Reconstitution of high-grade serous ovarian carcinoma from primary fallopian tube secretory epithelial cells

Abstract

Fallopian tube secretory epithelial cells (FTSECs) have been suggested to be the source of high-grade serous ovarian carcinoma (HGSOC). Although several genetic alterations are known to be involved in HGSOC development, the minimal requirements remain unclear. We aimed to identify oncogenic mutations indispensable for HGSOC development in a stepwise model, using immortalized FTSECs. FTSECs were isolated from clinical samples and immortalized by overexpression of cyclin D1, CDK4^R24C, and hTERT. Oncogenic mutations in the p53, c-Myc, and RAS/PI3K pathways were mimicked by lentiviral transduction. We found two distinct patterns of gene alteration essential for HGSOC development: p53/KRAS/AKT and p53/KRAS/c-Myc. Dominant-negative p53, alone or combined with oncogenic KRAS (KRASV12), constitutively active AKT (CA-AKT), and c-Myc, did not induce tumorigenesis in immortalized cells; however, overexpression of CA-AKT or c-Myc, along with dominant-negative p53 and KRASV12, conferred tumorigenic potential. Transformed FTSECs formed tumors in nude mice that were grossly, histologically, and immunohistochemically similar to human HGSOCs. Interestingly, mice harboring tumors with c-Myc amplifications displayed extensive metastases, consistent with the increased dissemination in their human counterparts. Thus, aberrant p53/KRASV12/c-Myc or p53/KRASV12/PI3K-AKT signaling was the minimum requirement for FTSEC carcinogenesis. The model based on this evidence could shed light on the early stages of HGSOC development.

Keywords: RAS/PI3K pathway; c-Myc; carcinogenesis; fallopian tube; high-grade serous ovarian carcinoma.

Figure 1. Fallopian tube secretory epithelial cell (FTSEC) isolation and characterization

(A) Tissue acquisition and purification, including dissection (upper-left panel), culturing in DMEM with 5% FBS and 1% PenStrep (upper-right panel), epithelial cell disruption (lower-right panel), and acquisition of adherent secretory cells (lower-left panel). (B) Western blot (left panel) and immunocytochemical (right panel) analyses of Pan-cytokeratin expression in immortalized FTSECs. (C) Western blot (left panel) and immunocytochemical (right panel) analyses of PAX8 expression in immortalized FTSECs. (D) Immunocytochemical analysis of Bcl2 (left panel) and FOXJ1 (right panel) expression in immortalized FTSECs.

Figure 2. Mutation, amplification, and pathway activation status of tumor samples from patients with high-grade serous ovarian carcinoma (HGSOC)

(A) Status of P53, PTEN, PIK3CA, KRAS, and BRAF mutations; PIK3CA, c-Myc, and KRAS amplification; and RAS/ERK and PI3K/AKT pathway activation in HGSOC tumors. (B) Analysis of p53 mutation, c-Myc amplification, and activation of the RAS-ERK and PI3/AKT pathways in 34 patients with HGSOC.

Figure 3. Anchorage-independent growth in soft agar and analysis of transformed phenotypes in mouse xenografts

(A) Growth of (1) immortalized FTSECs; immortalized FTSECs expressing (2) DN-p53, (3) DN-p53/KRAS^V12, (4) DN-p53/c-Myc, (5) DN-p53/Myr-AKT, (6) DN-p53/KRAS^V12/c-Myc, (7) and DN-p53/KRAS^V12/Myr-AKT; and (8) SKOV3 serous ovarian carcinoma cells (positive control) was evaluated in soft agar (left panel), and representative colony images (DN-p53/KRAS^V12: right-upper panel, DN-p53/KRAS^V12/c-Myc: right-middle panel, DN-p53/KRAS^V12/Myr-AKT: right-lower panel). (B) Tumor formation assay using fallopian tube secretory epithelial cells (FTSECs) transfected with different genetic elements. SKOV3 ovarian carcinoma cells were injected as a positive control (right panel). Subcutaneous injection of cells with DN-p53/KRAS^V12/c-Myc or DN-p53/KRAS^V12/Myr-AKT expression resulted in tumor formation in nude mice (left panel). (C) Hematoxylin and eosin (HE) staining and PAX8 immunostaining of tumors formed from FTSECs expressing DN-p53/KRAS^V12/c-Myc (left panels) or DN-p53/KRAS^V12/Myr-AKT (middle panels) or from immortalized ovarian surface epithelial cells transfected with DN-p53, KRAS^V12, c-Myc, and BCL2 (right panels).

Figure 4. Association between c-Myc amplification and metastasis in high-grade serous ovarian carcinoma (HGSOC)

(A) Tumors formed in nude mice after intraperitoneal injection of fallopian tube secretory epithelial cells (FTSECs) expressing DN-p53/KRAS^V12/c-Myc (left panel) or DN-p53/KRAS^V12/Myr-AKT (right panel). (B) Correlation between c-Myc amplification and degree of dissemination in patients with HGSOC. (C) Migration of FTSECs expressing DN-p53/KRAS^V12/c-Myc and DN-p53/KRAS^V12/Myr-AKT in wound healing assays (0 h, left panels; 24 h, right panels). (D) Matrigel invasion assay analysis of FTSECs expressing DN-p53/KRAS^V12/c-Myc (upper panel) or DN-p53/KRAS^V12/Myr-AKT (lower panel). Error bars indicate standard deviation.