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1.
Int J Mol Sci ; 22(21)2021 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-34769191

RESUMO

The balance between anti-tumor and tumor-promoting immune cells, such as CD4+ Th1 and regulatory T cells (Tregs), respectively, is assumed to dictate the progression of hepatocellular carcinoma (HCC). The transforming growth factor beta (TGFß) markedly shapes the HCC microenvironment, regulating the activation state of multiple leukocyte subsets and driving the differentiation of cancer associated fibroblasts (CAFs). The fibrotic (desmoplastic) reaction in HCC tissue strongly depends on CAFs activity. In this study, we attempted to assess the role of TGFß on transendothelial migration of Th1-oriented and Treg-oriented CD4+ T cells via a direct or indirect, CAF-mediated mechanisms, respectively. We found that the blockage of TGFß receptor I-dependent signaling in Tregs resulted in impaired transendothelial migration (TEM) of these cells. Interestingly, the secretome of TGFß-treated CAFs inhibited the TEM of Tregs but not Th1 cells, in comparison to the secretome of untreated CAFs. In addition, we found a significant inverse correlation between alpha-SMA and FoxP3 (marker of Tregs) mRNA expression in a microarray analysis involving 78 HCCs, thus suggesting that TGFß-activated stromal cells may counteract the trafficking of Tregs into the tumor. The apparent dual behavior of TGFß as both pro- and anti-tumorigenic cytokines may add a further level of complexity to the mechanisms that regulate the interactions among cancerous, stromal, and immune cells within HCC, as well as other solid tumors, and contribute to better manipulation of the TGFß signaling as a therapeutic target in HCC patients.

2.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073989

RESUMO

(1) Background: The transforming growth factor (TGF)-ß plays a dual role in liver carcinogenesis. At early stages, it inhibits cell growth and induces apoptosis. However, TGF-ß expression is high in advanced stages of hepatocellular carcinoma (HCC) and cells become resistant to TGF-ß induced suppressor effects, responding to this cytokine undergoing epithelial-mesenchymal transition (EMT), which contributes to cell migration and invasion. Metabolic reprogramming has been established as a key hallmark of cancer. However, to consider metabolism as a therapeutic target in HCC, it is necessary to obtain a better understanding of how reprogramming occurs, which are the factors that regulate it, and how to identify the situation in a patient. Accordingly, in this work we aimed to analyze whether a process of full EMT induced by TGF-ß in HCC cells induces metabolic reprogramming. (2) Methods: In vitro analysis in HCC cell lines, metabolomics and transcriptomics. (3) Results: Our findings indicate a differential metabolic switch in response to TGF-ß when the HCC cells undergo a full EMT, which would favor lipolysis, increased transport and utilization of free fatty acids (FFA), decreased aerobic glycolysis and an increase in mitochondrial oxidative metabolism. (4) Conclusions: EMT induced by TGF-ß in HCC cells reprograms lipid metabolism to facilitate the utilization of FFA and the entry of acetyl-CoA into the TCA cycle, to sustain the elevated requirements of energy linked to this process.


Assuntos
Carcinoma Hepatocelular/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Metaboloma/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Células Hep G2 , Humanos , Metaboloma/genética , Metabolômica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transcriptoma/genética
3.
Cell Death Dis ; 12(4): 316, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767160

RESUMO

The prognosis of locally advanced colorectal cancer (CRC) is currently unsatisfactory. This is mainly due to drug resistance, recurrence, and subsequent metastatic dissemination, which are sustained by the cancer stem cell (CSC) population. The main driver of the CSC gene expression program is Wnt signaling, and previous reports indicate that Wnt3a can activate p38 MAPK. Besides, p38 was shown to feed into the canonical Wnt/ß-catenin pathway. Here we show that patient-derived locally advanced CRC stem cells (CRC-SCs) are characterized by increased expression of p38α and are "addicted" to its kinase activity. Of note, we found that stage III CRC patients with high p38α levels display reduced disease-free and progression-free survival. Extensive molecular analysis in patient-derived CRC-SC tumorspheres and APCMin/+ mice intestinal organoids revealed that p38α acts as a ß-catenin chromatin-associated kinase required for the regulation of a signaling platform involved in tumor proliferation, metastatic dissemination, and chemoresistance in these CRC model systems. In particular, the p38α kinase inhibitor ralimetinib, which has already entered clinical trials, promoted sensitization of patient-derived CRC-SCs to chemotherapeutic agents commonly used for CRC treatment and showed a synthetic lethality effect when used in combination with the MEK1 inhibitor trametinib. Taken together, these results suggest that p38α may be targeted in CSCs to devise new personalized CRC treatment strategies.


Assuntos
Cromatina/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Células-Tronco Neoplásicas/metabolismo , Organoides/metabolismo , Processamento de Proteína Pós-Traducional/genética , beta Catenina/metabolismo , Neoplasias Colorretais/genética , Humanos , Prognóstico
4.
Oncol Rep ; 45(2): 776-785, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33416143

RESUMO

Liver cancer (LC) is an aggressive disease with a markedly poor prognosis. Therapeutic options are limited, and, until recently the only FDA­approved agent for first­line treatment of patients with LC was the multi­kinase inhibitor sorafenib, which exhibits limited activity and an increased overall survival (OS) of only 3 months over placebo. Therefore, the development of alternative therapeutic molecules for the treatment of LC is an urgent medical need. Antibody­drug conjugates (ADCs) are an emerging class of novel anticancer agents, which have been developed recently for the treatment of malignant conditions, including LC, and are being studied in preclinical and clinical settings. Our group has recently generated an ADC [EV20/monomethyl auristatin F (MMAF)] by coupling the HER3 targeting antibody (EV20) to MMAF via a non­cleavable maleimidocaproyl linker. This ADC was revealed to possess potent therapeutic activity in melanoma and breast carcinoma. In the present study, using western blot and flow cytometric analysis, it was reported that HER­3 receptor was highly expressed in LC and activated by its ligand NRG­1ß in a panel of LC cell lines, thus indicating that this receptor may serve as a suitable target for ADC therapy. A novel ADC [EV20­sss­valine­citrulline (vc)/MMAF] was generated, in which the cytotoxic payload MMAF was site­specifically coupled to an engineered variant of EV20 via a vc cleavable linker. Cytotoxicity assays were performed to investigate in vitro antitumor activity of EV20­sss­vc/MMAF and it was compared to EV20/MMAF, which revealed only modest activity in LC.EV20­sss­vc/MMAF exhibited a significant cell killing activity in several LC cell lines. Additionally, in vivo xenograft experiments revealed that EV20­sss­vc/MMAF inhibited growth of LC tumors. The present data indicated that EV20­sss­vc/MMAF is a worthy candidate for the treatment of HER­3 positive LC.


Assuntos
Imunoconjugados/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Oligopeptídeos/farmacologia , Receptor ErbB-3/antagonistas & inibidores , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Imunoconjugados/uso terapêutico , Fígado/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Oligopeptídeos/uso terapêutico , Receptor ErbB-3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cell Death Dis ; 11(11): 984, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-33199679

RESUMO

Sorafenib and regorafenib administration is among the preferential approaches to treat hepatocellular carcinoma (HCC), but does not provide satisfactory benefits. Intensive crosstalk occurring between cancer cells and other multiple non-cancerous cell subsets present in the surrounding microenvironment is assumed to affect tumor progression. This interplay is mediated by a number of soluble and structural extracellular matrix (ECM) proteins enriching the stromal milieu. Here we assess the HCC tumor expression of the ECM protein proteoglycan 4 (PRG4) and its potential pharmacologic activity either alone, or in combination with sorafenib and regorafenib. PRG4 mRNA levels resulted strongly correlated with increased survival rate of HCC patients (p = 0.000) in a prospective study involving 78 HCC subjects. We next showed that transforming growth factor beta stimulates PRG4 expression and secretion by primary human HCC cancer-associated fibroblasts, non-invasive HCC cell lines, and ex vivo specimens. By functional tests we found that recombinant human PRG4 (rhPRG4) impairs HCC cell migration. More importantly, the treatment of HCC cells expressing CD44 (the main PRG4 receptor) with rhPRG4 dramatically enhances the growth-limiting capacity of sorafenib and regorafenib, whereas not significantly affecting cell proliferation per se. Conversely, rhPRG4 only poorly potentiates drug effectiveness on low CD44-expressing or stably CD44-silenced HCC cells. Overall, these data suggest that the physiologically-produced compound PRG4 may function as a novel tumor-suppressive agent by strengthening sorafenib and regorafenib effects in the treatment of HCC.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Compostos de Fenilureia/uso terapêutico , Proteoglicanas/uso terapêutico , Piridinas/uso terapêutico , Sorafenibe/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/patologia , Compostos de Fenilureia/farmacologia , Proteoglicanas/farmacologia , Piridinas/farmacologia , Sorafenibe/farmacologia , Microambiente Tumoral
6.
Cancers (Basel) ; 12(3)2020 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-32182707

RESUMO

Different subsets of lymphocytes have the capacity to promote or counteract the progression of solid cancers, including hepatocellular carcinoma (HCC). Therefore, to determine the infiltrative ability and functional status of major immune cell subtypes into tumor may lead to novel insights from the perspective of immunotherapy. After obtaining single cell suspensions from freshly collected specimens of HCC tumor, along with paired peritumor tissues and peripheral blood mononuclear cells (PBMCs) from 14 patients, we flow-cytometrically identified and quantified the relative frequencies of lymphocyte subsets within the tissues of origin. We found that the recruitment in the tumor of cytotoxic cells, namely the terminally differentiated CD4+ and CD8+ T cells (TEFF), is impaired, whereas the effector memory CD4+ T cells (TEM) are more attracted in this site. Concerning the other subsets, the frequency of NK CD56hi and NKT CD56hi cells infiltration in the tumor is increased, whereas that of NKT CD56low is reduced. Although CD4+ and CD8+ T cells settled in the tumor show a higher degree of activation than the circulating counterpart, they occur with a more exhausted phenotype. Overall, these data demonstrate the prevalently immunosuppressive nature of HCC microenvironment, and prompt us to search for strategies to enhance the activity of anti-tumor immune cell subsets.

7.
Cell Death Differ ; 27(8): 2330-2343, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32042099

RESUMO

Intrahepatic cholangiocarcinoma (iCCA) is a deadly disease with rising incidence and few treatment options. An altered expression and/or activation of NOTCH1-3 receptors has been shown to play a role in iCCA development and progression. In this study, we established a new CCA patient-derived xenograft model, which was validated by immunohistochemistry and transcriptomic analysis. The effects of Notch pathway suppression by the Crenigacestat (LY3039478)-specific inhibitor were evaluated in human iCCA cell lines and the PDX model. In vitro, LY3039478 significantly reduced Notch pathway components, including NICD1 and HES1, but not the other Notch receptors, in a panel of five different iCCA cell lines. In the PDX model, LY3039478 significantly inhibited the Notch pathway and tumor growth to the same extent as gemcitabine. Furthermore, gene expression analysis of iCCA mouse tissues treated with LY3039478 revealed a downregulation of VEGFA, HES1, and MMP13 genes. In the same tissues, DLL4 and CD31 co-localized, and their expression was significantly inhibited in the treated mice, as it happened in the case of MMP13. In an in vitro angiogenesis model, LY3039478 inhibited vessel formation, which was restored by the addition of MMP13. Finally, RNA-sequencing expression data of iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database demonstrated that NOTCH1, HES1, MMP13, DLL4, and VEGFA genes were significantly upregulated in tumors compared with adjacent nontumorous tissues. These data were confirmed by our group, using an independent cohort of iCCA specimens. Conclusion: We have developed and validated a new iCCA PDX model to test in vivo the activity of LY3039478, demonstrating its inhibitory role in Notch-dependent angiogenesis. Thus, the present data provide new knowledge on Notch signaling in iCCA, and support the inhibition of the Notch cascade as a promising strategy for the treatment of this disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Benzazepinas/farmacologia , Neoplasias dos Ductos Biliares/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Colangiocarcinoma/patologia , Progressão da Doença , Metaloproteinase 13 da Matriz/metabolismo , Receptor Notch1/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Benzazepinas/uso terapêutico , Neoplasias dos Ductos Biliares/irrigação sanguínea , Neoplasias dos Ductos Biliares/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Colangiocarcinoma/irrigação sanguínea , Colangiocarcinoma/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Metaloproteinase 13 da Matriz/genética , Camundongos Nus , Microvasos/patologia , Neovascularização Patológica/tratamento farmacológico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor Notch1/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Transcriptoma/genética , Fator A de Crescimento do Endotélio Vascular/genética , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Cancers (Basel) ; 11(10)2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31600917

RESUMO

Transforming growth factor beta (TGF-ß) is a pleiotropic cytokine with dual role in hepatocellular carcinoma (HCC). It acts as tumor-suppressor and tumor-promoter in the early and late stage respectively. TGF-ß influences the tumor-stroma cross-talk affecting the tumoral microenvironment. Therefore, inhibiting the TGF- ß mediated pathway alone and/or in combination with chemotherapeutics represents an important therapeutic option. Experimental models to dissect the role of TGF-ß in HCC tumor progression as well as the effectiveness of specific inhibitors are tricky. HCC cell lines respond to TGF-ß according to their epithelial phenotype. However, the mesenchymal and more aggressive HCC cell lines in vitro, do not develop tumors when transplanted in vivo, thus hampering the understanding of molecular pathways that dictate outcome. In addition, in this model the native immune system is abolished, therefore the contribution of inflammation in hepatocarcinogenesis is unreliable. Different strategies have been set up to engineer HCC animal models, including genetically modified mice, chemically induced HCC, or hydrodynamic techniques. Patient-derived xenograft is currently probably the most fascinating model, keeping in mind that models cannot mirror all the reality. In this context, we discuss the different available HCC mouse models including our experimental model treated with inhibitor of TGF-ß receptor Type I kinase (Galunisertib) and a potential role of exosomes in TGF-ß moderated tumor progression of HCC. Unfortunately, no positive results were obtained in our treated orthotopic model because it does not reproduce the critical tumor-stroma interactions of the HCC.

9.
Cancers (Basel) ; 11(10)2019 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-31635301

RESUMO

Calcium is the most abundant element in the human body. Its role is essential in physiological and biochemical processes such as signal transduction from outside to inside the cell between the cells of an organ, as well as the release of neurotransmitters from neurons, muscle contraction, fertilization, bone building, and blood clotting. As a result, intra- and extracellular calcium levels are tightly regulated by the body. The liver is the most specialized organ of the body, as its functions, carried out by hepatocytes, are strongly governed by calcium ions. In this work, we analyze the role of calcium in human hepatoma (HCC) cell lines harboring a wild type form of the Epidermal Growth Factor Receptor (EGFR), particularly its role in proliferation and in EGFR downmodulation. Our results highlight that calcium is involved in the proliferative capability of HCC cells, as its subtraction is responsible for EGFR degradation by proteasome machinery and, as a consequence, for EGFR intracellular signaling downregulation. However, calcium-regulated EGFR signaling is cell line-dependent. In cells responding weakly to the epidermal growth factor (EGF), calcium seems to have an opposite effect on EGFR internalization/degradation mechanisms. These results suggest that besides EGFR, calcium could be a new therapeutic target in HCC.

10.
Cells ; 8(10)2019 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547567

RESUMO

The Transforming Growth Factor beta (TGFß) and Bone Morphogenic Protein (BMP) pathways intersect at multiple signaling hubs and cooperatively or counteractively participate to bring about cellular processes which are critical not only for tissue morphogenesis and organogenesis during development, but also for adult tissue homeostasis. The proper functioning of the TGFß/BMP pathway depends on its communication with other signaling pathways and any deregulation leads to developmental defects or diseases, including fibrosis and cancer. In this review we explore the cellular and physio-pathological contexts in which the synergism or antagonism between the TGFß and BMP pathways are crucial determinants for the normal developmental processes, as well as the progression of fibrosis and malignancies.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Neoplasias/genética , Organogênese/genética , Fator de Crescimento Transformador beta/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/genética , Progressão da Doença , Fibrose/genética , Fibrose/patologia , Redes Reguladoras de Genes/fisiologia , Humanos , Neoplasias/patologia , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/genética
11.
Semin Liver Dis ; 39(1): 53-69, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30586675

RESUMO

Therapeutic attempts to treat hepatocellular carcinoma (HCC) frequently result in a poor response or treatment failure. The efficacy of approved drugs and survival expectancies is affected by an ample degree of variability that can be explained at least in part by the enormous between-patient cellular and molecular heterogeneity of this neoplasm. Transforming growth factor-ß (TGF-ß) is hyperactivated in a large fraction of HCCs, where it influences complex interactive networks covering multiple cell types and a plethora of other local soluble ligands, ultimately establishing several malignancy traits. This cytokine boosts the invasiveness of cancerous epithelial cells through promoting the epithelial-to-mesenchymal transition program, but also skews the phenotype of immune cells toward a tumor-supporting status. Here, we discuss recent strategies pursued to offset TGF-ß-dependent processes that promote metastatic progression and immune surveillance escape in solid cancers, including HCC. Moreover, we report findings indicating that TGF-ß reduces the expression of the proinflammatory factors CCL4 and interleukin-1ß (IL-1ß in human ex vivo treated HCC tissues. While this is consistent with the anti-inflammatory properties of TGF-ß, whether it is an outright tumor promoter or suppressor is still a matter of some debate. Indeed, IL-1ß has also been shown to support angiogenesis and cell invasiveness in some cancers. In addition, we describe an inhibitory effect of TGF-ß on the secretion of CCL2 and CXCL1 by HCC-derived fibroblasts, which suggests the existence of an indirect stroma-mediated functional link between TGF-ß and downstream immunity.


Assuntos
Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Animais , Modelos Animais de Doenças , Progressão da Doença , Transição Epitelial-Mesenquimal , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Invasividade Neoplásica , Linfócitos T Reguladores/metabolismo
12.
Biochim Biophys Acta Mol Basis Dis ; 1865(1): 38-47, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30321589

RESUMO

Metabolic reprogramming is a common hallmark of cancer cells. Although some biochemical features have been clarified, there is still much to learn about cancer cell metabolism and its regulation. Aspartate-glutamate carrier isoform 1 (AGC1), encoded by SLC25A12 gene, catalyzes an exchange between intramitochondrial aspartate and cytosolic glutamate plus a proton across the mitochondrial membrane, so supplying aspartate to the cytosol. SLC25A12, expressed in brain, heart, and skeletal muscle, is silenced in normal liver. Here, we demonstrate that SLC25A12 gene is reactivated in hepatocellular carcinoma (HCC) HepG2 cell line through histone acetylation and CREB recruitment. Furthermore, SLC25A12 knockdown by small interfering RNA, impairs HepG2 cell proliferation by inducing cell cycle arrest. AGC1 sustains HCC cell growth by supplying cytosolic aspartate for nucleotide biosynthesis. In addition, SLC25A12-silenced HCC cells show a strong reduction of cell migration. Overall, we have provided evidence for molecular mechanisms controlling SLC25A12 gene expression in liver and pointing to an important role for AGC1 in HCC.


Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antiporters/metabolismo , Ácido Aspártico/metabolismo , Carcinoma Hepatocelular/metabolismo , Epigênese Genética , Ácido Glutâmico/metabolismo , Neoplasias Hepáticas/metabolismo , Isoformas de Proteínas/metabolismo , Regulação para Cima , Encéfalo/metabolismo , Carcinoma Hepatocelular/genética , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Coração , Células Hep G2 , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Músculo Esquelético/metabolismo
13.
Int J Mol Sci ; 19(5)2018 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-29701666

RESUMO

Transforming growth factor-β (TGF-β) is a cytokine essential for the induction of the fibrotic response and for the activation of the cancer stroma. Strong evidence suggests that a strong cross-talk exists among TGF-β and the tissue extracellular matrix components. TGF-β is stored in the matrix as part of a large latent complex bound to the latent TGF-β binding protein (LTBP) and matrix binding of latent TGF-β complexes, which is required for an adequate TGF-β function. Once TGF-β is activated, it regulates extracellular matrix remodelling and promotes a fibroblast to myofibroblast transition, which is essential in fibrotic processes. This cytokine also acts on other cell types present in the fibrotic and tumour microenvironment, such as epithelial, endothelial cells or macrophages and it contributes to the cancer-associated fibroblast (CAF) phenotype. Furthermore, TGF-β exerts anti-tumour activity by inhibiting the host tumour immunosurveillance. Aim of this review is to update how TGF-β and the tissue microenvironment cooperate to promote the pleiotropic actions that regulate cell responses of different cell types, essential for the development of fibrosis and tumour progression. We discuss recent evidences suggesting the use of TGF-β chemical inhibitors as a new line of defence against fibrotic disorders or cancer.


Assuntos
Carcinoma Hepatocelular/metabolismo , Microambiente Celular , Cirrose Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Matriz Extracelular/metabolismo , Humanos , Fator de Crescimento Transformador beta/genética
14.
Cell Death Dis ; 9(3): 373, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29515105

RESUMO

Cancer stem cells (CSCs) niche in the tumor microenvironment is responsible for cancer recurrence and therapy failure. To better understand its molecular and biological involvement in hepatocellular carcinoma (HCC) progression, one can design more effective therapies and tailored then to individual patients. While sorafenib is currently the only approved drug for first-line treatment of advanced stage HCC, its role in modulating the CSC niche is estimated to be small. By contrast, transforming growth factor (TGF)-ß pathway seems to influence the CSC and thus may impact hallmarks of HCC, such as liver fibrosis, cirrhosis, and tumor progression. Therefore, blocking this pathway may offer an appealing and druggable target. In our study, we have used galunisertib (LY2157299), a selective ATP-mimetic inhibitor of TGF-ß receptor I (TGFßI/ALK5) activation, currently under clinical investigation in HCC patients. Because the drug resistance is mainly mediated by CSCs, we tested the effects of galunisertib on stemness phenotype in HCC cells to determine whether TGF-ß signaling modulates CSC niche and drug resistance. Galunisertib modulated the expression of stemness-related genes only in the invasive (HLE and HLF) HCC cells inducing a decreased expression of CD44 and THY1. Furthermore, galunisertib also reduced the stemness-related functions of invasive HCC cells decreasing the formation of colonies, liver spheroids and invasive growth ability. Interestingly, CD44 loss of function mimicked the galunisertib effects on HCC stemness-related functions. Galunisertib treatment also reduced the expression of stemness-related genes in ex vivo human HCC specimens. Our observations are the first evidence that galunisertib effectiveness overcomes stemness-derived aggressiveness via decreased expression CD44 and THY1.


Assuntos
Carcinoma Hepatocelular/metabolismo , Receptores de Hialuronatos/metabolismo , Neoplasias Hepáticas/metabolismo , Pirazóis/farmacologia , Quinolinas/farmacologia , Western Blotting , Linhagem Celular Tumoral , Citometria de Fluxo , Células Hep G2 , Humanos , Receptores de Hialuronatos/genética , Técnicas In Vitro , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo
15.
Int J Mol Sci ; 20(1)2018 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-30597907

RESUMO

The epithelial mesenchymal transition (EMT) is a physiological multistep process involving epithelial cells acquiring a mesenchymal-like phenotype. It is widely demonstrated that EMT is linked to tumor progression and metastasis. The transforming growth factor (TGF)-ß pathways have been widely investigated, but its role in the hepatocarcinoma EMT is still unclear. While the biochemical pathways have been extensively studied, the alteration of biomechanical behavior correlated to cellular phenotype and motility is not yet fully understood. To better define the involvement of TGF-ß1 in the metastatic progression process in different hepatocarcinoma cell lines (HepG2, PLC/PRF/5, HLE), we applied a systematic morphomechanical approach in order to investigate the physical and the structural characteristics. In addition, we evaluated the antitumor effect of LY2157299, a TGF-ßR1 kinase inhibitor, from a biomechanical point of view, using Atomic Force and Confocal Microscopy. Our approach allows for validation of biological data, therefore it may be used in the future as a diagnostic tool to be combined with conventional biomolecular techniques.


Assuntos
Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Mecanotransdução Celular , Fator de Crescimento Transformador beta/metabolismo , Biomarcadores , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Módulo de Elasticidade , Humanos , Neoplasias Hepáticas/patologia , Microscopia de Força Atômica , Gradação de Tumores
16.
Cell Death Dis ; 8(8): e3017, 2017 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-28837142

RESUMO

In HCC, tumor microenvironment, heavily influenced by the underlying chronic liver disease, etiology and stage of the tissue damage, affects tumor progression and determines the high heterogeneity of the tumor. Aim of this study was to identify the circulating and tissue components of the microenvironment immune-mediated response affecting the aggressiveness and the ensuing clinical outcome. We analyzed the baseline paired HCC and the surrounding tissue biopsies from a prospective cohort of 132 patients at the first diagnosis of HCC for immunolocalization of PD-1/PD-L1, FoxP3, E-cadherin, CLEC2 and for a panel of 82 microRNA associated with regulation of angiogenesis, cell proliferation, cell signaling, immune control and autophagy. Original microarray data were also explored. Serum samples were analyzed for a panel of 19 cytokines. Data were associated with biochemical data, histopathology and survival. Patients with a more aggressive disease and shorter survival, who we named fast-growing accordingly to the tumor doubling time, at presentation had significantly higher AFP levels, TGF-ß1 and Cyphra 21-1 levels. Transcriptomic analysis evidenced a significant downregulation of CLEC2 and upregulation of several metalloproteinases. A marked local upregulation of both PD-1 and PD-L1, a concomitant FoxP3-positive lymphocytic infiltrate, a loss of E-cadherin, gain of epithelial-mesenchymal transition (EMT) phenotype and extreme poor differentiation at histology were also present. Upregulated microRNA in fast-growing HCCs are associated with TGF-ß signaling, angiogenesis and inflammation. Our data show that fast HCCs are characterized not only by redundant neo-angiogenesis but also by unique features of distinctively immunosuppressed microenvironment, prominent EMT, and clear-cut activation of TGFß1 signaling in a general background of long-standing and permanent inflammatory state.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Estudos Prospectivos , Transdução de Sinais , Análise de Sobrevida , Microambiente Tumoral
17.
Cell Death Dis ; 8(6): e2867, 2017 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-28594404

RESUMO

The aim of this study was to design a road map for personalizing cancer therapy in hepatocellular carcinoma (HCC) by using molecular pattern diagnostics. As an exploratory study, we investigated molecular patterns of tissues of two tumors from individual HCC patients, which in previous experiments had shown contrasting reactions to the phase 2 transforming growth factor beta receptor 1 inhibitor galunisertib. Cancer-driving molecular patterns encompass - inter alias - altered transcription profiles and somatic mutations in coding regions differentiating tumors from their respective peritumoral tissues and from each other. Massive analysis of cDNA ends and all-exome sequencing demonstrate a highly divergent transcriptional and mutational landscape, respectively, for the two tumors, that offers potential explanations for the tumors contrasting responses to galunisertib. Molecular pattern diagnostics (MPDs) suggest alternative, individual-tumor-specific therapies, which in both cases deviate from the standard sorafenib treatment and from each other. Suggested personalized therapies use kinase inhibitors and immune-focused drugs as well as low-toxicity natural compounds identified using an advanced bioinformatics routine included in the MPD protocol. The MPD pipeline we describe here for the prediction of suitable drugs for treatment of two contrasting HCCs may serve as a blueprint for the design of therapies for various types of cancer.


Assuntos
Carcinoma Hepatocelular , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas , Técnicas de Diagnóstico Molecular , Medicina de Precisão/métodos , Transcrição Genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Feminino , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/terapia , Masculino , Projetos Piloto
18.
Int J Mol Sci ; 18(3)2017 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-28245562

RESUMO

Mediterranean diet components, such as olive oil and ω-3 polyunsaturated fatty acids (ω-3 PUFAs), can arrest cell growth and promote cell apoptosis. Recently, olive oil has been demonstrated to modulate type-1 cannabinoid (CB1) receptor gene expression in both human colon cancer cells and rat colon. The aim of this study was to investigate a possible link between olive oil and ω-3 PUFAs effects and CB1 receptor expression in both intestinal and adipose tissue of ApcMin/+ mice. To confirm the role for the CB1 receptor as a negative modulator of cell proliferation in human colon cancer, CB1 receptor gene expression was also detected in tumor tissue and in surrounding normal mucosa of patients with colorectal cancer (CRC). Dietary ω-3 PUFAs significantly inhibited intestinal polyp growth in mice, correlating with CB1 receptor gene and protein expression induction. CB1 receptor gene up-regulation was also detected in adipose tissue, suggesting a close communication between cancer cells and the surrounding environment. Tissue CB1 receptor induction was associated with a concurrent inactivation of the Wnt/ß-catenin pathway. Moreover, there was a significant reduction in CB1 receptor gene expression levels in cancer tissue compared to normal surrounding mucosa of patients with CRC, confirming that in cancer the "protective" action of the CB1 receptor is lost.


Assuntos
Ácidos Graxos Ômega-3/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Receptor CB1 de Canabinoide/metabolismo , Ração Animal , Animais , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Ácidos Graxos Ômega-3/administração & dosagem , Expressão Gênica , Genes APC , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Camundongos , Camundongos Transgênicos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Receptor CB1 de Canabinoide/genética , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Cell Death Dis ; 8(2): e2634, 2017 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-28230858

RESUMO

Transforming growth factor-beta (TGF-ß) signaling has gained extensive interest in hepatocellular carcinoma (HCC). The small molecule kinase inhibitor galunisertib, targeting the TGF-ß receptor I (TGF-ßRI), blocks HCC progression in preclinical models and shows promising effects in ongoing clinical trials. As the drug is not similarly effective in all patients, this study was aimed at identifying new companion diagnostics biomarkers for patient stratification. Next-generation sequencing-based massive analysis of cDNA ends was used to investigate the transcriptome of an invasive HCC cell line responses to TGF-ß1 and galunisertib. These identified mRNA were validated in 78 frozen HCC samples and in 26 ex-vivo HCC tissues treated in culture with galunisertib. Respective protein levels in patients blood were measured by enzyme-linked immunosorbent assay. SKIL, PMEPA1 ANGPTL4, SNAI1, Il11 and c4orf26 were strongly upregulated by TGF-ß1 and downregulated by galunisertib in different HCC cell lines. In the 78 HCC samples, only SKIL and PMEPA1 (P<0.001) were correlated with endogenous TGF-ß1. In ex-vivo samples, SKIL and PMEPA1 were strongly downregulated (P<0.001), and correlated (P<0.001) with endogenous TGF-ß1. SKIL and PMEPA1 mRNA expression in tumor tissues was significantly increased compared with controls and not correlated with protein levels in the blood of paired HCC patients. SKIL and PMEPA1 mRNA levels were positively correlated with TGF-ß1 mRNA concentrations in HCC tissues and strongly downregulated by galunisertib. The target genes identified here may serve as biomarkers for the stratification of HCC patients undergoing treatment with galunisertib.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Pirazóis/farmacologia , Quinolinas/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/genética , Transcriptoma/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Perfilação da Expressão Gênica/métodos , Células Hep G2 , Humanos , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transcriptoma/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
20.
Hepatology ; 64(6): 2103-2117, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27639064

RESUMO

In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of α3ß1 and α6ß4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both α3ß1 and α6ß4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing α6ß4 but lacking the α3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B α3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. CONCLUSION: This study unveils a novel mechanism of sorafenib resistance depending on the α3ß1/Ln-332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016;64:2103-2117).


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteína-Tirosina Quinases de Adesão Focal/fisiologia , Células Estreladas do Fígado/fisiologia , Integrina alfa3/fisiologia , Laminina/fisiologia , Neoplasias Hepáticas/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Ubiquitinação , Humanos , Niacinamida/uso terapêutico , Sorafenibe , Células Tumorais Cultivadas
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