[Pemetrexed + Sorafenib] lethality is increased by inhibition of ERBB1/2/3-PI3K-NFκB compensatory survival signaling.

In the completed phase I trial NCT01450384 combining the anti-folate pemetrexed and the multi-kinase inhibitor sorafenib it was observed that 20 of 33 patients had prolonged stable disease or tumor regression, with one complete response and multiple partial responses. The pre-clinical studies in this manuscript were designed to determine whether [pemetrexed + sorafenib] -induced cell killing could be rationally enhanced by additional signaling modulators. Multiplex assays performed on tumor material that survived and re-grew after [pemetrexed + sorafenib] exposure showed increased phosphorylation of ERBB1 and of NFκB and IκB; with reduced IκB and elevated G-CSF and KC protein levels. Inhibition of JAK1/2 downstream of the G-CSF/KC receptors did not enhance [pemetrexed + sorafenib] lethality whereas inhibition of ERBB1/2/4 using kinase inhibitory agents or siRNA knock down of ERBB1/2/3 strongly promoted killing. Inhibition of ERBB1/2/4 blocked [pemetrexed + sorafenib] stimulated NFκB activation and SOD2 expression; and expression of IκB S32A S36A significantly enhanced [pemetrexed + sorafenib] lethality. Sorafenib inhibited HSP90 and HSP70 chaperone ATPase activities and reduced the interactions of chaperones with clients including c-MYC, CDC37 and MCL-1. In vivo, a 5 day transient exposure of established mammary tumors to lapatinib or vandetanib significantly enhanced the anti-tumor effect of [pemetrexed + sorafenib], without any apparent normal tissue toxicities. Identical data to that in breast cancer were obtained in NSCLC tumors using the ERBB1/2/4 inhibitor afatinib. Our data argue that the combination of pemetrexed, sorafenib and an ERBB1/2/4 inhibitor should be explored in a new phase I trial in solid tumor patients.

OSU-03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood-Brain Barrier: Implications for Anti-Cancer Therapies.

We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality.

Nexavar/Stivarga and viagra interact to kill tumor cells.

We determined whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with phosphodiesterase 5 (PDE5) inhibitors such as Viagra (sildenafil) to kill tumor cells. PDE5 and PDGFRα/ß were over-expressed in liver tumors compared to normal liver tissue. In multiple cell types in vitro sorafenib/regorafenib and PDE5 inhibitors interacted in a greater than additive fashion to cause tumor cell death, regardless of whether cells were grown in 10 or 100% human serum. Knock down of PDE5 or of PDGFRα/ß recapitulated the effects of the individual drugs. The drug combination increased ROS/RNS levels that were causal in cell killing. Inhibition of CD95/FADD/caspase 8 signaling suppressed drug combination toxicity. Knock down of ULK-1, Beclin1, or ATG5 suppressed drug combination lethality. The drug combination inactivated ERK, AKT, p70 S6K, and mTOR and activated JNK. The drug combination also reduced mTOR protein expression. Activation of ERK or AKT was modestly protective whereas re-expression of an activated mTOR protein or inhibition of JNK signaling almost abolished drug combination toxicity. Sildenafil and sorafenib/regorafenib interacted in vivo to suppress xenograft tumor growth using liver and colon cancer cells. From multiplex assays on tumor tissue and plasma, we discovered that increased FGF levels and ERBB1 and AKT phosphorylation were biomarkers that were directly associated with lower levels of cell killing by 'rafenib + sildenafil. Our data are now being translated into the clinic for further determination as to whether this drug combination is a useful anti-tumor therapy for solid tumor patients.

Parallel evolution of tetrodotoxin resistance in three voltage-gated sodium channel genes in the garter snake Thamnophis sirtalis.

Members of a gene family expressed in a single species often experience common selection pressures. Consequently, the molecular basis of complex adaptations may be expected to involve parallel evolutionary changes in multiple paralogs. Here, we use bacterial artificial chromosome library scans to investigate the evolution of the voltage-gated sodium channel (Nav) family in the garter snake Thamnophis sirtalis, a predator of highly toxic Taricha newts. Newts possess tetrodotoxin (TTX), which blocks Nav's, arresting action potentials in nerves and muscle. Some Thamnophis populations have evolved resistance to extremely high levels of TTX. Previous work has identified amino acid sites in the skeletal muscle sodium channel Nav1.4 that confer resistance to TTX and vary across populations. We identify parallel evolution of TTX resistance in two additional Nav paralogs, Nav1.6 and 1.7, which are known to be expressed in the peripheral nervous system and should thus be exposed to ingested TTX. Each paralog contains at least one TTX-resistant substitution identical to a substitution previously identified in Nav1.4. These sites are fixed across populations, suggesting that the resistant peripheral nerves antedate resistant muscle. In contrast, three sodium channels expressed solely in the central nervous system (Nav1.1-1.3) showed no evidence of TTX resistance, consistent with protection from toxins by the blood-brain barrier. We also report the exon-intron structure of six Nav paralogs, the first such analysis for snake genes. Our results demonstrate that the molecular basis of adaptation may be both repeatable across members of a gene family and predictable based on functional considerations.

Rapid evolution of enormous, multichromosomal genomes in flowering plant mitochondria with exceptionally high mutation rates.

Genome size and complexity vary tremendously among eukaryotic species and their organelles. Comparisons across deeply divergent eukaryotic lineages have suggested that variation in mutation rates may explain this diversity, with increased mutational burdens favoring reduced genome size and complexity. The discovery that mitochondrial mutation rates can differ by orders of magnitude among closely related angiosperm species presents a unique opportunity to test this hypothesis. We sequenced the mitochondrial genomes from two species in the angiosperm genus Silene with recent and dramatic accelerations in their mitochondrial mutation rates. Contrary to theoretical predictions, these genomes have experienced a massive proliferation of noncoding content. At 6.7 and 11.3 Mb, they are by far the largest known mitochondrial genomes, larger than most bacterial genomes and even some nuclear genomes. In contrast, two slowly evolving Silene mitochondrial genomes are smaller than average for angiosperms. Consequently, this genus captures approximately 98% of known variation in organelle genome size. The expanded genomes reveal several architectural changes, including the evolution of complex multichromosomal structures (with 59 and 128 circular-mapping chromosomes, ranging in size from 44 to 192 kb). They also exhibit a substantial reduction in recombination and gene conversion activity as measured by the relative frequency of alternative genome conformations and the level of sequence divergence between repeat copies. The evolution of mutation rate, genome size, and chromosome structure can therefore be extremely rapid and interrelated in ways not predicted by current evolutionary theories. Our results raise the hypothesis that changes in recombinational processes, including gene conversion, may be a central force driving the evolution of both mutation rate and genome structure.

Maternal food provisioning in relation to condition-dependent offspring odours in burrower bugs (Sehirus cinctus).

The sensory modalities used for communication among family members have at least partly evolved within an organism's pre-existing sensory context. Given the well-known general importance of chemical communication in insects, we hypothesized in sub-social insects with parental care that chemical signals emitted by larvae to influence parental care (i.e. solicitation pheromones) would have evolved. To test this hypothesis, we performed an experiment in the burrower bug Sehirus cinctus (Hemiptera: Cydnidae) where nymphs were hand-reared under high- or low-food conditions. These hand-reared clutches were used as a source of volatiles. The volatiles were collected for chemical analysis and delivered to caring mothers to quantify their behavioural response. As predicted, mothers exposed to volatiles from nymphs in poor condition provisioned significantly more food than those exposed to air (controls) or volatiles from high-condition nymphs. Chemical analysis revealed that nymphs emitted a blend of eight compounds of which alpha-pinene and camphene showed the strongest relationship with food treatment. Exposure to pure synthetic alpha-pinene and camphene did not affect maternal provisioning, however, suggesting that the functional significance of alpha-pinene and/or camphene may occur in a blend with other compounds. This study shows a clear effect of condition-dependent offspring odours on maternal food provisioning and identifies, for the first time, candidate compounds for a potential chemical offspring begging signal.