S100A7作为S100蛋白家族成员，最先在牛皮癣病人皮损及非皮损区被鉴定发现。在乳腺癌及多种鳞癌（Squamous cell carcinomas，SCCs）组织中也可见S100A7高表达。我们的前期研究表明，S100A7只选择性的在部分肺鳞癌、腺鳞混合癌及大细胞肺癌中表达，而在其他肺癌亚型中不表达。然而，对于S100A7在其他鳞癌组织及细胞中的表达特点，目前则少有报道。因此，在本研究中，我们首先对食管鳞癌、宫颈鳞癌、皮肤鳞癌、口腔鳞癌、膀胱鳞癌以及肺鳞癌这6种鳞癌组织类型，共计452例组织标本中的S100A7进行免疫组化检测。结果显示，在上述6种鳞癌组织中，S100A7均呈现异质性高表达，并发现S100A7表达与分化程度正相关。尤其在高分化鳞癌组织的角化珠部位，S100A7表达最强。此外，我们又选取了宫颈鳞癌细胞HCC94、咽鳞癌细胞FaDu、表皮癌细胞A431以及肺鳞癌脑转移细胞H226Br进行体外S100A7表达检测。结果显示，在A431、FaDu及H226Br细胞中，S100A7阳性细胞占比及其表达量远低于其相应类型的鳞癌组织。除HCC94细胞外，上述3种鳞癌细胞中S100A7阳性细胞占比极低（小于1%）。这一现象引发我们思考，是什么原因导致了体内外S100A7表达的巨大差异呢？有文献报道，悬浮培养可诱导皮肤角化细胞中S100A7在基因及蛋白水平表达上调。那么，在上述鳞癌细胞中，S100A7是否也能被诱导表达呢？为了证实此推测，我们将上述4种鳞癌细胞进行了不同天数的悬浮培养。结果显示，悬浮可明显提高细胞中S100A7阳性细胞占比及其基因和蛋白表达水平，且上述细胞经高密度培养后也获得了相似结果。另外，我们发现，悬浮或高密度培养对S100A7的诱导表达会随着细胞重新贴壁或恢复正常细胞密度培养而显著下降。这一结果提示，S100A7的诱导表达与细胞形态及细胞密度密切相关。 有报道，细胞在悬浮及高密度培养条件下，微丝骨架结构会发生破坏或重塑，从而激活Hippo信号通路。而Hippo通路中的关键蛋白YAP则是细胞形态及密度变化的感受器。基于上述报道，我们推测，Hippo通路可能介导了悬浮及高密度培养对S100A7的诱导表达。为此，我们选用 HCC94及FaDu细胞为实验模型，将细胞进行悬浮及高密度培养。结果显示，悬浮及高密度培养均可诱导上述细胞中S100A7的表达，同时还伴随着Hippo通路的激活，即上调了LATS1（T1079）、YAP（S127）磷酸化水平并导致YAP出核失活。免疫荧光结果也进一步证实了悬浮培养可诱导S100A7表达，促进YAP出核的结果。同时，我们观察到一个有趣的现象：当悬浮细胞恢复贴壁生长或高密度培养的细胞恢复到正常密度培养后，Hippo通路活性可随之降低，同时也伴随着S100A7表达的显著下降。上述结果提示，Hippo通路的激活或失活与S100A7表达密切相关。为了进一步明确Hippo通路与S100A7之间的关系，首先，我们在正常贴壁培养的HCC94及FaDu细胞中瞬时过表达LATS1或沉默YAP，以模拟Hippo通路激活状态。结果显示，激活Hippo通路可明显提高S100A7基因和蛋白表达水平。另一方面，我们在上述细胞中瞬时沉默LATS1、MST1或瞬时过表达YAP-S127A，以模拟Hippo通路抑制状态。所得结果与激活Hippo通路相反，抑制Hippo通路活性可明显抑制S100A7表达。上述结果表明，Hippo通路确实介导了S100A7的表达，并且核内YAP是S100A7的负调控分子。进一步研究发现，核内YAP可通过与共转录因子TEAD1结合从而间接抑制S100A7的诱导表达。接下来，我们又探究了细胞微丝骨架完整性对S100A7诱导表达的影响。首先，利用荧光染色并结合激光共聚焦显微观察，我们发现悬浮及高密度培养均可严重破坏HCC94细胞微丝骨架结构完整性。而后，我们采用已知的细胞微丝骨架破坏药物拉春库林B（Latrunculin B，LatB）、细胞松弛素D（Cytochalasin D，CytoD）以及Rho蛋白抑制剂肉毒杆菌毒素（Botulinum toxin C3，C3）处理正常贴壁培养的HCC94和FaDu细胞以模拟细胞骨架破坏情况。结果显示，上述药物在破坏细胞微丝骨架的同时，均可激活Hippo通路并显著上调S100A7的表达。相反，共同沉默微丝解聚因子CFL1、GSN及CAPZB后，则明显抑制了悬浮培养对S100A7的诱导表达作用。然而，对几乎没有内源S100A7表达的低分化宫颈鳞癌细胞SiHa及肺鳞癌H226来说，悬浮培养、过表达LATS1、瞬时沉默YAP或外加CytoD处理后，虽能激活Hippo通路，却并不能诱导S100A7在上述细胞中的表达。以上结果提示，S100A7的诱导表达可能与鳞癌细胞分化程度相关。后续体内检测结果也证实了这一推测。我们在对宫颈鳞癌及舌鳞癌共117例组织标本检测时发现，S100A7仅在高中分化的鳞癌组织中高表达，且与磷酸化YAP（S127）表达正相关而与核内YAP表达负相关。表明S100A7的诱导表达和Hippo通路的激活状态与鳞癌组织细胞的分化程度密切相关。功能上，虽然YAP负调控S100A7表达，但两者均可促进鳞癌细胞增殖。以上结果表明，在高分化HCC94及FaDu鳞癌细胞中，悬浮及高密度培养可通过破坏细胞微丝骨架结构，激活Hippo通路，从而诱导S100A7的表达。 进一步研究发现，另一条信号通路PI3K/AKT通过调控GSK3β活性，也参与调控了S100A7在细胞悬浮培养时的诱导表达。我们发现悬浮培养HCC94及FaDu细胞时，抑制了PI3K/AKT通路的活性，上调了GSK3β活性，同时显著诱导了S100A7的表达。如果PI3K/AKT通路确实参与调控了S100A7的诱导表达，那么干预这一信号通路的活性必然会影响S100A7表达。为了证明这一推测，首先，我们在上述细胞中瞬时导入组成型激活状态的AKT（myr-AKT）质粒。结果显示，过表达AKT可明显抑制GSK3β活性和S100A7表达。相反，加入PI3K特异性抑制剂LY294002，则会显著促进GSK3β活性及S100A7表达。但如果在过表达myr-AKT的悬浮细胞中，同时加入LY294002则会削弱过表达AKT对上述两个蛋白的抑制作用。由此证明，PI3K/AKT/GSK3β通路确实参与调控了悬浮细胞中S100A7的诱导表达。那么GSK3β是如何调控S100A7的呢？我们通过外加GSK3β抑制剂或转染其特异性siRNA的方式抑制其活性，反之则通过瞬时转染持续激活型的Flag-GSK3β-S9A突变质粒上调GSK3β活性，随后检测上述处理对S100A7表达的影响。结果显示，GSK3β可正向调控S100A7表达。上述结果表明GSK3β介导了PI3K/AKT对S100A7的抑制作用。由于Hippo/YAP/TEAD1通路也调控S100A7表达，故接下来，我们对PI3K/AKT/GSK3β与Hippo通路之间的关系进行了初步探究。结果显示，过表达myr-AKT并不影响YAP磷酸化水平，而沉默GSK3β则会上调YAP磷酸化，从而导致其失活。当采用GSK3β特异性抑制剂处理细胞时，发现抑制GSK3β会激活Hippo通路而导致YAP出核。上述结果表明，GSK3β可通过抑制Hippo通路激活YAP活性。但总的来看，抑制GSK3β始终会显著下调S100A7表达。反之，沉默YAP并不对GSK3β活性产生影响。综上所述，PI3K/AKT/GSK3β通路不仅可直接调控S100A7，GSK3β还可通过调节Hippo通路调控S100A7的表达。 在对S100A7生物学功能进行研究时发现，S100A7可通过正向调控GSK3β活性，发挥促进细胞增殖、抑制细胞分化的双重作用。在HCC94细胞中，沉默S100A7可进一步增强由GSK3β沉默引起的抑生长促分化现象；而在FaDu细胞中过表达S100A7则会削弱沉默GSK3β后对细胞增殖及分化的影响。 综上所述，在HCC94及FaDu鳞癌细胞中，悬浮或/和高密度培养通过激活Hippo通路和抑制PI3K/AKT/GSK3β通路，共同完成了对S100A7诱导表达的调控。而S100A7可通过正向调控GSK3β活性发挥促进细胞增殖、抑制细胞分化的双重作用。该研究结果为全面了解S100A7在鳞癌发生及发展中的生物学作用提供了新的数据， 也为今后研发以S100A7为靶点的抗肿瘤药物提供了新的思路。

S100A7, as a member of the S100 protein family, was first discovered and identified in the lesions and non lesions of psoriasis patients’ skin. It was also highly expressed in breast cancer and variety of squamous cell carcinomas (SCCs). Our previous work shows that S100A7 was only detected in part of squamous cell carcinomas, adenosquamous carcinoma and large cell lung carcinomas, but not in other lung caner subtypes. However, the expression characteristic of S100A7 in other SCC tissues and cells is not very known. In the present study, we examined the expression pattern of S100A7 using immunohistochemistry. The result show that in six kinds of SCC tissue arrays with 452 cases including esophageal SCC, cervical SCC, skin SCC, oral SCC, bladder SCC as well as lung SCC, S100A7 was heterogeneous expression (positive rate above 40%). In well differentiation SCC tissues, particularly in keratinize perals, S100A7 presented higher intensity. As a sharply contrast, in SCC cells lines, including HCC94, FaDu, A431 and H226Br cells, less than 1% S100A7 positive cells was detected except HCC94 cells, which is much lower than tissues. The opposite number of S100A7 positive cells in vitro and in vivo in SCCs suggests that there is a factor that triggers this difference. It was reported that S100A7 can be induced in normal primary keratinocytes (HEKn) cells in cell detachment culture. To determine whether S100A7 induction occurs also in SCC cells under this condition, we cultured HCC94, FaDu, A431 and H226Br cells in suspension for different days. Notably, the number of S100A7 positive cells as well as the mRNA and protein level were significantly increased under suspension condition, and then significantly decreased after the suspension cells were reattached to slides. Besides, the high cell density culture shows the similar phenomenon. These results indicate that S100A7 negative and positive cells bi-directionally transform, depending on cell shape and cell density. It has been reported that suspension and high density culture conditions can remodeling cell microfilament skeleton structure thus to stimulate Hippo pathway. Besides, the key protein YAP of the Hippo pathway is a receptor for cell morphology and density changes. Therefore, we speculate that Hippo pathway may mediate the induction of S100A7 by suspension and high density culture. We used HCC94 and FaDu cells as experimental model and culture them under cell suspension and high cell density conditions. The results showed that in HCC94 and FaDu cells, S100A7 is easily induced by both suspension and high density culture, which is accompanied by Hippo pathway activation reflected by an increase in LATS1 (T1079), YAP(S127) phosphorylation and a decrease in nuclear YAP. The immunofluorescence results further confirmed that suspension culture could induce S100A7 expression and YAP out of the nucleus. Interestingly, the correlations of S100A7 and YAP reverse after recovery of cell attachment or relief from dense culture. To ensure the relationship between Hippo pathway and S100A7, firstly, we transiently overexpressed LATS1 or silenced YAP to mimic the Hippo activation in HCC94 and FaDu cells under normal culture. The results show that Hippo pathway activation obviously promotes S100A7 mRNA and protein level. On the other hand, in the indicated cells, we inhibited the Hippo pathway via loss of LATS1, MST1 function or transiently overexpressed YAP-S127A. In contrast to the activation of the Hippo pathway, silencing the Hippo pathway inhibited S100A7 expression. The results indicated that S100A7 induction is positive regulated by Hippo pathway and is significantly repressed by nuclear YAP. Further research found that TEAD1 is required for YAP indirect transcription repression of S100A7. Next, we studied the effect of cellular microfilament skeleton integrity on S100A7 induced expression. Using fluorescence staining, we observed that suspension and high density culture could seriously abrogate F-actin structure of the cells. Then, we treated the HCC94 and FaDu cells with F-actin disrupting drugs including latrunculin B (Latrunculin B LatB), cytochalasin D (Cytochalasin D CytoD) and botulinum toxin (Botulinum toxin C3, C3) to simulate cytoskeleton destruction which result of Hippo pathway activation and induction of S100A7. Conversely, triple-silencing of F-actin-severing/capping proteins, including CFL1, GSN and CAPZB obviously suppressed S100A7 under suspension condition. However, in poorly differentiated SiHa cervical cells and NCI-H226 pulmonary cells, S100A7 is hardly induced even in suspension or activation of the Hippo pathway. The above results suggest that S100A7 induction may be related to the degree of cell differentiation. The detection results in vivo also confirm this speculation. The cervical and lingual SCC tissues array analyse show that S100A7 expression displays the positive correlation with pYAP (S127) and the negative correlation with nuclear YAP in the majority of well and moderate differentiated but not in poorly differentiated tissues. Functionally, S100A7 plays the similar effect as YAP on HCC94 and FaDu cells proliferation. Thus, our findings demonstrate that in well differentaited HCC94 and FaDu cells, suspension and high density culture condition induce S100A7 through the F-actin remodeling mediated activation of Hippo pathway. Further studies have found that another signal pathway, PI3K/AKT, also participates in the expression of S100A7 in suspension culture by regulating the activity of GSK3β. When HCC94 and FaDu cells were suspended, the GSK3β activity was up-regulated through the inhibition of PI3K/AKT pathway and thus induced S100A7 expression. Next, we transiently transfected with constitutively activated Akt (myr-Akt) plasmid and then suspended for 2 days. The results indicated that Akt overexpression could inhibit GSK3β and S100A7 which will be rescued by the addition of PI3K specific inhibitor LY294002. In order to further understand the relationship between GSK3β and S100A7, we inhibit or activate GSK3β through many different ways and examined S100A7 expression. The result suggested that GSK3β can regulate S100A7 positively. Further studies have shown that GSK3β mediate the inhibitory effect of PI3K/AKT on S100A7. Next, we preliminary explored the relationship between the PI3K/AKT/GSK3β and the Hippo/YAP/TEAD1 pathway. It was found that the expression of myr-AKT did not affect the level of YAP phosphorylation. On the contrary, silencing GSK3β could decrease YAP activity. Treated with GSK3β inhibitor further indicated that inhibition of GSK3β can decrease YAP activity via activation of Hippo pathway. However, in general, inhibition of GSK3β could significantly reduce the expression of S100A7. The above results showed that the PI3K/AKT/GSK3β pathway not only regulate S100A7 expression directly, but also can effect S100A7 through Hippo pathway. When studying the biological function of S100A7, we found that S100A7 can play a dual role in promoting cell proliferation and inhibiting cell differentiation by regulating GSK3β activity. Double silencing of S100A7 and GSK3β suppressed HCC94 cells proliferation more effective than each single deletion. Similarly, the proliferation of FaDu cells was significantly promoted by overexpression of S100A7 but inhibited by depletion of GSK3β, and slightly influenced by both combinations. In conclusion, in HCC94 and FaDu SCC cells, suspension or high density culture can induce S100A7 expression through activating Hippo pathway and inhibiting PI3K/AKT/GSK3β pathway. In addition, S100A7 can play a dual role in promoting cell proliferation and inhibiting cell differentiation through the positive regulation of GSK3β activity.The above results enriched the biological role of S100A7 in carcinogenesis and development of SCC, and also provided new targets for the treatment of SCC.