Activation Of Alternative Signaling Pathways Research Articles

Everolimus synergizes with gefitinib in non-small-cell lung cancer cell lines resistant to epidermal growth factor receptor tyrosine kinase inhibitors

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy is considered as one of the most important treatments for patients with advanced non-small-cell lung cancer (NSCLC). However, not all patients benefit from this therapy because of primary or acquired resistance, both of which are usually caused by the activation of alternative signaling pathways. Thus, a combination of different signaling pathway inhibitors is a promising strategy. We used the mammalian target of rapamycin (mTOR) inhibitor everolimus in combination with gefitinib in NSCLC cell lines to analyze the efficacy of this combination regimen and the underlying molecular mechanism. Acquired gefitinib-resistant cell lines, together with EGFR wild-type and mutant primary gefitinib-resistant NSCLC cell lines, were treated with everolimus alone, gefitinib alone, or the combination of the two drugs, and the effects were evaluated using cell proliferation assays. The effects of everolimus and gefitinib on the EGFR pathway in NSCLC cell lines were determined by Western blot analysis. Combined treatment resulted in synergistic antitumor effects in gefitinib-resistant cells A549 and H1975. The combination index (CI) of cells increased with increasing dose of everolimus. Everolimus demonstrated no apparent inhibition of phosphorylated Akt (p-Akt) and phosphorylated p44/42 MAPK (p-MAPK) in H1650 cells. Additionally, in gefitinib-resistant cell lines, the combination of gefitinib and everolimus not only showed stronger inhibition of phosphorylated mTOR and phosphorylated p70S6K expression than either drug alone but also reduced the levels of p-Akt and p-MAPK in both cell lines. Our data showed that the combination of everolimus and gefitinib exhibits dose-dependent synergism in primary and acquired gefitinib-resistant NSCLC cells. Thus, a preclinical rationale exists for the use of everolimus to enhance the efficacy of gefitinib in EGFR-TKI-resistant patients with NSCLC.

Novel Anti-cancer Compounds for Developing Combinatorial Therapies to Target Anoikis-Resistant Tumors

Anoikis, a cell death pathway induced by loss of normal cell-matrix attachment or upon adhesion to a non-native matrix, ensures the balance between proliferative potential of normal cells and maintenance of tissue integrity. Thereby, anoikis serves as a potential molecular barrier against oncogenic transformation of normal cells. Cancer cells acquire anoikis resistance for survival and distant metastatic progression. During the acquisition of anoikis resistance, tumors modulate multiple cell signaling parameters through changes in the expression of up-stream receptors and by dynamically calibrating the dependency on down-stream signaling cascades. Many compounds that target the tumor-acquired switches in integrins, tumor antigens, growth factors, metabolic pathways, oxidative and osmotic-stress signaling are in various phases of pre-clinical and clinical development. Combinatorial approaches maximize the therapeutic efficacy and minimize the activation of alternate signaling pathways, which will otherwise contribute to drug resistance. In this regard, an integrated analysis of the mechanisms of action of potential drugs and lead compounds that can target significant nodes of anoikis signaling networks will provide a rational frame-work for further development and clinical use of respective agents, by formulating more effective combinatorial therapies, in patients with distinct drug-sensitivity profiles.

Targeting of Insulin-Like Growth Factor Type 1 Receptor in Ewing Sarcoma: Unfulfilled Promise or a Promising Beginning?

Targeting of Insulin-Like Growth Factor Type 1 Receptor in Ewing Sarcoma: Unfulfilled Promise or a Promising Beginning?

Adaptive Changes Result in Activation of Alternate Signaling Pathways and Acquisition of Resistance to Aromatase Inhibitors

Hormone therapy is an effective approach for the treatment of breast cancer. Although the antiestrogen tamoxifen has had a major impact on the treatment of the disease, aromatase inhibitors (AIs), which reduce estrogen synthesis, have recently proved to be more effective. These agents are now used as first-line therapy for postmenopausal breast cancer. Nevertheless, despite the efficacy of these agents, resistance to treatment eventually may occur in some patients. In an effort to overcome this resistance and extend the benefits of AIs, investigators have studied the mechanisms involved in resistance to AIs. Adaptive changes that result in activation of alternate signaling pathways in AI-resistant tumors have been identified in xenograft and cell line models. Expression of estrogen receptor α and aromatase was shown to be decreased in tumors after long-term treatment with AIs. In contrast, increased expression was observed in tyrosine kinase receptors such as Her-2 and insulin-like growth factor receptor, as well as in downstream signaling proteins such as mitogen-activated protein kinase. Functional activation of the mitogen-activated protein kinase pathway and dependency on growth factor receptor signaling have been observed in AI-resistant cells and tumors.

A novel insight to the functional role of Stathmin 1 in IgE-mediated activation of RBL-2H3 cells.

IgE-mediated cell signaling, induced by cross-linking of high affinity receptor for IgE (FcεRI) in the presence of antigen (Ag), is a well known mechanism described for mast cell activation in allergy and hypersensitivity reactions, which induces a spectrum of cellular responses such as secretion and up-regulation of cell surface FcεRI. Although for several years IgE binding to FcεRI was considered to be a passive sensitization process, the outcomes of several recent studies have revealed a variety of different cellular responses to IgE binding compared to IgE plus Antigen binding. The present study applied a functional proteomics-based approach to investigate mast cell signaling events and provided new insights to FcεRI-mediated cell signaling in RBL-2H3.1 cells, and may point to the activation of alternative signaling pathways in response to IgE or IgE plus Ag. Comparative analysis by 2-D PAGE of RBL cells activated with IgE plus Ag for three and four hours compared to non-activated cells was followed by mass spectrometric protein identification and provided evidence for the induction of Stathmin 1 (STMN1) gene expression in response to IgE plus Ag activation.Complementary SDS-PAGE analysis showed a distinct up-regulation of STMN1 induction in response to challenge with IgE plus Ag compared to sensitization with IgE only. Phosphoproteomics analysis gave evidence for significant increase at phosphorylation of STMN1 on ser16 after 1min, though a slight rise at 5 min, and on ser38 after 1 and 5min sensitization with IgE and a similar result was observed for 1min IgE plus Ag-activation. IgE plus Ag-activation was also found to induce the phosphorylation of ser38 to a greater extent than sensitization with IgE. In contrast, IgE alone was more effective than IgE plus Ag at inducing phosphorylation of ser16. Collectively this study provides further insights into the role of stathmin 1 in FcRI-mediated activation of cells of mast cell lineage and might shed light on the diverse response of these cells to IgE or IgE plus Ag.

Phase I clinical trial of an allosteric AKT inhibitor, MK-2206, using a once weekly (QW) dose regimen in patients with advanced solid tumors.

3037^ Background: AKT is a key regulator of cellular proliferation, apoptosis and growth. A range of cancers have genetic aberrations that involve the PI3K/AKT pathway, leading to abnormal AKT hyperactivation and tumor progression. MK-2206 is a novel potent investigational AKT inhibitor, which was initially administered orally in a QOD schedule, where the maximum tolerated dose (MTD) was established at 60mg, based on safety and biomarker studies (Yap et al, ASCO 2010). In view of a long half-life (t1/2) (60-80h), a QW schedule was subsequently pursued to explore the potential of less frequent dosing to circumvent feedback activation of alternative signaling pathways in response to sustained and uninterrupted AKT inhibition. Methods: Eligible patients with advanced solid tumors were recruited in a phase I, multi-center, dose escalation study. Patients had regular safety assessments and blood sampling for pharmacokinetic (PK) studies. Pharmacodynamic (PD) and/or predictive biomarker analyses were undertaken on paired tumor samples (mandatory biopsies at MTD), platelet-rich plasma, hair follicles and circulating nucleic acids in plasma. Results: 30 patients (17 M; median age 60 yr; ECOG PS 0/1: 11/19) received MK-2206 at 90, 135, 200, 250 and 300 mg QW. Grade 3 rash was the dose limiting toxicity (DLT) at 300mg (n=3) and 250mg (n=2). Expansion of the 200mg QW resulted in grade 3 rash DLT in 4 of 15 patients. An intermediate dose of 150mg QW is being investigated. Common reversible MK-2206-related toxicities included rash (32.3%), fatigue (9.7%) and nausea, vomiting, diarrhea, pruritus, stomatitis, dry skin and dysguesia, each occurring at 6.5%. No MK-2206-related grade 5 toxicities were observed. PK analysis showed a median Tmax of 6 hr and a mean apparent terminal t1/2 of 78.6 hr. PD analysis of platelet-rich plasma at 200mg QW (n=9) showed suppression of pAKT up to 96 hours post-dose (p<0.001). Biomarker analyses of tumor samples are ongoing. Conclusions: MK-2206 has shown an acceptable toxicology profile, significant and sustained AKT blockade and a PK-PD profile allowing for QW dosing.

What is hot in experimental thyroidology?

Over the recent years, considerable progress has been made in elucidating processes that are critical for thyroid hormone (TH) synthesis and action. In this issue of Thyroid Research several German groups dedicated to thyroid research have assembled and discussed novel aspects of thyroid hormone action with the perspective to improve diagnosis and therapy of thyroid disorders by better understanding disturbed thyroid differentiation. The over-arching theme addressed in the contributions of this special issue relates to the main focus of current thyroid research and treatment of thyroid disesase, i.e. “What defines thyroid health?” [1]. Thereby the putative role of seemingly established players in TH synthesis and action are revisited. A report on a patient characterized by a novel germline TSH-receptor mutation links this case of non-autoimmune hyperthyroidism to new impliations in TSH-receptor structure [2]. The study highlights the importance of introducing novel diagnostic approaches to the clinics, because this particular mutation in the TSH-receptor is correlated to altered Gq-signalling pathways, thereby pointing to an important role of Gq signalling in the thyroid It is known only since recently that transport of TH is an active and regulated mechanism. The monocarboxylate transporter 8 (MCT8) is a crucially important transporter of TH and it transports triiodothyronine (T3) specifically well. Mutations in MCT8 are known to cause a severe syndrome of X-linked mental retardation known as Allan-Herndon Dudley syndrome. Based on homology modeling all of the currently reported pathogenic mutations in MCT8 are studied in the article by Kleinau et al. [3]. A focus is set on identification of those MCT8 mutations that are most sensitive for substrate transport with the goal to simulate in silico the potencies of future therapies based on TH transporter-specific inhibitors mimicking TH structurally. Besides MCT8 other transporters have been proposed to play important roles in TH transport (MCT10, OATP1C1, OATP1A2, OATP14, LAT1 and LAT2). The structure-function relationships of primary TH transporters like MCT8 and additional, so-called secondary TH transporters such as those listed above is reviewed by Kinne et al. [4] with special attention on their expression profiles, transport specificities and substrates. Similarities and differences in structure and molecular features of the known and suspected TH transporters are compared between man and mouse, in particular asking their crucial impact on TH actions in the central nervous system. TH actions in the brain and clinical consequences of non-classical, rather novel treatment protocols to relieve symptoms of severe mood and depression disorders is reviewed in the article by Pilhatsch et al. [5]. For a long time, only genomic actions of TH, namely of T3 were recognized by both, thyroid researchers and clinicians. However this picture has to be revised as non-classical, non-genomic TH actions were described which lead to activation of alternative signaling pathways via PI3K and MAPK. The impact and perspectives of these recent findings are summarized by Moller and Brocker-Preus [6] who comment on the outcomes of activation of alternative signaling pathways on transcriptional regulation of TH target cells. Non-classical and classical TH actions must be taken into consideration for better management of thyroid malignancies. Moreover, it is increasingly appreciated that disturbances in TH synthesis occur in thyroid tumorigenesis and that disturbed TH action affects carcinogenesis in general. Of the many novel aspects in thyroid carcinogenesis, it was the recent identification of miRNAs and their intricate involvement in the regulation of transcriptional, translational and epigentic control mechanisms that provides one of the most promising diagnostic and high-potential, future therapeutic tool. The aspects of miRNA relevance for thyroid pathologies are discussed by Braun and Huttelmaier [7]. Likewise, regulation of transcription and translation by RNA binding proteins is addressed in the article by Trojanowicz et al. The adenylate uridylate-rich element binding proteins AUF together with HuR was reported to regulate mRNA stability and could hence participate in thyroid carcinogenesis. RNA binding proteins may harbor multifunctional properties that not only affect thyrocyte differentiation but could also affect TH synthesis [8]. Finally, the characterization of protein transport pathways and the identification of altered trafficking in thyroid carcinoma cells are approached in the experimental study by Tedelind et al. [9] that investigated the cathepsins which are known as thyroglobulin-processing enzymes with important functions in TH biosynthesis. We believe that the papers collected for this special issue of Thyroid Research will have a stimulatory effect not only for thyroid researchers but will be interesting for those from associated disciplines.

Abstract P3-14-14: Activation of SRC and MAPK Mediates Resistance to Lapatinib in ER-/ERBB2+ Breast Cancer

Abstract Background Resistance to lapatinib represents a significant problem in the treatment of ERBB2+ breast cancer, especially after failure of prior trastuzumab therapy. A number of resistance mechanisms have been identified in recent years that limit the anti-tumor effects of lapatinib. These mechanisms include the activation of ERα signaling, and sustained activation of AKT either through coexpression of receptor tyrosine kinases (IGF-IR, AXL, MET, ERBB3), loss of PTEN or activating mutations in PIK3CA. However, these mechanisms have been shown to confer resistance primarily in ER+/ERBB2+ breast cancer cells; the mechanisms of resistance to lapatinib in ER-/ERBB2+ breast cancer (∼ 50% of ERBB2+ breast cancers) remain poorly understood. We hypothesized that resistance to lapatinib in ER-/ERBB2+ breast cancer cells can be mediated by activation of alternative signaling pathways which bypass the PI3K/AKT signaling axis. Materials and Methods To investigate the mechanisms of acquired resistance to lapatinib we generated ER-/ERBB2+ and ER+/ERBB2+ breast cancer cells resistant to lapatinib at the clinically relevant concentration of 2.6 uM (SK-lapR and BT-lapR). Resistance to lapatinib was achieved by exposure of SKBR3 and BT474 cells to increasing concentrations of the drug, and resistant cells were selected through serial passage. The activation status of multiple signaling pathways in lapatinib sensitive and resistant cells was determined by western blot analysis, and the role of individual signaling pathways in promoting resistance to lapatinib was investigated in cell proliferation assays using selective small molecule inhibitors. Results and Discussion Consistent with previous studies, we show that inhibition of ERα signaling using 100 nM fulvestrant in combination with lapatinib restored lapatinib sensitivity and effectively leads to growth arrest and cell death in ER+/ERBB2+ BT-lapR cells (P&amp;lt;0.001). However, none of the previously proposed mechanisms of resistance to lapatinib were apparent in our SK-lapR cells (ER-/ERBB2+). Strikingly, these cells which are stably resistant to lapatinib show no activation of AKT even in the absence of lapatinib. Rather, they exhibit increased phosphorylation of SRC kinase (3.9 fold, analyzed with ImageJ) and no inhibition of ERK1/2 after treatment with lapatinib compared to sensitive SKBR3 cells. Inhibition of SRC activity with 1uM AZD0530 in combination with lapatinib significantly reduced growth of SK-lapR cells compared to treatment with lapatinib alone (P&amp;lt;0.05). These data reveal potential novel mechanisms for lapatinib resistance in ER-/ERBB2+ breast cancer, and may indicate therapeutic strategies to prevent or overcome lapatinib resistance. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-14-14.

DNA damage-induced Leukaemic Stem and Progenitor Cell Kill with Mylotarg and Tipifarnib/Zarnestra Drug Combination In Acute Myeloid Leukemia (AML)

AbstractAbstract 1824Leukaemic stem and progenitor cells (LSPC) are relatively resistant to current chemotherapy and considered to contribute to disease progression and relapse in Acute Myeloid Leukemia (AML) patients following initial treatment. We have developed a model to study the in vitro targeting of AML LSPC by novel agents which have the potential to target LSPC whilst in a resistant niche microenvironment. Using this model, we showed that under ‘niche-like’ conditions CD34+CD38-CD123+ LSPC are less sensitive to cytarabine and to the Flt3 inhibitor AG1296. In contrast, LSPC when grown under niche support are extremely sensitive to Mylotarg (Gemtuzumab Ozogamicin), one of a few successful chemotherapeutic agents that is currently used in the clinic. We are now proposing that the combination of Mylotarg (10ng/ml) and the farnesyltransferase inhibitor, Tipifarnib/Zarnestra (5microM) is successful at further targeting the LSPC subset resulting in 70% LSPC kill (n=29 AML primary cells) after 48hr in vitro treatment compared to 31% and 12% LSPC kill in Mylotarg and Tipifarnib treated cells, respectively (p<0.0001). Moreover, stromal MS5 co-culture experiments (used to depict drug-induced cell survival in a niche-like setting) with the Tipifarnib/Mylotarg combination showed enhanced cytotoxicity even in such a resistant setting. Because activation of alternative signaling pathways maybe one mechanism that cancer cells use to evade drug-induced cell kill, we used a protein profiler assay to depict the expression of 46 known human phosphokinase proteins when treated with single and drug combination. Using the CD34+CD38- TF1A cell line, 34/46 (74%) of phosphokinase proteins that are essential for functional survival pathways (e.g. p38MAPK, ERK, JNK, AKT, STAT1/2/4/5, GSK3beta, beta-catenin, p53 etc.) were maximally reduced by the drug combination compared to housekeeping phosphoproteins, 12/46 (26%) proteins were unaffected by the drug combination and only 1/46 (the cell cycle checkpoint regulator and putative tumor suppressor Chk2) increased maximally with the drug combination. We next show that the drug combination's success is indeed characterized by the activation of DNA damage response (increase in H2AX foci by immunoflourescence), an induction of phosphoATM (by flow cytometry) leading to the activation of Chk2 and G2/M cell cycle arrest (using BrdU assay). Finally, we demonstrate that Tipifarnib may be acting as a potent P-glycoprotein inhibitor by allowing the retention of Mylotarg in cells that would otherwise efflux the drug out of the cell. In conclusion, the in vitro data presented here clearly shows why we think the Tipifarnib/Mylotarg combination can be successful in the treatment of AML and warrants further investigation to confirm its efficacy in in vivo models. Disclosures:No relevant conflicts of interest to declare.

W1726 ERK MAP Kinase Signalling and PEA3 are Synergistic in Predicting Adversity in Gastric Adenocarcinomas

Introduction The PEA3 sub family consists of 3 similar transcription factors, PEA3, ER81 and ERM. In gastric cancer cells PEA3 is activated by H pylori and it has been shown to regulate MMP-7 and invasion. PEA3 mRNA expression correlates with an adverse prognosis in gastric adenocarcinomas. We have previously shown that PEA3 and/or ER81 mRNA correlates with adverse the indicator, MMP-1 and MMP-7. Very high PEA3 protein expression in isolation is associated with tumour metastases. However tumours with PEA3 mRNA and lower protein expression did not correlate with prognostic markers. PEA3 is regulated by ERK MAP kinase signalling in oesophageal adenocarcinomas and we investigated if this pathway plays an important role in gastric adenocarcinomas. Methods Patients with gastric adenocarcinoma (37) and controls (11) were selected. Tissue microarrays were constructed for immunohistochemical studies of PEA3 and pERK. The t test and chi square test were used for statistical analysis. A p value Results In patients with gastric adenocarcinoma, active ERK signalling is present in 60%. From these, ERK signalling is associated with an advanced tumour stage (AJCC stage 1 67%, stage 2: 33%, stage 3: 21% stage 4: 80% p=0.04). Active signalling is present in stage 1 and 2 tumours (55%). Survival is significantly reduced in patients with active ERK signalling (41 vs 34 months P=0.05) on univariate analysis. Adenocarcinomas with PEA3 and pERK co-occurrence frequently have distant metastases 60% (T and N stage vs M stage p=0.048). Survival is worse in patients with combined pERK and PEA3 presence compared to PEA3 or pERK presence in isolation (12 vs 30 vs 63 months p=0.071). Conclusion Our results indicate that ERK MAP kinase signalling is an important marker of an advanced tumour stage and prognosis in gastric adenocarcinoma. The signalling pathway is frequently active in early stage (AJCC 1–3) tumours. Previous studies have shown poor results with MEK inhibition treatment in Pancreatic, Colon, Lung and Breast cancers. This may be due to the selection of heterogeneous tumours with an advanced disease stage or tumours with activation of alternative signalling pathways. Our results suggest consideration of trialling MEK inhibition treatment in patients with earlier stage disease in gastric adenocarcinoma. The more adverse outcome in patients with the combination of ERK MAP signalling and PEA3 indicates that a combined approach with inhibition of PEA3, could be considered in selected patients.

Abstract 1171: Gastrointestinal stromal tumors (GIST) cell proliferation inhibition with combined adenoviral and molecular targeted therapy

Abstract Introduction: GIST, the most common mesenchymal malignancies of the GI tract, are characterized by gain-of-function mutations in KIT and PDGFRα receptors. Imatinib and sunitinib are tyrosine kinase inhibitors (TKI) currently approved for the treatment of GIST. Recent evidence suggests that GIST cells acquire secondary resistance to TKI through activation of alternative signaling pathways. We hypothesize that gene therapy with replicative vector oncolysis (virotherapy) can be utilized to improve efficacy of TKI and overcome GIST resistance to treatment. Objectives: To determine if a panel of oncolytic adenoviruses in combination with imatinib and sunitinib, results in synergistic inhibition of GIST cell proliferation. Results: GIST882 and GIST-T1 cells were subjected to imatinib and sunitinib treatment in combination with a panel of oncolytic adenoviruses (Ad5, Ad5RGD, Ad5pK7, and Ad5 - Ad3 fiber knob), to assess their inhibitory effects on cell proliferation. The Ad5/3 vector had the best rate of gene transfer and cytolytic effect. The greatest anti-proliferative effects were observed with 100 viral particles/cell of the Ad5/3 vector combined with TKI concentrations of 0.1 µM in GIST-T1 cell line. The combination of TKI and oncolytic adenoviruses showed greater inhibition of GIST cell proliferation (78% decrease) when compared to TKI alone (58% decrease) and viral therapy alone (23% decrease, p&amp;lt;0.01). Successful ex vivo infection and oncolysis of GIST by Ad5/3 particles was confirmed in cells obtained from patient surgical specimens (n=4). Conclusions: The oncolytic adenovirus Ad5/3 infects and inhibits GIST cell proliferation both in vitro and ex vivo. Virotherapy with Ad5/3 has synergistic anti-proliferative effects with TKI and may help overcome GIST resistance to KIT targeted therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1171.

OC-051 Erk MAP kinase signalling and PEA3 are synergistic in predicting adversity in gastric adenocarcinomas

<h3>Introduction</h3> The PEA3 sub family consists of 3 similar transcription factors, PEA3, ER81 and ERM. In gastric cancer cells PEA3 is activated by H pylori and it has been shown to regulate MMP-7 and invasion. PEA3 mRNA expression correlates with an adverse prognosis in gastric adenocarcinomas. We have previously shown that PEA3 and/or ER81 mRNA correlates with adverse the indicator, MMP-1 and MMP-7. Very high PEA3 protein expression in isolation is associated with tumour metastases. However tumours with PEA3 mRNA and lower protein expression did not correlate with prognostic markers. PEA3 is regulated by ERK MAP kinase signalling in oesophageal adenocarcinomas and we investigated if this pathway plays an important role in gastric adenocarcinomas. <h3>Methods</h3> Patients with gastric adenocarcinoma (37) and controls (11) were selected. Tissue microarrays were constructed for immunohistochemical studies of PEA3 and pERK. The t test and chi square test were used for statistical analysis. A p value &lt;0.05 was judged to be significant. <h3>Results</h3> In patients with gastric adenocarcinoma, active ERK signalling is present in 60%. From these, ERK signalling is associated with an advanced tumour stage (AJCC stage 1 67%, stage 2: 33%, stage 3: 21% stage 4: 80% p=0.04). Active signalling is present in stage 1 and 2 tumours (55%). Survival is significantly reduced in patients with active ERK signalling (41 vs 34 months P=0.05) on univariate analysis. Adenocarcinomas with PEA3 and pERK co-occurrence frequently have distant metastases 60% (T and N stage vs M stage p=0.048). Survival is worse in patients with combined pERK and PEA3 presence compared to PEA3 or pERK presence in isolation (12 vs 30 vs 63 months p=0.071). <h3>Conclusion</h3> Our results indicate that ERK MAP kinase signalling is an important marker of an advanced tumour stage and prognosis in gastric adenocarcinoma. The signalling pathway is frequently active in early stage (AJCC 1–3) tumours. Previous studies have shown poor results with MEK inhibition treatment in Pancreatic, Colon, Lung and Breast cancers. This may be due to the selection of heterogeneous tumours with an advanced disease stage or tumours with activation of alternative signalling pathways. Our results suggest consideration of trialling MEK inhibition treatment in patients with earlier stage disease in gastric adenocarcinoma. The more adverse outcome in patients with the combination of ERK MAP signalling and PEA3 indicates that a combined approach with inhibition of PEA3, could be considered in selected patients.

KIT Inhibition Affects Heat Shock Protein (HSP) Activity: A Potential Rescue Mechanism towards KIT Tyrosine Kinase Inhibition and a Rationale for Dual KIT/HSP Inhibition.

Abstract 2753Poster Board II-729Activating mutations of the KIT class III receptor tyrosine kinases are associated with core binding factor leukemias (CBF AML), systemic mastocytosis (SM), gastrointestinal stromal tumors (GIST), melanomas, seminoma/dysgerminoma and sinonasal natural killer/T-cell non-Hodgkin lymphoma. Despite the encouraging therapeutic potential of KIT-tyrosine kinase inhibitors (TKI), resistance leading to disease progression occurs in many patients, specifically after TKI monotherapy. We hypothesized that resistance to therapy is promoted by activation of alternative signaling pathways which override TKI inhibition.To explore the downstream signaling pathways of class III receptor tyrosine kinases, we performed unbiased phoshoproteomic analyses of mutant FLT3 or KIT leukemia and mastocytosis cell lines before and after TKI treatment. Tantalizingly, immunoaffinity purification of phosphopeptides followed by tandem mass spectrometry following KIT-inhibition with Imatinib at IC90 (100nM) revealed a significant upregulation of phosphorylation levels of peptides identified as members of the heat shock protein (HSP) family. Of interest, mRNA GeneChip® Array analysis of hematopoietic Ba/F3 cells transfected with either a mutant KIT isoform (D816V) or a mutant FLT3 isoform (ITD) and treated with TKI revealed significant downregulation of HSP family members in the FLT3 model – but stable mRNA levels in the KIT model. Taken together, our phosphoproteome and mRNA data suggest a protective function of HSP in mutant-KIT tumors treated with TKI.Next we studied the antiproliferative and proapoptotic effects of the HSP90 inhibitor IPI-504, a 17-AGG-derivative, in mutant-KIT cell models. IPI-504 potently inhibited proliferation and induced apoptosis with an IC50 of 0.5 up to 5μM depending on the KIT isoform. Importantly, combination of IPI-504 with TKIs resulted in potentiation of the antiproliferative and proapoptotic effects achieved by either drug alone. Antitumor efficacy in combination therapy was observed even at HSP90 inhibitor concentrations that did not display antitumor activity if administered alone.In conclusion, our model suggests that inhibition of KIT affects heat shock protein activity serving to stabilize the functionality of targeted autoactivated receptor tyrosine kinases, which provides a potential mechanism for resistance to TKI therapy. Importantly, we provide a rationale to combine TKI with (low-dose) HSP-inhibitors such as IPI-504 to optimize TKI therapy. Disclosures:Normant:Infinity: Employment.

A phase I trial of liposomal doxorubicin (D), bevacizumab (A), and temsirolimus (T) in advanced malignancy

e13508 Background: Liposomal doxorubicin (D) and bevacizumab (A) are active drugs in multiple malignancies. Unfortunately, resistance mechanisms have been described for each that limit their efficacy following repeated exposure. These include aberrant activation of alternate signaling pathways, such as PI3K/AKT/mTOR and upregulation of hypoxia inducible factor (HIF-1α). We therefore added temsirolimus (T), an agent known to inhibit mTOR and to downregulate HIF-1α, to the combination of D and A. The objectives of this trial were to assess the safety of this combination, obtain preliminary efficacy data and to identify biologic correlates for treatment response. Methods: D, A and T were administered intravenously on a 21 day cycle. D, A and T were given day 1, and T was administered alone weeks 2 and 3. A standard 3+3 trial design was used for dose escalation; 6 dose levels are planned. Patient serum and biopsies were also collected pre- and post-treatment for batched analysis to assess biologic response correlates. Results: 6/12 treated patients are evaluable (remaining patients have not yet completed 1st restaging). 5 patients are male; median age (all patients) 53, range 12 to 59. Median number of prior treatments = 5, range 2 - 8. At first restaging, 2/2 patients with endometrial cancer were found to have ≥ 25% decrease in tumor size (RECIST), and one patient with colorectal carcinoma had a 15% decrease. 1 patient had stable disease. One of the patients with endometrial cancer and extensive intra-abdominal disease who showed rapid tumor regression (≥ 25% decrease at 2 months) developed an entero-colonic fistula, and chose hospice care. Grade 2 stomatitis and fatigue (CTCAEv3)have also been seen. The current doses being used are 20mg/m2 of D and 5mg/m2 of A every 21 days, and 25mg of T every 7 days (dose level 3). Conclusions: Preliminary data indicate that the combination of D, A and T has anti-tumor activity and is well tolerated at the doses used. Patients with intra-abdominal disease who experience rapid tumor regression may be at risk for fistula formation. Biomarker analysis for modulation of the mTOR signal (via PI3K mutations or other aberrations) and the HIF-1α pathway is ongoing. No significant financial relationships to disclose.

Stemming Resistance to HER-2 Targeted Therapy

Although the development of trastuzumab and lapatinib has improved the outlook for women with HER-2 positive breast cancer, resistance to HER-2 targeted therapy is a growing clinical dilemma. Recent evidence indicates that the HER-2 pathway may play an important role in the maintenance of cancer stem cells (CSCs). The success of HER-2 targeted therapies may, in part, be explained by their direct activity against HER-2 positive CSCs. Our understanding of the mechanisms involved in resistance to trastuzumab, including loss or blockade of the trastuzumab binding site, activation of alternative signaling pathways, and induction of epithelial-mesenchymal transition (EMT), suggests that CSCs may be at the root of resistance of HER-2 targeted therapy. A variety of novel HER-2 targeted approaches have demonstrated promising preliminary clinical activity. Future clinical trials should involve the integration of technologies to assess the impact of novel HER-2 targeted therapies on HER-2 positive CSCs.

Targeting heat-shock protein 90 improves efficacy of rapamycin in a model of hepatocellular carcinoma in mice

Hepatocellular carcinoma (HCC) remains associated with a poor prognosis, but novel targeted therapies in combination with anti-angiogenic substances may offer new perspectives. We hypothesized that simultaneous targeting of tumor cells, endothelial cells, and pericytes would reduce growth and angiogenesis of HCC, which represents a highly vascularized tumor entity. Recently, because of their anti-angiogenic properties, inhibitors of mammalian target of rapamycin (mTOR) have entered clinical trials for therapy of HCC. However, treatment with mTOR inhibitors may lead to paradoxical activation of Akt signaling in tumor cells via insulin-like growth factor-I receptor (IGF-IR)-dependent and IGF-IR-independent mechanisms. Because we have recently identified heat shock protein 90 (Hsp90) antagonists to impair both oncogenic and angiogenic signaling cascades in tumor cells, including Akt and IGF-IR, we sought to investigate whether Hsp90 blockade could improve growth-inhibitory and anti-angiogenic effects of the mTOR inhibitor rapamycin. Human HCC cells, a murine hepatoma cell line, endothelial cells (ECs), and vascular smooth muscle cells (VSMC) were employed in experiments. Results show that dual inhibition of mTOR and Hsp90 leads to effective disruption of oncogenic signaling cascades and substantially improves growth-inhibitory effects in vivo. Importantly, blocking Hsp90 abrogated the rapamycin-induced activation of Akt and of the downstream effector nuclear factor kappa-B (NF-kappaB) in HCC tumors. Furthermore, Hsp90 inhibition reduced the expression of platelet-derived growth factor-receptor-beta (PDGF-Rbeta) on VSMCs, and diminished vascular endothelial growth factor-receptor 2 (VEGFR-2) expression on ECs, which further improves the anti-angiogenic capacity of this regimen. Blocking Hsp90 disrupts rapamycin-induced activation of alternative signaling pathways in HCCs and substantially improves the growth-inhibitory effects of mTOR inhibition in vivo. Hence, the concept of targeting tumor cells, ECs, and VSMCs by blocking Hsp90/mTOR could prove valuable for treatment of HCC.

Vascular Endothelial Growth Factor Receptor-1 Contributes to Resistance to Anti–Epidermal Growth Factor Receptor Drugs in Human Cancer Cells

The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor-resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor-resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.

Tyrosine Kinase Inhibition by SU5614 Fails To Eradicate Leukemic Stem Cells in FLT3-ITD+ Acute Myeloid Leukemia: Role of the Microenvironment.

Activating mutations of the FLT3 receptor by internal tandem duplication (FLT3-ITD) are present in 30% of all cases of acute myeloid leukemia (AML) and are associated with poor prognosis. FLT3-ITD mutations are present in leukemic stem/progenitor cells and induce ligand-independent downstream signaling promoting oncogenesis through pathways involved in proliferation, differentiation and survival, making the mutated receptor an attractive therapeutic target for tyrosine kinase inhibition. Although tyrosine kinase inhibitors have been shown to be cytotoxic to FLT3-ITD+ leukemic blasts, the effects on more primitive leukemic stem cells have not been studied in detail. We examined the effect of the tyrosine kinase inhibitor SU5614 on leukemic CD34+ stem/progenitor cells from patients with newly diagnosed normal karyotype AML with wild-type FLT3 or mutated FLT3-ITD receptor. SU5614 was chosen because initial experiments comparing SU5614, PKC412 and imatinib had shown that SU5614 was the most potent in inducing cell cycle arrest without significant apoptosis in normal CD34+ stem/progenitor cells. CD34+ cells were isolated from bone marrow of AML patients at diagnosis by density gradient centrifugation and magnetic bead isolation. Cells were cultured for four days in serum-free medium with growth factors in the presence or absence of SU5614 (5 uM) in suspension culture or in stroma-contact cultures. Hematopoietic activity was assessed in colony-forming assays. Overall, untreated CD34+FLT3-ITD+ leukemic progenitors cells formed significantly fewer CFU than CD34+FLT3-WT leukemic progenitors. However, the percentage of more primitive LTC-IC was higher in FLT3-ITD+ samples. SU5614 induced cell cycle arrest in all FLT3-ITD+ as well as FLT3-WT samples whereas apoptosis was variable. FLT3-ITD+ committed progenitor cells were effectively reduced by SU5614 treatment in suspension culture while stroma contact exerted a significant protective effect. In contrast, committed progenitors from FLT3-WT AML were less susceptible to tyrosine kinase inhibition but also protected by adhesion to stroma. More importantly, primitive LTC-IC from FLT3-ITD+ AML were selectively spared from tyrosine kinase inhibition. Additional stromal contact led to expansion of LTC-IC in the presence of SU5614. PCR from single hematopoietic colonies of stromal contact cultures revealed both WT and FLT3-ITD products after treatment with SU5614, indicating LTC-IC were of leukemic origin. To further elucidate the mechanism by which stromal contact selectively protects FLT3-ITD+ LTC-IC, leukemic cell lines harboring either FLT3-ITD (MV4-11) or FLT3-WT (RS 4;11) were studied. As expected, SU5614 effectively inhibited constitutively active FLT3 in MV4-11 as well as ligand activated FLT3 in RS 4;11 cell lines independent of stromal contact. However, inhibition of downstream Akt activation by SU5614 in MV4-11 cells was completely abrogated in the presence of stroma. In contrast, stromal contact had no effect on Akt activation in FLT3-WT RS 4;11 cells. Activation of downstream Erk and Stat5 and inhibition by SU5614 were not affected by stromal contact in either cell line. In conclusion, our data suggest activation of alternate signaling pathways in FLT3+ leukemic stem cells allowing escape from dependence on FLT3 signaling and subsequently tyrosine kinase inhibition. In addition, protection of leukemic FLT3-ITD+ LTC-IC is mediated by stromal contact.

Uncoupling of the epidermal growth factor receptor from downstream signal transduction molecules guides the acquired resistance to gefitinib in prostate cancer cells

The clinical efficacy of ErbB1 kinase inhibitors is limited by the development of acquired autoresistance. The activation of alternative signaling pathways can contribute to gefitinib resistance. In this study, we demonstrate that the continuous in vitro exposure of the phosphatase and tensin homologue (deleted from chromosome 10)-negative prostate cancer (PC)3 cell line to gefitinib resulted in a sustained growth inhibition of 50% for about 2 months, but afterwards the surviving cells resumed their usual proliferation rate. During chronic treatment, gefitinib-treated cells developed drug resistance undergoing a G0/G1 cell cycle arrest, with a corresponding reduction in the G2/M cells without evident cell apoptosis, and thus a tyrosine kinase inhibitor-resistant (TKI-R) PC3 cell subline was isolated. TKI-R cells show i) an increment in basal ERK activation, ii) an epidermal growth factor-mediated and gefitinib insensitive ERK phosporylation, iii) increased levels of Her2/Neu, iv) a significant decrement in epidermal growth factor receptor (EGFR) expression, v) a very low sensitivity against EGFR TKIs and blocking antibodies, vi) a moderate increase in the sensitivity to growth inhibition by the Her2 inhibitor, AG825 or by 2C4, the humanized monoclonal antibody which blocks Her2 heterodymerization, vii) an increased expression of the neutrophine receptors, TrkA and TrkB, and viii) a significantly increased sensitivity to growth inhibition by the TrkA inhibitor, CEP701. Treatment with a mitogen-activated-protein kinase inhibitor abolished gefitinib resistance completely. Therefore, the ability of tumor cells to maintain high ERK activity under EGFR inhibition could represent a potential mechanism of the resistance to gefitinib.

G‐protein‐coupled receptor dephosphorylation at the cell surface

Agonist-induced desensitization of G-protein-coupled receptors (GPCRs) usually involves phosphorylation of the receptor by G-protein-coupled receptor kinases (GRKs) or second messenger-dependent protein kinases such as PKC and PKA. Phosphorylation by GRKs promotes arrestin binding to the receptor, which not only uncouples receptor and G protein but also targets the receptor to clathrin-coated pits for internalization. The internalized GPCR is either trafficked to lysosomes for degradation or alternatively is dephosphorylated by phosphatase enzymes in an acidic endosomal compartment before being recycled to the membrane for further rounds of agonist stimulation. These GPCR regulatory pathways were identified largely as a result of the seminal studies by the Lefkowitz laboratory in the U.S.A. (reviewed in Lefkowitz, 2004), and have reached the status of dogma in the field of GPCR biology. However, some new studies, including one in this issue of the British Journal of Pharmacology (Iyer et al., 2005), suggest that the role of internalization in GPCR dephosphorylation is less clear than previously thought. This work is particularly elegant because the authors have developed antibodies to detect β2-adrenoceptor phosphorylation by PKA at Ser262 in the receptor's third intracellular loop and by GRKs at Ser355,356 in the COOH-terminus (Tran et al., 2004). They show that when internalization of the phosphorylated β2-adrenoceptor is blocked, either by expression of a dominant-negative mutant form of dynamin or with hypertonic sucrose, the receptor still undergoes dephosphorylation of both PKA and GRK sites. Indeed, the rate of dephosphorylation of the receptor is the same whether or not the receptor is able to internalize. Furthermore, they show that the β2-adrenoceptor still undergoes dephosphorylation following selective phosphorylation of PKA site Ser262 subsequent to the addition of a very low concentration of isoprenaline (300 pM), which does not induce receptor internalization. These results show conclusively that the β2-adrenoceptor does not have to internalize in order to become dephosphorylated (Figure 1). Does this apply to other GPCRs? Apparently it does; a recent study (Gardiner et al., 2001) shows that the agonist-phosphorylated Gs-coupled D1 dopamine receptor undergoes the same rate of dephosphorylation whether or not it is able to undergo internalization. Furthermore, a very recent study shows that the agonist-activated Gq-coupled thyrotropin-releasing hormone receptor is rapidly dephosphorylated at the cell membrane in mouse embryo fibroblast cells from arrestin-knockout animals where these receptors are unable to undergo significant internalization (Jones & Hinkle, 2005). Figure 1 Dephosphorylation of β2-adrenoceptors. (a) In response to prolonged agonist treatment (isoprenaline; ISOP), the receptor at the cell membrane is phosphorylated by PKA and GRK, and arrestins bind to the receptor. (b) In the classical view, receptor ... How to reconcile these recent findings with the earlier results (Yu et al., 1993; Pippig et al., 1995; Krueger et al., 1997)? Iyer et al. (2005) suggest that the interpretation of earlier studies on the β2-adrenoceptor is made difficult because the 32P labelling technique reflects a combination of PKA and GRK phosphorylation of the receptor. This may be particularly relevant since the kinetics and agonist concentration dependence of PKA and GRK phosphorylation of the β2-adrenoceptor are different (Tran et al., 2004). However, although most of the data provided by Iyer et al. (2005) indicates that the β2-adrenoceptor undergoes dephosphorylation at the cell surface, they do report some experimental conditions (longer agonist treatment time in the presence of hypertonic sucrose) where dephosphorylation is inhibited. Thus, the experimental conditions used (e.g. agonist concentration, length of agonist treatment, presence of antagonist in the wash buffer, use of 32P labelling versus phosphosite-specifc antibodies) may explain some of the differences. In all likelihood, the β2-adrenoceptor is able to undergo dephosphorylation both at the cell membrane and intracellularly in endosomes. Important questions remain. For example, the work of Iyer et al. (2005) does not explain why inhibitors of internalization can block resensitization, as shown in the earlier studies (Yu et al., 1993; Pippig et al., 1995). Furthermore, do GPCRs that undergo dephosphorylation at the cell surface resensitize immediately, and does this provide for more rapid resensitization following agonist-induced desensitization than for an internalized receptor? Is the role of GPCR internalization more concerned with the activation of alternative signalling pathways, or with downregulation, than with dephosphorylation? Finally, what determines the activity and localization of phosphatase enzymes at the plasma membrane? One possibility here may be the family of A-kinase-anchoring proteins, which associate with at least some GPCRs and which, by acting as a scaffold protein, can localize both kinase and phosphatase enzymes to the vicinity of the receptor in the cell membrane (Fraser et al., 2000; Lin et al., 2000). In summary, Iyer et al. (2005) show that the β2-adrenoceptor can undergo dephosphorylation without having to undergo internalization. The functional consequences of this interesting finding for GPCR signalling remain to be explored in detail.