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

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

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

Gene expression levels of A279T-transduced cells normalized to wtTERT-transduced cells.

<p>*ND: Not detected.</p

Telomerase Variant A279T Induces Telomere Dysfunction and Inhibits Non-Canonical Telomerase Activity in Esophageal Carcinomas

<div><p>Background</p><p>Although implicated in the pathogenesis of several chronic inflammatory disorders and hematologic malignancies, telomerase mutations have not been thoroughly characterized in human cancers. The present study was performed to examine the frequency and potential clinical relevance of telomerase mutations in esophageal carcinomas.</p><p>Methods</p><p>Sequencing techniques were used to evaluate mutational status of <i>telomerase reverse transcriptase (TERT)</i> and <i>telomerase RNA component (TERC)</i> in neoplastic and adjacent normal mucosa from 143 esophageal cancer (EsC) patients. MTS, flow cytometry, time lapse microscopy, and murine xenograft techniques were used to assess proliferation, apoptosis, chemotaxis, and tumorigenicity of EsC cells expressing either wtTERT or TERT variants. Immunoprecipitation, immunoblot, immunofluorescence, promoter-reporter and qRT-PCR techniques were used to evaluate interactions of TERT and several TERT variants with BRG-1 and β-catenin, and to assess expression of cytoskeletal proteins, and cell signaling. Fluorescence in-situ hybridization and spectral karyotyping techniques were used to examine telomere length and chromosomal stability.</p><p>Results</p><p>Sequencing analysis revealed one deletion involving <i>TERC (TERC del 341-360)</i>, and two non-synonymous <i>TERT</i> variants [A279T (2 homozygous, 9 heterozygous); A1062T (4 heterozygous)]. The minor allele frequency of the A279T variant was five-fold higher in EsC patients compared to healthy blood donors (p<0.01). Relative to wtTERT, A279T decreased telomere length, destabilized TERT-BRG-1-β-catenin complex, markedly depleted β-catenin, and down-regulated canonical Wnt signaling in cancer cells; these phenomena coincided with decreased proliferation, depletion of additional cytoskeletal proteins, impaired chemotaxis, increased chemosensitivity, and significantly decreased tumorigenicity of EsC cells. A279T expression significantly increased chromosomal aberrations in mouse embryonic fibroblasts (MEFs) following Zeocin™ exposure, as well as Li Fraumeni fibroblasts in the absence of pharmacologically-induced DNA damage.</p><p>Conclusions</p><p>A279T induces telomere dysfunction and inhibits non-canonical telomerase activity in esophageal cancer cells. These findings warrant further analysis of A279T expression in esophageal cancers and premalignant esophageal lesions.</p></div

Effects of A279T on genomic stability in normal cells.

<p>A. SKY assay demonstrating that A279T-induces genomic instability in Zeocin™-treated MEF-1 cells. Upper panel: translocations, dicentric, and rearranged chromosomes are present in cells expressing A279T compared to wtTERT. Lower left panel: a multi-centric chromosome observed in cells harboring A279T. Lower right panel: a ring chromosome is formed and every chromosome is rearranged in cells transfected with A279T. See text for additional details. B. Upper panel: representative results of SKY analysis Li Fraumeni fibroblasts constitutively expressing wtTERT or A279T-TERT. Lower panel: close-up of chromosomes 1 and 16. C. Summary of results of two independent experiments demonstrating that A279T expression increases genomic instability in Li Fraumeni cells.</p

Effects of A279T on tumorigenicity and telomere length of esophageal cancer cells in vivo.

<p>A. A279T significantly inhibits growth of subcutaneous EsC2 xenografts in athymic nude mice. B. Representative results of telomere-specific FISH analysis of ExC2 xenografts depicting shortened telomere length in xenografts from EsC2-A279T cells compared to those derived from EsC2-TERT cells; Red: telomere; green: centromere; blue: DAPI.</p