Author of

• 18972

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  P2.02 - Biology/Pathology (ID 616)

  • Event: WCLC 2017
  • Type: Poster Session with Presenters Present
  • Track: Biology/Pathology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)

  • 23252

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    P2.02-054 - Thymidylate Synthase Promotes Epithelial-To-Mesenchymal Transition and Aggressiveness in NSCLC (ID 8332)

    09:30 - 09:30  |  Author(s): A. Siddiqui
    • Abstract

    Background:
    Thymidylate synthase (TS) is a nucleotide metabolism enzyme and a chemotherapeutic target. TS overexpression is associated with worst prognosis in NSCLC and is important for the pathological progression of tumors. We investigated the role of TS in epithelial-to-mesenchymal transition (EMT), a developmental process that allows the cancer cells to acquire features of aggressiveness like motility and chemoresistance.
    
    Method:
    Immunohistochemistry (IHC) on TS and EMT markers was performed in tissues from 61 NSCLC patients. EMT and cancer stemness markers were quantified in cultured NSCLC cell lines by western blotting. Migration assays were conducted using an automated wound-healing real-time quantification system. Spheres-forming assays were performed by growing cells in low-adherence plates. In addition, cells were stably infected with a lentiviral reporter plasmid in which the expression of m-Cherry fluorescent protein was driven by the full-length promoter sequence of the TS gene (TYMS).
    
    Result:
    A significant association between TS mRNA expression and the markers of EMT was found (p<0.01). This was confirmed at the protein level in cultured cell lines, and TS was found up-regulated following EMT induction by TGF-Beta. In addition, a strong association between TS and the powerful EMT driver ZEB1 was found by western blotting and validated by IHC in tissue specimens from NSCLC patients (p=0.02). Importantly, TS showed to regulate EMT, as a shRNA-mediated knockdown of TS could reduce in vitro the levels of ZEB1 and of other EMT markers, suppress the cells’ migratory and spheres-forming abilities and the chemoresistance. Furthermore, following infection with the fluorescent promoter reporter, we could FACS-sort two populations with distinct mesenchymal and epithelial-like phenotypes (TS-prom[high] and TS-prom[low], respectively) from NSCLC cells. Sorted cells, in fact, displayed differential expression of EMT markers (E-Cadherin, Vimentin and ZEB1), migratory ability and spheroids-forming properties (all p<0.001), while no differences were observed using a GAPDH promoter reporter as control.
    
    Conclusion:
    All together, these data indicated an unprecedented role for TS in governing cancer differentiation and aggressiveness. The lentiviral promoter reporter may lead to the identification of the regulatory elements and the transcription factors linking TS to EMT. With regards to possible translational implications, the cancer EMT status could be tested as a predictor of the response to TS-inhibiting drugs in NSCLC, and eventually used as a stratification criterion. In the longer run, these data could inspire the development of conceptually novel anti-TS agents that better suppress tumor's growth and aggressiveness.