ABSTRACT
Islet microvasculature provides key architectural and functional roles, yet the morphological features of islets from patients with type 1 diabetes are poorly defined. We examined islet and exocrine microvasculature networks by multiplex immunofluorescence imaging of pancreases from organ donors with and without type 1 diabetes (n=17 and n=16, respectively) and determined vessel diameter, density, and area. We also analyzed these variables in insulin-positive and insulin-negative islets of 7 type 1 diabetes donors. Control islet vessel diameter was significantly larger (7.6 ± 1.1 µm) compared with vessels in diabetic islets (6.2 ± 0.8 µm; p<0.001). Control islet vessel density (number/islet) was significantly lower (5.3 ± 0.6) versus diabetic islets (9.3 ± 0.2; p<0.001). Exocrine vessel variables were not significantly different between groups. Islets with residual beta-cells were comparable to control islets for both vessel diameter and density and were significantly different from insulin-negative islets within diabetic donors (p<0.05). Islet smooth muscle actin area had a significant positive correlation with age in both groups (p<0.05), which could negatively impact islet transplantation efficiency from older donors. These data underscore the critical relationship of islet beta-cells and islet vessel morphology in type 1 diabetes. These studies provide new knowledge of the islet microvasculature in diabetes and aging.
Subject(s)
Diabetes Mellitus, Type 1/pathology , Insulin-Secreting Cells/pathology , Microvessels/pathology , Actins/analysis , Adolescent , Adult , Child , Female , Fluorescent Antibody Technique/methods , Humans , Insulin/analysis , Insulin-Secreting Cells/ultrastructure , Islets of Langerhans/blood supply , Islets of Langerhans/pathology , Islets of Langerhans/ultrastructure , Male , Microscopy, Confocal/methods , Microvessels/ultrastructure , Tissue Donors , Young AdultABSTRACT
BACKGROUND: The LKB1 tumor suppressor gene is commonly inactivated in non-small cell lung carcinomas (NSCLC), a major form of lung cancer. Targeted therapies for LKB1-inactivated lung cancer are currently unavailable. Identification of critical signaling components downstream of LKB1 inactivation has the potential to uncover rational therapeutic targets. Here we investigated the role of INSL4, a member of the insulin/IGF/relaxin superfamily, in LKB1-inactivated NSCLCs. METHODS: INSL4 expression was analyzed using global transcriptome profiling, quantitative reverse transcription PCR, western blotting, enzyme-linked immunosorbent assay, and RNA in situ hybridization in human NSCLC cell lines and tumor specimens. INSL4 gene expression and clinical data from The Cancer Genome Atlas lung adenocarcinomas (n = 515) were analyzed using log-rank and Fisher exact tests. INSL4 functions were studied using short hairpin RNA (shRNA) knockdown, overexpression, transcriptome profiling, cell growth, and survival assays in vitro and in vivo. All statistical tests were two-sided. RESULTS: INSL4 was identified as a novel downstream target of LKB1 deficiency and its expression was induced through aberrant CRTC-CREB activation. INSL4 was highly induced in LKB1-deficient NSCLC cells (up to 543-fold) and 9 of 41 primary tumors, although undetectable in all normal tissues except the placenta. Lung adenocarcinomas from The Cancer Genome Atlas with high and low INSL4 expression (with the top 10th percentile as cutoff) showed statistically significant differences for advanced tumor stage (P < .001), lymph node metastasis (P = .001), and tumor size (P = .01). The INSL4-high group showed worse survival than the INSL4-low group (P < .001). Sustained INSL4 expression was required for the growth and viability of LKB1-inactivated NSCLC cells in vitro and in a mouse xenograft model (n = 5 mice per group). Expression profiling revealed INSL4 as a critical regulator of cell cycle, growth, and survival. CONCLUSIONS: LKB1 deficiency induces an autocrine INSL4 signaling that critically supports the growth and survival of lung cancer cells. Therefore, aberrant INSL4 signaling is a promising therapeutic target for LKB1-deficient lung cancers.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Intercellular Signaling Peptides and Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Transcriptome/genetics , A549 Cells , AMP-Activated Protein Kinase Kinases , Animals , Autocrine Communication/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/genetics , Cyclic AMP Response Element-Binding Protein/genetics , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , Male , Mice , Signal Transduction/genetics , Transcription Factors/genetics , Xenograft Model Antitumor AssaysABSTRACT
Immunohistochemistry (IHC) is a powerful immunology-based method that is used to study the location of proteins in cells and tissues. There have been numerous advancements in IHC technology that continually increase the sensitivity and specificity through which this method can be used to generate new discoveries. Similarly, Alpha-1 Antitrypsin (AAT) IHC can be used to study AAT protein expression within the human liver or exogenous AAT that is delivered through gene therapy. Here, we describe a highly sensitive method to detect the AAT antigen in formalin-fixed paraffin-embedded human or mouse tissues.
Subject(s)
Immunohistochemistry/methods , Staining and Labeling , alpha 1-Antitrypsin/metabolism , Formaldehyde , Humans , Paraffin EmbeddingABSTRACT
Periodic Acid-Schiff (PAS) with diastase (PAS-D) refers to the use of the PAS stain in combination with diastase, which is an enzyme that digests the glycogen. The purpose of using the PAS-D procedure is to differentiate glycogen from other PAS-positive elements in tissue samples. The PAS-D method is also used for periportal liver staining of AAT polymer inclusions that are seen in alpha-1 antitrypsin deficiency disease. Here, we describe the procedure of PAS-D staining in formalin-fixed, paraffin-embedded human liver tissues.
Subject(s)
Amylases/metabolism , Periodic Acid/metabolism , Staining and Labeling/methods , Humans , Liver/cytology , Liver/metabolism , Paraffin EmbeddingABSTRACT
The LKB1 tumor suppressor gene is frequently mutated and inactivated in non-small cell lung cancer (NSCLC). Loss of LKB1 promotes cancer progression and influences therapeutic responses in preclinical studies; however, specific targeted therapies for lung cancer with LKB1 inactivation are currently unavailable. Here, we have identified a long noncoding RNA (lncRNA) signature that is associated with the loss of LKB1 function. We discovered that LINC00473 is consistently the most highly induced gene in LKB1-inactivated human primary NSCLC samples and derived cell lines. Elevated LINC00473 expression correlated with poor prognosis, and sustained LINC00473 expression was required for the growth and survival of LKB1-inactivated NSCLC cells. Mechanistically, LINC00473 was induced by LKB1 inactivation and subsequent cyclic AMP-responsive element-binding protein (CREB)/CREB-regulated transcription coactivator (CRTC) activation. We determined that LINC00473 is a nuclear lncRNA and interacts with NONO, a component of the cAMP signaling pathway, thereby facilitating CRTC/CREB-mediated transcription. Collectively, our study demonstrates that LINC00473 expression potentially serves as a robust biomarker for tumor LKB1 functional status that can be integrated into clinical trials for patient selection and treatment evaluation, and implicates LINC00473 as a therapeutic target for LKB1-inactivated NSCLC.
