The DNA-sensing AIM2 inflammasome controls radiation-induced cell death and tissue injury

Acute exposure to ionizing radiation induces massive cell death and severe damage to tissues containing actively proliferating cells, including bone marrow and the gastrointestinal tract. However, the cellular and molecular mechanisms underlying this pathology remain controversial. Here, we show that mice deficient in the double-stranded DNA sensor AIM2 are protected from both subtotal body irradiation-induced gastrointestinal syndrome and total body irradiation-induced hematopoietic failure. AIM2 mediates the caspase-1-dependent death of intestinal epithelial cells and bone marrow cells in response to double-strand DNA breaks caused by ionizing radiation and chemotherapeutic agents. Mechanistically, we found that AIM2 senses radiation-induced DNA damage in the nucleus to mediate inflammasome activation and cell death. Our results suggest that AIM2 may be a new therapeutic target for ionizing radiation exposure

A Wnt-producing niche drives proliferative potential and progression in lung adenocarcinoma

The heterogeneity of cellular states in cancer has been linked to drug resistance, cancer progression and the presence of cancer cells with properties of normal tissue stem cells. Secreted Wnt signals maintain stem cells in various epithelial tissues, including in lung development and regeneration. Here we show that mouse and human lung adenocarcinomas display hierarchical features with two distinct subpopulations, one with high Wnt signalling activity and another forming a niche that provides the Wnt ligand. The Wnt responder cells showed increased tumour propagation ability, suggesting that these cells have features of normal tissue stem cells. Genetic perturbation of Wnt production or signalling suppressed tumour progression. Small-molecule inhibitors targeting essential posttranslational modification of Wnt reduced tumour growth and markedly decreased the proliferative potential of lung cancer cells, leading to improved survival of tumour-bearing mice. These results indicate that strategies for disrupting pathways that maintain stem-like and niche cell phenotypes can translate into effective anti-cancer therapies

Interactions of anti-cancer Ru(III)-complexes with serum proteins

Interactions of anti-cancer Ru(III)-complexes, KP1019, KP418, and NAMI-A, with human serum transferrin (hsTf) and albumin (hsA), and their speciation in serum were characterized. NAMI-A was distinguished from KP1019 and KP418 by the instability of its Ru(III) oxidation state. EPR studies suggested a ligand-exchange mediated binding of KP1019 and KP418 to hsTf and hsA. With hsA a hydrophobic interaction was also detected. KP1019 exhibited a different speciation profile in serum from that of KP418. UV-visible studies demonstrated a faster hsTf-binding ability of KP1019 over KP418. It was proposed that these differences might partially explain the disparate anti-cancer activities of KP1019 and KP418. EPR analysis of KP1019-binding to the His249Ala mutant of hsTf and to diferric-hsTf revealed a Ru(III)-state stabilizing role of the iron-binding site of hsTf in KP1019 binding. In addition, a procedure for expression and purification of full-length recombinant hsTf in P. pastoris was devised

Reciprocal regulation of glutamine metabolism and reactive oxygen species

Reactive oxygen species (ROS) are byproducts of normal cellular processes. While low or moderate levels of ROS promote and sustain oncogenic properties of cancer cells, excessive amounts are detrimental. Cancer cells counterbalance increased ROS production by engaging ROS-scavenging systems, which heavily rely on the antioxidants GSH and NADPH that can be synthesized from glutamine (GLN). Although GLN is not an essential amino acid, some cancer cells depend on exogenous GLN for survival, a phenotype known as GLN addiction. GLN plays versatile roles in cells from synthesis of macromolecules to redox balance. However, why GLN dependence for survival varies among different cancer cell types is not fully understood. This thesis tested the hypothesis that GLN addiction phenotype is ROS dependent. We first showed that loss of Hace1, a tumor suppressor that regulates ROS levels, results in increased GLN metabolism and GLN addiction. Inhibition of ROS reverses GLN addiction phenotype of Hace1 deficient cells, providing the first evidence that loss of a tumor suppressor leads to GLN addiction due to increased ROS levels. Using a panel of human cancer cell lines we established that GLN deprivation induces cell death in GLN addicted cells primarily by depleting intracellular antioxidant pools, resulting in increased ROS levels and oxidative damage. Furthermore GLN deprivation results in ROS-dependent elevation of glucose uptake in GLN addicted cells, which exacerbates oxidative stress causing cell death. Finally, we showed that GLN addicted cells are more sensitive to exogenous oxidants without GLN, and that AMPK mediated upregulation of ASCT2 expression and GLN uptake confers resistance to oxidative stress in GLN addicted cells. These studies establish the reciprocal regulation of GLN metabolism and oxidative stress in cancer cells.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine

Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by 13C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis

The DNA-sensing AIM2 inflammasome controls radiation-induced cell death and tissue injury.

Acute exposure to ionizing radiation induces massive cell death and severe damage to tissues containing actively proliferating cells, including bone marrow and the gastrointestinal tract. However, the cellular and molecular mechanisms underlying this pathology remain controversial. Here, we show that mice deficient in the double-stranded DNA sensor AIM2 are protected from both subtotal body irradiation-induced gastrointestinal syndrome and total body irradiation-induced hematopoietic failure. AIM2 mediates the caspase-1-dependent death of intestinal epithelial cells and bone marrow cells in response to double-strand DNA breaks caused by ionizing radiation and chemotherapeutic agents. Mechanistically, we found that AIM2 senses radiation-induced DNA damage in the nucleus to mediate inflammasome activation and cell death. Our results suggest that AIM2 may be a new therapeutic target for ionizing radiation exposure

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;Kras(G12D/+);Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5(+) stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.</p