RESUMEN
INTRODUCTION: Cancer stem cells (CSCs) are a theorized subset of cells within the tumor that is thought to drive disease recurrence and metastatic spread. The aim of this study is to investigate mRNA and protein levels of ganglioside GD2 synthase (GD2S), in breast cancer (BC) patients. METHODS: 65 PBMCs of preoperative BC patients without chemotherapy were compared to PBMCs after chemotherapy and controls. RESULTS: GD2S were significantly higher in BC patients after chemotherapy compared to pre-chemotherapy at both mRNA and protein. GD2S was higher in pre-chemotherapy blood samples compared to control samples. CONCLUSIONS: Higher expression of GD2S in BC samples compared to healthy control indicates the potential utility of GD2S as a marker of malignancy.
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Neoplasias de la Mama , N-Acetilgalactosaminiltransferasas , Recurrencia Local de Neoplasia , Células Madre Neoplásicas , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Femenino , Humanos , N-Acetilgalactosaminiltransferasas/sangre , Recurrencia Local de Neoplasia/metabolismo , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/metabolismo , ARN Mensajero/metabolismoRESUMEN
The semaphorins represent a large family of signaling molecules with crucial roles in neuronal and cardiac development. While normal semaphorin function pertains largely to development, their involvement in malignancy is becoming increasingly evident. One member, Semaphorin 3C (SEMA3C), has been shown to drive a number of oncogenic programs, correlate inversely with cancer prognosis, and promote the progression of multiple different cancer types. This report surveys the body of knowledge surrounding SEMA3C as a therapeutic target in cancer. In particular, we summarize SEMA3C's role as an autocrine andromedin in prostate cancer growth and survival and provide an overview of other cancer types that SEMA3C has been implicated in including pancreas, brain, breast, and stomach. We also propose molecular strategies that could potentially be deployed against SEMA3C as anticancer agents such as biologics, small molecules, monoclonal antibodies and antisense oligonucleotides. Finally, we discuss important considerations for the inhibition of SEMA3C as a cancer therapeutic agent.
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Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Próstata/efectos de los fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Semaforinas/farmacología , Semaforinas/uso terapéutico , Animales , Proliferación Celular/efectos de los fármacos , Humanos , MasculinoRESUMEN
FOXA1 is a pioneer transcription factor that is frequently mutated in prostate, breast, bladder, and salivary gland malignancies. Indeed, metastatic castration-resistant prostate cancer (mCRPC) commonly harbour FOXA1 mutations with a prevalence of 35%. However, despite the frequent recurrence of FOXA1 mutations in prostate cancer, the mechanisms by which FOXA1 variants drive its oncogenic effects are still unclear. Semaphorin 3C (SEMA3C) is a secreted autocrine growth factor that drives growth and treatment resistance of prostate and other cancers and is known to be regulated by both AR and FOXA1. In the present study, we characterize FOXA1 alterations with respect to its regulation of SEMA3C. Our findings reveal that FOXA1 alterations lead to elevated levels of SEMA3C both in prostate cancer specimens and in vitro. We further show that FOXA1 negatively regulates SEMA3C via intronic cis elements, and that mutations in FOXA1 forkhead domain attenuate its inhibitory function in reporter assays, presumably by disrupting DNA binding of FOXA1. Our findings underscore the key role of FOXA1 in prostate cancer progression and treatment resistance by regulating SEMA3C expression and suggest that SEMA3C may be a driver of growth and tumor vulnerability of mCRPC harboring FOXA1 alterations.
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Factor Nuclear 3-alfa del Hepatocito , Neoplasias de la Próstata Resistentes a la Castración , Semaforinas , Humanos , Masculino , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Mutación , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Factores de Transcripción/metabolismo , Semaforinas/genética , Semaforinas/metabolismoRESUMEN
Intratumoral androgen biosynthesis contributes to castration-resistant prostate cancer progression in patients treated with androgen deprivation therapy. The molecular mechanisms by which castration-resistant prostate cancer acquires the capacity for androgen biosynthesis to bypass androgen deprivation therapy are not entirely known. Here, we show that semaphorin 3C, a secreted signaling protein that is highly expressed in castration-resistant prostate cancer, can promote steroidogenesis by altering the expression profile of key steroidogenic enzymes. Semaphorin 3C not only upregulates enzymes required for androgen synthesis from dehydroepiandrosterone or de novo from cholesterol but also simultaneously downregulates enzymes involved in the androgen inactivation pathway. These changes in gene expression correlate with increased production of androgens induced by semaphorin 3C in prostate cancer model cells. Moreover, semaphorin 3C upregulates androgen synthesis in LNCaP cell-derived xenograft tumors, likely contributing to the enhanced in vivo tumor growth rate post castration. Furthermore, semaphorin 3C activates sterol regulatory element-binding protein, a transcription factor that upregulates enzymes involved in the synthesis of cholesterol, a sole precursor for de novo steroidogenesis. The ability of semaphorin 3C to promote intratumoral androgen synthesis may be a key mechanism contributing to the reactivation of the androgen receptor pathway in castration-resistant prostate cancer, conferring continued growth under androgen deprivation therapy. These findings identify semaphorin 3C as a potential therapeutic target for suppressing intratumoral steroidogenesis.
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Neoplasias de la Próstata Resistentes a la Castración , Semaforinas , Masculino , Humanos , Andrógenos/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Antagonistas de Andrógenos , Receptores Androgénicos/metabolismo , Colesterol/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
Cancer stem cells (CSCs) are thought to be a major player in tumor initiation, progression, and metastasis. Targeting CSCs for elimination presents a promising therapeutic strategy; however, this approach will require a stronger understanding of CSC biology and identification of CSC-specific markers. The present study was conducted to examine the correlation between DCLK1 and miR-137 and miR-15a levels in colorectal cancer. A total of 222 samples, including 181 colorectal cancer specimens, 24 adenomatosis, and 17 non-adenomatosis colonic polyps, were stained for DCLK1 expression using immunohistochemistry. Also, expression of miR-137 and miR-15a was assessed in colorectal cancer with high and low DCLK1 expression levels. Most colorectal cancer specimens (76%) showed strong expression of DCLK1, whereas only 21% of adenomatous and none of non-adenomatous colonic polyps showed strong DCLK1 expression. A significant difference in DCLK1 expression was found between colorectal cancer, adenomatous, and non-adenomatous colonic polyps (P < 0.001). Higher expression of DCLK1 was more frequently detected in colorectal cases with larger tumor size (P = 0.03), poor differentiation (P = 0.03), and lymph node involvement (P = 0.04). Comparison of miR-137 and miR-15a in colorectal cancer cases revealed a significant inverse correlation with DCLK1 expression (P = 0.03 and P = 0.04, respectively). DCLK1 may act as a candidate marker for colorectal cancer stem cells. The critical role of DCLK1 in colorectal cancer suggests that it may represent an early diagnostic marker and therapeutic target; however, further investigation is warranted.
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Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MicroARNs/genética , Células Madre Neoplásicas/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Progresión de la Enfermedad , Quinasas Similares a Doblecortina , Femenino , Estudios de Seguimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Células Madre Neoplásicas/metabolismo , Pronóstico , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The putative cardioprotective and chemopreventive properties of the red wine phenolic resveratrol (RES) have made it the subject of a growing body of clinical and basic research. We have begun investigations focusing on the effects of RES on the activity of the aryl hydrocarbon receptor (AHR) complex. Our evidence suggests that RES is a potent repressor of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible gene transcription in estrogen receptor (ER)-positive human breast, lung, and colon cancer cell lines. RES activates the transcription of the ER target genes to the same degree as estradiol (E(2)) in human MCF-7 breast cancer cells. Unlike E(2), which can only diminish TCDD-inducible CYP1A1 gene transcription by approximately 50%, RES can completely abrogate this response. Furthermore, 50% repression of TCDD-inducible transcription can be achieved with 100 nM RES, approximately 2.5 orders of magnitude lower than concentrations required for maximal inhibition, suggesting that multiple mechanisms are responsible for this effect. RES (100 nM) does not prevent ligand binding of a TCDD analog, nor does it prevent AHR from binding to its response element in the 5'-regulatory region of the CYP1A1 gene. Small inhibitory RNAs directed to ERα have demonstrated that RES-mediated repression of CYP1A1 depends on ERα. Whereas CYP1A1 protein levels in MCF-7 cells are refractory to the low-dose transcriptional effects of RES, a concomitant decrease in CYP1A1 protein levels is observed in Caco-2 cells. These results highlight a low-dose RES effect that could occur at nutritionally relevant exposures and are distinct from the high-dose effects often characterized.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Citocromo P-450 CYP1A1/antagonistas & inhibidores , Receptor alfa de Estrógeno/biosíntesis , Sustancias Protectoras/farmacología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Estilbenos/farmacología , Unión Competitiva , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Citocromo P-450 CYP1A1/genética , Relación Dosis-Respuesta a Droga , Receptor alfa de Estrógeno/genética , Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Regiones Promotoras Genéticas/efectos de los fármacos , Sustancias Protectoras/administración & dosificación , Unión Proteica , Interferencia de ARN , Ensayo de Unión Radioligante , Resveratrol , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estilbenos/administración & dosificaciónRESUMEN
Cancer stem cells (CSCs) are a theorized small subpopulation of cells within tumors thought to be responsible for metastasis, tumor development, disease progression, treatment-resistance, and recurrence. The identification, isolation, and biological characterization of CSCs may therefore facilitate the development of efficient therapeutic strategies targeting CSCs. This study aims to compare the biology and telomerase activity of CSCs to parental cells (PCs) in renal cancer. Renal CSCs were enriched from the ACHN cell line using a sphere culture system. Spheroid-derived cells (SDCs) and their adherent counterparts were compared with respect to their colony and sphere formation, expression of putative CSC markers, tumorigenicity in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and invasiveness. The expression of genes associated with CSCs, stemness, EMT, apoptosis, and ABC transporters was also compared between the two populations using quantitative real-time PCR (qRT-PCR). Finally, telomerase activity, hTERT expression, and sensitivity to MST-312, a telomerase inhibitor, was investigated between the two populations. We demonstrated that a subpopulation of ACHN cells was capable of growing as spheroids with many properties similar to CSCs, including higher clonogenicity, superior colony- and sphere-forming ability, and stronger tumorigenicity and invasiveness. In addition, SDCs demonstrated a higher expression of markers for CSCs, stemness, EMT, apoptosis, and ABC transporter genes compared to PCs. The expression of hTERT and telomerase activity in SDCs was significantly lower than PCs; however, the SDC population was more sensitive to MST-312 compared to PCs. These findings indicate that the SDC population exhibits stem-like potential and invasive characteristics. Moreover, the reduced expression of hTERT and telomerase activity in SDCs demonstrated that the expressions of hTERT and telomerase activity are not always higher in CSCs. Our results also showed that MST-312 treatment inhibited SDCs more strongly than PCs and may therefore be useful as a complementary targeted therapy against renal CSCs in the future.
RESUMEN
The androgen receptor (AR) is a hormone-activated transcription factor that regulates the development and progression of prostate cancer (PCa) and represents one of the most well-established drug targets. Currently clinically approved small molecule inhibitors of AR, such as enzalutamide, are built upon a common chemical scaffold that interacts with the AR by the same mechanism of action. These inhibitors eventually fail due to the emergence of drug-resistance in the form of AR mutations and expression of truncated AR splice variants (e.g. AR-V7) that are constitutively active, signalling the progression of the castration-resistant state of the disease. The urgent need therefore continues for novel classes of AR inhibitors that can overcome drug resistance, especially since AR signalling remains important even in late-stage advanced PCa. Previously, we identified a collection of 10-benzylidene-10H-anthracen-9-ones that effectively inhibit AR transcriptional activity, induce AR degradation and display some ability to block recruitment of hormones to the receptor. In the current work, we extended the analysis of the lead compounds, and used methods of both ligand- and structure-based drug design to develop a panel of novel 10-benzylidene-10H-anthracen-9-one derivatives capable of suppressing transcriptional activity and protein expression levels of both full length- and AR-V7 truncated forms of human androgen receptor. Importantly, the developed compounds efficiently inhibited the growth of AR-V7 dependent prostate cancer cell-lines which are completely resistant to all current anti-androgens.
Asunto(s)
Antagonistas de Andrógenos/farmacología , Variación Genética/genética , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Antagonistas de Andrógenos/síntesis química , Antagonistas de Andrógenos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Modelos Moleculares , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-ActividadRESUMEN
Prostate cancer (PCa) is a leading cause of death for men in North America. The androgen receptor (AR) - a hormone inducible transcription factor - drives expression of tumor promoting genes and represents an important therapeutic target in PCa. The AR is activated by steroid recruitment to its ligand binding domain (LBD), followed by receptor nuclear translocation and dimerization via the DNA binding domain (DBD). Clinically used small molecules interfere with steroid recruitment and prevent AR-driven tumor growth, but are rendered ineffective by emergence of LBD mutations or expression of constitutively active variants, such as ARV7, that lack the LBD. Both drug-resistance mechanisms confound treatment of this 'castration resistant' stage of PCa (CRPC), characterized by return of AR signalling. Here, we employ computer-aided drug-design to develop small molecules that block the AR-DBD dimerization interface, an attractive target given its role in AR activation and independence from the LBD. Virtual screening on the AR-DBD structure led to development of prototypical compounds that block AR dimerization, inhibiting AR-transcriptional activity through a LBD-independent mechanism. Such inhibitors may potentially circumvent AR-dependent resistance mechanisms and directly target CRPC tumor growth.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Multimerización de Proteína/efectos de los fármacos , Receptores Androgénicos/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Secuencia de Aminoácidos , Sitios de Unión/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Imidazoles/metabolismo , Imidazoles/farmacología , Masculino , Mutación , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Dominios Proteicos , Receptores Androgénicos/química , Receptores Androgénicos/genética , Homología de Secuencia de Aminoácido , Bibliotecas de Moléculas Pequeñas/metabolismo , Tiazoles/metabolismo , Tiazoles/farmacologíaRESUMEN
Despite the amenability of early-stage prostate cancer to surgery and radiation therapy, locally advanced and metastatic prostate cancer is clinically problematic. Chemical castration is often used as a first-line therapy for advanced disease, but progression to the castration-resistant prostate cancer phase occurs with dependable frequency, largely through mutations to the androgen receptor (AR), aberrant AR signaling, and AR-independent mechanisms, among other causes. Semaphorin 3C (SEMA3C) is a secreted signaling protein that is essential for cardiac and neuronal development and has been shown to be regulated by the AR, to drive epithelial-to-mesenchymal transition and stem features in prostate cells, to activate receptor tyrosine kinases, and to promote cancer progression. Given that SEMA3C is linked to several key aspects of prostate cancer progression, we set out to explore SEMA3C inhibition by small molecules as a prospective cancer therapy. A homology-based SEMA3C protein structure was created, and its interaction with the neuropilin (NRP)-1 receptor was modeled to guide the development of the corresponding disrupting compounds. Experimental screening of 146 in silicoâidentified molecules from the National Cancer Institute library led to the discovery of four promising candidates that effectively bind to SEMA3C, inhibit its association with NRP1, and attenuate prostate cancer growth. These findings provide proof of concept for the feasibility of inhibiting SEMA3C with small molecules as a therapeutic approach for prostate cancer.
RESUMEN
Growth factor receptor tyrosine kinase (RTK) pathway activation is a key mechanism for mediating cancer growth, survival, and treatment resistance. Cognate ligands play crucial roles in autocrine or paracrine stimulation of these RTK pathways. Here, we show SEMA3C drives activation of multiple RTKs including EGFR, ErbB2, and MET in a cognate ligand-independent manner via Plexin B1. SEMA3C expression levels increase in castration-resistant prostate cancer (CRPC), where it functions to promote cancer cell growth and resistance to androgen receptor pathway inhibition. SEMA3C inhibition delays CRPC and enzalutamide-resistant progression. Plexin B1 sema domain-containing:Fc fusion proteins suppress RTK signaling and cell growth and inhibit CRPC progression of LNCaP xenografts post-castration in vivo SEMA3C inhibition represents a novel therapeutic strategy for treatment of advanced prostate cancer.
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Proteínas del Tejido Nervioso/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/metabolismo , Semaforinas/metabolismo , Animales , Proliferación Celular , Humanos , Masculino , Ratones , Neoplasias de la Próstata Resistentes a la Castración/patología , Semaforinas/antagonistas & inhibidores , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Clostridium botulinum produces the potent botulinum neurotoxin, the causative agent of botulism. Based on distinctive physiological traits, strains of C. botulinum can be divided into four groups: however, only groups I and II are associated with human illness. Alignment of the flaA gene sequences from 40 group I and 40 group II strains identified a single BsrG1 restriction cut site that was present at base pair 283 in all group II flaA sequences and was not found in any group I sequence. The flaA gene was amplified by rapid colony PCR from 22 group I strains and 18 group II strains and digested with BsrGI restriction enzyme. Standard agarose gel electrophoresis with ethidium bromide staining showed two fragments, following restriction digestion of group II flaA gene amplicons with BsrGI, but only a single band of uncut flaA from group I strains. Combining rapid colony PCR with BsrGI restriction digest of the flaA gene at 60 degrees C is a significant improvement over current methods, such as meat digestion or amplified fragment length polymorphism, as a strain can be identified as either group I or group II in under 5 h when starting with a visible plated C. botulinum colony.
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Clostridium botulinum/genética , Flagelina/genética , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , ADN Bacteriano/análisis , Flagelina/metabolismo , Amplificación de Genes , Genes Bacterianos , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
The androgen receptor (AR) is a member of the nuclear receptor superfamily of transcription factors and is central to prostate cancer (PCa) progression. Ligand-activated AR engages androgen response elements (AREs) at androgen-responsive genes to drive the expression of gene batteries involved in cell proliferation and cell fate. Understanding the transcriptional targets of the AR has become critical in apprehending the mechanisms driving treatment-resistant stages of PCa. Although AR transcription regulation has been extensively studied, the signaling networks downstream of AR are incompletely described. Semaphorin 3C (SEMA3C) is a secreted signaling protein with roles in nervous system and cardiac development but can also drive cellular growth and invasive characteristics in multiple cancers including PCa. Despite numerous findings that implicate SEMA3C in cancer progression, regulatory mechanisms governing its expression remain largely unknown. Here we identify and characterize an androgen response element within the SEMA3C locus. Using the AR-positive LNCaP PCa cell line, we show that SEMA3C expression is driven by AR through this element and that AR-mediated expression of SEMA3C is dependent on the transcription factor GATA2. SEMA3C has been shown to promote cellular growth in certain cell types so implicit to our findings is the discovery of direct regulation of a growth factor by AR. We also show that FOXA1 is a negative regulator of SEMA3C. These findings identify SEMA3C as a novel target of AR, GATA2, and FOXA1 and expand our understanding of semaphorin signaling and cancer biology.
Asunto(s)
Factor de Transcripción GATA2/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/metabolismo , Semaforinas/metabolismo , Transcripción Genética , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular , Relación Dosis-Respuesta a Droga , Factor de Transcripción GATA2/genética , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-alfa del Hepatocito/genética , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/efectos de los fármacos , Receptores Androgénicos/genética , Elementos de Respuesta , Semaforinas/genética , Transducción de Señal , Congéneres de la Testosterona/farmacología , Transcripción Genética/efectos de los fármacosRESUMEN
Prostate cancer (PCa) is among the most commonly-occurring cancers worldwide and a leader in cancer-related deaths. Local non-invasive PCa is highly treatable but limited treatment options exist for those with locally-advanced and metastatic forms of the disease underscoring the need to identify mechanisms mediating PCa progression. The semaphorins are a large grouping of membrane-associated or secreted signalling proteins whose normal roles reside in embryogenesis and neuronal development. In this context, semaphorins help establish chemotactic gradients and direct cell movement. Various semaphorin family members have been found to be up- and down-regulated in a number of cancers. One family member, Semaphorin 3 C (SEMA3C), has been implicated in prostate, breast, ovarian, gastric, lung, and pancreatic cancer as well as glioblastoma. Given SEMA3C's roles in development and its augmented expression in PCa, we hypothesized that SEMA3C promotes epithelial-to-mesenchymal transition (EMT) and stem-like phenotypes in prostate cells. In the present study we show that ectopic expression of SEMA3C in RWPE-1 promotes the upregulation of EMT and stem markers, heightened sphere-formation, and cell plasticity. In addition, we show that SEMA3C promotes migration and invasion in vitro and cell dissemination in vivo.
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Transición Epitelial-Mesenquimal/genética , Células Madre Neoplásicas/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Semaforinas/genética , Animales , Biomarcadores , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Expresión Génica , Xenoinjertos , Humanos , Inmunofenotipificación , Masculino , Ratones , Invasividad Neoplásica , Neoplasias de la Próstata/patologíaRESUMEN
Human androgen receptor (AR) is a hormone-activated transcription factor that is an important drug target in the treatment of prostate cancer. Current small-molecule AR antagonists, such as enzalutamide, compete with androgens that bind to the steroid-binding pocket of the AR ligand-binding domain (LBD). In castration-resistant prostate cancer (CRPC), drug resistance can manifest through AR-LBD mutations that convert AR antagonists into agonists, or by expression of AR variants lacking the LBD. Such treatment resistance underscores the importance of novel ways of targeting the AR. Previously, we reported the development of a series of small molecules that were rationally designed to selectively target the AR DNA-binding domain (DBD) and, hence, to directly interfere with AR-DNA interactions. In the current work, we have confirmed that the lead AR DBD inhibitor indeed directly interacts with the AR-DBD and tested that substance across multiple clinically relevant CRPC cell lines. We have also performed a series of experiments that revealed that genome-wide chromatin binding of AR was dramatically impacted by the lead compound (although with lesser effect on AR variants). Collectively, these observations confirm the novel mechanism of antiandrogen action of the developed AR-DBD inhibitors, establishing proof of principle for targeting DBDs of nuclear receptors in endocrine cancers. Mol Cancer Ther; 16(10); 2281-91. ©2017 AACR.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Receptores Androgénicos/genética , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Antagonistas de Receptores Androgénicos/administración & dosificación , Andrógenos/genética , Andrógenos/metabolismo , Benzamidas , Línea Celular Tumoral , Cromatina/efectos de los fármacos , Cromatina/genética , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Nitrilos , Feniltiohidantoína/administración & dosificación , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Receptores Androgénicos/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies.
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Biomarcadores de Tumor/genética , Diferenciación Celular , Hipoxia/genética , Células Neuroendocrinas/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteína de Retinoblastoma/metabolismo , Apoptosis , Movimiento Celular , Proliferación Celular , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Masculino , Invasividad Neoplásica , Células Neuroendocrinas/metabolismo , Neoplasias de la Próstata/metabolismo , Proteína de Retinoblastoma/genética , Células Tumorales CultivadasRESUMEN
Human cutaneous melanoma is a devastating skin cancer because of its invasive nature and high metastatic potential. We used tissue microarray to study the role of human eukaryotic translation initiation factor 4E (eIF4E) in melanoma progression in 448 melanocytic lesions and found that high eIF4E expression was significantly increased in primary melanomas compared with dysplastic nevi (P<0.001), and further increased in metastatic melanomas (P<0.001). High eIF4E expression was associated with melanoma thickness (P=0.046), and poor overall and disease-specific 5-year survival of all, and primary melanoma patients, especially those with tumors ≥1 mm thick. Multivariate Cox regression analysis revealed that eIF4E is an independent prognostic marker. eIF4E knockdown (KD) in melanoma cells resulted in a significant increase in apoptosis (sub-G1 populations) and decrease in cell proliferation, and also resulted in downregulation of mesenchymal markers and upregulation of E-cadherin. In addition, eIF4E KD led to a decrease in melanoma cell invasion, matrix metalloproteinase-2 expression and activity, c-myc and BCL2 expression, and an increase in cleaved PARP and cleaved caspase-3 expression and chemosensitivity. Taken together, our data suggest that the eIF4E may promote melanoma cell invasion and metastasis, and may also serve as a promising prognostic marker and a potential therapeutic target for melanoma.
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Biomarcadores de Tumor/metabolismo , Factor 4E Eucariótico de Iniciación/metabolismo , Melanoma/mortalidad , Melanoma/patología , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Apoptosis/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Progresión de la Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Melanoma/metabolismo , Persona de Mediana Edad , Invasividad Neoplásica , Pronóstico , Análisis de Regresión , Neoplasias Cutáneas/metabolismo , Tasa de SupervivenciaRESUMEN
Localized hypoxia in solid tumors activates transcriptional programs that promote the metastatic transformation of cells. Like hypoxia-inducible hyper-vascularization, loss of the retinoblastoma protein (Rb) is a trait common to advanced stages of tumor progression in many metastatic cancers. However, no link between the role of Rb and hypoxia-driven metastatic processes has been established. We demonstrated that Rb is a key mediator of the hypoxic response mediated by HIF1α/ß, the master regulator of the hypoxia response, and its essential co-activator, the thyroid hormone receptor/retinoblastoma-interacting protein (TRIP230). Furthermore, loss of Rb unmasks the full co-activation potential of TRIP230. Using small inhibitory RNA approaches in vivo, we established that Rb attenuates the normal physiological response to hypoxia by HIF1α. Notably, loss of Rb results in hypoxia-dependent biochemical changes that promote acquisition of an invasive phenotype in MCF7 breast cancer cells. In addition, Rb is present in HIF1α-ARNT/HIF1ß transcriptional complexes associated with TRIP230 as determined by co-immuno-precipitation, GST-pull-down and ChIP assays. These results demonstrate that Rb is a negative modulator of hypoxia-regulated transcription by virtue of its direct effects on the HIF1 complex. This work represents the first link between the functional ablation of Rb in tumor cells and HIF1α-dependent transcriptional activation and invasion.
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Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Nucleares/metabolismo , Proteína de Retinoblastoma/metabolismo , Proteínas del Citoesqueleto , Regulación Neoplásica de la Expresión Génica/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Células MCF-7 , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , Proteína de Retinoblastoma/genéticaRESUMEN
Vertebrate opsin genes often occur in sets of tandem duplicates, and their expression varies developmentally and in response to environmental cues. We previously identified two highly conserved regions upstream of the long-wave sensitive opsin (LWS) gene cluster in teleosts. This region has since been shown in zebrafish to drive expression of LWS genes in vivo. In order to further investigate how elements in this region control opsin gene expression, we tested constructs encompassing the highly conserved regions and the less conserved portions upstream of the coding sequences in a promoter-less luciferase expression system. A â¼4500 bp construct of the upstream region, including the highly-conserved regions Reg I and Reg II, increased expression 100-fold, and successive 5' deletions reduced expression relative to the full 4.5 Kb region. Gene expression was highest when the transcription factor RORα was co-transfected with the proposed regulatory regions. Because these regions were tested in a promoter-less expression system, they include elements able to initiate and drive transcription. Teleosts exhibit complex color-mediated adaptive behavior and their adaptive significance has been well documented in several species. Therefore these upstream regions of LWS represent a model system for understanding the molecular basis of adaptive variation in gene regulation of color vision.
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Visión de Colores/genética , Opsinas de los Conos/genética , Secuencia Conservada/fisiología , Peces/genética , Regulación de la Expresión Génica , Receptores de Ácido Retinoico/metabolismo , Animales , Visión de Colores/fisiología , Opsinas de los Conos/metabolismo , Elementos de Facilitación Genéticos , Genes Reporteros , Luciferasas , Unión Proteica/genética , Células Fotorreceptoras Retinianas Conos , Receptor alfa de Ácido Retinoico , Factores de Transcripción/metabolismoRESUMEN
Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region.