UPP1 enhances bladder cancer progression and gemcitabine resistance through AKT

Abstract

UPP1, a crucial pyrimidine metabolism-related enzyme, catalyzes the reversible phosphorylation of uridine to uracil and ribose-1-phosphate. However, the effects of UPP1 in bladder cancer (BLCA) have not been elucidated. AKT, which is activated mainly through dual phosphorylation (Thr308 and Ser473), promotes tumorigenesis by phosphorylating downstream substrates. This study demonstrated that UPP1 promotes BLCA cell proliferation, migration, invasion, and gemcitabine resistance by activating the AKT signaling pathway in vitro and in vivo. Additionally, UPP1 promoted AKT activation by facilitating the binding of AKT to PDK1 and PDK2 and the recruitment of phosphatidylinositol 3,4,5-triphosphate to AKT. Moreover, the beneficial effects of UPP1 on BLCA tumorigenesis were mitigated upon UPP1 mutation with Arg94 or MK2206 treatment (AKT-specific inhibitor). AKT overexpression or SC79 (AKT-specific activator) treatment restored tumor malignancy and drug resistance. Thus, this study revealed that UPP1 is a crucial oncogene and a potential therapeutic target for BLCA and that UPP1 activates the AKT signaling pathway and enhances tumorigenesis and drug resistance to gemcitabine.

Keywords: AKT; UPP1; bladder cancer.; gemcitabine; metastasis.

Figure 1

UPP1 expression in BLCA. (A) Diagram of evidence sources of UPP1 as an essential prognostic biomarker. (B) Immunohistochemical staining of UPP1 in different stages of BLCA from the tissue microarray. (C) Relative expression in paired adjacent tissues and BLCA tissues from the tissue microarray. (D) Kaplan-Meier analysis of overall survival of samples in the tissue microarray. (E) The relative expression of UPP1 in BLCA cell lines was examined using qRT-PCR. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant (p > 0.05).

Figure 2

UPP1 deficiency suppresses BLCA cell proliferation, metastasis, and invasion in vitro and in vivo. (A) MTT assay results revealed that UPP1 knockdown inhibited the proliferation of UM-UC-3 cells. (B) Statistical chart of the colony formation assay results with UPP1 knockdown UM-UC-3 cells. (C) The statistical chart of the flow cytometric analysis revealed that UPP1 knockdown promoted cell cycle arrest at the G1 phase. The weight (D) and volume (E) of tumors derived from shNC-transfected or shUPP1-transfected UM-UC-3 cells. (F-G) Statistical chart of the results of the transwell assay with UPP1 knockdown UM-UC-3 cells. (H) Statistical chart of the wound healing assay results with UPP1 knockdown UM-UC-3 cells. Fluorescence intensity (I) and H&E staining (J) of the lung from the BALB/c nude mouse metastasis model injected with Vector-transfected and UPP1-transfected T24 cells. (K) Immunoblotting analysis revealed that UPP1 knockdown downregulated the expression of EMT-related and cell cycle-related proteins in UM-UC-3 cells. UPP1 overexpression promoted the expression of these proteins in T24 cells. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant (p > 0.05).

Figure 3

UPP1 knockdown upregulates cellular ROS levels and promotes apoptosis in BLCA through the AKT signaling pathway in vitro and in vivo. (A) Flow cytometric analysis revealed that UPP1 knockdown upregulated ROS levels in UM-UC-3 cells. (B) Statistical chart of the apoptosis rates of UM-UC-3 cells evaluated using flow cytometry. (C) GSEA based on TCGA dataset. (D) Immunoblotting analysis revealed that UPP1 knockdown suppressed the phosphorylation of AKT at Ser473 and Thr308, downregulated anti-apoptotic proteins, and upregulated pro-apoptotic proteins in UM-UC-3 cells. UPP1 overexpression reversed the expression trend of these proteins in T24 cells. (E) H&E and IHC staining of subcutaneous Vector-transfected and UPP1-transfected T24 cell-derived xenograft tumors. (F) Immunoblotting indicates that UPP1 overexpression could promote AKT phosphorylation and inhibit apoptosis of BLCA cells in vivo. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant (p > 0.05).

Figure 4

Inhibition of AKT rescues the stimulating effect of UPP1 overexpression in BLCA cells. (A) Frame diagram of rescue experiments. (B) MTT assay results revealed that treatment with MK2206 (10 μM) suppressed the UPP1-induced upregulation of T24 cell proliferation. (C) The colony formation assay results revealed that MK2206 (10 μM) mitigated the UPP1-induced upregulation of T24 cell proliferation. (D-E) The statistical chart of the transwell assay results indicates that MK2206 (10 μM) suppressed the UPP1-induced upregulation of migration and invasion in T24 cells. (F) Immunoblotting analysis revealed that UPP1-induced AKT activation was mitigated upon treatment with MK2206 (10 μM). *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant (p > 0.05).

Figure 5

AKT overexpression rescues the inhibitory effect of UPP1 knockdown in BLCA cells. (A) MTT assay results with UPP1 knockdown and AKT-WT-overexpressing SCaBER cells. (B) The results of the transwell assay with UPP1 knockdown and AKT-WT-overexpressing SCaBER cells. (C) Immunoblotting analysis of the effect of UPP1 knockdown and AKT-WT overexpression on the expression of related proteins in SCaBER cells. (D) The results of the MTT assay examine the effect of AKT-WT and AKT-CA overexpression in UPP1 knockdown UM-UC-3 cells. (E) The results of the transwell assay examining the effect of AKT-WT and AKT-CA overexpression in UPP1 knockdown UM-UC-3 cells. (F) Immunoblotting analysis of the expression of related proteins in UPP1 knockdown, AKT-WT-overexpressing, and AKT-CA-overexpressing UM-UC-3 cells. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant (p > 0.05).

Figure 6

UPP1 interacts with the C-terminus of AKT. (A) Immunofluorescence analysis of the cytoplasmic co-localization of UPP1-Flag and AKT-HA in UM-UC-3 cells. (B) Endogenous co-IP analysis indicates that UPP1 interacted with AKT in UM-UC-3, SCaBER, T24, and 5637 cell lines. (C) GST pull-down assay results revealed that UPP1 interacted with AKT in vitro. (D) Exogenous co-IP analysis demonstrated that UPP1 interacted with the C-terminus of AKT in HEK-293T cells.

Figure 7

UPP1 promotes AKT activation through direct interaction. (A) Schematic for site mutation of UPP1. (B) Quantitative exogenous co-IP analysis indicates that UPP1-R94A overexpression did not promote AKT phosphorylation in HEK-293T cells. (C) The results of MTT assay with T24 cells overexpressing UPP1-WT or UPP1-R94A. SC79 (20 μM) promoted the proliferation of T24 cells. (D) UPP1-WT or UPP1-R94A overexpression did not promote cell cycle transition in T24 cells. SC79 (20 μM) promoted cell cycle transition in T24 cells. (E) The results of the colony formation assay with T24 cells overexpressing UPP1-WT or UPP1-R94A. SC79 (20 μM) increased the colony numbers of T24 cells. (F) ROS levels in cells overexpressing UPP1-WT or UPP1-R94A. SC79 (20 μM) significantly downregulated the intercellular ROS level. (G-H) The results of the transwell assay with T24 cells overexpressing UPP1-WT or UPP1-R94A. SC79 (20 μM) promoted the metastasis of T24 cells. (I) The relative AKT activity in T24 cells overexpressing UPP1-WT or UPP1-R94A. (J) UPP1-WT promoted the binding of AKT to PDK1 in HEK-293T cells. (K) UPP1-WT facilitated phosphatidylinositol 3,4,5-triphosphate (PIP₃) recruitment to AKT. (L) UPP1-R94A did not promote PIP₃ recruitment to AKT. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant (p > 0.05).

Figure 8

UPP1 knockdown promotes gemcitabine chemosensitivity through the AKT/FOXO1/DCK signaling pathway. (A) MTT assay results revealed that UPP1 knockdown promoted gemcitabine chemosensitivity in SCaBER cells. (B) UPP1 knockdown significantly increased the apoptosis rate in SCaBER cells. (C) Immunoblotting analysis revealed that gemcitabine significantly downregulated AKT phosphorylation and upregulated pro-apoptotic protein expression in UPP1 knockdown SCaBER cells. UPP1 overexpression suppressed the gemcitabine-induced inhibition of AKT phosphorylation and downregulated pro-apoptotic protein expression in gemcitabine-treated 5637 cells. The effect of gemcitabine on the volume (D) and viability (E) of shUPP1-transfected and shNC-transfected UM-UC-3 cell-derived xenograft tumors. (F) Analysis of the protein levels in xenograft tumors of different groups. (G) Schematic showing the mechanism by which UPP1 regulates biological status via the AKT signaling pathway. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant (p > 0.05).