Best Radiobiology Paper Award

 

ESTRO 2025 Congress report

 

Head-and-neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide. Patients with early-stage disease are treated with established surgical and/or radiotherapy methods and are associated with good cure rates in general. However, in locally advanced disease, in many cases even current methods of disease management cannot prevent relapses.

Patients with locally advanced HNSCC typically receive primary chemoradiotherapy (RCTx) or surgery and postoperative cisplatin-based chemoradiotherapy (PORT-C). In addition to established treatment-determining factors, such as the tumour, node, metastasis (TNM) classification, other indicators have prognostic potential, including the level of human papillomavirus (HPV) infection, the tumour microenvironment, and hypoxia. Furthermore, local tumour control after fractionated radiation therapy may depend critically on tumour cell plasticity.

Tumour cell plasticity is promoted by epithelial-mesenchymal transition, inflammation, hypoxia, microenvironmental changes, and therapies, as well as by the acquisition of genetic mutations and epigenetic alterations. Plasticity regulates the molecular programmes that control the stem cell status of tumours. Cells that maintain tumours with self-renewal and differentiation properties are referred to as cancer stem cells (CSCs). CSCs have been identified in many cancer types, including HNSCC. Tumour cells have been shown to be plastic and can reversibly switch between CSC and non-CSC states (1). Nevertheless, tumour cell populations that have self-renewal and differentiation properties contribute significantly to tumour growth and therapy resistance, regardless of whether these populations are transient or permanent, well-defined cell subsets (Figure 1 A). Therefore, inactivation of all CSCs is important to achieve permanent tumour control.

Figure 1. A) CSC populations with self-renewal and differentiation properties contribute to tumour growth and therapy resistance. Inactivation of all CSCs is important if permanent tumour control is to be achieved. Created with BioRender. B) Interplay between homologous recombination (HR) proteins RAD54L and PSMC3IP and the CSC transcription factors Oct4 and MYC (1, 2) regulates stemness and radioresistance in HNSCC (3). CSC - cancer stem cells; DDR - DNA damage response; HR - homologous recombination. Adapted from (2).

Previous studies have shown that local tumour control after fractionated irradiation may depend on the number of CSCs present before therapy initiation as well as their intrinsic radioresistance. In HNSCC, CSCs can be protected from the effects of tumour therapy through several intrinsic and extrinsic mechanisms, such as increased expression of important DNA repair proteins (3). Current findings suggest that the role of DNA repair proteins extends far beyond their function in the repair of DNA damage (4). Our work shows that the role of the HR proteins RAD54L and PSMC3IP in the regulation of tumour radioresistance is not limited by their impact on DNA repair and the cell cycle. In addition, they are both able to influence CSC properties in vitro. The mechanisms that govern how these DNA repair factors can impact the CSC phenotype warrant further analysis. On the other hand, we have also found that CSC-related transcriptional factors Myc and Oct4 positively regulate RAD54L and PSMC3IP expression levels and therefore mediate the DNA repair mechanisms in tumour cells, including CSC populations (Figure 1 B).

We hypothesised that, as for many genes involved in HR-mediated DNA repair, deregulated (both high and low) expression of Oct4, MYC, PSMC3IP and RAD54L should result in DNA repair deficiency. To validate this hypothesis, we used the retrospective immunohistochemical (IHC) analyses of protein expression in tumour tissues taken from patients with locally advanced HNSCC who had been treated with PORT-C (n = 167) or primary RCT (n = 98), as well as the results of 2D and 3D radiobiological assays in several HPV-negative HNSCC cell lines. The IHC analyses revealed that deregulated (high or low) nuclear Oct4 protein expression was associated with better loco-regional control (LRC) compared with intermediate Oct4 expression (p = 0.015). Similar to the results of the Oct4 IHC analysis, both very high and very low levels of MYC and RAD54L expression were found to be associated with better patient prognosis (LRC and distant metastasis-free survival, correspondingly) compared with intermediate levels (p = 0.03).

The in vitro assays confirmed that the deregulated expression of CSC-related transcriptional factors such as Oct4 and its target genes, such as RAD54L and PSMC3IP, were associated with abnormal HR-mediated DNA repair and higher cell radiosensitivity. Next, we analysed whether this HR deficiency might be therapeutically exploitable. We used a combination of radiotherapy with synthetic lethality induced in Oct4-knockout HNSCC cells through the use of poly-adenosine diphosphate ribose polymerase (PARP)-inhibitor Olaparib. Similarly, RAD54L and PSMC3IP knockdown sensitised cells to the combination of Olaparib and radiotherapy compared with controls (Figure 2 A).

 

Figure 2. A) The deregulated (both high and low) expression of the CSC-related transcriptional factors and HR genes PSMC3IP and RAD54L results in DNA repair deficiency. It can be therapeutically exploited based on synthetic lethality through the targeting of the tumour DNA repair pathways (e.g., by PARP inhibition) in combination with radiotherapy. Adapted from (2). B) Clinical relevance and anticipated personalised treatment based on our hypothesis. Created with BioRender.

In conclusion, our study suggests that deregulated expression of the CSC-related transcriptional factors Oct4, MYC, and their target genes, such as RAD54L and PSMC3IP, are associated with abnormal HR-mediated DNA repair. Such expression can be used to predict high tumour sensitivity to DNA-damaging treatment such as radio(chemo)therapy in patients with HPV-negative HNSCC and to exploit therapeutic approaches based on synthetic lethality through the targeting of tumour DNA repair pathways (e.g., by PARP inhibition) in combination with radiotherapy (Figure 2).

In our future research, we aim to characterise the molecular mechanisms that underlie these distinct roles of RAD54L and PSMC3IP. We anticipate that these studies will uncover resistance mechanisms and identify novel therapeutic targets for tumour radiosensitisation.

Figure 3. This project is a result of collaborative work between Annett Linge (in the middle), the team of Anna Dubrovska (on the left), and the team of Kerstin Borgmann (on the right).

PD Dr med. habil. Annett Linge
Corresponding author

University Hospital Carl Gustav Carus Dresden

Department for Radiotherapy and Radiation Oncology

Dresden, Germany

 

References:

1. Peitzsch C, Nathansen J, Schniewind SI, Schwarz F, Dubrovska A. Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma: Identification, Characterization and Clinical Implications. Cancers (Basel). 2019 May 2;11(5):616. doi: 10.3390/cancers11050616.
2. Nathansen J, Lukiyanchuk V, Hein L, Stolte MI, Borgmann K, Löck S, Kurth I, Baumann M, Krause M, Linge A, Dubrovska A. Oct4 confers stemness and radioresistance to head and neck squamous cell carcinoma by regulating the homologous recombination factors PSMC3IP and RAD54L. Oncogene. 2021 Jun;40(24):4214-4228.
3. Schulz A, Meyer F, Dubrovska A, Borgmann K. Cancer Stem Cells and Radioresistance: DNA Repair and Beyond. Cancers (Basel). 2019 Jun 21;11(6):862.
4. Nathansen J, Meyer F, Müller L, Schmitz M, Borgmann K, Dubrovska A. Beyond the Double-Strand Breaks: The Role of DNA Repair Proteins in Cancer Stem-Cell Regulation. Cancers (Basel). 2021 Sep 26;13(19):4818.