Annual Report 2022

Laboratory of Intracellular Traffic and Oncology

Yuuki Obata, Miyuki Natsume, Eika Riku, Sho Ichikawa, Haruyo Tozaki, Hinako Sumiyoshi

Introduction

 Gain-of-function mutations in receptor-type tyrosine kinases (RTKs) are critical drivers for cancerization. Considering that in normal cells, wild-type RTKs are localized to the plasma membrane to send signals, 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 apparatus 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. To visualize precise localization of RTK, we use a super-resolution confocal fluorescence confocal microscopy.

Research Activities

Mechanism of the retention of KIT tyrosine kinase in the Golgi/trans-Golgi network (TGN) in gastrointestinal stromal tumor (GIST)

 We search for a small molecule that releases KIT from the Golgi/TGN of GIST cells. For that 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 and underwent degradation, indicating that PKD activity is essential for KIT retention in the Golgi/TGN. Human PKD consists of three members, PKD1, PKD2, and PKD3. Our knockdown assay showed that only PKD2, but not PKD1/PKD3, plays a critical role in Golgi retention of KIT in GIST cells. Identification of mediators between KIT and PKD2, as well as of PKD2 effectors, is under way.

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 that are accumulated in the Golgi/TGN in blood cancers. 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.

Education

 We are teaching one graduate student and two undergraduate students.

Future Prospects

 Analysis of the physical association of KIT with PKD2 and identification of PKD2 effectors are under way. Because loss-of-function of PKD2 does not affect the localization and signaling of FLT3-ITD and FGFR3-Y373C, further studies are required to understand the mechanism of Golgi retention of individual oncogenic signaling molecules. This study suggests that understanding the mechanism of intracellular trafficking and the induction of degradation will help us establish a novel method for the suppression of cancer cell proliferation.

List of papers published in 2022

Journal

1. Teranishi R, Takahashi T, Obata Y, Nishida T, Ohkubo S, Kazuno H, Saito Y, Serada S, Fujimoto M, Kurokawa Y, Saito T, Yamamoto K, Yamashita K, Tanaka K, Makino T, Nakajima K, Hirota S, Naka T, Eguchi H, Doki Y. Combination of pimitespib (TAS-116) with sunitinib is an effective therapy for imatinib-resistant gastrointestinal stromal tumors. International journal of cancer, 152:2580-2593, 2023

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 GIST cells. SSRN, 2023