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Supplementary Fig. 1 Confirmation of cell synchronisation at the G1/S boundary. H1299 cells with or without prior transfection with plasmids expressing GFP-tagged CDC6 or GFP alone were transfected twice with the indicated miRNA mimics or control RNA. Untreated cells and those treated with the transfection reagent alone were used for comparison. Some 48 hrs after the final transfection, cells were synchronised at the G1/S boundary with mimosine, and the cell synchrony was determined by flow cytometry. Supplementary Fig. 2 MicroRNA-26a/b inhibit pre-replicative complex formation and cell cycle progression by suppressing CDC6 in human lung cancer cells. (A) SPAC cells with or without prior transfection with plasmids expressing GFP-tagged CDC6 or GFP alone were transfected twice with the indicated miRNA mimics or control RNA. Untreated cells and those treated with the transfection reagent alone were used for comparison. Some 48 hrs post transfection, cells were synchronized at the G1/S boundary with mimosine, and chromatin binding assay was performed with an aliquot of each cell sample. (B) The remaining cells were then released into fresh medium and harvested at different time points for FACS analysis.

Cancer is characterized by mutations, genome rearrangements, epigenetic changes, and altered gene expression that enhance cell proliferation, invasion, and metastasis. To accommodate deregulated cellular proliferation, many DNA replication-initiation proteins are overexpressed in human cancers. However, the mechanism that represses the expression of these proteins in normal cells and the cellular changes that result in their overexpression are largely unknown. One possible mechanism is through miRNA expression differences. Here, it is demonstrated that miR26a and miR26b inhibit replication licensing and the proliferation, migration, and invasion of lung cancer cells by targeting CDC6. Importantly, miR26a/b expression is significantly decreased in human lung cancer tissue specimens compared with the paired adjacent normal tissues, and miR26a/b downregulation and the consequential upregulation of CDC6 are associated with poorer prognosis of patients with lung cancer. These results indicate that miR26a/b repress replication licensing and tumorigenesis by targeting CDC6.Implications: The current study suggests that miR26a, miR26b, and CDC6 and factors regulating their expression represent potential cancer diagnostic and prognostic markers as well as anticancer targets.Visual Overview: http://mcr.aacrjournals.org/content/12/11/1535/F1.large.jpg.Mol Cancer Res; 12(11); 1535–46. ©2014 AACR.

BackgroundDifferentiating multiple pulmonary lesions as multiple primary lung cancer (MLC) or intra-pulmonary metastasis (IPM) is critical. Lung cancer also has a high genetic heterogeneity, which influenced the treatment strategy. Genetic information may aid in tracing lineage information on multiple lung lesions. This study applied comprehensive genomic profiling to decipher the intrinsic genetics of multiple lung lesions.MethodsSixty-six lung adenocarcinomas (LUAD) tumor lesions (FFEP) archived from 30 patients were included in this study. The 508 cancer-related genes were evaluated by targeted next-generation sequencing (MGI-seq 2000).ResultsThe study included a total of 30 LUADs (66 samples). The majority of tumors demonstrated intra-tumoral heterogeneity. Two hundred twenty-four mutations were detected by sequencing the 66 samples. We investigated the driver gene mutations of NSCLC patients with multiple lesions. EGFR was the most frequently (48/198) mutated driver gene. The codons in EGFR mainly affected by mutations were p.L858R (18/66 [27.3%]) and exon 19del (8/66 [12.1%]). In addition, additional driver genes were found, including TP53, BRAF, ERBB2, MET, and PIK3CA. We also found that the inter-component heterogeneity of different lesions and more than two different mutation types of EGFR were detected in seven patients with two lesions (P3, P10, P24, P25, P28, P29, and P30). The TMB values of different lesions in each patient were different in 26 patients (except P4, P5, P14, and P30).ConclusionsComprehensive genomic profiling should be applied to distinguishing the nature of multiple lung lesions irrespective of radiologic and histologic diagnoses.

IntroductionRHPN2, a member of rhophilin family of rho-binding proteins, regulates actin cytoskeleton and vesicular trafficking, and promotes mesenchymal transformation in cancer. We have found that RHPN2 was significantly mutated in lung adenocarcinoma (LUAD). However, the role of RHPN2 in lung cancer is not fully understood.MethodsIn the present study, we investigated the expression of RHPN2 in 125 patients with LUAD by qRT-PCR and correlated its expression with clinical characteristics. The effects of RHPN2 on the proliferation and invasion of lung cancer cells were determined by CCK-8 and in vitro transwell assays, clonogenic assay, and xenograft mouse model. The RhoA pull down assay and Western blotting were performed to elucidate the mechanism of RNPN2 in tumorigenesis of lung cancer.ResultsRHPN2 was overexpressed in tumors from LUAD, and high levels of RHPN2 were associated with poor prognosis of LUAD patients. RHPN2 was required for proliferation and invasion of lung cancer cells. Intriguingly, overexpression of RHPN2 conferred the resistance to glutamine depletion in lung cancer cells. Mechanistic studies revealed that ectopic overexpression of RHPN2 promoted the stability of c-Myc protein via phosphorylation at Ser62 and increased c-Myc target glutamine synthetase (GS). Analysis of GS expression in clinical sample showed that the expression of GS was elevated in tumor cells. Kaplan-Meier analysis revealed that high levels of GS were significantly associated with worse overall survival time of the patients with LUAD.ConclusionsTaken together, this study suggested that RHPN2 was involved in tumorigenesis of lung cancer via modulating c-Myc stability and the expression of its target GS in lung adenocarcinoma, which links RHPN2 and glutamine metabolism.

Background: As the aging issue and increased elderly esophageal cancer (EC) patients, we sought to study the clinical characteristics, treatment modality and outcomes of EC patients 70 years or older compared with those younger than 70 years old. Methods: The national surveillance, epidemiology, and end results (SEER) database for the period from 1973 to 2013 was analyzed. The patient and treatment characteristics were compared between the age groups. Multivariate Cox proportional hazard regression analyses were also performed to identify independent prognostic factors. Propensity-score matching analyses (PSA) regarding survival after different treatments were also performed in locoregional EC. Results: Compared with the younger group, patients 70 years or older were characterized by having a higher proportion of patients with female gender, white race, localized disease, non-adenocarcinoma and without any treatment, as well as inferior overall survival (OS) [hazard ratio (HR), 1.324] and EC-specific survival (HR, 1.270). In addition, older patients shared same independent prognostic factors with younger patients, including age, histology and race. Specifically, compared with those receiving no intensive treatments, surgery alone (HR, 0.342), both surgery and RT (HR, 0.323) and RT only (HR, 0.525) were favorable among elderly patients, as confirmed by both multivariate adjustment and PSA. Conclusions: Compared to younger EC patients, those 70 years or older showed distinctive clinical characteristics and inferior survival. Despite showing a higher proportion of localized disease, patients 70 years or older were less likely to be subjected to surgery or/and RT. Thus, the role of intensive treatments, which were identified as favorable factors among elderly patients in this study, warrants further investigation.

BACKGROUND Small cell lung cancer (SCLC) is a subtype of lung cancer with poor prognosis. In this study, we aimed to build a nomogram to predict the survival of individual with SCLC by incorporating significant clinical parameters. METHODS The patients with SCLC were enrolled from the First Affiliated Hospital of Guangzhou Medical University (GMUFAH) between 2009 and 2013. We identified and incorporated the independent prognostic factors to build a nomogram to predict the survival of SCLC patients. The predictive accuracy and discriminative ability of the nomogram were evaluated by concordance index (C-index) and calibration curve. We also compared the accuracy of the built model with the 7th AJCC TNM and VALSG staging system. The nomogram was further validated in an independent cohort of 80 patients with SCLC from Cancer Center of Guangzhou Medical University (GMUCC) between 2009 and 2013. RESULTS A total of 275 patients with SCLC were included in the primary cohort, and seven independent prognostic factors were identified including age, N stage, metastasis status, histology, platelets to lymphocyte ratio (PLR), neuron specific enolase (NSE) and CYFRA21-1 as independent prognostic factors after using Cox regression model. A nomogram incorporating these prognostic factors was subsequently built. The calibration curves for possibilities of 1-, 2-year overall survival (OS) revealed optimal agreement between nomogram prediction and actual observation. The C-index of this nomogram was higher than that of TNM and VALSG staging system in both primary and validation cohort (nomogram vs. TNM, primary cohort 0.68 vs. 0.65, P

e21051 Background: Differentiating multiple pulmonary lesions as multiple primary lung cancer (MLC) or intra-pulmonary metastasis (IPM) is critical for clinics. Radiological or histological (dis)concordance may be informative, while, genetic information may aid tracing lineage information on multiple lung lesions. This study aims to apply comprehensive genomic profiling deciphering intrinsic genetics of multiple lung lesions. Methods: 32 patients, 69 FFPE samples were applied to perform high-through put sequencing. 636 cancer-related genes were captured and sequenced by MGI-seq 2000. Raw data was processed via in-house developed pipeline. Unsupervised clustering was applied to cluster samples based on genetic characters after variant calling and annotation. Patients were categorized into “Genomic related” and “Genomic unrelated” groups. Another cohort consisting of 402 patients with mono-lesion was used as control cohort. Results: No difference of age of onset was found between 32 patients and 403 patients with mono lesion (60.5 versus 60 years). Male had relatively lower incidence of multiple lung lesions (RR:0.46,95%CI: 0.20-0.93, P = 0.04). Eight patients were classified as “Genomic related” by presenting same mutation in driver or tumor suppressor genes. Six of eight patients were characterized to carry EGFR L858R, in addition, two patients presented co-mutation of RB1(c.1814+1G > T, p.E48Kfs*18) and TP53 mutations (p.H193R, p.R249S). Another two patients carried EGFR 19 Deletion. Two lesions from one patient with predefined diagnosis of intra-pulmonary metastasis showed biallelic loss of function mutations in same loci of NF1 gene (p.S2311*/ p.Q1360*) together with same KRAS mutation (p.G13C). Radiologically, no same lobe as well as left-right preference were revealed between “Genomic related” and “Genomic unrelated” groups. Histologically, there was no enrichment of patients with same histology subtype in “genomic related” group, verse visa. While, patients in “genomic related” group had higher ages at initial diagnosis (median age 69.5 vs 56.5 years, P = 0.04). Conclusions: Comprehensive genomic profiling should be applied to clinics distinguishing the nature of multiple lung lesion irrespective of radiological and histological diagnosis. [Table: see text]

e21024 Background: Alterations of TP53, EGFR, KRAS genes are of importance in LUAD etiology, vital prognostic markers as well as therapeutic targets as recently reported. In addition, in advanced or metastatic NSCLC, concomitant mutations of TP53 with EGFR or KRAS closely associated with prognosis and TKIs’ efficacy. While, distribution of concomitant mutations of TP53, EGFR and KRAS in early stage or resectable LUAD remained to be elucidated. Methods: 434 patients with defined pathological diagnosis of LUAD were recruited from 1st.Jan.2019-31st.Dec.2019, which made 468 FFPE blocks for the study. Histology and composition were given by authorized by pathologist. Genomic DNA from FFPE samples and peripheral blood was extracted. 636 cancer-related genes were specifically captured and sequenced by MGI-seq 2000. Association of concomitant status with clinical and genomic characteristics was further analyzed. Results: Generally, TP53, EGFR and KRAS mutation rate is 26.7%, 60.9% and 10% respectively. Among these samples, Subtypes of EGFR mutations significantly differed in between tumors rising in left and right lung (P = 0.048). Tumors with KRAS mutations occurred more easily in right lung than those with TP53 or EGFR mutations (P = 0.012, P = 0.043 respectively). In addition, histology subtype and clinical stage were closely associated with TP53, EGFR and KRAS mutation status (P < 0.001). 90 samples (19.2%) carried concomitant TP53 and EGFR mutation, 14 samples (3%) were TP53/KRAS co-mutated. LUAD samples with EGFR amplification had higher rate of concomitant TP53/EGFR mutations (RR:1.1,95%CI: 1.032 to 1.159,P = 0.0009). Moreover, KRAS G13 mutated samples more easily co-mutated with TP53 mutations compared with those with mutations in G12 loci (RR:1.33,95%CI: 1.24 to 1.37,P = 0.024). Further, samples with non-concomitant TP53 mutations had highest level of tumor mutation burden (muts/Mb) (median TMB = 9.74 vs 2.05 for TP53/EGFR/KRAS wild type, P < 0.0001). Samples with concomitant TP53/EGFR mutations and TP53/KRAS mutations displayed higher TMB level than their non-concomitant compartment (P < 0.001 and P = 0.05 respectively). Conclusions: TP53,EGFR and KRAS mutations and their concomitant mutation status displayed diverse correlation with spatial, clinical and genomic characteristics in resectable LUAD patients. This correlation may indicate complex biological heterogeneity of LUAD which may need further exploration.

e21023 Background: TMB is associated with mono-immunotherapy efficacy for patients with advanced or metastatic non-small-cell lung cancer (NSCLC). For early-stage NSCLC or EGFR mutated non-resectable NSCLC, TMB-H predicted better prognosis or poor response to TKIs. This research might develop potential predictive parameters for distinguishing NSCLC with high or low TMB. Methods: Samples of surgically resected, cancer tissues were collected from 499 patients with NSCLC: 29 (5.8%) lung squamous cell carcinoma (LUSC) and 468 (94.2%) lung adenocarcinoma (LUAD). The mutations and TMB were confirmed by target region capture sequencing (Oseq™-508). Results: According to the median value (3.08), TMB is divided into high (N = 244) and low (N = 255). Interactions between TMB and sex, age, location, histology, pathological subtype, lymph node, parabronchial lymph node, LVI, neuro-invasive, stage and mutation status were evaluated. The distribution of TMB-H was significantly (p < 0.001) correlated with sex, age, histology, pathological subtype, stage (TN stage) and partial mutations. Among all mutations, 6 genes (TP53 / FAT3 / KMT2D / TSHZ3 / NAV3 / EPHA3) were confirmed to be significantly related to TMB-H in NSCLC. We also found that TMB-H was significantly affected by mutation status of KRAS gene (P. G12X, p < 0.001) and missense mutations in FAT3 (p < 0.001). The Kruskal–Wallis H test results showed that the mutation types of FAT3, KRAS, TP53 and PIK3CA were closely related to TMB-H. Lasso linear regression analysis was applied and resulted the better predictors of TMB status including TSHZ3, KMT2D, TP53, gender and T stage. Conclusions: Instead of comprehensive genomic profiling to evaluate TMB, clinical characteristics and special mutation types may help to effectively screen and predicate patients with TMB-H status. [Table: see text]

e13542 Background: Environment factors are associated with lung cancer occurrence. Genetic susceptibility to lung cancer remains unclear. This study assessed germline mutations in Chinese non-small-cell lung cancer (NSCLC) patients. Methods: 506 FFPE samples were collected from 469 patients pathologically confirmed as lung adenocarcinoma. A 508-gene panel including 63 hereditary cancer genes was applied to detect mutations on MGI-seq 2000. Raw data was processed and analyzed. Variation pathogenicity was categorized following ACMG 2015 guideline. Results: 21 patients (4%) carried (likely) pathogenic (P or LP) mutations in 15 cancer predisposition genes. 11 germline variations included MUTYH (1/21), BLM (1/21), BRIP1(2/21), RAD50(1/21), PMS1(1/21), TP53(1/21), BRCA2(1/2), PALB2(1/21), NF1(1/21), CDH1(1/21) and MRE11A (1/21) genes. Nine likely pathogenic mutations were identified in MRE11A, RAD51C, RAD50, ATR, BRCA2, BLM, PMS1 and VHL genes. These mutations are involved in homologous recombination repair, mismatch repair, Fanconi anemia, and Li-Fraumeni syndrome. Germline mutations were commonly occurred in females (female vs Male: 13 vs 7) without statistical significance. No significant correlations of age of onset and TMB were observed between NSCLC patients with germline wild type and mutation. Other clinical characters like histology and clinical stage presented similar results. Somatic mutations with high frequency like EGFR, KRAS were distributed equally in patients with both germline mutation positive and negative. Conclusions: In this study, there is no significant correlation of germline susceptibility gene mutations with clinical and genetic characters of NSCLC patients. Further investigation on germline genetic aberrations of NSCLC is definitely needed to clarify germline impact on the etiology of NSCLC. [Table: see text]

e21049 Background: Inhibition of PI3K/AKT/mTOR pathway has emerged as a promising anticancer strategy. Measuring genetic alterations in PI3K/AKT/mTOR pathway are critical for clinical decision making for patients with lung cancer. This study aims to analyze PI3K/AKT/mTOR pathway-related gene mutations in non-small cell lung cancer (NSCLC) in a consecutive cohort. Methods: 536 surgically resected tumor tissues were collected. Target region capture sequencing for 508 cancer-related genes was conducted on MGI-seq 2000 platform. Results: PI3K/AKT/mTOR pathway genes were mutated in 120 samples (22.4%). Female or older patients displayed higher mutation incidence of PI3K/AKT/mTOR pathway (P = 0.06 for female, P = 0.05 for patients ≥60 y). In NSCLC samples, mutations mainly occurred in NF1/2(18/499), PTEN (7/499), MTOR (4/499), TSC1/2(18/499), PIK3CA (26/499), PDK1 (2/499), AKT1/2/3 (5/499) and KRAS (49/499) genes. Genetic alterations of AKT2 (p = 0.003), mTOR (P = 0.03), PTEN (P = 0.005) and PIK3CA (P < 0.001) are preferred to occur in squamous NSCLC. PIK3CA variants (p.E542K, p.E545K, p.Q546K) were more enriched in non-squamous NSCLC, while amplification of PIK3CA was more prevalent in squamous NSCLC (P = 0.038). Histologically, TSC1/2 mutations were more correlate with micro-invasion subtype in non-squamous NSCLC (P = 0.002). Meanwhile, PIK3CA alterations, especially, amplification occurred more often in keratinizing squamous NSCLC (P < 0.001). In addition, four patients with biallelic NF1 loss of function mutations were exclusively found in non-squamous NSCLC without co-occurrence of mutation in known driver genes like EGFR or TP53, which indicated biallelic loss of function of NF1 might be sufficient to drive oncogenesis of non-squamous NSCLC. Conclusions: PI3K/AKT/mTOR pathway alterations present a heterogenous distribution across various types of lung malignancies especially in NSCLC. As more small molecule inhibitors developed to target PI3K/AKT/mTOR pathway, this study might shed light on further clinical investigation of these lately developed therapeutic approaches. [Table: see text]

Serine incorporator 2 (SERINC2) is a member of the SERINC family of transmembrane proteins that incorporate serine into membrane lipids during synthesis. In the present study, the biological function of SERINC2 in lung adenocarcinoma cells was investigated. The data from a previous study and the publicly available Oncomine database were analysed regarding the expression levels of SERINC2 mRNA in lung adenocarcinoma. A lentiviral-based short hairpin RNA (shRNA) was used to suppress SERINC2 expression in lung cancer cells. The effect of SERINC2 expression on lung cancer proliferation was determined using cell counting kit-8 and colony formation assays. The influence on invasion and migration was examined in vitro using Transwell and wound-healing assays, respectively. Phosphorylated protein kinase B (p-AKT) expression levels were assessed by immunoblotting. According to a previous study and Oncomine, expression levels of SERINC2 mRNA are significantly upregulated in tumour tissues compared with those in healthy tissues in patients with lung adenocarcinoma. SERINC2-knockdown by lentiviral-based shRNA inhibited the proliferation, migration and invasion of the H1650 and A549 cells. In addition, p-AKT expression levels were significantly decreased following SERINC2-knockdown. In conclusion, SERINC2-knockdown suppresses lung adenocarcinoma proliferation, migration and invasion through a mechanism that may be associated with phosphatidylinositol 3-kinase/AKT signalling. Based on these findings, SERINC2 serves an important role in the progression of lung adenocarcinoma.

Background: Albumin and globulin are main components of serum protein. The level of albumin and globulin partially represents the nutrition status and immune system. Albumin-to-globulin ratio (AGR) has been reported as a prognostic factor in various cancers. We therefore performed a meta-analysis to elucidate the prognosis effect of AGR on survival outcomes in solid tumors. Method: Six electronic database were searched for the relevant articles that assessing the prognostic value of pre-treatment AGR in solid tumor patients. The primary outcome was overall survival (OS) and the secondary outcomes were cancer-specific survival (CSS), disease-free survival (DFS) and disease-metastasis-free survival (DMFS). The time-to-event outcomes were summarized in hazard ratio (HR) and 95% confidence interval (CI). Result: A total of 13890 solid tumor patients in 24 studies were included. The AGR higher than the cut-off values ranging from 1.15-1.75 was related to better OS (HR=0.58, 95%CI 0.537-0.626, p

Cancer is characterized by mutations, genome rearrangements, epigenetic changes, and altered gene expression that enhance cell proliferation, invasion, and metastasis. To accommodate deregulated cellular proliferation, many DNA replication-initiation proteins are overexpressed in human cancers. However, the mechanism that represses the expression of these proteins in normal cells and the cellular changes that result in their overexpression are largely unknown. One possible mechanism is through miRNA expression differences. Here, it is demonstrated that miR26a and miR26b inhibit replication licensing and the proliferation, migration, and invasion of lung cancer cells by targeting CDC6. Importantly, miR26a/b expression is significantly decreased in human lung cancer tissue specimens compared with the paired adjacent normal tissues, and miR26a/b downregulation and the consequential upregulation of CDC6 are associated with poorer prognosis of patients with lung cancer. These results indicate that miR26a/b repress replication licensing and tumorigenesis by targeting CDC6. Implications: The current study suggests that miR26a, miR26b, and CDC6 and factors regulating their expression represent potential cancer diagnostic and prognostic markers as well as anticancer targets. Visual Overview: http://mcr.aacrjournals.org/content/12/11/1535/F1.large.jpg. Mol Cancer Res; 12(11); 1535–46. ©2014 AACR.

Background ALK-tyrosine kinase inhibitor (TKI) is associated with a favarable survival benefit in ALK-fusion patients in advanced non-small cell lung cancer (NSCLC). However, almost 20% patients with TKI early resistance (progression-free survival is shorter than six months while treated by either crizotinib or second-line TKIs), show a poor survival with no more than one year. The mechanisms of early resistance to TKIs are unknown. Reliable biomarkers are required to predict the response to ALK-TKIs, especially in the early resistance. So we investigated genomic variations associated with responses to crizotinib in advanced NSCLC patients with ALK-fusion. Methods Advanced NSCLC primary tumor samples with ALK-fusion from 5 extreme poor and 5 extreme strong responders to crizotinib were subjected to whole-genome analysis. And we found 44 genomic variant sites with poor response through gene based pathway enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Then we randomly assigned 17 advanced NSCLC patients’ sample with ALK-fusion to validate the candidate variant sites using small sequence capture panel. Genomic data of > 0.2 gigabases/sample was generated at average of 828 sequencing depth which covering 44 relative genes. Results In total, 774 genomic variations were matched to crizotinib responses in clinical data; which were located within regions for DNA repaired, mitochondrial apoptosis and tumor angiogenesis-target genes. From them, 4 DNA damage repair (DDR)-related gene variations (TP53, MLH1, XPA, and MSH2) were associated with early resistance to crizotinib in ALK-fusion NSCLC patients, from which 5 variants were within the coding regions and 4 identified as non-synonymous single-nucleotide variants. Validation genotyping confirmed sequencing results and revealed patients genotype for rs28934575 in TP53 showed extreme shorter PFS (P Conclusions Thus, DDR deficiency may contribute to the early resistance to crizotinib in ALK-fusion patients. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

// Dakai Xiao 1, 2, 3, * , Hui Pan 1, 2, * , Fuqiang Li 4 , Kui Wu 4 , Xin Zhang 1, 3 , Jianxing He 1, 3 1 Department of Thoracic Surgery, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China 2 Research Center for Translational Medicine, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China 3 Guangzhou Institute of Respiratory Disease and State Key Laboratory of Respiratory Disease, Guangzhou, China 4 BGI-Shenzhen, Shenzhen, China * These authors contributed equally to this work Correspondence to: Jianxing He, e-mail: Drjianxing.he@gmail.com Keywords: lung adenocarcinoma, targeted NGS sequencing, mutation burden Received: December 21, 2015 Accepted: February 23, 2016 Published: March 19, 2016 ABSTRACT Gender-associated difference in incidence and clinical outcomes of lung cancer have been established, but the biological mechanisms underlying these gender-associated differences are less studied. Recently we have characterized the genomic landscape of lung adenocarcinoma derived from Chinese population (Reference [ 1 ]). In this study we evaluated the clinical significance of mutation burden in lung adenocarcinoma and found that the male tumors harbored statistically greater burden of genetic alterations than female counterparts (Male median 3 (range 0–34) vs female median = 2 (0–24), male to female ratio = 1.636, 95% CI = 1.343–1.992) after adjustment of age at surgery, stage, smoking status. Kaplan-Meier survival analysis revealed that greater burden of genetic alterations was associated with worse overall survival. Moreover, multivariable analysis demonstrated mutation burden was an independent prognostic factor for the patients. Taken together, our analysis demonstrated gender disparity of mutation burden and their prognostic value in lung adenocarcinoma. This gender difference in mutation burden might provide an explanation for the distinct difference in the clinical outcomes between sexes in lung adenocarcinoma.

International audience; A treatment strategy that combines arsenic trioxide (ATO) with the tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) appears to induce markedly more cell apoptosis than imatinib mesylate alone in chronic myeloid leukemia (CML). To understand the mechanisms underlying the synergistic/additive action of these agents, we applied cDNA microarrays, component plane presentation integrated self-organizing map (CPP-SOM), and methods of protein biochemistry to study cell apoptosis induced by imatinib mesylate, ATO, and the combination of the 2 agents in the CML cell line K562. Numerous features with temporospatial relationships were revealed, indicating the coordinated regulation of molecular networks from various aspects of proapoptotic and apoptotic activities in CML. Imatinib mesylate appears to induce mainly the intrinsic pathway of cell apoptosis, whereas ATO induces the endoplasmic reticulum (ER) stress-mediated pathway of cell apoptosis, and the combination of the 2 agents seems to more effectively induce the intrinsic, extrinsic, and ER stress-mediated pathways of cell apoptosis, which results in a more effective and efficient induction of programmed cell death in K562 cells. This finding appears to be supported also by data derived from bone marrow cells of 2 patients with CML, one in chronic phase and the other in blast-crisis phase of the disease.

Despite the therapeutic advances, lung cancer remains the leading cause of cancer-related death in the United States and worldwide. Metastasis and recurrence are considered to be responsible for the failure of treatment. Recent studies indicate Epithelial mesenchymal transition, an evolutionarily conserved process, plays an important role in the embryonic development and cancer progression and is involved in the metastasis, drug resistance and correlated with progression of many tumors. Of importance, EMT is also involved in the acquisition of stemness phenotype of tumor cells. Although a growing body of evidence supports the role of EMT in the progression of many cancers, and a number of signal pathways, transcriptional factors and microRNAs involved in EMT process have been identified. However, the role of EMT in lung cancer is elusive. In this review, we present the recent findings in EMT including the molecular mechanisms of EMT, and the involvement of EMT in cancer progression, cancer stem cells and drug resistance, especially focusing on the correlation of EMT and lung cancer.

Purpose: Angiogenesis has generated interest in oncology because of its important role in cancer growth and progression, particularly when combined with cytotoxic therapies, such as radiotherapy. Among the numerous pathways influencing vascular growth and stability, inhibition of protein kinase B(Akt) or protein kinase C(PKC) can influence tumor blood vessels within tumor microvasculature. Therefore, we wanted to determine whether PKC inhibition could sensitize lung tumors to radiation. Methods and Materials: The combination of the selective PKC{beta} inhibitor Enzastaurin (ENZ, LY317615) and ionizing radiation were used in cell culture and a mouse model of lung cancer. Lung cancer cell lines and human umbilical vascular endothelial cells (HUVEC) were examined using immunoblotting, cytotoxic assays including cell proliferation and clonogenic assays, and Matrigel endothelial tubule formation. In vivo, H460 lung cancer xenografts were examined for tumor vasculature and proliferation using immunohistochemistry. Results: ENZ effectively radiosensitizes HUVEC within in vitro models. Furthermore, concurrent ENZ treatment of lung cancer xenografts enhanced radiation-induced destruction of tumor vasculature and proliferation by IHC. However, tumor growth delay was not enhanced with combination treatment compared with either treatment alone. Analysis of downstream effectors revealed that HUVEC and the lung cancer cell lines differed in their response to ENZ and radiationmore » such that only HUVEC demonstrate phosphorylated S6 suppression, which is downstream of mTOR. When ENZ was combined with the mTOR inhibitor, rapamycin, in H460 lung cancer cells, radiosensitization was observed. Conclusion: PKC appears to be crucial for angiogenesis, and its inhibition by ENZ has potential to enhance radiotherapy in vivo.« less

Background The disturbance of zinc homeostasis featured with a significant decrease of cellular zinc level was well documented to associate with the development and progression of human prostate malignancy. We have previously reported that zinc treatment induces prostate malignant cell apoptosis through mitochondrial pathway. Metallothionein (MT) is a major receptor/donor of zinc in the cells. However, the studies on the expression of MT in association with the prostate pathological and malignant status are very limited, and the zinc regulation of MT isoform expression in prostate cells remains elusive. The goals of this study were to define the expression of endogenous MTs, the isoforms of MT 1, 2, 3 at both messenger ribonucleic acid (mRNA) and protein levels; and to investigate the zinc effect on MT expression in normal prostate, benign prostatic hyperplasia (BPH) and malignant PC-3 cells, and in relevant human tissues. Cellular MT proteins were detected by immunohistochemistry, fluorescence staining and Western blot analysis; reverse transcription polymerase chain reaction (RT-PCR) was used to determine the MT isoform-specific mRNAs. Results Our results demonstrated a significant suppression of endogenous levels of MT1/2 in malignant PC-3 cells (95% reduction compared to the normal prostate cells) and in human adenocarcinoma tissues (73% MT1/2 negative). A moderate reduction of MT1/2 expression was observed in BPH. Zinc treatment remarkably induced MT1/2 expression in PC-3 and BPH cells, which was accordant with the restored cellular zinc level. MT 3, as a growth inhibitory factor, was detected and up-regulated by zinc mainly in BPH cells. Conclusion This study provided evidence of the association of attenuated MT1/2 with prostate tumor progression, and the zinc induction of MT1/2 expression resulting in cellular zinc restoration. The results suggest the potential of MT1/2 as a candidate biomarker for prostate cancer and the utilization of zinc in prostate cancer prevention and treatment.

Purpose: Poly(ADP-ribose) polymerase-1 (PARP-1) is the founding member of a family of enzymes that catalyze the addition of ADP-ribose units to proteins that mediate DNA repair pathways. Ionizing radiation induces DNA strand breaks, suggesting that PARP-1 inhibition may sensitize tumor cells to radiation. Experimental Design: We investigated the combination of PARP-1 inhibition with radiation in lung cancer models. ABT-888, a novel potent PARP-1 inhibitor, was used to explore the effects of PARP-1 inhibition on irradiated tumors and tumor vasculature. Results: ABT-888 reduced clonogenic survival in H460 lung cancer cells, and inhibited DNA repair as shown by enhanced expression of DNA strand break marker histone γ-H2AX. Both apoptosis and autophagy contributed to the mechanism of increased cell death. Additionally, ABT-888 increased tumor growth delay at well-tolerated doses in murine models. For a 5-fold increase in tumor volume, tumor growth delay was 1 day for ABT-888 alone, 7 days for radiation alone, and 13.5 days for combination treatment. Immunohistochemical staining of tumor sections revealed an increase in terminal deoxyribonucleotide transferase–mediated nick-end labeling apoptotic staining, and a decrease in Ki-67 proliferative staining after combination treatment. Matrigel assay showed a decrease in in vitro endothelial tubule formation with ABT-888/radiation combination treatment, and von Willebrand factor staining of tumor sections revealed decreased vessel formation in vivo, suggesting that this strategy may also target tumor angiogenesis. Conclusions: We conclude that PARP-1 inhibition shows promise as an effective means of enhancing tumor sensitivity to radiation, and future clinical studies are needed to determine the potential of ABT-888 as a radiation enhancer.

Pancreatic cancer is characterized by its extremely poor prognosis and it is the 4th leading cancer death in both men and women. The limited scientific knowledge and lack of effective treatment of this cancer are critically urging to an extensive research development in the field. Emerging evidence shows that pancreatic cancer cells exhibit aberrant activation of class I phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling. To understand the molecular mechanism underlying the relationship between signaling activation and pancreatic cancer cell growth, we investigated the anti-cancer effect of a novel dual inhibitor of PI3K/mTOR, PI-103 {3-(4-(4-Morpholinyl)pyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl)phenol}, on pancreatic cancer cells. The cell proliferation experiment assessed by MTT assay showed that PI-103 (0.1-10μM) effectively inhibits the growth of p53-mutant pancreatic cancer cells including Panc-1, MIA paca-2, BxPC-3 and AsPC-1 cells. The growth inhibitory effect of PI-103 was further supported by clonogenic assay showing a complete blockage of colony formation of Panc-1 cells after 7 days treatment. Our results demonstrated that PI-103 induces autophagy in Panc-1 cells characterized by a significant increase of the conversion of LC3-1 to LC3-II, which was then attenuated by an autophagy inhibitor, 3-methyladenine (3-MA), and augmented using a thiol proteinase inhibitor, E-64d, by blocking the degradation of autolysosomes. Knockdown of the expression of beclin1 with siRNA attenuated LC3 conversion and the growth inhibitory effect of PI-103 as well as colony formation, suggesting a possible linkage of autophagy and cell growth inhibition. Furthermore, cell cycle analysis and apoptotic assay with Annexin V staining showed that PI-103 induced G0/G1 arrest significantly without an effective induction of apoptosis. Western blotting revealed that PI-103 regulated the expression of cell-cycle relevant proteins, including increased p27 and decreased CDK1 and cyclin B. In addition to the effect of PI-103 on autophagy and cell cycle arrest, to our surprise, a striking appearance of cytoplasmic vacuoles ranging from 1-4μm in diameter was observed in the cells treated with PI-103 at as early as 3 hrs. The origin of the vacuoles was explored using the markers with GFP-LAMP1 for late endosome and lysosome, and DsRed-FYVE-labeling for early endosome, respectively. The results showed the large vacuoles are LAMP1-positive lysosomes or late endosomes, but not early endosomes, which is leading to future studies. Taken altogether, our data show that PI-103 is a potent compound for inhibiting pancreatic cancer cell growth, and the anti-cancer effect may operate through the induction of autophagy and cell cycle arrest. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2497.

Loss of cellular zinc accumulation is one of the characteristic features of prostate cancer, compared to the high zinc level in normal prostate epithelial cells. We previously demonstrated that zinc exposure induces apoptosis in human prostate malignant cells via a mitochondrial pathway in both in vitro and in vivo models, suggesting an important role of zinc homeostasis in growth control of prostate cancer. However, it remains unclear whether the apoptotic effect of zinc via the targeting of mitochondrial function is the only mechanism leading to growth inhibition in prostate cancer. Recent progress in autophagy indicates that this evolutionarily conserved catabolic process mediates the degradation of cellular components including damaged mitochondria and could promote either cell survival or cell death. The goal of this study was to ascertain the zinc effect on autophagy and its relationship to prostate cancer cell growth. Zinc induced autophagy was characterized in human prostate cancer PC-3 cells by Western blot analysis of LC-3, sub-cellular localization of ectopically expressed GFP-LC3, and transmission electron microscopy (TEM). Reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) were measured by fluorescence labeling and FACS analysis. Cell viability was assessed by MTT assay. Our results showed that zinc (15µM) significantly increased the conversion of LC3-I to LC3-II in a time-dependent manner (2.5 fold increase at 24 hrs), and induced the formation of punctuate GFP-LC3 structures in the cytoplasm as revealed by a fluorescence microscopy. Blockade of autophagy flux by inhibiting autolysosomal degradation using a thiol proteinase inhibitor, E-64d, greatly augmented zinc-induced LC3-II conversion. Electron microscopy observation revealed typical morphologic ultra-structures for autophagy including the formation of autophagosomes and autophagosome-related cytoplasmic structures. Inhibition of class III PI3K by 3-MA (3-methyladenine) attenuated zinc-induced autophagy and the cell death as quantified by MTT assay. Furthermore, zinc treatment led to a striking increase in ROS production and alteration in MMP, which were then abrogated by anti-oxidative N-acetyl-cysteine (NAC) associated with a significant suppression of zinc-induced autophagy and cell death. Our data indicate that zinc exposure induces autophagy in prostate malignant cells and leads to autophagy-related cell death, which is most likely due to direct targeting of mitochondrial function by zinc. Thus, we have provided evidence that the inhibitory role of zinc in prostate cancer cell growth may operate through the convergence of apoptosis and autophagy pathways, both of which are downstream from mitochondrial stress signaling leading to nuclear degradation and cellular metabolic alteration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4856.

BackgroundPharmacological intervention of redox balance in cancer cells often results in oxidative stress-mediated apoptosis, attracting much attention for the development of a new generation of targeted therapy in cancer. However, little is known about mechanisms underlying the conversion from oxidative signaling to downstream activities leading cells to death.Methodology/principal findingsWe here report a systematic detection of transcriptome changes in response to oxidative signals generated in leukemia cells upon fenretinide treatment, implicating the occurrence of numerous stress-responsive events during the fenretinide induced apoptosis, such as redox response, endoplasmic reticulum stress/unfolded protein response, translational repression and proteasome activation. Moreover, the configuration of these relevant events is primarily orchestrated by stress responsive transcription factors, as typically highlighted by NF-E2-related factor-2 (NRF2) and heat shock factor 1 (HSF1). Several lines of evidence suggest that the coordinated regulation of these transcription factors and thus their downstream genes are involved in converting oxidative signaling into downstream stress-responsive events regulating pro-apoptotic and apoptotic activities at the temporal and spatial levels, typifying oxidative stress-mediated programmed death rather than survival in cancer cells.Conclusions/significanceThis study provides a roadmap for understanding oxidative stress-mediated apoptosis in cancer cells, which may be further developed into more sophisticated therapeutic protocols, as implicated by synergistic induction of cell apoptosis using proteasome inhibitors with fenretinide.

Pharmacological intervention affecting intracellular redox balance often results in oxidative stress-mediated apoptosis, which also represents a new direction for cancer therapeutic designs. However, mechanistic links between redox signals and their downstream effects on apoptosis have not yet been elucidated in cancer cells. Here, we report a detailed analysis of fenretinide-induced apoptosis in leukemia-derived cells through a systems approach, integrating experimental and computational methods together with advanced data mining tools. Robust transcriptome profiling reveals numerous stress-responsive events at the temporal and spatial levels, including oxidative stress, endoplasmic reticulum stress/unfolded protein response, translational repression, proteasome activation, and apoptosis induction. Moreover, stress-responsive transcription factors, as highlighted by NRF2 and HSF1, play prominent roles in the configuration of these relevant events. Several lines of evidence suggest that these stress-responsive regulators and thus their target genes are involved not only in converting oxidative signaling into downstream effects but also in coordinating the progression of cell apoptosis. This study provides a roadmap for understanding oxidative stress-mediated apoptosis in cancer cells, which may be further developed into more sophisticated therapeutic protocols, as implicated by synergistic induction of cell apoptosis using proteasome inhibitors with fenretinide.

A treatment strategy that combines arsenic trioxide (ATO) with the tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) appears to induce more cell apoptosis than STI571 alone in chronic myeloid leukemia (CML). To understand the mechanisms underlying the synergistic action of these agents, we applied cDNA microarrays, component plane presentation integrated self-organizing maps (CPP-SOM) and methods of protein biochemistry to study proapoptotic and apoptotic activities in programmed cell death induced by STI571, ATO and the combination of the two agents in detail. Numerous features with temporospatial relationship were revealed, indicating the coordinated regulation of molecular networks from various aspects of proapoptotic and apoptotic activities in CML. In consequence, STI571 induces the intrinsic pathway of cell apoptosis, whereas ATO induces the ER stress-mediated pathway of cell apoptosis, and the combination of the two agents induces the intrinsic, extrinsic and ER stress mediated pathways of cell apoptosis, which results in a more effective and efficient induction of programmed cell death in CML. The potential impact of this study on the development of more sophisticated therapy in human malignancy can be substantial.

Fenretinide (N-4-hydroxyphenyl retinamide, 4-HPR), a synthetic analogue of all trans retinoic acid (ATRA), has shown promising potentials in chemopreventive/therapeutic treatment of human malignancies due to its minimal toxicity, ability to control tumor growth in animal models, and synergism with a number of other anti-cancer agents. Clinical trials of fenretinide, as a preventive agent, have proven its efficacy in a number of tumor types including breast cancer, ovary carcinoma, prostate cancer, neuroblastoma and leukemia. In contrast to ATRA which induces cell differentiaion, fenretinide induces cell apoptosis in a variety of tumor cell lines. Although intensive studies have revealed that fenretinide may trigger apoptosis via induction of different signal molecules such as reactive oxygen species (ROS) and ceramide, mechanisms underlying fenretinide induced apoptosis are far from clear, which is largely due to the fact that a multitude of responses occur in cells upon fenretinide treatment. Accordingly, we have applied an approach integrated with cDNA microarray, advanced technology of data and literature mining, and methods of protein biochemistry to study the process of fenretinide-induced apoptosis in leukemia cell line NB4 in detail. Numerous novel features with the temporospatial relationship were revealed, indicating the coordinated regulation of molecular network from various aspects of cellular activities to program cell death. These features include changes in transcriptional regulation, translational regulation, signal transduction, metabolism and stress response. In consequence, cells treated with fenretinide appear to enter programmed cell death through complex cross-talk between organelles such as mitochondria and endoplsmic reticulum. This study may therefore have substantial impact on the development of more sophisticated protocols in leukemia and probably other malignancies as well.