The tumor-promoting fibrotic stroma rich in tumor-associated fibroblasts (TAF) is drawing increased therapeutic attention. Intriguingly, a trial with the antifibrotic drug nintedanib in nonsmall cell lung cancer reported clinical benefits in adenocarcinoma (ADC) but not squamous cell carcinoma (SCC), even though the stroma is fibrotic in both histotypes. Likewise, we reported that nintedanib inhibited the tumor-promoting fibrotic phenotype of TAFs selectively in ADC. Here we show that tumor fibrosis is actually higher in ADC-TAFs than SCC-TAFs in vitro and patient samples. Mechanistically, the reduced fibrosis and nintedanib response of SCC-TAFs was associated with increased promoter methylation of the profibrotic TGFb transcription factor SMAD3 compared with ADC-TAFs, which elicited a compensatory increase in TGFb1/SMAD2 activation. Consistently, forcing global DNA demethylation of SCC-TAFs with 5-AZA rescued TGFb1/SMAD3 activation, whereas genetic downregulation of SMAD3 in ADC-TAFs and control fibroblasts increased TGFb1/SMAD2 activation, and reduced their fibrotic phenotype and antitumor responses to nintedanib in vitro and in vivo. Our results also support that smoking and/or the anatomic location of SCC in the proximal airways, which are more exposed to cigarette smoke particles, may prime SCC-TAFs to stronger SMAD3 epigenetic repression, because cigarette smoke condensate selectively increased SMAD3 promoter methylation. Our results unveil that the histotype-specific regulation of tumor fibrosis in lung cancer is mediated through differential SMAD3 promoter methylation in TAFs and provide new mechanistic insights on the selective poor response of SCC-TAFs to nintedanib. Moreover, our findings support that patients with ADC may be more responsive to antifibrotic drugs targeting their stromal TGFb1/SMAD3 activation.Significance: This study implicates the selective epigenetic repression of SMAD3 in SCC-TAFs in the clinical failure of nintedanib in SCC and supports that patients with ADC may benefit from antifibrotic drugs targeting stromal TGFb1/ SMAD3.
BackgroundCellfood™ (CF) is a nutritional supplement containing deuterium sulphate, minerals, amino acids, and enzymes, with well documented antioxidant properties. Its organic and inorganic components are extracted from the red algae Lithothamnion calcareum, whose mineral extract has shown growth-inhibitory effect both on in vitro and in vivo models. The purpose of this study was to evaluate the antiproliferative effects of CF on leukemic cells. In fact, according to its capacity to modulate O2 availability and to improve mitochondrial respiratory metabolism, we wondered if CF could affect cancer cell metabolism making cells susceptible to apoptosis.MethodsThree leukemic cell lines, Jurkat, U937, and K562, were treated with CF 5 μl/ml up to 72 hours. Cell viability, apoptosis (i.e. caspase-3 activity and DNA fragmentation), hypoxia inducible factor 1 alpha (HIF-1α) concentration, glucose transporter 1 (GLUT-1) expression, lactate dehydrogenase (LDH) activity and lactate release in the culture medium were detected and compared with untreated cells.ResultsCF significantly inhibited leukemic cell viability by promoting cell apoptosis, as revealed by caspase-3 activation and DNA laddering. In particular, CF treated cells showed lower HIF-1α levels and lower GLUT-1 expression as compared to untreated cells. At the same time, CF was able to reduce LDH activity and, consequently, the amount of lactate released in the extracellular environment.ConclusionsWe supplied evidence for an antiproliferative effect of CF on leukemia cell lines by inducing cell death through an apoptotic mechanism and by altering cancer cell metabolism through HIF-1α and GLUT-1 regulation. Thanks to its antioxidative and proapoptotic properties, CF might be a good candidate for cancer prevention.
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