Leukemia Inhibitory Factor Promotes Aggressiveness of Chordoma

Abstract

Chordomas are rare tumors of the spine and skull base that are locally destructive and resistant to chemotherapy and radiation therapy, with a poor prognosis and limited therapeutic options. Chordoma patients have a long life expectancy with high mortality from the disease. Cancer stem cells, which are known to exist in chordomas, have extensive proliferative and self-renewal potential and are responsible for maintaining tumor heterogeneity along with chemotherapy and radiotherapy resistance. Leukemia inhibitory factor (LIF) has multiple functions in stem cell biology, the immune response, and cancer, and is potentially a key molecule that allows cancer stem cells to self-renew. The purpose of this study was to determine whether LIF increases the aggressive traits of chordoma cells and leads to a poor prognosis in patients. Chordoma cell lines were treated with LIF, and functional tests were done. Twenty skull base chordoma samples were checked for levels of LIF and a correlation with clinicopathological features. The whole transcriptome microarray was used to observe changes in gene expression. We observed increased migration, invasion, tumorosphere formation, colony formation, epithelial-mesenchymal transition, and chemoresistance accompanied by a dramatic elevation in inflammatory gene networks and pathways in chordomas. The expression of LIF was associated with tumor size and a poorer overall survival. Microarray and quantitative real-time polymerase chain reaction assessments suggest that LIF can facilitate tumor-promoting inflammation. Results indicate that LIF plays a role in maintaining cancer stem cells in chordomas.

Figure 1

LIF treatment increases invasion and migration abilities and induces EMT-related genes in chordomas. (a) Anti-LIFR antibody (green) was used to visualize the LIFR level of U-CH1 and MUG-Chor1 after LIF treatment of 1 and 8 weeks. The control group was treated with complete chordoma medium. (b) Representative images of migration and invasion assays at week 1. (c, d) LIF promoted the migration of U-CH1 and MUG-Chor1. Data were normalized with the control group. The highest rate occurred at week 1 and steadily decreased thereafter. (e) LIF treatment decreased the relative level of epithelial markers E-Cad and CK19 while increasing the late EMT marker ZEB2 level at weeks 1 and 3 for MUG-Chor1 and at week 5 for U-CH1 normalized with the control. Proinvasive MET was significantly increased at weeks 1 and 3, a pattern consistent with the invasive character at the time interval. *p < 0.05; **p < 0.01.

Figure 2

LIF promotes the anchorage-independent growth, tumorosphere-forming ability, and chemoresistance of chordoma cells, indicating an increase in cancer stem cell (CSC) character. (a) LIF treatment promoted anchorage-independent growth of U-CH1 and MUG-Chor1 cells in soft agar at weeks 5 and 8. The figure shows the relative number of colonies formed as a percentage of the control group. (b) The tumorosphere formation abilities of U-CH1 and MUG-Chor1 cells on attachment-free conditions increased after week 3 and peaked at week 8. Graph columns represent the average tumorosphere number for each well in a 96-well plate. (c) LIF treatment increased the chemoresistance of chordoma cells against paclitaxel and bortezomib at weeks 5 and 8. Chemoresistance was calculated as the percentage viability of LIF-treated cells over untreated cells for each week and each cell line. (d) Relative CSC marker levels against the control group were determined with real-time polymerase chain reaction (PCR). The timing of increased CSC marker expression was consistent with the results of the functional tests. (e, f) Flow cytometry results of CSC markers CD133, CD15, and the drug efflux protein ABCG2. The columns indicate the percentage increase of these surface markers after LIF treatment compared with the control group. In the histograms, pale red represents unstained cells, whereas dark red represents stained cells. *p < 0.05.

Figure 3

LIF escalates tumor inflammatory and antiapoptotic pathways in chordomas. (a) A heat map representing the differential expression of 21 inflammation-related genes after 3 weeks of LIF treatment. (b) TNFAIP2 levels were checked as a marker of activated inflammation after LIF treatment in chordoma cells by using real-time PCR. *p < 0.05, **p < 0.01.

Figure 4

LIF is correlated with TNFAIP2, KLF4, and MET genes; tumor size; and overall survival in chordoma patient samples. (a) Dot plot comparing the level of LIF expression in chordoma tumor samples against that in nucleus pulposus samples. The horizontal bar represents the average value. (b) Correlation of LIF level (x-axis) versus tumor size, and (c) LIF level (x-axis) versus TNFAIP2, KLF4, and MET. (d) Kaplan–Meier survival plot of our patient cohort. High- and low-LIF groups were defined according to the relative LIF expression levels. The high-LIF group had a poorer overall survival.