Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Pathways regulated by oncogenic PTEN/PI3K/AKT signalling in NSCLC

ABSTRACT: Hyperactivation of the phosphatydil-inositol-3' phosphate kinase (PI3K)/AKT pathway is observed in most NSCLCs, promoting proliferation, migration, invasion and resistance to therapy. AKT can be activated through several mechanisms that include loss of the negative regulator PTEN, activating mutations of the catalytic subunit of PI3K (PIK3CA) and/or mutations of AKT1 itself. However, number and identity of downstream targets of activated PI3K/AKT pathway are poorly defined. To identify the genes that are targets of constitutive PI3K/AKT signalling in lung cancer cells, we performed a comparative transcriptomic analysis of human lung epithelial cells (BEAS-2B) expressing active mutant AKT1 (AKT1-E17K), active mutant PIK3CA (PIK3CA-E545K) or that are silenced for PTEN. For each sample, 500 ng of total RNA were used to synthesize biotinylated cRNA with Illumina RNA Amplification Kit (Ambion, Austin, TX). Synthesis was carried out according to the manufacturersâ instructions. From each sample, technical triplicates were produced and 750 ng cRNA were hybridized for 18h to Human HT-12_V3_0_R1 Expression BeadChips (Illumina, San Diego, CA). Hybridized chips were washed and stained with streptavidin-conjugated Cy3 (GE Healthcare, Milan, Italy). BeadChips were dried and scanned with an Illumina Bead Array Reader (Illumina).

ORGANISM(S): Homo sapiens

SUBMITTER: Carmelo Laudanna

PROVIDER: E-MTAB-5286 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Hyperactivation of the PI3K/AKT pathway is observed in most NSCLCs, promoting proliferation, migration and resistance to therapy. AKT can be activated through several mechanisms that include loss of the negative regulator PTEN, activating mutations of the catalytic subunit of the positive regulator phosphatydil-inositol-3â phosphate kinase (PIK3CA) and/or mutations of AKT1 itself. However, whereas the effects of AKT activation on mRNAs and proteins in the cell are fairly clear, the role of miRNAs as downstream targets of activated PI3K/AKT signalling is still poorly defined so far. To identify miRNAs that are targets of constitutive signalling of PI3K/AKT in lung cancer cells, we performed miRNA profiling of human lung epithelial cells expressing active mutant AKT1 (E17K), active mutant PI3KCA (E545K) or that are silenced for PTEN. We identified 28 differentially expressed miRNAs (8 up-regulated, 20 down-regulated) that were common to BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells. Among the 8 up-regulated miRNAs that were common to all the alterations, the miRNA most consistently up-regulated by activation of the PI3K/AKT pathway in BEAS-2B cells was miR-196a. The results reported here demonstrate that miR-196a acts as oncogene downstream the PI3K/AKT pathway, mediating the proliferative, pro-migratory and tumorigenic activity of this pathway in NSCLC cells by targeting FoxO1 and p27 expression. By adoptive expression of miR-196a in BEAS-2B cells, we demonstrated that miR-196a stimulates anchorage-dependent and -independent proliferation, migration and tumorigenicity in BEAS-2B cells. On the other hand, by use of antimir-196a in NCI-H460 cells to silence the endogenous expression of miR-196a, we demonstrate that miR-196a plays a pivotal role in mediating the stimulation of proliferation, migration and tumorigenicity induced by aberrant activation of PI3K/AKT signalling. Accordingly, we found that miR-196a was significantly overexpressed in human NSCLC-derived cell lines (n=6) and primary lung cancer samples (n=28). Finally, based on predicted binding sites for miR-196a by microRNA analysis software, we found that miR-196a affects protein levels of FoxO1 and p27 in NSCLC cells.

Project description:Background: PIK3CA mutations are observed in >30% of breast cancers, which are more common in estrogen receptor (ERÎ±)-positive breast cancer compared with ERÎ±-negative breast cancer. AKT1, 2, and 3 isoforms, major isoforms downstream of PI3K, modulate ERÎ± activity. It is unknown whether PIK3CA mutation leads to preferential activation of specific AKT isoforms with an ability to modulate ERÎ± function. Methods: Gene expression arrays were performed on parental, AKT1 knockdown or AKT2 knockdown MCF-7 breast cancer cells with or without estradiol treatment for three hours. Results: AKT1 had a dominant role in ERÎ±:estradiol-dependent gene expression and proliferation. We have identified a unique gene expression signature that is dependent on ERÎ±, estradiol, AKT1 and the pioneer factor FOXA1. Overexpression of this signature was associated with better outcome in patients with ERÎ±-positive breast cancer. In contrast, AKT2 controlled global gene expression. Cells were maintained in phenol red free MEM with 5% charcoal dextran treated fetal calf serum for four days and then treated with ethanol or 0.1 nM estradiol for three hours. RNA was prepared using RNeasy kit (Qiagen, Valencia, CA, USA) Microarray with biological triplicates was performed using Illumina HumanHT-12 V4 expression beadchip. Genes that showed statistically insignificant signals in majority of samples were removed from the microarray data, and only those probes that showed statistically significant signal in at least half of samples of at least one group were retained. The probe level data was then collapsed to gene level data by retaining only the probes, which showed a maximal coefficient of variation across all samples. The data was imported into partek genomics suite for differential expression analysis. ANOVA analysis was performed to identify genes differentially expressed between AKT1KD vs. pLKO, AKT2KD vs. pLKO, and AKT1KD vs. AKT2KD groups. Stratagene Universal Human Reference RNA (Illumina Catalog # 740000) was used as quality control specimen

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Pathways regulated by oncogenic PTEN/PI3K/AKT signalling in NSCLC

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