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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Chemotherapy with platinum agents is the standard of care for non-small-cell lung cancer (NSCLC); however, novel molecular-targeted agents like gefitinib have been approved for advanced NSCLCs, including recurrent cases previously treated with platinum-based chemotherapy. Although these agents show antitumor activity through distinct mechanisms and elicit positive initial responses, tumors invariably develop resistance. Recent studies have revealed mechanisms by which both types of agents induce acquired resistance. However, little is known about whether first-line treatment with either type of agent affects cancer cell susceptibility and development of resistance against subsequent treatment with the other. Using in vitro drug-resistant NSCLC cell models, we provide evidence that acquired cisplatin resistance may reduce the sensitivity of cancer cells to subsequent treatment with a molecular-targeted agent. In addition, first-line cisplatin treatment influenced the mechanism by which cancer cells developed resistance to subsequent treatment with a molecular-targeted agent. The influence of cisplatin on acquisition of resistance to a molecular-targeted agent was associated with epithelial–mesenchymal transition (EMT)-like alterations such as increased expression of mesenchymal markers, morphological change, and AXL tyrosine kinase-mediated increased cell motility. Our findings indicate that the influence of platinum-based chemotherapy on molecular-targeted therapies and the involvement of EMT and EMT-related effectors should be considered when developing therapeutic strategies using antitumor agents, especially in the context of sequential therapy.

Treatment with cisplatin, a platinum-based agent that binds to and crosslinks DNA, is the standard of care for non-small-cell lung cancer (NSCLC), which is the leading cause of cancer-related mortality and accounts for one-third of all deaths from cancer worldwide. Despite the high efficacy of these agents, the ability of cancer cells to become resistant remains a significant impediment to successful chemotherapy. To overcome this issue, new molecular-targeted drugs exert antitumor effects through mechanisms different from those of platinum-based drugs, and these drugs have been approved for treatment of advanced NSCLC in patients who have previously received platinum-based chemotherapy. For example, gefitinib, the first approved tyrosine kinase inhibitor (TKI), is effective against tumors harboring epidermal growth factor receptor (EGFR)-activating mutations.[1-4] Although a proportion of NSCLC patients with EGFR mutations show a rapid and dramatic response to gefitinib, these patients eventually develop drug resistance in the same manner in which they develop resistance to platinum-based agents. Thus, understanding the mechanisms by which cancers acquire resistance to both molecular-targeted and platinum-based agents is critical for the development of more effective therapeutic strategies.

Studies indicate that multiple pathways contribute to the development of cancer drug resistance. For example, cisplatin resistance is associated with increased cisplatin efflux, inactivation of intracellular cisplatin, evasion of apoptotic pathways, replication checkpoint bypass, increased cell proliferation, and increased DNA damage repair.[5, 6] Recent studies also indicate that multiple resistance mechanisms may operate within an individual tumor to promote acquired resistance to EGFR TKIs in NSCLC patients. One of these potential mechanisms is secondary mutation of T790M, which increases the affinity of the oncogenic mutant EGFR for ATP, leading to the reduced efficacy of EGFR TKIs.[7-9] Another mechanism involves hepatocyte growth factor receptor (MET) amplification, which promotes cell survival through persistent AKT signaling by circumventing MET signaling when the EGFR signal is blocked in the presence of EGFR-TKIs.[10, 11]

Epidermal growth factor receptor TKI resistance in EGFR-mutant NSCLC patients may be associated not only with the previously described genotypic alterations, but also with non-genetic or epigenetic alterations, specifically the epithelial–mesenchymal transition (EMT).[12-14] The EMT process, which is important during embryogenesis,[15] is known to be involved in acquired resistance to chemotherapeutic agents like cisplatin.[16-18] Epithelial–mesenchymal transition is triggered by several extracellular factors, including components of the ECM and growth factors, and is mediated by the activation of EMT transcription factors, such as TWIST1, SNAIL, SLUG, ZEB1, and ZEB2.[19] After activation of the EMT program, epithelial cells undergo dramatic phenotypic changes, lose expression of E-cadherin and other components of epithelial cell junctions, adopt a mesenchymal cell phenotype, and acquire motility and invasive properties, allowing them to migrate through the ECM. Accumulating evidence suggests that in addition to acquired resistance to anticancer agents,[12-14, 16-18] aberrant activation of the EMT program contributes to tumor invasion and metastatic dissemination.[20, 21] AXL tyrosine kinase, in particular, is known to be involved in EMT-associated elevated motility and invasive properties.[22, 23]

Recent studies have elucidated the mechanisms by which cancer cells acquire resistance to either platinum-based or molecular-targeted agents. However, little information is available regarding mechanisms by which first-line treatment with either type of agent affects cancer cell susceptibility and resistance to subsequent treatment with the other. Here, we addressed this issue by focusing on the EMT program, which is commonly associated with acquired resistance to both types of agents, using in vitro drug-resistant models.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Cells and culture conditions

Human NSCLC cell lines HCC4006 (ATCC CRL-2871), NCI-H2170 (CRL-5928), HCC827 (ATCC CRL-2868), and NCI-H1993 (ATCC CRL-5909) were obtained from ATCC (Manassas, VA, USA). The human NSCLC cell line PC-9 was obtained from Immuno-Biological Laboratories (Gunma, Japan). All cell lines were maintained in RPMI-1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% FBS.

Antibodies and reagents

Anti-EGFR mAb (clone 19-1) was raised against the cytoplasmic domain of human EGFR as previously described.[24] Anti-TWIST1 (Twist2C1a), anti-E-cadherin (HECD-1), and anti-β-Actin (AC-15) mouse mAbs and anti-N-cadherin rabbit polyclonal antibody were purchased from Abcam (Cambridge, UK). Anti-ZEB1 rabbit polyclonal antibody and anti-phosphotyrosine mouse mAb (PY20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Vimentin rat mAb (280618) was purchased from R&D Systems (Minneapolis, MN, USA). Anti-SLUG (C19G7) and anti-AXL rabbit (C44G1) mAbs were purchased from Cell Signaling Technology (Danvers, MA, USA). Cisplatin was purchased from Wako Pure Chemical Industries (Osaka, Japan). Gefitinib and lapatinib were purchased from Tocris Bioscience (Ellisville, MO, USA). Small interfering RNAs targeting human AXL and a non-targeting control (Stealth RNAi Negative Control) were purchased from Invitrogen (Carlsbad, CA, USA).

Establishment of drug-resistant cell lines

Drug-resistant cell lines were established as described below. HCC4006, HCC827, and NCI-H2170 cells were subjected to elevated concentrations of cisplatin (range, 0.5–25 μM) in RPMI-1640 medium supplemented with 10% FBS. Cells were then subcultured in 25 μM cisplatin for an additional 1 month to establish stable cisplatin-resistant cell lines (HCC4006-CR, HCC827-CR, and NCI-H2170-CR, respectively). HCC4006, HCC4006-CR, HCC827, and HCC827-CR cells were further subjected to 2 weeks of continuous treatment with 1 μM gefitinib to select subpopulations of gefitinib-resistant cells (HCC4006-GR2w, HCC4006-CR-GR2w, HCC827-GR2w, and HCC827-CR-GR2w, respectively). In addition, NCI-H2170 and NCI-H2170-CR cells were subjected to 2 weeks of continuous treatment with 1 μM lapatinib to select subpopulations of lapatinib-resistant cells (NCI-H2170-LR2w and NCI-H2170-CR-LR2w, respectively).

Cell proliferation assay

Cells were plated at 2.5 × 105 cells per 100-mm dish in RPMI-1640 medium supplemented with 10% FBS. After 24 h, the culture medium was replaced with medium containing 10% FBS and 0.01% DMSO with or without 25 μM cisplatin, and thereafter, every 3 days. Cells were cultured at 37°C until they reached confluence or up to 12 days. Cells were counted every other day after the start of cisplatin treatment in multiple randomly selected microscopic visual fields to determine the number of surviving cells.

Western blot analysis

Cells were harvested and lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP40, 1% deoxycholic acid sodium salt, 0.1% SDS, 1 mM EDTA, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM PMSF, 2 μg/mL leupeptin, and 2 μg/mL aprotinin. Lysates were mixed with Laemmli sample buffer, boiled, subjected to SDS-PAGE (40 μg protein in 20 μL), then blotted onto PVDF membranes. Membranes were blocked and incubated with primary antibodies, followed by HRP-conjugated anti-mouse, anti-rabbit, or anti-rat IgG secondary antibodies (Dako, Glostrup, Denmark). Signals were visualized with ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chalfont St. Giles, UK) using ImageQuant LAS-4000 (GE Healthcare, Piscataway, NJ, USA).

Cell growth inhibition assay

Growth inhibition by chemical inhibitors was assessed by MTS assay using Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. In brief, cells were seeded in 96-well plates and allowed to attach for 24 h before the medium was replaced with medium containing 10% FBS and 0.01% DMSO with or without either inhibitor. After 72 h of incubation, absorbance was measured at 490 nm with a microplate reader.

RNA isolation and quantitative real-time PCR

Total RNA was isolated from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and cDNA was synthesized using the High Capacity RNA to cDNA Kit (Applied Biosystems, Foster City, CA, USA). Quantitative real-time PCR (qRT-PCR) was carried out using FastStart Universal SYBR Green Master (Rox; Roche Diagnostics, Indianapolis, IN, USA) on a 7300 Real-Time PCR System (Applied Biosystems). The sample was incubated at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. GAPDH was used as the reference gene, and the fold-change in gene expression was calculated using the 2-ΔΔCt method. Primer sequences are listed in Table S1.

Transwell migration assay

The Transwell migration assay was carried out in 8-μm polycarbonate membrane Transwell chambers (Corning, Corning, NY, USA). Cells (1 × 105 in RPMI-1640 medium supplemented with 0.1% BSA) were seeded on an upper chamber over a lower chamber that contained RPMI-1640 medium supplemented with 20% FBS. Cell migration through a serum gradient was allowed to proceed for 24 h at 37°C, after which the cells remaining on the upper-chamber side of the filter were removed with cotton swabs. The cells that migrated to the bottom of the Transwell chamber were fixed and stained with Diff-Quik (Sysmex, Kobe, Japan), and migrated (stained) cells were counted in multiple randomly selected microscopic visual fields.

RNA interference assay

Cells were detached from culture dishes by trypsin digestion and seeded on a 100-mm dish in a medium containing Lipofectamine RNAiMAX (35 μL; Invitrogen) and siRNAs (600 pM). For the MTS cell growth inhibition assay, transfected cells were further treated with gefitinib 24 h after transfection, and the MTS assay was carried out as described above. For the Transwell migration assay, the transfected cells were lifted with trypsin and distributed into the upper chamber 24 h after transfection. Knockdown efficiency was assessed 48 h after transfection by Western blot analysis.

Additional procedures

Descriptions of additional experimental procedures used and associated references are given in Documents S1 and S2, respectively.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Acquired cisplatin resistance in HCC4006 cells is associated with EMT-like alterations

The cisplatin-resistant cell line HCC4006-CR was generated from HCC4006, an NSCLC adenocarcinoma cell line characterized by EGFR exon 19 deletion and gefitinib sensitivity.[3] The growth capacity of HCC4006 was completely impaired by cisplatin. Conversely, HCC4006-CR cells showed a growth capacity comparable to controls even in the presence of cisplatin, although cisplatin did inhibit HCC4006-CR cell growth (Figs 1a,S1). HCC4006-CR cells expressed increased levels of N-cadherin, a mesenchymal marker, compared to HCC4006 cells (Fig. 1b). In addition, 10 of 11 randomly selected HCC4006-CR clones, established by limited dilution, expressed increased N-cadherin levels. Among these, clones 1, 4, 7, and 9 also expressed reduced levels of E-cadherin and increased levels of Vimentin compared with HCC4006 or HCC4006-CR cells (Fig. 1b). These results indicate that acquisition of cisplatin resistance involves an EMT-like mechanism, although the degree of involvement varies among individual HCC4006-CR cells.

image

Figure 1. Involvement of epithelial–mesenchymal transition in the acquisition of cisplatin resistance in HCC4006 non-small-cell lung cancer cells. (a) Cells were treated with 25 μM cisplatin, and surviving cells were counted at the indicated time points. (b) HCC4006 and HCC4006-CR cells and clones obtained by limited dilution were analyzed by Western blot for expression of epithelial–mesenchymal transition markers (E-cadherin, N-cadherin, and Vimentin). CR, cells subjected to elevated concentrations of cisplatin (range, 0.5–25 μM) and subcultured in 25 μM cisplatin for an additional 1 month to establish cisplatin resistance.

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First-line cisplatin treatment reduces HCC4006 cell sensitivity to gefitinib

HCC4006-CR cells showed partial resistance to gefitinib compared to HCC4006 cells in the MTS cell growth inhibition assay (36% vs. 15% viability at 1 μM gefitinib; Fig. 2a). To further examine the direct influence of cisplatin resistance on gefitinib sensitivity, a subpopulation of gefitinib-resistant cells was selected from parental HCC4006 and HCC4006-CR cells by short-term exposure to 1 μM gefitinib. Gefitinib sensitivities of these cells (HCC4006-GR2w and HCC4006-CR-GR2w) were then compared using the MTS cell growth inhibition assay. HCC4006-CR-GR2w cells were highly resistant to subsequent gefitinib treatment (Fig. 2a), although gefitinib treatment inhibited EGFR autophosphorylation in these cells (Fig. S2). In addition, HCC4006-CR-GR2w cells showed higher resistance than HCC4006-GR2w cells (75% vs. 55% viability at 1 μM gefitinib; Fig. 2a).

image

Figure 2. Effect of cisplatin resistance on susceptibility to gefitinib in HCC4006-CR non-small-cell lung cancer cells. (a) Cell viability was assessed by MTS assay 72 h after drug treatment at the indicated concentrations. The results reflect the mean percentage of growth in cells treated with gefitinib relative to untreated controls. (b) Quantitative real-time PCR analysis of epithelial–mesenchymal transition marker gene expression. (c) Western blot analysis of epithelial–mesenchymal transition marker protein expression. CR, cells subjected to elevated concentrations of cisplatin (range, 0.5-25 μM) and subcultured in 25 μM cisplatin for an additional 1 month to establish cisplatin resistance; GR2w, cells subjected to 2 weeks of continuous treatment with 1 μM gefitinib to select a gefitinib-resistant subpopulation.

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Epithelial–mesenchymal transition is associated with cisplatin-induced gefitinib resistance in HCC4006 cells

HCC4006-CR-GR2w cells expressed increased CDH2 (N-cadherin), VIM (Vimentin), ZEB1, ZEB2, and TWIST1 mRNA levels, but decreased CDH1 (E-cadherin) levels compared with HCC4006 and HCC4006-CR cells (Fig. 2b). HCC4006-CR-GR2w cells also expressed increased N-cadherin, Vimentin, ZEB1, and TWIST1 protein levels, and decreased E-cadherin levels compared to HCC4006 and HCC4006-CR cells (Fig. 2c). Moreover, HCC4006-CR-GR2w cells showed higher expression levels of mesenchymal marker genes (CDH2, VIM, ZEB1, ZEB2, and TWIST1) than did HCC4006-GR2w (Fig. 2b).

First-line cisplatin treatment influences the mechanism by which HCC827 cells acquire gefitinib resistance

To further investigate the influence of cisplatin resistance on the development of gefitinib resistance, the cisplatin-resistant cell line HCC827-CR was generated from HCC827, another gefitinib-sensitive adenocarcinoma cell line with EGFR exon 19 deletion (Fig. S1).[3] HCC827 and HCC827-CR cells were subjected to 2 weeks of continuous gefitinib treatment to select subpopulations of gefitinib-resistant cells (HCC827-GR2w and HCC827-CR-GR2w, respectively). HCC827-CR-GR2w cells showed partial resistance to gefitinib, whereas HCC827-CR cells were sensitive to gefitinib treatment (53% vs. 18% viability at 1 μM gefitinib; Fig. 3a). In addition, HCC827-CR-GR2w cells showed higher resistance than HCC827-GR2w cells (53% vs. 25% viability at 1 μM gefitinib; Fig. 3a). Contrary to a previous report,[10] MET amplification was not observed in either HCC827-CR or HCC827-CR-GR2w cells (Fig. S3). However, HCC827-CR-GR2w cells upregulated mRNA expression of multiple mesenchymal markers, including VIM, ZEB1, ZEB2, and TWIST1 genes relative to HCC827 and HCC827-CR cells (Fig. 3b) and protein expression of Vimentin and ZEB1 (Fig. 3c). Conversely, HCC827-GR2w cells underwent only minimal changes in the expression of EMT markers including VIM, ZEB1, and ZEB2 (Fig. 3b).

image

Figure 3. Effect of first-line cisplatin treatment on resistance to gefitinib in HCC827 non-small-cell lung cancer cells. (a) Cell viability was assessed by MTS assay 72 h after drug treatment at the indicated concentrations. The results reflect the mean percentage of growth in cells treated with gefitinib relative to untreated controls. (b) Quantitative real-time PCR analysis of epithelial–mesenchymal transition marker gene expression. (c) Western blot analysis of epithelial–mesenchymal transition marker protein expression. CR, cells subjected to elevated concentrations of cisplatin (range, 0.5-25 μM) and subcultured in 25 μM cisplatin for an additional 1 month to establish cisplatin resistance; GR2w, cells subjected to 2 weeks of continuous treatment with 1 μM gefitinib to select a gefitinib-resistant subpopulation.

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First-line cisplatin treatment also influences resistance to lapatinib in NCI-H2170 cells

Cisplatin treatment also influenced acquired resistance to another molecular-targeted agent, lapatinib. The cisplatin-resistant cell line NCI-H2170-CR was established from NCI-H2170, a squamous cell lung carcinoma cell line sensitive to human epidermal growth factor receptor 2 (HER2) inhibitors including lapatinib (Fig. S1).[25] NCI-H2170 and NCI-H2170-CR cells were subjected to 2 weeks of continuous treatment with 1 μM lapatinib to select subpopulations of lapatinib-resistant cells (NCI-H2170-LR2w and NCI-H2170-CR-LR2w, respectively). NCI-H2170 cells were sensitive to lapatinib, whereas NCI-H2170-CR-LR2w cells showed high levels of resistance to lapatinib (23% vs. 79% viability at 1 μM lapatinib; Fig. 4a). In addition, NCI-H2170-CR-LR2w cells showed higher resistance than NCI-H2170-LR2w cells (79% vs 54% viability at 1 μM lapatinib; Fig. 4a). NCI-H2170-CR-LR2w cells upregulated the expression of multiple mesenchymal markers including CDH2, VIM, and SLUG genes and their translational products N-cadherin and SLUG to a greater extent than did NCI-H2170 and NCI-H2170-CR cells (Fig. 4b,c). Conversely, no similar alterations in EMT marker expression were observed in NCI-H2170-LR2w cells (Fig. 4b).

image

Figure 4. Effect of first-line cisplatin treatment on resistance to lapatinib in NCI-H2170 non-small-cell lung cancer cells. (a) Cell viability was assessed by MTS assay 72 h after drug treatment at the indicated concentrations. The results reflect the mean percentage of growth in cells treated with lapatinib relative to untreated controls. (b) Quantitative real-time PCR analysis of epithelial–mesenchymal transition marker gene expression. (c) Western blot analysis of epithelial–mesenchymal transition marker protein expression. CR, cells subjected to elevated concentrations of cisplatin (range, 0.5-25 μM) and subcultured in 25 μM cisplatin for an additional 1 month to establish cisplatin resistance; LR2w, cells subjected to 2 weeks of continuous treatment with 1 μM lapatinib to select a lapatinib-resistant subpopulation.

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Drug-resistant cell lines show increased motility in accordance with EMT marker expression alterations

Consistent with previous reports indicating a correlation between cancer cell morphology/motility and EMT,[20, 21] the morphologies of drug-resistant cell lines differed according to their EMT marker expression. Specifically, HCC4006, HCC827, and NCI-H2170 cells maintained tight cell adhesion, whereas HCC4006-CR-GR2w, HCC827-CR-GR2w, and NCI-H2170-CR-LR2w cells showed a scattered phenotype and a reduced number of close cell–cell junctions (Fig. 5a). HCC4006-CR cells also showed a higher cell migration rate than HCC4006 cells; HCC4006-CR-GR2w cells showed the highest migration rate (Fig. 5b, left). Likewise, HCC827-CR and NCI-H2170-CR cells had a higher cell migration rate than HCC827 and NCI-H2170 cells, and HCC827-CR-GR2w and NCI-H2170-CR-LR2w cells had a higher rate than HCC827-CR and NCI-H2170-CR cells (Fig. 5b, middle and right, respectively). These findings indicate that migration capacity precisely correlates with EMT marker expression and the extent of morphological change.

image

Figure 5. Increased motility of drug-resistant cell lines mediated by AXL. (a) Cell morphology of drug-resistant cell lines and their parent cell lines shown under bright field microscopy. (b) Cell motility was assessed by a Transwell migration assay in which cells were allowed to migrate towards a serum gradient, and the number of migrated cells was counted after 24 h. (c) Expression of AXL in the indicated parental and drug-resistant cell lines derived from HCC4006, HCC827, and NCI-H2170 cells was assessed by Western blot. (d) Knockdown efficiency of AXL-targeting siRNAs on HCC4006-CR-GR2w cells was assessed by Western blot 48 h after transfection using an anti-AXL antibody. (e) Motility of AXL-knocked-down cells was assessed using a Transwell migration assay (left panel, HCC4006-CR-GR2w cells; right panel, HCC827-CR-GR2w cells). Cells were seeded in the upper chamber 24 h after siRNA transfection and allowed to migrate toward a serum gradient. The number of migrated cells was counted after 24 h. CR, cells subjected to elevated concentrations of cisplatin (range, 0.5-25 μM) and subcultured in 25 μM cisplatin for an additional 1 month to establish cisplatin resistance. GR2w, cells subjected to 2 weeks of continuous treatment with 1 μM gefitinib to select a gefitinib-resistant subpopulation. LR2w, cells subjected to 2 weeks of continuous treatment with 1 μM lapatinib to select a lapatinib-resistant subpopulation.

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AXL is a key mediator of migration capacity in resistant cell lines

Drug-resistant cell lines showed different levels of AXL protein, which is associated with acquisition of gefitinib resistance and EMT-associated increased cancer cell motility.[22, 23] Although both HCC4006-CR-GR2w and HCC827-CR-GR2w cells showed increased AXL protein levels and EMT-like alterations, the AXL protein level in HCC827-CR-GR2w cells was relatively lower than that in HCC4006-CR-GR2w cells (Fig. 5c). Conversely, AXL protein was undetectable in NCI-H2170-CR-LR2w cells (Fig. 5c). Moreover, HCC4006-CR-GR2w cells transfected with siRNAs targeting AXL (Fig. 5d) and treated with gefitinib showed substantial resistance to gefitinib despite slight siRNA-related inhibition of their growth (Fig. S4). In contrast, transfection with AXL-targeting siRNAs effectively prevented HCC4006-CR-GR2w cells from migrating through a serum gradient in a Transwell chamber (Fig. 5e, left), according to their knockdown efficiencies. Migration of HCC827-CR-GR2w cells was also inhibited by transfection with si-AXL-2 and si-AXL-3 (Fig. 5e, right). In contrast, AXL knockdown had little or no influence on EMT marker gene expression in either HCC4006-CR-GR2w or HCC827-CR-GR2w cells (Fig. S5).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

This study demonstrates that cisplatin resistance reduced the susceptibility of HCC4006 cancer cells to molecular-targeted drugs. Specifically, cisplatin-resistant HCC4006-CR cells showed partial loss of susceptibility to acute (72-h) gefitinib treatment, and highly gefitinib-resistant cells (HCC4006-CR-GR2w cells) could be obtained from HCC4006-CR cells with only 2 weeks of continuous treatment with 1 μM gefitinib. In addition, these cells showed a higher level of gefitinib resistance than did non-cisplatin-resistant HCC4006 cells that had been treated with gefitinib for 2 weeks (HCC4006-GR2w cells; Fig. 2a). These findings suggest that HCC4006-CR cells harbor highly gefitinib-resistant cells before undergoing gefitinib treatment and that the gefitinib resistance in HCC4006-CR-GR2w cells is likely induced by cisplatin treatment independent of gefitinib treatment.

The influence of cisplatin on the acquisition of molecular-targeted agent resistance was strongly associated with EMT-like alterations. During first-line cisplatin treatment and the subsequent 2-week gefitinib treatment, HCC4006 cells underwent EMT-like alterations in a stepwise fashion (Fig. 2b). Suda et al. demonstrated that HCC4006 cells acquire resistance to EGFR TKIs by an EMT-related mechanism,[13] whereas our results indicated that HCC4006-CR-GR2w cells showed higher levels of gefitinib resistance and EMT progression than did HCC4006-GR2w cells (Fig. 2a,b). This result suggests that cisplatin-induced EMT-like alterations accelerated HCC4006 cell acquisition of gefitinib resistance. Similar results were obtained using the HCC827 and NCI-H2170 cell models (Figs 3a,b, 4a,b). In addition, HCC4006-CR-derived clones showing EMT characteristics (clones 4, 7, and 9) showed higher resistance to gefitinib than did the other clones in the MTS assay (Fig. S6). Taken together, these findings indicate that cisplatin-induced EMT-like alterations may facilitate acquisition of resistance to a molecular-targeted therapy in a subset of cancer cells, highlighting the importance of monitoring EMT progression during sequential therapy.

The HCC827 cell line model provided more evidence that cisplatin resistance may influence the development of resistance to subsequent treatment with a molecular-targeted agent. Consistent with a previous report,[10] HCC827 cells developed MET-amplified resistance after a 5-month treatment with gefitinib (Fig. S3). MET amplification was also detected in HCC827-GR2w cells, although the level of amplification was relatively low (Fig. S3). These MET-amplified cells might be derived from a subpopulation of MET-amplified cells preexisting in the parental HCC827 cells.[11] However, our double agent-resistant cell line, HCC827-CR-GR2w, did not show MET amplification (Fig. S3), indicating that MET amplification might not be involved in gefitinib resistance in HCC827-CR-GR2w cells. HCC827 cells undergo EMT progression during sequential treatment with cisplatin followed by gefitinib (Fig. 3b,c). However, an EMT-like phenotype was not observed in HCC827-GR2w cells (Fig. 3b), suggesting that gefitinib resistance is associated with an EMT-like mechanism only in HCC827-CR-GR2w cells. Thus, cisplatin resistance might influence the mechanism by which a subset of cancer cells acquires resistance to subsequent treatment with a molecular-targeted agent by way of an EMT-related mechanism.

The proposed influence of cisplatin on the acquisition of molecular-targeted drug resistance through EMT-related mechanisms is further supported by data from the NCI-H2170 cell line, a lapatinib-sensitive squamous cell lung cancer cell line harboring an amplified HER2 gene.[25] NCI-H2170 cells do not show EMT-like alterations after lapatinib treatment; however, sequential treatment with cisplatin followed by lapatinib induces EMT-like phenotypes in these cells (Fig. 4b,c). These results indicate that lapatinib resistance may operate by an EMT-like mechanism in NCI-H2170-CR-LR2w cells and by a different mechanism in NCI-H2170-LR2w cells. Notably, our results show that a common EMT transcription factor, ZEB1, is intimately involved in drug resistance-related EMT in HCC4006 and HCC827 adenocarcinoma cell lines (Figs 2b,c, 3b,c), whereas another EMT transcription factor SLUG is involved in the development of drug resistance in the NCI-H2170 squamous carcinoma cell line (Fig. 4b,c). Thus, different mechanisms may drive drug resistance-related EMT depending on the cell type.

Although a previous report indicated that the expression level of phosphatase and tensin homolog (PTEN) was decreased after acquisition of cisplatin resistance in EGFR mutant lung cancer cells,[26] the expression level of PTEN protein in our drug-resistant cells was comparable to that of their parent cell lines (Fig. S7). This indicates that PTEN loss is not a characteristic of our resistant cell lines.

According to recent phase III studies, compared with first-line platinum-based standard chemotherapy, first-line treatment with EGFR inhibitors confers longer progression-free survival in patients with EGFR mutation-positive advanced NSCLC, whereas overall survival does not differ significantly between the two treatment groups.[27, 28] However, these studies aimed to determine the superiority of one first-line treatment over the other. Our results suggest that first-line treatment might influence the sensitivity of cancer cells to second-line treatment. Therefore, further clinical studies on sequential therapy are needed.

Our drug-resistant cells showed EMT-like alterations and markedly increased migration in comparison with their parental cell lines (Fig. 5b), indicating that both platinum-based and molecular-targeted agents permit the emergence of highly motile and metastatic cancer cells. Treatment of resistant AXL-positive cells (HCC4006-CR-GR2w and HCC827-CR-GR2w) with siRNAs targeting the AXL gene, which has been associated with EMT and cancer metastasis,[22, 23] impaired their migration activity compared to controls (Fig. 5e). Thus, AXL may be a useful therapeutic target in preventing the metastatic expansion of resistant cancers that have acquired resistance to both cisplatin and molecular-targeted drugs accompanied by increased AXL expression.

In conclusion, our results indicate that the potential influence of platinum-based chemotherapy on the acquisition of resistance to molecular-targeted therapies should be carefully considered when these treatments are used sequentially. As an EMT-like mechanism likely underlies the influence of cisplatin on molecular-targeted drug resistance and limits the efficacy of sequential therapy with both agents, future research should focus on the EMT process to develop more effective therapeutic strategies.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

This work was supported by a Grant-in-Aid for Scientific Research (23112513 to S.H.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank the members of the Higashiyama laboratory for their helpful insights.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cas12171-sup-0001-DocS1-S2-TableS1-FigS1-S7.docWord document1343K

Doc. S1. Supporting materials and methods.

Doc. S2. Supporting references.

Table S1. Primers for quantitative real-time PCR

Fig. S1. Growth of the established drug-resistant cell lines in the presence of cisplatin.

Fig. S2. Effect of gefitinib on autophosphorylation of EGFR in HCC4006, HCC4006-CR, and HCC4006-CR-GR2w cells.

Fig. S3. MET gene copy number in HCC827 and HCC827-derived resistant cell lines.

Fig. S4. Effect of AXL knockdown on gefitinib resistance of HCC4006-CR-GR2w cells.

Fig. S5. Influence of AXL knockdown on EMT marker expression in HCC4006-CR-GR2w and HCC827-CR-GR2w cells.

Fig. S6. Gefitinib sensitivity of HCC4006-CR-derived clones.

Fig. S7. Expression of PTEN protein in the established drug-resistant cell lines.

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