Oncogenic KEAP1 mutations activate TRAF2-NFκB signaling to prevent apoptosis in lung cancer cells
Ashik Jawahar Deen,
Simone Adinolfi,
Jouni Härkönen,
Tommi Patinen,
Xiaonan Liu,
Tuomo Laitinen,
Piia Takabe,
Kirsi Kainulainen,
Sanna Pasonen-Seppänen,
Lisa M. Gawriyski,
Uma Thanigai Arasu,
Ilakya Selvarajan,
Petri Mäkinen,
Hanna Laitinen,
Emilia Kansanen,
Minna U. Kaikkonen,
Antti Poso,
Markku Varjosalo,
Anna-Liisa Levonen
Affiliations
Ashik Jawahar Deen
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland
Simone Adinolfi
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland
Jouni Härkönen
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland; Department of Pathology, Hospital Nova of Central Finland, Jyväskylä, 40620, Finland
Tommi Patinen
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland
Xiaonan Liu
Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, 00014, Finland
Tuomo Laitinen
School of Pharmacy, University of Eastern Finland, Kuopio, 70211, Finland
Piia Takabe
Institute of Biomedicine, University of Eastern Finland, Kuopio, 70211, Finland
Kirsi Kainulainen
Institute of Biomedicine, University of Eastern Finland, Kuopio, 70211, Finland
Sanna Pasonen-Seppänen
Institute of Biomedicine, University of Eastern Finland, Kuopio, 70211, Finland
Lisa M. Gawriyski
Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, 00014, Finland
Uma Thanigai Arasu
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland
Ilakya Selvarajan
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland
Petri Mäkinen
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland
Hanna Laitinen
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland
Emilia Kansanen
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland; Science Service Centre, Kuopio University Hospital, Kuopio, 70211, Finland
Minna U. Kaikkonen
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland
Antti Poso
School of Pharmacy, University of Eastern Finland, Kuopio, 70211, Finland; Department of Internal Medicine VIII, University Hospital Tübingen, Tübingen, 72076, Germany
Markku Varjosalo
Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, 00014, Finland
Anna-Liisa Levonen
A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, 70211, Finland; Corresponding author.
The Kelch-like ECH-associated protein 1 (KEAP1) – Nuclear factor erythroid 2 -related factor 2 (NRF2) pathway is the major transcriptional stress response system in cells against oxidative and electrophilic stress. NRF2 is frequently constitutively active in many cancers, rendering the cells resistant to chemo- and radiotherapy. Loss-of-function (LOF) mutations in the repressor protein KEAP1 are common in non-small cell lung cancer, particularly adenocarcinoma. While the mutations can occur throughout the gene, they are enriched in certain areas, indicating that these may have unique functional importance. In this study, we show that in the GSEA analysis of TCGA lung adenocarcinoma RNA-seq data, the KEAP1 mutations in R320 and R470 were associated with enhanced Tumor Necrosis Factor alpha (TNFα) – Nuclear Factor kappa subunit B (NFκB) signaling as well as MYC and MTORC1 pathways. To address the functional role of these hotspot mutations, affinity purification and mass spectrometry (AP-MS) analysis of wild type (wt) KEAP1 and its mutation forms, R320Q and R470C were employed to interrogate differences in the protein interactome. We identified TNF receptor associated factor 2 (TRAF2) as a putative protein interaction partner. Both mutant KEAP1 forms showed increased interaction with TRAF2 and other anti-apoptotic proteins, suggesting that apoptosis signalling could be affected by the protein interactions. A549 lung adenocarcinoma cells overexpressing mutant KEAP1 showed high TRAF2-mediated NFκB activity and increased protection against apoptosis, XIAP being one of the key proteins involved in anti-apoptotic signalling. To conclude, KEAP1 R320Q and R470C and its interaction with TRAF2 leads to activation of NFκB pathway, thereby protecting against apoptosis.
