EGFR Downstream Signaling Pathways Research Articles

Arglabin, an EGFR receptor tyrosine kinase inhibitor, suppresses proliferation and induces apoptosis in prostate cancer cells

Evidence for clinical efficacy of a semisynthetic derivative of arglabin in anticancer treatment prompted us to examine molecular mechanisms and cellular targets of arglabin. Arglabin, a sesquiterpene lactone isolated from Artemisia glabella was cytotoxic to different human cancer cell lines including those derived from advanced triple-negative breast, lung, androgen-dependent and androgen-independent prostate carcinomas. Noteworthy, arglabin was less toxic to non-neoplastic prostate epithelial cells indicating selectivity for cancer cells. At the molecular level, prior to any biochemical signs of cellular toxicity, arglabin reduced levels of cell-surface sulphanyl groups and inhibited phosphorylation of the redox-sensitive receptor tyrosine kinase EGFR, the only active RTK in PC-3 prostate cancer cells among 49 TRKs analyzed by the assay. Henceforth, arglabin inhibited the EGFR downstream signaling pathways mTORC1 and mTORC2. Accordingly, arglabin induced autophagosome formation and autophagic flux, inhibited phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and impeded cell cycle progression and proliferation of PC-3 cells. In agreement with inhibition of the mTORC2 pathway, arglabin induced sustained actin polymerization, inhibited cell migration, and triggered apoptosis in vitro in 2D cell culture and colony formation assay and in vivo in prostate cancer xenografts grown on chick chorioallantoic membranes. Under physiological conditions, arglabin rapidly formed adducts with reduced glutathione (GSH). Moreover, thiol-based antioxidants GSH and β-mercaptoethanol abolished arglabin-induced cancer cell toxicity, whereas the non-thiol antioxidant trolox was ineffective pointing to a crucial role of interaction with cell-surface sulphanyl groups for arglabin cytotoxic activity against cancer cells.

The therapeutic inhibition of topoisomerase inhibitor and crizotinib combination in EGFR wild and mutant lung cancer cells

Combination therapy can enhance therapeutic effect by activation of multiple downstream pathways. The present study was aimed to investigate a novel strategy to successfully inhibit the EGFR pathway in EGFR wild and mutated types lung cancer by combination method. Topotecan (TPT) and crizotinib (CRI) were used to evaluate the effect on EGFR-wild, primary and secondary mutant non-small cell lung cancer (NSCLC) cell lines (H1299, HCC827 and H1975 cells). The combination group significantly inhibited the lung cancer growth with combination index (CI) < 1, and they synergistically induced the cell apoptosis by disrupting the balance of Bax and Bcl-xL, loss of mitochondrial membrane potential (MMP), and accumulation of reactive oxygen species (ROS). In addition, EGFR downstream signaling pathways including AKT, ERK, JNK, and p38 MAPK were regulated when treated with the combination regimen. Meanwhile, a nano-liposomes co-loaded CRI and TPT was prepared and exhibited strong cytotoxicity to the lung cancer cells especially H1299 and H1975 cells. The animal study confirmed the synergy between TPT and CRI from the results that they remarkable repressed the tumor growth with the inhibition rate of 81.32 %. The nano-liposomes of TPT and CRI achieved an optimal curative effect (71.52 % of inhibition rate) at 2 mg/kg. Moreover, the synergistic mechanism of the combination was consistent with the in vitro cell experiment by regulating EGFR signaling pathways. Collectively, we proposed a preclinical rationale and potential formulation for the use of a combination therapy consisting of the topoisomerase inhibitor TPT and the ALK-TKI CRI for treatment of lung cancers.

CD73 Is Regulated by the EGFR-ERK Signaling Pathway in Non-small Cell Lung Cancer.

Successful therapy of EGFR-mutant NSCLC remains a challenging task despite initial benefits with the usage of EGFR tyrosine kinase inhibitors. Cancer immunotherapy has shown promising results in certain tumors, but response rate in EGFR-mutant NCLC is low, because these tumors are thought to have weak immunogenicity. We used data from in vivo NSCLC databases as well as from in vitro cell culture experiments to investigate the regulation of CD73 by EGFR. EGFR expression was correlated with CD73 expression in patients' datasets, with EGFR-mutant tumors showing higher expression than their EGFR wildtype counterparts. Treatment of EGFR-mutant NSCLC cell lines with EGFR TKI reduced expression of CD73 at both mRNA and protein level. Among EGFR downstream signaling pathways, the Ras-Raf-ERK pathway was involved in the regulation of CD73 expression directly via ERK1/2 without the engagement of RSKs or MSKs. The results of this study may provide novel therapeutic strategies for the treatment of oncogene-driven NSCLC.

Role of the V1G1 subunit of V-ATPase in breast cancer cell migration

V-ATPase is a large multi-subunit complex that regulates acidity of intracellular compartments and of extracellular environment. V-ATPase consists of several subunits that drive specific regulatory mechanisms. The V1G1 subunit, a component of the peripheral stalk of the pump, controls localization and activation of the pump on late endosomes and lysosomes by interacting with RILP and RAB7. Deregulation of some subunits of the pump has been related to tumor invasion and metastasis formation in breast cancer. We observed a decrease of V1G1 and RAB7 in highly invasive breast cancer cells, suggesting a key role of these proteins in controlling cancer progression. Moreover, in MDA-MB-231 cells, modulation of V1G1 affected cell migration and matrix metalloproteinase activation in vitro, processes important for tumor formation and dissemination. In these cells, characterized by high expression of EGFR, we demonstrated that V1G1 modulates EGFR stability and the EGFR downstream signaling pathways that control several factors required for cell motility, among which RAC1 and cofilin. In addition, we showed a key role of V1G1 in the biogenesis of endosomes and lysosomes. Altogether, our data describe a new molecular mechanism, controlled by V1G1, required for cell motility and that promotes breast cancer tumorigenesis.

Prolidase Stimulates Proliferation and Migration through Activation of the PI3K/Akt/mTOR Signaling Pathway in Human Keratinocytes.

Recent reports have indicated prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR). Since this receptor is involved in the promotion of cell proliferation, growth, and migration, we aimed to investigate whether prolidase may participate in wound healing in vitro. All experiments were performed in prolidase-treated human keratinocytes assessing cell vitality, proliferation, and migration. The expression of downstream signaling proteins induced by EGFR, insulin-like growth factor 1 (IGF-1), transforming growth factor β1 (TGF-β1), and β1-integrin receptors were evaluated by Western immunoblotting and immunocytochemical staining. To determine collagen biosynthesis and prolidase activity radiometric and colorimetric methods were used, respectively. Proline content was determined by applying the liquid chromatography coupled with mass spectrometry. We found that prolidase promoted the proliferation and migration of keratinocytes through stimulation of EGFR-downstream signaling pathways in which the PI3K/Akt/mTOR axis was involved. Moreover, PEPD upregulated the expression of β1-integrin and IGF-1 receptors and their downstream proteins. Proline concentration and collagen biosynthesis were increased in HaCaT cells under prolidase treatment. Since extracellular prolidase as a ligand of EGFR induced cell growth, migration, and collagen biosynthesis in keratinocytes, it may represent a potential therapeutic approach for the treatment of skin wounds.

Cytoplasmic Chaperones Enhance Soluble Expression of Anti-EGFR huscFv in Escherichia coli.

Background: Overexpression of EGFR is associated with carcinogenesis in more than 70% of head and neck cancers. Anti-EGFR monoclonal antibodies bind to the extracellular domain of EGFR and block the EGFR downstream signaling pathway, which results in the suppression of the growth of the tumor cells. Escherichia coli is the preferred system for expressing various recombinant proteins, including single chain antibodies, but the formation of inclusion bodies negatively affects the efficacy of this system. Several strategies have been suggested to solve this problem, notably the utilization of molecular chaperones. Objectives: In this study, we attempted to increase the soluble expression of huscfv antibody via co-expression with the cytoplasmic chaperones. Materials and Methods: To achieve this purpose, chaperones plasmids pG-KJE8, pGro7, pKjE7, pTf16 and pG-Tf2 encoding cytoplasmic chaperones were co-expressed with the humanized anti-EGFR scFv construct in E. coli. Different temperatures, incubations times, and concentrations of IPTG were used to produce an active antibody with the highest solubility. Results were analyzed by SDS-PAGE. Soluble huscFv was purified by Ni-NTA column and the biologic activity of the recombinant protein was determined by ELISA. Result: The results indicated that the highest concentrations of humanized anti-EGFR scFv were obtained by co-expression of huscFv via chaperone plasmid pG-KJE8 with 0.2 mM concentration of inducer (IPTG), culture temperature of 25 °C, and 4 h incubation time after induction. Conclusion: In conclusion, co-expression with chaperones could be used as an efficient strategy to produce soluble active ScFvs in E. coli.

YH25448, an Irreversible EGFR-TKI with Potent Intracranial Activity in EGFR Mutant Non-Small Cell Lung Cancer.

Given that osimertinib is the only approved third-generation EGFR-TKI against EGFR activating and resistant T790M mutated non-small cell lung cancer (NSCLC), additional mutant-selective inhibitors with a higher efficacy, especially for brain metastases, with favorable toxicity profile are still needed. In this study, we investigated the antitumor efficacy of YH25448, an oral, mutant-selective, irreversible third-generation EGFR-TKI in preclinical models. Antitumor activity of YH25448 was investigated in vitro using mutant EGFR-expressing Ba/F3 cells and various lung cancer cell lines. In vivo antitumor efficacy, ability to penetrate the blood-brain barrier (BBB), and skin toxicity of YH25448 were examined and compared with those of osimertinib using cell lines and PDX model. Compared with osimertinib, YH25448 showed a higher selectivity and potency in kinase assay and mutant EGFR-expressing Ba/F3 cells. In various cell line models harboring EGFR activating and T790M mutation, YH25448 effectively inhibited EGFR downstream signaling pathways, leading to cellular apoptosis. When compared in vivo at equimolar concentrations, YH25448 produced significantly better tumor regression than osimertinib. Importantly, YH25448 induced profound tumor regression in brain metastasis model with excellent brain/plasma and tumor/brain area under the concentration-time curve value. YH25448 rarely suppressed the levels of p-EGFR in hair follicles, leading to less keratosis than osimertinib in animal model. The potent systemic and intracranial activity of YH25448 has been shown in an ongoing phase I/II clinical trial for advanced EGFR T790M mutated NSCLC (NCT03046992). Our findings suggest that YH25448 is a promising third-generation EGFR inhibitor, which may be more effective and better tolerated than the currently approved osimertinib.

USP22 promotes resistance to EGFR-TKIs by preventing ubiquitination-mediated EGFR degradation in EGFR-mutant lung adenocarcinoma

As a newly discovered deubiquitinating enzyme, ubiquitin-specific protease 22 (USP22) is predictive of therapeutic outcomes in individual cancer patients. However, its clinical effects on malignancy and its roles in conferring resistance to EGFR-TKIs (epidermal growth factor receptor-tyrosine kinase inhibitors) in lung adenocarcinoma (ADC) remain largely unknown. Here, we showed that USP22 promotes cell proliferation, migration and invasion, and contributes to resistance to EGFR-TKIs in EGFR mutant lung ADC cells. Mechanistically, USP22 deubiquitinates EGFR localized on late endosomes, prevents ubiquitination mediated EGFR degradation and enhances recycling of EGFR after EGF stimulation. Additionally, USP22 sustained the activation of multiple EGFR downstream signaling pathways, including STAT3, AKT/mTOR and MEK/ERK pathways, in lung ADC cell lines H1975 and PC9. Furthermore, USP22 stabilizes EGFR protein expression, which correlates with USP22 expression in EGFR-mutant lung ADC patient samples. We are the first to demonstrate that silencing USP22 counteracts EGFR-TKIs resistance both in vitro and in vivo. We propose USP22 as a potential therapeutic target for EGFR-TKIs-resistant lung ADC.

Abstract 2752: EGFR tyrosine kinase inhibitor limits tumor relapse through enhancing anti-tumor T cell responses

Abstract EGFR tyrosine kinase inhibitors (TKIs) have been clinically considered as the first line therapy for EGFR-driven tumor patients. Although the initial outcome of patients has significantly improved, almost all patients eventually relapse. Fully understanding the mechanisms of the anti-tumor effect of EGFR TKIs will help promote the clinical development of more efficient therapeutic strategies. The traditional view of the main mechanism is that the inhibitors can directly inhibit EGFR downstream signaling pathways to induce tumor cell death. Whether and how EGFR TKIs affect the host immune system is largely unknown. In current study, we have established syngeneic murine tumor models that are sensitive to EGFR TKIs treatment. To our surprise, we revealed that EGFR TKI treatment could control tumor growth and limit tumor relapse in T cells dependent manner. Mechanistically, EGFR TKI triggers type I IFN and its stimulation gene CXCL10 production through the Myd88 signaling pathway to enhance tumor specific T cells infiltration and activation. However, EGFR TKI cannot efficiently limit tumor relapse in advanced tumor models because of PD-L1 upregulation in the tumor microenvironment after treatment. We have observed that anti-PDL1 blockade can synergize with EGFR TKI to control advanced tumors. Our study will challenge the current regimen of EGFR TKI in clinic and provide new efficient therapeutic strategies for EGFR dependent cancer patients. Citation Format: Zhida Liu, Chuanhui Han, Aijun Shen, Chunbo Dong, Jian Qiao, Yang-Xin Fu. EGFR tyrosine kinase inhibitor limits tumor relapse through enhancing anti-tumor T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2752.

P3.01-038 STAT3 and Src-YAP1 Inhibition Results in Greater Necitumumab Sensitivity in Lung Squamous Cell Carcinoma: Topic: Functional Biology in Lung Cancer

The anti-EGFR monoclonal antibody (mAb), necitumumab, has been recently approved in combination with chemotherapy, as 1st-line treatment for advanced lung squamous cell carcinoma (LSCC) patients, but with minimal survival benefit. Evidence continues to accumulate that signal transducer and activator of transcription 3 (STAT3) is a promising molecular target for cancer therapies. STAT3 is activated by tyrosine phosphorylation in response to EGF and interleukin-6 (IL-6). In addition to STAT3, IL-6 activates the Src family kinases, and subsequently YES-associated protein 1 (YAP1). STAT3 and Src-YAP1 activation contributes to EGFR inhibitor resistance and concomitant targeting of EGFR and STAT3-Src may represent an effective treatment strategy for LSCC. RNA was isolated from six LSCC cell lines and the mRNA expression analysis of EGFR, STAT3, Src and YAP1 was performed by TaqMan based qRT-PCR. Cell viability was assessed by MTT (thiazolyl blue) assay after treatment with necitumumab and evodiamine, an alkaloid isolated from the dried, unripe Evodia rutaecarpa (Juss.) Benth fruit that exerts an anticancer effect by inhibiting STAT3 and Src. Western blotting was performed to assess the effect of necitumumab on EGFR downstream signaling pathways. We first evaluated the expression of EGFR in our panel of LSCC cell lines. We found that almost all of them homogeneously express high levels of EGFR. We then assessed the effect of necitumumab on EGFR downstream signaling in the SK-MES1 cell line. Treatment of SK-MES1 cells with 25ug/ml of necitumumab for seven days was unable to ablate STAT3, Src or YAP1 mRNA expression. Consistent with this, we found that necitumumab suppressed EGFR, ERK1/2 and AKT phosphorylation but increased STAT3 phosphorylation on the critical tyrosine residue 705 in a time and dose-dependent manner. We examined the growth inhibitory effect of the necitumumab and evodiamine combination. We performed an MTT cell proliferation assay on SK-MES1 cells and we used a constant ratio drug combination method to determine synergy, additivity, or antagonism. The combination of necitumumab and evodiamine resulted in a clear synergism in SK-MES1 cells as measured by the combination index (CI) analysis, with a CI of 0.74. Experiments in the rest of our LSCC cell lines are ongoing. Herein we have examined the role of STAT3 and Src-YAP1 in the context of treatment with the FDA-approved EGFR mAb, necitumumab. Our data provide initial evidence that co-activation of STAT3 and Src-YAP1 may limit the cellular response to EGFR inhibition in LSCC.

The underlying mechanisms of overcoming EGFR-TKIs resistance by casein kinase 2 inhibitor

Nowadays, EGFR-TKIs are important treatment strategy in lung cancer, but the resistance to EGFR-TKIs remains an unsolved issue preventing the patients from further benefits. Recent studies have shown that casein kinase (CK2) plays an important role in carcinogenesis and development of cancer. CK2 inhibitor has also demonstrated anti-tumor effects. Here we reviewed the mechanism of EGFR-TKIs and the potential reasons of resistance. Interestingly, there is a crosstalk between CK2 and EGFR downstream signaling pathways, therefore, it may be possible that CK2 inhibitor can overcome the EGFR-TKIs resistance.

New crossroads for potential therapeutic intervention in cancer - intersections between CDCP1, EGFR family members and downstream signaling pathways.

Signaling pathways regulated by the receptor CDCP1 play central roles in promoting cancer and in mediating resistance to chemo- and targeted-therapies. In this perspective we briefly summarize these findings as well as data demonstrating poorer outcomes for several malignancies that exhibit elevated CDCP1 expression. Promising data from preclinical studies suggest that CDCP1 targeted agents, including therapeutic antibodies, could be useful in the treatment of cancer patients selected on the basis of activation of CDCP1 and its signaling partners including EGFR, HER2, Met and Src.

H45 - Clinical applications of a next-generation sequencing panel in non-small cell lung cancer

Introduction: Molecular markers have become important predictors of response to targeted therapies in non-small cell lung cancer (NSCLC), and Next-Generation Sequencing (NGS) has recently emerged as a cost-effective method able to provide more comprehensive genomic information with potential repercussions in clinical practice. The aim of this study was to identify potential actionable mutations in advanced NSCLC. Here we report the findings obtained for an initial group of patients screened in our Lung Cancer Unit. Patients and methods: Twenty patients with advanced lung adenocarcinoma were evaluated. DNA was extracted from 4 cytological samples and 16 FFPE biopsies from primary or metastatic lesions. The specimens were analyzed for 22 cancer-associated genes using the Ion AmpliSeq Colon and Lung Cancer Panel v.1 (Life Technologies) and sequenced through Ion Torrent PGMTM (Life Technologies). Data were analyzed with the Ion Torrent Software Suite v.4.4 (Life Technologies) using the plugin Variant Caller (VC) v.4.4.2.1. Results: Patients' characteristics: median age 65 (42-81); 30% males, 70% females; never smoker 60%, former smoker/smoker 40%. Data analysis reported an average of one genetic variant per patient annotated in the Catalogue of Somatic Mutation in Cancer (COSMIC database) (range 0-5). Twenty-one mutations were diagnosed in 12 patients (55%) in the following genes: EGFR (4), KRAS (1), MET (4), TP53 (8), NRAS (1), BRAF (1), and PI3KCA (2). In addition to the known mutations in EGFR, we found a BRAF mutation resulting in an amino-acid substitution (K601E) potentially sensitive to BRAF or MEK inhibitors on the basis of preclinical studies on BRAF downstream signaling.. A potentially druggable mutation was found in NRAS gene (Q61R), which is sensitive to MEK inhibitors in in vitro models. Among EGFR-mutated patients, one case showed a PI3KCA missense mutation (E545K) synchronous with the EGFR exon 19 deletion (del746-750);notably, the E545K mutation has been described to activate EGFR downstream signaling pathway, resulting in TKIs resistance. Finally, 7 patients (35%) had a mutation of the gene encoding for TP53, which is reported as a prognostic marker of poor outcome in advanced NSCLC. Conclusion: Targeted NGS is a sensitive tool to screen multiple genes simultaneously and represents an attractive system to identify relevant mutations. The potential clinical significance of these variations needs to be elucidated in further studies.

Cetuximab in combination with icotinib overcomes the acquired resistance caused by EGFR T790M mutation in non-small cell lung cancer

The aim of this study was to investigate the effects of combination of icotinib and cetuximab on the acquired drug resistance caused by T790M mutation of EGFR in NSCLC, and provide experimental evidence for rational treatment of NSCLC. The effects of these two agents on cell proliferation, apoptosis, and EGFR-dependent signaling were evaluated using 3-(4, 5-dimethylthiazol-2-yl)- 5-diphenyltetrazolium bromide (MTT) assay, annexin V staining, and Western blotting. The expression of molecular markers of tumor proliferation PCNA and Ki-67 protein was further examined by immunohistochemistry, and the expression of EGFR-signaling-related proteins in tissue sections taken from H1975 tumor xenografts was assessed by Western blot assay. Sensitivity to EGFR inhibitors was detected in human H1975 tumor xenograft in nude mice. The in vitro experiment showed that the proliferative ability of H1975 cells was inhibited in a dose-dependent manner, along with the increasing doses of cetuximab and icotinib, and the combination of cetuximab with icotinib resulted in a more pronounced growth inhibition of the H1975 cells. The apoptosis rate of H1975 cells after treatment with 0.5 µmol/L icotinib and 1 µg/ml cetuximab was (22.03 ± 2.41)% and that after treatment with 5 µmol/L icotinib and 10 µg/ml cetuximab was (42.75 ± 2.49)%, both were significantly higher than that after treatment with the same dose of icotinib or cetuximab alone (P < 0.05). The nude mouse experiment showed that the transplanted tumor was growing to (614.5 ± 10.8) mm(3) in the blank control group and to (611.2 ± 8.7) mm(3) at 28 days after icotinib treatment, but (30.8 ± 2.0) mm(3) in the cetuximab treatment group and 0 mm(3) in the cetuximab combined with icotinib group. There was a significantly decreased expression of Ki-67 and PCNA proteins and down-regulation of phosphorylation of EGFR signaling-related proteins in the cetuximab combined with icotinib group. The combination of icotinib with cetuximab can exert synergistic inhibitory effect on the acquired drug resistance caused by T790M mutation of EGFR in NSCLC H1975 cells, interrupts the EGFR-downstream signaling pathway, and enhances the anticancer activity of chemotherapeutic drugs. Our results provide further experimental evidence for the clinical studies of combination of icotinib with cetuximab in the treatment of NSCLC patients associated with secondary drug resistance caused by T790M mutation of EGFR.

Transactivation of ErbB Receptors by Leptin in the Cardiovascular System: Mechanisms, Consequences and Target for Therapy

Many experimental and clinical studies have demonstrated that elevated leptin concentration in patients with obesity/metabolic syndrome contributes to the pathogenesis of cardiovascular disorders including arterial hypertension, atherosclerosis, restenosis after coronary angioplasty and myocardial hypertrophy. Receptor tyrosine kinases belonging to the ErbB family, especially ErbB1 (epidermal growth factor receptor) and ErbB2 are abundantly expressed in the blood vessels and the heart. EGFR is activated not only by its multiple peptide ligands but also by many other factors including angiotensin II, endothelin-1, norepinephrine, thrombin and prorenin; the phenomenon referred to as "transactivation". Augmented EGFR signaling contributes to abnormalities of vascular tone and renal sodium handling as well as vascular remodeling and myocardial hypertrophy through various intracellular mechanisms, in particular extracellular signal-regulated kinases (ERK) and phosphoinositide 3-kinase (PI3K). Recent experimental studies indicate that chronically elevated leptin transactivates the EGFR through the mechanisms requiring reactive oxygen species and cytosolic tyrosine kinase, c-Src. In addition, hyperleptinemia increases ErbB2 activity in the arterial wall. Stimulation of EGFR and ErbB2 downstream signaling pathways such as ERK and PI3K in the vascular wall and the kidney may contribute to the increase in vascular tone, enhanced tubular sodium reabsorption as well as vascular and renal lesions in hyperleptinemic obese subjects.

Abstract B86: Potent antitumor effect of TAS-102, a novel oral anti-neoplastic nucleoside agent, on both cetuximab-sensitive KRAS wild-type and cetuximab-resistant KRAS mutated colorectal cancer cells via trifluorothymidine incorporation into DNA.

Abstract Background: The KRAS gene is mutated in approximately 40% of colorectal cancer (CRC). Patients with KRAS mutant CRC do not benefit from treatment with anti-EGFR antibody cetuximab and panitumumab. TAS-102 is a novel oral anti-neoplastic nucleoside agent, consisting of trifluorothymidine (FTD) as the antitumor component and thymidine phosphorylase inhibitor which prevents degradation of FTD. In a previous report, TAS-102 showed a broad spectrum of antitumor effect against various types of tumors via FTD incorporation into DNA followed by possible DNA dysfunction. Here, we investigated TAS-102 antitumor potency against colorectal cancer cell lines with differing KRAS status. Materials and Methods: Cytotoxicity of FTD and cetuximab by 72h treatment was evaluated using cancer cell lines with varying of KRAS gene status. The antitumor effect of TAS-102 and cetuximab was compared in mice subcutaneously implanted with colorectal cancer cell lines with KRAS wild-type or mutation. In the same models, the amount of FTD incorporated into DNA was analyzed by HPLC in the TAS-102-treated tumors. EGFR downstream signaling pathways were analyzed by immunoblotting in the cetuximab-treated tumors. Results: KRAS wild-type cell lines were sensitive to cetuximab in vitro, while some of the KRAS mutants were not. Cetuximab showed antitumor effect against KRAS wild-type tumors with downregulation of EGFR downstream signaling pathways, and it didn't show antitumor effect against KRAS mutants. In contrast to cetuximab, FTD exhibited a broad spectrum of cytotoxicity in vitro irrespective of KRAS status. Additionally, TAS-102 exerted significant antitumor effect against KRAS wild-type and mutant tumors. FTD incorporation into DNA was observed in both tumor types. Conclusion: TAS-102 appears to exert significant antitumor effects on CRC cell lines irrespective of KRAS status which is consistent with preliminary clinical findings. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B86. Citation Format: Nozomu Tanaka, Yukari Yamada, Akio Fujioka, Keisuke Yamamura, Satoko Ito, Kenichi Matsuo, Teruhiro Utsugi. Potent antitumor effect of TAS-102, a novel oral anti-neoplastic nucleoside agent, on both cetuximab-sensitive KRAS wild-type and cetuximab-resistant KRAS mutated colorectal cancer cells via trifluorothymidine incorporation into DNA. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B86.

Lyn, a Src family kinase, regulates activation of epidermal growth factor receptors in lung adenocarcinoma cells

BackgroundActivation of receptors for growth factors on lung epithelial cells is essential for transformation into tumor cells, supporting their viability and proliferation. In most lung cancer patients, EGFR is constitutively activated without evidence of mutation. Defining mechanisms for constitutive activation of EGFR could elucidate additional targets for therapy of lung cancers.MethodsThe approach was to identify lung cancer cell lines with constitutively activated EGFR and use systematic selection of inhibitors to evaluate their effects on specific EGFR phosphorylations and downstream signaling pathways. Interactions between receptors, kinases, and scaffolding proteins were investigated by co-immunoprecipitation plus Western blotting.ResultsThe results revealed a dependence on Src family of tyrosine kinases for downstream signaling and cell growth. Lyn, a Src family kinase functional in normal and malignant B-lymphocytes, was a defining signal transducer required for EGFR signaling in Calu3 cell line. Src family kinase activation in turn, was dependent on PKCßII. Lyn and PKC exist in membrane complexes of RACK1 and in association with EGFR which pairs with other receptor partners. Silencing of Lyn expression with interfering siRNA decreased EGFR activation and cell viability.ConclusionsThe importance of Src family kinases and PKCßII in the initiation of the EGFR signaling pathway in lung tumor cells was demonstrated. We conclude that phosphorylation of EGFR is mediated through PKCßII regulation of Lyn activation, and occurs in association with RACK1 and Cbp/PAG proteins. We suggest that protein complexes in cell membranes, including lipid rafts, may serve as novel targets for combination therapies with EGFR and Src Family Kinase inhibitors in lung cancer.

Correlation of activated AKT and MAPK expression in liver metastases with clinical outcome in colorectal cancer patients receiving irinotecan/cetuximab treatment.

449 Background: An aberrant activation of the EGFR downstream signaling pathway via MAP-kinase and Akt could be responsible for resistance to anti-EGFR treatment. We tested the interaction between phosphorylated Akt and MAPK in primary colorectal tumours and corresponding metastases and clinical outcome in terms of response rate (RR), progression free survival (PFS) and overall survival (OS) to identify a group of patients more likely to benefit from EGFR-targeted treatment among those harbouring a K-RAS wild type status. Methods: Seventy-two advanced K-RAS wild type colorectal cancer patients treated with irinotecan-cetuximab were analysed. Primary tumour were available in all cases, whereas paired tumour samples from metastatic sites were available in 37 patients. Phosphorylated Akt and MAPK were analyzed by immunohistochemistry. Results: Akt resulted overexpressed in 31 primary tumours (43%) and 23 metastases (62%), whereas MAPK was over-expressed in 32 primary tumours (44%) and 20 metastases (54%). Akt altered expression in primary tumours correlated with a statistically significant worse median PFS (2.4 months vs. 6.5 months, p= 0.0006) and OS (7.8 months vs. 26.7 months, p &lt; 0.0001), without any significant correlation with RR. No significant correlation could be found between MAPK expression in primary tumours and RR, PFS or OS. In metastases Akt expression correlated with RR (9% vs, 58%, p= 0.004), PFS (2.3 months vs.9.2 months p &lt; 0.0001) and OS (6.1 months vs.26.7 months p &lt; 0.0001). Analogously MAPK expression in metastases correlated with RR (10% vs, 47%, p = 0.002), PFS (2.3 months vs.8.6 months p &lt; 0.0001) and OS (7.8 months vs.26 months p = 0.0004). At multivariate analysis Akt and MAPK status in metastases was able to independently predict PFS. Akt status in metastases independently correlated with RR as well. Conclusions: We suggest that Akt and MAPK expression in metastases may have a relevant role in determining the activity of anti-EGFR treatment strategies. Our observations seem also to indicate that for some molecular determinants of resistance the biological profile in metastases is prominent.

Cellular response to cetuximab in PTEN-silenced head and neck squamous cell carcinoma cell line

The implication of loss of PTEN expression in resistance to targeted therapy has already been described in many tumor types. The absence of response to anti-EGFR agents in PTEN-deficient tumors relies on persistent activation of signaling pathways downstream of pEGFR. To investigate the role of PTEN loss of expression in head and neck squamous cell carcinoma (HNSCC) response to cetuximab, we used siRNA in Cal 27 cells and then evaluated key signaling protein activation (pAKT and pERK 1/2) as well as cell viability and proliferation. PTEN silencing in Cal 27 cells led to a constitutive activation of signaling pathways evidenced by a strong increase in pAKT and pERK 1/2 expression. Moreover, PTEN-silenced cells did not show any significant changes either in cell viability or proliferation, only slight modifications on cell cycle. Additionally and unpredictably, our results indicated that PTEN silencing, led to a drastic reduction in pEGFR expression whereas total EGFR level did not significantly vary. Strikingly, despite this overactivation of signaling pathways ruling cell survival and proliferation in siPTEN cells, cetuximab fully exerted pAKT and pERK 1/2 inhibition of expression, similarly to its effect in untransfected Cal 27 cells. In conclusion, our study established that in Cal 27 cells, cetuximab keeps full ability to inhibit EGFR-dependent mechanisms, as shown by a decreased pAKT and pERK 1/2 level of expression, despite a strong PTEN silencing-induced overactivation. In Cal 27 cells, loss of PTEN expression does not lead to a loss of cetuximab efficacy in inhibiting EGFR-downstream signaling pathways, contrarily to data shown in previous works conducted in other tumor types.

Additional value of EGFR downstream signaling phosphoprotein expression to KRAS status for response to anti‐EGFR antibodies in colorectal cancer

KRAS mutations are a strong predictive marker of resistance to anti-epidermal growth factor receptor (EGFR) antibodies in advanced colorectal cancer (CRC) but only a subset of wild-type (WT) KRAS patients are responders, suggesting the existence of additional markers of resistance to this treatment. The activation of EGFR downstream signaling pathways may be one of these ones. In a series of 42 patients with advanced CRC treated with cetuximab/panitumumab, for whom KRAS status was previously determined, we retrospectively analyzed the intratumor expression of EGFR downstream signaling phosphoproteins of the RAS/MAPK and PI3K/AKT pathways (pERK1/2, pMEK1, pAKT, pP70S6K and pGSK3beta) using Bio-Plex phosphoprotein array. Association with tumor response, progression-free survival (PFS) and overall survival (OS) was assessed. The expression of all the phosphoproteins was higher in KRAS mutated tumors than in WT tumors. The expression of pP70S6K was lower in responders than in nonresponder patients. In univariate analysis, patients with high pMEK1 or pP70S6K expression had a shorter PFS than those with low expression. Patients with high pP70S6K expression also had a shorter OS. In multivariate analysis, PFS was shorter for patients with high pMEK1 or pP70S6K expression, independently of KRAS status, as OS for patients with high pP70S6K expression. Therefore, WT KRAS patients with high pP70S6K expression had a shorter survival than those with low expression. Our results suggest the importance of EGFR downstream signaling phosphoproteins expression in addition to KRAS status to define the subgroup of patients who will not benefit from anti-EGFR therapy.