Alteration in ATR protein level does not account for the inherent radiosensitivity of HPV-positive head and neck squamous cell carcinoma

TL;DR

This study shows that lower ATR protein levels in HPV-positive head and neck cancer cells do not cause their increased sensitivity to radiation, but instead work together with radiation effects.

Contribution

The study reveals that reduced ATR protein levels in HPV-positive cells are not responsible for their radiosensitivity but contribute additively to it.

Findings

HPV-positive cells have lower ATR protein levels but maintain functional ATR activity after radiation.

ATR knockdown increases radiosensitivity and residual DNA damage only in HPV-positive cells.

HPV-positive cells repair DNA breaks via homologous recombination despite low ATR levels.

Abstract

•HPV-positive HNSCC cells show lower ATR protein levels than HPV-negative.•Despite low protein levels, ATR is fully functional and activated in response to radiation.•HPV-positive but not HPV-negative cells are radiosensitized after ATR knockdown.•HPV-positive cells are able to repair one-ended DSBs by homologous recombination.

HPV-positive HNSCC cells show lower ATR protein levels than HPV-negative.

Despite low protein levels, ATR is fully functional and activated in response to radiation.

HPV-positive but not HPV-negative cells are radiosensitized after ATR knockdown.

HPV-positive cells are able to repair one-ended DSBs by homologous recombination.

Human papilloma virus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) cells are highly radiosensitive resulting from an elevated number of DNA double-strand breaks (DSB) remaining after irradiation. Partially this effect is due to a defective homologous recombination (HR). HPV-positive cells also show pronounced instability of chromosome 3, which codes for the kinase ataxia-telangiectasia and Rad3-related (ATR) protein, a central player of HR. If there is a contribution of ATR to the radiosensitivity of HPV-positive cells remains unclear, and this in-vitro study tested a functional involvement of ATR expression.

The study was performed with six HPV-negative and six HPV-positive HNSCC cell lines. Gene copy number and gene expression were determined via qRT-PCR, protein expression by Western Blot. Response of cells towards irradiation in dependence of ATR expression was tested after siRNA Knock-down (ATRKD). Clonogenic survival after photon irradiation was evaluated by colony formation assay and DSBs were visualized by γH2AX/53BP1 co-staining.

ATR gene copy number and expression were not altered. Protein level was almost two-fold lower in HPV-positive compared to HPV-negative cells, but fully functional as observed by active phosphorylation in response towards irradiation. ATRKD resulted in a further increase in both, radiosensitivity as well as number of residual DSBs, but only for HPV-positive cells.

Since the effect of ATRKD was compensated in HPV-negative but not in HPV-positive cells, these data revealed that the two-fold lower level of ATR in HPV-positive cells does not account for their enhanced inherent radiosensitivity, but acts additive to irradiation.

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