Forkhead Box L1 Is Frequently Downregulated in Gallbladder Cancer and Inhibits Cell Growth through Apoptosis Induction by Mitochondrial Dysfunction

<div><p>Background</p><p>Forkhead box L1 (FOXL1), considered as a novel candidate tumor suppressor, suppresses proliferation and invasion in certain cancers. However, the regulation and function of FOXL1 in gallbladder cancer (GBC) remains unclear.</p><p>Methods</p><p>FOXL1 expression at mRNA and protein levels in GBC tissues and cell lines were examined by RT-PCR, immunohistochemistry and western blot assay. FOXL1 expression in GBC cell lines was up-regulated by transfection with pcDNA-FOXL1. The effects of FOXL1 overexpression on cell proliferation, apoptosis, migration and invasion were evaluated in vitro or in vivo. In addition, the status of mediators involved in migration, invasion and apoptosis was examined using western blot after transfection with pcDNA-FOXL1.</p><p>Results</p><p>FOXL1 was frequently downregulated in GBC tissues and cell lines. Its higher expression is associated with better prognosis, while its lower expression is correlated with advanced TNM stage and poor differentiation. FOXL1 overexpression in NOZ cells significantly suppresses cell proliferation, migration and invasion in vitro and tumorigenicity in nude mice. FOXL1 overexpression disrupted mitochondrial transmembrane potential and triggered mitochondria-mediated apoptosis in NOZ cells. In addition, FOXL1 overexpression suppressed ZEB1 expression and induced E-cadherin expression in NOZ cells.</p><p>Conclusion</p><p>Our findings suggested that dysregulated FOXL1 is involved in tumorigenesis and progression of GBC and may serve as a predictor of clinical outcome or even a therapeutic target for patients with GBC.</p></div

FOXL1 overexpression promoted apoptosis in vitro and in vivo.

<p>(<b>A</b>) Apoptosis of NOZ cells in vitro was examined by Annexin V/PI double staining. Cells that stain negative for both Annexin V and PI are viable cells (lower left). Cells that stain positive for Annexin V and negative for PI are undergoing early apoptosis (lower right). Cells that stain positive for both Annexin V and PI are either in the end stage of apoptosis, or undergoing necrosis (upper right). The results shown are representative of three experiments. (<b>B</b>) The number of apoptotic NOZ cells (including early apoptosis, late apoptosis and necrosis) was significantly increased after transfection with pcDNA-FOXL1 (P<0.05). (<b>C</b>) Apoptosis of NOZ cells in vivo was determined using TUNEL assay. Apoptotic cells are visualized as intensely green fluorescent cells. The number of apoptotic cells was recorded under high-power fields (400×). Localization in nuclei was visualized by counterstaining with DAPI (blue). (<b>D</b>) FOXL1 overexpression significantly promoted apoptosis of NOZ cells in vivo (P<0.05). *indicates significant difference.</p

FOXL1 overexpression induced mitochondrial dysfunction and caspase-mediated apoptosis.

<p>(<b>A</b>) The loss of mitochondrial membrane potential (ΔΨm) is indicated by a decrease in the red/green fluorescence intensity ratio. X axis indicates green fluorescence intensity; Y axis indicates red fluorescence intensity. The results shown are representative of three experiments. (<b>B</b>) The proportion of red fluorescence-positive cells decreased while the proportion of green fluorescence-positive cells increased after transfection with pcDNA-FOXL1 (P<0.05). (<b>C</b>) Western blot analysis showed the level of cytosolic cytochrome c markedly increased, while the level of mitochondrial cytochrome c markedly decreased. (<b>D</b>) The levels of cleaved caspase-9, 3 and PARP and bax were elevated, while the level of bcl-2 was decreased. *indicates significant difference.</p

Overexpression of NOTCH-regulated Ankyrin Repeat Protein is associated with papillary thyroid carcinoma progression

<div><p>Papillary thyroid cancer (PTC) is one of the endocrine cancers with high clinical and genetic heterogeneity. NOTCH signaling and its downstream NOTCH-Regulated Ankyrin Repeat Protein (NRARP) have been implicated in oncogenesis of many cancers, but the roles in PTCs are less studied. In this study, we show that NRARP is frequently over-expressed in thyroid carcinoma. The over-activation of NRARP is highly and positively correlated with NOTCH genes. Moreover, we find that the expression of NRARP is highly associated with several epithelial mesenchymal transition (EMT) markers and contributes to poor survival outcomes. Therefore, these results indicate that NRARP is an important clinical biomarker in thyroid carcinoma and it promotes EMT induction as well as the progression of PTCs via NOTCH signaling activation.</p></div

Expression correlation between NRARP and NOTCH signaling.

<p>A) Enriched pathways that are positively correlated with NRARP. B) Scatter plot of NRARP expression and NOTCH1 expression in TCGA THCA dataset. Each point stands for one tumor patient, of which values in X-axis and Y-axis represent NOTCH1 and NRARP expression, respectively. C) Scatter plots of NRARP and NOTCH3 genes. D) Scatter plots of NRARP and NOTCH4. E) Protein interaction between NRARP and NOTCH genes.</p

Over-expression of NRARP in thyroid tumors.

<p>A) Up-regulated pathways in TCGA THCA dataset. B) Boxplot of NRARP expression profiles in TCGA THCA dataset. C) Boxplot of NRARP expression profiles in public dataset GSE64912. D) Genomic alteration frequency of NRARP in TCGA THCA dataset. Each cell stands for the tumor patient.</p

Proposed model regulatory model of NRARP in thyroid cancer patients.

<p>NRARP and NOTCH related genes are up-regulated in tumor patients and contribute to tumor progression. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167782#sec013" target="_blank">discussion</a> for details.</p