The Tribble with APL: A New Road to Therapy.
Carmody Ruaidhrí,Keeshan Karen
The t(15;17) translocation generates a PML-RARα fusion protein causative for acute promyelocytic leukemia (APL). Li et al. now identify the pseudokinase stress protein TRIB3 as an important factor in APL disease progression and therapy resistance. Targeting the interaction of TRIB3 and PML-RARα using peptide technology provides a novel therapeutic approach.
TRIB3 Interacts With β-Catenin and TCF4 to Increase Stem Cell Features of Colorectal Cancer Stem Cells and Tumorigenesis.
Hua Fang,Shang Shuang,Yang Yu-Wei,Zhang Hai-Zeng,Xu Tian-Lei,Yu Jiao-Jiao,Zhou Dan-Dan,Cui Bing,Li Ke,Lv Xiao-Xi,Zhang Xiao-Wei,Liu Shan-Shan,Yu Jin-Mei,Wang Feng,Zhang Cheng,Huang Bo,Hu Zhuo-Wei
BACKGROUND & AIMS:Activation of Wnt signaling to β-catenin contributes to the development of colorectal cancer (CRC). Expression of tribbles pseudo-kinase 3 (TRIB3) is increased in some colorectal tumors and associated with poor outcome. We investigated whether increased TRIB3 expression promotes stem cell features of CRC cells and tumor progression by interacting with the Wnt signaling pathway. METHODS:We performed studies with C57BL/6J-Apc/J mice injected with an adeno-associated virus vector that expresses a small hairpin RNA against Trib3 mRNA (Apc/J-Trib3) or a control vector (Apc/J-Ctrl). We created BALB/c mice that overexpress TRIB3 from an adeno-associated virus vector and mice with small hairpin RNA-mediated knockdown of β-catenin. The mice were given azoxymethane followed by dextran sodium sulfate to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by histology, gene expression profiling, immunohistochemistry, and immunofluorescence. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5)-positive (LGR5) and LGR5-negative (LGR5) HCT-8 CRC cells, with or without knockdown or transgenic expression of TRIB3, were sorted and analyzed in sphere-formation assays. We derived organoids from human and mouse colorectal tumors to analyze the function of TRIB3 and test the effect of a peptide inhibitor. Wnt signaling to β-catenin was analyzed in dual luciferase reporter, chromatin precipitation, immunofluorescence, and immunoblot assays. Proteins that interact with TRIB3 were identified by immunoprecipitation. CRC cell lines were grown in nude mice as xenograft tumors. RESULTS:At 10 weeks of age, more than half the Apc/J-Ctrl mice developed intestinal high-grade epithelial neoplasia, whereas Apc/J-Trib3 mice had no intestinal polyps and normal histology. Colon tissues from Apc/J-Trib3 mice expressed lower levels of genes regulated by β-catenin and genes associated with cancer stem cells. Mice with overexpression of Trib3 developed more tumors after administration of azoxymethane and dextran sodium sulfate than BALB/c mice. Mice with knockdown of β-catenin had a lower tumor burden after administration of azoxymethane and dextran sodium sulfate, regardless of Trib3 overexpression. Intestinal tissues from mice with overexpression of Trib3 and knockdown of β-catenin did not have activation of Wnt signaling or expression of genes regulated by β-catenin. LGR5 cells sorted from HCT-8 cells expressed higher levels of TRIB3 than LGR5 cells. CRC cells that overexpressed TRIB3 had higher levels of transcription by β-catenin and formed larger spheroids than control CRC cells; knockdown of β-catenin prevented the larger organoid size caused by TRIB3 overexpression. TRIB3 interacted physically with β-catenin and transcription factor 4 (TCF4). TRIB3 overexpression increased, and TRIB3 knockdown decreased, recruitment of TCF4 and β-catenin to the promoter region of genes regulated by Wnt. Activated β-catenin increased expression of TRIB3, indicating a positive-feedback loop. A peptide (P2-T3A6) that bound β-catenin disrupted its interaction with TRIB3 and TCF4. In primary CRC cells and HCT-8 cells, P2-T3A6 decreased expression of genes regulated by β-catenin and genes associated with cancer stem cells and decreased cell viability and migration. Injection of C57BL/6J-Apc/J mice with P2-T3A6 decreased the number and size of tumor nodules and colon expression of genes regulated by β-catenin. P2-T3A6 increased 5-fluorouracil-induced death of CRC cells and survival times of mice with xenograft tumors. CONCLUSION:TRIB3 interacts with β-catenin and TCF4 in intestine cells to increase expression of genes associated with cancer stem cells. Knockdown of TRIB3 decreases colon neoplasia in mice, migration of CRC cells, and their growth as xenograft tumors in mice. Strategies to block TRIB3 activity might be developed for treatment of CRC.
The anti-cancer drug ABTL0812 induces ER stress-mediated cytotoxic autophagy by increasing dihydroceramide levels in cancer cells.
Muñoz-Guardiola Pau,Casas Josefina,Megías-Roda Elisabet,Solé Sònia,Perez-Montoyo Héctor,Yeste-Velasco Marc,Erazo Tatiana,Diéguez-Martínez Nora,Espinosa-Gil Sergio,Muñoz-Pinedo Cristina,Yoldi Guillermo,Abad Jose L,Segura Miguel F,Moran Teresa,Romeo Margarita,Bosch-Barrera Joaquim,Oaknin Ana,Alfón Jose,Domènech Carles,Fabriàs Gemma,Velasco Guillermo,Lizcano Jose M
ABTL0812 is a first-in-class small molecule with anti-cancer activity, which is currently in clinical evaluation in a phase 2 trial in patients with advanced endometrial and squamous non-small cell lung carcinoma (NCT03366480). Previously, we showed that ABTL0812 induces TRIB3 pseudokinase expression, resulting in the inhibition of the AKT-MTORC1 axis and macroautophagy/autophagy-mediated cancer cell death. However, the precise molecular determinants involved in the cytotoxic autophagy caused by ABTL0812 remained unclear. Using a wide range of biochemical and lipidomic analyses, we demonstrated that ABTL0812 increases cellular long-chain dihydroceramides by impairing DEGS1 (delta 4-desaturase, sphingolipid 1) activity, which resulted in sustained ER stress and activated unfolded protein response (UPR) via ATF4-DDIT3-TRIB3 that ultimately promotes cytotoxic autophagy in cancer cells. Accordingly, pharmacological manipulation to increase cellular dihydroceramides or incubation with exogenous dihydroceramides resulted in ER stress, UPR and autophagy-mediated cancer cell death. Importantly, we have optimized a method to quantify mRNAs in blood samples from patients enrolled in the ongoing clinical trial, who showed significant increased and mRNAs. This is the first time that UPR markers are reported to change in human blood in response to any drug treatment, supporting their use as pharmacodynamic biomarkers for compounds that activate ER stress in humans. Finally, we found that MTORC1 inhibition and dihydroceramide accumulation synergized to induce autophagy and cytotoxicity, phenocopying the effect of ABTL0812. Given the fact that ABTL0812 is under clinical development, our findings support the hypothesis that manipulation of dihydroceramide levels might represents a new therapeutic strategy to target cancer. ABBREVIATIONS:4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase; ATG: autophagy related; ATF4: activating transcription factor 4; Cer: ceramide; DDIT3: DNA damage inducible transcript 3; DEGS1: delta 4-desaturase, sphingolipid 1; dhCer: dihydroceramide; EIF2A: eukaryotic translation initiation factor 2 alpha; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; NSCLC: non-small cell lung cancer; THC: Δ-tetrahydrocannabinol; TRIB3: tribbles pseudokinase 3; XBP1: X-box binding protein 1; UPR: unfolded protein response.
The New Antitumor Drug ABTL0812 Inhibits the Akt/mTORC1 Axis by Upregulating Tribbles-3 Pseudokinase.
Erazo Tatiana,Lorente Mar,López-Plana Anna,Muñoz-Guardiola Pau,Fernández-Nogueira Patricia,García-Martínez José A,Bragado Paloma,Fuster Gemma,Salazar María,Espadaler Jordi,Hernández-Losa Javier,Bayascas Jose Ramon,Cortal Marc,Vidal Laura,Gascón Pedro,Gómez-Ferreria Mariana,Alfón José,Velasco Guillermo,Domènech Carles,Lizcano Jose M
Clinical cancer research : an official journal of the American Association for Cancer Research
PURPOSE:ABTL0812 is a novel first-in-class, small molecule which showed antiproliferative effect on tumor cells in phenotypic assays. Here we describe the mechanism of action of this antitumor drug, which is currently in clinical development. EXPERIMENTAL DESIGN:We investigated the effect of ABTL0812 on cancer cell death, proliferation, and modulation of intracellular signaling pathways, using human lung (A549) and pancreatic (MiaPaCa-2) cancer cells and tumor xenografts. To identify cellular targets, we performed in silico high-throughput screening comparing ABTL0812 chemical structure against ChEMBL15 database. RESULTS:ABTL0812 inhibited Akt/mTORC1 axis, resulting in impaired cancer cell proliferation and autophagy-mediated cell death. In silico screening led us to identify PPARs, PPARα and PPARγ as the cellular targets of ABTL0812. We showed that ABTL0812 activates both PPAR receptors, resulting in upregulation of Tribbles-3 pseudokinase (TRIB3) gene expression. Upregulated TRIB3 binds cellular Akt, preventing its activation by upstream kinases, resulting in Akt inhibition and suppression of the Akt/mTORC1 axis. Pharmacologic inhibition of PPARα/γ or TRIB3 silencing prevented ABTL0812-induced cell death. ABTL0812 treatment induced Akt inhibition in cancer cells, tumor xenografts, and peripheral blood mononuclear cells from patients enrolled in phase I/Ib first-in-human clinical trial. CONCLUSIONS:ABTL0812 has a unique and novel mechanism of action, that defines a new and drugable cellular route that links PPARs to Akt/mTORC1 axis, where TRIB3 pseudokinase plays a central role. Activation of this route (PPARα/γ-TRIB3-Akt-mTORC1) leads to autophagy-mediated cancer cell death. Given the low toxicity and high tolerability of ABTL0812, our results support further development of ABTL0812 as a promising anticancer therapy. Clin Cancer Res; 22(10); 2508-19. ©2015 AACR.
Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO inactivation.
Salazar M,Lorente M,García-Taboada E,Pérez Gómez E,Dávila D,Zúñiga-García P,María Flores J,Rodríguez A,Hegedus Z,Mosén-Ansorena D,Aransay A M,Hernández-Tiedra S,López-Valero I,Quintanilla M,Sánchez C,Iovanna J L,Dusetti N,Guzmán M,Francis S E,Carracedo A,Kiss-Toth E,Velasco G
Cell death and differentiation
Tribbles pseudokinase-3 (TRIB3) has been proposed to act as an inhibitor of AKT although the precise molecular basis of this activity and whether the loss of TRIB3 contributes to cancer initiation and progression remain to be clarified. In this study, by using a wide array of in vitro and in vivo approaches, including a Trib3 knockout mouse, we demonstrate that TRIB3 has a tumor-suppressing role. We also find that the mechanism by which TRIB3 loss enhances tumorigenesis relies on the dysregulation of the phosphorylation of AKT by the mTORC2 complex, which leads to an enhanced phosphorylation of AKT on Ser473 and the subsequent hyperphosphorylation and inactivation of the transcription factor FOXO3. These observations support the notion that loss of TRIB3 is associated with a more aggressive phenotype in various types of tumors by enhancing the activity of the mTORC2/AKT/FOXO axis.
Rapalog-Mediated Repression of Tribbles Pseudokinase 3 Regulates Pre-mRNA Splicing.
Stefanovska Bojana,Vicier Cecile Edith,Dayris Thibault,Ogryzko Vasily,Scott Veronique,Bouakka Ibrahim,Delaloge Suzette,Rocca Anna,Le Saux Olivia,Trédan Olivier,Bachelot Thomas,André Fabrice,Fromigué Olivia
Rapalogs have become standard-of-care in patients with metastatic breast, kidney, and neuroendocrine cancers. Nevertheless, tumor escape occurs after several months in most patients, highlighting the need to understand mechanisms of resistance. Using a panel of cancer cell lines, we show that rapalogs downregulate the putative protein kinase TRIB3 (tribbles pseudokinase 3). Blood samples of a small cohort of patients with cancer treated with rapalogs confirmed downregulation of TRIB3. Downregulation of TRIB3 was mediated by LRRFIP1 independently of mTOR and disrupted its interaction with the spliceosome, where it participated in rapalog-induced deregulation of RNA splicing. Conversely, overexpression of TRIB3 in a panel of cancer cell lines abolished the cytotoxic effects of rapalogs. These findings identify TRIB3 as a key component of the spliceosome, whose repression contributes significantly to the mechanism of resistance to rapalog therapy. SIGNIFICANCE: Independent of mTOR signaling, rapalogs induce cytoxicity by dysregulating spliceosome function via repression of TRIB3, the loss of which may, in the long term, contribute to therapeutic resistance.
The TRIB3-SQSTM1 interaction mediates metabolic stress-promoted tumorigenesis and progression via suppressing autophagic and proteasomal degradation.
Hua Fang,Li Ke,Yu Jiao-Jiao,Hu Zhuo-Wei
Cancer and diabetes are 2 multifactorial chronic diseases with tremendous impact on health worldwide. Metabolic risk factors play a critical role in fueling a wide range of cancers, but with undefined mechanisms. We recently reported that TRIB3, a stress-induced protein, mediates a reciprocal antagonism between autophagic and proteasomal degradation systems and connects insulin-IGF1 to malignant promotion. We found that several human cancer tissues express higher TRIB3 and phosphorylated IRS1 (insulin receptor substrate 1), which correlates negatively with patient prognosis. Silencing of TRIB3 not only restores insulin-IGF1-suppressed autophagic flux, but also attenuates tumor growth and metastasis. TRIB3 physically interacts with the autophagic receptor SQSTM1, and this interaction hinders the binding of SQSTM1 to LC3 and ubiquitinated proteins, leading to SQSTM1 accumulation and clearance inhibition of ubiquitinated proteins. Interrupting the TRIB3-SQSTM1 interaction with an α-helical peptide derived from SQSTM1 attenuates tumor growth and metastasis through activating autophagic flux. Our findings indicate that TRIB3 links insulin-IGF1 to cancer development and progression through interacting with SQSTM1. Thus, interrupting the TRIB3-SQSTM1 interaction may provide a potential strategy against cancers in patients with diabetes.
Metformin reduces TRIB3 expression and restores autophagy flux: an alternative antitumor action.
Li Ke,Zhang Ting-Ting,Hua Fang,Hu Zhuo-Wei
Deregulation of metabolism during melanoma progression is tightly associated with the genetic and epigenetic alterations of metabolic regulators. Metformin, a macroautophagy/autophagy inducer, has beneficial effects of preventing and treating multiple cancers with an unclear mechanism. Enhanced pseudokinase TRIB3 was reported to link metabolic stressors to melanoma promotion by inhibiting autophagy and ubiquitin-proteasome degradation systems. Here, we discuss our recent findings regarding how metformin reduces TRIB3 expression to restore autophagic flux and suppress melanoma progression in non-diabetic and diabetic mice. We found that overexpression of TRIB3 reverses the metformin-activated autophagic flux, clearance of accumulated tumor-promoting factors and inhibition of tumor progression. Mechanistically, TRIB3 interacts with KAT5 (lysine acetyltransferase 5) and promotes the physical association of KAT5 and SMAD3, which enhances SMAD3 K333 acetylation in a phosphorylation-dependent manner, sustains SMAD3 transcriptional activity and induces TRIB3 expression. Metformin inhibits SMAD3 phosphorylation and impedes the KAT5-SMAD3 interaction, which attenuates the KAT5-mediated K333 acetylation of SMAD3 to suppress SMAD3 transcriptional activity and TRIB3 expression. Our finding defines a molecular mechanism by which metformin targets TRIB3 expression to induce autophagy and protect against melanoma progression.
TRIB3 confers radiotherapy resistance in esophageal squamous cell carcinoma by stabilizing TAZ.
Zhou Sha,Liu Shiliang,Lin Chuyong,Li Yue,Ye Liping,Wu Xianqiu,Jian Yunting,Dai Yuhu,Ouyang Ying,Zhao Lei,Liu Mengzhong,Song Libing,Xi Mian
Radioresistance becomes the major obstacle to reduce tumor recurrence and improve prognosis in the treatment of esophageal squamous cell carcinoma (ESCC). Thus new strategies for radioresistant ESCC are urgently needed. Herein, we reported that tribbles pseudokinase 3 (TRIB3) serves as a key regulator of radioresistance in ESCC. TRIB3 is overexpressed in ESCC tissues and cell lines. High expression of TRIB3 significantly correlates with poor radiotherapy response and prognosis in ESCC patients. Upregulation of TRIB3 in ESCC cells conferred radioresistance in vitro and in vivo by interacting with TAZ thus impeding β-TrCP-mediated TAZ ubiquitination and degradation. Conversely, silencing TRIB3 sensitized ESCC cells to ionizing radiation. More importantly, TRIB3 was significantly correlated with TAZ activation in ESCC biopsies, and patients with high expression of both TRIB3 and TAZ suffered the worst radiotherapy response and survival. Our study uncovers the critical mechanism of ESCC resistance to radiotherapy, and provides a new pharmacological opportunity for developing a mechanism-based strategy to eliminate radioresistant ESCC in clinical practice.