S100A14是EF-手型钙结合蛋白家族成员之一，在多种肿瘤中表达异常并与肿瘤发生发展密切相关。TCGA乳腺癌数据库分析表明：S100A14在乳腺癌中存在拷贝数扩增，在转移性乳腺癌数据集中拷贝数扩增比例高达27%；S100A14拷贝数扩增与其mRNA表达正相关；淋巴结转移的乳腺癌患者S100A14表达亦显著升高。GEO数据库进一步分析表明S100A14高表达的乳腺癌患者无进展生存差。我们进一步通过免疫组化实验在蛋白水平验证S100A14在乳腺癌配对组织和淋巴结中的表达，结果显示：S100A14在乳腺癌组织中表达显著上调，且与淋巴结转移正相关。为了明确S100A14在乳腺癌转移中的作用，我们通过一系列功能获得和缺失实验进行表型研究。结果表明：过表达S100A14显著增强乳腺癌细胞侵袭和迁移能力；敲除S100A14起到相反的作用。过表达S100A14的原位种植转移、实验性转移结果均显示：S100A14促进乳腺癌体内肺转移；而抑制S100A14表达后实验性肺转移能力和S100A14基因敲除MMTV-PyMT自发乳腺癌肺转移能力均显著降低。进一步研究发现S100A14促进乳腺癌细胞发生上皮间质转化，增加乳腺癌细胞的干性特征，并可通过分泌性蛋白形式发挥功能。

接下来，我们通过RNA-Seq和分泌蛋白组学对S100A14促乳腺癌转移的作用机制进行探索。结果表明：S100A14调节的差异基因及分泌蛋白富集在细胞运动、细胞因子和趋化因子信号通路。QRT-PCR、Western Blot和ELISA等实验证实过表达S100A14增加CCL2和CXCL5 mRNA、蛋白的表达及分泌；而敲除S100A14显著抑制MMTV-PyMT小鼠肿瘤组织中CCL2和CXCL5的表达及其在血清中的分泌量。分泌性S100A14亦可促进巨噬细胞的迁移，诱导M1到M2型极化。中和抗体阻断CCL2和CXCL5显著抑制S100A14对乳腺癌细胞和巨噬细胞侵袭迁移的促进作用。上述结果表明S100A14通过上调CCL2和CXCL5的表达与分泌促进了巨噬细胞的募集和活化，从而促进乳腺癌转移。GEO数据分析表明S100A14与CCL2和CXCL5 mRNA表达正相关。免疫组化检测发现S100A14、CCL2和CXCL5在乳腺癌中高表达，且S100A14/CCL2和S100A14/CXCL5表达呈正相关。ELSIA检测血清中S100A14含量发现：乳腺癌患者血清中S100A14含量显著高于健康人群；且淋巴结阳性患者S100A14血清含量显著高于无淋巴结转移患者；ROC曲线分析显示：AUC面积为0.883，检测灵敏度为75.7%，特异度为84.8%。

综上所述，S100A14在乳腺癌组织中表达显著上调，S100A14是促进乳腺癌侵袭转移的重要分子，通过促进CCL2和CXCL5表达和分泌发挥功能。S100A14具有作为乳腺癌诊断血清学标志物的潜在价值。深入系统地研究S100A14在乳腺癌转移中的功能和精确机制，为乳腺癌诊断和治疗提供重要的理论支持。

S100A14 is a member of the EF-hand calcium binding protein family. It had been reported that S100A14 expression is altered in multiple cancers and closely related to tumorigenesis and tumor progression. We found that S100A14 was amplified in breast cancer by analyzing TCGA data, and its percentage of copy number amplification was about 27% in the metastatic breast cancer dataset. There was a positive connection between copy number amplification of S100A14 and its mRNA expression. Meanwhile, the expression of S100A14 was positively correlated with lymph node metastasis. Importantly, the GEO data analysis indicated that breast cancer patients with high expression of S100A14 had poor metastasis-free survival. To confirm the protein expression of S100A14 in breast cancer tissues, we detected S100A14 expression in breast cancer tissues, matched normal tissues and lymph node tissues through immunohistochemistry (IHC) analysis. The results showed that S100A14 expression was significantly upregulated in breast cancer tissues than that in matched normal tissues. Moreover, the expression of S100A14 was obviously higher in metastatic lymph node tissues compared with non-metastatic lymph node tissues. To further verify the role of S100A14 in promoting breast cancer metastasis, we conducted a series of functional assays through gain or loss-of-function of S100A14 in breast cancer cell lines and mice. In vitro transwell assays showed that the ectopic overexpression of S100A14 significantly enhanced the migration and invasion capabilities of breast cancer cells. In contrast, knockout of S100A14 exhibited the opposite effects.In vivometastasis models in athymic nude mice via tail vein or mammary fat pad in situ injection and MMTV spontaneous breast cancer lung metastasis mouse model results further confirmed that S100A14 promoted lung metastasis of breast cancer. Overexpression of S100A14 also promoted epithelial-mesenchymal transition and increased stem cell properties of breast cancer cells. And S100A14 can exert its function via the extracellular secretion.

Next, we explored the possible molecular mechanism of S100A14 on breast cancer metastasis by RNA-Seq and secreted proteomics. Functional annotation analysis illustrated that differential expressed genes and secreted proteins regulated by S100A14 mainly enriched in cell motility, cytokines and chemokines mediated signaling pathways. Validation by qRT-PCR, Western Blot and ELISA demonstrated that overexpression of S100A14 increased the expression and secretion of CCL2 and CXCL5 in breast cancer cells, whereas knockout of S100A14 significantly inhibited the expression and serum secretion of CCL2 and CXCL5 in MMTV-PyMT mice. Importantly, delivery of anti-CCL2/CXCL5 antibodies significantly inhibited S100A14-enhanced breast cancer cells/macrophages migration and invasion, which indicated that S100A14 promotes breast cancer metastasis by upregulating the expression and secretion of CCL2 and CXCL5. GEO database analysis demonstrated that S100A14 mRNA expression was positively correlated with CCL2 and CXCL5 mRNA expression, respectively. The analysis of clinical breast cancer tissues by IHC also displayed the protein expression of S100A14 was positively correlated with the protein expression of CCL2 and CXCL5. Finally, we performed enzyme-linked immunosorbent assay (ELISA) to examine the serum S100A14 levels in breast cancer patients and normal healthy controls to assess whether S100A14 could be used as a serological marker for breast cancer diagnosis. We found that the serum levels of S100A14 in breast cancer patients were significantly higher than those in healthy controls, and the S100A14 serum levels in metastatic lymph node patientswere observably higher than those in non-metastatic lymph node patients. ROC analysis exhibited that the area under curve (AUC) is 0.883, the detection sensitivity and specificity were 75.7% and 84.8%, respectively.

In conclusion, S100A14 is significantly upregulated in breast cancer, it can promote breast cancer metastasisby increasing the expression and secretion of CCL2 and CXCL5. And S100A14 has the potential to serve as aserologicalmarker for diagnosis of breast cancer. This study comprehensively clarifies the role of S100A14 in breast cancer metastasis and explores the possible mechanism, which may provide important theoretical support for the diagnosis and treatment of breast cancer.