SIRT7 promotes lung cancer progression by destabilizing the tumor suppressor ARF

Significance We found that the NAD+-dependent deacetylase SIRT7 directly interacts and destabilizes the tumor suppressor ARF. Mechanistically, SIRT7 prevents association of ARF to Nucleophosmin and thereby facilitates ARF proteasomal-dependent degradation in lung cancer cells. The study unveils a unique mechanism by which SIRT7 promotes proliferation of non-small-cell lung cancer that may be exploited to increase cellular levels of ARF for antitumor therapies in cancers with intact ARF expression.

HY) were generated by employing a retroviral system.Phoenix-AMPHO cells were transfected with 10µg pMSCV plasmid containing SIRT7 WT or SIRT7 HY mutant cDNA together with 1µg of envelope vector (VSV-G/pMD2.G).Medium containing retroviral particles was filtered and polybrene (Sigma-Aldrich) was added to a final concentration of 8μg/mL.Target cells were trypsinized, re-suspended in retroviral particles-containing medium, and plated in new dishes for 48 hours.After incubation, cells were supplemented with fresh medium for additional 24 hours.Retrovirus-infected cells were selected using puromycin (5µg/mL; Sigma-Aldrich) for 48 hours.After selection, cells were grown in medium without puromycin for at least 4 passages before further use.SIRT7 KO H1299 cells were generated using CRISPR/Cas9 technology as described (4) using the following guide RNA: gRNA 1: CCGCTCCGAGCGCAAAGCGG and gRNA 2: CGAGAGCGCGGACCT GGTAA.

RNA sequencing
RNA was isolated using Direct-zol™ RNA miniprep kit (Zymo Research) following the manufacturer's instructions.To avoid contamination by genomic DNA, samples were treated by on-column DNase digestion (DNase-Free DNase Set, Qiagen).Total RNA and library integrity were verified on LabChip Gx Touch 24 (Perkin Elmer).1µg of total RNA was used as input for SMARTer Stranded Total RNA Sample Prep Kit -HI Mammalian (Clontech).Sequencing was performed with a NextSeq2000 instrument (Illumina) using v3 chemistry, 1x72bp single end setup.Trimmomatic version 0.39 was employed to trim reads after a quality drop below a mean of Q20 in a window of 20 nucleotides and keeping only filtered reads longer than 15 nucleotides (5).Reads were aligned versus Ensembl human genome version hg38 (Ensembl release 104) with STAR 2.7.10a (6).Aligned reads were filtered with Picard 2.27.1 to remove duplicates multi-mapping, ribosomal and mitochondrial reads.Gene counts were established with featureCounts 2.0.2 by aggregating reads overlapping exons on the correct strand excluding those overlapping multiple genes as previously described (7).The raw count matrix was normalized with DESeq2 version 1.30.1 (8).Contrasts were created with DESeq2 based on the raw count matrix.Genes were classified as significantly differentially expressed at average count > 5, multiple testing adjusted p-value < 0.05, and -0.585 < log2FC > 0.585.The Ensemble annotation was enriched with UniProt data (Activities at the Universal Protein Resource (UniProt).All downstream analyses were performed on the normalized gene count matrix.A global clustering heatmap of samples was created based on the euclidean distance of regularized log transformed gene counts.Dimension reduction analyses were performed by principal component analysis (PCA) on regularized log transformed counts using the R packages FactoMineR (9).

Analysis of public datasets
For correlation between relative SIRT7 mRNA and ARF protein levels in lung cancer cell lines, normalized SIRT7 gene expression and ARF protein data (ID: Q8N726) of 54 different lung cancer cell lines were obtained from public data available through the Cancer Dependency Portal (https://depmap.org/portal).P-values were obtained after a linear regression fit.To compare SIRT7 mRNA expression in healthy tissues and tumour samples, normalized RNA expression data of SIRT7 in TCGA lung adenocarcinoma data sets were downloaded from the Broad Institute Firehose Pipeline (http://gdac.broadinstitute.org).For correlation analysis between SIRT7 mRNA and CDKN2A protein levels in tumors, gene expression and protein data were obtained from a publicly available dataset of lung adenocarcinoma patients (10) using cBioportal (https://www.cbioportal.org/)for Cancer Genomics (11)(12)(13).The analysis was carried using log2 transformed normalized RPKM (reads per kilobase of exon per million reads mapped) values and protein abundance ratios.Correlation analysis was performed using the Spearman's method.Data for Nectin2, XRCC1, SUPT5H, SIPA1L3 and CCNE1 mRNA expression were derived from the TCGA lung adenocarcinoma data set (TCGA, Firehose Legacy) via the cBioPortal (11)(12)(13).Patients were grouped according to SIRT7 expression levels, using the mean SIRT7 mRNA expression and separated into groups harbouring WT CDKN2A (threshold -1, +1) or CDKN2A homozygous deletion (threshold -0.75, +0.75) based on CDKN2A copy number alteration data (CNA) from genomic sequencing of the same tumors.

Human samples
Tumor tissue specimens (T1-T4) as well as adjacent healthy tissues (H1-H4), based on the histopathologic analysis, were collected from patients with non-small cell lung cancer at the time of surgery before chemotherapy at the University Hospital Giessen (Giessen, Germany).H1-T1: female, aged 51; H2-T2 male aged 76; H3-T3 male aged 39; H4-T4 female aged 72.A written informed consent was obtained from all patients.

Protein modelling and multiple sequence alignment.
Since a crystal structure is not available for human ARF or ARF homologues, the AF-Q8N726-F1 model structure from Alphafold database was used (14).The structure was energy minimized using Gromacs5 (15).Pymol (16) was used to generate the corresponding Figure .Multiple sequence alignment of ARF orthologous sequences in mammals was performed using TCoffee (17,18).

Cell growth assay
Growth curve analysis was performed as described ( 19) differentially expressed genes (DEGs) based on Log2 of expression in scrambled and ARF knockdown H1299 cells (n=3) as assessed by RNA-sequencing (padj; fdr<0,05 Log2FC >+/-0.585,Mean >5).Arrows indicate genes commonly upregulated in in ARF KD and SIRT7-overexpressing H1299 cells.Red arrows indicate genes further validated in this study (see also Fig. S3 E). C. Principal component analysis.(PCA) of RNA-sequencing data for samples in A-B.D. Western blot analysis of ARF expression in stable H1299 cell lines expressing V5-tagged SIRT7 or an empty vector (Ev).Quantification of four independent experiments is shown on the right histogram.E. Heat map of top 50 most significant differentially expressed genes (DEGs) based on Log2 of expression in stable H1299 cells expressing empty vector and V5-tagged SIRT7 as in D. Arrows indicate genes that are also upregulated in ARF KD H1299 cells.Red arrows indicate genes further validated in this study (see also Figure S3 B).F. PCA of RNA-Sequencing data for samples as in D-E.G. GO term analysis of 159 genes that are significantly upregulated both after SIRT7 overexpression and ARF knockdown (Fold of change > 0,58) in cells as in A-C and D-F (see main Fig. 5A).GO term analysis is based on p values, according to the WikiPathway (WP) 2023 human obtained using Enrichr.The top 10 enriched terms for the input gene set are displayed based on the -log10 (p-value).The term at the top has the most significant overlap with the input query gene set.p-values are shown on the right.mRNA expression of indicated genes in scrambled (Scr.) and SIRT7 knockdown (KD) Calu-3 lung cancer cells.β-actin was used as loading control (n=6).C. Western blot analysis of Cyclin E1 (CCNE1) expression in scrambled and SIRT7 KD H1299 lung cancer cells after shRNA-mediated depletion of ARF expression.Quantification of relative CCNE1 levels ±SD is shown in the histogram on the right (n=11).D. RT-qPCR analysis of mRNA expression of CCNE1 in scrambled (Scr.) and SIRT7 knockdown (KD) Calu-3 lung cancer cells.β-actin was used as loading control (n=5).E. Western blot analysis of lung samples from WT and SIRT7 KO animals for indicated markers.Quantification of Cyclin E1 (CcnE1) relative levels ±SD is shown in the histogram on the right (n=4 WT and n=6 KO).F. RT-qPCR analysis of mRNA expression of CCNE1 in control (scrambled; Scr.shRNA) and SIRT7 KD (SIRT7 shRNA) ARFnegative H226 and H322 cells.GAPDH was used as loading control.Quantification of average mRNA levels ± SD is shown in the histograms (n=4).G. Chromatin immunoprecipitation analysis of H2BK20 acetylation at the promoter of indicated genes in scrambled, SIRT7 KD, ARF KD and SIRT7-ARF double knockdown (DKD) H1299 cells (n=5).H. Chromatin immunoprecipitation analysis of p14ARF enrichment at the promoter of indicated genes in cells as in G (n=5).

S5. SIRT7 catalytic activity is not required to stimulate expression of genes suppressed by ARF.
A. Representative Western blot analyses of expression of indicated targets in SIRT7 KO H1299 cells stably expressing exogenously introduced WT or catalytic inactive (HY) SIRT7 at physiological levels.B. RT-qPCR analysis of mRNA expression of indicated genes in SIRT7 knockout H1299 cells lung cancer cells re-expressing WT or the SIRT7 HY mutant as in A. β-actin was used as loading (n=5).

S6. SIRT7 and ARF protein levels inversely correlate in human lung
S2.The N-terminal domain of ARF is required for interaction with SIRT7 or NPM. A. Multiple sequence alignment of human ARF orthologous sequences from mammals.Residues Phe23 and Leu49 are highly conserved among mammals (red box).B. Schematic representation of ARF deletion mutants used to probe interactions with SIRT7 and ARF.Dashed boxes indicate the deleted regions.The amino acids contained in the N-terminal ARF mutant (∆N), middle domain mutant (∆M) and C-terminal domain mutant (∆N) are indicated.C. Coupled immunoprecipitation (anti-SIRT7 antibody) and Western blot analysis (anti-Flag antibody) of purified Flag-tagged SIRT7 and ARF wild type (WT) or deletion mutants as in B. Quantification of relative co-immunoprecipitated ARF is shown in the right histogram (n=5).D. Coupled immunoprecipitation (anti-NPM antibody) and Western blot analysis (anti-Flag antibody) of purified Flag-tagged NPM and ARF WT or deletion mutants as in B. Quantification of relative coimmunoprecipitated ARF is shown in the right histogram (n=4).
cancers. A. Plot illustrating the inverse correlation between CDKN2A protein and SIRT7 mRNA levels in human lung cancers as assessed by bioinformatics analyses.B. Representative immunofluorescence staining of SIRT7 and ARF in healthy human lung tissues and lung cancers.Scale bar 50µm.C. Immunofluorescence staining of SIRT7 and ARF in human lung tumors.Note that individual lung cancer cells displaying high levels of SIRT7 exhibit low levels of ARF in the nucleoli (yellow arrow) and vice versa (white arrow).Scale bar 10 µm.S7.SIRT7 depletion inhibits lung cancer cells growth in vitro and in vivo in an ARF-dependent manner.A.Western blot analysis of p14ARF levels in stable scrambled, SIRT7 KD, ARF KD and SIRT7/p14ARF dKD Calu-3 lung cancer cells.B. Growth curves of cells as in A (n=3; Two-way ANOVA).C. Growth curves of SIRT7 KO H1299 cells re-expressing WT or catalytic inactive SIRT7 HY mutant as in Fig.S5A.(n=3; Two-way ANOVA).D. Soft-agar colony formation assay of cells as in C. The average number of colonies of 3 independent experiments ± SD is shown in the histogram.E. Fluorescencebased imaging of tumor volumes in the mouse xenografts using scrambled SIRT7 KD, ARF KD and SIRT7/ARF DKD H1299 cells.F. Macroscopic images of excised tumors 34 days after injection of tumor cells in mouse xenografts as in E. G. RT-qPCR analysis of mRNA expression of indicated genes in tumors derived from xenografts models (n=4).