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      MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E‐mutant colorectal adenocarcinoma

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          Abstract

          The mitogen‐activated protein kinase ( MAPK) pathway plays a central role in colorectal cancers ( CRC). In particular, BRAF V600E‐mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 ( SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort ( N = 571) using tissue‐derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real‐time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway‐targeted therapies on SPINK1 secretion, we conducted in vitro studies using both wild‐type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT‐29 with a concomitant decrease in trypsin‐1 and ‐2 secretion. Notably, no SPINK1 increase or trypsin‐1 decrease was observed in BRAF wild‐type CRC cell line Caco‐2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 ( ATF‐4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF‐4 and trypsin, might be beneficial in the therapy of BRAF V600E‐mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.

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          Most cited references 24

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          The integrated stress response.

          In response to diverse stress stimuli, eukaryotic cells activate a common adaptive pathway, termed the integrated stress response (ISR), to restore cellular homeostasis. The core event in this pathway is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) by one of four members of the eIF2α kinase family, which leads to a decrease in global protein synthesis and the induction of selected genes, including the transcription factor ATF4, that together promote cellular recovery. The gene expression program activated by the ISR optimizes the cellular response to stress and is dependent on the cellular context, as well as on the nature and intensity of the stress stimuli. Although the ISR is primarily a pro-survival, homeostatic program, exposure to severe stress can drive signaling toward cell death. Here, we review current understanding of the ISR signaling and how it regulates cell fate under diverse types of stress.
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            Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma.

            Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.
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              Clinical Acquired Resistance to RAF Inhibitor Combinations in BRAF-Mutant Colorectal Cancer through MAPK Pathway Alterations.

              BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance.
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                Author and article information

                Contributors
                kati.rasanen@helsinki.fi
                Journal
                Mol Oncol
                Mol Oncol
                10.1002/(ISSN)1878-0261
                MOL2
                Molecular Oncology
                John Wiley and Sons Inc. (Hoboken )
                1574-7891
                1878-0261
                27 December 2017
                February 2018
                : 12
                : 2 ( doiID: 10.1002/mol2.2018.12.issue-2 )
                : 224-238
                Affiliations
                [ 1 ] Department of Clinical Chemistry Medicum Helsinki University Hospital University of Helsinki Finland
                [ 2 ] Minerva Foundation Institute for Medical Research Helsinki Finland
                [ 3 ] Department of Surgery Helsinki University Hospital University of Helsinki Finland
                [ 4 ] Department of Genomics BPARC Vietnam Military Medical University Hanoi Vietnam
                [ 5 ] Research Program Unit Translational Cancer Biology University of Helsinki Finland
                [ 6 ] Department of Pathology Helsinki University Hospital University of Helsinki Finland
                [ 7 ] Department of Women's and Children's Health Karolinska Institutet Stockholm Sweden
                Author notes
                [*] [* ] Correspondence

                K. Räsänen, Department of Clinical Chemistry, Medicum, University of Helsinki, 00014 Helsinki, Finland

                Tel: +358 947171725

                E‐mail: kati.rasanen@ 123456helsinki.fi

                [†]

                These authors contributed equally to this study.

                Article
                MOL212160
                10.1002/1878-0261.12160
                5792734
                29193645
                f8f4a1b5-9629-4e42-8901-fc5d7ee2e9bb
                © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

                This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                Page count
                Figures: 6, Tables: 4, Pages: 15, Words: 8291
                Product
                Funding
                Funded by: Orion Research Foundation
                Funded by: Ruth and Nils‐Erik Stenbäck Foundation
                Funded by: Finska Läkaresällskapet
                Funded by: Sigrid Jusélius Foundation
                Funded by: Finnish Cancer Foundation
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                mol212160
                February 2018
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.3.2 mode:remove_FC converted:01.02.2018

                Oncology & Radiotherapy

                biomarker, braf v600e, colorectal cancer, inhibitor, spink1, trametinib

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