Inhibition of androgen receptor transactivation function by adenovirus type 12 E1A undermines prostate cancer cell survival.

BACKGROUND: Mutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance. METHODS: Luciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays. RESULTS: Adenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12. CONCLUSION: Ad12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.

Stabilization of 4E-BP1 by PI3K kinase and its involvement in CHK2 phosphorylation in the cellular response to radiation.

Objectives: 4E-BP1 is a family member of eIF4E binding proteins (4E-BPs) which act as the suppressors of cap-dependent translation of RNA via competitively associating with cap-bound eIF4E. RNA translation regulation is an important manner to control the cellular responses to a series of stress conditions such as ionizing radiation (IR)-induced DNA damage response and cell cycle controlling. This study aimed to determine the mechanism of 4E-BP1 stabilization and its potential downstream target(s) in the response to IR. Methods: PI3Ks kinase inhibitors were used to determine the signaling control of 4E-BP1 phosphorylation and protein stability. shRNA strategy was employed to silence the expression of 4E-BP1 in HeLa and HepG2 cells, and determine its effect on the irradiation-induced CHK2 phosphorylation. The protein degradation/stability was investigated by western blotting on the condition of blocking novel protein synthesis by cycloheximide (CHX). Results: The phosphorylation of 4E-BP1 at Thr37/46 was significantly increased in both HepG2 and HeLa cells by ionizing radiation. Depression of 4E-BP1 by shRNA strategy resulted in an incomplete G2 arrest at the early stage of 2 hours post-irradiation, as well as a higher accumulation of mitotic cells at 10 and 12 hours post-irradiation as compared to the control cells. Consistently, the CHK2 phosphorylation at Thr68 induced by IR was also attenuated by silencing 4E-BP1 expression. Both PI3K and DNA-PKcs kinase inhibitors significantly decreased the protein level of 4E-BP1, which was associated with the accelerated degradation mediated by ubiquitination-proteasome pathway. Conclusion: PI3K kinase activity is necessary for maintaining 4E-BP1 stability. Our results also suggest 4E-BP1 a novel biological role of regulating cell cycle G2 checkpoint in responding to IR stress in association with controlling CHK2 phosphorylation.

Tyrosine 251 at the C-terminus of neuronal glycoprotein M6a is critical for neurite outgrowth.

Neuronal glycoprotein M6a is involved in neuronal plasticity, promoting neurite and filopodia outgrowth and, likely, synaptogenesis. Polymorphisms in the human M6a gene GPM6A have recently been associated with mental illnesses such as schizophrenia, bipolar disorders, and claustrophobia. Nevertheless, the molecular bases underlying these observations remain unknown. We have previously documented that, to induce filopodia formation, M6a depends on the association of membrane lipid microdomains and the activation of Src and mitogen-activated protein kinase kinases. Here, in silico analysis of the phosphorylation of tyrosine 251 (Y251) at the C-terminus of M6a showed that it could be a target of Src kinases. We examined whether phosphorylation of M6a at Y251 affects neurite and filopodia outgrowth and the targets involved in its signal propagation. This work provides evidence that the Src kinase family and the phosphatidylinositide 3-kinase (PI3K), but not Ras, participate in M6a signal cascade leading to neurite/filopodia outgrowth in hippocampal neurons and murine neuroblastoma N2a cells. Phosphorylation of M6a at Y251 is essential only for neurite outgrowth by the PI3K/AKT-mediated pathway and, moreover, rescues the inhibition caused by selective Src inhibitor and external M6a monoclonal antibody treatment. Thus, we suggest that phosphorylation of M6a at Y251 is critical for a specific stage of neuronal development and triggers redundant signaling pathways leading to neurite extension.

The Landscape of PIK3CA Mutations in Colorectal Cancer.

BACKGROUND: Colorectal cancer is one of the most common malignancies in both men and women. Despite progress in the treatment of the disease, metastatic colorectal cancer remains lethal with a median survival slightly surpassing 2 years and commonly for some cases a more aggressive course. New therapies are urgently needed based on a better understanding of the molecular pathogenesis of the disease. METHODS: The focus of this investigation is the PIK3CA gene, encoding the alpha catalytic subunit of the enzyme phosphatidylinositol-3 kinase (PI3K). Publicly available data from 3 extensive published series of colorectal carcinomas were analyzed to define the molecular landscape of colorectal adenocarcinomas with and without mutations of PIK3CA. An analysis for discovery of associations with alterations in other critical genes and pathways involved in colorectal cancer was performed. The total mutation burden (TMB) and copy number alteration burden of colorectal cancers with and without mutations of PIK3CA, as well as prognostic implications of alterations of the gene for survival, were examined. RESULTS: Mutations in PIK3CA are observed in 20% to 25% of colorectal cancers. PIK3CA represents one of the most frequently mutated oncogenes in these cancers. Mutations in PIK3CA are associated with higher rates of mutations in other genes of important cancer-associated pathways such as the tyrosine kinase receptors/K-Ras/BRAF/MAPK and the Wnt/ß-catenin pathway. In addition, PIK3CA mutated colorectal cancers display a higher TMB than nonmutated cancers. CONCLUSION: Frequent mutations of PIK3CA gene in colorectal carcinomas may represent an opportunity for targeted therapy combination development inhibiting both the PI3K kinase itself and associated pathway defects. Increased TMB may additionally confer immunotherapy sensitivity, which could be augmented by other targeted therapies.

PI3KCA Mutations in Uterine Cervix Carcinoma.

BACKGROUND: Squamous cervical carcinoma represents an infection-associated malignancy that produces a high mortality when metastatic or recurrent after primary local treatment. There is an urgent need for new therapies for this cancer. Molecular lesions in cervical cancer may provide opportunities for targeted therapies development. METHODS: Publicly available data from the Cancer Genome Atlas (TCGA) were analyzed to define the molecular landscape of squamous cervical carcinomas with and without mutations of PIK3CA, the gene encoding the alpha catalytic subunit of phosphatidylinositol 3 kinase (PI3K). Associations with alterations in other critical genes and pathways of cancer and the total mutation burden and copy number alteration burden of cervical cancers were examined. RESULTS: Mutations in PIK3CA are observed in 27.1% of squamous cervical cancers. PIK3CA represents the most frequently mutated gene in these cancers. Mutations in PIK3CA are associated with higher rates of mutations in other genes of important cancer-associated pathways such as the tyrosine kinase receptors/K-Ras/BRAF/MAPK and the Wnt/ß catenin pathway. In addition, PIK3CA mutated cervical cancers display a higher tumor mutation burden (TMB) than non-mutated cancers. CONCLUSION: Frequent mutations of PIK3CA gene in squamous cervical carcinomas may represent an opportunity for targeted therapies development both inhibiting the PI3K kinase and associated pathway defects. Increased TMB may additionally confer immunotherapy sensitivity.

NVP-BEZ235 overcomes gefitinib-acquired resistance by down-regulating PI3K/AKT/mTOR phosphorylation.

BACKGROUND: Patients harboring activating mutations in epidermal growth factor receptors (EGFR) are particularly sensitive to EGFR tyrosine kinase inhibitors (TKIs). However, most patients develop an acquired resistance after a period of about 10 months. This study focuses on the therapeutic effect of NVP-BEZ235, a dual inhibitor of phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR), in gefitinib-resistant non-small cell lung cancer. METHODS: H1975 cell line was validated as a gefitinib-resistant cell model by the nucleotide-sequence analysis. We used the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to detect the growth of H1975 cell line in vitro. H1975 cells' migration was detected by the migration assay. Xenograft models were used to investigate the growth of gefitinib-resistant non-small cell lung cancer in vivo. Western blot and immunohistochemical analysis were used to investigate the level of PI3K/protein kinase B(AKT)/mTOR signaling pathway proteins. RESULTS: We show that NVP-BEZ235 effectively inhibited the growth of H1975 cells in vivo as well as in vitro. Similarly, H1975 cell migration was reduced by NVP-BEZ235. Further experiments revealed that NVP-BEZ235 attenuated the phosphorylation of PI3K/AKT/mTOR signaling pathway proteins. CONCLUSION: Taken together, we suggest that NVP-BEZ235 inhibits gefitinib-resistant tumor growth by downregulating PI3K/AKT/mTOR phosphorylation.