[Show abstract][Hide abstract] ABSTRACT: Cetuximab, an anti-EGFR monoclonal antibody, is used to treat several cancers. However, many patients who initially respond to cetuximab acquire resistance. To examine mechanisms of acquired resistance, we developed a series of cetuximab-resistant (CtxR) clones derived from the cetuximab sensitive (CtxS) non-small cell lung cancer (NSCLC) cell line H226. Previous studies characterizing this model revealed that: 1) EGFR was robustly overexpressed in CtxR clones due to decreased EGFR ubiquitination and degradation and 2) CtxR clones expressed increased HER2 and HER3 activation resulting in constitutive activation of the PI3K/AKT signaling axis. These findings suggest that dual targeting HER family receptors would be highly beneficial in the CtxR setting.
Molecular Cancer 10/2014; 13(1):242. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The EGFR antibody cetuximab is used to treat numerous cancers, but intrinsic and acquired resistance to this agent is a common clinical problem. In this study we show that overexpression of the oncogenic receptor kinase AXL is sufficient to mediate acquired resistance to cetuximab in models of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC), where AXL was overexpressed, activated and tightly associated with EGFR expression in cells resistant to cetuximab (CtxR cells). Using RNAi methods and novel AXL targeting agents, we found that AXL activation stimulated cell proliferation, EGFR activation and MAPK signaling in CtxR cells. Notably, EGFR directly regulated the expression of AXL mRNA through MAPK signaling and the transcription factor c-Jun in CtxR cells, creating a positive feedback loop that maintained EGFR activation by AXL. Cetuximab-sensitive parental cells were rendered resistant to cetuximab by stable overexpression of AXL or stimulation with EGFR ligands, the latter of which increased AXL activity and association with the EGFR. In tumor xenograft assays, the development of resistance following prolonged treatment with cetuximab was associated with AXL hyperactivation and EGFR association. Furthermore, in an examination of patient-derived xenografts established from surgically resected HNSCCs, AXL was overexpressed and activated in tumors that displayed intrinsic resistance to cetuximab. Collectively, our results identify AXL as a key mediator of cetuximab resistance, providing a rationale for clinical evaluation of AXL targeting drugs to treat cetuximab-resistant cancers.
[Show abstract][Hide abstract] ABSTRACT: Triple-negative breast cancer (TNBC) is a subclass of breast cancers (i.e. estrogen receptor negative, progesterone receptor negative, and HER2 negative) that have poor prognosis and very few identified molecular targets. Strikingly, a high percentage of TNBC's overexpress the epidermal growth factor receptor (EGFR), yet EGFR inhibition has yielded little clinical benefit. Over the last decade, advances in EGFR biology have established that EGFR functions in two distinct signaling pathways: 1) classical membrane-bound signaling, and 2) nuclear signaling. Previous studies have demonstrated that nuclear EGFR (nEGFR) can enhance resistance to anti-EGFR therapies and is correlated with poor overall survival in breast cancer. Based on these findings we hypothesized that nEGFR may promote intrinsic resistance to cetuximab in TNBC. To examine this question, a battery of TNBC cell lines and human tumors were screened and found to express nEGFR. Knockdown of EGFR expression demonstrated that TNBC cell lines retained dependency on EGFR for proliferation, yet all cell lines were resistant to cetuximab. Further, Src Family Kinases (SFKs) influenced nEGFR translocation in TNBC cell lines and in vivo tumor models, where inhibition of SFK activity led to potent reductions in nEGFR expression. Inhibition of nEGFR translocation led to a subsequent accumulation of EGFR on the plasma membrane, which greatly enhanced sensitivity of TNBC cells to cetuximab. Collectively, these data suggest that targeting both the nEGFR signaling pathway, through the inhibition of its nuclear transport, and the classical EGFR signaling pathway with cetuximab may be a viable approach for the treatment of TNBC patients.
Molecular Cancer Therapeutics 03/2014; · 5.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Small cell lung cancer (SCLC) is a devastating disease and current therapies have not greatly improved the 5-year survival rates. Topoisomerase (Top) inhibition is a treatment modality for SCLC; however, the response is short lived. Consequently, our research has focused on improving SCLC therapeutics through the identification of novel targets. Previously, we identified MET to be overexpressed and functional in SCLC. Herein, we investigated the therapeutic potential of combinatorial targeting of MET using SU11274 and Top1 using 7-ethyl-10-hydroxycamptothecin (SN-38). MET and TOP1 gene copy numbers and protein expression were determined in 29 patients with limited (n=11) and extensive (n=18) disease. MET gene copy number was significantly increased (>6 copies) in extensive disease compared to limited disease (p=0.015). Similar TOP1 gene copy numbers were detected in limited and extensive disease. Immunohistochemical staining revealed significantly higher Top1 nuclear expression in extensive (0.93) versus limited (0.15) disease (p=0.04). Interestingly, a significant positive correlation was detected between MET gene copy number and Top1 nuclear expression (r=0.5). In vitro stimulation of H82 cells revealed HGF-induced nuclear co-localization of p-MET and Top1. Furthermore, activation of the HGF/MET axis enhanced Top1 activity, which was abrogated by SU11274. Combination of SN-38 with SU11274 dramatically decreased SCLC growth as compared to either drug alone. Collectively, these findings suggest that combinatorial inhibition of MET and Top1 is a potentially efficacious treatment strategy for SCLC.
Molecular Cancer Therapeutics 12/2013; · 5.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Kinases downstream of growth factor receptors have been implicated in radioresistance and are, therefore, attractive targets to improve radiotherapy outcome in head and neck squamous cell carcinoma (HNSCC) patients.
An antibody-based array was used to quantify the expression levels of multiple phospho-kinases involved in growth factor signaling in nine untreated or irradiated HNSCC lines. Radiosensitivity was assessed with clonogenic cell survival assays and correlated with the expression levels of the phospho-kinases. Inhibitors of the kinases that were associated with radiosensitivity were tested for their ability to increase radiosensitivity in the 3 most radioresistant HNSCC lines.
The basal expression of phosphorylated Yes, Src and STAT5A, and the expression after radiotherapy of phosphorylated AKT, MSK1/2, Src, Lyn, Fyn, Hck, and STAT6, were correlated with radiosensitivity in the panel of HNSCC lines. In combination with radiotherapy, inhibitors of AKT, p38 and Src Family Kinases (SFK) were variably able to reduce survival, whereas MEK1/2, STAT5 and STAT6 inhibition reduced survival in all cell lines. The combined effect of radiotherapy and the kinase inhibitors on cell survival was mostly additive, although also supra-additive effects were observed for AKT, MEK1/2, p38 and STAT5 inhibition.
Kinases of the AKT, MAPK, STAT and SFK pathways correlated with radiosensitivity in a panel of HNSCC lines. Particularly inhibitors against MEK1/2, STAT5 and STAT6 were able to decrease survival in combination with radiotherapy. Hence, inhibitors against these kinases have the potential to improve radiotherapy outcome in HNSCC patients and further research is warranted to confirm this in vivo.
Molecular Cancer 11/2013; 12(1):133. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in a variety of human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for head and neck and colorectal cancer treatment, but many patients treated with cetuximab don't respond or eventually acquire resistance. To determine how tumor cells acquire resistance to cetuximab, we previously developed a model of acquired resistance using the non-small cell lung cancer line NCI-H226. These cetuximab-resistant (Ctx(R)) cells exhibit increased steady-state EGFR expression secondary to alterations in EGFR trafficking and degradation and, further, retained dependence on EGFR signaling for enhanced growth potential. Here, we examined Sym004, a novel mixture of antibodies directed against distinct epitopes on the extracellular domain of EGFR, as an alternative therapy for Ctx(R) tumor cells. Sym004 treatment of Ctx(R) clones resulted in rapid EGFR degradation, followed by robust inhibition of cell proliferation and down-regulation of several mitogen-activated protein kinase pathways. To determine whether Sym004 could have therapeutic benefit in vivo, we established de novo Ctx(R) NCI-H226 mouse xenografts and subsequently treated Ctx(R) tumors with Sym004. Sym004 treatment of mice harboring Ctx(R) tumors resulted in growth delay compared to mice continued on cetuximab. Levels of total and phospho-EGFR were robustly decreased in Ctx(R) tumors treated with Sym004. Immunohistochemical analysis of these Sym004-treated xenograft tumors further demonstrated decreased expression of Ki67, and phospho-rpS6, as well as a modest increase in cleaved caspase-3. These results indicate that Sym004 may be an effective targeted therapy for Ctx(R) tumors.
Neoplasia (New York, N.Y.) 10/2013; 15(10):1196-206. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: HER3 is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In the present study, we investigated the capacity of the HER3 blocking antibody, U3-1287/AMG888, to modulate the in vitro and in vivo radiation response of human squamous cell carcinomas of the lung and head and neck. We screened a battery of cell lines from these tumors for HER3 expression and demonstrated that all cell lines screened exhibited expression of HER3. Importantly, U3-1287/AMG888 treatment could block both basal HER3 activity and radiation induced HER3 activation. Proliferation assays indicated that HER3 blockade could decrease the proliferation of both HNSCC cell line SCC6 and NSCLC cell line H226. Further, we demonstrated that U3-1287/AMG888 can sensitize cells to radiation in clonogenic survival assays, in addition to increasing DNA damage as detected via λ-H2AX immunofluorescence. To determine if U3-1287/AMG888 could enhance radiation sensitivity in vivo we performed tumor growth delay experiments using SCC6, SCC1483, and H226 xenografts. The results of these experiments indicated that the combination of U3-1287/AMG888 and radiation could decrease tumor growth in studies using single or fractionated doses of radiation. Analysis of HER3 expression in tumor samples indicated that radiation treatment activated HER3 in vivo and that U3-1287/AMG888 could abrogate this activation. Immunohistochemistry analysis of SCC6 tumors treated with both U3-1287/AMG888 and a single dose of radiation demonstrated that various cell survival and proliferation markers could be reduced. Collectively our findings suggest that U3-1287/AMG888 in combination with radiation has an impact on cell and tumor growth by increasing DNA damage and cell death. These findings suggest that HER3 may play an important role in response to radiation therapy and blocking its activity in combination with radiation may be of therapeutic benefit in human tumors.
Discovery medicine 09/2013; 16(87):79-92. · 3.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nuclear localized HER family receptor tyrosine kinases (RTKs) have been observed in primary tumor specimens and cancer cell lines for nearly two decades. Inside the nucleus, HER family members (EGFR, HER2, and HER3) have been shown to function as co-transcriptional activators for various cancer-promoting genes. However, the regions of each receptor that confer transcriptional potential remain poorly defined. The current study aimed to map the putative transactivation domains (TADs) of the HER3 receptor. To accomplish this goal, various intracellular regions of HER3 were fused to the DNA binding domain of the yeast transcription factor Gal4 (Gal4DBD) and tested for their ability to transactivate Gal4 UAS-luciferase. Results from these analyses demonstrated that the C-terminal domain of HER3 (CTD, amino acids distal to the tyrosine kinase domain) contained potent transactivation potential. Next, nine HER3-CTD truncation mutants were constructed to map minimal regions of transactivation potential using the Gal4 UAS-luciferase based system. These analyses identified a bipartite region of 34 (B1) and 27 (B2) amino acids in length that conferred the majority of HER3's transactivation potential. Next, we identified full-length nuclear HER3 association and regulation of a 122 bp region of the cyclin D1 promoter. To understand how the B1 and B2 regions influenced the transcriptional functions of nuclear HER3, we performed cyclin D1 promoter-luciferase assays in which HER3 deleted of the B1 and B2 regions was severely hindered in regulating this promoter. Further, the overexpression of HER3 enhanced cyclin D1 mRNA expression, while HER3 deleted of its identified TADs was hindered at doing so. Thus, the ability for HER3 to function as a transcriptional co-activator may be dependent on specific C-terminal TADs.
PLoS ONE 08/2013; 8(8):e71518. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Src family kinases (SFKs) have been implicated in resistance to both radiation and epidermal growth factor receptor (EGFR) inhibition. Therefore, we investigated whether inhibition of SFK through dasatinib (DSB) can enhance the effect of radiotherapy in two in vivo human head and neck squamous cell carcinoma (HNSCC) models. Response to DSB and/or radiotherapy was assessed with tumor growth delay assays in two HNSCC xenograft models, SCCNij153 and SCCNij202. Effects on EGFR signaling were evaluated with Western blot analysis, and effects on DNA repair, hypoxia, and proliferation were investigated with immunohistochemistry. DSB and radiotherapy induced a significant growth delay in both HNSCC xenograft models, although to a lesser extent in SCCNij202. DSB did not inhibit phosphorylated protein kinase B (pAKT) or phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) but did inhibit (phosphorylated) DNA-dependent protein kinase. Moreover, DSB reduced repair of radiation-induced DNA double-strand breaks as shown by an increase of p53-binding protein 1 (53BP1) staining 24 hours after radiation. This effect on DNA repair was only observed in the cell compartment where phosphorylated SFK (pSFK) was expressed: for SCCNij153 tumors in both normoxic and hypoxic areas and for SCCNij202 tumors only in hypoxic areas. No consistent effects of DSB on hypoxia or proliferation were observed. In conclusion, DSB enhances the effect of radiotherapy in vivo by inhibition of radiation-induced DNA repair and is a promising way to improve outcome in HNSCC patients.
[Show abstract][Hide abstract] ABSTRACT: The epidermal growth factor receptor (EGFR) has been one of the most targeted receptors in the field of oncology. While anti-EGFR inhibitors have demonstrated clinical success in specific cancers, most patients demonstrate either intrinsic or acquired resistance within one year of treatment. Many mechanisms of resistance to EGFR inhibitors have been identified, one of these being attributed to alternatively localized EGFR from the cell membrane into the cell's nucleus. Inside the nucleus, EGFR functions as a co-transcription factor for several genes involved in cell proliferation and angiogenesis, and as a tyrosine kinase to activate and stabilize proliferating cell nuclear antigen and DNA dependent protein kinase. Nuclear localized EGFR is highly associated with disease progression, worse overall survival in numerous cancers, and enhanced resistance to radiation, chemotherapy, and the anti-EGFR therapies gefitinib and cetuximab. In this review the current knowledge of how nuclear EGFR enhances resistance to cancer therapeutics is discussed, in addition to highlighting ways to target nuclear EGFR as an anti-cancer strategy in the future.
Radiotherapy and Oncology 07/2013; · 4.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (Ctx (R) ) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in Ctx (R) clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all Ctx (R) clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of Ctx (R) cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting.
Cancer biology & therapy 06/2013; 14(6):481-91. · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L and K506R) in a GFP tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1) and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets.
Cancer biology & therapy 05/2013; 14(7). · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Nuclear EGFR (nEGFR) has been identified in various human tumor tissues, including cancers of the breast, ovary, oropharynx, and esophagus, and has predicted poor patient outcomes. We sought to determine if protein expression of nEGFR is prognostic in early stage non-small cell lung cancer (NSCLC). METHODS: Resected stages I and II NSCLC specimens were evaluated for nEGFR protein expression using immunohistochemistry (IHC). Cases with at least one replicate core containing ≥5% of tumor cells demonstrating strong dot-like nucleolar EGFR expression were scored as nEGFR positive. RESULTS: Twenty-three (26.1% of the population) of 88 resected specimens stained positively for nEGFR. Nuclear EGFR protein expression was associated with higher disease stage (45.5% of stage II vs. 14.5% of stage I; p=0.023), histology (41.7% in squamous cell carcinoma vs. 17.1% in adenocarcinoma; p=0.028), shorter progression-free survival (PFS) (median PFS 8.7 months [95% CI 5.1-10.7 mo] for nEGFR positive vs. 14.5 months [95% CI 9.5-17.4 mo] for nEGFR negative; hazard ratio (HR) of 1.89 [95% CI 1.15-3.10]; p=0.011), and shorter overall survival (OS) (median OS 14.1 months [95% CI 10.3-22.7 mo] for nEGFR positive vs. 23.4 months [95% CI 20.1-29.4 mo] for nEGFR negative; HR of 1.83 [95% CI 1.12-2.99]; p=0.014). CONCLUSIONS: Expression of nEGFR protein was associated with higher stage and squamous cell histology, and predicted shorter PFS and OS, in this patient cohort. Nuclear EGFR serves as a useful independent prognostic variable and as a potential therapeutic target in NSCLC.
Lung cancer (Amsterdam, Netherlands) 04/2013; · 3.14 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND AND PURPOSE: EGFR-inhibitor Cetuximab (C225) improves the efficacy of radiotherapy in only a subgroup of HNSCC patients. Identification of predictive tumor characteristics is essential to improve patient selection. MATERIAL AND METHODS: Response to C225 and/or radiotherapy was assessed with tumor growth delay assays in 4 HNSCC xenograft models with varying EGFR-expression levels. Hypoxia and proliferation were quantified with immunohistochemistry and the expression of proteins involved in C225-resistance with Western blot. RESULTS: EGFR-expression did not predict response to C225 and/or radiotherapy. Reduction of hypoxia by C225 was only observed in SCCNij202, which was highly sensitive to C225. Proliferation changes correlated with response to C225 and C225 combined with radiotherapy, as proliferation decreased after C225 treatment in C225-sensitive SCCNij202 and after combined treatment in SCCNij185, which showed a synergistic effect to combined C225-radiotherapy. Furthermore, C225-resistant SCCNij153 tumors expressed high levels of (activated) HER3 and MET. CONCLUSIONS: EGFR-expression is needed for C225-response, but is not sufficient to predict response to C225 with or without radiotherapy. However, basal expression of additional growth factor receptors and effects on proliferation, but not hypoxia, correlated with response to combined C225-radiotherapy treatment and are potential clinically relevant predictive biomarkers.
Radiotherapy and Oncology 02/2013; · 4.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Only a minority of cancer patients benefits from the combination of EGFR-inhibition and radiotherapy in head and neck squamous cell carcinoma (HNSCC). A potential resistance mechanism is activation of EGFR and/or downstream pathways by stimuli in the microenvironment. The aim of this study was to find molecular targets induced by the microenvironment by determining the in vitro and in vivo expression of proteins of the EGFR-signaling network in 6 HNSCC lines. As hypoxia is an important microenvironmental parameter associated with poor outcome in solid tumors after radiotherapy, we investigated the relationship with hypoxia in vitro and in vivo. METHODS: Six human HNSCC cell lines were both cultured as cell lines (in vitro) and grown as xenograft tumors (in vivo). Expression levels were determined via western blot analysis and localization of markers was assessed via immunofluorescent staining. To determine the effect of hypoxia and pAKT-inhibition on cell survival, cells were incubated at 0.5% O2 and treated with MK-2206. RESULTS: We observed strong in vitro-in vivo correlations for EGFR, pEGFR and HER2 (rs=0.77, p=0.10, rs=0.89, p=0.03) and rs=0.93, p=0.02, respectively), but not for pAKT, pERK1/2 or pSTAT3 (all rs<0.55 and p>0.30). In vivo, pAKT expression was present in hypoxic cells and pAKT and hypoxia were significantly correlated (rs=0.51, p=0.04). We confirmed in vitro that hypoxia induces activation of AKT. Further, pAKT-inhibition via MK-2206 caused a significant decrease in survival in hypoxic cells (p<0.01), but not in normoxic cells. CONCLUSIONS: These data suggest that (p)EGFR and HER2 expression is mostly determined by intrinsic features of the tumor cell, while the activation of downstream kinases is highly influenced by the tumor microenvironment. We show that hypoxia induces activation of AKT both in vitro and in vivo, and that hypoxic cells can be specifically targeted by pAKT-inhibition. Targeting pAKT is thus a potential way to overcome therapy resistance induced by hypoxia and improve patient outcome.
BMC Cancer 10/2012; 12(1):463. · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epidermal growth factor receptor (EGFR) signaling is strongly implicated in glioblastoma (GBM) tumorigenesis. However, molecular agents targeting EGFR have demonstrated minimal efficacy in clinical trials, suggesting the existence of GBM resistance mechanisms. GBM cells with stem-like properties (CSCs) are highly efficient at tumor initiation and exhibit therapeutic resistance. In this study, GBMCSC lines showed sphere-forming and tumor initiation capacity after EGF withdrawal from cell culture media, compared with normal neural stem cells that rapidly perished after EGF withdrawal. Compensatory activation of related ERBB family receptors (ERBB2 and ERBB3) was observed in GBM CSCs deprived of EGFR signal (EGF deprivation or cetuximab inhibition), suggesting an intrinsic GBM resistance mechanism for EGFR-targeted therapy. Dual inhibition of EGFR and ERBB2 with lapatinib significantly reduced GBM proliferation in colony formation assays compared to cetuximab-mediated EGFR-specific inhibition. Phosphorylation of downstream ERBB signaling components (AKT, ERK1/2) and GBM CSC proliferation were inhibited by lapatinib. Collectively, these findings show that GBM therapeutic resistance to EGFR inhibitors may be explained by compensatory activation of EGFR-related family members (ERBB2, ERBB3) enabling GBM CSC proliferation, and therefore simultaneous blockade of multiple ERBB family members may be required for more efficacious GBM therapy.
Neoplasia (New York, N.Y.) 05/2012; 14(5):420-8. · 5.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR antibody that has been approved for use in oncology. Previously we investigated mechanisms of resistance to cetuximab using a model derived from the non-small cell lung cancer line NCI-H226. We demonstrated that cetuximab-resistant clones (Ctx(R)) had increased nuclear localization of the EGFR. This process was mediated by Src family kinases (SFKs), and nuclear EGFR had a role in resistance to cetuximab. To better understand SFK-mediated nuclear translocation of EGFR, we investigated which SFK member(s) controlled this process as well as the EGFR tyrosine residues that are involved. Analyses of mRNA and protein expression indicated upregulation of the SFK members Yes (v-Yes-1 yamaguchi sarcoma viral oncogene) and Lyn (v-yes-1 Yamaguchi sarcoma viral-related oncogene homolog) in all Ctx(R) clones. Further, immunoprecipitation analysis revealed that EGFR interacts with Yes and Lyn in Ctx(R) clones, but not in cetuximab-sensitive (Ctx(S)) parental cells. Using RNAi interference, we found that knockdown of either Yes or Lyn led to loss of EGFR translocation to the nucleus. Conversely, overexpression of Yes or Lyn in low nuclear EGFR-expressing Ctx(S) parental cells led to increased nuclear EGFR. Chromatin immunoprecipitation (ChIP) assays confirmed nuclear EGFR complexes associated with the promoter of the known EGFR target genes B-Myb and iNOS. Further, all Ctx(R) clones exhibited upregulation of B-Myb and iNOS at the mRNA and protein levels. siRNAs directed at Yes or Lyn led to decreased binding of EGFR complexes to the B-Myb and iNOS promoters based on ChIP analyses. SFKs have been shown to phosphorylate EGFR on tyrosines 845 and 1101 (Y845 and Y1101), and mutation of Y1101, but not Y845, impaired nuclear entry of the EGFR. Taken together, our findings demonstrate that Yes and Lyn phosphorylate EGFR at Y1101, which influences EGFR nuclear translocation in this model of cetuximab resistance.Oncogene advance online publication, 19 March 2012; doi:10.1038/onc.2012.90.
[Show abstract][Hide abstract] ABSTRACT: The treatment of metastatic colorectal cancer (mCRC) remains one of the largest hurdles in cancer therapeutics to date. The most advanced treatment option for mCRC patients are anti-epidermal growth factor receptor (EGFR) monoclonal antibodies (mAbs) that bind to and inhibit the activity of EGFR. While the use of anti-EGFR mABs has had great impact in the treatment of mCRC, it has now been widely accepted that mCRC tumors with a mutation in the small GTPase KRAS do not respond to these therapies. KRAS mutations allow for EGFR independent activation of various oncogenic signaling cascades. In attempts to inhibit KRAS mutant tumor growth, BRAF, MEK and farsenyltransferase inhibitors have been used, however, their clinical efficacy is still accruing in the setting of CRC. Recent data suggests that various other inhibitors, including inhibitors of Src family kinases (SFK) and hepatocyte growth factor receptor (MET), may have potential preclinical and clinical success in KRAS mutant tumors. Additionally, it is becoming increasingly clear that different KRAS missense mutations may have varied biological responses to cetuximab, suggesting that cetuximab may still be a potential therapeutic option in some KRAS mutant tumors. In this review, we highlight the importance for both improved multimodality approaches for treating KRAS mutant mCRC tumors and stratification of KRAS mutations in response to different treatment regimes in order to optimize the best possible care for mCRC patients.
[Show abstract][Hide abstract] ABSTRACT: There is presently great interest in mechanisms of acquired resistance to epidermal growth factor receptor (EGFR) inhibitors that are now being used widely in the treatment of a variety of common human cancers. To investigate these mechanisms, we established EGFR inhibitor-resistant clones from non-small cell lung cancer cells. A comparative analysis revealed that acquired resistance to EGFR inhibitors was associated consistently with the loss of p53 and cross-resistance to radiation. To examine the role of p53, we first knocked down p53 in sensitive parental cells and found a reduction in sensitivity to both EGFR inhibitors and radiation. Conversely, restoration of functional p53 in EGFR inhibitor-resistant cells was sufficient to resensitize them to EGFR inhibitors or radiation in vitro and in vivo. Further studies indicate that p53 may enhance sensitivity to EGFR inhibitors and radiation via induction of cell-cycle arrest, apoptosis, and DNA damage repair. Taken together, these findings suggest a central role of p53 in the development of acquired resistance to EGFR inhibitors and prompt consideration to apply p53 restoration strategies in future clinical trials that combine EGFR inhibitors and radiation.
Cancer Research 11/2011; 71(22):7071-9. · 9.28 Impact Factor