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Aberrant Expression of Select Pirna-Pathway Genes Does Not Aberrant expression of select piRNA-pathway genes BRIEF REPORT does not reactivate piRNA silencing in cancer cells Pavol Genzora, Seth C. Cordtsa, Neha V. Bokila, and Astrid D. Haasea,1 aNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Edited by Brigid L. M. Hogan, Duke University Medical Center, Durham, NC, and approved April 30, 2019 (received for review March 15, 2019) Germline genes that are aberrantly expressed in nongermline the slicer activity of PIWIL2 (HILI) and the RNA helicase DDX4/ cancer cells have the potential to be ideal targets for diagnosis and VASA (13) (Fig. 1E). We did not observe aberrant expression of therapy due to their restricted physiological expression, their DDX4/VASA in any nongermline cancer (Fig. 1 B–D), eliminating the broad reactivation in various cancer types, and their immunogenic possibility of reactivated ping-pong processing, and thus focused our properties. Among such cancer/testis genes, components of the analysis on potential PLD6/ZUC-dependent piRNA production. Be- PIWI-interacting small RNA (piRNA) pathway are of particular inter- cause of high sequence diversity and lack of sequence conservation, est, as they control mobile genetic elements (transposons) in germ piRNAs are best defined by their interaction with PIWI proteins, cells and thus hold great potential to counteract genome instability according to their initial definition as PIWI-interacting RNAs (11, 14). in cancer. Here, we systematically investigate the potential reac- Echoing the phenomenon of cancer/germline gene-expression in pri- mary tumor samples [The Cancer Genome Atlas (TCGA)], some tivation of functional piRNA-silencing mechanisms in the aberrant cancer cell lines exhibit aberrant expression of piRNA-pathway com- context. While we observe expression of individual piRNA-pathway ponents. These cell lines represent a trackable model to directly probe genes in cancer, we fail to detect the formation of functional piRNA- for the presence of bona fide piRNAs. For the molecular and func- silencing complexes. Accordingly, the expression of a PIWI protein tional characterization of aberrant PIWIL1 complexes, we chose the alone remains inconsequential to the cancer cell transcriptome. Our colon cancer cell line COLO205, which is the top available cell line data provide a framework for the investigation of complex aberrant that robustly expresses PIWIL1 but no other PIWIL gene (Fig. 1D). gene-expression signatures and establish that reactivation of piRNA To unambiguously detect the expression of PIWIL1 protein in silencing, if at all, is not a prevalent phenomenon in cancer cells. ’ COLO205, we inserted a FLAG (F-) tag in-frame with PIWI sstart CELL BIOLOGY codon into the endogenous genomic locus using CRISPR-Cas9 ge- RNA | piRNA | germline | cancer | transposon nome editing. Western blotting analysis showed expression of endog- enouslytaggedF-PIWIL1withanexpectedmolecularmassof∼100 obile genetic elements (transposons) threaten genomic kDa, in agreement with the presence of full-length, wild-type messenger Mintegrity and must be restrained (1). Transposon restric- RNA (Fig. 1F). To identify if aberrantly expressed PIWIL1 interacts tion is imperative, especially in germ cells, where transposition with small RNAs, we purified F-PIWIL1 and characterized associated RNAs by next-generation sequencing. RNA fragments copurifying with mutagenesis has the potential to change the identity of a species. F-PIWIL1 were indistinguishable from background pull-down both by Thus, germ cells employ a specialized small RNA-silencing path- — nucleotide size distribution and by genomic annotation, and mainly way PIWI-interacting small RNAs (piRNAs) and their PIWI contained background from the abundant class of microRNAs (Fig. — protein partners to establish lasting epigenetic restriction of 1G). None of our experiments detected potentially functional piRISCs transposons (2, 3). Recently, aberrant expression of select piRNA- in COLO205 cells. Accordingly, neither knockdown (Fig. 1H, Left) pathway genes has been observed in nongermline cancers, in line nor knockout (Fig. 1H, Right)ofPIWIL1 had any effect on gene ex- with the well-established phenomenon of cancer/germline genes, pression or abundance of transposon transcripts in the aberrant context. which describes the aberrant expression of germline-specific genes in Despite our effort, we did not find any evidence for the presence of nongermline cancers (4–6). Such cancer/germline genes have the bona fide piRNAs or a piRNA-independent function of PIWIL1 in potential to be ideal targets for diagnosis and therapy because of their cancer cells. Our careful experimental analyses support a recent report restricted physiological expression and their broad reactivation in that identified abundant RNA fragments as ubiquitous background of various cancer types. However, their function in the aberrant context small RNA-sequencing (RNA-seq) data (15). Such fragments have remains largely elusive. Three consequences of cancer/germline gene sometimes been misinterpreted as piRNAs and contaminate databases expression could be envisioned: (i) cancer cells could reactivate that attempt to catalog individual piRNA sequences from different experimental sources. While we do not wish to generalize from our ii germline programs such as piRNA-mediated transposon silencing; ( ) limited set of experiments in COLO205 cells, we aim to raise awareness individual germline genes could assume novel function in the aberrant for the experimental requirements to define piRNAs as “PIWI- context; and (iii) the aberrant expression of an individual germline interacting RNAs,” and caution against hasty interpretation of gene in the absence of cofactors could be nonfunctional and RNA fragments. The careful study of aberrant cancer/germline without biological consequence. Here, we systematically investi- gene expression holds great promise for identifying modulators of gate the potential aberrant reactivation of functional piRNA- cancer biology (16) and for discovering novel diagnostic markers and silencing mechanisms in cancer considering all three hypotheses. therapeutic targets (5). However, not every aberrant expression results in functional products with biological impact. Results and Discussion Biogenesis of piRNAs and formation of functional PIWI–piRNA- silencing complexes (piRISCs) requires the coordinated action of Author contributions: A.D.H. designed research; P.G., S.C.C., and N.V.B. performed re- biogenesis and effector factors (3, 7), most of which are only search; P.G. analyzed data; and P.G. and A.D.H. wrote the paper. expressed in the germline of healthy individuals (Fig. 1A) (8). To The authors declare no conflict of interest. evaluate the potential formation of functional piRISCs in cancer, we This open access article is distributed under Creative Commons Attribution-NonCommercial- first characterized the expression of conserved piRNA-pathway NoDerivatives License 4.0 (CC BY-NC-ND). genes in tumor samples (Fig. 1B) and in cancer cell lines (Fig. 1 C Data deposition: RNA sequencing data and small RNAsequencing data have been depos- and D) using available gene-expression data (9, 10). The human ited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo genome encodes four PIWI proteins—PIWI-like (PIWIL)1, -2, -3, (accession no. GSE128526). and -4 (11, 12)—that are either fueled by PLD6/ZUCCHINI(ZUC)- 1To whom correspondence may be addressed. Email: [email protected]. dependent piRNA biogenesis or “ping-pong” processing, which requires www.pnas.org/cgi/doi/10.1073/pnas.1904498116 PNAS Latest Articles | 1of2 Downloaded by guest on September 26, 2021 A B C D TDRD9 TPM PNLDC1 100 TDRKH 75 GTSF1 GPAT2 50 MAEL MOV10L1 25 DDX4 0 PLD6 PIWIL4 PIWIL3 PIWIL2 PIWIL1 1 on off 645 V [75] cancer cell lines KM−12 SNU−1 O AD [103] AN [222] AD [128] ACC [19] ACC UCS [14] CHOL [9] UVM [20] LIHC [92] GBM [38] KIRP [72] KICH [16] PAAD [44] PAAD LGG [129] DLBC [12] TGCT [37] ESCA [46] Liver [188] READ [23] SARC [64] CESC [76] Lung [607] THYM [30] UCEC [44] PCPG [44] COLO 201 COAD [71] COAD COLO 205 SKCM [25] KIRC [133] MESO [21] Heart [727] Skin [1362] Kidney [50] ST STES [149] BLCA [102] Colon [539] LU Testis [284] Nerve [498] Ovary [138] LUSC [125] THCA [125] BRCA [273] PRAD [124] HNSC [130] KIP COLO 206F Brain [2076] Bladder [11] Breast [306] Uterus [117] Fal. Tube [7] Spleen [169] Vagina [124] Blood [2561] Muscle [718] Thyroid [564] ADREAD [94] Pituitary [192] Prostate [159] Stomach [272] TPM Small Int. [143] Pancreas [268] GBMLGG [167] Adipose T.[886] Cervix Uteri [11] Adr. Gland [205] Esophag. [1111] Blood Vsl. [1041] CO >100 2575 Sal.y Gland [104] EGF H 6 coverage (reads) log Fraction SH1: p = .97/1 PIWIL1: chr12,130335888-130374332 6 .8 miRNA SH2: p = .99/1 ko1 10 Cytoplasm 100 5 15 ] SH3: p = 1/1 [ 6 mRNA .6 5 Nucleus TE intron .4 exon1 ] normalized counts +1 PLD6 x10 piRNA 60 tRNA 4 Δ 8 bp ] [ .2 5 4 PIWIL1 precursor snoRNA ] TPM 11° repeat p = 1 x10 75 0 rest 3 22°° coverage (reads) CDS 80 genes 50 [ PIWIL2/4 Transposon 2 10 2 25 KDa WB log n = 3 PIWIL1 ko1: p = .99/.99 IP PIWIL1 IP Control DDX4 1 % mRNA 40 100 F-PIWIL1 n = 4 ko2: p = .99/1 Read counts [ n = 1 SH1 SH3 PIWIL4 Flag exon1 0 0 SH2 padj <=.05 PIWIL2/4 sashimi (RPKM) 0 CRISPR/Cas9 50 Tubulin 20 25 30 35 01234560123456 read size PIWIL1 kd [SH1] (y), wt (x) PIWIL1 ko1 (y), wt (x) Fig. 1. Individual piRNA-pathway genes are aberrantly expressed in cancer without evidence for the reactivation of functional piRNA silencing. (A and B) Gene expression of core piRNA-pathway genes in human tissues (Genotype-Tissue Expression) (A) and tumor samples (TCGA) (B); red indicates “on”: 25th percentile RSEM >5. Abbreviations of tumor types are according to TCGA, with the number of individual samples indicated in brackets. (C) Expression of select piRNA-pathway genes in 645 human cancer cell lines (on: RPKM >5). (D) The top five cell lines ranked by aberrant PIWIL1 expression.
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