Differently expressed gene analysis in HeLa cells expressing high levels of phosphorylated PCNA
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE127276 | GEO | 2019/02/28
REPOSITORIES: GEO
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Project description:Activation of the epithelial-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers (CRCs) contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with CRC contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine Specific Demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer (CRC) and enhances cell migration. In this study we determine that LSD1 expression is significantly elevated in CRC patients with mutation of the catalytic subunit of PI3K, PIK3CA, compared to CRC patients with WT PIK3CA. LSD1 enhances activation of the AKT kinase in CRC cells through a non-catalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. Additionally, growth of PIK3CA mutant CRC cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA mutant cells.
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Project description:Epithelial-Mesenchymal Transition (EMT) is thought to contribute to cancer metastasis, but its underlying mechanisms are not well understood. To define early steps in this cellular transformation, we analyzed human mammary epithelial cells with tightly regulated expression of Snail-1, a master regulator of EMT. Following Snail-1 induction, epithelial markers were repressed within 6 hours and mesenchymal genes induced at 24 hours. Snail-1 binding to its target promoters was transient (6-48 hours) despite continued protein expression and it was followed by both transient and long-lasting chromatin changes. To generate a potent reversible EMT-inducing stimulus, we created a Snail-1 retroviral expression construct, using a fused estrogen receptor (ER) response element to mediate regulation by exogenous 4-hydroxy-tamoxifen (4-OHT). Since Snail-1 protein stability and nuclear localization are suppressed by GSK3-beta-mediated phosphorylation, we substituted the six targeted amino acids (ER-Snail-1(6SA)), thus conferring constitutive activity to the induced protein (Zhou et al., 2004, Pubmed ID 15448698). Infection of non-transformed, immortalized human mammary epithelial MCF10A cells with ER-Snail-1(6SA), followed by treatment with 4-OHT, triggered morphological and biomarker characteristics of EMT. At 0, 6, 48 and 120 hours after beginning exposure to 4-OHT in ethanol (or, for controls, ethanol only) we ChIPed Snail-1 and 6 histone marks. We perfomed two replicates of each, except we only had one replicate of H3K27Me3 at 6 hours.
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Project description:To screen the genes regulated by wt-Snail and non-acetylated Snail The successful development of cancer metastasis requires two major events: the reprogramming of cancer cells to increase their migration and tumor-initiation capabilities; and the remodeling of the tumor microenvironment to facilitate invasion and colonization of cancer cells. Epithelial-mesenchymal transition (EMT) is a crucial mechanism for reprogramming cancer cells to possess tumor initiation and migration capabilities1,2. However, the role of EMT in the interplay between tumor and host cells is largely unknown. The EMT regulator Snail is mainly known as a transcriptional repressor of the adhesion protein E-cadherin, whose repression is considered to be a key step in initiating metastasis3,4. We previously found that Snail can also act as an activator that induces the transcription of ERCC15 and IL86. Here we show that Snail is acetylated by CREB-binding protein (CBP) and that Snail and CBP co-occupy the promoters of target genes to activate transcription of the target genes. Furthermore, Snail activates the expression of a panel of cytokine genes, including TNFa (which forms a positive feedback loop with Snail to amplify the signal) and CCL2 and CCL5 (which facilitate the recruitment of macrophages by cancer cells). Our results demonstrate a novel function for Snail, providing new understanding of the recruitment of host cells to tumor sites during metastatic evolution.
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