DDX39, upregulated in lung squamous cell cancer, displays RNA helicase activities and promotes cancer cell growth

To explore differentially expressed genes involved in non-small cell lung cancer progression, we used the gene expression profile database of various human tissues and identified DDX39, a new member of the DEAD box RNA helicases, showing overexpression in human lung squamous cell carcinoma (LSCC) but not in lung adenocarcinoma (LAC). There existed three types of alternatively spliced DDX39 variants (DDX39-L, -S and -SS), of which only DDX39-L contains all the motifs required for RNA helicase activity. RT-PCR analysis verified the increased expression of DDX39-L in LSCC, but not LAC, cultured cells compared with normal bronchial epithelial cells. A high sequence similarity to UAP56 and punctate nuclear localization pattern of DDX39-L suggest that it plays a role in RNA splicing/export. Recombinant DDX39-L binds RNA, hydrolyzes NTPs in an RNA-dependent manner and unwinds double strand RNA bidirectionally, proving that DDX39 is an RNA helicase. Overexpression of DDX39-L stimulates colony formation of HeLa cells, probably through elevation of a translational level, indicating the biological significance of DDX39 in cancer pathogenesis. Thus, DDX39 is a novel RNA helicase capable of promoting cancer cell growth and, thereby, can be a potential target for development of a therapeutic strategy for LSCC.     

Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer

Identifying druggable targets in the Wnt-signaling pathway can optimize colorectal cancer treatment. Recent studies have identified a member of the RNA helicase family DDX3 (DDX3X) as a multilevel activator of Wnt signaling in cells without activating mutations in the Wnt-signaling pathway. In this study, we evaluated whether DDX3 plays a role in the constitutively active Wnt pathway that drives colorectal cancer.We determined DDX3 expression levels in 303 colorectal cancers by immunohistochemistry. 39% of tumors overexpressed DDX3. High cytoplasmic DDX3 expression correlated with nuclear β-catenin expression, a marker of activated Wnt signaling. Functionally, we validated this finding in vitro and found that inhibition of DDX3 with siRNA resulted in reduced TCF4-reporter activity and lowered the mRNA expression levels of downstream TCF4-regulated genes. In addition, DDX3 knockdown in colorectal cancer cell lines reduced proliferation and caused a G1 arrest, supporting a potential oncogenic role of DDX3 in colorectal cancer.RK-33 is a small molecule inhibitor designed to bind to the ATP-binding site of DDX3. Treatment of colorectal cancer cell lines and patient-derived 3D cultures with RK-33 inhibited growth and promoted cell death with IC50 values ranging from 2.5 to 8 μM. The highest RK-33 sensitivity was observed in tumors with wild-type APC-status and a mutation in CTNNB1.Based on these results, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes inhibition of Wnt signaling and may therefore be a promising future treatment strategy for a subset of colorectal cancers.     

Development and validation of prognostic nomograms for metastatic gastrointestinal stromal tumour treated with imatinib

PurposeMetastatic gastrointestinal stromal tumour (GIST) is generally an incurable disease with variable response to imatinib. We aimed to develop prognostic nomograms to predict overall survival (OS) and progression-free survival (PFS) for patients treated with imatinib.MethodsNomograms were developed in a training cohort (n = 330) of patients treated in a randomised trial (EORTC-ISG-AGITG 62005 phase III study) using Cox regression models, and validated in patients (n = 236) treated in routine clinical care from six referral centres. Nomogram performance was assessed by calculating the c statistic. A classification based on the nomograms’ scores was generated to group patients according to risk.ResultsNomogram risk factors for OS and PFS were size of the largest metastasis, tumour genotype, primary tumour mitotic count, haemoglobin and blood neutrophil count at commencement of imatinib. The nomograms predicted survival with a c statistic of 0.75 (training) and 0.62 (validation) for OS, and 0.69 (training) and 0.62 (validation) for PFS. When tested in the validation cohort, the nomograms discriminated well the high and intermediate risk from low risk patients (hazard ratio [HR] for OS 3.83, 95% confidence interval [CI] 1.71–8.56; and 2.48, 95% CI 1.12–5.50; for PFS 2.84, 95% CI 1.66–4.87; and 1.45, 95% CI 0.87–2.41, respectively).ConclusionThe nomograms predicted the risk of GIST progression and death with good discrimination of risk groups, and may be of value for patient counselling and risk stratification.     

胃癌中RNA解旋酶DDX19A的表达及临床意义

背景与目的：RNA解旋酶DDX19A（DEAD-box helicases 19A）是DDX蛋白超家族的成员之一,主要参与RNA的转运过程。该家族的许多成员参与肿瘤的发生及发展过程,然而关于DDX19A在胃癌中的作用,目前尚未见报道。结合生物信息学分析,探讨胃癌组织中DDX19A的表达情况及临床意义。方法：采用UALCAN数据库,转录水平分析胃癌及癌旁胃组织中DDX19A的表达差异,进一步分析其在胃癌中的表达及与临床分期的关系;采用Kaplan-Meier Plotter数据库分析DDX19A表达水平与胃癌患者总生存期（overall survival,OS）及无病生存期（disease-free survival,DFS）的相关性。采用蛋白质印迹法（Western blot）检测16对冻存的新鲜胃癌组织及配对癌旁胃黏膜中DDX19A蛋白的表达。收集2011—2015年承德医学院附属医院病理科存档的胃癌石蜡包埋标本109例、癌旁胃黏膜标本30例,所有患者临床病理学资料完整。免疫组织化学S-P四步法检测109例胃癌及30例癌旁胃黏膜中DDX19A蛋白的表达,分析其高表达与胃癌临床病理学特征及预后的关系。结果：UALCAN及Kaplan-Meier Plotter数据库检索结果显示,DDX19A mRNA在胃癌组织中的表达明显高于癌旁胃组织（P<0.01）,其高表达与胃癌临床分期及患者不良预后正相关（P<0.05）。Western blot结果显示,胃癌组织中DDX19A蛋白显著高表达（P<0.01）。免疫组织化学结果显示,胃癌组织中的DDX19A阳性表达率为68.8%（75/109）,显著高于其在癌旁胃黏膜中的表达（33.3%,10/30）,差异有统计学意义（P<0.01）。DDX19A高表达与胃癌分化程度、浸润深度、TNM分期密切相关（P<0.05）,而与患者性别、年龄、肿瘤大小、淋巴结转移无关（P>0.05）。69例随访资料完整的病例中,DDX19A高表达患者的OS明显短于低表达患者（P<0.05）。结论：DDX19A在胃癌组织中明显高表达,并且其高表达与胃癌临床分期、侵袭性及患者不良预后相关。DDX19A有望成为与胃癌预后相关的潜在生物标志物及治疗的新靶点。     

Validation of the Joensuu risk criteria for primary resectable gastrointestinal stromal tumour – The impact of tumour rupture on patient outcomes

Background

Approval of imatinib for adjuvant treatment of gastrointestinal stromal tumours (GIST) raised discussion about accuracy of prognostic factors in GIST and the clinical significance of the available risk stratification criteria.

Methods

We studied the influence of a new modification of the NIH Consensus Criteria (the Joensuu risk criteria), NCCN-AFIP criteria, and several clinicopathological factors, including tumour rupture, on relapse-free survival (RFS) in a prospectively collected tumour registry series consisting of 640 consecutive patients with primary, resectable, CD117-immunopositive GIST. The median follow-up time after tumour resection was 39 months. None of the patients received adjuvant imatinib.

Results

The median RFS time after surgery was 50 months. In univariable analyses, high Joensuu risk group, tumour mitotic count >5/50 HPF, size >5 cm, non-gastric location, tumour rupture (7% of cases; P = 0.0014) and male gender had adverse influence on RFS. In a multivariable analysis mitotic count >5/50HPF, tumour size >5 cm and non-gastric location were independent adverse prognostic factors. Forty, 151, 86 and 348 patients were assigned according to the Joensuu criteria to very low, low, intermediate and high risk groups and had 5-year RFS of 94%, 94%, 86% and 29%, respectively.

Conclusion

The Joensuu criteria, which include 4 prognostic factors (tumour size, site, mitotic count and rupture) and 3 categories for the mitotic count, were found to be a reliable tool for assessing prognosis of operable GIST. The Joensuu criteria identified particularly well high risk patients, who are likely the proper candidates for adjuvant therapy.     

Circulating tumour cells and circulating free nucleic acid as prognostic and predictive biomarkers in colorectal cancer

The detection of circulating tumour cells or circulating free tumour nucleic acids can potentially guide treatment and inform prognosis in colorectal cancer using minimally invasive “liquid biopsies”. Current literature supports the notion that high circulating tumour cell counts or presence of tumour nucleic acid correlate with inferior clinical outcomes for patients, but they are not yet part of routine clinical care. Future research evolves around the examination of the molecular phenotype of circulating tumour cells. The key unanswered areas include differentiating between circulating tumour cell presence and their proliferative capacity and dormancy, identifying tumour heterogeneity and understanding the epithelial–mesenchymal transition.     

胃肠道间质瘤临床特征及预后影响因素分析

目的:探讨胃肠道间质瘤临床特征及预后影响因素。方法:回顾性分析我院2011年3月至2014年1月收治的653例胃肠道间质瘤患者临床资料,归纳临床特征,采用统计学方法分析影响预后的危险因素。结果:男女性别比为1.17∶1,年龄分布以40岁以上人群为主(84.07%)。首发症状以消化道出血(37.98%)、腹痛及腹胀(32.01%)、消化道症状(14.85%)为主,部分患者无明显症状(4.44%)。肿瘤发生部位以小肠(20.06%)和胃部(58.35%)为主。581例(88.97%)患者行根治性切除,72例(11.03%)患者行姑息性切除。根治性手术患者中,137例(20.98%)患者行联合脏器切除。112例(17.15%)患者行淋巴结清扫,淋巴结清扫数平均为(23.45±6.12)枚。63例患者术后采用伊马替尼（400 mg/d）口服治疗。371例(56.81%)患者核分裂象≤5个/50 HPF,282例(43.18%)患者核分裂象＞5个/50 HPF。CD117阳性581例(88.97%),S-100阳性61例(9.34%),CD34阳性568例(86.98%)。患者1年、3年及5年总体生存率为96.71%、76.52%和61.83%。单因素分析发现核分裂象、肿瘤最大径、手术根治性及改良NIH危险度分级与胃肠道间质瘤患者5年生存率有关,多因素回归分析证实以上因素是影响患者5年生存率的独立危险因素(P＜0.05)。结论:胃肠道间质瘤主要分布在40岁以上人群,但临床症状不典型,可通过核分裂象、肿瘤最大径、手术根治性及改良NIH危险度分级评估预后。     

Pfetin as a prognostic biomarker of gastrointestinal stromal tumors revealed by proteomics.

PURPOSE: We aimed to develop prognostic biomarkers for gastrointestinal stromal tumors (GIST) using a proteomic approach. EXPERIMENTAL DESIGN: We examined the proteomic profile of GISTs using two-dimensional difference gel electrophoresis. The prognostic performance of biomarker candidates was examined using a large-scale sample set and specific antibodies. RESULTS: We identified 43 protein spots whose intensity was statistically different between GISTs with good and poor prognosis. Mass spectrometric protein identification showed that the 43 spots corresponded to 25 distinct gene products. Eight of the 43 spots derived from pfetin, a potassium channel protein, and four of the eight pfetin spots had a high discriminative power between the two groups. Western blotting and real-time PCR showed that pfetin expression and tumor metastasis were inversely related. The prognostic performance of pfetin was also examined by immunohistochemistry on 210 GIST cases. The 5-year metastasis-free survival rate was 93.9% and 36.2% for patients with pfetin-positive and pfetin-negative tumors, respectively (P < 0.0001). Univariate and multivariate analyses revealed that pfetin expression was a powerful prognostic factor among the clinicopathologic variables examined, including risk classification and c-kit- or platelet-derived growth factor receptor A mutation status. CONCLUSIONS: These results establish pfetin as a powerful prognostic marker for GISTs and may provide novel therapeutic strategies to prevent metastasis of GIST.     

Role of human tissue kallikrein in gastrointestinal stromal tumour invasion

Background:

Human tissue kallikrein (hK1) generates vasodilator kinins from kininogen and promotes angiogenesis by kinin-dependent and kinin-independent mechanisms. Here, we investigate the expression and functional relevance of hK1 in human gastrointestinal stromal tumour (GIST).

Methods:

Vascularisation and hK1 expression of GIST samples were assessed by immunohistochemistry. In two GIST cell lines, hK1 expression was assessed by PCR, and hK1 protein levels and activity were measured by ELISA and an amidolytic assay, respectively. The effect of hK1 silencing, inhibition or overexpression on GIST cell proliferation, migration and paracrine induction of angiogenesis was studied. Finally, local and systemic levels of hK1 were assessed in mice injected with GIST cells.

Results:

Human tissue kallikrein was detected in 19 out of 22 human GIST samples. Moreover, GIST cells express and secrete active hK1. Titration of hK1 demonstrated its involvement in GIST invasive behaviour, but not proliferation. Furthermore, hK1 released by GIST cells promoted endothelial cell migration and network formation through kinin-dependent mechanisms. Gastrointestinal stromal tumour implantation in nude mice resulted in local and systemic hK1 expression proportional to tumour dimension.

Conclusions:

Human tissue kallikrein is produced and released by GIST and participates in tumour invasion. Further studies are needed to validate hK1 as a diagnostic biomarker and therapeutic target in GIST.     

RNA helicase DDX5 is a p53-independent target of ARF that participates in ribosome biogenesis

The p19ARF tumor suppressor limits ribosome biogenesis and responds to hyperproliferative signals to activate the p53 checkpoint response. Although its activation of p53 has been well characterized, the role of ARF in restraining nucleolar ribosome production is poorly understood. Here we report the use of a mass spectroscopic analysis to identify protein changes within the nucleoli of Arf-deficient mouse cells. Through this approach, we discovered that ARF limited the nucleolar localization of the RNA helicase DDX5, which promotes the synthesis and maturation of rRNA, ultimately increasing ribosome output and proliferation. ARF inhibited the interaction between DDX5 and nucleophosmin (NPM), preventing association of DDX5 with the rDNA promoter and nuclear pre-ribosomes. In addition, Arf-deficient cells transformed by oncogenic RasV12 were addicted to DDX5, because reduction of DDX5 was sufficient to impair RasV12-driven colony formation in soft agar and tumor growth in mice. Taken together, our findings indicate that DDX5 is a key p53-independent target of the ARF tumor suppressor and is a novel non-oncogene participant in ribosome biogenesis.     

解旋酶DDX46 基因对食管鳞癌Eca-109细胞增殖及凋亡的影响

目的：探讨RNA解旋酶DDX46基因对食管鳞癌细胞株Eca-109增殖和凋亡的影响及可能的作用机制。方法：以人永生化食管鳞状上皮细胞Het-1A为对照,实时荧光定量PCR（qPCR）检测DDX46 mRNA在Eca-109细胞中的相对表达水平。应用shRNA干扰技术沉默Eca-109细胞DDX46基因后,分别采用MTT实验、克隆形成实验及流式细胞术检测细胞增殖情况、克隆形成能力以及细胞周期和细胞凋亡情况。并用Western blot检测DDX46基因沉默后Eca-109细胞DDX46蛋白和凋亡信号转导通路关键分子Caspase-3和PARP-1蛋白表达的变化。结果：与Het-1A细胞相比,Eca-109细胞DDX46 mRNA的表达水平显著升高（P < 0.01）。与对照组相比,靶向沉默DDX46基因后MTT实验显示Eca-109细胞活力明显减弱,增殖能力被显著抑制（P < 0.01）;克隆形成实验显示Eca-109细胞克隆形成能力被显著抑制（P < 0.01）;流式细胞术检测显示处于G1期的细胞增加,而处于S期的细胞减少（P < 0.05）,细胞周期停滞于G0/G1期;凋亡实验显示细胞凋亡率显著增加（P < 0.01）。Western blot检测显示,与对照组比较,DDX46-shRNA干扰使Eca-109细胞DDX46蛋白表达水平显著下降（P < 0.01）,而cleaved Caspase-3和cleaved PARP-1蛋白表达水平显著上升（P < 0.01）。结论：DDX46 mRNA在食管鳞癌Eca-109细胞中高表达,DDX46基因沉默可能通过激活细胞凋亡信号转导通路而发挥抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡的作用。     

长链非编码RNA作为食管癌潜在的预后生物标志物的系统评价及荟萃分析

目的通过系统回顾和荟萃分析探讨多种长链非编码RNA(lncRNA)在食管癌预后预测中的价值。方法在PubMed、Embase、Web of Science、中国知网和万方数据库中检索相关文献,检索关键词为"lncRNA""食管癌"。应用风险比(HR)及95%置信区间(CI)分析lncRNA表达情况与食管癌患者预后的关系,采用Stata 15.0软件进行数据分析。结果本研究共纳入25篇文献,3830例患者,22种不同的lncRNA与食管癌患者预后有关。通过荟萃分析确定了HOTAIR和MALAT1对食管癌患者的预后价值。合并效应量后的荟萃分析结果显示,HOTAIR过表达与食管癌患者生存期较短有关(HR=2.404,95%CI:1.661~3.479,P﹤0.01)。MALAT1的表达情况与食管癌患者的预后无明显相关性(HR=3.384,95%CI:0.948~12.082,P=0.060)。结论 lncRNA尤其是HOTAIR可能是食管癌的潜在预后标志物。     

MicroRNAs as diagnostic and prognostic biomarkers in colorectal cancer

MicroRNAs (miRNAs) are key regulators involved in various tumors. They regulate cell cycle, apoptosis and cancer stemness, metastasis and chemoresistance by controlling their target gene expressions. Here, we mainly discuss the potential uses of miRNAs in colorectal cancer (CRC) diagnosis. We also shed light on the important corresponding miRNA targets and on the major regulators of miRNAs. Furthermore, we discuss miRNA activity in assessing the prognosis and recurrence of CRC as well as in modulating responsiveness to chemotherapy. Based on the various pro-oncogenic/anti-oncogenic roles of miRNAs, the advantages of a therapeutic strategy based on the delivery of miRNA mimics are also mentioned. Together, miRNA seems to be an excellent tool for effectively monitoring and targeting CRC.     

Relative hypocalcaemia and muscle cramps in patients receiving imatinib for gastrointestinal stromal tumour

Purpose. Imatinib treatment causes muscle cramps in up to 40% of patients, but their pathogenesis is unknown. We present a case series illustrating an association between imatinib, relative hypocalcaemia, and the development of cramps. Patients. The index patient developed muscle spasms and cramps after receiving imatinib for gastrointestinal stromal tumour (GIST) for 5 months. The adjusted serum calcium had dropped to the lower limit of normal. The low serum calcium and muscle cramps improved on stopping imatinib and recurred on rechallenge. We reviewed the medical records of 16 further patients. Results. Two patients reported muscle cramps (12%). There was a rapid and sustained reduction in adjusted serum calcium in the first 6 months from 2.45 +/- 0.11 mmol/L (mean +/- SD) to 2.30 +/- 0.08 mmol/L (p = 0.025). Conclusion. Imatinib treatment of GIST is associated with reduction in serum calcium which may explain the development of neuromuscular symptoms. In patients receiving imatinib, serum electrolytes should be monitored and muscle cramps treated by correction of serum calcium, or an empirical trial of quinine sulphate.