Background

The tumor microenvironment (TME) is known to play a prominent role in the pathology of head and neck squamous cell carcinoma (HNSCC). Cancer-associated fibroblasts (CAFs) have been reported to regulate tumor progression, and serglycin (SRGN), one of the paracrine cytokines of CAFs, has been reported to play an important role in various signaling pathways. Hypoxia is a distinct feature of the HNSCC TME. Here, we investigated the mechanism underlying CAF-secreted SRGN leading to HNSCC progression under hypoxia.

Methods

Immunohistochemical staining was used to detect SRGN expression in clinical HNSCC samples, after which its relation with patient survival was assessed. CAFs were isolated and SRGN expression and secretion by CAFs under normoxia and hypoxia were confirmed using qRT-PCR and ELISA assays, respectively. HNSCC sphere-forming abilities, stemness-related gene expression, and chemoresistance were assessed with or without SRGN treatment. A Wnt/β-catenin pathway inhibitor (PNU-75,654) was used to block its activation, after which nuclear translocation of β-catenin in the presence of SRGN with or without PNU-75,654 was evaluated. shRNAs were used to stably knock down SRGN expression in CAFs. HNSCC tumor cells with or without (SRGN silenced) CAFs were inoculated submucosally in nude mice after which tumor weights and sizes were determined to assess the effects of CAFs and SRGN on tumor growth.

Results

We found that SRGN was expressed in both HNSCC tumor and stroma cells, and that high SRGN expression in the stroma cells, but not in the tumor cells, was significantly related to a poor patient survival. After the extraction of CAFs and normal fibroblasts (NFs) from paired tumor samples and adjacent normal tissues, respectively, we found that the expression of CAF-specific genes, including fibroblast activation protein (FAP) and alpha-smooth muscle actin (α-SMA), was clearly upregulated compared to the expression in NFs. The hypoxia marker HIF-1α was found to be expressed in tumor stroma cells. Hypoxyprobe immunofluorescence staining confirmed stromal hypoxia in an orthotopic tongue cancer mouse model. Using qRT-PCR and ELISA we found that a hypoxic TME upregulated SRGN expression and secretion by CAFs. SRGN markedly enhanced the sphere-forming ability, stemness-related gene expression and chemoresistance of HNSCC tumor cells. SRGN activated the Wnt/β-catenin pathway and promoted β-catenin nuclear translocation. An in vivo study confirmed that CAFs can accelerate HNSCC tumor growth, and that this effect can be counteracted by SRGN silencing.

Conclusions

Our data indicate that a hypoxic tumor stroma can lead to upregulation of SRGN expression. SRGN secreted by CAFs can promote β-catenin nuclear translocation to activate downstream signaling pathways, leading to enhanced HNSCC cell stemness, chemoresistance and accelerated tumor growth.

中文翻译：

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癌症相关成纤维细胞分泌缺氧诱导的serglycin通过激活Wnt/β-catenin通路促进头颈部鳞状细胞癌肿瘤细胞体外和体内生长

背景

众所周知，肿瘤微环境 (TME) 在头颈部鳞状细胞癌 (HNSCC) 的病理学中起重要作用。据报道，癌症相关成纤维细胞 (CAF) 可调节肿瘤进展，据报道，CAF 的旁分泌细胞因子之一 Serglycin (SRGN) 在各种信号通路中发挥重要作用。缺氧是 HNSCC TME 的一个显着特征。在这里，我们研究了 CAF 分泌的 SRGN 在缺氧条件下导致 HNSCC 进展的机制。

方法

免疫组织化学染色用于检测临床 HNSCC 样本中的 SRGN 表达，然后评估其与患者存活率的关系。分离 CAFs 并分别使用 qRT-PCR 和 ELISA 测定法确认常氧和缺氧条件下 CAFs 的 SRGN 表达和分泌。在有或没有 SRGN 治疗的情况下评估 HNSCC 球体形成能力、干细胞相关基因表达和化学抗性。使用 Wnt/β-catenin 通路抑制剂 (PNU-75,654) 阻断其活化，然后评估在 SRGN 存在或不存在 PNU-75,654 的情况下 β-catenin 的核易位。shRNA 用于稳定地抑制 CAF 中的 SRGN 表达。

结果

我们发现 SRGN 在 HNSCC 肿瘤和基质细胞中均表达，并且基质细胞中的高 SRGN 表达，但在肿瘤细胞中不表达，与患者生存率低显着相关。在分别从配对肿瘤样本和邻近正常组织中提取 CAFs 和正常成纤维细胞 (NFs) 后，我们发现 CAF 特异性基因的表达，包括成纤维细胞激活蛋白 (FAP) 和 α-平滑肌肌动蛋白 (α-SMA )，与 NFs 中的表达相比明显上调。发现缺氧标志物 HIF-1α 在肿瘤基质细胞中表达。Hypoxyprobe 免疫荧光染色证实了原位舌癌小鼠模型中的基质缺氧。使用 qRT-PCR 和 ELISA，我们发现缺氧 TME 上调了 CAF 的 SRGN 表达和分泌。SRGN 显着增强 HNSCC 肿瘤细胞的球形成能力、干性相关基因表达和化学抗性。SRGN 激活 Wnt/β-catenin 通路并促进 β-catenin 核转位。一个体内研究证实，CAFs 可以加速 HNSCC 肿瘤的生长，并且这种效应可以被 SRGN 沉默所抵消。

结论

我们的数据表明，缺氧的肿瘤基质可导致 SRGN 表达的上调。CAFs分泌的SRGN可以促进β-catenin核转位激活下游信号通路，从而增强HNSCC细胞干性、化学抗性和加速肿瘤生长。