Oncogenic Receptor Tyrosine Kinases Research Articles

Immunohistochemical expression of receptor-tyrosine-kinase in patients with gastric cancer: A multicenter study in Japan.

41 Background: Treatment of HER2 positive gastric cancer (GC) with trastuzumab, receptor-tyrosine-kinase (RTK) inhibitor, has led to significant gains in overall survival. Several other targeted therapies are currently undergoing preclinical and clinical testing in some malignancies and GC, directed against diverse oncogenic RTK, therefor it is important to define the expression pattern of target RTK in GC. A multicenter retrospective study was conducted to evaluate the correlation between the expression of some RTKs, HER2, EGFR and c-KIT, and clinicopathological features in GC. Methods: A total of 153 cases with GC which was surgically resected between 2000 and 2006 were registered from nine hospitals. The neoadjuvant treatment cases were excluded. Tumors were centrally examined for HER2, EGFR and c-KIT status with immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) for HER2. The relation between the expressions of HER2, EGFR and c-KIT and clinicopathological factors was examined by Fisher’s exact test. The hazard ratio (HR) for death was estimated, and overall survivals (OS) were compared between positive and negative cases of HER2, EGFR, and c-KIT using log-rank test. Results: The proportion of positive cases with IHC of HER2, EGFR and c-KIT was 7.8%, 14.4% and 11.1%, respectively. There was no significant correlation between the expression of HER2, EGFR and c-KIT and differentiation, location and depth. HER2-positive cases tended to be more frequent in differentiated type tumors. EGFR-positve cases were more frequent in lymph node metastasis (P=0.013). c-KIT-positive cases tended to be more frequent in T1-T2 tumors. OS in HER2-positive cases were clearly worse than in HER2-negative cases (HR 2.23 (95%CI, 1.16-4.38); P=0.014). EGFR-positive cases had a tendency to be worse than in EGFR-negative cases (HR 1.60 (95%CI, 0.94-2.71); P=0.082). c-KIT-negative cases were worse than in c-KIT-positive cases (HR 0.42 (95%CI, 0.17-1.02); P=0.048). Conclusions: This study showed that HER2 expression is a factor of poor prognosis and that EGFR-positive status and c-KIT-negative status tended to be associated with worse outcomes in resected GC.

Tumor angiogenesis is caused by single melanoma cells in a reactive oxygen species and NF-κB dependent manner

Melanomas have a high angiogenic potential, but respond poorly to medical treatment and metastasize very early. To understand the early events in tumor angiogenesis, animal models with high tumor resolution and blood vessel resolution are required, which provide the opportunity to test the ability of small molecule inhibitors to modulate the angiogenic tumor program. We have established a transgenic melanoma angiogenesis model in the small laboratory fish species Japanese medaka. Here, pigment cells are transformed by an oncogenic receptor tyrosine kinase in fish expressing GFP throughout their vasculature. We show that angiogenesis occurs in a reactive oxygen species (ROS)- and NF-κB-dependent, but hypoxia-independent manner. Intriguingly, we observed that blood vessel sprouting is induced even by single transformed pigment cells. The oncogenic receptor as well as human melanoma cells harboring other oncogenes caused the production of pro-angiogenic factors, most prominently angiogenin, through NF-κB signaling. Inhibiting NF-κB prevented tumor angiogenesis and led to the regression of existing tumor blood vessels. In conclusion, our high-resolution medaka melanoma model discloses that ROS and NF-κB signaling from single tumor cells causes hypoxia-independent angiogenesis, thus, demonstrating that the intrinsic malignant tumor cell features are sufficient to initiate and maintain a pro-angiogenic signaling threshold.

Identification of Caveolin-1 as a Potential Causative Factor in the Generation of Trastuzumab Resistance in Breast Cancer Cells

The oncogenic tyrosine kinase receptor ErbB2 is a prognostic factor and target for breast cancer therapeutics. In contrast with the other ErbB receptors, ErbB2 is hardly internalized by ligand induced mechanisms, indicating a prevalent surface expression. Elevated levels of ErbB2 in tumor cells are associated with its defective endocytosis and down regulation. Here we show that caveolin-1 expression in breast cancer derived SKBR-3 cells (SKBR-3/Cav-1) facilitates ligand induced ErbB2 endocytosis using an artificial peptide ligand EC-eGFP. Similarly, stimulation with humanized anti ErbB2 antibody Trastuzumab (Herceptin) was found to be internalized and co-localized with caveolin-1 in SKBR-3/Cav-1 cells. Internalized EC-eGFP and Trastuzumab in SKBR-3/Cav-1 cells were then delivered via caveolae to the caveolin-1 containing early endosomes. Consequently, attenuated Fc receptor mediated ADCC functions were observed when exposed to Trastuzumab and EC-Fc (EC-1 peptide conjugated to Fc part of human IgG). On the other hand, this caveolae dependent endocytic synergy was not observed in parental SKBR-3 cells. Therefore, caveolin-1 expression in breast cancer cells could be a predictive factor to estimate how cancer cells are likely to respond to Trastuzumab treatment.

Evaluation of regorafenib in colorectal cancer and GIST

Regorafenib is an orally administered multikinase inhibitor. It is administered on a continuous dosing schedule and shows non-specific binding to several intracellular kinases, with potent inhibitory activity against vascular endothelial growth factor receptors 1–3 (VEGFR1 [also known as FLT1], VEGFR2 [KDR], and VEGFR3 [FLT4]), PDGFRB, FGFR1, RAF, TIE2, and the mutant oncogenic kinases KIT, RET, and BRAF. 1 Wilhelm SM Dumas J Adnane L et al. Regorafenib (BAY 73-4506): a new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases with potent preclinical antitumor activity. Int J Cancer. 2011; 129: 245-255 Crossref PubMed Scopus (921) Google Scholar Commonly reported (>10%) toxic effects include fatigue, hand-foot skin reaction, diarrhoea, anorexia, voice changes, hypertension, oral mucositis, rash, and weight loss. 2 Grothey A Van Cutsem E Sobrero A et al. for the CORRECT Study GroupRegorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet. 2012; (published online Nov 22.)http://dx.doi.org/10.1016/S0140-6736(12)61900-X Google Scholar , 3 Demetri GD Reichardt P Kang Y-K et al. on behalf of all GRID study investigatorsEfficacy and safety of regorafenib for advanced gastrointestinal stromal tumours after failure of imatinib and sunitinib (GRID): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet. 2012; (published online Nov 22.)http://dx.doi.org/10.1016/S0140-6736(12)61857-1 Google Scholar Clinical trials are underway assessing regorafenib in the treatment of colorectal cancer, gastrointestinal stromal tumours (GIST), lung cancer, renal cell carcinoma, and hepatocellular carcinoma. Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trialRegorafenib is the first small-molecule multikinase inhibitor with survival benefits in metastatic colorectal cancer which has progressed after all standard therapies. The present study provides evidence for a continuing role of targeted treatment after disease progression, with regorafenib offering a potential new line of therapy in this treatment-refractory population. Full-Text PDF Efficacy and safety of regorafenib for advanced gastrointestinal stromal tumours after failure of imatinib and sunitinib (GRID): an international, multicentre, randomised, placebo-controlled, phase 3 trialThe results of this study show that oral regorafenib can provide a significant improvement in progression-free survival compared with placebo in patients with metastatic GIST after progression on standard treatments. As far as we are aware, this is the first clinical trial to show benefit from a kinase inhibitor in this highly refractory population of patients. Full-Text PDF

FLT3-driven redox-modulation of Ezrin regulates leukaemic cell migration

The concept of reactive oxygen species (ROS) being produced via the activation of specific oncogenes provides a basis for generating genomic instability and pro-survival signalling in tumour cells. The purpose of this study was to identify downstream targets of NADPH oxidase (Nox)-derived ROS signalling in acute myeloid leukaemia cells, by performing a proteomic analysis utilizing two-dimensional phosphotyrosine immunoblotting. The majority of the targets identified were cytoskeletal-associated proteins including Ezrin, a known regulator of the cytoskeleton, which was examined further. The study demonstrated that inhibition of Nox enzymes, using diphenyleneiodonium chloride in the acute myeloid leukaemia cell line MOLM-13, resulted in a decrease in Ezrin tyrosine phosphorylation and also triggered a shift in Ezrin sub-cellular localization as detected by immunofluorescence. The change in Ezrin localization coincided with altered cell morphology, observed using scanning electron microscopy and a decreased ability to migrate through a polycarbonate transwell membrane. Similar effects were observed upon inhibition of the oncogenic receptor tyrosine kinase FLT3 using the staurosporine derivate PKC412, implicating a role for FLT3 as an upstream regulator of Ezrin. Our results indicate that FLT3 drives production of ROS by Nox, which stimulates changes in Ezrin tyrosine phosphorylation and localization via redox regulation of Src. Furthermore, inhibition of FLT3 signalling leads to alterations in MOLM-13 cell morphology and has a significant influence on cell motility.

Combined Drug Action of 2-Phenylimidazo[2,1-b]Benzothiazole Derivatives on Cancer Cells According to Their Oncogenic Molecular Signatures

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by “RTK swapping” by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation.

Isolation and Functional Characterization of Peptide Agonists of PTPRJ, a Tyrosine Phosphatase Receptor Endowed with Tumor Suppressor Activity

PTPRJ is a receptor-type protein tyrosine phosphatase whose expression is strongly reduced in the majority of investigated cancer cell lines and tumor specimens. PTPRJ negatively interferes with mitogenic signals originating from several oncogenic receptor tyrosine kinases, including HGFR, PDGFR, RET, and VEGFR-2. Here we report the isolation and characterization of peptides from a random peptide phage display library that bind and activate PTPRJ. These agonist peptides, which are able to both circularize and form dimers in acqueous solution, were assayed for their biochemical and biological activity on both human cancer cells and primary endothelial cells (HeLa and HUVEC, respectively). Our results demonstrate that binding of PTPRJ-interacting peptides to cell cultures dramatically reduces the extent of both MAPK phosphorylation and total phosphotyrosine levels; conversely, they induce a significant increase of the cell cycle inhibitor p27(Kip1). Moreover, PTPRJ agonist peptides both reduce proliferation and trigger apoptosis of treated cells. Our data indicate that peptide agonists of PTPRJ positively modulate the PTPRJ activity and may lead to novel targeted anticancer therapies.

518 Genetic and Epigenetic Alterations of PTPRD in Hepatocellular Carcinoma

Introduction: Genomic instability is a hallmark of ovarian cancer. MicroRNAs (miRNAs) are small RNA molecules that negatively regulate gene expression, and are often dysregulated in cancer. The aim of this study is to determine whether copy number variations (CNV) lead to altered miRNA expression and downstream effects on target gene expression in serous epithelial ovarian cancer (SEOC). Materials and Methods: CN, mRNA and miRNA expression profiling was performed on 4 SEOC (OV167, OV202, OVCAR-3, PE01) and 1 normal (HOSE) cell lines using Affymetrix Cytogenetic 2.7M Arrays, Affymetrix GeneChip Gene 1.0 ST arrays and Exiqon MiRCURY LNA arrays respectively. Chromosomal positioning of miRNAs was performed using Bowtie v 0.12.7 [1]. Results and Discussion: We determined that 60% (361/605) of miRNAs assessed were in regions of CNV in at least 2 cancer cell lines. Of these miRNAs, 32% (115/361) had changes in expression levels that correlated with CNV. One of these, miR-23a, is predicted to target AXL, an oncogenic receptor tyrosine kinase upstream of the PI3K/mTOR signalling pathway. AXL is over-expressed in ovarian cancer, and an inverse relationship was identified between the expression of AXL and miR-23a in 2 of the 4 cell lines. Axl has been previously reported to be over-expressed in 73% of ovarian tumour samples [2]. Transient transfection of miRNA mimics (Ambion) into ovarian cancer cell lines confirmed AXL down regulation after miR-23a addition. Conclusion: We demonstrate that approximately one third of miRNAs that are located in regions of CNV have altered expression which correlates with the chromosomal change. We conclude that genomic instability may contribute to miRNA dysregulation in ovarian cancer.

PI3 Kinase in Cancer: From Biology to Clinic

The discovery and clinical development of small-molecule inhibitors of the phosphatidylinositide 3-kinase (PI3 kinase) family of lipid kinases have marked a remarkable 20-year journey that follows the progressive developments in cancer biology over the last few decades: from hypothesis-driven, basic cancer research that began with viral oncogenesis and developed in the 1960s and 70s, through the discovery of individual mutated oncogenes and tumor suppressor genes in 1970 and 80s and the linkage of these cancer genes to signal transduction pathways in the 1990s, to all large-scale genome-wide sequencing, functional screening, and network biology efforts today. Thus, PI3 kinase research began with the discovery in 1985 of a new type of enzyme activity associated with viral oncogenesis. It benefited greatly from the discovery of wortmannin and LY294002 as PI3 kinase inhibitors and chemical tools in late 1980s to mid-90s. Alongside these tools, genetic validation of PI3 kinase as a target initially involved activation by upstream oncogenic receptor tyrosine kinases and RAS mutation, together with overexpression and amplification of the p110α catalytic isoform of PI3 kinase and frequent loss of the tumor suppressor and negative regulator of PI3 kinase activity, PTEN. As PI3 kinase drug development began, further stimulus came from the discovery through genome sequencing of mutations in PIK3CA, which encodes p110α and is the most frequently mutated kinase in the human genome. From these beginnings, there are now many PI3 kinase inhibitors in clinical trials and more in preclinical development. We review progress, current challenges, and future opportunities in this article.

Abstract 2545: FLT3-driven redox signalling in acute myeloid leukemia

Abstract The concept of reactive oxygen species (ROS) being produced via the activation of specific oncogenes provides a basis for generating genomic instability and pro-survival signalling in tumor cells. The purpose of this study was to identify downstream targets of NADPH oxidase (Nox)-derived ROS signalling in acute myeloid leukemia cells, by performing a proteomic analysis utilizing two-dimensional phosphotyrosine immnoblotting. The majority of the targets identified were cytoskeletal-associated proteins including Ezrin, a known regulator of the cytoskeleton, which was examined further. The study demonstrated that inhibition of Nox enzymes, using diphenyleneiodonium chloride in the acute myeloid leukemia cell line MOLM-13, resulted in a decrease in Ezrin tyrosine phosphorylation and also triggered a shift in Ezrin sub-cellular localization as detected by immunofluorescence. The change in Ezrin localization coincided with altered cell morphology, observed using scanning electron microscopy and a decreased ability to migrate through a polycarbonate transwell membrane. Similar effects were observed upon inhibition of the oncogenic receptor tyrosine kinase FLT3 using the staurosporine derivate PKC412, implicating a role for FLT3 as an upstream regulator of Ezrin. Our results indicate that FLT3 drives production of ROS by Nox, which stimulates changes in Ezrin tyrosine phosphorylation and localization via redox regulation of Src. Furthermore, the modulation of Ezrin by FLT3 leads to subsequent alterations of MOLM-13 cell morphology and a significant influence on cell motility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2545. doi:1538-7445.AM2012-2545

Maximizing Human Epidermal Growth Factor Receptor 2 Inhibition: A New Oncologic Paradigm in the Era of Targeted Therapy

ERBB2 (HER2) amplification in breast cancer is a classic example of oncogene addiction in solid tumor oncology; most human epidermal growth factor receptor 2 (HER2) –positive tumors exhibit dependency on signaling pathways downstream of the HER2 receptor tyrosine kinase, even after developing resistance to anti-HER2 drugs. Trastuzumab is a monoclonal antibody that inhibits HER2 and demonstrates efficacy in both treatment-naive patients and those who progress after a prior trastuzumab-based regimen. Serial administration of anti-HER2 therapies in clinical practice has dramatically altered the natural course of HER2positive breast cancer. Previously considered to have a worse prognosis, women with HER2-amplified breast cancer who receive trastuzumab now experience survival outcomes similar to those with HER2-negative disease. Furthermore, recent studies suggest that dual HER2 inhibition (the administration of two anti-HER2 therapeutics simultaneously) may induce more durable tumor responses than sequential HER2-specific monotherapy. In Journal of Clinical Oncology, Cortes et al demonstrate the value of persistent HER2 blockade by reporting a clinical benefit rate of 41.2% after combined treatment with the anti-HER2 monoclonal antibodies trastuzumab and pertuzumab in patients with evidence of disease progression while receiving either of the two agents alone. This noteworthy observation suggests that the traditional oncologic paradigm—in which most drugs are used only once and discontinued permanently at the first sign of progression—may require modification in the era of targeted therapy. HER2 is a member of the HER family of transmembrane receptors, which also includes epidermal growth factor receptor, HER3 (ERBB3), and HER4 (ERBB4). In normal tissue, HER proteins mediate cell growth signals transmitted between stromal and epithelial cells. Unlike other members of the family, HER2 does not interact directly with the growth factor ligand. Instead, the receptor functions by a ligand-independent dimerization mechanism that primes HER2 for associations with both itself and other HER kinase family members. In HER2-amplified breast cancers, the overabundance of membrane HER2 further potentiates this ligand-independent mechanism, resulting in “addiction” to HER2 signaling for tumor cell survival. In particular, overexpression of HER2 results in increased HER2 homoand heterodimer formation and augmented signaling through intracellular cascades such as the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase pathways (Fig 1). Contrary to HER2, HER3 has an impaired intracellular kinase and lacks significant biologic activity when expressed alone. However, when HER3 undergoes heterodimerization with HER2, a potent oncogenic signal may result. The HER2:HER3 heterodimer functions as the most transforming and mitogenic receptor complex of the HER family of proteins and is a robust activator of PI3K pathway signaling. The expression of HER3 can be a rate-limiting step for HER2induced cell growth, because HER2-amplified breast tumors undergo apoptotic cell death when HER3 expression is suppressed. Clinically, patients with high levels of HER2:HER3 heterodimers and low levels of HER2 homodimers exhibit a diminished response to trastuzumab therapy. The dependence on HER2 and pathogenesis of HER2: HER3 heterodimers in HER2-amplified breast cancer provide a scientific rationale for targeting both HER2 and HER3 in the clinical setting (Fig 1). Sequential HER2 blockade aims to achieve an extended and diversified therapeutic blockade of this oncogenic receptor tyrosine kinase. However, as seen in the modest activity of singleagent treatment, demonstrated by Cortes et al and others, repetitive HER2-specific monotherapy is often insufficient to engender long-lasting responses in patients who progress while receiving trastuzumab-based regimens. Trastuzumab-DM1 and pan-HER agents are notable exceptions with reasonable singleagent activity, but these drugs are probably more synonymous to combination treatment than to monotherapy. Trastuzumab-DM1 harnesses the effects of HER2 blockade through its trastuzumab component and simultaneously delivers the cytotoxic agent DM1, and pan-HER drugs are known to affect multiple kinases. Several mechanisms of resistance to single-agent HER2 inhibition have been proposed, including alterations affecting the HER2 receptor, increased interactions with both HER proteins and other membrane-bound receptors, and alternative activation of oncogenic signaling networks such as the PI3K pathway. Dual anti-HER2 regimens, in contrast to monotherapy, show excellent synergy and improved outcome in patients previously JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 30 NUMBER 14 MAY 1

New Roles Opined for OPCML

OPCML, frequently inactivated in ovarian tumors, mediates its antitumor effect via binding to the extracellular domains of several important oncogenic receptor tyrosine kinases (RTK). This, in turn, leads to the downregulation of RTKs in tumor cells and results in significant inhibition of tumor growth.

RET: A Multi-Faceted Gene in Human Cancer

Receptor tyrosine kinases (RTK) are transmembrane (TM) proteins featuring an intracellular domain containing the tyrosine kinase (TK) enzyme. RTKs are often involved in cancer formation [13]. Notable examples are epidermal growth factor receptor (EGFR/ HER1) and anaplastic lymphoma kinase (ALK) in non-small cell lung carcinoma (NSCLC) [4], KIT in gastrointestinal stromal tumors (GIST) [5], FLT3 in acute myeloid leukemia (AML) [6], and HER2/ ERBB2/neu in breast cancer [7]. In some cases, cancer cells up-regulate expression of the RTK (as an example HER2 in breast cancer), its cognate growth factor or both, in other cases, structural alterations such as chromosomal rearrangements leading to the RTK recombination to heterologous genes (as an example EML4-ALK in lung adenocarcinoma) or point mutations (as EGFR, KIT or FLT3 mutations in NSCLC, GIST, or AML, respectively), lead to unchecked kinase and oncogenic activity [1-3]. This notion has stimulated the search for agents, such as monoclonal antibodies against the RTK extracellular domain (like trastuzumab for HER2 or cetuximab for EGFR) or ATP-competitive small molecule protein kinase inhibitors (PKIs) (like gefitinib and erlotinib for EGFR or crizotinib for ALK), to combat cancers driven by oncogenic RTKs [1-3]. The RET RTK was originally identified as an oncogene activated by a rearrangement occurred in vitro during transfection of NIH3T3 cells with human lymphoma DNA [8]. RET protein belongs to a cell-surface complex able to bind glial-derived neurotrophic factor (GDNF) ligands (GDNF, neurturin, artemin, and persephin) in conjunction with co-receptors of the GDNF receptor α family, designated GFRα 1-4 [9]. Binding to the ligand-co-receptor complex leads to RET dimerization and kinase activation. RET expression is tightly regulated during development and in the adulthood is limited to specific tissues, including neural crest-derived cells. RET is essential for the development of the enteric neurvous system and kidney, and germline loss-of-function mutations in RET cause Hirschsprung disease (aganglionic megacolon) and congenital anomalies of the kidney or lower urinary tract [10,11]. RET gene maps to chromosome 10q11.2. Fig. 1 shows that it is splitted in 21 coding exons. Exons 1-10 code for the extracellular region; exon 11 codes for the COOH-terminal part of the extracellular region, the TM domain, and the intracellular juxtamembrane domain. Finally, exons 12-21 code for the intracellular domain. An alternative splicing at exon 19 determine the synthesis of three RET protein isoforms with different C-terminal tails. In RET9 (1072 aa), exon 19 is unspliced; in RET51 (1114 aa), exon 19 is spliced to exon 20; in RET43 (1106 aa), exon 19 is spliced to to exon 21 [12-15]. RET9 and RET51 are the most abundant and well characterized isoforms (Fig. 1). RET protein features an extracellular portion (RET-EC) that

Phosphoproteomic studies of receptor tyrosine kinases: future perspectives

In the last decade, large-scale mass spectrometry-based phosphoproteomic studies of receptor tyrosine kinases (RTKs) have generated a compendium of signalling networks that are activated downstream of these receptors. In this article, a brief summary of previous phosphoproteomic studies on epidermal growth factor receptor (EGFR) signalling will be presented together with a perspective on the importance for the field to keep pace with new advances in RTK biology. Using examples drawn primarily from studies on the EGFR, c-Met and Flt3 receptors, areas in RTK biology which will greatly benefit from the power of phosphoproteomics will be discussed, including (a) validating oncogenic RTK mutants identified in cancer genome sequencing efforts, (b) spatial RTK signalling networks and (c) understanding crosstalk and co-activation between members of the RTK superfamily.

Tyrosine Phosphorylation of Lactate Dehydrogenase A Is Important for NADH/NAD+ Redox Homeostasis in Cancer Cells

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD(+). LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD(+). However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD(+) redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.

CBL Deletion Mutant Found in AML Patients Cause Transformation of Receptor Tyrosine Kinase Class III Expressing Cells by Activation of the AKT Pathway

Abstract▪2447▪This icon denotes a clinically relevant abstractWe examined the oncogenic potential of CBL deletion mutant found in AML patients in cytokine receptor and receptor tyrosine kinase (RTKs) expressing cells. In addition, we analyzed the interaction sites of FLT3/CBL and the critical pathways activated by CBL deletion mutants.RTK, CBL and AKT constructs were expressed in Ba/F3 cells via a retroviral expression vector. Stable protein expression after transduction and fluorescence-activated cell sorting (FACS) was confirmed by western blotting and cellsurface-marker expression of receptors by flow cytometry. Cell Proliferation and apoptosis assays were done in presence and absence of IL-3 or receptor-ligands.Coexpression of RTK III-WT (PDGFRA, PDGFRB, FLT3, KIT) and CBL deletion mutants cause IL-3 independent and ligand dependent growth of Ba/F3 cells. RTK III-WT/CBLΔexon8 cells show a more than 10 fold hyperproliferation in response to ligand stimulation. In contrast Non-class III receptor tyrosine kinases (EGFR, EPOR, MPL, IGF1R) and CSF1R show just a very weak hyperproliferation if coexpressed with the CBL deletion mutant. Selective protein tyrosine kinase inhibitors abrogate this proliferation. In cells coexpressing RTK-III receptor and CBLΔexon8 the receptor internalization is delayed and cells were protected from apoptosis after cytokine withdrawal. Ba/F3 cells after ligand stimulation and AML cell lines coexpressing CBL deletion mutants and FLT3 show an enhanced AKT phosphorylation. The PI3K inhibitor LY294002 and the AKT inhibitor MK2206 abolish the CBL mutant mediated hyperproliferation. Furthermore, a combined pharmacological inhibition of PI3K/AKT pathway and RTK shows an additive effect. The transforming potential of the CBL mutant is completely abolished by a mutated PTB domain of CBL (G306E) and decreased by mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutive active AKT mutant (E17K) recapitulates the CBL deletion mutant induced phenotype in Ba/F3 cells.CBL is a selective negative regulator of class III RTK receptors and the PI3K/AKT pathway is critical for the transforming potential of the CBL oncogene. An alternative mechanism for the constitutive activation of RTKs in tumors occurs through inactivation of a negative regulator. CBL mutants mirror the phenotype of oncogenic RTK and cause an enhanced AKT phosphorylation. Targeted inhibition of FLT3 and AKT might be of therapeutic value in AML patients carrying CBL deletion mutants. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract B2: Regorafenib, an oral multi-kinase inhibitor of angiogenic, stromal and oncogenic receptor tyrosine kinases, potently inhibits tumor growth and angiogenesis in patient-derived xenograft models of gastric cancer.

Abstract Background: Gastric cancer (GC) is a prevalent and deadly disease. Because the VEGFR and PDGFR signaling cascade plays a critical role in the development and progression of gastric cancer and VEGF expression is correlated with angiogenesis, tumor progression and poor prognosis in patients with GC, blocking these signaling pathways could thus have therapeutic efficacy. This study aims to investigate the potential of a combined therapeutic approach to target these pathways in preclinical models of GC with regorafenib an oral multi-kinase inhibitor targeting VEGFR1–3, PDGFR, FGFR, TIE-2, RET and KIT. Methods: Eight patient-derived GC xenograft models with different histotypes were treated orally with regorafenib or vehicle and tumor growth was monitored using caliper measurements. Tissue sections of vehicle- or regorafenib-treated xenografts were analyzed by immunohistochemistry for apoptosis, microvessel area, and cell proliferation using antibodies against cleaved PARP, CD31 and phospho-Histone3Ser10, respectively. Tumor lysates were analyzed by Western Blotting with respective antibodies for effects on marker proteins for proliferation, apoptosis and angiogenesis. Results: Regorafenib demonstrated significant tumor growth inhibition of 81%, 85% and 88% in the GC09-0109 model after oral administration of 5, 10 and 15 mg/kg/d, respectively. In addition potent tumor growth inhibition ranging from 72–96% was observed in seven other GC models at a dose of 10 mg/kg/d. Regorafenib significantly inhibited angiogenesis in all models by 4–12 fold compared to the vehicle-treated group, independent of their degree of vascularization, as indicated by reduced CD31 staining. Furthermore inhibition of cell proliferation by 2–4 fold was observed by reduced staining for p-histone 3Ser10. Increased staining for caspase-cleaved p85 PARP by 2 to 20 fold was detected, indicating that regorafenib induced apoptosis. Finally, regorafenib also induced tumor necrosis as detected by histological staining. Inhibition of angiogenesis, reduced cell proliferation and induction of apoptosis and necrosis was associated with decreased levels of p-ERK1/2, p-cdk2Thr14/Tyr15, p-VEGFR2Tyr951, p-p70S6KThr421/Ser424, p-S6RSer235/236 and p-eIF4ESer209 and elevation of cleaved caspase-3 and cleaved PARP. Conclusion: We show that regorafenib is an efficacious anti-tumor targeted agent in patient-derived gastric cancer preclinical models. Our findings support further investigation of regorafenib in the treatment of GC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B2.

Lrig1 Is an Estrogen-Regulated Growth Suppressor and Correlates with Longer Relapse-Free Survival in ERα-Positive Breast Cancer

Lrig1 is the founding member of the Lrig family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth-inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor α (ERα)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ERα-positive disease than in ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by showing that Lrig1 is a target of ERα. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen-regulated growth suppressor. In addition, we find that Lrig1 expression correlates with prolonged relapse-free survival in ERα-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we show that ErbB2 activation antagonizes ERα-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth-inhibitory role for Lrig1 in breast cancer.

Rapid Phospho-Turnover by Receptor Tyrosine Kinases Impacts Downstream Signaling and Drug Binding

Epidermal growth factor receptors (ErbB1-4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100-1000 times in the course of a typical immediate-early response to ligand. Rapid phospho-turnover is also observed for EGF-activated ErbB2 and ErbB3, unrelated RTKs, and multiple intracellular adaptor proteins and signaling kinases. Thus, the complexes formed on the cytoplasmic tail of active receptors and the downstream signaling kinases they control are highly dynamic and antagonized by potent phosphatases. We develop a kinetic scheme for binding of anti-ErbB1 drugs to receptors and show that rapid phospho-turnover significantly impacts their mechanisms of action.

Pro-neural miR-128 is a glioma tumor suppressor that targets mitogenic kinases

MicroRNAs (miRNAs) carry out post-transcriptional control of a multitude of cellular processes. Aberrant expression of miRNA can lead to diseases, including cancer. Gliomas are aggressive brain tumors that are thought to arise from transformed glioma-initiating neural stem cells (giNSCs). With the use of giNSCs and human glioblastoma cells, we investigated the function of miRNAs in gliomas. We identified pro-neuronal miR-128 as a candidate glioma tumor suppressor miRNA. Decreased expression of miR-128 correlates with aggressive human glioma subtypes. With a combination of molecular, cellular and in vivo approaches, we characterize miR-128's tumor suppressive role. miR-128 represses giNSC growth by enhancing neuronal differentiation. miR-128 represses growth and mediates differentiation by targeting oncogenic receptor tyrosine kinases (RTKs) epithelial growth factor receptor and platelet-derived growth factor receptor-α. Using an autochthonous glioma mouse model, we demonstrated that miR-128 repressed gliomagenesis. We identified miR-128 as a glioma tumor suppressor that targets RTK signaling to repress giNSC self-renewal and enhance differentiation.