RESUMO
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.
Assuntos
Trifosfato de Adenosina , Neoplasias Ósseas , Neoplasias da Mama , Conexina 43 , Osteócitos , Osteossarcoma , Osteossarcoma/patologia , Osteossarcoma/metabolismo , Animais , Osteócitos/metabolismo , Trifosfato de Adenosina/metabolismo , Humanos , Feminino , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Conexina 43/metabolismo , Camundongos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Neoplasias Ósseas/secundário , Linhagem Celular Tumoral , Microambiente Tumoral , Anticorpos/farmacologiaRESUMO
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.
Assuntos
Carcinoma Hepatocelular , Movimento Celular , Cobre , Neoplasias Hepáticas , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Cobre/metabolismo , Linhagem Celular Tumoral , Animais , Regulação Neoplásica da Expressão Gênica , Camundongos , Progressão da Doença , Masculino , Oxirredutases/metabolismo , Oxirredutases/genética , FemininoRESUMO
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.
RESUMO
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.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Humanos , Transcriptoma , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Carcinogênese/patologia , Células Acinares/metabolismo , Perfilação da Expressão Gênica , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama , Animais , Humanos , Feminino , Neoplasias da Mama/genética , Glândulas Mamárias Animais/metabolismo , Células-Tronco/metabolismo , Biomarcadores/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sirolimo/farmacologia , Sirolimo/metabolismo , Células Epiteliais/metabolismoRESUMO
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.
Assuntos
Diabetes Mellitus Tipo 2 , Hiperglicemia , Humanos , Animais , Camundongos , Glucagon/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hiperglicemia/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Hepatócitos/metabolismo , Fígado/metabolismo , Glucose/metabolismo , Gluconeogênese , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
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.
RESUMO
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.
Assuntos
Inibidor de Quinase Dependente de Ciclina p57 , Proteínas F-Box , Receptores Citoplasmáticos e Nucleares , Neoplasias do Colo do Útero , Animais , Biomarcadores/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/genética , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Feminino , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Ubiquitinação , Neoplasias do Colo do Útero/genéticaRESUMO
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.
Assuntos
Carcinoma Hepatocelular , Integrina alfa6 , Integrina alfa6beta4 , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Integrina alfa6beta4/genética , Integrina alfa6beta4/metabolismo , Integrina beta4/genética , Integrina beta4/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologiaRESUMO
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.
Assuntos
Neoplasias da Mama , Receptores Androgênicos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Proteínas Hedgehog , Humanos , Proteínas do Tecido Nervoso/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais , Proteína Gli3 com Dedos de Zinco/genética , Proteína Gli3 com Dedos de Zinco/metabolismoRESUMO
Although the Sonic hedgehog (SHH) signaling pathway has been implicated in promoting malignant phenotypes of prostate cancer, details on how it is activated and exerts its oncogenic role during prostate cancer development and progression is less clear. Here, we show that GLI3, a key SHH pathway effector, is transcriptionally upregulated during androgen deprivation and posttranslationally stabilized in prostate cancer cells by mutation of speckle-type POZ protein (SPOP). GLI3 is a substrate of SPOP-mediated proteasomal degradation in prostate cancer cells and prostate cancer driver mutations in SPOP abrogate GLI3 degradation. Functionally, GLI3 is necessary and sufficient for the growth and migration of androgen receptor (AR)-positive prostate cancer cells, particularly under androgen-depleted conditions. Importantly, we demonstrate that GLI3 physically interacts and functionally cooperates with AR to enrich an AR-dependent gene expression program leading to castration-resistant growth of xenografted prostate tumors. Finally, we identify an AR/GLI3 coregulated gene signature that is highly correlated with castration-resistant metastatic prostate cancer and predictive of disease recurrence. Together, these findings reveal that hyperactivated GLI3 promotes castration-resistant growth of prostate cancer and provide a rationale for therapeutic targeting of GLI3 in patients with castration-resistant prostate cancer (CRPC). IMPLICATIONS: We describe two clinically relevant mechanisms leading to hyperactivated GLI3 signaling and enhanced AR/GLI3 cross-talk, suggesting that GLI3-specific inhibitors might prove effective to block prostate cancer development or delay CRPC.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares , Neoplasias de Próstata Resistentes à Castração/genética , Proteínas Repressoras/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteína Gli3 com Dedos de Zinco/metabolismo , Animais , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Mutação , Receptores Androgênicos/metabolismoRESUMO
Osteocytes, the most abundant bone cell types embedded in the mineral matrix, express connexin 43 (Cx43) hemichannels that play important roles in bone remodeling and osteocyte survival. Estrogen deficiency decreases osteocytic Cx43 hemichannel activity and causes a loss in osteocytes' resistance to oxidative stress (OS). In this study, we showed that OS reduced the growth of both human (MDA-MB-231) and murine (Py8119) breast cancer cells. However, co-culturing these cells with osteocytes reduced the inhibitory effect of OS on breast cancer cells, and this effect was ablated by the inhibition of Cx43 hemichannels. Py8119 cells were intratibially implanted in the bone marrow of ovariectomized (OVX) mice to determine the role of osteocytic Cx43 hemichannels in breast cancer bone metastasis in response to OS. Two transgenic mice overexpressing dominant-negative Cx43 mutants, R76W and Δ130-136, were adopted for this study; the former inhibits gap junctions while the latter inhibits gap junctions and hemichannels. Under normal conditions, Δ130-136 mice had significantly more tumor growth in bone than that in WT and R76W mice. OVX increased tumor growth in R76W but had no significant effect on WT mice. In contrast, OVX reduced tumor growth in Δ130-136 mice. To confirm the role of OS, WT and Δ130-136 mice were administered the antioxidant N-acetyl cysteine (NAC). NAC increased tumor burden and growth in Δ130-136 mice but not in WT mice. Together, the data suggest that osteocytes and Cx43 hemichannels play pivotal roles in modulating the oxidative microenvironment and breast cancer growth in the bone.
RESUMO
During prostate cancer progression, cancerous epithelial cells can undergo epithelial-mesenchymal transition (EMT). EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. It is associated with biochemical changes such as epithelial cell markers E-cadherin and occludins being down-regulated, and mesenchymal markers vimentin and N-cadherin being upregulated. These changes in protein expression, specifically in the cell membrane, may be monitored via biophysical principles, such as changes in the refractive index (RI) of the cell membrane. In our previous research, we demonstrated the feasibility of using cellular RI as a unique contrast parameter to accomplish label-free detection of prostate cancer cells. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free biosensing system, which allows for ultra-sensitive quantification of the changes in cellular RI due to EMT. We induced prostate cancer cells to undergo EMT by exposing these cells to soluble Transforming Growth Factor Beta 1 (TGF-ß1). The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced cancer cells. Our study shows promising clinical potential in utilizing the PC-TIR biosensing system not only to detect prostate cancer cells, but also to evaluate changes in prostate cancer cells due to EMT.
RESUMO
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.
Assuntos
Neoplasias da Mama/genética , Mama/fisiologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Progressão da Doença , Dineínas/genética , Dineínas/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais CultivadasRESUMO
Gliomaassociated oncogene family zinc finger 2 (Gli2), a key component of the hedgehog signaling pathway, has been previously demonstrated to promote the malignant properties of prostate cancer in vitro. However, the role of Gli2 in the development of castrationresistant prostate cancer (CRPC) has yet to be fully elucidated. In the present study, Gli2 expression was knocked down in androgenresponsive prostate cancer cells using an inducible Gli2 short hairpin RNA. Suppression of Gli2 expression resulted in significant reduction of cell viability, increased the proportion of cells in the G0/G1 phases of the cell cycle and reduced the expression of genes associated with cell cycle progression. Gli2 knockdown sensitized both androgendependent and independent prostate cancer cells to the antiandrogen drug Casodex and prevented the outgrowth of LNCaP prostate cancer cells. In addition, Gli2 knockdown significantly suppressed the development of CRPC in a LNCaP xenograft mouse model, which was reversed by the reexpression of Gli2. In conclusion, to the best of our knowledge, the present study was the first occasion in which the essential role of Gli2 in the development of CRPC was demonstrated, providing a potential therapeutic target for the intervention of CRPC.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Proteínas Nucleares/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Proteína Gli2 com Dedos de Zinco/metabolismo , Antagonistas de Androgênios/farmacologia , Antagonistas de Androgênios/uso terapêutico , Anilidas/farmacologia , Anilidas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Interferente Pequeno/metabolismo , Compostos de Tosil/farmacologia , Compostos de Tosil/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína Gli2 com Dedos de Zinco/antagonistas & inibidores , Proteína Gli2 com Dedos de Zinco/genéticaRESUMO
PURPOSE: We evaluated the role of everolimus in the prevention of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) progression. EXPERIMENTAL DESIGN: The effects of everolimus on breast cancer cell invasion, DCIS formation, and DCIS progression to IDC were investigated in a 3D cell culturing model, intraductal DCIS xenograft model, and spontaneous MMTV-Her2/neu mouse model. The effect of everolimus on matrix metalloproteinase 9 (MMP9) expression was determined with Western blotting and IHC in these models and in patients with DCIS before and after a window trial with rapamycin. Whether MMP9 mediates the inhibition of DCIS progression to IDC by everolimus was investigated with knockdown or overexpression of MMP9 in breast cancer cells. RESULTS: Everolimus significantly inhibited the invasion of human breast cancer cells in vitro. Daily intragastric treatment with everolimus for 7 days significantly reduced the number of invasive lesions from intraductal DCIS foci and inhibited DCIS progression to IDC in the MMTV-Her2/neu mouse mammary tumor model. Mechanistically, everolimus treatment decreased the expression of MMP9 in the in vitro and in vivo models, and in breast tissues from patients with DCIS treated with rapamycin for 1 week. Moreover, overexpression of MMP9 stimulated the invasion, whereas knockdown of MMP9 inhibited the invasion of breast cancer cell-formed spheroids in vitro and DCIS in vivo. Knockdown of MMP9 also nullified the invasion inhibition by everolimus in vitro and in vivo. CONCLUSIONS: Targeting mTORC1 can inhibit DCIS progression to IDC via MMP9 and may be a potential strategy for DCIS or early-stage IDC therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Carcinoma Intraductal não Infiltrante/tratamento farmacológico , Everolimo/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/patologia , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Regulação para Baixo , Feminino , Humanos , Metaloproteinase 9 da Matriz/química , Camundongos , Camundongos Nus , Camundongos Transgênicos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: To investigate the role of IGF-1 signaling pathway in the treatment of uterine leiomyomas with mifepristone. PATIENTS AND METHODS: From October 2015 to December 2018, 50 patients with uterine leiomyoma were included in this study. Overexpression or siRNA of IGF-1 in primary human uterine leiomyoma cells were treated with or without mifepristone. MTT was used to evaluate cell viability in assays of cell proliferation and cytotoxicity. IGF-1 expression in the cells was measured with real-time RT-PCR and Western blotting and manipulated with lentivirus ectopic overexpression or siRNA silencing. RESULTS: Inhibition of cell viability by mifepristone was found dependent on drug concentration and treatment time. IGF-1 and phosphorylation-ERK1/2 expression were decreased, while phosphorylation-AKT expression was increased after mifepristone treatment. IGF-1 significantly promoted cell growth, while IGF-1 knockdown and mifepristone showed synergistic inhibition effects on cell growth. The overexpression of IGF-1 reversed the inhibition of cell growth and ERK1/2 phosphorylation but showed no effect on AKT phosphorylation. CONCLUSION: Our study for the first time demonstrated that IGF-1 signaling via ERK1/2 appears to be an important target of mifepristone in the treatment of uterine leiomyomas, which may provide a new approach to avoid leiomyoma re-growth after cessation of mifepristone.
Assuntos
Antineoplásicos/farmacologia , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Leiomioma/tratamento farmacológico , Mifepristona/farmacologia , Transdução de Sinais/efeitos dos fármacos , Neoplasias Uterinas/tratamento farmacológico , Adulto , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Leiomioma/metabolismo , Leiomioma/patologia , Células Tumorais Cultivadas , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologiaRESUMO
OBJECTIVE: To investigate dynein light chain Tctex-type 3 (DYNLT3) protein expression in ovarian epithelial lesions and explore the effects and related mechanisms of DYNLT3 in terms of the biological behavior of ovarian cancer. MATERIALS AND METHODS: Initially, expression of the DYNLT3 protein in ovarian epithelial lesions was detected by immunohistochemical staining, and the prognostic value of DYNLT3 mRNA expression in ovarian cancer patients was assessed using the Kaplan-Meier plotter database. Then, the mRNA and protein expression of DYNLT3 in IOSE80 normal ovarian epithelial cells and SKOV3 ovarian cancer cells was evaluated by quantitative real-time polymerase chain reaction and Western blotting respectively, and the proliferation, apoptosis, migration and invasion of SKOV3 cells after DYNLT3 over-expression and under-expression were investigated by CCK-8 assays and immunofluorescence staining, flow cytometry, wound healing assays and Transwell invasion assays, respectively. Furthermore, the expression of the proliferation-related proteins PCNA and Ki-67 and the invasion- and migration-related proteins Ezrin, Fascin, MMP2 and MMP9 in cells was examined by Western blotting. RESULTS: The protein expression of DYNLT3 gradually increased during the progression of ovarian epithelial lesions, and was related to the development of ovarian cancer. High expression of DYNLT3 mRNA was related to poor overall survival and progression free survival, especially in serous ovarian cancer patients. In addition, overexpression of DYNLT3 promoted SKOV3 cell proliferation, invasion and migration. The corresponding results were also verified by a DYNLT3 knockdown assay. Moreover, DYNLT3 increased cell proliferation, which was related to Ki-67 expression. Besides, DYNLT3 enhanced cell invasion and migration through regulating Ezrin, but not Fascin, MMP2 or MMP9. CONCLUSION: DYNLT3 exerts pro-tumoral effects on ovarian cancer through promoting cell proliferation, migration and invasion, possibly via regulating the protein expression of Ki-67 and Ezrin. DYNLT3 may be a potential prognostic predictor in ovarian cancer.
RESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Everolimo/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Ciclina D1/genética , Feminino , Humanos , Células MCF-7 , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteólise , Transdução de Sinais , Técnicas de Cultura de TecidosRESUMO
In vivo transplantation is the gold standard method for characterization of stem/progenitor cell self-renewal, tissue regeneration, and tumorigenesis. The method requires an enriched population of stem cells that represent a small fraction of a given tissue. An enriched population of stem/progenitor cells increases the likelihood of engraftment and reduces the number of recipient animals needed for in vivo transplantation. Methods for mammosphere formation by mammary epithelial stem and progenitor cells have been widely adopted for enriching stem/progenitor cells, allowing researchers to study genetic and epigenetic properties, interaction with other cell types, and differentiation and oncogenic transformation. The generation of mammospheres is complex, however, involving many steps and requiring particular skill. Here we describe a detailed mammosphere protocol, including isolation and culture of human primary mammary epithelial stem/progenitor cells and their differentiation and passage in 3D organoid culture. We also describe a protocol for ex vivo culture of fresh human breast tissue for use in assays of clinical treatment. Step-by-step instructions detail tissue handling through passage of the stem/progenitor cell-generated 3D organoids, which can be used to assess the properties, function, and neoplastic transformation of mammary stem/progenitor cells. © 2018 by John Wiley & Sons, Inc.