p53 and the role of autophagy in pancreatic cancer development

Autophagy is an intracellular catabolic process that involves the sequestration of proteins and whole organelles into specialized cargo vesicles (autophagosomes) and their delivery to lysosomes with subsequent degradation. Autophagy is active at low levels at any time in virtually all cells and can be induced upon a variety of different stimuli. The core function of autophagy is the degradation and recycling of intracellular material. However, how this impacts on cellular survival likely depends on the biological context. The role of autophagy in cancer is very complex and incompletely understood. It is therefore very surprising that few studies exists that employ genetically modified mouse models of human cancer to examine the role of autophagy in this context. This is even more true when considering, that pharmacological inhibition of autophagy is currently being used in several clinical trials to treat cancer of various origins. The goal of this study was to examine the role of autophagy in a mouse model of pancreatic cancer. To achieve this several mouse strains were crossed: a) Pdx1-Cre LSLKRasG12D/wt mice that develop Pancreatic Ductal Adenocarcinoma (PDAC) similar to humans initiated by oncogenic Ras and b) Atg5flox/flox or Atg7flox/flox mice that permit Cre-induced deletion of either one of the essential autophagy regulating genes 5 and 7 (Atg5, Atg7). Offspring allowed us to examine the role of autophagy in pancreatic function. Loss of autophagy in the pancreas leads to exocrine and endocrine tissue destruction and reduces survival in approx. 60% of animals. The early death in autophagy-deficient mice can be delayed by additional deletion of p53; the mortality rate however remains unchanged. Moribund mice show a diabetic phenotype with elevated blood glucose and fructosamine levels. In the absence of oncogenic Ras autophagy deletion does not lead to cancer formation or occurrence of pre-malignant lesions in mice aged up to 700d. In mice that express oncogenic Ras in the pancreas (Pdx1-CreKRasG12D/wt) additional, genetic deletion of autophagy leads to accumulation of pre-malignant Pancreatic Intraepithelial Neoplasias (PanINs) that unlike their autophagy proficient counterparts never progress to cancer. In this genetic context autophagy therefore serves as a tumour promotor. In stark contrast in mice expressing oncogenic Ras and lacking both copies of p53 (Pdx1-KRasG12D/wt p53-/-) inhibition of autophagy, either genetically by deletion of Atg5, Atg7 or pharmacologically by chloroquine, tumour onset is accelerated. Therefore in a p53-deficient situation autophagy is now a tumour suppressor. Tumours that developed from a p53-proficient background have increased autophagy compared to tumours that developed from a p53-null background. Furthermore p53-/- Atg7-/- tumours have increased glycolysis in vitro and in vivo and enhanced intracellular metabolites of the anabolic Pentose Phosphate Pathway (PPP) compared to p53-/- Atg7+/+ tumours. In summary it is the p53 status that determines the role of autophagy in PDAC development. In tumours developing from a p53-proficient background loss of autophagy completely prevents cancer development; whereas in tumours arising from p53-deficient tissue loss of autophagy accelerates tumour formation

DARPins detect the formation of hetero-tetramers of p63 and p73 in epithelial tissues and in squamous cell carcinoma

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p63$_{2}$/p73$_{2}$ hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p63$_{2}$/p73$_{2}$ hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma

The glutathione redox system is essential to prevent ferroptosis caused by impaired lipid metabolism in clear cell renal cell carcinoma

The publisher's final edited version of this article is available at Oncogene. 2018 Oct;37(40):5435-5450. doi: 10.1038/s41388-018-0315-z. Epub 2018 Jun 5. Non-commercial use onlyThis study was funded by Cancer Research UK, the German Research Foundation (FOR2314) and the German Cancer Aid (111917)

The role of autophagy in tumour development and cancer therapy

Autophagy is a catabolic membrane-trafficking process that leads to sequestration and degradation of intracellular material within lysosomes. It is executed at basal levels in every cell and promotes cellular homeostasis by regulating organelle and protein turnover. In response to various forms of cellular stress, however, the levels and cargoes of autophagy can be modulated. In nutrient-deprived states, for example, autophagy can be activated to degrade cargoes for cell-autonomous energy production to promote cell survival. In other contexts, in contrast, autophagy has been shown to contribute to cell death. Given these dual effects in regulating cell viability, it is no surprise that autophagy has implications in both the genesis and treatment of malignant disease. In this review, we provide a comprehensive appraisal of the way in which oncogenes and tumour suppressor genes regulate autophagy. In addition, we address the current evidence from human cancer and animal models that has aided our understanding of the role of autophagy in tumour progression. Finally, the potential for targeting autophagy therapeutically is discussed in light of the functions of autophagy at different stages of tumour progression and in normal tissues

A case of nodular lymphocyte predominant Hodgkin lymphoma with unexpected EBV-latency type

No abstract available

Epstein–Barr virus infection patterns in nodular lymphocyte‐predominant Hodgkin lymphoma

Aims To investigate Epstein‐Barr virus (EBV) latency types in 19 cases of EBV‐positive nodular lymphocyte‐predominant Hodgkin lymphoma (NLPHL), as such information is currently incomplete. Methods and results Immunohistochemistry (IHC) for CD20, CD79a, PAX5, OCT2, CD30, CD15, CD3 and programmed cell death protein 1 was performed. For EBV detection, in‐situ hybridisation (ISH) for EBV‐encoded RNA (EBER) was employed combined with IHC for EBV‐encoded latent membrane protein (LMP)‐1, EBV‐encoded nuclear antigen (EBNA)‐2, and EBV‐encoded BZLF1. In 95% of the cases, neoplastic cells with features of Hodgkin and Reed–Sternberg (HRS) cells were present, mostly showing expression of CD30. In all cases, the B‐cell phenotype was largely intact, and delineation from classic Hodgkin lymphoma (CHL) was further supported by myocyte enhancer factor 2B (MEF2B) detection. All tumour cells were EBER‐positive except in two cases. EBV latency type II was most frequent (89%) and type I was rare. Cases with latency type I were CD30‐negative. Five cases contained some BZLF1‐positive and/or EBNA‐2‐positive bystander lymphocytes. Conclusions As HRS morphology of neoplastic cells and CD30 expression are frequent features of EBV‐positive NLPHL, preservation of the B‐cell transcription programme, MEF2B expression combined with NLPHL‐typical architecture and background composition facilitate distinction from CHL. EBER ISH is the method of choice to identify these cases. The majority present with EBV latency type II, and only rare cases present with latency type I, which can be associated with missing CD30 expression. The presence of occasional bystander lymphocytes expressing BZLF1 and/or EBNA‐2 and the partial EBV infection of neoplastic cells in some cases could indicate that EBV is either not primarily involved or is only a transient driver in the pathogenesis of EBV‐positive NLPHL

Inhibition of focal adhesion kinase overcomes resistance of mantle cell lymphoma to ibrutinib in the bone marrow microenvironment

Mantle cell lymphoma and other lymphoma subtypes often spread to the bone marrow, and stromal interactions mediated by focal adhesion kinase frequently enhance survival and drug resistance of the lymphoma cells. To study the role of focal adhesion kinase in mantle cell lymphoma, immunohistochemistry of primary cases and functional analysis of mantle cell lymphoma cell lines and primary mantle cell lymphoma cells co-cultured with bone marrow stromal cells (BMSC) using small molecule inhibitors and RNAi-based focal adhesion kinase silencing was performed. We showed that focal adhesion kinase is highly expressed in bone marrow infiltrates of mantle cell lymphoma and in mantle cell lymphoma cell lines. Stroma-mediated activation of focal adhesion kinase led to activation of multiple kinases (AKT, p42/44 and NF-kappa B), that are important for prosurvival and proliferation signaling. Interestingly, RNAi-based focal adhesion kinase silencing or inhibition with small molecule inhibitors (FAKi) resulted in blockage of targeted cell invasion and induced apoptosis by inactivation of multiple signaling cascades, including the classic and alternative NF-kappa B pathway. In addition, the combined treatment of ibrutinib and FAKi was highly synergistic, and ibrutinib resistance of mantle cell lymphoma could be overcome. These data demonstrate that focal adhesion kinase is important for stroma-mediated survival and drug resistance in mantle cell lymphoma, providing indications for a targeted therapeutic strategy

Inhibition of focal adhesion kinase overcomes resistance of mantle cell lymphoma to ibrutinib in the bone marrow microenvironment

Mantle cell lymphoma and other lymphoma subtypes often spread to the bone marrow, and stromal interactions mediated by focal adhesion kinase frequently enhance survival and drug resistance of the lymphoma cells. To study the role of focal adhesion kinase in mantle cell lymphoma, immunohistochemistry of primary cases and functional analysis of mantle cell lymphoma cell lines and primary mantle cell lymphoma cells cocultured with bone marrow stromal cells (BMSC) using small molecule inhibitors and RNAi based focal adhesion kinase silencing was performed. We could show that focal adhesion kinase is highly expressed in bone marrow infiltrates of mantle cell lymphoma and in mantle cell lymphoma cell lines. Stroma-mediated activation of focal adhesion kinase led to activation of multiple kinases (AKT, p42/44 and NF-κB), that are important for prosurvival and proliferation signalling. Interestingly, RNAi based focal adhesion kinase silencing or inhibition with small molecule inhibitors (FAKi) resulted in blockage of targeted cell invasion and induced apoptosis by inactivation of multiple signalling cascades, including the classical and alternative NF-κB pathway. In addition, the combined treatment of ibrutinib and FAKi was highly synergistic, and ibrutinib resistance of mantle cell lymphoma could be overcome. These data demonstrate that focal adhesion kinase is important for stroma-mediated survival and drug-resistance in mantle cell lymphoma, providing indications for a targeted therapeutic strategy