S100A8和S100A9归属于S100蛋白家族，最先发现于噬中性粒细胞中，二者在细胞内通常以异源二聚体形式存在。据文献报道，S100A8/S100A9在多种癌组织中存在高表达，并发现其高表达与癌细胞的增殖、浸润和转移密切相关，但二者在癌细胞中表达调控机制鲜有报道。为此，我们将宫颈鳞癌细胞HCC94、表皮癌细胞A431和咽鳞癌细胞FaDu选定为研究对象，探究了S100A8/S100A9在上述鳞癌细胞中是否能被诱导表达，如果是，其诱导表达的分子机制是什么？ 

首先，我们对A431、 HCC94和FaDu细胞进行了细胞免疫化学检测，目的是确认S100A8/S100A9在体外培养的鳞癌细胞中的表达情况。结果显示，在正常细胞密度的培养条件下，上述三种鳞癌细胞只存在极少数量的S100A8/S100A9阳性细胞。这与文献报道的癌组织中高表达S100A8/S100A9不同。但有文献报道，当正常皮肤角化细胞处于高密度和悬浮培养时，S100A8/S100A9受到诱导表达，其基因和蛋白表达水平均明显提高。那当鳞癌细胞处于上述培养条件时，S100A8/S100A9是否也能受到诱导表达呢？为此，我们将上述三种鳞癌细胞进行了高密度和悬浮培养。结果显示，高密度和悬浮培养均可显著提高S100A8/S100A9的阳性细胞数占比。并发现当细胞进行高密度培养后恢复至正常培养密度时，S100A8/S100A9的阳性细胞数占比又恢复至正常培养密度的水平。上述结果表明，S100A8/S100A9在体外培养的鳞癌细胞中确实可被诱导表达，且二者诱导表达的水平与细胞密度和形态有关。进一步我们采用激光共聚焦显微镜观察了HCC94细胞在不同的培养条件下，S100A8/S100A9是否具有共定位现象。结果显示，无论在正常细胞密度和高密度以及悬浮培养时，S100A8/S100A9均呈现出共定位现象。为了证实这种诱导表达现象是否在体内也可出现，我们又对A431、HCC94和FaDu细胞的小鼠荷瘤组织进行了免疫组化检测，结果发现在上述三种细胞所形成的荷瘤组织中，S100A8/S100A9的阳性细胞数量占比明显增多，且呈共定位现象。这就说明S100A8/S100A9在体内也可被诱导表达。随后，我们又对257例鳞癌组织样本（包括肺鳞癌、食管鳞癌、宫颈鳞癌、口腔鳞癌和皮肤鳞癌）进行了S100A8/S100A9的免疫组化染色。结果显示，S100A8/S100A9在上述五种鳞癌组织中，不仅呈现出异质性高表达，而且具有共表达的现象。综上所述， 在上述三种鳞癌细胞中，S100A8/S100A9均可在体内外被诱导表达，并呈现出共定位或共表达现象。我们推测S100A8/S100A9的这种诱导表达可能与癌细胞的增殖、分化和抗凋亡相关。

那么S100A8/S100A9在鳞癌细胞中是如何受到诱导表达的？有文献报道，改变细胞的形态能够激活Hippo通路，从而改变其下游关键效应因子YAP的活性，最终调控相应基因的转录与表达。基于此报道，我们推测，在高密度和悬浮培养条件下，Hippo 通路的激活是否也参与了对S100A8/S100A9诱导表达的调控呢？为此，我们对 HCC94和FaDu细胞进行了高密度和悬浮培养。根据结果分析可知，高密度和悬浮培养可显著上调S100A8/S100A9的表达，同时还伴随着Hippo通路的激活，体现在 LATS1和磷酸化YAP蛋白水平表达的增加。当把高密度和悬浮培养的细胞，恢复至正常贴壁培养且密度适中的状态时，可发现，伴随着Hippo信号通路的活性显著下降，S100A8/S100A9的表达水平明显下调；以上现象提示，Hippo信号通路的活性与S100A8/S100A9的诱导表达之间是密切相关的。

为进一步研究Hippo通路的活性与S100A8/S100A9诱导表达之间的关系，我们采用将Hippo通路进行激活或抑制的两个策略，来确认S100A8/S100A9的诱导表达是否受Hippo通路调控。首先，我们利用瞬时过表达LAST1和使YAP蛋白沉默的方式使Hippo通路处于激活状态。根据结果可知，在Hippo通路激活后，S100A8/S100A9在基因和蛋白表达水平上显著上调。相反，为使Hippo通路处于抑制状态，我们瞬时沉默LATS1和MST，并且通过过表达YAP-S127A模拟Hippo通路抑制后YAP的入核状态，均观察到S100A8/S100A9基因和蛋白的表达量显著下调。我们从正反两个方面证实 Hippo通路确实参与S100A8/S100A9的诱导表达调控，并发现YAP在核内通过与共转录因子TEAD1结合，间接调控S100A8/S100A9的表达。另外， 细胞高密度和悬浮培养可导致微丝骨架的破坏或重组，从而激活Hippo通路。那么微丝骨架完整性改变是否也能诱导S100A8/S100A9的表达上调？为此，我们利用药物Lat B、Cyto D和C3破坏 F-actin，结果显示，鳞癌细胞经上述三种药物处理后，Hippo通路被激活，S100A8/S100A9的表达量显著上调。综上所述，细胞密度与形态的改变导致了细胞骨架的重塑，进而使Hippo通路激活，引起核内YAP与其共转录因子TEAD1的结合，最终诱导了S100A8/S100A9的表达。

那么在鳞癌细胞中，S100A8/S100A9的表达具有哪些生物学功能呢？我们对正常贴壁培养的FaDu和A431细胞进行沉默YAP及共沉YAP和S100A8/S100A9。结果显示，S100A8/S100A9和YAP具有相似的生物学功能，即促进鳞癌细胞的增殖，抑制其分化。对于高密度和悬浮培养的细胞，沉默S100A8/S100A9可显著提高细胞的凋亡比率，过表达YAP以及与YAP相作用的p73可显著提高细胞的凋亡比率。由此我们认为，当细胞在高密度和悬浮培养条件下时，细胞处于易凋亡状态，而S100A8/S100A9的诱导表达和YAP的失活，则是癌细胞克服凋亡，提高存活率的方式之一。

本论文揭示了在鳞癌细胞中S100A8/S100A9在体内外可受到诱导表达，并且二者的诱导表达呈现共表达或共定位现象。进一步研究发现二者的诱导表达受Hippo/YAP/TEAD1通路的调控。并发现S100A8/S100A9的表达具有促进鳞癌细胞增殖，但抑制其分化和抗凋亡的作用。上述研究结果为开发新药和鳞癌的治疗提供了新的思路和治疗靶点。

S100A8 and S100A9, two heterodimer-forming members of the S100 family, were originally discovered as immunogenic proteins expressed and secreted by neutrophils. Aberrant expression of S100A8/S100A9 complex was detected in a variety of cancer tissues, and it has been found that its high expression is closely related to the proliferation, invasion and metastasis of cancer cells. However, the regulatory mechanisms of the expression of S100A8/S100A9 in cancer cells are rarely reported. Therefore, we selected epidermal cancer cells A431, cervical squamous cell carcinoma cells HCC94, and pharyngeal squamous cell carcinoma cells FaDu as research objects to explore whether S100A8/S100A9 can be induced to express in the above-mentioned cancer cells. If so, what is the molecular mechanism of its induced expression?

We used Immunohistochemistry test the expression of S100A8 / S100A9 in A431, HCC94 and FaDu cells cultured in vitro. The results showed that less than 1% S100A8/S100A9-positive cells were detected in all tested cell lines under culture conditions. This is different from the high expression of S100A8 / S100A9 in cancer tissues reported in the literature. It has reported that S100A8/S100A9 expression can be induced in normal primary keratinocytes (HEKn) under dense culture and suspension culture. So whether S100A8/S100A9 may also induced by dense and suspension in SCC cells? Therefore, we cultured the above three squamous cell carcinomas under dense and suspension conditions. The results show that the percentage of S100A8/S100A9-positive cells was significantly increased through dense and suspension culture and then, decreased after the dense cells were reseeded at the pre-dense density and returned to the original ratio after three cell passage. These results reveal that S100A8/S100A9 is inducible in camcer cells cultured in vitro, which depends on cell shape and cell density. Furthermore, we observed the co-localization of S100A8/S100A9 in HCC94 cells under different culture conditions by using laser confocal microscopy. The results suggest that S100A8/S100A9 display co-localization in both normal, dense and suspension culture. Based on the above results, we hypothesized that S100A8/S100A9 may also be induced and co-expressed or co-localized in vivo. Therefore, we examined S100A8/S100A9 expression pattern in xenografts derived from FaDu, A431, and HCC94 cells by immunohistochemistry in two consecutive sections. The results showed that the percentage of S100A8 / S100A9-positive cells in three SCC cell lines’ corresponding xenografts increased significantly and co-localization, which means that  S100A8/S100A9-positive cells are co-inducible in vivo. Furthermore, we tested the expression of S100A8/S100A9 in 257 SCC specimens derived from SCC of lung, esophagus, cervix, oral cavity, and skin by immunochemistry. The results suggest that the expression characteristics of S100A8 / S100A9 were marked heterogeneity and display co-expression and co-localization in the above five squamous cell carcinoma tissues. In summary, S100A8 / S100A9 can be induced to express in vivo and in vitro in three types of squamous cancer cells, and exhibit co-localization or co-expression. We suggest that this induced expression of S100A8/S100A9 may be related to the proliferation, differentiation and anti-apoptosis of cancer cells.

So what is the expression mechanism of S100A8/S100A9 induced expression in cancer cells ? It has been reported that both suspension and dense can activate the Hippo pathway and change the activity of YAP—Hippo pathway’s downstream key effector, thereby regulating the transcription and expression of corresponding genes. Therefore, we speculate whether the activation of the Hippo pathway was also involved in the regulation of S100A8 / S100A9-induced expression under dense and suspension culture conditions. For this purpose, HCC94 and FaDu cells were cultured in dense and suspension conditions. The results showed that the above two culture conditions could significantly increased the expression of S100A8/S100A9, and also accompanied the activation of the Hippo pathway, which was reflected in the increase of LATS1 and YAP phosphorylation. Next, the decrease of S100A8/S100A9 co-expression also accompanied by inhibition of the Hippo pathway after recovery of cell attachment or relief from dense culture. These results indicate that the activity of the Hippo pathway is closely related to the induced expression of S100A8/S100A9.

In order to further research the relationship between the activity of the Hippo pathway and the expression of S100A8/S100A9, we adopted two strategies of activating and inhibiting the Hippo pathway to confirm whether the induced expression of S100A8/S100A9 is regulated by the Hippo pathway. Firstly, we simulated the state of Hippo pathway activation by over-expressing LAST1and silencing YAP. The results showed that the expression levels of S100A8/S100A9 genes and proteins were significantly up-regulated through activation of the Hippo pathway. On the contrary, when the Hippo pathway was inhibited by knockdown of MST1 or LATS1 and over-expressing YAP-S127A dramatically decreased S100A8/S100A9 expression as well as YAP phosphorylation in suspended or dense HCC94 cells. These results suggest that Hippo-YAP pathway may regulate S20018/S100A9 expression in SCC cells, and find the transcription of S100A8/S100A9 may be indirectly regulated by YAP/TEAD1 complex. In addition, it has reported that dense and suspension culture could activate the Hippo pathway by F-action cytoskeleton reorganization. So can the disruption of F-actin also induce the expression of S100A8/S100A9? therefore, we treated HCC94 and FaDu cells with three drugs, LatB, CytoD and C3, which could disrupt the F-action cytoskeleton. These results revealed that the Hippo-YAP pathway was activated after SCC cells were treated with three drugs, and the expression of S100A8/S100A9 were significantly up-regulated. In summary, changes of cell density and cell morphology caused the remodeling of action cytoskeleton, which activates the Hippo-YAP pathway, leads to the combination of nuclear YAP and co-transcription factor TEAD1, and finally induces the expression of S100A8/S100A9.

So what are the biological functions of S100A8/S100A9 express in SCC? We performed depletion of YAP with or without combined with S100A8/S100A9 in In normal adherent cultured FaDu and A431 cells. These results suggest that S100A8/S100A9 and YAP both play the similar role in promoting cell proliferation and inhibiting squamous differentiation. we next explore the function of S100A8/S100A9 in suspended and dense SCC cells. The results showed that silencing of S100A8/S100A9 together and overexpression of YAP-S127 and p73 significantly promoted cell apoptosis in suspended and dense cells. Therefore, we speculated that suspension and dense culture leads to cancer cell apoptosis, and the inactivation of YAP or the induction of S100A8/S100A9 in the abovementioned conditions may be a mechanism for cancer cells resisting apoptosis and maintaining survival.

This study uncovers for the first time that S100A8/S100A9 is inducible both in vitro and in vivo, and both proteins display co-expression and /or co-localization. Furthermore, S100A8/S100A9 co-induction is regulated by Hippo-YAP-TEAD1 pathway. Induced S100A8/S100A9 promoted SCC cells proliferation, inhibit cell differentiation and apoptosis, which provides new ideas and therapeutic targets for the development of new drugs and treatment of squamous cell carcinoma.