Activation of WNT/?-catenin signaling results in resistance to a dual PI3K/mTOR inhibitor in colorectal cancer cells harboring PIK3CA mutations.
ABSTRACT: PIK3CA is a frequently mutated gene in cancer, including about ~15 to 20% of colorectal cancers (CRC). PIK3CA mutations lead to activation of the PI3K/AKT/mTOR signaling pathway, which plays pivotal roles in tumorigenesis. Here, we investigated the mechanism of resistance of PIK3CA-mutant CRC cell lines to gedatolisib, a dual PI3K/mTOR inhibitor. Out of a panel of 29 CRC cell lines, we identified 7 harboring one or more PIK3CA mutations; of these, 5 and 2 were found to be sensitive and resistant to gedatolisib, respectively. Both of the gedatolisib-resistant cell lines expressed high levels of active glycogen synthase kinase 3-beta (GSK3?) and harbored the same frameshift mutation (c.465_466insC; H155fs*) in TCF7, which encodes a positive transcriptional regulator of the WNT/?-catenin signaling pathway. Inhibition of GSK3? activity in gedatolisib-resistant cells by siRNA-mediated knockdown or treatment with a GSK3?-specific inhibitor effectively reduced the activity of molecules downstream of mTOR and also decreased signaling through the WNT/?-catenin pathway. Notably, GSK3? inhibition rendered the resistant cell lines sensitive to gedatolisib cytotoxicity, both in vitro and in a mouse xenograft model. Taken together, these data demonstrate that aberrant regulation of WNT/?-catenin signaling and active GSK3? induced by the TCF7 frameshift mutation cause resistance to the dual PI3K/mTOR inhibitor gedatolisib. Cotreatment with GSK3? inhibitors may be a strategy to overcome the resistance of PIK3CA- and TCF7-mutant CRC to PI3K/mTOR-targeted therapies.
Project description:Elevated basal, ligand-independent, Wnt signaling in some canine breast cancer cells is not caused by classical mutations in APC, ?-Catenin or GSK3? but, at least partially, by enhanced LEF1 expression. We examined the expression and function of EGFR/HER-regulated pathways on the ligand-independent Wnt signaling.Twelve canine mammary tumor cell lines with previously reported differential basal Wnt activity were used. The expression levels of genes related to EGF-signaling were analyzed by cluster analysis. Cell lines with a combined overexpression of EGF-related genes and enhanced basal Wnt activity were treated with PI3K/mTor or cSRC inhibitors or transfected with a construct expressing wild-type PTEN. Subsequently, effects were measured on Wnt activity, cell proliferation, gene expression and protein level.High basal Wnt/LEF1 activity was associated with overexpression of HER2/3, ID1, ID2, RAC1 and HSP90 together with low to absent cMET and PTEN mRNA expression, suggesting a connection between Wnt- and HER-signaling pathways. Inhibition of the HER-regulated PI3K/mTor pathway using the dual PI3K/mTor inhibitor BEZ235 or the mTor inhibitor Everolimus® resulted in reduced cell proliferation. In the cell line with high basal Wnt activity, however, an unexpected further increased Wnt activity was found that could be greatly reduced after inhibition of the HER-regulated cSRC activity. Inhibition of the PI3K/mTor pathway was associated with enhanced expression of ?-Catenin, Axin2, MUC1, cMET, EGFR and HER2 and a somewhat increased ?-Catenin protein content, whereas cSRC inhibition was associated with slightly enhanced HER3 and SLUG mRNA expression. A high protein expression of HER3 was found only in a cell line with high basal Wnt activity.High basal Wnt activity in some mammary cancer cell lines is associated with overexpression of HER-receptor related genes and HER3 protein, and the absence of PTEN. Inhibition of the PI3K/mTor pathway further stimulated, however, canonical Wnt signaling, whereas the inhibitory effect with the cSRC inhibitor Src-I1 on the Wnt activity further suggested a connection between Wnt and HER2/3-signaling.
Project description:Activation of Wnt/?-catenin signaling is essential for colorectal carcinogenesis. Tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, is a positive regulator of the Wnt/?-catenin signaling. Accordingly, tankyrase inhibitors are under preclinical development for colorectal cancer (CRC) therapy. However, Wnt-driven colorectal cancer cells are not equally sensitive to tankyrase inhibitors, and cellular factors that affect tankyrase inhibitor sensitivity remain elusive. Here, we established a tankyrase inhibitor-resistant cell line, 320-IWR, from Wnt/?-catenin-dependent CRC COLO-320DM cells. 320-IWR cells exhibited resistance to tankyrase inhibitors, IWR-1 and G007-LK, but remained sensitive to a PARP-1/2 inhibitor, olaparib, and several anti-CRC agents. In 320-IWR cells, nuclear localization of active ?-catenin was decreased and expression of ?-catenin target genes was constitutively repressed, suggesting that these cells repressed the Wnt/?-catenin signaling and were dependent on alternative proliferation pathways. 320-IWR cells exhibited upregulated mTOR signaling and were more sensitive to mTOR inhibition than the parental cells. Importantly, mTOR inhibition reversed resistance to tankyrase inhibitors and potentiated their anti-proliferative effects in 320-IWR cells as well as in CRC cell lines in which the mTOR pathway was intrinsically activated. These results indicate that mTOR signaling confers resistance to tankyrase inhibitors in CRC cells and suggest that the combination of tankyrase and mTOR inhibitors would be a useful therapeutic approach for a subset of CRCs.
Project description:Tyrosine kinase inhibitors (TKIs) against EGFR and c-Met are initially effective when administered individually or in combination to non-small cell lung cancer (NSCLC) patients. However, the overall efficacies of TKIs are limited due to the development of drug resistance. Therefore, it is important to elucidate mechanisms of EGFR and c-Met TKI resistance in order to develop more effective therapies. Model NSCLC cell lines H1975 and H2170 were used to study the similarities and differences in mechanisms of EGFR/c-Met TKI resistance. H1975 cells are positive for the T790M EGFR mutation, which confers resistance to current EGFR TKI therapies, while H2170 cells are EGFR wild-type. Previously, H2170 cells were made resistant to the EGFR TKI erlotinib and the c-Met TKI SU11274 by exposure to progressively increasing concentrations of TKIs. In H2170 and H1975 TKI-resistant cells, key Wnt and mTOR proteins were found to be differentially modulated. Wnt signaling transducer, active ?-catenin was upregulated in TKI-resistant H2170 cells when compared to parental cells. GATA-6, a transcriptional activator of Wnt, was also found to be upregulated in resistant H2170 cells. In H2170 erlotinib resistant cells, upregulation of inactive GSK3? (p-GSK3?) was observed, indicating activation of Wnt and mTOR pathways which are otherwise inhibited by its active form. However, in H1975 cells, Wnt modulators such as active ?-catenin, GATA-6 and p-GSK3? were downregulated. Additional results from MTT cell viability assays demonstrated that H1975 cell proliferation was not significantly decreased after Wnt inhibition by XAV939, but combination treatment with everolimus (mTOR inhibitor) and erlotinib resulted in synergistic cell growth inhibition. Thus, in H2170 cells and H1975 cells, simultaneous inhibition of key Wnt or mTOR pathway proteins in addition to EGFR and c-Met may be a promising strategy for overcoming EGFR and c-Met TKI resistance in NSCLC patients.
Project description:<label>BACKGROUND</label>Aberrant activation of Wnt/?-catenin signaling pathway is considered to be an important issue in progression and metastasis of various human cancers, especially in colorectal cancer (CRC). MiR-452 could activate of Wnt/?-catenin signaling. But the mechanism remains unclear.<label>METHODS</label>The expression of miR-452 in CRC and normal tissues was detected by real-time quantitative PCR. The effect of miR-452 on CRC growth and invasion was conducted by functional experiments in vitro and in vivo. Bioinformatics and cell luciferase function studies verified the direct regulation of miR-452 on the 3'-UTR of the GSK3?, which leads to the activation of Wnt/?-catenin signaling.<label>RESULTS</label>MiR-452 was upregulated in CRC compared with normal tissues and was correlated with clinical significance. The luciferase reporter system studies affirmed the direct regulation of miR-452 on the 3'-UTR of the GSK3?, which activate the Wnt/?-catenin signaling. The ectopic upregulation of miR-452 significantly inhibited the expression of GSK3? and enhanced CRC proliferation and invasion in vitro and in vivo. Meanwhile, knockdown of miR-452 significantly recovered the expression of GSK3? and attenuated Wnt/?-catenin-mediated cell metastasis and proliferation. More important, T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors, which are crucial downstream molecules of the Wnt/?-catenin signaling pathway was verified as a valid transcription factor of miR-452's promoter.<label>CONCLUSIONS</label>Our findings first demonstrate that miR-452-GSK3?-LEF1/TCF4 positive feedback loop induce CRC proliferation and migration.
Project description:Colorectal cancer (CRC) is a heterogeneous disease with multiple underlying causative genetic mutations. Genetic mutations in the phosphatidylinositol-3 kinase (PI3K) and the mitogen activated protein kinase (MAPK) pathways are frequently implicated in CRC. Targeting the downstream substrate MEK in these mutated tumors stands out as a potential target in CRC. Several selective inhibitors of MEK have entered clinical trial evaluation; however, clinical activity with single MEK inhibitors has been rarely observed and acquired resistance seems to be inevitable. Amplification of the driving oncogene KRAS(13D), which increases signaling through the ERK1/2 pathway, upregulation of the noncanonical wingless/calcium signaling pathway (Wnt), and coexisting PIK3CA mutations have all been implicated with resistance against MEK inhibitor therapy in KRAS mutated CRC. The Wnt pathway and amplification of the oncogene have also been associated with resistance to MEK inhibitors in CRCs harboring BRAF mutations. Thus, dual targeted inhibition of MEK and PI3K pathway effectors (mTOR, PI3K, AKT, IGF-1R or PI3K/mTOR inhibitors) presents a potential strategy to overcome resistance to MEK inhibitor therapy. Many clinical trials are underway to evaluate multiple combinations of these pathway inhibitors in solid tumors.
Project description:Therapy directed against oncogenic FLT3 has been shown to induce response in patients with acute myeloid leukemia (AML), but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant AML cell lines by sustained treatment with the FLT3 inhibitor sorafenib. Parental cell lines carry the FLT3-ITD (tandem duplication) mutation and are highly responsive to FLT3 inhibitors, whereas resistant cell lines display resistance to multiple FLT3 inhibitors. Sanger sequencing and protein mass-spectrometry did not identify any acquired mutations in FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, whereas it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer FLT3 dependent. Gene expression analysis of sensitive and resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype, which was further supported by next-generation sequencing and phospho-specific-antibody array analysis. Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in resistant cell lines. Gedatolisib significantly extended survival of mice in a sorafenib-resistant AML patient-derived xenograft model. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD-dependent AML results in resistance to drugs targeting FLT3.
Project description:The canonical Wnt pathway transcriptional co-activator β-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated β-catenin levels in NPC cultures using the GSK3 inhibitor CHIR99021 (CHIR) to examine opposing developmental actions of β-catenin. Low CHIR-mediated maintenance and expansion of NPCs are independent of direct engagement of TCF/LEF/β-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/β-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, β-catenin's direct transcriptional role is restricted to the induction of NPCs, where rising β-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.
Project description:Although mutations in the phosphoinositide 3-kinase catalytic subunit (PIK3CA) are common in breast cancer, PI3K inhibitors alone have shown modest efficacy. We sought to identify additional pathways altered in PIK3CA-mutant tumors that might be targeted in combination with PI3K inhibitors. We generated two transgenic mouse models expressing the human PIK3CA-H1047R- and the -E545K hotspot-mutant genes in the mammary gland and evaluated their effects on development and tumor formation. Molecular analysis identified pathways altered in these mutant tumors, which were also targeted in multiple cell lines derived from the PIK3CA tumors. Finally, public databases were analyzed to determine whether novel pathways identified in the mouse tumors were altered in human tumors harboring mutant PIK3CA. Mutant mice showed increased branching and delayed involution of the mammary gland compared to parental FVB/N mice. Mammary tumors arose in 30% of the MMTV-PIK3CA-H1047R and in 13% of -E545K mice. Compared to MMTV-Her-2 transgenic mouse mammary tumors, H1047R tumors showed increased upregulation of Wnt/?-catenin/Axin2, hepatocyte growth factor (Hgf)/Stat3, insulin-like growth factor 2 (Igf-2), and Igf-1R pathways. Inhibitors of STAT3, ?-catenin, and IGF-1R sensitized H1047R-derived mouse tumor cells and PIK3CA-H1047R overexpressing human HS578T breast cancer cells to the cytotoxic effects of PI3K inhibitors. Analysis of The Cancer Genome Atlas database showed that, unlike primary PIK3CA-wild-type and HER-2<sup>+</sup> breast carcinomas, PIK3CA-mutant tumors display increased expression of AXIN2, HGF, STAT3, IGF-1, and IGF-2 mRNA and activation of AKT, IGF1-MTOR, and WNT canonical signaling pathways. Drugs targeting additional pathways that are altered in PIK3CA-mutant tumors may improve treatment regimens using PI3K inhibitors alone.
Project description:RAS proteins play critical roles in various cellular processes, including growth and transformation. RAS proteins are subjected to protein stability regulation via the Wnt/?-catenin pathway, and glycogen synthase kinase 3 beta (GSK3?) is a key player for the phosphorylation-dependent RAS degradation through proteasomes. GSK3?-mediated RAS degradation does not occur in cells that express a nondegradable mutant (MT) ?-catenin. Here, we show that ?-catenin directly interacts with RAS at the ?-interface region that contains the GSK3? phosphorylation sites, threonine 144 and threonine 148 residues. Exposure of these sites by prior ?-catenin degradation is required for RAS degradation. The introduction of a peptide that blocks the ?-catenin-RAS interaction by binding to ?-catenin rescues the GSK3?-mediated RAS degradation in colorectal cancer (CRC) cells that express MT ?-catenin. The coregulation of ?-catenin and RAS stabilities by the modulation of their interaction provides a mechanism for Wnt/?-catenin and RAS-ERK pathway cross-talk and the synergistic transformation of CRC by both APC and KRAS mutations.
Project description:<h4>Aim</h4>Differentiated embryonic chondrocyte gene 1 (DEC1) is involved in the neuronal differentiation and development. The aim of this study is to investigate the role of DEC1 in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP<sup>+</sup> )-induced PD model.<h4>Methods</h4>The location of DEC1 and tyrosine hydroxylase (TH)-positive neurons were detected by immunofluorescence. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse subacute model of PD was established to evaluate the change of DEC1 expression in midbrain. Then, SH-SY5Y cells were used to investigate the role of DEC1 in MPP<sup>+</sup> -induced neurotoxicity.<h4>Results</h4>We showed that the co-expressed DEC1 and TH neurons took up more than 80% of the expressed TH neurons in the midbrain of mice. DEC1/TH double-positive neurons decreased by 40.6% in SNpc and 28.8% in VTA of MPTP-injured mice. Consistently, DEC1, TH and dopamine transporter (DAT) expression decreased in the midbrain of MPTP mice. In SY-SY5Y cells, MPP<sup>+</sup> significantly suppressed DEC1 expression and increased the cleaved caspase 3/caspase 3 and Bax/Bcl-2. DEC1 overexpression relieved, whereas DEC1 knockdown aggravated MPP<sup>+</sup> -induced cytotoxicity. Likewise, DEC1 overexpression and knockdown inversely regulated the expression of ?-catenin and PI3Kp110? (PIK3CA), an essential role in Wnt/?-catenin and PI3K/Akt signaling pathways. Interestingly, LY294002, an inhibitor of PI3K/Akt signaling, aggravated, whereas LiCl, an activator of Wnt/?-catenin signaling, abolished the reduction in DEC1 by MPP<sup>+</sup> . It is established that these two pathways are interconnected by the phosphorylation status of GSK3?. DEC1 overexpression increased but MPP<sup>+</sup> and DEC1 knockdown decreased GSK3? phosphorylation.<h4>Conclusion</h4>Downregulation of DEC1 contributes to MPP<sup>+</sup> -induced neurotoxicity by suppressing PI3K/Akt/GSK3? pathway.