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1.
Mol Cancer Res ; 17(12): 2492-2507, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31537618

RESUMEN

The major obstacle in successfully treating triple-negative breast cancer (TNBC) is resistance to cytotoxic chemotherapy, the mainstay of treatment in this disease. Previous preclinical models of chemoresistance in TNBC have suffered from a lack of clinical relevance. Using a single high dose chemotherapy treatment, we developed a novel MDA-MB-436 cell-based model of chemoresistance characterized by a unique and complex morphologic phenotype, which consists of polyploid giant cancer cells giving rise to neuron-like mononuclear daughter cells filled with smaller but functional mitochondria and numerous lipid droplets. This resistant phenotype is associated with metabolic reprogramming with a shift to a greater dependence on fatty acids and oxidative phosphorylation. We validated both the molecular and histologic features of this model in a clinical cohort of primary chemoresistant TNBCs and identified several metabolic vulnerabilities including a dependence on PLIN4, a perilipin coating the observed lipid droplets, expressed both in the TNBC-resistant cells and clinical chemoresistant tumors treated with neoadjuvant doxorubicin-based chemotherapy. These findings thus reveal a novel mechanism of chemotherapy resistance that has therapeutic implications in the treatment of drug-resistant cancer. IMPLICATIONS: These findings underlie the importance of a novel morphologic-metabolic phenotype associated with chemotherapy resistance in TNBC, and bring to light novel therapeutic targets resulting from vulnerabilities in this phenotype, including the expression of PLIN4 essential for stabilizing lipid droplets in resistant cells.

2.
Cancer Res ; 79(10): 2761-2774, 2019 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-30877105

RESUMEN

The antitumor activity of bromodomain and extraterminal motif protein inhibitors (BETi) has been demonstrated across numerous types of cancer. As such, these inhibitors are currently undergoing widespread clinical evaluation. However, predictive biomarkers allowing the stratification of tumors into responders and nonresponders to BETi are lacking. Here, we showed significant antiproliferative effects of low dosage BETi in vitro and in vivo against aggressive ovarian and lung cancer models lacking SMARCA4 and SMARCA2, key components of SWI/SNF chromatin remodeling complexes. Restoration of SMARCA4 or SMARCA2 promoted resistance to BETi in these models and, conversely, knockdown of SMARCA4 sensitized resistant cells to BETi. Transcriptomic analysis revealed that exposure to BETi potently downregulated a network of genes involved in receptor tyrosine kinase (RTK) signaling in SMARCA4/A2-deficient cells, including the oncogenic RTK HER3. Repression of signaling downstream of HER3 was found to be an important determinant of response to BETi in SMARCA4/A2-deficient cells. Overall, we propose that BETi represent a rational therapeutic strategy in poor-prognosis, SMARCA4/A2-deficient cancers. SIGNIFICANCE: These findings address an unmet clinical need by identifying loss of SMARCA4/A2 as biomarkers of hypersensitivity to BETi.

3.
Autophagy ; 15(8): 1376-1390, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-30773992

RESUMEN

Patients with triple-negative breast cancer (TNBC) often have a poor prognosis largely due to lack of effective targeted therapy. Using a library of seleno-purines coupled to a high-throughput biochemical enzymatic assays we identified a potent pharmacological enhancer of autophagy (referred herein as SLLN-15) that selectively activated cytostatic macroautophagy/autophagy in TNBC preclinical models. SLLN-15 induced a dose-dependent anti-proliferative activity in the TNBC cell lines MDA-MB-231 and BT-20 via induction of autophagy and autophagic flux. This induction was associated with a selective inhibition of AKT-MTOR signaling. Conversely, rapamycin, a known autophagy inducer and MTOR inhibitor, was unable to duplicate SLLN-15's effect on TNBC cells. Inhibition of autophagy by siRNA-mediated targeting of the autophagy regulators, BECN1, ATG5 and ATG7 or using 3-methyladenine (3-MA), significantly protected against SLLN-15-induced inhibition of cell viability, further supporting that SLLN-15-induced inhibition of cancer cell proliferation was autophagy-dependent. SLLN-15-induced autophagy in TNBC cells was also associated with decreased AURKA expression, decreased AKT phosphorylation and subsequent blockage of the AKT-MTOR pathway. In vivo, oral SLLN-15 revealed a potent anticancer and anti-metastatic activity in mice bearing TNBC. Altogether, this study describes a novel regulator of mammalian autophagy, with potential utility as an experimental therapeutic for TNBCs. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; AURKA: aurora kinase A; AURKB: aurora kinase B; BECN1: beclin 1; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; ERBB2: erb-b2 receptor tyrosine kinase 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose) polymerase 1; PI: propidium iodide; SQSTM1/p62: sequestosome 1; TNBC: triple-negative breast cancer.

4.
Cancer Res ; 79(7): 1646-1657, 2019 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-30659022

RESUMEN

The mechanisms by which breast cancers progress from relatively indolent ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) are not well understood. However, this process is critical to the acquisition of metastatic potential. MAPK-interacting serine/threonine-protein kinase 1 (MNK1) signaling can promote cell invasion. NODAL, a morphogen essential for embryogenic patterning, is often reexpressed in breast cancer. Here we describe a MNK1/NODAL signaling axis that promotes DCIS progression to IDC. We generated MNK1 knockout (KO) or constitutively active MNK1 (caMNK1)-expressing human MCF-10A-derived DCIS cell lines, which were orthotopically injected into the mammary glands of mice. Loss of MNK1 repressed NODAL expression, inhibited DCIS to IDC conversion, and decreased tumor relapse and metastasis. Conversely, caMNK1 induced NODAL expression and promoted IDC. The MNK1/NODAL axis promoted cancer stem cell properties and invasion in vitro. The MNK1/2 inhibitor SEL201 blocked DCIS progression to invasive disease in vivo. In clinical samples, IDC and DCIS with microinvasion expressed higher levels of phospho-MNK1 and NODAL versus low-grade (invasion-free) DCIS. Cumulatively, our data support further development of MNK1 inhibitors as therapeutics for preventing invasive disease. SIGNIFICANCE: These findings provide new mechanistic insight into progression of ductal carcinoma and support clinical application of MNK1 inhibitors to delay progression of indolent ductal carcinoma in situ to invasive ductal carcinoma.


Asunto(s)
Carcinoma de Mama in situ/patología , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Nodal/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Carcinoma de Mama in situ/metabolismo , Neoplasias de la Mama/metabolismo , Sistemas CRISPR-Cas , Carcinoma Ductal de Mama/metabolismo , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Femenino , Xenoinjertos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Desnudos , Proteínas Serina-Treonina Quinasas/genética
5.
Oncogene ; 38(12): 2177-2191, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30459355

RESUMEN

Poly (ADP-ribosylation), known as PARylation, is a post-translational modification catalyzed by poly (ADP-ribose) polymerases (PARP) and primarily removed by the enzyme poly (ADP-ribose) glycohydrolase (PARG). While the aberrant removal of post-translation modifications including phosphorylation and methylation has known tumorigenic effects, deregulation of PARylation has not been widely studied. Increased hydrolysis of PARylation chains facilitates cancer growth through enhancing estrogen receptor (ER)-driven proliferation, but oncogenic transformation has not been linked to increased PARG expression. In this study, we find that elevated PARG levels are associated with a poor prognosis in breast cancers, especially in HER2-positive and triple-negative subtypes. Using both in vitro and in vivo models, we demonstrate that heightened expression of catalytically active PARG facilitates cell transformation and invasion of normal mammary epithelial cells. Catalytically inactive PARG mutants did not recapitulate these phenotypes. Consistent with clinical data showing elevated PARG predicts poor outcomes in HER2+ patients, we observed that PARG acts in synergy with HER2 to promote neoplastic growth of immortalized mammary cells. In contrast, PARG depletion significantly impairs the growth and metastasis of triple-negative breast tumors. Mechanistically, we find that PARG interacts with SMAD2/3 and significantly decreases their PARylation in non-transformed cells, leading to enhanced expression of SMAD target genes. Further linking SMAD-mediated transcription to the oncogenicity of PARG, we show that PARG-mediated anchorage-independent growth and invasion are dependent, at least in part, on SMAD expression. Overall, our study underscores the oncogenic impact of aberrant protein PARylation and highlights the therapeutic potential of PARG inhibition in breast cancer.


Asunto(s)
Carcinogénesis , Glicósido Hidrolasas/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , ADN/metabolismo , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Glicósido Hidrolasas/genética , Humanos , Ratones , Metástasis de la Neoplasia , Fenotipo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Análisis de Supervivencia
6.
Mol Cancer Ther ; 18(1): 139-146, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30373932

RESUMEN

TRAF2, a RING finger adaptor protein, plays an important function in tumor necrosis factor (TNF)- and TNF-like weak inducer of apoptosis (TWEAK)-dependent signaling, in particular during inflammatory and immune responses. We identified a functional interaction of TRAF2 with focal adhesion (FA) signaling involving the focal adhesion kinase (FAK) in the regulation of cell susceptibility to anoikis. Comparison of TRAF2-proficient (TRAF2+/+) versus TRAF2-deficient (TRAF2-/-), and FAK-proficient (FAK+/+) versus FAK-deficient (FAK-/-) mouse embryonic fibroblasts and their matched reconstituted cells demonstrated that TRAF2 interacts physically with the N-terminal portion of FAK and colocalizes to cell membrane protrusions. This interaction was found to be critical for promoting resistance to cell anoikis. Similar results were confirmed in the human breast cancer cell line MDA-MB-231, where TRAF2 and FAK downregulation promoted cell susceptibility to anoikis. In human breast cancer tissues, genomic analysis of The Cancer Genome Atlas database revealed coamplification of TRAF2 and FAK in breast cancer tissues with a predictive value for shorter survival, further supporting a potential role of TRAF2-FAK cooperative signaling in cancer progression.


Asunto(s)
Neoplasias de la Mama/genética , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Animales , Anoicis , Sitios de Unión , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Regulación hacia Abajo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Quinasa 1 de Adhesión Focal/química , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones , Transducción de Señal , Análisis de Supervivencia
7.
Nat Commun ; 9(1): 3598, 2018 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-30185791

RESUMEN

Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor-node-metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis.


Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Escamosas/mortalidad , Neoplasias de la Boca/mortalidad , Recurrencia Local de Neoplasia/diagnóstico , Proteómica/métodos , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/patología , Toma de Decisiones Clínicas , Femenino , Estudios de Seguimiento , Humanos , Inmunohistoquímica , Metástasis Linfática , Aprendizaje Automático , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/diagnóstico , Neoplasias de la Boca/patología , Recurrencia Local de Neoplasia/prevención & control , Péptidos/análisis , Valor Predictivo de las Pruebas , Pronóstico , Estudios Retrospectivos , Saliva/química , Tasa de Supervivencia
8.
J Clin Invest ; 128(10): 4525-4542, 2018 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-30222135

RESUMEN

The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full-length RNF8 correlated with poor prognosis for breast cancer patients. Our data revealed that in addition to its role in the repair of DSBs, RNF8 regulated Notch1 signaling and cell-fate determination of mammary luminal progenitors. Mechanistically, RNF8 acted as a negative regulator of Notch signaling by ubiquitylating the active NOTCH1 protein (N1ICD), leading to its degradation. Consistent with abnormal activation of Notch signaling and impaired repair of DSBs in Rnf8-mutant mammary epithelial cells, we observed increased risk of mammary tumorigenesis in mouse models for RNF8 deficiency. Notably, deficiency of RNF8 sensitized breast cancer cells to combination of pharmacological inhibitors of Notch signaling and poly(ADP-ribose) polymerase (PARP), suggesting implications for treatment of breast cancer associated with impaired RNF8 expression or function.


Asunto(s)
Carcinogénesis/metabolismo , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/metabolismo , Proteínas de Neoplasias/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/biosíntesis , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Roturas del ADN de Doble Cadena , Femenino , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glándulas Mamarias Animales/patología , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/patología , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Receptor Notch1/genética , Ubiquitina-Proteína Ligasas/genética
9.
J Steroid Biochem Mol Biol ; 177: 135-139, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-28847749

RESUMEN

Hormonal 1,25-dihydroxyvitamin D (1,25D) and its analogues have shown efficacy in some preclinical models of cancer. However, many models are resistant to the antiproliferative effects of 1,25D or its analogues in vitro or in vivo, and such compounds have failed in the clinic as monotherapies because of tumor resistance. Given the observed synergism between 1,25D analogues and histone deacetylase inhibitors (HDACi) in 1,25D-resistant cells, we previously developed a series of hybrid secosteroidal and easily assembled non-secosteroidal analogues that combined agonism for the vitamin D receptor and HDACi in a single backbone. These compounds displayed enhanced efficacy against 1,25D-resistant malignant cells in vitro. Structure/function studies led to synthesis of several non-secosteroidal variants in which HDACi potency was optimized without substantially sacrificing VDR agonism. Here, we present the first studies of efficacy in vivo of two of these compounds, DK-366 and DK-406, in the aggressive mouse 4T1 model of triple-negative breast cancer, a form of the disease for which treatment options are limited. 4T1 cells are resistant in vitro to the cytostatic and cytotoxic effects of 1,25D and the potent HDACi SAHA individually up to concentrations of 1µM and 50µM, respectively, whereas combinations of the two are efficacious. In vitro, DK-366 or -406 induced dose-dependent arrest of cell proliferation and cytotoxicity at 10-20µM. In vivo, the maximum tolerated dose (MTD) of DK-366 and DK-406 were 2.5 and 5.0mg/kg, respectively. Although the compounds induced hypercalcemia at elevated doses, consistent with VDR agonism in vivo, they both reduced tumor burden at doses below their MTD's. Moreover, in a separate experiment, DK-406 at 5mg/kg reduced 4T1 lung metastases by at least 50%. Under the same conditions, 1,25D (0.25µg/kg) and SAHA (25mg/kg) combined had no effect on tumor burden or on lung metastases. These experiments show that hybrid compounds are bioavailable and efficacious against a particularly aggressive model of metastatic breast cancer, providing strong support for the therapeutic potential of the hybrid concept.


Asunto(s)
Antineoplásicos , Inhibidores de Histona Desacetilasas , Receptores de Calcitriol/agonistas , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Vitamina D , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Ratones Endogámicos BALB C , Vitamina D/análogos & derivados , Vitamina D/farmacología , Vitamina D/uso terapéutico
10.
Oncotarget ; 8(43): 74736-74754, 2017 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-29088820

RESUMEN

Oral squamous cell carcinoma (OSCC) prognosis is related to clinical stage and histological grade. However, this stratification needs to be refined. We conducted a comparative proteome study in microdissected samples from normal oral mucosa and OSCC to identify biomarkers for malignancy. Fascin and plectin were identified as differently expressed and both are implicated in several malignancies, but the clinical impacts of aberrant fascin and plectin expression in OSCCs remains largely unknown. Immunohistochemistry and real-time quantitative PCR were carried out in ex vivo OSCC samples and cell lines. A loss-of-function strategy using shRNA targeting fascin was employed to investigate in vitro and in vivo the fascin role on oral tumorigenesis. Transfections of microRNA mimics were performed to determine whether the fascin overexpression is regulated by miR-138 and miR-145. We found that fascin and plectin are frequently upregulated in OSCC samples and cell lines, but only fascin overexpression is an independent unfavorable prognostic indicator of disease-specific survival. In combination with advanced T stage, high fascin level is also an independent factor of disease-free survival. Knockdown of fascin in OSCC cells promoted cell adhesion and inhibited migration, invasion and EMT, and forced expression of miR-138 in OSCC cells significantly decreased the expression of fascin. In addition, fascin downregulation leads to reduced filopodia formation and decrease on paxillin expression. The subcutaneous xenograft model showed that tumors formed in the presence of low levels of fascin were significantly smaller compared to those formed with high fascin levels. Collectively, our findings suggest that fascin expression correlates with disease progression and may serve as a prognostic marker and therapeutic target for patients with OSCC.

11.
J Clin Invest ; 127(11): 4179-4192, 2017 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-29035277

RESUMEN

Melanoma can be stratified into unique subtypes based on distinct pathologies. The acral/mucosal melanoma subtype is characterized by aberrant and constitutive activation of the proto-oncogene receptor tyrosine kinase C-KIT, which drives tumorigenesis. Treatment of these melanoma patients with C-KIT inhibitors has proven challenging, prompting us to investigate the downstream effectors of the C-KIT receptor. We determined that C-KIT stimulates MAP kinase-interacting serine/threonine kinases 1 and 2 (MNK1/2), which phosphorylate eukaryotic translation initiation factor 4E (eIF4E) and render it oncogenic. Depletion of MNK1/2 in melanoma cells with oncogenic C-KIT inhibited cell migration and mRNA translation of the transcriptional repressor SNAI1 and the cell cycle gene CCNE1. This suggested that blocking MNK1/2 activity may inhibit tumor progression, at least in part, by blocking translation initiation of mRNAs encoding cell migration proteins. Moreover, we developed an MNK1/2 inhibitor (SEL201), and found that SEL201-treated KIT-mutant melanoma cells had lower oncogenicity and reduced metastatic ability. Clinically, tumors from melanoma patients harboring KIT mutations displayed a marked increase in MNK1 and phospho-eIF4E. Thus, our studies indicate that blocking MNK1/2 exerts potent antimelanoma effects and support blocking MNK1/2 as a potential strategy to treat patients positive for KIT mutations.


Asunto(s)
Antineoplásicos/farmacología , Dasatinib/farmacología , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Animales , Línea Celular Tumoral , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Melanoma/enzimología , Melanoma/secundario , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Mutación Missense , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Transducción de Señal , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/patología , Ensayos Antitumor por Modelo de Xenoinjerto
12.
Oncotarget ; 8(33): 55511-55524, 2017 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-28903437

RESUMEN

Head and neck squamous cell carcinoma (HNSCC) is often diagnosed at advanced stages, incurring significant high mortality and morbidity. This review explored the risk stratification of miRNAs, and investigated the impact of miRNA networking in HNSCC prognostication. We performed a meta-analysis and a systematic literature search on online databases for papers published prior to December 1, 2016. The list of miRNAs was uploaded to MetacoreTM to construct a protein-protein interaction network, which was used to identify targets of the miRNAs and potential drugs. In addition, a representative network was further validated by immunohistochemistry in a cohort of 100 patients. We found 116 studies that included 8,194 subjects, in which the relationship between miRNA expression and prognosis of HNSCC were analyzed. Significant elevated expressions of 27 miRNAs and decreased expression of 26 miRNAs were associated with poor outcome. After excluding the studies causing heterogeneity, a fixed model was applied, which showed a statistically significant association between increased expression of miR-21 and poor survival (Pooled HR = 1.81,95% CI = 0.66-2.95, P < 0.005). We identified four networks affected by the miRNAs expression and enriched in genes related to metabolic processes and regulation of cell mitogenesis in response to extracellular stimuli. One network point out to 16 miRNAs directly or indirectly involved in the regulation of androgen-receptor (AR). Evaluation of AR protein expression in our cohort revealed that patients with upregulation of AR had poor survival rates (log-rank test, P < 0.005). This study showed that miRNAs have potential prognostic value to serve as screening tool for HNSCC during the follow-up. In addition, the implementation of a network-based analysis may reveal proteins with potential to be used as a biomarker.

13.
Sci Adv ; 3(5): e1601898, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28560323

RESUMEN

The repair of DNA double-strand breaks (DSBs) is mediated via two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. DSB repair is vital for cell survival, genome stability, and tumor suppression. In contrast to NHEJ, HR relies on extensive homology and templated DNA synthesis to restore the sequence surrounding the break site. We report a new role for the multifunctional protein CCCTC-binding factor (CTCF) in facilitating HR-mediated DSB repair. CTCF is recruited to DSB through its zinc finger domain independently of poly(ADP-ribose) polymers, known as PARylation, catalyzed by poly(ADP-ribose) polymerase 1 (PARP-1). CTCF ensures proper DSB repair kinetics in response to γ-irradiation, and the loss of CTCF compromises HR-mediated repair. Consistent with its role in HR, loss of CTCF results in hypersensitivity to DNA damage, inducing agents and inhibitors of PARP. Mechanistically, CTCF acts downstream of BRCA1 in the HR pathway and associates with BRCA2 in a PARylation-dependent manner, enhancing BRCA2 recruitment to DSB. In contrast, CTCF does not influence the recruitment of the NHEJ protein 53BP1 or LIGIV to DSB. Together, our findings establish for the first time that CTCF is an important regulator of the HR pathway.


Asunto(s)
Factor de Unión a CCCTC/metabolismo , Roturas del ADN de Doble Cadena/efectos de la radiación , Rayos gamma , Reparación del ADN por Recombinación/efectos de la radiación , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Factor de Unión a CCCTC/genética , Línea Celular Tumoral , Células HEK293 , Humanos , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo
14.
Oncotarget ; 8(19): 31199-31214, 2017 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-28415719

RESUMEN

Post-translational mechanisms regulating cell-matrix adhesion turnover during cell locomotion are not fully elucidated. In this study, we uncovered an essential role of Y118 site-specific tyrosine phosphorylation of paxillin, an adapter protein of focal adhesion complexes, in paxillin recruitment to autophagosomes to trigger turnover of peripheral focal adhesions in human breast cancer cells. We demonstrate that the Rab-7 GTPase is a key upstream regulator of late endosomal sorting of tyrosine118-phosphorylated paxillin, which is subsequently recruited to autophagosomes via the cargo receptor c-Cbl. Essentially, this recruitment involves a direct and selective interaction between Y118-phospho-paxillin, c-Cbl, and LC3 and is independent from c-Cbl E3 ubiquitin ligase activity. Interference with the Rab7-paxillin-autophagy regulatory network using genetic and pharmacological approaches greatly impacted focal adhesion stability, cell locomotion and progression to metastasis using a panel of human breast cancer cells. Together, these results provide novel insights into the requirement of phospho-site specific post-translational mechanism of paxillin for autophagy targeting to regulate cell-matrix adhesion turnover and cell locomotion in breast cancer cells.


Asunto(s)
Autofagosomas/metabolismo , Neoplasias de la Mama/metabolismo , Endosomas/metabolismo , Matriz Extracelular/metabolismo , Paxillin/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Autofagia , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular/genética , Progresión de la Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Asociadas a Microtúbulos , Metástasis de la Neoplasia , Fosforilación , Unión Proteica , Proteolisis , Transducción de Señal , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo
15.
Appl Microbiol Biotechnol ; 101(5): 1999-2019, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-27837314

RESUMEN

Probiotics have been shown to have beneficial properties in attenuating the risk of colorectal cancer (CRC) development. However, functional evidence to support such effects for some probiotic bacteria are relatively unknown. Here, we document a significant antioxidant, anti-proliferative and pro-apoptotic activities of Lactobacillus acidophilus ATCC 314 and Lactobacillus fermentum NCIMB 5221 on CRC cells, particularly when used in combination (La-Lf). Furthermore, a superior synergistic activity on the inhibition of tumor growth and modulation of cell proliferation and epithelial markers in the Apc Min/+ CRC mouse model was explored, based on the expression levels of Ki-67, E-cadherin, ß-catenin, and cleaved caspase-3 (CC3) proteins. The anti-cancer activity of La-Lf co-culture was significantly enhanced in vitro with significant reduced proliferation (38.8 ± 6.9 %, P = 0.009) and increased apoptosis (413 RUL, P < 0.001) towards cancer cells, as well as significant protection of normal colon cell growth from toxic treatment (18.6 ± 9.8 %, P = 0.001). La-Lf formulation (1010cfu/animal/day) altered aspects of intestinal tumorigenesis by significantly reducing intestinal tumor multiplicity (1.7-fold, P = 0.016) and downregulating cellular proliferation markers, including ß-catenin (P = 0.041) and Ki-67 (P = 0.008). In conclusion, La-Lf showed greater protection against intestinal tumorigenesis supporting a potential use as a biotherapeutic for the prevention of CRC.


Asunto(s)
Antioxidantes/uso terapéutico , Carcinogénesis/efectos de los fármacos , Extractos Celulares/uso terapéutico , Neoplasias Colorrectales/prevención & control , Lactobacillus acidophilus/metabolismo , Lactobacillus fermentum/metabolismo , Probióticos/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Células CACO-2 , Cadherinas/biosíntesis , Caspasa 3/biosíntesis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/terapia , Modelos Animales de Enfermedad , Humanos , Antígeno Ki-67/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , beta Catenina/biosíntesis
16.
Sci Rep ; 6: 36699, 2016 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-27819326

RESUMEN

Fascin 1 (FSCN1) is a cytoskeleton-associated protein recognized to function primarily in the regulation of cytoskeleton structure and formation of plasma membrane protrusions. Here we report a novel nuclear function for Fascin 1. Biochemical studies and genome wide localization using ChIP-seq identified phosphorylated Fascin 1 (pFascin) in complexes associated with transcription and that it co-localizes with histone H3 Lys4 trimethylation (H3K4me3) on chromatin. Gene expression profiling identified genes affected by Fascin 1 including SLC3A2, a gene encoding for a plasma membrane transporter that regulates intracellular amino acid levels. RbBP5, a subunit of the H3K4 histone methyltransferase (HMT) complex was found to interact with Fascin 1 supporting its role in H3K4me3 establishment at target genes. Moreover, we show that changes to SLC3A2 levels affect amino acid-mediated mTORC1 activation. These results reveal that Fascin 1 has a yet undiscovered nuclear function as an epigenetic modulator of genes essential for amino acid metabolism.


Asunto(s)
Proteínas Portadoras/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Regulación de la Expresión Génica , Expresión Génica , Proteínas de Microfilamentos/metabolismo , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Células HEK293 , Histonas/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fosforilación , Serina-Treonina Quinasas TOR/metabolismo , Transcriptoma
17.
Sci Rep ; 6: 29389, 2016 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-27388124

RESUMEN

APE1 is an essential DNA repair protein that also possesses the ability to regulate transcription. It has a unique cysteine residue C65, which maintains the reduce state of several transcriptional activators such as NF-κB. How APE1 is being recruited to execute the various biological functions remains unknown. Herein, we show that APE1 interacts with a novel partner PRDX1, a peroxidase that can also prevent oxidative damage to proteins by serving as a chaperone. PRDX1 knockdown did not interfere with APE1 expression level or its DNA repair activities. However, PRDX1 knockdown greatly facilitates APE1 detection within the nucleus by indirect immunofluorescence analysis, even though APE1 level was unchanged. The loss of APE1 interaction with PRDX1 promotes APE1 redox function to activate binding of the transcription factor NF-κB onto the promoter of a target gene, the proinflammatory chemokine IL-8 involved in cancer invasion and metastasis, resulting in its upregulation. Depletion of APE1 blocked the upregulation of IL-8 in the PRDX1 knockdown cells. Our findings suggest that the interaction of PRDX1 with APE1 represents a novel anti-inflammatory function of PRDX1, whereby the association safeguards APE1 from reducing transcription factors and activating superfluous gene expression, which otherwise could trigger cancer invasion and metastasis.


Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Interleucina-8/genética , FN-kappa B/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Neoplasias Gástricas/genética , Núcleo Celular/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Peróxido de Hidrógeno/farmacología , Invasividad Neoplásica , Metástasis de la Neoplasia , Estrés Oxidativo , Regiones Promotoras Genéticas , Neoplasias Gástricas/metabolismo , Activación Transcripcional
18.
Mol Biol Rep ; 43(4): 229-40, 2016 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-26907180

RESUMEN

MicroRNAs (miRNAs) are small non-coding RNAs that function in transcriptional and post-transcriptional regulation of gene expression. Several miRNAs have been implicated in regulating prostate cancer (PCa) progression. Deregulations of miRNA regulatory networks have been reported in ERG positive PCa, which accounts for ~50 % of PCa and have been suggested to affect tumor aggressiveness. The function of miR338-3p, its prognostic significance, and its association with ERG positive PCa has not been fully investigated. Using microarray expression profiling, we identified miRNA338-3p as among the top deregulated miRNAs associated with ERG status in PCa. We investigated miR338-3p function using in vitro and in vivo experimental models and its expression was assessed and validated in clinical samples and a public cohort of localized and metastatic prostate cancer. miR338-3p was significantly down-regulated with disease progression from benign prostate tissue to primary and metastatic lesions. In localized disease, patients with lower miR338-3p expression levels showed increased association to biochemical recurrence and several adverse pathological parameters compared to patients with higher miRNA338-3p tissue expression levels. Using in vitro PCa cell models, overexpression of miR338-3p resulted in a decrease in cell invasion and expression of chemokine signalling genes CXCL12, CXCR4, and CXCR7. In vivo, orthotropic implantation of PC3 cells stably expressing miR338-3p was associated with a significant decrease in tumor weights compared to control cells. miR338-3p has anti-proliferative and anti-invasive properties. It affects CXCR4 axis, and its down-regulation is associated with adverse clinical outcomes in PCa patients.


Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , MicroARNs/genética , Neoplasias de la Próstata/metabolismo , Animales , Línea Celular Tumoral , Quimiocina CXCL12/genética , Humanos , Masculino , Ratones , Ratones SCID , Metástasis de la Neoplasia , Pronóstico , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/patología , Receptores CXCR/genética , Receptores CXCR4/genética
20.
Oncotarget ; 6(26): 21950-63, 2015 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-26110570

RESUMEN

Metastatic oral squamous cell carcinoma (OSCC) is frequently associated with recurrent gene abnormalities at specific chromosomal loci. Here, we utilized array comparative genomic hybridization and genome-wide screening of metastatic and non-metastatic tongue tumors to investigate genes potentially contributing to OSCC progression to metastasis. We identified predominant amplifications of chromosomal regions that encompass the RAB5, RAB7 and RAB11 genes (3p24-p22, 3q21.3 and 8p11-12, respectively) in metastatic OSCC. The expression of these Rab GTPases was confirmed by immunohistochemistry in OSCC tissues from a cohort of patients with a follow-up of 10 years. A significant overexpression of Rab5, Rab7 and Rab11 was observed in advanced OSCC cases and co-overexpression of these Rabs was predictive of poor survival (log-rank test, P = 0.006). We generated a Rab interaction network and identified central Rab interactions of relevance to metastasis signaling, including focal adhesion proteins. In preclinical models, mRNA and protein expression levels of these Rab members were elevated in a panel of invasive OSCC cell lines, and their down-regulation prevented cell invasion at least in part via inhibition of focal adhesion disassembly. In summary, our results provide insights into the cooperative role of Rab gene amplifications in OSCC progression and support their potential utility as prognostic markers and therapeutic approach for advanced OSCC.


Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Proteínas de Unión al GTP rab/genética , Anciano , Carcinoma de Células Escamosas/enzimología , Línea Celular Tumoral , Movimiento Celular/fisiología , Progresión de la Enfermedad , Femenino , Adhesiones Focales/genética , Adhesiones Focales/patología , Neoplasias de Cabeza y Cuello/enzimología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/enzimología , Metástasis de la Neoplasia , Carcinoma de Células Escamosas de Cabeza y Cuello , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismo
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