PAK Group I的活性减弱促进肺癌细胞对EGFR-TKIs的敏感性

目的:探讨p21活化激酶(p21-activated kinase,PAK)是否影响肺癌细胞对EGFR-TKIs的敏感性.方法:肺癌细胞系中加入PAK Group I抑制剂(IPA3)后,应用MTT、流式细胞术观察肺癌细胞的增殖能力.结果:IPA3和吉非替尼虽然能在不同程度上抑制肺癌细胞的增殖,但二者的联合用药能明显增强对肺癌细胞增殖的抑制作用.结论:PAK Group I的活性减弱后能抑制肺癌细胞的增殖,促进肺癌细胞对EGFR-TKIs的敏感性.     

PAK2通过调节ERK信号通路的活性促进肺癌细胞顺铂耐药

目的:探讨p21激活激酶2(PAK2)在非小细胞肺癌（NSCLC）中是否存在异常表达,以及是否影响肺癌细胞的顺铂敏感性。方法:采用免疫组化和Western blot（WB）方法检测NSCLC中PAK2的蛋白表达模式。利用PAK2-siRNA下调肺癌细胞系中PAK2的表达后,通过WB和细胞功能学实验等方法,明确PAK2对NSCLC细胞系增殖和侵袭能力的影响,并检测顺铂耐药前后的肺癌细胞系中PAK2的表达改变,及其对肺癌细胞顺铂敏感性的影响。结果:PAK2在NSCLC中呈现明显的胞浆强表达,并且其异常表达与肺癌的恶性表型相关;PAK2在顺铂耐药肺癌细胞中表达增强,抑制ERK活性后,PAK2的表达上调不能显著促进肺癌细胞对顺铂耐药。结论:PAK2在NSCLC中发挥着重要的促癌基因功能,并通过调节ERK信号通路的活性介导肺癌细胞系对顺铂耐药性。     

非小细胞肺癌EGFR—TKI再治疗反应临床观察及机制探讨

目的：观察非小细胞肺癌（non-smallcelllungcancer,NSCLC）患者使用表皮生长因子受体酪氨酸激酶抑制剂（epidermalgrowthfactorreceptor_tyrosinekinaseinhibitor,EGFR-TKI）初始治疗失败后,经过一段时间的间歇期,再次应用EGFR—TKI治疗的临床疗效,并探讨其可能的分子机制。方法：回顾性分析2008—12-01—2012-05—30,我院19例初始用EGFR—TKI治疗失败后停止一段时间并再次应用EGFR-TKI治疗的NSCLC患者的临床资料。采用小剂量递增的方法,体外诱导EGFR-TKI耐药的细胞模型,研究“治疗再反应”现象可能的分子机制。结果：19例NSCLC患者中47．4％（9／19）再次应用EGFR-TKI仍可获得疾病控制,其中PR为15．8％（3／19）,SD为31．6％（6／19）。同时,体外诱导的耐药细胞株经顺铂治疗2个月后,再次给予厄洛替尼（Erlotinib）治疗,抑制率由3％升高至15％,P〈0．05,再次显示了部分的有效性。蛋白质印迹法检测自噬相关分子LC3B发现,耐药细胞株LC3B表达增加,再次Erlotinib治疗后LC3B表达水平相对未处理细胞明显下调。结论：EGFR-TKI治疗失败的NSCLC患者经过一段时间的间歇期再次应用EGFR—TKI,部分患者仍可获得较满意的疾病控制,该效应可能与细胞自噬有密切关系,这种再治疗反应有望成为NSCLC的一种有效治疗策略。     

Overexpression of Cathepsin L is associated with gefitinib resistance in non-small cell lung cancer

Lung cancer, the most common malignancy, is still the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) accounts for 80 % of all lung cancers. Recent studies showed Cathepsin L (CTSL) is overexpressed in various cancerous tissues; however, the association between CTSL expression and EGFR-TKI resistance remains unknown. In this study, we investigated the expression of CTSL in lung cancer specimens and matched normal tissues by quantitative real-time PCR and IHC. The functional role of CTSL in resistant PC-9/GR cell line was investigated by proliferation and apoptosis analysis compared with control PC-9 cells. Our results found that the level of CTSL expression was higher in NSCLC tissues compared with matched normal adjacent tissue samples, and CTSL was more highly expressed in PC-9/GR cells compared to PC-9 cells. Knocking-down of CTSL in PC-9/GR cells could decrease cell proliferation and potentiate apoptosis induced by gefitinib, suggesting CTSL may contribute to gefitinib resistance in NSCLC. CTSL might be explored as a candidate of therapeutic target for modulating EGFR-TKI sensitivity in NSCLC.     

Overexpression of P21-activated kinase 4 is associated with poor prognosis in non-small cell lung cancer and promotes migration and invasion

Background

P21-activated kinase 4 (PAK4), an effector of the Rho family protein Cdc42, is an important oncogene whose expression is increased in many human cancers and is generally positively correlated with advanced disease and decreased survival. However, little is known about the expression and biological function of PAK4 in human non-small cell lung cancer (NSCLC).

Methods

PAK4 expression in NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry, real-time PCR, and western blotting. Prognostic value of PAK4 expression was evaluated by Kaplan-Meier analysis and Cox regression. siRNA-mediated gene silencing and protein kinase assay was applied to demonstrate the role and the mechanism of PAK4 in lung cancer cell migration, invasion.

Results

The results showed that PAK4 was overexpressed in NSCLC cell lines and human NSCLC tissues. PAK4 expression was detected both in the membranes and cytoplasm of NSCLC cancer cells in vivo. Moreover, increased expression of PAK4 was associated with metastasis, shorter overall survival, advanced stage of NSCLC. Furthermore, PAK4 expression was positively correlated with phosphorylation of LIMK1 expression levels. Knockdown of PAK4 in NSCLC cell lines led to reduce the phosphorylation of LIMK1, which resulted in decrease of the cell migration and invasion. In addition, PAK4 bound to LIMK1 directly and activated it via phosphorylation.

Conclusions

These data demonstrate that PAK4 mediated LIMK1 phosphorylation regulates the migration and invasion in NSCLC. Therefore, PAK4 might be a significant prognostic marker and potential therapeutic molecular target in NSCLC.     

Inhibition of mitochondrial glutaminase activity reverses acquired erlotinib resistance in non-small cell lung cancer

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib has been approved based on the clinical benefit in non-small cell lung cancer (NSCLC) patients over the past decade. Unfortunately, cancer cells become resistant to this agent via various mechanisms, and this limits the improvement in patient outcomes. Thus, it is urgent to develop novel agents to overcome erlotinib resistance. Here, we propose a novel strategy to overcome acquired erlotinib resistance in NSCLC by inhibiting glutaminase activity. Compound 968, an inhibitor of the glutaminase C (GAC), when combined with erlotinib potently inhibited the cell proliferation of erlotinib-resistant NSCLC cells HCC827ER and NCI-H1975. The combination of compound 968 and erlotinib not only decreased GAC and EGFR protein expression but also inhibited GAC activity in HCC827ER cells. The growth of erlotinib-resistant cells was glutamine-dependent as proved by GAC gene knocked down and rescue experiment. More importantly, compound 968 combined with erlotinib down-regulated the glutamine and glycolysis metabolism in erlotinib-resistant cells. Taken together, our study provides a valuable approach to overcome acquired erlotinib resistance by blocking glutamine metabolism and suggests that combination of EGFR-TKI and GAC inhibitor maybe a potential treatment strategy for acquired erlotinib-resistant NSCLC.     

Anti-tumor activity of a novel EGFR tyrosine kinase inhibitor against human NSCLC in vitro and in vivo

Epidermal growth factor receptor (EGFR) is highly expressed in many human tumors including non-small cell lung cancer (NSCLC). Treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has led to dramatic clinical improvement in selected patients with NSCLC. However, intrinsic and acquired resistance to EGFR-TKI remains a common phenomenon. Novel EGFR-TKI, structurally different with erlotinib or gefitinib might be beneficial for patients with NSCLC. In this study, we examined the anti-tumor effect of a newly synthesized novel EGFR tyrosine kinase inhibitor N-(3-chloro-4-fluorophenyl)-N-(7-methoxy-6-(3-morpholinopropoxy) quinazolin-4-yl)-3,3-dimethylbutanamide (F90). In vitro studies in a panel of three different human NSCLC cell lines revealed that F90 inhibited cell proliferation with high potency and induced G0/G1 arrest of cell cycle and apoptosis. F90 markedly reduced phosphorylation of EGFR and inhibited activation of MAPK and Akt. Oral administration of F90 (80 mg/kg/day) to BALB/c nude mice bearing NSCLC cell lines xenografts significantly retarded tumor growth. In conclusion, F90 has potent anti-tumor activity on human lung cancer in vitro and in vivo.     

非小细胞肺癌中微小RNA与表皮生长因子受体-酪氨酸激酶抑制剂敏感性关系的研究进展

表皮生长因子受体(epidermal growth factor receptor,EGFR) -酪氨酸激酶抑制剂(tyrosine kinase inhibitor,TKI)被广泛应用于肿瘤的治疗,尤其是伴有EGFR基因突变的非小细胞肺癌(non-small cell lung cancer,NSCLC)的治疗,但EGFR- TKI耐药性的产生已成为制约其疗效的主要瓶颈.微小RNA (microRNAs,miRNAs)是一类非编码小分子RNA,在转录后水平调控基因的表达.近年来的研究发现,miRNAs在多个环节参与调节NSCLC对EGFR-TKI的敏感性,提示其可能在TKI耐药中扮演了重要角色,并有可能成为一种新的预测TKI敏感性的生物学标志物.因此,本文就有关NSCLC对EGFR-TKI的敏感性与miRNAs之间关系的最新研究进展进行综述.     

CCDC25在非小细胞肺癌细胞中高表达及其促细胞增殖的分子机制

目的:探讨CCDC25蛋白在非小细胞肺癌细胞中的表达及其对非小细胞肺癌增殖的影响。方法:应用GEPIA数据库分析CCDC25在肺癌组织中表达明显高于癌旁组织，结合Western blot检测和免疫荧光，检测其在非小细胞肺癌细胞系中的表达和定位。Western blot 检测YAP、p-YAP、LATS1、p-LATS1蛋白表达量。克隆形成实验和MTS实验检测CCDC25过表达对肿瘤细胞增殖能力的影响。结果:CCDC25在非小细胞肺癌细胞系中明显高表达，CCDC25过表达促进肿瘤细胞增殖能力(P<0.05)。CCDC25过表达抑制YAP蛋白的磷酸化促进YAP蛋白水平的升高(P<0.05)。结论:CCDC25可能通过调控YAP蛋白表达，进而促进非小细胞肺癌的演进。     

Notch-1 contributes to epidermal growth factor receptor tyrosine kinase inhibitor acquired resistance in non-small cell lung cancer in vitro and in vivo

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs in non-small cell lung cancer (NSCLC) patients who initially respond to TKI treatment but whose cancer then progresses. Recent studies have shown that Notch signal is associated with drug resistance. However, the exact mechanism of Notch during acquisition of resistance to EGFR-TKI in human lung cancer remains unclear. In the present study, we showed that the expression of Notch-1 was highly upregulated in EGFR-TKI acquired resistant lung cancer cells. More importantly, Notch-1 contributed to the acquisition of the epithelial–mesenchymal transition (EMT) phenotype, which was critically associated with acquired resistance to EGFR-TKI. Silencing of Notch-1 using siRNA resulted in mesenchymal–epithelial transition (MET), which was associated with impaired invasion and anchorage-independent growth of lung cancer and resensitisation to gefitinib in acquired resistant NSCLC cells. Finally, gefitinib treatment of Balb/c nu/nu with acquired resistant lung cancer xenografts in combination with Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine t-butyl ester (DAPT) resulted in effective tumour growth retardation, with decreased proliferative activity and increased apoptotic activity. Collectively, these data suggest that Notch-1 might play a novel role in acquired resistance to gefitinib, which could be reversed by inhibiting Notch-1.     

MiR-21 overexpression is associated with acquired resistance of EGFR-TKI in non-small cell lung cancer

Background and purpose

With the increasing use of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI) in patients with advanced non-small cell lung cancer (NSCLC), its acquired resistance has become a major clinical problem. Recent studies revealed that miR-21 was involved into the resistance of cytotoxic agents. The aim of this study was to investigate its role in the acquired resistance of NSCLC to EGFR-TKI.

Methods

EGFR-TKI-sensitive human lung adenocarcinoma cell line PC9 and the acquired resistant cell line, PC9R, were used. Lentiviral vectors were used to infect PC9 or PC9R to regulate the miR-21 expression. The expression of targeted proteins PTEN and PDCD4 was controlled by RNA interference. MicroRNA array, RT-PCR and TaqMan MicroRNA Assays were used to detect miR-21 expression. The MTT and Annexin V assays were used to determine proliferation and apoptosis. Western Blot and immunohistochemistry were used to analyze target protein expression (PTEN, PDCD4, Akt, p-Akt). We also constructed PC9R xenograft tumor model to observe the relationship between miR-21 and EGFR-TKI resistance in vivo and validated it in the clinical serum specimens of NSCLC patients treated with EGFR-TKI.

Result

MiR-21 was overexpressed in the EGFR-TKI resistant cell line PC9R relative to PC9. The level of miR-21 was reversely correlated with the expression of PTEN and PDCD4 and positive correlated with PI3K/Akt pathway. Inhibiting miR-21 with lentivirus vector induces apoptosis in PC9R cell line and inhibiting miR-21with ASO suppressed tumor growth in nude mice treated with EGFR-TKI. Furthermore, serum miR-21 expression in NSCLC patients treated with EGFR-TKI was significantly higher at the time of acquiring resistance than at baseline (p < 0.01).

Conclusion

miR-21 is involved in acquired resistance of EGFR-TKI in NSCLC, which is mediated by down-regulating PTEN and PDCD4 and activating PI3K/Akt pathway.     

The soybean-derived peptide lunasin inhibits non-small cell lung cancer cell proliferation by suppressing phosphorylation of the retinoblastoma protein

Lunasin, a soybean bioactive peptide, has both chemopreventive and chemotherapeutic activities. The aim of this study was to determine the chemotherapeutic potential of lunasin against human lung cancer. Treatment of non-small cell lung cancer (NSCLC) cells with highly purified soybean-derived lunasin caused limited, cell-line specific anti-proliferative effects on anchorage-dependent growth whereas two normal bronchial epithelial cell lines were unaffected. Lunasin''s antiproliferative effects were potentiated upon utilization of anchorage-independent conditions. Furthermore, NSCLC cell lines that were unaffected by lunasin in anchorage-dependent assays exhibited a dose-dependent inhibition in colony formation or colony size. Mouse xenograft studies revealed that 30 mg lunasin/kg body weight per day decreased NSCLC H1299 tumor volume by 63.0% at day 32. Mechanistic studies using cultured NSCLC H661 cells showed that lunasin inhibited cell cycle progression at the G1/S phase interface without inducing apoptosis. Immunoblot analyses of key cell-cycle proteins demonstrated that lunasin altered the expression of the G1 specific cyclin-dependent kinase complex components, increased levels of p27Kip1, reduced levels of phosphorylated Akt, and ultimately inhibited the sequential phosphorylation of the retinoblastoma protein (RB). These results establish for the first time that lunasin can inhibit NSCLC proliferation by suppressing cell-cycle dependent phosphorylation of RB.     

Everolimus synergizes with gefitinib in non-small-cell lung cancer cell lines resistant to epidermal growth factor receptor tyrosine kinase inhibitors

Purpose

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapy is considered as one of the most important treatments for patients with advanced non-small-cell lung cancer (NSCLC). However, not all patients benefit from this therapy because of primary or acquired resistance, both of which are usually caused by the activation of alternative signaling pathways. Thus, a combination of different signaling pathway inhibitors is a promising strategy. We used the mammalian target of rapamycin (mTOR) inhibitor everolimus in combination with gefitinib in NSCLC cell lines to analyze the efficacy of this combination regimen and the underlying molecular mechanism.

Methods

Acquired gefitinib-resistant cell lines, together with EGFR wild-type and mutant primary gefitinib-resistant NSCLC cell lines, were treated with everolimus alone, gefitinib alone, or the combination of the two drugs, and the effects were evaluated using cell proliferation assays. The effects of everolimus and gefitinib on the EGFR pathway in NSCLC cell lines were determined by Western blot analysis.

Results

Combined treatment resulted in synergistic antitumor effects in gefitinib-resistant cells A549 and H1975. The combination index (CI) of cells increased with increasing dose of everolimus. Everolimus demonstrated no apparent inhibition of phosphorylated Akt (p-Akt) and phosphorylated p44/42 MAPK (p-MAPK) in H1650 cells. Additionally, in gefitinib-resistant cell lines, the combination of gefitinib and everolimus not only showed stronger inhibition of phosphorylated mTOR and phosphorylated p70S6K expression than either drug alone but also reduced the levels of p-Akt and p-MAPK in both cell lines.

Conclusions

Our data showed that the combination of everolimus and gefitinib exhibits dose-dependent synergism in primary and acquired gefitinib-resistant NSCLC cells. Thus, a preclinical rationale exists for the use of everolimus to enhance the efficacy of gefitinib in EGFR-TKI-resistant patients with NSCLC.     

The tumor suppressor LKB1 regulates lung cancer cell polarity by mediating cdc42 recruitment and activity

The tumor suppressor LKB1 is mutated in 30% of non-small cell lung cancer (NSCLC) tumors and cell lines and is proposed to be a key regulator of epithelial cell polarity; however, how LKB1 regulates cancer cell polarity is not known. The experiments described herein show for the first time that LKB1 is a dynamic, actin-associated protein that rapidly polarizes to the leading edge of motile cancer cells. LKB1 proves to be essential for NSCLC polarity, because LKB1 depletion results in classic cell polarity defects, such as aberrant Golgi positioning, reduced lamellipodia formation, and aberrant morphology. To probe how LKB1 regulates these events, we show that LKB1 colocalizes at the cellular leading edge with two key components of the polarity pathway - the small rho GTPase cdc42 and its downstream binding partner p21-activated kinase (PAK). Importantly, LKB1 functionality is required for cdc42 polarization to the leading edge, maintaining active cdc42 levels, and downstream PAK phosphorylation. To do this, LKB1 interacts only with active form of cdc42 and PAK, but not with inactive cdc42. Taken together, these results show that LKB1 is a critical mediator of the NSCLC polarity program in lung cancer cells through a novel LKB1-cdc42-PAK pathway.     

表皮生长因子受体酪氨酸激酶抑制剂与非小细胞肺癌的治疗

 表皮生长因子受体酪氨酸激酶抑制剂（EGFR-TKI）是一类高效低毒的抗肿瘤分子靶向药物,因其独特的疗效和良好的耐受性在非小细胞肺癌（NSCLC）治疗中成为研究的热点,在NSCLC的一线、二线或三线的辅助治疗中均有一定地位。随着研究的深入细化,EGFR TKI的适应证不断扩大,术前新辅助治疗可能成为一种新的治疗模式。     

Targeting group II PAKs in cancer and metastasis

The p21 activated kinases (PAKs) play an essential role in cell signaling and control a variety of cellular functions including cell motility, survival, angiogenesis and mitosis. PAKs are important regulators in growth factor signaling, cytoskeletal reorganization and growth factor-mediated cell migration. Overexpression of PAKs has been detected in many cancers and linked to increased migration potential, anchorage independent growth and metastasis. Six isoforms of PAKs are expressed in human and based on their regulatory properties they have been classified into group I (PAK1–3) and group II (PAK4–6). Besides the well studied group I family, members of the group II PAKs also emerged as interesting targets for the development of new inhibitors for cancer therapy. The availability of high resolution crystal structures for all group II PAKs and their fundamentally different regulatory properties when compared with group I enzymes has opened new opportunities for rational drug designing strategies. In this review, we summarize the results of recent advances of the function of group II PAKs in tumorigenesis and metastasis as well as opportunities for exploring the unique catalytic domain dynamics of this protein family for the design of group II PAK specific inhibitors.     

Glypican-5 is a novel metastasis suppressor gene in non-small cell lung cancer

Glypican-5 (GPC5) may be a potential tumor suppressor gene in non-small cell lung cancer (NSCLC). The present study aimed to clarify the GPC5 expression pattern and to explore its potential functions in NSCLC. The expression of GPC5 gene was lower in lung cancer tissues compared with adjacent noncancerous tissues. The GPC5 gene expression in the lymph node metastasis group was remarkably lower than that in the non-metastasis group. The tissue microarray (TMA) study found that the overall survival rate of GPC5-positive group was significantly higher than that of GPC5-negative group in AC subgroup. Overexpressing GPC5 in NSCLC cell lines significantly suppressed their migration, invasion, and proliferation activities and also induced G1/S phase arrest of the cells in vitro. Our data suggest that GPC5 is a novel metastasis suppressor gene in NSCLC and may be a potential biomarker that predicts NSCLC metastasis.