Pathways that Suppress or Accelerate Premalignant Progression of Squamous Cancer

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Premalignant progression and malignant conversion are multistage processes and in the skin carcinogenesis model this progression occurs with predictable sequential expression of markers and genetic/epigenetic changes that define stages of progression. For example, a subset of skin papillomas is marked for progression as they erupt (high risk tumors), and they have a specific phenotypic and genetic signature. Elevated expression of s100A7a prevents progression from low risk to high risk tumors and this is associated with enhanced TGFbeta expression in tumor cells. Together these factors may stimulate a senescence pathway. In vitro, s100a7a overexpressing cells transformed with H-ras fail to upregulate cell cycle regulators such as cyclin D1 and consequently arrest at G0/G1 rather than progress to S phase and proliferate. The molecular underpinnings of this phenotype are currently under investigation and will provide clarity on pathways implicated in malignant progression. CLIC4, a p53 and TNFalpha regulated gene, is a metamorphic, multifunctional, redox regulated protein that is lost from cancer cells and highly expressed in cancer stroma during tumor progression. CLIC4 translocates to the nucleus where it is an integral component of the TGFbeta signaling pathway. In previous studies we have shown that overexpressing CLIC4 in tumor cells reduces tumor growth and increases tumor response to growth suppression by TGFbeta. Additional studies indicate that a contrasting role for CLIC4 exists in tumor stroma where it is required for TGFbeta induced myofibroblast conversion. Overexpression of CLIC4 in stromal cells enhances tumor growth in vivo and invasion in vitro. The interaction of CLIC4 with the phosphatase PPM1a is essential to its TGFbeta regulation by preventing dephosphorylation of p-Smads and p-p38 after activation of the TGFbeta receptor. CLIC4-null mice have a wound healing deficiency and fail to support lung metastasis in a mouse breast cancer model. Transcriptomic and proteomic analyses indicate that host CLIC4 is required to form a premetastatic niche potentially through its contributions to angiogenesis, cell attachment, and the formation of myofibroblasts. CLIC4 also contributes to deactivation of macrophages, and bone marrow derived macrophages from CLIC4 null mice produce supra-normal levels of TNFalpha, IL-6, IL1-beta, and iNOS in response to LPS. LPS treatment increases the phosphorylation of p38 in these cells, and inhibition of p38 decreases the levels of IL-6 in CLIC4-null macrophages. We are evaluating CLIC4 as a biomarker for cancer development and recurrence. Ovarian cancer cells, ovarian cancer stroma, breast cancer cells, and breast cancer stroma release CLIC4 into exosomes. Human data sets indicate that the elevated expression of CLIC4 in breast cancers is associated with early invasion and reduced patient survival and positively assoicated with increased expression of TGFbeta. Using breast cancer cells deleted of CLIC4 by CRISPR, we found that CLIC4 loss from tumor cells does not modify primary tumor growth or metastatic competence and CLIC4 in circulating exosomes is derived from the tumor cells. The mechanism for the loss of CLIC4 expression in the tumor epithelial cells of squamous cancers in multiple tissue sites remains unexplained. Current studies indicate that methylation of the CLIC4 gene is not altered and suggest the involvement of microRNA in regulating CLIC4 transcription/translation. We have identified microRNA 142-3p as capable of directly targeting the CLIC4 3'UTR, leading to protein downregulation. This microRNA is also upregulated in the circulation of patients with head and neck squamous cell carcinoma, where CLIC4 is often downregulated. CLIC4 also has redox enzymatic activity. The subcellular localization and protein expression of CLIC4 can be modified in response to oxidative stress in murine cancer cells. With the development of targeted based cancer therapy, the skin has evolved as a primary target for adverse events leading to treatment failure. Our long experience with combining skin and cancer biology place us in a unique position to evaluate these unexpected relationships. A subset of patients with NSCLC, HNSCC, mCRC, and pancreatic cancer are responding to therapy by several agents directed against the epidermal growth factor receptor (EGFR). Uniformly, patients develop a papulopustular folliculitis often accompanied by alopecia, xeroderma, and changes in nails and eyelashes. The discomfort and pruritis can be so severe that treatment may be terminated. To model this skin rash in the mouse, EGFR was ablated in the epidermis using cre-lox technology. The skin of double-transgenic mice (EGFR-null) reproduced the hallmarks of the skin lesions of patients undergoing chemotherapy with anti-EGFR agents: inflammation, pruritis, dry skin with neutrophilic pustules, and infiltration of mast cells, macrophages. and lymphocytes. We also documented changes in plasma cytokine/chemokine levels emanating from the skin lacking the EGFR. The mouse studies suggest that macrophages or mast cells may be fundamentally causative in the rash phenotype, and human adverse skin response to anti-EGFR drugs may be predicted based on circulating chemokine/cytokine levels before treatment begins. Macrophages play a pathogenic role in the development of EGFR-depleted skin lesions as treatment of null mice with clodronate to eliminate macrophages reduces the severity of the phenotype. The intense cutaneous inflammation elicited by systemic inhibition of EGFR activity suggests that the immune microenvironment in the tumor mass may also be modulated by these drugs. To test this, wildtype or EGFR-null mouse keratinocytes were transformed by oncogenic H-ras and grafted to syngeneic immune-competent hosts where they formed squamous cell carcinomas. After cancers formed, mice bearing wildtype cancers were treated for one week with gefitinib to inhibit EGFR systemically. Both tumor cell autonomous and systemic loss of EGFR activity indicated significant changes in T-reg infiltration into the tumor microenvironment and other immune mediated alterations, suggesting that local immune modulation by anti-EGFR drugs might contribute to their anti-tumor activity.