The Role of Protein Kinase D in Epidermal Tumorigenesis

描述（由申请人提供）： 非黑色素瘤皮肤癌（NMSC），基底细胞癌和鳞状细胞癌是最常见的癌症，发生频率超过所有癌症的总和，每年诊断出约100万例新病例。虽然NMSC，特别是基底细胞癌（BCC），在大多数情况下，基本上是可治愈的，但手术治疗通常会导致疤痕和毁容，并且复发的风险很高（第二次发生的机会为35-50%）。NMSC发展的主要风险因素是长期阳光照射（紫外线照射）和年龄：因此，这些癌症是退伍军人群体的主要问题。构成NMSC的大约80%的BCC产生于皮肤表皮中的基底角质形成细胞。我们以前的研究表明，蛋白激酶D（PKD）在人基底细胞癌的角质形成细胞中上调。此外，我们最近证明，PKD 1被激活后，暴露于紫外线B照射的小鼠角质形成细胞，并保护这些细胞从UVB诱导的细胞凋亡在体外。这些结果表明，UVB可能选择具有较高水平的促增殖PKD 1的细胞。或者，活性PKD 1可以允许UV损伤的细胞存活。PKD 1促进存活的这种能力将有利于防止低水平UVB暴露的过度凋亡，导致可以修复的最小DNA损伤。然而，如果PKD 1允许遭受不可修复的紫外线诱导的DNA损伤的细胞存活，这些具有DNA突变的角质形成细胞可能继续增殖并形成皮肤肿瘤。因此，促增殖或促存活机制可以提供PKD 1可以促进表皮肿瘤发生的手段。此外，这些结果表明，小分子PKD抑制剂可能是治疗非黑色素瘤皮肤癌的可行疗法。由于这些抑制剂可以局部应用，全身暴露最小，因此即使PKD 1在其他细胞类型中具有重要作用，它们也可能具有很少的副作用。此外，PKD抑制剂已被提议作为治疗胰腺癌的可能的新疗法。因此，了解PKD 1在表皮中的作用可能对确定这些药物全身治疗的表皮副作用的可能性很重要。例如，在使用PKD抑制剂治疗内部癌症的患者中，皮肤中PKD 1的抑制可导致增加的太阳敏感性，其中紫外线引发包括表皮的角质形成细胞的大量凋亡。 在这里提出的实验中，我们打算确定紫外线B光诱导的PKD 1活化保护角质形成细胞免于细胞死亡，从而促进其存活的机制。此外，我们将扩大我们的结果在小鼠角质形成细胞在体外，在小鼠体内。此外，我们将研究紫外线B光对PKD 1激活的人角质形成细胞在体外，在重建的人皮肤原位和在人体志愿者在体内的影响。具体而言，我们的目标是：（1）检验UVB激活的PKD 1定位于线粒体的假设，在线粒体的位置酶保护角质形成细胞免于UV诱导的凋亡，（2）检验PKD 1在体外人角质形成细胞中被激活并原位重建人表皮并促进UVB暴露后的存活的假设，（3）检验小鼠和人皮肤中的PKD 1在体内被UVB激活的假设和（4）检验UVB在体外和体内选择具有较高PKD 1水平的角质形成细胞的假设。这些研究的结果应该允许识别新的信号分子，如PKD 1或其下游通路，这些分子可能被靶向治疗和/或预防退伍军人和整个美国人口中的NMSC。此外，PKD抑制剂已被提议作为用于治疗其他类型的癌症（例如胰腺癌）的潜在新疗法。关于PKD在角质形成细胞对UVB反应中的作用的证据对于预测接受PKD抑制剂全身化疗的患者可能的太阳敏感性可能是重要的。

DESCRIPTION (provided by applicant): The non-melanoma skin cancers (NMSCs), basal and squamous cell carcinoma, are the most common cancers, occurring more often than all cancers combined, with approximately 1 million new cases diagnosed per year. Although the NMSCs, in particular basal cell carcinomas (BCCs) are, for the most part, essentially curable, surgical treatment often results in scarring and disfigurement, and the risk of recurrence is high (the chance of a second occurrence is 35-50%). The major risk factors for the development of NMSCs are chronic sun exposure (ultraviolet irradiation) and age: thus, these cancers are a major problem in the veteran population. BCCs, which make up approximately 80% of the NMSCs, arise from basal keratinocytes in the epidermis of the skin. Our previous studies have demonstrated that protein kinase D (PKD) is up-regulated in keratinocytes in human BCCs. In addition, we recently demonstrated that PKD1 is activated upon exposure of mouse keratinocytes to ultraviolet B irradiation and protects these cells from UVB-induced apoptosis in vitro. These results suggest that UVB might select for cells with higher levels of pro-proliferative PKD1. Alternatively, active PKD1 may allow survival of UV-damaged cells. This ability of PKD1 to promote survival would be beneficial in preventing excessive apoptosis with low levels of UVB exposure, causing minimal DNA damage that can be repaired. However, if PKD1 allows survival of cells that have suffered irreparable UV-induced DNA damage, these keratinocytes with DNA mutations could continue to proliferate and form skin tumors. Thus, either a pro-proliferative or pro-survival mechanism could provide a means by which PKD1 could contribute to epidermal tumorigenesis. Moreover, these results suggest that small-molecule PKD inhibitors might be a viable therapy for the treatment of non-melanoma skin cancers. Because these inhibitors can be applied topically, with minimal systemic exposure, they could potentially be used with few side effects even if PKD1 has important roles in other cell types. In addition, PKD inhibitors have been proposed as possible novel therapies for treatment of pancreatic cancer. Therefore, an understanding of the role of PKD1 in the epidermis may be important in determining the possibility of epidermal side effects of systemic treatment with these agents. For example, inhibition of PKD1 in the skin could result in increased sun sensitivity, with ultraviolet light triggering massive apoptosis of the keratinocytes comprising the epidermis, in patients on PKD inhibitors for the treatment of internal cancers. In the experiments proposed here, we intend to determine the mechanisms by which ultraviolet B light- induced PKD1 activation protects keratinocytes from cell death, thereby promoting their survival. In addition we will extend our results obtained in mouse keratinocytes in vitro, to the mouse in vivo. Further, we will investigate the effect of ultraviolet B light on PKD1 activation in human keratinocytes in vitro, in reconstituted human skin in situ and in human volunteers in vivo. In particular, our aims are to: (1) test the hypothesis that UVB-activated PKD1 localizes to the mitochondria, at which location the enzyme protects keratinocytes from UV-induced apoptosis, (2) test the hypothesis that PKD1 is activated in human keratinocytes in vitro and reconstituted human epidermis in situ and promotes survival upon UVB exposure, (3) test the hypothesis that PKD1 is activated by UVB in mouse and human skin in vivo and (4) test the hypothesis that UVB selects for keratinocytes with higher PKD1 levels in vitro and in vivo. The results of these studies should allow the identification of novel signaling molecules, such as PKD1 or its downstream pathways, which may be targeted to treat and/or prevent NMSCs in veterans and the U.S. population as a whole. In addition, PKD inhibitors have been proposed as potential novel therapies for the treatment of other types of cancers, such as pancreatic cancer. Evidence concerning the role of PKD in the keratinocyte response to UVB may be important for predicting possible sun sensitivity in patients undergoing systemic chemotherapy with PKD inhibitors.