United States

The adrenal gland is the site of steroid hormone synthesis. These hormones regulate many physiological processes including metabolism, blood pressure and volume, and sexual characteristic development. While signaling pathways, steroidogenic enzymes, and transcription factors have all been well characterized, little is known about the contribution of RNA-binding proteins (RBPs) in regulating steroidogenesis. We recently demonstrated that regulated RNA decay controls the timing of the steroidogenic gene expression response and identified 9 RBPs that regulate aldosterone production in an immortalized human cell line model for steroidogenesis, H295R. One of the RBPs we identified was Musashi-2 (MSI2), which is required for development of other steroidogenic tissues such as ovary and testis in mice. We found that MSI2 mRNA is enriched in human adrenal glands relative to other tissues, positively correlates in expression with mRNAs encoding steroidogenic enzymes, and abundantly expressed in H295R cells. Staining of normal human adrenal gland sections demonstrated that MSI2 protein is expressed in all three layers of the human adrenal cortex. Taken together, we hypothesized that MSI2 promotes steroidogenesis through regulating expression of target mRNAs encoding. Using the selective MSI2 inhibitor Ro-08-2750 (Ro) we found that aldosterone and cortisol production is decreased in a dosage dependent manner in H295R cells, which is consistent with siRNA knockdown of MSI2. We performed RNA-immunoprecipitation and sequencing and identified 1428 mRNA targets of MSI2 in H295R cells. A target of interest was Stearoyl-CoA Desaturase (SCD), which functions to convert saturated fatty acids to unsaturated fatty acids that promote formation of lipid droplets. Cholesterol esters in lipid droplets are a source of cholesterol used to produce all steroid hormones. Ro treatment of H295R cells resulted in a suppression of SCD mRNA and protein induction. Furthermore, inhibition of SCD with A939572 caused a dosage dependent decrease in aldosterone production. These data suggest that potentiation of stimulation dependent SCD induction is one mechanism by which MSI2 promotes steroidogenesis. It will be important to determine if and how MSI2 promotes steroidogenesis in primary human adrenal cells and mouse models. Since it is possible to deliver oligonucleotides to the adrenal cortex, disruption of MSI2-SCD mRNA regulatory interactions may open new therapeutic avenues for modulate aldosterone production.

ENDO 2022

Musashi-2 Regulatory Interactions Promote Adrenal Steroidogenesis

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

technical paper

Aberrant macrophage function is implicated in acute and chronic inflammatory settings, and central to diseases such as obesity, atherosclerosis and cancer. However, the transcriptional and epigenomic programs driving macrophage polarization toward distinct phenotypic states are not well understood. Macrophage phenotypes exist on a spectrum, with pro-inflammatory (M1-like) and homeostatic, tissue repair (M2-like) states viewed as polar opposites and relatively stable. The main driver of the M2-like program is a type-2 cytokine, IL4 which signals to STAT6 followed by the Krüppel-like factor 4 (KLF4), a master regulator for numerous M2-specific genes. Reportedly, glucocorticoid (GC) hormones also promote the M2-like state, although the extent to which the phenotypes elicited by such disparate signals overlap is unknown. Interestingly, we discovered that GRIP1, a well-known cofactor for nuclear receptors including GR, serves as a KLF4 coactivator, suggesting that GRIP1 could mediate transcriptional and phenotypic convergence of the IL4- and GC-driven pathways. Gene expression profiling on differentially polarized macrophages revealed a highly significant overlap between M2IL4 and M2GC transcriptomes which was mirrored by the H3K27ac de novo enhancer landscape. Notably, loss of GRIP1 blunted both up-regulation of M2- and downregulation of M1-associated genes irrespective of the signal driving M2 polarization. At the cellular level, polarization with either IL4 or GCs enhanced macrophage phagocytosis relative to non-polarized M0, and did so in a GRIP1-dependent manner. Finally, in the dextran sulphate sodium (DSS)-induced colitis model of intestinal inflammation and tissue damage, GRIP1-cKO mice lacking GRIP1 in myeloid cells, including macrophages, displayed more severe destruction of the colonic mucosa and greater immune cell infiltration into the mucosa and submucosa. Moreover, in total colon RNA, the abundance of many characteristic M2 transcripts was reduced in the cKO mice. Surprisingly, the expression of proinflammatory mediators such as TNF, IL1b and IL23 was unaffected by GRIP1 deletion, suggesting that the more severe disease in the cKO stems primarily from the failure of M2-like resolving macrophage to efficiently mitigate tissue destruction and repair the damage. Together, these findings point to GRIP1 as a crucial regulator of macrophage polarization to the homeostatic state in vitro and in vivo.

The Role Of Nuclear Receptor Coregulator Grip1 In Homeostatic Programming Of Macrophages

OBJECTIVE: Despite recent advances in diabetes technology, the distribution of healthcare resources is often unequal among racial and socio-economic groups. A recent multicenter study looking at technology use amongst young adults with T1D demonstrated that socioeconomic status (SES) was not the sole driver of racial/ethnic disparities and that adjustment for income, insurance level, and health literacy did not significantly attenuate disparities. Here we present a retrospective cohort study looking at racial/ethnic disparities in insulin pump and CGM use in adults with T1D at an urban university hospital system.
METHODS: We reviewed the charts of all adults aged 18 to 89 with T1D who attended the University of Maryland Center for Diabetes and Endocrinology (UMCDE) between 1/2019 and 12/2019. Age, gender, race/ethnicity, type of insurance, ZIP code, BMI, A1c, method of insulin delivery, and type of glucose monitoring device were obtained from each chart. We utilized the 2019 US Census data to determine the median income for each ZIP code. Patient charts were re-accessed in 2021 to check for changes in diabetes technology use from 1/2020 until 12/2021. Logistic regression modeling was done in PRISM to control for age, gender, race, insurance type, ZIP code, last known provider, BMI, and A1c. Hosmer-Lemeshow test was done to evaluate goodness of fit. Stratified analysis was done for potential confounders such as insurance type and income. The Breslow-Day test and Likelihood Ratio test for nested models were used to evaluate their degree and significance.
RESULT: A total of 532 adults were included in the study; 277 identified as non-Hispanic (NH) White, 204 identified as NH Black, 7 identified as Hispanic, and 44 identified as other racial/ethnic groups. NH White adults had a significantly higher insulin pump use when compared to NH Black (adjusted ratio 54.0% vs. 18.4%, OR 2.43 after regression, 95%CI [1.41-4.22]). Similarly, NH White adults had significantly higher CGM use when compared to NH Black (66.5% vs. 35.2%, OR 2.00 after regression, 95%CI [1.24-3.22]). Disparities between NH White and NH Black patients did not resolve throughout 2020-2021. Insurance type was a potential effect modifier for CGM prescriptions between NH White and NH Blacks (Crude OR 4.04, Commercial Strata OR 2.68, Medicaid 1.79, Medicare 2.79, Other/Self-pay 25.7, p=0.039 on Breslow-Day test). There was large heterogeneity in the rate of Pump or CGM prescriptions between providers.
CONCLUSION: NH White adults with T1D were significantly more likely to have been prescribed diabetes-related technologies when compared to other racial/ethnic groups, and this difference did not resolve over 2 years of follow-up. Future directions include a comprehensive survey of our population regarding patient-derived and patient-perceived factors driving disparities beyond socioeconomic status.

Racial and Ethnic Disparities in Diabetes Technology Use amongst Persons with Type 1 Diabetes

Background: Insulin pump use provides many advantages for the management of type 1 diabetes (T1D), but studies have shown racial, ethnic, and socioeconomic inequities in the use of this technology. As the prevalence of insulin pump use has increased over the past two decades, we aimed to determine whether these inequities have increased or diminished over time.
Methods: We used data from the population-based SEARCH for Diabetes in Youth study to perform a serial cross-sectional analysis to evaluate changes over time in insulin pump use in participants <20 years old with T1D by racial and ethnic group, health insurance, household income, and formal parental education across 4 phases of the study (Phase 1: 2001-2005, Phase 2: 2006-2010, Phase 3: 2011-2015 and Phase 4: 2016-2019). Data from the last study visit were analyzed for those with multiple visits within a phase. Multivariable generalized estimating equations with a binomial distribution were used to assess probability of insulin pump use, and models were further adjusted for the other predictors--age at visit, diabetes duration, sex and clinic site--with clustering for individuals.  An interaction effect between each primary predictor (race and ethnicity, health insurance, household income, education) and the phase variable was used to assess temporal changes.
Results: The prevalence of insulin pump use increased from 30% in Phase 1 to 58.3% in Phase 4 . Compared to those of non-Hispanic (NH) white race, odds of pump use in Hispanic participants was 0.08 (95% CI 0.01-0.63) in Phase 2 and 0.65 (95% CI 0.48-0.87) in Phase 4 with significantly reduced disparities over time (p=0.05). The disparities for Black participants and those of other races did not change over time, but was significantly lower than for white participants in all periods ( 0.28 (95%CI 0.21-0.37) and 0.43 (95%CI 0.26-0.71), respectively). Across all time periods, T hose with some high school/high school degree and those with some college had lower odds of pump use compared to those with at least a bachelor's degree 0.38 (95%CI 0.30-0.48) and 0.69 (95%CI 0.57-0.82). T hose with public insurance had lower odds for pump use compared to those with private insurance 0.84 (0.68-1.03). T hose with an income of < $25K, $25K-$49K, and $50K-74K had lower odds of pump use of 0.43 (95%CI 0.34-0.54), 0.57 (95%CI 0.46-0.71) and 0.80 (95%CI 0.65-0.97) compared to those with household incomes ≥ $75,000.
Conclusion: Over the past two decades, there have been no improvements in the ethnic, racial, and socioeconomic inequities in insulin pump use among youth with T1D.  Studies that evaluate barriers or test interventions to improve technology access are needed to address the persistent inequities in diabetes care.

Assessing Longitudinal Disparities in Insulin Pump Use Among Youth with Type 1 Diabetes

BACKGROUND:  Diabetic kidney disease (DKD) develops in approximately 40% of adults with diabetes and is the leading cause of chronic kidney disease (CKD). Less is known about early DKD, particularly in ethnically diverse populations that include Black, Hispanic/Latinx, and Asian Americans. Current guidelines recommend that patients with diabetes be screened annually for DKD. Albuminuria is the first sign of CKD and is associated with CKD progression, cardiovascular events, and death. This study characterizes ethnic differences in the prevalence of albuminuria in a diverse population of adults with diabetes with normal range eGFR.
METHODS:  This retrospective study was conducted in a large healthcare system where patients with diabetes are annually monitored for DKD. We identified adults with diabetes aged 45-74 years, eGFR ≥60 mL/min/m2 based on serum creatinine (using the CKD-EPI equation without race), and assessment of urine albumin-creatinine ratio (ACR), urine protein-creatinine ratio (PCR) and/or urine protein dipstick in 2015. Albuminuria was based on urine ACR (95%), otherwise, from urine PCR (1%) converted to ACR, and, if unavailable, from urine protein dipstick (4%) converted to ACR. The cohort was restricted to patients with diabetes onset after health plan enrollment to calculate diabetes duration. Hypertension was identified by clinical diagnosis. Modified Poisson regression with robust variance was used to examine the association of ethnicity and albuminuria, reporting relative risk (RR) with 95% confidence intervals (CI).
RESULTS: Among 79,184 adults with diabetes, eGFR ≥60, and urine albumin/protein assessment (age 60.5 ±7.7y, 54.0% male), 40.3% were non-Hispanic White, 9.6% Black, 26.3% Asian/Pacific Islander (PI), and 21.0% Hispanic/Latinx race/ethnicity. 69.9% had hypertension. Overall, 81.7%, 13.8%, and 4.6% had urine ACR <30 (normal), 30 to <300 (microalbuminuria), and ≥300 mg/g (macroalbuminuria), respectively, with albuminuria higher in males than females. By race/ethnicity, the prevalence of [micro/macroalbuminuria] was [15.7/5.4%] Asian/PI, [13.8/4.8%] Black, [13.1/4.5%] Hispanic/Latinx, and [12.8/4.1%] non-Hispanic Whites. Among Asian/PIs, prevalence ranged from [19.5/8.4%] Hawaiian/PI and [18.2/7.1%] Filipino to [14.5/3.6%] Chinese and [11.2/3.4%] South Asian. In multivariable analyses, adjusting for age, sex, diabetes duration, and hypertension, Asian/PI (RR 1.3, CI 1.2-1.3) was associated with higher risk of albuminuria compared to non-Hispanic White, but Black (RR 1.0) and Hispanic/Latinx (RR 1.0) were not. Among Asian/PIs, with Chinese as reference, Filipino (RR 1.3, CI 1.2-1.4) and Hawaiian/PI (RR 1.4, CI 1.3-1.6) had higher risk, South Asian had lower risk (RR 0.7, CI 0.7-0.8), and Japanese and Southeast Asian had similar risk of albuminuria.
CONCLUSIONS: The risk of albuminuria in adults with diabetes and normal range eGFR was higher in Asian/PI adults. Among Asian/PIs, the risk was higher among Filipino and Hawaiian/PI and lower in South Asian compared to Chinese adults, supporting the importance of disaggregating Asians when examining microvascular outcomes. Future studies should examine ACEI/ARB use and albuminuria progression.

Ethnic Differences in Albuminuria among Adults with Diabetes and Normal Range Estimated Glomerular Filtration Rate (eGFR).

Tirzepatide, a dual GIP/GLP-1 receptor agonist, demonstrated clinically meaningful reductions in HbA1c and body weight that were significantly greater compared with semaglutide in patients with type 2 diabetes on background metformin. In a pre-specified subgroup analysis, we evaluated the efficacy and safety of tirzepatide in the Hispanic or Latino population of SURPASS-2.
In this open-label, 40-week, Phase 3 study (N=1878; mean baseline HbA1c 8.28%; BMI 34.2 kg/m²; Hispanic or Latino ethnicity 70%) participants were randomized 1: 1: 1: 1 to tirzepatide (5, 10, 15 mg; doses were double-blinded) or semaglutide (1 mg). Efficacy analyses included all randomised patients who received ≥1 treatment dose (modified intention-to-treat [mITT] population) while patients were on treatment without rescue therapy and excluding patients who discontinued study drug due to inadvertent enrollment (efficacy estimand). Safety analyses were performed in the mITT population, including all data from the start of treatment to the end of the safety follow-up. The least square mean (LSM) change from baseline was calculated within each subgroup using a mixed model for repeated measurements (MMRM) with country, baseline value, treatment group, visit, and treatment-by-visit interaction as the fixed effect, and patient as the random effect. MMRM for weight change also included baseline HbA1c group (≤8.5%, >8.5%) as a covariate.
In patients of Hispanic or Latino ethnicity, the LSM change from baseline in HbA1c at 40 weeks was -2.11%, -2.46%, and -2.47% with tirzepatide 5, 10, and 15 mg, respectively, and -1.93% with semaglutide (all p<0.001). The respective changes in patients not of Hispanic or Latino ethnicity were -2.03%, -2.14%, -2.43%, and -1.69% (all p<0.001). Reductions in HbA1c were significantly greater for all tirzepatide doses compared with semaglutide in patients of both Hispanic or Latino and non-Hispanic or Latino ethnicity (all p≤0.027). In Hispanic or Latino patients, body weight change was -6.9 kg with tirzepatide 5 mg, -9.7 kg with 10 mg, -10.5 kg with 15 mg, and -5.6 kg with semaglutide (all p<0.001). In non-Hispanic or Latino patients, the changes from baseline in body weight were -9.9 kg, -11.6 kg, -17.1 kg, and -7.5 kg, for the tirzepatide doses and semaglutide, respectively, (all p<0.001). In both subgroups, reductions in body weight were significantly greater than semaglutide with all tirzepatide doses (all p≤0.013).
The most common adverse events were gastrointestinal in the tirzepatide and semaglutide arms, including nausea (Hispanic or Latino 11-17% and 13%; non-Hispanic or Latino 32-36% and 29%), diarrhoea (10-13% and 10%; 15-24% and 17%) and vomiting (4-7% and 7%; 10-16% and 12%).
Consistent with the primary SURPASS-2 results, in a Hispanic or Latino population with type 2 diabetes, tirzepatide demonstrated greater reductions from baseline in HbA1c and body weight than the selective GLP-1 receptor agonist semaglutide, with a comparable safety profile.

Efficacy and Safety of Tirzepatide Versus Semaglutide in a Hispanic or Latino Population: A Prespecified Subgroup Analysis of SURPASS-2

Introduction
Hyperglycemic emergencies, including diabetic ketoacidosis (DKA) and hyperglycemic hyperosmolar state (HHS), are common reasons for hospitalization among adults with diabetes. Household income is a known determinant of health and impacts access to medications and health services. Outpatient services and medications impact readmission rates as medication nonadherence and inadequate follow-up are common underlying precipitants of DKA and HHS. This study outlined the trends in readmissions following initial hospitalization for hyperglycemic emergencies.
Methods
This was a longitudinal trend analysis of the National Readmission Database (NRD), which is the largest readmission database in the US. The study period was from 2010 to 2018, and involved adults with DKA and HHS as the principal discharge diagnosis. We excluded elective and December admissions. Using the unique visit linkage variable within the NRD, we estimated the 30-day all-cause readmission rate (30ACR) stratified by the median household income for the patient's zip code quartile (MHOI). Outcomes included comparing trends in 30ACR in DKA and HHS between the low-income quartile (LIQ) and high-income quartile (HIQ) as well as trends in 30-day DKA and HHS specific readmission rates between the LIQ and HIQ. We estimated the estimated annual percent change (APC) via logistic trend regression adjusted for age categories, sex, and MHOI.
Results
From 2010 to 2018, there was a trend towards increasing readmission rates overall. Among DKA hospitalizations, patients in the LIQ had a higher 30ACR compared to the HIQ (17.1% vs 12.3% in 2010 and 21.7% vs 15.3% in 2018). The LIQ patients also had a higher DKA-specific readmission rate over the period (7.2% vs 5.1% in 2010 and 7.6% vs 10.4% in 2018). The adjusted APC for DKA 30ACR was 1.6%, with the overall odds of readmission higher in the LIQ (adjusted odds ratio [aOR]: 1.50, 95% CI: 1.42 - 1.58, p<0.001) compared to the HIQ. Among HHS hospitalizations, patients in the LIQ had a higher 30ACR compared to the HIQ (12.2% vs 10.4% in 2010 and 14.8% vs 12.4% in 2018). The LIQ patients also had a higher HHS-specific readmission rate over the period (0.8% vs 0.6% in 2010 and 1.2% vs 1.1% in 2018). The adjusted APC for HHS 30ACR was 1.9%, with the overall odds of readmission higher in the LIQ (adjusted odds ratio [aOR]: 1.13, 95% CI: 1.02 - 1.26, p=0.025) compared to the HIQ.
Conclusion
There seem to be rising readmission rates following hyperglycemic emergency hospitalizations with significant disparity based on median household income. This difference is was higher DKA. Though the study design limits the elaboration on specific income-related factors that are at play, these findings establish the need for further exploring the barriers that predispose patients to readmissions to decrease healthcare utilization costs.

Widening Economic Disparity in Rates of Readmissions Following Hospitalizations for Hyperglycemic Emergencies Among US Adults over a Decade.

Background: Despite newer diabetes medications and technology being available, therapeutic inertia persists and there are more people living with "uncontrolled" diabetes than meeting A1c targets. Here we evaluate how the ECHO© model for diabetes management changed prescribing practices among participating primary care providers (PCPS).
Methodology: Three unique diabetes ECHO programs evaluated comfort or perception of prescribing practice changes for local community PCPs (n=74) in four regions (Illinois, District of Columbia, New Mexico, and Washington). One site representing two regions collected pre- and post-program participant surveys (n=45) while two sites collected post-program surveys only (n=29), in which respondents reported perceptions of changes resulting from participation in ECHO. Participants reported their use of technology (professional and personal continuous glucose monitoring (CGM) and insulin pumps) and medications (insulin and non-insulin).
Results: On a 4-point Likert scale, PCPs' (n=45) average self-reported prescription use for newer diabetes medications with cardiovascular indications increased from 3.07 (sometimes) to 3.84 (sometimes-always).

Combating Therapeutic Inertia: Project ECHO for Diabetes Improves Primary Care Providers' Comfort and Use of Diabetes Medication and Technology

Breast cancer is the most common invasive cancer in females worldwide and accounts for 16% of all female cancers, 22.9% of invasive cancers in women, and 18.2% of all cancer-related deaths worldwide. About 80% of these cancers, once established, depend on available estrogens (i.e., estradiol) to grow. The production of estrogens occurs via a catalytic process governed by the enzyme Aromatase. This process occurs primarily in the ovaries in pre-menopause, while in post-menopause, this occurs in peripheral tissues, including the breast. In patients whose cancers have specific hormone receptors, such as Estrogen Receptor positive (ER+), Progesterone Receptor positive (PR+), and Human Epidermal Growth Factor 2 Receptor positive (HER2+), these tumors can continue receiving nutrients and signals to grow, which provides targets for therapy. Although many of the currently available drugs for breast cancer seek to suppress this process and thus reduce available estrogens, they have numerous harmful side effects and have high rates of therapy failure due to the development of resistance. Therefore, new therapies and targets for this devastating disease are needed. We observed that breast tumors had increased estrogen production in postmenopausal women, leading us to question the cause of this increase. Further, the complete mechanism of aromatase action and the possibility of targetable cofactors remained previously unknown. Thus, we set out to identify the mechanism of Aromatase action to provide prognostic and therapeutic insight into this distressing disease. As Aromatase is an endoplasmic reticulum (ER) resident protein, we purified ER fractions from breast tissues, separated through 1D, and then each ten kDa fractions were further separated and analyzed through Mass-spectrometry to determine the cofactors associated with aromatase activity. We observed an 11 mer peptide LEVVVDQPMER, which on cloning resulted in a 207 amino acid protein not present in the database. This previously undiscovered protein is colocalized with Aromatase in the ER. We saw that knocking down its expression by siRNA in wild-type breast cells (MCF-12A) or tumorigenic MCF-7 or T47-D cells increased estradiol synthesis significantly without affecting aromatase expression. Further biochemical experiments showed that Aromatase and the 207 amino acid protein interacted directly without affecting the expression of other ER-resident proteins. Additionally, our experiments showed that the new protein is not hormonally stimulated or transcriptionally inhibited and thus, regulates the aromatase catalytic activity of estradiol synthesis. Therefore, we have termed this new protein "Aromatase Interacting Partner in Breast" (AIPB).

Cloning, Sequencing, and Characterization of Aromatase Interacting Partner in Breast (AIPB). A New Protein Regulating Estradiol Synthesis.

Estrogen receptor positive (ER+) breast cancer is the most common subtype among all breast cancers and is responsible for most breast cancer related deaths. Endocrine therapies, such as selective estrogen receptor modulators (SERM) and aromatase inhibitors (AI), often given in combination with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, are initially effective but the majority of advanced ER+ breast cancers eventually become refractory to endocrine therapies. Therefore, understanding the underlying molecular mechanisms that enable or promote resistance to endocrine therapies may lead to novel therapeutic targets.Cancer cells have a high energy demand and as a result consume greater quantities of glucose to meet their energy and anabolic demands to support cell growth and proliferation. In this study, we used breast cancer cell models that are endocrine therapy sensitive and resistant and investigated the metabolic flux of glucose by growing the cells in the presence of the stable glucose isotope, UC13-Glucose. We measured the C13-labelled metabolites of glycolysis and the TCA cycle. Glucose consumption was higher in endocrine therapy resistant MCF7/LCC9 (LCC9) cells compared with parental MCF7 (MCF7) and estrogen independent, endocrine therapy sensitive MCF7/LCC1 (LCC1) cells. Notably, in LCC9 cells the m+4 and m+6 isotopomer of C-13 labelled citrate was absent. Further, in LCC9 cells, C-13 labelled fumarate was very low while the succinate: fumarate ratio was markedly higher. These results suggest that the TCA cycle is impaired in LCC9 cells and the conversion of succinate to fumarate is dysregulated. Succinate dehydrogenase (SDH) enzyme is responsible for catalytic conversion of succinate to fumarate. SDH is a multimeric protein comprised of four different subunits and SDH assembly factor 2 (SDHAF2), which is a tumor suppressor gene. Our study further showed that SDHAF2 protein was under expressed in LCC9 cells compared with LCC1 cells, likely contributing to lower SDH activity. Intriguingly, when SDH activity was inhibited using dimethylmalonate, the LCC9 were re-sensitized to both Fulvestrant and 4-hydroxytamoxifen. Data analysis of clinical samples shows that poor relapse free survival in ER+ breast cancer patients is correlated with lower expression levels of SDHAF2. Overall, this study suggests that targeting succinate metabolism may help to restore sensitivity to Fulvestrant and tamoxifen in endocrine therapy resistance in ER+ breast cancer cells.

Targeting Succinate Metabolism in Endocrine Therapy Resistant, ER Positive, Breast Cancer Cells

The transcription factor (TF) GRHL2 controls normal epithelial differentiation in different tissues. It is expressed in all breast cancer subtypes except those with a mesenchymal phenotype (claudin-low subtype), where its ectopic expression leads to induction of a mesenchymal-epithelial transition (MET) (1). Here we explored the mechanisms by which GRHL2 induces MET in claudin-low breast cancer cell lines using a combination of ChIP-seq and RNAseq analyses upon modulation of GRHL2 expression in mesenchymal and epithelial cell lines. Direct DNA binding by GRHL2 was enriched in proximity of genes positively regulated in mesenchymal cell lines, which include several epithelial TFs as well as an array of epithelial genes. Biotinylation and mass spectrometry analysis of membrane proteins confirmed surfaceome remodeling via induction of membrane proteins with roles in cell-cell contacts and adhesion. Repression of mesenchymal genes was mediated in part by direct induction of miRNAs targeting mesenchymal transcription factors. In addition, GRHL2 strongly induced expression of the cofactor VGLL1, whose expression is high in the triple-negative (TNBC) basal-like subtype. VGLL1 overexpression in mesenchymal cell lines and suppression in basal-like TNBC cells confirmed its role in mediating part of GRHL2 signaling, in particular via interaction with and modulation of TEAD transcription factors. In addition, GRHL2 also interacted with VGLL1 upon its induction in mesenchymal cells, recruiting it to some of its binding motifs, suggesting a multilayered cross-talk between GRHL2 and TEAD factors. Together, our results indicate that GRHL2 acts as a master regulator in the induction of TNBC epithelial differentiation via direct upregulation of epithelial transcription factors, miRNAs and TEAD cofactor VGLL1, resulting in both upregulation of epithelial genes and suppression of mesenchymal genes.(1) Cieply B. et al., Cancer Res. 2013 Oct. 1;73 (20): 6299-309

Musashi-2 Regulatory Interactions Promote Adrenal Steroidogenesis

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