Annual Report 2018
Division of Cancer Differentiation
Koji Okamoto, Daisuke Shiokawa, Yuki Obata, Hirokazu Ohata, Toshiaki Miyazaki, Kaoru Yamawaki, Hiroaki Sakai, Seiko Ogawa, Katsuya Yokoyama, Mayu Fukami, Masahiro Takahashi, Kohei Yamawaki
Introduction
The ability of cancer cells to metastasize to distant organs or to resist chemotherapy is essential for the refractory nature of cancers. Cancer stem cells (CSCs) are believed to be responsible for such capabilities of cancer cells. In order to understand the molecular basis of the refractory behavior of solid tumors, 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 them into immunocompromised mice. By integrating data from genomic and gene expression analyses and sensitivity data for chemotherapy compounds, we are in the process of establishing a comprehensive panel of the 3D culture cells. In addition, we use the established spheroids and xenograft tumors for singlecell gene expression analyses, drug-sensitivity experiments, and other research 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 the 3D culture from clinical specimens and their PDX models.
Research activities
1. OMICS analyses for cancer spheroids
Using the colon and ovarian cancerderived spheroids, we performed several OMICS analyses including RNA-seq, NCC Oncopanel, and metabolome analyses. In addition, we performed chemosensitivity analyses for ovarian cancer spheroids using more than one hundred chemotherapeutic compounds. Through integrative analyses of these data, we stratified the above-mentioned spheroids into several groups. This information will be instrumental for predicting efficacy of chemotherapeutic compounds.
2. 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. 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 can serve as a therapeutic target against CSCs. In particular, the elucidation of the detailed mechanism of mTORC1 activation in this specific subset of lysosomes will be instrumental for devising therapies that target tumorigenic populations of colon cancer in the future.
3. Understanding cancer heterogeneity and chemoresistance via single cell analyses of colon tumors
Recent studies have revealed that Lgr5 is a functional marker of cells with stem-like properties in both normal intestinal tissue and colon tumors and that the oncogenic activation of Lgr5-positive normal stem cells leads to colon cancer. Therefore, it is likely that colon cancer stem cells are derived from pools of Lgr5- positive cells. However, it is not clear whether Lgr5-positive stem cells are heterogeneous, and whether only a subset of these cells are capable of developing into tumors. We performed single-cell gene expression analyses of mouse colon epithelial stem cells and found that a subpopulation of tumor-specific Lgr5-positive stem cells were capable of forming tumors. These cells expressed a unique subset of Wnt target genes that were regulated by the long isoform of the transcription factor Tcf1. Expression of this long isoform was associated with nuclear accumulation of β-catenin and was induced during the early stages of colon tumorigenesis, as observed in mice and in human colorectal cancer tissue samples. Thus, the induction of the long isoform of Tcf1 preferentially activates a unique subset of Wnt target genes and is crucial for colon tumorigenesis.
4. 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), mis-localize to endosomes, lysosomes, or Golgi apparatus. Furthermore, imaging of phosphorylation by antibody suggests that these mutants are fully activated on intracellular compartments. Blockade of trafficking of mutant molecules to signal platforms may be a new strategy for inhibition of oncogenic signaling.
Education
Teaching students (one undergraduate students, three graduate students)
Future prospects
We intend to identify biological weaknesses of the refractory cancer 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 2018
Journal
1. Tominaga K, Minato H, Murayama T, Sasahara A, Nishimura T, Kiyokawa E, Kanauchi H, Shimizu S, Sato A, Nishioka K, Tsuji EI, Yano M, Ogawa T, Ishii H, Mori M, Akashi K, Okamoto K, Tanabe M, Tada KI, Tojo A, Gotoh N. Semaphorin signaling via MICAL3 induces symmetric cell division to expand breast cancer stem-like cells. Proc Natl Acad Sci USA, 116:625-630, 2019