RESUMEN
Chimeric antigen receptor (CAR) T cell therapies are medical breakthroughs in cancer treatment. However, treatment failure is often caused by CAR T cell dysfunction. Additional approaches are needed to overcome inhibitory signals that limit anti-tumor potency. Here, we developed bifunctional fusion "degrader" proteins that bridge one or more target proteins and an E3 ligase complex to enforce target ubiquitination and degradation. Conditional degradation strategies were developed using inducible degrader transgene expression or small molecule-dependent E3 recruitment. We further engineered degraders to block SMAD-dependent TGFß signaling using a domain from the SARA protein to target both SMAD2 and SMAD3. SMAD degrader CAR T cells were less susceptible to suppression by TGFß and demonstrated enhanced anti-tumor potency in vivo. These results demonstrate a clinically suitable synthetic biology platform to reprogram E3 ligase target specificity for conditional, multi-specific endogenous protein degradation, with promising applications including enhancing the potency of CAR T cell therapy.
Asunto(s)
Neoplasias , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Inmunoterapia Adoptiva/métodos , Ubiquitinación , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks multiple human proteins during infection and viral replication. To examine whether any viral proteins employ human E3 ubiquitin ligases, we evaluated the stability of SARS-CoV-2 proteins with inhibition of the ubiquitin proteasome pathway. Using genetic screens to dissect the molecular machinery involved in the degradation of candidate viral proteins, we identified human E3 ligase RNF185 as a regulator of protein stability for the SARS-CoV-2 envelope protein. We found that RNF185 and the SARS-CoV-2 envelope co-localize to the endoplasmic reticulum (ER). Finally, we demonstrate that the depletion of RNF185 significantly increases SARS-CoV-2 viral titer in a cellular model. Modulation of this interaction could provide opportunities for novel antiviral therapies.
RESUMEN
Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early-phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR/Cas9 screens implicated decreased translation initiation as protective following GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to the effects of GSPT1 degradation. We defined 2 Crbn amino acids that prevent Gspt1 degradation in mice, generated a knockin mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant acute myeloid leukemia.
Asunto(s)
Leucemia , Factores de Terminación de Péptidos , Animales , Muerte Celular , Células Madre Hematopoyéticas/metabolismo , Ratones , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , ProteolisisRESUMEN
Thalidomide analogs exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2-FGFR1 and ZMYM2-FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment.
Asunto(s)
Neoplasias Hematológicas , Talidomida , Proteínas de Unión al ADN , Neoplasias Hematológicas/tratamiento farmacológico , Humanos , Lenalidomida/farmacología , Proteínas Oncogénicas , Factores de Transcripción/metabolismoRESUMEN
Cell-based therapies are emerging as effective agents against cancer and other diseases. As autonomous "living drugs," these therapies lack precise control. Chimeric antigen receptor (CAR) T cells effectively target hematologic malignancies but can proliferate rapidly and cause toxicity. We developed ON and OFF switches for CAR T cells using the clinically approved drug lenalidomide, which mediates the proteasomal degradation of several target proteins by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif. We performed a systematic screen to identify "super-degron" tags with enhanced sensitivity to lenalidomide-induced degradation and used these degradable tags to generate OFF-switch degradable CARs. To create an ON switch, we engineered a lenalidomide-inducible dimerization system and developed split CARs that required both lenalidomide and target antigen for activation. Subtherapeutic lenalidomide concentrations controlled the effector functions of ON- and OFF-switch CAR T cells. In vivo, ON-switch split CARs demonstrated lenalidomide-dependent antitumor activity, and OFF-switch degradable CARs were depleted by drug treatment to limit inflammatory cytokine production while retaining antitumor efficacy. Together, the data showed that these lenalidomide-gated switches are rapid, reversible, and clinically suitable systems to control transgene function in diverse gene- and cell-based therapies.
Asunto(s)
Lenalidomida , Receptores Quiméricos de Antígenos , Linfocitos T , Humanos , Células Jurkat , Receptores de Antígenos de Linfocitos T , Ubiquitina-Proteína LigasasRESUMEN
Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.
Asunto(s)
Polimerizacion/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-6/química , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Microscopía por Crioelectrón , Humanos , Técnicas In Vitro , Ligandos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/ultraestructura , Solventes , Biología Sintética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines3-5, we identify CR8-a cyclin-dependent kinase (CDK) inhibitor6-as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues.
Asunto(s)
Ciclinas/deficiencia , Ciclinas/metabolismo , Proteolisis/efectos de los fármacos , Purinas/química , Purinas/farmacología , Piridinas/química , Piridinas/farmacología , Línea Celular Tumoral , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/química , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/química , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica/efectos de los fármacos , Purinas/toxicidad , Piridinas/toxicidad , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitinación/efectos de los fármacosRESUMEN
Protein ubiquitylation is involved in a plethora of cellular processes. While antibodies directed at ubiquitin remnants (K-É-GG) have improved the ability to monitor ubiquitylation using mass spectrometry, methods for highly multiplexed measurement of ubiquitylation in tissues and primary cells using sub-milligram amounts of sample remains a challenge. Here, we present a highly sensitive, rapid and multiplexed protocol termed UbiFast for quantifying ~10,000 ubiquitylation sites from as little as 500 µg peptide per sample from cells or tissue in a TMT10plex in ca. 5 h. High-field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) is used to improve quantitative accuracy for posttranslational modification analysis. We use the approach to rediscover substrates of the E3 ligase targeting drug lenalidomide and to identify proteins modulated by ubiquitylation in models of basal and luminal human breast cancer. The sensitivity and speed of the UbiFast method makes it suitable for large-scale studies in primary tissue samples.
Asunto(s)
Proteínas/metabolismo , Proteoma/análisis , Investigación Biomédica Traslacional/métodos , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Animales , Neoplasias de la Mama , Caseína Quinasa Ialfa , Femenino , Células HeLa , Humanos , Factor de Transcripción Ikaros , Espectrometría de Masas/métodos , Ratones , Mieloma Múltiple , Procesamiento Proteico-Postraduccional , Proteómica/métodos , Sensibilidad y Especificidad , Coloración y Etiquetado , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.
Asunto(s)
Proteolisis/efectos de los fármacos , Talidomida/análogos & derivados , Talidomida/química , Talidomida/farmacología , Ubiquitina-Proteína Ligasas/química , Neoplasias Hematológicas/enzimología , Humanos , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/efectos de los fármacosRESUMEN
Lenalidomide mediates the ubiquitination and degradation of Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and casein kinase 1α (CK1α) by facilitating their interaction with cereblon (CRBN), the substrate receptor for the CRL4CRBN E3 ubiquitin ligase. Through this mechanism, lenalidomide is a clinically effective treatment of multiple myeloma and myelodysplastic syndrome (MDS) with deletion of chromosome 5q [del(5q) MDS]. To identify the cellular machinery required for lenalidomide-induced CRL4CRBN activity, we performed a positive selection, genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) screen in a lenalidomide-sensitive myeloma cell line. CRBN was the top-ranking gene, with all CRBN-targeting guide RNAs (gRNAs) ranking as the 6 highest-scoring gRNAs. A counterscreen using an IKZF3 degron reporter to assay lenalidomide-induced protein degradation highlighted regulators of cullin-RING ligase neddylation and 2 E2 ubiquitin-conjugating enzymes as necessary for efficient lenalidomide-induced protein degradation. We demonstrated that loss of UBE2M or members of the constitutive photomorphogenesis 9 (COP9) signalosome results in altered neddylation of cullin 4A and impairs lenalidomide-dependent CRL4CRBN activity. Additionally, we established that UBE2D3 and UBE2G1 play distinct roles in substrate ubiquitination by CRL4CRBN, with UBE2D3 acting to prime targets via monoubiquitination and UBE2G1 functioning to extend polyubiquitin chains with lysine 48 linkages. The validation of UBE2D3 and UBE2G1 highlights the functional capacity of CRISPR-Cas9 screening to identify E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase complex pairings. More broadly, these findings establish key proteins required for lenalidomide-dependent CRL4CRBN function in myeloma and inform potential mechanisms of drug resistance.
Asunto(s)
Antineoplásicos/farmacología , Lenalidomida/farmacología , Mieloma Múltiple/tratamiento farmacológico , Ubiquitina-Proteína Ligasas/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/efectos de los fármacosRESUMEN
To identify novel coding association signals and facilitate characterization of mechanisms influencing glycemic traits and type 2 diabetes risk, we analyzed 109,215 variants derived from exome array genotyping together with an additional 390,225 variants from exome sequence in up to 39,339 normoglycemic individuals from five ancestry groups. We identified a novel association between the coding variant (p.Pro50Thr) in AKT2 and fasting plasma insulin (FI), a gene in which rare fully penetrant mutations are causal for monogenic glycemic disorders. The low-frequency allele is associated with a 12% increase in FI levels. This variant is present at 1.1% frequency in Finns but virtually absent in individuals from other ancestries. Carriers of the FI-increasing allele had increased 2-h insulin values, decreased insulin sensitivity, and increased risk of type 2 diabetes (odds ratio 1.05). In cellular studies, the AKT2-Thr50 protein exhibited a partial loss of function. We extend the allelic spectrum for coding variants in AKT2 associated with disorders of glucose homeostasis and demonstrate bidirectional effects of variants within the pleckstrin homology domain of AKT2.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Ayuno/metabolismo , Resistencia a la Insulina/genética , Insulina/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Población Blanca/genética , Negro o Afroamericano/genética , Alelos , Pueblo Asiatico/genética , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/metabolismo , Finlandia , Frecuencia de los Genes , Predisposición Genética a la Enfermedad , Genotipo , Hispánicos o Latinos/genética , Humanos , Oportunidad RelativaRESUMEN
Oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), occur with high frequency in breast cancer. The protein kinase Akt is considered to be the primary effector of PIK3CA, although mechanisms by which PI3K mediates Akt-independent tumorigenic signals remain obscure. We show that serum and glucocorticoid-regulated kinase 3 (SGK3) is amplified in breast cancer and activated downstream of PIK3CA in a manner dependent on the phosphoinositide phosphatase INPP4B. Expression of INPP4B leads to enhanced SGK3 activation and suppression of Akt phosphorylation. Activation of SGK3 downstream of PIK3CA and INPP4B is required for 3D proliferation, invasive migration, and tumorigenesis in vivo. We further show that SGK3 targets the metastasis suppressor NDRG1 for degradation by Fbw7. We propose a model in which breast cancers harboring oncogenic PIK3CA activate SGK3 signaling while suppressing Akt, indicative of oncogenic functions for both INPP4B and SGK3 in these tumors.
Asunto(s)
Neoplasias de la Mama/enzimología , Fosfatidilinositol 3-Quinasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Fosfatidilinositol 3-Quinasa Clase I , Activación Enzimática , Femenino , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Desnudos , Mutación , Células 3T3 NIH , Invasividad Neoplásica , Trasplante de Neoplasias , Fosfatidilinositol 3-Quinasas/fisiología , Procesamiento Proteico-Postraduccional , Proteolisis , Transducción de SeñalRESUMEN
Obesity is a risk factor for breast cancer, and low blood concentrations of adiponectin are associated with high incidence and poor prognosis of breast cancer. However, the molecular mechanisms underlying such inhibitory effects of adiponectin remain to be defined. By using MCF7 cells, we investigated the mechanisms underlying the inhibitory effects of adiponectin on breast cancer cells, particularly in the context of opposing IGF-1-induced proliferation. We found that adiponectin, at 20 and 40 µg/ml, reduced MCF7 cell growth in the absence and presence of IGF-1. These inhibitory effects were primarily the result of the significant increase of cells at G(1)/G(0) phase and concomitant decrease of cells at S phase. In addition, the percentage of apoptotic cells increased more than two-fold. Within 30-min of adiponectin addition, the phosphorylation of AMPKα was sharply elevated, and the level of IGF-1-activated Akt was decreased. Prolonged exposure to adiponectin resulted in reduction of cyclin D1 and cyclin E2 expression. Adiponectin also increased intracellular levels of cAMP and the activity of protein kinase-A (PKA). The inhibitors of PKA completely abolished the adiponectin's effects on cell growth. In conclusion, our studies revealed an important cellular mechanism underlying the relationship between reduced adiponectin concentrations and breast cancer development.
Asunto(s)
Adiponectina/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/fisiología , Transducción de Señal , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Ciclina D1/metabolismo , Ciclinas/metabolismo , Femenino , Humanos , Receptores de Adiponectina/análisis , Receptores de Adiponectina/fisiologíaRESUMEN
The Rictor/mTOR complex (also known as mTORC2) plays a critical role in cellular homeostasis by phosphorylating AGC kinases such as Akt and SGK at their hydrophobic motifs to activate downstream signaling. However, the regulation of mTORC2 and whether it has additional function(s) remain largely unknown. Here, we report that Rictor associates with Cullin-1 to form a functional E3 ubiquitin ligase. Rictor, but not Raptor or mTOR alone, promotes SGK1 ubiquitination. Loss of Rictor/Cullin-1-mediated ubiquitination leads to increased SGK1 protein levels as detected in Rictor null cells. Moreover, as part of a feedback mechanism, phosphorylation of Rictor at T1135 by multiple AGC kinases disrupts the interaction between Rictor and Cullin-1 to impair SGK1 ubiquitination. These findings indicate that the Rictor/Cullin-1 E3 ligase activity is regulated by a specific signal relay cascade and that misregulation of this mechanism may contribute to the frequent overexpression of SGK1 in various human cancers.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitinación , Secuencias de Aminoácidos , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Proteínas Cullin/genética , Humanos , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Neoplasias/genética , Neoplasias/metabolismo , Fosforilación/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina , Serina-Treonina Quinasas TOR , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Obesity is one of the well-established risk factors for endometrial cancer. Recent clinical studies have demonstrated that circulating adiponectin concentrations are inversely correlated with the incidence of endometrial carcinoma. Such epidemiological findings are consistent with the paradoxical observations that adiponectin levels are reduced in obesity. This study investigated the direct effects of adiponectin on two endometrial carcinoma cell lines, HEC-1-A and RL95-2. These cell lines express both variants of adiponectin receptors, adipo-R1 and adipo-R2. Adiponectin treatment leads to suppression of cell proliferation in both cell types, which is primarily due to the significant increase of cell populations at G(1)/G(0)-phase and to the induction of apoptosis. The inhibition of growth in these two cell lines appears to be mediated by different signaling pathways. Although adiponectin treatment markedly increases the phosphorylation (Thr172) of AMP-activated protein kinase alpha in both HEC-1-A and RL95-2 within 30 min, prolonged exposure (48 h) leads to inactivation of Akt as well as reduction of cyclin D1 protein expression in HEC-1-A cells. In contrast, similar treatment of RL95-2 cells with adiponectin, while having no effects on Akt activity and cyclin D1 expression, causes a decrease in cyclin E2 expression and the activity of mitogen-activated kinase (p42/44). We conclude that adiponectin exerts direct anti-proliferative effects on HEC-1-A and RL95-2 cells by inducing cell cycle arrest and apoptosis. Depending on the genotypes of the endometrial cancer cells, the inhibitory effects of adiponectin are associated with the reduction of different pro-growth regulators of cell cycle and signaling proteins. Our study thus provides a cellular mechanism underlying the linkages between endometrial cancer and obesity.