Primed nephron progenitor cells (NPCs) appear in metanephric mesenchyme by Ell.5 and differentiate in response to the inductive WNT9b signal from the ureteric bud. However, the NPC WNT-receptor complex is unknown. We obtained M15 cells from E10.5 mesonephric mesenchyme and systematically analyzed components required for canonical WNT9b-responsiveness. When M15 cells were transfected with a (β-catenin luciferase reporter plasmid, exposure to recombinant WNT9b resulted in minimal luciferase activity. We then analyzed mRNA-expression of WNT-pathway components and identified Fzdl-6 and Lrp6 transcripts but not RSPO1. When M15 cells were treated with recombinant RSPO1 the response to transfected WNT9b was augmented 4.8-fold. Co-transfection of M15 cells with Fzd5 (but no other Fzd family member) further increased the WNT9b signal to 16.8-fold and siRNA knockdown of Fzd5 reduced the signal by 52%. Knockdown of Lrp6 resulted in 60% WNT9b signal reduction. We confirmed Fzd5, Lrp6 and RSPO1 rrtRNA expression in CITED1(+) NPCs from E15.5 embryonic mouse kidney. Thus, while many WNT signaling-pathway components are present by E10.5, optimum responsiveness of Ell.5 cap mesenchyme requires that NPCs acquire RSPO1, FZD5 and LRP6.Summary StatementResponsiveness to the inductive WMT9b signal from ureteric bud is crucial for nephrogenesis. Here we analyze the molecules needed to prime nephron progenitor cells in embryonic mouse kidney.

Primed nephron progenitor cells (NPCs) appear in metanephric mesenchyme by E11.5 and differentiate in response to the inductive WNT9b signal from the ureteric bud. However, the NPC WNT-receptor complex is unknown. We obtained M15 cells from E10.5 mesonephric mesenchyme and systematically analyzed components required for canonical WNT9b-responsiveness. When M15 cells were transfected with a β-catenin luciferase reporter plasmid, exposure to recombinant WNT9b resulted in minimal luciferase activity. We then analyzed mRNA-expression of WNT-pathway components and identified Fzd1-6 and Lrp6 transcripts but not Rspo1 . When M15 cells were treated with recombinant RSPO1 the response to transfected WNT9b was augmented 4.8-fold. Co-transfection of M15 cells with Fzd5 (but no other Fzd family member) further increased the WNT9b signal to 16.8-fold and siRNA knockdown of Fzd5 reduced the signal by 52%. Knockdown of Lrp6 resulted in 60% WNT9b signal reduction. We confirmed Fzd5 , Lrp6 and Rspo1 mRNA expression in CITED1(+) NPCs from E15.5 embryonic mouse kidney. Thus, while many WNT signaling-pathway components are present by E10.5, optimum responsiveness of E11.5 cap mesenchyme requires that NPCs acquire RSPO1, FZD5 and LRP6.

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Background: Cancer cells release higher levels of cell-free DNA (cfDNA) into the circulatory system than normal cells. However, the molecular mechanisms underlying the process of programmed DNA elimination remain poorly understood. Emerging evidence has indicated that oncogenes such as HRAS can be released through extracellular vesicles (EVs). Moreover, studies have shown an active mechanism of DNA emission through autophagy. Autophagy is an evolutionarily conserved process that degrades and recycles cytoplasmic materials such as organelles and proteins in the lysosomes. Interestingly, Microtubule-associated protein 1 light chain-3 (LC3B) is known to degrade nuclear lamina components in HRAS-induced cells, potentially producing a pool of cytoplasmic chromatin. Cytoplasmic chromatin is ultimately degraded in lysosomes or re-routed outside of the cells. The aim of our study was to evaluate the role of autophagy proteins in circulating tumor (ctDNA) and EV-associated DNA emission in cancer cell lines harboring oncogenes (RAS and BRAF) as this aspect of the process is relatively understudied. Methods: We have assessed protein and RNA expression of autophagy genes (LC3B, ATG5 and ATG7) in IEC-18 cells that have been transduced with the HRAS oncogene (RAS3 cells) and human colorectal cancer cell lines SW620 and HCT116 using RT-qPCR and western blot (WB). All conditions were compared to their respective control cells (IEC-18, CCD-841 CoN and CCD18-Co cells. To determine the role of LC3 in nuclear membrane degradation, we employed transmission electron microscopy (TEM). We have further validated the role of autophagy genes in EV-DNA emission by shRNA knockdown using shRNA against highly expressed autophagy genes, such as LC3B in RAS3 cells. EV-DNA was measured using droplet digital PCR (ddPCR). Results: To determine whether autophagy expels oncogenic DNA via EVs in KRAS-mutant CRC cells, we focused on the role of ATG5, LC3B and ATG7, as this aspect of the process is understudied. Our data shows higher LC3B and ATG7 expression in transformed RAS3 cells, compared to control cell lines (IEC18). Moreover, ATG5 is overexpressed in SW620 and HT29 colorectal cancer cells compared to control cell lines. LC3 is known to degrade the nuclear membrane in HRAS-activated cells. Indeed, we observed degradation of the nuclear membrane in RAS3 cells leading to the accumulation of cytosolic DNA shown by TEM. Furthermore, overexpression of LC3B in IEC18 cells using GFP-tagged LC3B resulted in increased EV emission. These findings suggest that the autophagy axis may contribute to oncogenic DNA emission via EVs. Lastly, shRNA knockdown of LC3 resulted in reduction of EV-DNA emission (ddPCR). Impact: EV biogenesis and autophagy pathways play pivotal and interconnected roles during DNA processing and extracellular emission. Therefore, understanding the detailed mechanisms is critical for biomarker discovery and potential therapeutic strategies targeting this process. Citation Format: Thupten Tsering, Kyle Dickinson, Laura Montermini, Janusz Rak, Julia Burnier. Oncogenic regulation of autophagy and DNA emission [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1385.

The emergence of nephron progenitor cells (NPCs) in early embryonic life leads to the many rounds of nephrogenesis that result in a richly endowed kidney by the end of gestation. A delicate balance between NPC differentiation and self-renewal must be maintained to guarantee optimal nephron endowment. Genetic errors which disturb NPC cell fate can result in premature NPC depletion and renal hypoplasia/dysplasia or permit the B-catenin mutations that accompany malignant transformation into a Wilms tumor. Retention of a small population of NPCs scattered throughout the adult kidney are important for recovery from acute tubular injury later in life. In this review, we cover the role of NPCs in both mammalian kidney development and disease.