RESUMEN
Metabolism controls cellular phenotype and fate. In this report, we demonstrate that nicotinamide N-methyltransferase (NNMT), a metabolic enzyme that regulates developmental stem cell transitions and tumor progression, is highly expressed in human idiopathic pulmonary fibrosis (IPF) lungs, and is induced by the pro-fibrotic cytokine, transforming growth factor-ß1 (TGF-ß1) in lung fibroblasts. NNMT silencing reduces the expression of extracellular matrix proteins, both constitutively and in response to TGF-ß1. Furthermore, NNMT controls the phenotypic transition from homeostatic, pro-regenerative lipofibroblasts to pro-fibrotic myofibroblasts. This effect of NNMT is mediated, in part, by the downregulation of lipogenic transcription factors, TCF21 and PPARγ, and the induction of a less proliferative but more differentiated myofibroblast phenotype. NNMT confers an apoptosis-resistant phenotype to myofibroblasts that is associated with the downregulation of pro-apoptotic members of the Bcl-2 family, including Bim and PUMA. Together, these studies indicate a critical role for NNMT in the metabolic reprogramming of fibroblasts to a pro-fibrotic and apoptosis-resistant phenotype and support the concept that targeting this enzyme may promote regenerative responses in chronic fibrotic disorders such as IPF.
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Miofibroblastos , Nicotinamida N-Metiltransferasa , Humanos , Apoptosis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Fibroblastos/metabolismo , Fibrosis , Fibrosis Pulmonar Idiopática/metabolismo , Pulmón/metabolismo , Miofibroblastos/metabolismo , Nicotinamida N-Metiltransferasa/metabolismo , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Physical characteristics of solid tumors such as dense internal microarchitectures and pathological stiffness influence cancer progression and treatment. While it is routine to engineer culture substrates and scaffolds with elastic moduli that approximate tumors, these models often fail to capture characteristic internal microarchitectures such as densely compacted concentric ECM fibers at the stromal interface. Contractile mesenchymal cells can solve this engineering challenge by deforming, contracting, and compacting extracellular matrix (ECM) hydrogels to decrease tissue volume and increase tissue density. Here we demonstrate that allowing human fibroblasts of varying origins to freely contract collagen type I-containing hydrogels co-seeded with carcinoma cell spheroids produces a tissue engineered construct with structural features that mimic dense solid tumors in vivo. Morphometry and mechanical testing were conducted in tandem with biochemical analysis of proliferation and viability to confirm that dense carcinoma constructs engineered using this approach capture relevant physical characteristics of solid carcinomas in a tractable format that preserves viability and is amenable to extended culture. The reported method is adaptable to the use of multiple mesenchymal cell types and the inclusion of fibrin in the ECM combined with seeding of endothelial cells to produce prevascularized constructs. The physical dense carcinoma constructs engineered using this approach may provide more clinically relevant venues for studying cancer pathophysiology and the challenges associated with the delivery of macromolecular drugs and cellular immunotherapies to solid tumors.
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Carcinoma , Colágeno , Humanos , Colágeno/química , Hidrogeles/química , Células Endoteliales , Matriz Extracelular/metabolismo , Ingeniería de Tejidos/métodos , Carcinoma/metabolismoRESUMEN
Following metastatic spread, many hormone receptor positive (HR+) patients develop a more aggressive phenotype with an observed loss of the HRs estrogen receptor (ER) and progesterone receptor (PR). During metastasis, breast cancer cells are exposed to high magnitudes of fluid shear stress (FSS). Unfortunately, the role for FSS on the regulation of HR expression and function during metastasis is not fully understood. This study was designed to elucidate the impact of FSS on HR+ breast cancer. Utilizing a microfluidic platform capable of exposing breast cancer cells to FSS that mimics in situ conditions, we demonstrate the impact of FSS exposure on representative HR+ breast cancer cell lines through protein and gene expression analysis. Proteomics results demonstrated that 540 total proteins and 1473 phospho-proteins significantly changed due to FSS exposure and pathways of interest included early and late estrogen response. The impact of FSS on response to 17ß-estradiol (E2) was next evaluated and gene expression analysis revealed repression of ER and E2-mediated genes (PR and SDF1) following exposure to FSS. Western blot demonstrated enhanced phosphorylation of mTOR following exposure to FSS. Taken together, these studies provide initial insight into the effects of FSS on HR signaling in metastatic breast cancer.
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Neoplasias de la Mama , Regulación Neoplásica de la Expresión Génica , Receptores de Estrógenos , Receptores de Progesterona , Estrés Mecánico , Humanos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Femenino , Receptores de Progesterona/metabolismo , Receptores de Progesterona/genética , Línea Celular Tumoral , Receptores de Estrógenos/metabolismo , Receptores de Estrógenos/genética , Estradiol/farmacología , Fosforilación , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteómica/métodos , Células MCF-7 , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/genéticaRESUMEN
The significant increase in the incidence of obesity represents the next global health crisis. As a result, scientific research has focused on gaining deeper insights into obesity and adipose tissue biology. As a result of the excessive accumulation of adipose tissue, obesity results from hyperplasia and hypertrophy within the adipose tissue. The functional alterations in the adipose tissue are a confounding contributing factor to many diseases, including cancer. The increased incidence and aggressiveness of several cancers, including colorectal, postmenopausal breast, endometrial, prostate, esophageal, hematological, malignant melanoma, and renal carcinomas, result from obesity as a contributing factor. The increased morbidity and mortality of obesity-associated cancers are attributable to increased hormones, adipokines, and cytokines produced by the adipose tissue. The increased adipose tissue levels observed in obese patients result in more adipose stromal/stem cells (ASCs) distributed throughout the body. ASCs have been shown to impact cancer progression in vitro and in preclinical animal models. ASCs influence tumor biology via multiple mechanisms, including the increased recruitment of ASCs to the tumor site and increased production of cytokines and growth factors by ASCs and other cells within the tumor stroma. Emerging evidence indicates that obesity induces alterations in the biological properties of ASCs, subsequently leading to enhanced tumorigenesis and metastasis of cancer cells. As the focus of this review is the interaction and impact of ASCs on cancer, the presentation is limited to preclinical data generated on cancers in which there is a demonstrated role for ASCs, such as postmenopausal breast, colorectal, prostate, ovarian, multiple myeloma, osteosarcoma, cervical, bladder, and gastrointestinal cancers. Our group has investigated the interactions between obesity and breast cancer and the mechanisms that regulate ASCs and adipocytes in these different contexts through interactions between cancer cells, immune cells, and other cell types present in the tumor microenvironment (TME) are discussed. The reciprocal and circular feedback loop between obesity and ASCs and the mechanisms by which ASCs from obese patients alter the biology of cancer cells and enhance tumorigenesis will be discussed. At present, the evidence for ASCs directly influencing human tumor growth is somewhat limited, though recent clinical studies suggest there may be some link.
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Neoplasias de la Mama , Neoplasias Colorrectales , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Neoplasias de la Mama/patología , Carcinogénesis/patología , Transformación Celular Neoplásica/metabolismo , Neoplasias Colorrectales/patología , Citocinas/metabolismo , Humanos , Masculino , Obesidad/complicaciones , Obesidad/metabolismo , Células del Estroma/metabolismo , Microambiente TumoralRESUMEN
The liver is a major organ that is involved in essential biological functions such as digestion, nutrient storage, and detoxification. Furthermore, it is one of the most metabolically active organs with active roles in regulating carbohydrate, protein, and lipid metabolism. Hepatocellular carcinoma is a cancer of the liver that is associated in settings of chronic inflammation such as viral hepatitis, repeated toxin exposure, and fatty liver disease. Furthermore, liver cancer is the most common cause of death associated with cirrhosis and is the 3rd leading cause of global cancer deaths. LKB1 signaling has been demonstrated to play a role in regulating cellular metabolism under normal and nutrient deficient conditions. Furthermore, LKB1 signaling has been found to be involved in many cancers with most reports identifying LKB1 to have a tumor suppressive role. In this review, we use the KMPlotter database to correlate RNA levels of LKB1 signaling genes and hepatocellular carcinoma patient survival outcomes with the hopes of identifying potential biomarkers clinical usage. Based on our results STRADß, CAB39L, AMPKα, MARK2, SIK1, SIK2, BRSK1, BRSK2, and SNRK expression has a statistically significant impact on patient survival.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Quinasas de la Proteína-Quinasa Activada por el AMP , Proteínas Quinasas Activadas por AMP/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismoRESUMEN
BACKGROUND: Triple-negative breast cancers (TNBCs) are clinically aggressive subtypes of breast cancer. TNBC is difficult to treat with targeted agents due to the lack of commonly targeted therapies within this subtype. Androgen receptor (AR) has been detected in 12-55% of TNBCs. AR stimulates breast tumor growth in the absence of estrogen receptor (ER), and it has become an emerging molecular target in TNBC treatment. METHODS: Ceritinib is a small molecule inhibitor of tyrosine kinase and it is used in the therapy of non-small lung cancer patients. Enzalutamide is a small molecule compound targeting the androgen receptor and it is used to treat prostate cancer. Combination therapy of these drugs were investigated using AR positive breast cancer mouse xenograft models. Also, combination treatment of ceritinib and paclitaxel investigated using AR- and AR low mouse xenograft and patient derived xenograft models. RESULTS: We screened 133 FDA approved drugs that have a therapeutic effect of AR+ TNBC cells. From the screen, we identified two drugs, ceritinib and crizotinib. Since ceritinib has a well- defined role in androgen independent AR signaling pathways, we further investigated the effect of ceritinib. Ceritinib treatment inhibited RTK/ACK/AR pathway and other downstream pathways in AR+ TNBC cells. The combination of ceritinib and enzalutamide showed a robust inhibitory effect on cell growth of AR+ TNBC cells in vitro and in vivo. Interestingly Ceritinib inhibits FAK-YB-1 signaling pathway that leads to paclitaxel resistance in all types of TNBC cells. The combination of paclitaxel and ceritinib showed drastic inhibition of tumor growth compared to a single drug alone. CONCLUSIONS: To improve the response of AR antagonist in AR positive TNBC, we designed a novel combinational strategy comprised of enzalutamide and ceritinib to treat AR+ TNBC tumors through the dual blockade of androgen-dependent and androgen-independent AR signaling pathways. Furthermore, we introduced a novel therapeutic combination of ceritinib and paclitaxel for AR negative or AR-low TNBCs and this combination inhibited tumor growth to a great extent. All agents used in our study are FDA-approved, and thus the proposed combination therapy will likely be useful in the clinic.
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Neoplasias de la Mama Triple Negativas , Andrógenos/uso terapéutico , Animales , Línea Celular Tumoral , Humanos , Ratones , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Pirimidinas , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Sulfonas , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Melatonin, the primary hormone involved in circadian entrainment, plays a significant role in bone physiology. This study aimed to assess the role of MEK1/2 and MEK5 in melatonin-mediated actions in mouse and human mesenchymal stem cells (MSCs) and on bone using small-molecule inhibitors and CRISPR/Cas9 knockout approaches. Consistent with in vitro studies performed in mMSCs and hMSCs, nightly (25 mg/kg, i.p., 45 days) injections with PD184352 (MEK1/2 inhibitor) or Bix02189 (MEK5 inhibitor) or SC-1-151 (MEK1/2/5 inhibitor) demonstrated that MEK1/2 and MEK5 were the primary drivers underlying melatonin's actions on bone density, microarchitecture (i.e., trabecular number, separation, and connectivity density), and bone mechanical properties (i.e., ultimate stress) through increases in osteogenic (RUNX2, BMP-2, FRA-1, OPG) expression and decreases in PPARγ. Furthermore, CRISPR/Cas9 knockout of MEK1 or MEK5 in mMSCs seeded on PLGA scaffolds and placed into critical-size calvarial defects in Balb(c) mice (male and female) revealed that treatment with melatonin (15 mg/L; p.o., nightly, 90 days) mediates sex-specific actions of MEK1 and MEK5 in new bone formation. This study is the first to demonstrate a role for MEK1/2 and MEK5 in modulating melatonin-mediated actions on bone formation in vivo and in a sex-specific manner.
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Melatonina , Osteogénesis , Animales , Fenómenos Biomecánicos , Densidad Ósea , Huesos , Femenino , Humanos , Masculino , Melatonina/farmacología , Melatonina/fisiología , RatonesRESUMEN
Aromatase inhibitors (AIs) are standard treatment for estrogen-dependent postmenopausal breast tumors; however, resistance develops leading to tumor relapse and metastasis. We previously demonstrated that glyceollin inhibits proliferation, survival, and migration of hormone-independent letrozole-resistant breast cancer. Since many AI-resistant tumors remain hormone-dependent, identifying distinctions between estrogen-receptor-positive (ER+) and ER-negative (ER-) AI-resistant tumor response to therapy is critical. We hypothesize that treating ER+ letrozole-resistant T47D breast cancer cells (T47DaromLR) with a combination of 10 µM glyceollin and 0.5 µM lapatinib (a dual EGFR/HER2 inhibitor) will decrease cell proliferation through induction of apoptosis. The T47DaromLR cells were found to overexpress HER2 and MAPK while maintaining aromatase and ER levels compared to their letrozole-sensitive (T47Darom) counterparts. In the absence of estrogen stimulation, glyceollin ± lapatinib had no effect on the proliferation of the T47Darom cells, while glyceollin treatment caused 46% reduction in the proliferation of T47DaromLR cells, which was further diminished when combined with lapatinib. While neither agent influenced cell migration, glyceollin and lapatinib reduced S and G2/M phase cell entry and exclusively induced apoptosis by 1.29-fold in the T47DaromLR cells. Taken together, these results suggest that glyceollins and lapatinib may have potential as a novel combination therapeutic approach for hormone-dependent, letrozole-resistant tumors.
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Inhibidores de la Aromatasa , Neoplasias de la Mama , Apoptosis , Aromatasa , Inhibidores de la Aromatasa/farmacología , Inhibidores de la Aromatasa/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Estrógenos/farmacología , Estrógenos/uso terapéutico , Femenino , Humanos , Lapatinib/farmacología , Lapatinib/uso terapéutico , Letrozol/farmacología , Recurrencia Local de Neoplasia/tratamiento farmacológico , Nitrilos/uso terapéutico , Pterocarpanos , Triazoles/farmacologíaRESUMEN
Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT) when cells adopt a motile and invasive phenotype through loss of epithelial markers (CDH1), and acquisition of mesenchymal markers (VIM, CDH2). Although MAPK/ERK1/2 kinase inhibitors (MEKi) are useful antitumor agents in a clinical setting, including the Food and Drug Administration (FDA)-approved MEK1,2 dual inhibitors cobimetinib and trametinib, there are limitations to their clinical utility, primarily adaptation of the BRAF pathway and ocular toxicities. The MEK5 (HGNC: MAP2K5) pathway has important roles in metastatic progression of various cancer types, including those of the prostate, colon, bone and breast, and elevated levels of ERK5 expression in breast carcinomas are linked to a worse prognoses in TNBC patients. The purpose of this study is to explore MEK5 regulation of the EMT axis and to evaluate a novel pan-MEK inhibitor on clinically aggressive TNBC cells. Our results show a distinction between the MEK1/2 and MEK5 cascades in maintenance of the mesenchymal phenotype, suggesting that the MEK5 pathway may be necessary and sufficient in EMT regulation while MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Furthermore, additive effects on MET induction are evident through the inhibition of both MEK1/2 and MEK5. Taken together, these data demonstrate the need for a better understanding of the individual roles of MEK1/2 and MEK5 signaling in breast cancer and provide a rationale for the combined targeting of these pathways to circumvent compensatory signaling and subsequent therapeutic resistance.
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Movimiento Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , MAP Quinasa Quinasa 5/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Neoplasias de la Mama Triple Negativas/metabolismo , Femenino , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/genética , MAP Quinasa Quinasa 5/antagonistas & inhibidores , MAP Quinasa Quinasa 5/genética , Células MCF-7 , Proteínas Proto-Oncogénicas c-fos/genética , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
PURPOSE: The transcription factors ZEB1 and ZEB2 mediate epithelial-to-mesenchymal transition (EMT) and metastatic progression in numerous malignancies including breast cancer. ZEB1 and ZEB2 drive EMT through transcriptional repression of cell-cell junction proteins and members of the tumor suppressive miR200 family. However, in estrogen receptor positive (ER +) breast cancer, the role of ZEB2 as an independent driver of metastasis has not been fully investigated. METHODS: In the current study, we induced exogenous expression of ZEB2 in ER + MCF-7 and ZR-75-1 breast cancer cell lines and examined EMT gene expression and metastasis using dose-response qRT-PCR, transwell migration assays, proliferation assays with immunofluorescence of Ki-67 staining. We used RNA sequencing to identify pathways and genes affected by ZEB2 overexpression. Finally, we treated ZEB2-overexpressing cells with 17ß-estradiol (E2) or ICI 182,780 to evaluate how ZEB2 affects estrogen response. RESULTS: Contrary to expectation, we found that ZEB2 did not increase canonical epithelial nor decrease mesenchymal gene expressions. Furthermore, ZEB2 overexpression did not promote a mesenchymal cell morphology. However, ZEB1 and ZEB2 protein expression induced significant migration of MCF-7 and ZR-75-1 breast cancer cells in vitro and MCF-7 xenograft metastasis in vivo. Transcriptomic (RNA sequencing) pathway analysis revealed alterations in estrogen signaling regulators and pathways, suggesting a role for ZEB2 in endocrine sensitivity in luminal A breast cancer. Expression of ZEB2 was negatively correlated with estrogen receptor complex genes in luminal A patient tumors. Furthermore, treatment with 17ß-estradiol (E2) or the estrogen receptor antagonist ICI 182,780 had no effect on growth of ZEB2-overexpressing cells. CONCLUSION: ZEB2 is a multi-functional regulator of drug sensitivity, cell migration, and metastasis in ER + breast cancer and functions through non-canonical mechanisms.
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Neoplasias de la Mama , Transición Epitelial-Mesenquimal , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal/genética , Femenino , Fulvestrant , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genéticaRESUMEN
PURPOSE: Breast cancer remains a prominent global disease affecting women worldwide despite the emergence of novel therapeutic regimens. Metastasis is responsible for most cancer-related deaths, and acquisition of a mesenchymal and migratory cancer cell phenotypes contributes to this devastating disease. The utilization of kinase targets in drug discovery have revolutionized the field of cancer research but despite impressive advancements in kinase-targeting drugs, a large portion of the human kinome remains understudied in cancer. NEK5, a member of the Never-in-mitosis kinase family, is an example of such an understudied kinase. Here, we characterized the function of NEK5 in breast cancer. METHODS: Stably overexpressing NEK5 cell lines (MCF7) and shRNA knockdown cell lines (MDA-MB-231, TU-BcX-4IC) were utilized. Cell morphology changes were evaluated using immunofluorescence and quantification of cytoskeletal components. Cell proliferation was assessed by Ki-67 staining and transwell migration assays tested cell migration capabilities. In vivo experiments with murine models were necessary to demonstrate NEK5 function in breast cancer tumor growth and metastasis. RESULTS: NEK5 activation altered breast cancer cell morphology and promoted cell migration independent of effects on cell proliferation. NEK5 overexpression or knockdown does not alter tumor growth kinetics but promotes or suppresses metastatic potential in a cell type-specific manner, respectively. CONCLUSION: While NEK5 activity modulated cytoskeletal changes and cell motility, NEK5 activity affected cell seeding capabilities but not metastatic colonization or proliferation in vivo. Here we characterized NEK5 function in breast cancer systems and we implicate NEK5 in regulating specific steps of metastatic progression.
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Neoplasias de la Mama , Quinasas Relacionadas con NIMA , Animales , Neoplasias de la Mama/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Humanos , Ratones , Quinasas Relacionadas con NIMA/genética , Fenotipo , ARN Interferente PequeñoRESUMEN
Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.
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Supervivencia Celular/efectos de los fármacos , MAP Quinasa Quinasa 5/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias de la Mama Triple Negativas/metabolismo , Apoptosis/efectos de los fármacos , Benzodiazepinonas/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , MAP Quinasa Quinasa 5/antagonistas & inhibidores , Proteína Quinasa 7 Activada por Mitógenos/antagonistas & inhibidores , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridonas/farmacología , Pirimidinas/farmacología , Pirimidinonas/farmacologíaRESUMEN
Breast cancer affects women globally; the majority of breast cancer-related mortalities are due to metastasis. Acquisition of a mesenchymal phenotype has been implicated in the progression of breast cancer cells to an invasive, metastatic state. Triple-negative breast cancer (TNBC) subtypes have high rates of metastases, recurrence, and have poorer prognoses compared to other breast cancer types, partially due to lack of commonly targeted receptors. Kinases have diverse and pivotal functions in metastasis in TNBC, and discovery of new kinase targets for TNBC is warranted. We previously used a screening approach to identify intermediate-synthesis nonpotent, nonselective small-molecule inhibitors from the Published Kinase Inhibitor Set that reversed the mesenchymal phenotype in TNBC cells. Two of these inhibitors (GSK346294A and GSK448459A) are structurally similar, but have unique kinase activity profiles and exhibited differential biologic effects on TNBC cells, specifically on epithelial-to-mesenchymal transition (EMT). Here, we further interrogate these effects and compare activity of these inhibitors on transwell migration, gene (qRT-PCR) and protein (western blot) expressions, and cancer stem cell-like behavior. We incorporated translational patient-derived xenograft models in these studies, and we focused on the lead inhibitor hit, GSK346294A, to demonstrate the utility of our comparative analysis as a screening modality to identify novel kinase targets and signaling pathways to pursue in TNBC. This study introduces a new method for discovering novel kinase targets that reverse the EMT phenotype; this screening approach can be applied to all cancer types and is not limited to breast cancer.
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Biomarcadores de Tumor/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Apoptosis , Proliferación Celular , Transición Epitelial-Mesenquimal , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ratones , Estructura Molecular , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Fosforilación , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tamoxifen is used to prevent and treat estrogen receptor-positive (ER+) breast cancer (BC); however, its chronic use can increase uterine cancer risk and induce tamoxifen resistance. Novel melatonin-tamoxifen drug conjugates may be promising to treat BC and may help offset the adverse effects of tamoxifen usage alone due to the presence of melatonin. We synthesized and screened five drug conjugates (C2, C4, C5, C9, and C15 linked) for their effects on BC cell (MCF-7, tamoxifen-resistant MCF-7, mouse mammary carcinoma, MDA-MB-231, and BT-549) viability, migration, and binding affinity to melatonin receptor 1 (MT1R) and estrogen receptor 1 (ESR1). C4 and C5 demonstrated the most favorable pharmacological characteristics with respect to binding profiles (affinity for ESR1 and MT1R) and their potency/efficacy to inhibit BC cell viability and migration in four phenotypically diverse invasive ductal BC cell lines. C4 and C5 were further assessed for their actions against tamoxifen-resistant MCF-7 cells and a patient-derived xenograft triple-negative BC cell line (TU-BcX-4IC) and for their mechanisms of action using selective mitogen-activated protein kinase kinase MEK1/2, MEK5, and phosphoinositide 3-kinase (PI3K) inhibitors. C4 and C5 inhibited tamoxifen-resistant MCF-7 cells with equal potency (IC50 = 4-8 µM) and efficacy (â¼90% inhibition of viability and migration) but demonstrated increased potency (IC50 = 80-211 µM) and efficacy (â¼140% inhibition) to inhibit migration versus cell viability (IC50 = 181-304 mM; efficacy â¼80% inhibition) in TU-BcX-4IC cells. Unique pharmacokinetic profiles were observed, with C4 having greater bioavailability than C5. Further assessment of C4 and C5 demonstrates that they create novel pharmacophores within each BC cell that is context specific and involves MEK1/2/pERK1/2, MEK5/pERK5, PI3K, and nuclear factor κB. These melatonin-tamoxifen drug conjugates show promise as novel anticancer drugs and further preclinical and clinical evaluation is warranted.
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Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Receptor alfa de Estrógeno/metabolismo , Melatonina/administración & dosificación , Receptor de Melatonina MT1/metabolismo , Tamoxifeno/administración & dosificación , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Disponibilidad Biológica , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células MCF-7 , Melatonina/farmacocinética , Melatonina/farmacología , Ratones , Tamoxifeno/farmacocinética , Tamoxifeno/farmacologíaRESUMEN
BACKGROUND: Breast cancer is the second leading cause of cancer deaths in the USA. Triple-negative breast cancer (TNBC) is a clinically aggressive subtype of breast cancer with high rates of metastasis, tumor recurrence, and resistance to therapeutics. Obesity, defined by a high body mass index (BMI), is an established risk factor for breast cancer. Women with a high BMI have increased incidence and mortality of breast cancer; however, the mechanisms(s) by which obesity promotes tumor progression are not well understood. METHODS: In this study, obesity-altered adipose stem cells (obASCs) were used to evaluate obesity-mediated effects of TNBC. Both in vitro and in vivo analyses of TNBC cell lines were co-cultured with six pooled donors of obASCs (BMI > 30) or ASCs isolated from lean women (lnASCs) (BMI < 25). RESULTS: We found that obASCs promote a pro-metastatic phenotype by upregulating genes associated with epithelial-to-mesenchymal transition and promoting migration in vitro. We confirmed our findings using a TNBC patient-derived xenograft (PDX) model. PDX tumors grown in the presence of obASCS in SCID/beige mice had increased circulating HLA1+ human cells as well as increased numbers of CD44+CD24- cancer stem cells in the peripheral blood. Exposure of the TNBC PDX to obASCs also increased the formation of metastases. The knockdown of leptin expression in obASCs suppressed the pro-metastatic effects of obASCs. CONCLUSIONS: Leptin signaling is a potential mechanism through which obASCs promote metastasis of TNBC in both in vitro and in vivo analyses.
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Tejido Adiposo/citología , Transformación Celular Neoplásica/metabolismo , Leptina/biosíntesis , Células Madre/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Animales , Biopsia , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Humanos , Leptina/genética , Ratones , Obesidad/metabolismo , Neoplasias de la Mama Triple Negativas/etiología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options. Experimental preclinical models that recapitulate their tumors of origin can accelerate target identification, thereby potentially improving therapeutic efficacy. Patient-derived xenografts (PDXs), due to their genomic and transcriptomic fidelity to the tumors from which they are derived, are poised to improve the preclinical testing of drug-target combinations in translational models. Despite the previous development of breast and TNBC PDX models, those derived from patients with demonstrated health-disparities are lacking. METHODS: We use an aggressive TNBC PDX model propagated in SCID/Beige mice that was established from an African-American woman, TU-BcX-2 K1, and assess its metastatic potential and drug sensitivities under distinct in vitro conditions. Cellular derivatives of the primary tumor or the PDX were grown in 2D culture conditions or grown in mammospheres 3D culture. Flow cytometry and fluorescence staining was used to quantify cancer stem cell-like populations. qRT-PCR was used to describe the mesenchymal gene signature of the tumor. The sensitivity of TU-BcX-2 K1-derived cells to anti-neoplastic oncology drugs was compared in adherent cells and mammospheres. Drug response was evaluated using a live/dead staining kit and crystal violet staining. RESULTS: TU-BcX-2 K1 has a low propensity for metastasis, reflects a mesenchymal state, and contains a large burden of cancer stem cells. We show that TU-BcX-2 K1 cells have differential responses to cytotoxic and targeted therapies in 2D compared to 3D culture conditions insofar as several drug classes conferred sensitivity in 2D but not in 3D culture, or cells grown as mammospheres. CONCLUSIONS: Here we introduce a new TNBC PDX model and demonstrate the differences in evaluating drug sensitivity in adherent cells compared to mammosphere, or suspension, culture.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/genética , Neoplasias de la Mama Triple Negativas/genética , Animales , Biomarcadores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Inmunohistoquímica , Ratones , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adipose stem cells (ASCs) play an essential role in tumor microenvironments. These cells are altered by obesity (obASCs) and previous studies have shown that obASCs secrete higher levels of leptin. Increased leptin, which upregulates estrogen receptor alpha (ERα) and aromatase, enhances estrogen bioavailability and signaling in estrogen receptor positive (ERâº) breast cancer (BC) tumor growth and metastasis. In this study, we evaluate the effect of obASCs on ERâºBC outside of the ERα signaling axis using breast cancer models with constitutively active ERα resulting from clinically relevant mutations (Y537S and D538G). We found that while obASCs promote tumor growth and proliferation, it occurs mostly through abrogated estrogen signaling when BC has constitutive ER activity. However, obASCs have a similar promotion of metastasis irrespective of ER status, demonstrating that obASC promotion of metastasis may not be completely estrogen dependent. We found that obASCs upregulate two genes in both ER wild type (WT) and ER mutant (MUT) BC: SERPINE1 and ABCB1. This study demonstrates that obASCs promote metastasis in ER WT and MUT xenografts and an ER MUT patient derived xenograft (PDX) model. However, obASCs promote tumor growth only in ER WT xenografts.
Asunto(s)
Adipocitos/citología , Neoplasias de la Mama/metabolismo , Estrógenos/metabolismo , Obesidad/metabolismo , Células Madre/citología , Células Madre/metabolismo , Animales , Neoplasias de la Mama/genética , Línea Celular , Movimiento Celular/genética , Movimiento Celular/fisiología , Proliferación Celular/genética , Proliferación Celular/fisiología , Células Cultivadas , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Metástasis de la Neoplasia/genética , Obesidad/genética , Ovariectomía , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods. METHODS: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. RESULTS: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix. CONCLUSIONS: Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.
Asunto(s)
Proliferación Celular/genética , Claudinas/genética , Recurrencia Local de Neoplasia/genética , Neoplasias de la Mama Triple Negativas/genética , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Ratones , Recurrencia Local de Neoplasia/patología , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Investigation into the mechanisms driving cancer cell behavior and the subsequent development of novel targeted therapeutics requires comprehensive experimental models that mimic the complexity of the tumor microenvironment. Recently, our laboratories have combined a novel tissue culture model and laser direct-write, a form of bioprinting, to spatially position single or clustered cancer cells onto ex vivo microvascular networks containing blood vessels, lymphatic vessels, and interstitial cell populations. Herein, we highlight this new model as a tool for quantifying cancer cell motility and effects on angiogenesis and lymphangiogenesis in an intact network that matches the complexity of a real tissue. Application of our proposed methodology offers an innovative ex vivo tissue perspective for evaluating the effects of gene expression and targeted molecular therapies on cancer cell migration and invasion. J. Cell. Physiol. 231: 2333-2338, 2016. © 2016 Wiley Periodicals, Inc.
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Movimiento Celular , Rayos Láser , Modelos Biológicos , Neoplasias/patología , Especificidad de Órganos , Animales , Bioimpresión , Humanos , Ratas , Imagen de Lapso de TiempoRESUMEN
With the recognition of obesity as a global health crisis, researchers have devoted greater effort to defining and understanding the pathophysiological molecular pathways regulating the biology of adipose tissue and obesity. Obesity, the excessive accumulation of adipose tissue due to hyperplasia and hypertrophy, has been linked to an increased incidence and aggressiveness of colon, hematological, prostate, and postmenopausal breast cancers. The increased morbidity and mortality of obesity-associated cancers have been attributed to higher levels of hormones, adipokines, and cytokines secreted by the adipose tissue. The increased amount of adipose tissue also results in higher numbers of adipose stromal/stem cells (ASCs). These ASCs have been shown to impact cancer progression directly through several mechanisms, including the increased recruitment of ASCs to the tumor site and increased production of cytokines and growth factors by ASCs and other cells within the tumor stroma. Emerging evidence indicates that obesity induces alterations in the biologic properties of ASCs, subsequently leading to enhanced tumorigenesis and metastasis of cancer cells. This review will discuss the links between obesity and cancer tumor progression, including obesity-associated changes in adipose tissue, inflammation, adipokines, and chemokines. Novel topics will include a discussion of the contribution of ASCs to this complex system with an emphasis on their role in the tumor stroma. The reciprocal and circular feedback loop between obesity and ASCs as well as the mechanisms by which ASCs from obese patients alter the biology of cancer cells and enhance tumorigenesis will be discussed.