Project description:Mammalian cells possess intrinsic mechanisms to prevent tumorigenesis upon deleterious mutations, including oncogene-induced senescence (OIS). The molecular mechanisms underlying OIS are, however, complex, and remain to be characterized. In this study, we analyzed the changes in the nuclear (phospho)proteome during the progression of OIS. Interestingly, we found that most of the phosphosites regulated during OIS were Prolyl Isomerase Pin1 targets. Our results show that Pin1 is a key regulator of several PML-NB proteins, specifically regulating several proteins upon oncogene activation. We have found that the constitutive PML-NB proteins are significantly regulated when PIN1 is depleted. Furthermore, we show that PIN1 knockdown promotes cell proliferation, while diminishing the senescence phenotype and hallmarks of senescence such as p21. Thus, our data provides preliminary evidence that PML-NB proteins are involved in regulating OIS via phosphorylation, and that the prolyl isomerase PIN1 acts as a tumor suppressor in response to oncogenic ER:RasG12V activation.
Project description:We used IMR90 ER:RAS cells infected with an empty vector or an shRNA for ARID1B and induced senescence by addition of 4OHT. 6 days later RNA was collected for gene expression analysis. With a functional screen we previously identified ARID1B as a new regulator of cellular senescence. By performing gene expression analysis we confirmed this finding and showed that knockdown of ARID1B prevents the expression of genes induced during senescence.
Project description:In the present study, we analyze the effect of knocking down CASP4 in IMR90 ER:RAS primary lung cells five and eight days after the addition of 4-Hydroxytamoxifen by analyzing the transcriptome using Ampliseq RNA sequencing. We observed that caspase-4 targeting dampens the mRNA expression induction of critical senescence-associated secretory phenotype factors while increasing the transcription of E2F target genes and genes related to the G2/M checkpoint.
Project description:This experiment was designed to study oncogene-induced senescence (OIS). To this end we generated a series of cell lines derived from normal human diploid fibroblasts IMR90 forced to express the catalytic subunit of telomerase (hTERT). This cells were then subjected to further manipulation by orderly introducing defined genetic elements by retroviral transduction. The first cell line generated was ITV, which was obtained from the original cell line (IMR90 with hTERT) after introducing an empty vector. Subsequently, we introduced Mek:ER, which is a switchable version of the Mek kinase, a relevant downstream effector of Ras signaling during Ras-induced senescence, to generate ITM cells. We further modified this cell line by introducing SV40 small-t antigen (ST), papillomavirus oncoproteins E6 and E7 (E6/E7) or the combination of both (E6/E7 and ST). In this manner, we obtained ITMST, ITME6E7 and ITME6E7ST respectively. This cellular system allow us to have a representation of the different steps into neoplastic transformation. ITM and ITMST cells respond to Mek activation by inducing OIS. ITME6E7 and ITME6E7ST cells do not enter OIS after Mek activation. Mek activation is achieved by treating all cell cultures with 4-hydroxytamoxifen (4OHT) at 100 nM, in the absence of serum, and for 3 days. The gene expression profile of ITV cells served as a reference for all the expression values obtained with the rest of the cell lines. Thus, we ended up with the expression profiles of two cell lines representing oncogene-induced senescence (ITM and ITMST), and two cell lines representing bypass of oncogene-induced senescence, plus a reference profile provided by ITV, the cell line from which all the other cell lines were derived. Our final goal was to identify markers of the oncogene-induced senescence response by comparing the expression profiles of the cell lines entering OIS after Mek activation (that is, after 4OHT treatment) with the ones bypassing this response. Keywords: other
Project description:Oncogene-induced senescence (OIS) is an inherent and important tumor suppressor mechanism. However, if not timely removed via immune surveillance, senescent cells will also present a detrimental side. Although this has mostly been attributed to the senescence-associated-secretory-phenotype (SASP) of these cells, we recently suggested that “escape” from the senescent state represents another unfavorable outcome. Here, we exploit genomic and functional data from a prototypical human epithelial cell model carrying an inducible CDC6 oncogene to identify an early-acquired recurrent chromosomal inversion, which harbors a locus encoding the circadian transcription factor BHLHE40. This inversion alone suffices for activation of BHLHE40 upon CDC6 induction and for driving cell cycle re-entry and malignant transformation. In summary, we provide strong evidence in support of genomic instability underlying “escape” from oncogene-induced senescence.
Project description:By transcriptome analysis of IMR-90 human fibroblasts following oncogene-induced senescence (OIS) and replicative senescence (RS), we identified commonly regulated genes in both conditions.
Project description:We generated a humanized mouse model of oncogene-induced senescence in hematopoietic stem and progenitor cells by expressing the activated oncogene BRAF-V600E. Mice succumbed to bone marrow failure and multi-organ dissemination of aberrant macrophages and dendritic cells. We observed a myeloid-restricted hematopoiesis,and uncovered the activation of a senescence program, characterized by growth arrest and senescence-associated secretory phenotype (SASP), which involved also non-mutated bystander cells.