子宮頸癌に対する癌幹細胞の機能解析と癌幹細胞を標的とする治療の基礎的研究

学位の種別: 課程博士審査委員会委員 : （主査）東京大学教授 秋下 雅弘, 東京大学准教授 瀧田 順子, 東京大学准教授 野村 幸世, 東京大学講師 平田 哲也, 東京大学講師 石原 聡一郎University of Tokyo(東京大学

The oncogene KRAS promotes cancer cell dissemination by stabilizing spheroid formation via the MEK pathway

Abstract Background Peritoneal dissemination is a critical prognostic factor in ovarian cancer. Although stabilized spheroid formation promotes cancer cell peritoneal dissemination in ovarian cancer, the associated oncogenes are unknown. In this study, we assessed the role of the KRAS oncogene in ovarian cancer cell dissemination, focusing on the stability of cells in spheroid condition, as well as the modulation of intracellular signaling following spheroid transformation. Methods We used ID8, a murine ovarian cancer cell line, and ID8-KRAS, an oncogenic KRAS (G12 V)-transduced ID8 cell line in this study. Spheroid-forming (3D) culture and cell proliferation assays were performed to evaluate the growth characteristics of these cells. cDNA microarray analysis was performed to identify genes involved in KRAS-associated signal transduction in floating condition. A MEK inhibitor was used to evaluate the effect on cancer peritoneal dissemination. Results Cell viability and proliferation in monolayer (2D) cultures did not differ between ID8 and ID8-KRAS cells. However, the proportions of viable and proliferating ID8-KRAS cells in 3D culture were approximately 2-fold and 5-fold higher than that of ID8, respectively. Spheroid-formation was increased in ID8-KRAS cells. Analysis of peritoneal floating cells obtained from mice intra-peritoneally injected with cancer cells revealed that the proportion of proliferating cancer cells was approximately 2-fold higher with ID8-KRAS than with ID8 cells. Comprehensive cDNA microarray analysis revealed that pathways related to cell proliferation, and cell cycle checkpoint and regulation were upregulated specifically in ID8-KRAS cells in 3D culture, and that some genes partially regulated by the MEK-ERK pathway were upregulated only in ID8-KRAS cells in 3D culture. Furthermore, a MEK inhibitor, trametinib, suppressed spheroid formation in 3D culture of ID8-KRAS cells, although trametinib did not affect 2D-culture cell proliferation. Finally, we demonstrated that trametinib dramatically improved the prognosis for mice with ID8-KRAS tumors in an in vivo mouse model. Conclusions Our data indicated that KRAS promoted ovarian cancer dissemination by stabilizing spheroid formation and that the MEK pathway is important for stabilized spheroid formation. Disruption of spheroid formation by a MEK inhibitor could be a therapeutic target for cancer peritoneal dissemination

Intracellular signaling entropy can be a biomarker for predicting the development of cervical intraepithelial neoplasia.

While the mortality rates for cervical cancer have been drastically reduced after the introduction of the Pap smear test, it still is one of the leading causes of death in women worldwide. Additionally, studies that appropriately evaluate the risk of developing cervical lesions are needed. Therefore, we investigated whether intracellular signaling entropy, which is measured with microarray data, could be useful for predicting the risks of developing cervical lesions. We used three datasets, GSE63514 (histology), GSE27678 (cytology) and GSE75132 (cytology, a prospective study). From the data in GSE63514, the entropy rate was significantly increased with disease progression (normal < cervical intraepithelial neoplasia, CIN < cancer) (Kruskal-Wallis test, p < 0.0001). From the data in GSE27678, similar results (normal < low-grade squamous intraepithelial lesions, LSILs < high-grade squamous intraepithelial lesions, HSILs ≤ cancer) were obtained (Kruskal-Wallis test, p < 0.001). From the data in GSE75132, the entropy rate tended to be higher in the HPV-persistent groups than the HPV-negative group. The group that was destined to progress to CIN 3 or higher had a tendency to have a higher entropy rate than the HPV16-positive without progression group. In conclusion, signaling entropy was suggested to be different for different lesion statuses and could be a useful biomarker for predicting the development of cervical intraepithelial neoplasia

Additional file 1: Figure S1. of Expression of Par3 polarity protein correlates with poor prognosis in ovarian cancer

Par3 is sufficiently knocked down at 24, 48, and 72 h. JHOC cells were transfected with siPar3 or control siRNA (siControl). Total cell extracts were then taken after 24, 48, and 72 h. Par3 and ι-Tubulin protein were detected by western blotting. (PPTX 139 kb

Measurement of the signaling entropy in GSE27678.

<p>(A) Boxplot of the entropy rate from whole samples. The entropy rate increased according to the disease progression (Kruskal-Wallis test, <i>p</i> < 0. 001). Among them, the entropy rate from the cell line was highest, as described in the literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176353#pone.0176353.ref009" target="_blank">9</a>]. (B) Entropy rate from each platform. ○ represents the data obtained from the Affymetrix Human Genome U133A 2.0 Array, and * represents the data obtained from the Affymetrix Human Genome U133 Plus 2.0 Array. The distribution patterns of the entropy rate seemed slightly different among the platforms.</p

Measurement of the signaling entropy in GSE75132.

<p>The median of the entropy rate was higher in the HPV16-persistent (HPV16+) groups than the HPV-negative (HPV-) group. The group that was destined to progress to CIN3+, or the HPV16-persistent with progression group, tended to have a higher entropy rate than the HPV16-persistent without progression group.</p

Measurement of the signaling entropy in GSE63514.

<p>The entropy rate was significantly higher according to the disease progression (Kruskal-Wallis test, <i>p</i> < 0.0001). Cancer; invasive cancer.</p

Modification of the Tumor Microenvironment in KRAS or c-MYC-Induced Ovarian Cancer-Associated Peritonitis

<div><p>The most common properties of oncogenes are cell proliferation and the prevention of apoptosis in malignant cells, which, as a consequence, induce tumor formation and dissemination. However, the effects of oncogenes on the tumor microenvironment (TME) have not yet been examined in detail. The accumulation of ascites accompanied by chronic inflammation and elevated concentrations of VEGF is a hallmark of the progression of ovarian cancer. We herein demonstrated the mechanisms by which oncogenes contribute to modulating the ovarian cancer microenvironment. c-MYC and KRAS were transduced into the mouse ovarian cancer cell line ID8. ID8, ID8-c-MYC, or ID8-KRAS cells were then injected into the peritoneal cavities of C57/BL6 mice and the production of ascites was assessed. ID8-c-MYC and ID8-KRAS both markedly accelerated ovarian cancer progression <i>in vivo</i>, whereas no significant differences were observed in proliferative activity <i>in vitro</i>. ID8-KRAS in particular induced the production of ascites, which accumulated between approximately two to three weeks after the injection, more rapidly than ID8 and ID8-c-MYC (between nine and ten weeks and between six and seven weeks, respectively). VEGF concentrations in ascites significantly increased in c-MYC-induced ovarian cancer, whereas the concentrations of inflammatory cytokines in ascites were significantly high in KRAS-induced ovarian cancer and were accompanied by an increased number of neutrophils in ascites. A cytokine array revealed that KRAS markedly induced the expression of granulocyte macrophage colony-stimulating factor (GM-CSF) in ID8 cells. These results suggest that oncogenes promote cancer progression by modulating the TME in favor of cancer progression.</p></div

Granulocyte Macrophage Colony-stimulating Factor (GM-CSF) expression.

<p>(A) ID8, ID8-c-MYC, and ID8-KRAS cells were plated on 12-well culture plates at a concentration of 2×10⁶ cells/ml and cultured for 48 h, and total RNA was extracted followed by revers transcription. GM-CSF mRNA levels were assessed using a quantitative reverse transcription polymerase chain reaction. The expression of GM-CSF was normalized using PPIA mRNA as the internal standard. Expression levels were calculated by the comparative Ct method using PPIA as an endogenous reference gene. Data are the mean ± standard error of the mean (SEM) of three independent experiments. Data were analyzed using the Student’s t-test (*P ≤ 0.05, **P ≤ 0.01). (B) ID8, ID8-c-MYC, and ID8-KRAS cells were plated on 12-well culture plates at a concentration of 2×10⁶ cells/ml and cultured for 48 h. The concentrations of GM-CSF protein levels were assessed using specific ELISA. Error bars represent the mean ± SEM. A statistical analysis was performed with the Student’s t-test (*P ≤ 0.05, **P ≤ 0.01, n.s. indicates not significant.).</p