Annual Report 2019

Division of Cancer Differentiation

Koji Okamoto, Daisuke Shiokawa, Yuki Obata, Hirokazu Ohata, Toshiaki Miyazaki, Yutaro Mori, Hiroaki Sakai, Seiko Ogawa, Ayumi Yumura, Kohei Yamawaki, Mariko Niwa

Introduction

 Many clinical cancers show a variety of intertumoral and intratumoral heterogeneity. Such heterogeneity stems from the epigenetic hierarchy of cancer differentiation as well as genetic diversity as a result of the Darwinian evolution of cancer. In fact, the refractory nature of cancers, such as chemoresistance or the ability for metastasis, is profoundly associated with the heterogeneity of cancers, in which a subpopulation of cancer survives or metastasizes and leads to a fatal outcome. Our lab has focused on cancer heterogeneity that is associated with epigenetic diversity. In order to understand the molecular basis of such cancer heterogeneity, we are focusing on the in vitro 3D culture system that is established from clinical specimens of several solid tumors including colon, ovarian, and lung cancer. The established spheroid cells are then used for generation of xenograft tumors after transplanting into immunocompromised mice. In many cases, the generated xenografted tumors show cellular heterogeneity that is pathologically indistinguishable from the original tumors. By performing single-cell analyses using the xenograft models or clinical specimens, we are in the process of establishing a clear picture of cancer heterogeneity. In addition, we use the established spheroids and xenograft tumors for drug-sensitivity experiments, and other studies to understand the mechanism of chemoresistance.

The Team and What We Do

 We mainly perform basic research to understand the refractory nature of cancer and to devise the effective diagnosis and therapy. We specialize in deriving 3D cultures from clinical specimens and their PDX models and mainly perform single-cell analyses to investigate cellular heterogeneity of cancers.

Research activities

1. Understanding cancer heterogeneity and chemoresistance via single-cell analyses of xenografted colon tumors

 We performed single-cell gene expression analyses of xenografted tumors of human colon cancer spheroids. The xenografted tumors harbor Lgr5-positive cancer stem-like cells. The identified Lgr5-positive cells were classified into the slow-cycling and fast-cycling populations, and we demonstrated that the slow-cycling population showed enhanced chemoresistance. Further, we found that the identified slow-cycling CSC-like cells specifically expressed the signature genes including PROX1, a transcription factor. It is likely that the identified slow-cycling CSC-like cells will be a promising target for novel cancer chemotherapy in combination with conventional chemotherapeutic agents.

2. Understanding cancer heterogeneity and chemoresistance via single-cell nuclear analyses of clinical specimens of clear cell carcinoma

 Clear cell carcinoma is regarded as the deadliest type of ovarian cancer. Combined with a high frequency in Japan and lack of effective therapy for advance cases, devising an effective therapy against clear cell carcinoma is one of the unmet medical needs in Japan. In order to identify cell populations that are associated with chemoresistance of clear cell carcinoma, we performed single-cell nuclear RNA-seq analyses of frozen specimens from resistant and sensitive cases of this cancer. Through comparison of resistant and sensitive cases, we delineated a cell population that is associated with chemoresistance. We are in the process of identifying crucial genes that will be specifically expressed in the chemoresistant population and serve as therapeutic targets.

3. Delineation of essential pathways of colon cancer spheroid proliferation

 Using CSC-enriched colon spheroid cells, we found that elevated levels of NOX1, an NADPH oxidase, resulted in mTORC1 activation, which in turn facilitated the proliferation of spheroid cells, which may represent fast-cycling CSCs. The specific expression of NOX1 in CSC-like cells was verified in mouse xenograft models, where we observed the preferential expression of NOX1 in LGR5-positive cells. In addition, examination of tumor and non-tumor-derived organoids that were obtained from mouse carcinogenesis models indicated that colon tumorigenesis led to an acquired dependence on the activated NOX1-mTORC1 pathway for tumor growth. Further biological and biochemical analyses of the human spheroids revealed that NOX1 colocalized with mTORC1 in a VPS41-positive lysosome subset, in which NOX1-derived ROS mediates mTORC1 activation via oxidation of S100A9, a calcium-binding protein. We further showed that mTORC1 activation was dependent on calcium, thus outlining the molecular pathway by which NOX1-derived ROS and calcium signals converge on S100A9 to mediate proliferation of colon CSCs. Thus, NOX1-dependent mTORC1 activation may serve as a therapeutic target against fast-cycling CSCs.

4. OMICS analyses for cancer spheroids

 Using the colon and ovarian cancer-derived spheroids, we performed several OMICS analyses including RNA-seq, NCC Oncopanel, and metabolome analyses. In addition, we performed chemosensivity analyses for ovarian cancer spheroids using more than hundred chemotherapeutic compounds. Through integrative analyses of these data, we stratified these spheroids into several groups. This information will be instrumental for predicting efficacy of chemotherapeutic compounds.

5. Mislocalization of cancer-causing mutant molecules and organelle signaling

 We found by immunofluorescence assay that cancer-causing mutants, such as EGFR(Δex19), KIT(D816V), and K-RAS(G13D), mislocalize to endosomes, lysosomes, or the Golgi apparatus. Recently, we further reported that a mutant KIT in acute myeloid leukemia is autophosphorylated preferentially on the Golgi apparatus, where it can activate downstream molecules. Establishment of a new strategy of inhibition of oncogenic signals through blocking intracellular trafficking is currently underway.

Education

 Teaching students (two graduate students)

Future prospects

 We intend to identify biological weakness of refractory cancer by single-cell analyses and to translate the acquired knowledge into clinical purposes by targeting biological weaknesses of these cells to eradicate refractory cancer.

List of papers published in 2019

Journal

1. Namekawa T, Ikeda K, Horie-Inoue K, Suzuki T, Okamoto K, Ichikawa T, Yano A, Kawakami S, Inoue S. ALDH1A1 in patient-derived bladder cancer spheroids activates retinoic acid signaling leading to TUBB3 overexpression and tumor progression. Int J Cancer, 146:1099-1113, 2020

2. Shiba S, Ikeda K, Suzuki T, Shintani D, Okamoto K, Horie-Inoue K, Hasegawa K, Inoue S. Hormonal Regulation of Patient-Derived Endometrial Cancer Stem-like Cells Generated by Three-Dimensional Culture. Endocrinology, 160:1895-1906, 2019

3. Ohata H, Shiokawa D, Obata Y, Sato A, Sakai H, Fukami M, Hara W, Taniguchi H, Ono M, Nakagama H, Okamoto K. NOX1-Dependent mTORC1 Activation via S100A9 Oxidation in Cancer Stem-like Cells Leads to Colon Cancer Progression. Cell Rep, 28:1282-1295.e8, 2019

4. Obata Y, Hara Y, Shiina I, Murata T, Tasaki Y, Suzuki K, Ito K, Tsugawa S, Yamawaki K, Takahashi T, Okamoto K, Nishida T, Abe R. N822K- or V560G-mutated KIT activation preferentially occurs in lipid rafts of the Golgi apparatus in leukemia cells. Cell Commun Signal, 17:114, 2019

5. Mori Y, Yamawaki K, Ishiguro T, Yoshihara K, Ueda H, Sato A, Ohata H, Yoshida Y, Minamino T, Okamoto K, Enomoto T. ALDH-Dependent Glycolytic Activation Mediates Stemness and Paclitaxel Resistance in Patient-Derived Spheroid Models of Uterine Endometrial Cancer. Stem Cell Reports, 13:730-746, 2019

6. Spaety ME, Gries A, Badie A, Venkatasamy A, Romain B, Orvain C, Yanagihara K, Okamoto K, Jung AC, Mellitzer G, Pfeffer S, Gaiddon C. HDAC4 Levels Control Sensibility toward Cisplatin in Gastric Cancer via the p53-p73/BIK Pathway. Cancers (Basel), 11:1747; doi:10.3390/cancers11111747, 2019