Biomedicine & Pharmacotherapy 68 (2014) 307–313

Available online at

ScienceDirect www.sciencedirect.com

Original article

Fulvestrant increases gefitinib sensitivity in non-small cell lung cancer cells by upregulating let-7c expression Hua Shen a, Jinyuan Liu b, Rong Wang a, Xu Qian c, Ruitong Xu a, Tongpeng Xu a, Qi Li c, Lin Wang c, Zhumei Shi c, Jitai Zheng c, Qiudan Chen c, Yongqian Shu a,* a b c

Department of Oncology, First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of Cardiothoracic Surgery, First Affiliated Hospital of Nanjing Medical University, Nanjing, China Department of Pathology, Cancer Center, Nanjing Medical University, 210029 Nanjing, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 September 2013 Accepted 24 October 2013

Patients with non-small cell lung cancer (NSCLC) who have activating epidermal growth factor receptor (EGFR) mutations benefit from treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKIs), namely, gefitinib and erlotinib. However, these patients eventually develop resistance to EGFR-TKIs. About 50% of this acquired resistance may be the result of a secondary mutation in the EGFR gene, such as the one corresponding to T790M. In our previous study, we found that combined treatment with fulvestrant and gefitinib decreases the proliferation of H1975 NSCLC cells, compared to treatment with either fulvestrant or gefitinib alone; however, the molecular mechanism for the improved effects of the combination treatment are still unknown. In this study, we confirmed that fulvestrant increases the gefitinib sensitivity of H1975 cells and found that let-7c was most upregulated in the fulvestrant-treated cells. Our data revealed that let-7c increases gefitinib sensitivity by repressing RAS and inactivating the phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. Taken together, our findings suggest that let-7c plays an important role in fulvestrant-induced upregulation of gefitinib sensitivity in H1975 cells. ß 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Non-small cell lung cancer Epidermal growth factor receptor Gefitinib Resistance Fulvestrant

1. Introduction Lung cancer is one of the most common and fatal malignant diseases. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancer cases [1]. With recent improvements in our understanding of the molecular abnormalities in NSCLC, epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib have been used to treat patients with locally advanced or metastatic NSCLC [2]. Recent research showed that patients with activating EGFR mutations had significantly longer progression-free survival (PFS) rates when treated with EGFR-TKIs, compared to that with standard chemotherapy regimens [3]. Thus, EGFR-TKIs have been recommended as the first line therapy for NSCLC patients with EGFR mutations. However, most patients who initially respond to EGFRTKIs subsequently experience disease progression while on TKI treatment for about 3 to 8 months [4]. While KRAS mutations have been associated with some cases of primary resistance to EGFRTKIs, about 50% of patients who initially respond to EGFR-TKIs

* Corresponding author. Tel.: +86 138 5142 2201; fax: +86 021 6408 5875. E-mail address: [email protected] (Y. Shu). 0753-3322/$ – see front matter ß 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2013.10.007

cease responding because of an acquired EGFR mutation in exon 20, which is the most common mutation and results in a change from threonine to methionine at codon 790 (T790M) of EGFR [4,5]. Because the T790M mutation restores the affinity of ATP to the ATP-binding site of the mutated EGFR, oncogene addiction promoted by the EGFR pathway is presumed to be sustained in EGFR-TKI-resistant cells harboring the T790M mutation [6,7]. Thus, a method for suppressing the restored EGFR pathway is needed to overcome the resistance caused by the T790M mutation. Some reports of sex differences in lung cancer risk and disease presentation suggest that estrogen may be associated with the etiology of lung cancer [8]. There is increasing evidence for the fact that both estrogen receptor (ER)a and ERb are expressed in NSCLC cell lines, tumor tissues, and cells derived from normal lung. Additionally, Stabile et al. [9] found that ERb acted as a mitogen in NSCLC cells, both in vitro and in vivo. Other studies also showed that the bidirectional signaling between the estrogen pathway and the EGFR pathway may stimulate the growth of NSCLCs, in addition to promoting tumor-associated angiogenesis in NSCLC cell lines [10–12]. Moreover, a combination treatment with an anti-estrogen (fulvestrant) and gefitinib in in vitro and in vivo lung cancer models was shown to robustly inhibit cell proliferation, induce apoptosis, and affect downstream signaling pathways [12]. H1975 cells, an

308

H. Shen et al. / Biomedicine & Pharmacotherapy 68 (2014) 307–313

NSCLC cell line that carries the T790M mutation, are resistant to gefitinib. However, in our previous study, we found that combined treatment with fulvestrant and gefitinib decreased the proliferation of H1975 cells, compared to treatment with either fulvestrant or gefitinib alone, by up-regulating EGFR and ER expression levels [8]. Therefore, we hypothesized that fulvestrant may increase the TKI sensitivity of H1975 cells and that this increased sensitivity involves molecular mechanisms yet to be delineated. MicroRNAs (miRNAs) are known to intrinsically suppress mRNA by pairing with the 3’-untranslated region (UTR) of the target mRNAs [13]. As genes regulators, miRNAs can regulate about one thirds of genes encoded by the human genome and play important roles in mediating various cellular functions, including proliferation, growth, differentiation, and apoptosis [14]. In recent years, miRNAs have been shown to play crucial roles in carcinogenesis [15]. Moreover, some miRNAs can work as tumor suppressors in vitro [16] The overexpression of tumor suppressor miRNAs likely restricts the expression of the target proteins that function as carcinogenic factors [17]. As opposed to short interfering RNA, miRNAs regulate the functions of their target pathways at multiple levels [16]. Given the key role of miRNAs in regulating cellular pathways involved in carcinogenesis, we speculated that specific miRNAs may also play an important role in the fulvestrant-induced upregulation of gefitinib sensitivity in H1975 cells. In this study, we sought to identify miRNAs that may be involved in regulating the fulvestrant-mediated regulation of TKI sensitivity in NSCLC. We found that the let-7c miRNA was most upregulated in fulvestrant-treated H1975 cells. Our data also revealed that the upregulation of let-7c was associated with the increased gefitinib sensitivity of H1975 cells, and that this effect was mediated by repression of the RAS oncogene and inactivation of the phosphoinositide 3-kinase (PI3K)/AKT and mitogenactivated extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. 2. Materials and methods 2.1. Materials Gefitinib was a kind gift from AstraZeneca (Tocris, United Kingdom). Fulvestrant (Faslodex; ICI 182,780) was purchased from Cayman Chemical (Michigan, USA). Antibodies against RAS, phospho (p)-ERK1/2, p-AKT (Ser-473), and total AKT were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against ERK2 were purchased from Santa Cruz Biotechnology (CA, USA). Pre-let-7c and negative control (NC) precursor miRNAs were purchased from Ambion (Foster City, CA). The High Capacity RNA-to-cDNA Kit and Power SYBR Green PCR Master Mix were obtained from Applied Biosystems (Carlsbad, CA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Kangcheng Company (Shanghai, China). 2.2. Cell Culture The H1975 human non-small cell lung cancer cells were purchased from American Type Culture Collection, cultured in Dulbecco’s modified Eagle medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 ng/mL streptomycin. All cells were incubated at 37 8C in an atmosphere of 5% CO2. 2.3. Cell viability assay H1975 and H1975/fulvestrant cells were seeded into 96-well plates at a density of 4  103 cells/well and allowed to attach

overnight. In addition, H1975 were also seeded into 96-well plates and transfected with pre-let-7c and NC precursor miRNAs for 24 h before the next treatment step. After cellular adhesion, gefitinib was added at a final concentration of 2.5–40 mmol/L. Seventy-two hours after addition of the drug, cell viability was assessed using the Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories) according to the manufacturer’s instruction. Each sample was plated in triplicate and three independent repetitions of the experiment were performed. 2.4. RNA extraction, reverse transcription (RT)-PCR and quantitative real-time (qRT)-PCR Total RNA was extracted from cells with Trizol (Invitrogen). One microgram of RNA was used for cDNA synthesis using oligo(dT)15 and M-MLV reverse transcriptase (Ribo-Bio). Aliquots of cDNAs were amplified with primers specific for RAS, ERK, AKT, or GAPDH. The primer sequences are as follows: RAS forward primer, 5’TGGACGAATATGATCCAACAAT-3’ and reverse primer, 5’-TCCCTCATTGCACTGTACTCC-3’; AKT forward primer, 5’-GCAGCACGTGTACGAGAAGA-3’ and reverse primer, 5’-GGTGTCAGTCTCCGACGTG-3’; ERK forward primer, 5’-CCGTGACCTCAAGCCTTC-3’ and reverse primer, 5’-GCCAGGCCAAAGTCACAG-3’; and GAPDH forward primer, 5’-CCACCCATGGCAAATTCCATGGCA-3’ and reverse primer, 5’-TCTAGACGGCAGGTCAGGTCCACC-3’. The targeted genes and GAPDH were amplified by PCR for 30 cycles of: 95 8C for 1 min, 59 8C for 30 s, and 72 8C for 1 min. The relative mRNA levels were normalized to those of GAPDH mRNA levels and the data were analyzed using the Quality One analysis software package (Bio-Rad, USA). To analyze let-7c expression levels, total RNA was extracted from H1975 cells. The stem-loop RT-PCR assay was used to quantify the miRNAs expression levels, as described previously [18,19]. The following RT-PCR primers were used: hsalet-7c RT primer, 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAACCAT-3’; let-7c PCR forward primer, 5’CGCGCTGAGGTAGTAGGT-3’ and reverse primer, 5’-GTGCAGGGTCCGAGGT-3’; U6 RT primer, 5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATA-3’; U6 PCR forward primer, 5’-GCGCGTCGTGAAGCGTTC-3’ and reverse primer, 5’GTGCAGGGTCCGAGGT-3’. The PCR products were separated on 2.0% agarose gels, stained with ethidium bromide, and visualized under UV light. The RNA levels were normalized to those of U6 small nucleolar (sn)RNA. qRT-PCR was performed using SYBR Premix DimerEraser (Takara, Dalian, China) on a 7900HT system. The expression levels of let-7c were normalized to that of U6 snRNA, the internal control, and fold changes were calculated by relative quantification using the DDCt method [20]. 2.5. Transfection with miRNA precursors Pre-let-7c and miR-scrambled control (SCR) precursor miRNAs were purchased from Applied Biosystems (Carlsbad, CA). Cells at 50–70% confluence were transfected with 40 nmol/L of pre-let-7c or miR-SCR precursor miRNAs using Lipofectamine (Invitrogen, CA, USA) according to the manufacturer’s instructions. 2.6. Immunoblotting Immunoblotting was performed as described previously [11]. In brief, cells were treated as indicated and lysed with immunoprecipitation buffer (150 mM NaCl, 100 mM Tris [pH 8.0], 1% Triton X-100, 1% deoxycholic acid, 0.1% sodium dodecyl sulfate [SDS], 5 mM EDTA, and 10 mM NaF) supplemented with 1 mM sodium vanadate, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 mM leupeptin, 2 mM aprotinin, and

H. Shen et al. / Biomedicine & Pharmacotherapy 68 (2014) 307–313

309

2 mM pepstatin on ice for 30 min. The total cellular protein content was determined using Bio-Rad protein assay reagent (Richmond, CA). Aliquots of the protein extracts were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS), and incubated with antibodies against RAS, p-AKT, total AKT, p-ERK1/2, total ERK2, and GAPDH. Specific protein signals were detected by incubating the membranes with horseradish peroxidase-conjugated secondary antibodies and visualized with a chemiluminescence reagent (Pierce Biotechnology, Rockford, IL). 2.7. Statistical analysis All values are reported as the mean  SD of three independent experiments. The two-sided Student’s unpaired t-test was used for comparing data sets. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. Fulvestrant increases the sensitivity of H1975 NSCLC cells to gefitinib H1975 cells were treated with 3 mmol/L fulvestrant for 1 month; then, their sensitivity to gefitinib was compared with that of control H1975 cells that had not been treated with fulvestrant. The CCK-8 cell viability assay was used to examine the drug sensitivity. The results showed that fulvestrant increased the gefitinib sensitivity of H1975 cells (Fig. 1). 3.2. Let-7c is most upregulated in fulvestrant-treated H1975 cells To explore the molecular mechanism(s) involved in the fulvestrant-mediated increase in gefitinib sensitivity of NSCLC cells, we analyzed 10 potential TKI resistance related miRNAs between H1975/fulvestrant and H1975 cells using Quantitative RT-PCR. The results showed that both let-7 family and miR-200 family were up-regulated in the H1975/fulvestrant cells, compared with H1975 cells (Fig. 2). However, among the upregulated miRNAs, only let-7c was up-regulated more than 4 folds. So we hypothesized that let-7c may be involved in increasing gefitinib sensitivity induced by fulvestrant in H1975 cell lines.

Fig. 1. Gefitinib sensitivity of H1975 NSCLC cells was increased after treatment with 3 mmol/L fulvestrant for 1 month. The viability of the cells was assessed using the CCK-8 cell viability assay and the values for the fulvestrant-treated cells are expressed relative to that of the untreated control cells, which was set to 100%. The data shown are the mean  SD from three independent experiments. *P < 0.05, Student’s t-test, fulvestrant-treated vs. untreated H1975 cells.

Fig. 2. qRT-PCR analysis shows upregulation of let-7a,let-7b,let-7c, miR200a, miR200c in H1975/fulvestrant cells, compared with the levels in H1975 cells not treated with fulvestrant. The samples were assayed in triplicate and the experiment was repeated three times. The amount of miRNAs were normalized to that of U6 snRNA, the internal control, and the values were expressed as the relative levels of let-7c. Significant differences are indicated as follows: *P < 0.05, Student’s t-test, fulvestrant-treated vs. untreated H1975 cells.

3.3. Let-7c sensitizes H1975 cells to gefitinib Given the significance of the let-7 family of miRNAs in lung cancer [13], and of let-7c in particular, we further analyzed the role of let7-c in fulvestrant-mediated restoration of gefitinib sensitivity in NSCLC. To investigate the role of let-7c in modulating the gefitinib sensitivity of H1975 cells, we constructed an miR-let-7ccontaining let-7c expression plasmid. After transfection of the miR-let-7c plasmid into H1975 cells, we use qRT-PCR to examine the expression level of let-7c miRNA in the transfected cells. As expected, let-7c expression was increased in the let-7c-transfected cells, compared to that in cells transfected with the NC control miRNA precursor (Fig. 3A). To determine the effect of let-7c on the gefitinib sensitivity, we used the CCK-8 cell viability assay to measure the effect of gefitinib on the cells transfected with the let7c-expressing and control miRNA-expressing plasmids (Fig. 3B). The results of the cell viability assay showed that forced overexpression of let-7c increases the gefitinib sensitivity of H1975 cells (Fig. 3C). 3.4. Let-7c sensitizes H1975 cells to gefitinib by repressing RAS expression and by inactivating p-AKT and p-ERK To delineate the molecular mechanism(s) involved in let-7cinduced sensitization of H1975 cells to gefitinib, we used qRT-PCR and western blotting to monitor the changes in the mRNA and protein levels of key components of two signaling pathways involved in NSCLC cell proliferation, the PI3K/AKT and MEK/ERK signaling pathways. The results of the qRT-PCR analysis showed that forced overexpression of let-7c significantly reduces the expression of RAS mRNA in H1975 cells, compared with that in untreated and NC-treated cells (Fig. 4A). Moreover, there were no significant differences in the levels of ERK and AKT mRNAs (Fig. 4B and C). The western blot analysis showed that forced overexpression of let-7c significantly reduces the expression of RAS, pAKT, and p-ERK proteins in H1975 cells, compared with the levels untreated and NC-treated cells. However, there were no significant differences in the levels of total ERK and total AKT proteins in the three groups of cells (Fig. 5A). These data suggest that changes in the transcriptional regulation of RAS, AKT, and ERK mRNAs and the phosphorylation of AKT and ERK are likely involved in the let-7c-mediated sensitization of H1975 cells to gefitinib.

310

H. Shen et al. / Biomedicine & Pharmacotherapy 68 (2014) 307–313

Fig. 3. Let-7c sensitizes H1975 cells to gefitinib. (A) qRT-PCR analysis shows that transfection of H1975 cells with a let-7c expression vector increases let-7c expression (H1975/let-7c), compared with that in H1975 cells transfected with a control (NC) miRNA (H1975/NC). **P < 0.01, Student’s t-test, let-7c-transfected vs. NC-transfected H1975 cells. (B) The morphology of H1975, H1975/NC, and H1975/let-7c cells after treatment with gefitinib (2.5–40 mmol/L). The cell morphology was observed under a light microscope and images were captured at a magnification of 100. (C) CCK-8 cell viability assay shows that treatment of H1975 cells with either fulvestrant or forced overexpression of let-7c increases the gefitinib sensitivity of H1975 cells, compared with the sensitivity of untreated and NC-transfected cells to gefitinib.

A let-7c inhibitor restored the gefitinib resistance of H1975/ fulvestrant cells by increasing RAS expression and by activating AKT and ERK. To confirm the role of let-7c in modulating the fulvestrantinduced changes in the gefitinib sensitivity of H1975 cells, we transfected the H1975/fulvestrant cells with a let-7c inhibitor, or a control inhibitor (CI). We use qRT-PCR to examine the expression level of let-7c miRNA in the transfected cells. As expected, the let7c expression was attenuated in the let-7c inhibitor-treated cells, compared to that in cells transfected with CI (Fig. 6A). Then, we used the CCK-8 cell viability assay to measure the effect of gefitinib on the cells transfected with either the let-7c inhibitor or CI. The results showed that decreased expression of let-7c restored the gefitinib resistance of H1975/fulvestrant cells (Fig. 6B). Furthermore, the western blot analysis showed that decreased expression of let-7c significantly increased RAS, p-AKT, and p-ERK protein levels in H1975/fulvestrant cells, compared with the levels in untreated and CI-treated cells (Fig. 6C). 4. Discussion Fulvestrant is a steroidal anti-estrogen designed to have no agonist activity against ER [21]. Fulvestrant acts by promoting the

degradation and downregulation of ERa in tumor cells [22]. Currently, fulvestrant is used for treatment of advanced breast cancer that is resistant to other endocrine therapies. It is effective in tumors and cell lines that express ER, but are resistant to the selective estrogen receptor modulator (SERM) tamoxifen [23]. Although lung cancer was not considered as a target organ for SERMs, current and previous data show that estrogen and its receptors play an important role in the development of lung cancer [9,24]. Massarweh et al. [25] found that treatment with anastrozole and fulvestrant, combined with inhibition of the EGFR by gefitinib, can overcome endocrine resistance. However, in a randomized trial of combinations of anti-estrogens with the EGFRTKI gefitinib, the combination of anastrozole or fulvestrant with gefitinib yielded similar clinical benefits in the treatment of ER/ progesterone receptor-positive metastatic breast cancer to those achieved with gefitinib or endocrine therapy alone [26]. EGFR inhibitors have yielded greater clinical benefits for NSCLC patients; although about 10 to 25% of NSCLC patients, most of whom carry somatic EGFR mutations [27], initially respond dramatically to TKIs, resistance emerges over 8–10 months of treatment. Secondary EGFR mutations, most commonly the EGFR T790M mutation, are considered to be major causes of this acquired resistance [28]. In our previous study, we found that the

Fig. 4. The effect of let-7c over-expression on RAS, ERK, and AKT. (A) qRT-PCR analysis shows that transfection of an let-7c expression vector decreases RAS expression in H1975 cells, compared with that in untreated cells or in cells transfected with the NC vector. **P < 0.01. qRT-PCR analysis shows that over-expression of let-7c did not alter (B) ERK mRNA or (C) AKT mRNA expression in H1975 cells. Data shown are the mean  SD of three independent experiments.

H. Shen et al. / Biomedicine & Pharmacotherapy 68 (2014) 307–313

311

Fig. 5. Let-7c modulates RAS protein and its downstream targets. (A) Western blot analysis shows that forced over-expression of let-7c represses RAS expression and inactivates p-AKT and p-ERK, but does not alter total ERK or total AKT levels, in H1975 cells. Semi-quantitative densitometric analysis of the western blots indicates that forced over-expression of let-7c significantly decreases (B) RAS protein expression and (C, E) p-AKT and p-ERK protein levels, but does not alter (D) total AKT and (F) total ERK protein in H1975 cells.

combination of fulvestrant and gefitinib attenuates the proliferation of H1975 NSCLC cells, which carry the T790M mutation, compared with treatment with either fulvestrant or gefitinib alone [8]. Our results also showed that the fulvestrant-gefitinib combination treatment resulted in upregulation of ER and EGFR expression in H1975 cells. Therefore, we hypothesized that fulvestrant likely increases the gefitinib sensitivity or attenuates the gefitinib resistance of H1975 cells. The results of the cell viability assays in this study confirmed that fulvestrant (3 mmol/L) did indeed increase the gefitinib sensitivity of H1975 cells.miRNAs are noncoding small RNAs that may act as oncogenes or tumor suppressor genes [29]. Recent research has confirmed that miRNAs can modulate the drug resistance of cancer cells; for instance, Zhu et al. [30] found that mir-181b modulates multidrug resistance by targeting BCL2 in human cancer cell lines Another study found that mir-21 increases the resistance to platinum-based chemotherapy in NSCLC [31]. In light of these recent findings, we wondered whether miRNAs are involved in fulvestrant-induced sensitization of H1975 cells to gefitinib. Our microarray analysis revealed that the let-7 family of miRNAs was selectively upregulated in H1975 cells treated with fulvestrant, and that let-7c was the most upregulated among the let-7 family members. The let-7 family miRNAs have been shown to be tumor-suppressor genes in various types of cancers [14,15,32]. Ali et al. [32] found that loss of miRNAs of the let-7 family and of miR-143 expression correlated with increased RAS expression and activity in pancreatic cancer. Let-7i and miR-143 were also found to target RAS, and forced

re-expression of let-7i and miR-143 inhibited RAS activity [32]. Another recent study found that low expression of let-7 and high expression of K-RAS are correlated with the pathogenesis and prognosis of NSCLC [33]. As the tumor suppressor functions of let-7 miRNAs have been demonstrated in various cancers, we hypothesized that let-7c might also play an important role in modulating gefitinib sensitivity in H1975 NSCLC cells. To delineate the possible mechanism involved in this process, we transfected H1975 cells with an miR-let-7c-expressing plasmid. Our results confirmed the over-expression of let-7c in NSCLC cells, as well as the associated increase in the gefitinib sensitivity. Next, we investigated the signaling molecules involved in let7c- mediated upregulation of gefitinib sensitivity. The let-7 family can target the RAS gene to mediate its tumor suppressor function [34], and RAS is the upstream regulator of both the PI3K/AKT and MEK/ERK signaling pathways. The PI3K/AKT and ERK signaling pathways play crucial roles in the regulation of gefitinib sensitivity [35]. Persistent activity of the PI3K/AKT and/or RAS/ERK pathways is associated with gefitinib-resistance of NSCLC cell lines. Moreover, gefitinib-resistant NSCLC cell lines exhibit EGFR-independent activity of the PI3K/AKT or RAS/ERK pathways [36]. Preclinical studies also showed that continued activation of downstream signaling pathways, especially the PI3K/AKT pathway, is sufficient to confer resistance to EGFR-TKIs, due to bypass of EGFR inhibition [37]. In this study, we focused on the H1975 cell line, which carries the L858R and T790M mutations, the most common acquired

312

H. Shen et al. / Biomedicine & Pharmacotherapy 68 (2014) 307–313

Fig. 6. Inhibition of Let-7c expression restores gefitinib resistance in H1975/fulvestrant cells by increasing RAS expression and activating p-AKT and p-ERK. (A) qRT-PCR analysis shows that transfection of a let-7c inhibitor decreases let-7c expression in H1975/fulvestrant cells (H1975/fulv + let-7c inhibitor), compared with that in H1975/fulv cells transfected with control inhibitor (H1975/fulv + CI). **P < 0.01, Student’s t-test, let-7c inhibitor-transfected vs. CI-transfected H1975/fulv cells. (B) CCK-8 cell viability assay shows that decreased expression of let-7c restores the gefitinib resistance of H1975/fulv cells, compared with that of untreated and CI-transfected cells. (C) Western blot analysis indicates that decreased expression of let-7c increased RAS expression and activates p-AKT and p-ERK, but does not alter total ERK or total AKT levels, in H1975/ fulv cells. Semi-quantitative densitometric analysis of the western blots indicates that decreased expression of let-7c (D) significantly increases RAS protein expression and pAKT and p-ERK protein levels, but does not alter total AKT and total ERK protein in H1975 cells.

gefitinib-resistance-associated mutations. We found that the H1975 cells had activated the PI3K/AKT and MEK/ERK signaling pathways, which is characterized by an increase in the expression of p-AKT and p-ERK. This may be the molecular mechanism underlying the resistance of H1975 cells to gefitinib. In accordance with the results of a previous study [38], our data showed that forced overexpression of let-7c significantly reduced the expression of RAS, both at the mRNA and protein levels. We also found that overexpression of let-7c led to the inactivation of AKT and ERK1/2, proteins that are downstream of RAS. Consistent with these data, treatment of H1975/fulvestrant cells with a let-7c inhibitor restored gefitinib resistance by increasing RAS protein expression and by activating AKT and ERK. In conclusion, our study shows, for the first time, that overexpression of let-7c may be one of the molecular mechanisms for the fulvestrant-mediated upregulation of gefitinib sensitivity in H1975 NSCLC cells. Taken together, these data suggest that fulvestrant may be a promising agent for enhancing the efficacy of gefitinib in the context of acquired TKI resistance in NSCLC patients. However, it should be noted that our data are derived from an NSCLC cell line, which has been removed from the in vivo context and cannot represent TKI resistance acquired in patients comprehensively. Thus, future studies to assess the role of fulvestrant in modulating gefitinib resistance in vivo and in the clinical context are warranted to address the utility of fulvestrant in treatment of EGFR-TKIresistant NSCLC.

Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This work was supported by National Natural Science Foundation of China (81101705 and 81272532), by Jiangsu province clinical science and technology projects (clinical research center, BL2012008) and by The natural science foundation of Jiangsu Province (BK2011852). References [1] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277–300. [2] Shepherd FA, et al. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005;353:123–32. [3] Mok TS, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947–57. [4] Kris MG, et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA 2003;290:2149–58. [5] Toyooka S, Kiura K, Mitsudomi T. EGFR mutation and response of lung cancer to gefitinib. N Engl J Med 2005;352:2136. [6] Yun CH, Mengwasser KE, Toms AV, Woo MS, Greulich H, et al. The T790 M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci USA 2008;105:2070–5.

H. Shen et al. / Biomedicine & Pharmacotherapy 68 (2014) 307–313 [7] Godin-Heymann N, Bryant I, Rivera MN, Ulkus L, Bell DW, Riese 2nd DJ, et al. Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790 M drug resistance mutation. Cancer Res 2007;67:7319–26. [8] Xu R, Shen H, Guo R, Sun J, Gao W, Shu Y. Combine therapy of gefitinib and fulvestrant enhances antitumor effects on NSCLC cell lines with acquired resistance to gefitinib. Biomed Pharmacother 2012;66:384–9. [9] Stabile LP, Davis AL, Gubish CT, Hopkins TM, Luketich JD, Christie N, et al. Human non-small cell lung tumors and cells derived from normal lung express both estrogen receptor alpha and beta and show biological responses to estrogen. Cancer Res 2002;62:2141–50. [10] Marquez-Garban DC, Pietras RJ. Estrogen-signaling pathways in lung cancer. Adv Exp Med Biol 2008;617:281–9. [11] Shen H, Yuan Y, Sun J, Gao W, Shu YQ. Combined tamoxifen and gefitinib in non-small cell lung cancer shows antiproliferative effects. Biomed Pharmacother 2010;64:88–92. [12] Stabile LP, Lyker JS, Gubish CT, Zhang W, Grandis JR, Siegfried JM. Combined targeting of the estrogen receptor and the epidermal growth factor receptor in non-small cell lung cancer shows enhanced antiproliferative effects. Cancer Res 2005;65:1459–70. [13] Lai EC. Micro RNAs are complementary to 3’ UTR sequence motifs that mediate negative post-transcriptional regulation. Nat Genet 2002;30:363–4. [14] He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 2004;5:522–31. [15] Iorio MV, Croce CM. MicroRNAs in cancer: small molecules with a huge impact. J Clin Oncol 2009;27:5848–56. [16] Kefas B, et al. microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. Cancer Res 2008;68: 3566–72. [17] Tong AW, Nemunaitis J. Modulation of miRNA activity in human cancer: a new paradigm for cancer gene therapy? Cancer Gene Ther 2008;15:341–55. [18] Chen C, et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Re 2005;33:e179. [19] Wang X. A PCR-based platform for microRNA expression profiling studies. RNA 2009;15:716–23. [20] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–8. [21] Howell A, Bergh J. Insights into the place of fulvestrant for the treatment of advanced endocrine responsive breast cancer. J Clin Onco 2010;28: 4548–50. [22] Howell A. Pure oestrogen antagonists for the treatment of advanced breast cancer. Endocr Relat Cancer 2006;13:689–706. [23] Woode DR, Aiyer HS, Sie N, Zwart AL, Li L, Seeram NP, et al. Effect of berry extracts and bioactive compounds on fulvestrant (ICI 182,780) sensitive and resistant cell lines. Int J Breast Cancer 2012;147828.

313

[24] Bogush TA, Dudko EA, Beme AA, Bogush EA, Kim AI, Polotsky BE, et al. Estrogen receptors, antiestrogens, and non-small cell lung cancer. Biochemistry (Mosc) 2010;75:1421–7. [25] Massarweh S, et al. A phase II neoadjuvant trial of anastrozole, fulvestrant, and gefitinib in patients with newly diagnosed estrogen receptor positive breast cancer. Breast Cancer Res Treat 2011;129:819–27. [26] Carlson RW, O’Neill A, Vidaurre T, Gomez HL, Badve SS, Sledge GW. A randomized trial of combination anastrozole plus gefitinib and of combination fulvestrant plus gefitinib in the treatment of postmenopausal women with hormone receptor positive metastatic breast cancer. Breast Cancer Res Treat 2012;133:1049–56. [27] Lynch TJ, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39. [28] Paez JG, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–500. [29] Hua S, Xiaotao X, Renhua G, Yongmei Y, Lianke L, Wen G, et al. Reduced miR-31 and let-7 maintain the balance between differentiation and quiescence in lung cancer stem-like side population cells. Biomed Pharmacother 2012;66:89–97. [30] Zhu W, Shan X, Wang T, Shu Y, Liu P. miR-181b modulates multidrug resistance by targeting BCL2 in human cancer cell lines. Int J Cancer 2010;127: 2520–9. [31] Gao W, Lu X, Liu L, Xu J, Feng D, Shu Y. MiRNA-21: a biomarker predictive for platinum-based adjuvant chemotherapy response in patients with non-small cell lung cancer. Cancer Biol Ther 2012;13:330–40. [32] Ali S, Ahmad A, Aboukameel A, Bao B, Padhye S, Philip PA, et al. Increased Ras GTPase activity is regulated by miRNAs that can be attenuated by CDF treatment in pancreatic cancer cells. Cancer Lett 2012;319:173–81. [33] Xia XM, Jin WY, Shi RZ, Zhang YF, Chen J. Clinical significance and the correlation of expression between Let-7 and K-ras in non-small cell lung cancer. Oncol Lett 2010;1:1045–7. [34] Wang QZ, Lv YH, Gong YH, Li ZF, Xu W, Diao Y, et al. Double-stranded Let-7 mimics, potential candidates for cancer gene therapy. J Physiol Biochem 2012;68:107–19. [35] Gadgeel SM, Wozniak A. Preclinical Rationale for PI3 K/Akt/mTOR Pathway Inhibitors as Therapy for Epidermal Growth Factor Receptor Inhibitor-Resistant Non-Small-Cell Lung Cancer. Clin Lung Cancer 2013. [36] Janmaat ML, Rodriguez JA, Gallegos-Ruiz M, Kruyt FA, Giaccone G. Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells. Int J Cancer 2006;118:209–14. [37] Yamasaki F, et al. Acquired resistance to erlotinib in A-431 epidermoid cancer cells requires down-regulation of MMAC1/PTEN and up-regulation of phosphorylated Akt. Cancer Res 2007;67:5779–88. [38] Levy R, Biran A, Poirier F, Raz A, Kloog Y. Galectin-3 mediates cross-talk between K-Ras and Let-7c tumor suppressor microRNA. PLoS One 2011;6:e27490.

Fulvestrant increases gefitinib sensitivity in non-small cell lung cancer cells by upregulating let-7c expression.

Patients with non-small cell lung cancer (NSCLC) who have activating epidermal growth factor receptor (EGFR) mutations benefit from treatment with EGF...
2MB Sizes 0 Downloads 0 Views