BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (PtenΔ/Δ BRF1Tg) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In PtenΔ/Δ BRF1Tg tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis.
The BRF1 transcription factor functions together with TATA-binding protein and B double-prime 1 in a complex called TFIIIB. BRF1 is required to recruit RNA polymerase III (Pol III) to target genes and is rate-limiting for Pol III-mediated transcription . Increased protein synthesis plays a key role in oncogenesis and Pol III products, including transfer RNAs (tRNAs) and other short non-coding RNAs, such as the ribosomal component 5S RNA, are essential for protein synthesis. Major components of the ribosome (ribosomal proteins e.g. RPS-19, -21, -24 and ribosomal RNA) are upregulated in prostate cancer (PCa) [2, 3]. BRF1 expression is elevated and associated with poor prognosis in hepatocellular, breast and gastric cancers [4,5,6] but its role in PCa remains unclear.
PCa is the third most common cause of cancer-associated death in men worldwide and progression is unpredictable . Patients with similar tumour grade and histology can quickly progress to incurable, advanced metastatic disease or alternatively they can survive for decades with local, indolent, disease . Evasion of surveillance and recognition by the innate and adaptive immune system are implicated in PCa progression  and it has been proposed that oncogenic drivers can contribute to immunoresistance in PCa . There is an unmet need to better understand these biological processes in PCa which tends to resist available immune checkpoint inhibitors.
The complement pathway links innate and adaptive immunity to coordinate appropriate immune responses . Complement activation occurs via the classical, lectin or alternative pathways , culminating in the formation of the membrane attack complex (MAC) (composed of C5b-C9) which punctures cell membranes to cause cytolysis. The concentration of C7 at sites of complement activation is a limiting factor for MAC formation . Complement activation recruits neutrophils, monocytes and macrophages to sites of inflammation , promotes opsonisation of B-cell responses  and enhances T-cell responses (activation, differentiation, polarisation and apoptosis) [11, 12].
Here, we show that elevated BRF1 associates with poor prognosis in PCa and accelerates prostate tumorigenesis in a new genetically engineered mouse model. For the first time, we demonstrate that elevated BRF1 expression in the prostatic epithelium can impact upon the tumour microenvironment in vivo.
BRF1 is elevated in prostate cancer and confers a poor prognosis
BRF1 immunoreactivity was studied in 516 PCa cases and 134 benign prostatic hyperplasia (BPH) controls. Nuclear BRF1 immunoreactivity (Fig. 1a) was significantly upregulated in PCa (p = 0.0032) (Fig. S1a, Table S1) and in clinically significant disease (Gleason sum score = 7 and > 7: p = 0.0039 and 0.0091, respectively) (Fig. S1b, Table S2) when compared with BPH. Elevated BRF1 expression correlated with shorter disease-specific survival (5.39 vs. 7.81 years; p = 0.012) (Fig. 1b, Table S3). Utilising the Memorial Sloan-Kettering Cancer Center (MSKCC) (2010)  and The Cancer Genome Atlas (TCGA) (provisional) prostate adenocarcinoma datasets in cBioPortal [16, 17], elevated BRF1 expression also associated with poor outcome (Figs. 1c, d and S1c, d; Tables S4 and S5).
BRF1 drives in vitro cellular proliferation and cell cycle progression
BRF1 expression was assessed in human PCa cell lines (Fig. S1e). Ectopic overexpression of BRF1, using HA-BRF1 or eGFP-BRF1 plasmids, mediated a modest but significant increase in cell proliferation in PC3, PC3M and DU145 cells by WST1 assay (Figs. 1e, S2a and S3a; Table S6). Conversely, downregulation of BRF1 expression in PCa cells (with at least two out of three independent siRNAs) reduced proliferation (Figs. 1f, S2b and S3b; Table S7), with FACS analysis revealing reduced G1 subpopulation and increased G2 phase (Fig. 1g, h). Collectively, manipulation of BRF1 expression alters proliferation in vitro.
Increased BRF1 accelerates prostate tumourigenesis in vivo
An in vivo model with prostate epithelium-specific overexpression of BRF1 was generated by inter-crossing BRF1 mice  with the Pbsn (PB)-Cre4 strain  to generate PB-Cre4:BRF1 (herein referred to as BRF1Tg) mice (see Supplementary methods; Fig. 2a). Male BRF1Tg mice were aged to over 1 year, and no malignancy or alteration in prostate architecture was observed (n = 12 WT; n = 12 BRF1Tg) (Fig. 2b). We then crossed BRF1Tg with Ptenfl/fl  mice to generate prostate epithelium-specific double mutant PB-Cre4:Ptenfl/fl BRF1 (herein referred to as PtenΔ/Δ BRF1Tg) mice. Survival of PB-Cre4:Ptenfl/fl (herein referred to as PtenΔ/Δ) control mice was consistent with our previous report . PtenΔ/Δ BRF1Tg mice had significantly shorter disease-specific survival compared with PtenΔ/Δ siblings (median 256 vs 316 days, respectively; p < 0.0001) (Fig. 2c, Table S8), with comparable endpoint tumour weights (Fig. 2d, Table S9) despite being harvested earlier. Interestingly, whilst high BRF1 protein levels were observed in all PtenΔ/Δ BRF1Tg tumours, some tumours from PtenΔ/Δ mice also had elevated BRF1 expression (Fig. 2e). Quantitative real-time PCR (qPCR) was performed using primers specific for human BRF1 confirmed its overexpression in PtenΔ/Δ BRF1Tg prostate tumours (p = 0.0459) (Fig. 2f). PtenΔ/Δ and PtenΔ/Δ BRF1Tg tumours were found to be histologically similar (Fig. 2g). High BRF1 expression was found in the prostate epithelium of BRF1Tg (Fig. 2b) and PtenΔ/Δ BRF1Tg (Fig. 2h) mice as expected. Although levels of Ki67 and cleaved caspase-3, markers for proliferation and apoptosis, respectively, were similar (Fig. S4a, b), we observed a significant reduction in the expression of the cell cycle inhibitor p21 in PtenΔ/Δ BRF1Tg tumours (p = 0.0151) (Fig. 2h, i), consistent with the cell cycle data in Fig. 1g, h. Together these data suggest that increased expression of BRF1 alone does not drive PCa but can co-operate with Pten loss.
Pten Δ/Δ BRF1 Tg prostate tumours harbour alterations in immune system processes
RNA-Sequencing (RNA-Seq) identified 522 significantly altered genes (fold change > 1.5; p.adj < 0.05) between PtenΔ/Δ and PtenΔ/Δ BRF1Tg endpoint tumours, with 174 coding genes involved in immune processes (see heatmap in Fig. 3a; Table S10)—these are predominantly downregulated in PtenΔ/Δ BRF1Tg tumours. Gene enrichment analysis further identified immune system processes, inflammatory response and leukocyte chemotaxis amongst the most overrepresented biological processes (Fig. 3b and Table S11).
Untargeted proteomics analysis on the PtenΔ/Δ and PtenΔ/Δ BRF1Tg tumours revealed that the abundance of 300 proteins was significantly (fold change > 1.5; p.adj < 0.05) altered, with 52 of these being related to immune processes (Fig. 3c, Table S12). Pathway enrichment analysis subsequently revealed that 7 of the top 10 most overrepresented biological processes related to immune responses (Fig. 3d and Table S13).
Finally, analysis of the top 10 overrepresented pathways (Table S14) in both RNA-Seq and proteomics again pointed towards altered immune status as a key difference between PtenΔ/Δ and PtenΔ/Δ BRF1Tg prostate tumours, highlighting aspects of both the innate immune response and the adaptive immune response.
Infiltration of neutrophils, CD3 and CD4 positive T lymphocytes is significantly reduced in Pten Δ/Δ BRF1 Tg (compared with Pten Δ/Δ) tumours
Analysis of innate immune cells showed no significant difference in the level of macrophages but neutrophils were significantly reduced (p = 0.0421) in the stroma (defined in Fig. S5a) of PtenΔ/Δ BRF1Tg versus PtenΔ/Δ prostate tumours (Figs. 3e, f and S5c). Consistent with our previous report  and clinical PCa , stromal tertiary lymphoid aggregates were present in murine tumours (Fig. S5b), neither the total area of these structures nor markers of B-cell response were unaltered (Figs. S6 and S7a, b). PtenΔ/Δ BRF1Tg tumours contained significantly fewer stromal CD3 and CD4 T cells (p = 0.0438, 0.0394, respectively) but stromal CD8 or FOXP3 (a marker for regulatory T cells) T cells were not altered (Figs. 3g–i, S5d, c).
Collectively, we identified reduced infiltration of neutrophils and CD4 positive T lymphocytes in PtenΔ/Δ BRF1Tg prostate tumours, in keeping with reduced levels of immune/inflammatory response genes (Fig. 3a) and expression of Cd4 (RNA-Seq data: log2FoldChange = −1.8203; p.adj = 0.0067; Table S8) in prostate tumours from PtenΔ/Δ BRF1Tg mice.
Pten Δ/Δ BRF1 Tg tumours have reduced complement pathway activation
We generated an in vitro model system by engineering PC3 clones to stably express eGFP-tagged BRF1 (BRF1 CL4, 5, 6), or eGFP empty vector (Ctrl CL1, 2) as controls. Parental PC3 and CL1 and CL2 cells exhibited similar protein synthesis (Fig. S8a) and so CL2 was thereafter used as a representative control. Overexpression of BRF1 (Fig. 4a) resulted in significantly increased protein synthesis [p.adj = 0.0132 (CL4); 0.0024 (CL5); 0.0047 (CL6)] (Fig. 4b), demonstrating BRF1 expression promoted Pol III function as expected.
Stable isotope labelling by amino acids in cell culture followed by mass spectrometry (Fig. S8b) on culture media from BRF1 overexpressing PC3 cells revealed 33 common proteins which had > 1.2 fold change in abundance in the same direction in CL4, CL5 and 6 compared with Ctrl CL2 (Fig. 4c; Table S15). Reduced levels of complement factor D (CFD) in the secretome of BRF1 overexpressing cells was validated (Fig. 4d) and this was particularly interesting because the complement pathway, including C7, an ‘endpoint’ component of the MAC, was highlighted from RNA-Seq and proteomic data (Tables S10 and S14). Reduced C7 mRNA (p = 0.0244) (Fig. 4e) and CFD and C7 protein expression (Fig. 4f) was validated in murine prostate tumours. Taken together, these data indicate that complement pathway activation is reduced in PtenΔ/Δ BRF1Tg tumours.
Low C7 levels are clinically relevant in human prostate cancer
In clinical PCa cBioPortal data, alterations in BRF1 and C7 significantly co-occurred when interrogating all (p = 0.002) and metastatic (p < 0.0001) tumour cases in MSKCC (2010) dataset (Fig. 4g, h). Segregating cases revealed that patients with low C7 expression (Fig. S8c, d) have a poorer prognosis in both datasets (Fig. 4i, j; Tables S16 and 17), in keeping with mouse model data. Furthermore, an inverse correlation between BRF1 and C7 mRNA expression and a positive correlation between CFD and C7 mRNA expression was found in both MSKCC (2010) and TCGA (provisional) datasets (BRF1 vs C7: Pearson correlation coefficient, r = −0.307; p = 0.0001 and r = −0.173; p = 0.0001, respectively; C7 vs CFD: Pearson correlation coefficient, r = 0.490; p < 0.0001 and r = 0.495; p < 0.0001, respectively) (Figs. 4k, S8e–g).
Complement activation can enhance the differentiation and activation of T lymphocytes . In both MSKCC (2010) and TCGA (provisional) datasets, a positive relationship between C7 and the status of either CD3E [MSKCC: Pearson correlation coefficient, r = 0.324; p < 0.0001; TCGA: Pearson correlation coefficient, r = 0.139; p = 0.0019] or CD4 [MSKCC: Pearson correlation coefficient, r = 0.356; p < 0.0001; TCGA: Pearson correlation coefficient, r = 0.205; p < 0.0001] (Fig. S8h–k) was observed, consistent with mouse model data.
In summary (Fig. 4l), BRF1 expression is elevated in clinical PCa and its overexpression promotes prostate carcinogenesis in an established GEM model in vivo. Aberrant complement pathway activation, as indicated by altered secretion of CFD in vitro and reduced levels of CFD and C7 in vivo, is mechanistically consistent with the observed altered immune infiltrates, including neutrophils and CD4 positive T cells, within the tumour microenvironment upon elevation of BRF1.
For the first time, we show that BRF1 expression drives prostate carcinogenesis in vitro and in vivo. Recent work has highlighted that the repertoire of tRNAs expressed in oncogenic contexts specifically supports the production of proteins for a proliferative program . Increases in specific tRNAs such as the initiator methionine tRNAiMet are sufficient to promote migration and invasive behaviour in melanoma cells  and for fibroblasts to secrete collagen, which in turn enhanced tumour growth and angiogenesis , suggesting that BRF1 may play a major role in orchestrating altered programs of tRNA and thus protein synthesis. In support of this, we found that increased expression of BRF1 upregulated global protein synthesis in vitro and mediated a switch in cellular programme, manifesting with an altered secretome.
To our knowledge, this is the first study to report effects of BRF1 on the immune response in an in vivo model. A previous study identified a direct effect of Pol III activity in enhancing macrophage phagocytic function in vitro . Our study did not target Pol III activity directly in immune cells, but instead we show that elevated expression of BRF1 is associated with altered immune cell infiltration in an in vivo model of prostate carcinogenesis.
It is recognised that immune infiltration can have a significant influence upon clinical outcome in cancer patients . Our previous study found that improved survival upon genetic loss of Erk5 in PtenΔ/Δ PCa model was related to increased levels of the T cell recruiting cytokines Ccl5 and Cxcl10 and enhanced T cell infiltration (predominantly CD4+) within the tumours . Here, we report an association between reduced levels of CD4+ T lymphocytes, and also neutrophils, with poorer survival in PtenΔ/Δ BRF1Tg compared with PtenΔ/Δ mice.
The complement pathway has been reported to have both pro- and anti-tumourigenic effects . We observe that a low complement C7 environment is associated with poorer survival in our transgenic mouse model and human PCa. Furthermore, in both murine and human PCa, tumoral CD4+ T cell level positively correlated with complement C7 level. These data significantly contribute to our understanding of how the complement system may contribute to tumourigenesis.
In conclusion, our data highlight that elevated BRF1 expression promotes prostate carcinogenesis. We implicate a non-cell autonomous role for high Brf1 expression in the prostatic epithelium as impacting on the tumour microenvironment with altered immune infiltrates. Given the rapid pace of development of cancer therapies targeting the immune system, further research in this area is warranted.
Materials and methods
Detail for the following experimental procedures is provided in Supplementary Materials and Methods: IHC; ISH; image analysis of IHC and ISH staining; histoscore; use and analysis of clinical datasets; culture of human PCa cell lines; transient transfection of plasmids and siRNA’s; generation of PC3 eGFP empty vector and eGFP-PRF1 stable clones; western blotting; WST-1 cell proliferation assay; cell cycle analysis from BrdU labelling; generation of conditional human BRF1 expressing mice; mouse strains and breeding; quantitative real-time PCR (Table S18); isolation and quantification of RNA (Table S19); RNA-sequencing and bioinformatic analysis; SILAC labelling and preparation of secretome samples; proteomics; comparison of RNA-Seq and proteomics data and 35S-Methionine incorporation assay.
Human tissue microarray
Tissue microarrays (TMAs) consisting of cores of formalin-fixed, paraffin-embedded (FFPE) tissue from a cohort of 134 individuals with BPH and 516 patients with primary PCa were analysed for expression of BRF1 by immunohistochemistry. See Table S20 for demographic data at diagnosis: Age—median = 69.5 years (interquartile range (IQR) 63–75.5); Gleason sum score—median = 7 (IQR 6–8) (60.8% cases ≥ 7); Serum PSA levels—median = 15.55 ng/ml (IQR 7.025–44).
Statistical analyses, including: Mann–Whitney, Log-rank (Mantel–Cox), Pearson correlation coefficient, Welch’s t test (unpaired, 2 tailed) and Kaplan–Meier survival analysis, were performed using GraphPad Prism v7.02 or IBM SPSS Statistics 25 software. Bonferroni correction for multiple testing was performed as indicated. For all graphs, mean ± standard deviation (SD) or standard error of mean (SEM) (error bars) are presented and these are defined for each figure. In Forest plots, in both the upregulated and downregulated cases, the data were pooled (across all cell-lines), log-transformed, and a one-sample t-test was performed.
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This work was funded by Cancer Research UK (A15151, A10419 and A17196; HYL) and Prostate Cancer UK (PG10-10; HYL). SS's PhD was supported by Medical Research Council Clinical Research Training Fellowship. NAN's PhD was sponsored by Ministry of Higher Education, Malaysia and SLAI Fellowship from University of Malaya. We thank the Cancer Research UK Beatson Institute core research services, including the biological services unit and the histology department. We acknowledge Arnaud Blomme for helpful discussions and commenting on the manuscript. We are grateful to Bob White for involvement and useful discussions in the initial stage of this project, and to Owen Sansom for general support in the development of the BRF1 mouse model.
Conflict of interest
The authors declare that they have no conflict of interest.
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Loveridge, C.J., Slater, S., Campbell, K.J. et al. BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration. Oncogene 39, 1797–1806 (2020). https://doi.org/10.1038/s41388-019-1106-x
Disease Models & Mechanisms (2020)