Interacting Signaling Pathways that Initiate Squamous Cell Carcinogenesis

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

In the skin model activating mutations in H-ras, K-ras, and N-ras are sufficient to produce the clonal benign squamous papilloma expressing the phenotype of the initiated cell. Utilizing H-ras-transduced isolated primary keratinocytes, we have demonstrated that oncogenic Ras upregulates EGFR ligands, establishing a positive feedback loop through EGFR, thus increasing proliferation and simultaneously activating src kinases to tyrosine phosphorylate and inactivate PKC delta, thereby inhibiting PKC-dependent keratinocyte terminal differentiation. We recently uncovered the necessary role for a member of the AP-1 family regarding the transcriptional regulation of one of the EGFR ligands. Contemporaneous H-ras-mediated activation of phospholipase C and PKCalpha establishes a second feedback loop through IL-1alpha and its receptor, activating MyD88 and NFkappaB to establish a unique gene expression profile characteristic of both initiated cells and papillomas. Interruption of any of these interacting pathways by drugs, knockdown, or genetic deletion reduces or prevents tumor formation. An important downstream effector of these interacting pathways is the upregulation and release of chemokines such as CXCL1 and CXCL2 that act in a paracrine mode to attract inflammatory cells and stimulate angiogenesis and an autocrine mode to promote migration of initiated cells. Interruption of any of these pathways inhibits tumor formation. We have also discovered a fundamental function for Adam17, a disintegrin required for the release of EGFR ligands from their membrane attachment. Knockdown of Adam17 prevents the activation of EGFR in H-ras-transformed keratinocytes and reverses the oncogenic ras signature of ras-addicted tumor cell lines. H-ras mediates Adam17 activity via a yet-to-be-determined action on iRhom1, iRhom2, and p38 that are required for Adam17 membrane localization and maturation. Tissue array analysis of human skin tumors indicates that MET is abundantly expressed in progressing squamous carcinomas along with its ligand HGF. We induced tumors with DMBA and TPA on transgenic mice that overexpress HGF and noted a marked increase in papillomas and carcinomas relative to control groups. Further analysis revealed that activation of MET in epidermis is sufficient to initiate tumor formation after TPA promotion in the absence of DMBA. In isolated keratinocytes, activation of MET mimics the H-ras-initiated phenotype, reproducing the EGFR and other required feedback loops. However, the application of DMBA to mice with activated MET produces more carcinomas with K-ras mutations than application of DMBA to control mice where H-ras mutations dominate, suggesting that tissue context influences the selection of ras mutations in tumors. RNA profiling of initiated mouse keratinocytes produced by MET or H-ras activation has revealed signature gene expression changes common to both and not previously associated with tumor initiation. Current studies are extending the analysis to K-ras-initiated keratinocytes and using knockdown or overexpression approaches to test for critical elements in the common expression profiles essential for initiation and tumor formation. The pathways that have been defined for transforming mouse keratinocytes are also being tested in human keratinocytes transformed by the oncogenic H-, K-, and N- ras alleles and mouse keratinocytes with knock-in oncogenic ras alleles. These latter studies suggest that oncogenic allele dosage is an important determinant for tumor formation and that K-ras transcriptional profiles more closely align with keratinocytes harboring two oncogenic alleles while H-ras transcriptional profiles align more closely to normal keratinocytes. Since activated H-ras cane drive tumor formation in vivo, the result suggests that it is the response to tumor promotion that leads to selection of initiated cells bearing H-ras mutations. Keratinocytes and other epithelial cells may respond to oncogenic stimuli such as mutant ras alleles by activating a senescence program. Current studies indicate that this senescence program in H-ras expressing keratinocytes is mediated by the Rho pathway and depends on the activation of p19/Arf and p16 and the release of cytokines that have an inductive effect on senescence pathways. Antagonism of these pathways with a Rho kinase inhibitor provides an opportunity to evaluate the cellular biochemistry associated with premalignant progression and malignant conversion. While oncogenic ras can initiate the requirements for tumor formation, changes in the tumor microenvironment are required to enhance tumor growth. One such factor is IL-17, and current studies indicate that IL-17 is required for a tumor promoting microenvironment. IL-17 is provided to that environment through the migration of CD4+IL-17-producing cells from the bone marrow stimulated by IL-1 released from ras-transformed keratinocytes. Tumors do not form on initiated skin if CD4+IL-17 cells are prohibited from infiltrating the skin. Interestingly, IL-1a produced by keratinocytes and IL-17 released by T-cell have a common effector in RAS-keratinocytes, namely IkappaBzeta. Loss of IkappaBzeta alters the oncogenic RAS signature and reduces tumor formation in vivo. The IkappaBzeta-driven gene signature can predict overall survival in patients whose cancer is primarily driven by mutant RAS (COAD, PAAD, and LUAD). The canonical NF-kappaB pathway is important for regulating skin inflammation, and IkappaB kinases are upstream components of the NF-kappaB pathway. Recently, we tested two inhibitors of IKK for their activities as anti-skin inflammation agents. We found that ACHP and IKK16 are effective topical inhibitors for skin inflammation for prevention as well treatment of mouse skin inflammation from multiple pro-inflammatory stimuli including ultraviolet light, and activators of toll like receptors and can inhibit DMBA induced mouse skin carcinogenesis. More recent studies have also defined a role for IL-36 in the initiation phase of skin carcinogenesis. RAS transformation upregulates IL-36 release, producing both intrinsic effects on initiated keratinocytes and extrinsic effects on the tumor microenvironment. Studies are in progress testing the requirement for the IL-36 receptor for skin tumor formation.