Modulating Cancer Stem Cell Signaling in Thoracic Malignancies

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

Limited information is available pertaining to the potential utility of induced pluripotent stem cells (iPSC) for investigating molecular mechanisms mediating initiation and progression of thoracic malignancies and identifying novel therapeutic targets in these neoplasms. To examine this issue, we reprogrammed normal human small airway epithelial cells (SAEC) to pluripotency using lentiviral transduction of Yamanaka factors. The lung iPSC (Lu-iPSC) exhibited hallmarks of pluripotency in-vitro and in-vivo which coincided with complex alterations in DNA methylation, marked up-regulation of PRC-2-related genes, and modulation of 15,000 other transcripts. To explore the potential clinical relevance of the Lu-iPSC model, we compared transcriptome signatures in two Lu-iPSC clones, 14 small cell lung cancer (SCLC) lines, and 10 non-small cell lung cancer (NSCLC) lines relative to three SAEC cultures. RNA-seq analysis demonstrated that the Lu-iPSC transcriptomes overlap much more with SCLC than NSCLC. Infinium array analysis also indicated that DNA methylation signatures in the Lu-iPSC more closely aligned with SCLC compared to NSCLC. However, IPA did not provide useful information related to specific oncogenic pathways related to SCLC vs Lu-iPSC. To investigate how chromatin landscape contributes to SCLC biology, we expanded our study to perform DNase I hypersensitivity followed by deep sequencing (DNase-seq) to identify regulatory elements defined by the state of chromatin configuration among Lu-iPSC and SCLC relative to SAEC. More than 200,000 DNase 1 Hypersensitivity Sites (DHS) were identified in Lu-iPSC or SCLC but not in SAEC. Approximately 16% of these DHS were shared between Lu-iPSC and SCLC. The majority of DHS were outside promoters, encompassed by introns and intergenic regions, indicating that enhancers were the major contributors of genomic landscape in Lu-iPSC and SCLC. Integration of DHS and transcriptome data indicated that less than 5% of non-promoter differentially open regions (DOR) mapped to the nearest neighbor gene, indicating gene regulation by distant regulatory elements. A subset of DOR was unique to SCLC. Analysis of peak-to-gene links and gene-to-peak links across all samples showed that 95% of genes mapped to at least one open chromatin region, whereas each peak mapped to a mean number of 9 genes. Many of the predicted DOR-to-gene links occurred in clusters where multiple nearby peaks are predicted to be linked to the same gene, suggesting that these clusters function as part of a regulatory unit or enhancer. Bivariate analysis of Genomic Footprint (BaGFoot) identified NF1 family members as having the highest increase in digital footprinting and occupancy within open chromatin sites specifically in SCLC. A comprehensive manuscript pertaining to these studies is in final stages of preparation for peer review. Additional Sex Combs Like-3 (ASXL3), encoding a component of the polycomb deubiquitinase complex not previously described to be up-regulated in reprogrammed cells was identified as an essential mediator of pluripotency in human respiratory epithelia and a novel epigenetic target for small cell lung cancer (SCLC) therapy. ASXL3 over-expression correlated with gene amplification in SCLC. Knock-down of ASXL3 decreased the number and size of teratomas and inhibited growth of SCLC xenografts. Results of these studies were published in Cancer Research. Our recent analysis of single cell RNAs-seq data obtained from reprogramming of 5 human cell lines of different tissue origins revealed that ASXL3 up-regulation coincides with a transition state from normal to fully reprogrammed cells. Additional bioinformatics analysis indicates that ASXL3 is only up-regulated in the ASCL1 SCLC subtype; knock-out of ASCL1 or over-expression of myc which theoretically would drive SCLC from ASCL1 to either NeuroD1 or Yap subtypes does not appear to impact ASXL3 expression in SCLC. Studies are in progress using conditional ASXL3 knock-out as well as time course experiments using a FLAG-tagged sequence CRISPRed into the end of the ASXL3 coding region to examine if ASXL3 expression is essential for establishing and/or maintaining pluripotency during reprogramming in normal cells, elucidate the mechanisms by which ASXL3 expression is regulated in normal cells and cancer cells, identify ASXL3 binding partners, and define molecular and clinical implications of ASXL3 up-regulation in SCLC. Additional bioinformatics analysis indicates that ASXL3 is only up-regulated in the ASCL1 SCLC subtype; knock-out of ASCL1 or over-expression of myc which theoretically would drive SCLC from ASCL1 to either NeuroD1 or Yap subtypes does not appear to impact ASXL3 expression in SCLC. Several additional genes which were noted to be upregulated in a manner similar to ASXL3 in Lu-iPSC and subsequently found to be elevated in SCLC are also under investigation at this time. Based on our encouraging results pertaining to modeling of lung cancers, we have extended our reprogramming efforts to characterize and target epigenomic perturbations in esophageal cancer cells. Normal esophageal squamous epithelial cells (Heepic) as well as h-TERT immortalized metaplastic Barrett's esophageal cells (CP-A) were reprogrammed using established techniques. PCA demonstrated that the Eso-iPSC were fully reprogrammed; however, CP-A-iPSC were not. ASXL3 expression was much higher in the CP-A-iPSC compared to Eso-iPSC. Consistent with the notion that ASXL3 facilitates stemness, Eso-iPSC form teratomas in immunodeficient mice whereas CP-A-iPSC form high grade carcinomas. Also of note, whereas ASXL3 is not detected in 2D cultures of thoracic cancers (except SCLC), ASXL3 expression is upregulated when these cells are transitioned to non-adherent conditions. Lastly, ASXL3 is up-regulated in stem-like cells that we can now reproducibly isolate from primary surgical specimens irrespective of tissue histology. In related efforts, we isolated 24 separate stem-like clones from Lewis lung cancer cells stably transfected with a luciferase reporter construct. These clones exhibit distinct stem cell and cancer-testis gene expression profiles in-vitro, and unique, highly reproducible organ-specific metastases. ASXL3 expression is very high in many of the clones. As such, our unique models now enable us to systematically dissect epigenomic mechanisms contributing to pluripotency and metastatic potential in thoracic malignancies and to evaluate novel pharmacologic and immunologic regimens targeting CSC in these neoplasms. These efforts, which are a major focus of our current investigative work, will facilitate clinical development of novel therapies for thoracic cancers with potentially broad applicability for the treatment of other human malignancies.