Fig 2: Upregulated expression of NSE was positively correlated with distant metastasis of SCLC patients. (A) The relationship between serum NSE concentration and the prognosis of SCLC patient was evaluated by univariate and multivariate COX regression analysis. The correlations between NSE serum concentration and TNM stage (B), T stage (C), N stage (D), and M stage (E) were evaluated. (F) The correlations between NSE serum concentration and different sites of metastasis. (G) The expression difference of NSE in SCLC with or without distant metastasis was detected by immunohistochemistry (Left scale bars: 50 um, Right scale bars: 25 um). Data is represented as mean ± SD. The significance level is represented by ns, p < 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig 3: NSE induced EMT in SCLC cells. (A, B) NSE overexpression promoted the EMT process of SCLC. (A) Western blot assay was performed to measure the protein expression levels of the EMT-related markers (Snail, N-cadherin and E-cadherin). (B) qRT-PCR was performed to measure the mRNA expression levels of the EMT markers. (C, D) NSE knockdown represses the EMT process of SCLC. Protein levels (C) and mRNA levels (D) of EMT-related markers were measured using western blot or qRT-PCR, respectively. These results were repeated of three independent experiments.

Fig 4: A mouse embryonic fibroblast (MEF) immortalization system recapitulates the two-signature CNA patterns and glycolysis gene CNA enrichment observed in human tumors and mouse models Top: Copy number profiles of 59 samples from 42 independent mouse embryonic fibroblasts (MEF) sublines before, during, and after senescence recapitulate the two-signature CNA patterns observed in human tumors. Twenty-two sublines evolved to Sig. A patterns, 13 sublines exhibited Sig. B, and seven sublines remained diploid-like due to redox protection. PC1 scores for analysis of only signature A MEFs (PC1-Sig A), only signature B MEFs (PC1-Sig B), or all MEFs (PC1-Sig AB) are indicated on the left. Also indicated are a metric of the degree of senescence observed during immortalization (senescence score), protection by 3% O2-based hypoxia or catalase (+), or both (++) during immortalization, CNA profiling at passage 1 (P1), exogenous MYC expression (which enabled cells to bypass senescence), and Trp53 sequencing status (* indicates a non-severe mutation p.183D > E, blank indicates not sequenced, additional p53 sequencing information in Appendix Table S1). Signature A MEF lines profiled at more than one passage number are indicated by the start (earlier passage) and end points (later passage) of the upward “evolving signature A” arrows. For comparison to (C), the early (25) and late (49) passages of the H1 subline that were characterized by spectral karyotyping (SKY) are indicated on the left by triangles. The indicated chromosome 6 region includes loci for Bpgm, Hk2, Rpia, Eno2, Tpi1, Gapdh, Tigar, Ldhb, and Kras. Bottom: Summary signatures of amplification and deletion loci in copy number profiles (normalized PC1 loadings) are shown for MEF WT and p53-/- signature A (Sig. A) samples. A paired t-test analysis of signature A MEF lines profiled at more than one passage number revealed genomic regions associated with mid- to late-passage CNA evolution (log10 t-test P-value signed positive for amplifications, negative for deletions; labeled “Evolving”). Individual profiles of p53-/- signature A samples are not shown.Signature A MEFs have more DNA breakpoints and a larger degree of copy number alteration (integrated CNA score) than signature B MEFs. Signature A MEFs had a subset of cases with large numbers of breakpoints per chromosome (> 10) that were not observed in the signature B MEFs (hypergeometric P-value = 0.02, Student's t-test P-value = 4 × 10-5). Data are presented in box (median, first and third quartiles) and whisker (extreme value) plots.Spectral karyotyping (SKY) of the H1 MEF subline at passage 48. Whole chromosomal gains of varying extent are observed for all chromosomes with some chromosomes experiencing translocation, for example, t(3:16), and deletion, for example, del(4). Additional chromosome spreads and a summary table for P25 and P48 karyotypes are shown in Appendix Fig S8. CGH profiles for H1 cells are in (A) (indicated by the triangles in the SKY column) and Appendix Fig S6A and B.The amount of senescence demonstrated during MEF line derivation (senescence score, see Materials and Methods) is highly correlated with the degree of copy number alterations obtained (integrated CNA score) (Spearman rho = 0.66, P-value = 3 × 10-8). Single protection indicates protection by 3% O2 culture conditions or media supplementation with 250 U/ml catalase. Double protection indicates that cells were cultured at 3% O2 with catalase.Conserved amplification and deletion loci in copy number profiles across core and expanded signature A human tumor types (Hs Sig. A), corresponding mouse tumorigenesis model signatures (Mm models), and the MEF immortalization system (MEF Sig. A). The human and mouse signatures are as defined in Fig 2, and the wild-type MEF signatures are from (A). In the human consistent regions, the 52.3% consistently altered gene loci include bona fide tumor suppressor genes (p53), oncogenes (MYC, KRAS), telomere components (TERC, TERT), and core glycolysis genes (e.g., HK2). As in Fig 2D, genes listed in parentheses were consistently altered across the human tumors but not consistently altered in the mouse. The tissue-specific, non-lymphoid oncogenes from the COSMIC database that are in a conserved amplification or deletion region (TRRAP, CHD4) are listed for completeness (Forbes et al, 2015).Enrichment signal strength (negative of log permutation P-value) for the glycolysis and core glycolysis gene sets improve when MEF CNA signatures are added to the human and mouse CNA signatures from Fig 2. The degree of increased enrichment upon addition of the MEF signatures is quantitatively similar to adding mouse tumorigenesis model signatures. Adding all signatures into the analysis further strengthens the enrichment (last column). Table EV2 lists results for the enrichment analysis of the remaining MSigDB canonical pathways when human core and expanded signature A, mouse models, and MEF immortalization models are sequentially combined.Data information: See also Appendix Figs S5–S9.

Fig 5: NSE promoted EMT and tumor metastasis by activating the Wnt/ß-catenin pathway in vivo. H446 cells and H69 cells were transfected with Luciferase and GFP-NSE or sh1NSE or sh2NSE or vectors and then injected through the left cardiac ventricle of anesthetized nude mice. (A, B) The bioluminescence images analysis was conducted by using an IVIS imaging system in nude mice models of NSE-overexpressing H446 cells (A) and NSE-silencing H69 cells (B). (C, D) Body weight loss was observed in nude mice models of NSE-overexpressing H446 cells (C) and NSE-silencing H69 cells (D). (E, F) Kaplan–Meier plot of survival in the experiment of NSE-overexpressing group (E) and NSE-silencing group (F) (*p < 0.05, log-rank test). (G, H) qRT-PCR assay for the expression of NSE, ß-catenin and EMT markers in tumor tissues of nude mice models of NSE-overexpressing H446 cells (G) and NSE-silencing H69 cells (H). Data are presented as mean values ± SD. *p < 0.05 using the two-sided Student's t-test. ns: no significance.