Targeting the SIN1 mediated TTK/LDHA-H3K18la-GLUT3 axis disrupts metabolic-epigenetic crosstalk and suppresses progression in hyperglycolytic breast cancer.

Nutrient restriction reprograms cellular signaling and metabolic network to shape cancer phenotype. Lactate dehydrogenase A (LDHA) has a key role in aerobic glycolysis (the Warburg effect) through regeneration of the electron acceptor NAD+ and is widely regarded as a desirable target for cancer therapeutics. However, the mechanisms of cellular response and adaptation to LDHA inhibition remain largely unknown. Here, we show that LDHA activity supports serine and aspartate biosynthesis. Surprisingly, however, LDHA inhibition fails to impact human melanoma cell proliferation, survival, or tumor growth. Reduced intracellular serine and aspartate following LDHA inhibition engage GCN2-ATF4 signaling to initiate an expansive pro-survival response. This includes the upregulation of glutamine transporter SLC1A5 and glutamine uptake, with concomitant build-up of essential amino acids, and mTORC1 activation, to ameliorate the effects of LDHA inhibition. Tumors with low LDHA expression and melanoma patients acquiring resistance to MAPK signaling inhibitors, which target the Warburg effect, exhibit altered metabolic gene expression reminiscent of the ATF4-mediated survival signaling. ATF4-controlled survival mechanisms conferring synthetic vulnerability to the approaches targeting the Warburg effect offer efficacious therapeutic strategies.

CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have revolutionized the treatment of ER+ metastatic breast cancer (MBC), significantly prolonging survival. However, this combination is not curative, and tumors eventually acquire resistance. Following progression on this combination, patients are left with limited treatment options. A diverse array of mechanisms of resistance to CDK4/6i + antiestrogens have been described. However, laboratory models that capture this heterogeneity of resistance mechanisms are lacking. Patient-derived organoids (PDOs) provide a rapid, robust and reliable platform that recapitulates intra-tumor heterogeneity, partially mimics the cancer microenvironment, and accurately predicts drug response. We aspired to generate a platform of CDK4/6i-resistant breast cancer PDOs to serve as models for understanding acquired resistance to CDK4/6i + antiestrogens and identifying therapies to overcome resistance. We successfully established 16 PDOs out of 32 biopsies (50% efficiency) of metastates from patients with ER+ MBC progressing on CDK4/6i (palbociclib or abemaciclib) + antiestrogens (letrozole or fulvestrant; median response to combination = 9 months). Our collection includes PDOs derived from lobular (n=3) and inflammatory (n=2) breast cancers and reflects racial/ethnic diversity (50% white/not Hispanic; 18.8% Hispanic; 12.5% Black; 12.5% other/unknown). Next-gen sequencing reports were available for 10 patients from which organoids were established, revealing alterations associated with CDK4/6i and/or antiestrogen resistance, including ESR1 (n=2), HER2/ERBB2 (n=2), PTEN (n=2), CCNE1 (n=1), NF1 (n=1), and ARID1A (n=1). Furthermore, one biopsy and its derived organoid lost ER expression, and 5 harbored PIK3CA activating mutations. Thus far, we have performed targeted DNA-sequencing on 7 PDOs and found 13/15 (86.7%) concordance with driver mutations from tumor NGS reports. PDOs established from CDK4/6i-resistant biopsies maintained resistance to palbociclib or abemaciclib ± fulvestrant (500 nM each) in 3D cell viability assays (6 days of treatment). In contrast, control PDOs established from primary ER+ breast cancer surgical samples (n=2) were sensitive to each CDK4/6i ± fulvestrant (median viability for combination=25.6-31.5% for control vs 65.2-80.5% for resistant). GSEA analysis of RNA-seq data from control (n=2) and CDK4/6i-resistant (n=6) PDOs cultured in estrogen-depleted media ± 200 nM palbociclib revealed that palbociclib treatment resulted in downregulation of E2F target and G2M checkpoint signatures in control but not resistant PDOs. Next, we performed a high-throughput screen of 1,000 compounds in 3 resistant PDOs. One PDO showed exquisite sensitivity to G2/M cell cycle checkpoint components, including CDK1, PLK1, Aurora kinase, ATR, Chk1, and Wee1 inhibitors. Finally, treatment of 10 resistant PDOs with the CDK2/4/6 inhibitor PF-06873600 revealed that the CCNE1 (cyclin E1)-amplified PDO was highly sensitive (IC50=130 nM vs >1000 nM), supporting that CCNE1-amplified tumors are vulnerable to CDK2 inhibition. Conclusions: PDOs can be successfully established from ER+ MBC biopsies, maintain the resistant phenotype in culture, retain driver alterations found in tumors from which they were derived, and fail to suppress E2F targets following treatment with CDK4/6i. Therefore, these PDOs represent valuable models to understand and explore diverse mechanisms of CDK4/6i resistance and therapeutic vulnerabilities. Citation Format: Ariella B. Hanker, Sumanta Chatterjee, Yunguan Wang, Dan Ye, Dhivya R. Sudhan, Brian M. Larsen, Lauren C. Smith, Yilin Zhang, Vishal Kandagatla, Kuntal Majmudar, Ezequiel Renzulli, Saurabh Mendiratta, Kimberly Blackwell, Alana L. Welm, Sunati Sahoo, Nisha Unni, Cheryl M. Lewis, Tao Wang, Ameen A. Salahudeen, Carlos L. Arteaga. A platform of CDK4/6 inhibitor-resistant patient-derived breast cancer organoids illuminates mechanisms of resistance and therapeutic vulnerabilities [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-01.

miR-30a-5p suppresses breast tumor growth and metastasis through inhibition of LDHA-mediated Warburg effect

Background: Epithelial-mesenchymal transition (EMT) in breast cancer drives tumor invasion, metastasis and drug resistance. BRCA1 mutation carriers have a high risk for developing aggressive basal-like triple-negative breast cancers with EMT characteristics. It has been described that normal mammary epithelium of BRCA1-mutation carriers is comprised of aberrant luminal progenitor cell population resembling basal-like breast cancer cells. Yet, the role of BRCA1 in regulating epithelial cell plasticity in normal mammary gland remains largely obscure. Aim: Here, we used patient-derived normal and cancer organoid cultures from BRCA1-mutation carriers and noncarriers, to examine the effect of the BRCA1 mutation background on epithelial cell plasticity and the susceptibility to EMT. Methods and results: Mammary organoids were established from normal or cancer mammary tissues obtained from consenting patients undergoing lumpectomy or mastectomy. Isolated cells were plated in adherent basement membrane extract (BME) drops and overlaid with optimized organoid culture medium. EMT regulation in breast cancer is usually studied using cell lines and murine models. To determine the possibility to study EMT on patient-derived organoids, organoid culture media was optimized and established organoids were exposed to TGFβ to induce EMT. Morphological and phenotypic alterations were characterized using immunolabeling and visualization with confocal microscopy. Breast cancer organoids induced with TGFβ demonstrated EMT-like changes including the downregulation of E-Cadherin and upregulation of N-Cadherin. Moreover, breast cancer organoids showed typical cytoskeleton rearrangements. Here, the transformation from cortical actin into stress fibers formed in dedifferentiated mesenchymal cells, was visualized with actin staining. However, normal mammary organoids behaved differently. The cytoskeleton of BRCA-wild type (noncarriers) normal mammary organoids was not affected by the treatment. Curiously, normal mammary organoids derived from BRCA1-mutation carriers demonstrated EMT like changes upon exposure to TGFβ. To further determine mechanisms facilitating cell plasticity in BRCA1-mutation carriers, single cell RNA sequencing analysis on BRACA1-mutation carriers and noncarriers derived organoids is ongoing. Conclusion: The results suggest that BRCA1 germline mutation predisposes normal mammary epithelium dedifferentiation due to increased susceptibility to EMT. Citation Format: Rakefet Ruth Ben-Yishay, Naama Herman, Vered Noy, Eyal Mor, Aiham Mansur, Dana Ishay-Ronen. Normal mammary epithelium of BRCA1 mutation carriers demonstrates increased susceptibility to cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5847.

ANXA2P2/miR-9/LDHA axis regulates Warburg effect and affects glioblastoma proliferation and apoptosis

Introduction: Amplification and overexpression of c-Myc is very common in human cancers, including ovarian cancer. Heightened aerobic glycolysis (the Warburg effect) is a key metabolic hallmark of cancer and is thought to be under the control of c-Myc. JQ-1 is a selective small-molecure BET bromodomain inhibitor, and BET inhibition by JQ-1 leads to the downregulation of MYC transcription. Thus, JQ-1 functions as a novel c-Myc inhibitor and has been found to potently suppress tumor growth in hematologic malignancies. Thus, we aim to assess the impact of JQ-1 on the proliferation of human ovarian cancer cell lines and further explore its effects on cellular metabolism, especially the aerobic glycolysis pathway and its key enzyme—lactate dehydrogenase A (LDHA). Methods: Three human ovarian cancer cell lines (HEY and SKOV3) were used in this study. Cell proliferation was assessed by MTT assay after exposure to JQ-1 (generously provided by the laboratory of Dr. James E. Bradner). Cell cycle progression was evaluated by Cellometer. Apoptosis was assessed by Annexin V-FITC assay. Expression of c-Myc, p21, phosphorylated-LDHA and pan-LDHA were detected by Western blot analysis. Cellular glucose uptake, ATP production, reactive oxygen species (ROS), LDHA activity and lactate production were determined by ELISA assay using the corresponding commercial kits. Mitochondrial membrane potential changes were measured by JC-1 staining via Cellometer. Results: JQ-1 suppressed cellular proliferation (IC50 = 360 nM for HEY, p = 0.00001; IC50 = 3500 nM for SKOV3, p = 0.0007) via G1 cell cycle arrest in the ovarian cancer cell lines, but only caused moderate apoptosis. Western blot analysis demonstrated that JQ-1 increased p21 expression and decreased expression of c-Myc and phosphorylation of LDHA. In parallel, treatment with JQ-1 decreased LDHA activity, glucose uptake, mitochondrial membrane potential, lactate and ATP production and increased ROS levels in the ovarian cancer cell lines. Conclusions: We find that targeting c-Myc by JQ-1 significantly suppresses proliferation through G1 arrest in ovarian cancer cell lines. In addition, JQ-1 had multiple influences on cancer metabolism, particularly in the aerobic glycolysis pathway. JQ-1 reduced both the expression and activity of LDHA, inhibited lactate production and decreased the energy supply of the ovarian cancer cells. This evidence suggests that JQ-1 may be a promising targeted therapy for ovarian cancer through its effects on both cell proliferation and metabolism. Citation Format: Haifeng Qiu, Chunxiao Zhou, Amanda L. Jackson, Joshua E. Kilgore, Victoria L. Bae-Jump. JQ-1, a novel c-Myc inhibitor, suppressed cell proliferation and metabolism through the downregulation of lactate dehydrogenase A in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2418. doi:10.1158/1538-7445.AM2013-2418

S100A9 promotes glycolytic activity in HER2-positive breast cancer to induce immunosuppression in the tumour microenvironment

Hypoxia is a prevalent pathological process in chronic rhinosinusitis with nasal polyps (CRSwNP), leading to a cascade of pathological events, including epithelial-mesenchymal transition (EMT). However, the mechanisms underlying hypoxia-induced EMT remain unclear. This study aims to elucidate the mechanisms driving EMT under hypoxic conditions in CRSwNP. Transcriptome and proteome analyses of hypoxia-treated human nasal epithelial cells (HNECs) were performed to identify key molecules and pathways. The expression of hypoxia-inducible factor-1α (HIF-1α), pyruvate dehydrogenase kinase (PDK1), lactate dehydrogenase A (LDHA), and EMT markers was assessed in nasal tissues from CRSwNP patients. In vitro, cultured HNECs were exposed to hypoxia and lactate, or overexpressed PDK1, to evaluate changes in EMT markers. Hypoxia activated the glycolysis-related pathway in HNECs, with PDK1 and LDHA identified as significantly upregulated glycolysis-related enzymes. The expression of PDK1 and LDHA was closely correlated with HIF-1α and EMT markers in nasal tissues. Hypoxia induced an increase in PDK1 and LDHA expression, lactate production, and EMT occurrence in HNECs. PDK1 overexpression or lactate stimulation also triggered EMT, while PDK1 inhibition attenuated hypoxia-induced EMT in HNECs. This study is the first to reveal that hypoxia-induced activation of PDK1 plays a critical role in regulating EMT by promoting lactate production, thereby providing a potential therapeutic target for CRSwNP.

The ability of cancer cells to produce large amounts of lactate through aerobic glycosis (Warburg effect) is coupled to high rates of glucose uptake. Enhanced glucose uptake and glycolysis are closely correlated to increased breast tumor aggressiveness and poor prognosis. However, despite the importance of glucose uptake in supplying energy and preventing apoptosis of cancer cells, the majority of current efforts in searching for therapeutic agents targeting glucose metabolism have been aimed at modulating activities of different metabolic enzymes that are involved in glycolysis. Very limited studies have been done in developing novel therapeutic agents against glucose uptake in breast cancer cells. Ataxia-telangiectasia (A-T) is a monogenic, autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. The gene mutated in this disease, ATM (A-T, mutated), encodes a 370-kDa protein kinase. Although ATM is traditionally considered to be a nuclear protein that functions as a signal transducer in the cellular response to DNA damage, it is now known that ATM is also present in the cytoplasm and has important cytoplasmic functions. We previously discovered that ATM activates Akt, a main regulator of glucose uptake, by stimulating its phosphorylation at Ser473 following insulin treatment. We also found that ATM participates in insulin-mediated glucose uptake in muscle cells, and KU-55933, a specific inhibitor of ATM, strongly inhibits this process. Recently, we found that KU-55933 inhibits cell proliferation by inducing apoptosis in MDA-MB-231, a triple-negative breast cancer cell line. We have also found that KU-55933 inhibits migration of MDA-MB-231 by a cell invasion assay. Furthermore, we found that these cancer cells exhibit enhanced glucose uptake in response to insulin and the addition of KU-55933 leads to a dramatic reduction of insulin-mediated glucose uptake in these cells. To further test whether KU-55933's ability to induce apoptosis is linked to its inhibition of glucose uptake, we performed a cell death ELISA assay in MDA-MB-231 cells treated with KU-55933 and different concentrations of glucose. Our results show that KU-55933 induces apoptosis of MDA-MB-231 cells, resulting in a similar degree of cell death as glucose starvation, while cells treated with glucose in conjunction with KU-55933 have decreased apoptosis. Moreover, we performed a cell migration assay and found that KU-55933 strongly inhibits the migration of MDA-MB-231 cells (similar to that caused by glucose starvation), which is almost fully rescued by the extra glucose supplemented in the cell culture medium. We have also established a positional isotope labeling-based targeted metabolomics method that can directly measure the conversion from glucose to lactate through glycolysis in cancer cells. Our results show strong production of lactate from glucose in MDA-MB-231 cells even under normal aerobic growth conditions, and KU-55933 strongly inhibits this process. Our findings may lead to the development of KU-55933 and its analogs as a new generation of therapeutic agents against aggressive breast cancer. Citation Format: Yang D-Q, Harris B, Jiang S, Li Y, Freund D, Hegeman A, Cleary M. Inhibition of enhanced glucose uptake and glycolysis by KU-55933 as a novel strategy against aggressive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-05-05.

Sialyltransferases are enzymes that play a crucial role in regulating cancer progression by modifying glycoproteins through sialylation. In particular, the ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4) enzyme is known to be upregulated in breast cancer, but its specific biological functions have not been fully understood. This study aimed to investigate the impact and mechanisms of ST3GAL4 on aerobic glycolysis in breast cancer. We examined ST3GAL4 expression in tumor tissue samples and breast cancer cell lines and also manipulated ST3GAL4 expression in breast cancer cells using lentivirus transduction. The study evaluated cellular processes such as cell viability, cell cycle progression, and aerobic glycolysis by measuring parameters like extracellular acidification rate, glucose uptake, lactate production, and lactate dehydrogenase A (LDHA) expression. We found that ST3GAL4 expression was consistently increased in tumor tissues and breast cancer cell lines. High ST3GAL4 expression was associated with a poor prognosis for patients with breast cancer. Inhibiting ST3GAL4 expression decreased cell viability, disrupted cell cycle progression, and reduced aerobic glycolysis and LDHA expression. Furthermore, suppressing ST3GAL4 expression in animal models reduced tumor growth and cell proliferation. Conversely, overexpressing ST3GAL4 promoted cell viability and cell cycle progression, but these effects were reversed when an inhibitor of aerobic glycolysis was used. The study provided evidence in cells and animal models that ST3GAL4 promotes tumorigenesis in breast cancer by enhancing aerobic glycolysis. These findings suggest that targeting ST3GAL4 may be a potential strategy for the treatment of breast cancer.

Breast cancer is the most prevalent malignancy and the most significant contributor to mortality in female oncology patients. Potassium Two Pore Domain Channel Subfamily K Member 1 (KCNK1) is differentially expressed in a variety of tumors, but the mechanism of its function in breast cancer is unknown. In this study, we found for the first time that KCNK1 was significantly up-regulated in human breast cancer and was correlated with poor prognosis in breast cancer patients. KCNK1 promoted breast cancer proliferation, invasion, and metastasis in vitro and vivo. Further studies unexpectedly revealed that KCNK1 increased the glycolysis and lactate production in breast cancer cells by binding to and activating lactate dehydrogenase A (LDHA), which promoted histones lysine lactylation to induce the expression of a series of downstream genes and LDHA itself. Notably, increased expression of LDHA served as a vicious positive feedback to reduce tumor cell stiffness and adhesion, which eventually resulted in the proliferation, invasion, and metastasis of breast cancer. In conclusion, our results suggest that KCNK1 may serve as a potential breast cancer biomarker, and deeper insight into the cancer-promoting mechanism of KCNK1 may uncover a novel therapeutic target for breast cancer treatment.

Background: Only 14% of African American women with triple-negative breast cancers will be alive at one year. Early detection is greatly needed. Here we aimed to investigate whether activation of biologic pathways that predict aggressive triple-negative breast cancers are also activated in atypia in high-risk African American women. Aggressive cancers are known to consume glucose avidly and produce lactic acid (rather than fully metabolize glucose via the Tricarboxylic Acid (TCA) cycle). This shift toward lactate production, even in the presence of adequate oxygen, is termed the Warburg effect. The Warburg effect is thought to be a late event in breast cancer, however, our studies in high-risk African American women provide evidence that the Warburg effect occurs during cancer initiation. This is an important observation as glucose-signaling can be readily targeted for breast cancer prevention with minimal toxicity. Here we investigated the role of the Warburg effect in breast cancer initiation in young high-risk African American women. Methods and Results: Similar to fluorodeoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) is a fluorescent glucose analog that can be used to track glucose uptake and glycolysis. 2-NBDG spectroscopy provides a means to track glucose metabolism in live mammary epithelial cells from high-risk women. We used 2-NBDG spectroscopy to measure glucose uptake in ER-breast cancer and live atypical mammary epithelial cells from high-risk premenopausal women. We observe that both triple-negative breast cancer and a subset of atypia exhibits accumulation of 2-NBDG. There is growing recognition that phosphoprotein signaling networks (rather than single genes) play a key role in breast cancer initiation and progression. Our team used Reverse Phase Proteomic Microarray (RPPM) profiling to test for activation of phosphoprotein signaling networks in atypical RPFNA cytology from high-risk premenopausal women in our cohort. RPFNA were obtained from two independent sets of 39 and 38 high-risk premenopausal women; 45% of these women were African American. The signaling network most highly expressed in precancerous cells contained activated signaling proteins associated with the Warburg effect (AKT/mTOR/PI3K), insulin signaling (pACC, IRS1) and epithelial to mesenchymal transition (EMT) IL6/Stat3/vimentin. Conclusions: This is the first evidence that abnormal glucose uptake and the Warburg effect occurs during breast cancer initiation in high-risk African American premenopausal women. These studies demonstrate our ability to identify abnormal glucose and activated signaling networks associated with the Warburg effect in atypical mammary cells from high-risk African American women and provide an important target for breast cancer early detection and prevention. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):PR2.

BackgroundThe intratumor heterogeneity (ITH) of cancer cells plays an important role in breast cancer resistance and recurrence. To develop better therapeutic strategies, it is necessary to understand the molecular mechanisms underlying ITH and their functional significance. Patient-derived organoids (PDOs) have recently been utilized in cancer research. They can also be used to study ITH as cancer cell diversity is thought to be maintained within the organoid line. However, no reports investigated intratumor transcriptomic heterogeneity in organoids derived from patients with breast cancer. This study aimed to investigate transcriptomic ITH in breast cancer PDOs.MethodsWe established PDO lines from ten patients with breast cancer and performed single-cell transcriptomic analysis. First, we clustered cancer cells for each PDO using the Seurat package. Then, we defined and compared the cluster-specific gene signature (ClustGS) corresponding to each cell cluster in each PDO.ResultsCancer cells were clustered into 3–6 cell populations with distinct cellular states in each PDO line. We identified 38 clusters with ClustGS in 10 PDO lines and used Jaccard similarity index to compare the similarity of these signatures. We found that 29 signatures could be categorized into 7 shared meta-ClustGSs, such as those related to the cell cycle or epithelial–mesenchymal transition, and 9 signatures were unique to single PDO lines. These unique cell populations appeared to represent the characteristics of the original tumors derived from patients.ConclusionsWe confirmed the existence of transcriptomic ITH in breast cancer PDOs. Some cellular states were commonly observed in multiple PDOs, whereas others were specific to single PDO lines. The combination of these shared and unique cellular states formed the ITH of each PDO.

Organoids serve as an important preclinical model in multiple cancers including breast cancer, demonstrating preservation of biological features and feasibility of drug screening, with reasonable cost and high scalability. It has been reported that patient-derived organoids (PDOs) can be robustly derived from both primary and metastatic breast tumors, with high resemblance to source tissues in histological, genomic, and transcriptomic features, as well as consistency in drug response compared to xeno-transplantations or patients (Sachs et al, 2018). Since 2018, the Institute for Precision Medicine (IPM) has developed a panel of breast cancer organoids. The current inventory now consists of 128 PDOs from 94 primary tumors, 12 metastases, 12 rapid-autopsy tumors, and 10 normal breast tissues with a 60% success rate of culture; as well as 33 organoids from animal models including 11 patient-derived xenograft organoids (PDXOs), 15 rat (RDO) and 7 mouse (MDO) tumor organoids with 85% success rate. For PDOs, the protocol includes in-lab processing of fresh, deidentified tissue within 60 minutes of surgical operation, thanks to close collaboration with surgeons, consented patients, and an institutional biospecimen core. BC organoids have been comprehensively characterized in terms of morphology, histology, genomics, transcriptomics, and functional experiments. Key biological features are preserved. PDOs (n=56) exhibit a mutational spectrum consistent with human breast cancers. ER expression is detectable in a subset of cultures with robust estradiol response indicated by GREB1 expression; and PDOs from invasive lobular carcinoma (ILC) mostly showed more discohesive structures than PDOs from no special type (NST), with clear E-cadherin loss under immunofluorescence. We have also successfully constructed transcriptionally and genetic modified BC PDOs with shRNA and CRISPR, which showed replicable performance in proliferation assay, drug response, and signaling by western blot. Of note, longitudinal tracking of PDOs with single cell RNA-sequencing (scRNA-seq) revealed well-preserved heterogeneity compared to primary tumor, while transcriptomic clonality was mildly decreased in later passages from pilot data. Future work will include expansion of current collection from various sources, longitudinal characterization with scRNA-seq and DNA sequencing, as well as more functional studies. We aim to construct a robust resource of well-characterized and reliably-usable model bank for preclinical breast cancer research. Citation Format: Daniel Brown, Kai Ding, Fangyuan Chen, Jian Chen, Steffi Oesterreich, Peter Lucas, Priscilla McAuliffe, Jennifer Atkinson, ADRIAN LEE. Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-08.

Insulin stimulates glucose uptake in fat and muscle by redistributing GLUT4 glucose transporters from intracellular membranes to the cell surface. We previously proposed that, in 3T3-L1 adipocytes, TUG retains GLUT4 within unstimulated cells and insulin mobilizes this retained GLUT4 by stimulating its dissociation from TUG. Yet the relative importance of this action in the overall control of glucose uptake remains uncertain. Here we report that transient, small interfering RNA-mediated depletion of TUG causes GLUT4 translocation and enhances glucose uptake in unstimulated 3T3-L1 adipocytes, similar to insulin. Stable TUG depletion or expression of a dominant negative fragment likewise stimulates GLUT4 redistribution and glucose uptake, and insulin causes a 2-fold further increase. Microscopy shows that TUG governs the accumulation of GLUT4 in perinuclear membranes distinct from endosomes and indicates that it is this pool of GLUT4 that is mobilized by TUG disruption. Interestingly, in addition to translocating GLUT4 and enhancing glucose uptake, TUG disruption appears to accelerate the degradation of GLUT4 in lysosomes. Finally, we find that TUG binds directly and specifically to a large intracellular loop in GLUT4. Together, these findings demonstrate that TUG is required to retain GLUT4 intracellularly in 3T3-L1 adipocytes in the absence of insulin and further implicate the insulin-stimulated dissociation of TUG and GLUT4 as an important action by which insulin stimulates glucose uptake.