Radiation therapy for abdominal tumors is challenging because the small intestine is exquisitely radiosensitive. Unfortunately, there are no FDA-approved therapies to prevent or mitigate GI radiotoxicity. The EGLN protein family are oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs). Our group has previously shown that stabilization of HIF2 through genetic deletion or pharmacologic inhibition of the EGLNs mitigates and protects against GI radiotoxicity in mice by improving intestinal crypt stem cell survival. Here we aimed to elucidate the molecular mechanisms by which HIF2 confers GI radioprotection. We developed duodenal organoids from mice, transiently overexpressed non-degradable HIF2, and performed bulk RNA sequencing. Interestingly, HIF2 upregulated known radiation modulators and genes involved in GI homeostasis, including Wnt5a. Non-canonical Wnt5a signaling has been shown by other groups to improve intestinal crypt regeneration in response to injury. Here we show that HIF2 drives Wnt5a expression in multiple duodenal organoid models. Luciferase reporter assays performed in human cells showed that HIF2 directly activates the WNT5A promoter via a hypoxia response element. We then evaluated crypt regeneration using spheroid formation assays. Duodenal organoids that were pre-treated with recombinant Wnt5a had a higher cryptogenic capacity after irradiation, compared to vehicle-treated organoids. Conversely, we found that Wnt5a knockout decreased the cryptogenic potential of intestinal stem cells following irradiation. Treatment with recombinant Wnt5a prior to irradiation rescued the cryptogenic capacity of Wnt5a knockout organoids, indicating that Wnt5a is necessary and sufficient for duodenal radioprotection. Taken together, our results suggest that HIF2 radioprotects the GI tract by inducing Wnt5a expression.

Breast cancer (BC) affects approximately 1 in 8 women and is the second leading cause of cancer among women in the United States. BC tumors frequently overexpress Epidermal Growth Factor Receptor (EGFR) which contributes to BC progression and poor clinical outcomes. Studies associate the endocytosis related protein, Secretory Carrier Membrane Protein 3 (SCAMP3), with the regulation of EGFR recycling and degradation. Recently, we published that SCAMP3 is overexpressed in BC cells and tumors. Others have identified SCAMP3 as an indicator of poor prognosis in hepatocellular carcinoma. Therefore, we hypothesize that SCAMP3 promotes the trafficking of EGFR, induces EGFR signaling and BC progression. To evaluate whether SCAMP3 has a direct interaction with EGFR, we treated SUM-149 (EGFR+) and MDA-MB-468 (EGFR+++) BC cells with 100ng/mL EGF at different time points, then we evaluated the localization of both proteins by immunofluorescence. Results showed that SCAMP3 co-localizes with EGFR at the cytoplasm at early stimulation times and the perinuclear area after 30 mins in both cell lines. To evaluate if SCAMP3 has a role in BC progression, we knocked down SCAMP3 in SUM-149 cells using the CRISPR/Cas9 technique, and we assessed the effects in cell viability, colony, and sphere formation capacity and EGFR signaling. Knockdown (KO) and wild type (WT) cells were treated with the ligand at the same concentration described above for 30 min. Our results showed that SCAMP3 knockdown decreased cell viability, colony number and the area of formed spheres. The activation of ERK1/2 was also reduced in SCAMP3 KO cells. Taken together, SCAMP3 is involved in the internalization of EGFR, cell viability, proliferation, and tumor sphere formation. We conclude that SCAMP3 has an essential role in EGFR trafficking and BC progression through EGFR downstream signaling modulation. Citation Format: Ariana C. Acevedo Diaz, Jael Reyes- Chea, Michelle M. Martinez-Montemayor, Ivette J. Suárez-Arroyo. The role of SCAMP3 in EGFR trafficking and breast cancer cell response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3678.