Pathways that Suppress or Accelerate Premalignant Progression of Squamous Cancer

抑制或加速鳞状癌癌前进展的途径

• 批准号: 8157177
• 负责人: STUART H. YUSPA
• 金额: $ 123.28万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8157177
• 关键词: Malignant Neoplasms; Pathway interactions; Premalignant

CLIC4 is a highly conserved, multifunctional protein that causes growth arrest and terminal differentiation in skin keratinocytes and other cell types. We have reported that CLIC4 is reduced in virtually all major human cancer parenchyma and highly upregulated in virtually all human cancer stroma. Major insight into how CLIC4 functions in growth control came with our discovery that CLIC4 is a component of the TGFbeta pathway. In skin keratinocytes, TGFbeta causes CLIC4 to associate with Schnurri-2 and translocate to the nucleus. Nuclear CLIC4 prolongs TGFbeta signaling by inhibiting Smad2/3 dephosphorylation. TGFbeta is fundamental to the conversion of fibroblasts to myofibroblasts, an important event in skin wound healing, fibrosis and cancer development. We have now determined that CLIC4 participates in TGFbeta dependent myofibroblast conversion of dermal fibroblasts. Using primary dermal fibroblasts from CLIC4wt/wt and CLIC4fl/fl mice transduced with adenoviral Cre recombinase to ablate CLIC4 expression, we show that absence of CLIC4 inhibits myofibroblast conversion as measured by transcription and translation of alpha smooth muscle actin (alphaSMA). Expression of extracellular matrix components like the collagens, MMPs and thrombospondins that TGFbeta induces during fibroblast activation is reduced in TGFbeta treated adeno-Cre infected CLIC4 fl/fl fibroblasts. These cells also have higher cell motility compared to wild type controls. Fibroblasts deleted of CLIC4 have reduced activation of Smad2 and p38 upon TGFbeta stimulation. Smad2 siRNA and p38 kinase inhibitors prevented TGFbeta stimulation of myofibroblast conversion suggesting that CLIC4 might influence fibroblast differentiation through interaction with both Smad-dependent and Smad-independent TGFbeta pathways. Conversely, conditioned medium from fibroblasts that overexpress CLIC4 enhances p38 activation and migration of epithelial cells. Thus CLIC4 plays an important role in both epithelial and mesenchymal cell biology by altering TGFbeta signaling and is therefore an attractive target for modifying TGFbeta in both tissue compartments. Our previous studies have shown that the biological functions of CLIC4 relative to growth inhibition and cancer occur in the nucleus. CLIC4 has a functional nuclear localization signal but nuclear translocation of CLIC4 is defective in cancer cells. Thus it is important to understand the factors that control CLIC4 nuclear translocation. We have recently established that nitric oxide directly regulates nuclear translocation of CLIC4 by S-nitrosylation of specific cysteines in the protein. The modification induces a conformational change and results in enhanced association of the protein with nuclear import proteins Ran and importin alpha. Moreover, TNFalpha-induced nuclear translocation of CLIC4 is dependent on nitric oxide synthase (NOS)-activity. In macrophages iNOS (inducible nitric oxide synthase) induction and activity increased nuclear translocation and S-nitrosylation of CLIC4. Chemical inhibition or genetic ablation of iNOS decreases nuclear translocation and S-nitrosylation of CLIC4 in response to LPS and IFNgamma. Previous results showed that Ca2+-induced differentiation of keratinocytes enhances nitrosylation of CLIC4 as early as 3hrs. This is coincident with increased p-eNOS (S1176) that indicates enhanced eNOS activity. Chemical inhibition of eNOS activity using L-NAME inhibits Ca2+-induced nuclear translocation of CLIC4 and expression of K1 and K10. Expression of K10 can be increased under these conditions when an NO-donor is added back. We hypothesize that changes in redox potential in tumor cells are responsible for the altered nuclear localization of CLIC4. To that end, we have evaluated the expression and activity of thioredoxin reductase 1 (TR1) and thioredoxin in human and mouse tumor cell lines. Both human (SCC13 and HaCaT-Ras cells) and mouse (Pam212) carcinoma cells show high levels of TR1 protein and activity compared to normal cells. Our preliminary results suggest that pretreatment of Pam212 cells with auranofin, a TR1 inhibitor, followed by an NO-donor restores CLIC4 nuclear translocation. Indeed, Kras-transformed cells stably knocked down for Trx or TR1 show enhanced levels of CLIC4 translocation compared to the parental cell line. These results suggest that higher expression/activity of thioredoxin/thioredoxin reductase pathway may contribute to the aberrant translocation of CLIC4. The analysis of CLIC4 function in cancer pathogenesis should be facilitated by our recent production of CLIC4 knockout mice using recombineering technology. CLIC4 knockout mice are viable and fertile but develop skin erosions at high frequency after about 3 to 6 months. Knockout skin, fibroblasts and keratinocytes have TGFbeta signaling defects as evidenced by reduced Smad2 phosphorylation and increased c-Myc. Knockout keratinocytes also display defects in TGFbeta dependent transcript expression on PCR arrays. Knockout fibroblasts proliferate faster that WT cells and are less sensitive to TGFbeta induced growth arrest. Corneal epithelial wound healing is delayed in knockout mice suggesting the skin erosions might occur due to defects in wound healing. Currently additional mice are being generated targeting the CLIC4 knockout to epidermal cells and fibroblasts. Furthermore, a knockin mouse which would express Green Fluorescent Protein (GFP) under the control of CLIC4 promoter is undergoing construction. While these basic studies in tumor progression are ongoing we are also conducting studies to illuminate the mechanism of action of drugs that are designed to treat skin cancers. Ingenol-3-angelate (Ing3A), extracted from Euphorbia peplus, is currently in clinical trials for eradicating basal cell carcinoma (BCC), actinic keratosis and squamous cell carcinoma (SCC) in situ by topical application. Although structurally related to phorbol esters and a PKC activator, topical Ing3A, but not phorbol 12 myristate 13 acetate (PMA), inhibited the growth of subcutaneous tumors derived from PAM212 (mouse SCC) and B16 (mouse melanoma). Ing3A and PMA both induced acute neutrophilic inflammation on mouse skin, but only Ing3A caused subcutaneous hemorrhage and vascular damage. Both Ing3A and PMA activated Erk1/2 in epidermis, but Ing3A also activated Erk1/2 in skin dermal fibroblasts and endothelial cells. Pretreatment with topical cyclosporin A (CsA), verapamil or XR9576, modulators of P-glycoprotein (P-gp), prevented Ing3A induced hemorrhage but not neutrophil infiltration. CsA also impaired Ing3As anti cancer activity while the anti-inflammatory dexamethasone did not. In collaboration with Suresh Ambudkar we are examining the role of P-gp in the unusual properties of Ing3A. Ing3A, but not PMA, blocked photoaffinity labeling of human P-gp with [125I]-Iodoaryazidoprazosin and inhibited P-gp mediated drug resistance to HCT-15 cells. The intracellular levels of Ing3A were significantly lower in P-gp expressing cells and treatment with XR9576 increased the levels to those of cells that do not express P-gp, demonstrating that Ing3A binds to and is transported by P-gp. Taken together, our results suggest that P-gp mediated absorptive transport, dermal penetration and vascular damage contribute to the anti-cancer activity of Ing3A in vivo. These finding may serve as a paradigm for percutaneous absorption of other therapeutic agents and such studies are now in progress.

CLIC4是一种高度保守的多功能蛋白，可引起皮肤角质形成细胞和其他细胞类型的生长停滞和终末分化。我们报道说，几乎所有主要的人类癌实质中CLIC4均降低，并且在几乎所有人类癌基质中都高度上调。我们发现CLIC4是TGFBETA途径的组成部分，对CLIC4如何在生长控制中的功能进行了重大洞察力。在皮肤角质形成细胞中，TGFBETA导致Clic4与Schnurri-2缔合并转移到细胞核。核Clic4通过抑制Smad2/3去磷酸化来延长TGFBETA信号传导。 TGFBETA是将成纤维细胞转换为肌纤维细胞的基础，这是皮肤伤口愈合，纤维化和癌症发育的重要事件。现在，我们已经确定CLIC4参与皮肤成纤维细胞的TGFBETA依赖性成肌纤维细胞转换。使用clic4wt/wt的原代皮肤成纤维细胞以及用腺病毒CRE重组酶转导的clic4fl/fl小鼠，以消除clic4表达，我们表明，Clic4的缺失可抑制肌纤维细胞的转化，按照转录和转录的alpha平滑肌肌动蛋白的转录和转换（Anplosma）。在TGFBETA处理过的腺癌感染的CLIC4 FL/FL成纤维细胞中，降低了TGFBETA在成纤维细胞激活过程中TGFBETA诱导TGFBETA诱导TGFBETA诱导TGFBETA诱导的细胞外基质成分的表达。与野生型对照相比，这些细胞的细胞运动性也更高。 TGFBETA刺激后，clic4删除的成纤维细胞的激活降低。 Smad2 siRNA和p38激酶抑制剂阻止了TGFBETA刺激肌纤维细胞转换，这表明CLIC4可能通过与SMAD依赖性和独立于SMAD的TGFBETA途径相互作用而影响成纤维细胞分化。相反，过表达CLIC4的成纤维细胞的条件培养基增强了上皮细胞的激活和迁移。因此，CLIC4通过改变TGFBETA信号传导在上皮和间质细胞生物学中起重要作用，因此是修饰两个组织室中TGFBETA的有吸引力的靶标。我们先前的研究表明，CLIC4相对于生长抑制和癌症的生物学功能发生在细胞核中。 CLIC4具有功能性核定位信号，但CLIC4的核转运在癌细胞中有缺陷。因此，了解控制CLIC4核易位的因素很重要。我们最近确定，一氧化氮直接通过蛋白质中特定半胱氨酸的硝基化来直接调节clic4的核转运。该修饰会引起构象变化，并导致蛋白质与核进口蛋白的缔合性增强，并导致蛋白质的α和importin alpha。此外，TNFalpha诱导的Clic4核转运取决于一氧化氮合酶（NOS）活性。在巨噬细胞中，iNOS（诱导型一氧化氮合酶）诱导和活性增加了核转运和clic4的S-硝基化。 iNOS的化学抑制作用或遗传消融可降低CLIC4对LPS和IFNGAMMA的核转运和S-硝基化。先前的结果表明，CA2+诱导的角质形成细胞的分化早在3小时后会增强CLIC4的硝基基化。这与P-Enos（S1176）的增加相吻合，表明ENOS活性增强。 使用L-NAME对ENOS活性的化学抑制作用抑制Ca2+诱导的CLIC4的核转运和K1和K10的表达。在这些条件下，当添加无偏见的情况下，可以增加K10的表达。我们假设肿瘤细胞中氧化还原电位的变化导致CLIC4的核定位改变。为此，我们评估了人和小鼠肿瘤细胞系中硫氧还蛋白还原酶1（TR1）和硫氧还蛋白的表达和活性。与正常细胞相比，人（SCC13和HACAT-RAS细胞）和小鼠（PAM212）癌细胞均显示出高水平的TR1蛋白和活性。我们的初步结果表明，用Auranofin（TR1抑制剂）对PAM212细胞进行了预处理，然后进行无偏见恢复了Clic4核易位。 实际上，与父母细胞系相比，稳定击倒的KRAS转换细胞稳定地击倒了TRX或TR1的CLIC4易位水平增强。这些结果表明，硫氧还蛋白/硫氧还蛋白还原酶途径的较高表达/活性可能导致CLIC4的异常易位。癌症发病机理中CLIC4功能的分析应通过我们最近使用重组技术生产CLIC4敲除小鼠来促进。 CLIC4敲除小鼠可行且肥沃，但在大约3到6个月后以高频出现皮肤侵蚀。敲除皮肤，成纤维细胞和角质形成细胞具有TGFBETA信号传导缺陷，这可以通过降低的Smad2磷酸化和C-MYC增加证明。敲除角质形成细胞还在PCR阵列上的TGFBETA依赖性转录本表达式中显示缺陷。敲除成纤维细胞增殖的速度更快，对WT细胞的增殖速度更快，并且对TGFBETA诱导的生长停滞效果不太敏感。在敲除小鼠中，角膜上皮伤口愈合延迟，表明由于伤口愈合缺陷，可能发生皮肤侵蚀。目前，正在生成其他小鼠，将CLIC4敲除瞄准表皮细胞和成纤维细胞。此外，在CLIC4启动子控制下表达绿色荧光蛋白（GFP）的敲击小鼠正在进行构造。尽管这些肿瘤进展中的基础研究正在进行中，但我们也在进行研究，以阐明旨在治疗皮肤癌的药物的作用机理。从欣快的peplus中提取的Ingenol-3-Engelate（Ing3a）目前正在临床试验中，用于消除基底细胞癌（BCC），精骨角化病和鳞状细胞癌（SCC），并通过局部应用在现场进行。尽管在结构上与佛波酯和PKC激活剂，但无局部ING3A，但与凤梨12肉芽菌13醋酸盐（PMA）无关，但抑制了源自PAM212（小鼠SCC）和B16（小鼠黑色素瘤）的皮下肿瘤的生长。 ING3A和PMA都诱导了小鼠皮肤上的急性嗜中性炎症，但只有ING3A引起皮下出血和血管损伤。 ING3A和PMA都激活了表皮中的ERK1/2，但ING3A也激活了皮肤皮肤成纤维细胞和内皮细胞中的ERK1/2。局部环孢菌素A（CSA），Verapamil或XR9576，P-糖蛋白（P-GP）的预处理可阻止ING3A诱导的出血，但未诱导中性粒细胞浸润。 CSA还损害了ING3AS抗癌活性，而抗炎地塞米松则没有。与Suresh Ambudkar合作，我们正在研究P-gp在ING3A异常属性中的作用。 ING3A（而不是PMA）用[125i] - 碘氧羟基二丙酰瓜抑制了人类P-gp的光性标记，并抑制了对HCT-15细胞的P-gp介导的耐药性。 P-gp表达细胞的细胞内ING3A水平明显降低，用XR9576处理不表达p-gp的细胞的水平，表明ING3A与p-gp结合并转运。综上所述，我们的结果表明，P-gp介导的吸收性转运，皮肤渗透和血管损伤有助于ING3A在体内的抗癌活性。这些发现可能是对其他治疗剂的经皮吸收的范式，并且此类研究正在进行中。