# SP3-induced Timeless transcription contributes to cell growth of lung adenocarcinoma cells

## TL;DR

This study shows that SP3 promotes Timeless gene activity in lung cancer cells, which supports tumor growth and suggests a new treatment target.

## Contribution

The study is the first to demonstrate a direct interaction between SP3 and Timeless in lung adenocarcinoma.

## Key findings

- Timeless is overexpressed in lung adenocarcinoma and may serve as a biomarker.
- SP3 promotes Timeless transcription through a specific promoter fragment (F2).
- Reducing Timeless expression decreases cancer cell growth and increases drug sensitivity.

## Abstract

Timeless is well-known for its key role in replication checkpoints. Recent studies reveal the involvement of Timeless and specificity protein (SP) 1 in human malignancies. However, no evidence proved the interaction between SP3 and Timeless in lung adenocarcinoma (LUAD).

The expression and clinical significance of Timeless were analyzed using the LUAD dataset downloaded from the Cancer Genome Atlas (TCGA). Lentivirus-mediated Timeless knockdown in A549 cells was used to examine the role of Timeless in cell proliferation and pemetrexed (PEM) resistance. Transcription factors (TFs) bound to the Timeless promoter were identified by DNA pull-down technology with HPLC-MS/MS analysis and analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Dual-luciferase reporter assay was used to determine the activity of SP3 in Timeless transcription.

Timeless was overexpressed in LUAD samples, and it could serve as a potential diagnostic or prognostic biomarker for LUAD patients. shTimeless-mediated knockdown of Timeless reduced cell viability and proliferation and sensitized PEM-resistant A549 cells to PEM. Four fragments (F1: 1–373 bp), (F2: 374–962 bp), (F4: 1274–1645 bp), and (F5: 1646-2000bp) were confirmed as the TF binding profiles of the Timeless promoter. KEGG analysis showed that the TFs bound to the Timeless promoter had relevance to spliceosome, RNA transport, and mRNA surveillance pathways. SP3 promoted the transcription of Timeless via the F2 fragment (374–962 bp) binding motif.

Upregulation of Timeless mediated by SP3 promotes LUAD cell proliferation, providing evidence to support that targeting the SP3/Timeless axis may be a potential therapeutic strategy against LUAD.