Whole-animal mounts of Caenorhabditis elegans for 3D imaging using atomic force microscopy.

The 3D surface of Caenorhabditis elegans was imaged at nanometer resolution using atomic force microscopy (AFM). Oscillation of a medium stiffness silicon AFM cantilever at the upper second amplitude peak, typically 6 times above the fundamental frequency, vastly improved image quality on the moist, sticky, and soft worms. Whole-animal mounts of normal and double-headed mutants of the nematode worm were prepared and scanned. Well-preserved anatomical features including annuli, furrows, alae, and rows of never before seen nanometer-sized pores dotting the molted worm's outermost surface coat were resolved. Well-preserved anatomical features including annuli, furrows, alae, and rows of nanometer-sized pores or struts dotting the molted worm's outermost surface were resolved. This AFM method represents a simple and rapid new approach for nanometer-resolved 3D imaging and analysis of whole-animal specimens of C. elegans. FROM THE CLINICAL EDITOR: In this interesting article the authors describe a new AFM sampling method to allow better images on whole-animal mounts such as C. elegans. This method would generate more information and in the future may be useful for differentiating even individual animals with different genetic backgrounds.

Role of PAX8 in the regulation of MET and RON receptor tyrosine kinases in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancers (NSCLC) are highly heterogeneous at the molecular level and comprise 75% of all lung tumors. We have previously shown that the receptor tyrosine kinase (RTK) MET frequently suffers gain-of-function mutations that significantly promote lung tumorigenesis. Subsequent studies from our lab also revealed that PAX5 transcription factor is preferentially expressed in small cell lung cancer (SCLC) and promotes MET transcription. PAX8, however, is also expressed in NSCLC cell lines. We therefore investigated the role of PAX8 in NSCLC. METHODS: Using IHC analysis, PAX8 protein expression was determined in archival NSCLC tumor tissues (n = 254). In order to study the effects of PAX8 knockdown on NSCLC cellular functions such as apoptosis and motility, siRNA against PAX8 was used. Confocal fluorescence microscopy was used to monitor the localization of MET, RON and PAX8. The combinatorial effect of PAX8 knockdown and MET inhibition using SU11274 was investigated in NSCLC cell viability assay. RESULTS: Relative levels of PAX8 protein were elevated (≥ + 2 on a scale of 0-3) in adenocarcinoma (58/94), large cell carcinoma (50/85), squamous cell carcinoma (28/47), and metastatic NSCLC (17/28; lymph node). Utilizing early progenitors isolated from NSCLC cell lines and fresh tumor tissues, we observed robust overexpression of PAX8, MET, and RON. PAX8 knockdown A549 cells revealed abrogated PAX8 expression with a concomitant loss in MET and the related RON kinase expression. A dramatic colocalization between the active form of MET (also RON) and PAX8 upon challenging A549 cells with HGF was visualized. A similar colocalization of MET and EGL5 (PAX8 ortholog) proteins was found in embryos of C. elegans. Most importantly, knockdown of PAX8 in A549 cells resulted in enhanced apoptosis (~6 fold) and decreased cell motility (~45%), thereby making PAX8 a potential therapeutic target. However, the combinatorial approach of PAX8 knockdown and treatment with MET inhibitor, SU11274, had marginal additive effect on loss of NSCLC cell viability. CONCLUSION: PAX8 provides signals for growth and motility of NSCLC cells and is necessary for MET and RON expression. Further investigations are necessary to investigate the therapeutic potential of PA8 in NSCLC.

O-6-methylguanine-deoxyribonucleic acid methyltransferase methylation enhances response to temozolomide treatment in esophageal cancer.

BACKGROUND: World-wide, esophageal cancer is a growing epidemic and patients frequently present with advanced disease that is surgically inoperable. Hence, chemotherapy is the predominate treatment. Cytotoxic platinum compounds are mostly used, but their efficacy is only moderate. Newer alkylating agents have shown promise in other tumor types, but little is known about their utility in esophageal cancer. METHODS: We utilized archived human esophageal cancer samples and esophageal cancer cell lines to evaluate O-6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT) hypermethylation status and determined sensitivity to the alkylating drug temozolomide (TMZ). Immunoblot analysis was performed to determine MGMT protein expression in cell lines. To assess and confirm the effect of TMZ treatment in a methylated esophageal cancer cell line in vivo, a mouse flank xenograft tumor model was utilized. RESULTS: Nearly 71% (12/17) of adenocarcinoma and 38% (3/8) of squamous cell carcinoma (SCC) patient samples were MGMT hypermethylated. Out of four adenocarcinoma and nine SCC cell lines tested, one of each histology was hypermethylated. Immunoblot analyses confirmed that hypermethylated cell lines did not express the MGMT protein. In vitro cell viability assays showed the methylated Kyse-140 and FLO cells to be sensitive to TMZ at an IC50 of 52-420 µM, whereas unmethylated cells Kyse-410 and SKGT-4 did not respond. In an in vivo xenograft tumor model with Kyse-140 cells, which are MGMT hypermethylated, TMZ treatment abrogated tumor growth by more than 60%. CONCLUSION: MGMT methylation may be an important biomarker in subsets of esophageal cancers and targeting by TMZ may be utilized to successfully treat these patients.

Differential expression of RON in small and non-small cell lung cancers.

RON is a MET related receptor tyrosine kinase (RTK) and its natural ligand is macrophage stimulating protein (MSP). RON plays a very important role in the regulation of inflammation. Several studies have previously reported overexpression of RON in a variety of cancers including lung and identified numerous RON alternate splice forms that very likely contribute to tumor growth and metastasis. Here, we have analyzed the expression of total RON protein as well as its kinase-active form (phospho-RON) in 175 archival lung tumor FFPE (formalin fixed paraffin embedded) samples that included non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC), and their metastatic forms. The frequency and intensity of RON protein expression was much higher in lung tumors of neuroendocrine origin such as SCLC and in secondary tumors that metastasized to brain. In addition, the majority of the expressed RON protein was phospho-RON. We also identified 62, and 30 kDa isoforms of RON (GenBank accession numbers are JN689381 and JN689382) using RNA isolated from pooled lung cancer cell lines and RT-PCR. A majority of the NSCLC cell lines expressed a 150 kDa band that corresponded to the RON ß chain and 120 kDa band in the panel of SCLC cell lines tested. RON was expressed on the cell surface in NSCLC cell lines. Finally, knock down of RON expression resulted in a significant loss in viability as well as motility in lung cancer cells suggesting that RON is a potential therapeutic target.

The database of chromosome imbalance regions and genes resided in lung cancer from Asian and Caucasian identified by array-comparative genomic hybridization.

BACKGROUND: Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis. METHODS: Array-comparative genomic hybridization (array-CGH) was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR), chromogenic in situ hybridization (CISH), reverse transcriptase-qPCR (RT-qPCR), and immunohistochemistry (IHC) in more patients. RESULTS: We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian. CONCLUSIONS: Our study provides an invaluable database revealing common and differential imbalance regions at specific chromosomes among Asian and Caucasian lung cancer patients. Four validation methods confirmed our database, which would help in further studies on the mechanism of lung tumorigenesis.

Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer.

NOL7 is a putative tumor suppressor gene localized to 6p23, a region with frequent loss of heterozygosity in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells altered the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in 13 CC cell lines and 23 consecutive CC specimens by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Methylation of the NOL7 promoter was analyzed by bisulfite sequencing and mutations were identified through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 5' untranslated region and first exon of NOL7. However, bisulfite sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may affect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines were significantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression toward CC. NOL7 is inactivated in CC in accordance with the Knudson 2-hit hypothesis through loss of heterozygosity and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate tumor suppressor gene located on 6p23.

EphA2 mutation in lung squamous cell carcinoma promotes increased cell survival, cell invasion, focal adhesions, and mammalian target of rapamycin activation.

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Role of protein kinase C ß and vascular endothelial growth factor receptor in malignant pleural mesothelioma: Therapeutic implications and the usefulness of Caenorhabditis elegans model organism.

PURPOSE: To examine the role of both protein kinase C (PKC)-ß and vascular endothelial growth factor receptor (VEGFR)-2 in malignant pleural mesothelioma (MPM) using respective inhibitors, enzastaurin and KRN633. MATERIALS AND METHODS: MPM cell lines, control cells, and a variety of archived MPM tumor samples were used to determine the protein expression levels of PKC-ß, VEGFR-2, VEGF, and p-AKT. Effects of enzastaurin and KRN633 on phosphorylation status of key signaling molecules and viability of the mesothelioma cells were determined. The common soil nematode, Caenorhabditis elegans, was treated with enzastaurin to determine its suitability to screen for highly potent kinase inhibitors. RESULTS: PKC-ß1, PKC-ß2 and VEGFR-2/KDR were overexpressed in MPM cell lines and MPM tumor tissues. Enzastaurin treatment resulted in significant loss in viability of VEGF induced cell proliferation; however, the effect of KRN633 was much less. Enzastaurin also dramatically decreased the phosphorylation of PKC-ß, its downstream target p-AKT, and surprisingly, the upstream VEGFR-2. The combination of the two drugs at best was additive and similar results were obtained with respect to cell viability. Treatment of C. elegans with enzastaurin resulted in clear phenotypic changes and the worms were hypermotile with abnormal pattern and shape of eggs, suggesting altered fecundity. CONCLUSIONS: PKC-ß1 and VEGFR-2 are both excellent therapeutic targets in MPM. Enzastaurin was better at killing MPM cells than KRN633 and the combination lacked synergy. In addition, we show here that C. elegans can be used to screen for the next generation inhibitors as treatment with enzastaurin resulted in clear phenotypic changes that could be assayed.

Crizotinib (PF02341066) as a ALK /MET inhibitor- Special Emphasis as a Therapeutic Drug Against Lung Cancer.

There are a number of molecular abnormalities that can occur in normal cells to induce a malignant phenotype. Recently, the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) has been shown to have gain-of-function when partnered with different proteins. As an example, on chromosome 2p, with inversion, there is translocation with generation of EML4-ALK tyrosine kinase in lung cancer. In a phase I trial, EML4-ALK patients were selected to determine the response to a potent small molecule tyrosine kinase inhibitor crizotinib (previously identified as PF02341066). Dramatic durable responses were observed with crizotinib at 250 mg twice a day (orally). Interestingly, crizotinib also has activity against MET receptor tyrosine kinase. We have previously shown that MET can be overexpressed, sometimes mutated, or sometimes amplified in lung cancer. Thus, this review will emphasize the characteristics of crizotinib, and detail the clinical experience.

PAX5 is expressed in small-cell lung cancer and positively regulates c-Met transcription.

PAX5 is a nuclear transcription factor required for B cell development, and its expression was evaluated in upper aerodigestive malignancies and pancreatic cancer by immunoblotting. The PAX5 protein expression was relatively strong in small-cell lung cancer (SCLC, 11/12); however, its expression was not detected in non-SCLC (NSCLC, n=13), mesothelioma (n=7), pancreatic (n=6), esophageal (n=6) and head and neck cancer cell lines (n=12). In comparison, PAX8 and PAX3 expressions were absent or non-detectable in SCLC cell lines; however, PAX8 was expressed in most of the tested NSCLC cell lines (13/13) and also frequently in all the other cell lines. We also detected frequent expressions of PAX2 and PAX9 protein in the various cell lines. Utilizing neuroendocrine tumor samples, we found that the frequency as well as the average intensity of the expression of PAX5 increased from pulmonary carcinoid (9%, moderate and strong PAX5 expression, n=44), to large-cell neuroendocrine carcinoma (LCNC, 27%, n=11) to SCLC (33%, n=76). FISH analysis revealed no translocations of the PAX5 gene, but polyploidy in some SCLC tumor tissues (6/37). We determined that PAX5 could regulate the transcription of c-Met using luciferase-coupled reporter and chromatin immunoprecipitation analysis. In addition, the phospho-c-Met (active form) and PAX5 were both localized to the same intra-nuclear compartment in hepatocyte growth factor treated SCLC cells and interacted with each other. Finally, we determined the therapeutic translational potential of PAX5 using PAX5 knockdown SCLC cells in conjunction with Topoisomerase 1 (SN38) and c-Met (SU11274) inhibitors. Loss of endogenous PAX5 significantly decreased the viability of SCLC cells, especially when combined with SN38 or SU11274, and maximum effect was seen when both inhibitors were used. Therefore, we propose that PAX5 could be an important regulator of c-Met transcription and a potential target for therapy in SCLC.

Enterohemorrhagic Escherichia coli suppresses inflammatory response to cytokines and its own toxin.

Infection with the enteric pathogen enterohemorrhagic Escherichia coli (EHEC) causes a variety of symptoms ranging from nonbloody diarrhea to more severe sequelae including hemorrhagic colitis, altered sensorium and seizures, and even life-threatening complications, such as hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. The more severe consequences of EHEC infection are attributable to the production of Shiga toxin (Stx) and its subsequent effects on the vasculature, which expresses high levels of the Stx receptor, Gb3. Interestingly, the intestinal epithelium does not express Gb3. Despite the lack of Gb3 receptor expression, intestinal epithelial cells translocate Stx. The effect of Stx on intestinal epithelial cells is controversial with some studies demonstrating induction of inflammation and others not. This may be difficult to resolve because EHEC expresses both proinflammatory molecules, such as flagellin, and factor(s) that dampen the inflammatory response of epithelial cells. The goal of our study was to define the effect of Stx on the inflammatory response of intestinal epithelial cells and to determine whether infection by EHEC modulates this response. Here we show that Stx is a potent inducer of the inflammatory response in intestinal epithelial cells and confirm that EHEC attenuates the induction of IL-8 by host-derived proinflammatory cytokines. More importantly, however, we show that infection with EHEC attenuates the inflammatory response by intestinal epithelial cells to its own toxin. We speculate that the ability of EHEC to dampen epithelial cell inflammatory responses to Stx and cytokines facilitates intestinal colonization.

EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma.

Squamous cell carcinoma (SCC) and malignant pleural mesothelioma (MPM) are thoracic malignancies with very poor prognosis and limited treatment options. It is an established fact that most of the solid tumors have overexpression of EPHA2 receptor tyrosine kinase. EPHA2 is known to exhibit opposing roles towards cancer progression. It functions in inhibiting cancer survival and migration via a ligand and tyrosine kinase dependent signaling (Y772). Whereas it is known to promote tumor progression and cell migration through a ligand-independent signaling (S897). We analyzed the expression profile and mutational status of the ephrin receptor A2 (EPHA2) in SCC and MPM cell lines and primary patient specimens. The EPHA2 receptor was found to be either overexpressed, mutated or amplified in SCC and MPM. In particular, the EPHA2 mutants A859D and T647M were interesting to explore, A859D Y772 dead mutant exhibited lower levels of phosphorylation at Y772 compared to T647M mutant. Molecular Dynamics simulations studies suggested that differential changes in conformation might form the structural basis for differences in the level of EPHA2 activation. Consequently, A859D mutant cells exhibited increased proliferation as well as cell migration compared to controls and T647M mutant. Kinomics analysis demonstrated that the STAT3 and PDGF pathways were upregulated whereas signaling through CBL was suppressed. Considered together, the present work has uncovered the oncogenic characteristics of EPHA2 mutations in SSC and MPM reinstating the dynamics of different roles of EPHA2 in cancer. This study also suggests that a combination of doxazosin and other EPHA2 inhibitors directed to inhibit the pertinent signaling components may be a novel therapeutic strategy for MPM and Non-small cell lung cancer patients who have either EPHA2 or CBL alterations.

Focal adhesion kinase a potential therapeutic target for pancreatic cancer and malignant pleural mesothelioma.

The non-receptor cytoplasmic tyrosine kinase, Focal Adhesion Kinase (FAK) is known to play a key role in a variety of normal and cancer cellular functions such as survival, proliferation, migration and invasion. It is highly active and overexpressed in various cancers including Pancreatic Ductal Adenocarcinoma (PDAC) and Malignant Pleural Mesothelioma (MPM). Here, initially, we demonstrate that FAK is overexpressed in both PDAC and MPM cell lines. Then we analyze effects of two small molecule inhibitors PF-573228, and PF-431396, which are dual specificity inhibitors of FAK and proline rich tyrosine kinase 2 (PYK2), as well as VS-6063, another small molecule inhibitor that specifically inhibits FAK but not PYK2 for cell growth, motility and invasion of PDAC and MPM cell lines. Treatment with PF-573228, PF-431396 and VS-6063 cells resulted in a dose-dependent inhibition of growth and anchorage-independent colony formation in both cancer cell lines. Furthermore, these compounds suppressed the phosphorylation of FAK at its active site, Y397, and functionally induced significant apoptosis and cell cycle arrest in both cell lines. Using the ECIS (Electric cell-substrate impedance sensing) system, we found that treatment of both PF compounds suppressed adherence and migration of PDAC cells on fibronectin. Interestingly, 3D-tumor organoids derived from autochthonous KC (Kras;PdxCre) mice treated with PF-573228 revealed a significant decrease in tumor organoid size and increase in organoid cell death. Taken together, our results show that FAK is an important target for mesothelioma and pancreatic cancer therapy that merit further translational studies.

Genomic Heterogeneity as a Barrier to Precision Medicine in Gastroesophageal Adenocarcinoma.

Gastroesophageal adenocarcinoma (GEA) is a lethal disease where targeted therapies, even when guided by genomic biomarkers, have had limited efficacy. A potential reason for the failure of such therapies is that genomic profiling results could commonly differ between the primary and metastatic tumors. To evaluate genomic heterogeneity, we sequenced paired primary GEA and synchronous metastatic lesions across multiple cohorts, finding extensive differences in genomic alterations, including discrepancies in potentially clinically relevant alterations. Multiregion sequencing showed significant discrepancy within the primary tumor (PT) and between the PT and disseminated disease, with oncogene amplification profiles commonly discordant. In addition, a pilot analysis of cell-free DNA (cfDNA) sequencing demonstrated the feasibility of detecting genomic amplifications not detected in PT sampling. Lastly, we profiled paired primary tumors, metastatic tumors, and cfDNA from patients enrolled in the personalized antibodies for GEA (PANGEA) trial of targeted therapies in GEA and found that genomic biomarkers were recurrently discrepant between the PT and untreated metastases. Divergent primary and metastatic tissue profiling led to treatment reassignment in 32% (9/28) of patients. In discordant primary and metastatic lesions, we found 87.5% concordance for targetable alterations in metastatic tissue and cfDNA, suggesting the potential for cfDNA profiling to enhance selection of therapy.Significance: We demonstrate frequent baseline heterogeneity in targetable genomic alterations in GEA, indicating that current tissue sampling practices for biomarker testing do not effectively guide precision medicine in this disease and that routine profiling of metastatic lesions and/or cfDNA should be systematically evaluated. Cancer Discov; 8(1); 37-48. ©2017 AACR.See related commentary by Sundar and Tan, p. 14See related article by Janjigian et al., p. 49This article is highlighted in the In This Issue feature, p. 1.

Expression and mutational analysis of c-CBL and its relationship to the MET receptor in head and neck squamous cell carcinoma.

MET is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC) and degraded by c-CBL E3-ubiquitin ligase. We investigated genetic variations of c-CBL in HNSCC and the relationship between c-CBL and MET expression. High MET, low c-CBL expression was detected in 10 cell lines and 73 tumor tissues. Two novel mutations (L254S, L281F), and the single nucleotide polymorphism (SNP) P782L were identified from archival tumor tissues. 27.3% of loss of heterozygosity was found at CBL locus. Ectopic expression of wild-type c-CBL in SCC-35 cells downregulated MET expression and decreased cell viability. These results suggest MET overexpression is related to altered c-CBL expression, which may influence tumorigenesis.

FAK and paxillin, two potential targets in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating cancer in large part due to late diagnosis and a lack of effective screening tests. In spite of recent progress in imaging, surgery and new therapeutic options for pancreatic cancer, the overall five-year survival still remains unacceptably low. Numerous studies have shown that focal adhesion kinase (FAK) is activated in many cancers including PDAC and promotes cancer progression and metastasis. Paxillin, an intracellular adaptor protein that plays a key role in cytoskeletal organization, connects integrins to FAK and plays a key role in assembly and disassembly of focal adhesions. Here, we have reviewed evidence in support of FAK as a potential therapeutic target and summarized related combinatorial therapies.

PI3 Kinase Pathway and MET Inhibition is Efficacious in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive cancer that is commonly associated with prior asbestos exposure. Receptor tyrosine kinases (RTKs) such as MET and its downstream target PI3K are overexpressed and activated in a majority of MPMs. Here, we studied the combinatorial therapeutic efficacy of the MET/ALK inhibitor crizotinib, with either a pan-class I PI3K inhibitor, BKM120, or with a PI3K/mTOR dual inhibitor, GDC-0980, in mesothelioma. Cell viability results showed that MPM cells were highly sensitive to crizotinib, BKM120 and GDC-0980 when used individually and their combination was more effective in suppressing growth. Treatment of MPM cells with these inhibitors also significantly decreased cell migration, and the combination of them was synergistic. Treatment with BKM120 alone or in combination with crizotinib induced G2-M arrest and apoptosis. Both crizotinib and BKM120 strongly inhibited the activity of MET and PI3K as evidenced by the decreased phosphorylation of MET, AKT and ribosomal S6 kinase. Using a PDX mouse model, we showed that a combination of crizotinib with BKM120 was highly synergetic in inhibiting MPM tumor growth. In conclusion our findings suggest that dual inhibition of PI3K and MET pathway is an effective strategy in treating MPM as compared to a single agent.

Met gene amplification and protein hyperactivation is a mechanism of resistance to both first and third generation EGFR inhibitors in lung cancer treatment.

The 3rd generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs; e.g., AZD9291), which selectively and irreversibly inhibit EGFR activating and T790M mutants, represent very promising therapeutic options for patients with non-small cell lung cancer (NSCLC) that has become resistant to 1st generation EGFR-TKIs due to T790M mutation. However, eventual resistance to the 3rd generation EGFR-TKIs has already been described in the clinic, resulting in disease progression. Therefore, there is a great challenge and urgent need to understand how this resistance occurs and to develop effective strategies to delay or overcome the resistance. The current study has demonstrated that Met amplification and hyperactivation is a resistance mechanism to both 1st and 3rd generation EGFR-TKIs since both erlotinib- and AZD9291-resistant HCC827 cell lines possessed amplified Met gene and hyperactivated Met, and were cross-resistant to AZD9291 or erlotinib. Met inhibition overcame the resistance of these cell lines to AZD9291 both in vitro and in vivo, including enhancement of apoptosis or G1 cell cycle arrest. Hence, we suggest that Met inhibition is also an effective strategy to overcome resistance of certain EGFR-mutated NSCLCs with Met amplification to AZD9291, warranting the further clinical validation of our findings.

C. elegans and mutants with chronic nicotine exposure as a novel model of cancer phenotype.

We previously investigated MET and its oncogenic mutants relevant to lung cancer in C. elegans. The inactive orthlogues of the receptor tyrosine kinase Eph and MET, namely vab-1 and RB2088 respectively, the temperature sensitive constitutively active form of KRAS, SD551 (let-60; GA89) and the inactive c-CBL equivalent mutants in sli-1 (PS2728, PS1258, and MT13032) when subjected to chronic exposure of nicotine resulted in a significant loss in egg-laying capacity and fertility. While the vab-1 mutant revealed increased circular motion in response to nicotine, the other mutant strains failed to show any effect. Overall locomotion speed increased with increasing nicotine concentration in all tested mutant strains except in the vab-1 mutants. Moreover, chronic nicotine exposure, in general, upregulated kinases and phosphatases. Taken together, these studies provide evidence in support of C. elegans as initial in vivo model to study nicotine and its effects on oncogenic mutations identified in humans.

Unique fractal evaluation and therapeutic implications of mitochondrial morphology in malignant mesothelioma.

Malignant mesothelioma (MM), is an intractable disease with limited therapeutic options and grim survival rates. Altered metabolic and mitochondrial functions are hallmarks of MM and most other cancers. Mitochondria exist as a dynamic network, playing a central role in cellular metabolism. MM cell lines display a spectrum of altered mitochondrial morphologies and function compared to control mesothelial cells. Fractal dimension and lacunarity measurements are a sensitive and objective method to quantify mitochondrial morphology and most importantly are a promising predictor of response to mitochondrial inhibition. Control cells have high fractal dimension and low lacunarity and are relatively insensitive to mitochondrial inhibition. MM cells exhibit a spectrum of sensitivities to mitochondrial inhibitors. Low mitochondrial fractal dimension and high lacunarity correlates with increased sensitivity to the mitochondrial inhibitor metformin. Lacunarity also correlates with sensitivity to Mdivi-1, a mitochondrial fission inhibitor. MM and control cells have similar sensitivities to cisplatin, a chemotherapeutic agent used in the treatment of MM. Neither oxidative phosphorylation nor glycolytic activity, correlated with sensitivity to either metformin or mdivi-1. Our results suggest that mitochondrial inhibition may be an effective and selective therapeutic strategy in mesothelioma, and identifies mitochondrial morphology as a possible predictor of response to targeted mitochondrial inhibition.