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PACT-PKR Pathway Transactivates the Tumor Suppressor RhoB through C/EBPbeta in Cancer Cells with High Akt Activity

ABSTRACT: Activation of the PI3K/Akt/mTOR pathway in cancers can occur through loss of PTEN. Transcriptional profiling of pathway inhibitors identified the tumor suppressor RhoB as a gene markedly upregulated by lipid-based Akt inhibitors (LBAI). Here, we demonstrate that the C/EBPbeta full-length isoform LAP is responsible for transcriptional induction through its binding site within the RhoB proximal promoter. LBAI strongly transactivate RhoB by switching translation of C/EBPbeta from the truncated isoform LIP to LAP via PACT-mediated PKR activation in cancer cells with high Akt activity. Unlike PTEN commonly mutated, endogenous RhoB tumor-suppressive activity can be reconstituted by restoring its expression, which was noninvasively monitored by a RhoB promoter-driven luciferase reporter in living mice. LBAI administration increased luciferase activity and decreased the growth of human tumor xenografts. Increased PKR activation by LBAI leads to more robust RhoB induction and cytotoxicity than other PI3K/Akt/mTOR axis inhibitors, revealing a novel strategy for cancer therapy. H157 cells were plated 2 x 10^6 in T-75 flasks in RPMI 1640 containing 10% FBS and incubated for 24h. The medium was then changed to RPMI 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM of LY294002, OSU03012, PIA23, Perifosine, Miltefosine, API-2, DZ-50, or 100 nM of Wortmannin and Rapamycin for 6 hours, or an equal amount of DMSO as control. Following incubation, total RNA was extracted from cells in T-75 flasks using TRIzol reagent (Invitrogen). Two dye-swapped replicates were performed for each treatment.

ORGANISM(S): Homo sapiens

SUBMITTER: Dennis Phillip

PROVIDER: S-ECPF-GEOD-28993 | biostudies-other |

REPOSITORIES: biostudies-other

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PACT-PKR Pathway Transactivates the Tumor Suppressor RhoB through C/EBPbeta in Cancer Cells with High Akt Activity

Project description:Activation of the PI3K/Akt/mTOR pathway in cancers can occur through loss of PTEN. Transcriptional profiling of pathway inhibitors identified the tumor suppressor RhoB as a gene markedly upregulated by lipid-based Akt inhibitors (LBAI). Here, we demonstrate that the C/EBPbeta full-length isoform LAP is responsible for transcriptional induction through its binding site within the RhoB proximal promoter. LBAI strongly transactivate RhoB by switching translation of C/EBPbeta from the truncated isoform LIP to LAP via PACT-mediated PKR activation in cancer cells with high Akt activity. Unlike PTEN commonly mutated, endogenous RhoB tumor-suppressive activity can be reconstituted by restoring its expression, which was noninvasively monitored by a RhoB promoter-driven luciferase reporter in living mice. LBAI administration increased luciferase activity and decreased the growth of human tumor xenografts. Increased PKR activation by LBAI leads to more robust RhoB induction and cytotoxicity than other PI3K/Akt/mTOR axis inhibitors, revealing a novel strategy for cancer therapy. H157 cells were plated 2 x 10^6 in T-75 flasks in RPMI 1640 containing 10% FBS and incubated for 24h. The medium was then changed to RPMI 1640 with 0.1% FBS and the cells were incubated overnight. The following morning, cells were treated with 10 microM of LY294002, OSU03012, PIA23, Perifosine, Miltefosine, API-2, DZ-50, or 100 nM of Wortmannin and Rapamycin for 6 hours, or an equal amount of DMSO as control. Following incubation, total RNA was extracted from cells in T-75 flasks using TRIzol reagent (Invitrogen). Two dye-swapped replicates were performed for each treatment.

2015-01-29 | E-GEOD-28993 | biostudies-arrayexpress

PACT-PKR Pathway Transactivates the Tumor Suppressor RhoB through C/EBPbeta in Cancer Cells with High Akt Activity

2015-01-29 | GSE28993 | GEO

FGF14 Functions as a Tumor Suppressor through Inhibiting PI3K/AKT/mTOR Pathway in Colorectal Cancer.

Project description:We identified that Fibroblast Growth Factor 14 (FGF14) was preferentially methylated in colorectal cancer (CRC). In this study, we aimed to investigate the epigenetic regulation, biological function and molecular mechanism of FGF14 in CRC. The expression of FGF14 in CRC cell lines, normal human colon epithelial cell line, CRC tissues and paired adjacent normal tissues was detected by PCR and Western blot. The biological function of FGF14 in CRC was interrogated by cell viability assay, colony formation, flow cytometry, cell invasion and migration assay, as well as in vivo study. We found FGF14 was downregulated or silenced in all (10/10) CRC cell lines, while it was expressed in normal colonic tissues and normal human colon epithelial cell line. The expression of FGF14 was lower in primary CRCs as compared to their adjacent normal tissues. Significant higher methylation of FGF14 was observed in CRCs than that in normal tissues based on the data from TCGA database. The loss of FGF14 gene expression was restored by treatment with DNA methyltransferase inhibitor 5-Aza. Re-expression of FGF14 in CRC cell lines inhibited cell viability and colony formation, and induced cell apoptosis. FGF14 induced mitochondrial apoptosis and inhibited PI3K/AKT/mTOR pathway. In xenograft mouse model, overexpression of FGF14 significantly reduced tumor growth (P<0.001). In conclusion, FGF14 is a novel tumor suppressor, which suppresses cell proliferation and induces cell apoptosis via mediating PI3K/AKT/mTOR pathway.

| S-EPMC6959027 | biostudies-literature

Loss of G-Protein Pathway Suppressor 2 Promotes Tumor Growth Through Activation of AKT Signaling.

Project description:G Protein Suppressor 2 (GPS2) is a multifunctional protein that exerts important roles in inflammation and metabolism in adipose, liver, and immune cells. GPS2 has recently been identified as a significantly mutated gene in breast cancer and other malignancies and proposed to work as a putative tumor suppressor. However, molecular mechanisms by which GPS2 prevents cancer development and/or progression are largely unknown. Here, we have profiled the phenotypic changes induced by GPS2 depletion in MDA-MB-231 triple negative breast cancer cells and investigated the underlying molecular mechanisms. We found that GPS2-deleted MDA-MB-231 cells exhibited increased proliferative, migratory, and invasive properties in vitro, and conferred greater tumor burden in vivo in an orthotopic xenograft mouse model. Transcriptomic, proteomic and phospho-proteomic profiling of GPS2-deleted MBA-MB-231 revealed a network of altered signals that relate to cell growth and PI3K/AKT signaling. Overlay of GPS2-regulated gene expression with MDA-MB-231 cells modified to express constitutively active AKT showed significant overlap, suggesting that sustained AKT activation is associated with loss of GPS2. Accordingly, we demonstrate that the pro-oncogenic phenotypes associated with GPS2 deletion are rescued by pharmacological inhibition of AKT with MK2206. Collectively, these observations confirm a tumor suppressor role for GPS2 and reveal that loss of GPS2 promotes breast cancer cell proliferation and tumor growth through uncontrolled activation of AKT signaling. Moreover, our study points to GPS2 as a potential biomarker for a subclass of breast cancers that would be responsive to PI3K-class inhibitor drugs.

| S-EPMC7817781 | biostudies-literature

Metallothionein 1G functions as a tumor suppressor in thyroid cancer through modulating the PI3K/Akt signaling pathway.

Project description:BackgroundMT1G inactivation mediated by promoter methylation has been reported in thyroid cancer. However, the role of MT1G in thyroid carcinogenesis remains unclear. The aim of this study is to examine the biological functions and related molecular mechanisms of MT1G in thyroid cancer.MethodsMethylation-specific PCR (MSP) was performed to analyze promoter methylation of MT1G and its relationship with clinicopathological characteristics of papillary thyroid cancer (PTC) patients. Conventional and real-time quantitative RT-PCR assays were used to evaluate mRNA expression. The functions of ectopic MT1G expression were determined by cell proliferation and colony formation, cell cycle and apoptosis, as well as cell migration and invasion assays.ResultsMT1G expression was frequently silenced or down-regulated in thyroid cancer cell lines, and was also significantly decreased in primary thyroid cancer tissues compared with non-malignant thyroid tissues. Promoter methylation, along with histone modification, contributes to MT1G inactivation in thyroid tumorigenesis. Moreover, our data showed that MT1G hypermethylation was significantly positively associated with lymph node metastasis in PTC patients. Importantly, restoring MT1G expression in thyroid cancer cells dramatically suppressed cell growth and invasiveness, and induced cell cycle arrest and apoptosis through inhibiting phosphorylation of Akt and Rb.ConclusionsWe have for the first time revealed that MT1G appears to be functional tumor suppressor involved in thyroid carcinogenesis mainly through modulating the phosphatidylinositol-3-kinase (PI3K)/Akt pathway and partially through regulating the activity of Rb/E2F pathway in this study.

| S-EPMC3851544 | biostudies-literature

Loss of G-Protein Pathway Suppressor 2 promotes tumor growth through activation of AKT signaling

Project description:G Protein Suppressor 2 (GPS2) is a multifunctional protein that exerts important roles in inflammation and metabolism in adipose, liver and immune cells. GPS2 has recently been identified as a significantly mutated gene in breast cancer and other malignancies and proposed to work as a putative tumor suppressor. However, molecular mechanisms by which GPS2 prevents cancer development and/or progression are largely unknown. Here, we have profiled the phenotypic changes induced by GPS2 depletion in MDA-MB-231 triple negative breast cancer cells and investigated the underlying molecular mechanisms. We found that GPS2-deleted MDA-MB-231 cells exhibited increased proliferative, migratory, and invasive properties in vitro, and conferred greater tumor burden in vivo in an orthotopic xenograft mouse model. Transcriptomic, proteomic and phospho-proteomic profiling of GPS2-deleted MBA-MB-231 revealed a network of altered signals that relate to cell growth and PI3K/AKT signaling. Overlay of GPS2-regulated gene expression with MDA-MB-231 cells modified to express constitutively active AKT showed significant overlap, suggesting that sustained AKT activation is associated with loss of GPS2. Accordingly, we demonstrate that the pro-oncogenic phenotypes associated with GPS2 deletion are rescued by pharmacological inhibition of AKT with MK2206. Collectively, these observations confirm a tumor suppressor role for GPS2 and reveal that loss of GPS2 promotes breast cancer cell proliferation and tumor growth through uncontrolled activation of AKT signaling. Moreover, our study points to GPS2 as a potential biomarker for a subclass of breast cancers that would be responsive to PI3K-class inhibitor drugs.

2021-01-27 | GSE161283 | GEO

PACT-mediated regulation of endogenous dsRNA sensing by PKR

Project description:Protein kinase R (PKR), an innate immune sensor for double-stranded RNA (dsRNA), is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a crucial and controversial role in the PKR pathway. We demonstrate that PACT is an essential negative regulator of PKR against endogenous dsRNA ligands like inverted repeat Alu RNAs, which satisfy PKR’s selectivity for long dsRNA and robustly activate PKR in the absence of PACT. PACT employs an unusual inhibitory mechanism sensitive to dsRNA length and concentration, inhibiting PKR through low-affinity interactions most effective when both are bound to the same dsRNA molecule. Consequently, PACT’s inhibition of PKR is more robust when dsRNA is longer and less abundant, with minimal effect on abundant or short dsRNA. Thus, PACT functions as a rheostat, setting the PKR activation threshold for long endogenous dsRNA without altering its inherent activity.

2024-06-16 | GSE269684 | GEO

KLF4 Acts as a wt-CFTR Suppressor through an AKT-Mediated Pathway.

Project description:Cystic Fibrosis (CF) is caused by >2000 mutations in the CF transmembrane conductance regulator (CFTR) gene, but one mutation-F508del-occurs in ~80% of patients worldwide. Besides its main function as an anion channel, the CFTR protein has been implicated in epithelial differentiation, tissue regeneration, and, when dysfunctional, cancer. However, the mechanisms that regulate such relationships are not fully elucidated. Krüppel-like factors (KLFs) are a family of transcription factors (TFs) playing central roles in development, stem cell differentiation, and proliferation. Herein, we hypothesized that these TFs might have an impact on CFTR expression and function, being its missing link to differentiation. Our results indicate that KLF4 (but not KLF2 nor KLF5) is upregulated in CF vs. non-CF cells and that it negatively regulates wt-CFTR expression and function. Of note, F508del-CFTR expressing cells are insensitive to KLF4 modulation. Next, we investigated which KLF4-related pathways have an effect on CFTR. Our data also show that KLF4 modulates wt-CFTR (but not F508del-CFTR) via both the serine/threonine kinase AKT1 (AKT) and glycogen synthase kinase 3 beta (GSK3β) signaling. While AKT acts positively, GSK3β is a negative regulator of CFTR. This crosstalk between wt-CFTR and KLF4 via AKT/ GSK3β signaling, which is disrupted in CF, constitutes a novel mechanism linking CFTR to the epithelial differentiation.

| S-EPMC7408019 | biostudies-literature

MicroRNA-19a and microRNA-19b promote the malignancy of clear cell renal cell carcinoma through targeting the tumor suppressor RhoB.

Project description:Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma, which shows high aggressiveness and lacks biomarkers. RhoB acts as a tumor suppressor that inhibits the progression of ccRCC. In the present study, we examined the effects of oncogenic microRNAs, miR-19a and miR-19b, on RhoB expression in ccRCC cells. The results showed that both miR-19a and miR-19b could directly target the 3'untranslated region (3'UTR) of RhoB, resulting in the reduced expression of RhoB. With RT-PCR analysis, we detected the increased expression of miR-19a and miR-19b in ccRCC tissues compared to adjacent non-tumor renal tissues. These data also demonstrated an exclusive negative correlation between miR-19a/19b and RhoB expression in ccRCC specimens and cell lines. In addition, the knockdown of RhoB or overexpression of miR-19a and miR-19b in ccRCC cells could promote cell proliferation, migration and invasion. These data demonstrate the direct roles of miR-19a and miR-19b on the repression of RhoB and its consequences on tumorigenesis, cancer cell proliferation and invasiveness. These results suggest the potential clinical impact of miR-19a and miR-19b as molecular targets for ccRCC.

| S-EPMC5825063 | biostudies-literature

Long non-coding RNA DARS-AS1 promotes tumor progression by suppressing PACT-mediated PKR activation

Project description:Cancer cells evolve various mechanisms to overcome cellular stresses and maintain progression. Protein kinase R (PKR) and its protein activator (PACT) are the initial responders in monitoring diverse stress signals and lead to cell proliferation inhibition and cell apoptosis in consequence. However, the regulation of PACT-PKR pathway in cancer cells is largely unknown. Herein, we identify that the long non-coding RNA (lncRNA) aspartyl-tRNA synthetase antisense RNA 1 (DARS-AS1) is directly involved in the inhibition of the PACT-PKR pathway and promotes cancer cell proliferation. Using large-scale CRISPRi functional screening of 971 cancer-associated lncRNAs, we find that DARS-AS1 is associated with significantly enhanced cancer cell proliferation. Accordingly, knocking down DARS-AS1 inhibits cell proliferation of multiple cancer cell lines and promotes cancer cell apoptosis in vitro and significantly reduces tumor growth in vivo. Mechanistically, DARS-AS1 directly binds to the activator domain of PACT and prevents PACT-PKR interaction, thereby decreasing PKR activation, eIF2α phosphorylation and inhibiting apoptotic cell death. Clinically, DARS-AS1 is broadly expressed across multiple cancers and the increased expression of this lncRNA indicates poor prognosis. This study elucidates the lncRNA DARS-AS1 directed cancer-specific modulation of the PACT-PKR pathway and provides a novel target for cancer prognosis and therapeutic treatment.

2022-05-15 | GSE197980 | GEO

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