Abstract
Human cancers arising from different cell lineages display genotypes with characteristic features, but the lineage-specific factors that influence the differences in genetic profiles have not been identified1, 2. Such differences could be explained by models in which a cellular phenotype limits a cell’s vulnerability to certain mutations or by models in which observed mutations determine the ultimate cell phenotype. To evaluate the relative merits of these proposals, we are studying the initiation of cancers by genetically modifying cells at discrete stages of differentiation after derivation from human embryonic stem cells (hESCs). We have focused initially on small cell lung cancer (SCLC), the most aggressive type of human lung cancer, characterized by a poor prognosis, the rapid development of resistance to treatment, and nearly universal loss of function of multiple tumor suppressor genes, especially TP53 and RB 3-7. Earlier studies with mouse models indicate that the normal cell precursors to SCLCs are likely to be pulmonary neuro-endocrine cells (PNECs)8-10. Building on existing methods for sequentially differentiating hESCs in culture into several types of lung cells, we have now produced PNECs in significant numbers for the first time by blocking signaling through NOTCH receptors (using inhibitors of γ-secretase) and by interfering with expression of the RB gene (using inhibitory RNAs) in lung progenitor cells. In contrast, expression of mutated EGFR or KRAS genes, common in human lung adenocarcinomas that arise from the alveolar cell lineage, has no evident effects on the formation of PNECs. Although PNECs induced by blocking NOTCH and RB signaling do not form xenografts in immune-deficient mice, the cells form subcutaneous tumors resembling early stage human SCLC and bearing neuroendocrine markers if expression of the TP53 gene is also impaired. These findings imply that PNECs or their committed precursors are vulnerable to oncogenic mutations found specifically in human SCLC, resulting in a significant increase in number of PNECs and conversion to neoplasia. This experimental system provides opportunities to study tumor progression and cancer drug susceptibility and resistance, using human lung cells grown in culture.
ABBREVIATIONS
- PNECS
- pulmonary neuro-endocrine cells
- LUSD
- lung squamous cell carcinoma
- LUAD
- lung adenocarcinoma
- SCLC
- small cell lung cancer
- hESCs
- human embryonic stem cells
- SOX2
- SRY-Box 2
- SPB
- pulmonary-associated surfactant protein B
- SPC
- pulmonary-associated surfactant protein C
- CC10
- The amino acid sequence of Clara Cell 10 kDa secretory protein
- AT2
- alveolar type 2 cells
- EGFR
- Epidermal Growth Factor Receptor
- P63
- transformation-related protein 63
- FOXA2
- Forkhead Box A2
- shRNA
- short hairpin RNA
- HES1
- hairy and enhancer of split-1
- HEY1
- Hes Related Family BHLH Transcription Factor With YRPW Motif 1
- HES6
- hairy and enhancer of split-6
- NE
- neuroendocrine
- NKX2.1
- thyroid transcription factor-1 (TTF-1), also known as Nkx2.1
- NOTCH
- Notch homolog 1
- KRAS
- V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog
- EGFR
- Epidermal growth factor receptor
- TP53
- tumor protein P53
- RB
- retinoblastoma protein
- PTEN
- Phosphatase and tensin homolog
- FACS
- Fluorescence-activated cell sorting
- cDNA
- complementary DNA
- DAPT
- a γ-secretase inhibitor, blocking NOTCH signaling via preventing proteolytic cleavage of the Notch intracellular domain (NICD)
- CGRP
- calcitonin gene-related peptide, symbols: CALCA
- GRP
- gastrin-releasing peptide
- SYP
- Synaptophysin
- ASCL1
- Achaete-Scute Family BHLH Transcription Factor 1
- PGP9.5
- Protein Gene Product 9.5., also known as ubiquitin C-terminal hydrolase 1. (UCHL-1)
- NCAM1
- Neural Cell Adhesion Molecule 1
- CHGA
- Chromogranin A or parathyroid secretory protein 1
- CHGB
- Chromogranin B
- Oct4
- octamer-binding transcription factor 4
- NANOG
- homeobox transcription factor
- SSEA4
- stage-specific embryonic antigen-4
- RNAseq
- RNA sequencing
- tSNE
- t-Distributed Stochastic Neighbor Embedding analysis
- DBZ
- Diazepine inhibitor of γ-secretase, also known as YO-01027
- FACS
- fluorescence activated cell sorting
- Drop-seq
- a technique analyzing mRNA transcripts from droplets of individual cells in a highly parallel fashion
- 10X
- 10X Genomics single-cell 3′mRNA-sequence
- RUES2
- Rockefeller University Embryonic Stem Cell Line 2
- ES02
- HES2 human embryonic stem cell line (NIH codes:ES02).