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(NMSCs)、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，每年约有100万新诊断病例。虽然NMSCs，特别是基底细胞癌(BCC)基本上是可以治愈的，但手术治疗往往会导致疤痕和毁容，而且复发的风险很高(第二次发生的可能性为35-50%)。NMSCs发展的主要危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。基底细胞约占NMSCs的80%，起源于皮肤表皮的基底层角质形成细胞。我们以前的研究已经证明，蛋白激酶D(PKD)在人基底细胞癌的角质形成细胞中表达上调。此外，我们最近证实，在体外，小鼠角质形成细胞在紫外线B辐射下被激活，并保护这些细胞免受UVB诱导的细胞凋亡。这些结果表明，UVB可能选择具有较高水平的促增殖PKD1的细胞。另一种选择是，激活的PKD1可能允许紫外线损伤的细胞存活。这种PKD1促进存活的能力将有益于防止低水平UVB暴露下的过度凋亡，造成最小的DNA损伤，可以修复。然而，如果PKD1允许遭受不可修复的紫外线诱导DNA损伤的细胞存活，这些带有DNA突变的角质形成细胞可能会继续增殖并形成皮肤肿瘤。因此，无论是促进增殖的机制还是促进生存的机制，都可以提供一种手段，使PKD1能够促进表皮肿瘤的发生。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一种可行的疗法。由于这些抑制剂可以局部应用，全身暴露最少，即使PKD1在其他类型的细胞中具有重要作用，它们也可能使用时几乎没有副作用。此外，PKD抑制剂已被认为是治疗胰腺癌的可能的新疗法。因此，了解PKD1在表皮中的作用对于确定使用这些药物全身治疗的表皮副作用的可能性可能是重要的。例如，在服用PKD抑制剂治疗内癌的患者中，抑制皮肤中的PKD1可能会导致日光敏感度增加，紫外线会引发组成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PKD1激活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，我们还将在体外将我们在小鼠角质形成细胞中获得的结果推广到小鼠体内。进一步，我们将研究紫外线B光对体外培养的人角质形成细胞、原位重建的人皮肤和活体的人志愿者体内PKD1活性的影响。特别是，我们的目标是：(1)检验UVB激活的PKD1定位于线粒体的假说，该酶在该位置保护角质形成细胞免受紫外线诱导的凋亡；(2)检验在体外培养的人角质形成细胞和原位重组的人表皮中PKD1被激活并促进UVB照射后存活的假说；(3)在小鼠和人类皮肤中检验UVB激活PKD1的假说；以及(4)在体外和体内检验UVB选择PKD1水平较高的角质形成细胞的假说。这些研究的结果应该能够识别新的信号分子，如PKD1或其下游通路，这些分子可能有针对性地治疗和/或预防退伍军人和整个美国人口的NMSCs。此外，PKD抑制剂已被认为是治疗其他类型癌症的潜在新疗法，如胰腺癌。有关PKD在角质形成细胞对UVB反应中的作用的证据可能对预测接受PKD抑制剂全身化疗的患者可能的阳光敏感性很重要。

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.