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1.
Mol Pharmacol ; 100(1): 32-45, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33990407

RESUMEN

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is one of the leading causes of cancer-related deaths worldwide. The multitarget inhibitor sorafenib is a first-line treatment of patients with advanced unresectable HCC. Recent clinical studies have evidenced that patients treated with sorafenib together with the antidiabetic drug metformin have a survival disadvantage compared with patients receiving sorafenib only. Here, we examined whether a clinically relevant dose of metformin (50 mg/kg per day) could influence the antitumoral effects of sorafenib (15 mg/kg per day) in a subcutaneous xenograft model of human HCC growth using two different sequences of administration, i.e., concomitant versus sequential dosing regimens. We observed that the administration of metformin 6 hours prior to sorafenib was significantly less effective in inhibiting tumor growth (15.4% tumor growth inhibition) than concomitant administration of the two drugs (59.5% tumor growth inhibition). In vitro experiments confirmed that pretreatment of different human HCC cell lines with metformin reduced the effects of sorafenib on cell viability, proliferation, and signaling. Transcriptomic analysis confirmed significant differences between xenografted tumors obtained under the concomitant and the sequential dosing regimens. Taken together, these observations call into question the benefit of parallel use of metformin and sorafenib in patients with advanced HCC and diabetes, as the interaction between the two drugs could ultimately compromise patient survival. SIGNIFICANCE STATEMENT: When drugs are administered sequentially, metformin alters the antitumor effect of sorafenib, the reference treatment for advanced hepatocellular carcinoma, in a preclinical murine xenograft model of liver cancer progression as well as in hepatic cancer cell lines. Defective activation of the AMP-activated protein kinase pathway as well as major transcriptomic changes are associated with the loss of the antitumor effect. These results echo recent clinical work reporting a poorer prognosis for patients with liver cancer who were cotreated with metformin and sorafenib.


Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Diabetes Mellitus/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Metformina/administración & dosificación , Sorafenib/administración & dosificación , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Esquema de Medicación , Interacciones Farmacológicas , Quimioterapia Combinada , Femenino , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Metformina/farmacología , Ratones , Transducción de Señal/efectos de los fármacos , Sorafenib/farmacología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de Xenoinjerto
2.
Molecules ; 26(13)2021 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-34202689

RESUMEN

BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide. One of its subtypes is associated with defective mismatch repair (dMMR) genes. Saffron has many potentially protective roles against colon malignancy. However, these roles in the context of dMMR tumors have not been explored. In this study, we aimed to investigate the effects of saffron and its constituents in CRC cell lines with dMMR. METHODS: Saffron crude extracts and specific compounds (safranal and crocin) were used in the human colorectal cancer cell lines HCT116, HCT116+3 (inserted MLH1), HCT116+5 (inserted MSH3), and HCT116+3+5 (inserted MLH1 and MSH3). CDC25b, p-H2AX, TPDP1, and GAPDH were analyzed by Western blot. Proliferation and cytotoxicity were analyzed by MTT. The scratch wound assay was also performed. RESULTS: Saffron crude extracts restricted (up to 70%) the proliferation in colon cells with deficient MMR (HCT116) compared to proficient MMR. The wound healing assay indicates that deficient MMR cells are doing better (up to 90%) than proficient MMR cells when treated with saffron. CDC25b and TDP1 downregulated (up to 20-fold) in proficient MMR cells compared to deficient MMR cells, while p.H2AX was significantly upregulated in both cell types, particularly at >10 mg/mL saffron in a concentration-dependent manner. The reduction in cellular proliferation was accompanied with upregulation of caspase 3 and 7. The major active saffron compounds, safranal and crocin reproduced most of the saffron crude extracts' effects. CONCLUSIONS: Saffron's anti-proliferative effect is significant in cells with deficient MMR. This novel effect may have therapeutic implications and benefits for MSI CRC patients who are generally not recommended for the 5-fluorouracil-based treatment.


Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Crocus/química , Reparación de la Incompatibilidad de ADN/efectos de los fármacos , Inestabilidad de Microsatélites/efectos de los fármacos , Extractos Vegetales/farmacología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células HCT116 , Humanos , Extractos Vegetales/química
3.
J Cell Mol Med ; 24(14): 8091-8102, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32515546

RESUMEN

Membrane receptor intracellular trafficking and signalling are frequently altered in cancers. Our aim was to investigate whether clathrin-dependent trafficking modulates signalling of the ErbB receptor family in response to amphiregulin (AR), EGF, heparin-binding EGF-like growth factor (HB-EGF) and heregulin-1ß (HRG). Experiments were performed using three hepatocellular carcinoma (HCC) cell lines, Hep3B, HepG2 and PLC/PRF/5, expressing various levels of EGFR, ErbB2 and ErbB3. Inhibition of clathrin-mediated endocytosis (CME), by down-regulating clathrin heavy chain expression, resulted in a cell- and ligand-specific pattern of phosphorylation of the ErbB receptors and their downstream effectors. Clathrin down-regulation significantly decreased the ratio between phosphorylated EGFR (pEGFR) and total EGFR in all cell lines when stimulated with AR, EGF, HB-EGF or HRG, except in HRG-stimulated Hep3B cells in which pEGFR was not detectable. The ratio between phosphorylated ErbB2 and total ErbB2 was significantly decreased in clathrin down-regulated Hep3B cells stimulated with any of the ligands, and in HRG-stimulated PLC/PRF/5 cells. The ratio between phosphorylated ErbB3 and total ErbB3 significantly decreased in clathrin down-regulated cell lines upon stimulation with EGF or HB-EGF. STAT3 phosphorylation levels significantly increased in all cell lines irrespective of stimulation, while that of AKT remained unchanged, except in AR-stimulated Hep3B and HepG2 cells in which pAKT was significantly decreased. Finally, ERK phosphorylation was insensitive to clathrin inhibition. Altogether, our observations indicate that clathrin regulation of ErbB signalling in HCC is a complex process that likely depends on the expression of ErbB family members and on the autocrine/paracrine secretion of their ligands in the tumour environment.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Clatrina/metabolismo , Neoplasias Hepáticas/metabolismo , Receptor ErbB-2/metabolismo , Transducción de Señal , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Ligandos , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/patología , Receptor ErbB-3/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos
4.
Genes Chromosomes Cancer ; 56(9): 681-690, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28512763

RESUMEN

Colorectal cancers (CRCs) displaying microsatellite instability (MSI) most often result from MLH1 deficiency. The aim of this study was to assess the impact of MLH1 expression per se on tumor evolution after curative surgical resection using a xenograft tumor model. Transplantable tumors established with the human MLH1-deficient HCT116 cell line and its MLH1-complemented isogenic clone, mlh1-3, were implanted onto the caecum of NOD/SCID mice. Curative surgical resection was performed at day 10 in half of the animals. The HCT116-derived tumors were more voluminous compared to the mlh1-3 ones (P = .001). Lymph node metastases and peritoneal carcinomatosis occurred significantly more often in the group of mice grafted with HCT116 (P = .007 and P = .035, respectively). Mlh1-3-grafted mice did not develop peritoneal carcinomatosis or liver metastasis. After surgical resection, lymph node metastases only arose in the group of mice implanted with HCT116 and the rate of cure was significantly lower than in the mlh1-3 group (P = .047). The murine orthotopic xenograft model based on isogenic human CRC cell lines allowed us to reveal the impact of MLH1 expression on tumor evolution in mice who underwent curative surgical resection and in mice whose tumor was left in situ. Our data indicate that the behavior of MLH1-deficient CRC is not only governed by mutations arising in genes harboring microsatellite repeated sequences but also from their defect in MLH1 as such.


Asunto(s)
Carcinoma/genética , Neoplasias del Colon/genética , Homólogo 1 de la Proteína MutL/genética , Animales , Carcinoma/patología , Carcinoma/cirugía , Neoplasias del Colon/patología , Neoplasias del Colon/cirugía , Femenino , Células HCT116 , Humanos , Metástasis Linfática , Ratones , Ratones Endogámicos NOD , Ratones SCID , Homólogo 1 de la Proteína MutL/metabolismo , Mutación
5.
J Biol Chem ; 290(27): 16797-811, 2015 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-25947381

RESUMEN

Galectin-9 (gal-9) is a multifunctional ß-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca(2+)) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca(2+) mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca(2+) mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4(+) T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca(2+) mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4(+) T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex.


Asunto(s)
Apoptosis , Complejo CD3/metabolismo , Galectinas/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Complejo CD3/genética , Calcio/metabolismo , Citocinas/genética , Citocinas/metabolismo , Galectinas/genética , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/citología
6.
Hum Mutat ; 36(9): 894-902, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26077438

RESUMEN

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder associating macroglossia, abdominal wall defects, visceromegaly, and a high risk of childhood tumor. Molecular anomalies are mostly epigenetic; however, mutations of CDKN1C are implicated in 8% of cases, including both sporadic and familial forms. We aimed to describe the phenotype of BWS patients with CDKN1C mutations and develop a functional test for CDKN1C mutations. For each propositus, we sequenced the three exons and intron-exon boundaries of CDKN1C in patients presenting a BWS phenotype, including abdominal wall defects, without 11p15 methylation defects. We developed a functional test based on flow cytometry. We identified 37 mutations in 38 pedigrees (50 patients and seven fetuses). Analysis of parental samples when available showed that all mutations tested but one was inherited from the mother. The four missense mutations led to a less severe phenotype (lower frequency of exomphalos) than the other 33 mutations. The following four tumors occurred: one neuroblastoma, one ganglioneuroblastoma, one melanoma, and one acute lymphoid leukemia. Cases of BWS caused by CDKN1C mutations are not rare. CDKN1C sequencing should be performed for BWS patients presenting with abdominal wall defects or cleft palate without 11p15 methylation defects or body asymmetry, or in familial cases of BWS.


Asunto(s)
Síndrome de Beckwith-Wiedemann/diagnóstico , Síndrome de Beckwith-Wiedemann/genética , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/genética , Estudios de Asociación Genética , Impresión Genómica , Fenotipo , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Inhibidor p57 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Genotipo , Humanos , Masculino , Datos de Secuencia Molecular , Mutación , Linaje , Alineación de Secuencia
7.
Hepatogastroenterology ; 60(126): 1469-75, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23933939

RESUMEN

BACKGROUND: Evaluation of biomarkers and clinical factors associated with cancer-specific survival after curative resection for colorectal cancer liver metastases (LM). METHODOLOGY: All patients who had an R0 resection for LM between 2000-2006 were reviewed. Clinical and histological data were assessed; p53 expression was studied by IHC. ERCC1 codon 118 and XRCC1 codon 399 were analyzed by PCR-RFLP using BsrDI and HpaII, respectively. RESULTS: Out of 119 patients included (80 synchronous LM (67.2%), median number 2 (1-18)), 104 patients (87.4%) received chemotherapy before recurrence; 60 patients (50.4%) had a p53 negative tumor. ERCC1 distribution was: 31(26%) AAC/AAC, 44(37%) AAC/AAT and 44(37%) AAT/AAT. XRCC1 distribution was: 46(39%) CGG/CGG, 53(44.9%) CGG/CAG and 19(16.1%) CAG/CAG. Three and 5-years disease free survival (DFS) and overall survival (OS) were 31%, 22.7%, 77.4%, and 66.6%, respectively. Node ratio >0.2 (p = 0.0042), LM number >3 (p <0.0001), bilobar localization (p = 0.0074) and preoperative chemotherapy (p = 0.0036) were associated with a shorter DFS. None of the biomarkers was found to influence DFS. In multivariate analysis, a number of LM >3 was the only independent factor. No factor was found to influence OS. CONCLUSIONS: The studied biomarkers had no significant impact on prognosis. For routine practice, clinical factors remain the only usable available tools.


Asunto(s)
Neoplasias Colorrectales/patología , Hepatectomía , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Codón , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Femenino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Pronóstico , Proteína p53 Supresora de Tumor/análisis , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X
8.
Mol Biol Evol ; 26(1): 111-21, 2009 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18845548

RESUMEN

Simple sequence repeats (SSRs) are very common short repeats in eukaryotic genomes. "Long" SSRs are considered "hypermutable" sequences because they exhibit a high rate of expansion and contraction. Because they are potentially deleterious, long SSRs tend to be uncommon in coding sequences. However, several genes contain long SSRs in their exonic sequences. Here, we identify 1,291 human genes that host a mononucleotide SSR long enough to be prone to expansion or contraction, being called hypermutable hereafter. On the basis of Gene Ontology annotations, we show that only a restricted number of functions are overrepresented among those hypermutable genes including cell cycle and maintenance of DNA integrity. Using a probabilistic model, we show that genes involved in these functions are expected to host long SSRs because they tend to be long and/or are biased in nucleotide composition. Finally, we show that for almost all functions we observe fewer hypermutable sequences than expected under a neutral model. There are however interesting exceptions, for example, genes involved in protein and RNA transport, as well as meiosis and mismatch repair functions that have as many hypermutable genes as expected under neutrality. Conversely, there are functions (e.g., collagen-related genes) where hypermutable genes are more often avoided than in other functions. Our results show that, even though several functions harbor unusually long SSR in their exons, long SSRs are deleterious sequences in almost all functions and are removed by purifying selection. The strength of this purifying selection however greatly varies from function to function. We discuss possible explanations for this intriguing result.


Asunto(s)
Genoma Humano , Repeticiones de Microsatélite , Mutación , Exones , Humanos , Selección Genética
9.
Virchows Arch ; 477(1): 33-45, 2020 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-32447492

RESUMEN

Biliary tract carcinomas are divided into intrahepatic, perihilar, distal extrahepatic cholangiocarcinomas, and gallbladder adenocarcinomas. Therapies targeting ROS1, ALK, MET, and HER2 alterations are currently evaluated in clinical trials. We assessed ROS1 and ALK translocations/amplifications as well as MET and HER2 amplifications for each tumor subtype by fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) in 73 intrahepatic, 40 perihilar bile duct, 36 distal extrahepatic cholangiocarcinomas, and 45 gallbladder adenocarcinomas (n = 194). By FISH, we detected targetable alterations in 5.2% of cases (n = 10): HER2 and MET amplifications were found in 4.1% (n = 8) and 1.0% (n = 2), respectively. The HER2-amplified cases were mostly gallbladder adenocarcinomas (n = 5). The MET- and HER2-amplified cases were all positive by IHC. Fourteen cases without MET amplification were positive by IHC, whereas HER2 over-expression was detected by IHC only in HER2-amplified cases. We detected no ALK or ROS1 translocation or amplification. Several alterations were consistent with aneuploidy: 24 cases showed only one copy of ROS1 gene, 4 cases displayed a profile of chromosomal instability, and an over-representation of centromeric alpha-satellite sequences was found in five cases. We confirm a relatively high rate of HER2 amplifications in gallbladder adenocarcinomas and the efficacy of IHC to screen these cases. Our results also suggest the value of IHC to screen MET amplification. Contrary to initial publications, ROS1 rearrangements seem to be very rare in biliary tract adenocarcinomas. We confirm a relatively high frequency of aneuploidy and chromosomal instability and reveal the over-representation of centromeric alpha-satellite sequences in intrahepatic cholangiocarcinomas.


Asunto(s)
Adenocarcinoma/genética , Reordenamiento Génico/genética , Proteínas Tirosina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Quinasa de Linfoma Anaplásico/genética , Sistema Biliar/patología , Humanos , Hibridación Fluorescente in Situ/métodos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas c-met/genética
10.
Cell Oncol (Dordr) ; 43(2): 237-247, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31823290

RESUMEN

PURPOSE: The aim of this study was to investigate the association between expression of insulin-like growth factor-1 receptor (IGF1R) and its ligand, IGF-II, and disease-free survival (DFS) in patients with stage III colon cancer (CC). METHODS: In this retrospective study we included consecutive patients who underwent curative surgery for stage III CC. IGF1R and IGF-II/IGF2 status were evaluated in tumour samples by immunohistochemistry and quantitative real-time PCR (qRT-PCR). Associations of markers with DFS were analysed using Cox proportional hazards models. RESULTS: Hundred and fifty-one CC patients were included (median age, 66.6 years; female, 54.3%). Low levels of IGF1R and IGF-II protein expression were observed in 16.1% and 10.7% of the cases, respectively. No significant differences in clinicopathological characteristics between patients with tumours expressing low IGF1R or IGF-II protein levels and those with high levels were observed. A low IGF1R protein expression was found to be significantly associated with a shorter DFS (HR 3.32; 95% CI, 1.7-6.31; p = 0.0003), while no association was observed between IGF-II protein expression and DFS (HR 0.91; 95% CI, 0.28-2.96; p = 0.87). In a multivariate analysis, IGF1R protein status remained an independent prognostic factor for DFS (HR 2.73; 95% CI, 1.40-5.31; p = 0.003). Furthermore, we found that neither IGF1R nor IGF2 mRNA expression levels as measured by qRT-PCR correlated with the respective protein expression levels as assessed by immunohistochemistry. Neither of the mRNA expression levels was significantly associated with DFS. CONCLUSIONS: From our data we conclude that low IGF1R protein expression represents a poor prognostic biomarker in stage III colon cancer.


Asunto(s)
Neoplasias del Colon/genética , Regulación Neoplásica de la Expresión Génica , ARN Mensajero/genética , Receptor IGF Tipo 1/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Supervivencia sin Enfermedad , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , ARN Mensajero/metabolismo , Receptor IGF Tipo 1/biosíntesis , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
11.
Cancer Res ; 67(1): 398-407, 2007 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-17210723

RESUMEN

Obtaining representative human colon cancer cell lines from fresh tumors is technically difficult. Using 32 tumor fragments from patients with colon cancer, the present study shows that prior xenograft leads to more efficient cell line establishment compared with direct establishment from fresh tumors (P < 0.05). From 26 tumor specimens, we successfully established 20 tumor xenografts in nude mice (77%); among 19 of these xenografts, 9 (47%) led to cell lines, including four from liver metastases. Only 3 of 31 tumor specimens (9.7%) grew immediately in vitro, and all were derived from primary tumors. To compare major phenotypic and genotypic characteristics of human colon cancer cell lines derived from the same tumor fragment using two protocols, the two pairs of cell lines obtained from 2 of 32 tumor fragments were extensively studied. They displayed similar morphology and were able to form compact spheroids. Chemosensitivity to 5-fluorouracil, CPT11, and L-OHP differed between cell lines obtained from patient tumors and those derived from xenografts. Matched cell lines shared a common core of karyotype alterations and distinctive additional chromosomal aberrations. Expression levels of genes selected for their role in oncogenesis evaluated by real-time quantitative PCR were found to be statistically correlated whatever the in vitro culture model used. In conclusion, xenotransplantation in mice of tumor fragments before establishment of cell lines enables generation of more novel human cancer cell lines for investigation of colon cancer cell biology, opening up the opportunity of reproducing the diversity of this disease.


Asunto(s)
Línea Celular Tumoral , Neoplasias del Colon/patología , Animales , Procesos de Crecimiento Celular/fisiología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Ensayos de Selección de Medicamentos Antitumorales , Perfilación de la Expresión Génica , Humanos , Cariotipificación , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante Heterólogo
12.
Cancer Lett ; 450: 155-168, 2019 05 28.
Artículo en Inglés | MEDLINE | ID: mdl-30849481

RESUMEN

Hepatocellular carcinoma (HCC) is one of the most common and deadly neoplasms. Insulin receptor (IR) exists in two isoforms, IR-A and IR-B, the latter being predominantly expressed in normal adult hepatocytes while IR-A is overexpressed in HCC to the detriment of IR-B. This study evaluated the biological functions associated with IR-A overexpression in HCC in relation to expression of its ligand IGF-II. The value of INSRA:INSRB ratio which was increased in ˜70% of 85 HCC was associated with stem/progenitor cell features such as cytokeratin-19 and α-fetoprotein and correlated with shorter patient survival. IGF2 mRNA upregulation was observed in 9.4% of HCC and was not associated with higher INSRA:INSRB ratios. Ectopic overexpression of IR-A in two HCC cell lines presenting a strong autocrine IGF-II secretion loop or not stimulated cell migration and invasion. In cells cultured as spheroids, IR-A overexpression promoted gene programs related to stemness, inflammation and cell movement. IR-A also increased cell line tumorigenicity in vivo after injection to immunosuppressed mice and the sphere-forming cells made a significant contribution to this effect. Altogether, these results demonstrate that IR-A is a novel player in HCC progression.


Asunto(s)
Antígenos CD/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Células Madre Neoplásicas/patología , Receptor de Insulina/metabolismo , Animales , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Xenoinjertos , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Células Madre Neoplásicas/metabolismo , Isoformas de Proteínas
13.
Clin Cancer Res ; 24(17): 4282-4296, 2018 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-29716918

RESUMEN

Purpose: Cholangiocarcinoma (CCA) is a desmoplastic tumor of the biliary tree in which epidermal growth factor receptor (EGFR) is overexpressed and contributes to cancer progression. Although EGFR has been envisaged as a target for therapy, treatment with tyrosine kinase inhibitors (TKI) such as erlotinib did not provide therapeutic benefit in patients with CCA, emphasizing the need to investigate resistance mechanisms against EGFR inhibition.Experimental Design: Resistant CCA cells to EGFR inhibition were obtained upon long-time exposure of cells with erlotinib. Cell signaling, viability, migration, and spheroid growth were determined in vitro, and tumor growth was evaluated in CCA xenograft models.Results: Erlotinib-resistant CCA cells displayed metastasis-associated signatures that correlated with a marked change in cell plasticity associated with an epithelial-mesenchymal transition (EMT) and a cancer stem cell (CSC)-like phenotype. Resistant cells exhibited an upregulation of insulin receptor (IR) and insulin-like growth factor (IGF) 1 receptor (IGF1R), along with an increase in IGF2 expression. IR/IGF1R inhibition reduced EMT and CSC-like traits in resistant cells. In vivo, tumors developed from resistant CCA cells were larger and exhibited a more prominent stromal compartment, enriched in cancer-associated fibroblasts (CAF). Pharmacological coinhibition of EGFR and IR/IGF1R reduced tumor growth and stromal compartment in resistant tumors. Modeling of CCA-CAF crosstalk showed that IGF2 expressed by fibroblasts boosted IR/IGF1R signaling in resistant cells. Furthermore, IR/IGF1R signaling positively regulated fibroblast proliferation and activation.Conclusions: To escape EGFR-TKI treatment, CCA tumor cells develop an adaptive mechanism by undergoing an IR/IGF1R-dependent phenotypic switch, involving a contribution of stromal cells. Clin Cancer Res; 24(17); 4282-96. ©2018 AACR.


Asunto(s)
Colangiocarcinoma/tratamiento farmacológico , Factor II del Crecimiento Similar a la Insulina/genética , Receptor de Insulina/genética , Receptores de Somatomedina/genética , Animales , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Xenoinjertos , Humanos , Ratones , Miofibroblastos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Receptor IGF Tipo 1 , Transducción de Señal/efectos de los fármacos
14.
BMC Cancer ; 7: 225, 2007 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-18088404

RESUMEN

BACKGROUND: Carcinomas of unknown primary site (CUP) are epithelial malignancies revealed by metastatic lesions in the absence of any detectable primary tumor. Although they often adopt an aggressive clinical pattern, their basic biology remains poorly understood. Laboratory research on their biology have been hampered so far by the absence of cell lines representative of CUPs. METHODS: We attempted xenografts of CUP clinical specimens in immunodeficient mice and subsequent in vitro culture of transplanted malignant cells. Whenever possible, malignant xenografted or cultured cells were characterized by microsatellite genotyping, immunohistology, electron microscopy, multifish chromosome analysis and search of TP 53 gene mutations. RESULTS: Successful xenografts were achieved in 2 cases out of 4. One of them (Capi1) was lost after 3 passages whereas the other one (Capi3) has been adapted to in vitro culture and is currently available to the scientific community with reliable identification based on microsatellite genotyping. Both Capi1 and Capi3 have histological characteristics of adenocarcinomas and display intense expression of EMA, CEA and cytokeratin 7. Multifish chromosome analysis demonstrated a translocation involving chromosomes 4 and 21 in both specimens. Distinct rare missense mutations of the TP53 gene were detected in Capi1 (codon 312) and Capi3 (codon 181); the codon 181 mutation is consistent with a previously reported similar finding in a small series of CUP specimens. Finally, intense membrane expression of c-kit was recorded in Capi3. CONCLUSION: Our data suggest that xenografted tumors can be obtained from a substantial fraction of CUP clinical specimens. The hypothesis of a preferential association of CUPs with TP 53 mutations of codon 181 deserves further investigations. The Capi3 cell line will be a useful tool for assessment of novel c-kit inhibitors.


Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/secundario , Regulación Neoplásica de la Expresión Génica , Trasplante de Neoplasias/patología , Neoplasias Primarias Desconocidas/genética , Neoplasias Primarias Desconocidas/patología , Trasplante Heterólogo/patología , Adenocarcinoma/patología , Animales , Neoplasias Óseas/secundario , Línea Celular Tumoral , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 4/genética , Femenino , Genes Relacionados con las Neoplasias/genética , Genes p53/genética , Humanos , Inmunohistoquímica , Masculino , Neoplasias Mamarias Animales/secundario , Ratones , Ratones Desnudos , Ratones SCID , Repeticiones de Microsatélite/genética , Microscopía Electrónica , Mutación Missense/genética , Neoplasias Pleurales/secundario , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Neoplasias Cutáneas/secundario
15.
Nucleic Acids Res ; 33(1): 106-13, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-15642697

RESUMEN

Loss of a functional mismatch repair (MMR) system in colorectal cancer (CRC) cells is associated with microsatellite instability and increased sensitivity to topoisomerase inhibitors. In this study, we have investigated whether a defect in double-strand break (DSB) repair by non-homologous end-joining (NHEJ) could explain why MMR-deficient CRC cells are hypersensitive to camptothecin (CPT), a topoisomerase I inhibitor. To evaluate the efficiency and the fidelity of DSB repair, we have transiently transfected plasmids containing cohesive or non-complementary ends in cells with various MMR defects. We have observed that the repair efficiency of DSB with cohesive and non-complementary ends is comparable in all cell lines. In contrast to the MMR-proficient cell line HT29, the MMR-deficient cell lines were highly accurate in repairing DSB with cohesive ends, but this characteristic could not be directly assigned to the primary MMR deficiency. Furthermore, CPT treatment had no detectable effect on the repair of cohesive ends but significantly decreased the repair efficiency of non-complementary DSB. In conclusion, although our observations show that DSB repair efficiency by NHEJ decreases upon treatment with CPT, which possibly contributes to its cytotoxicity, it is quite unlikely that it accounts for the hypersensitivity of MMR-deficient cells to topoisomerase inhibitors.


Asunto(s)
Camptotecina/farmacología , Neoplasias Colorrectales/genética , Reparación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores de Topoisomerasa I , Disparidad de Par Base , Línea Celular Tumoral , Neoplasias Colorrectales/enzimología , Daño del ADN , Mutación del Sistema de Lectura , Humanos , Recombinación Genética
16.
Cancer Genomics Proteomics ; 14(5): 329-339, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28871000

RESUMEN

BACKGROUND/AIM: The aim of this study was to assess the incidence of MSI in a large series of human hepatocellular carcinomas (HCC) with various etiologies. MATERIALS AND METHODS: The MSI status was determined by polymerase chain reaction (PCR) using 5 mononucleotide and 13 CAn dinucleotide repeats. RESULTS: None of the 122 HCC samples displayed an MSI-High phenotype, as defined by the presence of alterations at more than 30% of the microsatellite markers analyzed. Yet, limited microsatellite instability consisting in the insertion or deletion of a few repeat motifs was detected in 32 tumor samples (26.2%), regardless of the etiology of the underlying liver disease. MSI tended to be higher in patients with cirrhosis (p=0.051), possibly reflecting an impact of the inflammatory context in this process. CONCLUSION: Based on a large series of HCC with various etiologies, our study allowed us to definitely conclude that MSI is not a hallmark of HCC.


Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Inestabilidad de Microsatélites , Repeticiones de Microsatélite/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/patología , Femenino , Humanos , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Adulto Joven
17.
Virchows Arch ; 449(2): 238-43, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16639607

RESUMEN

Hereditary nonpolyposis colon cancer (HNPCC) syndrome is the most frequent hereditary cancer syndrome predisposing to cancers of various locations, especially colon, endometrium, stomach, and upper urinary tract. Carcinomas of the kidney parenchyma are not considered as an HNPCC-related tumor. HNPCC tumors are characterized by microsatellite instability (MSI) due to a defect in mismatch repair (MMR) and carry somatic frameshift mutations in mononucleotide repeats within the coding regions of key genes. We report the first case of a papillary carcinoma of the kidney in an HNPCC patient who developed carcinomas of the upper urinary tract, endocervix, and colon. Whereas the HNPCC-related tumors demonstrated MSI phenotype, loss of MSH2 protein expression, and frameshift mutations in several of the 13 target genes analyzed, the kidney cancer displayed MSS phenotype, normal MMR protein expression, and no frameshift mutation in target genes. Our observations do not support the possibility that papillary carcinomas are part of HNPCC syndrome.


Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales/genética , Neoplasias Renales/genética , Neoplasias Primarias Secundarias/genética , Neoplasias Urológicas/genética , Neoplasias del Cuello Uterino/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Proteínas de Unión al ADN/genética , Femenino , Humanos , Inmunohistoquímica , Neoplasias Renales/patología , Proteína Homóloga de MRE11 , Repeticiones de Microsatélite , Persona de Mediana Edad , Neoplasias Primarias Secundarias/patología , Neoplasias Urológicas/patología , Neoplasias del Cuello Uterino/patología , Proteína X Asociada a bcl-2/genética
18.
Clin Cancer Res ; 11(17): 6212-7, 2005 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-16144923

RESUMEN

PURPOSE: The aim of our study was to assess whether the polymorphism of the nucleotide excision repair enzyme, excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1), had an effect on the tumor response in patients treated with standard chemotherapy regimens for a metastatic colorectal cancer. We have studied the synonymous polymorphism that causes a single nucleotide change C to T at codon 118 converting a codon of common usage (AAC) to a less used codon (AAT), both coding asparagine. This change results in a decreased ERCC1 gene expression, which impairs repair activity. EXPERIMENTAL DESIGN: Ninety-one patients with a median age of 55.1 years treated for a metastatic colorectal cancer were included in our retrospective study. The ERCC1 polymorphism was analyzed in the normal tissue of all patients. RESULTS: Twenty (22%) were homozygous for AAC codon (C/C genotype), 30 were (33%) homozygous for AAT codon (T/T genotype), and 41 (45%) were heterozygous (C/T genotype). The objective response rate to oxaliplatin in combination with 5-fluorouracil (5-FU) was significantly higher in the T/T genotype group compared with the C/T and the C/C genotype groups (61.9%, 42.3%, and 21.4%, respectively; P = 0.018). By contrast, no significant difference was observed when patients were treated with either 5-FU alone (45%, 29.2%, and 33.3%, respectively; P = 0.407) or in combination with irinotecan (46.1%, 25.0%, and 27.3%, respectively; P = 0.305). CONCLUSIONS: Our observations allowed us to define the first useful predictive criterion for oxaliplatin/5-FU response in patients with metastatic colorectal cancer.


Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Codón/genética , Neoplasias Colorrectales/tratamiento farmacológico , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Polimorfismo Genético/genética , Neoplasias Colorrectales/genética , Reparación del ADN , ADN de Neoplasias/genética , Femenino , Fluorouracilo/administración & dosificación , Humanos , Masculino , Persona de Mediana Edad , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos
19.
Cancer Res ; 63(20): 6935-41, 2003 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-14583494

RESUMEN

Gemcitabine [2',2'-difluoro-2'-deoxycytidine (dFdCyd)] is a potent ionizing radiation sensitizer in solid tumor cells in vitro and in vivo. Previously, we have demonstrated (Shewach et al., Cancer Res., 54: 3218-3223, 1994) a strong correlation between depletion of dATP (caused by dFdCyd diphosphate-mediated inhibition of ribonucleotide reductase) and radiosensitization. In addition, we and others (Latz et al., Int. J. Radiat. Oncol. Biol. Phys., 41: 875-882, 1998; Ostruszka and Shewach, Cancer Res., 60: 6080-6088, 2000) have shown that the accumulation of cells in S phase prior to irradiation is also important for radiosensitization with dFdCyd. This led us to hypothesize that the incorporation of incorrect nucleotides because of the dATP pool imbalance was important for radiosensitization with dFdCyd, and, therefore, cells deficient in mismatch repair (MMR) would exhibit greater radiosensitization. We tested this hypothesis by evaluating the ability of HCT116 colon carcinoma cell lines, which differ in MMR proficiency, to be radiosensitized by dFdCyd. The MMR-proficient cell line (HCT116 + ch3) was more sensitive to dFdCyd alone than were the MMR-deficient cell lines (HCT116, HCT116 + ch2, and HCT116 p53(-/-)). Interestingly, the MMR-proficient cells could not be radiosensitized at concentrations of dFdCyd IC(96)) enhanced cell killing with radiation. In contrast, the MMR-deficient cells were radiosensitized at concentrations of dFdCyd or=80% decrease in dATP within 4 h after drug addition, and this low dATP level was maintained for another 12-20 h. Although the IC(50) of dFdCyd was unable to sustain a >80% decrease in the dATP level in the MMR-proficient cells, the IC(90) did achieve this level of dATP depletion; however, it was unable to radiosensitize the MMR-proficient cells. Similar results were obtained with HCT116 cells, in which the MMR deficiency was corrected by transfection with a vector containing the hMLH1 cDNA. In addition, the deletion of p53 did not increase radiation enhancement ratios. These results demonstrate that MMR deficiency promotes radiosensitization with dFdCyd. We suggest that dATP depletion produces errors of replication in MMR-deficient cells, which, if left unrepaired, enhances cell death by ionizing radiation.


Asunto(s)
Disparidad de Par Base/fisiología , Reparación del ADN/fisiología , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , ADN de Neoplasias/metabolismo , Nucleótidos de Desoxiadenina/metabolismo , Células HCT116 , Humanos , Nucleótidos/metabolismo , Tolerancia a Radiación/genética , Gemcitabina
20.
Cancer Res ; 63(18): 5738-44, 2003 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-14522894

RESUMEN

The aim of our study was to assess the relationship between colorectal tumor responsiveness to irinotecan and microsatellite instability (MSI), a feature of colorectal tumors with DNA mismatch repair defect. Seventy-two patients with metastatic colorectal cancer were included in our retrospective study. A complete response to irinotecan was observed in 1 patient and a partial response in 10 patients, whereas 61 patients did not respond to this treatment. We analyzed the protein expression of hMLH1, hMSH2, and BAX by immunohistochemistry, determined the MSI phenotype, and looked for mutations in the coding repeats located in the transforming growth factor beta-RII, BAX, hMSH3, and hMSH6 genes. All 44 tumors analyzed expressed detectable levels of hMLH1; 1 tumor lacked hMSH2 staining, whereas 4 tumors showed a marked decrease in BAX expression. A better response to irinotecan was observed in the patients whose tumors have lost BAX expression (P < 0.001). Among the 7 tumors that displayed a MSI-H phenotype, 4 responded to irinotecan, whereas only 7 of the 65 MSI-L/ microsatellite stable tumors did (P = 0.009). Seven of the 72 tumors had inactivating mutations in the coding repeats of the target genes. Three tumors displayed a mutation in the poly-A10 tract of the transforming growth factor beta-RII gene, associated with a 1-bp deletion in the poly-A8 tract of hMSH3 in one tumor and with a 1-bp deletion in the poly-G8 tract of BAX in another. Four tumors displayed mutations in the poly-G8 repeat of BAX, whereas 2 mutations in hMSH6 and hMSH3 were characterized. Among the 7 tumors with mutations in these target genes, 5 responded to irinotecan, whereas only 6 of the other 65 tumors did (P < 0.001), indicating that MSI-driven inactivation of target genes modifies tumor chemosensitivity. Our observations allowed us to define the first useful predictive criteria for irinotecan response in patients with colorectal cancer.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Camptotecina/análogos & derivados , Camptotecina/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Enzimas Reparadoras del ADN , Inestabilidad Genómica , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Adaptadoras Transductoras de Señales , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Disparidad de Par Base/genética , Proteínas Portadoras , Neoplasias Colorrectales/metabolismo , Reparación del ADN/genética , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Exones , Femenino , Eliminación de Gen , Humanos , Irinotecán , Masculino , Persona de Mediana Edad , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas Nucleares , Valor Predictivo de las Pruebas , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteína X Asociada a bcl-2
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