Characterization of immune development of fetal and early-life of minipigs.

Fetal and child's immune systems differ from those of adults. Developing immune systems exhibit increased or decreased sensitivity to drugs, infection, or toxicants compared to adult immune systems. Understanding fetal and neonatal immune systems will help predict toxicity or the pathogenesis or prognosis of diseases. In this study, we evaluated whether the innate and adaptive immune system of fetal and young minipigs could respond to external stimuli compared to a medium-treated group and analyzed several immunological parameters for developmental immunotoxicity according to developmental stages. We performed a hematological analysis of fetal cord bloods and the bloods of neonatal and 4-week-old piglets. Splenocytes were isolated at each developmental stage and treated with lipopolysaccharide (LPS), R848, and concanavalin A (ConA). Various cytokines were measured in the cell supernatants. Total antibody production was also evaluated in serum. The percentage of lymphocytes was dominant in gestational weeks (GW) 10 and 12 and started to decrease from postnatal day (PND) 0. From PND0, the percentage of neutrophils increased. Interleukin (IL)-1ß, IL-6, and interferon (IFN)-α were induced from GW10 in response to LPS and R848 stimulation. Th1 cytokine induction was detected from PND0 upon ConA stimulation, whereas Th2 cytokine release was observed from GW10. IgM and IgG production was sustained at low levels at fetal stages and was significantly increased after birth. This study reconfirmed that the fetal immune system could respond to external stimuli and that hematological analysis, cytokine evaluation, and antibody subclass measurement can be useful parameters for developmental immunotoxicity using minipigs.

Identification and validation of stable reference genes for quantitative real time PCR in different minipig tissues at developmental stages.

BACKGROUND: Quantitative real time PCR (qPCR) is a powerful tool to evaluate mRNA expression level. However, reliable qPCR results require normalization with validated reference gene(s). In this study, we investigated stable reference genes in seven tissues according to four developmental stages in minipigs. Six candidate reference genes and one target gene (ACE2) were selected and qPCR was performed. BestKeeper, geNorm, NormFinder, and delta Ct method through the RefFinder web-based tool were used to evaluate the stability of candidate reference genes. To verify the selected stable genes, relative expression of ACE2 was calculated and compared with each other. RESULTS: As a result, HPRT1 and 18S genes had lower SD value, while HMBS and GAPDH genes had higher SD value in all samples. Using statistical algorithms, HPRT1 was the most stable gene, followed by 18S, ß-actin, B2M, GAPDH, and HMBS. In intestine, all candidate reference genes exhibited similar patterns of ACE2 gene expression over time, whereas in liver, lung, and kidney, gene expression pattern normalized with stable reference genes differed from those normalized with less stable genes. When normalized with the most stable genes, the expression levels of ACE2 in minipigs highly increased in intestine and kidney at PND28, which is consistent with the ACE2 expression pattern in humans. CONCLUSIONS: We suggest that HPRT1 and 18S are good choices for analyzing all these samples across the seven tissues and four developmental stages. However, this study can be a reference literature for gene expression experiments using minipig because reference gene should be validated and chosen according to experimental conditions.

The Impact of EGFR Tyrosine Kinase Inhibitor on the Natural Course of Concurrent Subsolid Nodules in Patients with Non-Small Cell Lung Cancer.

PURPOSE: The role of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) in the management of persistent subsolid nodules (SSNs) is unclear. This study aimed to investigate the impact of EGFR-TKIs on concurrent SSNs in patients with stage IV non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Patients who received an EGFR-TKI for at least 1 month for stage IV NSCLC and had concurrent SSN(s) that had existed for at least 3 months on chest computed tomography were included in this retrospective study. Size change of each nodule before and after EGFR-TKI therapies were evaluated using a cutoff value of 2 mm; increase (≥ 2 mm), decrease (≤ -2 mm), and no change (-2 mm < size change < +2 mm). RESULTS: A total of 77 SSNs, 51 pure ground-glass (66.2%) and 26 part-solid nodules (33.8%), were identified in 59 patients who received gefitinib (n=45) and erlotinib (n=14). Among 58 EGFR mutation analysis performed for primary lung cancer, 45 (77.6%) were EGFR mutant. The proportions of decrease group were 19.5% (15/77) on per-nodule basis and 25.4% (15/59) on per-patient basis. Four SSNs (5.2%) disappeared completely. On per-patient based multivariable analysis, EGFR exon 19 deletion positivity for primary lung cancer was associated with a decrease after initial EGFR-TKI therapy (adjusted odds ratio, 4.29; 95% confidence interval, 1.21 to 15.29; p=0.025). CONCLUSION: Approximately 20% of the concurrent SSNs decreased after the initial EGFR-TKI therapy. EGFR exon 19 deletion positivity for primary lung cancer was significantly associated with the size change of concurrent SSNs.

Potentiating Therapeutic Effects of Epidermal Growth Factor Receptor Inhibition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subset of breast cancer with aggressive characteristics and few therapeutic options. The lack of an appropriate therapeutic target is a challenging issue in treating TNBC. Although a high level expression of epidermal growth factor receptor (EGFR) has been associated with a poor prognosis among patients with TNBC, targeted anti-EGFR therapies have demonstrated limited efficacy for TNBC treatment in both clinical and preclinical settings. However, with the advantage of a number of clinically approved EGFR inhibitors (EGFRis), combination strategies have been explored as a promising approach to overcome the intrinsic resistance of TNBC to EGFRis. In this review, we analyzed the literature on the combination of EGFRis with other molecularly targeted therapeutics or conventional chemotherapeutics to understand the current knowledge and to provide potential therapeutic options for TNBC treatment.

Dual Inhibition of AKT and MEK Pathways Potentiates the Anti-Cancer Effect of Gefitinib in Triple-Negative Breast Cancer Cells.

There is an unmet medical need for the development of new targeted therapeutic strategies for triple-negative breast cancer (TNBC). With drug combination screenings, we found that the triple combination of the protein kinase inhibitors (PKIs) of the epidermal growth factor receptor (EGFR), v-akt murine thymoma viral oncogene homolog (AKT), and MAPK/ERK kinase (MEK) is effective in inducing apoptosis in TNBC cells. A set of PKIs were first screened in combination with gefitinib in the TNBC cell line, MDA-MB-231. The AKT inhibitor, AT7867, was identified and further analyzed in two mesenchymal stem-like (MSL) subtype TNBC cells, MDA-MB-231 and HS578T. A combination of gefitinib and AT7867 reduced the proliferation and long-term survival of MSL TNBC cells. However, gefitinib and AT7867 induced the activation of the rat sarcoma (RAS)/ v-raf-1 murine leukemia viral oncogene homolog (RAF)/MEK/ extracellular signal-regulated kinase (ERK) pathway. To inhibit this pathway, MEK/ERK inhibitors were further screened in MDA-MB-231 cells in the presence of gefitinib and AT7867. As a result, we identified that the MEK inhibitor, PD-0325901, further enhanced the anti-proliferative and anti-clonogenic effects of gefitinib and AT7867 by inducing apoptosis. Our results suggest that the dual inhibition of the AKT and MEK pathways is a novel potential therapeutic strategy for targeting EGFR in TNBC cells.

Activation of CXCL12-CXCR4 signalling induces conversion of immortalised embryonic kidney cells into cancer stem-like cells.

Cancer stem cells (CSCs) have been implicated in the growth and progression of several types of human cancer. The technology to derive and establish CSCs in vitro could be a critical tool for understanding cancer and developing new therapeutic targets. In this study, we derived expandable CD15+ induced CSCs (iCSCs) from immortalised 293FT human epithelial cells by co-culture with human bone marrow-derived mesenchymal stem cells (BM-MSCs) as feeder cells in vitro. The iCSCs converted through an epithelial-mesenchymal transition program acquired mesenchymal traits, the expression of stem cell markers, and epigenetic changes. Moreover, the iCSCs not only efficiently formed tumorspheres in vitro but also initiated tumours in immunocompromised mice injected with only 10 of the iCSCs. Furthermore, we showed that the expression of the chemokine CXCL12 and its receptor CXCR4 by the iCSCs resulted in the activation of the Fut4 gene through CXCR4/ERK/ELK-1-signalling pathways and the maintenance of the iCSCs in the undifferentiated state through CXCR4/AKT/STAT3-signalling. These findings suggest that immortalised 293FT cells may acquire potential oncogenicity through molecular and cellular alteration processes in microenvironments using BM-MSCs, and could represent a valuable in vitro model as a cancer stem cell surrogate for studying the pathophysiological properties of CSCs.

Mechanistic insights into differential requirement of receptor dimerization for oncogenic activation of mutant EGFR and its clinical perspective.

The epidermal growth factor receptor (EGFR), a member of the ErbB family (EGFR, ErbB2, ErbB3 and ErbB4), plays a crucial role in regulating various cellular responses such as proliferation, differentiation, and survival. As a result, aberrant activation of EGFR, mostly mediated through different classes of genomic alterations occurring within EGFR, is closely associated with the pathogenesis of numerous human cancers including lung adenocarcinoma, glioblastoma, and colorectal cancer. Thus, specific suppression of oncogenic activity of mutant EGFR with its targeted drugs has been routinely used in the clinic as a very effective anti-cancer strategy in treating a subset of tumors driven by such oncogenic EGFR mutants. However, the clinical efficacy of EGFR-targeted therapy does not last long due to several resistance mechanisms that emerge in the patients following the drug treatment. Thus, there is an urgent need for the development of novel therapeutic tactics specifically targeting mutant EGFR with the focus on the unique biological features of various mutant EGFR. Regarding this point, our review specifically emphasizes the recent findings about distinct requirements of receptor dimerization and autophosphorylation, which are critical steps for enzymatic activation of EGFR and signaling cascades, respectively, among wildtype and mutant EGFR and further discuss their clinical significance. In addition, the molecular mechanisms regulating EGFR dimerization and enzymatic activity by a key negative feedback inhibitor Mig6 as well as the clinical use for developing potential novel drugs targeting it are described in this review. [BMB Reports 2020; 53(3): 133-141].

Colorectal adenocarcinoma-derived EGFR mutants are oncogenic and sensitive to EGFR-targeted monoclonal antibodies, cetuximab and panitumumab.

Somatic mutations of epidermal growth factor receptor (EGFR) occur in ~3% of colorectal cancer (CRC) patients. Here, through systematic functional screening of 21 recurrent EGFR mutations selected from public data sets, we show that 11 colon cancer-derived EGFR mutants (G63R, E114K, R165Q, R222C, S492R, P596L, K708R, E709K, G719S, G724S and L858R) are oncogenic and able to transform cells in a ligand-independent manner. We demonstrate that cellular transformation by these mutants requires receptor dimerization. Importantly, the EGF-induced and constitutive oncogenic potential of these EGFR mutants are inhibited by cetuximab or panitumumab in vivo and in vitro. Taken together, we propose that a subset of EGFR mutations can serve as genomic predictors for response to anti-EGFR antibodies and that metastatic CRC patients with such mutations may benefit from these drugs as part of the first-line therapy.

Whole Transcriptome Analysis Identifies TNS4 as a Key Effector of Cetuximab and a Regulator of the Oncogenic Activity of KRAS Mutant Colorectal Cancer Cell Lines.

The targeting of activated epidermal growth factor receptor (EGFR) with therapeutic anti-EGFR monoclonal antibodies (mAbs) such as cetuximab and panitumumab has been used as an effective strategy in the treatment of colorectal cancer (CRC). However, its clinical efficacy occurs only in a limited number of patients. Here, we performed whole-transcriptome analysis in xenograft mouse tumors induced by KRASG12D mutation-bearing LS174T CRC cells following treatment with either cetuximab or PBS. Through integrated analyses of differential gene expression with TCGA and CCLE public database, we identified TNS4, overexpressed in CRC patients and KRAS mutation-harboring CRC cell lines, significantly downregulated by cetuximab. While ablation of TNS4 expression via shRNA results in significant growth inhibition of LS174T, DLD1, WiDr, and DiFi CRC cell lines, conversely, its ectopic expression increases the oncogenic growth of these cells. Furthermore, TNS4 expression is transcriptionally regulated by MAP kinase signaling pathway. Consistent with this finding, selumetinib, a MEK1/2 inhibitor, suppressed oncogenic activity of CRC cells, and this effect is more profound in combination with cetuximab. Altogether, we propose that TNS4 plays a crucial role in CRC tumorigenesis, and that suppression of TNS4 would be an effective therapeutic strategy in treating a subset of cetuximab-refractory CRC patients including KRAS activating mutations.

Early emergence of de novo EGFR T790M gatekeeper mutations during erlotinib treatment in PC9 non-small cell lung cancer cells.

The emergence of the T790M gatekeeper mutation in the Epidermal Growth Factor Receptor (EGFR) gene is an important mechanism that can lead to the acquired resistance to EGFR-targeted tyrosine kinase inhibitors such as erlotinib or gefitinib. These drugs have been used in treating a subset of non-small cell lung cancer (NSCLC) patients harboring EGFR activating mutations. Here we investigated the paths leading to the acquisition of the T790M mutation by establishing an erlotinib resistant PC9 cell model harboring ectopically introduced EGFR cDNA. We detected the emergence of T790M mutation within the EGFR cDNA in a subset of erlotinib resistant PC9 cell models through Sanger sequencing and droplet digital PCR-based methods, demonstrating that T790M mutation can emerge via de novo events following treatment with erlotinib. In addition, we show that the de novo T790M bearing erlotinib resistant PC9 cells are sensitive to the 3rd generation EGFR-targeted drug, WZ4002. Furthermore, GFP-based competition cell proliferation assays reveal that PC9 cells ectopically expressing EGFR mutant become more rapidly resistant to erlotinib than parental PC9 cells through the acquisition of the T790M mutation. Taken together, we believe that our findings expand upon the previous notion of evolutionary paths of T790M development, providing an important clue to designing a therapeutic strategy to overcome drug resistance.

Autophosphorylation of the carboxyl-terminal domain is not required for oncogenic transformation by lung-cancer derived EGFR mutants.

Aberrant activation of cancer-derived mutants of the epidermal growth factor receptor (EGFR) is closely associated with cancer pathogenesis and is thought to be mediated through multiple tyrosine phosphorylations within the C-terminal domain. Here, we examined the consequences of the loss of these C-terminal phosphorylation sites on cellular transformation in the context of lung-cancer-derived L858R, exon 19 deletion and exon 20 insertion mutant EGFR. Oncogenic EGFR mutants with substitution of the 10 potential C-terminal tyrosine autophosphorylation sites for phenylalanine (CYF10) were still able to promote anchorage-independent growth in soft agar at levels comparable to the parental L858R or exon19 deletion or exon 20 insertion mutants with intact autophosphorylation sites. Furthermore, these CYF10 mutants retained the ability to transform Ba/F3 cells in the absence of IL-3. Bead-based phosphorylation and immunoprecipitation analyses demonstrated that key EGFR-associated proteins-including Grb2 and PLC-γ-are neither phosphorylated nor bound to CYF10 mutants in transformed cells. Taken together, we conclude that tyrosine phosphorylation is not required for oncogenic activity of lung-cancer-derived mutant EGFR, suggesting these mutants can lead to cellular transformation by an alternative mechanism independent of EGFR phosphorylation.

MerTK is a novel therapeutic target in gastric cancer.

INTRODUCTION: The role of MerTK has not been assessed in gastric cancer (GC). The aim of this study was to identify a subgroup of GC patients with MerTK tumor overexpression, and to evaluate MerTK as a potential therapeutic target in this disease. METHODS: Protein and mRNA expression of MerTK were evaluated, and other various in vitro analyses including shRNA transfection, cell cycle anslysis, MTS assay and colony forming assay were carried out with GC cell lines and GC patient-derived cells (PDCs). RESULTS: shRNA-mediated knockdown of MerTK resulted in inhibition of cell growth, as well as increased cellular apoptosis in MerTK positive GC cells. Out of 192 GC patients, 16 (8.3%) patients showed strong protein expression and they had a significantly shorter overall survival compared to those with no MerTK expression. In 54 cases of GC PDCs, 4 cases (7.4%) showed mRNA overexpression, which was comparable to the protein expression rate. When we administered UNC1062, a novel MerTK-selective small molecular tyrosine kinase inhibitor, proliferation of MerTK overexpressing GC cells and PDCs were considerably inhibited. CONCLUSION: MerTK may be involved in GC carcinogenesis, and it could be a potential novel therapeutic target in GC patients.

Exome and transcriptome sequencing identifies loss of PDLIM2 in metastatic colorectal cancers.

BACKGROUND: Understanding the genomic determinants associated with metastasis in colorectal cancers (CRCs) provides crucial clues for improving patient care. PATIENTS AND METHODS: In this study, we performed whole-exome sequencing as well as RNA sequencing analyses on five pairs of primary and liver metastasized samples from CRC patients together with blood/normal control samples for each pair. RESULTS: We identified genomic deletions in the region of 8p21-23 (q value <0.01) from analysis of recurrent regions with copy number variations in both primary and matched metastatic lesions. Consistent with this result, we found significantly decreased expression levels of all 12 genes (ADAMDEC1, C8orf80, CLDN23, EPHX2, GFRA2, NEFL, NEFM, PDLIM2, PTK2B, SCARA5, SLC18A1 and STMN4) located within this region (adjusted P<0.01). Notably, the mRNA levels of PDLIM2, a key regulator of well-known cancer-associated genes including the proto-oncogene c-MYC, an early response gene IER3, and regulators of apoptosis such as BCL2, FAS, and FASLG, were highly downregulated in tumors compared to normal tissues. CONCLUSION: Taken together, our findings uncovered various genomic alterations potentially leading to metastasis in CRC and provide important insights into the development of potential therapeutic targets for preventing metastatic progression of CRC.

Analogues of Dehydroacetic Acid as Selective and Potent Agonists of an Ectopic Odorant Receptor through a Combination of Hydrophilic and Hydrophobic Interactions.

Identification of potent agonists of odorant receptors (ORs), a major class of G protein-coupled receptors, remains challenging due to complex receptor-ligand interactions. ORs are present in both olfactory and non-chemosensory tissues, indicating roles beyond odor detection that may include modulating physiological functions in non-olfactory tissues. Selective and potent agonists specific for particular ORs can be used to investigate physiological functions of ORs in non-chemosensory tissues. In this study, we designed and synthesized novel synthetic dehydroacetic acid analogues as agonists of odorant receptor 895 (Olfr895) expressed in bladder. Among the synthesized analogues, (E)-3-((E)-1-hydroxy-3-(piperidin-1-yl)allylidene)-6-methyl-2H-pyran-2,4(3H)-dione (10) exhibited extremely high agonistic activity for Olfr895 in Dual-Glo luciferase reporter (EC50 =9 nm), Ca2+ imaging, and chemotactic migration assays. Molecular docking and site-directed mutagenesis studies suggested that a combination of hydrophilic and hydrophobic interactions is central to the selective and specific binding of 10 to Olfr895. The design of agonists armed with both hydrophilic and hydrophobic portions could therefore lead to highly potent and selective ligands for ectopic ORs.

Integrated genomic approaches identify upregulation of SCRN1 as a novel mechanism associated with acquired resistance to erlotinib in PC9 cells harboring oncogenic EGFR mutation.

Therapies targeting the tyrosine kinase activity of Epidermal Growth Factor Receptor (EGFR) have been proven to be effective in treating a subset of non-small cell lung cancer (NSCLC) patients harboring activating EGFR mutations. Inevitably these patients develop resistance to the EGFR-targeted tyrosine kinase inhibitors (TKIs). Here, we performed integrated genomic analyses using an in vitro system to uncover alternative genomic mechanisms responsible for acquired resistance to EGFR-TKIs. Specifically, we identified 80 genes whose expression is significantly increased in the erlotinib-resistant clones. RNAi-based systematic synthetic lethal screening of these candidate genes revealed that suppression of one upregulated transcript, SCRN1, a secernin family member, restores sensitivity to erlotinib by enhancing inhibition of PI3K/AKT signaling pathway. Furthermore, immunohistochemical analysis revealed increased levels of SCRN1 in 5 of 11 lung tumor specimens from EGFR-TKIs resistant patients. Taken together, we propose that upregulation of SCRN1 is an additional mechanism associated with acquired resistance to EGFR-TKIs and that its suppression serves as a novel therapeutic strategy to overcome drug resistance in these patients.

Integrated genomic analyses identify frequent gene fusion events and VHL inactivation in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. We sequenced nine exomes and transcriptomes, and two genomes of GISTs for integrated analyses. We detected 306 somatic variants in nine GISTs and recurrent protein-altering mutations in 29 genes. Transcriptome sequencing revealed 328 gene fusions, and the most frequently involved fusion events were associated with IGF2 fused to several partner genes including CCND1, FUS, and LASP1. We additionally identified three recurrent read-through fusion transcripts: POLA2-CDC42EP2, C8orf42-FBXO25, and STX16-NPEPL1. Notably, we found intragenic deletions in one of three exons of the VHL gene and increased mRNAs of VEGF, PDGF-ß, and IGF-1/2 in 56% of GISTs, suggesting a mechanistic link between VHL inactivation and overexpression of hypoxia-inducible factor target genes in the absence of hypoxia. We also identified copy number gain and increased mRNA expression of AMACR, CRIM1, SKP2, and CACNA1E. Mapping of copy number and gene expression results to the KEGG pathways revealed activation of the JAK-STAT pathway in small intestinal GISTs and the MAPK pathway in wild-type GISTs. These observations will allow us to determine the genetic basis of GISTs and will facilitate further investigation to develop new therapeutic options.

High Expression of MicroRNA-196a Indicates Poor Prognosis in Resected Pancreatic Neuroendocrine Tumor.

There is limited data on miRNA expression in pancreatic neuroendocrine tumors (PanNETs). In this study, we aimed to identify miRNAs that could be potential prognostic biomarkers of PanNETs in patients who underwent curative surgery. For miRNA target screening, 2 primary PanNETs and corresponding liver metastases were screened for miRNA expression by the NanoString nCounter analysis. Candidate miRNAs were selected by ≥2-fold difference of expression between metastatic versus primary tumor. For miRNA target validation, quantitative real-time PCR was performed for candidate miRNAs on 37 PanNETs and matched nonneoplastic pancreata, and the miRNA levels were correlated with the clinicopathological features and patient survival data. Eight miRNAs (miRNA-27b, -122, -142-5p, -196a, -223, -590-5p, -630, and -944) were selected as candidate miRNAs. Only miR-196a level was significantly associated with stage, and mitotic count. When PanNETs were stratified into high (n = 10) and low (n = 27) miRNA-196a expression groups, miRNA-196a-high PanNETs were significantly associated with advanced pathologic T stage (50.0% vs 7.4%), N stage (50.0% vs 3.7%), higher mitotic counts (60.0% vs 3.7%), and higher Ki-67-labeling indices (60.0% vs 22.2%). In addition, high miRNA-196a expression was significantly associated with decreased overall survival (P = 0.046) and disease-free survival (P < 0.001) during a median follow-up of 37.9 months with the hazard ratio for recurrence of 16.267 (95% confidence interval = 1.732-153.789; P = 0.015). MiRNA-196a level may be a promising prognostic marker of recurrence in resected PanNETs, although further experimental investigation would be required.

Structure and mechanism of activity-based inhibition of the EGF receptor by Mig6.

Mig6 is a feedback inhibitor that directly binds, inhibits and drives internalization of ErbB-family receptors. Mig6 selectively targets activated receptors. Here we found that the epidermal growth factor receptor (EGFR) phosphorylates Mig6 on Y394 and that this phosphorylation is primed by prior phosphorylation of an adjacent residue, Y395, by Src. Crystal structures of human EGFR-Mig6 complexes reveal the structural basis for enhanced phosphorylation of primed Mig6 and show how Mig6 rearranges after phosphorylation by EGFR to effectively irreversibly inhibit the same receptor that catalyzed its phosphorylation. This dual phosphorylation site allows Mig6 to inactivate EGFR in a manner that requires activation of the target receptor and that can be modulated by Src. Loss of Mig6 is a driving event in human cancer; analysis of 1,057 gliomas reveals frequent focal deletions of ERRFI1, the gene that encodes Mig6, in EGFR-amplified glioblastomas.

Constitutive asymmetric dimerization drives oncogenic activation of epidermal growth factor receptor carboxyl-terminal deletion mutants.

Genomic alterations targeting the Epidermal Growth Factor Receptor (EGFR) gene have been strongly associated with cancer pathogenesis. The clinical effectiveness of EGFR targeted therapies, including small molecules directed against the kinase domain such as gefitinib, erlotinib and afatinib, have been proven successful in treating non-small cell lung cancer patients with tumors harboring EGFR kinase domain mutations. Recent large-scale genomic studies in glioblastoma and lung cancer have identified an additional class of oncogenic mutations caused by the intragenic deletion of carboxy-terminal coding regions. Here, we report that combinations of exonic deletions of exon 25 to 28 lead to the oncogenic activation of EGF receptor in the absence of ligand and consequent cellular transformation, indicating a significant role of C-terminal domain in modulating EGFR activation. Furthermore, we show that the oncogenic activity of the resulting C-terminal deletion mutants are efficiently inhibited by EGFR-targeted drugs including erlotinib, afatinib, dacomitinib as well as cetuximab, expanding the therapeutic rationale of cancer genome-based EGFR targeted approaches. Finally, in vivo and in vitro preclinical studies demonstrate that constitutive asymmetric dimerization in mutant EGFR is a key mechanism for oncogenic activation and tumorigenesis by C-terminal deletion mutants. Therefore, our data provide compelling evidence for oncogenic activation of C-terminal deletion mutants at the molecular level and we propose that C-terminal deletion status of EGFR can be considered as a potential genomic marker for EGFR-targeted therapy.

Pazopanib, a novel multitargeted kinase inhibitor, shows potent in vitro antitumor activity in gastric cancer cell lines with FGFR2 amplification.

Pazopanib is an orally bioavailable, ATP-competitive, multitargeted tyrosine kinase inhibitor mainly targeting VEGFR2 and PDGFR tyrosine kinases, but the biologic sequences of pazopanib activities beyond antiangiogenesis are poorly defined. We used a panel of 38 gastric cancer cell lines to test the efficacy of pazopanib. In a growth inhibition assay, genomic changes indicated that pazopanib had differential effects on cell growth. Treatment of the KATO-III, OCUM-2M, SNU-16, and HSC-39 gastric cancer cell lines harboring FGFR2 amplification with pazopanib resulted in marked decreases of cell survival with IC50 in ranges of 0.1 to 2.0 µmol/L, whereas the same treatment of those cell lines without FGFR2 amplification had no growth-inhibitory effects. In the ectopic FGFR2-expressing model, treatment with the indicated concentrations of pazopanib significantly inhibited cell growth and colony formation by FGFR2-expressing NIH 3T3 cells with wild-type (WT) FGFR2 and mutant FGFR2 (S252W). Pazopanib also selectively suppressed constitutive FGFR2 signaling and phosphorylation of downstream effectors. In cell-cycle analysis, FGFR2-amplified cells underwent cell-cycle arrest at the G1-S phase after pazopanib treatment, whereas there were no significant effects on cell-cycle progression in cells without FGFR2 amplification treated with pazopanib. In addition, pazopanib increased a substantial fraction of sub-G1 only in FGFR2-amplified cells. These findings show that the activation of FGFR2 signaling by amplification may be a critical mediator of cell proliferation in a small subset of gastric cancer patients and that pazopanib may provide genotype-correlated clinical benefits beyond the setting of highly vascular tumors.