Tumor Promoting Function of DUSP10 in Non-Small Cell Lung Cancer Is Associated With Tumor-Promoting Cytokines

Clinical specimens and survival analysis

A retrospective analysis of 195 cases from the First Affiliated Hospital of Zhejiang Chinese Medical University and the Sichuan Provincial People’s Hospital with NSCLC who were histologically diagnosed and treated with standard regiments from January 2014 to January 2022 in hospital were included. The tumor specimens consist of 113 adenocarcinoma, 57 squamous cell carcinoma, 20 adenosquamous carcinoma and 5 large cell lung carcinoma. Informed consent was obtained from patients before standard treatment for specimens to be used for this research. The study was approved by Institutional Review Board of the First Affiliated Hospital of Zhejiang Chines Medical University (2023-KL-031-02). All samples were re-evaluated by experienced pathologist according to the World Health Organization grading system and the General Rules for Clinical Lung Cancer.

Formalin-fixed, paraffin-embedded tissue sections of 4-μm thick obtained from 182 primary lung tumor tissues were immunostained using the Gene Tech EnVision™ Detection Kit (GK500710, Gene tech, Shanghai, China). After deparaffinization and dehydration through graded alcohols and xylene, endogenous peroxidase activity and non-specific Ag were blocked with 3% hydrogen peroxide. The sections were heated for 5 min twice at 100°C with Tris EDTA (pH 9.0) in a microwave oven for Ag retrieval. All sections were incubated with anti-DUSP10 Ab (Abcam #ab228987) at 37°C for 1 h. After washing, the sections were incubated with the ChemMate™ Envision™/HRP, Rabbit (ENV) reagent at 37°C for 30 min. Rinsed gently with PBS, the sections were visualized by the ChemMate TM DAB+ Chromogen, counterstained with hematoxylin, mounted in neutral gum, and analyzed using a bright field microscope.

The immunohistochemically stained tissue sections were reviewed and scored by two independent experienced pathologists blinded to the clinicopathological variables. For each section, 5 high power field were random selected and the staining was evaluated by the extent (the percentage of positive tumor cells or normal epithelial cells in relation to the whole tissue area: negative, 0; ≤10%,1; 10%–50%,2; ≥51%, 3) and the intensity (absent 0; weak 1; moderate 2; strong 3). The combined score (extensity×intensity) of 0–4 and 5–9 were defined as low and high expression levels of DUSP10 respectively.

Descriptive statistics comparing DUSP10 expression with the clinicopathological characteristics were analyzed by the chi-square test. Survival curves were calculated by the Kaplan–Meier method, and the differences were assessed by the log-rank test. Statistical analysis software (IBM SPSS Statistic 19.0; IBM Corp., Armonk, NY, USA) was used to perform the analyses, and a p-value of less than 0.05 (p<0.05) was considered statistically significant.

Animal experiment

Animal experiments were approved by the Institutional Animal Care and Use Committee of National University of Singapore. NSGS mice were obtained from Jackson Laboratory and were inbreed for all xenograft experiments in this study. For establishment of NSCLC cell line xenograft model, 5×10⁶ of H460 and HCC827 cells stably overexpressing DUSP10 were subcutaneously injected into the flanks of the animals. Tumor xenografts were allowed to develop over 3 wk before harvest.

Cell culture

The human NSCLC cell lines, H460 and HCC827 cells (American Type Culture Collection, Manassas, VA, USA), were maintained in complete Roswell Park Memorial Institute media, RPMI (Biowest, Nuaillé, France) containing 10% FBS and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), at 37°C with 5% CO₂. Cell harvest was done by incubating cells with 0.25% Trypsin-EDTA (Gibco, Grand Island, NY, USA) at 37°C for 5 min, till the adherent cells are detached from the surface. Detached cells were subsequently pelleted by centrifugation at 400 g for 3 min. Total live Quantification of live cell count was carried out using trypan blue (HyClone, Cramlington, UK) staining.

Generation of H460 and HCC827 stably overexpressing DUSP10 cell lines

Empty pcDNA plasmid vector (control) or pcDNA plasmid containing the full-length of human DUSP10 cDNA were transfected into both H460 and HCC827 cells using Lipofectamine® LTX and Plus™ Reagent (Invitrogen) according to the manufacturer’s instructions.

H460 and HCC827 cells that were positive for either the control vector or vector containing DUSP10 were identified based on geneticin (G418) (1.5 mg/ml) selection. The successfully transfected clones were selected and screened for DUSP10 overexpression by using quantitative real-time PCR (qPCR) to quantify DUSP10 mRNA level. Clones that stably overexpressed DUSP10 were maintained in complete RPMI medium with 1.5 mg/ml geneticin (G418).

Cell proliferation assay

H460- and HCC827-pcDNA control and H460- and HCC827-DUSP10 overexpression cells were seeded at a density of 0.08×10⁶ cells/well in 6-well plates and incubated overnight at 37°C with 5% CO₂. Subsequently, cells were incubated in serum free RPMI for 20 h at the same condition. After serum starvation, serum free RPMI was replaced with a complete RPMI for assessment of basal proliferation over a period of 5 days. At each time point, RPMI was removed and 500 μl crystal violet stain (0.5% crystal violet in 20% methanol) was added into the wells and was shaken for 5 min. Crytal violet stain was then removed, cells were washed with RO water and the plates were left to air-dry for 15 min. After drying, the stain was solubilized in 500 μl solubilisation buffer (0.1M sodium citrate in 50% absolute ethanol). Analysis of relative proliferation rate was done by measuring absorbance at 545nm using BioTek Synergy™ H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA).

Spheroid formation assay

H460- and HCC827-pcDNA control and H460- and HCC827-DUSP10 overexpression cells were seeded at a density of 10,000 cells/well in 0.35% (w/v) sterile low melting agarose, in 48-well plates, which were pre-coated with 0.5% (w/v) sterile low melting agarose. Complete RPMI medium was added to each well immediately after the agar were solidified. Fresh culture medium was replaced every three days during culture. Spheroids were left to develop over 2 wk before microscopic assessment to determine spheroid sizes. Olympus IX80 inverted microscope (Olympus, Tokyo, Japan) was used to capture images of the spheroids, which were measured using ImageJ software.

Wound healing assay

H460- and HCC827-pcDNA control and H460- and HCC827-DUSP10 cells were harvested and the cell suspensions were adjusted to 0.8×10⁶ cells/ml. A volume of 70 µL cell suspension was seeded into Ibidi culture insert (Ibidi, Fitchburg, WI, USA) forming a monolayer of cells. On the day of experiment, culture insert was removed before cells were treated with 10 μM of mitomycin-c (Sigma, St. Louis, MO, USA) for 2 h before imaging to determine the “wound” size at Time 0. Imaging was done after 24 h to determine percentage of “wound” closure for quantification of cell migration using Image J software (NIH, Bethesda, MD, USA).

Quantitative real-time polymerase chain reaction

Total RNA was extracted from cultured cells using TRizol (Invitrogen) and was used for cDNA synthesis using ImProm-II™ Reverse Transcription System (Promega, Madison, WI, USA). qPCR was performed with an Applied system 7900 Detection System using Fast SYBR Master Mix (Applied Biosystems, B.V., Singapore).

Western blot

Whole cell lysates were separated by 10% SDS-PAGE, and Western blotting was performed using antibodies against total and MAP kinases (Cell Signaling, Danvers, MA, USA), EGFR, c-Myc, COX2 (Santa Cruz, Santa cruz, CA, USA), and actin (Cell signaling). Immunoblots were developed with ECL donkey anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Chicago, IL, USA) and SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA). The blots were exposed to Amersham® Hyperfilm® ECL™ and MP Autoradiography Films (GE Healthcare).

Statistical analysis

Data were expressed as the mean±SE. Statistical analysis was performed using two tailed Student’s unpaired t-test. A p-value <0.05 was considered to be statistically significant.

Overexpression of DUSP10 in lung cancer cells resulted in increased cell growth and migration

To understand the function of DUSP10 in lung cancer, particularly NSCLC, we generated DUSP10 overexpression HCC827 cells. Six clones were selected from vector- and DUSP10-overexpression cells respectively to examine DUSP10 expression using qPCR. The results showed increased expression of DUSP10 in the DUSP10-overexpression clones compared to that in vector-expression ones (Fig. 1A), showing the successful generation of DUSP10-overexpression HCC827 cells. One vector clone and two DUSP10-overexpression clones (clone 4 and 5) were selected to examine the effect of DUSP10 in cell growth using MTT assay. It was found that the two DUSP10-overexpression clones had significantly increased proliferation compared to that of vector clone (Fig. 1B). Next, to confirm the effect of DUSP10 overexpression on HCC827 growth, we examined the expression of DUSP10 protein in the vector clone and the HCC827 DUSP10-oveexpression clone 5 using western blot analysis. It was found that DUSP10 protein was not expressed in the vector-expression cells and obvious DUSP10 protein was detected in the overexpression clone, confirming the successful generation of the DUSP10-overexpression cells (Fig. 1C). Next, vector- and DUSP10-overexpression cells were cultured in the absence or presence of epidermal growth factor (EGF) over a period of three days followed by examination of cell growth by crystal violet staining. The results showed that DUSP10-overexpression cells had significantly increased growth compared to vector cells with or without EGF (Fig. 1D). Together, these results suggested that DUSP10 may promote cell growth in lung cancer.

Figure 1
Establishment of DUSP10-overexpression HCC827 stable cell clones. (A) NSCLC cell line HCC827 was transfected with pcDNA3 vector or pcDNA3 containing the full-length of human DUSP10 cDNA followed by drug selection for stably transfected clones. Six clones each from pcDNA3- and pcDNA3-DUSP10 transfected cells were selected for examination of DUSP10 expression by qPCR. (B) One vector clone and two DUSP10-overexpression clones (clone #4 & 5) were selected to examine the impact of DUSP10 on cell proliferation by MTT assay. (C) Western blot analysis of DUSP10 protein expression in vector and DUSP10-overexpression HCC827 cells (clone #5). (D) Vector- and DUSP10-overexpression HCC827 cells were cultured with or without hEGF (100 ng/ml) for various days indicated to assess cell proliferation by crystal violet assay. Data are representative of three independent experiments with similar results.
^*p<0.05, ^**p<0.01, ^***p<0.001.

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To further exam the role of DUSP10 in lung cancer cells, we performed spheroid formation and wound healing assays. The results showed that overexpression of DUSP10 resulted increased sizes of spheroids (Fig. 2A). In addition, DUSP10-overexpression cells had greatly increased wound healing ability compared to that of vector cells (Fig. 2B), suggesting that overexpression of DUSP10 resulted in increased migration of lung cancer cells.

Figure 2
Overexpression of DUSP10 in NSCLC cells resulted in increased spheroid formation and wound healing. (A) Representative micrographs of multicellular spheroid aggregates, derived from vector- and DUSP10-overexpression HCC827 cells. Bar chart shows mean of spheroid size (n=30). (B) Representative micrographs showing wound closure in vector- and DUSP10-overexpression cells. Bar chart shows mean of percentage of wound closure (n=12). Results representative of three independent experiments.
^***p<0.001.

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To verify the function of DUSP10 in regulation of NSCLC cell growth and migration, we generated vector- and DUSP10-overexpression H460 cells (Supplementary Fig. 1A). The growth of vector- and DUSP10-overexpression cells with or without EGF over a period of 3 days was examined by crystal violet staining assay. In consistent with the role of DUSP10 in HCC827 cells, DUSP10-oveexpression H460 cells had increased growth compared to vector transfected cells with or without EGF treatment (Supplementary Fig. 1B). Next, spheroid formation and would healing assays were carried out. The results showed that overexpression of DUSP10 in H460 cells resulted in increased spheroid formation and wound healing (Supplementary Fig. 2A and B), which is consistent with the function of this molecule in HCC827 cells (Fig. 2). Together, these results demonstrated that DUSP10 promotes cell growth and migration in lung cancer.

DUSP10 regulates the activation of MAPKs and the expression of growth-related molecules

To understand the mechanism underlying the regulation of DUSP10 in lung cancer cells, we first examined the phosphorylation of ERK, JNK and p38 in both vector- and DUSP10-overexpression HCC827 cells with or without EGF stimulation. It was found that ERK was constitutively phosphorylated in vector-cells and its phosphorylation was slightly increased at 0.5 h, followed by reduction at 3 and 6 h upon EGF treatment (Fig. 3A and Supplementary Fig. 3). Slightly reduced phosphorylation of ERK was observed in DUSP10-overexpression cells without EGF stimulation. Upon EGF stimulation, ERK phosphorylation was greatly reduced at 0.5, 3 and 6 h in DUSP10-overexpression cells compared to that in vector-cells. JNK was not phosphorylated in both vector- and DUSP10-overexpression cells without stimulation (Fig. 3A and Supplementary Fig. 3). EGF stimulation induced JNK phosphorylation in both vector- and DUSP10-overexression cells at 0.5 h, but the level of phosphorylation was much lower in DUSP10-overexpression cells compared to that in vector cells. Phosphorylation of JNK was weaned off in both vector- and DUSP10-overexpression cells at 3 and 6 h. Interestingly, phosphorylation of p38 was found to be increased in DUSP10-overexpression cells compared to that in vector cells (Fig. 3A and Supplementary Fig. 3). Weakly phosphorylated p38 was detected in vector cells without EGF stimulation, increased phosphorylation was observed at 0.5 h upon EGF stimulation and the level of phosphorylation was further increased at 3 h followed by a reduction at 6 h upon EGF stimulation. In contrast, higher level of p38 phosphorylation was detected in DUSP10-overexpression cells compared to that in vector-cells without EGF stimulation. EGF stimulation resulted in a similar increase in p38 phosphorylation as that in vector cells, but the levels of phosphorylation were higher than that in vector-cells at each time point upon EGF stimulation. Together, these results suggest that DUSP10 negatively regulates ERK and JNK phosphorylation, but positively regulates p38 phosphorylation.

Figure 3
DUSP10 increases p38 activation and inhibits the activation of ERK and JNK as well as ERK-regulated growth promoting molecules. Vector- and DUSP10-overexpression HCC827 cells were stimulated with 100 ng/ml of EGF. Cells were harvested at the indicated time points to prepare protein lysates. (A) The levels of pERK, pJNK and pP38 as well as tERK, tJNK and tP38 were determined by western blot analysis. The expression of c-Myc, COX2 and actin was analyzed. (B) The proliferation of vector- and DUSP10-overexpression HCC827 cells with or without p38-specific inhibitor SB203580 was determined by MTT or crystal violet assays. (C) Representative micrographs showing wound closure in vector- and DUSP10-overexpression cells with or without SB203580 treatment. Bar chart shows mean of percentage of wound closure (n=12). Results representative of three independent experiments.
pERK, phospho-ERK; tERK, total-ERK.

^***p<0.001.

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Next, we examined the expression of cellular growth-related molecules including c-Myc and COX2. In consistent with reduced phosphorylation of ERK, the expression of both c-Myc and COX2 which are targets of ERK was reduced in DUSP10-overexpression cells compared to vector cells (Fig. 3A and Supplementary Fig. 3). These results demonstrated that DUSP10 inhibits ERK and ERK- regulated molecules which is consistent with our previous findings (11). However, the increased cell growth and migration in DUSP10-overexpression cells suggested that this molecule regulates lung cancer though other mechanisms, possibly through p38.

To test if p38 contributes to the increased growth and migration of lung cancer cells, we treated vector- and DUSP10-overexpression cells with p38 inhibitor SB203580 followed by examination of cell growth and migration. Western blot analysis showed the successful inhibition of p38 activation by the inhibitor (Supplementary Fig. 3B). Without p38 inhibition, DUSP10-overexpression HC827 cells had increased cell growth determined by both MTT and crystal violet assays (Fig. 3B), which was consistent with previous findings (Fig. 1). Upon inhibition, both the vector- and DUSP10-overexpression cells had reduced proliferation and the levels of proliferation became comparable between them. Similar results were obtained in vector- and DUSP10-overexpression H460 cells where overexpression increased cell growth and migration and p38 inhibition abrogated the differences in growth and migration between vector- and DUSP10-overexpression cells (Supplementary Fig. 4A and B). These results suggest that p38 plays an important role in NSCLC growth and development.

DUSP10 promotes lung cancer cell growth in vivo

To further understand the function of DUSP10 in lung cancer, vector- and DUSP10-overexpression cells were inoculated into NSG mice to access their growth. As shown in Fig. 4, DUSP10-overexpression cells formed significantly large tumors compared to these formed by vector-cells (Fig. 4A), demonstrating that DUSP10 promotes lung cancer cell growth in vivo.

Figure 4
DUSP10 promotes NSCLC growth in vivo. Vector- and DUSP10-overexpression HCC827 cells (A), or vector- and DUSP10-overexpression H460 cells (B) were inoculated into NSGS mice (n=5 for each cell type) respectively. After three weeks of growth, mice were sacrificed to assess tumor formation. Representative photographs of vector- and DUSP10-overexpression xenograft tumours and bar chart show the weights of the xenograft tumours. (C, D). Immunohistochemical analysis for p38 activation of vector- and DUSP10-overexpression HCC827 (C) and H460 (D) tumor sections. Representative images of phosphor-p38 and quantification of phosphor-p38 staining were shown (n=5). Scale bar = 100 µM. Results representative of two independent experiments with similar results.
^*p<0.05, ^**p<0.01, ^***p<0.001.

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To confirm that the tumor promoting function of DUSP10 in NSCLC in vivo, we inoculated vector- and DUSP10-overexpression H46 cells inoculated into NSG mice. The results showed that larger tumors were formed by DUSP10-oeverexpression H460 cells compared to those formed by vector cells (Fig. 4B), which was consistent with the results from vector- and DUSP10-overexpression HCC827 cells (Fig. 4A). These results further support the oncogenic function of DUSP10 in NSCLC.

Next, we performed immunohistochemical staining to evaluate the activation of p38 and ERK. Consistent with western blot results (Fig. 3A), increased p38 staining was observed in DUSP10-overexpression xenografts compared to that in vector-transfected xenografts (Fig. 4C and D), whereas ERK staining was found to be reduced in xenografts with DUSP10-overexpression compared to that in vector-transfected ones (Supplementary Fig. 5). These results suggested that the increased growth of DUSP10-overexpression NSCLC was associated with increased p38 activation in vivo.

Higher DUSP10 expression in lung cancer patients is associated with poorer prognosis

Next, we examined the protein expression of DUSP10 in tumor tissues from 195 NSCLC cancer patients using immunohistochemistry staining. Based the signal, the samples were assigned a score from 0 to 9. Score 0 to 4 indicate low DUSP10 protein expression and sore 5–9 indicate high DUSP10 expression (Fig. 5A). Kaplan-Meier analysis was carried out to compare the survival of patients between DUSP10 low and DUSP10 high groups. It was found that patients with high DUSP10 protein expression had significant lower survival compared with patients with low DUSP10 (Fig. 5B). This result demonstrates that DUSP10 promotes lung cancer development and influences the clinical outcome of NSCLC.

Figure 5
Higher DUSP10 levels in tumors were associated with poorer prognosis of NSCLC patients. (A) Formalin-fixed, paraffin-embedded tissue sections from NSCLC patients were immunostained with Ab against human DUSP10. DUSP10 signal was viewed and scored from 0 to 3, with no DUSP10 staining as score 0, weak staining as 1, moderate staining as 2 and strong staining as 3. Representative photographs of score 0–3 of DUSP10 staining were shown. (B) Kaplan-Meier survival curve and log-rank (Mantel-Cox) test showing the survival of 195 NSCLC patients stratified by low or high DUSP10 expression in tumours cells.

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Increased expression of IL-6 and TGFβ in DUSP10-overexpression lung cancer cells

Lung cancer cells are known to produce various cytokines which regulate tumor growth (13). In addition, p38 is known to be an important regulator of various cytokines. We therefore examine the regulation of cytokines by DUSP10 in lung cancer cells. Analysis of gene expression by qPCR confirmed that the expression of DUSP10 was significantly higher in DUSP10-overexpression HCC827 cells compared to that in vector cells (Fig. 6A). In addition, reduced expression of TNFα was observed in DUSP10-overexpression cells compared to that in vector cells, which is consistent with previous findings on the role of DUSP10 in the expression of these cytokines (14). Interestingly, the expression of TGFβ and IL-6 was found to be increased in DUSP10-overexpression cells compared to that in vector cells (Fig. 6A and B). To validate the role of p38 in regulation of these cytokines in NSCLC, SB203580 was used to treat vector- and DUSP10 overexpression cells followed by examination of TGFβ and IL-6 expression. The results confirmed the increased expression of these two cytokines in DUSP10-overexpression cells without p38 inhibition (Fig. 6C). Upon p38 inhibition, the expression of both cytokines was reduced in vector- and DUSP10-overexpression cells and the level of their expression become comparable between vector- and DUSP10-overexpression cells. These cytokines have been shown to play important roles in NSCLC progress (15, 16, 17, 18). Therefore, the tumor promoting function of DUSP10 in NCSLC is associated with increased p38 activation and the expression of p38-regulated tumor-promoting cytokines.

Figure 6
DUSP10 regulates tumor-promoting cytokines in NSCLC cells likely through p38. (A). Vector- and DUSP10-overexpression HCC827 cells were stimulated with 100 ng/ml EGF for 24 h before harvest to extract total RNA to prepare cDNA. The expression of the indicated cytokines was determined by qPCR. (B). Protein expression of TGFβ and IL-6 were assessed by ELISA. (C). Vector- and DUSP10-overexpression HCC827 cells were stimulated with 100 ng/ml EGF with or without SB20380 for 24 h before analysis of TGFβ and IL-6 by qPCR. Results representative of three independent experiments.
^*p<0.05, ^**p<0.01.

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