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Abstract 365: The impact of the RASSF1C-PIWIL1-piRNA pathway on DNA methylation

Introduction: Epigenomic regulation is becoming critically important in human cancers as epigenetically regulated genes can be used as biomarkers for diagnosis, prognosis, molecular classification of tumors, and predicting response to therapies. Hence, identification of epigenetic biomarkers for specific cancers is highly desirable for development of precision medicine tools. Recently we have identified a novel RASSF1C-PIWIL1-piRNA pathway that appears to promote lung cell proliferation and migration. PIWI-like proteins interact with piRNAs to form complexes that regulate gene expression at the transcriptional (epigenetic control) and translational (mRNA silencing) levels, leading to stimulation of cell stem renewal and proliferation. We have shown that RASSF1C modulates the PIWIL1-piRNA gene axis, suggesting the hypothesis that the RASSF1C-PIWI-piRNA pathway could potentially contribute to lung cancer stem cell development and progression, in part, through modulation of gene methylation of both oncogenic and tumor suppressor genes. Thus, we wanted to validate this hypothesis using a non-small cell lung cancer (NSCLC) cell model to determine if we can identify candidate genes targeted by the RASSF1C-PIWIL1-piRNA pathway through a gene methylation mechanism. Methods: We conducted a pilot study on the impact of over-expressing RASSF1C and knocking down RASSF1C and P1WIL1 expression on global gene DNA methylation in the NSCLC cell line H1299. DNA from cells over-expressing RASSF1C, RASSF1C-knockdown, PIWIL1-knockdwn, and control cells was used for the methylation study using the Reduced Representation Bisulfite Sequencing (RRBS) method. Results: Candidate Differentially Methylated Regions (DMR) were identified by comparing DNA methylation profiles of experimental and control cells. We found that over-expression of RASSF1C and knocking down RASSF1C and PIWIL1 modulated DNA methylation of genomic regions; and statistically significant candidate genes residing in hypo- and hyper-methylated regions in lung cancer cells were identified, including the Dual-specificity phosphatase 4 gene (DUSP4) that functions as a tumor suppressor. Conclusion: The RASSF1C-PIWI-piRNA pathway may play a critical role in epigenome regulation and genesis of lung cancer stem cells through modulation of key genes such as DUSP4. We found that RASSSF1C over-expression increases, while knock-down of RASSF1C or PIWIL1 decreases methylation/expression of the DUSP4 gene perhaps providing a potential mechanism through which the RASSF1C-PIWIL1-piRNA pathway could promote lung cancer stem cell development and progression. DUSP4 is an interesting target gene because knockdown of DUSP4 has recently been shown to enhance cancer stem cell formation. Citation Format: Yousef G. Amaar, Mark E. Reeves. The impact of the RASSF1C-PIWIL1-piRNA pathway on DNA methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 365.