Supplementary Material for: Tumor-intrinsic perinuclear LOXL2: Prognostic relevance and relationship with YAP1 activation status in oral squamous cell carcinoma

Introduction. Lysyl Oxidase-Like 2 (LOXL2) expression and function is frequently altered in different cancers, but scarcely explored in oral squamous cell carcinoma (OSCC). This prompted us to investigate the clinical relevance of LOXL2 expression pattern in OSCC and also a possible crosstalk with Hippo/YAP1 pathway signaling.
Methods. Immunohistochemical analysis of LOXL2 protein expression was performed in 158 OSCC patient samples, together with Yes-associated protein 1 (YAP1) activation status. Correlations with clinicopathological parameters and patient survival were assessed.
Results. Tumor cell-intrinsic LOXL2 expression showed two distinct expression patterns: diffuse cytoplasmic staining (64.6%), and heterogeneous perinuclear staining (35.4%). Remarkably, perinuclear LOXL2 staining was significantly associated with lymph node metastasis, advanced clinical stage and perineural invasion. Moreover, patients harboring tumors with perinuclear LOXL2 expression exhibited significantly poorer disease-specific survival (DSS) rates. Strikingly, we also found that perinuclear LOXL2 positivity gradually increased in relation to YAP1 activation, and patients harboring tumors with concomitant perinuclear LOXL2 and fully active YAP1 exhibited the worst DSS. Multivariate Cox analysis further revealed combined perinuclear LOXL2 and fully active YAP1 as a significant independent predictor of poor DSS.
Conclusion. Tumor-intrinsic perinuclear LOXL2 emerges as a clinically and biologically relevant feature associated with advanced disease, tumor aggressiveness, and poor prognosis in OSCC. Moreover, this study unprecedentedly uncovers a functional relationship between perinuclear LOXL2 and YAP1 activation with major prognostic implications. Notably, combined perinuclear LOXL2 and fully active YAP1 was revealed as independent predictor of poor prognosis. These findings encourage targeting oncogenic LOXL2 functions for personalized treatment regimens.

引言。
赖氨酰氧化酶样2（Lysyl Oxidase-Like 2，LOXL2）的表达与功能在多种癌症中常发生异常，但在口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）中的相关研究却较为匮乏。基于此，本研究旨在探讨LOXL2表达模式在OSCC中的临床相关性，以及其与Hippo/YAP1信号通路之间可能存在的串扰。

方法。
本研究对158例OSCC患者样本进行了LOXL2蛋白表达的免疫组化分析，同时检测了Yes相关蛋白1（Yes-associated protein 1，YAP1）的激活状态，并分析其与临床病理参数及患者生存情况的相关性。

结果。
肿瘤细胞内源性LOXL2表达存在两种截然不同的模式：弥漫性胞质染色（占比64.6%）与异质性核周染色（占比35.4%）。值得注意的是，核周LOXL2染色与淋巴结转移、临床晚期及神经周围侵犯显著相关。此外，携带有核周LOXL2表达肿瘤的患者，其疾病特异性生存期（disease-specific survival，DSS）显著更短。尤为关键的是，本研究还发现核周LOXL2阳性率随YAP1激活程度升高而逐渐增加；同时携带有核周LOXL2表达与完全激活型YAP1的肿瘤患者，其DSS最差。多因素Cox分析进一步证实，核周LOXL2与完全激活型YAP1的联合表达可作为不良DSS的独立显著预测因子。

结论。
肿瘤内源性核周LOXL2表达可作为OSCC中与疾病进展、肿瘤侵袭性及不良预后相关的临床及生物学特征。此外，本研究首次揭示了核周LOXL2与YAP1激活之间的功能关联，该关联具有重要的预后价值。值得注意的是，核周LOXL2与完全激活型YAP1的联合表达可作为不良预后的独立预测因子。上述研究结果提示，靶向致癌性LOXL2功能可为OSCC的个性化治疗方案提供新思路。