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AIMS/HYPOTHESIS: Glucagon-stimulated hepatic gluconeogenesis contributes to endogenous glucose production during fasting. Recent studies suggest that TGF-ß is able to promote hepatic gluconeogenesis in mice. However, the physiological relevance of serum TGF-ß levels to human glucose metabolism and the mechanism by which TGF-ß enhances gluconeogenesis remain largely unknown. As enhanced gluconeogenesis is a signature feature of type 2 diabetes, elucidating the molecular mechanisms underlying TGF-ß-promoted hepatic gluconeogenesis would allow us to better understand the process of normal glucose production and the pathophysiology of this process in type 2 diabetes. This study aimed to investigate the contribution of upregulated TGF-ß1 in human type 2 diabetes and the molecular mechanism underlying the action of TGF-ß1 in glucose metabolism. METHODS: Serum levels of TGF-ß1 were measured by ELISA in 74 control participants with normal glucose tolerance and 75 participants with type 2 diabetes. Human liver tissue was collected from participants without obesity and with or without type 2 diabetes for the measurement of TGF-ß1 and glucagon signalling. To investigate the role of Smad3, a key signalling molecule downstream of the TGF-ß1 receptor, in mediating the effect of TGF-ß1 on glucagon signalling, we generated Smad3 knockout mice. Glucose levels in Smad3 knockout mice were measured during prolonged fasting and a glucagon tolerance test. Mouse primary hepatocytes were isolated from Smad3 knockout and wild-type (WT) mice to investigate the underlying molecular mechanisms. Smad3 phosphorylation was detected by western blotting, levels of cAMP were detected by ELISA and levels of protein kinase A (PKA)/cAMP response element-binding protein (CREB) phosphorylation were detected by western blotting. The dissociation of PKA subunits was measured by immunoprecipitation. RESULTS: We observed higher levels of serum TGF-ß1 in participants without obesity and with type 2 diabetes than in healthy control participants, which was positively correlated with HbA1c and fasting blood glucose levels. In addition, hyperactivation of the CREB and Smad3 signalling pathways was observed in the liver of participants with type 2 diabetes. Treating WT mouse primary hepatocytes with TGF-ß1 greatly potentiated glucagon-stimulated PKA/CREB phosphorylation and hepatic gluconeogenesis. Mechanistically, TGF-ß1 treatment induced the binding of Smad3 to the regulatory subunit of PKA (PKA-R), which prevented the association of PKA-R with the catalytic subunit of PKA (PKA-C) and led to the potentiation of glucagon-stimulated PKA signalling and gluconeogenesis. CONCLUSIONS/INTERPRETATION: The hepatic TGF-ß1/Smad3 pathway sensitises the effect of glucagon/PKA signalling on gluconeogenesis and synergistically promotes hepatic glucose production. Reducing serum levels of TGF-ß1 and/or preventing hyperactivation of TGF-ß1 signalling could be a novel approach for alleviating hyperglycaemia in type 2 diabetes.
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Diabetes Mellitus Tipo 2 , Hiperglucemia , Humanos , Animales , Ratones , Glucagón/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hiperglucemia/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Hepatocitos/metabolismo , Hígado/metabolismo , Glucosa/metabolismo , Gluconeogénesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ratones Noqueados , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Mammary physiology is distinguished in containing adult stem/progenitor cells that are actively amending the breast tissue throughout the reproductive lifespan of women. Despite their importance in both mammary gland development, physiological maintenance, and reproduction, the exact role of mammary stem/progenitor cells in mammary tumorigenesis has not been fully elucidated in humans or animal models. The implications of modulating adult stem/progenitor cells in women could lead to a better understanding of not only their function, but also toward possible breast cancer prevention led us to evaluate the efficacy of rapamycin in reducing mammary stem/progenitor cell activity and malignant progression markers. METHODS: We analyzed a large number of human breast tissues for their basal and luminal cell composition with flow cytometry and their stem and progenitor cell function with sphere formation assay with respect to age and menopausal status in connection with a clinical study (NCT02642094) involving a low-dose (2 mg/day) and short-term (5-7 days) treatment of the mTOR inhibitor sirolimus. The expression of biomarkers in biopsies and surgical breast samples were measured with quantitative analysis of immunohistochemistry. RESULTS: Sirolimus treatment significantly abrogated mammary stem cell activity, particularly in postmenopausal patients. It did not affect the frequency of luminal progenitors but decreased their self-renewal capacity. While sirolimus had no effect on basal cell population, it decreased luminal cell population, particularly in postmenopausal patients. It also significantly diminished prognostic biomarkers associated with breast cancer progression from ductal carcinoma in situ to invasive breast cancer including p16INK4A, COX-2, and Ki67, as well as markers of the senescence-associated secretary phenotype, thereby possibly functioning in preventing early breast cancer progression. CONCLUSION: Overall, these findings indicate a link from mTOR signaling to mammary stem and progenitor cell activity and cancer progression. Trial registration This study involves a clinical trial registered under the ClinicalTrials.gov identifier NCT02642094 registered December 30, 2015.
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Neoplasias de la Mama , Animales , Humanos , Femenino , Neoplasias de la Mama/genética , Glándulas Mamarias Animales/metabolismo , Células Madre/metabolismo , Biomarcadores/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Sirolimus/farmacología , Sirolimus/metabolismo , Células Epiteliales/metabolismoRESUMEN
Integrin α6 (ITGA6) forms integrin receptors with either integrin ß1 (ITGB1) or integrin ß4 (ITGB4). How it functions to regulate hepatocellular carcinoma (HCC) progression is not well-elucidated. We found that ITGA6 RNA and protein expression levels are significantly elevated in human HCC tissues in comparison with paired adjacent nontumor tissues by RNA sequencing, RT-qPCR, Western blotting and immunofluorescence staining. Stable knockdown of ITGA6 with different ITGA6 shRNA expression lentivectors significantly inhibited proliferation, migration and anchorage-independent growth of HCC cell lines in vitro, and xenograft tumor growth in vivo. The inhibition of anchorage-dependent and -independent growth of HCC cell lines was also confirmed with anti-ITGA6 antibody. ITGA6 knockdown was shown to induce cell-cycle arrest at G0/G1 phase. Immunoprecipitation assay revealed apparent interaction of ITGA6 with ITGB4, but not ITGB1. Expression studies showed that ITGA6 positively regulates the expression of ITGB4 with no or negative regulation of ITGB1 expression. Finally, while high levels of ITGA6 and ITGB4 together were associated with significantly worse survival of HCC patients in TCGA data set, the association was not significant for high levels of ITGA6 and ITGB1. In conclusion, ITGA6 is upregulated in HCC tumors and has a malignant promoting role in HCC cells through integrin α6ß4 complex. Thus, integrin α6ß4 may be a therapeutic target for treating patients with HCC.
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Carcinoma Hepatocelular , Integrina alfa6 , Integrina alfa6beta4 , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrina beta4/genética , Integrina beta4/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologíaRESUMEN
BACKGROUND: Aging is a comorbidity of breast cancer suggesting that aging-associated transcriptome changes may promote breast cancer progression. However, the mechanism underlying the age effect on breast cancer remains poorly understood. METHOD: We analyzed transcriptomics of the matched normal breast tissues from the 82 breast cancer patients in The Cancer Genome Atlas (TCGA) dataset with linear regression for genes with age-associated expression that are not associated with menopause. We also analyzed differentially expressed genes between the paired tumor and non-tumor breast tissues in TCGA for the identification of age and breast cancer (ABC)-associated genes. A few of these genes were selected for further investigation of their malignancy-regulating activities with in vitro and in vivo assays. RESULTS: We identified 148 upregulated and 189 downregulated genes during aging. Overlapping of tumor-associated genes between normal and tumor tissues with age-dependent genes resulted in 14 upregulated and 24 downregulated genes that were both age and breast cancer associated. These genes are predictive in relapse-free survival, indicative of their potential tumor promoting or suppressive functions, respectively. Knockdown of two upregulated genes (DYNLT3 and P4HA3) or overexpression of the downregulated ALX4 significantly reduced breast cancer cell proliferation, migration, and clonogenicity. Moreover, knockdown of P4HA3 reduced growth and metastasis whereas overexpression of ALX4 inhibited the growth of xenografted breast cancer cells in mice. CONCLUSION: Our study suggests that transcriptome alterations during aging may contribute to breast tumorigenesis. DYNLT3, P4HA3, and ALX4 play significant roles in breast cancer progression.
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Neoplasias de la Mama/genética , Mama/fisiología , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Dineínas/genética , Dineínas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/metabolismo , Pronóstico , Tasa de Supervivencia , Células Tumorales CultivadasRESUMEN
Host restriction factors constitute a formidable barrier for viral replication to which many viruses have evolved counter-measures. Human SAMD9, a tumor suppressor and a restriction factor for poxviruses in cell lines, is antagonized by two classes of poxvirus proteins, represented by vaccinia virus (VACV) K1 and C7. A paralog of SAMD9, SAMD9L, is also encoded by some mammals, while only one of two paralogs is retained by others. Here, we show that SAMD9L functions similarly to SAMD9 as a restriction factor and that the two paralogs form a critical host barrier that poxviruses must overcome to establish infection. In mice, which naturally lack SAMD9, overcoming SAMD9L restriction with viral inhibitors is essential for poxvirus replication and pathogenesis. While a VACV deleted of both K1 and C7 (vK1L-C7L-) was restricted by mouse cells and highly attenuated in mice, its replication and virulence were completely restored in SAMD9L-/- mice. In humans, both SAMD9 and SAMD9L are poxvirus restriction factors, although the latter requires interferon induction in many cell types. While knockout of SAMD9 with Crispr-Cas9 was sufficient for abolishing the restriction for vK1L-C7L- in many human cells, knockout of both paralogs was required for abolishing the restriction in interferon-treated cells. Both paralogs are antagonized by VACV K1, C7 and C7 homologs from diverse mammalian poxviruses, but mouse SAMD9L is resistant to the C7 homolog encoded by a group of poxviruses with a narrow host range in ruminants, indicating that host species-specific difference in SAMD9/SAMD9L genes serves as a barrier for cross-species poxvirus transmission.
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Especificidad del Huésped/genética , Infecciones por Poxviridae/genética , Poxviridae/genética , Poxviridae/patogenicidad , Proteínas/fisiología , Proteínas Supresoras de Tumor/fisiología , Animales , Células Cultivadas , Chlorocebus aethiops , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mamíferos , Ratones , Ratones Noqueados , Células 3T3 NIH , Infecciones por Poxviridae/transmisión , Infecciones por Poxviridae/virología , Proteínas/genética , Homología de Secuencia , Proteínas Supresoras de Tumor/genética , Virus Vaccinia/genética , Virus Vaccinia/patogenicidad , Células VeroRESUMEN
Everolimus inhibits mammalian target of rapamycin complex 1 (mTORC1) and is known to cause induction of autophagy and G1 cell cycle arrest. However, it remains unknown whether everolimus-induced autophagy plays a critical role in its regulation of the cell cycle. We, for the first time, suggested that everolimus could stimulate autophagy-mediated cyclin D1 degradation in breast cancer cells. Everolimus-induced cyclin D1 degradation through the autophagy pathway was investigated in MCF-10DCIS.COM and MCF-7 cell lines upon autophagy inhibitor treatment using Western blot assay. Everolimus-stimulated autophagy and decrease in cyclin D1 were also tested in explant human breast tissue. Inhibiting mTORC1 with everolimus rapidly increased cyclin D1 degradation, whereas 3-methyladenine, chloroquine, and bafilomycin A1, the classic autophagy inhibitors, could attenuate everolimus-induced cyclin D1 degradation. Similarly, knockdown of autophagy-related 7 (Atg-7) also repressed everolimus-triggered cyclin D1 degradation. In addition, everolimus-induced autophagy occurred earlier than everolimus-induced G1 arrest, and blockade of autophagy attenuated everolimus-induced G1 arrest. We also found that everolimus stimulated autophagy and decreased cyclin D1 levels in explant human breast tissue. These data support the conclusion that the autophagy induced by everolimus in human mammary epithelial cells appears to cause cyclin D1 degradation resulting in G1 cell cycle arrest. Our findings contribute to our knowledge of the interplay between autophagy and cell cycle regulation mediated by mTORC1 signaling and cyclin D1 regulation.
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Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Everolimus/farmacología , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Ciclina D1/genética , Femenino , Humanos , Células MCF-7 , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteolisis , Transducción de Señal , Técnicas de Cultivo de TejidosRESUMEN
BACKGROUND: Europeans and American Indians were major genetic ancestry of Hispanics in the U.S. These ancestral groups have markedly different incidence rates and outcomes in many types of cancers. Therefore, the genetic admixture may cause biased genetic association study with cancer susceptibility variants specifically in Hispanics. For example, the incidence rate of liver cancer has been shown with substantial disparity between Hispanic, Asian and non-Hispanic white populations. Currently, ancestry informative marker (AIM) panels have been widely utilized with up to a few hundred ancestry-informative single nucleotide polymorphisms (SNPs) to infer ancestry admixture. Notably, current available AIMs are predominantly located in intron and intergenic regions, while the whole exome sequencing (WES) protocols commonly used in translational research and clinical practice do not cover these markers. Thus, it remains challenging to accurately determine a patient's admixture proportion without additional DNA testing. RESULTS: In this study we designed an unique AIM panel that infers 3-way genetic admixture from three distinct and selective continental populations (African (AFR), European (EUR), and East Asian (EAS)) within evolutionarily conserved exonic regions. Initially, about 1 million exonic SNPs from selective three populations in the 1000 Genomes Project were trimmed by their linkage disequilibrium (LD), restricted to biallelic variants, and finally we optimized to an AIM panel with 250 SNP markers, or the UT-AIM250 panel, using their ancestral informativeness statistics. Comparing to published AIM panels, UT-AIM250 performed better accuracy when we tested with three ancestral populations (accuracy: 0.995 ± 0.012 for AFR, 0.997 ± 0.007 for EUR, and 0.994 ± 0.012 for EAS). We further demonstrated the performance of the UT-AIM250 panel to admixed American (AMR) samples of the 1000 Genomes Project and obtained similar results (AFR, 0.085 ± 0.098; EUR, 0.665 ± 0.182; and EAS, 0.250 ± 0.205) to previously published AIM panels (Phillips-AIM34: AFR, 0.096 ± 0.127, EUR, 0.575 ± 0.290, and EAS, 0.330 ± 0.315; Wei-AIM278: AFR, 0.070 ± 0.096, EUR, 0.537 ± 0.267, and EAS, 0.393 ± 0.300). Subsequently, we applied the UT-AIM250 panel to a clinical dataset of 26 self-reported Hispanic patients in South Texas with hepatocellular carcinoma (HCC). We estimated the admixture proportions using WES data of adjacent non-cancer liver tissues (AFR, 0.065 ± 0.043; EUR, 0.594 ± 0.150; and EAS, 0.341 ± 0.160). Similar admixture proportions were identified from corresponding tumor tissues. In addition, we estimated admixture proportions of The Cancer Genome Atlas (TCGA) collection of hepatocellular carcinoma (TCGA-LIHC) samples (376 patients) using the UT-AIM250 panel. The panel obtained consistent admixture proportions from tumor and matched normal tissues, identified 3 possible incorrectly reported race/ethnicity, and/or provided race/ethnicity determination if necessary. CONCLUSIONS: Here we demonstrated the feasibility of using evolutionarily conserved exonic regions to infer admixture proportions and provided a robust and reliable control for sample collection or patient stratification for genetic analysis. R implementation of UT-AIM250 is available at https://github.com/chenlabgccri/UT-AIM250.
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Genoma Humano/genética , Estudio de Asociación del Genoma Completo/métodos , Hispánicos o Latinos/genética , Carcinoma Hepatocelular/etnología , Carcinoma Hepatocelular/genética , Etnicidad/genética , Exones/genética , Frecuencia de los Genes , Pruebas Genéticas , Genética de Población , Genotipo , Humanos , Neoplasias Hepáticas/etnología , Neoplasias Hepáticas/genética , Polimorfismo de Nucleótido Simple , Programas InformáticosRESUMEN
Advanced, recurrent, or persistent cervical cancer is often incurable. Therefore, in-depth insights into the molecular mechanisms are needed for the development of novel therapeutic targets and the improvement of current therapeutic strategies. In this study, we investigated the role of GLI2 and GLI3 in the regulation of the malignant properties of cervical cancer. We showed that down-regulation of GLI2, but not GLI3, with an inducible GLI2 shRNA inhibited the growth and migration of cervical cancer cell lines, which could be rescued by ectopic expression of GLI2. GLI2 appeared to support cell growth by regulating the mitosis, but not the apoptosis, of the cervical cancer cells. Mechanistically, these functions of GLI2 were in part mediated by the activation of AKT pathway. Knockdown of GLI2, but not GLI3, also inhibited xenograft growth of cervical cancer cells in vivo. Finally, analysis of TCGA data showed that high levels of GLI2, but not GLI3, conferred a poor prognosis in cervical cancer patients. These observations for the first time suggest that GLI2, but not GLI3, exerts a tumor-promoting role in cervical cancer and may be targeted as a novel therapeutic strategy.
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Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Proteína Gli2 con Dedos de Zinc/metabolismo , Proteína Gli3 con Dedos de Zinc/metabolismo , Animales , Movimiento Celular , Proliferación Celular , Femenino , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Ratones Desnudos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Neoplasias del Cuello Uterino/mortalidadRESUMEN
BACKGROUND: Medical therapeutic options remain quite limited for uterine fibroids treatment. Statins, competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, have anti-tumoral effects on multiple cancer types, however, little is known about their effects on uterine fibroids. METHODS: Initially, we conducted a retrospective study of 120 patients with uterine fibroids and hyperlipidemia from the Second Affiliated Hospital of Wenzhou Medical University. Then, we evaluated the effect of atorvastatin on proliferation and apoptosis both in immortalized uterine fibroids cells and primary uterine fibroids cells. Furthermore, the molecular mechanism by which atorvastatin suppressed uterine fibroids cell growth was explored. RESULTS: Our results showed that atorvastatin use for 1 or 2 years significantly suppressed growth of uterine fibroids. Atorvastatin inhibited the proliferation of immortalized and primary uterine fibroids cells in a dose and time-dependent manner and stimulated apoptosis of uterine fibroids cells by inducing caspase-3 activation, up-regulating Bim and down-regulating Bcl-2. Additionally, atorvastatin treatment suppressed phosphorylation of ERK1/2 and JNK. Furthermore, GGPP, a downstream lipid isoprenoid intermediate, significantly rescued the effect of atorvastatin. CONCLUSIONS: These results suggest that atorvastatin exerts anti-tumoral effects on uterine fibroids through inhibition of cell proliferation and induction of apoptosis in HMG-CoA-dependent pathway. Our results provide the first clinical and preclinical data on the use of atorvastatin as a promising nonsurgical treatment option for uterine fibroids.
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Atorvastatina/uso terapéutico , Leiomioma/tratamiento farmacológico , Neoplasias Uterinas/tratamiento farmacológico , Adulto , Apoptosis/efectos de los fármacos , Atorvastatina/farmacología , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Hiperlipidemias/complicaciones , Hiperlipidemias/tratamiento farmacológico , Leiomioma/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Persona de Mediana Edad , Fenotipo , Fosforilación/efectos de los fármacos , Fosfatos de Poliisoprenilo/farmacología , Fosfatos de Poliisoprenilo/uso terapéutico , Sesquiterpenos/farmacología , Sesquiterpenos/uso terapéutico , Neoplasias Uterinas/complicaciones , Neoplasias Uterinas/patologíaRESUMEN
BACKGROUND: p57(Kip2), a cyclin-dependent kinase inhibitor, is considered to be a candidate tumor suppressor gene that has been implicated in Beckwith-Wiedemann syndrome and sporadic cancers. In addition, decreased expression of p57(Kip2) protein has been frequently observed in pancreatic, lung, breast, bladder, gastrointestinal tract and prostate cancers. However, p57(Kip2) gene mutations are rare in these cancers suggesting that other unknown mechanisms might be at play in reducing its expression. The aim of this study was to investigate the molecular mechanism of down-regulation of p57(Kip2) in prostate cancer. FINDINGS: We observed a significant negative correlation between the expression of p57(Kip2) and microRNA-21 (miR-21) in prostate cancer samples and after androgen deprivation with castration in the CWR22 human prostate cancer xenograft model. We report that miR-21 targeted the coding region and decreased p57(Kip2) mRNA and protein levels in prostate cancer cells. Conversely, inhibition of endogenous miR-21 by an anti-miR-21 inhibitor strongly induced p57(Kip2) expression. Furthermore, we found that knockdown of p57(Kip2) reversed the effects of the anti-miR-21 inhibitor on cell migration and anchorage-independent cell growth. CONCLUSIONS: Our results indicate that miR-21 is able to downregulate p57(Kip2) expression by targeting the coding region of the gene and is also able to attenuate p57(Kip2) mediated functional responses. This is the first report demonstrating that p57(Kip2) is a novel target of miR-21 in prostate cancer and revealing a novel oncogenic function of this microRNA.
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Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , MicroARNs/metabolismo , Neoplasias de la Próstata/genética , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , MicroARNs/genética , Trasplante de Neoplasias , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismoRESUMEN
Six Transmembrane Epithelial Antigen of Prostate 2 (STEAP2) belongs to a family of metalloreductases, which indirectly aid in uptake of iron and copper ions. Its role in hepatocellular carcinoma (HCC) remains to be characterized. Here, we report that STEAP2 expression was upregulated in HCC tumors compared with paired adjacent non-tumor tissues by RNA sequencing, RT-qPCR, Western blotting, and immunostaining. Public HCC datasets demonstrated upregulated STEAP2 expression in HCC and positive association with tumor grade. Transient and stable knockdown (KD) of STEAP2 in HCC cell lines abrogated their malignant phenotypes in vitro and in vivo, while STEAP2 overexpression showed opposite effects. STEAP2 KD in HCC cells led to significant alteration of genes associated with extracellular matrix organization, cell adhesion/chemotaxis, negative enrichment of an invasiveness signature gene set, and inhibition of cell migration/invasion. STEAP2 KD reduced intracellular copper levels and activation of stress-activated MAP kinases including p38 and JNK. Treatment with copper rescued the reduced HCC cell migration due to STEAP2 KD and activated p38 and JNK. Furthermore, treatment with p38 or JNK inhibitors significantly inhibited copper-mediated cell migration. Thus, STEAP2 plays a malignant-promoting role in HCC cells by driving migration/invasion via increased copper levels and MAP kinase activities. Our study uncovered a novel molecular mechanism contributing to HCC malignancy and a potential therapeutic target for HCC treatment.
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Carcinoma Hepatocelular , Movimiento Celular , Cobre , Neoplasias Hepáticas , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/genética , Cobre/metabolismo , Línea Celular Tumoral , Animales , Regulación Neoplásica de la Expresión Génica , Ratones , Progresión de la Enfermedad , Masculino , Oxidorreductasas/metabolismo , Oxidorreductasas/genética , FemeninoRESUMEN
Bone tissue represents the most frequent site of cancer metastasis. We developed a hemichannel-activating antibody, Cx43-M2. Cx43-M2, directly targeting osteocytes in situ, activates osteocytic hemichannels and elevates extracellular ATP, thereby inhibiting the growth and migration of cultured breast and osteosarcoma cancer cells. Cx43-M2 significantly decreases breast cancer metastasis, osteosarcoma growth, and osteolytic activity, while improving survival rates in mice. The antibody's inhibition of breast cancer and osteosarcoma is dose dependent in both mouse and human cancer metastatic models. Furthermore, Cx43-M2 enhances anti-tumor immunity by increasing the population and activation of tumor-infiltrating immune-promoting effector T lymphocytes, while reducing immune-suppressive regulatory T cells. Our results suggest that the Cx43-M2 antibody, by activating Cx43 hemichannels and facilitating ATP release and purinergic signaling, transforms the cancer microenvironment from a supportive to a suppressive state. Collectively, our study underscores the potential of Cx43-M2 as a therapeutic for treating breast cancer bone metastasis and osteosarcoma.
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Adenosina Trifosfato , Neoplasias Óseas , Neoplasias de la Mama , Conexina 43 , Osteocitos , Osteosarcoma , Osteosarcoma/patología , Osteosarcoma/metabolismo , Animales , Osteocitos/metabolismo , Adenosina Trifosfato/metabolismo , Humanos , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Conexina 43/metabolismo , Ratones , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Línea Celular Tumoral , Microambiente Tumoral , Anticuerpos/farmacologíaRESUMEN
Animal studies have demonstrated the ability of pancreatic acinar cells to transform into pancreatic ductal adenocarcinoma (PDAC). However, the tumorigenic potential of human pancreatic acinar cells remains under debate. To address this gap in knowledge, we expand sorted human acinar cells as 3D organoids and genetically modify them through introduction of common PDAC mutations. The acinar organoids undergo dramatic transcriptional alterations but maintain a recognizable DNA methylation signature. The transcriptomes of acinar organoids are similar to those of disease-specific cell populations. Oncogenic KRAS alone do not transform acinar organoids. However, acinar organoids can form PDAC in vivo after acquiring the four most common driver mutations of this disease. Similarly, sorted ductal cells carrying these genetic mutations can also form PDAC, thus experimentally proving that PDACs can originate from both human acinar and ductal cells. RNA-seq analysis reveal the transcriptional shift from normal acinar cells towards PDACs with enhanced proliferation, metabolic rewiring, down-regulation of MHC molecules, and alterations in the coagulation and complement cascade. By comparing PDAC-like cells with normal pancreas and PDAC samples, we identify a group of genes with elevated expression during early transformation which represent potential early diagnostic biomarkers.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Humanos , Transcriptoma , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Carcinogénesis/patología , Células Acinares/metabolismo , Perfilación de la Expresión Génica , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
BACKGROUND: The incidence and mortality rates of hepatocellular carcinoma (HCC) among Hispanic individuals in the United States are much higher than in non-Hispanic white people. We conducted multi-omics analyses to elucidate molecular alterations in HCC among Hispanic patients. METHODS: Paired tumor and adjacent non-tumor samples were collected from 31 Hispanic HCCs in South Texas (STX-Hispanic) for genomic, transcriptomic, proteomic, and metabolomic profiling. Serum lipids were profiled in 40 Hispanic and non-Hispanic patients with or without clinically diagnosed HCC. RESULTS: Exome sequencing revealed high mutation frequencies of AXIN2 and CTNNB1 in STX Hispanic HCCs, suggesting a predominant activation of the Wnt/ß-catenin pathway. TERT promoter mutations were also significantly more frequent in the Hispanic cohort (Fisher's exact test, p < .05). Cell cycles and liver function were positively and negatively enriched, respectively, with gene set enrichment analysis. Gene sets representing specific liver metabolic pathways were associated with dysregulation of corresponding metabolites. Negative enrichment of liver adipogenesis and lipid metabolism corroborated with a significant reduction in most lipids in serum samples of HCC patients (paired t-test, p < .0001). Two HCC subtypes from our Hispanic cohort were identified and validated with the TCGA liver cancer cohort. Patients with better overall survival showed higher activity of immune and angiogenesis signatures, and lower activity of liver function-related gene signatures. They also had higher levels of immune checkpoint and immune exhaustion markers. CONCLUSIONS: Our study revealed specific molecular features of Hispanic HCC and potential biomarkers for therapeutic management. It provides a unique resource for studying Hispanic HCC.
RESUMEN
Background: The incidence and mortality rates of hepatocellular carcinoma (HCC) among Hispanics in the United States are much higher than those of non-Hispanic whites. We conducted comprehensive multi-omics analyses to understand molecular alterations in HCC among Hispanic patients. Methods: Paired tumor and adjacent non-tumor samples were collected from 31 Hispanic HCC in South Texas (STX-Hispanic) for genomic, transcriptomic, proteomic, and metabolomic profiling. Additionally, serum lipids were profiled in 40 Hispanic and non-Hispanic patients with or without clinically diagnosed HCC. Results: Exome sequencing revealed high mutation frequencies of AXIN2 and CTNNB1 in STX Hispanic HCCs, suggesting a predominant activation of the Wnt/ß-catenin pathway. The TERT promoter mutation frequency was also remarkably high in the Hispanic cohort. Cell cycles and liver functions were identified as positively- and negatively-enriched, respectively, with gene set enrichment analysis. Gene sets representing specific liver metabolic pathways were associated with dysregulation of corresponding metabolites. Negative enrichment of liver adipogenesis and lipid metabolism corroborated with a significant reduction in most lipids in the serum samples of HCC patients. Two HCC subtypes from our Hispanic cohort were identified and validated with the TCGA liver cancer cohort. The subtype with better overall survival showed higher activity of immune and angiogenesis signatures, and lower activity of liver function-related gene signatures. It also had higher levels of immune checkpoint and immune exhaustion markers. Conclusions: Our study revealed some specific molecular features of Hispanic HCC and potential biomarkers for therapeutic management of HCC and provides a unique resource for studying Hispanic HCC.
RESUMEN
Bone morphogenetic proteins (BMPs) are secreted signaling proteins - they transduce their signals by assembling complexes comprised of one of three known type II receptors and one of four known type I receptors. BMP-9 binds and signals through the type I receptor Alk1, but not other Alks, while BMP-2, -4, and -7 bind and signal through Alk3, and the close homologue Alk6, but not Alk1. The present results, which include the determination of the Alk1 structure using NMR and identification of residues important for binding using SPR, show that the ß-strand framework of Alk1 is highly similar to Alk3, yet there are significant differences in loops shown previously to be important for binding. The most pronounced difference is in the N-terminal portion of the ß4-ß5 loop, which is structurally ordered and includes a similarly placed but shorter helix in Alk1 compared to Alk3. The altered conformation of the ß4-ß5 loop, and to lesser extent ß1-ß2 loop, cause clashes when Alk1 is positioned onto BMP-9 in the manner that Alk3 is positioned onto BMP-2. This necessitates an alternative manner of binding, which is supported by a model of the BMP-9/Alk1 complex constructed using the program RosettaDock. The model shows that Alk1 is positioned similar to Alk3 but is rotated by 40 deg. The alternate positioning allows Alk1 to bind BMP-9 through a large hydrophobic interface, consistent with mutational analysis that identified several residues in the central portion of the ß4-ß5 loop that contribute significantly to binding and are nonconservatively substituted relative to the corresponding residues in Alk3.
Asunto(s)
Receptores de Activinas Tipo II/química , Proteínas Morfogenéticas Óseas/química , Factores de Diferenciación de Crecimiento/química , Receptores de Activinas Tipo II/genética , Receptores de Activinas Tipo II/metabolismo , Secuencia de Aminoácidos , Animales , Proteína Morfogenética Ósea 2/química , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/química , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Factor 2 de Diferenciación de Crecimiento , Factores de Diferenciación de Crecimiento/genética , Factores de Diferenciación de Crecimiento/metabolismo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Células 3T3 NIH , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Biomarkers are lacking for identifying the switch of transforming growth factor-ß (TGF-ß) from tumor-suppressing to tumor-promoting. Mutated p53 (mp53) has been suggested to switch TGF-ß to a tumor promoter. However, we found that mp53 does not always promote the oncogenic role of TGF-ß. Here, we show that endogenous mp53 knockdown enhanced cell migration and phosphorylation of ERK in DU145 prostate cancer cells. Furthermore, ectopic expression of mp53 in p53-null PC-3 prostate cancer cells enhanced Smad-dependent signaling but inhibited TGF-ß-induced cell migration by down-regulating activated ERK. Reactivation of ERK by the expression of its activator, MEK-1, restored TGF-ß-induced cell migration. Because TGF-ß is known to activate the MAPK/ERK pathway through direct phosphorylation of the adaptor protein ShcA and MAPK/ERK signaling is pivotal to tumor progression, we investigated whether ShcA contributed to mp53-induced ERK inhibition and the conversion of the role of TGF-ß during carcinogenesis. We found that mp53 expression led to a decrease of phosphorylated p52ShcA/ERK levels and an increase of phosphorylated Smad levels in a panel of mp53-expressing cancer cell lines and in mammary glands and tumors from mp53 knock-in mice. By manipulating ShcA levels to regulate ERK and Smad signaling in human untransformed and cancer cell lines, we showed that the role of TGF-ß in regulating anchorage-dependent and -independent growth and migration can be shifted between growth suppression and migration promotion. Thus, our results for the first time suggest that mp53 disrupts the role of ShcA in balancing the Smad-dependent and -independent signaling activity of TGF-ß and that ShcA/ERK signaling is a major pathway regulating the tumor-promoting activity of TGF-ß.
Asunto(s)
Genes p53 , Proteínas Adaptadoras de la Señalización Shc/genética , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación , Neoplasias de la Próstata/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Transducción de Señal , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Hedgehog signaling pathway has been previously elucidated to be inappropriately activated in many human cancers, including ovarian and breast cancer. However, mechanistic contribution of GLI3, one of the terminal effectors of the pathway, to ovarian and mammary cancer development is underexplored. In this study, we investigated whether GLI3 is necessary for the growth and migration of ovarian and breast cancer cells and further explored the underlying mechanism of GLI3-mediated oncogenesis. We report that GLI3 knockdown inhibited growth and migration of androgen receptor (AR)-positive ovarian and breast cancer cells, but not AR-negative ovarian and breast cancer cells. Furthermore, knockdown of AR expression was effective in inhibiting the growth and migration of AR-positive ovarian and breast cancer cells in the presence of GLI3, but not in GLI3 knockdown cells. Similarly, ectopic expression of AR promoted the growth and migration of AR-negative ovarian and breast cancer cells in the presence of GLI3, but not in GLI3 knockdown cells. GLI3 and AR co-immunoprecipitated each other. GLI3 expression was negatively associated with overall survival of ovarian or breast patients whose tumors expressed a high level of AR. Our findings suggest that GLI3 and AR not only physically interact, but also are mutually dependent for their malignancy-promoting activity in ovarian and breast cancer cells. GLI3-specific inhibitors may be novel therapeutics for AR-expressing ovarian and breast cancers.
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Neoplasias de la Mama , Receptores Androgénicos , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Proteínas Hedgehog , Humanos , Proteínas del Tejido Nervioso/metabolismo , Receptores Androgénicos/metabolismo , Transducción de Señal , Proteína Gli3 con Dedos de Zinc/genética , Proteína Gli3 con Dedos de Zinc/metabolismoRESUMEN
F-box only protein 22 (FBXO22) is a key subunit of the Skp1-Cullin 1-F-box protein (SCF) E3 ubiquitin ligase complex. Little is known regarding its biological function and underlying molecular mechanisms in regulating cervical cancer (CC) progression. In this study, we aim to explore the role and mechanism of FBXO22 in CC progression. The correlation between FBXO22 and clinicopathological characteristics of CC was analyzed by tissue microarray. MTT, colony formation, flow cytometry, Western blotting, qRT-PCR, protein half-life, co-immunoprecipitation, ubiquitination, and xenograft experiments were performed to assess the functions of FBXO22 and potential molecular mechanisms of FBXO22-mediated malignant progression in CC. The expression of FBXO22 protein in CC tissues was higher than that in adjacent non-tumor cervical tissues. Notably, high expression of FBXO22 was significantly associated with high histology grades, positive lymph node metastasis, and poor outcomes in CC patients. Functionally, ectopic expression of FBXO22 promoted cell viability in vitro and induced tumor growth in vivo, while knockdown of FBXO22 exhibited opposite effects. In addition, overexpression of FBXO22 promoted G1/S phase progression and inhibited apoptosis in CC cells. Mechanistically, FBXO22 physically interacted with the cyclin-dependent kinase inhibitor p57Kip2 and subsequently mediated its ubiquitination and proteasomal degradation leading to tumor progression. FBXO22 protein level was found negatively associated with p57Kip2 protein levels in patient CC samples. FBXO22 promotes CC progression partly through regulating the ubiquitination and proteasomal degradation of p57Kip2. Our study indicates that FBXO22 might be a novel prognostic biomarker and therapeutic target for CC.
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Inhibidor p57 de las Quinasas Dependientes de la Ciclina , Proteínas F-Box , Receptores Citoplasmáticos y Nucleares , Neoplasias del Cuello Uterino , Animales , Biomarcadores/metabolismo , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Femenino , Humanos , Receptores Citoplasmáticos y Nucleares/metabolismo , Ubiquitinación , Neoplasias del Cuello Uterino/genéticaRESUMEN
Bone metastasis is a common and devastating consequence of several major cancer types, including breast and prostate. Osteocytes are the predominant bone cell, and through connexin (Cx) 43 hemichannels release ATP to the bone microenvironment that can be hydrolyzed to adenosine. Here, we investigated how genes related to ATP paracrine signaling are involved in two common bone-metastasizing malignancies, estrogen receptor positive (ER+) breast and prostate cancers. Compared to other sites, bone metastases of both cancer types expressed higher levels of ENTPD1 and NT5E, which encode CD39 and CD73, respectively, and hydrolyze ATP to adenosine. ADORA3, encoding the adenosine A3 receptor, had a similar expression pattern. In primary ER+ breast cancer, high levels of the triplet ENTPD1/NT5E/ADORA3 expression signature was correlated with lower overall, distant metastasis-free, and progression-free survival. In ER+ bone metastasis biopsies, this expression signature is associated with lower survival. This expression signature was also higher in bone-metastasizing primary prostate cancers than in those that caused other tumor events or did not lead to progressive disease. In 3D culture, a non-hydrolyzable ATP analog inhibited the growth of breast and prostate cancer cell lines more than ATP did. A3 inhibition also reduced spheroid growth. Large-scale screens by the Drug Repurposing Hub found ER+ breast cancer cell lines were uniquely sensitive to adenosine receptor antagonists. Together, these data suggest a vital role for extracellular ATP degradation and adenosine receptor signaling in cancer bone metastasis, and this study provides potential diagnostic means for bone metastasis and specific targets for treatment and prevention.