Phosphorylation of RAB7 by TBK1/IKKε Regulates Innate Immune Signaling in Triple-Negative Breast Cancer

• Published in: Cancer research (Chicago, Ill.) Vol. 80; no. 1; pp. 44 - 56
• Main Authors: Ritter, Jessica L, Zhu, Zehua, Thai, Tran C, Mahadevan, Navin R, Mertins, Philipp, Knelson, Erik H, Piel, Brandon P, Han, Saemi, Jaffe, Jacob D, Carr, Steven A, Barbie, David A, Barbie, Thanh U
• Format: Journal Article
• Language: English
• Published: United States 01.01.2020
• Subjects: Breast - immunology; Breast - pathology; Cell Line, Tumor; Female; HEK293 Cells; Humans; I-kappa B Kinase - metabolism; Immunity, Innate; Membrane Proteins - agonists; Membrane Proteins - metabolism; Mutagenesis, Site-Directed; Mutation; Phosphorylation - genetics; Phosphorylation - immunology; Protein-Serine-Threonine Kinases - metabolism; Proteolysis; PTEN Phosphohydrolase - genetics; PTEN Phosphohydrolase - metabolism; rab GTP-Binding Proteins - genetics; rab GTP-Binding Proteins - immunology; rab GTP-Binding Proteins - metabolism; Serine - genetics; Serine - metabolism; Signal Transduction - immunology; Triple Negative Breast Neoplasms - immunology; Triple Negative Breast Neoplasms - pathology
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.CAN-19-1310

Triple-negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here, we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TANK-binding kinase 1 (TBK1)/IκB kinase ε (IKKε) on the same serine-72 (S72) site. An unbiased search for novel TBK1/IKKε substrates using stable isotope labeling with amino acids in cell culture phosphoproteomic analysis identified Rab7-S72 as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated stimulator of interferon genes (STING) degradation. Thus, convergence of PTEN loss and TBK1/IKKε activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFNβ. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyperresponsive to STING agonists. Together, these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKε in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic. SIGNIFICANCE: These findings identify Rab7 as a substrate for TBK1 for regulation of innate immune signaling, thereby providing important insight for strategies aimed at manipulating the immune response to enhance therapeutic efficacy in TNBC.