ROR1激活AKT/FOXO1信号介导非小细胞肺癌吉非替尼耐药

EGFR-TKI靶向治疗在非小细胞肺癌（non-small cell lung cancer, NSCLC）综合治疗中显示出重要作用;然而,耐药性却极大限制其临床治疗效果。受体酪氨酸激酶样孤儿受体（receptor tyrosine kinase-like orphan receptor 1, ROR1）是I型受体酪氨酸激酶家族中的成员,在肿瘤发生发展中发挥重要作用。本研究拟探讨ROR1介导非小细胞肺癌吉非替尼耐药的作用及机制。采用吉非替尼反复诱导非小细胞肺癌HCC827细胞,建立吉非替尼耐药细胞株HCC827/GR。应用荧光定量PCR和Western 印迹检测HCC827/GR内ROR1的表达。采用shRNA的方法体外检测ROR1敲除前后HCC827/GR对吉非替尼耐药的变化,采用体外检测ROR1过表达前后HCC827对吉非替尼耐药的变化。体内检测ROR1敲除前后HCC827/GR对吉非替尼耐药的变化。Western 印迹检测HCC827/GR内ROR1下游信号分子的活化。实时荧光定量PCR及Western 印迹结果显示,HCC827/GR耐药细胞中的ROR1 mRNA和蛋白质表达水平显著高于HCC827敏感细胞。体外干扰ROR1表达,可明显增强HCC827/GR耐药细胞对吉非替尼的敏感性 (IC50 15.3±3.69 vs. 4.2±1.38),增加吉非替尼诱导的细胞凋亡 (20.5±2.52 vs. 41.8±3.74)。体外过表达ROR1显著增强HCC827敏感细胞对吉非替尼的耐药性(IC50 0.8±0.52 vs. 2.2±0.87)。体内裸鼠移植瘤实验同样发现,干扰ROR1能增强HCC827/GR移植瘤对吉非替尼的敏感性。进一步研究发现,AKT/FOXO1信号在HCC827/GR耐药细胞中异常活化,而干扰ROR1能够抑制AKT的磷酸化,并上调FOXO1的表达。上述结果表明,ROR1参与非小细胞肺癌吉非替尼耐药,抑制ROR1能够逆转吉非替尼耐药,其机制与ROR1调控AKT/FOXO1信号有关。     

miR‐1‐3p and miR‐206 sensitizes HGF‐induced gefitinib‐resistant human lung cancer cells through inhibition of c‐Met signalling and EMT

Hepatocyte growth factor (HGF) overexpression is an important mechanism in acquired epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib resistance in lung cancers with EGFR activating mutations. MiR‐1‐3p and miR‐206 act as suppressors in lung cancer proliferation and metastasis. However, whether miR‐1‐3p and miR‐206 can overcome HGF‐induced gefitinib resistance in EGFR mutant lung cancer is not clear. In this study, we showed that miR‐1‐3p and miR‐206 restored the sensitivities of lung cancer cells PC‐9 and HCC‐827 to gefitinib in present of HGF. For the mechanisms, we demonstrated that both miR‐1‐3p and miR‐206 directly target HGF receptor c‐Met in lung cancer. Knockdown of c‐Met mimicked the effects of miR‐1‐3p and miR‐206 transfections Meanwhile, c‐Met overexpression attenuated the effects of miR‐1‐3p and miR‐206 in HGF‐induced gefitinib resistance of lung cancers. Furthermore, we showed that miR‐1‐3p and miR‐206 inhibited c‐Met downstream Akt and Erk pathway and blocked HGF‐induced epithelial‐mesenchymal transition (EMT). Finally, we demonstrated that miR‐1‐3p and miR‐206 can increase gefitinib sensitivity in xenograft mouse models in vivo. Our study for the first time indicated the new function of miR‐1‐3p and miR‐206 in overcoming HGF‐induced gefitinib resistance in EGFR mutant lung cancer cell.     

Gefitinib Inhibits Invasive Phenotype and Epithelial-Mesenchymal Transition in Drug-Resistant NSCLC Cells with MET Amplification

Despite the initial response, all patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). The EGFR-T790M secondary mutation is responsible for half of acquired resistance cases, while MET amplification has been associated with acquired resistance in about 5-15% of NSCLCs. Clinical findings indicate the retained addiction of resistant tumors on EGFR signaling. Therefore, we evaluated the molecular mechanisms supporting the therapeutic potential of gefitinib maintenance in the HCC827 GR5 NSCLC cell line harbouring MET amplification as acquired resistance mechanism. We demonstrated that resistant cells can proliferate and survive regardless of the presence of gefitinib, whereas the absence of the drug significantly enhanced cell migration and invasion. Moreover, the continuous exposure to gefitinib prevented the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and down-regulation of vimentin and N-cadherin. Importantly, the inhibition of cellular migration was correlated with the suppression of EGFR-dependent Src, STAT5 and p38 signaling as assessed by a specific kinase array, western blot analysis and silencing functional studies. On the contrary, the lack of effect of gefitinib on EGFR phosphorylation in the H1975 cells (EGFR-T790M) correlated with the absence of effects on cell migration and invasion. In conclusion, our findings suggest that certain EGFR-mutated patients may still benefit from a second-line therapy including gefitinib based on the specific mechanism underlying tumor cell resistance.     

Hypoxia Increases Gefitinib-Resistant Lung Cancer Stem Cells through the Activation of Insulin-Like Growth Factor 1 Receptor

Accumulating evidence indicates that a small population of cancer stem cells (CSCs) is involved in intrinsic resistance to cancer treatment. The hypoxic microenvironment is an important stem cell niche that promotes the persistence of CSCs in tumors. Our aim here was to elucidate the role of hypoxia and CSCs in the resistance to gefitinib in non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutation. NSCLC cell lines, PC9 and HCC827, which express the EGFR exon 19 deletion mutations, were exposed to high concentration of gefitinib under normoxic or hypoxic conditions. Seven days after gefitinib exposure, a small fraction of viable cells were detected, and these were referred to as “gefitinib-resistant persisters” (GRPs). CD133, Oct4, Sox2, Nanog, CXCR4, and ALDH1A1–all genes involved in stemness–were highly expressed in GRPs in PC9 and HCC827 cells, and PC9 GRPs exhibited a high potential for tumorigenicity in vivo. The expression of insulin-like growth factor 1 (IGF1) was also upregulated and IGF1 receptor (IGF1R) was activated on GRPs. Importantly, hypoxic exposure significantly increased sphere formation, reflecting the self-renewal capability, and the population of CD133- and Oct4-positive GRPs. Additionally, hypoxia upregulated IGF1 expression through hypoxia-inducible factor 1α (HIF1α), and markedly promoted the activation of IGF1R on GRPs. Knockdown of IGF1 expression significantly reduced phosphorylated IGF1R-expressing GRPs under hypoxic conditions. Finally, inhibition of HIF1α or IGF1R by specific inhibitors significantly decreased the population of CD133- and Oct4-positive GRPs, which were increased by hypoxia in PC9 and HCC827 cells. Collectively, these findings suggest that hypoxia increased the population of lung CSCs resistant to gefitinib in EGFR mutation-positive NSCLC by activating IGF1R. Targeting the IGF1R pathway may be a promising strategy for overcoming gefitinib resistance in EGFR mutation-positive NSCLC induced by lung CSCs and microenvironment factors such as tumor hypoxia.     

Pemetrexed induces ROS generation and cellular senescence by attenuating TS-mediated thymidylate metabolism to reverse gefitinib resistance in NSCLC

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) are strongly recommended for non-small-cell lung cancer (NSCLC) patients harbouring active EGFR mutations, while drug resistance makes exploring resistance mechanisms and seeking effective therapeutic strategies urgent endeavours. Thymidylate synthetase (TYMS or TS) is a dominant enzyme in thymidylate nucleotide metabolism. In this study, we found a positive correlation between TS expression and overall survival (OS) and disease-free survival (DFS) in lung adenocarcinoma. The examination of gene sets from 140 NSCLC patients received EGFR-TKI therapy demonstrated a negative correlation between high TS expression and the efficacy of EGFR-TKI therapy. 24 tissue specimens from NSCLC patients exhibited upregulated TS mRNA expression in NSCLC patients resistant to gefitinib. The NSCLC cell PC9 and HCC827 sensitive to gefitinib and relatively resistant PC9/GR and HCC827/GR cells were used to demonstrate the knockdown of TS restored the sensitivity of resistant cells to gefitinib. Furthermore, pemetrexed effectively suppressed TS-mediated thymidylate metabolism and induced ROS generation, DNA damage and cellular senescence, thereby hampering cancer progression and restoring sensitivity to gefitinib. Our findings illuminate the potential mechanism of TS-triggered gefitinib resistance and indicate inhibition of TS by pemetrexed can potentiate the effect of gefitinib in NSCLC. Pemetrexed combined with gefitinib has potent anti-progression potential in gefitinib-resistant NSCLC. This study suggests that NSCLC patients with both high TS expression and EGFR-driving mutations might benefit more from a combination strategy of EGFR-TKI and pemetrexed-based chemotherapy than EGFR-TKI monotherapy, which has profound clinical implications and therapeutic value.     

Effect of simvastatin on the resistance to EGFR tyrosine kinase inhibitors in a non-small cell lung cancer with the T790M mutation of EGFR

Although non-small cell lung cancer (NSCLC) tumors with activating mutations in the epidermal growth factor receptor (EGFR) are highly responsive to EGFR tyrosine kinase inhibitors (TKIs) including gefitinib and erlotinib, development of acquired resistance is almost inevitable. Statins show antitumor activity, but it is unknown whether they can reverse EGFR-TKIs resistance in NSCLC with the T790M mutation of EGFR. This study investigated overcoming resistance to EGFR-TKI using simvastatin. We demonstrated that addition of simvastatin to gefitinib enhanced caspase-dependent apoptosis in T790M mutant NSCLC cells. Simvastatin also strongly inhibited AKT activation, leading to suppression of β-catenin activity and the expression of its targets, survivin and cyclin D1. Both insulin treatment and AKT overexpression markedly increased p-β-catenin and survivin levels, even in the presence of gefitinib and simvastatin. However, inhibition of AKT by siRNA or LY294002 treatment decreased p-β-catenin and survivin levels. To determine the role of survivin in simvastatin-induced apoptosis of gefitinib-resistant NSCLC, we showed that the proportion of apoptotic cells following treatment with survivin siRNA and the gefitinib–simvastatin combination was greater than the theoretical additive effects, whereas survivin up-regulation could confer protection against gefitinib and simvastatin-induced apoptosis. Similar results were obtained in erlotinib and simvastatin-treated HCC827/ER cells. These findings suggest that survivin is a key molecule that renders T790M mutant NSCLC cells resistant to apoptosis induced by EGFR-TKIs and simvastatin. Overall, these data indicate that simvastatin may overcome EGFR-TKI resistance in T790M mutant NSCLCs via an AKT/β-catenin signaling-dependent down-regulation of survivin and apoptosis induction.     

Gefitinib resistance in HCC mahlavu cells: upregulation of CD133 expression, activation of IGF-1R signaling pathway, and enhancement of IGF-1R nuclear translocation

Hepatocellular carcinoma (HCC) is the major form of primary liver cancer which accounts for more than half million deaths annually worldwide. While the incidence of HCC is still on the rise, options of treatment are limited and the overall survival rate is poor. The acquisition of cancer drug resistance remains one of the key hurdles to successful treatment. Clearly, a thorough understanding of the underlying mechanisms is needed for new strategies to design novel treatments and/or to improve the current therapies. In the present study, we examined the expression of cancer stem cell (CSC) marker CD133, the activation of insulin-like growth factor 1 receptor (IGF-1R) signaling, and the nuclear translocation of IGF-1R in HCC Mahlavu cells under the treatment of gefitinib, a cancer drug that inhibits epidermal growth factor receptor (EGFR) pathway. Our results demonstrated that Mahlavu cells exhibited strong gefitinib resistance and the CD133 expression level was dramatically increased (from 3.88% to 32%) after drug treatment. In addition, the gefitinib treated cells displayed increased levels of phosphorylation in IGF-1R and Akt, indicating the intensified activation of this cancer-associated signaling pathway. Moreover, we revealed that IGF-1R underwent nuclear translocation in gefitinib treated cells using confocal microscopy. The IGF-1R nuclear translocation was enhanced under gefitinib treatment and appeared in a dose-dependent manner. Our findings suggest that increased IGF-1R nuclear translocation after gefitinib treatment may contribute to the drug resistance and IGF1-R activation, which might also associate with the upregulation of CD133 expression.     

Integrin beta1 over-expression associates with resistance to tyrosine kinase inhibitor gefitinib in non-small cell lung cancer

The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) such as gefitinib and erlotinib have been widely used in treating patients with advanced non-small cell lung cancer (NSCLC). However, acquired resistance to EGFR TKI almost occurs in every patient eventually. To identify its potential mechanism, we established a human NSCLC cell line PC9/AB2 which was 576-fold decrease in gefitinib sensitivity compared with its parental PC9 cell lines. No EGFR-T790M mutation or abnormal expression of c-Met protein was found in PC9/AB2 cells. Over-expression of integrin β1 was found, accompanied with increase of the cells' adhesion and migration. To further confirm the role of integrin β1 in gefitinib acquired resistance, we transferred its siRNA-expressing plasmid and its whole cDNA expressing plasmid into PC9/AB2 and into PC9 cells, respectively. The sensitivity of NSCLC cells to gefitinib was negatively correlated with integrin β1 expression levels. All these data suggest that up-regulation of integrin β1 might be an important factor for gefitinib resistance in NSCLC cell line PC9/AB2.     

FGFR1通过调控PI3K/AKT/mTOR信号通路介导非小细胞肺癌对吉非替尼的耐药

目的:探讨成纤维细胞生长因子受体1(FGFR1)诱导非小细胞肺癌(NSCLC)吉非替尼获得性耐药的机制。方法:用吉非替尼诱导PC9细胞构建耐药细胞株PC9/GR,用CCK-8、平板克隆形成、transwell技术检测细胞的增殖和迁移能力,用流式细胞术检测细胞的凋亡状况,qRT-PCR、免疫荧光和蛋白免疫印迹技术检测基因表达水平。进一步采用FGFR1抑制剂PD173074或si RNA-FGFR1处理PC9/GR细胞,检测细胞的增殖、迁移、克隆形成能力的变化及Akt、p-Akt、m TOR和p-mTOR表达的变化。结果:PC9/GR细胞的增殖、迁移及对吉非替尼的耐受能力显著增强;FGFR1在PC9/GR细胞中的表达水平显著升高;用PD173074处理PC9细胞后,其增殖、迁移能力及对吉非替尼的耐受能力显著下降;敲低FGFR1后Akt和m TOR的磷酸化水平显著下降。结论:FGFR1通过PI3K/AKT/mTOR信号通路介导非小细胞肺癌对吉非替尼的耐药。     

Reciprocal positive regulation between Cx26 and PI3K/Akt pathway confers acquired gefitinib resistance in NSCLC cells via GJIC-independent induction of EMT

Gefitinib efficiency in non-small-cell lung cancer (NSCLC) therapy is limited due to development of drug resistance. The molecular mechanisms of gefitinib resistance remain still unclear. In this study, we first found that connexin 26 (Cx26) is the predominant Cx isoform expressed in various NSCLC cell lines. Then, two gefitinib-resistant (GR) NSCLC cell lines, HCC827 GR and PC9 GR, from their parental cells were established. In these GR cells, the results showed that gefitinib resistance correlated with changes in cellular EMT phenotypes and upregulation of Cx26. Cx26 was detected to be accumulated in the cytoplasm and failed to establish functional gap-junctional intercellular communication (GJIC) either in GR cells or their parental cells. Ectopic expression of GJIC-deficient chimeric Cx26 was sufficient to induce EMT and gefitinib insensitivity in HCC827 and PC9 cells, while knockdown of Cx26 reversed EMT and gefitinib resistance in their GR cells both in vitro and in vivo. Furthermore, Cx26 overexpression could activate PI3K/Akt signaling in these cells. Cx26-mediated EMT and gefitinib resistance were significantly blocked by inhibition of PI3K/Akt pathway. Specifically, inhibition of the constitutive activation of PI3K/Akt pathway substantially suppressed Cx26 expression, and Cx26 was confirmed to functionally interplay with PI3K/Akt signaling to promote EMT and gefitinib resistance in NSCLC cells. In conclusion, the reciprocal positive regulation between Cx26 and PI3K/Akt signaling contributes to acquired gefitinib resistance in NSCLC cells by promoting EMT via a GJIC-independent manner.Lung cancer, of which non-small-cell lung cancer (NSCLC) is the most common form, remains the leading cause of cancer-related deaths worldwide.¹ Currently, gefitinib, as the first epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), is one of the most accepted therapies against NSCLC carrying EGFR mutations. However, almost all NSCLC patients who initially respond well to EGFR-TKIs eventually develop acquired resistance.² Development of effective therapeutic interventions to overcome gefitinib resistance is an urgent need.Epithelial-mesenchymal transition (EMT), during which cancer cells lose epithelial markers such as E-cadherin but gain mesenchymal markers such as vimentin, is known to be deeply involved in cancer progression and chemotherapy resistance. Specially in NSCLC, EMT plays pivotal roles in the acquired resistance to EGFR-TKIs such as gefitinib.^{3, 4} For example, restoring E-cadherin expression or silencing EMT regulator Slug increases gefitinib sensitivity in NSCLC cells with a mesenchymal phenotype.^{5, 6} Accumulating evidences indicate that constitutively activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling is a central feature of EMT in many cancers including NSCLC.^{7, 8} However, the exact mechanism for the acquired gefitinib resistance of NSCLC remains unclear.Connexins (Cxs) are a family of transmembrane proteins, which compose the intercellular gap junctions between the neighboring cells.⁹ Gap junctions directly connect the cytoplasms of adjacent cells, thereby mediating direct exchange of signaling molecules smaller than 1 kDa, such as ions, small metabolites, and second messengers. This process is termed gap-junctional intercellular communication (GJIC). Cx expression and/or GJIC are frequently reduced or loss in malignant cell lines and cancers, while restoration of Cx expression and/or GJIC retarded tumor growth and increased cytotoxicities of chemotherapeutics such as cisplatin and docetaxel.^{10, 11, 12, 13} Therefore, Cxs have long been deemed tumor suppressors. However, increasing new observations were apparently contradicting the ''dogma'' and became clear that Cxs and GJIC also contribute to cancer progression and chemoresistance. For example, Cx32 expression was detected in breast cancer and significantly increased in lymph node metastases compared with primary tumors, suggesting Cx32 may be a sign of more malignant phenotype of breast cancer.¹⁴ Besides, cytoplasmic accumulation of Cx32 exerted favorable effects for hepatocellular carcinoma (HCC) progression including invasion and metastasis by Cx linked, but GJIC-independent mechanism.¹⁵ Recently, Gielen et al.¹⁶ reported that increasing the level of Cx43 confers temozolomide resistance in human glioma cells whereas knockdown of Cx43 sensitizes them to temozolomide treatment via both GJIC-dependent and -independent mechanisms.Up to now, there are ~21 isoforms of Cxs that distribute in almost all human organs in tissue-specific patterns.¹⁷ Cx26, one of the most common isoforms of Cxs, is predominantly expressed in lung tissue.^{18, 19} Despite Cx26 has been considered as a potential tumor suppressor or chemotherapy sensitizer in some types of tumors,^{20, 21} Ito et al.²² found that Cx26 helps lung squamous cell carcinoma (SCC, one histological type of NSCLC), acquire aggressive phenotypes, lymph node metastasis, and poor prognosis, indicating that a potential role of Cx26 on the malignant development of SCC. However, the roles of Cx26 and its derived GJIC in the development of gefitinib resistance in NSCLC have not been explored.In this study, to clarify the potential role of Cx26 and its derived GJIC in gefitinib resistance in NSCLC, we first surveyed the expression of four major Cxs in different gefitinib-sensitive NSCLC cell lines and found a positive correlation between high level of Cx26 and gefitinib insensitivity in NSCLC cells. Such an association was further confirmed in established gefitinib-resistant (GR) HCC827 and PC9 cell lines both in vitro and in vivo. Importantly, we find a positive mutual regulation between Cx26 and PI3K/Akt pathway, which confers acquired gefitinib resistance in NSCLC cells by GJIC-independent induction of EMT.     

ALDH1A1诱导肺腺癌细胞发生上皮-间质转化及化疗耐药

目的:探索醛脱氢酶1A1(aldehyde dehydrogenase 1A1,ALDH1A1)在肺腺癌细胞(lung adenocarcinoma cell,LAC)化疗耐药中的作用及机制,为肺癌临床治疗和新型药物的研发提供实验依据。方法:采用慢病毒载体构建ALDH1A1高表达肺腺癌细胞模型,并通过流式细胞术和western blot技术对该细胞模型进行验证。通过CCK8法检测ALDH1A1高表达肺腺癌细胞对肺癌治疗药物顺铂(cisplatin,DDP)、紫杉醇(paclitaxcel)、厄洛替尼(erlotinib)和吉非替尼(gefitinib)的耐药性。通过检测肿瘤干细胞(cancer stem cell,CSC)分子标志物、上皮-间质转化(Epithelial-Mesenchymal Transition,EMT)分子标志物及细胞迁移能力探讨ALDH1A1高表达对肺腺癌细胞的干性和EMT特征的影响。双硫仑(disulfiram,DSF)是ALDH的抑制剂,我们通过CCK8法和transwell细胞迁移实验探究DSF对肺腺癌细胞体外生长和迁移能力的影响,体内实验探究DSF和厄洛替尼联合用药对HCC827-ALDH1A1细胞皮下异种移植瘤生长的影响。结果:ALDH1A1高表达诱导肺腺癌细胞对厄洛替尼、吉非替尼、紫杉醇和顺铂产生不同程度的耐药,干细胞标志物CD44、CD133蛋白表达上调,EMT间充质标志物vimentin蛋白表达上调,transwell实验结果显示ALDH1A1高表达肺腺癌细胞的迁移能力增强,使用ALDH靶向抑制剂DSF能选择性抑制ALDH1A1高表达肺腺癌细胞所增高的迁移能力并克服HCC827-ALDH1A1细胞皮下异种移植瘤的生长,延缓体内耐药。结论:ALDH1A1能诱导肺腺癌细胞对多种抗肺癌药物产生耐药并发生干细胞样转化,靶向抑制ALDH酶活性可克服由ALDH1A1高表达所产生的耐药,为肺癌的临床治疗提供新的思路。     

Effect of hepatocyte growth factor on methionine adenosyltransferase genes and growth is cell density-dependent in HepG2 cells

Hepatocyte growth factor (HGF) is a potent hepatocyte mitogen but its effect in liver cancer is conflicting. Methionine adenosyltransferase (MAT) is an essential enzyme encoded by two genes (MAT1A and MAT2A), while a third gene (MAT2beta) encodes for a subunit that regulates the MAT2A-encoded isoenzyme. MAT1A is silenced while MAT2A and MAT2beta are induced in hepatocellular carcinoma (HCC). The current work examined expression of HGF/c-met in HCC and whether HGF regulates MAT genes and growth in HepG2 cells. We found the mRNA levels of HGF and c-met are markedly increased in HCC. To study the influence of cell density, HepG2 cells were plated under high-density (HD) or low-density (LD) and treated with HGF (10 ng/ml). Cell density had a dramatic effect on MAT1A expression, being nearly undetectable at LD to a ninefold induction under HD. Cell density also determined the effect of HGF. At HD, HGF increased the mRNA levels of p21 and p27, while lowering the levels of MAT genes, cyclin A, and c-met. At LD, HGF increased the mRNA levels of cyclin A, MAT2A, MAT2beta, and c-met. Consistently, HGF inhibits growth under HD but stimulates growth under LD. HGF induced sustained high ERK activation under HD as compared to LD. In summary, HGF induces genes favoring growth and is mitogenic when HepG2 cells are plated under LD; however, the opposite occurs under HD. This involves cell density-dependent differences in HGF-induced ERK activation. This may explain why HGF is mitogenic only when there is loss of cell-cell contact in vivo.     

AUY922 Effectively Overcomes MET- and AXL-Mediated Resistance to EGFR-TKI in Lung Cancer Cells

The activation of bypass signals, such as MET and AXL, has been identified as a possible mechanism of EGFR-TKI resistance. Because various oncoproteins depend on HSP90 for maturation and stability, we investigated the effects of AUY922, a newly developed non-geldanamycin class HSP90 inhibitor, in lung cancer cells with MET- and AXL-mediated resistance. We established resistant cell lines with HCC827 cells harboring an exon 19-deletion mutation in of the EGFR gene via long-term exposure to increasing concentrations of gefitinib and erlotinib (HCC827/GR and HCC827/ER, respectively). HCC827/GR resistance was mediated by MET activation, whereas AXL activation caused resistance in HCC827/ER cells. AUY922 treatment effectively suppressed proliferation and induced cell death in both resistant cell lines. Accordingly, the downregulation of EGFR, MET, and AXL led to decreased Akt activation. The inhibitory effects of AUY922 on each receptor were confirmed in gene-transfected LK2 cells. AUY922 also effectively controlled tumor growth in xenograft mouse models containing HCC827/GR and HCC827/ER cells. In addition, AUY922 reduced invasion and migration by both types of resistant cells. Our study findings thus show that AUY922 is a promising therapeutic option for MET- and AXL-mediated resistance to EGFR-TKI in lung cancer.     

ANXA2 could act as a moderator of EGFR-directed therapy resistance in triple negative breast cancer

Triple negative breast cancer (TNBC) patients cannot benefit from EGFR-targeted therapy even though the EGFR is highly expressed, because patients exhibit resistance to these drugs. Unfortunately, the molecular mechanisms remain relatively unknown. ANXA2, highly expressed in invasive breast cancer cells, is closely related with poor prognosis, and acts as a molecular switch to EGFR activation. In this study, MDA-MB-231 cells and MCF7 cells were used. Our results showed that ANXA2 expression is inversely correlated with cell sensitivity to gefitinib. Knockdown of ANXA2 expression in MDA-MB-231 cells increased the gefitinib induced cell death. When ANXA2 was overexpressed in MCF7 cells, the gefitinib induced cell death was decreased. Furthermore, we demonstrated that phosphorylation of ANXA2 at Tyr23 is negatively correlated with the sensitivity of TNBC to gefitinib. Altogether, our results suggest a new role of ANXA2 in regulating sensitivity of TNBC MDA-MB-231 cells to the EGFR inhibitor gefitinib.     

Activation of Notch-1 enhances epithelial-mesenchymal transition in gefitinib-acquired resistant lung cancer cells

Despite an initial response to EGFR tyrosine kinase inhibitors (EGFR-TKI) in EGFR mutant lung cancer, most patients eventually become resistant and result in treatment failure. Recent studies have shown that epithelial to mesenchymal transition (EMT) is associated with drug resistance and cancer cell metastasis. Strong multiple gene signature data indicate that EMT acts as a determinant of insensitivity to EGFR-TKI. However, the exact mechanism for the acquisition of the EMT phenotype in EGFR-TKI resistant lung cancer cells remains unclear. In the present study, we showed that the expression of Notch-1 was highly upregulated in gefitinib-resistant PC9/AB2 lung cancer cells. Notch-1 receptor intracellular domain (N1IC), the activated form of the Notch-1 receptor, promoted the EMT phenotype in PC9 cells. Silencing of Notch-1 using siRNA reversed the EMT phenotype and restored sensitivity to gefitinib in PC9/AB2 cells. Moreover, Notch-1 reduction was also involved in inhibition of anoikis as well as colony-formation activity of PC9/AB2 cells. Taken together, these results provide strong molecular evidence that gefitinib-acquired resistance in lung cancer cells undergoing EMT occurs through activation of Notch-1 signaling. Thus, inhibition of Notch-1 can be a novel strategy for the reversal of the EMT phenotype thereby potentially increasing therapeutic drug sensitivity to lung cancer cells.     

SPARCL1通过MEK/ERK信号通路调节非小细胞肺癌细胞的增殖、凋亡和侵袭

摘要 目的：分析富含半胱氨酸的酸性分泌蛋白类似蛋白1（SPARCL1）对非小细胞肺癌（NSCLC）细胞增殖、凋亡、侵袭的影响,并探讨分裂原活化抑制剂（MEK）/细胞外调节蛋白激酶（ERK）通路在其中发挥的作用。方法：收集2019年9月~2021年6月期间本院接受手术治疗的84例NSCLC患者癌组织与相应癌旁组织,实时定量逆转录聚合酶链反应（qRT-PCR）法测定并比较各组织以及正常肺上皮细胞HBEpiC、NSCLC细胞A549、HCC827、H1299、H292中SPARCL1 信使RNA（mRNA）表达水平,选取A549、HCC827培养并分组,分为对照组、NC siRNA组、SPARCL1 siRNA组、U0126组（MEK/ERK特异性抑制剂）、SPARCL1 siRNA加U0126组,细胞计数法（CCK8）以及平板克隆法测定A549、HCC827细胞增殖,流式细胞仪测定A549、HCC827细胞凋亡,Transwell小室法测定A549、HCC827细胞侵袭能力,蛋白质印迹法（western blot）检测SPARCL1、p-MEK、MEK、p-ERK1/2、ERK1/2蛋白表达。结果：SPARCL1在NSCLC组织中mRNA表达水平低于癌旁组织（P＜0.05）;与HBEpiC细胞相比,NSCLC细胞A549、HCC827、H1299、H292细胞中SPARCL1 mRNA表达水平降低（P＜0.05）;与对照组相比,SPARCL1 siRNA组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率降低（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达升高（P＜0.05）,U0126组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率升高（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达降低（P＜0.05）;与SPARCL1 siRNA组相比,SPARCL1 siRNA加U0126组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率升高（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达降低（P＜0.05）。结论：SPARCL1可能通过调控MEK/ERK通路影响NSCLC A549、HCC827细胞增殖、侵袭与凋亡。     

Crizotinib sensitizes the erlotinib resistant HCC827GR5 cell line by influencing lysosomal function

In non-small cell lung cancer, sensitizing mutations in epidermal growth factor receptor (EGFR) or cMET amplification serve as good biomarkers for targeted therapies against EGFR or cMET, respectively. Here we aimed to determine how this different genetic background would affect the interaction between the EGFR-inhibitor erlotinib and the cMET-inhibitor crizotinib. To unravel the mechanism of synergy we investigated the effect of the drugs on various parameters, including cell cycle arrest, migration, protein phosphorylation, kinase activity, the expression of drug efflux pumps, intracellular drug concentrations, and live-cell microscopy. We observed additive effects in EBC-1, H1975, and HCC827, and a strong synergism in the HCC827GR5 cell line. This cell line is a clone of the HCC827 cells that harbor an EGFR exon 19 deletion and has been made resistant to the EGFR-inhibitor gefitinib, resulting in cMET amplification. Remarkably, the intracellular concentration of crizotinib was significantly higher in HCC827GR5 compared to the parental HCC827 cell line. Furthermore, live-cell microscopy with a pH-sensitive probe showed a differential reaction of the pH in the cytoplasm and the lysosomes after drug treatment in the HCC827GR5 in comparison with the HCC827 cells. This change in pH could influence the process of lysosomal sequestration of drugs. These results led us to the conclusion that lysosomal sequestration is involved in the strong synergistic reaction of the HCC827GR5 cell line to crizotinib–erlotinib combination. This finding warrants future clinical studies to evaluate whether genetic background and lysosomal sequestration could guide tailored therapeutic interventions.     

mTOR inhibitors radiosensitize PTEN‐deficient non‐small‐cell lung cancer cells harboring an EGFR activating mutation by inducing autophagy

Clinical resistance to gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), in patients with lung cancer has been linked to acquisition of the T790M resistance mutation in activated EGFR or amplification of MET. Phosphatase and tensin homolog (PTEN) loss has been recently reported as a gefitinib resistance mechanism in lung cancer. The aim of this study was to evaluate the efficacy of radiotherapy in non‐small‐cell lung cancer (NSCLC) with acquired gefitinib resistance caused by PTEN deficiency to suggest radiotherapy as an alternative to EGFR TKIs. PTEN deficient‐mediated gefitinib resistance was generated in HCC827 cells, an EGFR TKI sensitive NSCLC cell line, by PTEN knockdown with a lentiviral vector expressing short hairpin RNA‐targeting PTEN. The impact of PTEN knockdown on sensitivity to radiation in the presence or absence of PTEN downstream signaling inhibitors was investigated. PTEN knockdown conferred acquired resistance not only to gefitinib but also to radiation on HCC827 cells. mTOR inhibitors alone failed to reduce HCC827 cell viability, regardless of PTEN expression, but ameliorated PTEN knockdown‐induced radioresistance. PTEN knockdown‐mediated radioresistance was accompanied by repression of radiation‐induced cytotoxic autophagy, and treatment with mTOR inhibitors released the repression of cytotoxic autophagy to overcome PTEN knockdown‐induced radioresistance in HCC827 cells. These results suggest that inhibiting mTOR signaling could be an effective strategy to radiosensitize NSCLC harboring the EGFR activating mutation that acquires resistance to both TKIs and radiotherapy due to PTEN loss or inactivation mutations. J. Cell. Biochem. 114: 1248–1256, 2013. © 2012 Wiley Periodicals, Inc.