Phenotype specific analyses reveal distinct regulatory mechanism for chronically activated p53

This work was supported by the University of Cambridge; Cancer Research UK (C14303/A17197); Hutchison Whampoa. In addition, MasasN and TO were supported by the Human Frontier Science Program (RGY0078/2010); HK was supported by MEXT KAKENHI (Grant Numbers 25116005 and 26291071); KT was supported by the Japan Society for the Promotion of Science (24–8563).The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical ‘acute’ p53 binding profile, ‘chronic’ p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory ‘p53 hubs’ where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the ‘lipogenic phenotype’, a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.Publisher PDFPeer reviewe

Phenotype specific analyses reveal distinct regulatory mechanism for chronically activated p53.

The downstream functions of the DNA binding tumor suppressor p53 vary depending on the cellular context, and persistent p53 activation has recently been implicated in tumor suppression and senescence. However, genome-wide information about p53-target gene regulation has been derived mostly from acute genotoxic conditions. Using ChIP-seq and expression data, we have found distinct p53 binding profiles between acutely activated (through DNA damage) and chronically activated (in senescent or pro-apoptotic conditions) p53. Compared to the classical 'acute' p53 binding profile, 'chronic' p53 peaks were closely associated with CpG-islands. Furthermore, the chronic CpG-island binding of p53 conferred distinct expression patterns between senescent and pro-apoptotic conditions. Using the p53 targets seen in the chronic conditions together with external high-throughput datasets, we have built p53 networks that revealed extensive self-regulatory 'p53 hubs' where p53 and many p53 targets can physically interact with each other. Integrating these results with public clinical datasets identified the cancer-associated lipogenic enzyme, SCD, which we found to be directly repressed by p53 through the CpG-island promoter, providing a mechanistic link between p53 and the 'lipogenic phenotype', a hallmark of cancer. Our data reveal distinct phenotype associations of chronic p53 targets that underlie specific gene regulatory mechanisms.This work was supported by the University of Cambridge; Cancer Research UK (C14303/A17197); Hutchison Whampoa. In addition, MasasN and TO were supported by the Human Frontier Science Program (RGY0078/2010); HK was supported by MEXT KAKENHI (Grant Numbers 25116005 and 26291071); KT was supported by the Japan Society for the Promotion of Science (24–8563).This is the final version of the article. It first appeared at http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.100505

Scribble Deficiency Promotes Pancreatic Ductal Adenocarcinoma Development and Metastasis.

Perturbation of cell polarity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) progression. Scribble (SCRIB) is a well characterized polarity regulator that has diverse roles in the pathogenesis of human neoplasms. To investigate the impact of SCRIB deficiency on PDAC development and progression, Scrib was genetically ablated in well-established mouse models of PDAC. Scrib loss in combination with KrasG12D did not influence development of pancreatic intraepithelial neoplasms (PanIN) in mice. However, Scrib deletion cooperated with KrasG12D and concomitant Trp53 heterozygous deletion to promote invasive PDAC and metastatic dissemination, leading to reduced overall survival. Immunohistochemical and transcriptome analyses revealed that Scrib-null tumors display a pronounced reduction of collagen content and cancer associated fibroblast (CAF) abundance. Mechanistically, interleukin 1α (IL1α) levels were reduced in Scrib deficient tumors, and Scrib knockdown downregulated IL1α in mouse PDAC organoids (mPDOs), which impaired CAF activation. Furthermore, Scrib loss increased YAP activation in mPDOs and established PDAC cell lines, enhancing cell survival. Clinically, SCRIB expression was decreased in human PDAC, and SCRIB mislocalization was associated with poorer patient outcome. These results indicate that SCRIB deficiency enhances cancer cell survival and remodels the tumor microenvironment to accelerate PDAC development and progression, establishing the tumor suppressor function of SCRIB in advanced pancreatic cancer

Choice of the initial antiretroviral treatment for HIV-positive individuals in the era of integrase inhibitors

BACKGROUND: We aimed to describe the most frequently prescribed initial antiretroviral therapy (ART) regimens in recent years in HIV-positive persons in the Cohort of the Spanish HIV/AIDS Research Network (CoRIS) and to investigate factors associated with the choice of each regimen. METHODS: We analyzed initial ART regimens prescribed in adults participating in CoRIS from 2014 to 2017. Only regimens prescribed in >5% of patients were considered. We used multivariable multinomial regression to estimate Relative Risk Ratios (RRRs) for the association between sociodemographic and clinical characteristics and the choice of the initial regimen. RESULTS: Among 2874 participants, abacavir(ABC)/lamivudine(3TC)/dolutegavir(DTG) was the most frequently prescribed regimen (32.1%), followed by tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC)/elvitegravir(EVG)/cobicistat(COBI) (14.9%), TDF/FTC/rilpivirine (RPV) (14.0%), tenofovir alafenamide (TAF)/FTC/EVG/COBI (13.7%), TDF/FTC+DTG (10.0%), TDF/FTC+darunavir/ritonavir or darunavir/cobicistat (bDRV) (9.8%) and TDF/FTC+raltegravir (RAL) (5.6%). Compared with ABC/3TC/DTG, starting TDF/FTC/RPV was less likely in patients with CD4100.000 copies/mL. TDF/FTC+DTG was more frequent in those with CD4100.000 copies/mL. TDF/FTC+RAL and TDF/FTC+bDRV were also more frequent among patients with CD4<200 cells//muL and with transmission categories other than men who have sex with men. Compared with ABC/3TC/DTG, the prescription of other initial ART regimens decreased from 2014-2015 to 2016-2017 with the exception of TDF/FTC+DTG. Differences in the choice of the initial ART regimen were observed by hospitals' location. CONCLUSIONS: The choice of initial ART regimens is consistent with Spanish guidelines' recommendations, but is also clearly influenced by physician's perception based on patient's clinical and sociodemographic variables and by the prescribing hospital location

The role of microRNAs in the modulation of cancer-associated fibroblasts activity during pancreatic cancer pathogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is the deadliest of the common cancers. A major hallmark of PDAC is an abundant and dense fibrotic stroma, the result of a disproportionate deposition of extracellular matrix (ECM) proteins. Cancer-associated fibroblasts (CAFs) are the main mediators of PDAC desmoplasia. CAFs represent a heterogenous group of activated fibroblasts with different origins and activation mechanisms. microRNAs (miRNAs) are small non-coding RNAs with critical activity during tumour development and resistance to chemotherapy. Increasing evidence has revealed that miRNAs play a relevant role in the differentiation of normal fibroblasts into CAFs in PDAC. In this review, we discuss recent findings on the role of miRNAs in the activation of CAFs during the progression of PDAC and its response to therapy, as well as the potential role that PDAC-derived exosomal miRNAs may play in the activation of hepatic stellate cells (HSCs) and formation of liver metastasis. Since targeting of CAF activation may be a viable strategy for PDAC therapy, and miRNAs have emerged as potential therapeutic targets, understanding the biology underpinning miRNA-mediated tumour cell-CAF interactions is an important component in guiding rational approaches to treating this deadly disease

Titration of RAS alters senescent state and influences tumour initiation

Oncogenic RAS-induced senescence (OIS) is an autonomous tumour suppressor mechanism associated with pre-malignancy1,2. Achieving this phenotype typically requires a high level of oncogenic stress, yet the phenotype provoked by lower oncogenic dosage remains unclear. Here we develop oncogenic RAS-dose escalation models in vitro and in vivo, revealing a RAS-dose-driven non-linear continuum of downstream phenotypes. In a hepatocyte OIS model in vivo, ectopic expression of NRASG12V fails to induce tumours in part due to OIS-driven immune clearance3. scRNA-seq analyses reveal distinct hepatocyte clusters with typical OIS or progenitor-like features, corresponding to high- and intermediate-NRASG12V levels, respectively. Remarkably, when titered down, NRASG12V-expressing hepatocytes become immune-resistant, and develop tumours. Time-series monitoring at single-cell resolution identifies two distinct tumour types: early-onset aggressive undifferentiated and late-onset differentiated hepatocellular carcinoma (HCC). The molecular signature of each murine tumour type is associated with different progenitor features and enriched in distinct human HCC subclasses. Our results define the oncogenic dosage-driven OIS spectrum, reconciling the senescence and tumour initiation phenotypes in early tumorigenesis.Please see Acknowledgements section. Diabetes UK via BIRAX & the British Council; Cancer Research UK-Oregon Health and Science University Joint Award; NIH/NCI; Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)

Locus-specific induction of gene expression from heterochromatin loci during cellular senescence

Cellular senescence is a fate-determined state, accompanied by reorganization of heterochromatin. While lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3K9me3. The fate of these heterochromatic genes during the chromatin reorganization accompanying senescence is unclear. Here we show a small number of lineage-inappropriate genes are derepressed in senescent cells from H3K9me3 regions that gain open chromatin marks. DNA FISH experiments reveal that these gene loci, which are tightly condensed at the nuclear periphery in proliferative cells, are physically decompacted during senescence. Among these gene loci, NLRP3 is predominantly expressed in immune cells, such as macrophages, where it resides within an open topologically associated domain (TAD). In contrast, NLRP3 is derepressed in senescent fibroblasts, potentially due to the local disruption of the H3K9me3-rich TAD that contains it. The role of NLRP3 has been implicated in the amplification of inflammatory cytokine signalling in senescence and aging, underscoring the functional relevance of gene induction from ‘permissive’ H3K9me3 regions in senescent cells

Scribble Deficiency Promotes Pancreatic Ductal Adenocarcinoma Development and Metastasis

Perturbation of cell polarity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) progression. Scribble (SCRIB) is a well characterized polarity regulator that has diverse roles in the pathogenesis of human neoplasms. To investigate the impact of SCRIB deficiency in PDAC development and progression, Scrib expression was genetically ablated in well-established mouse models of PDAC. Scrib loss in combination with KrasG12D did not influence development of pancreatic intraepithelial neoplasms (PanIN) in mice. However, Scrib deletion cooperated with KrasG12D and concomitant Trp53 heterozygous deletion to promote invasive PDAC and metastatic dissemination, leading to reduced overall survival. Immunohistochemical and transcriptome analyses revealed that Scrib-null tumors display a pronounced reduction of collagen content and cancer associated fibroblasts (CAF) abundance. Mechanistically, interleukin 1 (IL1α) levels were reduced in Scrib-deficient tumors, and Scrib knockdown downregulated IL1α in mouse PDAC organoids (mPDOs), which impaired CAF activation. Furthermore, Scrib loss increased YAP activation in mPDOs and established PDAC cell lines, enhancing cell survival. Clinically, SCRIB expression was decreased in human PDAC, and SCRIB mislocalization was associated with poorer patient outcome. These results indicate that SCRIB deficiency enhances cancer cell survival and remodels the tumor microenvironment to accelerate PDAC development and progression, establishing the tumor suppressor function of SCRIB in advanced pancreatic cancer

Locus-specific induction of gene expression from heterochromatin loci during cellular senescence

Senescence is a fate-determined state, accompanied by reorganization of heterochromatin. Although lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3 lysine 9 trimethylation (H3K9me3). The fate of these heterochromatic genes during senescence is unclear. In the present study, we show that a small number of lineage-inappropriate genes, exemplified by the LCE2 skin genes, are derepressed during senescence from H3K9me3 regions in fibroblasts. DNA FISH experiments reveal that these gene loci, which are condensed at the nuclear periphery in proliferative cells, are decompacted during senescence. Decompaction of the locus is not sufficient for LCE2 expression, which requires p53 and C/EBPβ signaling. NLRP3, which is predominantly expressed in macrophages from an open topologically associated domain (TAD), is also derepressed in senescent fibroblasts due to the local disruption of the H3K9me3-rich TAD that contains it. NLRP3 has been implicated in the amplification of inflammatory cytokine signaling in senescence and aging, highlighting the functional relevance of gene induction from ‘permissive’ H3K9me3 regions in senescent cells

Phenotype-associated p53-responsive gene expression in IMR90 cells.

<p><b>(A)</b> Schematic of the p53-associated phenotypes. <b>(B)</b> Cell viability, senescence-associated ß-galactosidase activity (SA-ß-gal), and BrdU incorporation (mean ± SEM; n = 3) were measured for each condition as indicated in (A). In addition, DNA damage-induced senescence (DDIS) was included for comparison: cells were treated with etoposide (100 μM) for two days, and maintained for an additional five days in drug-free media. <b>(C, D)</b> Immunoblot analyses for the proteins indicated for total lysates and chromatin fractions from the cells as labeled. Cyclin A2, a cell cycle marker; HMGA proteins, senescence markers. d1 and d7 correspond to acDDR and DDIS, respectively (C). Core histones (C, D) and HMGA proteins (C) were stained with Coomassie blue. The arrow indicates non-specific bands (the Cyclin A2 blot in (C)). <b>(E)</b> Immunoblot analysis in the indicated cells for chromatin fractions for p53. sh and v, sh-p53#1 and corresponding lentiviral vector (a miR30 design), respectively. For acDDR, sh-p53 was introduced first for at least 5 days before administration of etoposide. For RIS and pApo, sh-p53 was introduced after the phenotype establishment. Core histones were stained with Coomassie blue. <b>(F)</b> Venn diagram showing the numbers of differentially expressed (DE) genes upon p53 depletion with lentivirus-mediated RNAi (sh-p53#1) compared to vector, in the indicated conditions. <b>(G)</b> Pathway heatmap for differentially expressed genes upon p53 depletion.</p