FAM171B stabilizes vimentin and enhances CCL2-mediated TAM infiltration to promote bladder cancer progression

Abstract

Background: Invasion and metastasis are the main causes of unfavourable prognosis in patients diagnosed with bladder cancer. The efficacy of immunotherapy in bladder cancer remains suboptimal due to the presence of an immunosuppressive microenvironment. The novel protein family with sequence similarity 171B (FAM171B) has been identified, but its precise role and mechanism in bladder cancer remain unclear.

Methods: In this study, we conducted an analysis to investigate the associations between FAM171B expression and the prognosis and clinicopathological stage of bladder cancer. To this end, we utilized RNA sequencing data from the TCGA and GEO databases, as well as tumor tissue specimens obtained from our clinical centre. RNA sequencing analysis allowed us to examine the biological function of FAM171B at the transcriptional level in bladder cancer cells. Additionally, we used immunoprecipitation and mass spectrometry to identify the protein that interacts with FAM171B in bladder cancer cells. The effects of FAM171B on modulating tumor-associated macrophages (TAMs) and vimentin-mediated tumor progression, as well as the underlying mechanisms, were clarified by phalloidin staining, immunofluorescence staining, ELISA, RNA immunoprecipitation, flow cytometry and a bladder cancer graft model.

Results: FAM171B expression exhibits strong positive correlation with poor survival outcomes and advanced clinicopathological stages in patients with bladder cancer. FAM171B significantly promoted bladder cancer growth and metastasis, accompanied by TAM accumulation in the microenvironment, in vivo and in vitro. Through studies of the molecular mechanism, we found that FAM171B contributes to tumor progression by stabilizing vimentin in the cytoplasm. Additionally, our research revealed that FAM171B enhances the splicing of CCL2 mRNA by interacting with heterogeneous nuclear ribonucleoprotein U (HNRNPU), ultimately leading to increased recruitment and M2 polarization of TAMs.

Conclusions: In this study, we identified FAM171B as a potent factor that promotes the progression of bladder cancer. These findings establish a solid theoretical foundation for considering FAM171B as a potential diagnostic and therapeutic biomarker for bladder cancer.

Keywords: Bladder cancer; CCL2; Macrophage; Vimentin.

Fig. 1

FAM171B expression is positively correlated with tumor progression and M2 TAM infiltration in BLCA patients. A Analysis of FAM171B expression in tumor tissue and paired normal tissue from TCGA dataset. B Representative IHC staining images of FAM171B in BLCA tissue array. C IHC score in different T stages from BLCA tissue array. D IHC score in different N stages from BLCA tissue array. E Relationship between FAM171B expression level and overall survival from TCGA dataset. F Relationship between FAM171B expression level and progression free survival from TCGA dataset. G Relationship between FAM171B expression level and disease specific survival from TCGA dataset. H Relationship between FAM171B expression level and disease-free survival from TCGA dataset. I Relationship between FAM171B expression level and overall survival from GEO dataset (GSE13507/GSE31684/GSE32894/GSE37815). J Analysis of FAM171B expression in different T stages from TCGA dataset. K Analysis of FAM171B expression in different N stages from TCGA dataset. L Analysis of FAM171B expression in different pathologic stages from TCGA dataset. M Lollipop plot demonstrating the correlation between FAM171B expression and different immune cell infiltration. N Heat map of the correlation between FAM171B expression and major monocyte/macrophage chemokines. O Immunoscore analysis of FAM171B and M1 macrophages in bladder cancer using QUANTISEQ algorithm. P Immunoscore analysis of FAM171B and M2 macrophages in bladder cancer using QUANTISEQ algorithm. Q Representative IHC staining images (FAM171B) and IF staining images (CD206, CD8 and α-SMA) in BLCA tissue array. R Proportion of immunofluorescence-positive cells (CD206, CD8 and α-SMA) in FAM171B high or low expression group from tissue array

Fig. 2

FAM171B promotes the malignant phenotype of bladder cancer cells. A The Chronos dependency score analysis of FAM171B in the 28 human bladder urothelial carcinoma cell lines in the CCLE database. The Chronos dependency score is based on data from a cell depletion assay. A lower Chronos score indicates a higher likelihood that the gene of interest is essential in a given cell line. B Protein levels of FAM171B in the knockdown and overexpression cell lines. C Representative colony formation images in different groups. D Colony formation analysis of T24 and MB49 cells after knockdown and overexpression of FAM171B. E Cell viability of T24 and MB49 after knockdown and overexpression of FAM171B. F Representative transwell images of T24 and MB49 cells after knockdown and overexpression of FAM171B. G Statistical results of the number of invasive cells in each group of T24 and MB49 cells. H Representative images of migration in T24 and MB49 cells after knockdown and overexpression of FAM171B. I Statistical results of the number of migrated cells in each group of T24 and MB49 cells

Fig. 3

RNA sequencing of T24 cells to investigate the function of FAM171B. A Heatmap of the difference in overall mRNA expression between the FAM171B overexpression T24 cells (F1, F2 and F3) and the control T24 cells (N1, N2 and N3). The Pearson’s correlation coefficient was represented by a color scale. B Volcano plot of the differential gene expression between the FAM171B overexpression group and the control group. The vertical gray lines corresponded to two-fold up- and down-regulation, and the horizontal gray line represented a p value of 0.01. The red points represented significantly up-regulated gene, and the blue points represented significantly down-regulated gene. C Heatmap of specific differential genes between the FAM171B overexpression T24 cells and the control cells. D, E, F Histogram of GO annotations for the differential genes in biological processes (D), cellular components (E) and molecular functions (F). X axis represented the number of genes. Y axis showed the name of GO terms. G Histogram of KEGG analysis for the differential genes. X axis represented the number of genes. Y axis showed the name of enriched pathways. The p-value was represented by a color scale. The statistical significance increased from purple (relatively lower significance) to orange (relatively higher significance). H Histogram of eggNOG functional classification statistics for differentially expressed genes. X axis represented the function of the gene. Y axis showed the frequency (the number of genes)

Fig. 4

FAM171B interacts with vimentin and HNRNPU in bladder cancer cells. A Mass spectrometry analysis of interacting proteins enriched by immunoprecipitation. Immunoprecipitation is performed by Anti-DYKDDDDK beads on T24 cells transfected with FLAG-FAM171B or FLAG-control. B Analysis of positive and background proteins based on Mass Spectrometry scores. C The top 16 proteins with the highest scores based on MS results. D Protein interaction network map of proteins bound to FAM171B based on MS results. The protein interactions data is from STRING database. E The predicted protein structure image of FAM171B based on the Alpha Fold. F Immunofluorescence analysis images revealed the localization of FAM171B in T24 cells. G Colocalization of FAM171B and vimentin was visualized by confocal microscope in T24 cells. Cytoplasmic staining of FAM171B and vimentin was mostly merged together. H Colocalization of FAM171B and HNRNPU was visualized by confocal microscope in T24 cells. Nuclear staining of FAM171B and HNRNPU was mostly merged together. I ImageJ was used for colocation analysis of FAM171B and vimentin. Pearson correlation analysis showed that r > 0.5. Manders’ colocation coefficient showed that M1 > 0.5 and M2 > 0.5. J Computational docking model between FAM171B and vimentin. K ImageJ was used for colocation analysis of FAM171B and HNRNPU. Pearson correlation analysis showed that r > 0.5. Manders’ colocation coefficient showed that M1 > 0.5 and M2 > 0.5. L Computational docking model between FAM171B and HNRNPU

Fig. 5

FAM171B stabilizes vimentin to promote the progression of bladder cancer. A Protein levels of FAM171B, vimentin, E-cadherin and N-cadherin in the FAM171B knockdown and overexpression T24 cell lines. B Protein levels of FAM171B and vimentin in the T24 cell lines in different groups. C Representative transwell images of T24 cells in different groups. D Statistical results of the number of invasive cells in each group of T24 cells. E Representative images of migration in T24 cells in different groups. F Statistical results of the number of migrated cells in each group of T24 cells. G Pictures of T24 cells in different groups. H Protein levels of FAM171B and vimentin in the T24 cells with or without FAM171B overexpression after treatmented with CHX (20 μg/ml) for various times. I Remaining vimentin protein level percentages in the T24 cells with or without FAM171B overexpression after treated with CHX for various times. J The western blot images showed the FAM171B regulated vimentin ubiquitination in T24 cells. Vimentin proteins were isolated from the FAM171B knockdown or overexpression T24 cells by Co-IP, and followed detected the ubiquitination of vimentin by western blot. input, whole cell lysate. K Representative IHC staining images of FAM171B and vimentin in FAM171B high or low expression group from tissue array. L Correlation analysis of IHC scores for FAM171B and vimentin from tissue array. M The pictures of tumors derived from human bladder cancer T24 cell line. N IHC staining pictures of FAM171B and vimentin expression and HE staining pictures in different groups. O The tumor volume in different groups. P The tumor weight in different groups. Q The average body weight of mice in different groups. R Analysis of FAM171B IHC score in tumors. S Analysis of vimentin IHC score in tumors

Fig. 6

FAM171B regulates CCL2 via HNRNPU and promotes TAM migration and infiltration in bladder cancer. A ELISA analysis of CCL2 secretion levels in MB49 cells with FAM171B overexpression or knockdown. B RIP results show that HNRNPU can immunoprecipitate with CCL2 precursor RNA. C qRT-PCR analysis of the relative RNA levels of precursor CCL2 and CCL2 in MB49 cells with HNRNPU knockdown or control. D ELISA analysis of CCL2 secretion levels in MB49 cells with HNRNPU knockdown or control. E Protein levels of FAM171B and HNRNPU in the MB49 cell lines in different groups. F ELISA analysis of CCL2 secretion levels in MB49 cells in different groups. G Analysis of migration of RAW264.7(mouse macrophages) toward MB49 cells in different groups. H Representative images of migration of RAW264.7(mouse macrophages) toward MB49 cells in co-culture chambers. I The pictures of tumors derived from mouse bladder cancer MB49 cell line. J IHC staining pictures of F4/80(macrophage marker) expression in different groups. K Representative images of flow cytometry used to evaluate F4/80 positive subpopulations. L The tumor weight in different groups. M The tumor volume in different groups. N The average body weight of mice in different groups. O Analysis of F4/80 positive population by IHC. P Analysis of F4/80 positive population by flow cytometry

Fig. 7

FAM171B regulates CCL2 expression to enhance TAM polarization towards the M2 phenotype. A Representative images of flow cytometry used to evaluate CD206 positive subpopulations in RAW264.7 co-cultured with MB49 cells in different groups. B Analysis of CD206 positive population by flow cytometry. C Representative images of immunostaining of Arg1 (M2 marker) in RAW264.7 co-cultured with MB49 cells. D Analysis of Arg1 positive population by immunostaining. E Analysis of EGF (M2 marker) secretion levels in RAW264.7 co-cultured with MB49 cells in different groups. F Analysis of IL-10 (M2 marker) secretion levels in RAW264.7 co-cultured with MB49 cells in different groups. G Representative images of flow cytometry used to evaluate CD206 and F4/80 positive subpopulations in MB49 subcutaneous graft tumor in different groups. H Analysis of CD206 and F4/80 positive population in MB49 subcutaneous graft tumor by flow cytometry. I Analysis of Arg1 and F4/80 positive population in MB49 subcutaneous graft tumor by immunostaining. J Representative images of immunostaining of Arg1 (M2 marker) and F4/80 (macrophage marker) in MB49 subcutaneous graft tumor in different groups

Fig. 8

Schematic illustration. Schematic illustration of the functional roles of FAM171B in stabilizing vimentin and enhancing M2-TAM infiltration by HNRNPU/CCL2 and leading the malignant progression of bladder cancer