CRISPR-KO screening to identify resistance/sensitation to PI3K pathway inhibition in PTEN-null breast cancer cell lines
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ABSTRACT:
SUBMITTER: Kosuke Yusa
PROVIDER: S-BSST189 | biostudies-other |
REPOSITORIES: biostudies-other
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| S-BSST190 | biostudies-other
Project description:Triple negative breast cancer (TNBC) has poorer prognosis compared to other types of breast cancers due to the lack of effective therapies and markers for patient stratification. Loss of PTEN tumor suppressor gene expression is a frequent event in TNBC, resulting in over-activation of the PI 3-Kinase (PI3K) pathway and sensitivity to its inhibition. However, PI3K pathway inhibitors show limited efficacy as monotherapies on these tumors. We report a whole genome screen to identify targets whose inhibition enhanced the effects of different PI3K pathway inhibitors on PTEN-null TNBC. This identified a signaling network that relies on both the G-protein-coupled receptor for thrombin (PAR1/F2R) and downstream G protein ?? subunits and also epidermal growth factor receptor (EGFR) for the activation of the PI3K isoform p110? and AKT. Compensation mechanisms involving these two branches of the pathway could bypass PI3K blockade, but combination targeting of both EGFR and PI3K? suppressed ribosomal protein S6 phosphorylation and exerted anti-tumor activity both in vitro and in vivo, suggesting a new potential therapeutic strategy for PTEN-null TNBC.
| S-SCDT-EMM-2020-11987 | biostudies-other
Project description:Triple-negative breast cancer (TNBC) has poorer prognosis compared to other types of breast cancers due to the lack of effective therapies and markers for patient stratification. Loss of PTEN tumor suppressor gene expression is a frequent event in TNBC, resulting in over-activation of the PI 3-kinase (PI3K) pathway and sensitivity to its inhibition. However, PI3K pathway inhibitors show limited efficacy as monotherapies on these tumors. We report a whole-genome screen to identify targets whose inhibition enhanced the effects of different PI3K pathway inhibitors on PTEN-null TNBC. This identified a signaling network that relies on both the G protein-coupled receptor for thrombin (PAR1/F2R) and downstream G protein βγ subunits and also epidermal growth factor receptor (EGFR) for the activation of the PI3K isoform p110β and AKT. Compensation mechanisms involving these two branches of the pathway could bypass PI3K blockade, but combination targeting of both EGFR and PI3Kβ suppressed ribosomal protein S6 phosphorylation and exerted anti-tumor activity both in vitro and in vivo, suggesting a new potential therapeutic strategy for PTEN-null TNBC.
| S-EPMC7411640 | biostudies-literature
Project description:Mutations and inactivation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) are observed in 15%-25% of cases of human T cell acute lymphoblastic leukemia (T-ALL). Pten deletion induces myeloproliferative disorders (MPDs), acute myeloid leukemia (AML), and/or T-ALL in mice. Previous studies attributed Pten-loss-related hematopoietic defects and leukemogenesis to excessive activation of phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling. Although inhibition of this signal dramatically suppresses the growth of PTEN-null T-ALL cells in vitro, treatment with inhibitors of this pathway does not cause a complete remission in vivo. Here, we report that focal adhesion kinase (Fak), a protein substrate of Pten, also contributes to T-ALL development in Pten-null mice. Inactivation of the FAK signaling pathway by either genetic or pharmacologic methods significantly sensitizes both murine and human PTEN-null T-ALL cells to PI3K/AKT/mTOR inhibition when cultured in vitro on feeder layer cells or a matrix and in vivo.
| S-EPMC7001526 | biostudies-literature
Project description:The PI3K pathway regulates cell metabolism, proliferation, and migration, and its dysregulation is common in cancer. We now show that both physiologic and oncogenic activation of PI3K signaling increase the expression of its negative regulator PTEN. This limits the duration of the signal and output of the pathway. Physiologic and pharmacologic inhibition of the pathway reduces PTEN and contributes to the rebound in pathway activity in tumors treated with PI3K inhibitors and limits their efficacy. Regulation of PTEN is due to mTOR/4E-BP1-dependent control of its translation and is lost when 4E-BP1 is deleted. Translational regulation of PTEN is therefore a major homeostatic regulator of physiologic PI3K signaling and plays a role in reducing the pathway activation by oncogenic PIK3CA mutants and the antitumor activity of PI3K pathway inhibitors. However, pathway output is hyperactivated in tumor cells with coexistent PI3K mutation and loss of PTEN function.
| S-EPMC8384339 | biostudies-literature
Project description:The mammalian signalling pathway involving class I PI3K (phosphoinositide 3-kinase), PTEN (phosphatidylinositol 3-phosphatase) and PKB (protein kinase B)/c-Akt has roles in multiple processes, including cell proliferation and apoptosis. To facilitate novel approaches for genetic, molecular and pharmacological analyses of these proteins, we have reconstituted this signalling pathway by heterologous expression in the unicellular eukaryote, Saccharomyces cerevisiae (yeast). High-level expression of the p110 catalytic subunit of mammalian PI3K dramatically inhibits yeast cell growth. This effect depends on PI3K kinase activity and is reversed partially by a PI3K inhibitor (LY294002) and reversed fully by co-expression of catalytically active PTEN (but not its purported yeast orthologue, Tep1). Growth arrest by PI3K correlates with loss of PIP2 (phosphatidylinositol 4,5-bisphosphate) and its conversion into PIP3 (phosphatidylinositol 3,4,5-trisphosphate). PIP2 depletion causes severe rearrangements of actin and septin architecture, defects in secretion and endocytosis, and activation of the mitogen-activated protein kinase, Slt2. In yeast producing PIP3, PKB/c-Akt localizes to the plasma membrane and its phosphorylation is enhanced. Phospho-specific antibodies show that both active and kinase-dead PKB/c-Akt are phosphorylated at Thr308 and Ser473. Thr308 phosphorylation, but not Ser473 phosphorylation, requires the yeast orthologues of mammalian PDK1 (3-phosphoinositide-dependent protein kinase-1): Pkh1 and Pkh2. Elimination of yeast Tor1 and Tor2 function, or of the related kinases (Tel1, Mec1 and Tra1), did not block Ser473 phosphorylation, implicating another kinase(s). Reconstruction of the PI3K/PTEN/Akt pathway in yeast permits incisive study of these enzymes and analysis of their functional interactions in a simplified context, establishes a new tool to screen for novel agonists and antagonists and provides a method to deplete PIP2 uniquely in the yeast cell.
| S-EPMC1198941 | biostudies-literature
Project description:The PI3K/Akt/PTEN axis is one of the most important signaling pathways in tumorigenesis. Recently, mutation of PIK3CA has been highlighted due to the similarities of mutational hotspots in both dogs and humans. PIK3CA H1047R (c.3140A > G) has been discovered as the most common mutational hot spot in canine mammary tumor in recent studies, while the feature of PIK3CA-mutated canine mammary tumor is obscure. A total of 83 mammary samples classified as normal (n = 13), adenoma (n = 25), low-grade carcinoma (n = 21), and high-grade carcinoma (n = 24) were included in this study. Genomic DNA from each sample was extracted, amplified by conventional PCR, and analyzed through Sanger sequencing. Analysis for the expression of PIK3CA, Akt, p-Akt, and PTEN was performed by immunohistochemistry, and of Akt2 by RNA in situ hybridization. PIK3CA H1047R mutation was detected in 14.3% (10/70) of tumor samples. Dysregulation of p-Akt, Akt2, and PTEN was observed in mammary tumor samples, but only PTEN dysregulation was associated with PIK3CA H1047R mutation. The present study showed that dysregulation of components in the PI3K/Akt/PTEN pathway is a feature of canine mammary tumors, but this dysregulation is not directly correlated to the PIK3CA H1047R mutation except for PTEN expression.
| S-EPMC8300234 | biostudies-literature
Project description:This study aimed to explore the role of the long non-coding RNA NOTCH1-associated lncRNA in T cell acute lymphoblastic leukemia (lncNALT) in the pathogenesis of hypertensive retinopathy (HR). LncNALT expression levels were determined using reverse transcription-quantitative polymerase chain reaction. The effects of lncNALT knockdown on the viability, proliferation, migration, and invasion of human retinal microvascular endothelial cells (RMECs) were determined via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 5-ethynyl-2'-deoxyuridine staining, and Transwell assays. Protein expression levels were determined using western blotting. We found that lncNALT expression levels were increased in RMECs treated with hydrogen peroxide (H2O2), while the knockdown of lncNALT rescued the viability, proliferation, migration, and invasion of RMECs treated with H2O2. Moreover, lncNALT interacted with ELAV like RNA binding protein 1 to affect the phosphatase and tensin homolog (PTEN) expression. Knockdown of lncNALT enhanced the viability, proliferation, migration, and invasion of RMECs via the PTEN/phosphoinositide 3-kinase (PI3K)/serine-threonine kinase (AKT) pathway. Taken together, knockdown of lncNALT enhanced the viability, proliferation, migration, and invasion of RMECs via the PTEN/PI3K/AKT pathway, suggesting that lncNALT could be a potential therapeutic target for patients with HR.
| S-EPMC10234215 | biostudies-literature
Project description:MDA-MB-468 cells were infected with a whole-genome shRNA library, treated with PI3Kbeta inhibitor (AZD8186), pan class I PI3K inhibitor (GDC0941) or AKT inhibitor (MK2206) for 6+6 days. At the end of the treatment genomic DNA was extracted. PCR amplified and shRNAs inserts were quantified by NGS.
2020-05-18 | GSE148785 | GEO
Project description:We intended to explore the effect of miR-202-5p and phosphatase and tensin homolog (PTEN) on doxorubicin (DOX) resistance of breast cancer cells. The result of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) reveals that miR-202-5p was highly expressed in drug-resistant breast cancer tissues, while PTEN was expressed less. MiR-202-5p directly targeted PTEN. Further, it was found that the overexpression of miR-202-5p promoted the DOX resistance and proliferation as well as decreased apoptosis of MCF-7 cells. The lower expression of miR-202-5p inhibited DOX resistance and proliferation as well as increased the apoptosis of MCF-7/DOX cells. In vivo experiments showed that mice with downregulated miR-202-5p had smaller tumor volume and lower Ki67 level. The overexpression of PTEN declined the proliferation of MCF7 cells, while miR-202-5p's overexpression could offset the function of overexpression of PTEN. The knockdown of PTEN promoted MCF7/DOX cell proliferation that could be counteracted by miR-202-5p silence. Moreover, we also revealed that downregulated miR-202-5p expression inhibited PI3k/Akt signaling pathway-related protein by regulating expression of PTEN.
| S-EPMC6606016 | biostudies-literature