50mg/kg EdU (Invitrogen) was administered intraperitoneally, followed by 100mg/kg BrdU (Invitrogen) injection 2 hours later, before assessing nucleoside incorporation by immunofluorescence 6 hours post-BrdU injection
50mg/kg EdU (Invitrogen) was administered intraperitoneally, followed by 100mg/kg BrdU (Invitrogen) injection 2 hours later, before assessing nucleoside incorporation by immunofluorescence 6 hours post-BrdU injection. Figure 4Candidates for proliferation and renewal regulation as identified by DESeq2 analysis of shRNA enrichment in non-dividing (EdU?) relative to dividing (EdU+) basal progenitors (for proliferation screen), or in basal progenitor relative to the differentiated suprabasal cells (for renewal screen). Positive fold-change value indicates shRNA enrichment in Rabbit polyclonal to ZBTB8OS the EdU? population (for proliferation screen) or in basal progenitors (for renewal screen). NIHMS1598458-supplement-7.xlsx (19K) GUID:?80AD7D91-5E25-41D8-AC7E-94AC899B091A 8: Supplemental Table S6: shRNA and primer sequences used in this study, Related to STAR Methods NIHMS1598458-supplement-8.xlsx (11K) GUID:?3F6AA42F-12DA-4058-BB6B-1700FBC0E9CB Data Availability StatementRibosome profiling sequencing data can be accessed at NCBI Gene Expression Omnibus (GSE 126660). All other data are available from the Lead Contact upon request. Summary Human skin tolerates a surprisingly high burden of oncogenic lesions. While adult epidermis can suppress the expansion of individual mutant clones, the mechanisms behind tolerance to oncogene activation across broader regions of tissue are unclear. Here, we uncover a dynamic translational mechanism that coordinates oncogenic HRAS-induced hyperproliferation with loss of progenitor self-renewal to restrain aberrant growth and tumorigenesis. We identify translation initiator eIF2B5 as a central co-regulator of HRAS proliferation and cell fate choice. By coupling ribosome profiling with genetic screening, we provide direct evidence that oncogene-induced loss of progenitor self-renewal is driven by eIF2B5-mediated translation of ubiquitination genes. Ubiquitin ligase FBXO32 specifically inhibits epidermal renewal without affecting overall proliferation, thus restraining HRAS-driven tumorigenesis while maintaining normal tissue growth. Thus, oncogene-driven translation is not necessarily inherently tumor promoting but instead can manage widespread oncogenic stress by steering progenitor fate to prolong normal tissue growth. Graphical Abstract eTOC Developing epidermis has remarkable ability to suppress aberrant growth despite widespread oncogenic insult. Cai et al. uncover translation initiation factor eIF2B5 as a central coordinator of HRAS progenitor behavior. Functional dissection of the oncogenic translatome reveals a dynamic translational mechanism that inhibits renewal during oncogenic hyperproliferation to restrain tumorigenesis. Introduction The skin possesses remarkable ability to tolerate genetic and structural abnormalities. Surprisingly, this extends to mutations in known cancer-driving genes, which are frequently found in physiologically normal human skin (Martincorena et al., 2015), suggesting that the tissue has adaptive mechanisms to restrain the expansion of mutant cell populations and protect against progression to Fingolimod cancer. We recently observed through direct intravital imaging that the adult epidermis can entirely resolve Fingolimod abnormal growth of mutant cell clones following activation of or -catenin (Brown et al., 2017). We further uncovered that oncogenic epidermal clones can be completely blocked from expansion and eventually expelled from the adult tissue through loss of growth-sustaining progenitor cells (Ying et al., 2018). However, these studies only examined the epidermis growth-restrictive potential in the context of individual clones arising from a single cell. The mechanisms behind tissue tolerance to widespread oncogene activation, as seen in oncogene-driven congenital overgrowth disorders (Keppler-Noreuil et al., 2016; Rauen, 2013) and in field cancerization where broad areas of genetically altered tissue are asymptomatic (Curtius et al., 2017), remain unexplored. Tissues can employ various cell-autonomous strategies to block the proliferation of single clones with somatic mutations, including apoptosis and senescence (Braig et al., 2005; Fearnhead et al., 1998). Neighboring wildtype (WT) cells can also facilitate oncogene tolerance through non-cell-autonomous events that restrict expansion or displace mutant clones from the tissue (Brown et al., 2017; Ying et al., 2018). An illustrative case is the recent finding that WT cells surrounding mutant clones maintain pro-renewal JNK signaling, allowing WT cells to outcompete and expel highly differentiating mutant clones (Ying et al., 2018). However, this mechanism for oncogene tolerance is not feasible when a large proportion of the tissue carries the same lesion, abolishing the growth-suppressive potential of WT neighbors. Furthermore, since the epidermis requires frequent cell turnover for its development and function (Fuchs and Raghavan, 2002; van der Flier and Clevers, 2009), extensive Fingolimod elimination of mutant cells or a complete proliferation block would significantly disrupt tissue architecture and integrity. How oncogenic epidermis preserves the rapid physiological growth needed for tissue development while restraining pathological overgrowth remains a fundamental question. The embryonic murine interfollicular epidermis (IFE) is an ideal system to explore oncogene-induced stem cell behaviors in the context of rapid tissue growth (Beronja et al., 2013; 2010; Williams et al., 2011). Its well-defined structure features frequently proliferating basal progenitor cells, which have the unique ability to self-renew, and their suprabasal differentiated progeny, which are post-mitotic and provide the.