ROEL FOR ALKALINE CERAMIDASE 1 (ACER1) IN SKIN CANCER

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 皮肤鳞状细胞癌（SSCC）是一种主要的皮肤癌类型，来源于恶性角质形成细胞，在美国每年诊断出超过100万例。该项目的长期目标是确定人类碱性神经酰胺酶1（ACER 1）在抑制SSCC中的作用，并将这一概念发展为预防和治疗SSCC的新方法。ACER 1是我们新克隆的神经酰胺酶，可催化神经酰胺水解产生游离脂肪酸和鞘氨醇（SPH）。我们发表的数据表明，ACER 1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起重要作用。原位杂交结果显示，ACER 1 mRNA在表皮的分化和分化细胞层中的表达水平比增殖基底细胞层高得多。细胞外Ca 2+是角质形成细胞生长停滞和分化的关键生理诱导物，可显著上调培养角质形成细胞中的ACER 1表达。这种上调被表皮生长因子（EGF）抑制，表皮生长因子促进角质形成细胞增殖但抑制分化。通过RNA干扰敲低ACER 1增加角质形成细胞增殖并抑制角质形成细胞分化以响应细胞外Ca 2+升高。我们的初步数据表明，ACER 1下调可能有助于SSCC细胞的过度增殖和去分化。在我们检测的所有SSCC细胞系中，ACER 1表达均受到抑制。小鼠碱性神经酰胺酶1（Acer 1）的表达在化学诱导的皮肤乳头状瘤中也显著下调。在SSCC细胞中恢复ACER 1的表达不仅抑制SSCC细胞的增殖，而且诱导分化标志物如角蛋白1的表达。我们的假设是，ACER 1通过其诱导角化细胞生长停滞和分化的能力，在SSCC的发展和进展中起着重要的作用，作为一个“肿瘤抑制因子”。我们将用以下具体目标来检验这一假设。 具体目标1确定ACER 1下调导致表皮增生 我们将确定在角质形成细胞中通过RNA干扰敲低ACER 1是否会导致严重联合免疫缺陷（SCID）小鼠再生皮肤移植物中的表皮增生。 具体目标2：确定ACER 1在SSCC发展中发挥肿瘤抑制作用 我们将使用在表皮中特异性表达人转基因ACER 1的ACER 1转基因小鼠，并研究在ACER 1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。 这项应用中的拟议研究非常重要，因为实验产生的结果将为预防和治疗SSCC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.