International Journal of Molecular Sciences Article (Phospho)proteomic Profiling of Microsatellite Unstable CRC Cells Reveals Alterations in Nuclear Signaling and Cholesterol Metabolism Caused by Frameshift Mutation of NMD Regulator UPF3A Malwina Michalak 1,2,3 , Eva-Maria Katzenmaier 1,2 , Nina Roeckel 1, † , Stefan M. Woerner 2,4 , Vera Fuchs 1,2 , Uwe Warnken 5 , Yan P. Yuan 6 , Peer Bork 2,6,7 , Gabriele Neu-Yilik 2,3 , Andreas Kulozik 2,3 , Magnus von Knebel Doeberitz 1,2,8 , Matthias Kloor 1,2,8 , Jürgen Kopitz 1,2,8 and Johannes Gebert 1,2,8, * 1 Department of Applied Tumor Biology, Institute of Pathology, Heidelberg University Hospital, Im Neuenheimer Feld 224, 69120 Heidelberg, Germany; malwina.michalak@med.uni-heidelberg.de (M.M.); Eva-Maria.Katzenmaier@med.uni-heidelberg.de (E.-M.K.); Nina.blessing@roche.com (N.R.); Vera.Fuchs@med.uni-heidelberg.de (V.F.); magnus.knebel-doeberitz@med.uni-heidelberg.de (M.v.K.D.); Matthias.Kloor@med.uni-heidelberg.de (M.K.); juergen.kopitz@med.uni-heidelberg.de (J.K.) 2 Molecular Medicine Partnership Unit, Medical Faculty of the University of Heidelberg and European Molecular Biology Laboratory, 69120 Heidelberg, Germany; stefan.woerner@uni-heidelberg.de (S.M.W.); Peer.Bork@embl.org (P.B.); Gabriele.Neu-Yilik@med.uni-heidelberg.de (G.N.-Y.); Andreas.Kulozik@med.uni-heidelberg.de (A.K.) 3 Department of Pediatric Oncology, Hematology and Immunology, Children’s Hospital, University of Heidelberg, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany 4 Department of Internal Medicine I, Endocrinology and Metabolism, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany 5 Clinical Cooperation Unit Neurooncology, DKFZ (German Cancer Research Center), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; u.warnken@dkfz-heidelberg.de 6 Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany; yuan@embl.de 7 Max-Delbrück-Centre for Molecular Medicine, Robert-Rössle-Straße 10, 13125 Berlin, Germany 8 Clinical Cooperation Unit Applied Tumor Biology, DKFZ (German Cancer Research Center) Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany * Correspondence: johannes.gebert@med.uni-heidelberg.de; Tel.: +49-6221-564223 † Current address: Roche Diagnostics GmbH, MMQXAD, Nonnenwald 2, 82377 Penzberg, Germany. Received: 9 June 2020; Accepted: 20 July 2020; Published: 23 July 2020   Abstract: DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in Int. J. Mol. Sci. 2020, 21, 5234; doi:10.3390/ijms21155234 www.mdpi.com/journal/ijms