The tumor promoting roles of lymphocytes during RAS initiated inflammation and skin carcinogenesis
Open Access
- Author:
- Gunderson, Andrew Joseph
- Graduate Program:
- Immunology and Infectious Diseases
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 16, 2012
- Committee Members:
- Adam Bleier Glick, Dissertation Advisor/Co-Advisor
Wendy Hanna Rose, Committee Member
Robert Paulson, Committee Member
Todd Schell, Committee Member
Avery August, Special Member - Keywords:
- tumor immunology
RAS
inflammation
skin cancer
lymphocytes - Abstract:
- The pathological progression of neoplastic tissues requires the concerted activities of normal stromal cells to orchestrate a series of growth promoting effects similar to that found in healing wounds but without resolution. Bone marrow derived leukocytes are instrumental in initiating and mediating many of these processes through various mechanisms employed during periods of inflammation. Recently, studies using chemical and genetic tumor models have demonstrated novel tumor promoting roles for immune cells of the lymphoid lineage, a seemingly counterintuitive discovery. To model and study the contributions of lymphocytes to oncogene induced pre-malignant inflammation and tumor development, we targeted a Harvey-RASG12V transgene to basal (K14) and suprabasal (Inv) layers of squamous epithelia under inducible control of a tetracycline transactivator. Reduced doses of doxycycline (dox) induced expression of RAS transgene in InvtTA/tetORas (InvRas) mice leading to outgrowth of benign papillomatous lesions. RAS expression on a lymphocyte deficient Rag1-/- genetic background greatly blunted tumor onset and total burdens indicating lymphocyte involvement in the promotion of RAS induced skin carcinogenesis. Antibody depletion of CD8+ T cells in Rag1+/+ mice partially but significantly reduced the early onset of papillomas while Rag1-/- reconstitution of purified CD8+ T cells briefly stimulated lesional growth similar to WT groups that could not be sustained over the course of the study. Examination of the acute inflammatory response to RAS expression revealed diverse myelocytic infiltration into cutaneous tissues predominated by intraepidermal infiltration of cytotoxic Ly6G+ neutrophils. Conversely, when acute RAS expression was observed on Rag1 -/- mice, epidermal proliferation and skin inflammation was greatly attenuated. Neutrophilia, microabcesses, and keratinocyte proliferation levels similar to Rag1+/+ mice could be rescued by reconstitution of transgenic Rag1-/- mice with total CD3+ T or CD8+ T cells. Depleting CD8+ but not CD4+ T cells diminished or ablated all of these pathologies as well as reduced Th17 and γδ-17 cell activation, demonstrating the requirement for CD8+ T cells in driving global RAS inflammatory responses. Neutralization of IFN-γ blocked neutrophilic inflammation and keratinocyte proliferation caused by CD8+ repletion of Rag1-/- mice thereby implicating this cytokine as a mechanism of action. RAS expression driven by an epithelial basal layer promoter (K14Ras) also activated severe systemic and skin inflammation but was characterized more predominantly by the expansion of Ly6Chi inflammatory monocytes capable of suppressing T cell proliferation ex vivo. This immunosuppressive behavior could be ameliorated by titration of RAS transgene quantities that correlated linearly with reduced GM-CSF expression in keratinocytes. Strikingly, the acquisition of immunosuppressive functionality in these myeloid subsets could also be abrogated by crossing RAS transgenes onto the Rag1-/- background. Depletion of CD4+ and/or CD8+ T cells was ineffective at reproducing this phenomenon. However, B cell reconstitution into K14Ras/Rag1-mice completely recovered Ly6Chi mediated blockade of T cell proliferation suggesting B cells were the lymphocyte mediating these activities. B cell depletion, in contrast, did not reverse these effects because of an anti-CD20 resistant Breg population that likely stimulates MDSC phenotypes through the paracrine activities of IL-10. Finally, we show that the unique inflammatory responses observed in basal and suprabasal RAS expressing mice may be caused by inherently different signaling properties in differentiated and proliferating keratinocytes. RAS activation of ERK1/2 was greatly inhibited in differentiated keratinocytes in vitro and in vivo, whereby MKK4 activation was increased. As AP-1 blockade during RAS activation revealed a repressive role for AP-1 in cytokine transcription, we hypothesize that increased AP-1 activation due to enhanced JNK signaling in post-mitotic keratinocytes may dampen RAS mediated immunosuppressive pathways in mutated non-dividing epidermal layers. In turn, this disparity in the ability to provoke regulatory myeloid cells that inhibit anti-tumor immunity may provide a reasonable explanation for the increased malignant potential in basal/stem cell driven epithelial cancers.