Key Considerations for Studying the Effects of High-Fat Diet on the Nulligravid Mouse Endometrium.

The obesity epidemic continues to increase, with half of US women predicted to be obese by 2030. Women with obesity are at increased risk for not only cardiovascular and liver disease, but also reproductive disorders. Although mouse models are useful in studying the effects of obesity, there is inconsistency in obesity-induction methods, diet composition, and mouse strains, and studies using female mice are limited. In this study, we sought to compare the effects of a 45% high-fat diet (HFD) versus a 60% HFD on the uterine estrous cycle of nulligravid C57BL/6J mice. For 22 weeks, we placed a total of 20 mice on either a 60% HFD, 45% HFD, or each HFD-matched control diet (CD). Both HFDs produced significant weight gain, with 60% HFD and 45% HFD gaining significant weight after 2 weeks and 15 weeks, respectively. Additionally, both HFDs led to glucose intolerance, fatty liver, and adipocyte hypertrophy. Mice fed 60% HFD displayed hyperphagia in the first 12 weeks of HFD treatment. Moreover, 60% HFD-treated mice had a longer estrous cycle length and an increased percentage of estrus stage samplings compared to CD-treated mice. Estrous cycle stage-controlled 60% HFD-treated mice displayed an increased estrogen-to-progesterone ratio and decreased ovarian corpora lutea compared to CD-treated mice, which may underlie the observed estrous cycle differences. There was no significant difference between diets regarding endometrial morphology or the percent of endometrial CD45+ immune cells. Our results indicate that consideration is needed when selecting a HFD-induced obesity mouse model for research involving female reproductive health.

Comprehensive proteogenomic characterization of rare kidney tumors.

Non-clear cell renal cell carcinomas (non-ccRCCs) encompass diverse malignant and benign tumors. Refinement of differential diagnosis biomarkers, markers for early prognosis of aggressive disease, and therapeutic targets to complement immunotherapy are current clinical needs. Multi-omics analyses of 48 non-ccRCCs compared with 103 ccRCCs reveal proteogenomic, phosphorylation, glycosylation, and metabolic aberrations in RCC subtypes. RCCs with high genome instability display overexpression of IGF2BP3 and PYCR1. Integration of single-cell and bulk transcriptome data predicts diverse cell-of-origin and clarifies RCC subtype-specific proteogenomic signatures. Expression of biomarkers MAPRE3, ADGRF5, and GPNMB differentiates renal oncocytoma from chromophobe RCC, and PIGR and SOSTDC1 distinguish papillary RCC from MTSCC. This study expands our knowledge of proteogenomic signatures, biomarkers, and potential therapeutic targets in non-ccRCC.

Loss of ZNRF3/RNF43 Unleashes EGFR in Cancer.

ZNRF3 and RNF43 are closely related transmembrane E3 ubiquitin ligases with significant roles in development and cancer. Conventionally, their biological functions have been associated with regulating WNT signaling receptor ubiquitination and degradation. However, our proteogenomic studies have revealed EGFR as the most negatively correlated protein with ZNRF3/RNF43 mRNA levels in multiple human cancers. Through biochemical investigations, we demonstrate that ZNRF3/RNF43 interact with EGFR via their extracellular domains, leading to EGFR ubiquitination and subsequent degradation facilitated by the E3 ligase RING domain. Overexpression of ZNRF3 reduces EGFR levels and suppresses cancer cell growth in vitro and in vivo, whereas knockout of ZNRF3/RNF43 stimulates cell growth and tumorigenesis through upregulated EGFR signaling. Together, these data highlight ZNRF3 and RNF43 as novel E3 ubiquitin ligases of EGFR and establish the inactivation of ZNRF3/RNF43 as a driver of increased EGFR signaling, ultimately promoting cancer progression. This discovery establishes a connection between two fundamental signaling pathways, EGFR and WNT, at the level of cytoplasmic membrane receptor, uncovering a novel mechanism underlying the frequent co-activation of EGFR and WNT signaling in development and cancer.

Frozen tissue coring and layered histological analysis improves cell type-specific proteogenomic characterization of pancreatic adenocarcinoma.

BACKGROUND: Omics characterization of pancreatic adenocarcinoma tissue is complicated by the highly heterogeneous and mixed populations of cells. We evaluate the feasibility and potential benefit of using a coring method to enrich specific regions from bulk tissue and then perform proteogenomic analyses. METHODS: We used the Biopsy Trifecta Extraction (BioTExt) technique to isolate cores of epithelial-enriched and stroma-enriched tissue from pancreatic tumor and adjacent tissue blocks. Histology was assessed at multiple depths throughout each core. DNA sequencing, RNA sequencing, and proteomics were performed on the cored and bulk tissue samples. Supervised and unsupervised analyses were performed based on integrated molecular and histology data. RESULTS: Tissue cores had mixed cell composition at varying depths throughout. Average cell type percentages assessed by histology throughout the core were better associated with KRAS variant allele frequencies than standard histology assessment of the cut surface. Clustering based on serial histology data separated the cores into three groups with enrichment of neoplastic epithelium, stroma, and acinar cells, respectively. Using this classification, tumor overexpressed proteins identified in bulk tissue analysis were assigned into epithelial- or stroma-specific categories, which revealed novel epithelial-specific tumor overexpressed proteins. CONCLUSIONS: Our study demonstrates the feasibility of multi-omics data generation from tissue cores, the necessity of interval H&E stains in serial histology sections, and the utility of coring to improve analysis over bulk tissue data.