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: 8348877
• 负责人: STUART H. YUSPA
• 金额: $ 117.89万
• 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/8348877
• 关键词: Ablation; Address; Adult; Antioxidants; Attention; Benign; Biological Assay; Biology; Blood Vessels; CLIC4 gene; CXCL1 gene; Cardiac; Cells; Cervix Uteri; Collaborations; Conditioned Culture Media; Corneal Injury; Cosmetics; Cyclosporine; Dermal; Development; Dorsal; Down-Regulation; Drug Transport; Drug toxicity; Embryonic Development; Endothelial Cells; Epithelial Cells; Fibroblasts; Genes; Growth; Hair follicle structure; Head and Neck Squamous Cell Carcinoma; Hemorrhage; Immune response; Immunomodulators; In Vitro; Knockout Mice; Laboratories; MAPK14 gene; Malignant Conversion; Malignant Neoplasms; Malignant neoplasm of prostate; Mediating; Mediator of activation protein; Medical; Modeling; Molecular; Molecular Target; Morbidity - disease rate; Mus; Myocardial; Myofibroblast; Normal Cell; Nuclear; Nude Mice; Organ; Oxidation-Reduction; Pathway interactions; Patients; Phagocytosis; Phorbol; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Phosphoric Monoester Hydrolases; Plasma; Premalignant; Prostate; Protein Isoforms; Protein Kinase C; Proteins; Reporting; Research; Resistance; Role; Signal Transduction; Skin; Skin Cancer; Skin Carcinogenesis; Skin Neoplasms; Spermatocytes; Spermatogonia; Therapeutic Agents; Thick; Tissues; Topical application; Transforming Growth Factor beta; United States; Work; Wound Healing; Xenograft procedure; absorption; attenuation; base; cancer cell; carcinogenesis; cell type; chemical carcinogen; in vitro Model; in vivo; inhibitor/antagonist; keratinocyte; kidney cortex; mRNA Expression; macrophage; melanoma; migration; neoplastic cell; novel therapeutics; overexpression; protein expression; research study; selenoprotein; skin disorder; sperm cell; tumor; tumor progression

CLIC4 is a metamorphic, multifunctional, redox regulated protein that is lost from cancer cells during tumor progression. To evaluate the function of CLIC4 in vivo we have generated CLIC4 floxed mice and ablated the gene en toto or in skin keratinocytes. The null mice have spontaneous skin wounds and reduced skin wound healing in full thickness and abrasion experiments. Further, they have defective corneal wound healing. CLIC4 is a redox sensitive protein. Collaboration with the Hatfield laboratory indicates that selenoprotein antioxidants have a marked effect on keratinocyte viability and skin integrity. We are addressing the role of CLIC4 in selenoprotein function. CLIC4 null keratinocytes are less responsive to TGF-beta induced migration in vitro and adhere with less efficiency on plates coated with matrix secreted by wild type keratinocytes. Skin tumor induction with chemical carcinogens did not demonstrate differences in benign tumor formation between CLIC4 null and intact mice, and results for malignant conversion are pending. Currently, mice lacking CLIC4 in fibroblasts are being generated by crossing with FSP1 Cre mice. CLIC4-GFP knockin mice have been generated to study the expression of CLIC4 during embryonic development and in adult tissues and cell types. CLIC4 expression is high in hair follicles, and CLIC4 is highly expressed in the renal cortex as opposed to the medulla. CLIC4 is also highly expressed in spermatozoa and a diminishing gradient is seen in spermatocytes and spermatogonia. CLIC4 is highly expressed in endothelial cells in blood vessels of all organs studied including cardiac blood vessels, but myocardial expression is low. CLIC4 mRNA expression is particularly high in macrophages upon stimulation with IFNgamma and LPS. In the absence of CLIC4, macrophages are defective in phagocytosis. Furthermore, the expression of IL-1beta, iNOS and CXCL1 is prolonged in null compared to wildtype macrophages suggesting attenuation of these immunomodulators is misregulated in the absence of CLIC4. TGF-beta regulates the dissipation of macrophage activity, and TGF-beta signaling is lower in CLIC4 null macrophages, with lower Smad4 levels. Overexpression of nuclear targeted CLIC4 or HA-CLIC4 in stimulated macrophages downregulates active IL-1beta protein suggesting CLIC4 is essential for TGF-beta-mediated downregulation of the immune response. In the past year, we have furthered our study of CLIC4 and pSmad2 protein expression in cancer. In separate collaborations with Dr. Carter Van Waes and Dr. Addie Alkhas we have shown that CLIC4 and nuclear CLIC4 are reduced in HNSCC and SCC of the cervix. Collaborations with Mark Simpson and Joshua Webster have demonstrated that expression of exogenous CLIC4 in skin SCC orthografts causes growth inhibition and activation of TGF-beta signaling in tumor cells. Furthermore, conditional overexpression of exogenous CLIC4 in mouse skin reduces the de novo tumor yield in initiation-promotion studies. While we have focused attention on CLIC4 in tumor cells, we are also cognizant of the role of CLIC4 and TGF-beta in the tumor stroma. CLIC4 is essential for TGF-beta to induce myofibroblast conversion in the formation of a cancer stroma. Conditioned media from tumor cells induces CLIC4 expression in fibroblasts via TGF-beta signaling. Tumor cells secrete several folds greater amounts of TGF-beta compared to normal cells. Conditioned media from fibroblasts overexpressing CLIC4 enhances migration and invasion of epithelial cells via TGF-beta signaling. These conditioned media secrete more TGF-beta than control fibroblasts. Thus CLIC4 enhances crosstalk between tumor cells and the tumor microenvironment through TGF-beta signaling. Myofibroblast conversion via a CLIC4 pathway is Smad and p38 dependent. p38alpha is the major isoform responsible for myofibroblast conversion and contrary to a previous report, changes in ROS do not alter conversion in dermal fibroblasts. Absence of CLIC4 enhances interaction of phospho p38 with PPM1a, its specific phosphatase. PPM1a interacts with CLIC4, and it is likely that this interaction interrupts PPM1a association with its other substrates thereby explaining how CLIC4 could potentially enhance both pSmad and p38 activation. We have continued to study the contribution of active transdermal transport of drugs through keratinocyte P-gp in collaboration with the Blumberg and Ambudkar laboratories. Cyclosporin A is a well-known P-gp substrate. Cyclosporin A is detected in plasma of nude and haired mice after topical application of cyclosporin A at 16 mg/kg. A stable level of plasma cyclosporin A (110-140 nmol/L) is detected when cyclosporin A is applied daily for 7 consecutive days to the dorsal skin of nude mice at 16 mg/kg. XR9576, a P-gp inhibitor, partially blocks skin P-gp mediated cyclosporin A absorption. We used an efflux assay and a P-gp mediated drug toxicity resistance assay to identify P-gp substrates among a number of phorbol ester derivates and other natural compounds that activate protein kinase C. Phorbol 12,13-dihexanoate, phorbol 12,13-dibutyrate and phorbol 12,13-dibenzoate are substrates of P-gp. However, when topically applied to the dorsal skin of nude mice, all three phorbol ester derivates failed to induce hemorrhage presumeably because of their weak activity as a protein kinase C activator. Our cancer studies have also extended into understanding the activity of RasGRP3 in melanoma and prostate cancer in collaboration with the Blumberg laboratory. RasGRP3, a Ras activator, is elevated in these tumors from patients. Experimentally, overexpression of rasGRP3 in tumor cells enhances prostate or melanoma xenografts while ablation of rasGRP3 in these same cells inhibits tumor formation.

CLIC4是一种变质，多功能，氧化还原调节的蛋白，在肿瘤进展过程中因癌细胞而丢失。为了评估clic4在体内的功能，我们已经产生了clic4 floxed小鼠，并消融了基因en toto或皮肤角质形成细胞。无效的小鼠具有自发的皮肤伤口，并在全厚度和磨损实验中减少了皮肤伤口的愈合。此外，它们具有缺陷的角膜伤口愈合。 CLIC4是一种氧化还原敏感蛋白。与Hatfield实验室的合作表明，硒蛋白抗氧化剂对角质形成细胞的生存能力和皮肤完整性有明显影响。我们正在解决CLIC4在硒蛋白功能中的作用。 CLIC4 NULL角质形成细胞对TGF-Beta诱导的体外迁移的反应较低，并且在用野生型角质形成细胞分泌的基质上的板上效率较小，并粘附在板上。用化学致癌物诱导皮肤肿瘤并未表现出CLIC4 NULL和完整小鼠之间良性肿瘤形成的差异，而恶性转化率的结果也在等待中。当前，通过与FSP1 Cre小鼠交叉产生缺乏成纤维细胞中CLIC4的小鼠。 CLIC4-GFP敲击蛋白已经生成以研究在胚胎发育以及成年组织和细胞类型中CLIC4的表达。 CLIC4表达在毛囊中很高，而Clic4在肾皮质中高度表达，而不是髓质。 CLIC4在精子中也高度表达，并且在精子细胞和精子中观察到梯度减少。 CLIC4在所研究的所有器官的血管中高度表达，包括心脏血管，但心肌表达较低。在用IFNGAMMA和LPS刺激后，CLIC4 mRNA表达在巨噬细胞中特别高。在没有Clic4的情况下，巨噬细胞在吞噬作用中有缺陷。此外，与野生型巨噬细胞相比，null的IL-1BETA，INOS和CXCL1的表达延长了，这表明在没有CLIC4的情况下，这些免疫调节剂的衰减被误导。 TGF-beta调节巨噬细胞活性的耗散，而在clic4 null巨噬细胞中，TGF-β信号传导较低，SMAD4水平较低。刺激的巨噬细胞中核靶向clic4或ha-clic4的过表达下调活性IL-1Beta蛋白表明CLIC4对于TGF-BETA介导的免疫反应的下调至关重要。在过去的一年中，我们进一步研究了癌症中CLIC4和PSMAD2蛋白表达的研究。在与Carter Van Waes博士和Addie Alkhas博士的单独合作中，我们表明Clic4和核Clic4在子宫颈的HNSCC和SCC中减少了。与马克·辛普森（Mark Simpson）和约书亚·韦伯斯特（Joshua Webster）的合作表明，皮肤SCC正交移植中外源性CLIC4的表达会导致肿瘤细胞中TGF-β信号传导的生长抑制和激活。此外，在起始促进研究中，小鼠皮肤中外源性CLIC4的有条件过表达可降低从头肿瘤的产量。尽管我们将注意力集中在肿瘤细胞中的CLIC4上，但我们也认识到Clic4和TGF-β在肿瘤基质中的作用。 CLIC4对于TGF-β在形成癌基质中诱导肌纤维细胞转化率至关重要。来自肿瘤细胞的条件培养基通过TGF-β信号传导诱导成纤维细胞中的CLIC4表达。与正常细胞相比，肿瘤细胞分泌几倍更多的TGF-β。来自成纤维细胞过表达CLIC4的条件培养基可通过TGF-β信号传导增强上皮细胞的迁移和侵袭。这些条件介质比对照成纤维细胞分泌更多的TGF-beta。因此，CLIC4通过TGF-β信号传导增强了肿瘤细胞与肿瘤微环境之间的串扰。通过CLIC4途径的肌纤维细胞转换为SMAD，p38依赖。 p38alpha是负责肌纤维细胞转换的主要同工型，与先前的报告相反，ROS的变化不会改变皮肤成纤维细胞的转化率。 CLIC4的不存在会增​​强磷酸p38与PPM1A（其特异性磷酸酶）的相互作用。 PPM1A与CLIC4相互作用，并且这种相互作用很可能会中断PPM1A与其他底物的关联，从而解释了Clic4如何可能增强PSMAD和p38激活。我们继续研究与Blumberg和Ambudkar实验室合作，通过角质形成细胞P-gp通过角质形成细胞P-gp进行主动透皮运输的贡献。环孢菌素A是众所周知的P-gp底物。在局部施用环孢菌素A以16 mg/kg的局部施用后，在裸鼠和头发小鼠的血浆中检测到环孢菌素A。当每天连续7天将环孢菌素A连续7天应用于16 mg/kg的裸鼠背皮时，检测到稳定的血浆环孢菌素A（110-140 nmol/L）。 XR9576是一种P-gp抑制剂，部分阻断了皮肤P-gp介导的环孢菌素A吸收。 We used an efflux assay and a P-gp mediated drug toxicity resistance assay to identify P-gp substrates among a number of phorbol ester derivates and other natural compounds that activate protein kinase C. Phorbol 12,13-dihexanoate, phorbol 12,13-dibutyrate and phorbol 12,13-dibenzoate are substrates of P-gp.但是，当局部应用于裸鼠的背皮，这三个佛波酯衍生物都无法诱导出血，因为它们作为蛋白激酶C活化剂的活性弱。 我们的癌症研究也扩展到与Blumberg实验室合作了解RasGRP3在黑色素瘤和前列腺癌中的活性。 RAS活化剂Rasgrp3在患者的这些肿瘤中升高。在实验上，肿瘤细胞中RasGRP3的过表达增强了前列腺或黑色素瘤异种移植，而在这些相​​同细胞中rasgrp3消融会抑制肿瘤的形成。