RESUMO
Colorectal cancer primarily metastasizes to the liver and globally kills over 600,000 people annually. By functionally screening 661 microRNAs (miRNAs) in parallel during liver colonization, we have identified miR-551a and miR-483 as robust endogenous suppressors of liver colonization and metastasis. These miRNAs convergently target creatine kinase, brain-type (CKB), which phosphorylates the metabolite creatine, to generate phosphocreatine. CKB is released into the extracellular space by metastatic cells encountering hepatic hypoxia and catalyzes production of phosphocreatine, which is imported through the SLC6A8 transporter and used to generate ATPfueling metastatic survival. Combinatorial therapeutic viral delivery of miR-551a and miR-483-5p through single-dose adeno-associated viral (AAV) delivery significantly suppressed colon cancer metastasis, as did CKB inhibition with a small-molecule inhibitor. Importantly, human liver metastases express higher CKB and SLC6A8 levels and reduced miR-551a/miR-483 levels relative to primary tumors. We identify the extracellular space as an important compartment for malignant energetic catalysis and therapeutic targeting.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Hepáticas/secundário , MicroRNAs/metabolismo , Metástase Neoplásica/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Creatina Quinase Forma BB/metabolismo , Metabolismo Energético , Matriz Extracelular , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica/patologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismoRESUMO
BACKGROUND: Clinical therapy for metastatic colorectal cancer (CRC) remains limited, especially when the tumor harbors a KRAS mutation. This study aimed to identify prognostic biomarkers in CRC that are accessible for therapeutic inhibition. METHODS: Conditioned media from human CRC epithelial cells and myofibroblasts were screened by cytokine arrays for tumorigenic factors. The protein and mRNA expressions of these factors were determined by immunohistochemistry and gene microarrays in human CRC tissues. Prognostic biomarkers were determined by correlation of mRNA expression to overall survival in stage IV CRC patients. Inhibition of CXCL1 was performed with specific neutralizing antibody and lentiviral shRNAs. Malignant growth was assessed by soft agar growth assays and xenograft tumor growth in immunocompromised mice. RESULTS: CXCL1 was highly secreted by KRAS mutant human CRC cells and myofibroblasts in a complementary adaptive response to serum deprivation. Elevated CXCL1 level promoted anchorage-independent growth of murine fibroblasts and human CRC cells. Inhibition of CXCL1 by neutralizing antibody and specific shRNAs decreased CRC tumor growth. Highly elevated CXCL1 expression significantly correlated with decreased overall survival in stage IV CRC patients (hazard ratio 0.28; 95% CI 0.11-0.72). CONCLUSIONS: High CXCL1 expression is a poor prognostic biomarker in metastatic CRC. CXCL1 inhibition suppressed tumorigenic growth of KRAS mutant CRC cells.
Assuntos
Quimiocina CXCL1/metabolismo , Neoplasias Colorretais/metabolismo , Epitélio/metabolismo , Miofibroblastos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL1/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio/efeitos dos fármacos , Epitélio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Células NIH 3T3 , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Adulto JovemRESUMO
There are well demonstrated differences in tumor cell metabolism between right sided (RCC) and left sided (LCC) colon cancer, which could underlie the robust differences observed in their clinical behavior, particularly in metastatic disease. As such, we utilized liquid chromatography-mass spectrometry to perform an untargeted metabolomics analysis comparing frozen liver metastasis (LM) biobank samples derived from patients with RCC (N = 32) and LCC (N = 58) to further elucidate the unique biology of each. We also performed an untargeted RNA-seq and subsequent network analysis on samples derived from an overlapping subset of patients (RCC: N = 10; LCC: N = 18). Our biobank redemonstrates the inferior survival of patients with RCC-derived LM (P = 0.04), a well-established finding. Our metabolomic results demonstrate increased reactive oxygen species associated metabolites and bile acids in RCC. Conversely, carnitines, indicators of fatty acid oxidation, are relatively increased in LCC. The transcriptomic analysis implicates increased MEK-ERK, PI3K-AKT and Transcription Growth Factor Beta signaling in RCC LM. Our multi-omic analysis reveals several key differences in cellular physiology which taken together may be relevant to clinical differences in tumor behavior between RCC and LCC liver metastasis.
Assuntos
Carcinoma de Células Renais , Neoplasias do Colo , Neoplasias Renais , Neoplasias Hepáticas , Humanos , Multiômica , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias Hepáticas/genética , Redes e Vias MetabólicasRESUMO
Loss of imprinting (LOI) of insulin-like growth factor 2 (IGF2) is a common event in many cancers and typically activates the maternally silenced allele. The resulting biallelic IGF2 expression correlates strongly with the hypomethylation of a differentially methylated region (DMR) near its promoter. It has also been shown that IGF2 undergoes overexpression in human malignancies; nevertheless, this phenomenon and its link to aberrant DMR methylation have not been reported in colorectal cancer (CRC). The aim of this study was to determine the relationship between IGF2 LOI, overexpression and DMR hypomethylation in CRC. By analyzing IGF2 and H19 methylation in 97 primary CRC and 64 matched normal colorectal tissues, we have shown a significant correlation between IGF2 LOI and DMR hypomethylation of IGF2 and H19. Additionally, when analyzing Affymetrix expression data of 167 primary CRC tumors and 32 normal tissues, 15% of tumors showed marked IGF2 elevation. We further investigated if substantially elevated IGF2 levels were linked to IGF2 or H19 hypomethylation, but found no significant correlation. However, we demonstrated that noticeable IGF2 overexpression, rather than LOI, negatively correlated with CRC microsatellite instability. These observations indicate that IGF2 expression, particularly when transcribed at significantly high levels, is a result of mechanisms unrelated to LOI. Our results suggest that IGF2 participates in CRC tumorigenesis through 2 different forms of aberrant gene expression.
Assuntos
Neoplasias Colorretais/genética , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , Metilação de DNA , Humanos , Polimorfismo Genético , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Observational studies of patients with primary colorectal cancer have identified KRAS mutation as a marker of poor prognosis. To examine more directly whether KRAS mutations are associated with accelerated metastatic progression, we evaluated KRAS mutation as well as Ki-67 expression in patients with colorectal liver metastases not treated with cetuximab. METHODS: KRAS mutation status was assessed in a series of resected or sampled colorectal liver metastases. In a subset of these tumors, Ki-67 antigen expression was assessed by immunohistochemical stains. Median follow-up after liver resection or biopsy was 2.3 years. RESULTS: KRAS mutation in the liver metastasis was detected in 27% of the 188 patients. High Ki-67 expression in the liver metastasis was identified in 62% of 124 patients analyzed. Both markers were associated with multiple liver metastases and shorter time interval to their detection. KRAS mutation and high Ki-67 expression were independent predictors of poor survival after colon resection (hazard ratio [HR] 1.9 [95% confidence interval (95% CI), 1.1-3.4], HR 2.6 [95% CI, 1.4-4.8], respectively). Tumors with high Ki-67 expression were less likely to be selected for liver resection, and KRAS mutation was independently associated with poor survival after liver resection (HR 2.4 [95% CI, 1.4-4.0]). CONCLUSIONS: KRAS mutation is associated with more rapid and aggressive metastatic behavior of colorectal liver metastases. These data suggest an important role for KRAS activation in colorectal cancer progression and support continued efforts to develop KRAS pathway inhibitors for this disease.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Hepáticas/genética , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Progressão da Doença , Feminino , Seguimentos , Humanos , Antígeno Ki-67/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteínas Proto-Oncogênicas p21(ras) , Taxa de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: In colorectal cancer (CRC), microsatellite instability (MSI) is a valuable marker of defective DNA mismatch repair that identifies cancers with distinct phenotypic properties, including favorable survival. However, the optimal assay for MSI status is unknown. We have evaluated a simplified 3-marker assay for MSI and compared it with the 5-marker (NCI) assay to see if technical variations in MSI testing are important. MATERIALS AND METHODS: DNA samples from 357 CRCs were evaluated for MSI using the 5 microsatellite markers recommended for the NCI assay (BAT 25, BAT26, D2S123, D5S346, and D17S250). Results were compared with a simplified 3-marker assay (BAT25, BAT26, and D2S123). CRCs identified as MSI were evaluated for their clinical, pathological, and genetic characteristics. RESULTS: The 5-marker assay identified 96 cancers as MSI. Only 56 of these were MSI by the 3-marker assay (3-marker+ group), leaving 40 cases identified as MSI only by NCI criteria (3-marker- group). The remaining 261 cancers were microsatellite stable (MSS). The 3-marker+ MSI tumors had features characteristic of MSI tumors: more proximal, poorly differentiated, associated with hereditary nonpolyposis colorectal cancer (HNPCC), more BRAF mutations, fewer KRAS mutations, better 5-year disease-specific survival, more frequent mismatch repair (MMR) protein loss, and less likely to be metastatic on presentation (P < .05). Chromosomal arm loss was observed only in 3-marker- MSI and MSS cancers (P < .05). CONCLUSION: The 3-marker MSI assay outperforms the traditional 5-marker assay for identifying patients with favorable prognosis and homogeneous clinical and genetic features. More accurate MSI testing should improve prognostic and predictive scoring systems for colorectal cancer.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Enzimas Reparadoras do DNA/genética , Mutação em Linhagem Germinativa/genética , Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Adenocarcinoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Bioensaio , Neoplasias Colorretais/patologia , Hibridização Genômica Comparativa , Reparo do DNA , Feminino , Seguimentos , Testes Genéticos , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Estudos Prospectivos , Taxa de Sobrevida , Adulto JovemRESUMO
INTRODUCTION: We examined two genetic markers established early in colorectal tumor development, microsatellite instability (MSI) and mutation of the KRAS proto-oncogene, to see if these genetic changes influence metastatic disease progression and survival. PATIENTS AND METHODS: MSI and KRAS mutation status were assessed in 532 primary adenocarcinomas (stage I-IV) from patients treated by colon resection. Median follow-up was 4.1 years (range 0-13.3 years) overall, 5.4 years for survivors. RESULTS: MSI and KRAS mutation were detected in 12 and 36% of cases, respectively. MSI was more common in early-stage disease (I, 15%; II, 21%; III, 10%; IV, 2%; P = 0.0001). Prevalence of KRAS mutation did not vary with stage (I, 36%; II, 34%; III, 35%; IV, 40%; P = ns). Disease-specific survival was far superior for MSI tumors than for microsatellite stability (MSS) tumors (5-year survival 92 vs. 59%, P < 0.0001). KRAS mutation was a marker of poor survival (5-year survival 55 vs. 68%, P = 0.0002). Using Cox regression analysis MSI, KRAS mutation, and stage were strong independent predictors of survival in the entire patient population. A high-mortality group with MSS/KRAS-mutant tumors was identified within the stage I and II cohort. CONCLUSIONS: MSI and KRAS mutation provide fundamental genetic signatures influencing tumor behavior across patient subsets and stages of tumor development.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Mutação/genética , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Progressão da Doença , Feminino , Seguimentos , Marcadores Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas p21(ras) , Reto/metabolismo , Reto/patologia , Taxa de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
Mdm2 is the main regulator of p53 and is amplified in approximately 7% of all human cancers. MDM2 gene amplification as well as expression has been correlated to an increased tumorigenic potential. We have analyzed the prevalence of MDM2 gene amplifications and SNP309 in 284 colorectal tumors using a relatively new highly sensitive PCR/ligase detection reaction method in relation to TP53 mutational status and genomic instability. We found MDM2 to be amplified in 9% of the 284 colorectal cancers analyzed and a significantly higher proportion of tumors with high MDM2 gene amplification retained a wild-type p53 gene (P = 0.058). MDM2 gene amplification was significantly correlated to advanced tumor stage. Several small-molecule MDM2 antagonists have already been identified that either physically inhibit the p53-MDM2 binding or the E3 ligase function of MDM2. Our results suggest that MDM2 is a promising target for this type of cancer therapy in a substantial subgroup of colorectal cancers.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Amplificação de Genes , Mutação/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Padrões de ReferênciaRESUMO
PURPOSE: Aberrant promoter methylation and genomic instability occur frequently during colorectal cancer development. CpG island methylator phenotype (CIMP) has been shown to associate with microsatellite instability, and BRAF mutation and is often found in the right-side colon. Nevertheless, the relative importance of CIMP and chromosomal instability (CIN) for tumorigenesis has yet to be thoroughly investigated in sporadic colorectal cancers. EXPERIMENTAL DESIGN: We determined CIMP in 161 primary colorectal cancers and 66 matched normal mucosae using a quantitative bisulfite/PCR/ligase detection reaction (LDR)/Universal Array assay. The validity of CIMP was confirmed in a subset of 60 primary tumors using MethyLight assay and five independent markers. In parallel, CIN was analyzed in the same study cohort using Affymetrix 50K Human Mapping arrays. RESULTS: The identified CIMP-positive cancers correlate with microsatellite instability (P = 0.075) and the BRAF mutation V600E (P = 0.00005). The array-based high-resolution analysis of chromosomal aberrations indicated that the degree of aneuploidy is spread over a wide spectrum among analyzed colorectal cancers. Whether CIN was defined by copy number variations in selected microsatellite loci (criterion 1) or considered as a continuous variable (criterion 2), CIMP-positive samples showed a strong correlation with low-degree chromosomal aberrations (P = 0.075 and P = 0.012, respectively). Similar correlations were observed when CIMP was determined by MethyLight assay (P = 0.001 and P = 0.013, respectively). CONCLUSION: CIMP-positive tumors generally possess lower chromosomal aberrations, which may only be revealed using a genome-wide approach. The significant difference in the degree of chromosomal aberrations between CIMP-positive and the remainder of samples suggests that epigenetic (CIMP) and genetic (CIN) abnormalities may arise from independent molecular mechanisms of tumor progression.
Assuntos
Aberrações Cromossômicas , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Ilhas de CpG , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Mutação , Instabilidade Cromossômica , Epigênese Genética , Feminino , Genoma Humano , Humanos , Masculino , Repetições de Microssatélites , Fenótipo , Proteínas Proto-Oncogênicas B-raf/genéticaRESUMO
Tumor cells with genomic amplification of MET display constitutive activation of the MET tyrosine kinase, which renders them highly sensitive to MET inhibition. Several MET inhibitors have recently entered clinical trials; however, as with other molecularly targeted agents, resistance is likely to develop. Therefore, elucidating possible mechanisms of resistance is of clinical interest. We hypothesized that collateral growth factor receptor pathway activation can overcome the effects of MET inhibition in MET-amplified cancer cells by reactivating key survival pathways. Treatment of MET-amplified GTL-16 and MKN-45 gastric cancer cells with the highly selective MET inhibitor PHA-665752 abrogated MEK/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT signaling, resulting in cyclin D1 loss and G(1) arrest. PHA-665752 also inhibited baseline phosphorylation of epidermal growth factor receptor (EGFR) and HER-3, which are transactivated via MET-driven receptor cross-talk in these cells. However, MET-independent HER kinase activation using EGF (which binds to and activates EGFR) or heregulin-beta1 (which binds to and activates HER-3) was able to overcome the growth-inhibitory effects of MET inhibition by restimulating MEK/MAPK and/or PI3K/AKT signaling, suggesting a possible escape mechanism. Importantly, dual inhibition of MET and HER kinase signaling using PHA-665752 in combination with the EGFR inhibitor gefitinib or in combination with inhibitors of MEK and AKT prevented the above rescue effects. Our results illustrate that highly targeted MET tyrosine kinase inhibition leaves MET oncogene-"addicted" cancer cells vulnerable to HER kinase-mediated reactivation of the MEK/MAPK and PI3K/AKT pathways, providing a rationale for combined inhibition of MET and HER kinase signaling in MET-amplified tumors that coexpress EGFR and/or HER-3.
Assuntos
Antineoplásicos/farmacologia , Receptores ErbB/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Receptor ErbB-3/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/enzimologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais , Sulfonas/farmacologia , Sulfonas/uso terapêutico , TransfecçãoRESUMO
The c-Met proto-oncogene encodes a receptor tyrosine kinase (TK) that promotes invasive tumor growth and metastasis. Recent studies show that the presence of c-Met gene amplification is predictive for selective c-Met TK inhibitors in gastric cancer and lung cancer. In this study, we utilized a highly quantitative PCR/ligase detection reaction technique to quantify c-Met gene copy number in primary colorectal cancer (CRC) (N=247), liver metastases (N=147), and paired normal tissues. We identified no differences in c-Met gene copy number between normal colonic mucosa and liver tissue. However, mean c-Met gene copy number was significantly elevated in CRC compared with normal mucosa (P<0.001), and in liver metastases compared with normal liver (P<0.001). Furthermore, a significant increase in c-Met was seen in liver metastases compared with primary CRC (P<0.0001). c-Met gene amplification was observed in 2% (3/177) of localized cancers, 9% (6/70) of cancers with distant metastases (P<0.02), and 18% (25/147) of liver metastases (P<0.01). Among patients treated by liver resection, there was a trend toward poorer 3-year survival in association with c-Met gene amplification (P=0.07). Slight increases in c-Met copy number can be detected in localized CRCs, but gene amplification is largely restricted to Stage IV primary cancers and liver metastases. c-Met gene amplification is linked to metastatic progression, and is a viable target for a significant subset of advanced CRC.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Amplificação de Genes , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Proteínas Proto-Oncogênicas c-met/genética , Linhagem Celular Tumoral , Feminino , Dosagem de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Proto-Oncogene MasRESUMO
Several studies have verified the existence of multiple chromosomal abnormalities in colon cancer. However, the relationships between DNA copy number and gene expression have not been adequately explored nor globally monitored during the progression of the disease. In this work, three types of array-generated data (expression, single nucleotide polymorphism, and comparative genomic hybridization) were collected from a large set of colon cancer patients at various stages of the disease. Probes were annotated to specific chromosomal locations and coordinated alterations in DNA copy number and transcription levels were revealed at specific positions. We show that across many large regions of the genome, changes in expression level are correlated with alterations in DNA content. Often, large chromosomal segments, containing multiple genes, are transcriptionally affected in a coordinated way, and we show that the underlying mechanism is a corresponding change in DNA content. This implies that whereas specific chromosomal abnormalities may arise stochastically, the associated changes in expression of some or all of the affected genes are responsible for selecting cells bearing these abnormalities for clonal expansion. Indeed, particular chromosomal regions are frequently gained and overexpressed (e.g., 7p, 8q, 13q, and 20q) or lost and underexpressed (e.g., 1p, 4, 5q, 8p, 14q, 15q, and 18) in primary colon tumors, making it likely that these changes favor tumorigenicity. Furthermore, we show that these aberrations are absent in normal colon mucosa, appear in benign adenomas (albeit only in a small fraction of the samples), become more frequent as disease advances, and are found in the majority of metastatic samples.
Assuntos
Aberrações Cromossômicas , Neoplasias Colorretais/genética , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Cromossomos Humanos Par 20/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , DNA de Neoplasias/genética , Dosagem de Genes , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Estadiamento de NeoplasiasRESUMO
c-Met, a receptor tyrosine kinase responsible for cellular migration, invasion, and proliferation, is overexpressed in human cancers. Although ligand-independent c-Met activation has been described, the majority of tumors are ligand dependent and rely on binding of hepatocyte growth factor (HGF) for receptor activation. Both receptor and ligand are attractive therapeutic targets; however, preclinical models are limited because murine HGF does not activate human c-Met. The goal of this study was to develop a xenograft model in which human HGF (hHGF) is produced in a controllable fashion in the mouse. Severe combined immunodeficient mice were treated with adenovirus encoding the hHGF transgene (Ad-hHGF) via tail vein injection, and transgene expression was determined by the presence of hHGF mRNA in mouse tissue and hHGF in serum. Ad-hHGF administration to severe combined immunodeficient mice resulted in hHGF production that was (a) dependent on quantity of virus delivered; (b) biologically active, resulting in liver hypertrophy; and (c) sustainable over 40 days. In this model, the ligand-dependent human tumor cell line SW1417 showed enhanced tumor growth, whereas the ligand-independent cell lines SW480 and GTL-16 showed no augmented tumor growth. This novel xenograft model is ideal for investigating c-Met/HGF-dependent human tumor progression and for evaluating c-Met targeted therapy.
Assuntos
Fator de Crescimento de Hepatócito/genética , Neoplasias/patologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Transplante Heterólogo , Adenoviridae , Animais , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Fator de Crescimento de Hepatócito/sangue , Humanos , Ligantes , Camundongos , Camundongos SCID , Neoplasias/genética , Fosforilação , Transgenes , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stem cells of the small and large intestine are marked by expression of the Wnt target gene LGR5, a leucine-rich-repeat-containing G protein-coupled receptor. Previous studies reported increased expression of LGR5 in human colorectal cancer (CRC) compared to normal tissue either by immunohistochemistry or in situ hybridization (ISH). However, as these studies were semi-quantitative they did not provide a numerical estimate of the magnitude of this effect. While we confirm that LGR5+ cells are exclusively located at the base of normal human small and large intestinal crypts, representing approximately 6% of total crypt cells, we show this cell population is 10-fold expanded in all grades of CRC, representing as much as 70% of the cells of tumor crypt-like structures. This expansion of the LGR5 compartment coincides with maintenance of crypt-like glandular structure (adenomas, and well and moderately differentiated adenocarcinomas), and is reduced in poorly differentiated CRC, where crypt-like glandular architecture is lost, accompanied by reduced epithelial terminal differentiation. Altogether these results indicate that LGR5+ cell expansion is a hallmark of CRC tumorigenesis occurring during progression to adenoma, supporting CRC as a stem cell disease with implications for CRC therapy.
Assuntos
Adenocarcinoma/genética , Adenoma/genética , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/genética , Receptores Acoplados a Proteínas G/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Adenoma/diagnóstico , Adenoma/metabolismo , Adenoma/patologia , Contagem de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Intestino Grosso/metabolismo , Intestino Grosso/patologia , Intestino Grosso/ultraestrutura , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Intestino Delgado/ultraestrutura , Análise em Microsséries , Gradação de Tumores , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/metabolismoRESUMO
INTRODUCTION/HYPOTHESIS: Over-expression of the c-Met receptor tyrosine kinase has been described in a variety of cancers and implicated in tumor progression. Unlike some solid tumors, current evidence indicates that c-Met activation in colon cancer is unrelated to gene mutation, is ligand dependent, and occurs via a paracrine fashion. We hypothesize that over-expression of the c-Met receptor and its ligand, hepatocyte growth factor (HGF) in the tumor microenvironment is associated with tumor progression and metastases. METHODS: Primary tumor c-Met and HGF mRNA expression was analyzed in 60 colon adenocarcinomas. Receptor and ligand expression was analyzed for correlation and association with clinicopathologic features and outcome. RESULTS: Compared to adjacent normal mucosa, 69% and 48% of tumors showed a greater than 2- and greater than 10-fold elevation in c-Met mRNA, respectively. Elevated HGF mRNA was noted in 47% of tumors with 19% having a greater than 10-fold increase. Tumor c-Met expression was correlated with HGF expression, and a cohort of 33 patients could be defined with both low c-Met and HGF expression. Compared with the 27 tumors with either high c-Met or HGF, the cohort with low c-Met and HGF expression had fewer nodal and distant metastases as well as improved overall survival (HR=2.3, p<0.05). CONCLUSION: Evaluation of the c-Met receptor in context of ligand, HGF, allows identification of a metastatic phenotype that correlates with advanced stage and poor survival. c-Met and HGF co-expression in the tumor microenvironment could be useful in the molecular staging of colon cancer and viable therapeutic targets.
Assuntos
Adenocarcinoma/patologia , Biomarcadores Tumorais/análise , Neoplasias do Colo/patologia , Fator de Crescimento de Hepatócito/biossíntese , Proteínas Proto-Oncogênicas c-met/biossíntese , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Idoso , Neoplasias do Colo/metabolismo , Neoplasias do Colo/mortalidade , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de SobrevidaRESUMO
Genetic variability in KRAS and EGFR predicts response to cetuximab in irinotecan refractory colorectal cancer. Whether these markers or others remain predictive in combination biologic therapies including bevacizumab is unknown. We identified predictive biomarkers from patients with irinotecan refractory metastatic colorectal cancer treated with cetuximab plus bevacizumab. Patients who received cetuximab plus bevacizumab for irinotecan refractory colorectal cancer in either of two Phase II trials conducted were identified. Tumor tissue was available for 33 patients. Genomic DNA was extracted and used for mutational analysis of KRAS, BRAF, and p53 genes. Fluorescence in situ hybridization was performed to assess EGFR copy number. The status of single genes and various combinations were tested for association with response. Seven of 33 patients responded to treatment. KRAS mutations were found in 14/33 cases, and 0 responded to treatment (p = 0.01). EGFR gene amplification was seen in 3/33 of tumors and in every case was associated with response to treatment (p < 0.001). TP53 and BRAF mutations were found in 18/33 and 0/33 tumors, respectively, and there were no associations with response to either gene. EGFR gene amplification and KRAS mutations are predictive markers for patients receiving combination biologic therapy of cetuximab plus bevacizumab for metastatic colorectal cancer. One marker or the other is present in the tumor of half of all patients allowing treatment response to be predicted with a high degree of certainty. The role for molecular markers in combination biologic therapy seems promising.
Assuntos
Neoplasias Colorretais/genética , Receptores ErbB/deficiência , Amplificação de Genes/genética , Mutação/genética , Proteínas ras/genética , Adulto , Idoso , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bevacizumab/administração & dosagem , Biomarcadores Tumorais/genética , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Cetuximab/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Análise Mutacional de DNA/métodos , Feminino , Genes p53/genética , Humanos , Irinotecano , Masculino , Pessoa de Meia-IdadeRESUMO
Although small and large intestines possess seemingly similar Wnt-driven leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)+ adult epithelial stem cells, we report here that the two organs exhibit distinct mechanisms of tissue response to ionizing radiation. Employing Lgr5-lacZ transgenic mice and Lgr5 in situ hybridization, we found colonic epithelial stem cells (CESC) markedly more radioresistant in vivo than small intestinal crypt base columnar stem cells (CBC; D0 = 6.0 ± 0.3 Gy vs. 1.3 ± 0.1, respectively; P < 0.01). Accordingly, CESCs survived 30 Gy exposure, while CBCs were completely depleted after 15 Gy. EdU incorporation studies indicated that after 19 Gy, CBCs exited growth arrest at 12 hours, resuming normal mitotic activity despite 60% of this population displaying residual γH2AX foci, indicative of persistent unrepaired DNA damage. Checkpoint recovery before complete double-strand break (DSB) repair represents the sine qua non of a newly defined potentially lethal pathophysiology termed checkpoint adaptation. In the small intestinal mucosa, checkpoint adaptation resulted in CBCs succumbing to an 8-fold increase in the incidence of highly lethal chromosomal aberrations and mitotic catastrophe by 48 hours postradiation. In contrast, Lgr5+ CESCs displayed delayed checkpoint recovery at 48 hours post-19 Gy, coordinated with complete DSB repair and regeneration of colonic mucosa originating, at least in part, from surviving CESCs. The discovery that small intestinal CBCs succumb to checkpoint adaptation is the first demonstration that this aberrant cell-cycle response may drive mammalian tissue radiosensitivity. Cancer Res; 77(8); 2124-33. ©2017 AACR.
Assuntos
Colo/efeitos da radiação , Mucosa Intestinal/efeitos da radiação , Intestino Delgado/efeitos da radiação , Receptores Acoplados a Proteínas G/biossíntese , Células-Tronco/efeitos da radiação , Animais , Colo/citologia , Colo/metabolismo , Hibridização In Situ , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Tolerância a Radiação/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Irradiação Corporal TotalRESUMO
Colorectal cancer is the third most frequently diagnosed cancer worldwide. Prevention of colorectal cancer initiation represents the most effective overall strategy to reduce its associated morbidity and mortality. Activating KRAS mutation (KRASmut) is the most prevalent oncogenic driver in colorectal cancer development, and KRASmut inhibition represents an unmet clinical need. We apply a systems-level approach to study the impact of KRASmut on stem cell signaling during human colon cancer initiation by performing gene set enrichment analysis on gene expression from human colon tissues. We find that KRASmut imposes the embryonic stem cell-like program during human colon cancer initiation from colon adenoma to stage I carcinoma. Expression of miR145, an embryonic SC program inhibitor, promotes cell lineage differentiation marker expression in KRASmut colon cancer cells and significantly suppresses their tumorigenicity. Our data support an in vivo plasticity model of human colon cancer initiation that merges the intrinsic stem cell properties of aberrant colon stem cells with the embryonic stem cell-like program induced by KRASmut to optimize malignant transformation. Inhibition of the embryonic SC-like program in KRASmut colon cancer cells reveals a novel therapeutic strategy to programmatically inhibit KRASmut tumors and prevent colon cancer.
Assuntos
Adenoma/patologia , Carcinogênese/genética , Neoplasias Colorretais/patologia , Células-Tronco Embrionárias/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células 3T3 , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Colo/patologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genéticaRESUMO
INTRODUCTION: Colorectal cancer (CRC) diagnosed before age 30 years is a fatal disease whose biology remains poorly understood. To understand its pathogenesis, we compared molecular and clinical data in surgically treated early-age onset and adult onset patients. MATERIALS AND METHODS: Clinical data and tumor tissue were collected retrospectively for 94 patients with early-age onset CRC (age ≤30 years) and compared to 275 adult CRC patients (age ≥50 years). Tumor morphology, microsatellite instability (MSI) and stability (MSS), KRAS and BRAF mutations, and mismatch repair (MMR) expression (MSH2, MLH1, MSH6, PMS2) were assessed. RESULTS: Early-age CRC was distinguished from adult CRC by advanced stage presentation (P<0.001), frequent high grade cancers (P<0.001), and poor prognosis (P<0.001). MSI was associated with favorable survival and MMR loss in both groups. Compared to adults, MSI in early-onset CRC was more prevalent (P<0.01), not tightly linked to MLH1/PMS2 loss, and never associated with BRAFV600E mutations (P<0.01). MSS/BRAFV600E genotype had poor prognosis and was more prevalent in early-age CRC (9% vs. 3%). DISCUSSION: Specific genetic subtypes are found at different frequencies in early-age onset and adult onset CRC. Complete absence of the indolent MSI/BRAFV600E genotype and enrichment in the unfavorable MSS/BRAFV600E genotype help explain the poor prognosis of early onset CRC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/diagnóstico , DNA de Neoplasias/genética , Mutação , Estadiamento de Neoplasias , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Criança , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Reparo de Erro de Pareamento de DNA , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Estudos Retrospectivos , Taxa de Sobrevida/tendências , Estados Unidos/epidemiologia , Adulto JovemRESUMO
Colorectal cancer metastasis to the liver is a major cause of cancer-related death; however, the genes and pathways that govern this metastatic colonization event remain poorly characterized. Here, using a large-scale in vivo RNAi screen, we identified liver and red blood cell pyruvate kinase (PKLR) as a driver of metastatic liver colonization. PKLR expression was increased in liver metastases as well as in primary colorectal tumors of patients with metastatic disease. Evaluation of a murine liver colonization model revealed that PKLR promotes cell survival in the tumor core during conditions of high cell density and oxygen deprivation by increasing glutathione, the primary endogenous antioxidant. PKLR negatively regulated the glycolytic activity of PKM2, the major pyruvate kinase isoenzyme known to regulate cellular glutathione levels. Glutathione is critical for metastasis, and we determined that the rate-limiting enzyme of glutathione synthesis, GCLC, becomes overexpressed in patient liver metastases, promotes cell survival under hypoxic and cell-dense conditions, and mediates metastatic liver colonization. RNAi-mediated inhibition of glutathione synthesis impaired survival of multiple colon cancer cell lines, and pharmacological targeting of this metabolic pathway reduced colonization in a primary patient-derived xenograft model. Our findings highlight the impact of metabolic reprogramming within the niche as metastases progress and suggest clinical potential for targeting this pathway in colorectal cancer.