FOXH1 expression is upregulated in lung cancer
The Cancer Genome Atlas (TCGA) is a large-scale cancer genomics program that has molecularly characterized 33 primary cancer types including 3 subtypes of lung cancer. In order to test whether FOXH1 is differentially expressed in lung cancer samples, the normalized gene expression data from TCGA were extracted, and FOXH1 gene expression levels in lung cancer samples were compared with normal tissues using t-tests. The results showed that FOXH1 expression levels are significantly higher in lung adenocarcinoma (p<0.05), papillary lung carcinoma(p<0.05) and squamous cell lung carcinoma (p<0.05) compare to normal samples. The mean fold change of FOXH1 expression in lung adenocarcinoma, papillary lung adenocarcinoma and squamous cell lung carcinoma were 0.168, 0.258, and 0.225, respectively (Fig 1a). Next, to determine whether FOXH1 is a prognostic marker for lung cancer, we included lung adenocarcinoma samples with patient’s cancer progression and survival data available in TCGA dataset. The lung cancer patients were stratified into FOXH1 high and FOXH1 low groups using the median of FOXH1 gene expression levels in lung cancer samples. Both progression free survival and overall survival were assessed by Kaplan-Meier plots and logrank tests (Fig 1b). Patients whose samples have low FOXH1 expression level have significantly longer progression free survival and overall survival. Poorer prognosis observed for lung adenocarcinoma patients with higher FOXH1 expression in tumor implies FOXH1 is a prognosis marker and can be a potential treatment target for lung cancer.
The immunohistochemistry staining was applied to determined FOXH1 protein expression levels in the paraffin-embedded lung cancer tissues. Representatively, immunohistochemical detection of FOXH1 in the lung cancer and normal lung tissues according to different samples of lung cancer revealed that the immunoreactivity of FOXH1 was barely expressed in surrounding non-tumor and normal lung tissue compared with the tumor. FOXH1 protein expression was highly detected in the nuclei of tumors (Fig 1c). To establish the functional role of FOXH1 in lung cancer, its expression levels were examined in various lung cancer cell lines and the 16HBE cell line derived from normal lung epithelial cells. As shown in Fig 1d, the expression of FOXH1 in all the lung cancer cell lines was higher than that in 16HBE normal cell line. While A549, PC-9 and H1299 cells had significantly higher FOXH1 expression, the FOXH1 expression in H460 and H1975 cells were only marginally higher. Since A549 and PC9 cell lines simulate lung cancer tissue in terms of the FOXH1 expression level, they were selected for the further experiments.
Knockdown of FOXH1 suppresses growth of lung cancer cells
To test the hypothesis that FOXH1 is an oncogene whose elevated expression in lung cancer cells drives the tumor growth and proliferation, effects of FOXH1 knockdown on cellular growth and cell cycles of lung cancer cells were examined in subsequent experiments. The two lung cancer cell lines, PC9 and A549, were transfected with either a sh-RNA specifically targeting FOXH1 (sh-FOXH1) or a negative control sh-RNA (sh-NC), then a Western blot assay was applied. As shown in Fig 2a, FOXH1 was effectively knocked down in both sh-FOXH1 transfected lung cancer cell lines and its expression level reduced >95% 72 hours after transfection compared to cells transfected with negative control sh-RNA (sh-NC). And the FOXH1 expression level stayed low in the following 14 days. CCK8 assays revealed that the proliferation of lung cancer cells was suppressed by FOXH1 silencing while the negative control cells proliferated at the same speed as the un-transfected cells (Fig 2b). We also examined the FOXH1 depleted cells in the cell colony formation assays and the soft agar colony formation assays that are extensively applied for evaluating the cellular proliferation in vitro. The colony forming capability possessed by A549 and PC9 cells was markedly decreased upon FOXH1 depletion (Fig 2c) and the soft agar colony formation assay showed a significant decrease in colony counts when the A549 and PC9 cells were depleted of FOXH1 (Fig 2d). These results indicated that knockdown of FOXH1suppresses growth of lung cancer cells.
FOXH1 down-regulation inhibits lung cancer cell proliferation in vivo
To confirm the oncogenic effect of FOXH1 in vivo, a xenograft tumor-bearing model was established by inoculating sh-NC or sh-FOXH1 transfected A549 cells into the nude mice. Thirty-six days after inoculation, the xenografted tumors were harvested and the tumor sizes were measured. As shown in Fig 3a, the tumors induced by sh-FOXH1 transfected A549 cells were significantly smaller than those induced by sh-NC transfected A549 cells in xenografted mice (Fig 3a). Tumors induced by the sh-FOXH1 transfected cells grew significantly slower compared with those in the control mice (Fig 3b). IHC analysis of tumor tissue verified that FOXH1 was barely expressed in the tumor induced by sh-FOXH1transfected cells (Fig 3c). Additionally, we also examined the expression of Ki-67 antigen, a cellular proliferation marker in mouse tumors. As shown in Fig 3c, the proportion of the Ki-67 positive cells was significantly decreased in the tumor induced by FOXH1 depleted A549 cells. Collectively, these results suggested that FOXH1 depletion efficiently suppresses tumor growth in vivo.
Knockdown of FOXH1 suppresses lung cancer cell invasion and migration
Since cancer cell invasion and migration are essential for cancer advancement and malignancy, we investigated how FOXH1 expression affects the mobility of lung cancer cells.
Effects of FOXH1 on cell proliferation and migration were investigated through wound healing assay. FOXH1-suppressed A549 and PC9 cells showed a marked decrease proliferative and migration ability compared to the sh-NC control (Fig 4a). Cell cycle progression was further assessed via flow cytometry. Knockdown of FOXH1 led to an increase in G1 phase from 64.5% to 74.88% (A549 cells) or 55.85% to 66.18% (p <0.05) (PC9 cells), and decrease in G2 phase in A549 and PC9 cells from 10.52% to 6.40% or 23.30% to 13.55% (p <0.05), and decrease in S phase in A549 and PC9 cells from 24.89% to 18.73% or 20.85% to 20.28% (Fig 4b) indicating the FOXH1 knockdown inhibits cell cycle progression. In the transwell invasion assay, suppression of FOXH1 led to cell invasion decrease from 108 to 16 (p <0.001) in A549 cells and from 162 to 19 (p <0.001) in PC9 cells, relative to corresponding control groups (Fig 4c). These results clearly suggest that knockdown of FOXH1 inhibits migration, proliferation and invasion of lung cancer cells.
Knockdown of FOXH1 decreased EMT marker alterations in PC9 and A549 cells
As cellular invasion and migration are the main phenotypes of epithelial-mesenchymal transition (EMT), a process that involves in tumor metastatic expansion or cancer advancement, we further assessed whether FOXH1 knockdown affects EMT-specific molecules in lung cancer cells using the A549 and PC9 cell lines that stably express sh-FOXH1 and sh-NC.
Western blot analysis results of A549 and PC9 cells with FOXH1 knockdown are shown in Fig 5. The mesenchymal markers Matrix metalloproteinase-2 (MMP2), Vimentin, N-cadherin, Snail and Slug were significantly down-regulated, whereas the epithelial markers E-cadherin was notably upregulated by FOXH1 depletion in both lung cancer cell lines (Figs 5a and b). No changes were found in the negative control cell lines. Our findings indicate that FOXH1 can regulate the metastatic potential of lung cancer cells via activation of EMT.
Knockdown of FOXH1 suppresses β-Catenin signaling in lung cancer
Wnt/β-catenin signaling pathway is essential in regulating multiple processes, including cell proliferation, migration, and invasion in various cancer types, it may also play an important role in EMT. To further explore and clarify the molecular mechanisms underlying FOXH1-mediated tumor promotion and malignancy in lung cancer, effects of FOXH1 on Wnt/β-catenin signaling pathway were assessed. First, we evaluated expression of β-catenin signaling molecules β-catenin, cyclin D1 and p-GSK-3β via western blot analysis.
As shown in Fig 6a, the protein levels of β-catenin and its two downstream targets, cyclin D1 and p-GSK-3β, were significantly decreased in A549 and PC9 cells transfected with sh-FOXH1. Next, we used a Dual-Luciferase Reporter (DLR) assay for further determination of changes in β-catenin activity induced by FOXH1. TCF/LEF-dependent transcriptional activity of β-catenin in A549 and PC9 cells was measured after transfection with TOP/FOP flash reporter plasmids. Compare to the sh-NC control group, sh-FOXH1 induced a significant decrease in TOP luciferase reporter activity (Fig 6b), thus a decreased Wnt/β-catenin activity. Based on the results above, we conclude FOXH1 exerts its activity upstream of the β-catenin, and consequently, its silencing inhibits activation of Wnt/β-catenin signaling in lung cancer.