Influence of REST Deficiency on Obesity-Related Endometrial cancer and Chromogranin A Expression in Mice

Obesity increases the risk of endometrial cancer (EC). It was previously demonstrated that the transcription regulator, REST, may be a putative tumor suppressor in EC. Chromogranin A (CHGA) is elevated in various cancers, including neuroendocrine tumors. Interestingly, REST knockdown has been shown to upregulate CHGA in breast cancer and may play a role in carcinogenesis. The objective of this study was to determine if Rest deficiency augments obesity related EC in mice and if Chga is altered in this pathway. To determine the impact of diet-induced obesity and uterine Rest deficiency on the development of uterine cancer, uterine Rest deficient mice (Restd/d) and control mice (Restfl/fl) were placed on high-fat/high-sucrose (HFHS) or control diets (n=6 mice/diet/genotype). After 24 weeks, total body weight and body fat were measured, and glucose tolerance tests were performed. Endometrial histology was evaluated by H&E staining while immunohistochemistry (IHC) for Chga was quantified using H-Scores. Chga expression was quantitated by qRT-PCR and data is represented as fold change (FC). Data were analyzed by unpaired t-test or ANOVA (or the non-parametric equivalent) where applicable with significance defined as p<0.05. After 24 weeks, total body weight and body fat content were significantly higher in mice on HFHS diet compared to control diet regardless of genotype. Uterine weight was higher in Restd/d mice compared to Restfl/fl mice, regardless of diet. Glucose levels were significantly higher after 30 min in Restd/d/HFHS diet (521mg/dL vs 366mg/dL, p=0.03) and Restfl/fl/HFHS diet (509mg/dL vs 366mg/dL, p=0.04) compared to Restfl/fl/control diet mice. Histologically, all Restfl/fl uterine sections had small glands with gland to stroma ratio <2:1 and no overcrowding of glands. Restd/d mice demonstrated large/dilated glands with some degree of overcrowding and gland to stroma ratio >2:1 in n=3/6 on the control diet and n=6/6 on the HFHS diet. No myoinvasion was seen in any of the groups. Stromal cell Chga expression by IHC was higher in Restd/d/HFHS diet compared to Restfl/fl/control diet mice (H-score: 0.29 vs 0.0, p<0.01), Restfl/fl/HFHS diet (H-score: 0.29 vs 0.04, p<0.01), and Restd/d/control diet (H-score: 0.29 vs 0.08, p<0.01). Chga mRNA expression was higher in Restd/d/control diet (FC:392,4X, p<0.05) and Restd/d/HFHS diet (FC:525,6X, p<0.01) compared to Restfl/fl/control diet mice. In conclusion, Restd/d mice on HFHS diet exhibited elevated Chga expression as well as uterine pathology with large/dilated glands and gland to stroma ratio >2:1, without myoinvasion, mimicking endometrial hyperplasia. In previous work high levels of CgA may cause vascular abnormalities leading to cancer cell multiplication; it can inhibit capillary tube formation and angiogenesis. These data support the notion that REST deficiency may have an influence on obesity related uterine pathology potentially by upregulation of CHGA. Interestingly, carcinoma was not detected in Restd/d mice. It is possible that an extended observation period or additional mutational factors may be necessary for the development of EC.