Annual Report 2023
Laboratory of Intracellular Traffic and Oncology
Yuuki Obata, Miyuki Natsume, Sho Ichikawa, Takuma Okamoto, Naoe Takayama, Hinako Sumiyoshi
Introduction
Gain-of-function mutations in receptor protein-tyrosine kinases (RTKs) are critical drivers for development of cancer. Considering that in normal cells, wild-type RTKs are localized to the plasma membrane (PM) to bind their ligands, RTK mutants have been thought to be distributed on the cell surface membrane. However, we found that mutant KIT RTK is aberrantly localized in the intracellular compartments such as the Golgi/trans-Golgi network (TGN), endoplasmic reticulum (ER) and endosomes. It predominantly causes downstream activations in the endomembranes. Our investigations have also identified the presence of other mutant proteins in organelles. Therefore, mislocalized growth signals in organelles are a characteristic feature of cancer-causing RTK mutants. Our primary aim is to clarify the molecular mechanism underlying RTK retention in organelles and to develop a new strategy for the suppression of oncogenic RTK signaling.
The Team and What We Do
We mainly perform spatio-temporal analyses of tyrosine phosphorylation signaling in cancer cells with fluorescent imaging technics and biochemical assays, such as immunoblotting, pulldown, and immunoprecipitation. To visualize precise localization of RTK, we use a super-resolution confocal fluorescence confocal microscopy.
Research Activities
Mechanism of the retention of KIT RTK in the Golgi/TGN in gastrointestinal stromal tumor (GIST)
We search for a small molecule that releases KIT from the Golgi/TGN of GIST cells. For the purpose, we treated GIST cells with more than 20 compounds followed by immunofluorescence confocal microscopic analyses. As a result, we identified the inhibitor of protein kinase D (PKD), CRT0066101 (CRT). In cells treated with CRT, KIT migrated from the Golgi/TGN to lysosomes via the PM and underwent degradation, indicating that PKD activity is essential for KIT retention in the Golgi/TGN region. Human PKD consists of three members, PKD1 (PKCµ), PKD2, and PKD3 (PKCν). Our knockdown assay showed that only PKD2, but not PKD1/PKD3, plays a critical role in Golgi retention of KIT in GIST cells. Furthermore, we identified PI4-kinaseIIIβ and AP1 (adaptor protein 1) as downstream effectors of PKD2. GIST cells bearing a targeted drug-resistant KIT mutant gave similar results. Blockade of these molecules with knockdown caused release of KIT from the Golgi/TGN region, indicating that pharmacological inhibition of PKD2 and PI4KIIIβ is a novel strategy for suppression of KIT signaling in GIST cells (Cell Reports, 2023).
Mislocalization of RTKs other than KIT in cancer cells is independent of PKD2 activity
We hypothesized that Golgi retention of RTKs other than KIT is also dependent on PKD2 activity. Therefore, we examined whether PKD2 knockdown affected the localization of FLT3-ITD and FGFR3-Y373C, which are accumulated in the Golgi/TGN area in acute myelogenous leukemia and multiple myeloma, respectively. Localization of these RTK mutants, however, was not altered by PKD2 knockdown, indicating that Golgi retention of individual RTKs is regulated by different molecular mechanisms. At present, the investigation of cause of Golgi retention of RTKs other than KIT is under way.
Molecular mechanism of ER export of RTKs
Inhibitors of protein export from the ER, such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, indicating that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER–Golgi protein trafficking. M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. Single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, suggesting that BFA/M-COPA treatment cannot be mimicked by knockdown of only one ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Thus, these results suggest that RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export (bioRxiv, 2023). Trafficking inhibition from the ER would be a promising strategy for the suppression of growth signaling of RTK.
Education
We are teaching one graduate student and two undergraduate students.
Future Prospects
Because loss-of-function of PKD2 does not affect the localization and signaling of RTKs other than KIT, such as FLT3-ITD and FGFR3-Y373C, further studies are required to understand the mechanism of Golgi retention of individual oncogenic signaling molecules. Furthermore, we found that blockade of ER export of RTK mutants markedly suppresses growth signaling. These studies suggest that understanding the mechanism of intracellular trafficking will help us establish a novel method for the suppression of cancer cell proliferation.
List of papers published in 2023
Journal
1. Natsume M, Niwa M, Ichikawa S, Okamoto T, Tsutsui H, Usukura D, Murata T, Abe R, Shimonaka M, Nishida T, Shiina I, Obata Y. Brefeldin A and M-COPA block the export of RTKs from the endoplasmic reticulum via simultaneous inactivation of ARF1, ARF4, and ARF5. The Journal of biological chemistry, 300:107327, 2024
2. Obata Y, Kurokawa K, Tojima T, Natsume M, Shiina I, Takahashi T, Abe R, Nakano A, Nishida T. Golgi retention and oncogenic KIT signaling via PLCγ2-PKD2-PI4KIIIβ activation in gastrointestinal stromal tumor cells. Cell reports, 42:113035, 2023