Sam68 is a known sequence-specific RNA binding protein that regulates alternative splicing events during the cell cycle and apoptosis. Sam68 has also been shown to influence transcription, but the ...molecular mechanism remains undefined. Herein we identify Sam68 as a transcriptional coactivator of the p53 tumor suppressor in response to DNA damage. Using CRISPR/Cas9 generated isogenic HCT116 Sam68
cell lines wild type or deficient for p53, we show that Sam68 is required for the efficient transactivation of p53 target genes. Consistently, Sam68 depletion caused defects in DNA damage-induced cell cycle arrest and apoptosis mediated by p53. Mechanistically, we demonstrate that Sam68 physically interacted with p53 in an RNA-dependent manner, and that this interaction was essential for the coactivator function of Sam68. Furthermore, we show that both Sam68 and p53 were recruited to promoters of p53-responsive genes, suggesting interdependence. Finally, Sam68 acted in concert with the p53 long noncoding RNA (lncRNA) target PR-lncRNA-1 for p53 recruitment, implicating a positive-feedback mechanism in which lncRNAs induced by the Sam68/p53 complex can enhance p53 transcriptional activity. These findings define a hitherto novel mechanism of action for Sam68 in governing p53 transcriptional activation, and represent the first report of Sam68 in the regulation of tumor suppressor activities.
Abstract
Background
Young Ghanaian women experience high rates of unmet need for contraception and unintended pregnancy, and face unique barriers to accessing sexual and reproductive health services. ...This study provides a comprehensive national analysis of young women’s contraceptive and abortion practices and needs.
Methods
In 2018, we conducted a nationally representative survey of women aged 15–49, including 1039 women aged 15–24. We used descriptive statistics, multivariable logistic and multinomial regression to compare young versus older (25–49 year-old) women’s preferred contraceptive attributes, reasons for discontinuing contraception, quality of counseling, use of Primolut N-tablet, method choice correlates, and friends’ and partners’ influence. We also examined youth’s self-reported abortion incidence, abortion methods, post-abortion care, and barriers to safe abortion.
Results
Among Ghanaian 15–24 year-olds who had ever had sex, one-third (32%) were using contraception. Compared to older women, they had higher desires to avoid pregnancy, lower ever use of contraception, more intermittent sexual activity, and were more likely to report pregnancies as unintended and to have recently ended a pregnancy. Young contraceptors most commonly used condoms (22%), injectables (21%), withdrawal (20%) or implants (20%); and were more likely than older women to use condoms, withdrawal, emergency contraception, and N-tablet. They valued methods for effectiveness (70%), no risk of harming health (31%) nor future fertility (26%), ease of use (20%), and no effect on menstruation (19%). Infrequent sex accounted for over half of youth contraceptive discontinuation. Relative to older women, young women’s social networks were more influential on contraceptive use. The annual self-reported abortion rate among young women was 30 per thousand. Over half of young women used abortion methods obtained from non-formal providers. Among the third of young women who experienced abortion complications, 40% did not access treatment.
Conclusions
Young people’s intermittent sexual activity, desire for methods that do not harm their health, access barriers and provider bias, likely contribute to their greater use of coital-dependent methods. Providers should be equipped to provide confidential, non-discriminatory counseling addressing concerns about infertility, side effects and alternative methods. Use of social networks can be leveraged to educate around issues like safe abortion and correct use of N-tablet.
The KH-type RNA binding protein Sam68 is required for adipogenesis. We have previously shown that Sam68-deficient mice have a lean phenotype and are protected against dietary-induced obesity due to ...defects in mTOR and S6K1 alternative splicing. Herein we profiled the transcriptome of Sam68 wild type and deficient 3T3-L1 mouse preadipocytes. We identified 652 protein-coding genes and 9 ncRNAs that were significantly altered with the loss of Sam68. As expected, downregulated genes were significantly associated with GO terms linked to cell migration, motility, and fat cell differentiation, while upregulated genes were mostly associated with GO terms linked to neurogenesis. Of the lncRNAs, we identified
,
, as well as two new lncRNAs (
and
) that were regulated by Sam68, and contained consensus Sam68 binding sites. RNA stability assays showed that Sam68-deficiency decreased the half-life of
and increased the half-lives of
and
, while the stability of
was unaffected. Depletion of
and
in wild type 3T3-L1 cells led to defects in adipogenesis, whereas depletion of
in Sam68-deficient 3T3-L1 cells partially rescued the adipogenesis defect observed in these cells. Collectively, our findings define a new role for Sam68 as a regulator of lncRNAs during adipogenic differentiation.
COVID-19 pandemic has spread rapidly and caused a shortage of global medical resources. The efficiency of COVID-19 diagnosis has become highly significant. As deep learning and convolutional neural ...network (CNN) has been widely utilized and been verified in analyzing medical images, it has become a powerful tool for computer-assisted diagnosis. However, there are two most significant challenges in medical image classification with the help of deep learning and neural networks, one of them is the difficulty of acquiring enough samples, which may lead to model overfitting. Privacy concerns mainly bring the other challenge since medical-related records are often deemed patients' private information and protected by laws such as GDPR and HIPPA. Federated learning can ensure the model training is decentralized on different devices and no data is shared among them, which guarantees privacy. However, with data located on different devices, the accessible data of each device could be limited. Since transfer learning has been verified in dealing with limited data with good performance, therefore, in this paper, We made a trial to implement federated learning and transfer learning techniques using CNNs to classify COVID-19 using lung CT scans. We also explored the impact of dataset distribution at the client-side in federated learning and the number of training epochs a model is trained. Finally, we obtained very high performance with federated learning, demonstrating our success in leveraging accuracy and privacy.
The COVID-19 pandemic in the United States caused disruptions in care seeking and delivery during the spring of 2020, including for contraceptive care. We examined how some individuals experienced ...and responded to barriers to accessing contraceptive care by conducting a content analysis of relevant Reddit posts. We collected 2666 posts by scraping relevant subreddits from February 1, 2020, to April 15, 2020, and filtering by selected keywords. Among the 101 posts on contraception and the COVID-19 pandemic, we explored three main themes: barriers to accessing general healthcare during the early pandemic, problems and concerns specific to contraceptive use, and attempts to navigate the obstacles to contraceptive care or use-related concerns. The Reddit posts demonstrated the disruptive force the early pandemic had on contraceptive care and provided a unique window into the concerns posters expressed on Reddit during this time. Many posters asked questions related to accessing contraception and side effects and sought reassurance from these online forums. Our results suggest that there were barriers to accessing reliable, high-quality, and evidence-based information about contraception during this disruption in care. The findings also underscore that conversational and interactive means of seeking out information are important modes for learning about and discussing contraception for some and may be especially helpful during clinic closures and other restrictions on access.
Abstract
Targeting of mutated oncogenes has led to the identification of new targeted therapies. However, druggable oncogenes do not occur in most cancers. Systematic identification of signaling ...pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 793 cancer cell lines to identify selective co-essentiality modules and found that a ubiquitination ligase complex composed of UBA6, BIRC6, KCMF1 and UBR4, which encode an E1, E2 and two heterodimeric E3 subunits, respectively, is required for the survival of a subset of epithelial tumors, particularly subtypes of breast cancer. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization and upregulation of the heme-regulated inhibitor (HRI), a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy.
Citation Format: Lisa D Cervia, Tsukasa Shibue, Benjamin Gaeta, Ashir A Borah, Lisa Leung, Naomi Li, Nancy Dumont, Alfredo Gonzalez, Nolan Bick, Mariya Kazachkova, Joshua M Dempster, John M Krill-Burger, Federica Piccioni, Namrata D Udeshi, Meagan E Olive, Steven A Carr, David E Root, James M McFarland, Francisca Vazquez, William C Hahn. A ubiquitination cascade regulating the integrated stress response and survival in carcinomas abstract. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-01.
Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 ...cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy.
We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells. See related commentary Leli and Koumenis, p. 535. This article is highlighted in the In This Issue feature, p. 517.
Abstract
Systematic identification of signaling pathways required for the viability of cancer cells will facilitate the development of novel cancer therapies. We used gene essentiality measurements ...in 726 cancer cell lines to identify selective co-essentiality modules and found a functional ubiquitination cascade containing UBA6, BIRC6, KCMF1 and UBR4, which encode an E1, E2, and two heterodimeric E3 subunits, respectively, as a vulnerability in a subset of epithelial tumors. Suppressing BIRC6 in cancer cell lines that are dependent on this ubiquitination cascade led to a strong reduction in cell fitness in vitro, and to potent tumor regression and metastasis suppression in vivo. Mechanistically, BIRC6 suppression resulted in selective and robust activation of the integrated stress response (ISR) signaling via upregulation of the heme-regulated inhibitor (HRI). Using proteomic profiling, we found that HRI itself is a key degradation target of the UBA6/BIRC6/KCMF1/UBR4 cascade. These observations demonstrate a protein ubiquitination cascade regulating ISR and highlight the potential of this cascade as a novel therapeutic target for a subset of epithelial cancers.
Citation Format: Lisa D. Cervia, Tsukasa Shibue, Benjamin Gaeta, Ashir Borah, Lisa Leung, Naomi Li, Nancy Dumont, Alfredo Gonzalez, Nolan Bick, Mariya Kazachkova, Joshua Dempster, John M. Krill-Burger, Namrata Udeshi, Meagan Olive, Steven A. Carr, David E. Root, Federica Piccioni, James M. McFarland, Francisca Vazquez, William C. Hahn. A ubiquitination cascade regulates the integrated stress response and epithelial cancer survival abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1950.
RNA binding proteins are becoming increasingly appreciated as fundamental players in both normal development and disease pathogenesis. Of particular interest is the STAR family RNA binding protein ...Sam68, which has been implicated in a variety of biological processes, including cell proliferation, migration, apoptosis, and differentiation. Notably, Sam68 expression and activities are frequently dysregulated in human cancers; however, its role appears to be complex, with reports indicating both pro-tumorigenic and tumor suppressive functions. In the first part of my thesis, we identify Sam68 as a transcriptional coactivator of the p53 tumor suppressor. Using CRISPR/Cas9 generated isogenic HCT116 Sam68-/- cell lines, we show that Sam68 is required for the efficient transactivation of p53 target genes and effector functions in response to DNA damage. Mechanistically, we demonstrate that Sam68 physically interacts with p53 in an RNA-dependent manner, and show that Sam68 acts in concert with the p53 lncRNA target PR-lncRNA-1 to enhance p53 promoter recruitment.In the second part of my thesis, we report using two classical mouse cancer models, that the physiological role of Sam68 in tumor development can be influenced by status of the p53 tumor suppressor. We demonstrate that in mice expressing wild type p53, Sam68-deficiency resulted in a higher incidence and malignancy of carcinogen-induced tumors, suggesting a tumor suppressive role for Sam68. In marked contrast, Sam68-haploinsufficiency significantly delayed the onset of tumors in mice lacking p53 and prolonged their survival, indicating that Sam68 specifically accelerates the development of p53-deficient tumors.Additionally, our lab has previously shown that Sam68-deficient mice have a lean phenotype and are protected against dietary-induced obesity due to defects in mTOR and S6K1 alternative splicing. The third part of my thesis extends beyond those findings, and defines a new role for Sam68 as a regulator of lncRNAs in preadipocytes. We profiled the transcriptome of Sam68 wild type and deficient 3T3-L1 mouse preadipocytes, and identified Hotair, Mir155hg, as well as two new lncRNAs (SR-lncRNA-1 and SR-lncRNA-2) as Sam68 targets. Mechanistically, these lncRNAs contained consensus Sam68 binding sites, and RNA stability assays show that Sam68 largely modulated their expressions via transcript stability. Furthermore, loss-of-function studies demonstrate that Sam68-regulated lncRNAs have critical roles in adipogenesis. Collectively, the work presented in my thesis provides considerable insight into previously unknown functions of Sam68 in gene expression regulation. More importantly, my work defines Sam68 as a new, yet pivotal liaison between transcriptional and lncRNA networks that eventually determines cell fate.
The expression and activities of RNA binding proteins are frequently dysregulated in human cancer. Their roles, however, appears to be complex, with reports indicating both pro-tumorigenic and tumor ...suppressive functions. Here we show, using two classical mouse cancer models, that the role of KH-type RNA binding protein, Sam68, in tumor development can be influenced by the status of the p53 tumor suppressor. We demonstrate that in mice expressing wild type p53, Sam68-deficiency resulted in a higher incidence and malignancy of carcinogen-induced tumors, suggesting a tumor suppressive role for Sam68. In marked contrast, Sam68-haploinsufficiency significantly delayed the onset of tumors in mice lacking p53 and prolonged their survival, indicating that Sam68 accelerates the development of p53-deficient tumors. These findings provide considerable insight into a previously unknown relationship between Sam68 and the p53 tumor suppressor in tumorigenesis.
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