Clinicopathologic features of TDO2 overexpression in renal cell carcinoma

Abstract

Background: Tryptophan 2,3-dioxygenase (TDO2) is the primary enzyme catabolizing tryptophan. Several lines of evidence revealed that overexpression of TDO2 is involved in anoikis resistance, spheroid formation, proliferation, and invasion and correlates with poor prognosis in some cancers. The aim of this research was to uncover the expression and biofunction of TDO2 in renal cell carcinoma (RCC).

Methods: To show the expression of TDO2 in RCC, we performed qRT-PCR and immunohistochemistry in integration with TCGA data analysis. The interaction of TDO2 with PD-L1, CD44, PTEN, and TDO2 expression was evaluated. We explored proliferation, colony formation, and invasion in RCC cells line affected by knockdown of TDO2.

Results: RNA-Seq and immunohistochemical analysis showed that TDO2 expression was upregulated in RCC tissues and was associated with advanced disease and poor survival of RCC patients. Furthermore, TDO2 was co-expressed with PD-L1 and CD44. In silico analysis and in vitro knockout of PTEN in RCC cell lines revealed the ability of PTEN to regulate the expression of TDO2. Knockdown of TDO2 suppressed the proliferation and invasion of RCC cells.

Conclusion: Our results suggest that TDO2 might have an important role in disease progression and could be a promising marker for targeted therapy in RCC. (199 words).

Keywords: Cancer stem cell; PD-L1; PTEN; Renal cell carcinoma; TDO2.

Fig. 1

TDO2 expression and association with progression and prognosis. a, b TDO2 expression in TCGA RCC. a TDO2 expression in normal and tumor tissues. b TDO2 expression associated with clinicopathologic features; Kaplan-Meier analysis of RCC patients with high and low TDO2 expression. Unit: log2(count+ 1), Wilcox/Kruskal-Wallis test. c Validating TDO2 expression in 12 pairs of normal and tumor tissue samples by qRT-PCR. The data are displayed as mean ± SD (n = 3). d Immunohistochemical staining of TDO2 in a RCC sample, original magnification × 40. Normal kidney showed weak cytoplasmic staining (upper right corner, original magnification × 400), whereas tumor cells showed strong cytoplasmic staining with TDO2 (lower right corner, original magnification × 400). (e) Survival analysis of RCC patients with high and low TDO2 expression by Kaplan-Meier method

Fig. 2

Correlation between TDO2 and PD-L1 expression. a Immunohistochemistry of TDO2 and PD-L1. Left picture, cytoplasmic staining of TDO2. Right picture, membranous staining of PD-L1. Original magnifications × 100. b Correlation between PD-L1 staining and TDO2 expression score. Wilcox test. c, d In silico analysis of the correlation between TDO2 and PD-L1 and T-cell exhaustion signature genes. c TDO2 expression correlated with PD-L1 expression. d TDO2 expression correlated with T-cell exhaustion signature genes (CLTA4, PDCD1, GZMB, LAG3). Unit: log2(count+ 1). Spearman rank correlation test

Fig. 3

Correlation of TDO2 expression with PTEN alteration. a TDO2 expression correlated with mutation of STED2, PTEN and ARID1A genes by TIMER2.0. b qRT-PCR results of TDO2 expression in RCC cell lines. c Western blot analysis of the expression of PTEN and TDO2 in three RCC cell lines. GAPDH served as an internal control. The western blot bands were cropped and full-length blots/gels were presented in Fig. S5, Fig. S6. d, e The levels of TDO2 expression affected by PTEN knockout. d qRT-PCR results of TDO2 expression in PTEN knockout and its parental cells. e Western blot analysis of the expression of PTEN and TDO2 in PTEN knockout and its parental cells. GAPDH served as an internal control. The western blot bands were cropped and full-length blots/gels were presented in Fig. S7, Fig. S8. The bars indicate the mean ± SD (n = 3)

Fig. 4

Effect of knockdown of TDO2 expression on proliferation, colony formation and invasion of RCC cells. a TDO2 mRNA levels in 786-O cells transfected with siRNA1, siRNA2, and siRNA negative control. The bars indicate the mean ± SD (n = 3). *p < 0.01. b MTT assay of 786-O cells transfected with siRNA1, siRNA2, and siRNA negative control. c Images of colony formation in 786-O cells transfected with siRNA1, siRNA2, and siRNA negative control at 14 days, and quantification of the average number of clones. The error bars indicate SE (n = 3). *p < 0.01. d Images of invasion assay in 786-O cells transfected with siRNA1, siRNA2, and siRNA negative control at 24 h, and quantification of the average number of invaded cells. The error bars indicate SE (n = 3). *p < 0.01. e Western blot analysis of the expression of TDO2, Akt, pAkt, Erk1/2, and pErk1/2 in 786-O cells transfected with siRNA1, siRNA2, and siRNA negative control. GAPDH served as an internal control. The western blot bands were cropped and full-length blots/gels were presented in Fig. S9, Fig. S10