ABSTRACT
Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy typified by a highly stromal and weakly immunogenic tumor microenvironment that promotes tumor evolution and contributes to therapeutic resistance. Here, we demonstrate that PDA tumor cell-derived proinflammatory cytokine IL1ß is essential for the establishment of the protumorigenic PDA microenvironment. Tumor cell-derived IL1ß promoted the activation and secretory phenotype of quiescent pancreatic stellate cells and established an immunosuppressive milieu mediated by M2 macrophages, myeloid-derived suppressor cells, CD1dhiCD5+ regulatory B cells, and Th17 cells. Loss of tumor cell-derived IL1 signaling in tumor stroma enabled intratumoral infiltration and activation of CD8+ cytotoxic T cells, attenuated growth of pancreatic neoplasia, and conferred survival advantage to PDA-bearing mice. Accordingly, antibody-mediated neutralization of IL1ß significantly enhanced the antitumor activity of α-PD-1 and was accompanied by increased tumor infiltration of CD8+ T cells. Tumor cell expression of IL1ß in vivo was driven by microbial-dependent activation of toll-like receptor 4 (TLR4) signaling and subsequent engagement of the NLRP3 inflammasome. Collectively, these findings identify a hitherto unappreciated role for tumor cell-derived IL1ß in orchestrating an immune-modulatory program that supports pancreatic tumorigenesis. SIGNIFICANCE: These findings identify a new modality for immune evasion in PDA that depends on IL1ß production by tumor cells through TLR4-NLRP3 inflammasome activation. Targeting this axis might provide an effective PDA therapeutic strategy.
Subject(s)
Carcinogenesis/immunology , Carcinoma, Pancreatic Ductal/immunology , Interleukin-1beta/metabolism , Pancreatic Neoplasms/immunology , Tumor Escape/immunology , Animals , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor/transplantation , Disease Models, Animal , Drug Synergism , Epithelial Cells , Female , Humans , Inflammasomes/immunology , Inflammasomes/metabolism , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/immunology , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Mice , Mice, Transgenic , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pancreatic Ducts/cytology , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Primary Cell Culture , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Signal Transduction/immunology , Toll-Like Receptor 4/metabolism , Tumor Escape/drug effects , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
We recently showed that E6 protein of human papillomavirus (HPV) 16, a mucosal high-risk α-PV type, can potentiate Wnt/ß-catenin/TCF signaling. Here we investigated the transcriptional activities of E6 proteins of cutaneous HPV types from the ß and α genera. Results from reporter-gene assays showed that similar to HPV16 E6, E6 of HPV10, a cutaneous α-HPV type that is prevalent in skin warts, efficiently enhances and stimulates Wnt/ß-catenin/TCF transcription. HPV10 E6 also effectively elevated the expression levels of ß-catenin and promoted its nuclear accumulation. E6 proteins of ß-HPV types 8, 24, 38 and 49, which are prevalent in skin cancer, exhibited lower activities in all tested functions. The differences in activity correlated with E6's competence to interact with the ubiquitin ligase E6AP. This study reveals a role for E6 proteins of diverse cutaneous HPV types in potentiation of Wnt/ß-catenin signaling, irrespective of their carcinogenic potential.
Subject(s)
Host-Pathogen Interactions , Oncogene Proteins, Viral/metabolism , Papillomaviridae/physiology , Skin Neoplasms/virology , Warts/virology , Wnt Signaling Pathway , Humans , Papillomaviridae/isolation & purification , Transcription, Genetic , Wnt Proteins/biosynthesis , beta Catenin/biosynthesisABSTRACT
Hyperactive Wnt signaling is a common feature in human colorectal cancer (CRC) cells. A central question is the identification and role of Wnt/ß-catenin target genes in CRC and their relationship to genes enriched in colonic stem cells, since Lgr5+ intestinal stem cells were suggested to be the cell of CRC origin. Previously, we identified the neural immunoglobulin-like adhesion receptor L1 as a Wnt/ß-catenin target gene localized in cells at the invasive front of CRC tissue and showed that L1 expression in CRC cells confers enhanced motility and liver metastasis. Here, we identified the clusterin (CLU) gene that is also enriched in Lgr5+ intestinal stem cells, as a gene induced during L1-mediated CRC metastasis. The increase in CLU levels by L1 in CRC cells resulted from transactivation of CLU by STAT-1. CLU overexpression in CRC cells enhanced their motility and the reduction in CLU levels in L1 overexpressing cells suppressed the ability of L1 to confer increased tumorigenesis and liver metastasis. Genes induced during L1-mediated CRC cell metastasis and enriched in intestinal stem cells might be important for both CRC progression and colonic epithelium homeostasis.
Subject(s)
Clusterin/genetics , Colonic Neoplasms/pathology , Neural Cell Adhesion Molecule L1/metabolism , Stem Cells/metabolism , Animals , Chromatin Immunoprecipitation , Fluorescent Antibody Technique , HCT116 Cells , Heterografts , Humans , Immunoblotting , Mice , Mice, Nude , Neoplasm Invasiveness , Real-Time Polymerase Chain Reaction , TranscriptomeABSTRACT
The present study investigated the roles of E6 and E6AP in the Wnt pathway. We showed that E6 levels are markedly reduced in cells in which Wnt signaling is activated. Coexpression of wild-type or mutant E6AP (C820A) in Wnt-activated cells stabilized E6 and enhanced Wnt/ß-catenin/TCF transcription. Expression of E6AP alone in nonstimulated cells elevated ß-catenin level, promoted its nuclear accumulation, and activated ß-catenin/TCF transcription. A knockdown of E6AP lowered ß-catenin levels. Coexpression with E6 intensified the activities of E6AP. Further experiments proved that E6AP/E6 stabilize ß-catenin by protecting it from proteasomal degradation. This function was dependent on the catalytic activity of E6AP, the kinase activity of GSK3ß and the susceptibility of ß-catenin to GSK3ß phosphorylation. Thus, this study identified E6AP as a novel regulator of the Wnt signaling pathway, capable of cooperating with E6 in stimulating or augmenting Wnt/ß-catenin signaling, thereby possibly contributing to HPV carcinogenesis.
Subject(s)
Oncogene Proteins, Viral/metabolism , Repressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Wnt Proteins/metabolism , Wnt Signaling Pathway/physiology , Cells, Cultured , Cloning, Molecular , Gene Expression Regulation/physiology , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Keratinocytes , Plasmids , Proteasome Endopeptidase Complex , Protein Stability , TCF Transcription Factors/genetics , TCF Transcription Factors/metabolism , Ubiquitin-Protein Ligases/genetics , Wnt Proteins/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
In the present study, E6E7 and E6 proteins of human papillomaviruses (HPVs) associated with skin warts and cancer were compared for their transforming and carcinogenic abilities in primary human keratinocytes (PHKs). We show that E6E7 of cancer associated beta HPV types, notably 49 and 24, were able to extend the life span and enhance the clonogenic efficiency of PHKs when maintained in serum free/low calcium medium. Activities of the beta HPV E6E7 were lower than those of HPV16 E6E7. In contrast, E6 proteins from HPV types detected in skin warts or cancer, notably 10, 49 and 38, attenuated UVB induced protective responses in PHKs including cell death, proliferation arrest and accumulation of the proapoptotic proteins, p53, bax or bak. Together, this investigation revealed functional differences and commonalities between HPVs associated with skin warts and cancer, and allowed the identification of specific properties of beta HPVs supporting their involvement in skin carcinogenesis.