ROEL FOR ALKALINE CERAMIDASE 1 (ACER1) IN SKIN CANCER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 皮肤鳞状细胞癌(SSCC)是一种主要的皮肤癌类型，由恶性角质形成细胞衍生而来，在美国每年诊断出100多万例。该项目的长期目标是明确人类碱性神经酰胺酶1(ACER1)在抑制SSCC中的作用，并将这一概念发展成预防和治疗SSCC的新方法。ACER1是我们新克隆的神经酰胺酶，它催化神经酰胺的水解生成游离脂肪酸和鞘氨醇(SPH)。我们发表的数据表明，ACER1是一种皮肤特异性神经酰胺酶，在表皮角质形成细胞的生长停滞和分化中发挥重要作用。原位杂交结果显示，ACER1mRNA在表皮分化和分化细胞层的表达水平明显高于增殖的基底细胞层。细胞外钙离子是角质形成细胞生长停滞和分化的关键生理诱导剂，显著上调角质形成细胞ACER1的表达。这种上调被表皮生长因子(EGF)抑制，EGF促进角质形成细胞的增殖，但抑制分化。通过RNA干扰抑制ACER1基因的表达可促进角质形成细胞的增殖，并抑制角质形成细胞的分化，以响应细胞外钙的升高。我们的初步数据表明，ACER1下调可能与SSCC细胞的过度增殖和去分化有关。在我们检测的所有SSCC细胞系中，ACER1的表达都被抑制。在化学诱导的皮肤乳头状瘤中，小鼠碱性神经酰胺酶1(Acer1)的表达也显著下调。ACER1在SSCC细胞中的恢复表达不仅抑制了SSCC细胞的增殖，还诱导了角蛋白1等分化标记物的表达。我们的假设是，ACER1通过诱导角质形成细胞的生长停滞和分化，在SSCC的发生发展中起着重要的肿瘤抑制作用。我们将通过以下具体目标来检验这一假设。 特异性目标1确定ACER1下调会导致表皮增生 我们将确定在严重联合免疫缺陷(SCID)小鼠上，角质形成细胞中通过RNA干扰敲除ACER1是否会导致再生的人类皮肤移植物的表皮增生。 特异性目的2证实ACER1在SSCC发生中发挥肿瘤抑制作用 我们将使用在表皮中特异表达人类转基因ACER1的ACER1转基因小鼠，并研究ACER1转基因小鼠中化学诱导的皮肤肿瘤形成是否被抑制或减少。 这方面的研究具有重要意义，因为实验结果将为预防和治疗鳞状细胞癌提供重要的分子基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The skin squamous cell carcinoma (SSCC), which is derived from malignant keratinocytes, is a major type of skin cancer, more than 1 million cases of which are diagnosed annually within the US. The long-term goals of this project are to define the role of the human alkaline ceramidase 1 (ACER1) in suppressing SSCC, and to develop this concept into novel approaches for the prevention and treatment of SSCC. ACER1 is our newly cloned ceramidase that catalyzes the hydrolysis of ceramide to generate free fatty acid and sphingosine (SPH). Our published data demonstrate that ACER1 is a skin-specific ceramidase that plays an important role in the growth arrest and differentiation of epidermal keratinocytes. In situ hybridization revealed that ACER1 mRNA is expressed at much higher levels in the differentiating and differentiated cell layers of the epidermis than the proliferating basal cell layer. ACER1 expression in cultured keratinocytes is markedly upregulated by extracellular Ca2+, a key physiological inducer of the growth arrest and differentiation of keratinocytes. This upregulation is inhibited by epidermal growth factor (EGF), which promotes keratinocyte proliferation but suppresses differentiation. ACER1 knockdown by RNA interference increases keratinocyte proliferation and suppresses keratinocyte differentiation in response to extracellular Ca2+ elevation. Our preliminary data suggest that ACER1 downregulation may contribute to both hyperproliferation and dedifferentiation of SSCC cells. ACER1 expression is suppressed in all SSCC cell lines that we examined. The expression of the mouse alkaline ceramidase 1 (Acer1) is also substantially down-regulated in chemically induced skin papillomas. Restored expression of ACER1 in SSCC cells not only inhibit the proliferation of SSCC cells but also induces the expression of differentiation markers, such as keratin 1. Our hypothesis is that ACER1 plays an important role as a "tumor suppressor" in SSCC development and progression through its ability to induce the growth arrest and differentiation of keratinocytes. We will test this hypothesis with the following Specific Aims. Specific Aim 1 Establish that ACER1 downregulation leads to epidermal hyperplasia We will determine whether ACER1 knockdown by RNA interference in keratinocytes causes epidermal hyperplasia in regenerated human skin grafts on a severe combined immunodeficiency (SCID) mouse. Specific Aim 2 Establish that ACER1 plays a tumor suppressor role in SSCC development We will use ACER1 transgenic mice that express the human transgene ACER1 specifically in the epidermis and investigate whether the chemically induced formation of skin tumors is inhibited or reduced in ACER1 transgenic mice. The proposed research in this application is of great importance as the results generated from the experiments will provide an important molecular foundation for the prevention and treatment of SSCC.