Oncogenic signaling in the adult Drosophila prostate-like accessory gland leads to activation of a conserved pro-tumorigenic program, in the absence of proliferation

The Drosophila melanogaster male accessory gland (AG) is crucial for reproductive success, producing seminal fluid proteins that impact female post-mating behavior and physiology. To understand the regulatory networks governing accessory gland function, we conducted a bulk RNA sequencing (RNA-Seq) study to analyze the effects of overexpressing key regulatory genes. Specifically, we compared Gal4-induced Yki+Dp and Myc+E2F+Dp overexpression to controls not expressing these transgenes. We found upregulated and downregulated genes in each condition compared to controls, affecting immune response, cell cycle, stress response, and reproductive functions. In both Yki constitutively active, Dp and dMyc, E2F+DP conditions, several genes known to be involved in wound healing and tumorigenic models in flies were upregulated, including JNK signaling targets Ets21C, MMP1, puckered, and Jak/STAT signaling pathway ligands upd2 and upd3. Conversely, accessory gland-specific genes, such as many Accessory Gland Proteins (Acps), and genes involved in fertility, mating behavior, and sperm competition were downregulated. There was a strong overlap between the Yki constitutively active, Dp and dMyc, E2F+Dp gene expression signatures, converging on genes commonly misregulated in proliferative tumor models in various larval tissues, including imaginal discs and brains. This study provides insights into the molecular mechanisms by which key regulatory genes influence the function and integrity of the Drosophila male accessory gland. Overall design: We induced adult-specific transgene expression in the accessory gland (AG) using the Gal4-UAS and Flp-FRT recombinant systems. This system includes a Gal4 gene under the control of the actin promoter, with a stop cassette flanked by FRT sites upstream of the Gal4 coding sequence. The flippase enzyme is controlled by a heat-shock promoter. The flies were exposed to 37°C for 20 min on the day of eclosion, leading to flippase expression, to remove the stop cassette, allowing Gal4 to drive transgene expression. UAS transgenes used in this study include GFP-NLS, dMyc with E2F+Dp, a wild-type Yki, a constitutively active Yki with Dp, and a P35 apoptosis inhibitor. All samples, including the control sample, had UAS-P35 and GFP-NLS expression in the background. All samples were virgin males, kept at 25°C after transgene activation and dissected for RNA isolation 10 days of age. Each sample contained 80-100 dissected accessory glands with the ejaculatory duct and testis removed. Samples were visually inspected for GFP expression. Glands with no or low GFP expression were excluded from downstream analysis. We performed three independent replicates per sample. RNA was extracted from the accessory glands of adult males, followed by sequencing on the Illumina platform. The resulting data were analyzed to identify differentially expressed genes between the overexpression conditions and WT.