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Cell Stemness Is Maintained Upon Concurrent Expression of RB and the Mitochondrial Ribosomal Protein S18-2

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Cell Stemness Is Maintained Upon Concurrent Expression of RB and the Mitochondrial Ribosomal Protein S18-2 Cell stemness is maintained upon concurrent expression of RB and the mitochondrial ribosomal protein S18-2 Muhammad Mushtaqa,b, Larysa Kovalevska (Ковалевська)c, Suhas Darekara, Alexandra Abramssond, Henrik Zetterbergd, Vladimir Kashubaa,e, George Kleina, Marie Arsenian-Henrikssona, and Elena Kashubaa,c,1 aDepartment of Microbiology, Tumor and Cell Biology, Biomedicum, Karolinska Institutet, SE-171 65, Stockholm, Sweden; bDepartment of Biotechnology, Faculty of Life Sciences and Informatics, Balochistan University of Information Technology, Engineering and Management Sciences, 87300 Quetta, Pakistan; cLaboratory of Molecular Mechanisms of Cell Transformation, RE Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology of National Academy of Sciences of Ukraine, UA-03022 Kyiv, Ukraine; dInstitute of Neuroscience and Physiology, Sahlgrenska Academy at the University of Gothenburg, SE-405 30 Gothenburg, Sweden; and eDepartment of Molecular Oncogenetics, Institute of Molecular Biology and Genetics of National Academy of Sciences of Ukraine, UA-03143 Kyiv, Ukraine Edited by Tak W. Mak, University of Toronto, Toronto, Canada, and approved May 8, 2020 (received for review December 28, 2019) Stemness encompasses the capability of a cell for self-renewal and S18-2–overexpressing cells included high telomerase activity, differentiation. The stem cell maintains a balance between pro- anchorage-independent growth, and the ability to form aggressive liferation, quiescence, and regeneration via interactions with the tumors in severe combined immunodeficiency mice (SCID) mice. microenvironment. Previously, we showed that ectopic expression However, the mechanism via which S18 immortalizes and trans- of the mitochondrial ribosomal protein S18-2 (MRPS18-2) led to forms cells remains unexplored. Recently, other researchers have immortalization of primary fibroblasts, accompanied by induction used S18-2 as a tool to immortalize chicken embryonic liver cells (6). of an embryonic stem cell (ESC) phenotype. Moreover, we demon- The use of a model system of cell immortalization, i.e., in- strated interaction between S18-2 and the retinoblastoma- fection of primary B cells by the Epstein–Barr virus, led to the associated protein (RB) and hypothesized that the simultaneous targeting of the normally cytoplasmic S18-2 to the nucleus by expression of RB and S18-2 is essential for maintaining cell stem- interacting with the transforming viral protein EBNA-6 in the ness. Here, we experimentally investigated the role of S18-2 in cell CELL BIOLOGY immortalized cells. Furthermore, we showed that S18-2 forms a stemness and differentiation. Concurrent expression of RB and S18- 2 resulted in immortalization of Rb1−/− primary mouse embryonic bridge between the EBNA-6 and RB proteins (7). fibroblasts and in aggressive tumor growth in severe combined The retinoblastoma-associated protein (RB) exerts its function immunodeficiency mice. These cells, which express both RB and primarily by inhibiting the E2F family of transcription factors S18-2 at high levels, exhibited the potential to differentiate into (TFs) through direct binding, thus regulating cell-cycle progres- various lineages in vitro, including osteogenic, chondrogenic, and sion (8). The RB-E2F1 interaction relies on the phosphorylation adipogenic lineages. Mechanistically, S18-2 formed a multimeric status of RB, which is mediated by cyclin-dependent kinases protein complex with prohibitin and the ring finger protein 2 (CDKs) (9). Importantly, S18-2 can bind RB directly, regardless (RNF2). This molecular complex increased the monoubiquitination of the phosphorylation status of this protein (10). Consequently, of histone H2ALys119, a characteristic trait of ESCs, by enhanced E3- E2F1 is released from a complex with RB, thereby promoting ligase activity of RNF2. Furthermore, we found enrichment of KLF4 cell-cycle progression (7). at the S18-2 promoter region and that the S18-2 expression is pos- itively correlated with KLF4 levels. Importantly, knockdown of S18-2 Significance in zebrafish larvae led to embryonic lethality. Collectively, our find- ings suggest an important role for S18-2 in cell stemness and differ- We here describe the essential roles of RB and MRPS18-2 (S18- entiation and potentially also in cancerogenesis. 2) in homeostasis of cell stemness. Mouse primary cells expressing both S18-2 and RB (designated as RH18RB) exhibited stemness and differentiation | cell immortalization | embryogenesis | a stem cell phenotype. As a proof of principle, we demonstrated tumorigenesis decreased expression of stem-cell–related genes in human mes- enchymal stem cells upon down-regulation of S18-2 and RB. No- temness refers to the molecular processes underlying the tably, loss of the S18-2 protein resulted in embryonic lethality in Sfundamental stem cell (SC) properties of self-renewal and zebrafish. To reveal putative molecular mechanisms, we demon- generation of differentiated daughter cells. Highly organized and strated that S18-2, in a multimeric complex with RB, enhanced the strictly controlled signaling systems regulate these two charac- E3-ubiquitin ligase activity of RNF2 toward histone H2A. Finally, teristics of SCs. The self-renewal of these cells is controlled through based on our observation that RH18RB cells generated tumors in the activation of a defined set of genes that sustain a balance be- severe combined immunodeficiency mice, S18-2 could be a tween self-renewal and differentiation (1). Under normal physio- promising target in the development of cancer therapies. logical conditions, controlled shifts in the balance of such signaling pathways induce differentiation. Abnormalities in signaling cascades Author contributions: M.M., S.D., V.K., and E.K. designed research; M.M., L.K., S.D., A.A., and E.K. performed research; H.Z., G.K., and E.K. contributed new reagents/analytic tools; can initiate and promote cellular transformation (2). M.M., V.K., M.A.-H., and E.K. analyzed data; and M.M., M.A.-H., and E.K. wrote the paper. We showed previously that the ectopic expression of the mi- The authors declare no competing interest. tochondrial ribosomal protein S18-2 (MRPS18-2, herein re- This article is a PNAS Direct Submission. ferred to as S18-2) (see SI Appendix, Table S1, for gene names) This open access article is distributed under Creative Commons Attribution-NonCommercial- led to the immortalization of primary rat fibroblasts. Surpris- NoDerivatives License 4.0 (CC BY-NC-ND). ingly, and importantly, these cells exhibited an embryonic stem 1To whom correspondence may be addressed. Email: [email protected]. cell (ESC) phenotype (3, 4). Furthermore, terminally differen- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ tiated skin fibroblasts were transformed into malignant cells doi:10.1073/pnas.1922535117/-/DCSupplemental. upon S18-2 overexpression (5). The common features of these First published June 22, 2020. www.pnas.org/cgi/doi/10.1073/pnas.1922535117 PNAS | July 7, 2020 | vol. 117 | no. 27 | 15673–15683 Downloaded by guest on September 28, 2021 Because of the constitutive activity of CDKs in ESCs, RB is dishes (Fig. 1 A, Middle). RH18 cells did not show this phenotype hyperphosphorylated, and only a very small fraction of RB binds (Fig. 1 A, Lower). to E2F1, suggesting a different mode of action of RB in SCs and The sublines generated were injected subcutaneously (s.c.) their differentiated counterparts (11). Conversely, deletion of into SCID mice in order to analyze their in vivo growth ability. RB1 led to the loss of SC self-renewal characteristics (11). As expected, only the RH18 and RH18RB cells gave rise to Generally, ESCs proliferate rapidly and have a distinct cell cycle tumors. The RH18RB cells generated tumors that were growing with truncated gap phases (12). They may remain in a quiescent faster and that produced a larger tumor mass compared to the state but reenter the cell cycle upon induction of proliferation via RH18 cells (Fig. 1 B, Left). Staining of tissue sections showed that extrinsic signals (13). The quiescent state must be finely regulated; the tumors produced by RH18RB cells could be described as otherwise, ESCs can be directed toward differentiation or senes- aggressive fibrosarcomas (Fig. 1 B, Right). In contrast, RH18 cells cence (14). However, the molecular mechanisms underlying the formed tumors with large necrotic areas (indicated with black function of RB in SCs are largely unknown (15). To study the role arrows, Fig. 1 B, Right). These tumor cells showed epithelial cell- of RB in cell stemness, we developed a model of mouse embryonic like features (indicated with red arrows, Fig. 1 B, Right). fibroblasts (MEFs) derived from homozygous knockout Rb1 em- We next picked four clones each from the RH18 and RH18RB − − bryos. The Rb1 / MEFs exhibited rapid proliferation with an cultures for further studies. To confirm cell immortalization, one of the clones of both RH18 and RH18RB cells were passaged for anchorage-dependent growth pattern. After passage 11, the pro- more than 350 population doublings; thus, these two clones were liferative rate of the cells diminished, and they became senescent − − cultured in vitro for more than 2 y. The remaining three clones of (16). The rationale of the present work was to use the Rb1 / each cell type were kept in culture for 2 mo (SI Appendix, Fig. MEF model to analyze the manner in which high expression levels S1 E and F). In contrast, control RH and RHRB cells became of RB and S18-2 cooperate to control cell fate. We hypothesized senescent quite rapidly and died after 4 to 6 wk, and no cultures that the simultaneous expression of these two proteins at a high could be passaged beyond this time point. The RHRB cells level supports stemness (17). showed contact inhibition and a significantly higher percentage of senescent cells (up to 12% β-galactosidase–positive cells) Results β – −/− compared with RH cultures (with 2% -galactosidase positive Overexpression of S18-2 Leads to Immortalization of Rb1 MEFs. To cells). In contrast, very few senescent cells were detected in the analyze whether expression of RB is needed for S18-2-induced RH18 and RH18RB cultures (Fig.
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