Progress on the role and mechanism of ribosome assembly regulator PNO1 in tumor

Abstract

PNO1 (Dim2 or Rrp2 or YOR145), a highly conserved regulator of ribosome assembly from yeast to mammals, is involved in forming the 90S pre-ribosome and plays an essential role in the late stage of 40S small subunit maturation. Recent studies have found that PNO1 is involved in the progression of a variety of tumors and is highly expressed in colorectal, lung, esophageal, glioma, and breast cancer and is associated with poor prognosis. In tumors, PNO1 mainly promotes cell proliferation, invasion, and autophagy and inhibits apoptosis by regulating the P53 pathway, FAK/AKT pathway, Notch signaling pathway, and MAPK signaling pathway. The high expression of PNO1 in various tumors may be reduced by the regulation of early B cell factor 1 (EBF1), transcription factor MYC, miR-340-5p, and the drug celecoxib inhibiting tumor proliferation, invasion and migration, and autophagy, promoting apoptosis. This article reviews the structure and function of PNO1, related molecular pathways, and its regulatory role in tumor formation and discusses its possibility as a molecular target for tumor therapy.

KEYWORDS:

• PNO1
• biological function
• signal pathway
• tumor
• cell proliferation
• invasion

Introduction

Cancer cells are characterized by the uncontrolled and massive proliferation of cells (Evan and Vousden 2001), which requires extensive protein synthesis, thus increasing ribosome biogenesis (Ruggero 2012). Ribosome biogenesis, involving the de novo synthesis of ribosomes, is a complex and essential process within cells, including transcription and processing of ribosomal RNA, production of ribosomal proteins, and assembly of nuclear export of ribosomal subunits. Biogenesis in eukaryotes is complex and depends on more than 200 assembly factors. During the process of ribosomal maturation, assembly factors such as helicase, ATPase, GTPase, and kinase are inserted at different times and released from the ribosomal particles. Studies have shown that ribosomal biogenesis affects cell growth and proliferation by affecting the ability of cells to synthesize proteins. Many diseases, such as cancer, are relevant to the disorder of this vital process (Ruggero and Pandolfi 2003; Algra and Rothwell 2012; Cao et al. 2016). Over-expressed ribosomal assembly factors and specific ribosomal proteins promote ribosomal biogenesis, regulate the progression of some human malignant tumors, and are linked to poor prognosis (Pogue-Geile et al. 1991; Kondoh et al. 2001; Kasai et al. 2003; Bee et al. 2006; Lai and Xu 2007; Wang et al. 2009; Bee et al. 2011; Song et al. 2011). If it is supposed that up-regulated ribosomal biogenesis can promote the proliferation of cancer cells, then down-regulated ribosomal biogenesis will lead to cell cycle arrest, inhibit cell proliferation, induce apoptosis, and achieve the objective of inhibiting tumor growth (Boon et al. 2001; Deisenroth and Zhang 2010; Stumpf and Ruggero 2011; Armistead and Triggs-Raine 2014).

PNO1 (Dim2/Rrp2/YOR145), a highly conserved regulator of ribosome assembly from yeast to mammals, is involved in the transition from pre-90S to 40S and the final stage of maturation of the 40S small subunit in the cytoplasm (Turowski et al. 2014; Sturm et al. 2017; Ameismeier et al. 2020). In both eukaryotes and prokaryotes, the PNO1 family exhibits significant conservation. Nucleus-localized PNO1 regulates the biosynthesis of ribosomes and proteasomes, mainly concentrated in the nucleolus. Studies showed that PNO1 was mainly expressed in the liver and lung, and a small amount was expressed in the thymus and testis, while no expression was found in the heart and brain, skeletal muscle, small intestine, and colon (Tone and Toh 2002; Miura et al. 2004; Vanrobays et al. 2004; Zhou et al. 2004; Lin et al. 2020). In 2002, research showed that PNO1 acts as a molecular chaperone to form a complex with NOB1 and localize in the nucleus (Tone and Toh 2002). At the last stage of the maturation of the 40S small subunit in the cytoplasm, RKIO1 (a member of the atypical eukaryotic protein kinase family, which is highly conserved and participates in the development of all systems) shifts and digests PNO1 to dissociate it from the endonuclease NOB1, which stimulates the cleavage of NOB1 at the 3′ end of pre-18SrRNA, and plays an essential role in ribosomal biogenesis (Senapin et al. 2003; Vanrobays et al. 2008; Shen et al. 2019). Recent studies have found that PNO1 is involved in the progression of a variety of tumors and is highly expressed in colorectal cancer, lung cancer, esophageal cancer, glioma, and breast cancer, and is associated with poor prognosis (Shen et al. 2019; Liu et al. 2020; Chen et al. 2021; Wang et al. 2021 may). Therefore, a promising anticancer strategy might inhibit ribosome biosynthesis by targeting the ribosome assembly factor PNO1.

Main text

The structure and function of PNO1

PNO1, the human homologous protein of the NOB1 gene, also known as Dim2, or Rrp2, or YOR145 (Zemp and Kutay 2007), is a highly conserved regulator of ribosome assembly from yeast to mammals. The human PNO1 gene is located on chromosome 2q14 and consists of five introns and seven exons (Miura et al. 2004). The full-length cDNA sequence of PNO1 is 1637 bp, containing a 759 bp open reading frame, encoding a protein of 252 or 248 amino acids (Miura et al. 2004; Woolls et al. 2011). KH domains exist in many different proteins involved in countless different biological processes, including splicing, transcriptional regulation, and translational control (Valverde et al. 2008), whereas PNO1 contains two conserved KH domains (KH1, KH2) in sequence, in which KH2 contains the GXXG motifs necessary for RNA/DNA binding, but KH1 lacks the typical GXXG RNA-binding motifs and instead participates in protein–protein interactions (Woolls et al. 2011; Zheng et al. 2014). The KH1 domain of PNO1 provides a binding site for the endonuclease NOB1 (Sturm et al. 2017). Ribosomes are where cells synthesize proteins, and are known as ‘the protein factory of cells,’ responsible for translating the genetic code in mRNA and catalyzing protein synthesis in organisms. Ribosomes are composed of two ribonucleoprotein subunits, large and small. In eukaryotes, ribosome biosynthesis begins with forming a large precursor particle, the 90S pre-ribosome, from which pre-40S and pre-60S particles are generated (Dragon et al. 2002; Grandi et al. 2002). In 90S biogenesis, PNO1 localizes to the pre-90S ribosome by interacting with the UTP-B module to receive and transmit information about the correct assembly state of the nascent ribosome. UTP-B is a module with ‘antenna’-like properties which can glean information on the assembly state from unusual regions of 90S granules. Following proper 90S assembly and coordinated pre-rRNA processing, the information can be transported from UTP-B by PNO1 to nearby Utp14 (cofactor and activator of Dhr1 helicase activity which catalyzes the removal of U3 snoRNP from 90S) (Sturm et al. 2017). In this way, the Dhr1 helicase can induce dissociation of U3 snoRNP from their pre-rRNA binding sites by unwinding the RNA heteroduplex, resulting in a pre-90S to 40S transition (Martin et al. 2013) (Figure 1).

Figure 1. Model of PNO1 (Dim2) involvement during the transition from the 90S to the 40S pre-subunit (Turowski et al. 2014; Sturm et al. 2017).

PNO1 is not only involved in early ribosome maturation but also plays an important role in the late stage of small subunit maturation. On the one hand, it is involved in the final assembly of the 40S small subunit (Ameismeier et al. 2020). The formation of the small subunit ends with the final 18S rRNA precursor 18S-E being cleaved by the endonuclease NOB1 at position 3 (D site 18), which flanks residues of the internal transcribed spacer 1 (ITS1) (a combination of transcription factors, which has the characteristics of conservative phylogeny and may play a role in ribosomal maturation) and all remaining biogenesis factors are rapidly released (Woolford and Baserga 2013; Cerezo et al. 2019). Previous structures of the human 40S precursor indicate that PNO1 binds to the 3′ end of the 18S rRNA with its cognate protein NOB1 (Ameismeier et al. 2018 jun), and NOB1 exists in an inactive conformation (Sloan et al. 2019). During the final stage of small subunit maturation, with the participation of the eukaryotic translation initiation factor 1A domain protein (EIF1AD) and the mature 60S subunit, the atypical kinase RIOK1 (a member of the atypical eukaryotic protein kinase family, which is highly conserved and participates in the development of all systems) coordinates the conformational maturation of 18S rRNA and the release of PNO1, thereby activation of the endonuclease NOB1 cleaves 20S pre-rRNA to generate 18S rRNA (Ameismeier et al. 2020) (Figure 2).

Figure 2. Model for the involvement of PNO1 in the final stage of 40S small subunit maturation (Turowski et al. 2014; Sturm et al. 2017).

On the other hand, PNO1, together with NOB1 and the kinase RIOK1, establish a quality checkpoint that prevents premature translation of immature ribosomes. PNO1 stabilizes NOB1 structure and prevents mRNA recruitment to the 40S ribosome of immature 20S pre-rRNA, thus preventing premature release of the immature 40S subunit into the translation pool. Kinase RIOK1 utilizes the energy generated by ATP hydrolysis to release PNO1 and NOB1 from nascent ribosomes, thereby regulating their entry into the translation pool in an ATPase-dependent manner. RIOK1-NOB1-PNO1 plays a vital role by ensuring that only fully mature ribosomes enter the translation pool (Parker et al. 2019) (Figure 3).

Figure 3. RIOK1-NOB1-PNO1 checkpoint mechanism model (Parker et al. 2019).

The role and mechanism of PNO1 in tumor

Recent studies have shown that PNO1 is not only involved in the normal physiological function of ribosomes but also is closely related to the occurrence and development of tumors. High expression of PNO1 is considered a marker of poor prognosis in various tumors and promotes cell proliferation, invasion, and autophagy mainly by regulating the p53 pathway, FAK/AKT pathway, Notch signal pathway, and MAPK signal pathway (Table 1 and Figure 4).

Figure 4. Regulation of PNO1-related pathways. (EBF1: early B cell factor 1; MYC: transcription factor) (Dai et al. 2019; Shen et al. 2019; Liu et al. 2020; Chen et al. 2021; Han et al. 2021; Wang et al. 2021 may).

Table 1. The role and related pathways of PNO1 in different tumors.

Tumor	Cellular level	Clinical level	Mechanism/Pathway
Colorectal cancer	Promotes cell proliferation, and inhibits cell apoptosis	Poor prognosis with high expression	EBF1/PNO1/p53 axis (Shen et al. 2019)
Glioma	Promotes cell proliferation, migration, and invasion	Poor prognosis with high expression	MYC/PNO1/THBS1/FAK/Akt axis (Chen et al. 2021)
Lung adenocarcinoma	Promotes cell proliferation and invasion	Poor prognosis with high expression	miR-340-5p/PNO1/Notch axis, EMT (epithelial-mesenchymal transition) (Liu et al. 2020)
Urinary bladder cancer	Promotes cell proliferation, inhibits cell apoptosis	Poor prognosis with high expression	CD44, PTGS2, cyclin D1, CDK1, IL-8, FRA1, mTOR, p70 S6 kinase, p38, Caspase-3 protein, etc. (Lin et al. 2020)
Esophageal cancer	Promotes cell proliferation, migration, and invasion and inhibits cell apoptosis	Poor prognosis with high expression	AKT1, Twist, MYC, mTOR, matrix metalloproteinase 2 (MMP2), NF-κB p65 and CTNNB1 (Wang et al. 2021 may)
Hepatocellular carcinoma	Promotes cell proliferation and invasion	Poor prognosis with high expression	Celecoxib—AKT/mTOR (Dai et al. 2019)
Hepatocellular carcinoma	Promotes cell proliferation and autophagy, and inhibits cell apoptosis	Poor prognosis with high expression	MAPK signaling pathway (Han et al. 2021)
Breast and lung cancer	Promotes cell proliferation	Poor prognosis with high expression	Shen et al. (2019)

PNO1 regulates the p53 pathway through the nuclear stress pathway to promote tumor cell proliferation and inhibit apoptosis

PNO1 regulates cell proliferation mainly by regulating P53-related protein in colorectal cancer. Activation of the well-known tumor suppressor p53 induces the transcription of various genes leading to cell cycle arrest, inhibition of cell proliferation, and induction of apoptosis (Morgado-Palacin et al. 2012; Liu et al. 2016; Shen et al. 2019). Various cellular injuries can activate p53, including the blockade of ribosome biogenesis (Zhang and Lu 2009; Deisenroth and Zhang 2010). Experiments have demonstrated that PNO1 knockdown reduces the number of 18S rRNA, 40S subunit, 60S subunit, and 80S ribosomes, resulting in defects in ribosome biosynthesis (Shen et al. 2019), which triggers a p53-dependent cellular stress response called ‘nucleolar stress’ or ‘ribosomal stress’ (Deisenroth and Zhang 2010). Nuclear stress promotes the binding of the ribosomal proteins RPL1/(rp)L11 and RPL5/(rp)L5 as well as 5S rRNA to MDM2. It inhibits its ubiquitin ligase activity on p53, resulting in reduced degradation and ubiquitination of p53, promoting p53 accumulation (Deisenroth and Zhang 2010; Golomb et al. 2014; Goudarzi et al. 2014), thereby inhibiting cell proliferation. In addition, inhibition of endogenous PNO1 increases the percentage of colorectal cancer cells apoptosis and increases the activities of caspase-3 and caspase-9. PNO1 knockdown also increases the percentage of cells in the G0-G1 phase and decreases the percentage of cells in the S phase. Inhibition of endogenous PNO1 can induce apoptosis during the G1-S transition phase of the cell cycle and inhibit tumor growth in vivo and in vitro (Shen et al. 2019). Early B cell factor 1 (EBF1) in B cell development helps drive DNA demethylation and chromatin remodeling, thereby controlling the transcription of various genes (Liao 2009; Bohle et al. 2013). In colorectal cancer, EBF1 overexpression significantly decreases the levels of PNO1 mRNA and protein and increases the levels of p53 and p21 protein (Shen et al. 2019), inhibiting tumor proliferation by inhibiting PNO1-mediated p53/p21 signaling pathway activation. Furthermore, EBF1 overexpression induces cell cycle arrest in the G0/G1 phase and increases apoptosis (Shen et al. 2020).

PNO1 promotes glioma cell proliferation and metastasis by regulating the FAK pathway

In many tumor cells, tyrosine kinase FAK promotes the development of the malignant phenotype of the tumor through over-expression (Zhou et al. 2019). Thrombospondin 1 (THBS1) is one of the critical components of the extracellular matrix and is involved in regulating the development of tumors, including gliomas (Gahtan et al. 1999; Adams and Lawler 2004 jun; Firlej et al. 2011). In glioma cells U251, co-immunoprecipitation experiments showed that PNO1 interacts with THBS1 and regulates the expression of THBS1, leading to the phosphorylation of FAK and Akt, participating in the activation of the FAK/Akt pathway and significantly promoting the proliferation and metastasis of glioma cells (Chen et al. 2021). The proliferation and metastasis of PNO1 overexpression in glioma can be attenuated or even reversed by simultaneous silencing of THBS1. Transcription factor MYC is a key integrator of growth-regulatory and oncogenic signaling pathways (Kress et al. 2015). In glioma cells, overexpression of MYC increases the activity of the PNO1 promoter. In contrast, MYC knockdown significantly reduces the expression levels of PNO1 and THBS1 mRNA and protein, resulting in the phosphorylation of FAK and Akt, significantly reduced cell viability, anti-apoptosis capacity, and invasion capacity through the PNO1/THBS1/FAK/Akt signaling pathway (Chen et al. 2021).

PNO1 promotes tumor cell proliferation, migration, and invasion by regulating the Notch signaling pathway

Cell differentiation, proliferation, apoptosis, and metastasis in colorectal cancer, lung cancer, breast cancer, and other tumors are regulated by the Notch signaling pathway. Cancer cells can also enhance the ability of invasion and migration, stem cell-like characteristics, and therapeutic resistance through epithelial–mesenchymal transformation (EMT) (Yuan et al. 2014; Jin et al. 2017; Wang et al. 2018). Results on the GSEA dataset (Gene Set Enrichment Analysis) suggest that PNO1 may act as an oncogene to promote lung adenocarcinoma (LUAD) progression through the Notch signaling pathway and that PNO1 expression is positively correlated with EMT (Liu et al. 2020). In LUAD, PNO1 knockdown decreases the protein levels of Notch2 (Notch pathway ligand), Notch4 (Notch intracellular domain), and Hey1 (Notch target gene). In addition, PNO1 can induce the up-regulation of EMT-related marker E-cadherin, and the down-regulation of EMT-related marker N-cadherin and vimentin are also regulated by PNO1. Downregulation of PNO1 can inhibit the proliferation, migration, and invasion of LUAD cells by inhibiting the Notch signaling pathway that regulates EMT. The xenotransplantation model and lung metastasis experiment showed that the proliferation and metastasis of LUAD in vivo might be encouraged by the expression of PNO1 (Liu et al. 2020). There is a precise molecular upstream mechanism in LUAD, negative regulation of PNO1 by miR-340-5p. By directly binding to PNO1 3′ untranslatable region (UTR), miR-340-5p inhibits the expression of PNO1 in LUAD and plays an essential role in the progression of LUAD through the Notch signaling pathway (Liu et al. 2020).

PNO1 regulates tumor cell proliferation, invasion, and apoptosis by inducing AKT/mTOR, NF-κB and other signaling

AKT signaling pathway regulates many processes under physiological and pathological conditions, including metabolism, proliferation, cell survival, growth, and angiogenesis (Lien et al. 2016; Manning and Toker 2017). In in vitro and in vivo experiments, PNO1 knockdown has been shown to significantly reduce protein kinase B (AKT)/rapamycin (mTOR) signaling, reduced tumor volume, tumor weight, and lung metastasis, and significantly inhibit the growth and metastasis of hepatoma cells (Dai et al. 2019). In esophageal cancer tissues, knockdown of PNO1 inhibits cell proliferation, migration, and invasion. It promotes cell apoptosis, which may function by regulating the expression of AKT1, Twist, MYC, mTOR, matrix metalloproteinase 2 (MMP2), NF-κB p65 and CTNNB1 (β-catenin 1). Furthermore, in nude mice, smaller tumor volumes are observed after PNO1 knockdown (Wang et al. 2021 may). Celecoxib may also exert its anti-tumor activity by inhibiting PNO1. Celecoxib is a member of the cyclooxygenase-2 (COX-2) selective non-steroidal anti-inflammatory drug (NSAID) family. In tumors, celecoxib can inhibit proliferation (Gao et al. 2016), apoptosis (Toriyama et al. 2016), angiogenesis (Gao et al. 2016 Oct), and invasion (Behr et al. 2015) and helps slow the progression of liver, lung, breast, and prostate tumors (Stasinopoulos et al. 2013; Vosooghi and Amini 2014; Suri et al. 2016). In hepatocellular carcinoma, celecoxib significantly reduces the level of PNO1 in tumor tissue, inhibits the growth of hepatoma cells in vitro and in vivo (Dai et al. 2019), and in a mouse xenograft tumor model, celecoxib has shown efficacy against hepatocellular carcinoma (Chu et al. 2018). AKT/mTOR signaling helps mediate the oncogenic effects of PNO1 (Dai et al. 2019).

PNO1 promotes autophagy and inhibits apoptosis of tumor cells by activating the MAPK signal pathway

Autophagy can double regulate the progress of cancer (Poillet-Perez and White 2019). On the one hand, autophagy can promote tumorigenesis and angiogenesis by providing nutrition and energy. On the other hand, autophagy can inhibit inflammation and promote genomic stability, thus serving as an inhibitor of early cancer development (Wu et al. 2012; White 2015; Amaravadi et al. 2016; Singh et al. 2018). Autophagy has served as the entry point to treat cancer in clinical trials (Barnard et al. 2014; Chude and Amaravadi 2017 jun 16). The principal pathway of cancer autophagy may include Erk/MAPK signaling pathway (Gao et al. 2019; Yue et al. 2019; Xiong et al. 2020). Activation of the Erk/MAPK signal pathway can promote the proliferation, invasion, and metastasis of hepatoma cells (Liu et al. 2016; Chen et al. 2019; Li et al. 2019). In hepatocellular carcinoma, it has been found that the over-expression of PNO1 inhibits the apoptosis of hepatocellular carcinoma cells by activating the MAPK signal pathway to promote autophagy, and the down-regulation of PNO1 interferes with the downstream of autophagy-related markers p62, Beclin-1, LC3B, Atg5 and Atg7 (Han et al. 2021).

Perspectives

The above results not only highlight the potential carcinogenic role of PNO1 and the possibility of being used as a biomarker for early diagnosis and prognosis of tumors, but also prove the rationality of finding small molecular inhibitors targeting PNO1 as a new cancer treatment strategy, and are expected to provide new targets for individualized cancer therapy and prognostic value for the identification of patients.

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Competing interests

The authors report there are no competing interests to declare.

Acknowledgment

Thanks to all the working partners and funding agencies who participated and contributed to this project.

Author contributions

Wanyi Liu, Yongqiu Zeng, and Guicheng Kuang were involved in drafting the paper and the conception, design, analysis, and interpretation of data. Binbin Yang, Hanlin Liu, Yangpin Lv, and Yang Xiong revised the paper critically for intellectual content. All authors agree on the final approval of the version to be published and that all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data sharing does not apply to this article as no new data were created or analyzed in this study.

Additional information

Funding

This work was supported by Undergraduate Innovation and Entrepreneurship project funding (Grant number S202010632243). Corresponding author Yongqiu Zeng has received research support from Undergraduate Innovation and Entrepreneurship project funding.