cancers Article A Comprehensive Molecular and Clinical Analysis of the piRNA Pathway Genes in Ovarian Cancer Eunice Lee 1 , Noor A. Lokman 2,3 , Martin K. Oehler 2,3,4 , Carmela Ricciardelli 2, * ,† and Frank Grutzner 1, * ,†   Citation: Lee, E.; Lokman, N.A.; Oehler, M.K.; Ricciardelli, C.; Grutzner, F. A Comprehensive Molecular and Clinical Analysis of the piRNA Pathway Genes in Ovarian Cancer. Cancers 2021, 13, 4. https:// dx.doi.org/10.3390/cancers13010004 Received: 15 October 2020 Accepted: 18 December 2020 Published: 22 December 2020 Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2020 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). 1 Department of Molecular and Biomedical Sciences, Robinson Research Institute, University of Adelaide, Adelaide, SA 5000, Australia; eunicehsiuyee.lee@adelaide.edu.au 2 Discipline of Obstetrics and Gynaecology, Robinson Research Institute, Adelaide Medical School, University of Adelaide, Adelaide, SA 5000, Australia; noor.lokman@adelaide.edu.au (N.A.L.); martin.oehler@adelaide.edu.au (M.K.O.) 3 Future Industries Institute, University of South Australia, Mawson Lakes, SA 5095, Australia 4 Department of Gynaecological Oncology, Royal Adelaide Hospital, Adelaide, SA 5005, Australia * Correspondence: carmela.ricciardelli@adelaide.edu.au (C.R.); frank.grutzner@adelaide.edu.au (F.G.); Tel.: +61-8-8313-8255 (C.R.); +61-8-8313-4812 (F.G.) † Authors contributed equally to the work. Simple Summary: Although ovarian cancer (OC) is one of the most lethal gynecological cancers, its development and progression remain poorly understood. The piRNA pathway is important for transposon defense and genome stability. piRNA maturation and function involve a number of genes known as the piRNA pathway genes. These genes have recently been implicated in cancer development and progression but information about their role in OC is limited. Our work aimed to provide a better understanding of the roles of piRNA pathway genes in OC. Through analyzing changes in the abundance of 10 piRNA pathway genes, we discovered gene expression differences in benign vs. cancer, chemosensitive vs. chemoresistant and post hormone treatment in OC samples and cells. Furthermore, we observed the differential effects of these genes on patient survival and OC cell invasion. Overall, this work supports a role of the piRNA pathway genes in OC progression and encourages further study of their clinical relevance. Abstract: Ovarian cancer (OC) is one of the most lethal gynecological malignancies, yet molecular mechanisms underlying its origin and progression remain poorly understood. With increasing reports of piRNA pathway deregulation in various cancers, we aimed to better understand its role in OC through a comprehensive analysis of key genes: PIWIL1-4, DDX4, HENMT1, MAEL, PLD6, TDRD1,9 and mutants of PIWIL1 (P1Δ17) and PIWIL2 (PL2L60). High-throughput qRT-PCR (n = 45) and CSIOVDB (n = 3431) showed differential gene expression when comparing benign ovarian tumors, low grade OC and high grade serous OC (HGSOC). Significant correlation of disparate piRNA pathway gene expression levels with better progression free, post-progression free and overall survival suggests a complex role of this pathway in OC. We discovered PIWIL3 expression in chemosensitive but not chemoresistant primary HGSOC cells, providing a potential target against chemoresistant disease. As a first, we revealed that follicle stimulating hormone increased PIWIL2 expression in OV-90 cells. PIWIL1, P1Δ17, PIWIL2, PL2L60 and MAEL overexpression in vitro and in vivo decreased motility and invasion of OVCAR-3 and OV-90 cells. Interestingly, P1Δ17 and PL2L60, induced increased motility and invasion compared to PIWIL1 and PIWIL2. Our results in HGSOC highlight the intricate role piRNA pathway genes play in the development of malignant neoplasms. Keywords: ovarian cancer; piRNA pathway; patient survival; invasion; follicle stimulating hormone; chemoresistance; therapeutic targets Cancers 2021, 13, 4. https://dx.doi.org/10.3390/cancers13010004 https://www.mdpi.com/journal/cancers