Pre-clinical and retrospective studies of patients using statins to reduce plasma cholesterol have suggested that statins may be useful to treat cancer. However, prospective clinical trials have yet to demonstrate significant efficacy. We have previously shown that this is in part because a hydrophobic statin with a long half-life is necessary. Pitavastatin, the only statin with this profile, has not undergone clinical evaluation in oncology. The target of pitavastatin, hydroxymethylglutarate coenzyme-A reductase (HMGCR), was found to be over-expressed in all ovarian cancer cell lines examined and upregulated by mutated TP53, a gene commonly altered in ovarian cancer. Pitavastatin-induced apoptosis was blocked by geranylgeraniol and mevalonate, products of the HMGCR pathway, confirming that pitavastatin causes cell death through inhibition of HMGCR. Solvent extracts of human and mouse food were also able to block pitavastatin-induced apoptosis, suggesting diet might influence the outcome of clinical trials. When nude mice were maintained on a diet lacking geranylgeraniol, oral pitavastatin caused regression of Ovcar-4 tumour xenografts. However, when the animal diet was supplemented with geranylgeraniol, pitavastatin failed to prevent tumour growth. This suggests that a diet containing geranylgeraniol can limit the anti-tumour activity of pitavastatin and diet should be controlled in clinical trials of statins.

A subclade of connexins comprising Cx26, Cx30, and Cx32 are directly sensitive to CO 2 . CO 2 binds to a carbamylation motif present in these connexins and causes their hemichannels to open. Cx26 may contribute to CO 2 -dependent regulation of breathing in mammals. Here, we show that the carbamylation motif occurs in a wide range of non-mammalian vertebrates and was likely present in the ancestor of all gnathostomes. While the carbamylation motif is essential for connexin CO 2 -sensitivity, it is not sufficient. In Cx26 of amphibia and lungfish, an extended C-terminal tail prevents CO 2 -evoked hemichannel opening despite the presence of the motif. Although Cx32 has a long C-terminal tail, Cx32 hemichannels open to CO 2 because the tail is conformationally restricted by the presence of proline residues. The loss of the C-terminal tail of Cx26 in amniotes was an evolutionary innovation that created a connexin hemichannel with CO 2 -sensing properties suitable for the regulation of breathing.

Statins are widely used to treat hypercholesterolaemia. However, by inhibiting the production of mevalonate, they also reduce the production of several isoprenoids that are necessary for the function of small GTPase oncogenes such as Ras. As such, statins offer an attractive way to inhibit an "undruggable" target, suggesting that they may be usefully repurposed to treat cancer. However, despite numerous studies, there is still no consensus whether statins are useful in the oncology arena. Numerous preclinical studies have provided evidence justifying the evaluation of statins in cancer patients. Some retrospective studies of patients taking statins to control cholesterol have identified a reduced risk of cancer mortality. However, prospective clinical studies have mostly not been successful. We believe that this has occurred because many of the prospective clinical trials have been poorly designed. Many of these trials have failed to take into account some or all of the factors identified in preclinical studies that are likely to be necessary for statins to be efficacious. We suggest an improved trial design which takes these factors into account. Importantly, we suggest that the design of clinical trials of drugs which are being considered for repurposing should not assume it is appropriate to use them in the same way as they are used in their original indication. Rather, such trials deserve to be informed by preclinical studies that are comparable to those for any novel drug.

Connexin26 (Cx26) mutations underlie human pathologies ranging from hearing loss to keratitis ichthyosis deafness (KID) syndrome. Cx26 hemichannels are directly gated by CO 2 and contribute to the chemosensory regulation of breathing. The KID syndrome mutation A88V is insensitive to CO 2, and has a dominant negative action on the CO 2 sensitivity of Cx26WT hemichannels, and reduces respiratory drive in humans. We have now examined the effect of further human mutations of Cx26 on its sensitivity to CO 2 . Mutated Cx26 subunits, carrying one of A88S, N14K, N14Y, M34T, or V84L, were transiently expressed in HeLa cells. The CO 2‐dependence of hemichannel activity, and their ability to exert dominant negative actions on cells stably expressing Cx26WT, was quantified by a dye‐loading assay. The KID syndrome mutation, N14K, abolished the sensitivity of Cx26 to CO 2. Both N14Y and N14K exerted a powerful dominant negative action on the CO 2 sensitivity of Cx26WT. None of the other mutations (all recessive) had a dominant negative action. A88S shifted the affinity of Cx26 to slightly higher levels without reducing its ability to fully open to CO 2. M34T did not change the affinity of Cx26 for CO 2 but reduced its ability to open in response to CO 2. V84L had no effect on the CO 2‐sensitivity of Cx26. Some pathological mutations of Cx26 can therefore alter the CO 2 sensitivity of Cx26 hemichannels. The loss of CO 2 sensitivity could contribute to pathology and consequent reduced respiratory drive could be an unrecognized comorbidity of these pathologies.

The Cx26 mRNA has not been reported to undergo alternative splicing. In expressing a series of human keratitis ichthyosis deafness (KID) syndrome mutations of Cx26 (A88V, N14K and A40V), we found the production of a truncated mRNA product. These mutations, although not creating a cryptic splice site, appeared to activate a pre-existing cryptic splice site. The alternative splicing of the mutant Cx26 mRNA could be prevented by mutating the predicted 3′, 5′ splice sites and the branch point. The presence of a C-terminal fluorescent protein tag (mCherry or Clover) was necessary for this alternative splicing to occur. Strangely, Cx26 A88V could cause the alternative splicing of co-expressed WT Cx26—suggesting a trans effect. The alternative splicing of Cx26 A88V caused cell death, and this could be prevented by the 3′, 5′ and branch point mutations. Expression of the KID syndrome mutants could be rescued by combining them with removal of the 5′ splice site. We used this strategy to enable expression of Cx26 A40V-5′ and demonstrate that this KID syndrome mutation removed CO 2 sensitivity from the Cx26 hemichannel. This is the fourth KID syndrome mutation found to abolish the CO 2 -sensitivity of the Cx26 hemichannel, and suggests that the altered CO ­2 -sensitivity could contribute to the pathology of this mutation. Future research on KID syndrome mutations should take care to avoid using a C-terminal tag to track cellular localization and expression or if this is unavoidable, combine this mutation with removal of the 5′ splice site.

CO2 readily combines with H2O to form and H+. Because an increase of only 100 nM in the concentration of H+ (a decrease of 0.1 unit of pH) in blood can prove fatal, the regulated excretion of CO2 during breathing is an essential life-preserving process. In rodents and humans, this vital process is mediated in part via the direct sensing of CO2 via connexin26 (Cx26). CO2 binds to hemichannels of Cx26 causing them to open and allow release of the neurotransmitter ATP. If Cx26 were to be a universal and important CO2 sensor across all homeothermic animals, then a simple hypothesis would posit that it should exhibit evolutionary adaptation in animals with different homeostatic set points for the regulation of partial pressure of arterial CO2 (PaCO2). In humans and rats, PaCO2 is regulated around a set point of 40 mmHg. By contrast, birds are able to maintain cerebral blood flow and breathing at much lower levels of PaCO2. Fossorial mammals, such as the mole rat, live exclusively underground in burrows that are both hypoxic and hypercapnic and can thrive under very hypercapnic conditions. We have therefore compared the CO2 sensitivity of Cx26 from human, chicken, rat and mole rat (Heterocephalus glaber). We find that both the affinity and cooperativity of CO2 binding to Cx26 have been subjected to evolutionary adaption in a manner consistent with the homeostatic requirements of these four species. This is analogous to the evolutionary adaptation of haemoglobin to the needs of O2 transport across the animal kingdom and supports the hypothesis that Cx26 is an important and universal CO2 sensor in homeotherms.

A moderate increase in P CO 2 (55 mmHg) closes Cx26 gap junctions. r This effect of CO 2 is independent of changes in intra-or extracellular pH. r The CO 2-dependent closing effect depends on the same residues (K125 and R104) that are required for the CO 2-dependent opening of Cx26 hemichannels. r Pathological mutations of Cx26 abolish the CO 2-dependent closing of the gap junction. r Elastic network modelling suggests that the effect of CO 2 on Cx26 hemichannels and gap junctions is mediated through changes in the lowest entropy state of the protein.

Cx26 hemichannels are opened by modest levels of CO2 (PCO2 55 mmHg) acting via a novel carbamylation mechanism to bias the hemichannel to the open state. In the hemichannel, CO2 nonenzymatically carbamylates Lys125, and the resulting carbamate forms a salt bridge to Arg104 of the neighbouring subunit. By contrast, similar modest levels of CO2 cause Cx26 gap junction closure.Gap junctions of Cx31, a beta connexin that lacks the carbamylation motif, are insensitive to these levels of CO2. Mutations of the carbamylation motif, Lys125Arg and Arg104Ala, which abrogate hemichannel opening in Cx26, also prevent gap junction closing. Elastic network modelling suggests that the lowest entropy state, when CO2 is bound, is in the open configuration for the hemichannel and the closed configuration for the gap junction Surprisingly, we conclude that the opposing actions of CO2 on Cx26 gap junctions and hemichannels results from carbamylation of the same residues.