Involvement of TRPC Channels in Lung Cancer Cell Differentiation and the Correlation Analysis in Human Non-Small Cell Lung Cancer

The canonical transient receptor potential (TRPC) channels are Ca²⁺-permeable cationic channels controlling the Ca²⁺ influx evoked by G protein-coupled receptor activation and/or by Ca²⁺ store depletion. Here we investigate the involvement of TRPCs in the cell differentiation of lung cancer. The expression of TRPCs and the correlation to cancer differentiation grade in non-small cell lung cancer (NSCLC) were analyzed by real-time PCR and immunostaining using tissue microarrays from 28 patient lung cancer samples. The association of TRPCs with cell differentiation was also investigated in the lung cancer cell line A549 by PCR and Western blotting. The channel activity was monitored by Ca²⁺ imaging and patch recording after treatment with all-trans-retinoic acid (ATRA). The expression of TRPC1, 3, 4 and 6 was correlated to the differentiation grade of NSCLC in patients, but there was no correlation to age, sex, smoking history and lung cancer cell type. ATRA upregulated TRPC3, TRPC4 and TRPC6 expression and enhanced Ca²⁺ influx in A549 cells, however, ATRA showed no direct effect on TRPC channels. Inhibition of TRPC channels by pore-blocking antibodies decreased the cell mitosis, which was counteracted by chronic treatment with ATRA. Blockade of TRPC channels inhibited A549 cell proliferation, while overexpression of TRPCs increased the proliferation. We conclude that TRPC expression correlates to lung cancer differentiation. TRPCs mediate the pharmacological effect of ATRA and play important roles in regulating lung cancer cell differentiation and proliferation, which gives a new understanding of lung cancer biology and potential anti-cancer therapy.     

肿瘤靶向治疗在NSCLC中的应用

肺癌已成为21世纪威胁人类健康最主要原因之一,全球每年因非小细胞肺癌(NSCLC)死亡的人数超过100万人.传统的抗肿瘤药物极大改善了NSCLC患者的预后.由于缺乏选择性和很强的细胞毒性,多中心研究表明化疗方案疗效已经达到治疗平台,应致力于研发作用机制不同于化疗的药物.肿瘤靶向治疗通过针对特异性的靶点来杀死和抑制肿瘤细胞,避免了对正常细胞的伤害,已成为当前肿瘤研究的热点,并已在临床肿瘤治疗中取得了一定疗效.随着肿瘤分子生物学与细胞生物学的发展,人们对肺癌癌变、侵袭转移的分子机理以及生物信号传导通路的认识加深,新的靶向治疗药物不断出现.本文就近年靶向治疗技术的进展及分子靶向药物在NSCLC中的应用作一综述.     

miR-32在非小细胞肺癌中的表达

目的:基于芯片数据库分析的基础上,探讨miR-32在非小细胞肺癌患者中的表达水平。方法:在GEO和Array Expression数据库中搜索关键词microRNA和肺癌检索出两个样本量最大的芯片数据,分别是GEO数据库中编码为GSE61741和Array Expression数据库中编码为E-TABM-22的芯片数据,对两组数据进行分析找出有差异的microRNA。另外收集2010.02-2012.09期间在吉林大学附属中日联谊医院胸外科进行肺癌手术切除的32名患者的肺癌组织及配对的癌旁正常肺组织标本,检测标本中miR-32的表达水平。结果:综合分析GSE61741和E-TABM-22的芯片数据发现有共同差异的microRNA共有8个,其中上调的有2个,分别是hsa-miR-192与hsa-miR-197,下调的有6个,分别是hsa-miR-126、hsa-miR-199b-3p、hsa-miR-219-1-3p、hsa-miR-26a、hsa-miR-32与hsa-miR-9。为了进一步探讨miR-32在癌症中的作用,我们对GSE61741中其他癌症患者及健康者血浆中miR-32的芯片数据进行分析,发现miR-32在结肠癌、神经胶质瘤、肾癌、前列腺癌、Wilm's瘤中的表达水平均显著低于健康者(P0.01)。对32例及E-TABM-22芯片数据中65例NSCLC患者癌及配对的癌旁正常肺组织中miR-32的表达水平分析发现癌组织中miR-32的表达水平显著下调(P0.001)。结论:miR-32在非小细胞肺癌组织中低表达,提示miR-32可能为新的NSCLC诊断标记分子。     

Phosphosignature Predicts Dasatinib Response in Non-small Cell Lung Cancer

Targeted drugs are less toxic than traditional chemotherapeutic therapies; however, the proportion of patients that benefit from these drugs is often smaller. A marker that confidently predicts patient response to a specific therapy would allow an individual therapy selection most likely to benefit the patient. Here, we used quantitative mass spectrometry to globally profile the basal phosphoproteome of a panel of non-small cell lung cancer cell lines. The effect of the kinase inhibitor dasatinib on cellular growth was tested against the same panel. From the phosphoproteome profiles, we identified 58 phosphorylation sites, which consistently differ between sensitive and resistant cell lines. Many of the corresponding proteins are involved in cell adhesion and cytoskeleton organization. We showed that a signature of only 12 phosphorylation sites is sufficient to accurately predict dasatinib sensitivity. Four of the phosphorylation sites belong to integrin β4, a protein that mediates cell-matrix or cell-cell adhesion. The signature was validated in cross-validation and label switch experiments and in six independently profiled breast cancer cell lines. The study supports that the phosphorylation of integrin β4, as well as eight further proteins comprising the signature, are candidate biomarkers for predicting response to dasatinib in solid tumors. Furthermore, our results show that identifying predictive phosphorylation signatures from global, quantitative phosphoproteomic data is possible and can open a new path to discovering molecular markers for response prediction.     

非小细胞肺癌免疫治疗的研究进展

肺癌(lung cancer)是全球发病率及死亡率最高的恶性肿瘤之一,非小细胞肺癌(non-small cell lung cancer,NSCLC)占肺癌的85%,其五年生存率只有15%,传统的抗肿瘤治疗方法(手术、放疗和化疗等)在抑制肿瘤进展中的作用有限,即使有手术机会,也有40%以上患者出现局部复发或远处转移。目前多学科治疗较大程度提高了晚期NSCLC的生存期,研究表明,免疫治疗(immunotherapy)可改善肺癌的预后,有望成为肺癌的重要辅助治疗方式。其中,治疗性肿瘤疫苗(vaccination)如MAGE-A3、L-BLP25、Belagenpum atucel-L等、免疫检查点抑制剂(immune checkpoint inhibition)如ipilimumab、nivolumab、pembrolizumab等得到广泛关注。一系列临床试验表明免疫治疗可以使非小细胞肺癌的死亡率得到缓解,本文就其原理、临床试验、不良反应及有待解决问题的临床研究作系统综述。     

小细胞肺癌药物治疗的研究进展

小细胞肺癌(small cell lung cancer,SCLC)约占肺癌患者总数的15%-20%,与吸烟关系密切,约60%-70%的患者就诊时已处于广泛期,预后很差,内科药物治疗仍然为其主要的治疗手段。由于其细胞生物学行为复杂,恶性程度高,易出现复发及耐药,通过多药联合、优化给药顺序、改变用药方法及调整药物剂量强度并没有获得预期的疗效,且毒副反应较多,其死亡率依然高居各种肿瘤之首,寻求有效的治疗方案变得迫切而棘手。近年来,随着SCLC内科治疗药物研究的不断深入,一些研究热点药物如bcl-2抑制剂、Hedgehog信号通路抑制剂、ipilimumab、贝伐单抗等已在Ⅱ期或Ⅲ期试验中显示出良好的抗肿瘤活性,为SCLC的药物治疗提供了新的方向。本文就目前针对SCLC的药物治疗进展情况作一综述。     

Pharmacogenomics of Cisplatin Sensitivity in Non-small Cell Lung Cancer

Cisplatin, a platinum-based chemotherapeutic drug, has been used for over 30 years in a wide variety of cancers with varying degrees of success. In particular, cisplatin has been used to treat late stage non-small cell lung cancer （NSCLC） as the standard of care. However, therapeutic outcomes vary from patient to patient. Considerable efforts have been invested to identify biomark- ers that can be used to predict cisplatin sensitivity in NSCLC. Here we reviewed current evidence for cisplatin sensitivity biomarkers in NSCLC. We focused on several key pathways, including nucleotide excision repair, drug transport and metabolism. Both expression and germline DNA variation were evaluated in these key pathways. Current evidence suggests that cisplatin-based treatment could be improved by the use of these biomarkers.     

Selectins Mediate Small Cell Lung Cancer Systemic Metastasis

Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC) patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181). However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.     

长链非编码RNA SNHG1促进肺癌细胞增殖

近年来,大量证据表明长链非编码RNA (long non-coding RNAs, lncRNAs) 在肿瘤发生发展中发挥重要的作用。本研究通过生物信息学预测发现,核仁小分子RNA宿主基因1(small nucleolar RNA host gene 1,SNHG1)无蛋白质编码功能,且主要定位于细胞质。qRT-PCR结果显示,SNHG1在肺癌细胞中的表达量显著高于正常肺上皮细胞。在肺癌细胞NCI-H1299中,敲低SNHG1发现,该细胞增殖能力明显下降;在正常肺上皮细胞BEAS-2B中,过表达SNHG1可显著促进该细胞的增殖能力。深入研究发现,SNHG1可上调CDK4和下调p27kip1在蛋白质水平的表达,而对其mRNA水平表达量无影响。此外,在肺癌临床组织中也发现SNHG1高表达,且高表达的SNHG1与肺癌瘤体大小、TNM分期和远端转移正相关,而与患者年龄及吸烟史无关。综上所述,SNHG1在肺癌中高表达,通过上调CDK4和下调p27kip1推进肺癌进程。     

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44^high) and a main population which was either weakly positive or negative for CD44 (CD44^low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44^highCD90⁺ sub-population. Moreover, these CD44^highCD90⁺ cells revealed mesenchymal morphology, increased expression of mesenchymal markers N-Cadherin and Vimentin, increased mRNA levels of the embryonic stem cell related genes Nanog and Oct4 and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44^highCD90⁺ population a good candidate for the lung CSCs. Both CD44^highCD90⁺ and CD44^highCD90⁻ cells in the PLCCL derived from SCC formed spheroids, whereas the CD44^low/− cells were lacking this potential. These results indicate that CD44^highCD90⁺ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44^high sub-population.     

RBP2 Induces Epithelial-Mesenchymal Transition in Non-Small Cell Lung Cancer

RBP2 has been found to actively participate in cancer progression. It inhibits the senescence of cancer cells, mediates cancer cell proliferation and promotes cancer metastasis. It is also essential to drug tolerance. However, the effects of RBP2 on epithelial-mesenchymal transition are still unknown. In this study, we analyzed the effects of RBP2 on epithelial-mesenchymal transition in non-small cell lung cancer. The results showed that RBP2 down-regulated the expression of E-cadherin by inhibiting the promoter activity of E-cadherin and up-regulated the expression of N-cadherin and snail via the activation of Akt signaling, and the overexpression of RBP2 induced epithelial-mesenchymal transition in non-small cell lung cancer cells. Our study further indicated thatRBP2 may be a potential target for anti-lung cancer therapy.     

小细胞肺癌分子标记物研究进展

肺癌仍然是现在最常见的恶性肿瘤之一。小细胞肺癌(Smallcelllungcancer,SCLC)是肺癌中恶性程度最高的一种类型,与吸烟密切相关,其较早发生远处转移和播散导致预后差,目前的主要治疗手段有手术、化学治疗、放射治疗。但其具有初始化放疗敏感,却很快耐受的特点,导致了它总体预后不良,生存期短。如何寻求一种有效的疾病预后、疗效判断标记物,显得尤为重要。本文主要介绍近年来在小细胞肺癌中分子标记物的研究进展,包括神经内分泌的相关蛋白、凋亡蛋白抑制剂(Survivin)、相关酶类及膜蛋白,这些分子标记物与小细胞肺癌疾病的进展、预后密切相关,能够为临床的疾病治疗评估提供潜在可行的方法。但是,这些标记物仍存在特异性不高的问题,最终应用于临床实践,仍需要更多的临床研究。     

Different Biomarkers in Non-Small Cell Lung Cancer in Blood Vessel Invasion

In clinical settings, lung cancer is divided into small cell lung cancer and non-small cell lung cancer, and chemotherapy is depended on the difference. Using the same chemotherapy treatment, different effects and prognosis can be seen among squamous-cell carcinoma and adenocarcinoma. These differences indicate that there may be various methods of invasion and immunity between squamous-cell carcinoma and adenocarcinoma. Blood vessel invasion and tumor immune escape play very important roles in the progression and metastasis of cancer, and CD105 and integrins are novel therapeutic targets. We assessed the possible association of CD105 expression and integrins with TNM classification in patients with two types of NSCLC. A total of 72 patients with resected Non-Small Cell Lung Cancer (NSCLC) were reviewed retrospectively. Integrin β1, β2, β3, and α5β1 are assayed by immunofluorescence and integrin α5β1 using immunoblot. Intratumoral microvessel density was determined with an anti-CD34 mAb and an anti-CD105 mAb. Invasive ability was assayed with MMP2 and MMP9 using immunofluorescence. The expressions of all integrins, CD105, and CD34 are low in the normal lung tissue and highly expressed in the cancer niche compared to the adjacent tissues. CD105 is highly expressed in the adenocarcinoma niche compared to the squamous-cell carcinoma in NSCLC. The expressions of both MMP2 and MMP9 are low in the normal lung tissue and highly expressed in adjacent tissues. This study shows that blood vessel invasion appears to be an independent negative prognosticator in surgically managed types of NSCLC. However, adequately designed large prospective studies are warranted to confirm the present findings.     

Chloroquine Enhances Gefitinib Cytotoxicity in Gefitinib-Resistant Nonsmall Cell Lung Cancer Cells

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib, are effective for non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, these patients eventually develop resistance to EGFR-TKI. The goal of the present study was to investigate the involvement of autophagy in gefitinib resistance. We developed gefitinib-resistant cells (PC-9/gef) from PC-9 cells (containing exon 19 deletion EGFR) after long-term exposure in gefitinib. PC-9/gef cells (B4 and E3) were 200-fold more resistant to gefitinib than PC-9/wt cells. Compared with PC-9/wt cells, both PC-9/gefB4 and PC-9/gefE3 cells demonstrated higher basal LC3-II levels which were inhibited by 3-methyladenine (3-MA, an autophagy inhibitor) and potentiated by chloroquine (CQ, an inhibitor of autophagolysosomes formation), indicating elevated autophagy in PC-9/gef cells. 3-MA and CQ concentration-dependently inhibited cell survival of both PC-9wt and PC-9/gef cells, suggesting that autophagy may be pro-survival. Furthermore, gefitinib increased LC3-II levels and autolysosome formation in both PC-9/wt cells and PC-9/gef cells. In PC-9/wt cells, CQ potentiated the cytotoxicity by low gefitinib (3nM). Moreover, CQ overcame the acquired gefitinib resistance in PC-9/gef cells by enhancing gefitinib-induced cytotoxicity, activation of caspase 3 and poly (ADP-ribose) polymerase cleavage. Using an in vivo model xenografting with PC-9/wt and PC-9/gefB4 cells, oral administration of gefitinib (50 mg/kg) completely inhibited the tumor growth of PC-9/wt but not PC-9/gefB4cells. Combination of CQ (75 mg/kg, i.p.) and gefitinib was more effective than gefitinib alone in reducing the tumor growth of PC-9/gefB4. Our data suggest that inhibition of autophagy may be a therapeutic strategy to overcome acquired resistance of gefitinib in EGFR mutation NSCLC patients.     

Pokemon在小细胞肺癌中的表达及临床意义

目的:检测Pokemon在小细胞肺癌(SCLC)中的表达情况,并探讨其在SCLC发生、发展中的作用及临床意义。方法:应用SP免疫组化方法检测Pokemon在52例SCLC肿瘤组织及20例病灶旁正常组织中的表达。结果:Pokemon在正常肺组织中不表达,52例SCLC组织中,35例呈阳性表达,占67.3%。Pokemon的表达与SCLC患者的性别、年龄、吸烟、淋巴结转移情况均无关(P0.05),而与TNM分期显著相关(P0.05)。Pokemon阳性表达者生存期明显短于阴性表达者,二者比较差异有统计学意义(P0.05)。单因素分析结果显示TNM分期、Pokemon的表达与SCLC患者的预后相关;多因素分析结果显示仅Pokemon的表达与SCLC患者的预后显著相关(P=0.013)。结论:Pokemon在SCLC组织中呈高表达,预示SCLC患者的预后较差,Pokemon表达可能作为SCLC的独立预后预测因素。     

Antibody Repertoire in Paraneoplastic Cerebellar Degeneration and Small Cell Lung Cancer

The goal of this study is to determine whether patients with paraneoplastic cerebellar degeneration (PCD) and small-cell lung cancer (SCLC) have a specific repertoire of antibodies, if SOX1 antibodies (SOX1-ab) can predict the presence of SCLC, and if antibodies to cell surface antigens occur in this syndrome. Antibody analysis was done using immunohistochemistry on rat brain, immunoblot with recombinant antigens, screening of cDNA expression libraries, and immunolabeling of live neurons in 39 patients with PCD and SCLC. VGCC-ab were measured by RIA, and SOX1-ab, Hu-ab, and ZIC4-ab by immunoblot. Lambert-Eaton myastenic syndrome (LEMS) was present in 10 of 23 patients with electrophysiological studies. At least one antibody was detected in 72% of patients. The individual frequencies were: 49% SOX1-ab, 44% VGCC-ab, 31% Hu-ab, and 13% ZIC4-ab. SOX1-ab occurred in 76% of patients with VGCC-ab and 27% of those without VGCC-ab (p = 0.0036). SOX1-ab were not found in 39 patients with sporadic late-onset cerebellar ataxia, 23 with cerebellar ataxia and glutamic acid decarboxylase antibodies, and 73 with PCD and cancer types other than SCLC (31 without onconeural antibodies, 25 with Yo-ab , 17 with Tr-ab). Five patients (13%) had antibodies against unknown neuronal cell surface antigens but none of them improved with immunotherapy. One serum immunoreacted against the axon initial segment of neurons and another serum against ELKS1, a protein highly expressed in the cerebellum that interacts with the beta4-subunit of the VGCC. In conclusion, 72% of patients with PCD and SCLC had one or more antibodies that indicate the presence of this tumor. In these patients, VGCC-ab and SOX1-ab occur tightly associated. SOX1-ab are predictors of SCLC in ataxia patients with a specificity of 100% and sensitivity of 49%. Unlike limbic encephalitis with SCLC, antibodies to cell surface antigens other than VGCC-ab, are infrequent and do not predict response to treatment.