Out-FOXing Pancreatic Cancer
Kaplan-Meier analysis showing a higher FOXL1 expression in tumors is associated with better survival in patients with pancreatic ductal adenocarcinoma. FOXL1 gene expression values were determined by qRT-PCR and dichotomized into high and low groups using median value. Survival profiles were compared using the log-rank test.
Pancreatic cancer is one of the most lethal cancer types worldwide with increasing incidence and mortality rates in the United States. Consequently, it is projected to become the second leading cause of cancer death by 2020. Poor patient outcomes are due to a combination of diagnosis at an advanced stage and a lack of effective treatments. However, a better understanding of the molecular pathways at work in pancreatic cancers may lead to the identification of novel therapeutic targets.
S. Perwez Hussain, Ph.D., of CCR’s Laboratory of Human Carcinogenesis, and his colleagues decided to investigate the role of Forkhead Box L1 (FOXL1), a transcription factor that has been implicated in normal and tumor growth in the gastrointestinal tract, in pancreatic carcinogenesis. The researchers examined FOXL1 expression in 45 tumor samples from patients with resected pancreatic ductal adenocarcinoma (PDAC). They found that patients whose tumors had higher levels of FOXL1 gene expression survived longer than those with lower levels. In fact, FOXL1 expression and tumor grade, a measure of tumor differentiation, were each associated with patient prognosis, and a model including both FOXL1 and grade was superior to grade alone at predicting prognosis.
Using immunohistochemical staining to look at FOXL1 protein in the tumor samples, the investigators observed FOXL1 predominantly in cell nuclei, suggesting its active involvement in regulating gene expression. They also verified that patients with higher FOXL1 gene expression had higher FOXL1 protein levels. FOXL1 protein was more abundant in normal tissue and early stage tumors, and lower FOXL1 correlated with metastases to the lymph nodes and beyond. These results suggest that FOXL1 may negatively regulate PDAC progression and aggressiveness.
To understand how FOXL1 might affect pancreatic tumor biology, Hussain and colleagues expressed high levels of the protein in pancreatic cancer cell lines that normally have low FOXL1 levels. The overexpression of FOXL1 slowed cell proliferation and limited colony formation in vitro. In contrast, reducing FOXL1 expression with siRNA enhanced the growth of pancreatic cancer cells. When the scientists examined their activity in vivo, the cells overexpressing FOXL1 formed smaller tumors that grew more slowly than those generated by control cells, demonstrating that FOXL1 suppresses pancreatic tumor growth.
The researchers wondered whether FOXL1 might limit pancreatic cancer cell growth by stimulating apoptosis. In support of this idea, they found that cells overexpressing FOXL1 had a higher activity of effector caspases-3 and -7, while siRNA-mediated FOXL1 knock down blunted the action of these caspases. Because FOXL1 is a transcription factor and might induce apoptosis by altering gene expression, the investigators compared the expression of a panel of apoptosis-related genes with or without FOXL1 overexpression. They observed a 2.5-fold increase in expression of the TNF-Related Apoptosis-Inducing Ligand (TRAIL) gene in FOXL1-overexpressing cells and a corresponding doubling of TRAIL protein levels. The scientists determined that FOXL1 enhanced TRAIL expression by directly binding the TRAIL promoter. Treating the FOXL1-overexpressing cells with antibodies against TRAIL significantly reduced the activity of caspases-3 and -7, confirming that FOXL1 increases apoptosis in pancreatic cancer cells by inducing TRAIL expression.
Motility and invasiveness are also important characteristics of tumor cells. To see whether FOXL1 might affect these aspects of pancreatic cancer cells, the researchers performed scratch healing and Matrigel assays. Compared to controls, fewer cells overexpressing FOXL1 moved to fill the wound generated during the scratch assay. Likewise, fewer FOXL1-overexpressing cells invaded into the Matrigel. In contrast, cells with siRNA-mediated knock down of FOXL1 had enhanced Matrigel invasion. These results indicate that, in addition to reducing pancreatic cancer cell growth, FOXL1 suppresses the migration and invasion of these cells.
The investigators noted that levels of FOXL1 were inversely related to the expression of ZEB1, an activator of the epithelial-mesenchymal transition. They then tested whether FOXL1 might regulate ZEB1 expression. Overexpression of FOXL1 reduced ZEB1 mRNA levels, and FOXL1 knock down enhanced ZEB1 expression. The scientists identified four potential FOXL1 binding sites in the ZEB1 promoter and found that FOXL1 could directly and efficiently bind one of these sites. Using a reporter assay, they demonstrated that FOXL1 binding to this site reduced gene transcription. To determine whether ZEB1 was necessary for the FOXL1-mediated reduction in pancreatic cancer cell invasion, the researchers knocked down FOXL1 and ZEB1. Loss of both proteins prevented the increased invasion induced by knock down of FOXL1 alone.
Taken together, these results show that FOXL1 is an important regulator of pancreatic cancer cells. By inducing expression of TRAIL, FOXL1 can stimulate apoptosis and reduce tumor growth. In addition, FOXL1 suppresses the expression of ZEB1, limiting pancreatic cancer cell invasiveness. While further studies are needed, FOXL1 may be a potential target for treating this deadly disease.Summary Posted: 07/2013
Zhang G, He P, Gaedcke J, Ghadimi BM, Ried T, Yfantis HG, Lee DH, Hanna N, Alexander HR, Hussain SP. FOXL1, a Novel Candidate Tumor Suppressor, Inhibits Tumor Aggressiveness and Predicts Outcome in Human Pancreatic Cancer. Cancer Research. June 27, 2013 PubMed Link