Not surprisingly, the death of a cell is a complex and well controlled process. For several decades, apoptosis, the first genetically programmed death process to be identified has taken centre stage as the principal mechanism of programmed cell death (type I cell death) in mammalian tissues. Apoptosis has been extensively studied and its contribution to the pathogenesis of disease well documented. However, apoptosis does not function alone in determining the fate of a cell. More recently, autophagy, a process in which de novo formed membrane enclosed vesicles engulf and consume cellular components, has been shown to engage in complex interplay with apoptosis. As a result, cell death has been subdivided into the categories apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). The boundary between Type I and II cell death is not completely clear and as we will discuss in this review and perhaps a discrete difference does not exist, due to intrinsic factors among different cell types and crosstalk among organelles within each cell type. Apoptosis may begin with autophagy and autophagy can often end with apoptosis, inhibition or a blockade of caspase activity may lead a cell to default into Type II cell death from Type I.

Patients whose NSCLC tumors become afatinib resistant presently have few effective therapeutic options to extend their survival. Afatinib resistant NSCLC cells were sensitive to clinically relevant concentrations of the irreversible pan-HER inhibitor neratinib, but not by the first generation ERBB1/2/4 inhibitor lapatinib. In multiple afatinib resistant NSCLC clones, HDAC inhibitors reduced the expression of ERBB1/3/4, but activated c-SRC, which resulted in higher total levels of ERBB1/3 phosphorylation. Neratinib also rapidly reduced the expression of ERBB1/2/3/4, c-MET and of mutant K-/N-RAS; K-RAS co-localized with phosphorylated ATG13 and with cathepsin B in vesicles. Combined exposure of cells to [neratinib + HDAC inhibitors] caused inactivation of mTORC1 and mTORC2, enhanced autophagosome and subsequently autolysosome formation, and caused an additive to greater than additive induction of cell death. Knock down of Beclin1 or ATG5 prevented HDAC inhibitors or neratinib from reducing ERBB1/3/4 and K-/N-RAS expression and reduced [neratinib + HDAC inhibitor] lethality. Neratinib and HDAC inhibitors reduced the expression of multiple HDAC proteins via autophagy that was causal in the reduced expression of PD-L1, PD-L2 and ornithine decarboxylase, and increased expression of Class I MHCA. In vivo, neratinib and HDAC inhibitors interacted to suppress the growth of 4T1 mammary tumors, an effect that was enhanced by an anti-PD-1 antibody. Our data support the premises that neratinib lethality can be enhanced by HDAC inhibitors, that neratinib may be a useful therapeutic tool in afatinib resistant NSCLC, and that [neratinib + HDAC inhibitor] exposure facilitates anti-tumor immune responses.

We focused on the ability of the pan-histone deacetylase (HDAC) inhibitors AR42 and sodium valproate to alter the immunogenicity of melanoma cells. Treatment of melanoma cells with HDAC inhibitors rapidly reduced the expression of multiple HDAC proteins as well as the levels of PD-L1, PD-L2 and ODC, and increased expression of MHCA. In a cell-specific fashion, melanoma isolates released the immunogenic protein HMGB1 into the extracellular environment. Very similar data were obtained in ovarian and H&NSCC PDX isolates, and in established tumor cell lines from the lung and kidney. Knock down of HDAC1, HDAC3, HDAC8 and HDAC10, but not HDAC6, recapitulated the effects of the HDAC inhibitors on the immunotherapy biomarkers. Using B16 mouse melanoma cells we discovered that pre-treatment with AR42 or sodium valproate enhanced the anti-tumor efficacy of an anti-PD-1 antibody and of an anti-CTLA4 antibody. In the B16 model, both AR42 and sodium valproate enhanced the anti-tumor efficacy of the multi-kinase inhibitor pazopanib. In plasma from animals exposed to [HDAC inhibitor + anti-PD-1], but not [HDAC inhibitor + anti-CTLA4], the levels of CCL2, CCL5, CXCL9 and CXCL2 were increased. The cytokine data from HDAC inhibitor plus anti-PD-1 exposed tumors correlated with increased activated T cell, M1 macrophage, neutrophil and NK cell infiltration. Collectively, our data support the use of pan-HDAC inhibitors in combination with kinase inhibitors or with checkpoint inhibitor antibodies as novel melanoma therapeutic strategies.

Prior studies demonstrated that the irreversible ERBB1/2/4 inhibitor neratinib caused plasma membrane-associated mutant K-RAS to localize in intracellular vesicles, concomitant with its degradation. Herein, we discovered that neratinib interacted with the chemically distinct irreversible ERBB1/2/4 inhibitor afatinib to reduce expression of ERBB1, ERBB2, K-RAS and N-RAS; this was associated with greater-than-additive cell killing of pancreatic tumor cells. Knock down of Beclin1, ATG16L1, Rubicon or cathepsin B significantly lowered the ability of neratinib to reduce ERBB1 and K-RAS expression, and to cause tumor cell death. Knock down of ATM-AMPK suppressed vesicle formation and knock down of cathepsin B-AIF significantly reduced neratinib lethality. PKG phosphorylates K-RAS and HMG CoA reductase inhibitors reduce K-RAS farnesylation both of which remove K-RAS from the plasma membrane, abolishing its activity. Neratinib interacted with the PKG activator sildenafil and the HMG CoA reductase inhibitor atorvastatin to further reduce K-RAS expression, and to further enhance cell killing. Neratinib is also a Ste20 kinase family inhibitor and in carcinoma cells, and hematopoietic cancer cells lacking ERBB1/2/4, it reduced K-RAS expression and the phosphorylation of MST1/3/4/ Ezrin by ~30%. Neratinib increased LATS1 phosphorylation as well as that of YAP and TAZ also by ~30%, caused the majority of YAP to translocate into the cytosol and reduced YAP/TAZ #

The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70, HSP90, GRP94, GRP58, HSP27, HSP40 and HSP60. OSU-03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1, receptors for Ebola/Marburg/Hepatitis A, Lassa fever, and Hepatitis B viruses, respectively. Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya, Mumps, Measles, Rubella, RSV, CMV, and Influenza viruses. OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU-03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU-03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus, Dna K and bacterial phosphodiesterases are novel antibiotic targets, and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections. J. Cell. Physiol. 230: 1661–1676, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

We performed proteomic studies using the GRP78 chaperone-inhibitor drug AR-12 (OSU-03012) as bait. Multiple additional chaperone and chaperone-associated proteins were shown to interact with AR-12, including: GRP75, HSP75, BAG2; HSP27; ULK-1; and thioredoxin. AR-12 down-regulated in situ immuno-fluorescence detection of ATP binding chaperones using antibodies directed against the NH2-termini of the proteins but only weakly reduced detection using antibodies directed against the central and COOH portions of the proteins. Traditional SDS-PAGE and western blotting assessment methods did not exhibit any alterations in chaperone detection. AR-12 altered the sub-cellular distribution of chaperone proteins, abolishing their punctate speckled patterning concomitant with changes in protein co-localization. AR-12 inhibited chaperone ATPase activity, which was enhanced by sildenafil; inhibited chaperone – chaperone and chaperone – client interactions; and docked in silico with the ATPase domains of HSP90 and of HSP70. AR-12 combined with sildenafil in a GRP78 plus HSP27 –dependent fashion to profoundly activate an eIF2α/ATF4/CHOP/Beclin1 pathway in parallel with inactivating mTOR and increasing ATG13 phosphorylation, collectively resulting in formation of punctate toxic autophagosomes. Over-expression of [GRP78 and HSP27] prevented: AR-12 –induced activation of ER stress signaling and maintained mTOR activity; AR-12 –mediated down-regulation of thioredoxin, MCL-1 and c-FLIP-s; and preserved tumor cell viability. Thus the inhibition of chaperone protein functions by AR-12 and by multi-kinase inhibitors very likely explains why these agents have anti-tumor effects in multiple genetically diverse tumor cell types.

Epidemiological studies have clearly demonstrated a link between dietary carotenoids and the reduced incidence of certain diseases, including some cancers. However recent intervention studies (e.g. ATBC, CARET and others) have shown that beta-carotene supplementation has little or no beneficial effect and may, in fact, increase the incidence of lung cancers in smokers. This presents a serious dilemma for the scientific community - are carotenoids at high concentrations actually harmful in certain circumstances? Currently, a significant number of intervention studies are on-going throughout the world involving carotenoids (of both natural and synthetic origin). Our approach has been to study the ability of supplementary carotenoids in protecting cells against oxidatively-induced DNA damage (as measured by the comet assay), and membrane integrity (as measured by ethidium bromide uptake). Both lycopene and beta-carotene only afforded protection against DNA damage (induced by xanthine/xanthine oxidase) at relatively low concentrations (1-3 microM). These levels are comparable with those seen in the plasma of individuals who consume a carotenoid-rich diet. However, at higher concentrations (4-10 microM), the ability to protect the cell against such oxidative damage was rapidly lost and, indeed, the presence of carotenoids may actually serve to increase the extent of DNA damage. Similar data were obtained when protection against membrane damage was studied. This would suggest that supplementation with individual carotenoids to significantly elevate blood and tissue levels is of little benefit and, may, in fact, be deleterious. This in vitro data presented maybe significant in the light of recent intervention trials.

Studies focused on the killing of activated B-RAF melanoma cells by the histone deacetylase (HDAC) inhibitor AR42. Compared to other tumor cell lines, PDX melanoma isolates were significantly more sensitive to AR42-induced killing. AR42 and the multi-kinase inhibitor pazopanib interacted to activate: an eIF2α–Beclin1 pathway causing autophagosome formation; an eIF2α–DR4/DR5/CD95 pathway; and an eIF2α-dependent reduction in the expression of c-FLIP-s, MCL-1 and BCL-XL. AR42 did not alter basal chaperone activity but increased the ability of pazopanib to inhibit HSP90, HSP70 and GRP78. AR42 and pazopanib caused HSP90/HSP70 dissociation from RAF-1 and B-RAF that resulted in reduced ‘RAF’ expression. The drug combination activated a DNA-damage-ATM-AMPK pathway that was associated with: NFκB activation; reduced mTOR S2448 and ULK-1 S757 phosphorylation; and increased ULK-1 S317 and ATG13 S318 phosphorylation. Knock down of PERK, eIF2α, Beclin1, ATG5 or AMPKα, or expression of IκB S32A S36A, ca-mTOR or TRX, reduced cell killing. AR42, via lysosomal degradation, reduced the protein expression of HDACs 2/5/6/10/11. In vivo, a 3-day exposure of dabrafenib/trametinib resistant melanoma cells to the AR42 pazopanib combination reduced tumor growth and enhanced survival from ∼25 to ∼40 days. Tumor cells that had adapted through therapy exhibited elevated HGF expression and the c-MET inhibitor crizotinib enhanced AR42 pazopanib lethality in this evolved drug-resistant population.