Abstract 2568: Investigation of Fanconi Anemia pathway downstream genes

Abstract

Introduction: A key response mechanism to DNA damage is the Fanconi Anemia repair pathway (FA), which involves homologous recombination DNA repair and is activated through mono- ubiquitination of FANCD2. FA deficiency is considered to increase the sensitivity of tumors to particular DNA-targeted agents, and may prove to be a target of cancer treatment. We hypothesize that FA deficient tumors have a low growth rate and reduced ability for DNA repair compared to FA functioning tumors. Given that genetic modifications can interfere with FA functionality, we aim to explore the association between the FA pathways and downstream genes that influence tumor growth. To date, few studies have examined gene expression associated with FA deficiency in lung cancer cells. Identification of the FA downstream genes may provide insight on DNA repair networks that impact cancer treatment. Methods: To generate FANCD2 knockdown cells, human lung cancer cell lines A549 and H1299 were transduced with FANCD2-specific short hairpin RNA expressing and puromycin-resistant lentiviral particles or control shRNA lentiviral particles. The cells were cultured in growth medium, and successful FANCD2 knockdown was confirmed by western immunoblot analysis. RNA deep sequencing was completed with Illumina RNA-Seq. We compared gene expression between knockdown FANCD2 and control samples across three cell lines and ranked significant gene expression changes, defined as a five-fold change in upregulation or downregulation. The fold change was calculated by dividing FANCD2 deficient expression by FANCD2 efficient expression. Results and discussion: 13436 genes were evaluated across three cell lines and 17 genes demonstrated gene expression change by at least 5-fold with FANCD2 knockdown in three cell lines. FANCD2 knockdown resulted in 14 downregulated genes and 3 upregulated genes. The downregulated genes RP11-618G20.1, RP5-1021I20.4, RP11-219A15.1, XXbac-BPG32J3.20, and BMS1P17 demonstrated significant expression change across three cell lines. Of the 14 downregulated genes, 13 genes had literature supporting oncogenic function. FA downstream genes confers oncogenic function. As FANCD2 is considered to promote cell proliferation, downregulation of oncogenic genes expression was expected with FANCD2 knockdown. However, the literature suggested that the 3 upregulated genes with FANCD2 knockdown also have oncogenic function. These genes may have functioning beyond the scope of carcinogenesis which may explain gene upregulation with FANCD2 knockdown. Pinpointing genes related to FA pathway deficiency may provide insight into genetic phenomena that drive cancer. Our results provide a starting point for developing targets to specific downstream genes associated with FA deficient tumors, which may prove to limit cancer progression. Further investigation is needed to determine how FANCD2 interacts with these genes to promote cell proliferation. Citation Format: Bianca Nguyen, Li Gao, Abeer Almiman, Shirley Tang, Kathleen Dotts, Miguel A. Villalona-Calero, Wenrui Duan. Investigation of Fanconi Anemia pathway downstream genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2568.

Fanconi anemia pathway as a prospective target for cancer intervention

“…FANCC regulates nuclear translocation of β-catenin and works as a transcriptional repressor of β-catenin downstream gene DKK1 [102]. Besides these case studies, An RNA-seq study revealed a collection of genes with altered expression among which a large portion are implicated in oncogenic processes [103]. Further studies are needed to explore the potential transcriptional regulation role of FA proteins especially in cancer contexts where FA gene expression is found to be high.…”