Page 18 - Read Online

Page 780 Fabbrizi et al. Cancer Drug Resist 2020;3:775-90 I http://dx.doi.org/10.20517/cdr.2020.49

of HIF1-a proportionately increased in both HPV-positive and HPV-negative HNSCC cell lines, with the
[41]
HPV-positive cells still retaining higher levels of the protein .

Whilst the general impact of hypoxia in mediating cellular radioresistance is well acknowledged, the
molecular mechanisms affected specifically in HPV-positive in comparison to HPV-negative HNSCC cell
lines, and the relationship to radiosensitivity have not been studied in detail. Hypoxia, particularly severe
hypoxia (< 0.1% oxygen), promotes genetic instability through activation of ATM/ATR leading to cell cycle
arrest, and downregulation of NHEJ and HR DSB repair pathways at a transcriptional and epigenetic level
thus conferring a more resistant cellular phenotype [42-46] . Under hypoxia-induced replication stress, ATR is
specifically required for phosphorylation of downstream proteins, including checkpoint kinase 1 (CHK1),
p53, and gH2AX [45,47] . It has been demonstrated using three HPV-positive and three HPV-negative HNSCC
cells, that a short term incubation (1 h) in severe hypoxia (0% oxygen) prior to irradiation, led to increased
[48]
radioresistance of both sets of cell lines with observed oxygen enhancement ratios of 2.3-2.9 . This was
associated with an upregulation in the expression levels of hypoxia-responsive genes, the magnitude of
which varied between the cell lines used. More recently, in three HPV-negative HNSCC cell lines it was
shown that hypoxia (0.1% oxygen) during irradiation increases cell survival and reduces the levels of
residual gH2AX foci post-irradiation. Interestingly though, when cells were exposed to a prolonged period
of 4-5 days in hypoxia (1% oxygen) prior to irradiation, ATM-deficient HNSCC cells showed increased
radiosensitivity associated with a delayed phosphorylation of DNA-Pkcs and delayed NHEJ pathway
[49]
activation, whilst the other two HPV-negative HNSCC cell lines displayed increased radioresistance .
This demonstrated a cell line-specific response following long term hypoxia treatment prior to irradiation.
HPV-negative HNSCC usually contain a high frequency of p53 mutations, therefore the concomitant roles
played by p53 mutants and HIF-1 in a hypoxia context could partially explain the higher radioresistance
capacity observed in these tumors. However, more detailed cellular investigations are required.

HNSCC RADIOSENSITISATION STRATEGIES
Enhancing the intrinsic radiosensitivity of HNSCC prior to RT by targeting key cellular pathways is an
approach for combating radioresistance, with the goal of yielding more effective treatment of the tumour.
Several targets have been identified and explored in vitro, including the epidermal growth factor receptor
(EGFR), PARPs, and several proteins involved in DSB repair and cell cycle regulation (key targets and
pathways summarised in Table 1). Hypoxia has also been investigated, and particularly strategies aimed at
increasing the oxygen concentration in the inner cancer mass to improve RT outcome.

Epidermal growth factor receptor
EGFR is a member of the ErbB/HER family of tyrosine kinase receptors. It has been linked to cancer
progression in HNSCC, and overexpression of EGFR is associated with poor prognosis and its level
has been suggested to be a good predictor for patient outcome in HNSCC [68-70] . At a cellular level,
EGFR has six known ligands, including epidermal growth factor, transforming growth factor-a, and
amphiregulin. When activated EGFR affects four major signaling pathways, namely the MAPK, PI3K/
[71]
AKT/mTOR, PLCg/PKC, and the JAK/STAT pathways . In addition, activated EGFR increases the
expression of COX2 and its downstream product PGE2, which creates a positive feedback in reactivating
EGFR [71,72] . Interestingly, nuclear EGFR has been suggested to induce proliferating cell nuclear antigen
and DNA-Pkcs phosphorylation, which ultimately triggers cell proliferation and promotes the repair of
[73]
DNA damage caused by chemoradiotherapy . Therefore, the inhibition of EGFR has been suggested
to be a possible therapeutic target in HNSCC. To date, two main approaches in inhibiting EGFR have
been explored, namely monoclonal antibody (mAb)-based drugs and small molecule tyrosine kinase
inhibitors (TKIs). mAb-based drugs act by binding the extracellular domain of EGFR and inhibiting its
dimerization, which ultimately leads to its degradation after cell internalization. The TKI compounds
target the intracellular tyrosine kinase domain of EGFR and compete with ATP binding to eliminate EGFR