OCT4-mediated upregulation of DUSP6 promotes metastasis in non-small-cell lung cancer

Abstract

The roles of cancer stem cells and Octamer-binding transcription factor 4 (OCT4) have been implicated in human tumorigenesis and metastasis. However, the role of OCT4 in the metastasis of non-small-cell lung cancer (NSCLC) remains undetermined, especially regarding stem cell-related pathways. Previous research has reported that dual-specificity phosphatase 6 (DUSP6), a mitogen-activated protein kinase (MAPK) phosphatase, is associated with cancer cells that display anti-apoptotic, migratory, and drug-resistance phenotypes. However, the regulation of DUSP6 in NSCLC is unclear. This study focused on the role of OCT4 in NSCLC, particularly its interaction with DUSP6. Here, we show a positive correlation between OCT4 and DUSP6 expression in NSCLC cells. Overexpression of OCT4 increased, whereas knockdown of OCT4 reduced DUSP6 expression. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays revealed that OCT4 transactivated DUSP6 expression by directly binding to the DUSP6 promoter, indicating that DUSP6 is a downstream target of OCT4. Furthermore, knockdown of DUSP6 in OCT4-overexpressing A549 human NSCLC cells decreased cell migration in vitro and reduced tumor growth and pulmonary metastasis in NOD/SCID mice. In conclusion, our findings highlight the importance of the OCT4-DUSP6 pathway in NSCLC progression. Furthermore, the OCT4-DUSP6 axis is a potential therapeutic target for NSCLC.

Keywords: dual-specificity phosphatase 6 (DUSP6); metastasis; non-small-cell lung cancer (NSCLC); octamer-binding transcription factor 4 (OCT4).

Figure 1

Expression of OCT4 and DUSP6 is positively correlated in lung adenocarcinoma (LUAD) cells. (A) Immunohistochemical (IHC) staining and quantification of paraffin-embedded tumor parts and normal tissue sections for OCT4 and DUSP6 expression (× 200 magnification, scale bar = 50 μm). IHC intensity was quantified using the ImageJ software. Values shown are levels of immunointensity in individual specimens, with the mean level in normal tissue arbitrarily set to 100, in three randomly selected fields in each section (n=3). (B) A positive correlation between the expression of POU5F1 (OCT4) and DUSP6 in the expression profiles of 226 pathological stage I-II lung adenocarcinomas (NCBI Gene Expression Omnibus: GSE31210). The correlation was assessed using Pearson's correlation coefficient (r=0.1268, P=0.0439). (C) Immunoblot analysis and quantification of endogenous expression levels of OCT4 and DUSP6 in eight lung cancer cell lines. Expression of β-actin served as the loading control. (D) Positive correlation between OCT4 and DUSP6 expression levels in cancer cell lines. Band intensity was quantified using the ImageJ software, and correlations were assessed using Pearson's correlation coefficient (r=0.8265, P=0.0017). (E, F) Increased expression of DUSP6 mRNA (E) and protein (F) in lung cancer cells overexpressing OCT4 as examined by RT-PCR (E, left), quantitative real-time PCR (qPCR) (E, right), and immunoblotting (F). A549 and H1299 cells were transduced with lentiviral vectors encoding OCT4 or luciferase, followed by puromycin selection. Expression of GAPDH (E) and β-actin (F) served as the internal control. (G, H) Knockdown of OCT4 by OCT4 shRNA reduces DUSP6 expression. A549 cells were transduced with lentiviral vectors expressing shRNAs specific to OCT4 (shOCT4 #2 and shOCT4 #5) or to luciferase (shLuc). Levels of OCT4 and DUSP6 mRNA and protein were examined by RT-PCR (G, left), qPCR (G, right), and immunoblotting (H). (I, J) Enhanced migratory and invasive capability of OCT4-overexpressing A549 cells, or reduced that of OCT4-knockdown A549 cells. Migration of A549/OCT4 and A549/Vector cells or A549/shLuc and A549/shOCT4#5 cells was detected by the Boyden chamber assay. Cells that migrated through the membrane of the lower surface in the Boyden chamber were stained with Giemsa solution and visualized by light microscopy (× 200 magnification, scale bar = 200 μm) (I, J, left) and quantified (I, J, right). Migratory cells in three fields in each membrane were counted. All quantification values and error bars of (A) and (E-J) shown were mean ± SEM. SEM, standard error of the mean.

Figure 2

OCT4 upregulates DUSP6 expression by transactivating the DUSP6 promoter in lung cancer cells. (A) Reporter assay of the DUSP6 promoter in lung cancer cell lines. A549, H1299, and CL1-5 cells were co-transfected with pFRL2-hDUSP6p-Luc and pCMV-tag2B-hOCT4 (or pCMV-tag2B). (B) Identification of OCT4 binding sites on the human DUSP6 promoter. Schematic representation of a series of deletion constructs containing different lengths of the DUSP6 promoter. H1299 cells were co-transfected with different deletion reporter constructs of pFRL2-hDUSP6p-Luc and pCMV-tag2B-hOCT4 (or pCMV-tag2B). (C) A549 cells were co-transfected with pFRL2-hDUSP6p-Luc carrying a point mutation within the -423 ~ -416 ORE and pCMV-tag2B-hOCT4 (or pCMV-tag2B). Numbering is relative to the translational start site at +1. The white box indicates putative OREs. Cell lysates were harvested 48 h after transfection, and their firefly and Renilla luciferase activities were determined using a dual-light luciferase reporter assay system. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light units (RLU). Data are expressed relative to the activity of the full-length promoter construct (B) or the wild-type (WT) promoter construct (C) obtained from three independent experiments (B). (D) ChIP assay showing direct binding of OCT4 to the ORE located at -423 to -416 within the human DUSP6 promoter. Cross-linked chromatin of H1299 cells was immunoprecipitated with mouse anti-human OCT4 antibody or normal mouse IgG in combination with protein G agarose beads, followed by PCR amplification of the DUSP6 promoter region encompassing OCT4 binding sites.

Figure 3

Overexpression of OCT4 in A549 cells promotes tumor growth and metastasis and increases DUSP6 expression in mice. (A) Tumor volumes of mice bearing OCT4-overexpressing A549 tumors. NOD/SCID mice were subcutaneously inoculated with 1 × 10⁶ cells of A549 cells overexpressing OCT4 (n=4 for OCT4 #4 and OCT4 #5) or vector control (n=4) A549 cells on day 0. Tumor volumes of the mice were measured. Tumor volumes were plotted through day 120 (primary tumor endpoint), and lungs were collected at day 156 to permit the development of spontaneous metastases in this slow‑metastasizing A549 model. (B) Number of metastatic nodules in the lung of individual mice. (C) Representative gross and histologic (hematoxylin and eosin stain, × 100 magnification, scale bar = 1.0 mm) appearances of lungs on day 156. Arrows denote tumor nodules. (D) Immunohistochemical detection of OCT4 and DUSP6 in paraffin-embedded lung tissue sections (× 100 magnification, scale bar = 100 μm).

Figure 4

The OCT4-DUSP6 axis promotes the migratory and invasive capability of A549 cells. (A) OCT4-DUSP6 axis upregulates the expression of MMP2 and MMP9. A549 cells were transduced with lentiviral vectors encoding OCT4 or GFP (control), followed by puromycin selection. Subsequently, the cells were further transduced with lentiviral vectors encoding shRNA specific to DUSP6 (shDUSP6 #2 and shDUSP6 #4) or luciferase (shLuc). Detection and quantification of OCT4, DUSP6, MMP2, and MMP9 protein expression levels were examined by immunoblotting (n=3). Expression of β-actin served as the loading control. (B) The OCT4-DUSP6 axis regulates MMP2 and MMP9 expression independently of the inhibition of ERK1/2-MAPK activity. OCT4 or GFP-transduced A549 cells were treated with DMSO or ERK inhibitor (LY3214996, 2μM) for 48 h. Total protein extracts were detected and quantified for OCT4, DUSP6, NICD, MMP2, and MMP9 expression (n=3). (C) The OCT4-DUSP6 axis regulates MMP2 and MMP9 expression through the Notch pathway. A549 cells were transduced with lentiviral vectors encoding OCT4 or GFP, followed by further transduction with lentiviral vectors encoding shRNA specific to NOTCH (shNOTCH) or luciferase (shLuc). Detection and quantification of OCT4, DUSP6, NICD, MMP2, and MMP9 protein expression levels were examined by immunoblotting (n=3). (D) Migratory and invasive capabilities of DUSP6-knockdown and Luc control A549/OCT4 cells. Cells that migrated through the membrane to the lower surface in the Boyden chamber were stained with Giemsa solution and visualized by light microscopy (× 200 magnification, scale bar = 200 μm) (B) and quantified (C). Migratory cells in three fields in each membrane were counted. All quantification values and error bars shown were mean ± SEM, and P-values less than 0.05 were shown in the quantitative charts. NICD, Notch intracellular domain; SEM, standard error of the mean.

Figure 5

Knockdown of DUSP6 in OCT4-overexpressing A549 cells decreases tumor growth and metastasis in mice. (A) Tumor volumes of mice bearing DUSP6-knockdown or vector control A549/OCT4 tumors. NOD/SCID mice were subcutaneously inoculated with 1 × 10⁶ cells of DUSP6-knockdown (n=6 for OCT4_shDUSP6#2; n=7 for OCT4_shDUSP6#4) or vector control A549/OCT4 cells (n=7 for OCT4_shLuc). Tumor volumes of the mice were measured. (B) Number of metastatic nodules in the lung of individual mice. (C) Representative gross and histologic (hematoxylin and eosin stain, × 200 magnification, scale bar = 1.0 mm) appearances of lungs on day 86. Arrows denote tumor nodules. (D) A schematic representation of the OCT4-DUSP6 axis involved in lung cancer progression. OCT4 overexpression in NSCLC cells upregulates DUSP6. DUSP6 has been shown to function as a phosphatase for Notch1, thereby regulating Notch1 transmembrane/intracellular region (NTM) stability and Notch1 intracellular domain (NICD) transcriptional activity, resulting in driving tumor aggressiveness through MMP2 and MMP9 upregulation. The pathways in gray color have been reported previously.