated signaling pathways and tumorigenesis.     

Cell-cell and cell-extracellular matrix adhesion molecules communicate with growth factor receptors: an interactive signaling Web

The review article by Weiner and Zagzag in this volume of Cancer Investigation highlights some of the more current data relating to the important interactions that can occur between cell surface growth factor receptor tyrosine kinases (RTKs) and the cell adhesion kinases (CAKs). This is an important biologic and clinical topic because oncogenic activation of RTKs, such as the epidermal growth factor receptor, c-erb B-2. or the fibroblast growth factor receptor-1, by gene amplification and/or by overexpression can lead to malignant transformation in vitro and is involved in the pathogenesis of several different types of human carcinomas (1,2). In addition. a decrease in cell adhesion is an initial prerequisite for engaging the initial stages of tumor cell invasion and metastasis. Selective therapeutic targeting of RTKs or effectors in the metastatic cascade have already shown to be effective in the treatment of some types of human tumors (3).     

The molecular rationale of Src inhibition in colorectal carcinomas

Src has been one of the most studied proto‐oncogenes. The cellular Src (c‐Src) holds a critical role in several human malignancies and has emerged as a key factor that promotes tumor progression during the multistep process of colorectal cancer (CRC) pathogenesis. The robust activation of Src in CRC of aggressive phenotype and poor prognosis seems to be a subsequent event of a strong link between its deregulated activity and the tumor's cell adhesion properties, invasiveness and metastatic potential. The rarely detected genetic defects drive interest in signaling networks that control Src kinase activity and integrate the association of Src with receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). Therefore, a dynamic crosstalk is being formed with oncogenic capacity and therapeutic applications, because Src inhibition seems to sensitize previously unresponsive cancer cells to chemotherapy and anti‐EGFR inhibitors. The present review explores the molecular basis behind Src inhibition in colorectal carcinomas. Furthermore, preclinical studies and clinical trials of Src inhibitors and combination regimens are discussed, providing new insights for further investigation and new therapeutic strategies.     

ErbB receptors and epithelial-cadherin-catenin complex in human carcinomas

The ErbB family of receptor tyrosine kinases have important roles in maintaining normal epithelial cell function. The ErbBs are involved in the interaction between cells and cell-matrix adhesion molecules and have proven critical in maintaining the integrity of the epithelial cell environment. Deregulation of these tyrosine receptors has been associated with several human diseases. In particular, the expression or activation of epidermal growth factor receptor (EGFR) and ErbB2 is altered in many epithelial tumors. Epithelial (E)-cadherin is another major molecule expressed by epithelial cells. To create efficient cell-cell adhesion, E-cadherin couples its cytoplasmic domain to catenins and the actin cytoskeleton. The loss of intercellular adhesion appears to be a fundamental aspect of the neoplastic phenomena. In addition, EGFR and ErbB2 signaling associated with the E-cadherin-catenin complex has been demonstrated in normal and cancer cells. This signaling is involved in regulating cell adhesion and the invasive growth of cancers. This article provides an overview of the interaction between the ErbB tyrosine receptors and the E-cadherin-catenin complex in human carcinomas.     

Targeted Inhibition of Multiple Receptor Tyrosine Kinases in Mesothelioma

The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR) and MET are activated in subsets of mesothelioma, suggesting that these kinases might represent novel therapeutic targets in this notoriously chemotherapy-resistant cancer. However, clinical trials have shown little activity for EGFR inhibitors in mesothelioma. Despite the evidence for RTK activation in mesothelioma pathogenesis, it is unclear whether transforming activity is dependent on an individual kinase oncoprotein or the coordinated activity of multiple kinases. Using phospho-RTK and immunoblot assays, we herein demonstrate activation of multiple RTKs (EGFR, MET, AXL, and ERBB3) in individual mesothelioma cell lines but not in normal mesothelioma cells. Inhibition of mesothelioma multi-RTK signaling was accomplished using combinations of RTK direct inhibitors or by inhibition of the RTK chaperone, heat shock protein 90 (HSP90). Multi-RTK inhibition by the HSP90 inhibitor 17-allyloamino-17-demethoxygeldanamycin (17-AAG) had a substantially greater effect on mesothelioma proliferation and survival compared with inhibition of individual activated RTKs. HSP90 inhibition also suppressed phosphorylation of downstream signaling intermediates (AKT, mitogen-activated protein kinase, and S6); upregulated the p53, p21, and p27 cell cycle checkpoints; induced G₂ phase arrest; induced caspase 3/7 activity; and led to an increase in the sub-G₁ apoptotic population. These compelling proapoptotic and antiproliferative responses indicate that HSP90 inhibition warrants clinical evaluation as a novel therapeutic strategy in mesothelioma.     

Tyrosine 1045 Codon Mutations in Exon 27 of EGFR are Infrequent in Oral Squamous Cell Carcinomas

Background: The activation and inactivation of receptor tyrosine kinases are tightly regulated to ensure faithfulreplication of cells. After having transduced extracellular growth activating signals, activated EGFR is subjectedto downregulation either by clathrin mediated endocytosis or c-Cbl mediated proteasome degradation dependingon the ligand concentration. c-Cbl is an ubiquitin ligase which requires a phosphorylated tyrosine residue atposition 1045 in the cytoplasmic domain of EGFR to interact and add ubiquitin molecules. While activatingmutations in exons 19 and 21 have been associated with the development of several cancers, the status of mutationsat tyrosine 1045 coding exon 27 of EGFR remain to be investigated. Consistently, defective phosphorylation at1045 has been associated with sustained phosphorylation of EGFR in non-small lung carcinomas. Hence in thepresent study we investigated the genetic status of the tyrosine 1045 coding site within exon 27 of EGFR geneto explore for possible occurrence of mutations in this region, especially since no studies have addressed thisissue so far. Materials and Methods: Tumor chromosomal DNA isolated from thirty five surgically excised oralsquamous cell carcinoma tissues was subjected to PCR amplification with intronic primers flanking the tyrosine1045 coding exon 27 of EGFR gene. The PCR amplicons were subsequently subjected to direct sequencing toelucidate the mutation status. Results: Sequence analysis identified no mutations in the tyrosine 1045 codon ofEGFR in any of the thirty five samples that were analyzed. Conclusions: The lack of identification of mutation inthe tyrosine 1045 codon of EGFR suggests that mutations in this region may be relatively rare in oral squamouscell carcinomas. To the best of our knowledge, this study is the first to have explored the genetic status of exon27 of EGFR in oral squamous cell carcinoma tissue samples.     

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

The overexpression or mutation of tyrosine kinases (TKs), such as the epidermal growth factor receptor (EGFR), can lead to the development of cancer. The most common mutation of the EGFR in glioblastomas is the deletion of exons 2-7 known as the EGFRvIII. This mutant receptor cannot bind EGF but, instead, is constitutively active. The Cbl family of ubiquitin ligases (Cbl, Cbl-b, and Cbl-c) targets the activated EGFR for degradation. As the EGFRvIII is transforming, we investigated whether it could be downregulated by the Cbl proteins. The overexpression of all three Cbl proteins resulted in the ubiquitination and degradation of the EGFRvIII. As with the wild-type EGFR, the TK-binding domain and the RING finger of Cbl-b are sufficient for the downregulation of the EGFRvIII. Also, we found that Cbl-b is recruited to the EGFRvIII and inhibits the transformation of NIH 3T3 cells by the EGFRvIII. Mutation of the Cbl-binding site (Y1045F) in the EGFRvIII inhibits its ubiquitination and downregulation by Cbl-b and enhances its ability to transform. Furthermore, the EGFR TK inhibitor, AG 1478, prevents the downregulation of the EGFRvIII by the Cbl proteins and antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor. In conclusion, the EGFRvIII does not transform by escaping regulation by Cbl proteins and this activation-induced downregulation of the EGFRvIII has an important role in mediating the toxicity of anti-EGFRvIII immunotoxins.     

AMP-activated kinase (AMPK) regulates activity of HER2 and EGFR in breast cancer

AMP-activated Protein Kinase (AMPK) activity retards growth of many types of cancers. Investigating effects of AMPK activation on breast cancer cell signaling and survival, we found that breast cancer cell lines with amplification and over-expression of HER2 or EGFR are 2- to 5-fold more sensitive to cytotoxic effects of AICAR, a canonical pharmacological activator of AMPK, than breast cancer cell lines lacking HER2 or EGFR overexpression. Paralleling effects on cell survival, AICAR leads to dose- and time-dependent inhibition of HER2 and EGFR in HER2-amplified breast cancer cells, with activation of AMPK and suppression of HER2/EGFR activity preceding commitment to cell death. Transfection of constitutively active AMPKα also leads to decreased HER2 and EGFR phosphorylation, reduced downstream signaling associated with these receptor tyrosine kinases (RTKs), and reduced breast cancer cell growth, confirming effects of AMPK activity on HER2/EGFR. Ensuing co-immunoprecipitation experiments demonstrated an interaction of HER2 with AMPK and an in vitro phosphorylation assay found that HER2 and EGFR contain sequences that are potential substrates for AMPK. Our results lead us to postulate that AMPK regulates HER2 and EGFR activity in HER2-amplified breast cancer cells and thus activation of AMPK might provide therapeutic benefit in such cancers.     

Feedback inhibition by RALT controls signal output by the ErbB network

The ErbB-2 interacting protein receptor-associated late transducer (RALT) was previously identified as a feedback inhibitor of ErbB-2 mitogenic signals. We now report that RALT binds to ligand-activated epidermal growth factor receptor (EGFR), ErbB-4 and ErbB-2.ErbB-3 dimers. When ectopically expressed in 32D cells reconstituted with the above ErbB receptor tyrosine kinases (RTKs) RALT behaved as a pan-ErbB inhibitor. Importantly, when tested in either cell proliferation assays or biochemical experiments measuring activation of ERK and AKT, RALT affected the signalling activity of distinct ErbB dimers with different relative potencies. RALT deltaEBR, a mutant unable to bind to ErbB RTKs, did not inhibit ErbB-dependent activation of ERK and AKT, consistent with RALT exerting its suppressive activity towards these pathways at a receptor-proximal level. Remarkably, RALT deltaEBR retained the ability to suppress largely the proliferative activity of ErbB-2.ErbB-3 dimers over a wide range of ligand concentrations, indicating that RALT can intercept ErbB-2.ErbB-3 mitogenic signals also at a receptor-distal level. A suppressive function of RALT deltaEBR towards the mitogenic activity of EGFR and ErbB-4 was detected at low levels of receptor occupancy, but was completely overcome by saturating concentrations of ligand. We propose that quantitative and qualitative aspects of RALT signalling concur in defining identity, strength and duration of signals generated by the ErbB network.     

ErbB/HER receptor activation and preclinical efficacy of lapatinib in vestibular schwannoma

Vestibular schwannomas (VS) arising sporadically or in patients with neurofibromatosis type 2 (NF2) consistently lack expression of Merlin, a tumor suppressor. Conventional treatment options include surgery and radiotherapy but there is no validated medical option. Recent evidence suggests that Merlin deficiency may result in abnormal activation of receptor tyrosine kinases (RTKs) and downstream signaling, promoting tumor growth. Although small-molecule RTK inhibitors are widely available for clinical use, no such therapy has been validated in patients with VS. To screen for RTK activation, surgical VS specimens from patients with and without NF2 were analyzed by phospho-RTK profiling arrays. Downstream signaling pathway activation was analyzed by phospho-MAPK arrays. Activated RTKs and downstream kinases were validated immunohistochemically in corresponding formalin-fixed, paraffin-embedded tissues. Phospho-RTK arrays and immunohistochemistry showed consistent overexpression and activation of EGFR family receptors and evidence of ERK1/2 downstream signaling was observed in all samples analyzed (n = 11). Based on the findings, the small-molecule EGFR/ErbB2 kinase inhibitor lapatinib was selected for evaluation of target inhibition and treatment efficacy in our in vitro human schwannoma model. EGFR/ErbB2 targeted therapy with lapatinib inhibited ErbB2 phosphorylation and survivin upregulation, as well as downstream ERK1/2 and AKT activation, resulting in decreased proliferation. We conclude that EGFR family receptor activation is a consistent feature of both sporadic and NF2-related VS. Molecular targeted therapy with lapatinib downregulates survivin and has antiproliferative activity in a preclinical VS model. Based on these findings, a clinical trial with lapatinib for the treatment of VS is currently underway.     

Inverse relationship between E-cadherin and p27Kip1 expression in renal cell carcinoma

The cell-cell adhesion system plays a pivotal role in the maintenance of tissue structure and cell-cell communication. E-cadherin is a major adhesion protein of the epithelial cells, and E-cadherin expression may be involved in the regulation of cell proliferation or differentiation. To address the relationship between cell-cell adhesion and cell proliferation, we focused on the alteration of p27Kip1 (p27), a cyclin-dependent kinase inhibitor, by E-cadherin-mediated adhesion. In an immunohistochemical study of 76 cases of renal cell carcinoma (RCC), the p27-labeling index (LI) was 67% in E-cadherin-reduced RCC, but only 28% in E-cadherin-preserved RCC. E-cadherin-expressing cells rarely expressed p27 in various cancers including those of the breast, colon, liver and prostate. In a subconfluent monolayer culture, the E-cadherin levels were increased steadily in the E-cadherin positive RCC cell line ACHN, whereas the p27 levels were decreased. Subsequent exposure of ACHN cells to the E-cadherin-specific function-blocking antibody reduced the growth associated with the increase in p27 and the decrease in phosphorylated epidermal growth factor receptor (EGFR). In the E-cadherin negative RCC cell line Caki-1, these effects were not observed. These results suggest that E-cadherin-mediated adhesion may be involved in the contact stimulation for cell proliferation in part through the downregulation of p27 and the activation of EGFR in human cancers.     

Multipoint targeting of the PI3K/mTOR pathway in mesothelioma

Background:

Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with those of asbestos-associated disease. We previously demonstrated co-activation of multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET, and AXL in mesothelioma cell lines, suggesting that these kinases could serve as novel therapeutic targets. Although clinical trials have not shown activity for EGFR inhibitors in mesothelioma, concurrent inhibition of various activated RTKs has pro-apoptotic and anti-proliferative effects in mesothelioma cell lines. Thus, we hypothesised that a coordinated network of multi-RTK activation contributes to mesothelioma tumorigenesis.

Methods:

Activation of PI3K/AKT/mTOR, Raf/MAPK, and co-activation of RTKs were evaluated in mesotheliomas. Effects of RTK and downstream inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, apoptosis, activation of signalling intermediates, expression of cell-cycle checkpoints, and cell-cycle alterations.

Results:

We demonstrate activation of the PI3K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, but not in non-neoplastic mesothelial cells. The AKT activation, but not MAPK activation, was dependent on coordinated activation of RTKs EGFR, MET, and AXL. In addition, PI3K/AKT/mTOR pathway inhibition recapitulated the anti-proliferative effects of concurrent inhibition of EGFR, MET, and AXL. Dual targeting of PI3K/mTOR by BEZ235 or a combination of RAD001 and AKT knockdown had a greater effect on mesothelioma proliferation and viability than inhibition of individual activated RTKs or downstream signalling intermediates. Inhibition of PI3K/AKT was also associated with MDM2-p53 cell-cycle regulation.

Conclusions:

These findings show that PI3K/AKT/mTOR is a crucial survival pathway downstream of multiple activated RTKs in mesothelioma, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention.     

Enhanced activation of epidermal growth factor receptor caused by tumor-derived E-cadherin mutations

Mutations of the tumor suppressor E-cadherin and overexpression of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) are among the most frequent genetic alterations associated with diffuse-type gastric carcinoma. Accumulating evidence suggests a functional relationship between E-cadherin and EGFR that regulates both proteins. We report that somatic mutation of E-cadherin is associated with increased activation of EGFR followed by enhanced recruitment of the downstream acting signaling components growth factor receptor binding protein 2 and Shc, and activation of Ras. Reduced complex formation of mutant E-cadherin - with an in frame deletion of exon 8 in the extracellular domain resulting in reduced adhesion and increased motility - with EGFR was observed compared with wild-type E-cadherin. We conclude that reduced binding of mutant E-cadherin to EGFR in a multicomponent complex or reduced stability of the complex may enhance EGFR surface motility, thereby facilitating EGFR dimerization and activation. Furthermore, reduced surface localization due to enhanced internalization of mutant E-cadherin compared with the wild-type protein was observed. The internalization of EGFR was decreased in response to epidermal growth factor stimulation in cells expressing mutant E-cadherin, suggesting that mutation of E-cadherin also influences the endocytosis of EGFR. Moreover, we show increased activation of EGFR in gastric carcinoma samples with mutant E-cadherin lacking exons 8 or 9. In summary, we describe activation of EGFR by mutant E-cadherin as a novel mechanism in tumor cells that explains the enhanced motility of tumor cells in the presence of an extracellular mutation of E-cadherin.