Regulation Of Epidermal Growth Factor Receptor Research Articles

The Ankrd 13 family of UIM-bearing proteins regulates EGF receptor endocytosis from the plasma membrane

The mechanism of ubiquitin-dependent endocytosis of cell surface proteins is not completely understood. Here we examine the role of the ankyrin repeat domain (Ankrd) 13A, 13B, and 13D proteins, which constitute a functionally unknown family of ubiquitin-interacting motif (UIM)-bearing proteins, in the process. Stimulation of human HeLa cells with epidermal growth factor (EGF) rapidly induced direct binding of Ankrd 13 proteins to ubiquitinated EGF receptor (EGFR) via the UIMs. The binding was inhibited when the Ankrd 13 proteins underwent UIM-dependent monoubiquitination, suggesting that their activity is regulated by ubiquitination of themselves. Ankrd 13 proteins bound specifically to Lys-63-linked ubiquitin chains, which was consistent with a previous report that EGFR mainly undergoes Lys-63-linked polyubiquitination. Ankrd 13 proteins were anchored, via the central region and UIMs, to the plasma membrane, where they colocalized with EGFR. Finally, overexpression of wild-type as well as truncated-mutant Ankrd 13 proteins strongly inhibited rapid endocytosis of ubiquitinated EGFR from the surface in EGF-treated cells. We conclude that by binding to the Lys-63-linked polyubiquitin moiety of EGFR at the plasma membrane, Ankrd 13 proteins regulate the rapid internalization of ligand-activated EGFR.

Negative Feedback Mechanisms Surpass the Effect of Intrinsic EGFR Activation during Skin Chemical Carcinogenesis

The negative feedback regulation of epidermal growth factor receptor (EGFR) and other tyrosine kinase receptors, including receptor dephosphorylation and endocytosis followed by degradation, is becoming recognized as a major determinant of receptor function. To evaluate the significance of the negative regulation of EGFR during carcinogenesis in vivo, we subjected the mutant mouse line Dsk5, in which the intrinsic activation of the receptor due to a point mutation is normally counterbalanced by increased posttranslational receptor down-regulation, to skin chemical carcinogenesis. Dsk5 mice showed reduced tumor numbers and tumor burden compared with control littermates, and Dsk5-derived tumors showed a reduction in the activation and total levels of EGFR. Furthermore, the transcript levels of several molecules known to act as negative regulators of EGFR were significantly increased in Dsk5-derived tumors. Another intriguing observation was the appearance of tumors with sebaceous differentiation in the ears of Dsk5 mice after chemical carcinogenesis. Further studies are necessary to reveal whether these tumors represent a cell type-specific evasion from EGFR negative feedback machinery. In conclusion, this study reveals that several negative feedback regulators contribute to suppression of the intrinsic activation of mutant EGFR during skin carcinogenesis, stressing the potential exploitation of negative regulators as either therapeutic targets or diagnostic tools in cancer and other diseases.

EGFR regulation of colon cancer stem-like cells during aging and in response to the colonic carcinogen dimethylhydrazine

One of the most consistent pathological conditions in the gastrointestinal tract with advancing age is malignancy, particularly gastrointestinal cancers, the incidence of which increases sharply with aging. Although the reasons for the age-related rise in colorectal cancer are not fully understood, we hypothesize that aging increases susceptibility of the colon to carcinogen(s)/toxicant(s), leading to an increase in cancer stem-like cells (CSLCs) that express cancer stem cell markers, in the colonic mucosa. The current study demonstrates that aging is associated with increased expression of several colon CSLC markers [CD44, CD166, and aldehyde dehydrogenase 1 (ALDH-1)] and a higher proportion of cells expressing these markers. Aging is also accompanied by increased expression of miR-21 in colon. These increases are further increased in response to the colonic carcinogen dimethylhydrazine (DMH). Aging is also associated with increased tyrosine-phosphorylated epidermal growth factor receptor (EGFR). Inhibition of EGFR using the EGFR inhibitor cetuximab abrogated the age-related increase in CD166 and ALDH-1 as well as miRNA (miR)-21. Our results provide new evidence that aging and DMH are associated with increases in CSLC biomarkers and miR21, each of which have been linked to colorectal cancer. EGFR inhibition attenuates these changes, indicating a role for EGFR in age- and mutagen-associated changes in CSLCs.

The regulatory role of the juxtamembrane region in the activity of the epidermal growth factor receptor

Although the EGFR (epidermal growth factor receptor) was discovered over 30years ago, its mechanism of activation is still the subject of intense study. There are many published studies on the mechanism of EGFR activation and regulation, including biochemical and biophysical analyses and crystallographic structures of EGFR in different activation states and conformations, mutated at various amino acids or bound to different pharmacological inhibitors. The cumulative biochemical, biophysical and structural data have led to a nearly complete account of the mechanism of activation of EGFR. The role of the JXM (juxtamembrane) domain in EGFR structure and activity has only recently begun to be elucidated through biochemical, biophysical and structural studies. In the present article, I review the studies that have highlighted the role of the JXM domain in EGFR activation.

Overexpression of phospho mutant forms of transglutaminase 2 downregulates epidermal growth factor receptor

Simultaneous upregulation of transglutaminase 2 (TG2) and epidermal growth factor receptor (EGFR) have been reported in a number of systems. Moreover, TG2 has been identified as a downstream target gene for EGF/EGFR. However, it is not known whether the relationship between EGFR and TG2 is only one-way or collaborative. Using embryonic fibroblasts derived from TG2 null mice (MEFtg2−/−), co-overexpressing native TG2 and EGFR, here we report that TG2 differentially regulates EGFR protein in the presence and absence of EGF. In the absence of EGF, TG2 facilitates EGFR downregulation whereas in the presence of EGF, TG2 has opposite effect on EGFR and facilitates Akt phosphorylation. TG2 mediated ligand-independent downregulation of EGFR was not observed in MEFtg2−/− cells overexpressing Ser212Ala phospho mutant form of TG2 suggesting a role of TG2 phosphorylation in this process. However, similar to native TG2, Ser212Ala-TG2 mutant was also able to attenuate ligand-dependent down regulation of EGFR in MEFtg2−/− cells. Interestingly, overexpression of Ser216Ala-TG2 mutant led to downregulation of EGFR in MEFtg2−/− cells irrespective of the ligand. These results were further confirmed in breast cancer cells expressing high levels of EGFR. Collectively, data presented here suggests that the relationship between EGFR and TG2 is collaborative and phosphorylation of TG2 play a key role in it. Phospho mutant forms of TG2 reported in this study may be utilized as a part of a novel strategy to downregulate EGFR in cancers with enhanced EGFR signaling.

β3 Integrin–EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells

Active RhoA localizes to plasma membrane, where it stimulates formation of focal adhesions and stress fibers. RhoA activity is inhibited by p190RhoGAP following integrin-mediated cell attachment to allow sampling of new adhesive environments. p190RhoGAP is itself activated by Src-dependent tyrosine phosphorylation, which facilitates complex formation with p120RasGAP. This complex then translocates to the cell surface, where p190RhoGAP down-regulates RhoA. Here we demonstrate that the epidermal growth factor receptor (EGFR) cooperates with β3 integrin to regulate p190RhoGAP activity in mouse mammary gland epithelial cells. Adhesion to fibronectin stimulates tyrosine phosphorylation of the EGFR in the absence of receptor ligands. Use of a dominant inhibitory EGFR mutant demonstrates that fibronectin-activated EGFR recruits p120RasGAP to the cell periphery. Expression of an inactive β3 integrin subunit abolishes p190RhoGAP tyrosine phosphorylation, demonstrating a mechanistic link between β3 integrin-activated Src and EGFR regulation of the RhoA inhibitor. The β3 integrin/EGFR pathway also has a positive role in formation of filopodia. Together our data suggest that EGFR constitutes an important intrinsic migratory cue since fibronectin is a key component of the microenvironment in normal mammary gland development and breast cancer. Our data also suggest that EGFR expressed at high levels has a role in eliciting cell shape changes associated with epithelial-to-mesenchymal transition.

The von Hippel-Lindau Tumor Suppressor Protein Promotes c-Cbl-Independent Poly-Ubiquitylation and Degradation of the Activated EGFR

Somatic mutations or reduced expression of the von Hippel-Lindau (VHL) tumor suppressor occurs in the majority of the clear cell renal cell carcinoma (ccRCC) and is a causal factor for the pathogenesis of ccRCC. pVHL was reported to suppress the oncogenic activity of Epidermal Growth Factor Receptor (EGFR) by reducing the expression of the EGFR agonist TGF-α and by reducing the translation efficiency of EGFR itself. Furthermore, it was reported that pVHL down-regulates activated EGFR by promoting efficient lysosomal degradation of the receptor. These modes of negative regulation of EGFR by pVHL were dependent on Hypoxia Inducible Factor (HIF). In this study, we report that HIF was not the only factor stabilizing the activated EGFR in VHL-deficient ccRCC cells. Down-regulation of endogenous HIF in these cells had little effect on the turnover rates of the activated EGFR. Furthermore, neither pretreatment with lysomomal inhibitors pretreatment nor down-regulation of c-Cbl, a major E3 ubiquitin ligase that targets the activated EGFR for lysosomal degradation, significantly increased the stabilities of EGFR in VHL-expressing ccRCC cells. In contrast, pretreatment with proteasomal inhibitors extended EGFR lifetime and led to similar EGFR half-lives in VHL-expressing and VHL-deficient ccRCC cells. Down-regulation of c-Cbl in VHL-deficient ccRCC cells revealed that the c-Cbl and pVHL collaborated to down-regulate the activated EGFR. Finally, we found that pVHL promoted the poly-ubiquitylation of the activated EGFR, and this function was c-Cbl-independent. Thus these results indicate that pVHL limits EGFR signaling by promoting c-Cbl-independent poly-ubiquitylation of the activated receptor, which likely results in its degradation by proteasome.

Acceleration of clear cell renal cell carcinoma growth in mice following bevacizumab/Avastin treatment: the role of CXCL cytokines

The anti-VEGF targeted antibody bevacizumab (BVZ) has been approved for treating renal cell carcinomas (RCCs). Although BVZ increases the progression-free survival of patients with metastatic RCC, the effect on overall survival is poor. To gain insight into the limited efficacy of BVZ on overall survival, we analyzed patient samples of RCC for angiogenic factors that may participate in escape from anti-VEGF therapy. Our study shows that the level of vascular endothelial growth factor (VEGF) in tumors was increased compared with normal tissue. The level of interleukin-8/CXCL8, a pro-angiogenic member of the CXCL family of cytokines, was also increased in tumors. These observations gave us a good reason to analyze the combined effects of BVZ and anti-CXCL8 antibodies on tumor growth. Surprisingly, we report that BVZ accelerates the growth of RCC in nude mice with in vivo selection of tumor cells with an increased growth capacity. Downregulation of receptor tyrosine phosphatase-κ, a phosphatase implicated in EGF receptor regulation, may partly explain this phenomenon. Modification of the vascular network and development of lymphatic vessels through VEGF-C production and compensatory production of pro-angiogenic CXCL cytokines were also observed. The apparent normalization of the vascular network prompted us to associate BVZ with the chemotherapeutic agent paclitaxel. While efficient in vitro, paclitaxel did not reverse the anti-VEGF effects in vivo. Anti-CXCL8-targeting antibodies were promising as they decreased intra-tumor VEGF production; decreased the pro-angiogenic CXCL/anti-angiogenic CXCL ratio and did not induce lymphangiogenesis. These observations hold clinical implication as they highlight putative markers implicated in escape from BVZ treatment. They also recommend proceeding with caution in the use of anti-VEGF therapy alone for treatment of RCC.

The cannabinoid receptor inverse agonist AM251 regulates the expression of the EGF receptor and its ligands via destabilization of oestrogen‐related receptor α protein

AM251 is an inverse agonist of the cannabinoid 1 receptor (CB(1)R) that can exert 'off-target' effects in vitro and in CB(1)R knock-out mice. AM251 is also potent at modulating tumour cell growth, suggesting that growth factor-mediated oncogenic signalling could be regulated by AM251. Since dysregulation of the EGF receptor has been associated with carcinogenesis, we examined AM251 regulation of EGF receptor (EGFR) expression and function. The various biological functions of AM251 were measured in CB(1)R-negative human cancer cells. Pharmacological and genetic approaches were used to validate the data. The mRNA levels for EGFR and its associated ligands, including HB-EGF, were induced several fold in PANC-1 and HCT116 cells in response to AM251. This event was associated with enhanced expression of EGFR on the cell surface with concomitant increase in EGF-induced cellular responses in AM251-treated cells. Exposure to XCT790, a synthetic inverse agonist of the orphan nuclear oestrogen-related receptor α (ERRα), also induced EGFR and HB-EGF expression to the same extent as AM251, whereas pretreatment with the ERRα-selective agonist, biochanin A, blunted AM251 actions. AM251 promoted the degradation of ERRα protein without loss of the corresponding mRNA. Knock-down of ERRα by siRNA-based approach led to constitutive induction of EGFR and HB-EGF levels, and eliminated the biological responses of AM251 and XCT790. Finally, AM251 displaced diethylstilbestrol prebound to the ligand-binding domain of ERRα. AM251 up-regulates EGFR expression and signalling via a novel non-CB(1)R-mediated pathway involving destabilization of ERRα protein in selected cancer cell lines.

Clinical Significance of miR-146a in Gastric Cancer Cases

The profiles of microRNAs change significantly in gastric cancer. MiR-146a is reported to be a tumor suppressor in pancreatic cancer, breast cancer, and prostate cancer. We investigated the clinical significance of miR-146a in gastric cancer, in particular focusing on hypothetical miR-146a target genes, such as epidermal growth factor receptor (EGFR) and interleukin-1 receptor-associated kinase (IRAK1). We examined miR-146a levels in 90 gastric cancer samples by q-real-time (qRT)-PCR and analyzed the association between miR-146a levels and clinicopathologic factors and prognosis. The regulation of EGFR and IRAK1 by miR-146a was examined with miR-146a-transfected gastric cancer cells. Moreover, we analyzed the association between miR-146a levels and the G/C single nucleotide polymorphism (SNP) within pre-miR-146a seed sequences in 76 gastric cancer samples, using direct sequencing of genomic DNA. In 90 clinical samples of gastric cancer, miR-146a levels in cancer tissues were significantly lower than those in the corresponding noncancerous tissue (P < 0.001). Lower levels of miR-146a were associated with lymph node metastasis and venous invasion (P < 0.05). Moreover, a lower level of miR-146a was an independent prognostic factor for overall survival (P = 0.003). Ectopic expression of miR-146a inhibited migration and invasion and downregulated EGFR and IRAK1 expression in gastric cancer cells. In addition, G/C SNP within the pre-miR-146a seed sequence significantly reduced miR-146a levels in the GG genotype compared with the CC genotype. MiR-146a contains an SNP, which is associated with mature miR-146a expression. MiR-146a targeting of EGFR and IRAK1 is an independent prognostic factor in gastric cancer cases.

MUC5AC Expression through Bidirectional Communication of Notch and Epidermal Growth Factor Receptor Pathways

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)-mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.

Usp18 Regulates Epidermal Growth Factor (EGF) Receptor Expression and Cancer Cell Survival via MicroRNA-7

Epidermal growth factor receptor (EGFR) is involved in development and progression of many human cancers. We have previously demonstrated that the ubiquitin-specific peptidase Usp18 (Ubp43) is a potent regulator of EGFR protein expression. Here we report that the 3'-untranslated region (3'-UTR) of the EGFR message modulates RNA translation following cell treatment with Usp18 siRNA, suggesting microRNA as a possible mediator. Given earlier evidence of EGFR regulation by the microRNA miR-7, we assessed whether miR-7 mediates Usp18 siRNA effects. We found that Usp18 depletion elevates miR-7 levels in several cancer cell lines because of a transcriptional activation and/or mRNA stabilization of miR-7 host genes and that miR-7 acts downstream of Usp18 to regulate EGFR mRNA translation via the 3'-UTR. Also, depletion of Usp18 led to a decrease in protein levels of other known oncogenic targets of miR-7, reduced cell proliferation and soft agar colony formation, and increased apoptosis. Notably, all of these phenotypes were reversed by a specific inhibitor of miR-7. Thus, our findings support a model in which Usp18 inhibition promotes up-regulation of miR-7, which in turn inhibits EGFR expression and the tumorigenic activity of cancer cells.

P130Cas Attenuates Epidermal Growth Factor (EGF) Receptor Internalization by Modulating EGF-Triggered Dynamin Phosphorylation

BackgroundEndocytosis controls localization-specific signal transduction via epidermal growth factor receptor (EGFR), as well as downregulation of that receptor. Extracellular matrix (ECM)-integrin coupling induces formation of macromolecular complexes that include EGFR, integrin, Src kinase and p130Cas, resulting in EGFR activation. In addition, cell adhesion to ECM increases EGFR localization at the cell surface and reduces EGFR internalization. The molecular mechanisms involved are not yet well understood.Methodology/Principal FindingsWe investigated the molecular mechanism by which p130Cas affects the endocytic regulation of EGFR. Biochemical quantification revealed that cell adhesion to fibronectin (FN) increases total EGFR levels and its phosphorylation, and that p130Cas is required for this process. Measurements of Texas Red-labeled EGF uptake and cell surface EGFR revealed that p130Cas overexpression reduces EGF-induced EGFR internalization, while p130Cas depletion enhances it. In addition, both FN-mediated cell adhesion and p130Cas overexpression reduce EGF-stimulated dynamin phosphorylation, which is necessary for EGF-induced EGFR internalization. Coimmunoprecipitation and GST pull-down assays confirmed the interaction between p130Cas and dynamin. Moreover, a SH3-domain-deleted form of p130Cas, which shows diminished binding to dynamin, inhibits dynamin phosphorylation and EGF uptake less effectively than wild-type p130Cas.Conclusions/SignificanceOur results show that p130Cas plays an inhibitory role in EGFR internalization via its interaction with dynamin. Given that the EGFR internalization process determines signaling density and specificity in the EGFR pathway, these findings suggest that the interaction between p130Cas and dynamin may regulate EGFR trafficking and signaling in the same manner as other endocytic regulatory proteins related to EGFR endocytosis.

Abstract 4691: Inhibition of ubiquitin specific protease Usp18 reduces cancer cell proliferation by down regulating multiple proto-oncogenes including EGFR

Abstract A greater understanding of the mechanisms by which specific oncogenic proteins are regulated is critical to developing improved targeted therapies. To identify novel regulators of oncogenic cell surface receptors we screened for enzymes whose inhibition promoted down-regulation of these receptors. One of these screens involved exposing cancer cells to an RNAi library targeting deubiquitinase enzymes and subsequently analyzing the cell-surface levels of the proto-oncogene, epidermal growth factor receptor (EGFR) in squamous cell carcinoma cells. This screen ultimately led to the discovery that depletion of the ubiquitin specific protease Usp18 from numerous cancer cell lines dramatically reduced protein and activity levels of EGFR, by as much as 90%. Furthermore, the activity of Usp18 was critical to its ability to control EGFR protein levels. Interestingly, this study also determined that Usp18 does not directly target EGFR proteins but instead controls EGFR protein synthesis. The work to be presented here shows that Usp18 executes this function by regulating the levels of microRNA-7 (miR-7). Acute depletion (72h) of Usp18 from several cancer cell lines, including squamous cell carcinoma (SCC2) and glioma (T98; U87), leads to an increase (up to 2-fold) in miR-7. Since miR-7 has previously been shown to be inhibitory to EGFR mRNA translation we hypothesized that miR-7 functions intermediary to Usp18 regulation of EGFR. This idea was confirmed when we observed that addition of a miR-7 specific inhibitor to Usp18 siRNA experiments completely reversed EGFR down-regulation. These findings illuminate for the first time the Usp18/miR-7/EGFR pathway in cancer cells. We have also found that Usp18 depletion promotes down-regulation of the proto-oncogene cyclin D1. Thus, acute depletion of Usp18 in numerous cancer cell lines leads to the down-regulation of multiple proto-oncogenes. To determine if chronic Usp18 inhibition also promotes down-regulation of EGFR and cyclin D1 we generated clonal SCC2 and U87 cells which express inducible Usp18 shRNA via lentiviral integration. Long term Usp18 depletion (5-15 days) also promotes proto-oncogene down-regulation. Furthermore, we observed a &amp;gt;50% reduction in cellular proliferation, matching observations made with acute depletion. We also hypothesized that since miR-7 regulates EGFR mRNA translation at the 3’ UTR the mutational status of EGFR would not counter the capabilities of Usp18 inhibition. Indeed, depletion of Usp18 from cancer cell lines encoding mutated EGFR also leads to dramatic down-regulation of the receptor (e.g., 75% in EGFR-T790M expressing H1975 cells). Thus, Usp18 inhibition may represent a unique therapeutic opportunity, even in tumors which express a heterogenic population of EGFR. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4691. doi:10.1158/1538-7445.AM2011-4691

Abstract 1189: MiR-145 inhibits cell proliferation of human lung adenocarcinoma by targeting EGFR and NUDT1

Abstract OBJECTIVE: MicroRNAs (miRNAs) are emerging as important modulators in cellular pathways and appear to play a key role in tumorigenesis. MiR-145 is downregulated in several human malignancies, including lung cancer, but the responsible molecular mechanisms remain unclear. We previously reported that restoration of miR-145 inhibits cancer cell growth in lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutation. The present study is undertaken to confirm and extend our previous findings through the identification of target genes for miR-145, and to analyze its biological functions in lung adenocarcinoma. METHODS: Systemic bioinformatic analysis was performed to investigate the potential targets of miR-145 in lung cancer, using publicly available algorithms. The associations of miR-145 and its targeted genes’ expressions were analyzed by mRNA and miRNA microarray profiling in tumor specimens from patients with lung adenocarcinoma. Expressions of miR-145 and its gene targets in human lung adenocarcinoma cells were determined using quantitative RT-PCR. Further validation was performed by Western blot analysis. RESULTS: This study showed that miR-145 targets EGFR and nucleoside diphosphate linked moiety X-type motif 1 (NUDT1 or MTH1) in lung adenocarinoma cells. A significant negative correlation was shown between expressions of miR-145 vs EGFR mRNA (r = -0.72; P = 6.3E-8) and NUDT1 mRNA (r = -0.78; P = 6.3E-8), indicating that miR-145 either directly or indirectly suppresses EGFR and NUDT1 transcriptions. The mRNA expressions of EGFR and NUDT1 were significantly downregulated by 60% and 50% after miR-145 transfection in human lung adenocarcinoma cells (P &amp;lt; 0.05). The enforced expression of miR-145 in three lung adenocarcinoma cell lines (NCI-H1975, HCC827, and NCI-H358) led to a decrease (2.5-, 5-, and 2-fold) of EGFR protein and the abolishing of NUDT1 protein expressions. Our results demonstrated miR-145 in the negative regulation of EGFR and NUDT1 expressions at both mRNA and protein levels. Further analysis showed that miR-145 has the ability to significantly inhibit cell proliferation on transfected lung adenocarcinoma cells over three time points (24, 48, and 72 hours). Proliferation of cells was elevated in a time-dependent fashion after transfection with pEP-miR-Null control (by 3%, 27%, and 58%). Conversely, transfection of miR-145 into the cells resulted in gradual reduction of cell viability (by 22%, 37%, and 34%). Upregulation of miR-145 appeared to be an important gene regulation mechanism for the proliferation of lung adenocarcinoma cells and it correlated strongly with the downregulation of EGFR and NUDT1. DISCUSSION: Our findings provided new insight into the complex regulating pathway comprising miR-145, EGFR, and NUDT1. Understanding miR-145's targets and its regulating pathways may lead to new therapeutic strategies for lung adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1189. doi:10.1158/1538-7445.AM2011-1189

C-Jun N-terminal Kinase 2 (JNK2) Enhances Cell Migration through Epidermal Growth Factor Substrate 8 (EPS8)

Membrane-bound receptors induce biochemical signals to remodel the actin cytoskeleton and mediate cell motility. In association with receptor tyrosine kinases, several downstream mitogen-induced kinases facilitate cell migration. Here, we show a role for c-Jun N-terminal kinase 2 (JNK2) in promoting mammary cancer cell migration through inhibition of epidermal growth factor substrate 8 (EPS8) expression, a key regulator of EGF receptor (R) signaling and trafficking. Using jnk2(-/-) mice, we found that EPS8 expression is higher in polyoma middle T antigen (PyVMT)jnk2(-/-) mammary tumors and jnk2(-/-) mammary glands compared with the respective jnk2(+/+) controls. The inverse relationship between the jnk2 and eps8 expression was also associated with cancer progression in that patients with basal-type breast tumors expressing high jnk2 and low eps8 experienced poor disease-free survival. In mammary tumor cell lines, the absence of jnk2 greatly reduces cell migration that is rescued by EPS8 knockdown. Subsequent studies show that JNK2 enhances formation of the EPS8-Abi-1-Sos-1 complex to augment EGFR activation of Akt and ERK, whereas the absence of JNK2 promotes ESP8/RN-Tre association to inhibit endocytotic trafficking of the EGFR. Together, these studies unveil a critical role for JNK2 and EPS8 in receptor tyrosine kinase signaling and trafficking to convey distinctly different effects on cell migration.

Role of Hyaluronan-Mediated CD44 Signaling in Head and Neck Squamous Cell Carcinoma Progression and Chemoresistance

Head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy that may involve the oral cavity, pharynx, larynx, and paranasal sinuses. The mechanisms of tumor progression underlying the clinical behavior of HNSCC remain unclear. CD44 comprises a family of transmembrane receptors that can give rise to multiple CD44 variant isoforms. Hyaluronan (HA), a major extracellular matrix component is the primary ligand for CD44 receptors. HA and CD44 signaling play an important role in HNSCC progression. Several CD44 variant isoforms (including v3-, v6-, and v10-containing isoforms) are associated with advanced disease, possibly through unique growth factor interactions with binding domains in the inserted variant regions of the cytoplasmic domain of CD44. In HNSCC, HA mediates the formation of a complex including CD44 and the epidermal growth factor receptor (EGFR) which is overexpressed in a large proportion of HNSCCs. Downstream effectors under EGFR regulation are activated, promoting promote cell growth and tumor survival. The leukemia-associated Rho-guanine nucleotide exchange factor (LARG) also associates with CD44 and EGFR to promote several Ras and RhoA pathway effectors, leading to cell migration, growth, and tumor survival. The secretion of matrix metalloproteinases, necessary for tumor cell invasion, is also regulated by these HA/CD44-mediated pathways. Finally, EGFR-mediated pathways play major roles in the HA/CD44 promotion of chemoresistance in HNSCC. Understanding HA/CD44-mediated signaling pathways may lead to improved treatment of HNSCC.

Epstein-Barr Virus LMP1 Activates EGFR, STAT3, and ERK through Effects on PKCδ

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that infects more than 90% of the world's adult population and is linked to multiple malignancies, including Burkitt lymphoma, Hodgkin disease, and nasopharyngeal carcinoma (NPC). The EBV oncoprotein LMP1 induces transcription of the epidermal growth factor receptor (EGFR), which is expressed at high levels in NPC. EGFR transcription is induced by LMP1 through a p50 NFκB1-Bcl-3 complex, and Bcl-3 is induced by LMP1-mediated activation of STAT3. This study reveals that LMP1, through its carboxyl-terminal activation domain 1 (LMP1-CTAR1), activates both STAT3 and EGFR in a serum-independent manner with constitutive serine phosphorylation of STAT3. Upon treatment with EGF, the LMP1-CTAR1-induced EGFR was additionally phosphorylated and STAT3 became phosphorylated on tyrosine, concomitant with upregulation of a subset of STAT3 target genes. The kinase responsible for LMP1-CTAR1-mediated serine phosphorylation of STAT3 was identified to be PKCδ using specific RNAi, a dominant negative PKCδ, and the PKCδ inhibitor rottlerin. Interestingly, inhibition of PKCδ also inhibited constitutive phosphorylation of EGFR and LMP1-CTAR1-induced phosphorylation of ERK. Inhibition of PKCδ blocked LMP1-CTAR1-mediated transformation of Rat-1 cells, likely through the inhibition of ERK activation. These findings indicate that LMP1 activates multiple distinct signaling pathways and suggest that PKCδ functions as a master regulator of EGFR, STAT3, and ERK activation by LMP1-CTAR1.

MiR-145 inhibits cell proliferation of human lung adenocarcinoma by targeting EGFR and NUDT1

MicroRNAs (miRNAs) are emerging as important modulators in cellular pathways and they appear to play a key role in tumorigenesis. MiR-145 is downregulated in several human malignancies, including lung cancer, but the responsible molecular mechanisms remain unclear. We previously reported that restoration of hsa-miR-145 inhibits cancer cell growth in lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutation. This study showed that hsa-miR-145 targets EGFR and nucleoside diphosphate linked moiety X-type motif 1 (NUDT1 or MTH1) in lung adenocarinoma cells. The mRNA expressions of EGFR and NUDT1 were significantly downregulated after miR-145 transfection in human lung adenocarcinoma cells. Our results demonstrated miR-145 in the negative regulation of EGFR and NUDT1 expressions at both mRNA and protein levels. Further analysis showed that miR-145 has the ability to inhibit cell proliferation on transfected lung adenocarcinoma cells over three time points (24, 48 and 72 hours). Upregulation of miR-145 appeared to be an important gene regulation mechanism for the proliferation of lung adenocarcinoma cells and it correlated strongly with the downregulation of EGFR and NUDT1. Interestingly, our study revealed that the altered proliferation in lung cancer cells is not accompanied by changes in apoptosis. Our findings provided new insight into the complex regulating pathway comprising of miR-145, EGFR, NUDT1 and other unknown factors which function in cell proliferation but not in apoptosis. Understanding miR-145’s targets and its regulating pathways may lead to new therapeutic strategies for lung adenocarcinoma.

ErbB2 Stabilizes Epidermal Growth Factor Receptor (EGFR) Expression via Erk and Sprouty2 in Extracellular Matrix-detached Cells

Epithelial cells are dependent on extracellular matrix (ECM) attachment for maintenance of metabolic activity and suppression of apoptosis. Here we show that loss of ECM attachment causes down-regulation of epidermal growth factor receptor (EGFR) and β1 integrin protein and mRNA expression and that ErbB2, which is amplified in 25% of breast tumors, reverses these effects of ECM deprivation. ErbB2 rescue of β1 integrin mRNA and protein in suspended cells is dependent on EGFR, however, the rescue of EGFR expression does not require β1 integrin. We show that there is a significant decrease in the stability of EGFR in ECM-detached cells that is reversed by ErbB2 overexpression. Rescue of both EGFR and β1 integrin protein by ErbB2 is dependent on Erk activity and induction of its downstream target Sprouty2, a protein known to regulate EGFR protein stability. Interestingly, expression of EGFR and β1 integrin protein is more dependent on Erk/Sprouty2 in ECM-detached ErbB2-overexpressing cells when compared with ECM-attached cells. These results provide further insight into the ErbB2-driven anchorage independence of tumor cells and provide a new mechanism for regulation of EGFR and β1 integrin expression in ECM-detached cells.