Effect of Trp53 and Rb1 knockout and Myc overexpression on fallopian tube epithelial organoids
Refractory high-grade serous carcinoma (HGSC) is one of the most severe clinical problems in gynecology, but we have barely grasped the underlying mechanism of aggressiveness and chemo-resistance. We performed gene set variation analysis and comprehensive log-rank tests on TCGA advanced serous ovarian cancer datasets. As a result, regulation of small GTPase signaling prominently correlated with poor prognosis uniquely in BRCA1/2-unmutated HGSC patients, and eventually, pathway clustering analysis discovered aberrant RAS/PI3K crosstalk as peculiar poor prognostic signature of BRCA1/2-unmutated HGSC patients. To elucidate the mechanism of poor progression triggered by RAS/PI3K, we utilized our syngeneic HGSC organoid models newly established from murine fallopian tube epithelium by making Myc overexpressing, and knocking out Trp53 and Rb1. After that, we knocked out Nf1 and Pten to induce aberrant RAS/PI3K crosstalk in the primitive HGSC model. In-vivo evaluation and growth comparison indicated that aberrant RAS/PI3K crosstalk transformed Brca1/2-unmutated HGSC modeling cells into aggressive phenotypes. Chemo-sensitivity assay also showed that the genetic manipulation made Brca1/2-unmutated HGSC modeling cells more tolerant to anti-tumor agents. To investigate biological processes that induce the aggressive and chemo-resistant phenotype, we performed gene set enrichment analysis among our HGSC models. The result showed that aberrant RAS/PI3K crosstalk induces cell cycle acceleration and downregulation of apoptosis in the HGSC cells, which backed up aggressive phenotype and chemo-resistance in our HGSC models. In conclusion, aberrant RAS/PI3K crosstalk transforms Brca1/2-unmutated HGSC modeling cells more aggressive and chemo-resistant, which was well recapitulated by our HGSC modeling organoids. The integration of our computational approach and experimental models will lead us to detect peculiar therapeutic targets against refractory HGSC. Overall design: To investigate the biological changes caused by TRP53 and RB1 knockout and MYC overexpression in fallopian tube epithelium, we established murine fallopian tube epithelial organoids and performed a transcriptomic analysis. Genotypes were as follows; RPM: Rb1-/-Trp53-/-MycOE, RPMN: Rb1-/-Trp53-/-MycOE + Nf1KO, RPMP: Rb1-/-Trp53-/-MycOE + PtenKO, RPMNP: Rb1-/-Trp53-/-MycOE + Nf1KO + PtenKO. A control sample was normal fallopian tube epithelial organoids established from an Rb1fl/flTrp53fl/flMycLSL/LSL mouse.