ATR-binding lncRNA ScaRNA2 promotes cancer resistance through facilitating efficient DNA end resection during homologous recombination repair

Background Our previous study first showed that ATR-binding long noncoding RNA (lncRNA) is necessary for ATR function and promotes cancer resistance. However, the specific lncRNAs instrumental in ATR activation remain largely unclear, which limits our comprehensive understanding of this critical biological process. Methods RNA immunoprecipitation (RIP) followed by RNA sequencing was employed to identify ATR-binding lncRNAs, which were further validated using RIP-qPCR assays. Immunofluorescence staining and Western blotting were applied to detect the activation of DNA damage repair factors. After the effect of scaRNA2 on cellular sensitivity to DNA-damaging reagents was determined, the effects of scaRNA2 on radiotherapy were investigated in patient-derived organoids and xenograft preclinical models. The clinical relevance of scaRNA2 was also validated in tissues isolated from rectal cancer patients. Results ScaRNA2 was identified as the most enriched ATR-binding lncRNA and was found to be essential for homologous recombination (HR) mediated DNA damage repair. Furthermore, scaRNA2 knockdown abrogated the recruitment of ATR and its substrates in response to DNA damage. Mechanistically, scaRNA2 was observed to be necessary for Exo1-mediated DNA end resection and bridged the MRN complex to ATR activation. Knockdown of scaRNA2 effectively increased the sensitivity of cancer cells to multiple kinds of DNA damage-related chemoradiotherapy. Preclinically, knockdown of scaRNA2 improved the effects of radiotherapy on patient-derived organoids and xenograft models. Finally, an increase in scaRNA2 colocalized with ATR was also found in clinical patients who were resistant to radiotherapy. Conclusions ScaRNA2 was identified as the most abundant lncRNA bound to ATR and was demonstrated to bridge DNA end resection to ATR activation; thus, it could be applied as a potent target for combined cancer treatments with chemoradiotherapy. Supplementary Information The online version contains supplementary material available at 10.1186/s13046-023-02829-4.


Fig. S2 Bioinformatics analysis of RNA sequencing in normal and scaRNA2 knockdown cells
A. Heat map of upregulated genes and downregulated genes in sg-ctrl and scaRNA2 knockdown cells.B. GO analysis of the biological processes affected by scaRNA2.

Fig. S7
Fig. S7 ScaRNA2 is necessary for the DNA damage responses after CPT and ETO treatment.A-B.HCT116 cells with/without scaRNA2 knockdown were treated with 100 μg/mL ETO for 4 h or 1 µM CPT for 1 h, and proteins involved in the DNA damage response were detected with western blotting assays at the indicated time points.The specific sites for phosphorylated proteins are indicated.GAPDH was used as a negative control.C-K.Quantification of the relative density based on gray value to determine protein expression or phosphorylation.GAPDH was used as the internal control, and normalized gray value of phosphorylated protein = the grey value of phosphorylated protein/gray value of total protein.*P < 0.05, **P < 0.01, ***P < 0.001

Fig. S9
Fig. S9 Overexpression of scaRNA2 inhibits apoptosis activation by DNA damage.A-L.Protein levels involved in the cell apoptosis signaling pathway in Vector and scaRNA2 overexpressing HCT116 cells at the indicated times after 8 Gy irradiation (A), 1 µM CPT for 1 h (B) or 100 μg/mL ETO for 4 h (C).

Fig. S10
Fig. S10 ScaRNA2 knockdown inhibited DNA damage checkpoint activation in HCT116 cells.A-F.Analysis of the cell cycle phase distribution in the G0/G1, S or G2/M phase by flow cytometry in sg-ctrl and scaRNA2 knockdown HCT116 cells at the indicated times after 8 Gy irradiation (A-B), 1 µM CPT for 1 h (C-D) or 100 μg/mL ETO for 4 h (E-F).*P < 0.05, **P < 0.01, ***P<0.001compared with the sg-ctrl group at the same dose of treatment.

Fig. S11
Fig. S11 ScaRNA2 knockdown inhibited DNA damage checkpoint activation in HT29 cells.A-F.Analysis of the cell cycle phase distribution in the G0/G1, S or G2/M phase by flow cytometry in sg-ctrl and scaRNA2 knockdown HT29 cells at the indicated times after 8 Gy irradiation (A-B), 1 µM CPT for 1 h (C-D) or 100 μg/mL ETO for 4 h (E-F).*P < 0.05, **P < 0.01, ***P<0.001compared with the sg-ctrl group at the same dose of treatment.

Fig. S12
Fig. S12 Knockdown of scaRNA2 inhibits the progression of cell cycle into G2/M after irradiation.A-D.Protein levels and quantitative analysis involved in the cell cycle signaling pathway in sg-ctrl and scaRNA2 knockdown HCT116 (A-B) or HT29 (C-D) cells at the indicated times after 8 Gy irradiation.*P<0.05,**P<0.01,***P < 0.001 compared with the sg-ctrl group at the same dose of treatment.

Fig. S13
Fig. S13 Overexpression of scaRNA2 promoted cell cycle progression after irradiation.A-D.Protein levels and quantitative analysis involved in the cell cycle signaling pathway in Vector and scaRNA2 overexpressing HCT116 (A-B) or HT29 (C-D) cells at the indicated times after 8 Gy irradiation.*P<0.05,**P<0.01,***P < 0.001 compared with the NC group at the same dose of treatment.

Fig. S15
Fig. S15 Schematic diagram of local irradiation field and shielding of cell-derived xenografts (CDX, A) and patient-derived xenografts (PDX, B).

Fig. S16
Fig. S16 Knockdown of scaRNA2 sensitized colorectal cancer to radiotherapy.A. The knockdown efficacy of was confirmed with RT-PCR after intratumoral injection with scaRNA2 knockdown lentivirus or sg-ctrl lentivirus.***P < 0.001 compared with the sg-ctrl group.B. Tumor images of xenografts after 15 Gy local irradiation in four groups at the end of the experiment (n = 5).

Fig. S17
Fig. S17 Scanned image of tissues microarray including CRC patients included in our study.RNA FISH and immunofluorescence staining were performed to detect the expression of scaRNA2 and ATR, respectively.