FOX-A1 contributes to acquisition of chemoresistance in human lung adenocarcinoma via transactivation of SOX5

Abstract

Background: Chemoresistance is a major obstacle for the effective treatment of lung adenocarcinoma (LAD). Forkhead box (FOX) proteins have been demonstrated to play critical roles in promoting epithelial-mesenchymal transition (EMT) and chemoresistance. However, whether FOX proteins contribute to the acquisition of EMT and chemoresistance in LAD remains largely unknown.

Methods: FOX-A1 expression was measured in LAD cells and tissues by qRT-PCR. The expression levels of EMT markers were detected by western blotting and immunofluorescence assay. The interaction between Sex determining region Y-box protein 5 (SOX5) and FOX-A1 was validated by chromatin immunoprecipitation sequence (ChIP-seq) and Chromatin immunoprecipitation (ChIP) assay. Kaplan-Meier analysis and multivariate Cox regression analysis were performed to analyze the significance of FOX-A1 and SOX5 expression in the prognosis of LAD patients.

Findings: FOX-A1 was upregulated in docetaxel-resistant LAD cells. High FOX-A1 expression was closely associated with a worse prognosis. Upregulation of FOX-A1 in LAD samples indicated short progression-free survival (PFS) and overall survival (OS). SOX5 is a new and direct target of FOX-A1 and was positively regulated by FOX-A1 in LAD cell lines. Knockdown of FOX-A1 or SOX5 reversed the chemoresistance of docetaxel-resistant LAD cells by suppressing cell proliferation, migration and EMT progress.

Interpretation: These data elucidated an original FOX-A1/SOX5 pathway that represents a promising therapeutic target for chemosensitizing LAD and provides predictive biomarkers for evaluating the efficacy of chemotherapies.

Keywords: Chemoresistance; FOX-A1; Lung adenocarcinoma; SOX5.

Fig. 1

FOX-A1 is upregulated in docetaxel-resistant LAD cells and insensitive LAD tissues and correlated with a poor prognosis of LAD patients. a, Relative expression of Forkhead box (Fox) genes in parental (SPC-A1 and H1299) and docetaxel-resistant LAD cells (SPC-A1/DTX and H1299/DTX) was determined by quantitative real-time PCR (qRT-PCR). GAPDH was used as an internal control. ^⁎⁎P < 0.01. Data are representative of at least three independent experiments (means±standard deviation). b, Western blotting was used to detect protein expression of FOX-A1 in parental and docetaxel-resistant LAD cells. GAPDH was used as an internal control. ^⁎⁎P < 0.01. Data are representative of at least three independent experiments (means±standard deviation). c, FOX-A1 gene expression was determined by qRT-PCR in parental LAD cells treated with different concentrations of DTX for 48 h. U6 was used as an internal control. ^⁎P < 0.05, ^⁎⁎P < 0.01. Data are representative of at least three independent experiments (means±standard deviation). d, qRT-PCR detection of FOX-A1 gene expression in parental LAD cells treated with DTX at different time points. U6 was used as an internal control. ^⁎P < 0.05, ^⁎⁎P < 0.01. Data are representative of at least three independent experiments (means±standard deviation). e, FOX-A1 expression detected by qRT-PCR in insensitive LAD tissues (CR + PR; n = 24) and sensitive LAD tissues (SD + PD; n = 28). GAPDH was used as an internal control. The cut-off value (0.959) of FOX-A1 mRNA expression in tumor tissues was determined by a receiver operating characteristic (ROC) curve.^⁎⁎P < 0.01. f, FOX-A1 expression levels in normal lung tissues (n = 18) and LAD tissues (n = 52). FOX-A1 expression was detected by qRT-PCR. GAPDH was used as an internal control. ^⁎⁎P < 0.01. g, Kaplan-Meier analysis of the correlation between FOX-A1 expression and progression-free survival (PFS) of LAD patients. ^⁎⁎P < 0.01. h, Kaplan-Meier analysis of the association between FOX-A1 expression and overall survival (OS) of LAD patients.^⁎⁎P < 0.01.

Fig. 2

FOX-A1 contributes to docetaxel resistance of LAD cells in vitro. a, Western blotting detection of FOX-A1 in docetaxel-resistant LAD cells transfected with sh-FOX-A1#1, sh-FOX-A1#2, sh-FOX-A1#3 or control. β-actin was used as a control. b, A CCK-8 assay was conducted to detect the IC₅₀ values of DTX in sh-control (or sh-FOX-A1#1)-transfected docetaxel-resistant LAD cells. ^⁎P < 0.05. c, A colony formation assay was performed to detect the proliferation ability of sh-control (or sh-FOX-A1#1)-transfected docetaxel-resistant LAD cells treated without (or with) DTX (50 μg/L). ^⁎P < 0.05, ^⁎⁎P < 0.01. d, Flow cytometric analysis of the early apoptosis rate of sh-control (or sh-FOX-A1#1)-transfected docetaxel-resistant LAD cells treated without (or with) DTX (50 μg/L). ^⁎⁎P < 0.01. e, Western blotting was used to detect cleaved caspase-3 (C-caspase-3) and caspase-3 in sh-control (or sh-FOX-A1#1)-transfected docetaxel-resistant LAD cells. β-actin was used as an internal control. Data represent the average of three independent experiments (means±standard deviation).

Fig. 3

FOX-A1 promotes migration, invasion and EMT of docetaxel-resistant LAD cells. a, A transwell assay was conducted to detect the migration and invasion of docetaxel-resistant LAD cells treated with sh-control or sh-FOX-A1#1 vectors. Migrated and invaded cells were calculated in five random fields of view at 100× magnification. A histogram displays the average number of migrated and invaded cells per field of view. Scar bar: 50 μm. ^⁎⁎P < 0.01. Data are presented as means±standard deviation. b, Western blotting was performed to detect the protein expression of an epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin and Vimentin) in docetaxel-resistant LAD cells treated with sh-control or sh-FOX-A1#1 vectors. GAPDH was used as an internal control. ^⁎⁎P < 0.01. Data are presented as means±standard deviation. c, Immunofluorescence staining was used to detect the expression of an epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin and Vimentin) in docetaxel-resistant LAD cells (SPC-A1/DTX) treated with sh-control or sh-FOX-A1#1 vectors. Scar bar: 50 μm.

Fig. 4

Suppression of FOX-A1 enhances the in vivo chemosensitivity of docetaxel-resistant LAD cells to DTX. a, Tumor growth was assessed by the tumor volume in nude mice that were subcutaneously transplanted with sh-control (or sh-FOX-A1#1)-transfected SPC-A1/DTX cells combined with DTX treatment. The data are displayed as means±standard deviation. Representative photographs of tumors are provided at 30 days after inoculation. b-c, Tumor volume and weight of xenograft tumors at the end of the treatment period. ^⁎⁎P < 0.01. d, H&E staining, immunohistochemical staining of FOX-A1, proliferating cell nuclear antigen (PCNA) and Ki67 staining and TUNEL staining were performed using tumors collected at 30 days after inoculation. Scar bar: 50 μm. e-f, The positive rate of PCNA and Ki67 expression in tumors developed from sh-control (or sh-FOX-A1#1)-transfected SPC-A1/DTX cells combined with DTX treatment. ^⁎⁎P < 0.01. g, The level of E-cadherin, N-cadherin and Vimentin in sh-FOX-A1#1 group and sh-control group. h, Kaplan-Meier analysis of the OS of nude mice that were subcutaneously transplanted with sh-control (or sh-FOX-A1#1)-transfected SPC-A1/DTX cells combined with DTX treatment.^⁎⁎P < 0.01. Data are presented as means±standard deviation.

Fig. 5

SOX5 is identified as a direct target of FOX-A1. a-b, FOX-A1 upregulated SOX5 expression. SPC-A1 and SPC-A1/DTX cells were infected with pcDNA-FOX-A1 (or pcDNA-control) or sh-FOX-A1#1 (or sh-control), and the mRNA and protein expression of SOX5 were measured by qRT-PCR and western blotting. GAPDH was used as an internal control. ^⁎⁎P < 0.01. c, FOX-A1 transactivated SOX5 promoter activity. A SOX5 promoter luciferase construct, (−1990/0)SOX5, was cotransfected with pcDNA-FOX-A1 (or pcDNA-control), and the promoter activity was measured using a luciferase reporter assay. ^⁎⁎P < 0.01. d, Three FOX-A1 binding sites were identified in the SOX5 promoter. Serially truncated and site-mutated regions of the SOX5 promoter were constructed (left) and cloned into a pGL3-basic vector. These luciferase reporter vectors were then transfected into SPC-A1/DTX cells co-transfected with Renilla luciferase and pcDNA-FOX-A1 (or pcDNA-control) vectors. Then, the relative luciferase activity was detected using a dual-luciferase reporter assay (right). ^⁎⁎P < 0.01. e, FOX-A1 bound to the SOX5 promoter in vivo. Chromatin immunoprecipitation (ChIP) assays were performed using SPC-A1/DTX cells with antibodies directly against FOX-A1 or IgG control. qRT-PCR was performed to analyze the immunoprecipitated DNA with primers for amplifying the sequences containing the putative FOX-A1-binding sites. ^⁎⁎P < 0.01. f, FOX-A1 increased FOX-A1 binding to the SOX5 promoter in vivo. ChIP assays were performed with antibodies directed against FOX-A1 in pcDNA-FOX-A1 (or pcDNA-control)-transfected SPC-A1/DTX cells. Immunoprecipitated DNA was detected using qRT-PCR with primers for amplifying the sequences containing the putative FOX-A1-binding sites. ^⁎⁎P < 0.01. Data are presented as means±standard deviation.

Fig. 6

SOX5 is responsible for docetaxel resistance of LAD cells in vitro. a, Western blotting detection of SOX5 in docetaxel-resistant LAD cells transfected with sh-SOX5#1, sh-SOX5#2, sh-SOX5#3 or control. β-actin was used as an internal control. b, A CCK-8 assay was performed to detect the IC₅₀ values of DTX in sh-control (or sh-SOX5#3)-transfected docetaxel-resistant LAD cells. ^⁎P < 0.05. c, A colony formation assay was conducted to detect the proliferation ability of sh-control (or sh-SOX5#3)-transfected docetaxel-resistant LAD cells treated without (or with) DTX (50 μg/L). ^⁎⁎P < 0.01. d, Flow cytometric analysis of the early apoptosis rate of sh-control (or sh-SOX5#3)-transfected docetaxel-resistant LAD cells treated without (or with) DTX (50 μg/L). ^⁎⁎P < 0.01. e, Western blotting was used to detect cleaved caspase-3 (C-caspase-3) and caspase-3 in sh-control (or sh-SOX5#3)-transfected docetaxel-resistant LAD cells. β-actin was used as an internal control. Data represent the average of three independent experiments (means±standard deviation).

Fig. 7

FOX-A1 is involved in the docetaxel resistance of LAD cells partially in a SOX5-dependent manner. a, CCK-8 assay analysis of the IC₅₀ values of DTX in sh-control (or sh-FOX-A1#1)-transfected docetaxel-resistant LAD cells co-transfected with pcDNA-SOX5. ^⁎P < 0.05, ^⁎⁎P < 0.01. b, A colony formation assay was performed to detect the proliferation ability of sh-control (or sh-FOX-A1#1)-transfected docetaxel-resistant LAD cells co-transfected with pcDNA-SOX5. ^⁎P < 0.05, ^⁎⁎P < 0.01. c, Flow cytometric analysis of the early apoptosis rate of sh-control (or sh-FOX-A1#1)-transfected docetaxel-resistant LAD cells co-transfected with pcDNA-SOX5. ^⁎P < 0.05, ^⁎⁎P < 0.01. d, Western blotting was used to detect cleaved caspase-3 (C-caspase-3) and caspase-3 in sh-control (or sh-FOX-A1#1)-transfected docetaxel-resistant LAD cells co-transfected with pcDNA-SOX5. Data represent the average of three independent experiments (means±standard deviation).

Fig. 8

Proposed model of the regulation of FOX-A1 by SOX5 between parental and docetaxel-resistant LAD cells. Compared with parental LAD cells, FOX-A1 was upregulated in docetaxel-resistant LAD cells, which resulted in the upregulation of its target gene SOX5 and subsequently contributed to cell proliferation, EMT, migration, invasion and chemoresistance.

Supplementary Fig. 1

Upregulation of SOX5 is correlated with the patients' prognosis. a, Peak signals detected from the ChIP sequecing revealed that SOX5 might be a potential target of FOX-A1 (The small figure is an enlargement of the first red peak in large plot). b, The GO terms enriched in the subset of most significant candidate genes identified by ChIP-Seq. c, Top 20 enriched pathway. d, SOX5 expression in sensitive and insensitive LAD tissues. ^⁎⁎P < .01. e, The level of SOX5 in paired normal and LAD tissues.^⁎⁎P < .01. f-g, The PFS and OS of LAD patients with high or low level of SOX5 were analyzed by Kaplan-Meier method. ^⁎⁎P < .01. h, Expression correlation between SOX5 and FOX-A1 in LAD tissues. ^⁎⁎P < .01. Data represent the average of three independent experiments (means±standard deviation).

Supplementary Fig. 2

Suppression of SOX5 represses migration and invasion and reverses EMT to MET in docetaxel-resistant LAD cells. a, A transwell assay was conducted to detect the migration and invasion of docetaxel-resistant LAD cells treated with sh-control or sh-SOX5#3 vectors. Migrated and invaded cells were calculated in five random fields of view at 100× magnification. The histogram displays the average number of migrated and invaded cells per field of view. Scar bar: 50 μm. ^⁎⁎P < .01. b, Western blotting detection of the protein expression of an epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin and Vimentin) in docetaxel-resistant LAD cells treated with sh-control or sh-SOX5#3. GAPDH was used as an internal control. ^⁎⁎P < .01. c, Immunofluorescence staining was used to investigate the expression of an epithelial marker (E-cadherin) and mesenchymal markers (N-cadherin and Vimentin) in docetaxel-resistant LAD cells (SPC-A1/DTX) treated with sh-control or sh-SOX5#3. Scar bar: 50 μm. Data represent the average of three independent experiments (means±standard deviation).

Supplementary Fig. 3

SOX5 is involved in the chemoresistance of docetaxel-resistant LAD cells in vivo. a, Tumor growth was assessed by the tumor volume in nude mice subcutaneously transplanted with sh-control (or sh-SOX5#3)-transfected SPC-A1/DTX cells combined with DTX treatment. The data are displayed as means±standard deviation. Representative photographs of tumors were obtained at 30 days after inoculation. b-c, Tumor volume and weight of xenograft tumors at the end of the treatment period. ^⁎⁎P < .01. d, H&E staining, PCNA and Ki67 staining and TUNEL staining were performed with tumors collected at 30 days after inoculation. Scar bar, 50 μm. e-f, The positive rate of PCNA and Ki67 expression in tumors that had developed from sh-control (or sh-SOX5#3)-transfected SPC-A1/DTX cells combined with DTX treatment. g, The level of E-cadherin, N-cadherin and Vimentin in sh-SOX5#3 group and sh-control group. h, Kaplan-Meier analysis of the OS of nude mice subcutaneously transplanted with sh-control (or sh-SOX5#3)-transfected SPC-A1/DTX cells combined with DTX treatment. ^⁎⁎P < .01. Data are presented as means±standard deviation.

Supplementary Fig. 4

FOX-A1 promotes the LAD cell proliferation and suppressed cell apoptosis. a, Overexpression efficiency of FOX-A1 in parental LAD cells. b, The IC₅₀ value of parental cells to docetaxel was examined in response to the overexpression of FOX-A1. ^⁎P < .05. c, Colony forming capability of cells transfected with pcDNA-FOX-A1 vector or empty vector. ^⁎⁎P < .01. d, Apoptosis rate in parental cells transfected with pcDNA-FOX-A1 vector or empty vector. ^⁎⁎P < .01. Data are presented as means±standard deviation.

Supplementary Fig. 5

SOX5 promotes LAD cell proliferation and suppressed cell apoptosis. a, SOX5 expression in parental cells and docetaxel-resistant LAD cells. ^⁎P < .05. b, SOX5 was overexpressed in two parental LAD cells. c, IC₅₀ value of parental cells in response to overexpression of SOX5. ^⁎P < .05. d, Colony forming ability in cells treated with empty vector and pcDNA-SOX5. ^⁎⁎P < .01. e, The apoptosis of parental cells was detected after transfection with empty vector and SOX5 expression vector. ^⁎⁎P < .01. Data are presented as means±standard deviation.

Supplementary Fig. 6

High expression level of FOX-A1 and SOX5 in LAD tissues. a, The expression level of FOX-A1 in insensitive tissues and sensitive tissues. ^⁎⁎P < .01. b, The level of FOX-A1 expression in LAD tissues and normal tissues. β-actin was internal control. ^⁎⁎P < .01. c-d, The expression level of SOX5 in different tissues by normalizing to β-actin. ^⁎⁎P < .01. Data are presented as means±standard deviation.

Supplementary Fig. 7

a, Resistance of SPC-A1/DTX or H1299/DTX cells to cisplatin or paclitaxel was determined after silencing of FOX-A1. ^⁎P < .05. b, FOX-A1 expression in normal cell line or ten NSCLC cell lines. ^⁎P < .05, ^⁎⁎P < .01. c, Sensitivity of eight NSCLC cell lines to docetaxel was examined after overexpression of FOX-A1. ^⁎P < .05, ^⁎⁎P < .01. d, Body weight of nude mice injected with different cell lines. ^⁎P < .05. e, FOX-A1 or SOX5 expression in docetaxel-resistant LAD cell lines transfected with miR-NC or miR-451 mimics. MiR-451 expression in FOX-A1 or SOX5-downregulated cell lines. N·S: no significance. Data are presented as means±standard deviation.

Supplementary Fig. 8

a, FOX-A1 expression in H1299 and SPC-A1 cell lines treated with different dose of Cisplatin. ^⁎P < .05. FOX-A1 expression in cells treated with cisplatin at different time point was measured. ^⁎P < .05. b, FOX-A1 expression in H1299 and SPC-A1 cell lines treated with different doses of PTX. ^⁎P < .05, ^⁎⁎P < .01. FOX-A1 expression in cells treated with PTX at different time point was measured.^⁎P < .05, ^⁎⁎P < .01. c, ABCB1 protein level in FOX-A1-overexpressed parental cell lines or in FOX-A1-downregulated docetaxel-resistant cell lines. Data are presented as means ± standard deviation.