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The colon differs regionally in local luminal environment, excretory function, and gene expression. Polycistronic microRNA (miR)-143 and miR-145 are downregulated early in colon cancer. We asked if these microRNAs (miRNAs) might be differentially expressed in the proximal vs. the distal colon, contributing to regional differences in protein expression. Primary transcripts and mature miR-143 and miR-145 were quantified by real-time PCR, putative targets were measured by Western blotting, and DNA methylation was assessed by sequencing bisulfite-treated DNA in proximal and distal normal colonic mucosa as well as colon cancers. Putative targets of these miRNAs were assessed following transfection with miR-143 or miR-145. Mean expression of mature miR-143 and miR-145 was 2.0-fold (P < 0.001) and 1.8-fold (P = 0.03) higher, respectively, in proximal than distal colon. DNA methylation or primary transcript expression of these miRNAs did not differ by location. In agreement with increased expression of miR-143 and miR-145 in proximal colon, predicted targets of these miRNAs, apoptosis inhibitor 5 (API5), ERK5, K-RAS, and insulin receptor substrate 1 (IRS-1), which are cell cycle and survival regulators, were expressed at a lower level in proximal than distal colon. Transfection of HCA-7 colon cancer cells with miR-145 downregulated IRS-1, and transfection of HT-29 colon cancer cells with miR-143 decreased K-RAS and ERK5 expression. In conclusion, miR-143 and miR-145 and the predicted target proteins API5, ERK5, K-RAS, and IRS-1 display regional differences in expression in the colon. We speculate that differences in these tumor suppressors might contribute to regional differences in normal colonic gene expression and modulate site-specific differences in malignant predisposition.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Colon/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , MicroARNs/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas ras/metabolismo , Adulto , Ciclo Celular , Línea Celular Tumoral , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Proteínas Proto-Oncogénicas p21(ras) , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Adulto JovenRESUMEN
Diffuse large B-cell lymphomas in humans are associated with chromosomal rearrangements (â¼40%) and/or mutations disrupting autoregulation (â¼16%) involving the BCL6 gene. Studies of lymphoma development in humans and mouse models have indicated that lymphomagenesis evolves through the accumulation of multiple genetic alterations. Based on our prior studies, which indicated that carcinogen-induced DNA mutations enhance the incidence of lymphomas in our mouse model expressing a human BCL6 transgene, we hypothesized that mutated genes are likely to play an important cooperative role in BCL6-associated lymphoma development. We used retroviral insertional mutagenesis in an effort to identify which genes cooperate with BCL6 in lymphomagenesis in our BCL6 transgenic mice. We identified PIM1 as the most frequently recurring cooperating gene in our murine BCL6-associated lymphomas (T- and B-cell types), and we observed elevated levels of PIM1 mRNA and protein expression in these neoplasms. Further, immunohistochemical staining, which was performed in 20 randomly selected BCL6-positive human B- and T-cell lymphomas, revealed concurrent expression of BCL6 and PIM1 in these neoplasms. As PIM1 encodes a serine/threonine kinase, PIM1 kinase inhibition may be a promising therapy for BCL6/PIM1-positive human lymphomas.
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Proteínas de Unión al ADN/genética , Linfoma/genética , Linfoma/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Proteínas Proto-Oncogénicas c-pim-1/genética , Animales , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Mutagénesis Insercional/genética , Proteínas Proto-Oncogénicas c-bcl-6 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retroviridae/genética , Análisis de SupervivenciaRESUMEN
The BCL6 gene, which is expressed in certain B- and T-cell human lymphomas, is involved with chromosomal rearrangements and mutations in a number of these neoplasms. Lymphomagenesis is believed to evolve through a multi-step accumulation of genetic alterations in these tumors. We used retroviral insertional mutagenesis in transgenic mice expressing the human BCL6 transgene in order to identify genes that cooperate with BCL6 during lymphomatous transformation. We previously reported PIM1 as the most frequently recurring cooperating gene in this model. We now report three newly identified cooperating genes-GFI1B, EVI5, and MYB-that we identified in the lymphomas of retroviral-injected BCL6 transgenic mice (but not in retroviral-injected non-transgenic controls); mRNA and protein expression of GFI1B and EVI5 were decreased in the murine tumors, whereas MYB mRNA and protein expression were increased or decreased. These findings correlated with protein expression in human lymphomas, both B- and T-cell. Improved therapy of lymphomas may necessitate the development of combinations of drugs that target the alterations specific to each neoplasm.
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Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B/genética , Linfoma de Células T/genética , Proteínas Oncogénicas v-myb/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Proteínas de Ciclo Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Proteínas de Unión al ADN/metabolismo , Femenino , Proteínas Activadoras de GTPasa , Vectores Genéticos , Humanos , Inmunohistoquímica , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Masculino , Ratones , Ratones Transgénicos , Proteínas Oncogénicas v-myb/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Retroviridae/genética , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
We conducted a 45 patient prospective study of reduced-intensity conditioning (RIC) and transplantation of unrelated umbilical cord blood (UCB) and CD34(+) stem cells from a haploidentical family member. Median age was 50 years; weight was 80 kg. Fifty-eight percent had active disease. Neutrophil engraftment occurred at 11 days (interquartile range [IQR], 9-15) and platelet engraftment at 19 days (IQR, 15-33). In the majority of patients, early haploidentical engraftment was replaced by durable engraftment of UCB by 100 days, with regular persistence of minor host and/or haplo-hematopoiesis. Percentage of haplochimerism at day 100 correlated with the haplo-CD34 dose (P = .003). Cumulative incidence of acute GVHD (aGVHD) was 25% and chronic GVHD (cGVHD) was 5%. Actuarial survival at 1 year was 55%, progression-free survival (PFS) was 42%, nonrelapse mortality (NRM) was 28%, and relapse was 30%. RIC and haplo-cord transplantation results in fast engraftment of neutrophils and platelets, low incidences of aGVHD and cGVHD, low frequency of delayed opportunistic infections, reduced transfusion requirements, shortened length of hospital stay, and promising long-term outcomes. UCB cell dose had no impact on time to hematopoietic recovery. Therefore, UCB selection can prioritize matching, and better matched donors can be identified rapidly for most patients. This study is registered at http://clinicaltrials.gov as NCI clinical trial no. NCT00943800.
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Trasplante de Células Madre de Sangre del Cordón Umbilical , Supervivencia de Injerto , Enfermedad Injerto contra Huésped/epidemiología , Trasplante de Células Madre de Sangre Periférica , Acondicionamiento Pretrasplante , Adulto , Células Madre Adultas/metabolismo , Células Madre Adultas/trasplante , Anciano , Antígenos CD34/metabolismo , Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/prevención & control , Haplotipos , Humanos , Illinois/epidemiología , Incidencia , Masculino , Persona de Mediana Edad , Trasplante de Células Madre de Sangre Periférica/efectos adversos , Proyectos Piloto , Inducción de Remisión , Análisis de Supervivencia , Trasplante Homólogo , Adulto JovenRESUMEN
OBJECTIVES: To investigate and characterize JAK2 mutations in myelodysplastic syndrome (MDS), we present three cases with diverse JAK2 mutations and review the literature. METHODS: The institutional SoftPath software was used to find MDS cases between January 2020 and April 2022. The cases with a diagnosis of a myelodysplastic/myeloproliferative overlap syndrome including MDS/MPN with ring sideroblasts and thrombocytosis were excluded. The cases with molecular data by next generation sequencing looking for gene aberrations commonly seen in myeloid neoplasms were reviewed for the detection of JAK2 mutations including variants. A literature review on the identification, characterization, and significance of JAK2 mutations in MDS was performed. RESULTS: Among 107 cases of the MDS reviewed, a JAK2 mutation was present in three cases, representing 2.8% of the overall cases. A JAK2 V617F mutation was found in one case representing slightly less than 1% of all the MDS cases. In addition, we found JAK2 R564L and JAK2 I670V point mutation variants to be associated with a myelodysplastic phenotype. CONCLUSIONS: JAK2 mutations in MDS are rare and represent less than 3% of cases. It appears that JAK2 variant mutations in MDS are diverse and further studies are needed to understand their role in the phenotype and prognosis of the disease.
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Colon cancer growth requires growth-promoting interactions between malignant colonocytes and stromal cells. Epidermal growth factor receptors (EGFR) are expressed on colonocytes and many stromal cells. Furthermore, EGFR is required for efficient tumorigenesis in experimental colon cancer models. To dissect the cell-specific role of EGFR, we manipulated receptor function on stromal cells and cancer cells. To assess the role of stromal EGFR, HCT116 human colon cancer cells were implanted into immunodeficient mice expressing dominant negative (DN) Egfr(Velvet/+) or Egfr(+/+). To assess the role of cancer cell EGFR, HCT116 transfectants expressing inducible DN-Egfr were implanted into immunodeficient mice. To dissect EGFR signals in vitro, we examined colon cancer cells in monoculture or in cocultures with fibroblasts for EGFR transactivation and prostaglandin synthase 2 (PTGS2) induction. EGFR signals were determined by blotting, immunostaining and real-time PCR. Tumor xenografts in Egfr(Velvet/+) mice were significantly smaller than tumors in Egfr(+/+) mice, with decreased proliferation (Ki67) and increased apoptosis (cleaved caspase-3) in cancer cells and decreased stromal blood vessels. Mouse stromal transforming growth factor alpha (TGFA), amphiregulin (AREG), PTGS2 and Il1b and interleukin-1 receptor 1 (Il1r1) transcripts and cancer cell beta catenin (CTNNB1) and cyclin D1 (CCND1) were significantly lower in tumors obtained from Egfr(Velvet/+) mice. DN-EGFR HCT116 transfectants also formed significantly smaller tumors with reduced mouse Areg, Ptgs2, Il1b and Il1r1 transcripts. Coculture increased Caco-2 phospho-active ERBB (pERBB2), whereas DN-EGFR in Caco-2 cells suppressed fibroblast PTGS2 and prostaglandin E2 (PGE2). In monoculture, interleukin 1 beta (IL1B) transactivated EGFR in HCT116 cells. Stromal cell and colonocyte EGFRs are required for robust EGFR signals and efficient tumor growth, which involve EGFR-interleukin-1 crosstalk.
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Neoplasias del Colon/metabolismo , Receptores ErbB/metabolismo , Células del Estroma/metabolismo , Animales , Apoptosis , Transformación Celular Neoplásica , Técnicas de Cocultivo , Neoplasias del Colon/patología , Células HCT116 , Humanos , Interleucina-1beta/metabolismo , Ratones , Transfección , Trasplante HeterólogoRESUMEN
In this paper, we are working toward a problem of great importance to global health: determination of viral HIV and hepatitis C (HCV) loads under point-of-care and resource limited settings. While antiretroviral treatments are becoming widely available, viral load must be evaluated at regular intervals to prevent the spread of drug resistance and requires a quantitative measurement of RNA concentration over a wide dynamic range (from 50 up to 10(6) molecules/mL for HIV and up to 10(8) molecules/mL for HCV). "Digital" single molecule measurements are attractive for quantification, but the dynamic range of such systems is typically limited or requires excessive numbers of compartments. Here we designed and tested two microfluidic rotational SlipChips to perform multivolume digital RT-PCR (MV digital RT-PCR) experiments with large and tunable dynamic range. These designs were characterized using synthetic control RNA and validated with HIV viral RNA and HCV control viral RNA. The first design contained 160 wells of each of four volumes (125 nL, 25 nL, 5 nL, and 1 nL) to achieve a dynamic range of 5.2 × 10(2) to 4.0 × 10(6) molecules/mL at 3-fold resolution. The second design tested the flexibility of this approach, and further expanded it to allow for multiplexing while maintaining a large dynamic range by adding additional wells with volumes of 0.2 nL and 625 nL and dividing the SlipChip into five regions to analyze five samples each at a dynamic range of 1.8 × 10(3) to 1.2 × 10(7) molecules/mL at 3-fold resolution. No evidence of cross-contamination was observed. The multiplexed SlipChip can be used to analyze a single sample at a dynamic range of 1.7 × 10(2) to 2.0 × 10(7) molecules/mL at 3-fold resolution with limit of detection of 40 molecules/mL. HIV viral RNA purified from clinical samples were tested on the SlipChip, and viral load results were self-consistent and in good agreement with results determined using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test. With further validation, this SlipChip should become useful to precisely quantify viral HIV and HCV RNA for high-performance diagnostics in resource-limited settings. These microfluidic designs should also be valuable for other diagnostic and research applications, including detecting rare cells and rare mutations, prenatal diagnostics, monitoring residual disease, and quantifying copy number variation and gene expression patterns. The theory for the design and analysis of multivolume digital PCR experiments is presented in other work by Kreutz et al.
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VIH/química , Hepacivirus/química , Técnicas Analíticas Microfluídicas , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Viral , VIH/genética , Hepacivirus/genética , ARN Viral/genética , RotaciónRESUMEN
Because ADAM17 promotes colonic tumorigenesis, we investigated potential miRNAs regulating ADAM17; and examined effects of diet and tumorigenesis on these miRNAs. We also examined pre-miRNA processing and tumour suppressor roles of several of these miRNAs in experimental colon cancer. Using TargetScan, miR-145, miR-148a, and miR-152 were predicted to regulate ADAM17. miR-143 was also investigated as miR-143 and miR-145 are co-transcribed and associated with decreased tumour growth. HCT116 colon cancer cells (CCC) were co-transfected with predicted ADAM17-regulating miRNAs and luciferase reporters controlled by ADAM17-3'UTR. Separately, pre-miR-143 processing by colonic cells was measured. miRNAs were quantified by RT-PCR. Tumours were induced with AOM/DSS in WT and transgenic mice (Tg) expressing pre-miR-143/miR-145 under villin promoter. HCT116 transfection with miR-145, -148a or -152, but not scrambled miRNA inhibited ADAM17 expression and luciferase activity. The latter was suppressed by mutations in ADAM17-3'UTR. Lysates from colonocytes, but not CCC, processed pre-miR-143 and mixing experiments suggested CCC lacked a competency factor. Colonic miR-143, miR-145, miR-148a, and miR-152 were downregulated in tumours and more moderately by feeding mice a Western diet. Tg mice were resistant to DSS colitis and had significantly lower cancer incidence and tumour multiplicity. Tg expression blocked up-regulation of putative targets of miR-143 and miR-145, including ADAM17, K-Ras, XPO5, and SET. miR-145, miR-148a, and miR-152 directly suppress colonocyte ADAM17 and are down-regulated in colon cancer. This is the first direct demonstration of tumour suppressor roles for miR-143 and miR-145 in an in vivo model of colonic tumorigenesis.
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Colitis , Neoplasias del Colon , MicroARNs , Animales , Neoplasias del Colon/genética , Metilación de ADN , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Humanos , Inflamación , Carioferinas , Ratones , MicroARNs/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: KRAS mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect KRAS mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for KRAS mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes. METHODS: We developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in KRAS codons 12 and 13 using the Roche LightCycler® instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (Tc) of 81°C increased the sensitivity of the assay >10-fold for the majority of KRAS mutations. RESULTS: We compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant KRAS mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background. CONCLUSIONS: We have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for KRAS mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic accuracy compared to traditional PCR and direct sequencing.
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Laboratory testing plays a critical role in the diagnosis and monitoring of patients with neurologic disorders. Although common tests are often performed in a central hospital laboratory, an increasing number of essential but esoteric tests are performed at reference laboratories or other outside health care facilities. In this article, we analyze recent trends in neurologic disease testing within the overall context of reference laboratory testing and discuss strategies to facilitate the provision of high-quality, cost-effective laboratory services.
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Técnicas de Laboratorio Clínico , Laboratorios de Hospital/normas , Enfermedades del Sistema Nervioso/diagnóstico , Técnicas de Laboratorio Clínico/economía , Técnicas de Laboratorio Clínico/normas , Técnicas de Laboratorio Clínico/estadística & datos numéricos , Control de Costos , Humanos , Revisión de Utilización de RecursosRESUMEN
STUDY OBJECTIVE: Triage quickly identifies critically ill patients, facilitating timely interventions. Many emergency departments (EDs) use emergency severity index (ESI) or abnormal vital sign triggers to guide triage. However, both use fixed thresholds, and false activations are costly. Prior approaches using machinelearning have relied on information that is often unavailable during the triage process. We examined whether deep-learning approaches could identify critically ill patients only using data immediately available at triage. METHODS: We conducted a retrospective, cross-sectional study at an urban tertiary care center, from January 1, 2012-January 1, 2020. De-identified triage information included structured (age, sex, initial vital signs) and textual (chief complaint) data, with critical illness (mortality or ICU admission within 24 hours) as the outcome. Four progressively complex deep-learning models were trained and applied to triage information from all patients. We compared the accuracy of the models against ESI as the standard diagnostic test, using area under the receiver-operator curve (AUC). RESULTS: A total of 445,925 patients were included, with 60,901 (13.7%) critically ill. Vital sign thresholds identified critically ill patients with AUC 0.521 (95% confidence interval [CI] = 0.519-0.522), and ESI <3 demonstrated AUC 0.672 (95% CI = 0.671-0.674), logistic regression classified patients with AUC 0.803 (95% CI = 0.802-0.804), 2-layer neural network with structured data with AUC 0.811 (95% CI = 0.807-0.815), gradient tree boosting with AUC 0.820 (95% CI = 0.818-0.821), and the neural network model with textual data with AUC 0.851 (95% CI = 0.849-0.852). All successive increases in AUC were statistically significant. CONCLUSION: Deep-learning techniques represent a promising method of augmenting triage, even with limited information. Further research is needed to determine if improved predictions yield clinical and operational benefits.
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Widespread indications for use of molecular diagnostics in various aspects of clinical medicine have driven proliferation of testing. The rapid adoption and continuous technological evolution of molecular diagnostics have often strained the development and maintenance of a functional underlying framework of coding, coverage, and reimbursement policies, thereby presenting challenges to various stakeholders, including molecular professionals, payers, and patients. A multidisciplinary working group convened by the Association for Molecular Pathology Economic Affairs Committee was tasked to describe the complex landscape of molecular pathology economics and highlight opportunities for member engagement. In this article, on the basis of review and synthesis of government regulations and procedures, published payer policy documents, peer-reviewed literature, and expert consensus, the Working Group navigates the ecosystem of molecular pathology economics in terms of stakeholders, coding systems and processes, coverage policy determination, and pricing mechanisms. The composition and interrelatedness of various working groups and committees are emphasized to highlight the functional underpinnings of the system. Molecular professionals must be conversant in the language and complex inner workings of molecular pathology economics to lead successful, viable laboratories and advocate effectively for policy development on their behalf. This overview is provided to be a resource to molecular professionals as they navigate the reimbursement landscape.
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Cobertura del Seguro/economía , Reembolso de Seguro de Salud/economía , Técnicas de Diagnóstico Molecular/economía , Costos y Análisis de Costo/métodos , Regulación Gubernamental , Humanos , Laboratorios de Hospital/economía , Medicare/economía , Participación de los Interesados , Estados UnidosRESUMEN
BACKGROUND: Heat shock protein 70 (Hsp70) regulates protein biosynthesis and refolding of denatured proteins. Since Hsp70 participates in recovery from stress injury, we examined the effect of Hsp70 genetic deletion in the azoxymethane (AOM)/dextran sulfate sodium (DSS) model of inflammation and colon cancer. METHODS: Hsp70 mutant mice (Hsp70.1(-/-)/70.3(-/-)) and wild-type (WT) littermates received AOM and three cycles of DSS and were killed 24 weeks later. Tumors were graded for histology and immunostained for p53, adenomatous polyposis coli, beta-catenin, cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNOS) and sequenced for p53 mutations. RESULTS: Elevated adenomas developed in 4/10 WT mice with no dysplasia in adjacent mucosa. In contrast, 7/8 Hsp70 knock out (KO) mice developed chronic mucosal inflammation and multifocal areas of flat dysplasia and 4/8 progressed to invasive carcinomas arising in a background of flat dysplastic mucosa. These differences in the incidence of flat dysplasia and invasive cancers were significant (P < 0.05). Nuclear p53 was stronger in Hsp70 KO tumors compared with WT tumors, and sequencing confirmed p53 mutations in 2/5 tumors from Hsp70(-/-) versus 0/5 in WT mice. In Hsp70 WT tumors, beta-catenin was predominantly nuclear, compared with membranous beta-catenin in Hsp70(-/-) tumors, suggesting that Hsp70 regulates beta-catenin in colonic tumorigenesis. Cox-2 and iNOS levels were increased in tumors from Hsp70(-/-) mice compared with Hsp70 WT tumors. CONCLUSIONS: Hsp70-deleted mice treated with AOM/DSS develop flat invasive colonic tumors that mimic many histological and molecular features of ulcerative colitis colon cancer. This model will be useful to dissect the role of Hsp70 in inflammatory bowel disease colon cancer.
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Colitis/patología , Neoplasias del Colon/patología , Proteínas HSP70 de Choque Térmico/fisiología , Animales , Secuencia de Bases , Western Blotting , Neoplasias del Colon/metabolismo , Cartilla de ADN , Proteínas HSP70 de Choque Térmico/genética , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Invasividad Neoplásica , Proteína p53 Supresora de Tumor/genéticaRESUMEN
PURPOSE: Colonic carcinogenesis deranges growth-regulating epidermal growth factor receptors (EGFR). We previously showed that EGFR signals were up-regulated in human aberrant crypt foci (ACF), putative colon cancer precursors. The azoxymethane model of colon cancer recapitulates many aspects of human colonic tumors. Recent studies indicate that flat dysplastic ACF with increased beta-catenin are tumor precursors in this model. We asked, therefore, if EGFR signals are required for flat dysplastic ACF development and cancer progression. EXPERIMENTAL DESIGN: Rats received azoxymethane or saline, and standard chow or chow supplemented with gefitinib, an EGFR inhibitor, for 44 weeks. EGFR signals were quantified in normal colon, flat ACF, and tumors by computerized analysis of immunostains and Western blots. K-ras mutations were assessed by PCR and mRNA for egfr ligands by quantitative real-time PCR. RESULTS: EGFR inhibition with gefitinib decreased the incidence of flat dysplastic ACF from 66% to 36% and tumors from 71% to 22% (P < 0.05). This inhibitor also reduced the overexpressions of cyclin D1 and Cox-2 in flat ACF. Furthermore, in flat ACF, EGFR blockade decreased the up-regulation of c-Jun, FosB, phosphorylated active signal transducers and activators of transcription 3, and CCAAT/enhancer binding protein-beta, potential regulators of cyclin D1 and Cox-2. In colonic tumors, EGFR blockade significantly decreased angiogenesis, proliferation, and progression while also increasing apoptosis (P < 0.05). Gefitinib also inhibited the activations of extracellular signal-regulated kinase, Src, and AKT pathways in tumors. CONCLUSIONS: We have shown for the first time that EGFR promotes the development of flat dysplastic ACF and the progression of malignant colonic tumors. Furthermore, we have mechanistically identified several transcription factors and their targets as EGFR effectors in colonic carcinogenesis.
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Neoplasias del Colon/etiología , Receptores ErbB/fisiología , Lesiones Precancerosas/etiología , Animales , Azoximetano/toxicidad , Proteína beta Potenciadora de Unión a CCAAT/fisiología , Neoplasias del Colon/prevención & control , Ciclina D , Ciclinas/análisis , Progresión de la Enfermedad , Receptores ErbB/antagonistas & inhibidores , Gefitinib , Masculino , Lesiones Precancerosas/prevención & control , Quinazolinas/uso terapéutico , Ratas , Ratas Endogámicas F344 , Factor de Transcripción STAT3/fisiología , Factor de Transcripción AP-1/fisiologíaRESUMEN
Colonic carcinogenesis involves the progressive dysregulation of homeostatic mechanisms that control growth. The epidermal growth factor (EGF) receptor (EGFR) regulates colonocyte growth and differentiation and is overexpressed in many human colon cancers. A requirement for EGFR in colonic premalignancy, however, has not been shown. In the current study, we used a specific EGFR antagonist, gefitinib, to investigate this role of the receptor in azoxymethane colonic premalignancy. The azoxymethane model shares many clinical, histologic, and molecular features of human colon cancer. Mice received azoxymethane i.p. (5 mg/kg/wk) or saline for 6 weeks. Animals were also gavaged with gefitinib (10 mg/kg body weight) or vehicle (DMSO) thrice weekly for 18 weeks, a dose schedule that inhibited normal receptor activation by exogenous EGF. Compared with control colonocytes [bromodeoxyuridine (BrdUrd), 2.2+/-1.2%], azoxymethane significantly increased proliferation (BrdUrd, 12.6+/-2.8%), whereas gefitinib inhibited this hyperproliferation (BrdUrd, 6.2+/-4.0%; <0.005). Azoxymethane significantly induced pro-transforming growth factor-alpha (6.4+/-1.3-fold) and increased phospho-(active) EGFR (5.9+/-1.1-fold), phospho-(active) ErbB2 (2.3+/-0.2-fold), and phospho-(active) extracellular signal-regulated kinase (3.3+/-0.4-fold) in premalignant colonocytes. Gefitinib inhibited activations of these kinases by >75% (P<0.05). Gefitinib also significantly reduced the number of large aberrant crypt foci and decreased the incidence of colonic microadenomas from 75% to 33% (P<0.05). Gefitinib concomitantly decreased cell cycle-regulating cyclin D1 and prostanoid biosynthetic enzyme cyclooxygenase-2 in microadenomas, suggesting that these regulators are key targets of EGFR in colonic carcinogenesis. These results show for the first time that EGFR signaling is required for early stages of colonic carcinogenesis. Our findings suggest, moreover, that inhibitors of EGFR might be useful in chemopreventive strategies in individuals at increased risk for colonic malignancies.
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Adenoma/enzimología , Transformación Celular Neoplásica/metabolismo , Neoplasias del Colon/enzimología , Receptores ErbB/metabolismo , Adenoma/inducido químicamente , Adenoma/genética , Adenoma/prevención & control , Animales , Azoximetano/antagonistas & inhibidores , Carcinógenos , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/genética , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/genética , Neoplasias del Colon/prevención & control , Ciclina D1/antagonistas & inhibidores , Ciclina D1/biosíntesis , Ciclooxigenasa 2/biosíntesis , Inhibidores de la Ciclooxigenasa 2/farmacología , Modelos Animales de Enfermedad , Receptores ErbB/antagonistas & inhibidores , Gefitinib , Genes ras/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos A , Mutación/efectos de los fármacos , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , beta Catenina/biosíntesis , beta Catenina/genéticaRESUMEN
Colorectal cancer is a leading cause of cancer deaths. The renin-angiotensin system (RAS) is upregulated in colorectal cancer, and epidemiologic studies suggest RAS inhibitors reduce cancer risk. Because vitamin D (VD) receptor negatively regulates renin, we examined anticancer efficacy of VD and losartan (L), an angiotensin receptor blocker. Control Apc+/LoxP mice and tumor-forming Apc+/LoxP Cdx2P-Cre mice were randomized to unsupplemented Western diet (UN), or diets supplemented with VD, L, or VD+L, the latter to assess additive or synergistic effects. At 6 months, mice were killed. Plasma Ca2+, 25(OH)D3, 1α, 25(OH)2D3, renin, and angiotensin II (Ang II) were quantified. Colonic transcripts were assessed by qPCR and proteins by immunostaining and blotting. Cancer incidence and tumor burden were significantly lower in Cre+ VD and Cre+ L, but not in the Cre+ VD+L group. In Apc+/LoxP mice, VD increased plasma 1,25(OH)2D3 and colonic VDR. In Apc+/LoxP-Cdx2P-Cre mice, plasma renin and Ang II, and colonic tumor AT1, AT2, and Cyp27B1 were increased and VDR downregulated. L increased, whereas VD decreased plasma renin and Ang II in Cre+ mice. VD or L inhibited tumor development, while exerting differential effects on plasma VD metabolites and RAS components. We speculate that AT1 is critical for tumor development, whereas RAS suppression plays a key role in VD chemoprevention. When combined with L, VD no longer increases active VD and colonic VDR in Cre- mice nor suppresses renin and Ang II in Cre+ mice, likely contributing to lack of chemopreventive efficacy of the combination.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/fisiología , Neoplasias del Colon/prevención & control , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Losartán/farmacología , Vitamina D/farmacología , Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Animales , Apoptosis , Proliferación Celular , Neoplasias del Colon/etiología , Neoplasias del Colon/patología , Quimioterapia Combinada , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Células Tumorales Cultivadas , Vitaminas/farmacologíaRESUMEN
BACKGROUND: With proven single-agent activity and favorable toxicity profile of MEK-1/2 inhibition in advanced leukemia, investigation into combination strategies to overcome proposed resistance pathways is warranted. Resistance to MEK inhibition is secondary to upstream hyperactivation of RAS/RAF or activation of the PI3K/PTEN/AKT/mTOR pathway. This phase II multi-institution Cancer Therapy Evaluation Program-sponsored study was conducted to determine efficacy and safety of the combination of the ATP-competitive pan-AKT inhibitor GSK2141795, targeting the PI3K/AKT pathway, and the MEK inhibitor trametinib in RAS-mutated relapsed/refractory acute myeloid leukemia (AML). PATIENTS AND METHODS: The primary objective was to determine the proportion of patients achieving a complete remission. Secondary objectives included assessment of toxicity profile and biologic effects of this combination. Twenty-three patients with RAS-mutated AML received the combination. Two dose levels were explored (dose level 1: 2 mg trametinib, 25 mg GSK2141795 and dose level 2: 1.5 mg trametinib, 50 mg GSK2141795). RESULTS: Dose level 1 was identified as the recommended phase II dose. No complete remissions were identified in either cohort. Minor responses were recognized in 5 (22%) patients. The most common drug-related toxicities included rash and diarrhea, with dose-limiting toxicities of mucositis and colitis. Longitudinal correlative assessment of the modulation of MEK and AKT pathways using reverse phase protein array and phospho-flow analysis revealed significant and near significant down-modulation of pERK and pS6, respectively. Combined MEK and AKT inhibition had no clinical activity in patients with RAS-mutated AML. CONCLUSION: Further investigation is required to explore the discrepancy between the activity of this combination on leukemia cells and the lack of clinical efficacy.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Genes ras , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Mutación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Diaminas/administración & dosificación , Femenino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Masculino , Persona de Mediana Edad , Pirazoles/administración & dosificación , Piridonas/administración & dosificación , Pirimidinonas/administración & dosificación , Resultado del Tratamiento , Adulto JovenRESUMEN
Aberrant crypt foci (ACF) are collections of abnormal colonic crypts with heterogeneous molecular and pathologic characteristics. Large and dysplastic ACF are putative precursors of colon cancer with neoplastic risk related to increased proliferation. In this study, we examined the role of epidermal growth factor receptor (EGFR) signaling in regulating ACF proliferation. Using magnification chromoendoscopy, we collected large ACF with endoscopic features of dysplasia and separately biopsied adjacent mucosa. Transcript levels were measured by real-time PCR, proteins were assessed by Western blotting, and levels were expressed as fold changes of adjacent mucosa. K-ras and B-Raf mutations were assessed by PCR and Ras activation by the ratio Ras-GTP / (Ras-GTP + Ras-GDP). At the RNA level, 38% of ACF were hyperproliferative, with proliferating cell nuclear antigen (PCNA) mRNA >/=2-fold of adjacent mucosa. Hyperproliferative ACF had significantly increased mRNA levels of EGFR (6.0 +/- 1.7-fold), transforming growth factor-alpha (14.4 +/- 5.0-fold), heparin-binding EGF-like growth factor (4.5 +/- 1.4-fold), cyclin D1 (4.6 +/- 0.7-fold), and cyclooxygenase-2 (COX-2; 9.3 +/- 4.2-fold; P < 0.05). At the protein level, 46% of ACF were hyperproliferative (PCNA, 3.2 +/- 1.2-fold). In hyperproliferative ACF, 44% possessed significant increases in four EGFR signaling components: EGFR (9.5 +/- 1.3-fold), phosphoactive ErbB2 (2.6 +/- 0.4-fold), phosphoactive extracellular signal-regulated kinase (3.7 +/- 1.1-fold), and cyclin D1 (3.4 +/- 0.8-fold; P < 0.05). Ras was activated in 46% of ACF (3.2 +/- 0.4-fold; P < 0.05), but K-ras mutations were present in only 7% of ACF. In contrast to COX-2 mRNA, the protein was not increased in hyperproliferative ACF. In summary, we have shown that ACF with up-regulated PCNA possess increased EGFR signaling components that likely contribute to the enhanced proliferative state of dysplastic-appearing ACF.
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Neoplasias del Colon/metabolismo , Receptores ErbB/metabolismo , Lesiones Precancerosas/metabolismo , Procesos de Crecimiento Celular/fisiología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Ciclina D1/biosíntesis , Ciclina D1/genética , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Femenino , Genes ras , Humanos , Masculino , Persona de Mediana Edad , Mutación , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Antígeno Nuclear de Célula en Proliferación/biosíntesis , Antígeno Nuclear de Célula en Proliferación/genética , Proteínas Proto-Oncogénicas B-raf/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
This commentary highlights the article by Hata et al that examines markers for assessing pancreatic neoplastic progression.
Asunto(s)
Quiste Pancreático/diagnóstico , Quiste Pancreático/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Patología Molecular/métodos , Telómero/genética , Humanos , Telomerasa/metabolismo , Homeostasis del Telómero/genéticaRESUMEN
Genomic medicine is transforming patient care. However, the speed of development has left a knowledge gap between discovery and effective implementation into clinical practice. Since 2010, the Training Residents in Genomics (TRIG) Working Group has found success in building a rigorous genomics curriculum with implementation tools aimed at pathology residents in postgraduate training years 1-4. Based on the TRIG model, the interprofessional Undergraduate Training in Genomics (UTRIG) Working Group was formed. Under the aegis of the Undergraduate Medical Educators Section of the Association of Pathology Chairs and representation from nine additional professional societies, UTRIG's collaborative goal is building medical student genomic literacy through development of a ready-to-use genomics curriculum. Key elements to the UTRIG curriculum are expert consensus-driven objectives, active learning methods, rigorous assessment and integration.