RUNX2 enhances bladder cancer progression by promoting glutamine metabolism

Abstract

Bladder cancer is a prevalent malignancy within the urinary system. Prior research has suggested that glutamine metabolism plays a crucial role in driving bladder cancer progression. However, the precise molecular mechanism governing glutamine metabolism in bladder cancer is still inadequately understood. The research revealed a significant correlation between high levels of RUNX2 and SLC7A6 and advanced clinical stage, as well as poor prognosis, in bladder cancer patients. Furthermore, manipulating the levels of RUNX2 through overexpression or silencing demonstrated a significant impact on glutamine and bladder cancer progression. Mechanically, RUNX2 regulates the transcription of SLC7A6, resulting in enhanced glutamine metabolism and promoting the progression of bladder cancer. Overall, this research affirms the crucial function of RUNX2 as a key transcription factor to promoting glutamine and cancer development through modulation of SLC7A6. Targeting RUNX2 could represent a promising therapeutic approach for addressing aberrant glutamine metabolism in bladder cancer.

Keywords: Bladder cancer; Glutamine metabolism; RUNX2.

Fig. 1

RUNX2 plays a crucial effect in regulating glutamine metabolism in bladder cancer. (A) The Venn diagram presenting a comprehensive analysis of crucial glutamine metabolism genes and potentially associated TFs in bladder cancer based on the TCGA-BLCA and Animal TFDB databases, respectively. (B-C) The expression of RUNX2 between the tumor and normal (B), as well as between the Ⅲ-Ⅳ and Ⅰ-Ⅱ stage (C) in TCGA-BLCA, respectively. (D) The K-M curve illustrating the overall survival of RUNX2 according to TCGA-BLCA (cutoff value = 10.774787). (E) RT-qPCR assay revealing the mRNA level of three TFs between normal urothelial cell line and bladder cancer cell lines (n = 5). (F) Western blot indicating the protein level of RUNX2 between normal urothelial cell line and bladder cancer cell lines (n = 5). (G-H) RT-qPCR (G, n = 5) and western blot (H, n = 5) revealing the expression of RUNX2, SLC1A6, and SLC7A6 in HT-1376 and SW1710, respectively. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 2

RUNX2 promotes glutamine uptake and metabolism by regulating the expression of SLC7A6 in HT-1376 cells. (A-J) The relative Gln (A, F),α-KG (B, G), ATP (C, H), GSH (D, I), and ROS (E, J, captured with fluorescence microscopy, scale bar: 100 μm) levels in HT-1376 cells were evaluated by using the appropriate assay kits (n = 5), respectively. * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 3

RUNX2 enhances bladder cancer progression. (A-D) The CCK-8 and Edu assays revealing the cell viability and proliferation ability of HT-1376 (A, C, Scale bar: 100 μm) and SW1710 (B, D, Scale bar: 100 μm), respectively (n = 5). (E-F) The transwell migration and invasion assays revealing the cell migration and invasion ability of HT-1376 (E, Scale bar: 100 μm) and SW1710 (F, Scale bar: 100 μm), respectively (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 4

RUNX2 stimulates bladder cancer progression rely on SLC7A6. (A-D) CCK-8 and Edu assays assessing the cell viability and proliferation ability of HT-1376 (A, C, Scale bar: 100 μm) and SW1710 (B, D, Scale bar: 100 μm) cells, respectively (n = 5). (E-H) The migration and invasion assays revealing the cell migration and invasion capability of HT-1376 (E, G, Scale bar: 100 μm) and SW1710 (F, H, Scale bar: 100 μm) cells, respectively (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 5

RUNX2 regulates the transcriptional activity of SLC7A6. (A-B) The subcellular localization of the RUNX2 protein in cells through the HPA database (www.proteinatlas.org). (C-D) Dual-luciferase assay assessing the relative activity of luciferase reporters in HT-1376 (C) and SW1710 (D) cells, respectively (n = 3). (E) ​A diagram displaying the luciferase reporter constructs with either wild-type (WT) or mutant binding sites of RUNX2 in the promoter of SLC7A6 based on the AnimalTFDB 4.0. (F) A dual-luciferase assay was conducted to evaluate the luciferase reporter activity within the promoter region of SLC7A6 in HT-1376 cells (n = 3). (G) ChIP-qPCR were carried out to evaluate the endogenous binding of RUNX2 to the SLC7A6 promoter using immunoglobulin G (IgG, negative control) or anti-RUNX2 antibody in HT1376 cells (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 6

Suppressing of RUNX2 inhibits bladder cancer progression in vivo. (A-D) The picture of subcutaneous tumor xenograft (A), HE staining of xenograft tumor (B, scale bar: 100 μm), growth curve (C), weight (D), and Ki-67 IHC staining and percentage (E, scale bar: 100 μm) founded by injecting SW-1710 cells (n = 5 per group). (F) HE staining and the metastatic counts of lung tissue were conducted to assess the effects of RUNX2 depletion in the lung aggressiveness assay (n = 5). (G) K-M survival curves indicating the survival rates in the lung metastasis assay (n = 5 per group). * P < 0.05, ** P < 0.01, *** P < 0.001.

Fig. 7

Elevated expression of RUNX2 and its target gene SLC7A6 correlates with a negative prognosis in bladder cancer. (A-D) RT-qPCR (A, n = 20), western blot (B, n = 20), and IHC (C-D, n=147) were used to assess the expression levels of RUNX2 and SLC7A6 in bladder cancer tissues compared to adjacent normal tissues, respectively. (E) Scatter plots revealing the correlation between the expression of RUNX2 and SLC7A6 based on the IHC results. (F-G) The survival curve of RUNX2 (F, cutoff value= 0.47363) and SLC7A6 (G, cutoff value= 0.45748) based on the IHC results. (H) A schematic diagram: RUNX2 enhances the transcription and expression of SLC7A6 by directly binding to its promoter, promotes the uptake of glutamine, resulting in increasing intracellular levels of Gln,α-KG, ATP, and GSH levels, while decreasing intracellular ROS levels, ultimately facilitating the malignant progression of bladder cancer. * P < 0.05, ** P < 0.01, *** P < 0.001.