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Correspondence: Zhi-Chao Wang, Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China.
MAPK/extracellular signal–regulated kinase kinase (MEK) 1/2 inhibitors (MEKis) have recently achieved surprising success in treating unresectable plexiform neurofibromas (PNFs). However, few studies have investigated the mechanisms of MEKi resistance in patients with PNF. We determined the efficacy of six different MEKis for treating PNFs, explored drug resistance mechanisms, and identified potential combination therapies to overcome resistance. By screening drug efficacy among six MEKis in human NF1-deficient PNF cell lines, TAK-733 was found to reduce PNF cell viability the most. We then cultured the TAK-733‒resistant cells and explored the potential targets for further treatment. Both high-throughput drug screening and RNA sequencing analyses of MEKi-resistant PNF cells identified cyclin-dependent kinase inhibitors as potential agents for PNFs. Dinaciclib, a cyclin-dependent kinase inhibitor, showed synergistic effects on MEKi-resistant cells. Coadministration of dinaciclib and TAK-733 significantly reduced cell viability and inhibited sphere formation and colony formation. Dinaciclib did not affect MEK signaling but decreased the expression of several prosurvival proteins, including survivin and cyclin-dependent kinase 1, to induce apoptosis and inhibit mitosis. TAK-733/dinaciclib combination therapy induced tumor reduction in PNF patient‒derived xenografts mouse models. Therefore, the combination of MEKi and cyclin-dependent kinase inhibitor may be promising for treating inoperable PNFs, especially when drug resistance exists. Our findings provide evidence for future clinical trials with MEKi-resistant patients with PNF.
MAPK/extracellular signal–regulated kinase (ERK) kinase (MEK) 1/2 inhibitors (MEKis) have demonstrated promising results in inoperable pediatric patients with PNF with a significant reduction in PNF volume and functional improvements. A phase I trial of selumetinib as a treatment for 24 inoperable pediatric patients with PNF showed a positive response in >70% of patients, with >20% decrease in tumor volume (
). Several other MEKis, including trametinib, cobimetinib, and TAK-733, also exhibit antitumor effects by interrupting growth signals for tumor cell proliferation and are currently in clinical trials (
). Resistance to MEKi can also be found in various types of cancers, including malignant peripheral nerve sheath tumor, the malignant transformation of PNFs, and other tumors such as melanoma or colorectal cancer (
). Investigating the underlying mechanisms of drug resistance is vital for designing combination therapies. There are currently few studies investigating the resistance mechanisms of MEKi in PNFs.
In this study, we first evaluated phosphorylated ERK (p-ERK) and phosphorylated MEK (p-MEK) expression in PNFs and its association with clinicopathological parameters. The efficiency of MEKi was tested in PNF models both in vitro and in vivo. We also employed a high-throughput drug screening system to identify potential combination agents, especially for patients in whom drug resistance may present. To further validate our findings in clinical specimens, we compared the feasibility and efficacy of combination therapies with those of MEKi monotherapy using a PNF patient‒derived xenograft (PNF-PDX) mouse model. This study provides evidence for a therapeutic option in the emergence of MEKi resistance in patients with PNF and informs the design of future clinical trials with patients with PNF.
p-MEK/p-ERK expression heterogeneity was observed in PNF tissues and cell lines
To evaluate cancer molecular heterogeneity, we employed a PNF tissue microarray containing two tissue cores per tumor. Figure 1a displays the different expression intensities of different markers, including p-MEK, p-ERK, MEK, and ERK, in the PNF tissue microarray. The expression of these markers was assessed as a score ranging from 0 to 6 on the basis of the intensity and area of positive staining with different proportions. A total of 94 of 141 (66.66%) of PNF tumor areas showed a relatively strong (staining scores ranging from 3 to 6) p-MEK expression (Figure 1b). More information about the relationships between the expression of p-MEK/p-ERK and the clinical characteristics, including sex, tumor sites, and size, is presented in Supplementary Tables S1 and S2. The expression of p-ERK was moderately correlated with that of p-MEK (r = 0.4601, P < 0.001) (Figure 1c), so as the expression of ERK and MEK (Figure 1d). The expression levels of p-MEK and p-ERK in tumor tissue derived from different individuals were compared to assess tumoral heterogeneity (Figure 1e). Moreover, the p-MEK and p-ERK protein levels in different clinical PNF tumor tissues (Figure 1f) and cell lines (Figure 1g) were also heterogeneous, which may provide an explanation for the differences in treatment efficacy.
PNF cell lines but not normal Schwann cells were responsive to MEKi
To evaluate how PNF cells respond to different MEKi in vitro, we screened the effectiveness of selumetinib, trametinib, PD0325901, TAK-733, cobimetinib, and refametinib (all purchased from MedChemExpress, Monmouth Junction, NJ) in three NF1-deficient PNF cell lines (ipNF05.5, ipNF9511.bc, and ipNF95.6) and one Schwann cell line (Ipn02.3 2λ) (all purchased from ATCC, Manassas, VA) as a control after 72 hours of incubation. The MEKis TAK-733, trametinib, and cobimetinib were determined to be more effective for inhibiting PNF cell viability than the other three inhibitors with <5 μM half-maximal inhibitory concentration values (Figure 2a and b). In addition, they exhibited a more sensitive trend to NF1-deficient PNFs, with a lower inhibitory concentration level, than the normal Schwann cell control. We then chose trametinib and TAK-733 for further evaluation in vitro. Treatment with high concentrations induced morphological changes in the cells, resulting in a poached egg shape (Figure 2c). In addition, apoptotic rate was significantly increased in both 10 μM and 1 μM concentrations of TAK-733 (P < 0.05) (Figure 2d and f). Cell cycle assays displayed the blockade of S phase (Figure 2e and g). TUNEL assays additionally proved that MEKis induced apoptosis in PNF cells, and this inhibition was stronger when the concentration of MEKis increased (Figure 2h and i). Inhibition of colony formation and growth (as evidenced by fewer colonies in the trametinib-treated groups than in the control group) were also observed in MEKi-treated cells (Figure 2j and k), which was in line with the decreased ratio of living cells in Cell Counting Kit-8 assay (Dojindo Laboratories, Kumamoto, Japan).
MEKi-related proliferation inhibition and apoptosis induction occurred in a time- and dose-dependent manner
The therapeutic effects of TAK-733 and trametinib were further evaluated in vitro, and the expression levels of MEK/ERK signaling pathway proteins in the PNF cell line ipNF9511.bc are analyzed in Supplementary Figure S1. The results showed that TAK-733 was more effective in reducing the level of p-ERK than trametinib because its expression could not be detected in ipNF9511.bc cells treated with 0.003 μM TAK-733 after both 4- and 24-hour incubations. In contrast, the reduction in p-MEK levels induced by trametinib remained for less than 24 hours when the concentration was lower than 0.01 μM. The p-MEK reduction observed with a higher dose of trametinib treatment (0.03–0.1 μM) was equivalent to that seen with 0.003 μM TAK-733, and this result indicates that TAK-733 induces strong inhibition of p-MEK expression than trametinib. Similarly, TAK-733 also induced potent decreases in the levels of p-ERK, a member of the MEK/ERK signaling pathway. Moreover, a significant decrease in the expression of cyclin D1 was observed with a lower concentration of TAK-733, and this pattern was not seen with trametinib. This result was in line with the results from the cell cycle assays regarding the S phase blockade induced in TAK-733‒treated PNF cells (Figure 2e and g).
To address the mechanism of drug resistance after MEKi monotherapy and to further evaluate the potential synergistic effects in reducing tumor growth, we established drug-resistant PNF cell lines from parental ipNF05.5 and ipNF9511.bc cells by chronic treatment with low-concentration TAK-733. Drug resistance to TAK-733 was obtained, and both ipNF05.5-resistant and ipNF9511.bc-resistant cells exhibited significantly higher viability than parental cells according to Cell Counting Kit-8 assay (Figure 3a). The results of cell scratch migration showed a 10% increment in tumor cell migration ratio after the induction of resistance (Figure 3b). In the long-term colony formation assay, we observed that resistant PNF cells can grow and form more colonies under DMSO and low concentrations (0.1, 0.01 μM) of TAK-733 (Figure 3c). Similarly, as shown in the flow cytometry analysis, the ratios of apoptotic cells were significantly higher than those of control cells (Figure 3d and g). More resistance-induced PNF cells were observed in the S phase of the cell cycle after TAK-733 treatment (Figure 3e and h). As for the sphere-forming assay, the size of spheroids that formed by resistant PNF cells displayed no significant change under 1 μM TAK-733. However, the size of spheroids that formed by nonresistant PNF cells was significantly reduced by treatment with 1 μM TAK-733, relative to the size of resistant spheroids (Figure 3f and i). The mechanism of acquired drug resistance and possible treatment options were then further explored.
Quantitative high-throughput drug screening identified potential therapeutic agents for PNFs
Long-term monotherapy can induce drug resistance. To select the possible therapeutic targets for PNF, a panel of 5,558 compounds assembled from MedChemExpress Chemicals, Selleck Chemicals (Shanghai, China), CSNpharm Chemicals (Shanghai, China), and other chemical companies was used to screen in both TAK-733‒resistant (Figure 4a) and ‒nonresistant (Supplementary Figure S2) ipNF9511.bc cells. This panel included various inhibitors of well-explored oncogenic targets, such as cyclin-dependent kinases (CDKs), histone deacetylases, and sodium ion‒potassium ion ATPases. The pharmacological outcomes in the various tested drugs illustrated their drug sensitivity profiles, and the details are displayed in the Supplementary Materials. We further performed RNA sequencing (RNA-seq) on both ipNF05.5 and ipNF9511.bc cells before and after induction of TAK-733 resistance, and the overall differences in gene transcription levels were visualized with heatmaps to explore the mechanisms underlying PNF cell resistance to TAK-733 (Figure 4b). We also performed Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analysis of each parental and resistant cell line pair, and there were substantial overlap pathways across comparisons (Figure 4c and d). The analysis identified pronounced differences in cell cycle signaling between parental and resistant cell lines. That is, cell cycle gene sets were significantly upregulated in both resistant cell lines, and specifically, CDK1 gene activation was observed in both resistant cell lines (Figure 4e and g). The CDK inhibitor (CDKi) dinaciclib (the red triangle in Figure 4a), was thus selected as the compound with the most potential for combination therapy for PNF and was thus further analyzed.
The CDKi dinaciclib was identified as a promising agent in combination with TAK-733 on the basis of in vitro evaluation in PNF cell cultures
The western blot results confirmed the pivotal role of CDK1 in treating PNFs with TAK-733 resistance, with resistant tumor cells having higher CDK1 expression than parental cells (Figure 5a). The expression of survivin was also higher in these cells than in parental cells, suggesting that the resistant cells developed inhibited apoptosis after the induction of resistance (Figure 5a). This result is consistent with the previous drug screening results and RNA-seq analysis because CDK1 was one of the identified targets of dinaciclib. Dinaciclib may become a promising therapeutic agent for PNFs, especially when resistance occurs. In vitro studies proved that for resistant ipNF9511.bc cells, the combination therapy with dinaciclib resulted in a significant reduction in cell viability (Figure 5b) and spheroid formation (Figure 5c and d), an increase in cell apoptosis (Figure 5e and f), and G2/M phase blockade (Figure 5g and h). For resistant ipNF05.5 cells, combination therapy can significantly inhibit cell viability but not spheroid formation and apoptosis. It is noteworthy that higher concentrations of dinaciclib may cause substantial decreases in the viability of PNF cells (Figure 5b).
The CDKi dinaciclib in combination with TAK-733 demonstrated efficacy in treating PNF-PDX mice
To evaluate in vivo efficacy, we generated a patient‒derived xenograft model by grafting freshly extracted PNF tissue into the hypervascular armpit areas of NOD scid gamma mice; the procedure of the model establishment is displayed in Supplementary Figure S3. On confirmation of successful grafting, mice were first given trametinib (1 mg/kg), TAK-733 (30 mg/kg), or control medium daily by gavage for 4 weeks. After administration, TAK-733 significantly reduced the tumor weight by 50–60% compared with the control treatment. Trametinib also showed a trend for decreasing tumor weight, although it was not statistically significant (Figure 6a and b). Then, we evaluated the effectiveness of combination therapy with the CDKi dinaciclib (the combination group). After intraperitoneal injection of dinaciclib (20 mg/kg) three times a week over a 4-week treatment cycle, the tumors in the combination group were significantly reduced compared with those in the TAK-733 monotherapy group (Figure 6c and d). After administration, mice in the combination group had a significant reduction in their weight after the second-week treatment, compared with those in the TAK-733 monotherapy group (Figure 6e). We also evaluated the expression of specific markers in tumor sections to determine the pathological characteristics of PNFs before and after combination therapy. Both kinds of MEKis induced a robust decrease in p-ERK and Ki-67, suggesting that they were potent inhibitors of the growth of PNF cells (Figure 6f). The expression of p-ERK, Ki-67, c-Myc, and cyclin D1 decreased dramatically (Figure 6g). After identifying the different expression intensity of CDK1 (Figure 6h), we analyzed the expression and correlation between CDK1 and p-ERK with a tissue microarray (Figure 6i and j), and the result showed a moderate correlation (r = 0.4921, P < 0.001). H&E staining of main organs was performed and captured after the intervention of different chemical agents at the end of therapy. No obvious toxicity was observed to the main organs after a drug treatment of 1 month (Supplementary Figure S4). Information about the relationships between the expression of CDK1 and clinical characteristics is presented in Supplementary Table S3.
Although PNFs comprise various cell types, such as Schwann cells, fibroblasts, and mast cells, Nf1-deficient Schwann cell precursors have been reported to drive the formation of PNFs in mouse models (
). For this reason, medical agents used to treat PNFs must target Schwann cells. We compared the expression of several phenotypic biomarkers and confirmed the similarities and representativeness between the PNF cell line ipNF9511.bc and the PNF primary cells (Supplementary Figure S5). Currently, the most common models used to study PNF treatment are genetically engineered mouse models with Nf1 ablation driven by the Cre/lox system, and these models feature multiple histologically identical neurofibromas (
). Other models, including porcine models with Nf1 exon 41 or 42 deletion at the animal level and NF1−/− pluripotent stem cells at the cell level have the capacity to mimic many features in NF1-related processes (
). On the basis of clinical considerations, we developed PNF-PDX NOD scid gamma mouse models, which are an easy and repeatable tool for in vivo benign tumor studies, compared with the relatively long and complicated methods for generating conditional models. PNF-PDX mouse models also retain most of the original PNF characteristics of the clinical patients from whom the tumor tissue was derived, and in our mice, the expression of S100-β and other protein markers remained the similar expression for as long as 4 weeks (Supplementary Figure S6).
The mechanisms underlying the agents used in the various clinical trials related to PNFs are shown in Supplementary Figure S7. We also presented our findings in this figure. MEKis have been proven to be effective and tolerable in both preclinical assessments in NF1 mouse models and children with NF1 with inoperative, diffuse PNFs. However, there are potential drawbacks with continuous usage, such as gastrointestinal toxicities, elevation of creatinine kinase, and skin toxicities (
). Differences in response to these drugs are another challenge affecting clinical outcomes. On the basis of the data from this study, 66.67% of PNF tumor areas showed medium to high p-MEK expression, and this may correlate with the 70% positive response rate to the MEKi selumetinib (
). In addition, the variable expression levels of p-MEK/p-ERK in PNF tissues derived from different patients with NF1 and PNF cell lines in our results emphasize the heterogeneity present in these tumors, and such heterogeneity is another reason for the differences in MEKi efficacy. Moreover, drug resistance remains a problem. It is noteworthy that in the phase 2 trial of selumetinib, four patients were observed to have disease progression after confirmed partial response (patient identification: 1019010, 1019023, 2019002, and 2019003), indicating acquired resistance in these patients. The limited efficacy of monotherapies is due to the adaption and evolution of tumor cells, and small subpopulations of tumor cells can persist in the presence of drugs and eventually develop further mutations that allow them to grow and become the dominant populations (
Our research discussed the increased possibilities of resistance to MEKi in patients with NF1 with PNFs receiving long-term treatment. In both in vitro and in vivo studies, TAK-733 induced a stronger reduction in tumor growth and in markers related to cancer proliferation, such as Ki-67 and c-Myc, than trametinib. In this research, CDKi was proven to be a promising therapeutic agent for PNFs by both high-throughput drug screening and in vivo and in vitro experiments. Cancers display an uncontrolled cell cycle. CDKis have critical roles in regulating cell proliferation and transcription in tumor cells (
). Not long before this research, the CDK4/6 inhibitor palbociclib was determined to inhibit the growth of both malignant peripheral nerve sheath tumor cells and orthotopic xenografts, providing perspectives on CDKi in neurofibroma treatment (
We examined the transcriptional profiles of PNF cells by RNA-seq and confirmed that tumor resistance to MEKi monotherapy may arise owing to CDK1 hyperactivation. CDK1, as a key determinant of mitotic progression, is needed for cell proliferation, as proven by mouse-knockout models (
). The combination of both MEKi and CDKi significantly suppressed tumor growth in resistant cells and in vivo PNF-PDX models. This combination therapy showed strong inhibition of p-ERK expression, as evidenced by comparing the protein level before and after the induction of resistance cells and mouse models. The Ki-67 expression in the combination group was also observed lower than in the monotherapy group.
It is worth noting that other MEKis showed limited effectiveness in reducing cell viability. This could be because traditional two-dimensional culture methods lacked the interactions between tumor and supporting infrastructure that presented in vivo, and instead, three-dimensional cell models provided a better pathomimetic recapitulation of PNF biology for more precise medical studies (
). In addition, significant weight loss in mice and viability inhibition in cell culture were found in the combination group compared with those in the TAK-733 monotherapy group. A reduced dinaciclib dose may be better tolerated in mice and may be a more appropriate dose for improved preclinical/clinical treatment, reducing adverse drug reactions and obtaining the best outcomes of combination therapy.
Although contrasting results can be observed between preliminary drug studies and studies in model animals, which may be linked to tumor heterogeneity; the results mentioned earlier suggest that MEKi have good therapeutic effects in PNFs. We showed that drug resistance to the MEKi TAK-733 is likely caused by genetic differences leading to evasion of cell cycle arrest. In this study, we developed comprehensive preclinical models for testing MEKi monotherapy and MEKi/CDKi combination therapy: PNF tumor cell lines and PNF-PDX mouse models. By considering the mechanisms of resistance to MEKi in individual tumors, we identified this promising combination therapy to maximize tumor control. Our results provide a scientific basis for future clinical trials and the development of precision medicine to treat PNFs in patients with NF1.
Materials and Methods
The human NF1-deficient PNF cell lines, including ipNF05.5, 9511.bc, 95.6, Schwann cell lines Ipn02.3 2λ, and NF1-deficient malignant peripheral nerve sheath tumor cell line S462, were purchased from the ATCC.
Human PNFs samples and xenograft study
Generally, patient‒derived xenograft models were established by transplanting a fresh PNFs tumor biopsy (cut into 3 × 3 × 3 mm tumor mass) from a patient with NF1 into the armpit of immunocompromised humanized NOD scid gamma mice aged 4 weeks under general anesthesia. Mice were divided into different treatment groups randomly (n = 4 per group) and were assigned to receive mono/combined drug therapies or vehicle by oral gavage or intraperitoneal injection. One week after operation, humanized NOD scid gamma mice were treated daily with trametinib (1 mg/kg), TAK-733 (30 mg/kg), or the drug diluted vehicle (10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline) by oral gavage. As for the combination therapy group, dinaciclib (20 mg/kg) was injected intraperitoneally three times a week. After 4 weeks of different treatment, mice were killed. Tumor xenografts were isolated and their weights were measured and photographed. Tissues were fixed overnight in 4% paraformaldehyde for further studies. All studies related to human or mice were designed in accordance with the Declaration of Helsinki and were approved by the Human Research Ethics Committee and Animal Care and Use Committee of Shanghai Ninth People’s Hospital Shanghai Jiao Tong University School of Medicine (Shanghai, China). Written informed consent for the use of the tissues for research was obtained from patients at the time of procurement of tumor specimens.
A library of 5,558 compounds was assembled from MedChemExpress Chemicals, Selleck Chemicals, CSNpharm Chemicals, and other chemical companies. Drug screening was performed as previously reported (
) and was used to culture the PNF cell line, ipNF9511.bc, at a density of 2,000 cells/well into 17 × 384-well plates and diluted at a final concentration of 200 μM. After 3 days of incubation, Celltiter Glo (Promega, Madison, WI) was added to examine the effect of inhibitors to evaluate cell viability. Agent selections for further combination strategies were determined by analyzing individual compound efficacy.
RNA isolation and RNA-seq
RNA isolation was performed using the Qiagen RNeasy Plus Mini kit (Qiagen, Hilden, Germany). RNA-seq libraries of poly (A) RNA from 500 ng total RNA were obtained from PNFs cell samples and were generated using the cBot Cluster Generation System of TruSeq PE Cluster Kit v3-cBot-HS for Illumina multiplexed sequencing (Illumina, San Diego, CA). Sequencing was performed from Illumina on a Novaseq 6000 with a paired-end module (Illumina). All the clustering and sequencing procedures were performed by Novogene Experimental Department. RNA-seq raw data of our samples were deposited to Gene Expression Omnibus (identification: GSE173577).
All the data are presented as mean ± SEM. All the graphs were plotted and analyzed with GraphPad Prism 8 Software (GraphPad Software, San Diego, CA) using Student’s t-test. P < 0.05 was considered statistically significant.
We thank Qing-Feng Li for help in providing surgical tissues and clinical data and in supervising this manuscript. This work was supported by grants from Youth Doctor Collaborative Innovation Team Project (QC201803) of Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine; the Shanghai Youth Top-Notch Talent Program (201809004); Chenguang Program supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission ( 19CG18 ); Science and Technology Commission of Shanghai Municipality ( 19JC1413 ); Shanghai Rising Star Program ( 20QA1405600 ); Innovative research team of high-level local universities in Shanghai ( SSMU-ZDCX20180700 ); and Shanghai Municipal Key Clinical Specialty ( shslczdzk00901 ). For additional correspondence queries, please contact Zhi-Chao Wang ( [email protected] ).
All cells were cultured in DMEM (Gibco, Waltham, MA), supplemented with 10% fetal bovine serum (Gibco), glutamine, and ×1 penicillin/streptomycin (Gibco) at 37 °C in a double-distilled water humidified atmosphere with 5% carbon dioxide. To establish a TAK-733‒resistant plexiform neurofibromas (PNFs) cell subline, ipNF05.5 and 9511.bc cells were incubated in a one-sixth half-maximal inhibitory concentration of TAK-733 for 2 weeks at first. After cells stably growing, the drug concentration was elevated gradually over 6 months to a final dose of five times the previous half-maximal inhibitory concentration and withdrawn for two passages before functional studies. Selumetinib, trametinib, TAK-733, cobimetinib, refametinib, and PD0325901 were all purchased from MedChemExpress Chemicals (Monmouth Junction, NJ). Antibodies against CDK1 (ab133327), survivin (ab76424), and S100-β (ab52642) were purchased from Abcam (Cambridge, United Kingdom). Antibodies against GAPDH (#5174), MAPK/extracellular signal–regulated kinase (ERK) kinase (#4694), phosphorylated MAPK/ERK kinase (#9154), ERK (#4696), phosphorylated ERK (#4370), protein kinase B (#4691), phosphorylated protein kinase B (#4060), caspase-3 (#9662), cleaved caspase-3 (#9661), cyclin D1 (#55506), Ki-67 (#9449), and c-Myc (#18583) were purchased from Cell Signaling Technology (Danvers, MA).
Immunohistochemistry, histological analysis, and immunocytochemistry
A total of 141 tumor samples diagnosed as PNFs were collected and constructed as tissue microarray with two cores and 1 mm in diameter. Sections were cut into 4-μm paraffin-embedded sections and stained by either H&E or immunohistochemistry. For immunohistochemistry, after deparaffinization and dehydration, the pretreated slides were incubated in ×1 EDTA buffer (Sangon Biotech, Shanghai, China) and treated in a microwave oven at 95 °C for 25 minutes. The staining procedures strictly followed the instructions provided by UltraSensitive SP IHC Kit (KIT-9730) purchased from Maxim Biomedical (Fuzhou, China). For each tissue sample, the percentage of phosphorylated MAPK/ERK kinase, phosphorylated ERK, and CDK1-positive tumor cells was estimated depending on the staining intensity and on distribution separately by two pathologists, and the score was recorded semiquantitatively as 0, 1+, 2+ or 3+. For statistical analysis, the staining results were added ranging from 0 to 6 accordingly. For immunocytochemistry, cells were cultured on sterile glass coverslips, and the assay was performed as previously described (
Protein lysis was extracted from PNFs tumors or cell lines by RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with complete protease/phosphatase inhibitor cocktails (MedChemExpress). After being separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane, samples were blocked with 5% nonfat milk for 1 hour at room temperature and incubated with different primary antibodies, diluted according to the manufacturer’s instructions, at 4 °C overnight. Followed by incubation with horseradish peroxidase‒conjugated secondary antibodies for 1 hour, the immunoreactive bands were visualized by ImageQuant LAS 4000 Imager after using enhanced chemiluminescence‒immunoprecipitation buffer.
Cell viability assay
A total of 2,500 cells were seeded in each well of 96-well plates in the presence or absence of various concentrations of MAPK/ERK kinase 1/2 inhibitor. Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan) was added (1:10) to each well of different treatment after 72 hours of treatment and further incubated for 1.5 hours. Fluorescence was measured using a multiwell plate reader.
Generation of tumor spheroids and colony formation assay
For sphere assay, cells were seeded at a density of 500 cells in each well in 100 μl complete DMEM media in round bottom ultralow attachment 96-well plates (Corning, Corning, NY). One day after, different concentrations of drugs were added and incubated for an additional 5 days. As for colony assay, 800 cells were seeded in the presence or absence of inhibitors in each well of six-well plate. Growth medium was changed every 3 days. Colonies were formed within 10 days, stained by crystal violet (Beyotime Biotechnology), and imaged for calculating colony amount when cell counts were >50.
Apoptosis assay and cell cycle assay
A total of 1 × 106 cells were seeded in each well of six-well plates in the presence or absence of different concentrations of inhibitors. Apoptosis ratio was analyzed using the Annexin V and PI Kit (BD Biosciences, Franklin Lakes, NJ), and cell cycles were stained with RNA stain (Thermo Fisher Scientific, Waltham, MA). Results were tested by flow cytometry after seeding for 24 hours.
Four percent paraformaldehyde-fixed PNF cells on glass slides were washed with PBS and permeabilized by 0.5% Triton X-100 (Beyotime Biotechnology) for 10 minutes, followed by incubation with 100 μl of TUNEL reaction mixture according to the instructions from Yeasen Biotechnology (Shanghai, China). Nuclei were then stained with DAPI (Abcam) and observed under a fluorescence microscope.
Supplementary Table S1Clinical Parameters with p-MEK Expression