SAT-353 The Synthetic Vitamin D Analog EB1089 Restricts Adrenocortical Tumor Proliferation in NCI-H295 Xenograft Model

Abstract

The vitamin D (VD) receptor (VDR) is underexpressed in adult and pediatric adrenocortical tumors (ACT), especially in carcinomas (ACC). VDR activation by VD represses B-catenin signaling in different types of tumors that like ACT, present aberrant B-catenin expression. EB1089 is a potent synthetic VD analog that presents lower calcemic effects and is well tolerated at therapeutic doses. Since there is no effective adjuvant target-therapy for patients with ACT, we evaluated the effects of EB1089 in adrenocortical tumor proliferation. Methods:In vitro, we treated NCI-H295 (B-catenin mutant/activated) and Y1 (normal B-catenin) ACC cells with EB1089 (10-7M) and evaluated VDR and B-catenin signaling (qPCR) and cell proliferation (MTS). In vivo, we bilaterally inoculated subcutaneously 10 female NGS mice (8 week-old) with NCI-H295 cells (2.5x106 / 100 mcL). We assessed body weight (BW) and tumor volume (TuV; d2D/2) weekly. Once the TuV reached 100 mm3, we randomized the animals for EB1089 (n=5; 0.8mcg/kg BW) or vehicle [Control group (CG) n=5; 80% propylene glycol-20% PBS] treatment (sc injection; 5 days/week during 5 weeks). At the end of the protocol, mice were sacrificed and each tumor excised, weighted and fixed for pathological evaluation (H&E). Blood samples were taken for serum calcium (colorimetry), cortisol and testosterone (RIA) assessment. Data were presented as percentage or mean ± SEM. Results: In NCI-H295 cells the treatment with EB1089 (48h) increased VDR mRNA expression (47%; p=0.01) and activated its target gene CYP24A1 (p<0.0001). B-catenin expression and signaling decreased, as shown by reduced CTNNB1 (-24%; p=0.001), MYC (-40%; p=0.0001), CCND1 (-38%; p=0.02), and DKK3 (-40%; p=0.02) mRNA expression. Cell proliferation reduced after 96h (-15%; p=0.001). None of these observations were present in Y1 cells. In vivo, EB1089 significantly restricted tumor growth starting after 2 weeks of treatment (TuV: 284 ± 52 vs. 156 ± 20 mm3; p=0.05). At the end of the experiment TuV was ~50% lower in EB1089 xenografts (683 ± 345 vs. 368 ± 211 mm3; p=0.03). TuV and tumor weight correlated positively (r=0.88; p<0.0001). Both, EB1089 and CG tumors presented highly proliferative areas (pleomorphism, mitosis and few apoptotic bodies). However, EB1089 xenografts presented large necrotic areas. BW (p=0.98) and calcium levels (9.96 ± 0.4 vs. 11.3 ± 0.3 mg/dL; p=0.06) did not differ between groups. EB1089 mice presented reduced serum testosterone (76.7 ± 11.6 vs. 39 ± 5.6 ng/dL; p=0.02) and undetectable cortisol levels (CG: 2 ± 1.2 mcg/dL). Conclusion: The activation of VDR signaling by EB1089 inhibited B-catenin activated ACT proliferation. Furthermore, in vivo, EB1089 reduced tumor steroid secretion and did not result in important VD side effects like significant hypercalcemia and weight loss. VD analogs have antitumor activity and may emerge as an adjuvant therapy for patients with ACT.