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.     

Mechanistic insights into acquired drug resistance in epidermal growth factor receptor mutation‐targeted lung cancer therapy

Oncogenic mutation of epidermal growth factor receptor kinase domain is strongly associated with clinical response to tyrosine kinase inhibitors in non‐small‐cell lung carcinoma. Despite an initial encouraging response, patients eventually develop drug resistance and relapse. Great efforts have been made to identify the molecular mechanisms of drug resistance. With the recognition of cancer as a whole complex system, here it is proposed that cancer may evolve drug resistance in a cancer‐cell‐autonomous manner as well as a non‐cancer‐cell‐autonomous manner. The former mainly arises at three levels: the robustness of the epidermal growth factor receptor signaling network; cancer epigenetic changes; or cancer genetic alteration, which may be dependent on the therapeutics methods and treatment duration. As cancer stroma plays an essential role in lung cancerigenesis, we further discuss the potential mechanisms for drug resistance development in a non‐cancer‐cell‐autonomous manner, which may arise from the interaction between cancer cells and cancer stroma, including stromal cells and extracellular matrix. (Cancer Sci 2010)     

Role of tumor-derived fibroblasts in the growth of primary cultures of human breast-cancer cells: Effects of epidermal growth factor and the somatostatin analogue octreotide

In the present study we have investigated the role of human breast-cancer-derived fibroblasts in the proliferation of primary cultures of epithelial cells derived from the same tumor. For this purpose, a co-culture system, using Transwell tissue-culture inserts with microporous membranes was employed. Fibroblasts and epithelial cells were enriched according to differences in their density on Percoll density gradients. The co-culture system was first established using MCF-7 breast cancer cells and a human fibroblast line (HF cells). Insulin, 17β-estradiol, EGF and HF cells all significantly stimulated the growth of MCF-7 breast cancer cells. The stimulatory effects of insulin, E2 and EGF were additive to the stimulatory effect of HF cells. These data suggest that (unique) factor(s), other than the above-mentioned growth-promoting compounds, are responsible for the growth-promoting effects of fibroblasts. In half of the human breast cancers investigated, tumor-derived fibroblasts stimulated tumor-derived epithelial cell proliferation. EGF significantly stimulated epithelial cell proliferation in 4 out of 6 cultures. The stimulatory effects of fibroblasts and EGF were additive or synergistic, and were observed in the additional presence of FCS, again suggesting production of unique factor(s) by the fibroblasts, in one culture the fibroblasts significantly inhibited epithelial tumor-cell proliferation. Conversely, the epithelial cells significantly stimulated proliferation of fibroblasts in 3 out of 3 cultures. The somatostatin analogue octreotide significantly inhibited epithelial cell proliferation by 46% in one tumor-cell culture in the absence, but not in the presence, of fibroblasts. In one culture, octreotide significantly inhibited the proliferation of fibroblasts co-cultured with epithelial cells. © 1995 Wiley-Liss, Inc.     

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.     

Biological effect of epidermal growth factor on the in vitro growth of human tumors

The effect of epidermal growth factor (EGF) on the in vitro growth of 186 malignant human tumor specimens (45 melanomas, 32 sarcomas, and 56 lung, 16 gynecological, 14 breast, 12 genitourinary, and 11 gastrointestinal carcinomas) was evaluated in the cellular adhesive matrix human tumor culture system supplemented with transferrin, insulin, hydrocortisone, and estradiol. EGF increased tumor growth by at least 50% in 81% of the 186 tumors and by over 100% in 54%. The enhanced growth induced by EGF was related to an accelerated cellular division independent of tumor type and not to an increase in the actual number of clonogenic units. The drug concentrations of cell cycle-independent Adriamycin and cisplatin needed to achieve a 90% tumor cell kill were not altered by the responsiveness of the tumor to EGF.     

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.     

Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective therapies for non-small cell lung cancer patients whose tumors harbor somatic mutations in EGFR. All patients, however, ultimately develop resistance to these agents. Thus, there is a great need to understand how patients become resistant to develop effective therapies for these cancers. Studies over the last few years have identified two different EGFR tyrosine kinase inhibitor resistance mechanisms, a secondary mutation in EGFR, EGFR 790M, and amplification of the MET oncogene. These findings have led to clinical trials using newly designed targeted therapies that can overcome these resistance mechanisms and have shown promise in laboratory studies. Ongoing research efforts will likely continue to identify additional resistance mechanisms, and these findings will hopefully translate into effective therapies for non-small cell lung cancer patients.     

上皮间质转化在非小细胞肺癌EGFR-TKIs获得性耐药中的作用研究

目的 探讨上皮间质转化（EMT）在非小细胞肺癌（NSCLC）对表皮生长因子受体 酪氨酸激酶抑制剂（EGFR TKIs）获得性耐药中的作用及可能机制。方法 选用EGFR基因19号外显子突变型吉非替尼耐药细胞PC9／AB和EGFR野生型厄洛替尼耐药细胞H460/ER,通过基因转染获得E-cadherin稳定过表达细胞PC9／AB-CDH1和H460ER-CDH1。四甲基偶氮唑盐（MTT）法检测细胞的增殖情况,划痕实验及Transwell侵袭实验检测细胞迁移和侵袭能力,实时荧光定量PCR（qRT-PCR）和蛋白印迹法检测EMT相关分子、EGFR信号通路分子的mRNA和蛋白表达水平。结果 PC9／AB和H460/ER细胞未发生T790M突变和c-Met基因扩增,但发生了EMT,表现为E-cadherin表达降低和Vimentin表达增加。通过基因转染提高E-cadherin表达水平能逆转PC9／AB和H460／ER耐药细胞的EMT,使其对EGFR TKIs的敏感性增加, PC9／AB-CDH1细胞较PC9／AB对吉非替尼的敏感性增加约11.4倍,其半数抑制浓度（IC50）分别为（0.70±0.22） μmol／L和（8.68±0.44）μmol／L,差异有统计学意义（P＜0.05）; H460／ER-CDH1较H460／ER对厄洛替尼的敏感性增加约6.1倍,其IC50分别为（7.51±1.12）μmol／L和（53.72±12.95）μmol／L,差异有统计学意义（P＜0.05）。同时,逆转EMT后,细胞的EGFR、p-EGFR的mRNA和蛋白表达量均增加,差异有统计学意义（P＜0.05）。结论 阻断耐药细胞的EMT可逆转NSCLC对EGFR-TKIs的获得性耐药,EMT在NSCLC对EGFR-TKIs的获得性耐药中起重要作用,其机制可能与EGFR磷酸化水平降低有关。     

Histological transformation after acquired resistance to epidermal growth factor tyrosine kinase inhibitors

Non-small-cell lung cancer patients with sensitive epidermal growth factor receptor mutations generally respond well to tyrosine kinase inhibitors (TKIs). However, acquired resistance will eventually develop place after 8–16 months. Several mechanisms contribute to the resistance including T790M mutation, c-Met amplification, epithelial mesenchymal transformation and PIK3CA mutation; however, histological transformation is a rare mechanism. The patterns and mechanisms underlying histological transformation need to be explored. We searched PubMed, EMBASE and search engines Google Scholar, Medical Matrix for literature related to histological transformation. Case reports, cases series, and clinical and basic medical research articles were reviewed. Sixty-one articles were included in this review. Cases of transformation to small-cell lung cancer, squamous cell carcinoma, large-cell neuroendocrine carcinoma and sarcoma after TKI resistance have all been reported. As the clinical course differed dramatically between cases, a new treatment scheme needs to be recruited. The mechanisms underlying histological transformation have not been fully elucidated and probably relate to cancer stem cells, driver genetic alterations under selective pressure or the heterogeneity of the tumor. When TKI resistance develops, we recommend that patients undergo a second biopsy to determine the reason, guide the next treatment and predict the prognosis.     

The major vault protein mediates resistance to epidermal growth factor receptor inhibition in human hepatoma cells

To better understand the response of HCC to EGFR inhibition, we analyzed factors connected to the resistance of HCC cells against gefitinib. Sensitive HCC3 cells co-expressed EGFR and ErbB3 but lacked kinase-domain mutations in EGFR. Interestingly, expression of MVP was restricted to resistant cell lines, whereas ABCB1 and ABCC1 showed no association with gefitinib resistance. Moreover, ectopic MVP expression in HCC3 cells decreased gefitinib sensitivity, increased AKT phosphorylation and reduced the expression of inflammatory pathway-associated genes, whereas silencing of MVP in Hep3B and HepG2 cells increased sensitivity. These findings suggest MVP as a novel player in resistance against EGFR inhibition.     

Radiation response of human lung cancer cells with inherent and acquired resistance to cisplatin.

We have derived sublines of three human lung cancer cell lines with acquired resistance to cisplatin. The cisplatin resistant sublines of NCI-H69 (small cell), COR-L23 (large cell), and MOR (adenocarcinoma) show 5.3 fold, 3.1 fold, and 3.8 fold resistance, respectively, determined in a 6-day MTT assay. Although the parent lines show a wide range of glutathione content per cell, the sublines each show similar values to their corresponding parent line. Radiation response curves have been obtained using a soft agar clonogenic assay. Values obtained for the parent lines (95% CL in parentheses) were: NCI-H69: Do = 0.99 Gy (0.87-1.16), n = 2.9 (1.6-5.2), GSH = 14 ng/10(4) cells; COR-L23: Do = 1.23 Gy (1.05-1.49), n = 1.3 (0.7-2.2), GSH = 47 ng/10(4) cells; MOR: Do = 1.66 Gy (1.48-1.88), n = 3.0 (1.9-4.8), GSH = 86 ng/10(4) cells. The cisplatin resistant variants of NCI-H69 and COR-L23 showed 31% and 63% increases, respectively, in Do compared to their parent lines, whereas no change in radiation response was seen in MOR. In this panel of lines, therefore, although there is a correlation between glutathione content and radiosensitivity of the parent cell lines, acquired resistance to cisplatin is not accompanied by increased glutathione content. However, two of the three cisplatin resistant lines do show a significantly reduced radiosensitivity.     

尼卡地平和表皮生长因子对胶质瘤细胞生长的影响

目的：观察钙通道拮抗剂尼卡地平（ｎｉｃａｒｄｉｐｉｎｅ）和表皮生长因子（ｒｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ,ＥＧＦ）对恶性胶质瘤细胞系Ｕ２５１ＭＧ生长的作用。方法：在含１０％小牛血清（ｆｅｔａｌ ｂｏｖｉｎｓ ｓｅｒｕｍ,ＦＢＳ）和无血清培养基中,予不同浓度的ＥＧＦ、尼卡地平和ＥＧＦ（１０ｎｇ／ｍｌ）干预,应用ＭＴＴ比色法观察它们的细胞生长效应。结果：在无血清培养基中,ＥＧＦ具有强大的     

Effects of progesterone on the response to epidermal growth factor and other growth factors in cultured human meningioma cells

The presence of receptors for progesterone in a large proportion of human meningioma tissues is well established. The occurrence of increased rates of growth of meningiomas in situ during pregnancy suggests the existence of a relationship between high progesterone levels and the growth of meningiomas. However, experiments with cultured meningioma tissue (cells or explants) have shown only minimal effects of progesterone. It has been shown recently that many meningiomas have receptors for epidermal growth factor. In this paper we have investigated the response of cultured human meningioma cells to epidermal growth factor and other growth factors and the modulation of this response by progesterone and the progesterone-receptor blocking agent mifepristone (RU 38486). The results suggest that the presence of progesterone in the culture medium increases the sensitivity of meningioma cells to mitogenic stimuli, whereas mifepristone can counteract the stimulating effects of progesterone.     

Primary and acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer: an update

Epidermal growth factor receptor (EGFR) is a critical target in the treatment of nonsmall cell lung cancer (NSCLC). The mutations involving EGFR are more prevalent in patients of Asian ancestry, women, never smokers, and those with adenocarcinoma histology. Primary mechanism of resistance to EGFR-TKIs includes in frame insertion mutation in exon 20, de novo T790M mutation also on exon 20, activating mutations in KRAS, loss of PTEN, and amplification of c-MET whereas acquired resistance results from development of secondary alteration in ATP domain of T790M. There are many novel targeting agents in development to overcome resistance to EGFR TKIs.     

ALDH-positive lung cancer stem cells confer resistance to epidermal growth factor receptor tyrosine kinase inhibitors

Molecular targeting therapeutics, such as EGFR tyrosine kinase inhibitors (TKIs), are important treatment strategies for lung cancer. Currently, the major challenge confronting targeted cancer therapies is the development of resistance. Cancer stem cells (CSCs) represent a rare population of undifferentiated tumorigenic cells responsible for tumor initiation, maintenance and spreading. Resistance to conventional chemotherapeutic drugs is a common characteristic of CSCs. However, the issue of whether CSCs contribute to EGFR TKI resistance in lung cancer is yet to be established. In the current study, we explored the association of ALDH1A1 expression with EGFR TKI resistance in lung cancer stem cells. ALDH1A1-positive lung cancer cells displayed resistance to gefitinib, compared to ALDH1A1-negative lung cancer cells. Moreover, PC9/gef cells (gefitinib-resistant lung cancer cells) presented a higher proportion of ALDH1A1-positive cells, compared to PC9 cells (gefitinib-sensitive lung cancer cells). Clinical sample studies were consistent with results from cell culture model systems showing that lung cancer cells with resistance to EGFR TKI and chemotherapy drugs contain significantly increased proportions of ALDH1A1-positive cells. These findings collectively suggest that ALDH1A1 positivity in cancer stem cells confers resistance to EGFR TKI in lung cancer.     

Expression of epidermal growth factor receptor gene in cultured human lung cancer cells

The expression and organization of epidermal growth factor (EGF) receptor gene in cultured human lung cancer cell lines (5 adenocarcinomas, 3 squamous cell carcinomas, 2 small cell carcinomas and 1 large cell carcinoma) have been studied. Two (PC-8 and PC-9) of the adenocarcinomas overproduced EGF receptor mRNA and protein, and exhibited gene amplification, the magnitude of which was comparable to that of A431 cells. Six cell lines (3 adenocarcinomas, 2 squamous cell carcinomas and 1 small cell carcinoma) expressed EGF receptor gene and its product to a significant level without gene amplification, and the other three cell lines were found to be negative as regards expression.     

Recurrent growth factor starvation promotes drug resistance in human leukaemic cells

Multi-drug resistance can be induced by various environmental stresses including an exposure to chemical drugs and X-ray irradiation. In addition, hypo-nutritive conditions are known to promote multi-drug resistance in solid tumours. To understand the importance of nutritive conditions in the development of drug resistance in non-solid tumours and to know whether a transient malnutrition could induce a permanent reduction in drug sensitivity, leukaemic cells were transiently cultured under growth factor-starved conditions. Granulocyte-macrophage colony-stimulating factor-dependent human leukaemic MO7e cells were cultured in the absence of granulocyte-macrophage colon-stimulating factor for 2 weeks, during which the majority of the cells died, and the minor viable cells were expanded in the presence of granulocyte-macrophage colon-stimulating factor for following 1 week. This procedure was repeated three times, and the surviving cells were cloned by limiting dilution. These clones underwent G1 arrest in the absence of granulocyte-macrophage colon-stimulating factor, while parental cells underwent apoptosis. Interestingly, activities of the downstream targets of granulocyte-macrophage colon-stimulating factor receptor were regulated in a granulocyte-macrophage colon-stimulating factor-independent manner, indicating that the ligand-independent activation of granulocyte-macrophage colon-stimulating factor receptor had not taken place. Moreover, the 4--7-fold increases in IC(50) for etoposide and the 2--6-fold increase in IC(90) for doxorubicin was observed. Furthermore, Bcl-2 protein expression was significantly up-regulated in the clones while no significant changes in Bax, Bcl-(xL), P-glycoprotein and Hsp70 protein expression and no consistent changes in p53 expression were detected. We propose that recurrent growth factor starvation, which may occur in vivo when stromal function is damaged after intensive chemotherapy or bone marrow occupation by malignant cells, causes selection of drug resistant leukaemia cells that will expand when the growth factor supply recovers.     

Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib.

PURPOSE: Non-small cell lung cancers carrying activating mutations in the gene for the epidermal growth factor receptor (EGFR) are highly sensitive to EGFR-specific tyrosine kinase inhibitors. However, most patients who initially respond subsequently experience disease progression while still on treatment. Part of this "acquired resistance" is attributable to a secondary mutation resulting in threonine to methionine at codon 790 (T790M) of EGFR. EXPERIMENTAL DESIGN: We sequenced exons 18 to 21 of the EGFR gene to look for secondary mutations in tumors with acquired resistance to gefitinib in 14 patients with adenocarcinomas. Subcloning or cycleave PCR was used in addition to normal sequencing to increase the sensitivity of the assay. We also looked for T790M in pretreatment samples from 52 patients who were treated with gefitinib. We also looked for secondary KRAS gene mutations because tumors with KRAS mutations are generally resistant to tyrosine kinase inhibitors. RESULTS: Seven of 14 tumors had a secondary T790M mutation. There were no other novel secondary mutations. We detected no T790M mutations in pretreatment specimens from available five tumors among these seven tumors. Patients with T790M tended to be women, never smokers, and carrying deletion mutations, but the T790M was not associated with the duration of gefitinib administration. None of the tumors had an acquired mutation in the KRAS gene. CONCLUSIONS: A secondary T790M mutation of EGFR accounted for half the tumors with acquired resistance to gefitinib in Japanese patients. Other drug-resistant secondary mutations are uncommon in the EGFR gene.     

Binding of phorbol dibutyrate and epidermal growth factor to cultured human epidermal cells

Primary cell cultures of normal human epidermal keratinocytes and melanocytes and human cell lines established from a primary melanoma (SK-PM-4) and metastatic melanomas (HO#1, SK-MEL21, and SK-MEL37) contain specific and saturable receptors for the tumor promoter phorbol dibutyrate (PDBu). Scatchard analyses of the keratinocytes revealed two classes of binding sites: 1) a high-affinity class (affinity constant = 37 nM; 1.3 X 10(6) sites/cell) and a low-affinity class (affinity constant = 4,880 nM; 7 X 10(7) sites/cell). The melanoma cultures, likewise, showed high- and low-affinity classes of PDBu binding sites. However, the affinity constant values and total numbers of sites in the melanoma cells were lower than the corresponding values in the keratinocytes. The binding of [3H]PDBu to human keratinocytes was inhibited by the tumor promoters 12-O-tetradecanoylphorbol 13-acetate and teleocidin but not by phorbol, which lacks tumor-promoting activity. Human serum also inhibited binding. Specific receptors for epidermal growth factor (EGF) were demonstrated in the keratinocytes and primary melanoma cultures. In contrast, three metastatic melanoma cultures gave negligible levels of EGF binding. Among the various cell types, the extent of [3H]PDBu binding did not correlate with the extent of EGF binding, indicating that these two substances occupy distinctly separate types of receptors.