Ras and TGFβ cooperatively regulate epithelial cell plasticity and metastasis

Multistep carcinogenesis involves more than six discrete events also important in normal development and cell behavior. Of these, local invasion and metastasis cause most cancer deaths but are the least well understood molecularly. We employed a combined in vitro/in vivo carcinogenesis model, that is, polarized Ha-Ras–transformed mammary epithelial cells (EpRas), to dissect the role of Ras downstream signaling pathways in epithelial cell plasticity, tumorigenesis, and metastasis. Ha-Ras cooperates with transforming growth factor β (TGFβ) to cause epithelial mesenchymal transition (EMT) characterized by spindle-like cell morphology, loss of epithelial markers, and induction of mesenchymal markers. EMT requires continuous TGFβ receptor (TGFβ-R) and oncogenic Ras signaling and is stabilized by autocrine TGFβ production. In contrast, fibroblast growth factors, hepatocyte growth factor/scatter factor, or TGFβ alone induce scattering, a spindle-like cell phenotype fully reversible after factor withdrawal, which does not involve sustained marker changes. Using specific inhibitors and effector-specific Ras mutants, we show that a hyperactive Raf/mitogen-activated protein kinase (MAPK) is required for EMT, whereas activation of phosphatidylinositol 3-kinase (PI3K) causes scattering and protects from TGFβ-induced apoptosis. Hyperactivation of the PI3K pathway or the Raf/MAPK pathway are sufficient for tumorigenesis, whereas EMT in vivo and metastasis required a hyperactive Raf/MAPK pathway. Thus, EMT seems to be a close in vitro correlate of metastasis, both requiring synergism between TGFβ-R and Raf/MAPK signaling.

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Effect of bypass kinase pathways on acquired CDK4/6 inhibitor resistance.

Journal of Clinical Oncology ◽

10.1200/jco.2018.36.6_suppl.379 ◽

2018 ◽

Vol 36 (6_suppl) ◽

pp. 379-379

Author(s):

Renee De Leeuw ◽

Christopher McNair ◽

Matthew Joseph Schiewer ◽

Neermala Poudel Neupane ◽

Michael Augello ◽

...

Keyword(s):

Mapk Pathway ◽

Early Stage ◽

Mapk Signaling ◽

Cross Resistance ◽

Mek Inhibitors ◽

Mesenchymal Transition ◽

Inhibitor Resistance ◽

Tumor Types

379 Background: Cyclin Dependent Kinase-4/6 (CDK4/6) kinase inhibitors have shown clinical benefit in treatment of solid tumor types, including breast cancer. However, resistance is common, and the underpinning mechanisms of action are not well understood. Given the dependence of CDK4/6 inhibitors on retinoblastoma tumor suppressor (RB) function for activity, this class of agents may be particularly effective in tumor types for which RB loss is infrequent or occurs late in tumor progression. Methods: Here, models of acquired palbociclib resistance were generated in early stage, RB positive cancers, wherein it was shown acquired palbociclib resistance resulted in cross-resistance to other CDK4/6 inhibitors under clinical testing. Results: Cells showing acquired resistance exhibited aggressive in vitro and in vivo phenotypes without genetic loss of RB or RB pathway members, including enhanced proliferative capacity, migratory potential, and characteristics of epithelial to mesenchymal transition. Further analyses through integration of RNA sequencing and phospho-proteomics identified activation of the MAPK signaling pathway as a mediator of CDK4/6 inhibitor resistance, capable of bypassing CDK4/6 activity. However, this altered kinase dependence resulted in sensitization to MEK inhibitors, suggestive of new clinical opportunities in CDK4/6 resistant tumors. Conclusions: In sum, the studies herein not only identify activation of the MAPK pathway as capable of bypassing the CDK4/6 requirement and promoting aggressive tumor characteristics, but nominate MEK inhibitors as potential mechanisms to treat or prevent CDK4/6 inhibitor resistance.

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Persistent ERK/MAPK Activation Promotes Lactotrope Differentiation and Diminishes Tumorigenic Phenotype

Molecular Endocrinology ◽

10.1210/me.2014-1168 ◽

2014 ◽

Vol 28 (12) ◽

pp. 1999-2011 ◽

Cited By ~ 12

Author(s):

Allyson Booth ◽

Tammy Trudeau ◽

Crystal Gomez ◽

M. Scott Lucia ◽

Arthur Gutierrez-Hartmann

Keyword(s):

Mapk Pathway ◽

Expression System ◽

Mapk Signaling ◽

Tumor Formation ◽

Cell Phenotype ◽

Mapk Activation

The signaling pathways that govern the lactotrope-specific differentiated phenotype, and those that control lactotrope proliferation in both physiological and pathological lactotrope expansion, are poorly understood. Moreover, the specific role of MAPK signaling in lactotrope proliferation vs differentiation, whether activated phosphorylated MAPK is sufficient for prolactinoma tumor formation remain unknown. Given that oncogenic Ras mutations and persistently activated phosphorylated MAPK are found in human tumors, including prolactinomas and other pituitary tumors, a better understanding of the role of MAPK in lactotrope biology is required. Here we directly examined the role of persistent Ras/MAPK signaling in differentiation, proliferation, and tumorigenesis of rat pituitary somatolactotrope GH4 cells. We stimulated Ras/MAPK signaling in a persistent, long-term manner (over 6 d) in GH4 cells using two distinct approaches: 1) a doxycycline-inducible, oncogenic V12Ras expression system; and 2) continuous addition of exogenous epidermal growth factor. We find that long-term activation of the Ras/MAPK pathway over 6 days promotes differentiation of the bihormonal somatolactotrope GH4 precursor cell into a prolactin-secreting, lactotrope cell phenotype in vitro and in vivo with GH4 cell xenograft tumors. Furthermore, we show that persistent activation of the Ras/MAPK pathway not only fails to promote cell proliferation, but also diminishes tumorigenic characteristics in GH4 cells in vitro and in vivo. These data demonstrate that activated MAPK promotes differentiation and is not sufficient to drive tumorigenesis, suggesting that pituitary lactotrope tumor cells have the ability to evade the tumorigenic fate that is often associated with Ras/MAPK activation.

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Dual-specificity phosphatases are targets of the Wnt/β-catenin pathway and candidate mediators of β-catenin/Ras signaling interactions

Biological Chemistry ◽

10.1515/hsz-2012-0130 ◽

2012 ◽

Vol 393 (10) ◽

pp. 1183-1191 ◽

Cited By ~ 11

Author(s):

Eva Zeller ◽

Katharina Mock ◽

Moritz Horn ◽

Sabine Colnot ◽

Michael Schwarz ◽

...

Keyword(s):

Mapk Pathway ◽

Growth Factor Receptor ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Ras Signaling ◽

Dual Specificity ◽

Murine Liver ◽

Dual Specificity Phosphatases

Abstract The Wnt/β-catenin and the Ras/mitogen-activated protein kinase (MAPK) pathways play important roles in cancer development. Both pathways have been studied discretely, but the mechanisms of possible crosstalk are still not fully understood. We have previously shown that β-catenin and MAPK signaling interfere with each other in murine liver in vivo and in vitro. Here, we show that dual specificity phosphatases (Dusps) 6 and 14, known to play an essential role in regulating MAPK pathway activity via feedback mechanisms, are up-regulated by activation of β-catenin in murine liver cells, whereas the epidermal growth factor receptor, an upstream effector in the Ras/MAPK cascade, is down-regulated by β-catenin. In addition, we identified a β-catenin-binding site within the Dusp6 promoter, which is responsible for the activation of the promoter by β-catenin signaling, and demonstrated reduced inducibility of MAPK signaling in cultured mouse hepatoma cells following β-catenin activation. Thus, β-catenin is able to inhibit activation of the Egfr/Ras/MAPK signaling cascade, both at the receptor level and by interfering with MAPK activity via Dusps.

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DDRE-07. DIPG HARBOUR ALTERATIONS TARGETABLE BY MEK INHIBITORS, WITH ACQUIRED RESISTANCE MECHANISMS OVERCOME BY COMBINATORIAL INHIBITION

Neuro-Oncology ◽

10.1093/neuonc/noaa215.252 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii62-ii62

Author(s):

Elisa Izquierdo ◽

Diana Carvalho ◽

Alan Mackay ◽

Sara Temelso ◽

Jessica K R Boult ◽

...

Keyword(s):

Drug Exposure ◽

Mapk Pathway ◽

Synergistic Effects ◽

Acquired Resistance ◽

Mek Inhibitor ◽

Resistance Mechanisms ◽

Genetic Alterations ◽

Mesenchymal Transition

Abstract The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 16-50nM) in samples, which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and of a patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones (62-188-fold, GI50 2.4–5.2µM) in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted an observed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

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OMRT-11. The effect of microenvironment on glioblastoma stem cells therapeutic resistance

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab070.035 ◽

2021 ◽

Vol 3 (Supplement_2) ◽

pp. ii9-ii9

Author(s):

Tamara Lah Turnsek ◽

Barbara Breznik ◽

Bernarda Majc ◽

Metka Novak ◽

Andrej Porčnik ◽

...

Keyword(s):

Stem Cells ◽

Epithelial To Mesenchymal Transition ◽

Combined Treatment ◽

Cell Plasticity ◽

Glioblastoma Stem Cells ◽

Mesenchymal Transition ◽

Non Invasive ◽

Differential Gene

Abstract Epithelial-to-mesenchymal transition (EMT) is an essential molecular and cellular process in physiologic processes and invasion of various types of carcinoma and glioblastoma (GBM) cells. EMT is activated and regulated by specific endogenous triggers in complex network of intercellular interactions and signaling pathways. The hallmark of cancer-linked EMT are intermediate states that show notable cell plasticity, characteristic of cancer stem cells (CSCs), including glioblastoma stem cells – GSCs. GSCs resistance to irradiation (IR) and temozolomide (TMZ) chemotherapy is responsible for early relapses, even at distant brain sites. As GSCs are mostly homing to their “niches” as slowly-dividing GSC-subtype, mimicking a proneural-like non- invasive phenotype PN-genotype, we assume that this, by undergoing an EMT-like transition, GSCs are-reprogrammed to an invasive mesenchymal (MES) GBs/GSCs phenotype in a processes, called PMT (1). However, it is not known, if and by which environmental cues within the niche, this transition of GSCs is induced in vivo. In this work, we are presenting the transriptome data obtained when we exposed GSC spheroids to irradiation alone, TMZ alone and to the combined treatment in vitro and compared their differential genetic fingerprints related to EMT/PMT transition to the GSCs PMT transition, when embedded in their natural microenvironment in the GBM organoid model. The differential gene expression upon GSCs therapeutic perturbation (when alone and vs in the tumoroid microenvironment) will reveal the effects of the major candidate genes, associated with micronevironmendt stromal cells and matrix are contributing their observed EMT/PMT transition of GSCs in vivo. •1. Majc, B., Sever, T., Zarić, M, Breznik, B., Turk, B, Lah Turnšek, T. Epithelial- to-mesenchymal transition as the driver of changing carcinoma and glioblastoma microenvironment. DOI: 10.1016/j.bbamcr.2020.118782

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LncRNA MALAT1 exhibits positive effects on nucleus pulposus cell biology in vivo and in vitro by sponging miR-503

10.21203/rs.2.17571/v2 ◽

2020 ◽

Author(s):

Hongyu Zheng ◽

Tingting Wang ◽

Xiangmin Li ◽

Wei He ◽

Zhiqiang Gong ◽

...

Keyword(s):

Nucleus Pulposus ◽

Disc Degeneration ◽

Cell Biology ◽

Mapk Pathway ◽

Critical Role ◽

Mapk Signaling ◽

Positive Effects ◽

Lncrna Malat1

Abstract Background: Intervertebral disc degeneration (IDD) is characterized by the loss of nucleus pulposus cells (NPCs) and phenotypic abnormalities. Accumulating evidence suggests that long noncoding RNAs (lncRNAs) are involved in the pathogenesis of IDD. In this study, we aimed to investigate the functional effects of lncRNA MALAT1 on NPCs in IDD and the possible mechanism governing these effects. Results: We validated the decreased expression of MALAT1 in the IDD tissues, which was associated with decreased Collagen II and Aggrecan expression. In vitro, overexpressed MALAT1 could attenuate the effect of IL-1β on NPC proliferation, apoptosis, and Aggrecan degradation. In vivo, MALAT1 overexpression attenuated the severity of disc degeneration in IDD model rats. Our molecular study further demonstrated that MALAT1 could sponge miR-503, modulate the expression of miR-503, and activate downstream MAPK signaling pathways. The effects of MALAT1 on NPCs were partially reversed/aggregated by miR-503 mimics/inhibitor treatment. Conclusion: Our data suggested that the MALAT1-miR-503-MAPK pathway plays a critical role in NPCs, which may be a potential strategy for alleviating IDD.

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Anti-Tumor Activity of ASTX029, a Dual Mechanism Inhibitor of ERK1/2, in Preclinical AML Models

Blood ◽

10.1182/blood-2020-139175 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 7-8

Author(s):

Christopher J. Hindley ◽

Lynsey Fazal ◽

Joanne M. Munck ◽

Vanessa Martins ◽

Alpesh D. Shah ◽

...

Keyword(s):

Cell Lines ◽

Mapk Pathway ◽

Mapk Signaling ◽

Activating Mutations ◽

Ic50 Value ◽

Ras Family ◽

Dual Mechanism ◽

Anti Tumor Activity

Oncogenic mutations in genes such as the RAS family (KRAS, NRAS or HRAS) or receptor tyrosine kinases (RTKs) drive tumor growth through aberrant activation of the mitogen activated protein kinase (MAPK) signaling pathway. Acute myeloid leukemia (AML) patients frequently exhibit activating mutations in MAPK pathway members, such as NRAS and KRAS, suggesting that these malignancies may be driven by aberrant activation of the MAPK pathway. Targeting of the MAPK pathway has been clinically validated in solid tumors, with agents targeting BRAF and MEK approved for the treatment of BRAF-mutant melanoma. However, there is currently no approved therapy directly targeting activated RAS family members and resistance to MAPK pathway inhibitors is frequently associated with reactivation of MAPK signaling. ERK1/2 (ERK) is a downstream node in the MAPK pathway and therefore represents an attractive therapeutic target for inhibition of MAPK signaling in these settings. We have recently described in vivo anti-tumor activity in MAPK-activated solid tumor models following treatment with ASTX029, a highly potent ERK inhibitor developed using fragment-based drug design. ASTX029 has a distinctive ERK binding mode which confers dual mechanism inhibition of ERK, inhibiting both the catalytic activity of ERK and its phosphorylation by MEK. Here, we demonstrate that ASTX029 is also active in AML models and potently inhibits in vitro and in vivo MAPK signaling and growth in these models. Using a panel of 15 AML cell lines, we investigated sensitivity to ASTX029 in vitro. We observed that 8 cell lines bearing mutations leading to increased MAPK pathway signaling were sensitive to treatment with ASTX029 with an average IC50 value of 47 nM, in contrast to an average IC50 value of 1800 nM for cell lines without activating mutations. The phosphorylation of RSK, a direct substrate of ERK, was suppressed for up to 24 h following treatment with ASTX029 in vitro. We have previously demonstrated good oral bioavailability for ASTX029 and once daily dosing resulted in significant tumor growth inhibition in AML cell line xenograft models. To confirm target engagement in vivo, we examined MAPK signaling in xenograft tissue and observed inhibition of the phosphorylation of RSK and of ERK itself, consistent with the dual mechanism of action proposed for ASTX029. In summary, the ERK inhibitor, ASTX029, has potent activity against MAPK-activated tumor models, including AML models, and is now being tested in a Phase 1/2 clinical trial in advanced solid tumors (NCT03520075). These data highlight its therapeutic potential for the treatment of AML in patients with mutations leading to MAPK pathway activation and support further investigation in these patient populations. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Fazal:Astex Pharmaceuticals: Current Employment. Munck:Astex Pharmaceuticals: Current Employment. Martins:Astex Pharmaceuticals: Current Employment. Shah:Astex Pharmaceuticals: Current Employment. Wilsher:Astex Pharmaceuticals: Current Employment. Wallis:Astex Pharmaceuticals: Current Employment. Keer:Astex Pharmaceuticals, Inc.: Current Employment. Lyons:Astex Pharmaceuticals: Current Employment.

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MEK-inhibitor-mediated rescue of skeletal myopathy caused by activating Hras mutation in a Costello syndrome mouse model

Disease Models & Mechanisms ◽

10.1242/dmm.049166 ◽

2021 ◽

Author(s):

William E. Tidyman ◽

Alice F. Goodwin ◽

Yoshiko Maeda ◽

Ophir D. Klein ◽

Katherine A. Rauen

Keyword(s):

Mouse Model ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Costello Syndrome ◽

Skeletal Myopathy ◽

Mitogen Activated Protein

Costello syndrome (CS) is a congenital disorder caused by heterozygous activating germline HRAS mutations in the canonical Ras/mitogen-activated protein kinase (Ras/MAPK) pathway. CS is one of the RASopathies, a large group of syndromes due to mutations within various components of the Ras/MAPK pathway. An important part of the phenotype that greatly impacts quality of life is hypotonia. To gain a better understanding of the mechanisms underlying hypotonia in CS, a mouse model with an activating HrasG12V allele was utilized. We identified a skeletal myopathy that was due in part to an inhibition of embryonic myogenesis and myofiber formation, resulting in a reduction of myofiber size and number that led to reduced muscle mass and strength. In addition to hyperactivation of the Ras/MAPK and PI3K/AKT pathways, there was a significant reduction of p38 signaling, as well as global transcriptional alterations consistent with the myopathic phenotype. Inhibition of Ras/MAPK pathway signaling using a MEK inhibitor rescued the HrasG12V myopathy phenotype both in vitro and in vivo, demonstrating that increased MAPK signaling is the main cause of the muscle phenotype in CS.

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RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis

Journal of Cell Science ◽

10.1242/jcs.113.16.2837 ◽

2000 ◽

Vol 113 (16) ◽

pp. 2837-2844 ◽

Cited By ~ 3

Author(s):

V. Jullien-Flores ◽

Y. Mahe ◽

G. Mirey ◽

C. Leprince ◽

B. Meunier-Bisceuil ◽

...

Keyword(s):

Signal Transduction ◽

Growth Factor ◽

Egf Receptor ◽

The Other ◽

Ras Signaling ◽

Receptor Endocytosis ◽

Epidermal Growth ◽

Two Hybrid

RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

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