Background:the treatment of squamous cell carcinomas of the oral cavity (OSCCs) is limited by the lack of reliable diagnostic/prognostic, and predictive markers, as well as by intrinsic tumor cell heterogeneity. 5-azacytidine (5-AZA) offers opportunities for cancer cell reprogramming to develop new target-specific treatments. The protein annexin A1 (ANXA1) is downregulated in head and neck squamous cell carcinoma (HNSCC), correlated with pathological differentiation grade.Objectives:this work aimed to further investigate the role of ANXA1 in OSCC progression based on 5-AZA activity.Methods:we used CAL27 and CAL33 cell lines, which differ in drug sensitivity and differentiation status.Results:CAL27 showed a higher expression of the stemness markers compared to CAL33 cells, but this positivity was lost after treatment with 5-AZA. This drug also decreased CAL27 cell motility, promoting a less aggressive phenotype. Moreover, 5-AZA increased ANXA1 expression only in CAL27. After siRNA-mediated downmodulation, we witnessed a significant rise in cell motility and the inversion of E-/N-cadherin expression, which was reverted again by 5-AZA. To investigate the role of exogenous ANXA1 derived from the tumor microenvironment, we treated CAL27 with Ac2-26, an ANXA1 mimetic peptide. Interestingly, we found that this peptide alone showed impacts similar to 5-AZA in reversing the aggressive phenotype. All these effects were not evidenced in CAL33 cells. Finally, to prove the loop of the exogenous protein, we detected increased expression of its receptors, formyl peptide receptors (FPRs), and their activation, leading to oncosuppressor effects.Conclusions:we propose that ANXA1 mediates the effects of 5-AZA only in poorly differentiated stemlike CAL27 cell lines. This suggests the relevance of ANXA1 as a diagnostic/prognostic biomarker in OSCCs, paving the way for personalized therapies to overcome treatment difficulties.
背景：口腔鳞状细胞癌（OSCCs）的治疗因缺乏可靠的诊断/预后及预测标志物，以及肿瘤细胞固有的异质性而受限。5-氮杂胞苷（5-AZA）为癌细胞重编程提供了开发新型靶向治疗的可能。膜联蛋白A1（ANXA1）在头颈部鳞状细胞癌（HNSCC）中表达下调，且与病理分化程度相关。 目的：本研究旨在基于5-AZA的作用机制，进一步探讨ANXA1在OSCC进展中的功能。 方法：采用具有不同药物敏感性和分化状态的CAL27与CAL33细胞系进行研究。 结果：与CAL33细胞相比，CAL27细胞干性标志物表达更高，但经5-AZA处理后该阳性表达消失。该药物同时降低了CAL27细胞的运动能力，促使其向侵袭性较低的表型转化。此外，5-AZA仅能提升CAL27细胞中ANXA1的表达水平。通过siRNA介导的基因下调后，我们观察到细胞运动能力显著增强，E-钙黏蛋白/N-钙黏蛋白表达发生逆转，而5-AZA处理可再次逆转此现象。为探究肿瘤微环境来源的外源性ANXA1的作用，我们使用ANXA1模拟肽Ac2-26处理CAL27细胞。值得注意的是，该肽单独使用即能产生与5-AZA类似的逆转侵袭表型的效果。上述现象在CAL33细胞中均未出现。最后，为验证外源性蛋白的作用环路，我们检测到其受体——甲酰肽受体（FPRs）表达上调及其激活过程，进而产生肿瘤抑制效应。 结论：我们认为ANXA1仅在低分化的干细胞样CAL27细胞系中介导5-AZA的作用效果。这提示ANXA1可作为OSCC诊断/预后的生物标志物，为克服治疗难题的个体化疗法开辟了新途径。
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