LRPPRC regulates redox homeostasis via the circANKHD1/FOXM1 axis to enhance bladder urothelial carcinoma tumorigenesis

Abstract

Reactive oxygen species (ROS) which are continuously generated mainly by mitochondria, have been proved to play an important role in the stress signaling of cancer cells. Moreover, pentatricopeptide repeat (PPR) proteins have been suggested to take part in mitochondrial metabolism. However, the mechanisms integrating the actions of these distinct networks in urothelial carcinoma of the bladder (UCB) pathogenesis are elusive. In this study, we found that leucine rich pentatricopeptide repeat containing (LRPPRC) was frequently upregulated in UCB and that it was an independent prognostic factor in UCB. We further revealed that LRPPRC promoted UCB tumorigenesis by regulating the intracellular ROS homeostasis. Mechanistically, LRPPRC modulates ROS balance and protects UCB cells from oxidative stress via mt-mRNA metabolism and the circANKHD1/FOXM1 axis. In addition, the SRA stem-loop interacting RNA binding protein (SLIRP) directly interacted with LRPPRC to protect it from ubiquitination and proteasomal degradation. Notably, we showed that LRPPRC modulated the tumorigenesis of UCB cells in a circANKHD1-FOXM1-dependent manner. In conclusion, LRPPRC exerts critical roles in regulating UCB redox homeostasis and tumorigenesis, and is a prognostic factor for UCB; suggesting that LRPPRC may serve as an exploitable therapeutic target in UCB.

Keywords: FOXM1; LRPPRC; ROS; Urothelial carcinoma of the bladder; circRNA.

Graphical abstract

Fig. 1

LRPPRC is overexpressed in UCBs and correlates with patient poor prognosis. (A) Bioinformatics analysis of PPR family members in UCB. LRPPRC was screened as a candidate that had potential oncogenic functions in UCB. N, non-neoplastic bladder tissues; T, tumor tissue. (B) The expression levels of LRPPRC (levels of mRNA expression) in paired UCB tissues and non-neoplastic tissues in the TCGA and SYSUCC cohort. (C) Prognostic significance of LRPPRC in 408 UCB patients (TCGA cohort) assessed by Kaplan–Meier analyses. (D) Western blot analysis of LRPPRC expression in 10 UCB tissues. N, non-neoplastic bladder tissues; T, tumor tissue. Expression of α-Tubulin was used as a loading control. (E) Western blot analysis of LRPPRC expression in 7 human UCB cell lines (ie, T24, J82, TCCSUP, UM-UC-3, SW-870, 5637 and RT4) and a non-tumorigenic cell line, SV-HUC-1. (F) Quantification of immunohistochemical analysis of LRPPRC expression (IHC score) in 30 primary UCBs and matched adjacent non-neoplastic tissues. (G) Representative images of IHC staining of LRPPRC in non-neoplastic bladder tissues and UCB tumor tissues. The normal bladder tissue and one UCB tissue (case 56) had low expression of LRPPRC, whereas another UCB tissue (case 31) had high expression of LRPPRC. The IHC scores were quantified by the HALO image analysis platform. Scale bar, 100 μm. (H) Kaplan-Meier analysis indicating an association between LRPPRC high-expression and poor OS rates in UCB patients (SYSUCC cohort, n = 224 cases).

Fig. 2

LRPPRC regulates cell proliferation and apoptosis in UCB cells. (A) Western blotting reveals that LRPPRC was efficiently knocked down and overexpressed in corresponding cells. (B-C) CCK-8 assays of the cell growth ability of the indicated UCB cells. (D–E) Colony formation assays of the indicated UCB cells. Columns: mean ± standard deviation (SD) of three independent experiments. (F–G) EDU assays of the indicated UCB cells. Columns: mean ± standard deviation (SD) of three independent experiments. (H-I) Apoptosis of UCB cells with modulation of LRPPRC expression was detected by flow cytometric analysis as indicated. Columns: mean ± standard deviation (SD) of three independent experiments. (J) Down expression of LRPPRC in the T24 cell line substantially repressed tumor formation compared with T24-NC cells. Left, images of the xenograft tumors formed in Balb/c nude mice injected with T24-NC or T24-shLRPPRC cells. Right, weights of xenograft tumors (n = 5). **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 3

LRPPRC regulates apoptosis and oxidative stress in UCB cells. (A) Volcano plot comparing the global gene expression profiles of T24 cells transfected with LRPPRC-sh2 and sh-NC. (B) GSEA analysis showing LRPPRC-related KEGG pathways in T24 cells (T24-NC vs T24-shLRPPRC). (C) GO analysis was performed using genes with |log2(fold change)|>1 by comparing T24-NC with T24-shLRPPRC. The five most involved GO terms are displayed. (D) 12 mRNAs encoded by mitochondrial (mtRNAs, mt-CO1,mt-CO2,mt-CO3,mt-CYB, mt-ND1,mt-ND2, mt-ND3, mt-ND4, mt-ND4L, mt-ND6, mt-ATP6, and mt-ATP8) and FOXM1 were downregulated in T24-shLRPPRC cells compared with T24-NC cells. (E) The expression of 12 mt-mRNAs, FOXM1 and PRDX3 in T24-shLRPPRC cells were assessed by qRT-PCR and presented as fold change relative to T24-NC cells. Columns: mean ± standard deviation (SD) of three independent experiments, ****P < 0.0001. (F) LRPPRC expression was positively correlated with the expression of FOXM1 (left) and PRDX3 (right) in UCB patients derived from the TCGA database. (G) Western blots comparing LRPPRC-silenced and LRPPRC-overexpressing UCB cells with their respective control cells are shown for relative expression of FOXM1, PRDX3, MnSOD and Catalase. α-Tubulin expression was used as a loading control. (H) ΔΨm of T24-NC and T24-shLRPPRC was assessed by JC-1 staining (Top). JC-1 monomers (green) and aggregates (red) were detected by confocal microscopy. Scale bar, 10 μm. Mitochondrial ROS (mROS) and cytosolic ROS (cROS) were detected using fluorescent dyes of MitoSOX and DCFH-DA (Bottom). Scale bar, 10 μm. Fluorescence intensity was analyzed by Image J. Columns: mean ± standard deviation (SD) of three independent experiments, ***P < 0.001. (I) Western blots comparing LRPPRC-silenced and LRPPRC-overexpressing UCB cells with their respective control cells for relative expression of BCL-2, BAX, cleaved-caspase3, cleaved-caspase9, cleaved-PARP and caspase8. α-Tubulin expression was used as a loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

SLIRP forms a complex with LRPPRC to enhance its stability and to prevent LRPPRC from degradation. (A) LRPPRC interaction partners were detected in UM-UC-3 and T24 cells. Silver staining is used for the detection of differential protein bands. The band of LRPPRC and SLIRP are indicated by arrows. (B) Mass spectrometry identified SLIRP, which was pulled down from T24 cell lysates by LRPPRC (Top). The interaction between LRPPRC and SLIRP was confirmed by co-immunoprecipitation in T24 cells (Bottom). (C) Confocal staining presented the co-localization of LRPPRC and SLIRP in T24 cells. (D) Western blots revealed the regulatory relationship between LRPPRC and SLIRP. (E) siNC and siSLIRP were transfected in T24 cells, and 48 h later, the cells were treated with 10 μM of MG132 for various number of times and the cell lysates were immunoblotted as indicated. (F) T24-NC and T24-siSLIRP cells were treated with 50 μg/mL cycloheximide. Whole-cell lysates were harvested at the indicated times and the cell lysates were immunoblotted as indicated. (G) Flag-LRPPRC and HA-ubiquitin were transfected to T24-NC and T24-siSLIRP cells. The cells were treated with 10 μM MG132 for 8 h before harvest. Cell lysates were immunoprecipitated with Flag antibody and immunoblotted as indicated. (H) Colony formation assays show that knockdown of LRPPRC inhibited UCB cell proliferation capacity which was reversed by SLIRP overexpression. Error bars: mean ± SD of three independent experiments. ***P < 0.001; ****P < 0.0001.

Fig. 5

LRPPRC-induced upregulation of circANKHD1 regulates the expression of FOXM1 by reducing the inhibitory effect of miR-671-5p and miR-507 via sponge activity. (A) Clustered heatmap for the circRNA expression profiles of T24 cells transfected with LRPPRC-sh2 and sh-NC. The circRNAs are classified according to Pearson correlation analysis. The numerical data represent the serial number of circRNAs in circBase. (B) qRT-PCR for circANKHD1 in UCB cells treated with two LRPPRC shRNAs (Left). qRT-PCR for circANKHD1 in UCB cells transfected with control vector or LRPPRC overexpression plasmid (Right). Data are presented as fold change relative to T24-NC, TCC-SUP-NC or UM-UC-3-Vec cells ±SD of three experiments, **P < 0.01. (C) qRT-PCR for the abundance of circANKHD1 in UCB cells, compared with a non-tumorigenic cell, SV-HUC-1 (Left). Data are presented as fold change relative to SV-HUC-1. LRPPRC is positively associated with circANKHD1 in UCB cells (Right). (D) Verification that circANKHD1 is a circRNA, using divergent and convergent primers. Left, schematic illustration of circANKHD1 locus with specific primers. RT-PCR products with divergent primers showing circularization of circANKHD1. Right, Sanger sequencing to confirm the specific back splicing site of circANKHD1. (E) qRT-PCR for the abundance of circANKHD1 and ANKHD1 mRNA with the treatment of Rnase R in T24 cells. Data are presented as fold change relative to Mock T24 cells ±SD of three experiments, ****P < 0.0001. (F) qRT-PCR for the half-life analysis of circANKHD1 and ANKHD1 mRNAs with the treatment of actinomycin-D in T24 cells. Data are presented as fold change relative to the mRNA expression level at the time point of 0-h ± SD of three experiments, *P < 0.05. (G) Cytoplasmic and nuclear mRNA Fractionation experiment showing that circANKHD1 is mainly located in the cytoplasm. β-actin and U3 were applied as positive controls in the cytoplasm and nucleus, respectively. Data shown are the means ± SD of three experiments. (H) RNA fluorescence in situ hybridization for circANKHD1. Nuclei were stained with DAPI. Scale bar, 10 μm. (I) A schematic drawing showing the putative binding sites of the miRNAs associated with circANKHD1 and FOXM1. (J) Luciferase reporter assay for the luciferase activity of LUC-circANKHD1 or LUC-circANKHD1-mutant in T24 cells co-transfected with miRNA mimics. Data shown are the means ± SD of three experiments. **P < 0.01. (K) The expression levels of FOXM1 and PRDX3 were enhanced bycircANKHD1 overexpression but were inhibited by miR-671-5p or miR-507 mimics as observed in WB.

Fig. 6

LRPPRC promotes proliferation and attendances apoptosis is partially dependent on circANKHD1 in UCB cells. (A) qRT-PCR analysis verified that the expression of circANKHD1 and FOXM1, were repressed by the knockdown of LRPPRC and was greatly increased after transfected with ectopic circANKHD1 plasmid (Left). Data shown are presented as fold change relative to T24-NC cells ±SD of three experiments,**P < 0.01,***P < 0.001, ****P < 0.0001. Western blot assay showed that the reduction of FOXM1, PRDX3, MnSOD and Catalase, were largely reversed after the overexpression of circANKHD1 (Right). (B) ΔΨm of T24-NC, T24-shLRPPRC and T24-shLRPPRC-OE-circANKHD1 was assessed by JC-1 staining. JC-1 monomers (green) and aggregates (red) were detected by confocal microscopy. Scale bar, 10 μm. Mitochondrial ROS (mROS) and cytosolic ROS (cROS) were detected through fluorescent dyes of MitoSOX and DCFH-DA (Right). Scale bar, 10 μm. Fluorescence intensity was analyzed by Image J. Columns: mean ± standard deviation (SD) of three independent experiments, ***P < 0.001. (C) The suppressed colony formation of LRPPRC-silenced T24 cells were rescued by the overexpression of circANKHD1. Data shown are the means ± SD of three experiments. ***P < 0.001. (D) Overexpression of circANKHD1 reversed the increased apoptosis cells resulting from LRPPRC knockdown. Data shown are the means ± SD of three experiments. ****P < 0.0001. (E) Western blot assay showed that overexpression of circANKHD1 reversed the up-regulated expression of the intrinsic apoptosis markers cleaved-caspase3, cleaved-caspase9, cleaved-PARP and BAX, and the down-regulated expression of BCL-2, without affecting the expression of caspase8. (F) Representative images of organoids derived from UCB patients treated with shNC, shLRPPRC or shLRPPRC-OE-circANKHD1, Scale bar, 100 μm (Top). qRT-PCR (Middle) and western blotting assays (Bottom, Left) validated that the LRPPRC-knockdown induced downexpression of FOXM1 was rescued after the overexpression of circANKHD1 in LRPPRC-silenced organoid UCB cells. qRT-PCR data are presented as fold change relative to NC organoids ±SD of three experiments, ****P < 0.0001. The suppressed proliferation of LRPPRC-silenced organoids was rescued by the overexpression of circANKHD1 (Bottom, Right). (G) Orthotopic xenograft bladder models implanted with LRPPRC-NC, LRPPRC-silenced or LRPPRC-silenced-OE-circANKHD1 T24 cells in Balb/c nude mice. The bioluminescent images of orthotopic xenograft bladder tumors were imaged by the IVIS 200 imaging system 6 weeks after cells were implanted (Left). The inhibition effect to orthotopic xenograft bladder tumors, induced by LRPPRC-deletion could be restored by overexpression of circANKHD1 in vivo. Data shown are the means ± SD, **P < 0.01, *P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

LRPPRC regulates the circANKHD1/FOXM1 axis to promote cell growth in UCB. (A–B) The expression of FOXM1 and PRDX3 decreased after the knockdown of LRPPRC and were restored by the overexpression of circANKHD1 in orthotopic xenograft bladder tumors. Representative images of hematoxylin and eosin staining and IHC staining of FOXM1 and PRDX3 of orthotopic xenograft bladder tumors. Scale bar, 100 μm (A). IHC score of FOXM1 (Left) or PRDX3 (Right) in orthotopic xenograft bladder tumors as indicated. Data shown are the means ± SD, n = 5 (B). (C–D) The expression level of LRPPRC was positively correlated with that of circANKHD1 and FOXM1, and the expression level of circANKHD1 was positively correlated with that of FOXM1 in UCBs of the SYSUCC cohort. Representative images of IHC staining of LRPPRC, FOXM1 and PRDX3 in two UCB tissues with low or high expression of the three proteins. Scale bar, 100 μm (C). Spearman's correlation demonstrating that LRPPRC expression is positively correlated with circANKHD1 (detected by qRT-PCR) and FOXM1 (D). (E) Proposed model for the regulatory landscape of the LRPPRC/SLIPR/circANKHD1/FOXM1 signaling axis in promoting the pathogenesis of UCB. SLIRP forms a stable complex with LRPPRC to protect it from degradation, LRPPRC modulates ROS balance and protects UCB cells from oxidative stress via mt-mRNA metabolism and the circANKHD1/FOXM1 axis, thereby resulting in UCB cell growth.