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- Materials and Methods
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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, 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. 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.
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- Materials and Methods
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- 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, 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, 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. 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. 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, 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.