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BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is aggressive and has high rates of relapse, conferring poor long-term survival after curative resection. Little is known about the genomic evolution that occurs during ICC relapse. APPROACH AND RESULTS: We conducted whole-exome sequencing of 30 paired primary and relapsed tumors from 10 patients with ICC who received curative resection. We sought to identify frequently altered genes, infer tumor subclonal architectures, and track genomic evolution from primary to relapsed tumors. We examined functional effects and the mechanism of action of SLIT2, a gene specifically mutated in relapsed tumors, on tumor growth and metastasis and the tumor microenvironment. Our results indicated that relapsed ICCs were genetically derived from intrahepatic dissemination of primary tumors. However, they acquired additional mutations while maintaining most drivers, such as TP53 and IDH1. Multiregion sequencing suggested polyclonal seeding of ICC dissemination. Four of 10 relapsed ICCs acquired SLIT2 mutations that were not present in the corresponding primary tumors. Validation in an expanded sample revealed SLIT2 mutations in 2.3% (1/44) of primary ICCs and 29.5% (13/44) of relapsed ICCs. Biofunctional investigations revealed that inactivating mutation of SLIT2 resulted in activation of PI3K-Akt signaling in ICC cells, directly enhanced neutrophil chemotaxis, mediated tumor-associated neutrophil infiltration, and contributed to ICC growth and metastasis. CONCLUSIONS: We characterized genomic evolution during ICC relapse and identified SLIT2 as a driver of tumor dissemination and tumor-associated neutrophil infiltration.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Péptidos y Proteínas de Señalización Intercelular , Proteínas del Tejido Nervioso , Neoplasias de los Conductos Biliares/genética , Neoplasias de los Conductos Biliares/patología , Colangiocarcinoma/genética , Colangiocarcinoma/patología , Evolución Molecular , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mutación , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/patología , Proteínas del Tejido Nervioso/genética , Fosfatidilinositol 3-Quinasas , Pronóstico , Microambiente Tumoral/genéticaRESUMEN
Infiltrating immune cells in the tumor microenvironment (TME) influence tumor progression and patient prognosis, making them attractive therapeutic targets for immunotherapy research. A deeper understanding of immune cell distributions in the TME in hepatocellular carcinoma (HCC) is needed to identify interactions among different immune cell types that might impact the effectiveness of potential immunotherapies. We performed multiplex immunohistochemistry using a tissue microarray of samples from 302 patients with HCC to elucidate the spatial distributions of immune cell subpopulations (CD3+ , CD4+ , CD8+ , CD66b+ , and CD68+ ) in HCC and normal liver tissues. We analyzed the associations between different immune subpopulations using Pearson's correlation. G(r) functions, K(r) functions and Euclidean distance were applied to characterize the bivariate distribution patterns among the immune cell types. Cox regression and Kaplan-Meier analysis were used to evaluate the associations between tumor infiltration by different immune cells and patient outcomes after curative surgery. We also analyzed the relationship between the spatial distribution of different immune cell subpopulations with HCC patient prognosis. We found that the immune cell spatial distribution in the HCC TME is heterogeneous. Our study provides a theoretical basis for HCC immunotherapy.
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Carcinoma Hepatocelular/inmunología , Neoplasias Hepáticas/inmunología , Antígenos CD/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Femenino , Humanos , Inmunohistoquímica , Inmunoterapia , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Linfocitos Infiltrantes de Tumor/metabolismo , Masculino , Persona de Mediana Edad , Infiltración Neutrófila , Pronóstico , Microambiente Tumoral/inmunologíaRESUMEN
Recent studies suggested that the immune microenvironment and mutational landscape are associated with the response to immune-based therapy in several types of cancer. The roles of those factors in Chinese HCC remain largely unknown. In this study, we obtained 182 FFPE samples of HCC cohort that were previously subjected to NGS (49 WGS, 18 WES, and 115 targeted sequencing). We performed immunohistochemistry to detect CD3, CD4, CD8, CD57, Foxp3, CD68, CD66b, and PD-L1 expression in the samples. We identified diverse associations between the mutational landscape and the immune microenvironment in the HCC samples. High mutational burden and an aristolochic acid-dominated mutational signature were both correlated with elevated tumoral PD-L1 expression and CD3+ T-cell infiltration and high numbers of CD68+ TAMs and CD66b+ TANs. CD4+ and CD8+ T cells exhibited lower infiltration levels in tumors with mutations in AXIN1/CTNNB1 and in tumors with aflatoxin-dominant mutational signatures. Moreover, tumors with TP53 mutations had less CD8+ T-cell infiltration and more Foxp3+ Treg-cell infiltration than those without TP53 mutations. Kaplan-Meier survival analysis revealed that the presence of CD8+, Foxp3+, CD66b+, or CD68+ immune cells; tumoral PD-L1 expression alone; or the presence of CD8+ or Foxp3+ cells combined with TP53 mutation were predictive of recurrence and poor overall survival after curative resection. In conclusion, the association between the mutational landscape and the immune microenvironment warrants further analysis to determine its impact on patient outcomes to guide personalized immune-based therapy for Chinese patients with HCC.
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Carcinoma Hepatocelular/epidemiología , Neoplasias Hepáticas/epidemiología , Microambiente Tumoral/inmunología , Secuenciación Completa del Genoma/métodos , Pueblo Asiatico , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/mortalidad , Femenino , Humanos , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/mortalidad , Masculino , Análisis de SupervivenciaRESUMEN
BACKGROUND AND AIMS: There is growing evidence that single-stranded, circular RNA (circRNA) plays a key role in the development of certain cancers, including hepatocellular carcinoma (HCC). It is less clear, however, what role circRNA plays in HCC metastasis. APPROACH AND RESULTS: In this study, through circRNA sequencing, we identified a circRNA: circASAP1 (a circRNA derived from exons 2 and 3 of the ASAP1 gene, hsa_circ_0085616), which is associated with pulmonary metastasis after curative resection in patients with HCC. CircASAP1 was overexpressed in HCC cell lines with high metastatic potential and in metastatic HCCs. In vitro, circASAP1 promoted cell proliferation, colony formation, migration, and invasion, and in vivo, it enhanced tumor growth and pulmonary metastasis. Mechanism studies showed that circASAP1 acts as a competing endogenous RNA for microRNA 326 (miR-326) and microRNA 532-5p (miR-532-5p), both of which are tumor suppressors in HCC. We found that mitogen-activated protein kinase (MAPK) 1 and colony stimulating factor (CSF)-1 were direct common targets for microRNA 326 (miR-326) and microRNA 532-5p (miR-532-5p), which were regulated by circASAP1. CircASAP1 promotes HCC cell proliferation and invasion by regulating miR-326/miR-532-5p-MAPK1 signaling and, furthermore, mediates tumor-associated macrophage infiltration by regulating the miR-326/miR-532-5p-CSF-1 pathway. Clinical HCC samples exhibited a positive correlation between circASAP1 expression and levels of CSF-1, MAPK1, and CD68+ tumor-associated macrophages, all of which were predictive of patient outcomes. CONCLUSION: We identified circASAP1 as a key regulator of HCC metastasis that acts on miR-326/miR-532-5p-MAPK1/CSF-1 signaling and serves as a prognostic predictor in patients with HCC.
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Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma Hepatocelular , Neoplasias Hepáticas , Neoplasias Pulmonares , Factor Estimulante de Colonias de Macrófagos/metabolismo , MicroARNs/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Metástasis de la Neoplasia/genética , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/cirugía , Femenino , Hepatectomía/efectos adversos , Hepatectomía/métodos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/cirugía , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Masculino , Ratones , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , ARN Circular/metabolismoRESUMEN
Tumor-associated neutrophils (TANs) play a crucial role in tumor development and progression in the cancer microenvironment. Despite increased understanding of TAN contributions to hepatocellular carcinoma (HCC) progression and prognosis, the direct interaction between TANs and HCC cells is not fully understood. In this study, we tested the effect of TANs on HCC cells in vitro and in vivo and investigated the mechanism of interaction between them. Our results showed that TANs secreted bone morphogenetic protein 2 and transforming growth factor beta 2 and triggered microRNA 301b-3p (miR-301-3p) expression in HCC cells, subsequently suppressed gene expression of limbic system-associated membrane protein (LSAMP) and CYLD lysine 63 deubiquitinase (CYLD), and increased stem cell characteristics in HCC cells. These TAN-induced HCC stem-like cells were hyperactive in nuclear factor kappa B signaling, secreted higher levels of chemokine (C-X-C motif) ligand 5 (CXCL5), and recruited more TAN infiltration, suggesting a positive feedback loop. In clinical HCC samples, increased TANs correlated with elevated miR-301b-3p, decreased LSAMP and CYLD expression, and increased nuclear p65 accumulation and CXCL5 expression, all of which predicted patient outcome. Conclusion: Our work identified a positive feedback loop governing cancer stem-like cells and TANs in HCC that controls tumor progression and patient outcome.
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Carcinoma Hepatocelular/genética , Quimiocina CXCL5/genética , Progresión de la Enfermedad , Neoplasias Hepáticas/genética , Neutrófilos/metabolismo , Animales , Biopsia con Aguja , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Retroalimentación , Humanos , Inmunohistoquímica , Técnicas In Vitro , Neoplasias Hepáticas/patología , Ratones , Ratones SCID , MicroARNs/genética , Células Madre Neoplásicas/metabolismo , Pronóstico , Transducción de Señal/genética , Microambiente TumoralRESUMEN
BACKGROUND: Plasmacytoid dendritic cells (pDCs) are present in various primary and metastatic human neoplasms; however, their clinical significance in intrahepatic cholangiocarcinoma is not clear. METHODS: To evaluate pDCs' distributions in and around tumors as well as their potential function and predictive value for prognosis in patients undergoing curative resection, we performed immunohistochemistry to examine the expression of pDC marker BDCA2, and CD3, CD4, CD8 and Foxp3 in intratumoral and peritumoral tissues from 359 patients with intrahepatic cholangiocarcinoma and compared with prognostic and clinicopathologic factors. RESULTS: Results showed that patients with high numbers of BDCA2+ pDCs in peritumoral tissues were more likely to have elevated levels of carbohydrate antigen 19-9 and gamma-glutamyl transferase, larger and more tumors, advanced tumor-node-metastasis staging, more vascular/bile duct invasion, and lymphatic metastasis in association with greater chance of recurrence and shorter overall survival. Peritumoral tissues with larger numbers of pDCs also showed increased Foxp3+ regulatory T cell infiltration, both of which were found to be independent factors for predicting time to recurrence and overall survival. By contrast, patient outcomes were not associated with the presence of intratumoral pDCs. CONCLUSIONS: Peritumoral pDC infiltration may indicate an immune tolerogenic peritumor microenvironment and can be used to predict a poor prognosis for patients undergoing curative resection for intrahepatic cholangiocarcinoma.
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BACKGROUND & AIMS: Early recurrence of hepatocellular carcinoma (HCC) after curative resection is common. However, the association between genetic mechanisms and early HCC recurrence, especially in Chinese patients, remains largely unknown. METHODS: We performed whole-genome sequencing (49 cases), whole-exome sequencing (18 cases), and deep targeted sequencing (115 cases) on 182 primary HCC samples. Focusing on WNK2, we used Sanger sequencing and qPCR to evaluate all the coding exons and copy numbers of that gene in an additional 554 HCC samples. We also explored the functional effect and mechanism of WNK2 on tumor growth and metastasis. RESULTS: We identified 5 genes (WNK2, RUNX1T1, CTNNB1, TSC1, and TP53) harboring somatic mutations that correlated with early tumor recurrence after curative resection in 182 primary HCC samples. Focusing on WNK2, the overall somatic mutation and copy number loss occurred in 5.3% (39/736) and 27.2% (200/736), respectively, of the total 736 HCC samples. Both types of variation were associated with lower WNK2 protein levels, higher rates of early tumor recurrence, and shorter overall survival. Biofunctional investigations revealed a tumor-suppressor role of WNK2: its inactivation led to ERK1/2 signaling activation in HCC cells, tumor-associated macrophage infiltration, and tumor growth and metastasis. CONCLUSIONS: Our results delineate genomic events that characterize Chinese HCCs and identify WNK2 as a driver of early HCC recurrence after curative resection. LAY SUMMARY: We applied next-generation sequencing and conducted an in-depth genomic analysis of hepatocellular carcinomas from a Chinese patient cohort. The results delineate the genomic events that characterize hepatocellular carcinomas in Chinese patients and identify WNK2 as a driver associated with early tumor recurrence after curative resection.
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Carcinoma Hepatocelular , Hepatectomía , Neoplasias Hepáticas , Recurrencia Local de Neoplasia/genética , Proteínas Serina-Treonina Quinasas/genética , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/cirugía , China , Femenino , Genes Supresores de Tumor , Predisposición Genética a la Enfermedad , Hepatectomía/efectos adversos , Hepatectomía/métodos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Mutación , Recurrencia Local de Neoplasia/patología , Proteína 1 Compañera de Translocación de RUNX1/genética , Transducción de Señal , Secuenciación del Exoma , beta Catenina/genéticaRESUMEN
Plasmacytoid dendritic cells (pDCs) are present in various primary and metastatic human neoplasms; however, their clinical significance in hepatocellular carcinoma (HCC) is unclear. In this study, we investigated the distribution, prognostic value, and potential function of pDCs in HCC patients undergoing curative resection. We performed immunohistochemical analyses of whole tumor sections from 224 patients to assess the expression of BDCA2, CD3, CD4, CD8, Foxp3, granzyme B, IL-17, and CD34. The findings were validated using tissue microarrays from another two independent cohorts totaling 841 HCC patients undergoing curative resection. Our results demonstrated that high numbers of BDCA2+ pDCs within tumors correlated with high alpha-fetoprotein levels, greater vascular invasion, advanced tumor-node-metastasis stage, shorter overall survival, and a higher recurrence rate. However, patient outcomes were not associated with pDCs in peritumoral stromal or nontumor tissues. Furthermore, an increase in intratumoral pDCs was associated with increased intratumoral infiltration of Foxp3+ regulatory T cells and IL-17-producing cells and correlated with tumor vascular density. Univariate and multivariate analyses revealed that the presence of intratumoral pDCs alone or in combination with regulatory T and/or IL-17-producing cells was an independent predictor of time to recurrence and overall survival. In conclusion, our study demonstrated that intratumoral infiltration by pDCs is a novel indicator for poor prognosis in patients with HCC, possibly through the induction of an immune tolerogenic and inflammatory tumor microenvironment comprising regulatory T and IL-17-producing cells. An assessment of the combination of these cells represents a superior predictor of patient outcome.
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Carcinoma Hepatocelular/diagnóstico , Células Dendríticas/inmunología , Interleucina-17/metabolismo , Neoplasias Hepáticas/diagnóstico , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/mortalidad , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Factores de Transcripción Forkhead/metabolismo , Hepatectomía , Humanos , Lectinas Tipo C/metabolismo , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/mortalidad , Masculino , Glicoproteínas de Membrana/metabolismo , Persona de Mediana Edad , Metástasis de la Neoplasia , Pronóstico , Receptores Inmunológicos/metabolismo , Análisis de Supervivencia , alfa-Fetoproteínas/metabolismoRESUMEN
BACKGROUND: Due to the phenotypic and molecular diversity of hepatocellular carcinomas (HCC), it is still a challenge to determine patients' prognosis. We aim to identify new prognostic markers for resected HCC patients. METHODS: 274 patients were retrospectively identified and samples collected from Zhongshan hospital, Fudan University. We analyzed the gene expression patterns of tumors and compared expression patterns with patient survival times. We identified a "9-gene signature" associated with survival by using the coefficient and regression formula of multivariate Cox model. This molecular signature was then validated in three patients cohorts from internal cohort (n = 69), TCGA (n = 369) and GEO dataset (n = 80). RESULTS: We identified 9-gene signature consisting of ZC2HC1A, MARCKSL1, PTGS1, CDKN2B, CLEC10A, PRDX3, PRKCH, MPEG1 and LMO2. The 9-gene signature was used, combined with clinical parameters, to fit a multivariable Cox model to the training cohort (concordance index, ci = 0.85), which was successfully validated (ci = 0.86 for internal cohort; ci = 0.78 for in silico cohort). The signature showed improved performance compared with clinical parameters alone (ci = 0.70). Furthermore, the signature predicted patient prognosis than previous gene signatures more accurately. It was also used to stratify early-stage, HBV or HCV-infected patients into low and high-risk groups, leading to significant differences in survival in training and validation (P < 0.001). CONCLUSIONS: The 9-gene signature, in which four were upregulated (ZC2HC1A, MARCKSL1, PTGS1, CDKN2B) and five (CLEC10A, PRDX3, PRKCH, MPEG1, LMO2) were downregulated in HCC with poor prognosis, stratified HCC patients into low and high risk group significantly in different clinical settings, including receiving adjuvant transarterial chemoembolization and especially in early stage disease. This new signature should be validated in prospective studies to stratify patients in clinical decisions.
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Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/diagnóstico , Transformación Celular Viral/genética , Hepacivirus/fisiología , Virus de la Hepatitis B/fisiología , Neoplasias Hepáticas/diagnóstico , Transcriptoma , Adulto , Anciano , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/cirugía , Carcinoma Hepatocelular/virología , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Hepacivirus/patogenicidad , Hepatitis B/complicaciones , Hepatitis B/diagnóstico , Hepatitis B/genética , Hepatitis B/cirugía , Virus de la Hepatitis B/patogenicidad , Hepatitis C/complicaciones , Hepatitis C/diagnóstico , Hepatitis C/genética , Hepatitis C/cirugía , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/cirugía , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Factores de RiesgoRESUMEN
AIM: The MAGE family member H1 (MAGEH1) belongs to melanoma-associated antigen (MAGE) superfamily. The role of MAGEH1 in hepatocellular carcinoma (HCC) is largely undefined. MATERIALS & METHODS: We used quantitative reverse transcription PCR and immunohistochemistry to detect MAGEH1 expression in HCC tissues. CCK-8 assay, wound healing migration assay and Transwell Matrigel invasion assay were used to measure HCC cell proliferation, migration and invasion ability. RESULTS: MAGEH1 expression was downregulated in HCC tumor tissues compared with adjacent normal liver tissues and in samples from patients with tumor recurrence. MAGEH1 reduced HCC cell proliferation, migration and invasion ability. Low MAGEH1 expression was significantly correlated with poor prognosis in HCC patients. CONCLUSION: MAGEH1 may serve as a potential biomarker and a new prognostic factor for HCC.
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Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Recurrencia Local de Neoplasia/diagnóstico , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/cirugía , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Hígado/patología , Hígado/cirugía , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/cirugía , Masculino , Proteínas Asociadas a Microtúbulos/genética , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Recurrencia Local de Neoplasia/epidemiología , Pronóstico , Tasa de SupervivenciaRESUMEN
BACKGROUND & AIMS: Neutrophils can either promote or inhibit tumor progression, depending on the tumor microenvironment, via release of cytokines. Neither the factors produced by tumor-associated neutrophils (TANs) nor their effects on tumor progression have been characterized. We investigated the roles of TANs in progression of hepatocellular carcinoma (HCC) using cell lines and immune cells isolated from patients. METHODS: We performed studies with HepG2, PLC/PRF/5, MHCC97H, and HCCLM3 human and Hepa1-6 and H22 mouse HCC cell lines; expression of chemokines and cytokines were knocked down with small hairpin RNAs. Cells were analyzed in chemotaxis assays and as growth as tumors in mice. HCC tissues and peripheral blood were collected from 20 patients undergoing curative resection or 20 healthy individuals (controls) in 2012 at Zhongshan Hospital in China. TANs and peripheral blood neutrophils (PBNs) were isolated and exposed to conditioned media from HCC cell lines; reverse-transcription polymerase chain reaction was used to quantify the expression of cytokines and chemokines. We collected neutrophils from another 60 patients undergoing curative resection for HCC in 2012 to measure the production of C-C motif chemokine ligand 2(CCL2) and CCL17. Patients were followed up until March 15, 2014. For immunohistochemical analyses, we collected HCC tissues and paired, adjacent, nontumor cirrhotic liver tissues from 832 HCC patients undergoing curative resection from 2006 through 2008. All patients were followed up until March 15, 2013. To study the effects of sorafenib, we collected clinical and pathology data from 46 patients who underwent curative resection in 2010. RESULTS: CCL2 and CCL17 were the cytokines most highly expressed by TANs and HCC cell-activated PBNs. Levels of CCL2 and CCL17 messenger RNAs and proteins were significantly higher in TANs than in PBNs, and increased in patients with HCC recurrence. CCL2 and CCL17 messenger RNA and proteins also increased when PBNs were exposed to conditioned media from HCC cell lines. Immunohistochemical analysis of a tissue microarray showed that CCL2+ and CCL17+ cells, which also expressed the neutrophil marker CD66b, were distributed throughout the HCC stroma, but not in tumor cells or the adjacent nontumor liver cells. The number of CCL2+ or CCL17+ TANs correlated with tumor size, microvascular invasion, tumor encapsulation, tumor differentiation, and stage. Patients whose tumors had lower levels of CCL2+ or CCL17+ cells had longer survival times than those with higher numbers of these cells. TAN-conditioned media, as well as recombinant CCL2 and CCL17, increased the migratory activity of the macrophages and T-regulatory (Treg) cells from patients or mice with HCC to a greater extent that PBN-conditioned media. Neutralizing antibodies against CCL2 and CCL17, or their receptors C-C chemokine receptor 2 and C-C chemokine receptor 4, reduced the migratory activities of macrophage and Treg cells. HCC cell lines injected into mice formed larger tumors when they were co-injected with TANs and formed more pulmonary metastases; these tumors were infiltrated by Ly6G+ cells, F4/80+ macrophages, and Foxp3+ Treg cells. In a phosphokinase array of human PBNs, levels of phosphorylated AKT and P38 increased after exposure to conditioned media from all 4 HCC cell types. Pharmacologic inhibitors of AKT and P38 inhibited secretion of CCL2 and CCL17 by these PBNs. In tumor-bearing mice, sorafenib increased the numbers of TANs and levels of CCL2 and CCL17 in tumors. HCC tissues from patients who received sorafenib before surgery contained more TANs than tissues from patients who did not receive sorafenib. In knockdown cells, HCC cell-derived CXCL5 was the strongest effector of neutrophil migration under hypoxic conditions. In mice, the combination of sorafenib and TAN depletion inhibited tumor growth and neovascularization to a greater extent than sorafenib alone. CONCLUSIONS: TANs recruit macrophages and Treg cells to HCCs to promote their growth, progression, and resistance to sorafenib.
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Antineoplásicos/inmunología , Carcinoma Hepatocelular/inmunología , Resistencia a Antineoplásicos/inmunología , Neoplasias Hepáticas/inmunología , Niacinamida/análogos & derivados , Compuestos de Fenilurea/inmunología , Animales , Antineoplásicos/administración & dosificación , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Macrófagos/inmunología , Ratones , Infiltración Neutrófila , Neutrófilos/inmunología , Niacinamida/administración & dosificación , Niacinamida/inmunología , Compuestos de Fenilurea/administración & dosificación , Sorafenib , Linfocitos T Reguladores/inmunologíaRESUMEN
UNLABELLED: MicroRNAs (miRNAs) play a critical role in regulation of tumor metastasis. However, the role of these molecules in hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we employed miRNA-sequencing and identified 22 miRNAs involved in HCC metastasis. One of these, miR-28-5p, was down-regulated in HCCs. This down-regulation correlated with tumor metastasis, recurrence, and poor survival. Biofunctional investigations revealed that miR-28-5p deficiency promoted tumor growth and metastasis in nude mice without altering the in vitro biological characteristics of HCC cells. Through gene expression profiles and bioinformatics analysis, we identified interleukin-34 (IL-34) as a direct target of miR-28-5p, and the effects of miR-28-5p deficiency on HCC growth and metastasis was dependent on IL-34-mediated tumor-associated macrophage (TAM) infiltration. Moreover, we found that TAMs induced by miR-28-5p-IL-34 signaling inhibit miR-28-5p expression on HCC cells by transforming growth factor beta 1, resulting in an miR-28-5p-IL-34-macrophage-positive feedback loop. In clinical HCC samples, miR-28-5p levels were inversely correlated with IL-34 expression and the number of TAMs. Patients with low miR-28-5p expression, high IL-34 levels, and high numbers of TAMs had a poor prognosis with shorter overall survival and time to recurrence. CONCLUSION: A miR-28-5p-IL-34-macrophage feedback loop modulates HCC metastasis and serves as a novel prognostic factor as well as a therapeutic target for HCC.
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Carcinoma Hepatocelular/secundario , Interleucinas/fisiología , Neoplasias Hepáticas/patología , Macrófagos/fisiología , MicroARNs/fisiología , Animales , Línea Celular Tumoral , Proliferación Celular , Retroalimentación Fisiológica , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Factor de Crecimiento Transformador beta1/fisiología , Microambiente TumoralRESUMEN
A high preoperative peripheral blood neutrophil-to-lymphocyte ratio (NLR) has been reported to be a predictor of poor survival in patients with various cancers. The aim of this study was to evaluate the predictive significance of the NLR in patients undergoing hepatectomy for intrahepatic cholangiocarcinoma (ICC). From 2005 to 2011, 322 patients who underwent hepatectomy for ICC were enrolled in this retrospective study. Clinicopathological parameters, including NLR, were evaluated to identify predictors of overall and recurrence-free survival after hepatectomy. The best cutoff for NLR was 2.49, and 177 of 322 patients (54.9 %) had an NLR ≥ 2.49. The 5-year survival rate after hepatectomy was 51.1 % in patients with NLR < 2.49 and 24.8 % in those with NLR ≥ 2.49 (P = 0.0001). Univariate analyses revealed that NLR was significantly associated with recurrence-free survival (RFS) and overall survival (OS; both P < 0.05). Multivariable analyses revealed that elevated NLR independently predicted poorer OS (P = 0.003, hazard ratio [HR] = 1.600). In summary, our results indicate that elevated NLR is a promising independent predictor of poor survival after hepatectomy in patients with ICC.
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Neoplasias de los Conductos Biliares/patología , Conductos Biliares Intrahepáticos/patología , Carcinoma Hepatocelular/patología , Colangiocarcinoma/patología , Linfocitos/patología , Recurrencia Local de Neoplasia/patología , Neutrófilos/patología , Adulto , Anciano , Neoplasias de los Conductos Biliares/sangre , Neoplasias de los Conductos Biliares/cirugía , Conductos Biliares Intrahepáticos/cirugía , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/cirugía , Colangiocarcinoma/sangre , Colangiocarcinoma/cirugía , Supervivencia sin Enfermedad , Femenino , Hepatectomía , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/cirugía , Periodo Preoperatorio , PronósticoRESUMEN
Calpain small subunit 1 (Capn4) has been identified as a major gene that promotes metastasis of hepatocellular carcinoma (HCC). However, the mechanism by which Capn4 promotes progression of HCC is not understood. In this study, we found that Capn4 expression was increased in highly metastatic HCC cell lines and in tumour tissue from HCC patients compared to healthy patient tissue. Over-expression of Capn4 in HCC cells enhanced tumour cell growth in vitro and increased invasiveness, tumourigenicity and lung metastasis in vivo. Protein microarray analyses showed that expression of multiple proteins was regulated by Capn4. Interestingly, Capn4 was found to physically associate with FAK and promoted hyperactivity of the FAK-Src signalling pathway via increased phosphorylation of specific tyrosine residues of FAK, Src and p130Cas. Knock-down of Capn4 expression suppressed the malignant behaviour of HCC cells and inhibited the FAK-Src signalling pathway. Furthermore, Capn4-mediated invasion and metastasis of HCC cells required up-regulation of matrix metalloproteinase-2 (MMP2) through activation of this signalling pathway. Our clinical data revealed that Capn4 expression correlated well with the levels of phospho-FAK, and over-expression of both Capn4 and phospho-FAK correlates with the poorest survival outcomes in HCC. In conclusion, our data showed that Capn4 can contribute to HCC growth and metastasis via activation of the FAK-Src signalling pathway and MMP2.
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Calpaína/metabolismo , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Transducción de Señal/fisiología , Anciano , Animales , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Femenino , Técnica del Anticuerpo Fluorescente , Quinasa 1 de Adhesión Focal/metabolismo , Xenoinjertos , Humanos , Inmunoprecipitación , Estimación de Kaplan-Meier , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Masculino , Ratones , Ratones Desnudos , Microscopía Confocal , Persona de Mediana Edad , Invasividad Neoplásica/patología , Modelos de Riesgos Proporcionales , Análisis de Matrices Tisulares , Transfección , Familia-src Quinasas/metabolismoRESUMEN
CXCL5 is a member of the CXC-type chemokine family that may play a role in carcinogenesis and cancer progression. This study investigates the biological function and clinical significance of CXCL5 in intrahepatic cholangiocarcinoma (ICC). We demonstrated that CXCL5 was overexpressed in ICC cell lines and tumor samples compared with paired normal tissues. CXCL5 had a direct chemoattractant effect on neutrophils in vitro through PI3K-Akt and extracellular signal-regulated kinase 1/2 signaling pathways. In animal studies, CXCL5 promoted tumor growth and metastasis without altering in vitro proliferative and invasive ability of ICC cells, and this effect was mediated by the recruitment of intratumoral infiltrative neutrophils by tumor-derived CXCL5. Immunohistochemical analysis of ICC samples showed that overexpression of CXCL5 correlated strongly with intratumoral neutrophil infiltration, shorter overall survival and high tumor recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoral neutrophils, was an independent prognostic indicator for ICC. In conclusion, our data showed that CXCL5 promotes ICC growth and metastasis by recruiting intratumoral neutrophils. CXCL5 alone or combined with intratumoral neutrophils is a novel prognostic predictor for ICC patients and a potential therapeutic target.
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Quimiocina CXCL5/fisiología , Colangiocarcinoma/fisiopatología , Neoplasias Hepáticas/fisiopatología , Linfocitos Infiltrantes de Tumor/patología , Metástasis de la Neoplasia , Neutrófilos/fisiología , Colangiocarcinoma/patología , Humanos , Neoplasias Hepáticas/patología , Regulación hacia ArribaRESUMEN
Subsequently to the publication of the above article, an interested reader drew to the authors' attention that a possible error had been identified in the selection of images in Figs. 1 and/or 7. After having consulted their original data, the authors realized that an erroneous image appeared on p. 593, in Fig. 7F [the 'HepG2 / IL8 (5 ng/ml)' data panel], where part of this figure panel was overlapping with an image on p. 589 in Fig. 1C (the 'HepG2 Cocultured' data panel). After a thorough review and verification of the data by all the authors, they have confirmed that the original data presented in the paper were accurate, and the error was solely due to the selection of an incorrect image during figure arrangement. The authors confirm that this mistake in image selection did not affect the overall conclusions reported in the article. A corrected version of Fig. 7, including the correct data for the 'HepG2 / IL8 (5 ng/ml)' panel in Fig. 7F, is shown on the next page. The authors are grateful to the Editor of International Journal of Oncology for granting them the opportunity to publish this Corrigendum. All the authors agree to the publication of this Corrigendum, and apologize to the readership for any inconvenience caused. [International Journal of Oncology 46: 587596, 2015; DOI: 10.3892/ijo.2014.2761].
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BACKGROUND: The tumor microbiome has been characterized in several malignancies; however, no previous studies have investigated its role in intrahepatic cholangiocarcinoma (ICC). Hence, we explored the tumor microbiome and its association with prognosis in ICC. METHODS: One hundred and twenty-one ICC tumor samples and 89 adjacent normal tissues were profiled by 16S rRNA sequencing. Microbial differences between tumor and adjacent nontumoral liver tissues were assessed. Tumor microbial composition was then evaluated to detect its association with prognosis. Finally, a risk score calculated by the tumor microbiota was accessed by the least absolute shrinkage and selector operator method (Lasso) to predict prognosis of ICC. RESULTS: The tumor microbiome displayed a greater diversity than that in adjacent nontumoral liver tissues. Tumor samples were characterized by a higher abundance of Firmicutes, Actinobacteria, Bacteroidetes, and Acidobacteriota. Higher tumor microbial α diversity was associated with lymph node metastasis and predicted shortened overall survival (OS) and recurrence-free survival (RFS). A total of 11 bacteria were selected to generate the risk score by Lasso. This score showed potential in predicting OS, and was an independent risk factor for OS. CONCLUSION: In conclusion, our study characterized the tumor microbiome and revealed its role in predicting prognosis in ICC.
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Neoplasias de los Conductos Biliares , Colangiocarcinoma , Humanos , ARN Ribosómico 16S/genética , Pronóstico , Colangiocarcinoma/patología , Conductos Biliares Intrahepáticos/patología , Neoplasias de los Conductos Biliares/patología , Estudios RetrospectivosRESUMEN
Background: Intrahepatic cholangiocarcinoma (ICC) is a highly desmoplastic tumor with poor prognosis even after curative resection. We investigated the associations between the composition of the ICC stroma and immune cell infiltration and aimed to develop a stromal-immune signature to predict prognosis in surgically treated ICC. Patients and methods: We recruited 359 ICC patients and performed immunohistochemistry to detect α-smooth muscle actin (α-SMA), CD3, CD4, CD8, Foxp3, CD68, and CD66b. Aniline was used to stain collagen deposition. Survival analyses were performed to detect prognostic values of these markers. Recursive partitioning for a discrete-time survival tree was applied to define a stromal-immune signature with distinct prognostic value. We delineated an integrated stromal-immune signature based on immune cell subpopulations and stromal composition to distinguish subgroups with different recurrence-free survival (RFS) and overall survival (OS) time. Results: We defined four major patterns of ICC stroma composition according to the distributions of α-SMA and collagen: dormant (α-SMAlow/collagenhigh), fibrogenic (α-SMAhigh/collagenhigh), inert (α-SMAlow/collagenlow), and fibrolytic (α-SMAhigh/collagenlow). The stroma types were characterized by distinct patterns of infiltration by immune cells. We divided patients into six classes. Class I, characterized by high CD8 expression and dormant stroma, displayed the longest RFS and OS, whereas Class VI, characterized by low CD8 expression and high CD66b expression, displayed the shortest RFS and OS. The integrated stromal-immune signature was consolidated in a validation cohort. Conclusion: We developed and validated a stromal-immune signature to predict prognosis in surgically treated ICC. These findings provide new insights into the stromal-immune response to ICC.
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Heterogeneous ribonucleoprotein (hnRNP) A1 is a member of the A/B subfamily of ubiquitously expressed hnRNPs, which have a wide variety of functions in gene expression and signal transduction. To investigate the biological function and clinical significance of hnRNP A1 in hepatocellular carcinoma (HCC), we measured hnRNP A1 expression in four HCC cell lines and two independent cohorts of HCC patients. We found that hnRNP A1 was overexpressed in the highly metastatic HCC cell lines and in tumor tissues of patients with recurrent HCC. Knockdown of hnRNP A1 in highly metastatic HCC cells caused a significant decrease in cell invasion, while upregulation of hnRNP A1 in poorly metastatic HCC cells led to a significant increase in their invasive capacity. We found that this effect may occur through the regulation of CD44v6 expression by hnRNP A1 in HCC cells. Both quantitative reverse transcription-polymerase chain reaction (qRT-RCR) and immunohistochemistry revealed that hnRNP A1 was upregulated in HCC tissues and coincided with overexpression of CD44v6. HCC patients with high hnRNP A1 tended to have higher levels of CD44v6, shorter overall survival (OS) and higher rates of tumor recurrence. Multivariate analyses revealed that hnRNP A1 alone or in combination with CD44v6 were independent prognostic indicators for OS and time to recurrence and have potential as therapeutic targets. In conclusion, overexpression of hnRNP A1 promotes HCC invasion by regulating the level of CD44v6 and indicates a poor prognosis for HCC patients after curative resection.