Functional analysis of receptor tyrosine kinase mutations in lung cancer identifies oncogenic extracellular domain mutations of ERBB2.

We assessed somatic alleles of six receptor tyrosine kinase genes mutated in lung adenocarcinoma for oncogenic activity. Five of these genes failed to score in transformation assays; however, novel recurring extracellular domain mutations of the receptor tyrosine kinase gene ERBB2 were potently oncogenic. These ERBB2 extracellular domain mutants were activated by two distinct mechanisms, characterized by elevated C-terminal tail phosphorylation or by covalent dimerization mediated by intermolecular disulfide bond formation. These distinct mechanisms of receptor activation converged upon tyrosine phosphorylation of cellular proteins, impacting cell motility. Survival of Ba/F3 cells transformed to IL-3 independence by the ERBB2 extracellular domain mutants was abrogated by treatment with small-molecule inhibitors of ERBB2, raising the possibility that patients harboring such mutations could benefit from ERBB2-directed therapy.

Interleukin-6 upregulates paraoxonase 1 gene expression via an AKT/NF-κB-dependent pathway.

The aim of this study is to investigate the relationship between paraoxonase 1 (PON1) and atherosclerosis-related inflammation. In this study, human hepatoma HepG2 cell line was used as a hepatocyte model to examine the effects of the pro-inflammatory cytokines on PON1 expression. The results showed that IL-6, but not TNF-α and IL-1ß, significantly increased both the function and protein level of PON1; data from real-time RT-PCR analysis revealed that the IL-6-induced PON1 expression occurred at the transcriptional level. Increase of IκB kinase activity and IκB phosphorylation, and reduction of IκB protein level were also observed in IL-6-treated HepG2 cells compared with untreated culture. This event was accompanied by increase of NF-κB-p50 and -p65 nuclear translocation. Moreover, treatment with IL-6 augmented the DNA binding activity of NF-κB. Furthermore, pharmacological inhibition of NF-κB activation by PDTC and BAY 11-7082, markedly suppressed the IL-6-mediated PON1 expression. In addition, IL-6 increased the levels of phosphorylated protein kinase B (PKB, AKT). An AKT inhibitor LY294002 effectively suppressed IKK/IκB/NF-κB signaling and PON1 gene expression induced by IL-6. Our findings demonstrate that IL-6 upregulates PON1 gene expression through an AKT/NF-κB signaling axis in human hepatocyte-derived HepG2 cell line.

Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing.

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.

Using Body Composition Analysis for Improved Nutritional Intervention in Septic Patients: A Prospective Interventional Study.

The study aimed to determine whether using body composition data acquired through bio-electrical impedance analysis (BIA) to adjust diet formulas could improve outcomes in septic patients. There were 132 septic patients in medical intensive care units enrolled in the prospective, randomized, double-blind, interventional study. For the intervention group, dietitians had access to BIA data for adjusting diet formulas according to body composition variables on days 1, 3, and 8. The patients were also stratified based on nutritional risk using the modified Nutrition Risk in Critically ill (mNUTRIC) score. Patients with intervention were more likely to achieve caloric and protein intake goals compared to the control group, especially in the low-risk group. The intervention did not significantly affect mortality, but the survival curves suggested potential benefits. The high-risk group had longer ICU stays and mechanical ventilation duration, which were mitigated by the intervention. Certain body composition variables (e.g., extracellular water to total body water ratio and phase angle) showed differences between high-risk and low-risk groups and may be related to patient outcomes. Non-invasive body composition assessment using BIA can help dietitians adjust diet formulas for critically ill septic patients. Body composition variables may be associated with sepsis outcomes, but further research with larger patient numbers is needed to confirm these findings.

Drug-sensitive FGFR2 mutations in endometrial carcinoma.

Oncogenic activation of tyrosine kinases is a common mechanism of carcinogenesis and, given the druggable nature of these enzymes, an attractive target for anticancer therapy. Here, we show that somatic mutations of the fibroblast growth factor receptor 2 (FGFR2) tyrosine kinase gene, FGFR2, are present in 12% of endometrial carcinomas, with additional instances found in lung squamous cell carcinoma and cervical carcinoma. These FGFR2 mutations, many of which are identical to mutations associated with congenital craniofacial developmental disorders, are constitutively activated and oncogenic when ectopically expressed in NIH 3T3 cells. Inhibition of FGFR2 kinase activity in endometrial carcinoma cell lines bearing such FGFR2 mutations inhibits transformation and survival, implicating FGFR2 as a novel therapeutic target in endometrial carcinoma.

RET fusions observed in lung and colorectal cancers are sensitive to ponatinib.

Genomic studies are revolutionizing clinical oncology, but bridging the lab and the bedside requires the ability to efficiently interrogate rare genetic lesions in unexpected pathological settings using preclinical models. Oncogenes can exhibit intrinsic drug resistance to targeted therapy in different cells of origin, adding complexity to clinical interpretations of genomic findings. Here, we capitalize on the flexibility of engineered cell systems to rapidly profile known multi-kinase inhibitors that harbor rearranged during transfection (RET) kinase activity across multiple RET fusions. Identifying ponatinib as the most potent RET inhibitor tested, we used ponatinib to gauge therapeutic responsiveness in RET fusion-positive patient-derived xenograft (PDX) models. Using a genomics guided outlier approach, we identified 4 RET fusion PDX models with 3 different fusion partners (KIF5B, CCDC6, and NCOA4) in both non-small cell lung cancer and colorectal cancer. By comparing ponatinib activity in RET fusion-positive and RET fusion-negative PDX models alongside a standard of care chemotherapeutic agent, we show that RET fusions in colorectal tumors are therapeutically responsive to RET inhibition. Finally, we suggest that coupling engineered cell systems and genomics guided PDX model selection provides a rapid workflow to triage rare genomics findings.

Antcin-H Isolated from Antrodia cinnamomea Inhibits Renal Cancer Cell Invasion Partly through Inactivation of FAK-ERK-C/EBP-ß/c-Fos-MMP-7 Pathways.

Antcin-H, a natural triterpene, is purified from a famous anticancer medicinal mushroom, Antrodia cinnamomea, in Taiwan. This study showed that antcin-H inhibited the growth of human renal carcinoma 786-0 cells; the IC50 value (for 48 h) was 170 µM. Besides, the migration and invasion of 786-0 cells were suppressed by antcin-H under noncytotoxic concentrations (<100 µM); these events were accompanied by inhibition of FAK and Src kinase activities, decrease of paxillin phosphorylation, impairment of lamellipodium formation, and upregulation of TIMPs and downregulation of MMPs, especially MMP-7 expression. Luciferase reporter assay showed that antcin-H repressed the MMP-7 promoter activity, in parallel to inhibiting c-Fos/AP-1 and C/EBP-ß transactivation abilities. Moreover, antcin-H suppressed the activity of ERK1/2 and decreased the binding ability of C/EBP-ß and c-Fos on the upstream/enhancer region of MMP-7 promoter. Overall, this study demonstrated that the anti-invasive effect of antcin-H in human renal carcinoma 786-0 cells might be at least in part by abrogating focal adhesion complex and lamellipodium formation through inhibiting the Src/FAK-paxillin signaling pathways and decreasing MMP-7 expression through suppressing the ERK1/2-AP-1/c-Fos and C/EBP-ß signaling axis. Our findings provide the evidence that antcin-H may be an active component existing in A. cinnamomea with anticancer effect.

Missense mutations of the BRAF gene in human lung adenocarcinoma.

Mutations of the BRAF protein serine/threonine kinase gene have recently been identified in a variety of human cancers, most notably melanomas. We sought to determine the frequency of BRAF mutations in human lung cancer pathogenesis. Analysis of BRAF sequence from 127 primary human lung adenocarcinomas revealed mutations in two tumor specimens, one in exon 11 (G465V), and a second in exon 15 (L596R). These specimens belong to the same adenocarcinoma subgroup as defined by clustering of gene expression data. BRAF may provide a target for anticancer chemotherapy in a subset of lung adenocarcinoma patients.

An integrated view of copy number and allelic alterations in the cancer genome using single nucleotide polymorphism arrays.

Changes in DNA copy number contribute to cancer pathogenesis. We now show that high-density single nucleotide polymorphism (SNP) arrays can detect copy number alterations. By hybridizing genomic representations of breast and lung carcinoma cell line and lung tumor DNA to SNP arrays, and measuring locus-specific hybridization intensity, we detected both known and novel genomic amplifications and homozygous deletions in these cancer samples. Moreover, by combining genotyping with SNP quantitation, we could distinguish loss of heterozygosity events caused by hemizygous deletion from those that occur by copy-neutral events. The simultaneous measurement of DNA copy number changes and loss of heterozygosity events by SNP arrays should strengthen our ability to discover cancer-causing genes and to refine cancer diagnosis.

High-resolution single-nucleotide polymorphism array and clustering analysis of loss of heterozygosity in human lung cancer cell lines.

Chromosomal loss of heterozygosity (LOH) is a common mechanism for the inactivation of tumor suppressor genes in human epithelial cancers. Hybridization to single-nucleotide polymorphism (SNP) arrays is an efficient method to detect genome-wide cancer LOH. Here, we survey LOH patterns in a panel of 33 human lung cancer cell lines using SNP array hybridization containing 1500 SNPs. We compared the LOH patterns generated by SNP array hybridization to those previously obtained by 399 microsatellite markers and find a high degree of concordance between the two methods. A novel informatics platform, dChipSNP, was used to perform hierarchical tumor clustering based on genome-wide LOH patterns. We demonstrate that this method can separate non-small-cell and small-cell lung cancer samples based on their shared LOH. Furthermore, we analysed seven human lung cancer cell lines using a novel 10 000 SNP array and demonstrate that this is an efficient and reliable method of high-density allelotyping. Using this array, we identified small regions of LOH that were not detected by lower density SNP arrays or by standard microsatellite marker panels.

Oncogenic transformation by inhibitor-sensitive and -resistant EGFR mutants.

BACKGROUND: Somatic mutations in the kinase domain of the epidermal growth factor receptor tyrosine kinase gene EGFR are common in lung adenocarcinoma. The presence of mutations correlates with tumor sensitivity to the EGFR inhibitors erlotinib and gefitinib, but the transforming potential of specific mutations and their relationship to drug sensitivity have not been described. METHODS AND FINDINGS: Here, we demonstrate that EGFR active site mutants are oncogenic. Mutant EGFR can transform both fibroblasts and lung epithelial cells in the absence of exogenous epidermal growth factor, as evidenced by anchorage-independent growth, focus formation, and tumor formation in immunocompromised mice. Transformation is associated with constitutive autophosphorylation of EGFR, Shc phosphorylation, and STAT pathway activation. Whereas transformation by most EGFR mutants confers on cells sensitivity to erlotinib and gefitinib, transformation by an exon 20 insertion makes cells resistant to these inhibitors but more sensitive to the irreversible inhibitor CL-387,785. CONCLUSION: Oncogenic transformation of cells by different EGFR mutants causes differential sensitivity to gefitinib and erlotinib. Treatment of lung cancers harboring EGFR exon 20 insertions may therefore require the development of alternative kinase inhibition strategies.

The Topoisomerase 1 Inhibitor Austrobailignan-1 Isolated from Koelreuteria henryi Induces a G2/M-Phase Arrest and Cell Death Independently of p53 in Non-Small Cell Lung Cancer Cells.

Koelreuteria henryi Dummer, an endemic plant of Taiwan, has been used as a folk medicine for the treatment of hepatitis, enteritis, cough, pharyngitis, allergy, hypertension, hyperlipidemia, and cancer. Austrobailignan-1, a natural lignan derivative isolated from Koelreuteria henryi Dummer, has anti-oxidative and anti-cancer properties. However, the effects of austrobailignan-1 on human cancer cells have not been studied yet. Here, we showed that austrobailignan-1 inhibited cell growth of human non-small cell lung cancer A549 and H1299 cell lines in both dose- and time-dependent manners, the IC50 value (48 h) of austrobailignan-1 were 41 and 22 nM, respectively. Data from flow cytometric analysis indicated that treatment with austrobailignan-1 for 24 h retarded the cell cycle at the G2/M phase. The molecular event of austrobailignan-1-mediated G2/M phase arrest was associated with the increase of p21Waf1/Cip1 and p27Kip1 expression, and decrease of Cdc25C expression. Moreover, treatment with 100 nM austrobailignan-1 for 48 h resulted in a pronounced release of cytochrome c followed by the activation of caspase-2, -3, and -9, and consequently induced apoptosis. These events were accompanied by the increase of PUMA and Bax, and the decrease of Mcl-1 and Bcl-2. Furthermore, our study also showed that austrobailignan-1 was a topoisomerase 1 inhibitor, as evidenced by a relaxation assay and induction of a DNA damage response signaling pathway, including ATM, and Chk1, Chk2, γH2AX phosphorylated activation. Overall, our results suggest that austrobailignan-1 is a novel DNA damaging agent and displays a topoisomerase I inhibitory activity, causes DNA strand breaks, and consequently induces DNA damage response signaling for cell cycle G2/M arrest and apoptosis in a p53 independent manner.

Adherence to phosphate binder therapy is the primary determinant of hyperphosphatemia incidence in patients receiving peritoneal dialysis.

We investigated the major determinant of hyperphosphatemia incidence among patients receiving peritoneal dialysis. Seventy-six patients aged 25-55 years who had received peritoneal dialysis for more than 3 months were recruited. The patients were divided into three groups according to their serum phosphorus levels (Group 1, ≥ 6 mg/dL; Group 2, 5.9-4.8 mg/dL; and Group 3, <4.8 mg/dL). Renal dietitians interviewed the patients to determine their phosphate intake and adherence to phosphate binder therapy. No statistical differences in demographics or phosphate intake were identified among the groups. However, adherence to phosphate binders was greater in Group 3 than in Groups 1 and 2 (96.3% vs. 21.4% and 52.4%, respectively; P < 0.001). Multivariate analysis showed that adherence to phosphate binder therapy was the only significant contributor to serum phosphorus levels (P= 0.0001). Adherence to diet was better than adherence to phosphate binder therapy among patients receiving peritoneal dialysis, and the latter determined the incidence of hyperphosphatemia.

Destruxin B Isolated from Entomopathogenic Fungus Metarhizium anisopliae Induces Apoptosis via a Bcl-2 Family-Dependent Mitochondrial Pathway in Human Nonsmall Cell Lung Cancer Cells.

Destruxin B, isolated from entomopathogenic fungus Metarhizium anisopliae, is one of the cyclodepsipeptides with insecticidal and anticancer activities. In this study, destruxin B was extracted and purified by ion-exchange chromatography, silica gel chromatography, and semipreparative high-performance liquid chromatography. The potential anticancer effects and molecular mechanisms of destruxin B in human nonsmall cell lung cancer cell lines were characterized. Our results showed that destruxin B induced apoptotic cell death in A549 cells. This event was accompanied by the activation of caspase-2, -3, and -9. Moreover, destruxin B increased the expression level of proapoptotic molecule, PUMA, while decreased antiapoptotic molecule Mcl-1. Additionally, the translocation of Bax from cytosol to mitochondrial membrane was observed upon destruxin B treatment. Knockdown of Bax by shRNA effectively attenuated destruxin-B-triggered apoptosis in A549 cells. Interestingly, similar toxic effects and underlying mechanisms including caspase activation, upregulation of PUMA, and downregulation of Mcl-1 were also observed in a p53-null lung cancer H1299 cell line upon destruxin B treatment. Taken together, our findings suggest that destruxin-B-induced apoptosis in human nonsmall cell lung cancer cells is via a Bcl-2 family-dependent mitochondrial pathway.

Luteolin attenuates TGF-ß1-induced epithelial-mesenchymal transition of lung cancer cells by interfering in the PI3K/Akt-NF-κB-Snail pathway.

AIMS: Luteolin is a natural flavonoid that possesses a variety of pharmacological activities, such as anti-inflammatory and anti-cancer abilities. Whether luteolin regulates the transformation ability of lung cancer cells remains unclear. The current study aims to uncover the effects and underlying mechanisms of luteolin in regulation of and epithelial-mesenchymal transition of lung cancer cells. MAIN METHODS: The lung adenocarcinoma A549 cells were used in this experiment; the cells were pretreated with luteolin followed by administration with TGF-ß1. The expression levels of various cadherin and related upstream regulatory modules were examined. KEY FINDINGS: Pretreatment of luteolin prevented the morphological change and downregulation of E-cadherin of A549 cells induced by TGF-ß1. In addition, the activation of PI3K-Akt-IκBa-NF-κB-Snail pathway which leads to the decline of E-cadherin induced by TGF-ß1 was also attenuated under the pretreatment of luteolin. SIGNIFICANCE: We provide the mechanisms about how luteolin attenuated the epithelial-mesenchymal transition of A549 lung cancer cells induced by TGF-ß1. This finding will strengthen the anti-cancer effects of flavonoid compounds via the regulation of migration/invasion and EMT ability of various cancer cells.

Dietary supplements usage among elderly Taiwanese during 2005-2008.

This study describes dietary supplement consumption practices among the Taiwanese population over the age of 65. Data for the analyses were derived from the 2005-2008 Nutrition and Health Survey in Taiwan. Data from a total of 914 participants (456 men and 458 women) was collected in the study to delineate patterns of supplement usage. The results indicated that the percentage of individuals taking supplements was 45.7% for men and 52.2% for women. There were no significant differences in supplement use by gender, age group, geographic stratum, current employment status, household monthly income, self-reported health status or marital status, except for higher education and adequate perceived financial resources. Half of both men and women chose to take only one supplement. In addition, as the number of supplements taken increased, the number of people decreased. The elderly with higher education levels were more likely to take two kinds of supplements. The top five supplements consumed from highest to lowest were: glucosamine, multivitamins and minerals, calcium, fish oil and vitamin B complex. The major reason for supplements use for men was to supplement an unbalanced diet, and that for women was to prevent joint degeneration. The main factor influencing choice of supplements in the elderly was receiving the supplement as a gift from another person. Note that mean intakes of vitamins A, C, E, B-1, B-2, B-6, B-12, biotin, niacin, and pantothenic acid from supplements over-exceeded DRIs in Taiwan.

Chemogenomic profiling provides insights into the limited activity of irreversible EGFR Inhibitors in tumor cells expressing the T790M EGFR resistance mutation.

Reversible epidermal growth factor receptor (EGFR) inhibitors are the first class of small molecules to improve progression-free survival of patients with EGFR-mutated lung cancers. Second-generation EGFR inhibitors introduced to overcome acquired resistance by the T790M resistance mutation of EGFR have thus far shown limited clinical activity in patients with T790M-mutant tumors. In this study, we systematically analyzed the determinants of the activity and selectivity of the second-generation EGFR inhibitors. A focused library of irreversible as well as structurally corresponding reversible EGFR-inhibitors was synthesized for chemogenomic profiling involving over 79 genetically defined NSCLC and 19 EGFR-dependent cell lines. Overall, our results show that the growth-inhibitory potency of all irreversible inhibitors against the EGFR(T790M) resistance mutation was limited by reduced target inhibition, linked to decreased binding velocity to the mutant kinase. Combined treatment of T790M-mutant tumor cells with BIBW-2992 and the phosphoinositide-3-kinase/mammalian target of rapamycin inhibitor PI-103 led to synergistic induction of apoptosis. Our findings offer a mechanistic explanation for the limited efficacy of irreversible EGFR inhibitors in EGFR(T790M) gatekeeper-mutant tumors, and they prompt combination treatment strategies involving inhibitors that target signaling downstream of the EGFR.

Predicting drug susceptibility of non-small cell lung cancers based on genetic lesions.

Somatic genetic alterations in cancers have been linked with response to targeted therapeutics by creation of specific dependency on activated oncogenic signaling pathways. However, no tools currently exist to systematically connect such genetic lesions to therapeutic vulnerability. We have therefore developed a genomics approach to identify lesions associated with therapeutically relevant oncogene dependency. Using integrated genomic profiling, we have demonstrated that the genomes of a large panel of human non-small cell lung cancer (NSCLC) cell lines are highly representative of those of primary NSCLC tumors. Using cell-based compound screening coupled with diverse computational approaches to integrate orthogonal genomic and biochemical data sets, we identified molecular and genomic predictors of therapeutic response to clinically relevant compounds. Using this approach, we showed that v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations confer enhanced Hsp90 dependency and validated this finding in mice with KRAS-driven lung adenocarcinoma, as these mice exhibited dramatic tumor regression when treated with an Hsp90 inhibitor. In addition, we found that cells with copy number enhancement of v-abl Abelson murine leukemia viral oncogene homolog 2 (ABL2) and ephrin receptor kinase and v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) kinase family genes were exquisitely sensitive to treatment with the SRC/ABL inhibitor dasatinib, both in vitro and when it xenografted into mice. Thus, genomically annotated cell-line collections may help translate cancer genomics information into clinical practice by defining critical pathway dependencies amenable to therapeutic inhibition.

Allele-dependent variation in the relative cellular potency of distinct EGFR inhibitors.

Targeted cancer therapies impede cancer cell growth by inhibiting the function of activated oncogene products. Patients with non-small cell lung cancer and somatic mutations of EGFR can have a dramatic response to treatment with erlotinib and gefitinib; different somatic mutations are associated with different times to progression and survival. In this study, the relative and absolute potencies of two distinct EGFR tyrosine kinase inhibitors, erlotinib and an investigational irreversible inhibitor, HKI-272, were found to vary significantly in a panel of Ba/F3 cells transformed by representative EGFR somatic mutations. HKI-272 more potently inhibited the primary exon 20 insertion mutants, the secondary erlotinib-resistance mutants including T790M and many erlotinib-sensitive mutants including L858R. In contrast, erlotinib is a more potent inhibitor of the major exon 19 deletion mutants than is HKI-272. Analyses of EGFR autophosphorylation patterns confirmed the mutation-specific variation in relative potency of these tyrosine kinase inhibitors. Our finding that distinct EGFR inhibitors are more effective in vitro for different mutant forms of the protein suggests that tyrosine kinase inhibitor treatment could be tailored to specific EGFR mutations. More broadly, these results imply that the development and deployment of targeted therapies should focus on inhibition of specific cancer-causing mutations, not only on the mutated target.

TT viral infection through blood transfusion: retrospective investigation on patients in a prospective study of post-transfusion hepatitis.

AIM:To investigate the role of blood transfusion in TT viral infection(TTV).METHODS:We retrospectively studied serum samples from 192 transfusion recipients who underwent cardiovascular surgery and blood transfusion between July 1991 and June 1992. All patients had a follow-up every other week for at least 6 months after transfusion. Eighty recipients received blood before screening donors for hepatitis C antibody (anti-HCV), and 112 recipients received screened blood. Recipients with alanine aminotransferase level > 2.5 times the upper normal limit were tested for serological markers for viral hepatitis A, B, C, G, Epstein-Barr virus and cytomegalovirus. TTV infection was defined by the positivity for serum TTV DNA using the polymerase chain reaction method.RESULTS: Eleven and three patients, who received anti-HCV unscreened and screened blood, respectively, had serum ALT levels > 90IU/L. Five patients (HCV and TTV 1; HCV, HGV, and TTV 1; TTV 2; and CMV and TTV 1) were positive for TTV DNA, and four of them had sero conversion of TTV DNA.CONCLUSION:TTV can be transmitted via blood transfusion.Two recipients infected by TTV alone may be associated with the hepatitis. However, whether TTV was the causal agent remains unsettled, and further studies are necessary to define the role of TTV infection in chronic hepatitis.