1 EBF1-mediated upregulation of ribosome assembly factor PNO1 contributes to cancer progression by negatively regulating the p53 signaling pathway

Conflict of Interest: The authors disclose no potential conflicts of interest. propidium Abstract The RNA-binding protein PNO1 is critical for ribosome biogenesis, but its potential role in cancer remains unknown. In the present study, on-line data mining, cDNA and tissue microarrays indicated that PNO1 expression was higher in colorectal cancer (CRC) tissue than in noncancerous tissue, and its overexpression was associated with worse patient survival. Gain-of-function and loss-of-function studies demonstrated that PNO1 knockdown suppressed growth of CRC cells in vitro and in vivo , while PNO1 overexpression promoted CRC cell proliferation in vitro. In CRC cells expressing wild-type p53, PNO1 knockdown enhanced expression of p53 and its downstream gene p21, and reduced cell viability; these effects were prevented by p53 knockout and attenuated by the p53 inhibitor PFT-α. Moreover, PNO1 knockdown in HCT116 cells decreased levels of 18S rRNA, of 40S and 60S ribosomal subunits, and of the 80S ribosome. It also reduced global protein synthesis, increasing nuclear stress and inhibiting MDM2-mediated ubiquitination and p53 degradation. Overexpressing EBF1 suppressed PNO1 promoter activity and decreased PNO1 mRNA and protein, inhibiting cell proliferation and inducing cell apoptosis through the p53/p21 pathway. In CRC tissues, the expression of EBF1 correlated inversely with PNO1. Data mining of online breast and lung cancer databases showed increased PNO1 expression and association with poor patient survival; PNO1 knockdown reduced cell viability of cultured breast and lung cancer cells. Taken together, these findings indicate that PNO1 is overexpressed in CRC and correlates with poor patient survival, and that PNO1 exerts oncogenic effects, at least in part, by altering ribosome biogenesis. Significance: This study identifies the ribosome assembly factor PNO1 as a potential oncogene involved in tumor growth and progression of CRC. 23 levels were associated with poorer overall survival and relapse-free survival of patients with breast or lung cancer, as well as shorter time to first progression (Supplementary Fig. S2A, P < 0.05). In cultures of A549 lung cancer cells and MCF-7 breast cancer cells, PNO1 knockdown using siRNA transfection (Supplementary Fig. S2B-C) significantly decreased cell viability (Supplementary Fig. S2D) and cell survival (Supplementary Fig. S2E). These data suggest that PNO1 may play oncogenic roles in CRC as well as other kinds of cancers. the essential role of PNO1 ribosome biogenesis mammalian cells, Further to be done explore functional correlation between PNO1 and NOB1 in cells. regulating the p53 signaling pathway. These findings justify the search for small-molecule inhibitors targeting PNO1 as a novel therapeutic strategy in CRC.


Introduction
Colorectal cancer (CRC) is the third most common cancer and fourth leading cause of cancer-related death worldwide, with 1.2 million new cases and over 600,000 deaths each year (1,2). Despite recent progress in treatment, outcomes in CRC remain poor.
Therefore, a better understanding of the molecular mechanisms of CRC is urgently required, as is the discovery of new diagnostic and prognostic biomarkers. In the present study, cDNA microarray analysis of paired cancerous and noncancerous tissues from CRC patients was followed by high-content screening using lentivirus-delivered short hairpin (sh)RNA interference in CRC cells. This led to the finding that ribosome assembly factor PNO1 was overexpressed in cancer tissues, while PNO1 knockdown inhibited the growth of CRC cells.
The ribosome is a supramolecular ribonucleoprotein complex responsible for translating mRNA into proteins. Ribosome biogenesis is a complicated, well-orchestrated process that involves the transcription and processing of ribosomal RNAs, the production of ribosomal proteins, as well as the assembly and nuclear export of ribosome subunits. In eukaryotes, ribosome biogenesis is facilitated by the coordinated function of over 200 assembly factors including helicases, ATPases, GTPases and kinases, which join and are released from pre-ribosomal particles at different times during ribosome maturation. Since ribosome biogenesis determines the capacity of a cell to synthesize proteins and hence plays a crucial role in cell growth and proliferation, dysregulation of this vital process is associated with many diseases including cancer (3)(4)(5).
Cancer cells are characterized by an uncontrolled increase in cell proliferation (6), which requires extensive protein synthesis and thus increased ribosome biogenesis (7).
In this way, a promising anti-cancer strategy may be to suppress ribosome biogenesis by targeting ribosome assembly factors. The RNA-binding protein "partner of NOB1" (PNO1), also known as Dim2 or Rrp2, is a ribosome neogenesis factor highly conserved from yeast to mammals. The human PNO1 gene is located on chromosome 2q14 and includes seven exons (22). The full-length cDNA sequence of PNO1 (1637 bp) contains a 759-bp open reading frame encoding a protein of 252 or 248 residues, which contains a conserved C-terminal K homolog (KH) domain responsible for RNA binding (23,24). Previous studies in yeast have revealed that PNO1 participates in ribosome biogenesis. PNO1 is one of six assembly factors required for cytoplasmic maturation of the 20S pre-rRNA to 18S rRNA; it binds to NOB1 and increases NOB1 affinity for RNA, stimulating NOB1 to cleave at the 3' end of pre-18S rRNA. Loss of PNO1 results in a decrease of 18S rRNA and defective assembly of pre-40S ribosomal subunits (25)(26)(27).
In contrast to extensive studies of the yeast homologue of PNO1 called Yor145, little is known about the functions of PNO1 in mammalian cells. In particular, a role in cancer progression has never been reported. Therefore in the present study we applied a combination of high-throughput "omics" technologies, on-line data mining, biochemistry and molecular biology to evaluate PNO1 expression in human cancers, its association with cancer prognosis, as well as its potential oncogenic activity and the underlying mechanisms. Research.
on April 30, 2019. © 2019 American Association for Cancer cancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Summary of the experimental design
A cDNA microarray was used to identify differentially expressed genes (DEGs) in paired cancerous and non-cancerous tissues from CRC patients. Cell-based high-content screening and shRNA-mediated knockdown led to identification of PNO1. Prognostic value of PNO1 was analyzed in both clinical CRC samples and on-line databases. Effects of PNO1 on tumor growth in vitro and in vivo, as well as on cell proliferation and cell apoptosis, were assessed in CRC cells after PNO1 knockdown and/or overexpression. A cDNA microarray was used after PNO1 knockdown in order to identify downstream regulatory mechanisms. Bioinformatics analysis of DEGs identified the p53/p21 pathway as one of the most enriched signaling pathways. Effects of p53/p21 signaling on oncogenic activities of PNO1 were investigated in HCT116 cells that were exposed to a p53 inhibitor or in which the p53 gene was deleted. The putative mechanism by which PNO1 knockdown activated p53 signaling was evaluated in CRC cells by examining ribosome biogenesis and protein synthesis, nuclear morphology and levels of p53 in nucleus, as well as ubiquitination and degradation of p53. Regulation of PNO1 by EBF1 was examined. Potential roles of PNO1 in breast and lung cancers were also examined.

High-content screening for cell growth
Growth of cultured cells was assessed using multi-parametric high-content screening.
At 72 h after shRNA transduction, cells were seeded into 96-well plates at a density of 2000 cells/well in 100 μl of medium. Cell growth was monitored every day for 5 days using the Cellomics® ArrayScan®V(TI) high-content image analysis platform and analyzed using HCS TM Studio Cell Analysis Software (Thermo Fisher Scientific).

Quantitative real-time PCR and tissue cDNA array analysis
Total RNA was extracted from the cell line samples or tissue samples using RNAiso

Western blot analysis
Proteins were extracted using RIPA lysis buffer (Thermo Fisher Scientific), separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and then blocked with a blocking buffer (Thermo Fisher Scientific) prior to overnight incubation at 4 ˚C with a primary antibody (Supplementary Table S4). Membranes were washed extensively and incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse secondary antibody. The blots were visualized using a chemiluminescence method (Thermo Fisher Scientific), and band intensities were quantified relative to intensity of β-actin using ImageJ software. Levels of target protein were expressed relative to levels in control cells, defined as 1.00.

Tissue microarray and survival analysis
Tissue microarray (TMA) slides (Shanghai Outdo Biotech) contained 90 pairs of tissue samples and were hybridized with primary antibody against PNO1 (1:500) using standard techniques (29). The 90 pairs of specimens were obtained from Taizhou Hospital of Zhejiang Province from July 2007 to October 2008; all subjects had reliable information on survival, and there was no data censoring prior to 7 years of follow-up.
Images were captured using a Nano Zoomer 2.0 HT slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) and processed using Nano Zoomer Digital Pathology View 1.6 software. Immunohistochemical score was determined independently by two experienced pathologists blinded to the clinical and pathological data. PNO1 expression was scored as described in the "Immunohistochemical staining" section. PNO1 expression was considered high for scores of 4-12 and low for scores of 0-3.
The relationship between PNO1 expression (low or high) and patient overall survival was analyzed using Kaplan-Meier analysis and assessed for significance using the log-rank test.

Cell transfection
Three non-overlapping anti-PNO1 short interfering (si)RNA oligonucleotides  Table S2). Cells were transfected with siRNAs or si-Ctrl at a concentration of 10 nM using Lipofectamine RNAiMax (Thermo Fisher Scientific).

Cell proliferation assay
Cell proliferation was determined using CFDA or CCK-8 assays. In the CFDA assay, cells were transfected with anti-PNO1 or control siRNA for 24-72 h, washed with phosphate-buffered saline (PBS) and incubated in fresh culture medium until the indicated time points, when 200 L CFDA (25 M; Thermo Fisher Scientific) was added to each well. Plates were incubated for an additional 2 h at 37 ˚C in the dark.
Fluorescence intensity was measured at 480 nm using a microplate reader (Tecan, Männedorf, Switzerland). In the CCK-8 assay, cells were transfected with siRNA or transduced with lentivirus and cultured for the indicated duration, when 10 L CCK-8 (Cell Counting Kit-8, Dojindo, Kumamoto, Japan) was added to each well. Plates were incubated for an additional 2 h at 37 ˚C, and absorbance was measured at 450 nm.

Colony formation assay
For cell survival analysis, transduced cells were seeded into 12-well plates (500 cells/well) and incubated in humidified air containing 5% CO 2 at 37 ˚C for 10-14 days to allow colony formation. Medium was replaced every 2-3 days. Cells were washed with PBS, fixed with 4% paraformaldehyde and stained with crystal violet. After staining, photographs were taken and numbers of colonies counted. Data were normalized to results for control cells. were seeded in plates containing medium with 0.6% solidified agarose. After incubation for 2 to 3 weeks, photographs were taken and numbers of colonies counted.
Three independent experiments were performed, and data were normalized to results for control cells.

Apoptosis analysis
Cells were washed with ice-cold PBS followed by binding buffer, and then stained for 15 min with Annexin V-APC (KeyGEN, Jiangsu, China). The percentage of apoptosis was analyzed by fluorescence-activated cell sorting (FACS Caliber).

Caspase activity assay
The activity of caspases-3 and -9 was determined by commercial colorimetric assays

Northern blot analysis of 18S rRNA
Extracted total RNA (10 μg per sample) was fractionated on a 1.2% agarose-formaldehyde gel and transferred to Hybond Nþ membranes (Amersham). for 30 min, then incubated at 50 ˚C for 20 min with stabilized streptavidin-horseradish peroxidase conjugate (1:300). Membranes were exposed to a phosphor storage screen and visualized using the Chemi Doc XRS+ System (Bio-Rad, Hercules, CA, USA).

Statistical analysis
Statistical analysis was performed using SPSS 20.0 (IBM, Chicago, IL, USA). Data are presented as mean ± SD. Differences between two groups were assessed for significance using the independent Student's t test, and differences among three or more groups were assessed using one-way ANOVA. Kaplan-Meier survival differences were assessed using the log-rank test. Correlation between PNO1 and EBF1 expression was analyzed using Pearson rank correlation. P < 0.05 was considered significant.

Data availability
The authors declare that all data supporting the findings of this study are available  Table S5). High-content screening in which lentivirus was used to deliver shRNAs into RKO cells in culture showed that PNO1, CDCA5, NUF2 and DACH1 strongly inhibited cancer cell growth (Fig. 1D-F). Considering the critical role of ribosome assembly in oncogenesis (8)(9)(10)(11)(12)(13)(14)(15)(16), we focused on PNO1 in subsequent studies.

PNO1 is highly expressed in CRC and associated with poor prognosis
Quantitative PCR of an independent sample of 50 CRC cases previously collected from our laboratory showed that levels of PNO1 mRNA were elevated in CRC ( Fig.   2A), and these results were confirmed by Western blot (Fig. 2B;  Outdo Biotech, Shanghai, China) showed increased PNO1 expression in CRC primary lesions (P < 0.05 vs. non-cancerous surrounding tissues; Fig. 2E), as well as an association between higher PNO1 expression and poorer overall survival (P < 0.05; Fig. 2F-G).

PNO1 promotes CRC cell proliferation
In cultured RKO and HCT116 cells, which constitutively express relatively high levels of endogenous PNO1 (Supplementary Fig. S1A-B), PNO1 knockdown based on lentivirus-delivered shRNA or on transfected siRNA ( Fig. 3A-B, Supplementary   Fig. S1C-D) decreased cell viability (Fig. 3 C, Supplementary Fig. S1E) and colony formation (Fig. 3D, Supplementary Fig. S1F). It also increased the percentage of cells in G0/G1 phase, with a concomitant decrease in the percentage of cells in S phase (Fig. 3E). PNO1 knockdown increased the percentage of cells under going apoptosis (Fig. 3F), and it increased activity of caspases-3 and -9 ( Fig. 3G-H).
In contrast, ectopic PNO1 expression in HT-29 and HCT-8 cells (Fig. 3I-J), which constitutively express low levels of endogenous PNO1, increased cell viability and colony formation (Fig. 3K-L). Xenografts of wild-type HCT116 or RKO cells grew significantly faster in nude mice than xenografts in which PNO1 was knocked down prior to inoculation (Fig. 4A-B).  (30)(31)(32)(33)(34). KEGG Pathway Enrichment Analysis of these DEGs indicated that p53 pathways were among the 10 most enriched signaling pathways (Fig. 5C); one of the genes most strongly up-regulated in response to PNO1 knockdown was the downstream effector p21 (Fig. 5B).

Oncogenic activities of PNO1 depend on p53 signaling
Microarray analysis (Fig. 5D) and quantitative PCR (Fig. 5E) showed a slight increase in p53 mRNA. PNO1 knockdown increased luciferase activity in the p53-driven luciferase reporter assay (Fig. 5F). Western blot analysis confirmed increased expression of both p53 and p21 at the protein level (Fig. 5G). The inhibitory effects of PNO1 knockdown were blocked when p53 was knocked out (Fig. 5H&I), and the inhibitory effects were attenuated when cells were treated with the p53 inhibitor PFT-α (Fig. 5J).

PNO1 knockdown activates p53 through the ribosomal stress pathway by inhibiting MDM2-mediated ubiquitination and degradation of p53
Consistently with studies in yeast (26)(27), we confirmed that PNO1 knockdown in HCT116 cells significantly decreased levels of 18S rRNA (Fig. 6A), 40S and 60S subunits of the 80S ribosome (Fig. 6B), and global protein synthesis (Fig. 6C). To determine whether these effects on the nucleolus were related to nucleolar stress, we stained cells for p53 and the major nucleolar protein nucleolin/C23. PNO1 knockdown or treatment with actinomycin D (as a positive control) resulted in translocation of nucleolin from the nucleolus to the nucleoplasm (Fig. 6D, left panel) and an increase in nuclear p53 immunoreactivity (Fig. 6D, right panel). Western blot analysis and co-immunoprecipitation (co-IP) assay indicated that PNO1 knockdown led to a reduction in degradation of p53 (Fig. 6E) and ubiquitination of p53 (Fig. 6F).
Moreover, co-IP analysis indicated that PNO1 knockdown increased the binding of RPL11 to MDM2 (Fig. 6G).
EBF1 displayed the most potent regulatory effect on PNO1 transcription in HEK293T cells based on a luciferase reporter system (Fig. 7B), and this regulatory effect was confirmed in RKO cells (Fig. 7C). EBF1 overexpression reduced PNO1 expression at the mRNA and protein levels (Fig. 7D&E), as well as reduced cell viability (Fig. 7F) and induced cell cycle arrest (Fig. 7G) and apoptosis (Fig. 7H). In microarray experiments with 14 paired samples of CRC and non-cancerous tissue, PNO1 expression was significantly higher in CRC than in non-cancerous tissue (Fig. 1C), whereas the converse was true for EBF1 expression (Fig. 7A); their expression exhibited a significant inverse correlation (Fig. 7I), which was consistent with the analyses in two CRC cohorts from the R2 Bioinformatic Platform (Fig. 7A&J-K).
Therefore, decreased expression of the negative transcription factor EBF1 may be one of the key reasons for PNO1 overexpression in CRC.

Discussion
The key finding of this report is that the ribosome assembly factor PNO1 may play a Activation of the well-known tumor suppressor p53 induces transcription of various genes, including the p21 gene, which can lead to cell cycle arrest and apoptosis as well as inhibit cell proliferation (36)(37)(38)(39)(40). Various cellular insults can activate p53, including disruption of ribosome biogenesis (41)(42)(43). In the present study, we demonstrated that PNO1 knockdown enhanced expression of p53 and its downstream gene p21 in wild-type HCT116 cells as well as reduced cell viability. These effects were blocked by p53 knockout and attenuated by the p53 inhibitor PFT-α.
Ribosome biogenesis is a very complex process (44)(45)(46), highly regulated by more than 200 ribosome assembly factors, including RIO1 and NOB1. While studies of the yeast homologue of PNO1 showed that it plays an essential role in the processing of Using RNA profiling and Northern blotting, we found that PNO1 knockdown in HCT116 cells decreased amounts of 18S rRNA, 40S subunits, 60S subunits and the 80S ribosome, leading thereby to significant inhibition of global protein synthesis.
These results demonstrate the essential role of PNO1 in ribosome biogenesis in mammalian cells, specifically in human CRC cells. Further studies need to be done to explore functional correlation between PNO1 and NOB1 in mammalian cells.
Deficiency in ribosome biogenesis elicits a p53-dependent cellular stress response referred to as "nucleolar stress" or "ribosomal stress" (47). We confirmed that the effect of PNO1 knockdown on the nucleolus is consistent with nucleolar stress and activation of p53. Moreover, PNO1 knockdown resulted in translocation of nucleolin from the nucleolus to the nucleoplasm, which is an indicator of increased nucleolar stress, and it resulted in an increase in levels of p53 in the nucleus. A p53-driven luciferase reporter assay and quantitative PCR analysis indicated that PNO1 knockdown slightly increased luciferase activity and p53 mRNA expression. PNO1 knockdown led to a reduction in the degradation and ubiquitination of p53. These studies suggest that both the increase in p53 mRNA expression and decrease in MDM2-mediated degradation and ubiquitination of p53 contribute to the increase in p53 after PNO1 knockdown. Therefore, further study is needed to explore precisely how PNO1 knockdown activates p53. The ribosome protein RPL11 binds to MDM2 and inhibits its ubiquitin ligase activity toward p53, resulting in p53 accumulation (40,47,48). We therefore detected the binding of RPL11 and MDM2 in HCT116 after PNO1 knockdown and found that this knockdown significantly increased the binding of RPL11 to MDM2, suggesting that ribosomal stress increases RPL11 binding to MDM2 and decreases ubiquitination and degradation of p53; this may contribute to In parallel with our efforts to explore downstream effectors of PNO1 oncogenicity, we searched for upstream regulators of PNO1. Web-based screening of transcription factor binding sites identified EBF1 as a potential transcription factor of PNO1. We found that EBF1 overexpression significantly decreased levels of PNO1 mRNA and protein in CRC cells, while it increased levels of p53 and p21 protein. Moreover, EBF1 over-expression in RKO cells significantly decreased cell viability, arrested the cell cycle at G0/G1 phase and induced cell apoptosis. We also found that EFB1 expression correlated inversely with that of PNO1 based on data from our CRC cDNA microarray analysis as well as from two on-line CRC cohorts. These results suggest that EBF1 suppression may help drive CRC by triggering PNO1 overexpression. This would be consistent with previous studies that have proposed EBF1 as a tumor suppressor in hematological malignancies (49,50). How EBF1 regulates PNO1 expression should be investigated in further studies. In B cell development, EBF1 helps drive DNA demethylation and chromatin remodeling, which controls the transcription of various genes [49][50][51] . Whether EBF1 regulates PNO1 expression in the same way should be explored, and more generally the potential roles of EBF1 in solid malignancies should be clarified.
In conclusion, this is the first demonstration of the significance of PNO1 in CRC. Our data suggest that the protein plays a role in tumorigenesis and prognosis. We have also shown, for the first time, that mammalian PNO1 is critical for ribosome biogenesis in cancer cells, suggesting that it works as a ribosome assembly factor like its yeast homologue. PNO1 transcription appears to be regulated by the transcription factor EBF1, and PNO1 exerts its oncogenic effects at least in part by negatively