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SDIRSACR Oncology Insights

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Acknowledgments and funding: This study was funded by the Horizon Europe STEPUPIORS Project (HORIZON-WIDERA-
2021-ACCESS-03, European Commission, Agreement No. 101079217) and the Ministry of Science, Technological
Development and Innovation of the Republic of Serbia (Agreement No. 451-03-136/2025-03/200043).

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Estrogen receptor modulation and Hedgehog-GLI crosstalk in Head and neck squamous cell carcinoma

Josipa Jelačić1, Jacqueline-Katrin Kranjčević , Nina Milutin1, Ozren Vugrinec2, Ivan Mumlek3, Ana Kvolik Pavić3, Dinko
1
Leović2, Vedran Zubčić3, Maja Sabol1, Petar Ozretić1

1Ruđer Bošković Institute, Division of Molecular Medicine, Zagreb, Croatia
2Zagreb University Hospital Centre, Department of Otorhinolaryngology and Head and Neck Surgery, Zagreb, Croatia
3University Hospital Osijek, Department of Maxillofacial and Oral Surgery, Osijek, Croatia

Keywords: squamous cell carcinoma of head and neck; receptors, estrogen; arsenic trioxide; tamoxifen

Background: Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous malignancy of the upper
aerodigestive tract. Despite therapeutic progress, it remains associated with high mortality and impaired quality of
life. Key risk factors include tobacco, alcohol, and HPV infection. A higher male prevalence suggests a role for sex
hormones, notably estrogen and its receptors, in modulating disease risk, possibly due to male-specific risks or female
hormonal protection. To elucidate the involvement of estrogen receptors (ER) in HNSCC pathogenesis, we performed
a comprehensive analysis of both nuclear and membrane-associated estrogen receptors, with particular emphasis on
their interaction with the Hedgehog-GLI signaling pathway.
Materials and Methods: Using quantitative real-time PCR (qPCR), we analyzed the mRNA expression levels of ESR1,
ESR2, GPER1, and SCN2A in 93 primary HNSCC tumors, 26 positive lymph nodes, and 42 healthy oral mucosa samples.
We then compared ER expression patterns with clinical parameters, including patient age, sex, tumor stage and
grade, HPV status, and primary tumor site. Given the differential expression of ESR1 and ESR2 in tumor versus normal
tissues, we investigated the impact of modulating ER activity using estradiol and tamoxifen, along with inhibition of the
Hedgehog-GLI pathway via arsenic trioxide, on cellular behavior in HNSCC.
Results: We established CAL-27 cell lines stably expressing ESR1 or ESR2 using CRISPR-Cas9-mediated gene editing.
Tamoxifen treatment significantly reduced viability and colony formation in ESR1-KI cells, while ESR2-KI cells exhibited
increased resistance compared to the parental line. Conversely, ESR2-KI cells showed enhanced sensitivity to arsenic
trioxide and its combination with tamoxifen. Functional assays revealed that tamoxifen also reduced colony formation
in ESR1-KI cells and migration capacity in both KI cell lines. Furthermore, single and combined treatments significantly
reduced the number of cells in the G2/M phase of the cell cycle in both KI cell lines, while in the ESR2-KI cell line
combined treatment increased the number of apoptotic cells.
Conclusions: In summary, our results suggest that elevated ESR1 and ESR2 expression influences the biological
characteristics of HNSCC cells. Further investigations are required to fully elucidate their potential as biomarkers or
therapeutic targets in HNSCC.

Acknowledgments and funding: We would like to thank the Croatian Science Foundation [IP-2018-01-4889 and DOK-
2021-02-5821] and the Terry Fox Foundation for financing our research and PhD student J. Č.

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