Jerry Krcek

INSL5 is a novel marker for human enteroendocrine cells of the large intestine and neuroendocrine tumours

We report for the first time the distribution of human INSL5 and its cognate leucine rich G-protein coupled receptor RXFP4 in the large intestine and in neuroendocrine/carcinoid tissues. Immunoreactive INSL5 was uniquely expressed by enteroendocrine cells (EECs) located within the colonic mucosa, whereas colonocytes were immunopositive for RXFP4. INSL5+ and RXFP4+ cells were also detected in human neuroendocrine/carcinoid tissues. We employed a recently described Insl5 knockout mouse model and 2 mouse models of induced colitis to address the relevance of Insl5 in EEC development and in acute inflammation of the colon. We identified INSL5 as a specific marker for synaptophysin+ EECs in the mucosa of the normal human and mouse colon. Insl5 was not essential for the development of mouse synaptophysin+ EECs. The mouse models of chemically induced colitis (dextran sulfate sodium and dinitrobenzene-sulfonic acid) failed to show changes in the numbers of Insl5+ EECs at inflammatory sites during the acute phase of colitis. In conclusion, we showed that INSL5 is a novel marker of colorectal EECs and provide first evidence for the presence of a potentially autocrine/paracrine INSL5-RXFP4 signaling system in the normal human and mouse colon and in rare human neuroendocrine tumours.

Platinum (IV) coiled coil nanotubes selectively kill human glioblastoma cells

UNLABELLED Malignant glioma are often fatal and pose a significant therapeutic challenge. Here we have employed α-helical right handed coiled coils (RHCC) which self-assemble into tetrameric nanotubes that stably associate with platinum (Pt) (IV) compound. This Pt(IV)-RHCC complex showed superior in vitro and in vivo toxicity in human malignant glioma cells at up to 5 fold lower platinum concentrations when compared to free Pt(IV). Pt(IV)-RHCC nanotubes activated multiple cell death pathways in GB cells without affecting astrocytes in vitro or causing damage to normal mouse brain. This Pt(IV)-RHCC nanotubes may serve as a promising new therapeutic tool for low dose Pt(IV) prodrug application for highly efficient and selective treatment of human brain tumors. FROM THE CLINICAL EDITOR The prognosis of malignant glioma remains poor despite medical advances. Platinum, one of the chemotherapeutic agents used, has significant systemic side effects. In this article, the authors employed α-helical right handed coiled coil (RHCC) protein nanotubes as a carrier for cisplatin. It was shown that the new compound achieved higher tumor kill rate but lower toxicity to normal cells and thus may hold promise to be a highly efficient treatment for the future.

C1q-tumour necrosis factor-related protein 8 (CTRP8) is a novel interaction partner of relaxin receptor RXFP1 in human brain cancer cells

We report a novel ligand–receptor system composed of the leucine‐rich G‐protein‐coupled relaxin receptor, RXFP1, and the C1q‐tumour necrosis factor‐related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1–3. In structural homology studies and computational docking experiments we delineated the N‐terminal region of the globular C1q region of CTRP8 and the leucine‐rich repeat units 7 and 8 of RXFP1 to mediate this new ligand–receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro‐migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8–RXFP1‐induced increased GB motility. The CTRP8–RXFP1‐mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8‐induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co‐immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8–RXFP1 signalling pathway by a combined inhibitor and peptide‐based strategy to block GB dissemination within the brain. Copyright

Dovitinib enhances temozolomide efficacy in glioblastoma cells

The multikinase inhibitor and FDA‐approved drug dovitinib (Dov) crosses the blood–brain barrier and was recently used as single drug application in clinical trials for GB patients with recurrent disease. The Dov‐mediated molecular mechanisms in GB cells are unknown. We used GB patient cells and cell lines to show that Dov downregulated the stem cell protein Lin28 and its target high‐mobility group protein A2 (HMGA2). The Dov‐induced reduction in pSTAT3Tyr705 phosphorylation demonstrated that Dov negatively affects the STAT3/LIN28/Let‐7/HMGA2 regulatory axis in GB cells. Consistent with the known function of LIN28 and HMGA2 in GB self‐renewal, Dov reduced GB tumor sphere formation. Dov treatment also caused the downregulation of key base excision repair factors and O6‐methylguanine‐DNA‐methyltransferase (MGMT), which are known to have important roles in the repair of temozolomide (TMZ)‐induced alkylating DNA damage. Combined Dov/TMZ treatment enhanced TMZ‐induced DNA damage as quantified by nuclear γH2AX foci and comet assays, and increased GB cell apoptosis. Pretreatment of GB cells with Dov (‘Dov priming’) prior to TMZ treatment reduced GB cell viability independent of p53 status. Sequential treatment involving ‘Dov priming’ and alternating treatment cycles with TMZ and Dov substantially reduced long‐term GB cell survival in MGMT+ patient GB cells. Our results may have immediate clinical implications to improve TMZ response in patients with LIN28+/HMGA2+ GB, independent of their MGMT methylation status.

Structural commonality of C1q TNF‐related proteins and their potential to activate relaxin/insulin‐like family peptide receptor 1 signalling pathways in cancer cells

We established the role of the GPCR relaxin/insulin‐like family peptide receptor 1 (RXFP1 receptor) as a novel active receptor in human glioblastoma (GB), a fatal brain tumour. We identified C1q/TNF‐related protein 8 (CTRP8) as a novel agonist of the RXFP1 receptor. CTRP8 enhanced the motility and matrix invasion of GB, and this involved PKC‐mediated up‐regulation of cathepsin B, a marker for poor prognosis in GB patients. We conclude that the absence of relaxin isoforms does not preclude the activation of the RXFP1 receptor, as the least known member of the CTRP family, CTRP8, can effectively target and activate RXFP1 receptors.

Structural commonality of C1q Tumor Necrosis Factor‐related proteins and their potential to activate RXFP1 signaling pathways in cancer cells

We established the role of the GPCR relaxin/insulin‐like family peptide receptor 1 (RXFP1 receptor) as a novel active receptor in human glioblastoma (GB), a fatal brain tumour. We identified C1q/TNF‐related protein 8 (CTRP8) as a novel agonist of the RXFP1 receptor. CTRP8 enhanced the motility and matrix invasion of GB, and this involved PKC‐mediated up‐regulation of cathepsin B, a marker for poor prognosis in GB patients. We conclude that the absence of relaxin isoforms does not preclude the activation of the RXFP1 receptor, as the least known member of the CTRP family, CTRP8, can effectively target and activate RXFP1 receptors.

C1q/TNF‐related peptide 8 (CTRP8) promotes temozolomide resistance in human glioblastoma

The C1q/TNF‐related peptide 8 (CTRP8) has recently emerged as a novel ligand of the G protein‐coupled receptor RXFP1 in the fatal brain tumor glioblastoma (GBM). We previously demonstrated that the CTRP8‐RXFP1 ligand–receptor system promotes motility and matrix invasion of patient GBM and U87 MG cells by specific phosphorylation of PI3 kinase and protein kinase C. Here, we demonstrate a novel role for CTRP8 in protecting human GBM cells against the DNA alkylating damage of temozolomide (TMZ), the standard chemotherapy drug used to treat GBM. This DNA protective role of CTRP8 required a functional RXFP1‐STAT3 signaling cascade in GBM cells. We identified N‐methylpurine DNA glycosylase (MPG), a monofunctional glycosylase that initiates base excision repair pathway by generating an apurinic/apyrimidinic (AP) site, as a new CTRP8‐RXFP1‐STAT3 target in GBM. Upon TMZ exposure, treatment with CTRP8 reduced the formation of AP sites and double‐strand DNA breaks in GBM cells. This CTRP8 effect was independent of cellular MGMT levels and was associated with decreased caspase 3/7 activity and increased survival of human GBM. CTRP8‐induced RXFP1 activation caused an increase in cellular protein levels of the anti‐apoptotic Bcl members and STAT3 targets Bcl‐2 and Bcl‐XL in human GBM. Collectively, our results demonstrate a novel multipronged and clinically relevant mechanism by which the CTRP8‐RXFP1 ligand–receptor system exerts a DNA protective function against TMZ chemotherapeutic stress in GBM. This CTRP8‐RXFP1‐STAT3 axis is a novel determinant of TMZ responsiveness/chemoresistance and an emerging new drug target for improved treatment of human GBM.