酪氨酸激酶抑制剂在非小细胞肺癌中的耐药机制

EGFR-TKIs在EGFR突变型非小细胞肺癌治疗中是非常有效的。然而在非小细胞肺癌EGFR突变患者中约有20%天然耐药,即使治疗有效,在10~16月左右会产生继发耐药。迄今为止,研究发现可能的继发性耐药机制包括EGFR二次突变、旁路激活、下游信号激活、小细胞转化及上皮-间质转化（EMT）等。近期,有研究发现BIM基因与TKI耐药相关。本文就有关耐药机制的近期研究进展作一综述。     

The insulin‐like growth factor 1 receptor causes acquired resistance to erlotinib in lung cancer cells with the wild‐type epidermal growth factor receptor

Epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor (TKI) therapy often provides a dramatic response in lung cancer patients with EGFR mutations. In addition, moderate clinical efficacy of the EGFR‐TKI, erlotinib, has been shown in lung cancer patients with the wild‐type EGFR. Numerous molecular mechanisms that cause acquired resistance to EGFR‐TKIs have been identified in lung cancers with the EGFR mutations; however, few have been reported in lung cancers with the wild‐type EGFR. We used H358 lung adenocarcinoma cells lacking EGFR mutations that showed modest sensitivity to erlotinib. The H358 cells acquired resistance to erlotinib via chronic exposure to the drug. The H358 erlotinib‐resistant (ER) cells do not have a secondary EGFR mutation, neither MET gene amplification nor PTEN downregulation; these have been identified in lung cancers with the EGFR mutations. From comprehensive screening of receptor tyrosine kinase phosphorylation, we observed increased phosphorylation of insulin‐like growth factor 1 receptor (IGF1R) in H358ER cells compared with parental H358 cells. H358ER cells responded to combined therapy with erlotinib and NVP‐AEW541, an IGF1R‐TKI. Our results indicate that IGF1R activation is a molecular mechanism that confers acquired resistance to erlotinib in lung cancers with the wild‐type EGFR.     

Clinical outcomes and secondary epidermal growth factor receptor (EGFR) T790M mutation among first-line gefitinib,erlotinib and afatinib-treated non-small cell lung cancer patients with activating EGFR mutations

Gefitinib, erlotinib and afatinib are approved for first-line treatment of advanced non-small cell lung cancer (NSCLC) bearing an activating epidermal growth factor receptor (EGFR) mutation. However, the clinical outcomes among the three EGFR tyrosine kinase inhibitors (TKIs) are still controversial. We aimed to evaluate clinical outcomes and secondary EGFR T790M mutation among the three EGFR TKIs. From May 2014 to January 2016, a total of 301 patients received treatment with gefitinib, erlotinib or afatinib, for first-line treatment of advanced NSCLC with an activating EGFR mutation, based on their clinicians’ choice. The median overall survival (OS) was 37.0 months. Although the baseline characteristics of patients were unequal, progression-free survival and OS did not differ among the 3 groups. Multivariate analysis found that gefitinib (adjusted odds ratio [aOR] 3.29, 95% confidence interval [CI], 1.15–9.46, p = 0.027), EGFR TKI treatment duration more than 13 months (aOR 3.16, 95% CI, 1.20–8.33, p = 0.020), male (aOR 3.25, 95% CI, 1.10–9.66, p = 0.034), initial liver metastasis (aOR 4.97, 95% CI 1.18–20.96, p = 0.029) and uncommon EGFR mutation (aOR 0.14, 95% CI, 0.02–0.97, compared to EGFR deletion 19, p = 0.047) were independent factors for secondary T790M mutation. In real-world practice, choosing first line EGFR TKI based on the patients’ clinical characteristics yielded good clinical outcomes. First-line gefitinib, longer EGFR TKI treatment duration, male, initial liver metastasis and uncommon EGFR mutations may be independent factors for secondary EGFR T790M mutation.     

Phase I/II trial of vorinostat (SAHA) and erlotinib for non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations after erlotinib progression

Objectives

Vorinostat or suberoylanilide hydroxamic acid (SAHA) is a novel histone deacetylase inhibitor with demonstrated antiproliferative effects due to drug-induced accumulation of acetylated proteins, including the heat shock protein 90. We prospectively studied the activity of vorinostat plus erlotinib in EGFR-mutated NSCLC patients with progression to tyrosine kinase inhibitors.

Patients and methods

We conducted this prospective, non-randomized, multicenter, phase I/II trial to evaluate the maximum tolerated dose, toxicity profile and efficacy of erlotinib and vorinostat. Patients with advanced NSCLC harboring EGFR mutations and progressive disease after a minimum of 12 weeks on erlotinib were included. The maximum tolerated dose of vorinostat plus erlotinib was used as recommended dose for the phase II (RDP2) to assess the efficacy of the combination. The primary end point was progression-free-survival rate at 12 weeks (PFSR_12w). Pre-treatment plasma samples were required to assess T790M resistant mutation.

Results

A total of 33 patients were enrolled in the phase I–II trial. The maximum tolerated dose was erlotinib 150 mg p.o., QD, and 400 mg p.o., QD, on days 1–7 and 15–21 in a 28-day cycle. Among the 25 patients treated at the RDP2, the most common toxicities included anemia, fatigue and diarrhea. No responses were observed. PFSR_12w was 28% (IC95%: 18.0–37.2); median progression-free survival (PFS) was 8 weeks (IC 95%: 7.43–8.45) and overall survival (OS) 10.3 months (95% CI: 2.4–18.1).

Conclusion

Full dose of continuous erlotinib with vorinostat 400 mg p.o., QD on alternative weeks can be safely administered. Still, the combination has no meaningful activity in EGFR-mutated NSCLC patients after TKI progression.     

The novel phosphoinositide 3‐kinase–mammalian target of rapamycin inhibitor,BEZ235, circumvents erlotinib resistance of epidermal growth factor receptor mutant lung cancer cells triggered by hepatocyte growth factor

Acquired resistance to epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR–TKIs), such as gefitinib and erlotinib, is a critical problem in the management of patients with EGFR mutant lung cancer. Several mechanisms have been reported involved in this acquired resistance, including hepatocyte growth factor (HGF) activation of an alternative pathway. PI3K and mTOR are downstream molecules of receptor tyrosine kinases, such as EGFR and Met, and are thought to be ideal targets for controlling various tumor types. We assessed whether BEZ235, a dual inhibitor of PI3K and mTOR, could overcome the EGFR–TKI resistance induced by HGF in an EGFR mutant lung cancer model. Exogenous and endogenous HGF triggered resistance to erlotinib in the PC‐9 and HCC827, EGFR mutant lung cancer cell lines. BEZ235 alone inhibited the viability of PC‐9 and HCC827 cells in vitro, irrespective of the presence or the absence of HGF. Using a xenograft model of severe combined immunodeficient mice with HGF‐gene‐transfected PC‐9 cells (PC‐9/HGF), we found that BEZ235 inhibited tumor growth, whereas erlotinib did not. BEZ235 monotherapy also inhibited the phosphorylation of Akt and p70S6K/S6RP, downstream molecules of PI3K and mTOR, respectively, as well as suppressing tumor‐cell proliferation and angiogenesis of PC‐9/HGF tumors. These results suggest that BEZ235, even as monotherapy, may be useful in managing HGF‐induced EGFR–TKI resistance in EGFR mutant lung cancer.     

The impact of epidermal growth factor receptor gene status on gefitinib-treated Japanese patients with non-small-cell lung cancer

We investigated the relationships between genetic factors and clinical outcome in Japanese non-small-cell lung cancer (NSCLC) patients treated with gefitinib. Ninety-eight NSCLC patients who had been treated with gefitinib, were screened for mutations in epidermal growth factor receptor (EGFR) exons 18-21, KRAS exon2, and polymorphisms including the CA simple sequence repeat in intron1 (CA-SSR1) and single nucleotide polymorphisms in the promoter region (-216G/T and -191C/A), using a PCR-based assay and direct sequencing. The EGFR copy number status was also evaluated using a fluorescence in situ hybridization assay. EGFR and KRAS mutations were found in 38 (38.8%) and 8 (8.2%) of the 98 patients, respectively. A high EGFR copy number status was identified in 31 (41.3%) of the 75 assessable patients. Drug-sensitive EGFR mutations limited to exon19 deletions and L858R were independent predictive factors of a stronger sensitivity to gefitinib (p = 0.0002), the overall survival (OS) (p = 0.0036), and prolonged progression-free survival (PFS) (p < 0.0001). The EGFR copy number status was not related to a sensitivity to gefitinib and prolonged OS and PFS. Regarding polymorphisms, patients with a short CA-SSR1 showed a prolonged OS as compared with those with a long length in patients with a drug-sensitive EGFR mutation, although this difference was not significant (p = 0.13). Thus, drug-sensitive EGFR mutations predict a favorable clinical outcome and a high EGFR copy number may not be related to clinical benefits in gefitinib-treated Japanese patients with NSCLC. Our findings also suggest that the CA-SSR1 length may influence the clinical outcome in patients with a drug-sensitive EGFR mutation.     

靶向NSCLC EGFR exon20插入突变的新抗原疫苗筛选和功能研究

目的:筛选具有免疫源性HLA-A^*02限制性表皮生长因子受体(epidermal growth factor receptor,EGFR)exon20插入突变编码的优势表位肽,为携带EGFR exon20插入突变的非小细胞肺癌(non-small cell lung cancer,NSCLC)提供新的免疫治疗手段。方法:通过IEDB、NetMHC 4.0和SYFPEITHI软件,筛选EGFR exon20插入突变编码的HLA-A^*02限制性表位,针对细胞毒性T淋巴细胞表位集中区域设计多肽疫苗,通过体外实验验证其免疫活性。结果:V769_D770insASV为EGFR exon20插入突变最高频突变位点(19.35%),经软件预测其编码的多肽YVMASVASV与HLA-A^*02具有较强的结合力。围绕核心序列设计两条优势表位多肽E-ASV-10和E-ASV-19,体外可诱导HLA-A^*02限制性T细胞的扩增和活化,上调4-1BB⁺CD25⁺...     

Enhanced gefitinib‐induced repression of the epidermal growth factor receptor pathway by ataxia telangiectasia‐mutated kinase inhibition in non‐small‐cell lung cancer cells

The epidermal growth factor receptor (EGFR) tyrosine kinase signaling pathways regulate cellular activities. The EGFR tyrosine kinase inhibitors (EGFR‐TKIs) repress the EGFR pathway constitutively activated by somatic EGFR gene mutations and have drastically improved the prognosis of non‐small‐cell lung cancer (NSCLC) patients. However, some problems, including resistance, remain to be solved. Recently, combination therapy with EGFR‐TKIs and cytotoxic agents has been shown to improve the prognosis of NSCLC patients. To enhance the anticancer effects of EGFR‐TKIs, we examined the cross‐talk of the EGFR pathways with ataxia telangiectasia‐mutated (ATM) signaling pathways. ATM is a key protein kinase in the DNA damage response and is known to phosphorylate Akt, an EGFR downstream factor. We found that the combination of an ATM inhibitor, KU55933, and an EGFR‐TKI, gefitinib, resulted in synergistic cell growth inhibition and induction of apoptosis in NSCLC cell lines carrying the sensitive EGFR mutation. We also found that KU55933 enhanced the gefitinib‐dependent repression of the phosphorylation of EGFR and/or its downstream factors. ATM inhibition may facilitate the gefitinib‐dependent repression of the phosphorylation of EGFR and/or its downstream factors, to exert anticancer effects against NSCLC cells with the sensitive EGFR mutation.     

Combined epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor and chemotherapy in non-small-cell lung cancer: Chemo-refractoriness of cells harboring sensitizing-EGFR mutations in the presence of gefitinib

Background

Combined epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) with chemotherapy is believed to be more effective in treating non-small-cell lung cancer (NSCLC) with sensitizing-EGFR mutation (SEM). This hypothesis failed to be realized clinically and needs to be examined in vitro.

Materials and methods

Using the tetrazolium colorimetric assay and classical isobole method, we investigated the combination effects of 6 gefitinib-chemotherapeutic doublets (gefitinib/cisplatin, gemcitabine, pemetrexed, paclitaxel, docetaxel, or vinorelbine) in a panel of 15 NSCLC cell lines.

Results

Upon treatment with the 6 gefitinib-chemotherapeutic doublets, the 12 cell lines that did not harbor SEM displayed a broad spectrum of group results, from obvious synergism to robust antagonism. The values of group mean combination index (mCIs) ranged from 0.769 to 1.201. In contrast, the 3 cell lines with SEM showed a tendency toward consistent antagonism to the tested doublets, impressively, with a narrow range of higher group mCIs (0.993–1.141). In the presence of gefitinib, the SEM or gefitinib-sensitive group was more chemo-refractory than the non-SEM (index of chemo-refractoriness (RI): 69.33 versus 42.67; P = 0.036) or gefitinib-resistant group (68.25 versus 40.64, P = 0.0108), respectively. The results of using the gefitinib/drug combinations with the gefitinib-sensitive non-SEM cell line H322 and the gefitinib-resistant EGFR mutant H820 shared patterns similar to those with the SEM and non-SEM cell lines, respectively.

Conclusion

Gefitinib-treated EGFR-TKI-sensitive NSCLC cells showed a wide spectrum of chemo-refractoriness, suggesting that concomitantly combined EGFR-TKI-chemotherapy might not be a good treatment strategy for NSCLC harboring SEM.     

Association of epidermal growth factor receptor mutations in lung cancer with chemosensitivity to gefitinib in isolated cancer cells from Japanese patients

Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are reported to be associated with clinical responsiveness of lung cancer to gefitinib, an EGFR tyrosine kinase inhibitor. To elucidate the association between somatic mutations and the pharmacological actions of gefitinib, the chemosensitivity of isolated cancer cells from the lungs of Japanese patients to gefitinib was examined by the collagen gel-droplet embedded culture drug sensitivity test in vitro. In 30 specimens isolated from non-small-cell lung cancer patients, mutations were observed in eight tumour specimens (27%) and chemosensitivity to gefitinib was observed in seven specimens (23%). However, somatic mutations were not predominantly associated with chemosensitivity to gefitinib in vitro. Both mutation and chemosensitivity frequencies in this study were higher than those reported in studies from the United States, indicating a possible ethnic difference. Moreover, both frequencies were much higher in females than in males. Since a gender difference in chemosensitivity to gefitinib was observed in isolated cancer cells in vitro, this suggests that gefitinib works in part through the suppression of EGFR signalling, but that other factors, including sex-related factors, may participate in gefitinib action.     

Glucose metabolism‐targeted therapy and withaferin A are effective for epidermal growth factor receptor tyrosine kinase inhibitor‐induced drug‐tolerant persisters

In pathway‐targeted cancer drug therapies, the relatively rapid emergence of drug‐tolerant persisters (DTPs) substantially limits the overall therapeutic benefit. However, little is known about the roles of DTPs in drug resistance. In this study, we investigated the features of epidermal growth factor receptor–tyrosine kinase inhibitor‐induced DTPs and explored a new treatment strategy to overcome the emergence of these DTPs. We used two EGFR‐mutated lung adenocarcinoma cell lines, PC9 and II‐18. They were treated with 2 μM gefitinib for 6, 12, or 24 days or 6 months. We analyzed the mRNA expression of the stem cell‐related markers by quantitative RT‐PCR and the expression of the cellular senescence‐associated proteins. Then we sorted DTPs according to the expression pattern of CD133 and analyzed the features of sorted cells. Finally, we tried to ablate DTPs by glucose metabolism targeting therapies and a stem‐like cell targeting drug, withaferin A. Drug‐tolerant persisters were composed of at least two types of cells, one with the properties of cancer stem‐like cells (CSCs) and the other with the properties of therapy‐induced senescent (TIS) cells. The CD133^high cell population had CSC properties and the CD133^low cell population had TIS properties. The CD133^low cell population containing TIS cells showed a senescence‐associated secretory phenotype that supported the emergence of the CD133^high cell population containing CSCs. Glucose metabolism inhibitors effectively eliminated the CD133^low cell population. Withaferin A effectively eliminated the CD133^high cell population. The combination of phloretin and withaferin A effectively suppressed gefitinib‐resistant tumor growth.     

Gefitinib as first-line treatment for patients with advanced non-small-cell lung cancer with activating epidermal growth factor receptor mutation: Review of the evidence

Gefitinib is a small molecule tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR). Since 2004, it was clear that a substantial proportion of non-small-cell lung cancers (NSCLC) obtaining objective response when treated with gefitinib harbour activating mutations in the EGFR gene. Consequently, EGFR mutation has been widely studied, together with other molecular characteristics, as a potential predictive factor for gefitinib efficacy. As of August 2010, four East Asian randomized phase III trials comparing gefitinib to platinum-based chemotherapy in patients with advanced non-small-cell lung cancer (NSCLC) eligible for first-line treatment have been reported or published. Two of these trials were conducted without a molecular selection in patients with clinical characteristics (adenocarcinoma histology, never or light smoking) characterized by higher prevalence of EGFR mutation. In patients selected for the presence of tumor harbouring EGFR mutation, the administration of first-line gefitinib, as compared to standard chemotherapy, was associated with longer progression-free survival, higher objective response rate, a more favourable toxicity profile and better quality of life. The relevant improvement in progression-free survival with first-line administration of gefitinib has been confirmed in the other two randomized trials, dedicated to cases with EGFR mutation. In July 2009, European Medicines Agency granted marketing authorization for gefitinib for the treatment of locally advanced or metastatic NSCLC with sensitizing mutations of the EGFR gene, across all lines of therapy. Gefitinib currently represents the best first-line treatment option for this molecularly selected subgroup of patients.     

Outcomes of Malaysian patients with advanced lung adenocarcinoma and unknown epidermal growth factor receptor mutation status treated with gefitinib

Aims: To evaluate the response and progression‐free survival (PFS) of Malaysian patients with advanced lung adenocarcinoma and unknown epidermal growth factor receptor (EGFR) mutation status treated with gefitinib. Methods: A retrospective analysis of consecutive patients with EGFR mutation unknown stage III or IV lung adenocarcinoma with EGFR mutation unknown treated with gefitinib until disease progression. Results: Of 71 patients, none had complete response while 26 (36.6%) had partial response and 26 (36.6%) had stable disease. Multivariate analysis showed the independent predictor of response to gefitinib was Eastern Cooperative Oncology Group (ECOG) performance status 1 (odds ratio [OR] 5.39, 95% confidence interval [CI 1.64–17.74]P = 0.006). The median PFS was 6.5 months and was significantly longer in female than male patients (39.0 vs 21.2 weeks; P < 0.001), never smokers vs smokers (32.3 vs 8.3 weeks, P = 0.001), and stage III versus stage IV disease (44 vs 24 weeks, P = 0.021). In a multivariate Cox proportional hazards model with age group, gender, ethnicity, smoking history, disease stage, ECOG performance status and prior cytotoxic chemotherapy as covariates, the independent predictors of longer median PFS were female gender (HR 95% CI 0.38 [0.22–0.66]; P < 0.001) and stage III disease (HR 95% CI 0.54 [0.30–0.98], P = 0.042). Conclusion: In our patients with EGFR mutation unknown advanced lung adenocarcinoma treated with gefitinib, the response rate was 36.6% and the median PFS was significantly longer in female patients, never smokers and patients with stage III disease.     

Coexpression of epidermal growth factor receptor with related factors is associated with a poor prognosis in non-small-cell lung cancer

The epidermal growth factor receptor (EGFR) is commonly expressed in non-small-cell lung cancer (NSCLC) and promotes a host of mechanisms involved in tumorigenesis. However, EGFR expression does not reliably predict prognosis or response to EGFR-targeted therapies. The data from two previous studies of a series of 181 consecutive surgically resected stage I-IIIA NSCLC patients who had survived in excess of 60 days were explored. Of these patients, tissue was available for evaluation of EGFR in 179 patients, carbonic anhydrase (CA) IX in 177 patients and matrix metalloproteinase-9 (MMP-9) in 169 patients. We have previously reported an association between EGFR expression and MMP-9 expression. We have also reported that MMP-9 (P=0.001) and perinuclear (p)CA IX (P=0.03) but not EGFR expression were associated with a poor prognosis. Perinuclear CA IX expression was also associated with EGFR expression (P<0.001). Multivariate analysis demonstrated that coexpression of MMP-9 with EGFR conferred a worse prognosis than the expression of MMP-9 alone (P<0.001) and coexpression of EGFR and pCA IX conferred a worse prognosis than pCA IX alone (P=0.05). A model was then developed where the study population was divided into three groups: group 1 had expression of EGFR without coexpression of MMP-9 or pCA IX (number=21); group 2 had no expression of EGFR (number=75); and group 3 had coexpression of EGFR with pCA IX or MMP-9 or both (number=70). Group 3 had a worse prognosis than either groups 1 or 2 (P=0.0003 and 0.027, respectively) and group 1 had a better prognosis than group 2 (P=0.036). These data identify two cohorts of EGFR-positive patients with diametrically opposite prognoses. The group expressing either EGFR and or both MMP-9 and pCA IX may identify a group of patients with activated EGFR, which is of clinical relevance with the advent of EGFR-targeted therapies.     

Analysis of the prognostic value of soluble epidermal growth factor receptor plasma concentration in advanced non-small-cell lung cancer patients

Background: Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial malignancies including lung cancer. A soluble fragment of the EGFR extracellular domain (sEGFR) can be detected in the blood of patients who have non–small-cell lung cancer (NSCLC), but its clinical/ prognostic role must be further elucidated. Methods: sEGFR concentration was retrospectively determined by enzyme-linked immunosorbent assay in plasma samples from 308 advanced NSCLC patients (before treatment) and 109 healthy controls and correlated with clinico-pathological variables. Results: The concentration of sEGFR was lower in NSCLC patients than in controls (P < .0001). sEGFR behaves as a sensitive but not specific screening biomarker. No significant associations were observed between sEGFR concentration and demographic/clinical characteristics such as gender, Eastern Cooperative Oncology Group performance status, stage, and number or location of the metastatic sites. sEGFR was lower in patients with progressive disease or in squamous cell carcinoma compared with adenocarcinoma, but these differences were not significant. Patients with sEGFR ≤ 34.56 ng/mL showed a shorter overall survival (median 9.1 versus 12.2 months, P = .019) than others. Moreover, in multivariate analysis, sEGFR remained a significant independent prognostic marker. Conclusion: Low baseline sEGFR is associated with reduced survival in advanced NSCLC. Therefore, our findings in this large cohort of patients suggest that the determination of sEGFR concentration provides valuable prognostic information.     

A phase II trial of gefitinib as first-line therapy for advanced non-small cell lung cancer with epidermal growth factor receptor mutations

Retrospective analysis has shown that activating mutations in exons 18–21 of the epidermal growth factor receptor (EGFR) gene are a predictor of response to gefitinib. We conducted a phase II trial to evaluate the efficacy and safety of gefitinib as first-line therapy for advanced non-small cell lung cancer (NSCLC) with EGFR mutations. Patients with stage IIIB or IV chemotherapy-naïve NSCLC with EGFR mutation were treated with 250 mg gefitinib daily. For mutational analysis, DNA was extracted from paraffin-embedded tissues and EGFR mutations were analysed by direct sequence of PCR products. Twenty (24%) of the 82 patients analysed had EGFR mutations (deletions in or near E746-A750, n=16; L858R, n=4). Sixteen patients were enrolled and treated with gefitinib. Twelve patients had objective response and response rate was 75% (95% CI, 48–93%). After a median follow-up of 12.7 months (range, 3.1–16.8 months), 10 patients demonstrated disease progression, with median progression-free survival of 8.9 months (95% CI, 6.7–11.1 months). The median overall survival time has not yet been reached. Most of the toxicities were mild. This study showed that gefitinib is very active and well tolerated as first-line therapy for advanced NSCLC with EGFR mutations.     

MEK inhibitors reverse resistance in epidermal growth factor receptor mutation lung cancer cells with acquired resistance to gefitinib

Lung adenocarcinoma cells harboring epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib. Acquired resistance to EGFR-TKIs develops after prolonged treatments. The study was prompt to explore effective strategies against resistance to EGFR-TKIs. We established gefitinib resistant PC-9 cells which harbor EGFR exon 19 deletion. Known mechanisms for intrinsic or acquired EGFR-TKI resistance, including KRAS mutation, HER2 mutation, EGFR T790M mutation and MET gene amplification, were studied, and we did not observe any known mechanisms for intrinsic or acquired resistance to EGFR-TKIs in the resistant cells. In the parental PC-9 cells, labeled as PC-9/wt, gefitinib completely inhibited EGF-induced phosphorylation of EGFR, AKT and ERK. Gefitinib inhibited EGFR phosphorylation, but was unable to block EGF-induced phosphorylation of ERK in resistant cells, labeled as PC-9/gef cells, including PC-9/gefB4, PC-9/gefE3, and PC-9/gefE7 subclones. We detected NRAS Q61K mutation in the PC-9/gef cells but not the PC-9/wt cells. MEK inhibitors, either AZD6244 or CI1040, inhibited ERK phosphorylation and sensitized gefitinib-induced cytotoxicity in PC-9/gef cells. Whereas MEK inhibitors or gefitinib alone did not activate caspases in PC-9/gef cells, combination of gefitinib and AZD6244 or CI1040 induced apoptosis. Our in vivo studies showed that gefitinib inhibited growth of PC-9/wt xenografts but not PC-9/gef xenografts. Furthermore, combination of a MEK inhibitor and gefitinib inhibited growth of both PC-9/wt xenografts and PC-9/gefB4 xenografts. To conclude, persistent activation of ERK pathway contributes to the acquired gefitinib-resistance. Combined treatment of gefitinib and MEK inhibitors may be therapeutically useful for acquired gefitinib-resistance lung adenocarcinoma cells harboring EGFR mutations.     

Induction of Cbl‐dependent epidermal growth factor receptor degradation in Ling Zhi‐8 suppressed lung cancer

Activating mutation of epidermal growth factor receptor (EGFR) is correlated with malignant lung tumor. In our study, we demonstrated that recombinant LZ‐8 (rLZ‐8), a medicinal mushroom Ganoderma lucidum protein, induced cell cycle arrest and apoptosis by downregulating the expression of wild‐type and mutated EGFR and inhibiting EGFR downstream effectors, AKT and ERK1/2 in lung cancer cells. We showed that rLZ‐8 effectively inhibited lung cancer progression and suppressed EGFR expression of lung tumor lesions in mouse model. Functional studies revealed that rLZ‐8 reduced the amount of EGFR in cell membranes by altering EGFR localization to enhance the EGF‐induced degradation of EGFR. Mechanistically, we demonstrated that rLZ‐8 bound to EGFR to induce EGFR autophosphorylation at tyrosine1045 and trigger ubiquitination by inducing the formation of EGFR/Cbl complexes, resulting in the degradation of EGFR; however, Cbl‐shRNA abolished rLZ‐8‐induced EGFR degradation. We provide the first evidence showing that rLZ‐8 inhibits growth and induces apoptosis of lung cancer cells by promoting EGFR degradation. The current findings therefore suggest a novel anti‐cancer function of rLZ‐8 that targeting EGFR overexpression or mutation as well as EGFR‐dependent processes in cancer cells.