Abstract 5193: The RNP biogenesis factor Thoc1 is required for proliferative intestinal stem cell viability.

Abstract Thoc1 encodes an RNA binding protein (Thoc1) which interacts with the tumor suppressor retinoblastoma (Rb1). Thoc1 levels have been found to be elevated in multiple cancers relative to normal tissue. Additionally cancer cells but not most normal cells undergo apoptosis following Thoc1 deletion, suggesting Thoc1 may be a good therapeutic target. In normal tissue, Thoc1 is needed for embryonic inner cell mass viability and Thoc1 hypomorphic mice are small and sterile. Based on these data we hypothesize highly proliferative cells, possibly those with an extended replicative potential, such as stem cells, require Thoc1. To test this, we examined the effect of Thoc1 deletion in the intestine since it contains well-defined stem cells Thoc1 was deleted in the adult mouse using the Tamoxifen/CreER system with Rosa26 driving CreER expression. Mice were treated with 2 mg/day Tamoxifen for 1, 2, 3, 4, 5, or 6 days. Thoc1 deletion completely disrupts small intestine (SI) structure. Specifically, Thoc1 deletion impairs SI crypt cell function and viability, which leads to a complete loss of SI epithelial cell turnover. The SI crypts contain rapidly proliferating intestinal stem (ISC) and progenitor cells that continuously feed and replace the differentiated SI villi epithelium every 3-5 days. Within the proliferative crypts, multiple ISC populations exist. These include the active, homeostatic Lgr5+ ISCs and the reserve, more quiescent mTert+ and Bmi1+ ISCs. Lgr5+ ISCs are the most actively proliferating ISC population, whereas mTert+ and Bmi1+ ISCs are believed to become proliferative upon tissue insult. QRT-PCR data of ISC (Lgr5, mTert, Bmi1) marker transcripts from crypt purified epithelial cells suggests Thoc1 loss initially induces ISC populations to proliferate and expand (transcripts increase), likely a direct response to the initial Thoc1 deletion. However, once active it appears proliferating ISCs become susceptible to Thoc1 deletion, as ISC marker transcripts ultimately approach zero on day 6 in test mice. In addition, ISC fate mapping studies show recombined ISCs in test mice are less likely to give rise to long lived stable progeny than recombined ISCs in control mice. Despite a similar structure to the SI, Thoc1 deletion does not appear to affect the colon. The colon too contains ISCs that give rise to all cells in the tissue; however, colon ISCs are more quiescent versus the SI ISC, which suggests actively proliferating stem/progenitor cell types may specifically require Thoc1. The results of this study suggest actively proliferating stem cell types require Thoc1, most likely to support the coordinated gene expression necessary for rapid proliferation and differentiation. This finding is significant as it sheds light on Thoc1 function and may help explain why Thoc1 levels are increased in various cancers, as proliferating stem cells and cancer cells share similar traits. Citation Format: Laura B. Pitzonka, Sumana Ullas, David Goodrich. The RNP biogenesis factor Thoc1 is required for proliferative intestinal stem cell viability. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5193. doi:10.1158/1538-7445.AM2013-5193</jats:p

Abstract 1850: The role of <i>Thoc1</i> in tissue homeostasis in the adult mouse

Abstract Thoc1 encodes a protein (Thoc1) that interacts with the retinoblastoma protein (Rb). Thoc1 functions in an evolutionary conserved protein complex called THO that is involved in nuclear ribonucleoprotein particle (RNP) formation. Loss of THO function compromises the packaging of nascent RNA, which causes defects in transcription, RNA processing and mRNA export. In mice, Thoc1 is required for embryonic development. It is currently unknown whether Thoc1 is required in adult mice for normal tissue homeostasis. We hypothesize that different tissues will vary in their dependence on Thoc1, possibly based on their proliferative potential. To test this hypothesis, we conditionally deleted Thoc1 in adult mice in a wide variety of tissues using the Tamoxifen/CreER recombination system, and assessed the consequences on tissue homeostasis. Thoc1F/F:Rosa26CreER+/− (test) and Thoc1+/+:Rosa26CreER+/− (control) adult mice were treated with 2 mg/day Tamoxifen for 6 days. Slowly proliferating (liver, kidney, colon) and rapidly proliferating (small intestinal (S.I.)) tissue were collected 24 hours after the last treatment. Histological analysis shows Thoc1 loss disrupts the architecture of the small intestine (S.I.) but does not affect the other tissues examined. The functional unit of the S.I. is the crypt-villus axis. S.I. crypts contain rapidly proliferating stem and progenitor cells that continuously feed the differentiated, non-proliferative villus epithelium. The crypts proliferate so rapidly that the entire S.I. epithelium is replaced every three to five days. Interestingly, Thoc1 loss only causes apoptosis in the S.I. crypts, suggesting disruption of villi architecture following Thoc1 loss is a result of altered crypt function, not a direct effect of Thoc1 loss. Supporting this, the crypts of mice in which Thoc1 has been deleted also have a loss of cell proliferation. While the colon and S.I. have similar tissue architecture, the colon's rate of turnover is much slower. Therefore, it is not surprising the colon is not affected by Thoc1 loss. These findings support the hypothesis that tissues with a high cell proliferative potential require Thoc1. To explore this further, we examined how Thoc1 deletion would affect the highly proliferative hematopoietic system using the same test and control mice as previously listed. Further supporting the hypothesis, Thoc1 loss decreases the number of lymphocytes in the blood, but does not affect spleen and thymus (slowly proliferating tissues) histology. The results of this study suggest highly proliferative tissues that turnover rapidly require Thoc1, most likely to support the gene expression and cell growth necessary for rapid cell division. This hypothesis is significant as it predicts cancer cells will be more sensitive to Thoc1 loss than normal cells, a prediction verified by preliminary results in our lab. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1850. doi:1538-7445.AM2012-1850</jats:p