Pancreatic ductal adenocarcinomas contain a subset of exclusively tumorigenic cancer stem cells (CSCs), which are capable of repopulating the entire heterogeneous cancer cell populations and are ...highly resistant to standard chemotherapy. Here we demonstrate that metformin selectively ablated pancreatic CSCs as evidenced by diminished expression of pluripotency-associated genes and CSC-associated surface markers. Subsequently, the ability of metformin-treated CSCs to clonally expand in vitro was irreversibly abrogated by inducing apoptosis. In contrast, non-CSCs preferentially responded by cell cycle arrest, but were not eliminated by metformin treatment. Mechanistically, metformin increased reactive oxygen species production in CSC and reduced their mitochondrial transmembrane potential. The subsequent induction of lethal energy crisis in CSCs was independent of AMPK/mTOR. Finally, in primary cancer tissue xenograft models metformin effectively reduced tumor burden and prevented disease progression; if combined with a stroma-targeting smoothened inhibitor for enhanced tissue penetration, while gemcitabine actually appeared dispensable.
Background/Aims The transforming growth factor-beta (TGF-β) induces apoptosis in hepatocytes through an oxidative stress process. Here, we have analyzed the role of different NADPH oxidase isoforms ...in the intracellular signalling induced by TGF-β in hepatocytes, to later explore whether this mechanism is altered in liver tumor cells. Methods Primary cultures of rat and human hepatocytes, HepG2 and Hep3B cells were used in in vitro studies to analyze the TGF-β response. Results TGF-β-induced apoptosis in rat hepatocytes does not require Rac-dependent NADPH oxidases. TGF-β upregulates the Rac-independent Nox4, which correlates with its pro-apoptotic activity. Regulation of Nox4 occurs at the transcriptional level and is counteracted by intracellular survival signals. siRNA targeted knock-down of Nox4 attenuates NADPH oxidase activity, caspase activation and cell death in rat hepatocytes. NOX4 upregulation by TGF-β is also observed in human hepatocytes, coincident with apoptosis. In human hepatocellular carcinoma (HCC) cell lines, NOX4 upregulation by TGF-β is only observed in cells that are sensitive to its cytotoxic effect, such as Hep3B cells. siRNA targeted knock-down of NOX4 in these cells impairs TGF-β-induced apoptosis. Conclusions Upregulation of NOX4 by TGF-β is required for its pro-apoptotic activity in hepatocytes. Impairment of this TGF-β-induced response might confer apoptosis resistance in HCC cells.
The multikinase inhibitor sorafenib is the only effective drug in advanced cases of hepatocellular carcinoma (HCC). However, response differs among patients and effectiveness only implies a delay. We ...have recently described that sorafenib sensitizes HCC cells to apoptosis. In this work, we have explored the response to this drug of six different liver tumor cell lines to define a phenotypic signature that may predict lack of response in HCC patients. Results have indicated that liver tumor cells that show a mesenchymal‐like phenotype, resistance to the suppressor effects of transforming growth factor beta (TGF‐β) and high expression of the stem cell marker CD44 were refractory to sorafenib‐induced cell death in in vitro studies, which correlated with lack of response to sorafenib in nude mice xenograft models of human HCC. In contrast, epithelial‐like cells expressing the stem‐related proteins EpCAM or CD133 were sensitive to sorafenib‐induced apoptosis both in vitro and in vivo. A cross‐talk between the TGF‐β pathway and the acquisition of a mesenchymal‐like phenotype with up‐regulation of CD44 expression was found in the HCC cell lines. Targeted CD44 knock‐down in the mesenchymal‐like cells indicated that CD44 plays an active role in protecting HCC cells from sorafenib‐induced apoptosis. However, CD44 effect requires a TGF‐β‐induced mesenchymal background, since the only overexpression of CD44 in epithelial‐like HCC cells is not sufficient to impair sorafenib‐induced cell death. In conclusion, a mesenchymal profile and expression of CD44, linked to activation of the TGF‐β pathway, may predict lack of response to sorafenib in HCC patients.
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Sorafenib remains the standard of care for advanced hepatocellular carcinoma (HCC), despite marked variability in patient response. According to this study, a lack of response to sorafenib may be predicted by certain phenotypic signatures of liver tumor cells, specifically activation of the TGF‐ß pathway, a mesenchymal phenotype, and expression of CD44. By contrast, sensitivity to sorafenib‐induced apoptosis is associated with expression of the stem‐related proteins EpCAM or CD133 in epithelial‐like cells and knockdown of CD44 in mesenchymal‐like cells. The findings suggest that CD44 targeting could be a valuable strategy to deploy in combination with sorafenib therapy.
The NADPH oxidase NOX4 has emerged as an important source of reactive oxygen species in signal transduction, playing roles in physiological and pathological processes. NOX4 mediates transforming ...growth factor-β-induced intracellular signals that provoke liver fibrosis, and preclinical assays have suggested NOX4 inhibitors as useful tools to ameliorate this process. However, the potential consequences of sustained treatment of liver cells with NOX4 inhibitors are yet unknown. The aim of this work was to analyze whether NOX4 plays a role in regulating liver cell growth either under physiological conditions or during tumorigenesis. In vitro assays proved that stable knockdown of NOX4 expression in human liver tumor cells increased cell proliferation, which correlated with a higher percentage of cells in S/G2/M phases of the cell cycle, downregulation of p21(CIP1/WAF1), increase in cyclin D1 protein levels, and nuclear localization of β-catenin. Silencing of NOX4 in untransformed human and mouse hepatocytes also increased their in vitro proliferative capacity. In vivo analysis in mice revealed that NOX4 expression was downregulated under physiological proliferative situations of the liver, such as regeneration after partial hepatectomy, as well as during pathological proliferative conditions, such as diethylnitrosamine-induced hepatocarcinogenesis. Xenograft experiments in athymic mice indicated that NOX4 silencing conferred an advantage to human hepatocarcinoma cells, resulting in earlier onset of tumor formation and increase in tumor size. Interestingly, immunochemical analyses of NOX4 expression in human liver tumor cell lines and tissues revealed decreased NOX4 protein levels in liver tumorigenesis. Overall, results described here strongly suggest that NOX4 would play a growth-inhibitory role in liver cells.
Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is an extremely lethal disease due to late diagnosis, aggressiveness and lack of effective therapies. Considering ...its intrinsic heterogeneity, patient stratification models based on transcriptomic and genomic signatures, with partially overlapping subgroups, have been established. Besides molecular alterations, PDAC tumours show a strong desmoplastic response, resulting in profound metabolic reprogramming involving increased glucose and amino acid consumption, as well as lipid scavenging and biosynthesis. Interestingly, recent works have also revealed the existence of metabolic subtypes with differential prognosis within PDAC, which correlated to defined molecular subclasses in patients: lipogenic subtype correlated with a classical/progenitor signature, while glycolytic tumours associated with the highly aggressive basal/squamous profile. Bioinformatic analyses have demonstrated that the representative genes of each metabolic subtype are up-regulated in PDAC samples and predict patient survival. This suggests a relationship between the genetic signature, metabolic profile, and aggressiveness of the tumour. Considering all this, defining metabolic subtypes represents a clear opportunity for patient stratification considering tumour functional behaviour independently of their mutational background.
Background & Aims Although smoking is a leading risk factor for pancreatic ductal adenocarcinoma (PDAC), little is known about the mechanisms by which smoking promotes initiation or progression of ...PDAC. Methods We studied the effects of nicotine administration on pancreatic cancer development in Kras +/LSLG12Vgeo ;Elas-tTA/tetO-Cre (Ela-KRAS) mice, Kras +/LSLG12D ;Trp53+/LSLR172H;Pdx-1-Cre (KPC) mice (which express constitutively active forms of KRAS), and C57/B6 mice. Mice were given nicotine for up to 86 weeks to produce blood levels comparable with those of intermediate smokers. Pancreatic tissues were collected and analyzed by immunohistochemistry and reverse transcriptase polymerase chain reaction; cells were isolated and assayed for colony and sphere formation and gene expression. The effects of nicotine were also evaluated in primary pancreatic acinar cells isolated from wild-type, nAChR7a−/− , Trp53−/− , and Gata6−/− ; Trp53−/− mice. We also analyzed primary PDAC cells that overexpressed GATA6 from lentiviral expression vectors. Results Administration of nicotine accelerated transformation of pancreatic cells and tumor formation in Ela-KRAS and KPC mice. Nicotine induced dedifferentiation of acinar cells by activating AKT–ERK–MYC signaling; this led to inhibition of Gata6 promoter activity, loss of GATA6 protein, and subsequent loss of acinar differentiation and hyperactivation of oncogenic KRAS. Nicotine also promoted aggressiveness of established tumors as well as the epithelial–mesenchymal transition, increasing numbers of circulating cancer cells and their dissemination to the liver, compared with mice not exposed to nicotine. Nicotine induced pancreatic cells to acquire gene expression patterns and functional characteristics of cancer stem cells. These effects were markedly attenuated in K-Ras+/LSL-G12D ; Trp53+/LSLR172H ; Pdx-1-Cre mice given metformin. Metformin prevented nicotine-induced pancreatic carcinogenesis and tumor growth by up-regulating GATA6 and promoting differentiation toward an acinar cell program. Conclusions In mice, nicotine promotes pancreatic carcinogenesis and tumor development via down-regulation of Gata6 to induce acinar cell dedifferentiation.
Malignant cells are commonly characterised by being capable of invading tissue, growing self-sufficiently and uncontrollably, being insensitive to apoptosis induction and controlling their ...environment, for example inducing angiogenesis. Amongst them, a subpopulation of cancer cells, called cancer stem cells (CSCs) shows sustained replicative potential, tumor-initiating properties and chemoresistance. These characteristics make CSCs responsible for therapy resistance, tumor relapse and growth in distant organs, causing metastatic dissemination. For these reasons, eliminating CSCs is necessary in order to achieve long-term survival of cancer patients. New insights in cancer metabolism have revealed that cellular metabolism in tumors is highly heterogeneous and that CSCs show specific metabolic traits supporting their unique functionality. Indeed, CSCs adapt differently to the deprivation of specific nutrients that represent potentially targetable vulnerabilities. This review focuses on three of the most aggressive tumor types: pancreatic ductal adenocarcinoma (PDAC), hepatocellular carcinoma (HCC) and glioblastoma (GBM). The aim is to prove whether CSCs from different tumour types share common metabolic requirements and responses to nutrient starvation, by outlining the diverse roles of glucose and amino acids within tumour cells and in the tumour microenvironment, as well as the consequences of their deprivation. Beyond their role in biosynthesis, they serve as energy sources and help maintain redox balance. In addition, glucose and amino acid derivatives contribute to immune responses linked to tumourigenesis and metastasis. Furthermore, potential metabolic liabilities are identified and discussed as targets for therapeutic intervention.
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical ...properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size.
•Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death.•Physico-chemical characterization of the tumour microenvironment of PDAC is lacking.•Patient-derived PDAC xenografts decellularized using a design of experiments approach.•Potential therapeutic strategy based on the size of the therapeutic agent.•Availability of certain molecular groups as cells are eliminated from the tumour.
The objective of this work was to analyze the possible association between cyclooxygenase-2 (COX-2) and NADPH oxidases (NOX) in liver cells, in response to various proinflammatory and toxic insults. ...First, we observed that treatment of Chang liver (CHL) cells with various COX-2 inducers increased reactive oxygen species (ROS) production concomitant with GSH depletion, phorbol 12-myristate 13-acetate (PMA) being the most effective treatment. Moreover, early changes in the oxidative status induced by PMA were inhibited by glutathione ethyl ester, which also impeded COX-2 induction. In fact, CHL cells expressed NOX1 and NOX4, although only NOX4 expression was up-regulated in the presence of PMA. Knock-down experiments suggested that PMA initiated a pathway in which NOX1 activation controlled COX-2 expression and activity, which, in turn, induced NOX4 expression by activation of the prostaglandin receptor EP4. In addition, CHL cells overexpressing COX-2 showed higher NOX4 expression and ROS content, which were decreased in the presence of the COX-2 inhibitor DFU. Interestingly, we found that addition of prostaglandin E2 (PGE2) also induced NOX4 expression and ROS production, which might promote cell adhesion. Finally, we determined that NOX4 induction by PGE2 was dependent on ERK1/2 signaling. Taken together, these results indicate that NOX proteins and COX-2 are reciprocally regulated in liver cells.
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► We wanted to analyze if COX-2 and NOX proteins are interconnected in liver cells. ► Using PMA as COX-2 inducer, we found that ROS production by NOX1 was necessary to up-regulate COX-2. ► COX-2 –produced PGE2 activated the PG receptor EP4 to induce NOX4 expression. ► Exogenous PGE2 also induced NOX4, found to be ERK1/2 dependent. ► NOX4 downstream COX-2 might promote cell adhesion to different substrates.
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