TP53 mutant cell lines selected for resistance to MDM2 inhibitors retain growth inhibition by MAPK pathway inhibitors but a reduced apoptotic response
Emergence of resistance to molecular targeted therapy constitutes a limitation to clinical benefits in cancer treatment. Cross-resistance commonly happens with chemotherapeutic agents but might not with targeted agents.
In the current study, TP53 wild-type cell lines with druggable MAPK pathway mutations [BRAFV600E (WM35) or NRAS Q61K (SJSA-1)] were compared with their TP53 mutant sublines (WM35-R, SN40R2) derived by selection for resistance to MDM2/p53 binding antagonists.
The continued presence of the druggable MAPK pathway targets in the TP53 mutant (TP53MUT) WM35-R and SN40R2 cells was confirmed. Trametinib and vemurafenib were tested on the paired WM35/WM35-R and SJSA-1/SN40R2 cells and similar growth inhibitory effects on the paired cell lines was observed. However, apoptotic responses to trametinib and vemurafenib were greater in WM35 than WM35-R, evidenced by FACS analysis and caspase 3/7 activity, indicating that these MAPK inhibitors acted on the cells partially through p53-regulated pathways. SiRNA mediated p53 knockdown in WM35 replicated the same pattern of response to trametinib and vemurafenib as seen in WM35-R, confirming that p53 plays a role in trametinib and vemurafenib induced apoptosis. In contrast, these differences in apoptotic response between WM35 and WM35-R were not seen with the SJSA-1/SN40R2 cell line pair. This is likely due to p53 suppression by overexpressed MDM2 in SJSA-1.
The TP53MUT cells selected by resistance to MDM2 inhibitors nevertheless retained growth inhibitory but not apoptotic response to MAPK pathway inhibitors.
KeywordsMelanoma p53 MDM2 Nutlin-3 RG7388 HDM201 Trametinib Vemurafenib
fluorescence-activated cell sorting
Dulbecco’s modified Eagle’s medium
multidrug resistance protein 1
multidrug-resistance-associated protein 1
standard error of mean
SiRNA of p53
siRNA for control
serial tandem repeat
quantitative real-time polymerase chain reaction
RAS and RAF oncogenes are frequently mutated in human cancer leading to a constitutively activated MAPK pathway which is critical for oncogenesis, tumour proliferation and survival [1, 2]. These genetic alterations provide important targets druggable by low molecular weight compounds which have been evaluated in clinical trials and become licensed treatments. For example, the dual blockade of the MAPK pathway by the combination of BRAF and MEK inhibitors has become the standard treatment for unresectable or metastatic melanoma harboring a BRAFV600 mutation [3, 4]. Vemurafenib  and trametinib  are the first BRAF and MEK inhibitors respectively to be approved for BRAF-mutated cancer (melanoma). Trametinib strongly inhibits MEK1/2 kinase activities and was shown not to inhibit another 98 kinase activities . Preclinical study of trametinib showed efficacy against cancer cells with either BRAF or RAS mutations and even on cancer cells with neither BRAF nor RAS mutations ; therefore, cancers without BRAF mutations are included in clinical trials of trametinib.
Tumour protein p53, encoded by the tumour suppressor gene TP53, is a transcription factor activating target genes that mediate various functions, including deoxyribonucleic acid (DNA) repair, metabolism, cell cycle arrest, apoptosis, senescence and autophagy [9, 10, 11]. Loss of wild-type (WT) p53 function by either TP53 mutations, or overexpression of its negative regulators such as MDM2, causes cancer cell development, survival, and proliferation . MDM2-p53 binding antagonists are designed to occupy the p53-binding pocket of MDM2 and therefore stabilize p53 through prevention of MDM2-mediated ubiquitylation and proteasomal degradation. MDM2-p53 binding antagonists cause cell cycle arrest, apoptosis, and growth inhibition of cancer cells resulting from activation of the p53 pathway in p53WT (p53-wild type) cancer cells . The first small molecule MDM2 inhibitor, Nutlin-3, showed efficacy in vitro and with tumour xenograft (SJSA-1) models in nude mice . RG7388  and HDM201  are new generations of MDM2 inhibitors which are more potent and specific than Nutlin-3 and clinical trials are ongoing to investigate their efficacy in clinical settings.
Emergence of resistance to molecular targeted therapy constitutes a limitation to maintained clinical benefits in cancer treatment. Cross-resistance commonly happens with chemotherapeutic agents. After selection for resistance to a single drug, cells frequently also show cross-resistance to other structurally and mechanistically unrelated drugs by increasing the activity of efflux pumps (p-glycoprotein, PGP, also known as multidrug resistance protein 1, MDR-1; multidrug-resistance-associated protein 1, MRP-1) or reducing drug influx, or other mechanisms .
Unlike cytotoxic agents, resistance to targeted therapy usually results from mutation of the target gene or activation of pro-survival signaling pathways . Because of the different resistance mechanisms involved, cross-resistance might play less of a role in targeted therapy than with cytotoxic agents. The evidence for cross-resistance of targeted therapies, particularly for MDM2 inhibitors is lacking. Michaelis et al. selected p53 mutant (P53MUT) cells (UKF-NB-3r Nutlin10μM) by continuous exposure to Nutlin-3 and found the resistant cells displayed a multi-drug resistant phenotype which was resistant to various cytotoxic drugs and irradiation . By contrast, another study showed the resistant cells (S_N40R2, N_N20R1) selected by Nutlin-3 retained sensitivity to ionizing radiation . Previous studies have focused on the cross-resistance between MDM2 inhibitors and cytotoxic agents and irradiation, but to date no reported studies have investigated the response to alternative effective small molecule targeted agents in cells selected for resistance to MDM2 inhibitors.
The aim of the current study was to select cell lines with resistance to MDM2/p53 binding antagonists from cancer cells with druggable targets of the MAPK pathway resulting from either BRAFV600E (WM35) or NRASQ61K (SJSA-1) mutation and to examine whether the MDM2 inhibitor-resistant cell lines retain sensitivity to the MAPK pathway inhibitors, vemurafenib and trametinib.
Cell lines and reagents
Two parental and MDM2 inhibitor resistant cell line pairs, WM35/WM35-R and SJSA-1/SN40R2, were routinely cultured using Dulbecco’s modified Eagle’s medium (DMEM) medium and RPMI-1640 medium (Sigma, Dorset, UK) respectively, which were supplemented with 10% (v/v) foetal calf serum. All the cell lines were authenticated by serial tandem repeat (STR) profiling (WM35, WM35-R by NewGene, Newcastle, UK; SJSA-1, SN40R2 by LGC Standards). Nutlin-3 was purchased from NewChem (Newcastle, UK), RG7388 and HDM201 were obtained by custom synthesis via Astex Pharmaceuticals. Trametinib and vemurafenib were obtained from Cambridge Bioscience. All compounds were initially dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and used to dose cells at a final concentration of 0.5% DMSO, optimised to give minimal cytotoxic effects on cells, and 0.5% DMSO only solvent controls were included in all experiments.
Growth inhibition assay
Cells were seeded in 96-well plates overnight and treated with indicated drugs for 72 h. The cells were fixed using Carnoy’s fixative followed by Sulforhodamine B (SRB) assay . A spectrophotometer (Spectramax250 Molecular Devices) was used to measure absorbance at 570 nm. The GI50 value was determined by the concentration of a compound which can reduce the growth of the cell population by 50% compared to untreated control cultures after subtraction of baseline seeded cell amount prior to the start of treatment .
Cells lysates were harvested by scraping and suspension in lysis buffer (62.5 mM Tris HCl/pH 6.8, 10% glycerol and 2% SDS), heated and sonicated. The protein concentrations of the cell lysates were estimated using a Pierce® BCA Protein Assay kit. Equal quantities of protein were loaded onto and separated by SDS–polyacrylamide gels (4–20% Mini-PROTEAN® TGX™ Gel, BioRad). The separated proteins were transferred and immobilized onto Amersham™ nitrocellulose membranes (GE Healthcare Life Science). Primary antibodies against p53 (DO-7) (#M7001, Dako), MDM2 (Ab-1) (#OP46, Merck Millipore), p21WAF1 (EA10) (#OP64, Calbiochem), p-ERK (E-4) (sc-7383, Santa Cruz), ERK (K-23) (sc-94, Santa Cruz), GAPDH (14C10) (#2118, Cell Signaling Technology), BRAFV600E (VE1, Spring Bioscience), Actin (A4700, Sigma) and secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (#P0447/P0448, Dako) were used. All antibodies were diluted in 5% (w/v) non-fat milk or BSA in TBS-tween (20 mM Tris/pH 6.8, 137 mM NaCl, 0.1% tween-20). Protein signals were visualized using enhanced chemiluminescence (GE Healthcare Life Sciences) and X-ray film (Fujifilm).
RNA extraction and qRT-PCR (quantitative real-time polymerase chain reaction)
MDM2: F-AGTAGCAGTGAATCTACAGGGA, R-CTGATCCAACCAATCACCTGAAT
CDKN1A: F-TGTCCGTCAGAACCCATGC, R-AAAGTCGAAGTTCCATCGCTC
PUMA: F-ACCTCAACGCACAGTACGA, R-CTGGGTAAGGGCAGGAGTC
BAX: F-CCCGAGAGGTCTTTTTCCGAG, R-CCAGCCCATGATGGTTCTGAT
PIG-3: F-AGCGAGGAAGTCTGATCACC, R-CGTGGAGAAGTGAGGCAGAA
AEN: F-CTTCCAGGCGCTCAAGTATGT, R-GGGCCAGGTCCTTTAGAGAGA
FDXR: F-CAGCATTGGGTATAAGAGCCG, R-GGCCTGGCACATCCATAACC
TNFRSF10B: F-ATGGAACAACGGGGACAGAAC, R-CTGCTGGGGAGCTAGGTCT
TP53INP1: F-TCTTGAGTGCTTGGCTGATACA, R-GGTGGGGTGATAAACCAGCTC
TP53: F-CAGCACATGACGGAGGTTGT, R-TCATCCAAATACTCCACACGC
GAPDH: F-CAATGACCCCTTCATTGACC, R-GATCTCGCTCCTGGAAGAT.
A total of 10 ng of the cDNA samples per 10 µL final reaction volume, with the standard cycling parameters (stage 1: 50 °C for 2 min, stage 2: 95 °C for 10 min, then 40 cycles of 95 °C for 15 s, and 60 °C for 1 min), were set and carried out on an ABI 7900HT sequence detection system. GAPDH was used as endogenous control and samples of cells exposed to DMSO carrier were used as the calibrator for each independent repeat, with the formula 2ΔΔCt used to calculate fold-changes. Analysis was carried out using SDS 2.3 software (Applied Biosystems).
Fluorescence-activated cell sorting (FACS)
After treatment, floating and adhered cells were pooled and fixed using 70% cold ethanol. Samples were incubated in 250 μL PBS with 40 μg/mL propidium iodide (Sigma-Aldrich), 20 μg/mL RNAse A (Sigma-Aldrich) for 20 min in the dark at room temperature, then were analyzed on a FACSCaliburTM flow cytometer using CellQuest Pro software (Becton–Dickinson, Oxford, UK). Cell cycle distribution based on DNA content was determined using Cyflogic (CyFlo Ltd, Turku, Finland).
Caspase 3/7 activity assay
Melanoma cells were seeded in white 96-well plates and treated after 24 h. Caspase-3/7 enzymatic activities were measured using a FLUOstar Omega plate reader (BMG Labtech) after adding a 1:1 ratio of CaspaseGlo 3/7 reagent (Promega) to growth media and incubating for 30 min. All values were expressed as a ratio of signal relative to solvent control.
DNA sequencing and mutation-specific PCR
NRAS: F-5′-CCACACCCCCAGGATTCTTAC-3′, R-5′-AGTGTGGTAACCTCATTTCCC-3′
TP53(exon10): F-5′-CATGTTGCTTTTGTACCGTCA-3, R-5′-TGAAGGCAGGATGAGAATGGA-3′.
Mutations detected by sequencing were then investigated by mutation specific PCR.
Forward wild type: 5′-CTGTTGCTGCAGATCCGTGG-3′,
Forward mutant: 5′-CTGTTGCTGCAGATCCGTGT-3′,
Following PCR, products were analysed on 2% agarose gels containing ethidium bromide and were visualised by UV light (G:BOX imaging system).
siRNAs and transfection
SiRNA of p53 (SiP53)
SiRNA of control (SiControl)
Data were presented as mean ± standard error of mean (SEM) unless otherwise stated. Statistical tests were carried out using GraphPad Prism 6 software and all p-values represent paired t-tests of at least three independent repeats. A p-value less than 0.05 was considered as statistically significant.
Selection for resistant cells
SN40R2 and WM35-R cell lines with resistance to MDM2/p53 binding antagonists were generated by continuously exposing SJSA-1 osteosarcoma cells  and WM35 cutaneous melanoma cells to either Nutlin-3 or RG7388 respectively. WM35 parental cells were cultured in medium with 0.5 μM RG7388 in 175 cm-squared flasks and then RG7388 was escalated to 1, 2, 3, 5 μM gradually within 3 months. Paired WM35/WM35-R and SJSA-1/SN40R2 have BRAFV600E and NRASQ61K mutations which activate the MAPK pathway and render the cells druggable by MEK or BRAF inhibitors.
WM35-R cells are resistant to other MDM2 inhibitors
SN40R2 cells selected from SJSA-1 are resistant to all MDM2 inhibitors tested
Growth inhibition of paired WM35 and WM35-R cells by trametinib and vemurafenib
The effect of trametinib or vemurafenib treatment on cell cycle distribution and apoptosis for paired WM35 and WM35-R
WM35-R showed much lower induction of p53-dependent gene transcripts than WM35 after 24 h trametinib treatment
SiRNA-mediated knockdown of p53 reduced sensitivity and caspase 3/7 activity after trametinib and vemurafenib
The response of the paired SJSA-1 and SN40R2 cell lines to trametinib
Cell lines selected for resistance to MDM2 inhibitors had TP53 mutations and were cross-resistant to other MDM2 inhibitors
Most in vitro studies reported have shown that cells selected for resistance to MDM2 inhibitors harbour p53 mutations and show a deficiency of p53-dependent apoptosis in response to MDM2 inhibitor treatment [19, 20, 23]. One recent study used piggyBac transposon insertional mutagenesis in a cohort of allografts with an underlying CDKN2A deletion, to anticipate resistance mechanisms which might occur during treatment with the MDM2-p53 inhibitor HDM201. The most frequent mechanisms conferring resistance converged on direct (TP53 mutations) or indirect (gain-of-function of MDM4, TP63, TP73) loss-of-function inactivation of the p53 protein. In addition, activation of the anti-apoptotic B-cell lymphoma-extra large (Bcl-xL) gene was observed . Although MDM2 inhibitors (Nutlin-3, RG7388) were previously reported as a modulators of MDR-1(p-glycoprotein) [25, 26] and MDR-1-overexpressed cells showed decreased sensitivity to Nutlin-3 , there are no reports that selection for resistance to MDM2 inhibitors results from amplification and/or overexpression of genes encoding multidrug resistance proteins.
Consistent with previous reports, both cell lines selected for resistance to MDM2 inhibitors investigated in the current study have TP53 mutations. The selection and characterisation of SN40R2, which has been reported previously by our group  and of WM35-R described here, are the first reported p53MUT cell lines which have been selected in vitro for resistance to RG7388 [22, 27], a clinically relevant more potent and specific MDM2 inhibitor than Nutlin-3 [19, 20, 23] and MI-63  which were used in previous studies. In contrast to another study which was based on p53 mutation which had been generated de novo , the current study investigated the selection of cells resistant to MDM2 inhibitors from a parental culture of WM35 melanoma cells. This resulted in the selection of a p53 mutated subpopulation. Most reported TP53 mutations are located in the DNA binding domain, however the WM35-R resistant sub-line has an uncommonly reported p53G334V point missense mutation in a critical residue the tetramerization domain of p53, which consists of a α-strand (Glu326–Arg333), a tight turn (Gly334), and a β-helix (Arg335–Gly356) . In the wild-type tetramerization domain, Gly334 facilitates the formation of a sharp turn connecting the β-strand with the α-helix and adopts a backbone conformation that would be energetically unfavorable for a non-glycine residue, indicating that mutation of Gly334 is expected to result in structural distortions . The Gly334 mutant p53 protein exhibits a global decrease in DNA binding and transactivation activity . In the current study, WM35-R with such a p53G334V mutation lost its transactivation activity, evidenced by no induction of downstream targets of p53 and consequent cross-resistance to other MDM2 inhibitors. For the other resistant cell line, SN40R2, detailed characteristics and discussions have been addressed in our previous publication .
TP53 MUT cells selected for resistance to MDM2 inhibitors retain sensitivity to growth inhibition by MAPK pathway inhibitors but a reduced p53-dependent apoptotic response to them
To investigate whether cross-resistance occurs between MDM2/p53 binding antagonists and MAPK pathway inhibitors, trametinib and vemurafenib, paired WM35/WM35-R and SJSA-1/SN40R2 cells were tested and showed similar growth inhibitory effects for the paired cell lines. This showed that a growth inhibitory response to MAPK pathway inhibition was maintained in the TP53 mutant cell lines selected for resistance to MDM2 inhibitors. However, more p53-regulated apoptosis was found in WM35 than WM35-R, supported by sub-G1 fraction on FACS analysis and caspase 3/7 activity, indicating that trametinib and vemurafenib acted on the cells partially through a p53-regulated pro-apoptotic pathway, particularly at higher doses. The interaction between the MAPK pathways and the p53/MDM2/MDMX network are tightly regulated . A murine model study showed that the RAS/RAF pathway activated the transcription of both MDM2 and its inhibitor p19ARF . The level of p53 was determined by opposing effects of RAF-induced p19ARF and MDM2. In the absence of p19ARF or when the induction of MDM2 exceeds that of p19ARF (a common situation in many human cancers), the RAS/RAF/MEK/ERK pathway attenuates p53. Another study supported this finding by using a RAS inhibitor, farnesylthiosalicylic acid (FTS), which increased p53 expression through both downregulation of MDM2 and transcriptional activation of p53 . Consistent with these observations, in the current study trametinib treatment induced transcripts of TP53 as well as CDKN1A and p53-regulated pro-apoptotic genes in WM35. CDKN1A only slightly increased in WM35 but not in WM35-R indicating that trametinib-induced G1 arrest was mainly p53/p21-independent, which is consistent with a previous report that MEK inhibition in melanoma cells resulted in p27 regulated G1 arrest rather than via p21 .
Differences between WM35 and SJSA-1
In contrast to the different responses between WM35 and WM35-R, the SJSA and SN40R2 cell lines did not show such differences. Little apoptosis was found in the parental TP53 wild-type SJSA-1 cells, which was different to WM35. SJSA-1 is a MDM2 amplified osteosarcoma cell line in which the function of p53 is suppressed by overexpressed MDM2, whereas WM35 has no MDM2 amplification and lower expression of MDM2 protein (Additional file 1: Figure S6). This possibly explains why little p53-dependent apoptosis was found after trametinib treatment of SJSA-1 and SN40R2. The MDM2 suppression of p53 rather than RAS mutation is a main driver target in SJSA-1, evidenced by the significant induction of caspase 3/7 activity after MDM2 inhibitor treatment (Additional file 1: Figure S7). The combination of MAPK and MDM2 inhibitors as a strategy for cancers with wild-type p53 and mutations of RAS/RAF merits further exploration.
In conclusion, there was restricted cross-resistance between MDM2/p53 binding antagonists and MAPK inhibitors indicating that resistant cancer cells potentially selected by MDM2 inhibitors can nevertheless be treated with MAPK inhibitors if the cells have druggable mutations driving the MAPK pathway.
CW, LP, JL conceived and designed the project; CW, TK, YH performed experiments and analyzed data; CW, TK, YH, LP, JL interpreted the results and wrote the paper. All authors read and approved the final manuscript.
The authors thank and gratefully acknowledge Newcastle University/Astex Pharmaceuticals Alliance and Cancer Research UK who funded the Drug Development Programme at the Newcastle University Northern Institute for Cancer research for their support and encouragement, including help with sourcing the HDM201 and RG7388 by custom synthesis.
The authors declare that they have no competing interests.
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Not applicable—established melanoma cell line models.
All work was performed at the Newcastle University Northern Institute for Cancer Research which receives Centre funding from Cancer Research UK. This work was supported by Grant (CMRPG3I0451 to C-EW) from Chang Gung Memorial Hospital, Taiwan.
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