Page 70 - SRPSKO DRUŠTVO ISTRAŽIVAČA RAKA

Serbian Association for Cancer Research SDIRSACR

Proteomic analysis performed by Biocentar (Zagreb, Croatia) identified several proteins involved in the UFMylation
pathway in CSCs. This was further studied by bioinformatics, transient transfection and RNA silencing, western blot,
immunocytochemistry, RNA isolation, real time PCR and cytotoxicity assay.The RNA and proteins were isolated from
tissue of 43 HNSCC patients admitted to the Clinical Hospital Dubrava (Zagreb, Croatia) and collected during surgery
by a Mini kit for RNA and protein purification (Macherey-Nagel, Dueren, Germany). Procedures that followed were
conducted according to the ethical protocols approved by the Institutional Ethics Committee. Informed patient consent
was obtained prior to surgery.
Results: We have performed proteomic analysis on 4 sets of samples: adherent cells, untreated tumor spheres, tumor
spheres treated with poly (I:C), and tumor spheres treated with poly (A:U). We found 51 changed proteins in tumors
spheres compared to adherent cells, 46 changed proteins in tumor spheres treated with poly (A:U) compared to
untreated tumor spheres, and 22 changed proteins in poly (I: C)-treated tumor spheres compared to the untreated
tumor spheres. By using STRING analysis, we have determined many different protein interactions. However, in this
study we have focused on the UFMylation pathway, as several of the changed proteins belonged to this pathway:
UFSP2 and DDRGK1 as enzymes involved in this system, and RPL9 and RPL29 as target proteins of UFMylation. We have
first confirmed our proteomic analysis results by western blot. Given the observed overexpression of UFSP2 in tumor
spheres when compared to adherent cells, we proceeded with a more in-depth investigation of this phenomenon.
Bioinformatic analysis revealed that high expression of UFM1 is linked with worse overall and disease-free survival, and
it correlated with the main EMT proteins in HNSCC. Our experiments showed that UFM1 was also strongly expressed
in tumor spheres compared to the adherent cells.Its inhibition by siRNA reduced tumor sphere growth, downregulated
CD133, and also reduced the expression of EMT markers fibronectin and vimentin. Our bioinformatic analysis, using
FunRich, showed that the Sp1 transcription factor regulates nearly 50% of the proteins we found altered in our proteomic
study. Therefore, we investigated mithramycin, Sp1 inhibitor, as a potential therapeutic agent targeting specifically
CSCs. We have established that mithramycin reduced cell survival of three HNSCC lines Detroit 562, FaDu and Cal27,
through apoptosis induction as evidenced by PARP cleavage. The UFMylation process was disrupted in mithramycin-
treated tumor spheres after 72 h, as there is almost no visible UFM1 and UFM1 conjugates expression.Mithramycin
also reduced the expression of other UFMylation proteins DDRGK1, UBA5, and UFC1, but also the expression of RPL26,
which is a target for UFMylation (9). Finally, we have analyzed HNSCC patients tissue samples to establish the level of
expression of UFSP2 and DDRGK1.
Conclusions: Our findings indicate that UFMylation is crucial for HNSCC cancer stem cells (CSCs). We propose that
inhibiting UFM1 or other UFMylation pathway proteins (DDRGK1, UBA5, UFC1) could be a reliable strategy to delay
HNSCC progression by targeting CSCs. Such inhibition is achievable with Sp1 inhibitors like mithramycin or its analogs.
This research highlights UFM1's potential as a prognostic biomarker and a therapeutic target for HNSCC, specifically in
eliminating CSCs through apoptosis.

References

1. Vukovic Đerfi K, Vasiljevic T, Matijevic Glavan T. Recent Advances in the Targeting of Head and Neck Cancer Stem
Cells. Applied Sciences. 2023; 13(24):13293.
2. Jing Y, Mao Z, Chen F. UFMylation System: An Emerging Player in Tumorigenesis. Cancers (Basel). 2022 Jul
19;14(14):3501.
3. Glavan TM, Pavelic J. The exploitation of Toll-like receptor 3 signaling in cancer therapy. Curr Pharm Des.
2014;20(42):6555-64.
4. Salaun B, Coste I, Rissoan MC, Lebecque SJ, Renno T. TLR3 can directly trigger apoptosis in human cancer cells. J
Immunol. 2006 Apr 15;176(8):4894-901.
5. Matijevic Glavan T, Cipak Gasparovic A, Vérillaud B, Busson P, Pavelic J. Toll-like receptor 3 stimulation triggers
metabolic reprogramming in pharyngeal cancer cell line through Myc, MAPK, and HIF. Mol Carcinog. 2017
Apr;56(4):1214-1226.
6. Paone A, Galli R, Gabellini C, Lukashev D, Starace D, Gorlach A, et al. Toll-like receptor 3 regulates angiogenesis and
apoptosis in prostate cancer cell lines through hypoxia-inducible factor 1 alpha. Neoplasia. 2010 Jul;12(7):539-49.
7. Jia D, Yang W, Li L, Liu H, Tan Y, Ooi S, et al. β-Catenin and NF-κB co-activation triggered by TLR3 stimulation
facilitates stem cell-like phenotypes in breast cancer. Cell Death Differ. 2015 Feb;22(2):298-310.
8. Vasiljevic T, Zapletal E, Tarle M, Bozicevic Mihalic I, Gouasmia S, Provatas G, et al. Targeting DAMPs by Aspirin
Inhibits Head and Neck Cancer Stem Cells and Stimulates Radio-Sensitization to Proton Therapy. Cancers. 2025;
17(13):2157.
9. Derfi KV, Vasiljevic T, Dragicevic T, Glavan TM. Mithramycin targets head and neck cancer stem cells by inhibiting
Sp1 and UFMylation. Cancer Cell Int. 2024 Dec 19;24(1):412.

55