Suppression of Hedgehog signalling promotes pro-tumourigenic integrin expression and function.

Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. αvß6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported αvß6 up-regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and αvß6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased αvß6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin-dependent TGF-ß1 activation. Gli1 inhibited αvß6 expression by suppressing TGF-ß1-induced Smad2/3 activation, blocking a positive feedback loop maintaining high αvß6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between αvß6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and αvß6 inversely correlates in tumours in vivo, and Hh targeting up-regulates TGF-ß1/Smad2/3-dependent αvß6 expression, promoting pro-tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting.

Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.

Oral submucous fibrosis (OSF) is a premalignant, fibrosing disorder of the mouth, pharynx, and oesophagus, with a malignant transformation rate of 7-13%. OSF is strongly associated with areca (betel) nut chewing and worldwide, over 5 million people are affected. As αvß6 integrin is capable of promoting both tissue fibrosis and carcinoma invasion, we examined its expression in fibroepithelial hyperplasia and OSF. αvß6 was markedly up-regulated in OSF, with high expression detected in 22 of 41 cases (p < 0.001). We investigated the functional role of αvß6 using oral keratinocyte-derived cells genetically modified to express high αvß6 (VB6), and also NTERT-immortalized oral keratinocytes, which express low αvß6 (OKF6/TERT-1). VB6 cells showed significant αvß6-dependent activation of TGF-ß1, which induced transdifferentiation of oral fibroblasts into myofibroblasts and resulted in up-regulation of genes associated with tissue fibrosis. These experimental in vitro findings were confirmed using human clinical samples, where we showed that the stroma of OSF contained myofibroblasts and that TGF-ß1-dependent Smad signalling was detectable both in keratinocytes and in myofibroblasts. We also found that arecoline, the major alkaloid of areca nuts, up-regulated keratinocyte αvß6 expression. This was modulated through the M(4) muscarinic acetylcholine receptor and was suppressed by the M(4) antagonist, tropicamide. Arecoline-dependent αvß6 up-regulation promoted keratinocyte migration and induced invasion, raising the possibility that this mechanism may support malignant transformation. Over 80% of OSF-related oral cancers examined had moderate/high αvß6 expression. These data suggest that the pathogenesis of OSF may be epithelial-driven and involve arecoline-dependent up-regulation of αvß6 integrin.

Regulation of lymphatic-blood vessel separation by endothelial Rac1.

Sprouting angiogenesis and lymphatic-blood vessel segregation both involve the migration of endothelial cells, but the precise migratory molecules that govern the decision of blood vascular endothelial cells to segregate into lymphatic vasculature are unknown. Here, we deleted endothelial Rac1 in mice (Tie1-Cre(+);Rac1(fl/fl)) and revealed, unexpectedly, that whereas blood vessel morphology appeared normal, lymphatic-blood vessel separation was impaired, with corresponding edema, haemorrhage and embryonic lethality. Importantly, normal levels of Rac1 were essential for directed endothelial cell migratory responses to lymphatic-inductive signals. Our studies identify Rac1 as a crucial part of the migratory machinery required for endothelial cells to separate and form lymphatic vasculature.

Mitochondrial involvement in aspirin-induced apoptosis in yeast.

We have previously reported that aspirin induces apoptosis in manganese superoxide dismutase (MnSOD)-deficient Saccharomyces cerevisiae cells when cultivated on the non-fermentable carbon source ethanol. Here, we investigated the role of mitochondria in aspirin-induced apoptosis. We report that aspirin had an inhibitory effect on cellular respiration, and caused the release of most of the mitochondrial cytochrome c and a dramatic drop in the mitochondrial membrane potential (DeltaPsi(m)). Also, aspirin reduced the intracellular cytosolic pH in the MnSOD-deficient cells growing in ethanol medium, but this did not seem to be the initial trigger that committed these cells to aspirin-induced apoptosis. Furthermore, loss of DeltaPsi(m) was not required for aspirin-induced release of cytochrome c, since the initial release of cytochrome c occurred prior to the disruption of the DeltaPsi(m). It is thus possible that cytochrome c release does not involve the early onset of the mitochondrial permeability transition, but only an alteration of the permeability of the outer mitochondrial membrane.

Metabolic aspects of aspirin-induced apoptosis in yeast.

We have previously shown that aspirin induces apoptosis in manganese superoxide dismutase (MnSOD)-deficient Saccharomyces cerevisiae cells cultivated in ethanol medium, and that it exhibits a significant antioxidant effect until the onset of overt apoptosis. We here report that glucose-6-phosphate dehydrogenase activity in these cells is not inhibited by aspirin. However, the reducing power, as measured by the NADPH/NADP(+) concentration ratio, is significantly lower than in wild-type cells. With aspirin, the levels of NADPH, NADP(+) and catalase in MnSOD-deficient cells decrease significantly after 72 h of cultivation, without significant decrease of the NADPH/NADP(+) ratio. This ratio is higher when the cells are grown in glycerol or acetate medium. This seems to prevent loss in viability and induction of apoptosis on treatment with aspirin. Additionally, the glutathione (GSH) level is maintained, but the level of oxidized glutathione (GSSG) increases, leading to a significant decrease in the GSH/GSSG ratio in aspirin-treated cells. This decrease in the GSH/GSSG ratio is much less in cells grown in glycerol medium, while there is an increase in the GSH/GSSG ratio of cells grown in acetate medium. Consequently, the decreased reducing power may be linked to apoptotic induction by aspirin. This occurs independently of the level of reactive oxygen species which, as shown in our previous studies, do not play a primary role in the apoptosis of cells exposed to aspirin. The protective effect of MnSOD appears to be related to the cellular reducing power.

Aspirin commits yeast cells to apoptosis depending on carbon source.

The effect of aspirin on the growth of a wild-type Saccharomyces cerevisiae strain (EG103), containing both copper,zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD), a strain deficient in MnSOD (EG110) and a strain deficient in CuZnSOD (EG118) was measured in media containing different carbon sources. Aspirin inhibited the fermentative growth of all three strains in glucose medium. It inhibited the non-fermentative growth of the MnSOD-deficient strain very drastically in ethanol medium and had no effect on this strain in glycerol or acetate medium. The non-fermentative growth of the other two strains was not affected by aspirin. The growth inhibition of strain EG110 was associated with early necrosis in glucose medium and late apoptosis in ethanol medium. The apoptosis was preceded by a pronounced loss of cell viability. The growth inhibitory effect of aspirin was not reversed by the antioxidants N-acetylcysteine and vitamin E. Furthermore, aspirin itself appeared to act as an antioxidant until the onset of overt apoptosis, when a moderate increase in the intracellular oxidation level occurred. This suggested that reactive oxygen species probably do not play a primary role in the apoptosis of cells exposed to aspirin.