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Fabbrizi et al. Cancer Drug Resist 2020;3:775-90 I http://dx.doi.org/10.20517/cdr.2020.49 Page 779

As for the mechanism involved, several reports have shown that there is a defect in expression of key DSB
repair effector proteins and/or defects in the efficiency of the DSB repair pathways post-irradiation. Firstly,
five HPV-positive HNSCC cell lines were demonstrated to harbour more persistent levels of 53BP1 and
gH2AX foci and display a marked G2/M arrest in response to radiation compared to five HPV-negative
[24]
HNSCC cell lines . This finding was replicated in another study comparing two HPV-positive with
two HPV-negative HNSCC cell lines, where the HPV-positive HNSCC cell lines demonstrated delayed
resolution of gH2AX and 53BP1 foci and delayed repair of radiation-induced DSBs post-irradiation revealed
[25]
through the neutral comet assay . A third study attributes the deficiency in DSB repair in two HPV-positive
versus one HPV-negative HNSCC cell line to decreased BRCA2, DNA-Pkcs, and 53BP1 protein expression
in HPV-positive HNSCC cells, which ultimately led to NHEJ and HR signaling deficiency as demonstrated
[26]
through the lack of DNA-Pkcs and BRCA2/RAD51 foci, respectively post-irradiation . It is interesting
to note that there is evidence that HPV-positive HNSCC cells also show upregulation of several proteins
[25]
involved in BER and SSB repair, including PARP-1, PNKP, Pol b, and XRCC1 . Whilst this does not
appear to relate to the observed increased radiosensitivity, it suggests a compensatory mechanism of
increased BER activity through loss of DSB capacity. These data are supported by analysis of the TCGA
database, which demonstrated increased gene expression of at least XRCC1 and PARP-1 in HPV-positive
HNSCC. Interestingly, it was further shown that shRNA-mediated downregulation of multiple BER genes
enhanced the radiosensitivity of one HPV-positive HNSCC cell line and not of a corresponding HPV-negative
[27]
HNSCC cell line .
In addition to defective DSB repair pathways, differentially regulated cell cycle control in HPV-positive
HNSCC cells is well established, primarily due to the impact of the E6 and E7 HPV genes. The E6 and
E7 proteins can bind the tumour suppressor protein p53 and retinoblastoma (Rb) protein, respectively,
facilitating their ubiquitylation-dependent proteasomal degradation resulting in the cells losing their ability
[28]
to perform cell cycle arrest or to undergo apoptosis . The oncoprotein E7 also leads to the accumulation
[29]
of p16, which is used as a biomarker of HPV-positive HNSCC . p16 overexpression in HPV-positive
HNSCC cells causes initiation of cell cycle progression, but independently has been suggested to have a
direct impact on DDR, specifically through HR by reducing RAD51 foci formation post-irradiation .
[30]
Interestingly, p16 has been recently demonstrated to cause downregulation in the protein levels of the
E3 ubiquitin ligase TRIP12 leading to deficiencies in DSB repair. Targeting TRIP12 using siRNA in
HPV-negative HNSCC cells led to an impairment of HR by inhibiting the recruitment of BRCA1 to DSBs
post-irradiation . Cumulatively, these data clearly show the impact of HPV in reducing the efficiency of
[31]
the cellular DDR in HNSCC cells.

Hypoxia
The existence of hypoxia (low oxygen) in human tumors, an important source of radioresistance, was
first established in the 1950s [32,33] . This biological effect is caused by the physical interference of the lack
of oxygen with the radiation effects on creating DNA damage, but also through the alteration of cellular
pathways and processes. Oxygen is a particularly powerful radiosensitiser. However, it has been shown that
HNSCC can reach levels of moderate and severe hypoxia which will ultimately lead to radioresistance and
poor prognosis [34,35] . In terms of cellular pathways, the major player mediating the hypoxic response are
the hypoxia-inducible factors (HIFs). The HIF-a subunits contain proline residues which are hydroxylated
by prolyl hydroxylase domain (PHD) enzymes allowing the von Hippel-Lindau tumour suppressor
protein to label it for ubiquitylation and rapid proteasomal degradation under normoxic conditions [36-38] .
During hypoxia, the PHD enzymes are inhibited, leading to HIF-a stabilization which in turn leads to
transcriptional activation of many hypoxia-related genes [39,40] . Interestingly, it has been observed that five
HPV-positive HNSCC cell lines show higher levels of HIF1-a (but also PHD2 enzyme) compared to two
HPV-negative HNSCC cells under conditions of normoxia, possibly due to the HPV oncoprotein inhibiting
PHD2-dependent hydroxylation of HIF1-a. Under hypoxic (1% oxygen) conditions, relative protein levels