B-cell chronic lymphocytic leukemia (B-CLL) cells are resistant to apoptosis, and consequently accumulate to the detriment of normal B cells and patient immunity. Because current therapies fail to eradicate these apoptosis-resistant cells, it is essential to identify alternative survival pathways as novel targets for anticancer therapies. Overexpression of cell-surface G protein-coupled receptors drives cell transformation, and thus plays a critical role in malignancies. In this study, we identified neurotensin receptor 2 (NTSR2) as an essential driver of apoptosis resistance in B-CLL. NTSR2 was highly expressed in B-CLL cells, whereas expression of its natural ligand, neurotensin (NTS), was minimal in both B-CLL cells and patient plasma. Surprisingly, NTSR2 remained in a constitutively active phosphorylated state, caused not by a mutation-induced gain-of-function but rather by an interaction with the oncogenic tyrosine kinase receptor TrkB. Functional and biochemical characterization revealed that the NTSR2–TrkB interaction acts as a conditional oncogenic driver requiring the TrkB ligand brain-derived neurotrophic factor (BDNF), which unlike NTS is highly expressed in B-CLL cells. Together, NTSR2, TrkB and BDNF induce autocrine and/or paracrine survival pathways that are independent of mutation status and indolent or progressive disease course. The NTSR2–TrkB interaction activates survival signaling pathways, including the Src and AKT kinase pathways, as well as expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL. When NTSR2 was downregulated, TrkB failed to protect B-CLL cells from a drastic decrease in viability via typical apoptotic cell death, reflected by DNA fragmentation and Annexin V presentation. Together, our findings demonstrate that the NTSR2–TrkB interaction plays a crucial role in B-CLL cell survival, suggesting that inhibition of NTSR2 represents a promising targeted strategy for treating B-CLL malignancy.

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.

BackgroundObesity is a major public health problem of our time as a risk factor for cardiometabolic disease and the available pharmacological tools needed to tackle the obesity pandemic are insufficient. Neurotensin (NTS) is a 13 amino acid peptide, which is derived from a larger precursor hormone called proneurotensin or Long Form NTS (LF NTS). NTS modulates neuro-transmitter release in the central system nervous, and facilitates intestinal fat absorption in the gastrointestinal tract. Mice lacking LF NTS are protected from high fat diet (HFD) induced obesity, hepatic steatosis and glucose intolerance. In humans, increased levels of LF NTS strongly and independently predict the development of obesity, diabetes mellitus, cardiovascular disease and mortality. With the perspective to develop therapeutic tools to neutralize LF NTS, we developed a monoclonal antibody, specifically inhibiting the function of the LF NTS (LF NTS mAb). This antibody was tested for the effects on body weight, metabolic parameters and behavior in mice made obese by high-fat diet.MethodsC57bl/6j mice were subjected to high-fat diet (HFD) until they reached an obesity state, then food was switched to chow. Mice were treated with either PBS (control therapy) or LF NTS mAb at the dose of 5 mg/kg once a week (i.v.). Mice weight, plasma biochemical analysis, fat and muscle size and distribution and behavioral tests were performed during the losing weight period and the stabilization period.ResultsObese mice treated with the LF NTS mAb lost weight significantly faster than the control treated group. LF NTS mAb treatment also resulted in smaller fat depots, increased fecal cholesterol excretion, reduced liver fat and larger muscle fiber size. Moreover, mice on active therapy were also less stressed, more curious and more active, providing a possible explanation to their weight loss.ConclusionOur results demonstrate that in mice subjected to HFD-induced obesity, a blockade of LF NTS with a monoclonal antibody results in reduced body weight, adipocyte volume and increased muscle fiber size, possibly explained by beneficial effects on behavior. The underlying mechanisms as well as any future role of LF NTS mAb as an anti-obesity agent warrants further studies.

Introduction De-differentiation is a highly lethal feature of aggressive breast cancers (BC), and is achieved through the epithelial-to-mesenchymal transition (EMT) and the cancer stem cell (CSC) programs. Targeting the mechanisms controlling BC de-differentiation can lead to more effective therapeutics. Recent studies indicated that nucleotide metabolism can regulate cancer stemness and EMT. Here we investigated the expression of the nucleotide metabolism enzyme and drug target thymidylate synthase (TS) in the BC subtypes and analysed its impact on BC de-differentiation. Material and methods Cells with TS knockdown and overexpression were tested in vitro and in vivo. Proteins were analysed by western blot, FACS and ELISA. Differential gene expression in TS-deficient cells was determined by RNA-seq. Immunohistochemistry (IHC) was used to stain samples from patients with different BC subtypes. Results and discussions TS mRNA expression was found to be significantly differentially expressed among the BC subtypes, exhibiting the highest levels in aggressive triple-negative BC (TNBC). shRNA-mediated TS knockdown in TNBC cell lines (n=3) increased the population of differentiated cells (CD24 high ) and strongly attenuated the stem-like phenotype, like the formation of mammospheres from single cells and the migration in a cell culture wound. TS-deficient cells also showed an altered ability to form metastasis in vivo, consistent with previous observations in EMTrepressed BC cells. A rescue experiment performed by overexpressing either a wild-type or catalytically inactive TS indicated that the enzymatic activity was essential for the maintenance of the BCSC phenotype. Along with a strong repression of EMT-signature genes, RNA-seq profiling indicated a reduction of inflammatory and NF-kB signalling pathways in TS deficient cells, which dramatically reduced IL-1b production and secretion. A TS-specific gene signature was generated, which significantly associated with worst survival in BC patients. IHC staining on FFPE samples from a series of BC patients (n=120) confirmed higher TS expression in tumours that were poorly differentiated and in TNBC. Conclusion We discovered a novel role for the TS enzyme in the maintenance of a de-differentiated and stem-like state of BC. These findings may not only open the possibility to study in-depth the role of nucleotide metabolism at the crossroad between proliferation and differentiation, but may provide the rationale for novel drug combinations with TS-inhibiting agents for the treatment of BC. Introduction Renal cell carcinoma (RCC) is characterised by high lethality in advanced stages. Vastly resistant to radio-and chemotherapy, RCCs respond to targeted therapies such as tyrosine kinase inhibitors, mTOR antagonists and immune checkpoint inhibitors. However, limited response rates and emerging resistance mechanisms demand new treatment strategies. SPOT-004Scaffolding protein NEDD9 (neural precursor cell expressed, developmentally down regulated 9) is frequently overexpressed in ...