Introduction
Platinum-based chemotherapy has been the primary treatment for cancer patients diagnosed with small-cell lung cancer (SCLC) treatment following initial diagnosis. In the United States, 82% of SCLC patients were treated with either carboplatin or cisplatin in combination with etoposide (IntrisiQ, 2008). Despite high initial response rates of 40–90% [
1], the majority of patients develop treatment resistance. Effective second-line treatment for recurrent SCLC is a major unmet medical need. There is no standard chemotherapy for second-line platinum-refractory (no response to initial therapy) or platinum-resistant SCLC (early relapse after initial therapy).
Currently, the understanding of platinum resistance mechanisms is largely based on studies of cisplatin. Resistance is multi-factorial and can vary between cell lines and types and this finding is consistent with variable responses to platinum therapies between patients with similar tumor types. Several mechanisms of resistance have been hypothesized and are related directly or indirectly to the platinum DNA binding. Potential mechanisms of resistance include processes that alter the net intracellular accumulation of platinum due to influx and efflux transporters, the inactivation of platinum drugs once inside the cell by thiol-containing proteins such as glutathione or metallothionine, the removal of platinum adducts from DNA by nucleotide excision repair, the status of DNA mismatch repair and bypass of DNA adducts by DNA polymerase, and the mechanisms that control apoptosis (for review, see [
2]).
Gene expression analysis has been used recently to identify individual genes and pathways whose transcriptional regulation contributes to platinum resistance. To enable physicians to provide the most appropriate care for individual patients, biomarkers for patient stratification for treatment as well as real-time monitoring of response are needed; gene expression signatures may provide this much-needed tool. A small number of recent publications report gene expression analysis of platinum resistance in ovarian cancer, osteosarcoma, esophageal cancer, and Hodgkin’s lymphoma. For ovarian cancer, potential gene signatures for resistance or correlation have been identified by profiling tumor samples from platinum responder and non-responder patients [
3] and by profiling the response to carboplatin in an ovarian cell line [
4]. Despite the potential clinical benefit, to date, no studies have been reported characterizing gene expression in SCLC cell lines (resistant or responsive to platinum agents) or SCLC clinical samples.
Picoplatin (AMD473, JM473, ZD0473) is a new generation platinum-based analog designed to overcome platinum resistance [
5]. In vitro preclinical studies confirmed the ability of picoplatin to retain activity in the presence of elevated levels of glutathione and also demonstrated that picoplatin can overcome mechanisms which decrease accumulation of cisplatin [
6,
7]. The suggestive in vitro findings were substantiated by in vivo preclinical observations: picoplatin has a greater anti-tumor effect on cisplatin-resistant ovarian tumors relative to other platinum agents [
8].
Limited preclinical data are available regarding the activity of picoplatin in SCLC. The only publication documents picoplatin cytotoxicity against two SCLC lines, H69 and SBC-3, and against cisplatin-resistant derivatives of these two lines [
7]. The following report describes the development of novel SCLC cell lines resistant to cisplatin and carboplatin followed by their characterization and the pharmacologic evaluation of picoplatin compared to other platinums in these tumor cells.
Materials and methods
Cell culture and reagents
Small-cell lung cancer lines, DMS53 and DMS114, were obtained from American Type Culture Collection (Manassas, VA). DMS53 and DMS114 are cultured in Waymouth’s MB 752/1 medium supplemented with 10% FBS. Injection grade cisplatin (1 mg/ml) and carboplatin (10 mg/ml) were obtained from Sicor (Irvine, CA) and oxaliplatin (5 mg/ml) was obtained from Sanofi-Aventis (Bridgewater, NJ). Picoplatin (0.5 mg/ml) was provided by Poniard Pharmaceuticals (South San Francisco, CA).
Drug selection
Drug-resistant sub-cultures were generated by repeated cycling in increasing selection drug concentration according to the following schedule: cells were treated in supplemented media with selection drug for 7 days. Cells were then gently washed and allowed to recover for 21 days in fresh supplemented media without drug. During this time, cells were tested for drug resistance. Drug concentrations used for selection were increased incrementally until cell lines acquired resistance. For DMS114, selection was initiated at 2 μM cisplatin and increased to 5 μM in 1 μM increments, and at 4 μM carboplatin and increased to 11 μM in 1 μM increments. Similarly in DMS53, cisplatin selection was from 3 to 6 μM, carboplatin from 5 to 11 μM, and oxaliplatin from 1 to 5 μM. These cell lines were then cycled at maintenance drug concentrations to retain resistance. In some cases, cell lines acquired durable resistance to the selection drug after 10–12 rounds of selection and no longer required cycling to maintain resistance. In cell lines where continued cycling is necessary to maintain drug resistance, cells in the recovery period (7–21 days after removal of selection drug) were used for experiments.
Cytotoxicity assay
An MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used to assess cytotoxicity. The CellTiter 96 AQueous Cell Proliferation Assay (Promega, Madison, WI) was performed essentially according to the manufacturer’s protocol. Briefly, 104 cells per well were seeded in flat bottom 96-well plates in supplemented media and allowed to adhere overnight. Following the addition of a serial dilution of drug, cells were incubated for 4 days. 20 μl of MTS reagent was added to each well, and plates were incubated for an additional 1–2 h at 37°C before spectrometric measurement of absorbance at 490 nm. Media-only background was subtracted from all samples. Data were analyzed using GraphPad Prism 5. Fifty percent inhibitory concentration (IC50) values were calculated based on normalized log transformed data using a non-linear regression algorithm. The resistance factor (RF) serves as a measure of drug resistance in resistant cells and is defined as the IC50 drug concentration of the resistant line divided by that of the parental line.
Cellular platinum accumulation
Total cellular platinum accumulation was determined by ICP-MS essentially as described by Kitada et al. [
9]. Briefly, 10
6 cells were seeded in supplemented media in 6-well plates and allowed to adhere overnight, washed with PBS, and treated with drug in serum-free media for 24 h. Cells were then washed three times with PBS, lysed and sonicated in 1 ml H
2O. A volume of 0.5 ml of sample was then reacted with aqua regia, digested at 95°C, and diluted to a final volume of 5 ml with H
2O. Samples were analyzed by conventional ICP-MS and quantified against a certified standard using multi-point weighted linear regression (CanTest, Burnaby, Canada).
Gene expression profiling by microarray
In supplemented Waymouth’s medium, 10
6 carboplatin-resistant (7–21 days into the drug cycling recovery period) and parental DMS53 cells were allowed to adhere overnight and treated with drug for 24 h. RNA was extracted using the RNeasy total RNA purification kit (Qiagen, Valencia, CA) according to manufacturer protocol. Total RNA quality was determined using a Pico Chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Four independent batches of RNA were prepared for gene expression profiling to allow for adequate statistical power. Sample preparation, labeling, and array hybridizations were performed according to standard protocols by the UCSF Shared Microarray Core Facilities and Agilent Technologies (
http://www.arrays.ucsf.edu and
http://www.agilent.com). Equal amounts of Cy3-labeled cRNA target were hybridized to Agilent human whole genome 4 × 44 K Ink-jet arrays (Agilent). Arrays were scanned using the Agilent microarray scanner (Agilent) and raw signal intensities were extracted with Feature Extraction 10.1.1 software (Agilent). Coefficients were included to adjust for batch specific differences. Subsequent gene ontology and functional clustering analyses were performed using The Database for Annotation, Visualization and Integrated Discovery (DAVID 2008; National Institute of Allergy and Infectious Disease (NIAID), NIH; [
10,
11]).
Discussion
Picoplatin is an investigational agent that has been evaluated in >1100 cancer patients to date. Picoplatin has been and continues to be evaluated as an anti-tumor agent as a single agent or in combination with other cytotoxics in a wide variety of tumor types. Two independent Phase II monotherapy studies of picoplatin in SCLC demonstrated a median overall survival of at least 27 weeks and were comparable to other therapeutic options [
43,
44]. In the SPEAR (Study of Picoplatin Efficacy After Relapse) pivotal Phase III trial conducted by Poniard Pharmaceuticals, the efficacy and safety of picoplatin in combination with best supportive care (BSC) vs BSC alone was evaluated in a total of 401 SCLC patients who were refractory to or who progressed within 6 months of first-line platinum-based therapy. Picoplatin was well-tolerated with manageable hematologic toxicity. Although the analysis of overall survival (321 events) did not achieve statistical significance (
P = 0.0895) in the intent-to-treat population, a statistically significant imbalanced use of post-study chemotherapy in favor of the BSC arm may have impacted this result (
P = 0.012). In 273 patients who did not receive post-study chemotherapy, there is statistically significant difference in overall survival in favor of the picoplatin arm (
P = 0.0345). More importantly, picoplatin demonstrated significant survival improvement in 294 refractory patients who never responded or relapsed within 45 days (
P = 0.0173). Picoplatin addresses a major unmet need in the treatment of second-line small-cell lung cancer as there is currently no FDA approved therapy for patients who are refractory or relapse within 45 days of first-line platinum-based therapy [
22].
In this study, we demonstrated the ability of picoplatin to overcome platinum drug resistance in SCLC using newly generated cisplatin- and carboplatin-resistant SCLC cell lines. These observations were consistent with a study by Kawamura et al. [
7], which showed continued sensitivity to picoplatin in two cisplatin-resistant SCLC and two cisplatin-resistant NSCLC lines. A number of resistance mechanisms to platinum drugs have been postulated [
2]. However, no reports of studies related to platinum resistance have been done in small-cell lung cancer. In this study, we identified decreased cellular platinum accumulation after drug treatment as a potential mechanism that could contribute to cisplatin and carboplatin resistance in these cell lines. In both platinum-resistant cell lines, picoplatin treatment resulted in high levels of cellular platinum that were indistinguishable from those of parental cells. Similar results were observed in human ovarian cancer lines [
6]. In addition, picoplatin appears to retain cytotoxic activity against both these platinum-resistant lines.
Gene expression microarray analysis has been useful to identify signatures of drug resistance and provide potential clinical biomarkers. A comprehensive study of the carboplatin-resistant cell line using gene expression profiling revealed additional pathways that may potentially contribute to carboplatin resistance. Functional annotation clustering showed significant upregulation of HOX-containing genes, genes involved in glutathione biosynthesis, and MAGE genes, many of which have been implicated in cancer.
Our study shows that homeodomain (HOX)-containing gene functional clusters are highly overrepresented in carboplatin-resistant DMS53 cells (Table
2, Cluster 1). HOX genes are well known for their role in development and hematopoiesis [
14,
15]. Their involvement in cancer was initially identified in hematologic malignancies such as myeloid lymphocytic leukemia where chromosomal translocation of HOX with Nup98 is thought to contribute to tumorigenesis [
16]. More recently, a significant number of studies suggest that the expression of HOX genes is dysregulated in multiple solid tumor types including SCLC [
17‐
19] and is involved in progression, invasion, metastasis, and DNA repair [
20‐
22]. For instance, the disruption of HOX interaction with PBX leads to increased apoptosis in NSCLC, RCC, and melanoma cells [
23‐
25]. HOXA9 and HOXB9, both upregulated in carboplatin-resistant DMS53 cells, were found to be frequently overexpressed in lung cancer [
26]. Interestingly, HOXB7, also upregulated in carboplatin-resistant DMS53, is believed to be involved in DNA repair [
22]. Activation of HOX gene clusters has been linked to poor survival and drug resistance in glioblastoma and resembles a self-renewal signature in leukemia [
27]. Little else is known regarding the role of HOX genes in drug resistance.
Increased glutathione levels and glutathione-S-transferase activity have frequently been implicated in platinum neutralization and resistance, and the correlation between increased glutathione levels and drug resistance has been well documented in multiple tumor types [
28‐
33]. However, a causal relationship between the two has thus far not been convincingly demonstrated to date. We found that glutathione biosynthetic and metabolic processes were overrepresented in carboplatin-resistant DMS53 relative to parental DMS53 cells. Consistent with these data, total cellular glutathione level was shown to be significantly higher in carboplatin-resistant DMS53 cells in a separate set of experiments (Supplementary Fig. S2). Together, these data show that upregulation of the glutathione pathway is associated with carboplatin resistance and that drug neutralization by glutathione may contribute to drug resistance in SCLC. Although the results of our study do not establish a causal relationship between increased glutathione and drug resistance, they do suggest that the glutathione biosynthetic pathway may be regulated by the development of drug resistance in SCLC. Interestingly, picoplatin retained cytotoxic activity against carboplatin-resistant DMS53 cells, and cellular accumulation remained high in both drug-resistant and parental cells, which suggests that picoplatin is able to circumvent glutathione mediated inactivation.
Expression of MAGE genes has been linked to SCLC and suggested as possible biomarkers for detection as well as potential targets for immune-based therapeutic modalities [
42,
43]. The first melanoma-associated antigen (MAGE-A1) was isolated from a melanoma cell line and found to be recognized by cytotoxic T lymphocytes [
44]. In subsequent years, numerous additional MAGE genes were identified. MAGE genes are subclassified into four groups (A-D) based on sequence homology and divided into two subgroups (I, containing groups A-C, and II, containing group D) based on expression pattern. Subgroup I, also called cancer/testis antigens, are expressed in testis and frequently in tumor cells and have become the subject of substantial interest in tumor immunotherapy. Although the functions of MAGE proteins have remained largely elusive, they are believed to be involved in embryonic development and aspects of tumor transformation and progression. Our data suggest that upregulation of several MAGE genes (MAGE A8, A9, A11, and B2) may be related to the acquisition of resistance to carboplatin in SCLC cells. If confirmed, these observations could potentially have utility in predicting SCLC resistance to platinum and guiding patient treatment decisions.
Interestingly, our results also point to the upregulation of a number of genes in the VCX/Y and PNMA families, the functions of which are currently unknown. Further studies will be required to assess whether these differentially regulated genes play a crucial role in conferring resistance to carboplatin.
Our gene expression data suggest a possible mechanism for resistance to carboplatin in SCLC cells and begin to reveal a distinct mode of action for picoplatin. Further studies will be required to confirm our findings in additional platinum-resistant SCLC cell lines and establish a potential signature for platinum resistance. Since the kinetics of DNA binding of picoplatin and cell cycle/death response to picoplatin appear to be somewhat slow relative to other platinums [
5], gene expression profiling of platinum-treated tumor cells over time may prove to be useful. Based on our finding that picoplatin treatment can overcome resistance to other platinum drugs and the observation that picoplatin elicits a gene expression profile that is distinct from that of carboplatin in SCLC, we believe that additional studies along this line of inquiry could help to determine the mechanism of action for picoplatin and potentially have clinical application by the establishment of gene expression signature of carboplatin resistance in SCLC.
The primary objective of this work was to determine whether picoplatin could overcome resistance to treatment by cisplatin and carboplatin in small-cell lung cancer. We found that picoplatin retains cytotoxic activity against cisplatin and carboplatin-resistant SCLC lines. In addition, our results demonstrated that platinum-resistant cells accumulated less platinum and differential gene expression. Picoplatin treatment resulted in high levels of platinum accumulation in all resistant and parental lines tested, suggesting that mechanisms controlling cellular cisplatin and carboplatin levels were not effective in controlling cellular picoplatin levels. Finally, we showed by gene expression profiling that picoplatin treatment triggers a distinct gene expression pattern relative to treatment with carboplatin and that this pattern is similar in parental and carboplatin-resistant SCLC cell line. Together our data suggest that picoplatin action is distinct from those of the other platinums tested and could have clinical benefit in the treatment of small-cell lung cancer resistant to cisplatin and carboplatin therapy.