Loss of CPSF2 expression is associated with increased thyroid cancer cellular invasion and cancer stem cell population, and more aggressive disease

Abstract

Purpose: Identification of molecular factors that promote thyroid cancer progression have important clinical implications for therapy and prognostication in patients with papillary thyroid cancer (PTC). The aim of this study was to validate and determine the function of dysregulated genes that were associated increased mortality in patients with PTC. Experiemental Design: We selected the cleavage and polyadenylation specificity factor subunit 2 (CPSF2) gene from the top 5 significantly dysregulated genes associated with PTC-associated mortality from our previous study. We used 86 PTC samples enriched for aggressive disease (recurrence and mortality) by quantitative RT-PCR (qRT-PCR). In vitro functional studies of the validated gene were performed.

Results: Decreased CPSF2 gene expression was associated with shorter disease-free survival (P = .03), large tumor size (T3 and T4) (P = .03), tumor recurrence (P < .01), and mortality (P < .01), independent of BRAF V600E mutation status. CPSF2 knockdown increased cellular invasion by 1.8- to 3.2-fold (P < .01) and increased markers of thyroid cancer stem cells (CD44 and CD133 expression). Immunohistochemistry showed an inverse correlation between CD44 protein expression in PTC samples and CPSF2 expression.

Conclusion: Decreased CPSF2 expression is associated with increased cellular invasion and cancer stem cell population, and more aggressive disease in PTC.

Figure 1.

CPSF2 mRNA expression was significantly lower in PTC-associated mortality using additional independent samples (n = 26) (A) CPSF2 mRNA expression levels of combined samples (n = 86) by clinical characteristics. Significantly lower CPSF2 mRNA expression in PTC with advanced T stage (T3 and T4) (B), tumor recurrence (C), mortality (D). Outliers are shown in small circles and stars. Y axis indicates the mRNA expression levels and X axis indicates adverse clinical status of patients with PTC. There was a significant correlation between CPSF2 mRNA expression levels and disease-free interval (E) and a significantly shorter disease-free survival was observed in patients with lower CPSF2 mRNA expression than average expression of the cohort, shown in green graph. Blue graph represents disease-free survival of patients with CPSF2 mRNA expression above than average expression of the cohort. Y-axis represents cumulative survival and X-axis represents disease-free interval in months (F).

Figure 2.

Immunohistochemistry stain of PTC samples showed a significantly lower expression of CPSF2 protein in PTC at advanced T stage (T3 and T4) than early T stage (T1 and T2). Pathological TNM (Tumor, Node, Metastasis) cancer staging system was used. Error bars indicate SEM.

Figure 3.

CPSF2 knockdown significantly increased cell invasion in TPC-1 (s28827 and s28828 CPSF2 siRNAs), FTC-133, and XTC-1 (both CPSF2 siRNAs). Negative control is shown on the first bar from the left and two groups treated with CPSF2 siRNAs (s28827 [s27] and s28828 [s28]) are shown on the right. Error bars represent SEM.

Figure 4.

CPSF2 expression was increased in TPC-1 and FTC-133 cells treated with CPSF2 siRNAs for 7 and 6 d, respectively (A). The population of CD44- and CD133- enriched thyroid cancer cells was increased when TPC-1 and FTC-133 cells were treated with CPSF2 siRNAs for 6 d (B). Unstained cells are shown red solid lines, cells treated with negative control (Neg), s28827 siRNA (s27) and s28828 (s28) siRNA are shown in dotted, dashed, and solid black lines, respectively. CD44 expression (membranous stain) in PTC samples inversely correlated with CPSF2 expression (nuclear and cytoplasmic stain) (C).

Figure 5.

CPSF2 protein expression in 8505C cells treated with selective BRAFV600E inhibitor (PLX4032) (A). Phospho-MEK 1/2 protein expression when TPC-1 cells were treated with CPSF2 siRNAs for 6 and 8 d (B).