Modeling high-grade serous ovarian carcinogenesis from the fallopian tube

Abstract

High-grade serous ovarian carcinoma (HGSOC) is a lethal disease for which improved screening and treatment strategies are urgently needed. Progress in these areas is impeded by our poor understanding of HGSOC pathogenesis. Most ovarian cancer research is based on the hypothesis that HGSOC arises from ovarian surface epithelial cells. However, recent studies suggest that >50% of high-grade serous carcinomas involving the ovary likely arise from fallopian tube epithelium. Therefore, limiting HGSOC research to modeling based on ovarian surface epithelium alone is inadequate. To address the need for a fallopian tube-based model of HGSOC, we have developed a system for studying human fallopian tube secretory epithelial cell (FTSEC) transformation. Our model is based on (i) immortalization of FTSECs isolated from primary samples of normal, nondiseased human fallopian tubes, (ii) transformation of FTSECs with defined genetic elements, and (iii) xenograft-based tumorigenic assays. We use our model to show that FTSECs immortalized with human telomerase reverse transcriptase (hTERT) plus SV40 large T and small T antigens are transformed by either oncogenic Ras (H-Ras(V12)) or c-Myc expression, leading to increased proliferation, clonogenicity, and anchorage-independent growth. Additionally, we demonstrate that FTSECs remain susceptible to c-Myc-mediated transformation in the absence of viral oncoproteins, by replacing SV40 large T and small T antigens with sh-p53, mutant CDK4 (CDK4(R24C)), and sh-PP2A-B56γ. Importantly, all transformed FTSECs gave rise to high-grade Müllerian carcinomas that were grossly, histologically, immunophenotypically, and genomically similar to human HGSOC. With this model, we will now be able to assess the transformative effects of specific genetic alterations on FTSECs in order to characterize their respective roles in HGSOC development.

Fig. 1.

Isolation and immortalization of primary human FTSECs. (A) Dissociated fallopian tube epithelial cells proliferate rapidly in culture (magnification: 40×). (B) Proliferative cells express secretory (Pax8) but not ciliated (FoxJ1) lineage markers, identifying them as FTSECs (magnification: 400×). (C) FT33 cell morphology before (Left) and after (Right) immortalization with SV40 TAg (magnification: 40×).

Fig. 2.

Transformation of SV40 TAg-immortalized FTSECs by H-Ras^V12 or c-Myc. (A) Immortal FT33-TAg cells transduced with H-Ras^V12 or c-Myc (Left; magnification: 40×) were analyzed by Western blotting (Right) to validate ectopic gene expression (Ras and Myc) and the retention of lineage markers (Pax8 and CK-7). (B–D) Expression of H-Ras^V12 or c-Myc dramatically accelerated FT33-TAg cell proliferation (B), enhanced colony formation (C), and enabled anchorage-independent growth in soft agar (D). The ovarian carcinoma cell line OVCAR-8 was used as a positive control.

Fig. 3.

H-Ras^V12 and c-Myc–transformed FTSECs produce high-grade serous tumors. (A) i.p. injection of either FT33-TAg-Ras or FT33-TAg-Myc cells produced widespread tumor formation in immunodeficient mice. (B) Both cell lines gave rise to high-grade Müllerian carcinomas, determined by H&E staining (magnification: 200×). (C) Tumors were analyzed by immunohistochemistry to validate ectopic and lineage-specific gene expression (magnification: 600×).

Fig. 4.

FTSECs can be immortalized and transformed in the absence of viral oncogenes. (A) Immortal FT33-shp53-R24C cells transduced with sh-PP2A-B56γ and c-Myc (Left; magnification: 40×) were analyzed by Western blotting (Right) to confirm expression of exogenous genetic alterations (p53, CDK4, and Myc) and endogenous lineage markers (Pax8 and CK-7). (B–D) Transduction of FT33-shp53-R24C cells with sh-PP2A-B56γ and c-Myc dramatically accelerated cell proliferation (B), increased colony formation (C), and enabled anchorage-independent growth (D). OVCAR-8 was used as a positive control.

Fig. 5.

FTSECs transformed with nonviral genetic alterations form high-grade serous carcinomas. (A) i.p. injection of FT33-shp53-R24C-shPP2A-Myc led to extensive tumor formation in immunodeficient mice. (B) Tumors were high-grade with epithelial features (Left and Center) and histologically consistent with HGSOC (Right) as determined by H&E staining (magnification: 200×). (C) Expression of ectopic and lineage-specific gene expression in tumor tissue was validated by immunohistochemistry analysis (magnification: 600×). (D) Tumors exhibited severe genomic instability as indicated by changes in DNA copy number. The y axis represents gain (positive) or loss (negative) of copy number along the length of each chromosome (shown on the x axis) as determined by aCGH analysis. Additional data are given in Fig. S10.