BRIX1 promotes ribosome synthesis and enhances glycolysis by selected translation of GLUT1 in colorectal cancer

Ribosome biogenesis protein BRX1 homolog (BRIX1) is critically required for the synthesis of the 60S ribosome subunit. However, the role and mechanism of BRIX1 in colorectal cancer (CRC) remain unclear.


| BACKGROUND
Colorectal cancer (CRC) is one of the most malignant tumors of the digestive tract with the highest incidence and fatality rates 1 globally.As CRC onset is hidden and there are no specific symptoms in the early stages, >50% of patients with CRC are in advanced stages (stage III or IV) when they are diagnosed.In addition, the overall prognosis of patients with advanced CRC is considerably worse than that of patients with CRC in the earlier stages. 2,3Therefore, studying and exploring the molecular mechanism underlying CRC development is a long-term theme.These efforts are conducive to the development of new methods for diagnosing and treating CRC.
Ribosomes, considered the general factory of protein synthesis, are the basic elements maintaining normal life activities in cells.In normal human cells, ribosome synthesis is a dynamic, orderly and precisely regulated process that ensures the normal operation of ribosomal functions and meets the needs of different life activities 4 ; however, in malignant tumor cells, ribosome synthesis is substantially activated to meet the needs of a large number of structural and functional proteins that are required for rapid cell division and proliferation. 5,6Pathologically, the increase in nucleolar organization owing to the abnormal activation of ribosome synthesis is a characteristic phenotype of malignant tumor cells and indicates the degree of tumor malignancy and prognosis. 7Additionally, several studies have reported the presence of ribose in malignant tumor cells.The number of ribose bodies is significantly higher than that of normal cells.
Ribosome biogenesis protein BRX1 homolog (BRIX1) is a vital component in the synthesis of the 60S subunit of ribosome, which is located in the nucleus.Malignant tumor studies have reported that BRIX1 expression is elevated in non-small cell lung cancer 8 ; however, the role and mechanism of BRIX1 in CRC remain unclear.
0][11] Changes in the tumor microenvironment determine the direction of tumor cell metabolic reprogramming. 12Abnormal glucose metabolism is a common phenomenon in the metabolic reprogramming of CRC cells. 13In 1920, Warburg reported that the glycolytic ability of liver cancer cells was considerably higher than that of normal liver cells.Notably, liver cells exhibit high glycolysis even in the presence of sufficient oxygen, known as the Warburg effect.Subsequent studies have reported that the abnormal activation of glycolysis is essential for the proliferation and growth of tumor cells. 14,15However, the related mechanisms regulating glycolysis need to be further explored.

| Cell culture
All cell lines used in this study were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).All cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin.

| Microarray data analysis
GSE21510 and GSE18105 were searched and downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih. gov/geo/).The analysis of differentially expressed genes (DEGs) was conducted using both fold change (FC) and Student's t-test.A FC of >2 and a p-value of <0.05 were statistically significant.The function of DEGs was assessed using Kyoto Encyclopedia of Gene and Genome (KEGG) pathway enrichment, where a p-value <0.05 was statistically significant.

| Small-interfering RNA transfection
Small-interfering RNAs (siRNAs) for GLUT1 were purchased from GenePharma (Shanghai GenePharma Co. Ltd, Shanghai, China).The sequences are shown in Table S1, and the experimental method was performed as previously described. 16

| Lentivirus transfection
Full-length human BRIX1 cDNA was transfected into CRC cell lines using a lentivirus to generate Lentivirus-BRIX1 (BRIX1 overexpression).Lentivirus-NC was used as the negative control (Vector).In addition, one short-hairpin RNA (shRNA) sequence against BRIX1 was transfected into CRC cell lines to generate shRNA-BRIX1 while sh-NC was used as the negative control.The sequences are shown in Table S1.

| RNA isolation and RT-qPCR
Trizol was used to extract RNA, and total RNA was reverse transcribed to cDNA with PrimeScriptTM (TAKARA).18S RNA served as an internal control.Primer sequences are shown in Table S1.The relative expression of the target gene was calculated using the À44Ct method.

| Immunohistochemistry
All tissues were paraffin-embedded and sectioned into 4 m thick blocks.All of the sections were dewaxed with xylene and hydrated with alcohol.Sodium citrate was used for antigen retrieval, and 0.3% hydrogen peroxide was used to block endogenous peroxidase.After blocking non-specific sites with bovine serum albumin, all sections were incubated with appropriate primary and secondary antibodies.
We used a 3,3-diaminobenzidine kit for visualization, and hematoxylin was used to stain the nuclei.All the sections were dehydrated with alcohol and sealed with neutral resin.The immunohistochemistry staining score was calculated based on pixel intensity.Staining was scored as follows according to the staining intensity: no staining, 1; weak staining, 2; moderate staining, 3; and strong staining, 4.

| Live metabolic analyses
Seahorse XF96 Flux Analyzer (Seahorse Bioscience, Agilent) was used to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in the CRC cell lines.Briefly, the CRC cells, including HCT-116, SW480 and HT29 cells, were seeded into an XF96-well plate.The media were replaced with assay media 1 h before the assay.For the ECAR assay (Seahorse Cat.#103020-100), 10 mM glucose, 1 μM oligomycin and 50 mM 2-deoxyglucose were added to the wells.For the OCR test (Seahorse Catalog no.103015-100), 1 μM oligomycin, 1 μM FCCP, 0.5 μM rotenone and 0.5 μM actinycin A were added to the wells at specific timepoints.
Both measurements were normalized via the quantitation of the total protein.The experiments were performed in triplicate and repeated twice.

| Glucose and lactate measurement
Amplex ® Red Glucose/Glucose Oxidase Assay Kit (Invitrogen, catalog no.A22189) was used to measure the glucose uptake.Glucose consumption was calculated using the net content of the original glucose concentration after measuring the glucose concentration in the media.
Lactate Assay Kit (BioVision, catalog no.ABIN411683) was used to measure lactate production.Total proteins were used for the normalization of the obtained results.The experiments were performed in triplicate and repeated twice.

| CCK8 assay
Cells (1 Â 10 3 ) were seeded into 96-well plates.Detection reagent (10 μL of CCK8 reagent + 90 μL of DMEM) was added to each plate and incubated at 37 C for 1 h.The absorbance was measured at 450 nm with a microplate reader.Each experiment was performed independently in triplicate.

| Polysome fractionation and RNA isolation
Cellular extracts were centrifuged at 10,000 rpm for 5 min at 4 C, and the supernatant was loaded onto 10-50% sucrose gradients containing 0.1 mg/mL heparin and 2 mM dithiothreitol (DTT) and centrifuged at 37,000 rpm for 2.5 h at 4 C (SW40 rotor).The sucrose gradient was subsequently fractionated with a gradient fractionation system connected to a ultraviolet detector to monitoring absorbance at 252 nm.RNA was isolated from polysomal fractions using the Pure-Link RNA Mini Kit (Invitrogen).

| Animal model
For the generation of an orthotopic model of CRC, all nude mice were anesthetized with sevoflurane.After opening the abdominal cavity,

| Ribosome-related pathways are primarily enriched in CRC
The KEGG pathway analysis revealed that ribosome biosynthesis in eukaryotes and ribosome pathways were primarily enriched in the CRC samples of the GEO datasets (GSE21510 and GSE18105) (Figure 1A).Overall, 55 genes were significantly expressed during ribosome biosynthesis in eukaryotes and ribosome pathways.We added these 55 genes into a protein-protein interaction network to select the key genes (degree > 30) involved in the aberrant activation of ribosome biosynthesis in CRC and found eight key genes, CMSS1, BRIX1, RPS7, RPS20, RPS25, WDR36, WDR43 and UTP14A, which could be of significance in the aberrant activation of ribosome biosynthesis (Figure 1B).Further analysis of mRNA expression in The Cancer Genome Atlas (TCGA) dataset revealed elevated expression levels of these eight genes in CRC samples compared with those in normal samples (Figure 1C,D).These results indicate that the abnormal activation of ribosome is a significant feature in CRC.In addition, we detected rRNAs in paired CRC and adjacent normal tissues and the results showed that rRNA expression levels were elevated in the CRC tissues compared with those in the adjacent healthy tissues (Figure 1E).Similarly, the rRNA expression levels were significantly higher in the CRC cell lines than in a normal colorectal cell line (Figure S1A-D).

| BRIX1 expression predicts ribosome activation in CRC
GO analysis from TCGA dataset (Figure 2A) showed that BRIX1 primarily enriched ribosome assembly and ribosome biogenesis (Figure 2B), indicating abnormal ribosome activation in CRC.The ribosome quantity was higher in CRC tissues with a higher expression of BRIX1 than that in CRC tissues with a low expression of BRIX1 (Figure 2C).We then knocked down the expression of BRIX1 in HCT-116 and SW480 cell lines (Figure 2D).BRIX1 knockdown produced a significant decrease in rRNA levels in the shBRIX1 CRC cells (Figure 2E).Moreover, BRIX1 knockdown significantly decreased nascent RNA synthesis in CRC cells (Figure 2F).
Thus, BRIX1 plays a vital role in regulating ribosome activation in CRC.

| BRIX1 determines a high glycolytic state in CRC cells
The PET-CT analysis showed that the glucose uptake in patients with CRC with high BRIX1 expression was significantly higher than that of patients with low BRIX1 expression (Figure 3A), indicating that BRIX1 is related to glucose metabolism.In vitro knockdown of BRIX1 significantly decreased the ECAR level in CRC cells (Figure 3B) but had no significant effect on the OCR level (Figure 3C).In addition, knocking down BRIX1 significantly reduced the glucose uptake capacity and reduced the production of lactic acid in CRC cells (Figure 3D-E).However, BRIX1 overexpression significantly increased the ECAR level in CRC cells but had no significant effect on the OCR level (Figure 3F).
Moreover, BRIX1 overexpression significantly promoted the glucose uptake capacity and increased the production of lactic acid in CRC cells (Figure 3G).

| BRIX1 regulates glycolysis in CRC cells via selected translation of GLUT1
The above experiments indicate that BRIX1 controls the glycolysis in CRC cells by regulating glucose uptake.GLUT1 is a key transporter for glucose uptake.BRIX1 and GLUT1 expression were not correlated in the TCGA dataset (Figure 4A), and in vitro BRIX1 knockdown did not affect GLUT1 mRNA expression (Figure 4B).However, BRIX1 knockdown significantly downregulated GLUT1 protein levels (Figure 4D).Similarly, BRIX1 overexpression only regulated the protein expression of BRIX1 but not mRNA expression (Figure 4C, E).
The relative translation rate of an mRNA can be inferred from the number of ribosomes (polysomes) it recruits and can be quantitatively analyzed via sucrose gradients to purify the mRNAs associated with translationally active ribosomes (Figure 4F).Using this approach, we collected polysome fractions, which revealed that BRIX1 knockdown decreased the mRNA level of GLUT1 in polysome fractions (Figure 4G).Conversely, BRIX1 overexpression markedly increased the mRNA level of GLUT1 in polysome fractions (Figure S2A-E).
GLUT1 knockdown by siRNAs substantially inhibited glycolysis enhancement and glucose uptake owing to BRIX1 overexpression in CRC cells (Figure 4H).Moreover, GLUT1 knockdown inhibited the increased production of lactic acid via BRIX1 overexpression in CRC cells (Figure 4I).

| BRIX1 plays a tumor-promoting role in CRC by regulating GLUT1-related glycolysis
As the effect of BRIX1 on CRC remains unclear, we knocked down BRIX1 in CRC cells (HCT-116 and SW480).The in vitro experiments revealed that BRIX1 knockdown significantly inhibited the proliferation of CRC cells (Figure 5A).Orthotopic transplantation experiments also showed that BRIX1 knockdown significantly inhibited the growth of transplanted tumors (Figures 5B-C and S3).Furthermore, BRIX1 overexpression significantly promoted the proliferation of CRC cells (HT29) (Figure 5D).The siRNA knockdown of GLUT1 considerably inhibited the effect of BRIX1 overexpression on the growth of CRC cells (Figure 5D).Similarly, blocking glycolysis using galactose substantially reversed the effect of BRIX1 on the growth of CRC cells (Figure 5D).

| Upregulated BRIX1 expression correlated with clinical features and associated with poor prognosis
We then analyzed the expression of BRIX1 from patients with CRC, and the results showed that the mRNA and protein expression of BRIX1 were both significantly increased in these samples compared with those in NC samples (Figure 6A-D).The high expression of BRIX1 was closely related to the clinical characteristics of patients with CRC, such as tumor size, T stage and pathological stage (Figure 6E).In addition, data analysis showed that the overall survival of patients with CRC in the BRIX1 high-expression group was significantly lower than that of patients with CRC in the BRIX1 lowexpression group (Figure 6F-G).These results all indicate that abnormally elevated BRIX1 expression plays an important role in CRC.Therefore, BRIX1 plays a tumor-promoting role in CRC by regulating glycolysis via the enhancement of GLUT1 translation (Figure 6H).

| DISCUSSION
In human cells, ribosomes are relatively conserved organelles comprising four types of rRNAs (5S, 5.8S, 18S and 28S rRNA) and a large number of ribosome proteins (RPs).The ribosome synthesis process includes rDNA transcription, precursor ribosomal RNA (pre-rRNA) formation, rRNA splicing and processing, and ribosomal subunit assembly and transport. 4In the nucleolus region, rDNA is transcribed and synthesized to 47S pre-rRNA (47S pre-rRNA) by RNA polymerase Pol I and the upstream binding factor.Three types of rRNAs (5.8S, 18S and 28S) are formed through modification and splicing while 5S rRNA is transcriptionally processed in the nucleoplasm by RNA polymerase Pol III. 17,18Ribosome proteins are transcribed by RNA polymerase Pol II, further mature and are transported to the nucleoli following translation for processing and modification.Subsequently, 18S rRNA and 33 types of RPs assemble to form the 40S small subunit of the ribosome, while 5S, 5.8S and 28S rRNAs assemble with 47 types of RPs to form the 60S large subunit of the ribosome.After further processing, assembly and transport, the large and small subunits are combined with mRNAs in the cytoplasm.Finally, mature ribosomal executive protein translation function is formed. 4rDNA transcription to form rRNA is crucial and directly determines the rate of ribosome synthesis. 19,20The transcriptional process of rRNA involves RNA polymerase Pol I/III activation, upstream binding factor recruitment, non-coding RNAs, nucleolin and ribosomal-related protein regulation, and precursor RNA splicing and processing; these processes form a complex molecular regulatory network, among which the interactions between related molecules are particularly important. 20,21Herein, we The yeast protein homologous to BRIX1 is the ribosome biogenesis protein Brx1 (Brx1), which plays an important role in the transcription of rRNA in yeast. 22Herein, bioinformatics analysis revealed that BRIX1 was associated with ribosome function.BRIX1 knockdown significantly inhibited ribosome synthesis in CRC cells.Interestingly, related clinical studies reported that the glucose uptake value, as revealed via PET-CT, and the BRIX1 expression in patients with CRC were significantly increased.Subsequent experiments revealed that BRIX1 knockdown significantly reduced the glycolytic ability of CRC cells.4][25] Further experiments revealed that BRIX1 only affects the protein expression level of GLUT1 and not that of other enzymes and that BRIX1 does not affect the mRNA level of GLUT1, thus indicating that the ribosome function regulated by BRIX1 has some connection with GLUT1 translation.
Ribosomes behave as dynamic and heterogeneous complexes rather than static and homogeneous organelles.The protein translation carried out by ribosomes is a selective process owing to ribosomal heterogeneity. 26Ribosome heterogeneity is a broad concept, including changes in ribosomal composition and modification of rRNA and RPs. 27Differences in the types of RPs of ribosomes in eukaryotic cells have been identified, and these differences result in ribosomes being selective in translating mRNAs. 27,28For example, ribosomes containing RPS25/eS25 or RPL10a/uL1 ribosomal proteins have different translation capabilities for hundreds of mRNAs. 28In this study, we found that BRIX1 also promoted the translation of GLUT1 mRNA.
A close relationship exists between the selective translation of ribosomes and cell metabolism. 29,30The metabolic state of cells can directly affect the production of ribosomes to exert their physiological effects.The activation of the mTOR pathway affects the translation of downstream proteins by regulating the level of cellular glucose metabolism. 31Furthermore, the ribosome selectively regulates the process of mRNA translation into proteins and regulates the metabolic state of the cell.The selective translation of ribosomes can regulate the level of glucose metabolism in tumor cells. 32,33In our study, BRIX1 promoted the translation of GLUT1 mRNA into protein.This partly explains the selective action of ribosomes on glycolysis.Thus, BRIX1 regulated ribosome function and promoted glycolysis by selected translation of GLUT1, which promoted CRC.

| CONCLUSION
Herein, we analyzed the changes of ribosome-related pathways in CRC and screened the key genes.By combining BRIX1 expression with clinical data, we identified a correlation between BRIX1 and glucose uptake.Furthermore, BRIX1 regulated the ribosome function and promoted glycolysis by enhancing translation of GLUT1, which promoted CRC.This study explored the possibility of ribosome selectively regulating tumor cell metabolism, which provides theoretical support for targeted ribosome therapy in CRC.

F I G U R E 3
BRIX1 induces a high glycolytic state in colorectal cancer (CRC) cells.(A) PET-CT images of patients with CRC who exhibited low or high expression of BRIX1 (n = 15).(B) Extracellular acidification rate (ECAR) of HCT-116 or SW480 cells in the sh-NC and sh-BRIX1 groups was detected using the Seahorse Bioscience XFp Analyzer.Glc, Glucose; O, oligomycin; 2-DG, 2-deoxy-D-glucose.(C) The O 2 consumption rate (OCR) of HCT-116 or SW480 cells in the sh-NC and sh-BRIX1 groups was detected via a Seahorse Bioscience XFp Analyzer.O, Oligomycin; F, FCCP; A&R, antimycin A/rotenone.(D) The ECAR and OCR of HT29 cells in the vector and BRIX1 OV groups were detected via a Seahorse Bioscience XFp Analyzer.(E) Glucose uptake of HCT-116 or SW480 cells in the sh-NC and sh-BRIX1 groups.(F) Lactic acid formation of HCT-116 or SW480 cells in the sh-NC and sh-BRIX1 groups.(G) Glucose uptake and lactic acid formation of HT29 cells in the vector and BRIX1 OV groups.Measurement data are presented as the mean ± SD.Student's t-test was used for statistical analysis.***p < 0.001.

F I G U R E 5
BRIX1 plays a tumor-promoting role in colorectal cancer (CRC) by regulating GLUT1-related glycolysis.(A) Viability of HCT-116 and SW480 cells transfected with sh-BRIX1 or sh-NC as analyzed via CCK8 assay.(B) Comparison of total flux between sh-NC and sh-BRIX1 groups (n = 6).(C) Comparison of orthotopic tumor volume and weight between sh-NC and sh-BRIX1 groups.(D) Viability of HT29 cells in the vector and BRIX1 OV groups treated with siGLUT1 and galactose as analyzed using CCK8 assay.Measurement data are presented as the mean ± SD.Student's t-test was used for statistical analysis.*p < 0.05, ***p < 0.001.first analyzed the enrichment of ribosomal function in CRC to show that ribosomal pathways were significantly enriched in CRC, ranking among the top three enriched pathways.Further analysis revealed that BRIX1 may be the core gene involves in ribosomal pathway changes in CRC.

F I G U R E 6
Upregulated BRIX1 expression shows a close correlation with clinical features and is associated with poor prognosis of colorectal cancer (CRC).(A) mRNA expression of BRIX1 in CRC and paired adjacent tissues in the GDS4382 dataset (n = 17).(B) mRNA expression of BRIX1 in CRC and paired adjacent tissues in the GSE18105 dataset (n = 20).(C) mRNA expression of BRIX1 in CRC and paired adjacent tissues in the GSE20510 dataset (n = 17).(D) BRIX1 protein expression in CRC tissues from tissue microarray (n = 392).(E) Clinical feature analysis in CRC tissues with low or high expression of BRIX1.(F) Overall survival analysis based on the mRNA expression of BRIX1 from The Cancer Genome Atlas (TCGA) dataset.(G) Overall survival analysis of the protein expression of BRIX1 based on the prognostic information of patients with CRC from the tissue microarray data.(H) BRIX1 plays a tumor-promoting role in CRC via the regulation of glycolysis by selecting GLUT1 translation.Measurement data are presented as the mean ± SD.Student's t-test was used for statistical analysis.***p < 0.001.