Glucocorticoid Receptor Signaling Research Articles

Abstract A011: TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer

Abstract Background: A major obstacle in the treatment of metastatic castrate resistant prostate cancer (mCRPC) is acquired resistance to androgen deprivation therapy (ADT). It is now recognized that ADT, particularly the 2nd generation androgen receptor (AR) antagonists, such as enzalutamide, orchestrate plasticity changes/molecular alterations leading to therapy resistance. It is hypothesized in the field that the emergence of castrate resistance to 2nd generation ADT is driven by a switch/bypass from AR signaling to the glucocorticoid receptor (GR) signaling. Therefore, identifying the molecular mechanisms that concurrently drive the loss of AR and gain of GR signaling may be crucial in devising novel and effective therapeutic modalities against mCRPC. We have previously reported that TBX2, a T-box transcription factor (TF) with both repressor and activator functions, is over-expressed in CRPC and that TBX2 drives PCa bone metastatic progression. In agreement with our findings, a recent report showed that TBX2 is a key TF that drives plasticity associated with CRPC. In this study, we investigated the molecular mechanisms by which TBX2 drives plasticity associated with enzalutamide resistance. Methods: We genetically modulated TBX2 using the dominant negative (DN), sh-RNA, and overexpression (OE) approaches. RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we performed chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM). Results: Using publicly available databases, we observed a negative correlation between TBX2 and AR; and a positive correlation between TBX2 and GR. Strikingly, blocking TBX2 expression in wildtype PC3 human PCa cells that do not express AR mRNA or protein led to a marked elevation of AR signature as assessed by RNA-seq analysis. These results were confirmed/validated at the protein level using additional human PCa cell lines. Further, these results were validated using all three approaches to genetically modulate TBX2, i.e. DN, shRNA, and OE. Further, ChIP and SDM analyses confirmed that TBX2 directly binds and transcriptionally represses AR. Conversely, genetic modulation of TBX2 was positively associated with GR expression, and TBX2 was found to directly bind to the GR promoter. Further, we found that knock-down of GR in LNCaP cells overexpressing TBX2 (LNCaPTBX2OE) reversed enzalutamide resistance that is associated with elevated TBX2 expression. Conclusions: Our studies suggest that TBX2 acts as the molecular switch that on one hand represses AR and on the other hand activates GR. Further our study paves the way for potential therapeutic strategies against the AR/GR switch/bypass in CRPC wherein AR and GR signaling could be co-inhibited through targeting TBX2. Citation Format: Sayanika Dutta, Girijesh Patel, Hamed Khedmatgozar, Daniel Latour, Manisha Tripathi, Srinivas Nandana. TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A011.

Phase I trial of enzalutamide (Enz) plus the glucocorticoid receptor antagonist relacorilant (Rela) for patients with metastatic castration resistant prostate cancer.

5062 Background: Resistance to androgen receptor (AR) targeted therapies is common in mCRPC. Glucocorticoid receptor (GR) expression increases with AR inhibition in patients (pts) and blockade of GR signaling inhibits CRPC growth in preclinical models when combined with AR blockade. We thus conducted a phase I study of the combination of rela and enz in patients with mCRPC. Methods: The study used a 6+3 design to assess safety and pharmacokinetics (PK) of the 2-drug combination. The starting dose of rela 100mg/day was chosen based on current clinical studies indicating this dose was both safe for daily dosing and pharmacologically active and the starting dose for Enz was 120mg based on anticipated drug-drug interactions. Escalating doses of rela were combined with Enz 120 using pre-specified dose escalation rules. An additional 12 patients were to be assessed at the MTD to refine safety and PK profiles. Primary endpoint was safety of the combination and to establish safe and pharmacologically active doses of both agents. Key secondary endpoints were tumor response per RECIST 1.1 criteria and progression-free survival (PFS; defined as either PSA progression, radiographic progression or death). PSA was measured after 12 weeks or at the last time at which PSA data was available, including times <12 weeks. Results: 35 pts were enrolled. Pts had a median age of 74 (range 62-85) and baseline PSA of 89.5 (range 7.3-2391). 100% had prior potent AR signaling inhibitor (abi or enz) with 89% receiving prior enz, 94% prior abi, 83% prior both. 69% of pts received prior docetaxel. Median PSA change was 0.1%. Enz 120mg + Rela 100mg and Enz 120+Rela 150 were found to be safe and tolerable and achieved desirable Enz PK. DLTs were noted in 3 pts at the 200mg Rela dose, which was deemed to have exceeded the MTD, which was thus defined as 150mg Rela+120mg Enz. Most common ≥ grade 3 treatment related adverse events (TRAEs) were fatigue (11% of pts), anemia (9%), abdominal pain (6%) and pericardial effusion (6%). Median PFS was 2.5 months for 21 of 35 evaluable pts. Best response per RECIST 1.1 were Not Evaluable (40%), SD (34%) & PD (26%). No CR or PR were noted. Most common reasons for discontinuation from trial included progressive disease/hospice (82%), AEs (12%), physician discretion (3%) & death (3%). 2 pts remain on trial as of data cutoff (12/31/22). Conclusions: The combination of enzalutamide and relacorilant was safe & largely well tolerated. The majority of pts were discontinued from trial due to progressive disease or hospice. Further analyses of secondary & correlative endpoints such as translational/endocrine biomarkers, GR target gene expression and circulating tumor cells are ongoing. Clinical trial information: NCT03674814 .

Adolescent stress impairs postpartum social behavior via anterior insula-prelimbic pathway in mice

Adolescent stress can be a risk factor for abnormal social behavior in the postpartum period, which critically affects an individual social functioning. Nonetheless, the underlying mechanisms remain unclear. Using a mouse model with optogenetics and in vivo calcium imaging, we found that adolescent psychosocial stress, combined with pregnancy and delivery, caused hypofunction of the glutamatergic pathway from the anterior insula to prelimbic cortex (AI-PrL pathway), which altered PrL neuronal activity, and in turn led to abnormal social behavior. Specifically, the AI-PrL pathway played a crucial role during recognizing the novelty of other mice by modulating “stable neurons” in PrL, which were constantly activated or inhibited by novel mice. We also observed that glucocorticoid receptor signaling in the AI-PrL pathway had a causal role in stress-induced postpartum changes. Our findings provide functional insights into a cortico-cortical pathway underlying adolescent stress-induced postpartum social behavioral deficits.

SOX2 expression in prostate cancer drives resistance to nuclear hormone receptor signaling inhibition through the WEE1/CDK1 signaling axis

The development of androgen receptor signaling inhibitor (ARSI) drug resistance in prostate cancer (PC) remains therapeutically challenging. Our group has described the role of sex determining region Y-box 2 (SOX2) overexpression in ARSI-resistant PC. Continuing this work, we report that NR3C1, the gene encoding glucocorticoid receptor (GR), is a novel SOX2 target in PC, positively regulating its expression. Similar to ARSI treatment, SOX2-positive PC cells are insensitive to GR signaling inhibition using a GR modulating therapy. To understand SOX2-mediated nuclear hormone receptor signaling inhibitor (NHRSI) insensitivity, we performed RNA-seq in SOX2-positive and -negative PC cells following NHRSI treatment. RNA-seq prioritized differentially regulated genes mediating the cell cycle, including G2 checkpoint WEE1 Kinase (WEE1) and cyclin-dependent kinase 1 (CDK1). Additionally, WEE1 and CDK1 were differentially expressed in PC patient tumors dichotomized by high vs low SOX2 gene expression. Importantly, pharmacological targeting of WEE1 (WEE1i) in combination with an ARSI or GR modulator re-sensitizes SOX2-positive PC cells to nuclear hormone receptor signaling inhibition in vitro, and WEE1i combined with ARSI significantly slowed tumor growth in vivo. Collectively, our data suggest SOX2 predicts NHRSI resistance, and simultaneously indicates the addition of WEE1i to improve therapeutic efficacy of NHRSIs in SOX2-positive PC.

Effect of SGLT2 Inhibition on the transcriptomic and proteomic profile of non-diabetic Dahl SS Rats

Sodium-Glucose Cotransporter-2 (SGLT2) inhibitors are new, highly effective drugs for treating type 2 diabetes mellitus. SGLT2 inhibitors also substantially reduced the risk of hospitalization for heart failure. Previous studies in rodents and humans have shown that SGLT2 inhibitors lower blood pressure levels, cardiovascular risk events, and kidney diseases in subjects with and without diabetes mellitus. However, the mechanisms behind those effects are still unknown. Our previous study showed that SGLT2 inhibition blunted the development of salt-sensitive (SS) hypertension and caused glucosuria and natriuresis in Dahl SS rats without affecting the expression and activity of Na + channels and changes in the renin-angiotensin-aldosterone system. Here, we aim to investigate the effect of SGLT2 treatment on non-diabetic SS rats using multi-omics analysis, which may provide insights into the underlying mechanisms. 8-week-old male SS rats were treated with or without SGLT2 inhibitor (dapagliflozin, 2 mg/kg/day in drinking water) for 3 weeks. Kidney cortex and medulla tissue were collected for RNA-Seq analysis. Proteomics analysis was performed on kidney cortex tissue and plasma. The data were further processed by Ingenuity Pathway Analysis (IPA).RNA-Seq analysis indicated that despite SLGT2 mainly being expressed in the kidney cortex, dapagliflozin treatment altered the expression of more genes in the medulla (855 in the cortex vs 2244 differentially expressed genes (DEGs) in the medulla). Dapagliflozin treatment also caused significant changes in multiple genes related to inflammation and hypertension, with IPA analysis predicting the downregulation of both functions. In hypertension network Adrb3, Retn, Foxn4 were the most upregulated genes, and Ptgs1, Nos1, Oxtr – downregulated. In addition, disease and biological function enrichment analysis revealed that the altered genes were mainly related to the following functions: lipid metabolism, small molecule biochemistry, amino acid metabolism, cell cycle and morphology, and molecular transport. Interestingly, lipid metabolism was predicted to be downregulated in the cortex but upregulated in the medulla.Proteomic analysis identified 450 differentially expressed proteins (DEPs) in the kidney cortex and 22 in plasma. Disease and biological function enrichment analysis on proteomics data revealed mostly the same biological functions as RNA-Seq data. In addition, canonical pathways analysis based on RNA-Seq and proteomics data revealed that endothelin-1 and oxytocin signaling pathways were the most downregulated pathways in the kidney cortex and medulla under the dapagliflozin treatment. Urea cycle and glucocorticoid receptor signaling were the most changed canonical pathway in proteomic data. In conclusion, our results indicated that inhibition of SGLT2 by dapagliflozin altered various genes and proteins in the kidney of Dahl SS rats, which may provide insights for future mechanistic studies. This research was supported by National Institutes of Health grants R35 HL135749 (to AS). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Human neutrophils are a key target of glucocorticoids

Abstract Neutrophils play central roles in immunity and inflammation. Glucocorticoids (GCs) are the cornerstone of anti-inflammatory and immunosuppressive therapy. While GCs trigger an acute rise in circulating neutrophils, little is known about the mechanisms by which GCs modulate neutrophil biology. We studied the response of 9 primary human cells to in vitro treatment with GCs. Surprisingly, neutrophils were transcriptionally the most responsive cell type, and they express proportionally the most glucocorticoid receptor (GR) transcripts. Long-read RNA-seq revealed 42 full-length GR transcript isoforms in neutrophils, twice as many as had been reported previously in all other cell types. At the protein level, neutrophils also have a unique and diverse set of GR isoforms. In contrast to the widely accepted model of GR signaling, in which GR binds GC molecules in the cytoplasm and then translocates to the nucleus, GR localization in neutrophils is almost exclusively nuclear, even in the absence of GC treatment. To validate our findings in vivo, we then performed a study of healthy volunteers, with blood collected before and serially after GC infusion. We generated a pathway-level map of the human neutrophil transcriptome at baseline and in response to GCs. The in vivo transcriptional response of human neutrophils to GCs was as strong as that observed in vitro, with a predominance of upregulation. Among the most strongly induced genes are several that are either not expressed, or expressed at very low levels, in human neutrophils at baseline. Some of the proteins encoded by these genes may have putative effects on neutrophil function, including SAMSN1 and ZBTB16. These findings indicate that human neutrophils are a key target for GC action.

Intact Recognition Memory and Altered Hippocampal Glucocorticoid Receptor Signaling in Fkbp5-deficient Mice Following Acute Uncontrollable Stress.

The FK506 binding protein 5 (FKBP5) is a co-chaperone that regulates the activity of the glucocorticoid receptor (GR) and has been reported to mediate stress resilience. This study aimed to determine the effects of Fkbp5 deletion on acute stress-induced recognition memory impairment and hippocampal GR signaling. Wild-type and Fkbp5-knockout mice were subjected to acute uncontrollable stress induced by restraint and electrical tail shock. First, we assessed the cognitive status of mice using a novel object recognition task. Next, we measured plasma corticosterone, GR levels, and the levels of GR phosphorylation at serine 211 in the hippocampus. Wild-type mice exhibited stress-induced memory impairments, whereas Fkbp5-knockout mice did not. Plasma corticosterone and GR levels did not differ between the non-stressed wild-type and Fkbp5-knockout mice, but the levels of phosphorylated GR were lower in Fkbp5-knockout mice than in wild-type mice. Wild-type and Fkbp5-knockout mice showed increased nuclear GR levels following stress, indicating GR translocation. However, cytosolic phosphorylated GR levels were lower in the hippocampi of Fkbp5-knockout mice following stress than in those of wild-type mice. These results suggest that FKBP5 deficiency increases resilience to acute stress by altering GR signaling.

EJE Prize 2023: genes on steroids-genomic control of hepatic metabolism by the glucocorticoid receptor.

Glucocorticoids are essential hormones produced by the adrenal cortex with prominent circadian rhythmicity and in times of stress. Glucocorticoids maintain liver homeostasis through coordinated activities that control the major pathways of energy metabolism. Glucocorticoids activate the glucocorticoid receptor (GR), a nuclear hormone receptor that regulates the transcription of hundreds of genes in response to ligand. This review aims to provide a comprehensive overview of glucocorticoid receptor signaling impact on glucose, amino acid, and lipid metabolism in the liver. We integrate fundamental and current findings elucidating key GR-regulated pathways from a physiologic, biochemical, and molecular point-of-view. Here, we focus on the transcriptional regulation of well-characterized hepatic GR target genes, and on those GR co-factors that coordinate nutritional and hormonal signals.

The crucial role of muscle glucocorticoid signaling in accelerating obesity and glucose intolerance via hyperinsulinemia.

Metabolic crosstalk from skeletal muscle to multiple organs is important for maintaining homeostasis, and its dysregulation can lead to various diseases. Chronic glucocorticoid administration often induces muscle atrophy and metabolic disorders such as diabetes and central obesity; however, the detailed underlying mechanism remains unclear. We previously reported that the deletion of glucocorticoid receptor (GR) in skeletal muscle increases muscle mass and reduces fat mass through muscle-liver-fat communication under physiological conditions. In this study, we show that muscle GR signaling plays a crucial role in accelerating obesity through the induction of hyperinsulinemia. Fat accumulation in liver and adipose tissue, muscle atrophy, hyperglycemia, and hyperinsulinemia induced by chronic corticosterone (CORT) treatment improved in muscle-specific GR knockout (GRmKO) mice. Such CORT-induced fat accumulation was alleviated by suppressing insulin production (streptozotocin injection), indicating that hyperinsulinemia enhanced by muscle GR signaling promotes obesity. Strikingly, glucose intolerance and obesity in ob/ob mice without CORT treatment were also improved in GRmKO mice, indicating that muscle GR signaling contributes to obesity-related metabolic changes, regardless of systemic glucocorticoid levels. Thus, this study provides new insight for the treatment of obesity and diabetes by targeting muscle GR signaling.

Glucocorticoid Receptor and β-Catenin Interact in Prostate Cancer Cells and Their Co-Inhibition Attenuates Tumorsphere Formation, Stemness, and Docetaxel Resistance.

Therapy resistance hinders the efficacy of anti-androgen therapies and taxane-based chemotherapy for advanced prostate cancer (PCa). Glucocorticoid receptor (GR) signaling mediates resistance to androgen receptor signaling inhibitors (ARSI) and has also been recently implicated in PCa resistance to docetaxel (DTX), suggesting a role in therapy cross-resistance. Like GR, β-catenin is upregulated in metastatic and therapy-resistant tumors and is a crucial regulator of cancer stemness and ARSI resistance. β-catenin interacts with AR to promote PCa progression. Given the structural and functional similarities between AR and GR, we hypothesized that β-catenin also interacts with GR to influence PCa stemness and chemoresistance. As expected, we observed that treatment with the glucocorticoid dexamethasone promotednuclear accumulation of GR and active β-catenin in PCa cells. Co-immunoprecipitation studies showed that GR and β-catenin interact in DTX-resistant and DTX-sensitive PCa cells. Pharmacological co-inhibition of GR and β-catenin, using the GR modulator CORT-108297 and the selective β-catenin inhibitor MSAB, enhanced cytotoxicity in DTX-resistant PCa cells grown in adherent and spheroid cultures and decreased CD44+/CD24- cell populations in tumorspheres. These results indicate that GR and β-catenin influence cell survival, stemness, and tumorsphere formation in DTX-resistant cells. Their co-inhibition could be a promising therapeutic strategy to overcome PCa therapy cross-resistance.

Dexamethasone-stimulated glucocorticoid receptor signaling positively regulates the endoplasmic reticulum-associated degradation (ERAD) mechanism in hepatocellular carcinoma cells

Hepatocellular carcinoma is one of the most common types of primary liver cancer in adults and also it is the third leading cause of cancer-related deaths worldwide. Although there are various treatment options such as surgery, radiation, targeted drug therapy, immunotherapy and chemotherapy, most hepatocellular carcinomas are highly resistant to systemic treatments. Today, the molecular pathogenesis of hepatocellular carcinoma remains largely obscure. Therefore, there is a need for detailed research for the characterization of molecular signaling networks related to the development of hepatocellular carcinoma. Recent studies have attention to the hormonal regulation of hepatocellular carcinoma cells mediated by systemic hormones such as glucocorticoids. However, glucocorticoid-mediated regulation of endoplasmic reticulum-associated degradation (ERAD) and unfolded protein response (UPR), which are known to be important survival mechanisms for cancer cells remains unknown in hepatocellular carcinoma. In the present study, we showed that dexamethasone-induced glucocorticoid receptor signaling mediated advanced regulation of ERAD and UPR signaling in hepatocellular carcinoma cells. Our findings indicated that glucocorticoid signaling positively regulated mRNA and protein levels of ERAD components and also protein kinase RNA-like ER Kinase (PERK) and inositol-requiring enzyme 1⍺ (IRE1⍺) branches of UPR signaling are accompanied by the glucocorticoid signaling. In addition, putative glucocorticoid response elements (GREs) were determined in the promoter regions of ERAD members in in-silico analyses. Additionally, silencing of ERAD components significantly reduced the tumorigenic features of hepatocellular carcinoma cells, including cell proliferation, metastasis, invasion and 3D tumor formation. Collectively, these results reveal a novel pattern of regulation of ERAD components by glucocorticoid-mediated in human hepatocellular carcinoma cells.

Abstract 1445: TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer

Abstract Background: A major obstacle in the treatment of metastatic castrate resistant prostate cancer (mCRPC) is acquired resistance to androgen deprivation therapy (ADT). It is now recognized that ADT, particularly the 2nd generation androgen receptor (AR) antagonists, such as enzalutamide, orchestrate plasticity changes/molecular alterations leading to therapy resistance. It is hypothesized in the field that the emergence of castrate resistance to 2nd generation ADT is driven by a switch/bypass from AR signaling to the glucocorticoid receptor (GR) signaling. Therefore, identifying the molecular mechanisms that concurrently drive the loss of AR and gain of GR signaling may be crucial in devising novel and effective therapeutic modalities against mCRPC. We have previously reported that TBX2, a T-box transcription factor (TF) with both repressor and activator functions, is over-expressed in CRPC and that TBX2 drives PCa bone metastatic progression. In agreement with our findings, a recent report showed that TBX2 is a key TF that drives plasticity associated with CRPC. In this study, we investigated the molecular mechanisms by which TBX2 drives plasticity associated with enzalutamide resistance. Methods: We genetically modulated TBX2 using the dominant negative (DN), sh-RNA, and overexpression (OE) approaches. RNA-seq was performed, and qRT-PCR, Western blot and immunohistochemical (IHC) analyses were used for validation. Further, we performed chromatin immunoprecipitation (ChIP) and site directed mutagenesis (SDM). Results: Using publicly available databases, we observed a negative correlation between TBX2 and AR; and a positive correlation between TBX2 and GR. Strikingly, blocking TBX2 expression in wildtype PC3 human PCa cells that do not express AR mRNA or protein led to a marked elevation of AR signature as assessed by RNA-seq analysis. These results were confirmed/validated at the protein level using additional human PCa cell lines. Further, these results were validated using all three approaches to genetically modulate TBX2, i.e. DN, shRNA, and OE. Further, ChIP and SDM analyses confirmed that TBX2 directly binds and transcriptionally represses AR. Conversely, genetic modulation of TBX2 was positively associated with GR expression, and TBX2 was found to directly bind to the GR promoter. Further, we found that knock-down of GR in LNCaP cells overexpressing TBX2 (LNCaPTBX2OE) reversed enzalutamide resistance that is associated with elevated TBX2 expression. Conclusions: Our studies suggest that TBX2 acts as the molecular switch that on one hand represses AR and on the other hand activates GR. Further our study paves the way for potential therapeutic strategies against the AR/GR switch/bypass in CRPC wherein AR and GR signaling could be co-inhibited through targeting TBX2. Citation Format: Sayanika Dutta, Girijesh Patel, Hamed Khedmatgozar, Daniel Latour, Manisha Tripathi, Srinivas Nandana. TBX2 acts as a molecular switch to downregulate androgen receptor and upregulate glucocorticoid receptor signaling in castrate resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1445.

Abstract 1933: Transcriptomic profiling of Hispanic colorectal cancer disparities

Abstract Colorectal cancer (CRC) was reported as the third most diagnosed cancer worldwide with approximately 1.93 million new cases reported in 2020. Within the U.S., the incidence and death rates differ by ethnicity. Among Hispanics, CRC accounted for 12% and 8% of all estimated new cases of cancer and 11% and 9% of all cancer deaths in men and women, respectively, in 2018. Though CRC is preventable, and the overall survival rate is approximately 90% when detected at early stages, only about 39% of CRC patients are diagnosed in the early stages, mostly because screening rates in the US are low, especially among Hispanics. Access to health care for Hispanics is limited when compared to non-Hispanic Whites (NHWs); as a result, they tend to present with later-stage CRC, which likely accounts for their inferior survival after CRC. Essential to improving treatment, prognosis, and detection strategies is the identification and validation of new ethnicity-specific transcriptomic profiles. Here, we have conducted RNA sequencing from Hispanic and NHW CRC (n=10 each) and normal adjacent tissues (NATs; n=3 each), to identify the differentially expressed genes (DEGs) in these two populations. The Illumina sequencing reads were mapped against the reference human genome, and the read counts were normalized using the median of ratios method using DESeq2 software. The transcriptomic analysis revealed that as compared to the respective NATs, 7222 and 7873 genes were observed to be differentially expressed in NHW and Hispanic CRC tissues. Among these, 213 and 363 genes were unique to the NHW and Hispanic CRC cohorts, respectively. Within the Hispanic cohort, gender-based analyses revealed that 504 and 266 genes were differentially expressed in the female and male donors (n=5 each), respectively, as compared to the NHW counterparts. Further, 271 and 475 genes were found to be differentially expressed in early (stage I/II; n=5) and late (stage III/IV; n=3) stages of Hispanic CRC tissues, respectively, as compared to the NHW counterparts. Pathway enrichment analysis of the unique DEGs from the Hispanic and NHW cohorts was conducted using Ingenuity Pathway Analysis software. The genes involved in cysteine biosynthesis, ferroptosis, and GABA receptor signaling pathways were seen to be enriched in the NHW cohort, whereas the genes involved in glucocorticoid receptor (GR) signaling, bile, and androgen biosynthesis pathways were enriched in the Hispanic cohort. Previous studies have implicated, GR signaling to promote tumor heterogeneity and invasion in CRC via CDK1, or bile acids to regulate numerous genes including p53, CDK1, cyclooxygenase 2, IL8, and various miRNAs leading to CRC metastasis. As each pathway likely regulates specific cellular behaviors, the DEGs identified from this study will constitute potential ethnicity-specific biomarkers or attractive targets for the development of novel therapeutic interventions. Citation Format: Soumya Nair, Aditi Kulkarni, Sourav Roy. Transcriptomic profiling of Hispanic colorectal cancer disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1933.

Abstract 3132: CLUMPS-PTM: Spatial clustering of post-translational modifications across cancer types

Abstract Post-translational modifications (PTMs) play key roles in regulating cell signaling in health and disease processes. Recent advances in mass spectrometry enable comprehensive investigation of PTMs. However, identifying functionally relevant PTMs and protein domains implicated in disease remains a key challenge. We present CLUMPS-PTM, an algorithm for spatial clustering of PTMs to propose functional domains. We apply CLUMPS-PTM to the Clinical Proteomic Tumor Analysis Consortium (CPTAC) dataset (one of the largest proteogenomic pan-cancer PTM datasets) comprising 1110 samples spanning 11 tumor types. First, we map over 14K phospho-sites and 13K acetyl-sites detected in the CPTAC dataset to protein data bank (PDB) files. We expand this to the predicted AlphaFold proteome and recover additional 12K phospho-sites and 10K acetyl-sites exceeding model confidence (pLDDT) of 70%. Consistent with previous research, phospho-sites are found on residues with lower AlphaFold prediction confidence than acetyl-sites due to their abundance in unstructured domains (p-value < 1e-4). To identify functionally relevant protein domains in the pan-cancer context, we characterize the 1110 samples by applying SignatureAnalyzer, a Bayesian non-negative matrix factorization method, to discover multi-omic patterns. This analysis identifies strong signatures associated with DNA damage response (DDR) and stratifies the CPTAC cohort into DDR-high (n=442) and DDR-low (n=668) groups. Next, we applied CLUMPS-PTM to differentially modified phospho/acetyl-sites between these groups. We identified 22 proteins (including GMPS, NCBP1, and ANPEP (CD13)) with significant clusters of differentially phosphorylated sites (q-value < 0.1) in DDR-high samples. ANPEP is a zinc-dependent aminopeptidase with a significant cluster (q-value = 0.0129) of differentially phosphorylated sites in the ERAP1-C domain that faces peptidase active sites known to have proliferative activity via the MAPK pathway. CLUMPS-PTM also identified a significant cluster of acetyl-sites on ARID1A (a SWI/SNF chromatin remodeler known to be mutated in cancer) in DDR-high samples (q-value = 0.085). The ARID1A cluster is on the C-terminal tail of the protein within the glucocorticoid receptor (GR) domain, an upstream regulator of proteins that increase catabolism, reduce inflammation, and increase cell survival. Increased acetylation may block a ubiquitination site at the C-terminus of the protein, resulting in increased protein levels, likely due to reduced ARID1A degradation, and increased GR signaling. Overall, CLUMPS-PTM is an open-source tool that allows near proteome-wide spatial analysis with the growing availability of PTM data. We anticipate that it will be useful to the broader proteomic community for the discovery of novel targets and generation of insights into functional mechanisms. Citation Format: Shankara K. Anand, Yifat Geffen, Yo Akiyama, Francois Aguet, Gad Getz. CLUMPS-PTM: Spatial clustering of post-translational modifications across cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3132.

Abstract 62: Untangling the connection between stress and metastatic breast cancer

Abstract There is emerging evidence that exposure to chronic stress is associated with increased risk of metastasis and poor survival in cancer patients. However, the underlying molecular and cellular mechanisms responsible for the effects of stress on tumor progression remain unclear. To determine how stress promotes metastasis, we established new mouse models in which multiple chronic stress exposure promoted breast cancer metastasis to the lungs. We found that chronic stress generates a pro-metastatic lung microenvironment with e.g., reduced T cell infiltration and increased extracellular matrix (ECM) deposition and neutrophil infiltration. Among them, neutrophils were required for these changes as their depletion abolished the pro-metastatic effects of stress and normalized the lung tumor microenvironment. Mechanistically, we analyzed neutrophils from chronically stressed mice and identified dramatically altered transcriptomes, including increased expression of several glucocorticoid receptor regulated genes. Phenotypically, neutrophils from stress exposed mice exhibited increased ROS production, and an increased ability to inhibit T cell activation and to form spontaneous neutrophil extracellular traps (NETs). The stress-associated changes to neutrophils were dependent on glucocorticoid receptor (GR) signaling as neutrophil-specific GR knockout mice showed no increase in NET-formation, a normalized lung microenvironment and no increase in lung metastasis upon stress exposure. Among the changes in the neutrophils caused by stress exposure, we found that the formation of pro-metastatic NETs was critical as directly digesting NETs with DNase I prevented stress-induced metastasis and normalized the lung microenvironment. In sum, this work shows that chronic stress exposure causes a glucocorticoid-mediated increase in NET formation and that NETs are critical drivers in the establishment of a stress-induced, metastasis-promoting microenvironment. Targeting NETs could be an important strategy to prevent metastatic recurrence in cancer patients, many of whom will experience stress due to their cancer diagnosis and subsequent treatments. Citation Format: Xue-Yan He, David Ng, Yuan Gao, Evdokia Michalopoulou, Shanu George, Jose M. Adrover, Lijuan Sun, Jean Albrengues, Ledong Wan, Xiao Han, Christopher Vakoc, Linda Van Aelst, Mikala Egeblad. Untangling the connection between stress and metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 62.

MP01-02 PROTEOMIC ANALYSIS OF SEMINAL FLUID IN RHESUS MACAQUES EXPOSED TO DELTA-9-TETRAHYDROCANNABINOL

You have accessJournal of UrologyCME1 Apr 2023MP01-02 PROTEOMIC ANALYSIS OF SEMINAL FLUID IN RHESUS MACAQUES EXPOSED TO DELTA-9-TETRAHYDROCANNABINOL Jasper C. Bash, Lyndsey E. Shorey-Kendrick, Carol B. Hannah, Charles A. Easley, Eliot Spindel, Jamie O. Lo, and Jason C. Hedges Jasper C. BashJasper C. Bash More articles by this author , Lyndsey E. Shorey-KendrickLyndsey E. Shorey-Kendrick More articles by this author , Carol B. HannahCarol B. Hannah More articles by this author , Charles A. EasleyCharles A. Easley More articles by this author , Eliot SpindelEliot Spindel More articles by this author , Jamie O. LoJamie O. Lo More articles by this author , and Jason C. HedgesJason C. Hedges More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cannabis use has been associated with male infertility, but the etiology is not well understood. Seminal fluid is comprised of multiple factors (e.g. proteins), that influence fertility. Our objective was to determine the impact of chronic delta-9-tetrahydrocannabinol (THC) use on the seminal fluid proteome in a rhesus macaque (RM) model. METHODS: 6 adult RMs were given a daily THC edible over 280 days (∼4 spermatogenesis cycles) with serial non-sedated semen collection. Differential expression of proteins in the seminal fluid at baseline (pre-THC), high-THC dose (2.5 mg/7 kg/day), and post-THC discontinuation for 140 days (∼2 spermatogenesis cycles) was evaluated. Proteomic analysis was performed by liquid chromatography tandem mass spectrometry followed by statistical analysis to identify differentially expressed proteins. RESULTS: Proteomic analysis resulted in 1,395 quantifiable proteins (Figure 1A). The average percent identity was 88.6% (SD=14.9%). All RM proteins were matched to human proteins with 99.4% of the RM proteins (1,386) exceeding an identity cutoff of 44.3% (average minus 3 SDs). 7 proteins reached FDR significance in any contrast, thus we focused downstream analyses on nominally significant candidate proteins. Ingenuity Pathway Analysis (Figure 1B) revealed that high-THC exposure was associated with dysregulation of pathways related to “Remodeling of Epithelial Adherens Junctions”, “Axonal Guidance Signaling”, and “Glucocorticoid Receptor Signaling”. THC discontinuation for 140 days was associated with decreased “LXR/RXR Activation”, “Acute Phase Response Signaling”, and “Production of Nitric Oxide and ROS in Macrophages”. 5 proteins dysregulated with THC and significantly restored to baseline included products of CHIT1, FGG, MMP9, TGFBI, and CFB genes (Figure 1D). CHIT1 has been associated with oligozoospermia and MMP9 protein with sperm motility. CONCLUSIONS: This study demonstrated the translational strength of the RM model to study male reproductive health; there was significant seminal fluid proteome homology between humans and RMs. THC-exposed RMs are likely to exhibit proteins in their seminal ejaculate that are involved in regulatory processes that could potentially contribute to male infertility. Source of Funding: OHSU Faculty SEED Award and ASRM Pilot and Exploratory Grant © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jasper C. Bash More articles by this author Lyndsey E. Shorey-Kendrick More articles by this author Carol B. Hannah More articles by this author Charles A. Easley More articles by this author Eliot Spindel More articles by this author Jamie O. Lo More articles by this author Jason C. Hedges More articles by this author Expand All Advertisement PDF downloadLoading ...

Eosinophilic esophagitis-associated epithelial remodeling may limit esophageal carcinogenesis.

Under homeostatic conditions, esophageal epithelium displays a proliferation/differentiation gradient that is generated as proliferative basal cells give rise to suprabasal cells then terminally differentiated superficial cells. This proliferation/differentiation gradient is often perturbed in esophageal pathologies. Basal cell hyperplasia may occur in patients with gastroesophageal reflux disease (GERD), a condition in which acid from the stomach enters the esophagus, or eosinophilic esophagitis (EoE), an emerging form of food allergy. While GERD is a primary risk factor for esophageal cancer, epidemiological data suggests that EoE patients do not develop esophageal cancer. In order to investigate the impact of EoE and esophageal cancer specifically on the cellular landscape of esophageal epithelium, we perform single cell RNA-sequencing in murine models of EoE and esophageal cancer, specifically esophageal squamous cell carcinoma (ESCC). We further evaluate modules of co-expressed genes in EoE- and ESCC-enriched epithelial cell clusters. Finally, we pair EoE and ESCC murine models to examine the functional relationship between these pathologies. In mice with either EoE or ESCC, we find expansion of cell populations as compared to normal esophageal epithelium. In mice with EoE, we detect distinct expansion of 4 suprabasal populations coupled with depletion of 2 basal populations. By contrast, mice with ESCC display unique expansion of 2 basal populations and 1 suprabasal population, as well as depletion of 2 suprabasal populations. Senescence, glucocorticoid receptor signaling, and granulocyte-macrophage colony-stimulating factor pathways are associated with EoE-enriched clusters while pathways associated with cell proliferation and metabolism are identified in ESCC-enriched clusters. Finally, our in vivo data demonstrate that exposure to EoE inflammation limits tumor burden of esophageal carcinogenesis. Our findings provide the first functional investigation of the relationship between EoE and esophageal cancer and suggest that esophageal epithelial remodeling events occurring in response to EoE inflammation may limit esophageal carcinogenesis. This investigation may have future implications for leveraging allergic inflammation-associated alterations in epithelial biology to prevent and/or treat esophageal cancer.

Regulation of Energy Metabolism and Anti-Inflammatory Activities of Mastiha Fractions from Pistacia lentiscus L. var. chia

Pistacia lentiscus L. var. chia resin (Chios Mastiha), the first natural chewing gum, is widely used in Mediterranean cuisine and has been used in traditional medicine from ancient times. Regarding its chemical composition, Chios Mastiha is known to be rich in triterpenes. Triterpenes have a similar structure to glucocorticoids (GCs), the steroid hormones that exert strong anti-inflammatory activities and play crucial roles in the regulation of cellular metabolism. To simplify the characterization of the bioactive compounds of Mastiha resin, three different polarity fractions were isolated and were further analyzed regarding their main chemical composition and an assessment of their biological activities. The biological assessment focused on the evaluation of the potential anti-proliferative, anti-inflammatory, and apoptotic activities as well as the possible interference of the three different polarity Mastiha fractions with the glucocorticoid receptor signaling, with the aim of characterizing the biochemical mechanisms of the actions of the Mastiha fraction. Applying MTT cell viability assay, luciferase/β-galactosidase assay, and Western blot analysis showed that Chios Mastiha apolar, medium-polar, and polar fractions reduced the HEK293 cell viability in a dose-dependent manner, possibly by mitochondrial-mediated induction of apoptosis. Medium-polar and polar Mastiha fractions also suppressed the GR and NF-κΒ transcriptional activation and the p65 protein levels. These activities were accompanied by the modulation of protein levels of regulatory molecules playing a crucial role in cellular energy homeostasis, such as GR, phosphoenolpyruvate carboxykinase (PEPCK), and/or peroxisome proliferator-activated receptor alpha (PPARα), and by the induction of phosphorylation and the activation of the AMP-activated protein kinase (AMPK). The medium-polar fraction was found to be enriched in triterpenes, such as lupeol, 24Z-masticadienonic acid methyl ester, and 24Z-isomasticadienonic acid methyl ester, and it was the most active one, so we propose that triterpenes in medium-polar fraction are possibly the bioactive compounds responsible for Mastiha's regulatory actions on energy metabolism and anti-inflammatory activities via interference with GR, NF-κΒ, and AMPK signaling. This highlights its potential applications in many fields of pharmaceutical, cosmetic, and nutraceutical interest.

Protective effects and mechanisms of the Erzhi formula on glucocorticoid induced primary cortical neuron injury.

High concentrations of glucocorticoids (GC) can cross the blood-brain barrier into the brain parenchyma, triggering a stress state that can lead to a range of physiological changes. This study investigated whether Erzhi formula has neuroprotective effects against glucocorticoid damage by establishing a dexamethasone-induced primary cortical neuron injury model in vitro. The results showed that Erzhi formula could reduce dexamethasone-induced apoptosis in primary cultured cortical neurons and improve synaptic damage. Further, network pharmacological analysis revealed that Erzhi formula may exert antidepressant effects by multi-component, multi-target, and multi-pathway characteristics, in which Salidroside, Biochanin-A and other ingredients are key components, HSD11B1, NR3C1, and other proteins are key targets, and steroid metabolism may be a key process in its action. Moreover, our study found that the neuroprotective effect of Erzhi formula might be related to the 11β-HSD1-GC/glucocorticoid receptor (GR) signaling pathway. The Erzhi formula could significantly inhibit the activity of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) in vitro using homogeneous time-resolved fluorescence. In addition to providing evidence for the pharmacological effects of the Erzhi formula, the present study lays down the foundation for subsequent experiments.

Cortisol/glucocorticoid receptor: a critical mediator of the ovulatory process and luteinization in human periovulatory follicles.

Do cortisol/glucocorticoid receptors play an active role in the human ovary during ovulation and early luteinization? The ovulatory hCG stimulation-induced glucocorticoid receptor signaling plays a crucial role in regulating steroidogenesis and ovulatory cascade in human periovulatory follicles. Previous studies reported an increase in cortisol levels in the human follicular fluid after the LH surge or ovulatory hCG administration. However, little is known about the role of cortisol/glucocorticoid receptors in the ovulatory process and luteinization in humans. This study was an experimental prospective clinical and laboratory-based study. An in vivo experimental study was accomplished utilizing the dominant ovarian follicles from 38 premenopausal women undergoing laparoscopic sterilization. An in vitro experimental study was completed using the primary human granulosa/lutein cells (hGLC) from 26 premenopausal women undergoing IVF. This study was conducted in a private fertility clinic and academic medical centers. Dominant ovarian follicles were collected before the LH surge and at defined times after hCG administration from women undergoing laparoscopic sterilization. Primary hGLC were collected from women undergoing IVF. hGLC were treated without or with hCG in the absence or presence of RU486 (20 µM; dual antagonist for progesterone receptor and glucocorticoid receptor) or CORT125281 (50 µM; selective glucocorticoid receptor antagonist) for 12 or 36 h. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and ovulatory cascade was studied with RT-quantitative PCR and western blotting. The production of cortisol, corticosterone, and progesterone was assessed by hormone assay kits. hCG administration upregulated the expression of hydroxysteroid 11-beta dehydrogenase 1 (HSD11B1), nuclear receptor subfamily 3 group C member 1 (NR3C1), FKBP prolyl isomerase 5 (FKBP5), and FKBP prolyl isomerase 4 (FKBP4) in human ovulatory follicles and in hGLC (P < 0.05). RU486 and CORT125281 reduced hCG-induced increases in progesterone and cortisol production in hGLC. The expression of genes involved in glucocorticoid receptor signaling, steroidogenesis, and the key ovulatory process was reduced by RU486 and/or CORT125281 in hGLC. N/A. The role of cortisol/glucocorticoid receptors demonstrated using the hGLC model may not fully reflect their physiological roles in vivo. Successful ovulation and luteinization are essential for female fertility. Women with dysregulated cortisol levels often suffer from anovulatory infertility. Deciphering the functional role of glucocorticoid receptor signaling in human periovulatory follicles enhances our knowledge of basic ovarian physiology and may provide therapeutic insights into treating infertility in women. This study was supported by P01HD71875 (to M.J., T.E.C., and M.B.) and R01HD096077 (to M.J.) from the Foundation for the National Institutes of Health and the BTPSRF of the University of Kentucky Markey Cancer Center (P30CA177558). The authors report no competing interests. N/A.