The Evolution of Single Cell-derived Colorectal Cancer Cell Lines is Dominated by the Continued Selection of Tumor Specific Genomic Imbalances, Despite Random Chromosomal Instability

Intratumor heterogeneity is a major challenge in cancer treatment. To decipher patterns of chromosomal heterogeneity, we analyzed six colorectal cancer cell lines by multiplex interphase FISH (miFISH). The mismatch repair deficient cell lines DLD-1 and HCT116 had the most stable copy numbers, whereas aneuploid cell lines (HT-29, SW480, SW620 and H508) displayed a higher degree of instability. We subsequently assessed the clonal evolution of single cells in two CRC cell lines, SW480 and HT-29, which both have aneuploid karyotypes but different degrees of chromosomal instability. The clonal compositions of the single cell-derived daughter lines, as assessed by miFISH, differed for HT-29 and SW480. Daughters of HT-29 were stable, clonal, with little heterogeneity. Daughters of SW480 were more heterogeneous, with the single cell-derived daughter lines separating into two distinct populations with different ploidy (hyper-diploid and near-triploid), morphology, gene expression and tumorigenicity. To better understand the evolutionary trajectory for the two SW480 populations, we constructed phylogenetic trees which showed ongoing instability in the daughter lines. When analyzing the evolutionary development over time, most single cell-derived daughter lines maintained their major clonal pattern, with the exception of one daughter line that showed a switch involving a loss of APC. Our meticulous analysis of the clonal evolution and composition of these colorectal cancer models shows that all chromosomes are subject to segregation errors, however, specific net genomic imbalances are maintained. Karyotype evolution is driven by the necessity to arrive at and maintain a specific plateau of chromosomal copy numbers as the drivers of carcinogenesis

Current Trends in Diagnosis, Treatment and Prognosis of Canine Insulinoma

The most common pancreatic tumour in dogs is the insulinoma. Canine insulinomas are considered to be malignant in more than 95% of the cases because they almost always tend to metastasise. The diagnosis of an insulinoma requires the demonstration of the simultaneous occurrence of hypoglycaemia and blood insulin levels that are within or above the high end of the reference interval. The staging of canine insulinomas is preferably conducted by contrast-enhanced computed tomography. In general, surgical treatment is the most effective because of it results in long survival times, but many dogs also require medical treatment at some point. The prognosis of canine insulinomas is guarded as clinical hypoglycaemia almost always returns due to metastasis or tumour regrowth. This review aims to provide a summary and update the current recommendations in the veterinary literature for the diagnosis, treatment and prognostication of canine insulinomas

Gene expression profiling of primary canine insulinomas and their metastases

Abstract The gene expression profile of 10 primary canine insulinomas was compared with that of their accompanying metastases using microarray analysis and quantitative real time-PCR. Analysis of microarray data revealed 84 genes that were differentially expressed between primary insulinomas and their metastases, along with 243 genes differentially expressed between a low-metastatic and a high-metastatic subset of primary insulinomas. The genes differently expressed between primary insulinomas and their metastases clustered together in nine signalling pathways. Comparing the low-metastatic to the high-metastatic subset of primary insulinomas, 26 pathways appeared to be significantly influenced. The acinar enzymes pancreatic lipase (PNLIP) and chymotrypsinogen B1 (CTRB1) were amongst the most down-regulated genes in the malignant group of primary insulinomas and in metastases. Immunofluorescence demonstrated co-localisation of insulin and PNLIP in tumour cells. Different subsets of canine insulinomas can be identified on the basis of their gene expression profile. Canine insulinomas appear to contain amphicrine cells, which exhibit both endocrine and exocrine cell features. Copyright © 2013 Elsevier Ltd. All rights reserved. PMID:23428643[PubMed - as supplied by publisher

Identification of CD90 as Putative Cancer Stem Cell Marker and Therapeutic Target in Insulinomas

The long-term prognosis after surgical resection of malignant insulinoma (INS) is poor. Novel adjuvant therapies, specifically targeting cancer stem cells (CSCs), are warranted. Therefore, the goal of this study was to characterize and target putative INS CSCs. Using fluorescence-activated cell sorting, human INS cell line CM and pancreatic carcinoid cell line BON1 were screened for the presence of stem cell-associated markers. CD90, CD166, and GD2 were identified as potential CSC markers. Only CD90(+) INS cells had an increased tumor-initiating potential in athymic nude mice. Anti-CD90 monoclonal antibodies decreased the viability and metastatic potential of injected cells in a zebrafish embryo INS xenograft model. Primary INS stained positive for CD90 by immunohistochemistry, however also intratumoral fibroblasts and vascular endothelium showed positive staining. The results of this study suggest that anti-CD90 monoclonals form a potential novel adjuvant therapeutic modality by targeting either INS cells directly, or by targeting the INS microenvironment

Identification of Novel Targets for Lung Cancer Therapy Using an Induced Pluripotent Stem Cell Model

RATIONALE: Despite extensive studies, the genetic and epigenetic mechanisms that mediate initiation and progression of lung cancers have not been fully elucidated. Previously, we have demonstrated that via complementary mechanisms, including DNA methylation, polycomb repressive complexes, and noncoding RNAs, cigarette smoke induces stem-like phenotypes that coincide with progression to malignancy in normal respiratory epithelia as well as enhanced growth and metastatic potential of lung cancer cells. OBJECTIVES: To further investigate epigenetic mechanisms contributing to stemness/pluripotency in lung cancers and potentially identify novel therapeutic targets in these malignancies, induced pluripotent stem cells were generated from normal human small airway epithelial cells. METHODS: Lung induced pluripotent stem cells were generated by lentiviral transduction of small airway epithelial cells of OSKM (Yamanaka) factors (octamer-binding transcription factor 4 [Oct4], sex-determining region Y box 2 [SOX2], Kruppel-like factor 4 [KLF4], and MYC proto-oncogene, bHLH transcription factor [MYC]). Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation sequencing analysis were performed. RESULTS: The lung induced pluripotent stem cells exhibited hallmarks of pluripotency, including morphology, surface antigen and stem cell gene expression, in vitro proliferation, and teratoma formation. In addition, lung induced pluripotent stem cells exhibited no chromosomal aberrations, complete silencing of reprogramming transgenes, genomic hypermethylation, upregulation of genes encoding components of polycomb repressive complex 2, hypermethylation of stem cell polycomb targets, and modulation of more than 15,000 other genes relative to parental small airway epithelial cells. Additional sex combs like-3 (ASXL3), encoding a polycomb repressive complex 2-associated protein not previously described in reprogrammed cells, was markedly upregulated in lung induced pluripotent stem cell as well as human small cell lung cancer lines and specimens. Overexpression of the additional sex combs like-3 gene correlated with increased genomic copy number in small cell lung cancer lines. Knock-down of the additional sex combs like-3 gene inhibited proliferation, clonogenicity, and teratoma formation by lung induced pluripotent stem cells and significantly diminished in vitro clonogenicity and growth of small cell lung cancer cells in vivo. CONCLUSIONS: Collectively, these studies highlight the potential utility of this lung induced pluripotent stem cell model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and suggest that additional sex combs like-3 is a novel target for small cell lung cancer therapy

ASXL3 is a Novel Pluripotency Factor in Human Respiratory Epithelial Cells and a Potential Therapeutic Target in Small Cell Lung Cancer

In this study, we generated induced pluripotent stem cells (iPSC) from normal human small airway epithelial cells (SAEC) to investigate epigenetic mechanisms of stemness and pluripotency in lung cancers. We documented key hallmarks of reprogramming in lung iPSC (Lu-iPSC) that coincided with modulation of more than 15,000 genes relative to parental SAEC. Of particular novelty, we identified the PRC2-associated protein, ASXL3 which was markedly upregulated in Lu-iPSC and small cell lung cancer (SCLC) lines and clinical specimens. ASXL3 overexpression correlated with increased genomic copy number in SCLC lines. ASXL3 silencing inhibited proliferation, clonogenicity and teratoma formation by Lu-iPSC, and diminished clonogenicity and malignant growth of SCLC cells in-vivo. Collectively, our studies validate the utility of the Lu-iPSC model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis, and highlight ASXL3 as a novel candidate target for SCLC therapy