Conditional Inactivation of p53 in Mouse Ovarian Surface Epithelium Does Not Alter MIS Driven Smad2-Dominant Negative Epithelium-Lined Inclusion Cysts or Teratomas

Abstract

Epithelial ovarian cancer is the most lethal gynecological malignancy among US women. The etiology of this disease, although poorly understood, may involve the ovarian surface epithelium or the epithelium of the fallopian tube fimbriae as the progenitor cell. Disruptions in the transforming growth factor beta (TGFβ) pathway and p53 are frequently found in chemotherapy-resistant serous ovarian tumors. Transgenic mice expressing a dominant negative form of Smad2 (Smad2DN), a downstream transcription factor of the TGFβ signaling pathway, targeted to tissues of the reproductive tract were created on a FVB background. These mice developed epithelium-lined inclusion cysts, a potential precursor lesion to ovarian cancer, which morphologically resembled oviductal epithelium but exhibited protein expression more closely resembling the ovarian surface epithelium. An additional genetic “hit” of p53 deletion was predicted to result in ovarian tumors. Tissue specific deletion of p53 in the ovaries and oviducts alone was attempted through intrabursal or intraoviductal injection of Cre-recombinase expressing adenovirus (AdCreGFP) into p53 flox/flox mice. Ovarian bursal cysts were detected in some mice 6 months after intrabursal injection. No pathological abnormalities were detected in mice with intraoviductal injections, which may be related to decreased infectivity of the oviductal epithelium with adenovirus as compared to the ovarian surface epithelium. Bitransgenic mice, expressing both the Smad2DN transgene and p53 flox/flox, were then exposed to AdCreGFP in the bursa and oviductal lumen. These mice did not develop any additional phenotypes. Exposure to AdCreGFP is not an effective methodology for conditional deletion of floxed genes in oviductal epithelium and tissue specific promoters should be employed in future mouse models of the disease. In addition, a novel phenotype was observed in mice with high expression of the Smad2DN transgene as validated through qPCR analysis, characterized by teratoma-like lesions implicating Smad signaling in teratoma development.

Activin A stimulates migration of the fallopian tube epithelium, an origin of high-grade serous ovarian cancer, through non-canonical signaling

“…The finding that TGFβ1 increased phospho-Smad2/3 levels, but did not increase migration, suggested that the increased migration was Smad independent. Confirming this, a previously validated dominant negative Smad2 (Smad2DN) construct [37,38] did not abrogate the migratory effect of activin A (Figure 2A). The Smad2DN construct blocked phosphorylation of Smad2/3 (Figure 2B).…”

“…The Smad2-dominant negative (Smad2DN) construct was a gift from Teresa Woodruff at Northwestern University and has been previously validated [37,38]. Cells were transfected with 500 ng/ml Smad2DN plasmid or empty vector (pcDNA3) with ViaFect (E4981, Promega, Madison, WI) following the manufacturer’s instructions.…”

“…Immunohistochemical staining for phospho-Smad2/3 was carried out as previously described [38]. Reproductive tracts were fixed in 4% PFA, embedded in paraffin, and sectioned at 5 µm.…”

Mutant p53 expression in fallopian tube epithelium drives cell migration

“…10 million cells suspended in PBS were injected into the peritoneal cavity via IP injection. All attenuated adenovirus was used at a concentration of 10 10 plaque forming units (pfu)/ml (Iowa Gene Transfer Vector Core, Iowa City, IA) and in vivo administration was performed as previously described …”

Serum Inhibin A and Inhibin B Levels in Epithelial Ovarian Cancer Patients

“…It has been found that activin, via Smad2/3 receptors, activates PI3 Akt pathway, existing in epithelial ovarian carcinomas, and affects the overexpression of antiapoptotic genes [28], [29]. Complicated interactions among inhibin, activin, estrogens and NF-κB signal pathway have been proven [30].…”