Probes for INS

Stressful life events induce abnormalities in emotional and cognitive behaviour. The endogenous opioid system plays an essential role in stress adaptation and coping strategies. In particular, the µ-opioid receptor (μR), one of the major opioid receptors, strongly influences memory processing in that alterations in μR signalling are associated with various neuropsychiatric disorders. However, it remains unclear whether μR signalling contributes to memory impairments induced by acute stress. Here, we utilized pharmacological methods and cell-type-selective/non-cell-type-selective μR depletion approaches combined with behavioural tests, biochemical analyses, and in vitro electrophysiological recordings to investigate the role of hippocampal μR signalling in memory-retrieval impairment induced by acute elevated platform (EP) stress in mice. Biochemical and molecular analyses revealed that hippocampal μRs were significantly activated during acute stress. Blockage of hippocampal μRs, non-selective deletion of μRs or selective deletion of μRs on GABAergic neurons (μRGABA) reversed EP-stress-induced impairment of memory retrieval, with no effect on the elevation of serum corticosterone after stress. Electrophysiological results demonstrated that stress depressed hippocampal GABAergic synaptic transmission to CA1 pyramidal neurons, thereby leading to excitation/inhibition (E/I) imbalance in a μRGABA-dependent manner. Pharmaceutically enhancing hippocampal GABAAreceptor-mediated inhibitory currents in stressed mice restored their memory retrieval, whereas inhibiting those currents in the unstressed mice mimicked the stress-induced impairment of memory retrieval. Our findings reveal a novel pathway in which endogenous opioids recruited by acute stress predominantly activate μRGABA to depress GABAergic inhibitory effects on CA1 pyramidal neurons, which subsequently alters the E/I balance in the hippocampus and results in impairment of memory retrieval.

Human colorectal tumors bear recurrent mutations in genes encoding proteins operative in the WNT, MAPK, TGF-β, TP53 and PI3K pathways. Although these pathways influence intestinal stem cell niche signaling, the extent to which mutations in these pathways contribute to human colorectal carcinogenesis remains unclear. Here we use the CRISPR-Cas9 genome-editing system to introduce multiple such mutations into organoids derived from normal human intestinal epithelium. By modulating the culture conditions to mimic that of the intestinal niche, we selected isogenic organoids harboring mutations in the tumor suppressor genes APC, SMAD4 and TP53, and in the oncogenes KRAS and/or PIK3CA. Organoids engineered to express all five mutations grew independently of niche factors in vitro, and they formed tumors after implantation under the kidney subcapsule in mice. Although they formed micrometastases containing dormant tumor-initiating cells after injection into the spleen of mice, they failed to colonize in the liver. In contrast, engineered organoids derived from chromosome-instable human adenomas formed macrometastatic colonies. These results suggest that 'driver' pathway mutations enable stem cell maintenance in the hostile tumor microenvironment, but that additional molecular lesions are required for invasive behavior.

LGR5 is expressed in various tumors and has been identified as a putative intestinal stem cell marker. Here we investigated LGR5 expression in colorectal neuroendocrine neoplasms and analyzed the correlation with pathological characteristics. We evaluated the clinicopathological features of 8 neuroendocrine tumor (NET) grade 1 (NET G1), 4 NET Grade 2 (NET G2), and 8 NET Grade 3 (NET G3; also termed neuroendocrine carcinoma, or NEC) cases. We examined LGR5 expression using an RNAscope, a newly developed RNA in situ hybridization technique, with a tissue microarray of the neuroendocrine neoplasm samples. LGR5 staining in individual tumor cells was semi-quantitatively scored using an H-score scale. We also performed a combination of LGR5 RNA in situ hybridization and synaptophysin immunohistochemistry. All cases contained tumor cells with some LGR5-positive dots. For all cases, H-scores showed a positive correlation with nuclear beta-catenin expression. In the NEC group, there was a strong positive correlation between H-score and beta-catenin expression. Our findings suggest that LGR5 may serve as a stem cell marker in NEC, as is the case in colon adenocarcinoma. The positive correlation between H-score and beta-catenin expression suggests that LGR5 expression might be affected by beta-catenin expression in neuroendocrine neoplasms and especially in NEC.

Estrogen signalling pathways are emerging targets for lung cancer therapy. Unravelling the contribution of estrogens in lung cancer development is a pre-requisite to support the development of sex-based treatments and to identify patients who could potentially benefit from anti-estrogen treatments. In this study, we highlight the contribution of lymphatic and blood endothelia in the sex-dependent modulation of lung cancer. The orthotopic graft of syngeneic lung cancer cells into immunocompetent mice showed that lung tumours grew faster in female mice than in males. Moreover, estradiol (E2) promoted tumour development in female mice and increased lymph/angiogenesis and levels of VEGFA and bFGF in lung tumours of females through an estrogen receptor (ER) alpha-dependent pathway. Furthermore, while treatment with ERbeta antagonist was inefficient, ERalpha antagonist (MPP) and tamoxifen decreased lung tumour volumes, altered blood and lymphatic vasculature and reduced VEGFA and bFGF levels in females, but not in males. Finally, the quantification of lymphatic and blood vasculature of lung adenocarcinoma biopsies from patients aged between 35 to 55 years old revealed more extensive lymphangiogenesis and angiogenesis in tumour samples issued from women than from men. In conclusion, our findings highlight an E2/ERalpha-dependent modulation of lymphatic and blood vascular components of lung tumour microenvironment. Our study has potential clinical implication in a personalised medicine perspective by pointing to the importance of estrogen status or supplementation on lung cancer development that should be considered to adapt therapeutic strategies.