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SDIRSACR Oncology Insights

followed by targeted miR-21 silencing and pharmacological treatment with JQ1.
Results: The combined intervention resulted in enhanced JQ1 sensitivity, marked by increased apoptotic activity,
reduced invasive potential, and cell cycle disruption. Molecular analysis revealed that these effects were mediated
through modulation of apoptotic regulators and components of the WNT signaling cascade.
Conclusions: Our data highlight a potent synergistic interaction between miR-21 inhibition and BET targeting, suggesting
a novel combinatorial approach to eliminate CSCs and improve therapeutic efficacy in OSCC.

Acknowledgments and funding: This research was funded by the Science Fund of the Republic of Serbia, #GRANT NO
7750038, Oral cancer—new approaches in prevention, control, and post-operative regeneration—an in vitro study—
ORCA–PCR.

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The role of UFMylationin head and neck cancer stem cells

Matijević Glavan T. 1

1 Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, Zagreb

Keywords: neoplasms, proteomics, therapeutics

Background: Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer, associated
with tobacco and alcohol use or viral infections with human papilloma virus (HPV) or Epstein-Barr virus (EBV). The
recurrence and treatment resistance, commonly seen in head and neck squamous cell carcinoma (HNSCC), may be
due to cancer stem cells (CSCs). Tumors contain a subpopulation of cells called cancer stem cells (CSCs). These cells
have the unique abilities to self-renew, differentiate, and initiate new tumors, and they are often linked to resistance
to treatments like chemotherapy and radiotherapy. Both the tumor microenvironment and genetic alterations can
drive CSC plasticity.CSC markers include CD44, CD133, CD24, CD90, and aldehyde dehydrogenase1 (ALDH1). Cancer
cells acquire these stem-like traits through epithelial-mesenchymal transition (EMT), a process driven by transcription
factors like Twist1/2, ZEB1/2, and Snai1/2 (1).
The UFMylation pathway is similar to ubiquitination, regulating a variety of biological processes by post-translationally
attaching to proteins. Ubiquitin Fold Modifier- 1 (UFM1) is a ubiquitin-like modifier that is post-translationally attached
to lysine residues on substrates through a system of enzymes conserved in most eukaryotes: Ubiquitin Like Modifier
Activating Enzyme 5 (UBA5), Ubiquitin-Fold Modifier Conjugating Enzyme 1 (UFC1), and UFM1 Specific Ligase 1 (UFL1)
classified as the E1, E2, and E3 enzymes respectively, while UFM1 Specific Peptidase 1 (UFSP1) and UFM1 Specific
Peptidase 2 (UFSP2) are the UFM1-specific proteases.It is important to note the reversibility of UFMylation: UFSPs are
capable of cleaving UFM1 from the substrate protein, thereby enabling the recycling of UFM1 and its corresponding
substrates. Despite associations between mutations in UFM1 pathway genes and various diseases, the functional
importance of UFMylation is still unclear. It is known that UFMylation is associated with ER homeostasis, DNA damage
response, p53 stabilization, protein biogenesis and regulation, autophagy etc. Researchers are still trying to understand
UFMylation's involvement in cancer, as studies have yielded contradictory findings depending on the specific cancer
type. A large number of amplifications and deletions of genes encoding UFMylationproteins have been found in tumors
(2).Toll-like receptors (TLRs) are a key component of the immune system. These evolutionarily conserved pattern
recognition receptors (PRRs) recognize molecular patterns originating from pathogens, subsequently activating immune
responses. TLR3 binds dsRNA or its synthetic analogs poly (I:C) and poly (A:U) (3).The role of TLR3 in cancer is still not
clear: some studies suggest it could be a target for cancer therapy as its activation leads to apoptosis (4), but other
studies reveal its unfavorable role resulting inthe promotion of tumor progression by either metabolic reprogramming
(5), or increased angiogenesis and migration (5, 6). Additionally, one study so far has associated TLR3 activation with
stem cell-like phenotypes in breast cancer cells (7), and we have recently published that TLR3 activation promotes
stemness in HNSCC lines (8).
Our proteomic analysis of CSCs, followed by further investigations, shows that UFMylation likely plays a significant role
in HNSCC CSCs.
Materials and Methods: HNSCC-derived cancer cell lines Detroit 562, FaDu, and Cal27, and tumor spheres were used
as a model for CSCs research. Tumor spheres were produced by cultivation of the cells in MEBM™ Mammary Epithelial
Cell Growth Basal Medium (Lonza), supplemented with B-27™ Supplement (ThermoFisher Scientific), 20 ng/ mL of
Epidermal growth factor (EGF), and 10 ng/mL of Fibroblast growth factor (FGF) (Peprotech, UK) in low adhesion dishes.

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