Inhibition of ribosome assembly factor PNO1 by CRISPR/Cas9 technique suppresses lung adenocarcinoma and Notch pathway: Clinical application

Abstract Growth is crucially controlled by the functional ribosomes available in cells. To meet the enhanced energy demand, cancer cells re‐wire and increase their ribosome biogenesis. The RNA‐binding protein PNO1, a ribosome assembly factor, plays an essential role in ribosome biogenesis. The purpose of this study was to examine whether PNO1 can be used as a biomarker for lung adenocarcinoma and also examine the molecular mechanisms by which PNO1 knockdown by CRISPR/Cas9 inhibited growth and epithelial–mesenchymal transition (EMT). The expression of PNO1 was significantly higher in lung adenocarcinoma compared to normal lung tissues. PNO1 expression in lung adenocarcinoma patients increased with stage, nodal metastasis, and smoking. Lung adenocarcinoma tissues from males expressed higher PNO1 than those from females. Furthermore, lung adenocarcinoma tissues with mutant Tp53 expressed higher PNO1 than those with wild‐type Tp53, suggesting the influence of Tp53 status on PNO1 expression. PNO1 knockdown inhibited cell viability, colony formation, and EMT, and induced apoptosis. Since dysregulated signalling through the Notch receptors promotes lung adenocarcinoma, we measured the effects of PNO1 inhibition on the Notch pathway. PNO1 knockdown inhibited Notch signalling by suppressing the expression of Notch receptors, their ligands, and downstream targets. PNO1 knockdown also suppressed CCND1, p21, PTGS‐2, IL‐1α, IL‐8, and CXCL‐8 genes. Overall, our data suggest that PNO1 can be used as a diagnostic biomarker, and also can be an attractive therapeutic target for the treatment of lung adenocarcinoma.

therefore it becomes incurable. 2 Despite recent advances in lung cancer, the 5-year survival rate of patients with lung cancer is 16%. 1 Therefore, it is urgent to identify new molecular biomarkers to predict the prognosis of lung cancer patients and develop novel molecular targeted therapy for lung adenocarcinoma.
The ribosome, conserved from yeast to mammals, is a ribonucleoprotein complex that regulates translation machinery during protein synthesis. 3,4 The higher energy demand of cancer cells is associated with increased ribosome biogenesis and mutation of ribosomal proteins. Therefore, ribosome represents an attractive anti-cancer therapy target. The RNA-binding protein "partner of NOB1" (PNO1, also known as Dim2, Rrp20) is a ribosome assembly factor. 3,5 The PNO1 gene is located in human chromosome 2p14, comprising seven exons and six introns, and plays a crucial role in ribosome biogenesis and promotes the maturation of small ribosomal subunits. 5 PNO1 cleaves 18S mediated by binding to NOB1. 6 In spite of its role in ribosome biogenesis, the mechanism by which PNO1 regulates oncogenesis is not well understood. Since PNO1 is highly expressed in cancer cells, [7][8][9][10][11] it can be used as a diagnostic biomarker and also can be an attractive target for cancer therapy. Therefore, understanding the expression and biological function of PNO1 is crucial for effectively managing lung adenocarcinoma.
Cancer cells generally possess unlimited replicative potential, divide faster, and display increased biosynthesis and metabolic activity to meet enhanced energy demand. 12 Thus, frequently dividing cancer cells will require enhanced global protein synthesis. One of the mechanisms by which protein synthesis can be controlled through an increase in mRNA translation requires ribosomes. 13 Ribosomes are produced in the nucleolus and act as molecular machines where they translate mRNA into protein. Recent studies have demonstrated the dysregulated ribosome biogenesis during carcinogenesis. 14,15 Increased size and numbers of nucleoli are frequently observed in most cancers, requiring malignant cells to acquire enhanced ribosomal biogenesis. This suggests that increased ribosome biogenesis may play an essential role in cancer initiation and progression. Therefore, inhibition of ribosome biogenesis may provide an attractive therapeutic strategy for the treatment of cancer. To address this, we have inhibited the expression of PNO1 by CRISPR/ Cas9 technology and examined the inhibitory effects of PNO1 on the mechanism of lung carcinogenesis.
There are four Notch receptors, referred to as Notch-1, Notch-2, Notch-3, and Notch-4. 19,22 The Notch receptors are transmembrane proteins consisting of extracellular and intracellular domains. 19 The binding of ligand to the receptor causes a conformational change leading to ADAM-mediated ectodomain shedding and subsequent γsecretase-mediated proteolysis within the transmembrane domain, resulting in the release of Notch intracellular domain (NICD). 23 Subsequently, NICD translocates to the nucleus and associates with the RBPjκ to form an active protein complex (Mastermind, histone acetyltransferase, and p300), leading to the induction of target genes such as Hes1 and Hey1. The oncogenic role of Notch in lung cancer has been demonstrated. [24][25][26] Therefore, inhibition of the Notch signalling pathway represents a novel therapeutic strategy for managing lung adenocarcinoma.
The main objective of this paper is to assess whether PNO1 can be used as a diagnostic marker for lung cancer and examine the molecular mechanisms by which PNO1 inhibition regulates lung cancer growth and epithelial-mesenchymal transition (EMT) in lung cancer. The expression of PNO1 was significantly higher in lung cancer cells than in adjacent normal tissue, suggesting it can be used as a diagnostic marker for lung cancer. PNO1/CRISPR/Cas9 inhibited growth and EMT of lung cancer cells by suppressing the Notch pathway. PNO1 knockdown inhibited those genes which play significant roles in cell proliferation, cell cycle, apoptosis, and EMT. In addition, PNO1/CRISPR/Cas9 also inhibited Notch signalling pathway, which plays a crucial role in lung carcinogenesis. In conclusion, PNO1 can be used as a diagnostic biomarker for lung adenocarcinoma, and inhibition of PNO1 by CRISPR/Cas9 technology can be a useful strategy for the treatment of lung adenocarcinoma.

| Cell culture
Human lung cancer cells (A549 and H460) were purchased from American Type Culture Collection (ATCC, Manassas, VA). A549 and H460 cell lines both express wild-type p53. 27,28 Lung cancer cells were grown in RPMI 1640 culture medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO 2 .

| Cell viability assay
Cell viability was measured as described elsewhere. 29 In brief, cells (1.5 × 10 4 ) transduced with NTC or CRISPR/Cas9 were grown in a cell culture medium for various time points. Cell viability was measured by CellTiter-Glo® Luminescent Cell Viability Assay (Promega), which determines the number of viable cells based on the quantitation of the ATP present, which signals the presence of metabolically active cells.

| Apoptosis assays
Apoptosis was measured as described elsewhere. 30 In brief, cells transduced with either NTC or CRISPR/Cas9 lentiviral particles were grown in a cell culture medium at various times. Apoptosis was measured by TUNEL assay as per manufacturer's instructions (Thermo Fisher Scientific, Suwanee, GA).

| Motility assay
Cell motility assay was performed as we described elsewhere. 31,32 In brief, cells (NTC and CRISPR/Cas9) were grown to a conflu-

| Transwell migration assay
Transwell migration assays were performed as we described elsewhere. 33 In brief, 1 × 10 5 cells (NTC and CRISPR/Cas9) in 200 μl of medium with 1% FBS were plated in the top chamber onto the noncoated membrane (6.5-mm diameter, 8μm pores; Corning Costar, Corning, NY) and allowed to migrate in the lower chamber towards 10% FBS (as chemoattractant)-containing medium. After 48 h of incubation at 37°C in 5% CO 2 , we fixed the cells with methanol, stained with crystal violet, and counted under an inverted microscope.

| Western blot analysis
Western blot analysis was performed as we described elsewhere. 36 In brief, cell lysates were prepared using RIPA lysis buffer containing 1 X protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Cell lysates containing 40-60 μg of protein were loaded and separated on 10% Tris-HCl gel. Proteins from the gel were transferred on polyvinylidene difluoride (PVDF) membranes and subsequently blocked in blocking buffer [5% nonfat dry milk in 1 X Tris Buffer Saline (TBS)] and incubated overnight with primary antibodies (1:500 or 1:1000 dilution). Membranes were washed three times with TBS-T for 10, 5, and 5 min each. After washing, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase at 1:5000 dilution in TBS for 1 h at room temperature. Membranes were again washed three times in TBS-T (5 min each time) and developed using ECL Substrate. Protein bands were visualized on X-ray film using an enhanced chemiluminescence system.

| Quantitative real-time PCR
Total RNA was extracted from cells using the TRIzol reagent as per the manufacturer's instructions (Thermo Fisher Scientific, Suwanee, GA). After checking the RNA quality and concentration, qRT-PCR was performed as described elsewhere. 32,37 Briefly, cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Primers specific for each of the signalling molecules were designed using NCBI/Primer-BLAST and used to generate the PCR products. For the quantification of gene amplification,

qRT-PCR was performed using an ABI 7300 Sequence Detection
System in the presence of SYBR-Green.

| Statistical analysis
The mean and SD were calculated for each group of the experiment. Differences between groups were analysed by anova or t-tests using PRISM statistical analysis software (GrafPad Software, Inc.).
Significant differences among groups were considered at p < 0.05.

| The Cancer Genome Atlas (TCGA) reveals the differential expression of PNO1 which increase with smoking in lung adenocarcinoma
PNO1 has been associated with cancer progression and poor survival. 8,9,38 We first examined the expression of PNO1 using the Cancer Genome Atlas (TCGA) data bank by UALCAN (The University of Alabama at Birmingham Cancer Data Analysis Portal). As shown in Figure 1A, the expression of PNO1 was significantly higher in lung adenocarcinoma than in normal lung tissues. We next examined whether the expression of PNO1 changed during various stages of lung adenocarcinoma ( Figure 1B). PNO1 expression was significantly higher in all stages (stages 1-4) of lung adenocarcinoma development compared to normal tissues. The highest expression of PNO1 was observed in stage 3. We next examined the PNO1 expression at different stages of nodal metastasis. PNO1 expression was significantly higher in all stages (N0-N3) of nodal metastasis compared to normal tissues ( Figure 1C). The highest expression of PNO1 was observed at N2 metastasis. We next examined whether Tp53 mutant status plays any role on the expression of PNO1 in lung adenocarcinoma ( Figure 1D). PNO1 expression was significantly higher in both Tp53 mutant and wild-type lung adenocarcinoma compared to normal lung tissues. However, lung adenocarcinoma expressing mutant Tp53 showed significantly higher PNO1 expression than those expressing wild-type Tp53. We next measured the expression of PNO1 in both male and female lung adenocarcinoma patients ( Figure 1E). PNO1 expression was significantly higher in both male and female lung adenocarcinoma patients than those in normal lung tissues.
However, lung adenocarcinoma from male patients showed significantly higher PNO1 expression than those from female patients. We next compared the expression of PNO1 among smokers with lung adenocarcinoma ( Figure 1F). Lung adenocarcinoma patients were classified into 4 groups; non-smokers, smokers, reformed smokers (<15 years), and reformed smoker (>15 years). PNO1 expression was higher in lung adenocarcinoma tissues compared to normal tissues.
Smokers with lung cancer and reformed smokers (>15 years) showed significantly higher expression of PNO1 than non-smokers and reformed smokers (<15 years). These data suggest that constant smoking for more than 15 years can have significant effects on PNO1 expression.

| PNO1/CRISPR/Cas9 inhibits colony formation and cell viability, and induces apoptosis in lung cancer cells
Since PNO1 expression has been associated with lung cancer progression and poor survival, 8,9,38 we examined the effects of PNO1 inhibition on lung cancer growth. The PNO1 expression was inhib- Since PNO1/CRISPR/Cas9 inhibited cell proliferation in both A549 and H460 cells, we next sought to examine the effects of inhibiting PNO1 expression on lung cancer cell apoptosis ( Figure 2D,H).

PNO1/CRISPR/Cas9 significantly induced apoptosis in both A549
and H460 cells compared to NTC control. These data suggest that PNO1 can be a therapeutic target for lung cancer.

| PNO1/CRISPR/Cas9 inhibits cell motility, migration, and invasion in lung adenocarcinoma
Epithelial-mesenchymal transition (EMT) is a biological process that converts epithelial cells into mesenchymal cells. 39,40 During EMT, cells undergo genetic changes that allow them to lose polarity, leave the primary site and migrate to a distant location (secondary site) to reestablish, differentiate, proliferate, and survive. 41 We next measured the effects of PNO1/CRISPR/Cas9 on cell motility. PNO1/ CRISPR/Cas9 inhibited cell motility of both A549 and H460 cells ( Figure 3A). Since inhibition of PNO1 suppressed cell motility, we next measured the effects of PNO1/CRISPR/Cas9 on cell migration and invasion. PNO1/CRISPR/Cas9 inhibited cell migration and invasion of both A549 and H460 cells ( Figure 3B,C). These data suggest that inhibition of PNO1 can be beneficial for suppressing lung cancer cell metastasis.

| PNO1/CRISPR/Cas9 regulates the expression of markers of epithelial-mesenchymal transition in lung adenocarcinoma
Epithelial-mesenchymal transition (EMT) occurring during tumour progression is highly deregulated. 41 Transcription factors such as Snail, Slug, and ZEB1 are involved in the orchestration of EMT. 41,42 Since PNO1 knockdown inhibited cell motility, migration, and invasion, we next examined the molecular mechanisms of These data suggest that PNO1 knockdown may inhibit inflammation by suppressing inflammatory cytokines in lung adenocarcinoma.

| PNO1/CRISPR/Cas9 inhibits Notch signalling pathway and targets lung cancer cells
Since the Notch signalling pathway plays a crucial role in lung carcinogenesis, 25,49 we sought to measure the effects of PNO1 knockdown on the components of the Notch pathway and its target genes.
PNO1/CRISPR/Cas9 inhibited the expression of Notch1, Notch2, Notch3 Jagged1, and DLL1 in lung cancer A549 and H460 cells ( Figure 8A,B). Similarly, PNO1/CRISPR/Cas9 inhibited the expression of Notch target genes Hes1 and Hey1 in lung cancer A549 and H460 cells ( Figure 8A,B). We next confirmed the effects of PNO1 knockdown on the expression of some of the proteins of the Notch F I G U R E 2 PNO1/CRISPR/Cas9 inhibits colony formation and cell proliferation, and induces apoptosis in lung cancer cells. (A, E) mRNA and protein expression of PNO1. Lung cancer (A549 and H460) cells were infected with lentiviral particles expressing either non-targeting control (NTC) or PNO1/CRISPR/Cas9, and the mRNA and protein expression of PNO1 was measured by the qRT-PCR (left) and Western blot analysis (right), respectively. β-Actin was used as a loading control. (B, F) Colony formation. Lung cancer (A549 and H460) cells were infected with lentiviral particles expressing either NTC or PNO1/CRISPR/Cas9. The number of colonies formed at 21 days were counted. Data represent mean (n = 4) ± SD. * = significantly different from NTC, p < 0.05. (C, G) Cell viability. A549 and H460 cells were infected with lentiviral particles expressing either NTC or PNO1/CRISPR/Cas9. Cell viability was measured as described in Materials and Methods. Data represent mean (n = 4) ± SD. * = significantly different from NTC, p < 0.05. (D, H) Apoptosis. A549 and H460 cells were infected with lentiviral particles expressing either NTC or PNO1/CRISPR/Cas9. Apoptosis was measured as described in Materials and Methods. Data represent mean (n = 4) ± SD. * = significantly different from NTC, p < 0.05.

pathway. PNO1/CRISPR/Cas9 inhibited the protein expression of
Notch1, Notch2, Notch3, and Hey1 in both A549 and H460 cell lines ( Figure 8C,D). These data suggest that PNO1 knockdown can inhibit lung carcinogenesis by targeting the Notch signalling pathway.

| DISCUSS ION
Despite extensive research on lung adenocarcinoma, clinical outcomes remain very poor. Therefore, novel therapeutic technologies are urgently needed for the management of the disease. Considering the role of genetics and epigenetics in carcinogenesis, gene therapy provides an attractive approach in cancer treatment research.
Gene therapy causes fewer side effects to patients compared to conventional methods such as chemotherapy and radiotherapy.
Furthermore, the gene therapy approach offers a persistent cure compared to traditional therapy which generally ends up in drug resistance and relapse. PNO1/CRISPR/Cas9 technique could be an effective strategy for genome editing and thus treating patients. 50 This system comprises Cas9 (RNA-guided DNA endonuclease) and gRNA. Specifically, this system has been used to alter site-specific mutagenesis, gene expression, and epigenetics and to target RNAs and specific DNA sequences. 51,52 In the present study, inhibition of

Cancer cells rely on ribosome biogenesis which increases in
cancer cells to cope with a rise in protein synthesis and sustain unrestricted growth. 53,54 The oncogenic role of PNO1 in cancer has recently been reported. [7][8][9]11,38,55 In hepatocellular carcinoma, celecoxib inhibited PNO1 expression and tumour growth through modulation of AKT/mTOR signalling pathway. 7 In colorectal cancer, EBF1 over-expression down-regulated PNO1 expression and transcription, and up-regulated the expression of p53 and p21 proteins. 10 In lung adenocarcinoma, higher expression of PNO1 has been associated with poor survival. 9 According to TCGA data, PNO1 expression F I G U R E 4 Effects of PNO1/CRISPR/Cas9 on the expression of E-Cadherin, OVOL1, and N-Cadherin in lung cancer cells. (A-C) Lung cancer A549 cells expressing either non-targeting control (NTC) or PNO1/CRISPR/Cas9 were seeded. After 24 h, cells were harvested, and RNA was extracted to measure the mRNA expression of E-cadherin, OVOL1, and N-Cadherin by qRT-PCR. Data represent mean ± SD. * = significantly different from control, p < 0.05. (D-F) Lung cancer H460 cells expressing either NTC or PNO1/CRISPR/Cas9 were seeded. After 24 h, cells were harvested, and RNA was extracted to measure the mRNA expression of E-cadherin, OVOL1, and N-Cadherin by qRT-PCR. Data represent mean ± SD. * = significantly different from control, p < 0.05. (G) Protein expression of cadherins in A549 cells. Cell lysates were collected from A549/NTC and A549/PNO1/CRISPR/Cas9 cells and protein expression of E-cadherin and N-cadherin was measured by the Western blot analysis. (H) Protein expression of cadherins in H460 cells. Cell lysates were collected from H460/NTC and H460/PNO1/CRISPR/Cas9 cells and protein expression of E-cadherin and N-Cadherin was measured by the Western blot analysis. β-Actin was used as a loading control.
in lung adenocarcinoma patients increased with stage of development, nodal metastasis, and smoking. Lung adenocarcinoma tissues from males expressed higher PNO1 than those from females, suggesting the influence of sex on PNO1 expression. Furthermore, lung adenocarcinoma tissues with mutant Tp53 expressed higher PNO1 than those with wild-type Tp53, suggesting the influence of Tp53 status on PNO1 expression. In the present study, PNO1 was overexpressed in lung adenocarcinoma, and its inhibition by CRISPR/Cas9 technology inhibited cell proliferation, motility, migration, and invasion, and induced apoptosis. Our data are in agreement with others where PNO1 has been shown to promote cell proliferation and migration, and PNO1 knockdown inhibited tumorigenesis. 7,8,10,11,38 These data suggest that PNO1 can be used as a diagnostic and prognostic biomarker for lung cancer, and its inhibition can be used for the treatment of cancer.
The NOTCH signalling pathway plays a crucial role in lung development, growth, differentiation, and tissue regeneration. 25,49 Improper activation of the NOTCH pathway has been associated with lung adenocarcinoma. 25,49 In the present study, we have demonstrated that PNO1 knockdown inhibited Notch signalling by suppressing the expression of Notch receptors (Notch1, Notch2, and Notch3), their ligands (Jagged 1 and DLL1), and downstream targets Hes-1 and Hey1. Similarly, another study has demonstrated the oncogenic role of PNO1 where PNO1 promoted lung adenocarcinoma progression through the Notch signalling pathway. 9 Overall, these data suggest that PNO1 is an oncogenic factor, and its inhibition shown as an unfavourable prognostic factor in non-small cell lung cancer (NSCLC). 56,57 In support of this concept, the expression of Vimentin and Snail has also been linked with the malignant phenotype of NSCLC. 56,57 During carcinogenesis, Snail generally acts F I G U R E 6 Effects of PNO1/CRISPR/Cas9 on the expression of CCND1, p21, and PTGS-2 in lung cancer cells. (A-C) Lung cancer A549 cells expressing either non-targeting control (NTC) or PNO1/CRISPR/Cas9 were seeded. After 24 h, cells were harvested, and RNA was extracted to measure the expression of CCND1, p21, and PTGS-2 by qRT-PCR. Data represent mean ± SD. * = significantly different from control, p < 0.05. (D-F) Lung cancer H460 cells expressing either NTC or PNO1/CRISPR/Cas9 were seeded. After 24 h, cells were harvested, and RNA was extracted to measure the expression of CCND1, p21, and PTGS-2 by qRT-PCR. Data represent mean ± SD. * = significantly different from control, p < 0.05. (G) Protein expression of CCND1, p21, and PTGS-2 in A549 cells. Cell lysates were collected from A549/ NTC and A549/PNO1/CRISPR/Cas9 cells and protein expression of CCND1, p21, and PTGS-2 were measured by the Western blot analysis. (H) Protein expression of CCND1, p21, and PTGS-2 in H460 cells. Cell lysates were collected from H460/NTC and H460/PNO1/CRISPR/ Cas9 cells and protein expression of CCND1, p21, and PTGS-2 were measured by the Western blot analysis. β-Actin was used as a loading control.
as an inducer, while Twist and Zeb ½ are principally involved in retaining the invasive mesenchymal phenotype. 58 In the present study, PNO1 knockdown inhibited EMT by inducing a cadherin switch, inducing OVAL1 and inhibiting Snail, Slug, and Zeb1, and indicating the significance of PNO1 knockdown for suppressing EMT and metastasis.
A direct link between ribosome biogenesis and cell cycle regulation has been reported. Impaired ribosome biogenesis induces a checkpoint control that prevents cell cycle progression. 59 In another study, the p53 pathway as a mediator (p53-dependent) of the signalling link between ribosome biogenesis and the cell cycle was demonstrated. 60 Transgenic mice model overexpressing Myc has demonstrated the importance of p53 in the inhibition of cell proliferation in response to obstructed ribosome biogenesis. 61 Downregulation of ribosome biogenesis by haploinsufficiency reduces cell proliferation and extends tumour formation in p53 wildtype but not in p53 null mice. Inhibition of ribosome biogenesis suppresses cell proliferation by blocking the G 1 /S phase transition through the p21-mediated suppression of pRb phosphorylation. 60 Interestingly, the suppression of ribosome biogenesis resulted in cell cycle arrest in a p53-independent manner. 62,63 In the present study, inhibition of PNO1-induced p21 and suppressed CCND1 in lung cancer cells. Overall, these data links ribosome biogenesis and cell cycle regulation in both p53-dependent and independent manners. Chronic inflammation has been linked with an increased rate of ribosome biogenesis. 64,65 During inflammation, elevated levels of prostaglandins, inflammatory cytokines, and chemokines have been observed. [66][67][68][69] These inflammatory signals are sufficient to trigger cancer initiation. In the present study, inhibition of PNO1 downregulated PTGS-2, IL-1α, IL-8, and CXCL-8 in A549 and H460 cells. Our data suggest that the inhibition of PNO1 can inhibit lung adenocarcinoma by suppressing the production of inflammatory cytokines and chemokines.
In conclusion, our study has demonstrated that inhibition of

ACK N O WLE D G E M ENTS
We thank our lab members for the critical reading of the manuscript.

CO N FLI C T S O F I NTE R E S T
All the authors have declared that no competing interests exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.