Context Obesity is a disease with deleterious effects on the female reproductive tract, including the endometrium.Objective We sought to understand the effects of excess adipose on the benign endometrium.Methods A physiologic in vitro coculture system was developed, consisting of multicellular human endometrial organoids, adipose spheroids, and menstrual cycle hormones. Native human endometrial tissue samples from women with and without obesity were also analyzed. Benign endometrial tissues from premenopausal women ages 33 to 53 undergoing hysterectomy were obtained following written consent at Northwestern University Prentice Women's Hospital, Chicago, Illinois. Gene expression, protein expression, chromatin binding, and expression of DNA damage and oxidative damage markers were measured.Results Under high adiposity conditions, endometrial organoids downregulated endometrial secretory phase genes, suggestive of an altered progesterone response. Progesterone specifically upregulated the metallothionein (MT) gene family in the epithelial cells of endometrial organoids, while high adiposity significantly downregulated the MT genes. Silencing MT genes in endometrial epithelial cells resulted in increased DNA damage, illustrating the protective role of MTs. Native endometrium from women with obesity displayed increased MT expression and oxidative damage in the stroma and not in the epithelium, indicating the cell-specific impact of obesity on MT genes.Conclusion Taken together, the in vitro and in vivo systems used here revealed that high adiposity or obesity can alter MT expression by decreasing progesterone response in the epithelial cells and increasing oxidative stress in the stroma.

We recently identified HAPSTR1 (C16orf72) as a key component in a novel pathway which regulates the cellular response to molecular stressors, such as DNA damage, nutrient scarcity, and protein misfolding. Here, we identify a functional paralog to HAPSTR1: HAPSTR2. HAPSTR2 formed early in mammalian evolution, via genomic integration of a reverse transcribed HAPSTR1 transcript, and has since been preserved under purifying selection. HAPSTR2, expressed primarily in neural and germline tissues and a subset of cancers, retains established biochemical features of HAPSTR1 to achieve two functions. In normal physiology, HAPSTR2 directly interacts with HAPSTR1, markedly augmenting HAPSTR1 protein stability in a manner independent from HAPSTR1's canonical E3 ligase, HUWE1. Alternatively, in the context of HAPSTR1 loss, HAPSTR2 expression is sufficient to buffer stress signaling and resilience. Thus, we discover a mammalian retrogene which safeguards fitness.

Uterine fibroids (UFs) are the most common benign tumor in reproductive age women. UFs negatively impair endometrial receptivity (ER). Yet, mechanism isn't fully clear. ER is influenced by hormones, cytokines, growth factors, microRNAs as well as immune cells, mainly natural killer (NK) cells, which plays key role in implantation. Our objective is to explore UF effects on ER using 3D organoid system. Stem cells isolated from UFs or normal myometrium (MyoN) tissues were used to develop 3D organoids. RNA-seq was employed to comparatively profile their transcriptomes. Organoids were stained for inflammatory markers TNF-α and NF-kB (p65) by immunohistochemistry (IHC). Organoids secretome was analyzed for 48-cytokine array as well as TGF-β1 using multiplex ELISA. Exosomal miRNA was extracted from organoids secretome and differential gene expression of miR223-3p, 494-3p and 150 was measured. UFs organoid secretome was exposed to (1) 3D endometrial organoids established from primary epithelial and stromal cells and then ER related markers insulin growth factor binding protein 1 (IGFBP1) and prolactin (PRL) were measured using qRT-PCR. (2) cultured CD56+ CD16- NK cells and cell proliferation was measured using XTT assay after 24hr. Also, NK cells were treated with TGF-b1 (10ng/ml) or its receptor inhibitor LY-364947 (2μM) and viability was measured. UFs organoids were treated with VitaminD3 (VitD3 100nM) or green tea extract (EGCG 100μM) for 48hr and IHC, exosomal miRNA expression, cytokine array and effect on NK cell viability was explored using same markers. Unpaired student t-test was used for statistical significance detection. RNA-seq analysis showed enhanced inflammatory signaling in UFs organoids compared to MM using DisGeNET (adjp=0.00001, gene ratio 40/407). UF organoids secreted higher levels of many cytokines including IL-6, IL-8, and TNF-α than MyoN organoid and showed higher expression of TNF-α, and p-NF-kB using IHC (p<0.05). UF organoids secreted higher exosomal miR-223-3p, miR-494-3p, and miR-150 vs. MyoN (p<0.05) which are associated with recurrent implantation failure, infertility and impaired ER. Importantly, UF organoid secretome downregulated IGFBP1 and PRL expression in endometrial organoids compared to untreated control (p<0.05). UFs organoids secreted more TGF-b1 compared to MyoN Which might justify NK cells growth inhibition following UF organoid secretome, compared to MyoN, since TGF-β1 treatment inhibited NK cell growth while its inhibitor restored it (p<0.05). Treating UFs organoids with VitD3 or EGCG induced reduction of IL-6, IL-8, TNF-α and miRs (223-3p, 494-3p and 150) secretion. Moreover, it restored NK cells viability compared to untreated UFs secretome (p<0.05). UFs may negatively impact ER directly through secretion of cytokines and miRNA that interfere with implantation and indirectly via disrupting NK cells viability. VitD3/EGCG might offer beneficial effects by interrupting such negative crosstalk.

Pancreatic ductal adenocarcinoma (PDA) cells reprogram their transcriptional and metabolic programs to survive the nutrient-poor tumor microenvironment. Through in vivo CRISPR screening, we discovered islet-2 (ISL2) as a candidate tumor suppressor that modulates aggressive PDA growth. Notably, ISL2, a nuclear and chromatin-associated transcription factor, is epigenetically silenced in PDA tumors and high promoter DNA methylation or its reduced expression correlates with poor patient survival. The exogenous ISL2 expression or CRISPR-mediated upregulation of the endogenous loci reduces cell proliferation. Mechanistically, ISL2 regulates the expression of metabolic genes, and its depletion increases oxidative phosphorylation (OXPHOS). As such, ISL2-depleted human PDA cells are sensitive to the inhibitors of mitochondrial complex I in vitro and in vivo. Spatial transcriptomic analysis shows heterogeneous intratumoral ISL2 expression, which correlates with the expression of critical metabolic genes. These findings nominate ISL2 as a putative tumor suppressor whose inactivation leads to increased mitochondrial metabolism that may be exploitable therapeutically.

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers. Uncovering mechanisms responsible for the heterogeneous clinical features of this disease is an essential step toward developing improved and more specific therapeutic approaches. Here, we sought to identify transcriptional regulators of aggressive PDAC growth through in vivo CRISPR screening of epigenetic and transcription factors in an orthotopic model. We identified the ISL LIM homeobox 2 ( ISL2 ) gene as a tumor suppressor whose depletion enhances the proliferation of human PDAC cells in vitro and in vivo and cooperates with activated KRAS to initiate PDAC in a murine model. Conversely, the upregulation of ISL2 expression through CRISPR-mediated locus-specific epigenetic editing results in reduced cell proliferation. Importantly, ISL2 is epigenetically silenced through DNA methylation in ~60% of PDAC tumors, which correlates with poor patient outcome. Functional studies showed that ISL2 loss rewires metabolic gene expression, and consequently potentiates oxidative phosphorylation while reducing glycolysis. This metabolic shift creates selective vulnerability to small molecule inhibitors of mitochondrial respiration and fatty acid oxidation. Collectively, these findings reveal ISL2 as a novel tumor suppressor whose inactivation drives metabolic reprogramming in an aggressive PDAC subset and point to potential therapeutic vulnerabilities in these tumors.

Certain aspects of diagnosis, prognosis, and treatment of cancer patients are still important challenges to be addressed. We developed a pipeline to uncover patterns of alternative polyadenylation (APA), a hidden complexity in cancer transcriptomes, to further accelerate efforts to discover novel cancer genes and pathways. Here, we found a significant shift in usage of poly(A) signals in six common tumor types compared to normal tissues. We further defined specific subsets of APA events to efficiently classify cancer types/subtypes. Triple negative breast cancers, for example, have specific 39UTR length alterations where the significant majority are shortening events (70%, 113 of 165) of mostly proliferation-related transcripts compared with normal breast tissue. Such shortening events correlate with increased protein levels and relapse free survival of patients, suggesting functional significance of isoform variability. In line with this isoform diversity, we also detected deregulated expression of mRNA polyadenylation complex proteins in breast cancer cells. Of note, APA proteins are responsive to proliferative signals including estrogen and epidermal growth factor, suggesting a potential explanation to 39-end isoform diversity in cancer cells. Overall, our study offers a computational and experimental approach for use of APA in novel gene discovery and classification in common tumor types, with important implications in basic research, biomarker discovery, and precision medicine approaches. Citation Format: Oguzhan Begik, Melda Ercan, Harun Cingoz, Tolga Can, Merve Oyken, Ayse Elif Erson-Bensan. Deregulated APA and cancer specific APA isoforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2360.