Cigarette smoke metabolically promotes cancer,via autophagy and premature aging in the host stromal microenvironment

Cigarette smoke has been directly implicated in the disease pathogenesis of a plethora of different human cancer subtypes, including breast cancers. The prevailing view is that cigarette smoke acts as a mutagen and DNA damaging agent in normal epithelial cells, driving tumor initiation. However, its potential negative metabolic effects on the normal stromal microenvironment have been largely ignored. Here, we propose a new mechanism by which carcinogen-rich cigarette smoke may promote cancer growth, by metabolically “fertilizing” the host microenvironment. More specifically, we show that cigarette smoke exposure is indeed sufficient to drive the onset of the cancer-associated fibroblast phenotype via the induction of DNA damage, autophagy and mitophagy in the tumor stroma. In turn, cigarette smoke exposure induces premature aging and mitochondrial dysfunction in stromal fibroblasts, leading to the secretion of high-energy mitochondrial fuels, such as L-lactate and ketone bodies. Hence, cigarette smoke induces catabolism in the local microenvironment, directly fueling oxidative mitochondrial metabolism (OXPHOS) in neighboring epithelial cancer cells, actively promoting anabolic tumor growth. Remarkably, these autophagic-senescent fibroblasts increased breast cancer tumor growth in vivo by up to 4-fold. Importantly, we show that cigarette smoke-induced metabolic reprogramming of the fibroblastic stroma occurs independently of tumor neo-angiogenesis. We discuss the possible implications of our current findings for the prevention of aging-associated human diseases and, especially, common epithelial cancers, as we show that cigarette smoke can systemically accelerate aging in the host microenvironment. Finally, our current findings are consistent with the idea that cigarette smoke induces the “reverse Warburg effect,” thereby fueling “two-compartment tumor metabolism” and oxidative mitochondrial metabolism in epithelial cancer cells.     

X-ray reflectivity studies of cPLA2{alpha}-C2 domains adsorbed onto Langmuir monolayers of SOPC

X-ray reflectivity is used to study the interaction of C2 domains of cytosolic phospholipase A(2) (cPLA(2)alpha-C2) with a Langmuir monolayer of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) supported on a buffered aqueous solution containing Ca(2+). The reflectivity is analyzed in terms of the known crystallographic structure of cPLA(2)alpha-C2 domains and a slab model representing the lipid layer to yield an electron density profile of the lipid layer and bound C2 domains. This new method of analysis determines the angular orientation and penetration depth of the cPLA(2)alpha-C2 domains bound to the SOPC monolayer, information not available from the standard slab model analysis of x-ray reflectivity. The best-fit orientation places the protein-bound Ca(2+) ions within 1 A of the lipid phosphate group (with an accuracy of +/-3 A). Hydrophobic residues of the calcium-binding loops CBL1 and CBL3 penetrate deepest into the lipid layer, with a 2 A penetration into the tailgroup region. X-ray measurements with and without the C2 domain indicate that there is a loss of electrons in the headgroup region of the lipid monolayer upon binding of the domains. We suggest that this is due to a loss of water molecules bound to the headgroup. Control experiments with a non-calcium buffer and with domain mutants confirm that the cPLA(2)alpha-C2 binding to the SOPC monolayer is Ca(2+)-dependent and that the hydrophobic residues in the calcium-binding loops are critical for membrane binding. These results indicate that an entropic component (due to water loss) as well as electrostatic and hydrophobic interactions contributes to the binding mechanism.     

Synthesis of linear and cyclic phosphopeptides as ligands for the N-terminal SH2-domain of protein tyrosine phosphatase SHP-1.

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.     

Activity of substituted thiophene sulfonamides against malarial and mammalian cyclin dependent protein kinases

Cyclin dependent protein kinases (CDKs) are pursued as drug targets for several eukaryotic pathogens. In this study, we identified thiophene and benzene sulfonamides as potent inhibitors of Pfmrk, a Plasmodium falciparum CDK with sequence homology to human CDK7. Several of the compounds demonstrated inhibitor selectivity for CDK7 over CDK1, CDK2, and CDK6. The compounds are moderate antimalarial agents against drug resistant parasites and possess encouraging in vitro therapeutic indices as determined against human cell lines. One particular sub-class of compounds, bromohydrosulfonylacetamides, was specific for Pfmrk with IC₅₀ values in the sub-micromolar range. These compounds represent the most potent Pfmrk inhibitors reported and provide support for further characterization and derivation as potential antimalarial agents.     

Disruption of Paneth and goblet cell homeostasis and increased endoplasmic reticulum stress in Agr2−/− mice

Anterior Gradient 2 (AGR2) is a protein disulfide isomerase that plays important roles in diverse processes in multiple cell lineages as a developmental regulator, survival factor and susceptibility gene for inflammatory bowel disease. Here, we show using germline and inducible Agr2−/− mice that Agr2 plays important roles in intestinal homeostasis. Agr2−/− intestine has decreased goblet cell Mucin 2, dramatic expansion of the Paneth cell compartment, abnormal Paneth cell localization, elevated endoplasmic reticulum (ER) stress, severe terminal ileitis and colitis. Cell culture experiments show that Agr2 expression is induced by ER stress, and that siRNA knockdown of Agr2 increases ER stress response. These studies implicate Agr2 in intestinal homeostasis and ER stress and suggest a role in the etiology of inflammatory bowel disease.     

Synergistic activity of Paenibacillus sp. BP-23 cellobiohydrolase Cel48C in association with the contiguous endoglucanase Cel9B and with endo- or exo-acting glucanases from Thermobifida fusca

Cellobiohydrolase Cel48C from Paenibacillus sp. BP-23, an enzyme displaying limited activity on most cellulosic substrates, was assayed for activity in the presence of other bacterial endo- or exocellulases. Significant enhanced activity was observed when Cel48C was incubated in the presence of Paenibacillus sp. BP-23 endoglucanase Cel9B or Thermobifida fusca cellulases Cel6A and Cel6B, indicating that Cel48C acts synergistically with them. Maximum synergism rates on bacterial microcrystalline cellulose or filter paper were obtained with a mixture of Paenibacillus cellulases Cel9B and Cel48C, accompanied by T. fusca exocellulase Cel6B. Synergism was also observed in cell extracts from recombinant clone E. coli pUCel9-Cel48 expressing the two contiguous Paenibacillus cellulases Cel9B and Cel48C. The enhanced cellulolytic activity displayed by the cellulase mixtures assayed could be used as an efficient tool for biotechnological applications like pulp and paper manufacturing.