Detecting ALK,ROS1 and RET Fusion Genes in Cell Block Samples

Whether Cell block (CB) samples are applicable to detect anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and ret proto-oncogene (RET) fusion genes in lung adenocarcinoma is still unknown. In this study, 108 cytological samples that contained lung adenocarcinoma cells were collected, and made into CB. The CB samples all contained at least 30% lung adenocarcinoma cells. In these patients, 48 harbored EGFR mutation. Among the 50 EGFR wild type patients who detected fusion genes, 14 carried EML4-ALK fusion (28%), 2 had TPM3-ROS1 fusion (4%), and 3 harbored KIF5B-RET fusion (6%). No double fusions were found in one sample. Patients with fusion genes were younger than those without fusion genes (p = 0.032), but no significant difference was found in sex and smoking status (p > 0.05). In the thirty-five patients who received first-line chemotherapy, patients with fusion gene positive had disease control rate (DCR) (72.7% VS 50%, p > 0.05) and objective response rate (ORR) (9.1% VS 4.2%, p > 0.05) compared with those having fusion gene negative. The median progression free survival (mPFS) were 4.0 and 2.7 months in patients harbored fusion mutations and wild type, respectively (p > 0.05). We conclude that CB samples could be used to detect ALK, ROS1 and RET fusions in NSCLC. The frequency distribution of three fusion genes is higher in lung adenocarcinoma with wild-type EGFR, compared with unselected NSCLC patient population. Patients with fusion genes positive are younger than those with fusion gene negative, but they had no significantly different PFS in first-line chemotherapy.     

KIF26B在非小细胞肺癌发生发展过程中的表达与功能研究

驱动蛋白与肿瘤的发生有密切联系，但对 KIF26B驱动蛋白在非小细胞肺癌的表达和相关功能作用的研究甚少。为了探索KIF26B在非小细胞肺癌中的表达水平及潜在机制，通过干扰KIF26B后探索对非小细胞肺癌增殖、侵袭、迁移、细胞周期、凋亡以及相关蛋白表达量的影响。对mRNA TCGA 数据库信息分析得出，KIF26B基因在非小细胞肺癌中高表达。qRT-PCR 检测 KIF26B在几株常见非小细胞肺癌细胞系中的表达水平，筛选出 KIF26B在A549 和 NCI-H292细胞系中高表达。利用 RNA干扰技术(RNA interference, RNAi)敲低 A549 和 NCI-H292细胞的 KIF26B基因，通过CCK8、采用实时细胞分析仪、平板克隆及 Transwell 实验检测敲低 KIF26B基因后的生物学功能，免疫印迹法检测蛋白表达水平。结果显示，敲低KIF26B后A549 和 NCI-H292细胞增殖明显降低，侵袭及迁移能力明显减弱。敲低KIF26B后阻碍了A549 和 NCI-H292细胞从G1期向S期的转变，同时凋亡细胞明显增多，与之相关的细胞周期蛋白 D1、Bcl-2、E-cadherin和Vimentin的表达水平显著下调，同时活化的半胱天冬酶-3（active Caspase-3）和其剪切底物 PARP1 的剪切体（cleaved PARP1）表达水平显著上调。结果表明KIF26B可能作为非小细胞肺癌发生的促癌基因，参与了非小细胞肺癌的发生及发展过程。KIF26B有望成为非小细胞肺癌治疗的潜在靶点。     

RET, ROS1 and ALK fusions in lung cancer

Through an integrated molecular- and histopathology-based screening system, we performed a screening for fusions of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1, receptor tyrosine kinase (ROS1) in 1,529 lung cancers and identified 44 ALK-fusion-positive and 13 ROS1-fusion-positive adenocarcinomas, including for unidentified fusion partners for ROS1. In addition, we discovered previously unidentified kinase fusions that may be promising for molecular-targeted therapy, kinesin family member 5B (KIF5B)-ret proto-oncogene (RET) and coiled-coil domain containing 6 (CCDC6)-RET, in 14 adenocarcinomas. A multivariate analysis of 1,116 adenocarcinomas containing these 71 kinase-fusion-positive adenocarcinomas identified four independent factors that are indicators of poor prognosis: age ≥ 50 years, male sex, high pathological stage and negative kinase-fusion status.     

Identification of KIF4A and its effect on the progression of lung adenocarcinoma based on the bioinformatics analysis

Background: Lung adenocarcinoma (LUAD) is the most frequent histological type of lung cancer, and its incidence has displayed an upward trend in recent years. Nevertheless, little is known regarding effective biomarkers for LUAD.Methods: The robust rank aggregation method was used to mine differentially expressed genes (DEGs) from the gene expression omnibus (GEO) datasets. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to extract hub genes from the protein–protein interaction (PPI) network. The expression of the hub genes was validated using expression profiles from TCGA and Oncomine databases and was verified by real-time quantitative PCR (qRT-PCR). The module and survival analyses of the hub genes were determined using Cytoscape and Kaplan–Meier curves. The function of KIF4A as a hub gene was investigated in LUAD cell lines.Results: The PPI analysis identified seven DEGs including BIRC5, DLGAP5, CENPF, KIF4A, TOP2A, AURKA, and CCNA2, which were significantly upregulated in Oncomine and TCGA LUAD datasets, and were verified by qRT-PCR in our clinical samples. We determined the overall and disease-free survival analysis of the seven hub genes using GEPIA. We further found that CENPF, DLGAP5, and KIF4A expressions were positively correlated with clinical stage. In LUAD cell lines, proliferation and migration were inhibited and apoptosis was promoted by knocking down KIF4A expression.Conclusion: We have identified new DEGs and functional pathways involved in LUAD. KIF4A, as a hub gene, promoted the progression of LUAD and might represent a potential therapeutic target for molecular cancer therapy.     

Proteomic detection of cancer in asbestosis patients using SELDI-TOF discovered serum protein biomarkers

Objectives: To identify biomarkers for cancer in asbestosis patients.

Methods: SELDI-TOF and CART were used to identify serum biomarker profiles in 35 asbestosis patients who subsequently developed cancer and 35 did not develop cancer.

Results: Three polypeptide peaks (5707.01, 6598.10, and 20,780.70?Da) could predict the development of cancer with 87% sensitivity and 70% specificity. The first two peaks were identified as KIF18A and KIF5A, respectively, and are part of the Kinesin Superfamily of proteins.

Conclusions: We identified two Kinesin proteins that can be potentially used as blood biomarkers to identify asbestosis patients at risk of developing lung cancer.     

The Motor Protein KIF14 Inhibits Tumor Growth and Cancer Metastasis in Lung Adenocarcinoma

The motor protein kinesin superfamily proteins (KIFs) are involved in cancer progression. The depletion of one of the KIFs, KIF14, might delay the metaphase-to-anaphase transition, resulting in a binucleated status, which enhances tumor progression; however, the exact correlation between KIF14 and cancer progression remains ambiguous. In this study, using loss of heterozygosity and array comparative genomic hybridization analyses, we observed a 30% loss in the regions surrounding KIF14 on chromosome 1q in lung adenocarcinomas. In addition, the protein expression levels of KIF14 in 122 lung adenocarcinomas also indicated that approximately 30% of adenocarcinomas showed KIF14 down-regulation compared with the expression in the bronchial epithelial cells of adjacent normal counterparts. In addition, the reduced expression of KIF14 mRNA or proteins was correlated with poor overall survival (P = 0.0158 and <0.0001, respectively), and the protein levels were also inversely correlated with metastasis (P<0.0001). The overexpression of KIF14 in lung adenocarcinoma cells inhibited anchorage-independent growth in vitro and xenograft tumor growth in vivo. The overexpression and silencing of KIF14 also inhibited or enhanced cancer cell migration, invasion and adhesion to the extracellular matrix proteins laminin and collagen IV. Furthermore, we detected the adhesion molecules cadherin 11 (CDH11) and melanoma cell adhesion molecule (MCAM) as cargo on KIF14. The overexpression and silencing of KIF14 enhanced or reduced the recruitment of CDH11 in the membrane fraction, suggesting that KIF14 might act through recruiting adhesion molecules to the cell membrane and modulating cell adhesive, migratory and invasive properties. Thus, KIF14 might inhibit tumor growth and cancer metastasis in lung adenocarcinomas.     

Impact of comorbidity on survival by tumour location: Breast,colorectal and lung cancer (2000–2014)

BackgroundTo assess the impact of comorbidity, measured by the Charlson Comorbidity Index (CCI), on survival in breast, colorectal and lung cancer.MethodsWe identified 3455 breast cancer, 3336 colorectal cancer and 2654 lung cancer patients through the Hospital del Mar cancer registry. The prevalence of comorbidities according to the CCI was calculated. Kaplan-Meier curves and the log-rank test were used to compare survival curves for each cancer location. Cox regression was used to calculate survival hazard ratios and 1-, 3- and 5-year mortality rate ratios adjusted by age, sex, CCI, place of first consultation, stage, treatment and period of diagnosis.ResultsThe overall unadjusted 5-year follow-up survival proportion was 82.6% for breast cancer, 55.7% for colorectal cancer, and 16.3% for lung cancer. Overall survival was associated with CCI ≥ 3 in breast cancer (HR: 2.33 95%CI: 1.76–3.08), colorectal cancer (HR: 1.39; 95%CI: 1.13–1.70) and lung cancer (HR: 1.22; 95%CI: 1.06–1.40). In breast cancer, the higher the CCI, the higher the adjusted mortality rate ratio and differences were greater in 5-year than in 1-year follow-up survival.ConclusionsComorbidity is a significant predictor of overall survival in cancer patients; however, it has a stronger impact on survival in breast cancer than in colorectal and lung cancer.     

Identification of Cytoskeleton-Associated Proteins Essential for Lysosomal Stability and Survival of Human Cancer Cells

Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, i.e. increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.     

非小细胞肺癌组织KIF2A、KIF2C、KIF20A mRNA表达与临床病理特征和预后的关系

摘要 目的：探讨非小细胞肺癌（NSCLC）组织驱动蛋白超家族成员2A（KIF2A）、驱动蛋白超家族成员2C（KIF2C）、驱动蛋白超家族成员20A（KIF20A）信使核糖核酸（mRNA）表达与临床病理特征和预后的关系。方法：选择2016年9月至2019年9月天津医科大学总医院手术切除的NSCLC患者106例,取其癌组织及其对应的癌旁组织,应用荧光定量聚合酶链式反应（RT-qPCR）检测组织中KIF2A、KIF2C、KIF20A mRNA表达,分析其与临床病理特征的关系。应用 Pearson相关性分析NSCLC组织中KIF2A、KIF2C、KIF20A mRNA表达间的关系。随访3年,应用Kaplan-Meier生存曲线分析KIF2A、KIF2C、KIF20A mRNA表达与患者预后关系。结果：NSCLC癌组织中KIF2A、KIF2C、KIF20A mRNA表达水平显著高于癌旁组织（P<0.05）。低分化、淋巴结转移、临床分期Ⅲ A 期NSCLC癌组织中KIF2A、KIF2C、KIF20A mRNA表达水平显著高于中高分化、无淋巴结转移及临床分期I、II期NSCLC癌组织（P<0.05）。Pearson相关分析显示,NSCLC癌组织中KIF2A mRNA表达与KIF2CmRNA、KIF20A mRNA表达呈正相关,KIF2C mRNA表达与KIF20A mRNA表达呈正相关（P<0.05）。Kaplan-Meier法分析显示KIF2A mRNA低表达组、KIF2C mRNA低表达组、KIF20A mRNA低表达组3年生存率分别为（84.78%,86.27%,81.48%）显著高于KIF2A mRNA高表达组、KIF2C mRNA高表达组、KIF20A mRNA高表达组（59.62%,55.32%,59.09%）（P<0.05）。结论：KIF2A、KIF2C、KIF20A mRNA在NSCLC组织中存在高表达,且与低分化、淋巴结转移、临床分期及预后有关。     

Polymorphisms of arylamine N-acetyltransferase2 and risk of lung and colorectal cancer

The arylamine N-acetyltransferase 2 (NAT2) enzymes detoxify a wide range of naturally occurring xenobiotics including carcinogens and drugs. Point mutations in the NAT2 gene result in the variant alleles M1 (NAT2 *5A), M2 (NAT2*6A), M3 (NAT2*7) and M4 (NAT2 *14A) from the wild-type WT (NAT2 *4) allele. The current study was aimed at screening genetic polymorphisms of NAT2 gene in 49 lung cancer patients, 54 colorectal cancer patients and 99 cancer-free controls, using PCR-RFLP. There were significant differences in allele frequencies between lung cancer patients and controls in the WT, M2 and M3 alleles (p < 0.05). However, only M2 and M3 allele frequencies were different between colorectal cancer patients and controls (p < 0.05). There was a marginal significant difference in the distribution of rapid and slow acetylator genotypes between lung cancer patients and controls (p = 0.06 and p = 0.05, respectively), but not between colorectal cancer patients and controls (p = 1.0 and p = 0.95, respectively). Risk of lung cancer development was found to be lower in slow acetylators [odds ratio (OR): 0.51, 95% confidence interval (95% CI): 0.25, 1.02, p-value = 0.07]. No effect was observed in case of colorectal cancer. Our results showed that NAT2 genotypes and phenotypes might be involved in lung cancer but not colorectal cancer susceptibility in Jordan.     

KIF2C exerts an oncogenic role in nonsmall cell lung cancer and is negatively regulated by miR‐325‐3p

Nonsmall cell lung cancer (NSCLC) is one of the leading causes of cancer‐related death worldwide. Kinesin family member 2C (KIF2C), a modulator in microtubule depolymerization, bipolar spindle formation, and chromosome segregation, has been reported to take roles in cancer biology, but its role in NSCLC remains unclear. This study was intended to investigate the expression and function of KIF2C in NSCLC. Our results demonstrated that KIF2C was up‐regulated in NSCLC tissues and cell lines. The high expression of KIF2C in NSCLC tissues was significantly correlated with higher T stage (0.0078), worse differentiation status (0.0049), and lymph node metastasis (P < .0001). We also proved that the high expression level of KIF2C predicted worse prognosis of the patients. After knockdown of KIF2C, the proliferation and metastasis of NSCLC cells were inhibited. Luciferase reporter assay suggested that KIF2C was a target gene of miR‐325‐3p, which was reported to be a tumour suppressor in NSCLC. In conclusion, this study proved an oncogenic role of KIF2C in NSCLC and partly clarified the mechanism of its high expression. Our findings provided a useful insight into the mechanism of NSCLC progression and offered clues to novel therapy strategies.     

Depletion of Kinesin 5B Affects Lysosomal Distribution and Stability and Induces Peri-Nuclear Accumulation of Autophagosomes in Cancer Cells

Background

Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of the ErbB2 (ΔN-ErbB2).

Methodology/Principal Findings

Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic ΔN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase endocytosis functioned, however, normally in these cells. Both HeLa and MCF-7 cells appeared to express similar levels of the KIF5B isoform but the death phenotype was weaker in KIF5B-depleted MCF-7 cells. Surprisingly, KIF5B depletion inhibited the rapamycin-induced accumulation of autophagosomes in MCF-7 cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm.

Conclusions/Significance

Our data identify KIF5B as a cancer relevant lysosomal motor protein with additional functions in autophagosome formation.     

Activation of signal pathways and the resistance to anti-EGFR treatment in colorectal cancer

Colorectal cancer is the third most common cancer with a 5-year survival rate of less than 10%. It is caused by alterations of multiple signal pathways which are affected by both genetic and environmental factors. In some cases, EGFR is important in the carcinogenesis of colorectal cancer suggesting anti-EGFR therapy may be a potential treatment option. However, in other cases it is not effective, which may be related to its down-stream targeted gene mutations. KRAS is highly emphasized in the literature but other mutations like Src, PIK3CA, and BRAF may also be important. Furthermore, obesity may decrease the effectiveness of anti-EGFR treatment as it increases the risk factors for colorectal cancer. Using next-generation sequencing technology, it may be possible to identify all gene mutations in an individual with colorectal cancer. Therefore, gene mutations affecting anti-EGFR therapy in colorectal cancer patients can be identified.     

Comparative Screening of K-ras Mutations in Colorectal Cancer and Lung Cancer Patients Using a Novel Real-Time PCR with ADx-K-ras Kit and Sanger DNA Sequencing

We determined frequency/types of K-ras mutations in colorectal/lung cancer. ADx-K-ras kit (real-time/double-loop probe PCR) was used to detect somatic tumor gene mutations compared with Sanger DNA sequencing using 583 colorectal and 244 lung cancer paraffin-embedded clinical samples. Genomic DNA was used in both methods; mutation rates at codons 12/13 and frequency of each mutation were detected and compared. The data show that 91.4% colorectal and 59.0% lung carcinoma samples were detected conclusively by DNA sequencing, whereas 100% colorectal and lung samples were detected by ADx-K-ras kit. K-ras gene mutations were detected in 32.9–27.4% colorectal samples using kit and sequencing methods, respectively. Whereas 10.6–8.3% lung cancer samples were positively detected by kit and sequencing methods, respectively. Notably, 172/677 showed mutations and 467/677 showed wild type by both methods; 38 samples showed mutations with kit but wild type with sequencing. Mutations in colorectal samples were as follows: GGT → GAT/codon-12 (35.1%); GGC → GAC/codon-13 (26.6%); GGT → GTT/codon-12 (18.2%); and GGT → GCT/codon-12 (1.6%). Mutations in lung samples were as follows: GGT > GTT/codon-12 (40.9%) and GGT > GCT/codon-12 (4.5%). In conclusion, K-ras mutations involved 32.2% colorectal and 10.6% lung samples among this cohort. ADx-K-ras real-time PCR showed higher detection rates (P < 0.05). The kit method has good clinical applicability as it is simple, fast, less prone to contamination and hence can be used effectively and reliably for clinical screening of somatic tumor gene mutations.