Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype

Abstract

Background: We previously demonstrated that ovarian high grade serous carcinomas (OHGSeCa) and ovarian clear cell carcinomas (OCCCa) with an HNF-1β+/p53+/ARID1A+ immunophenotype were associated with the worst unfavorable prognosis. To clarify the molecular mechanisms underlying this finding, we focused on alterations in the p53 signaling pathway in these tumors. Methods: Changes in cell phenotype and function following knockdown of wild-type p53 (p53-KD) were assessed using OCCCa cells expressing endogenous HNF-1β and ARID1A. The prognostic significance of molecules that were deregulated following p53-KD was also examined using 129 OCCCa/OHGSeCa cases. Results: p53-KD cells had increased expression of Snail, phospho-Akt (pAkt), and pGSK3β, and decreased E-cadherin expression, leading to epithelial-mesenchymal transition (EMT)/cancer stem cell (CSC) features. The cells also exhibited acceleration of cell motility and inhibition of cell proliferation and apoptosis. Next generation sequencing revealed that fibronectin (FN) expression was significantly increased in the p53 KD-cells, in line with our observation that wild-type p53 (but not mutant p53) repressed FN1 promoter activity. In addition, treatment of OCCCa cells with FN significantly increased cell migration capacity and decreased cell proliferation rate, independent of induction of EMT features. In clinical samples, FN/p53 scores were significantly higher in OCCCa/OHGSeCa with the HNF-1β+/p53+/ARID1A+ immunophenotype when compared to others. Moreover, high FN/high p53 expression was associated with the worst overall survival and progression-free survival in OCCCa/OHGSeCa patients. Conclusion: These findings suggest that upregulation of FN following loss of p53 function may impact the biological behavior of OCCCa/OHGSeCa, particularly in tumors with an HNF-1β+/p53+/ARID1A+ immunophenotype, through alterations in cell mobility and cell proliferation. The accompanying induction of EMT/ CSC properties and inhibition of apoptosis due to p53 abnormalities also contribute to the establishment and maintenance of tumor phenotypic characteristics.

Linking Oxidative Stress and DNA Damage to Changes in the Expression of Extracellular Matrix Components

“…These studies thus indicate p53 as a negative regulator of FN1 expression. In accordance with this notion, overexpression of wild type p53 in ovarian carcinoma cells was able to repress the activity of the FN1 promoter, while a mutant p53 failed to do so ( Yokoi et al, 2020 ), showing that p53 can act as a transcription factor directly regulating FN1 transcription. Another indication of an effect of DNA damage on the expression of ECM components came from a study on the CDK inhibitor p21 ( Yosef et al, 2017 ), a negative regulator of the cell cycle, which can be induced by p53 and drive cell cycle arrest ( Williams and Schumacher, 2016 ).…”

“…p53 has been implicated in the regulation of FN1 expression. Depletion of p53 was shown to increase FN1 mRNA expression, leading to increased cell motility and decreased apoptosis ( Yokoi et al, 2020 ; Figure 1C ), while treatment with the p53 activator RITA caused a decrease in fibronectin levels ( You et al, 2017 ). These studies thus indicate p53 as a negative regulator of FN1 expression.…”

PTEN overexpression and nuclear beta-catenin stabilization promote morular differentiation through induction of epithelial-mesenchymal transition and cancer stem cell-like properties in endometrial carcinoma

“…Transfection was carried out using LipofectAMINE PLUS (Invitrogen), in duplicate or triplicate, and luciferase activity was assayed as described previously [19][20][21].…”

“…IHC was performed using a combination of the microwave oven heating and polymer immunocomplex (Envision, Dako) methods as described previously [19][20][21]. For evaluation of IHC ndings, scoring of cytoplasmic, membranous, or nuclear immunoreactivities in morular, premorular, and the surrounding carcinoma (Sur Ca) components, respectively, was performed on the basis of the percentage of immunopositive cells and the immunointensity with multiplication of the values of the two parameters as described previously [19][20][21]. Nuclear Ki-67 immunopositivity was also counted in at least 200 cells from the three lesions, respectively, and the LIs were then calculated as a percentage.…”

“…Ampli cation by RT-PCR was carried out in the exponential phase to allow comparison among cDNA synthesized from identical reactions using speci c primers (Supplementary Table S1). Primers for the GAPDH gene were also used as described previously [19][20][21]. The intensity of individual signals was measured using ImageJ software version 1.41 (NIH, Bethesda, MD, USA).…”