The role of peroxiredoxin 1 in prostate cancer
Riddell, Jonah R.
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Prostate cancer (CaP) is one of the leading causes of cancer related deaths in men. Treatment options remain limited for patients diagnosed with high grade prostatic disease, metastatic CaP and androgen deprivation therapy (ADT) resistant CaP. The risk of CaP and CaP tumor progression is linked with the establishment of a chronic inflammatory state. One promoter of a chronic inflammatory microenvironment is toll-like receptor 4 (TLR4) activation; TLR4 is linked with CaP risk and tumor growth via an unknown endogenous agonist. TLR4 is a pattern recognition receptor that recognizes bacterial components including lipopolysaccharide (LPS), endogenous stress danger signals. Recent evidence indicates an evolutionary homolog of Prx1/2; thus, we hypothesized that Prx1 interacts with TLR4 to mediate CaP tumor growth . This thesis provides evidence that mammalian Prx1 interacts with the TLR4/MD-2/CD14 complex to induce nuclear factor κB (NF-κB) activation and cytokine secretion in vivo and in vitro independent of LPS contamination. Addition of Prx1 to dendritic cells induced expression of maturation markers. Also, Prx1 colocalizes and immuneprecipitates with TLR4. Studies support that Prx1 interacts with TLR4 but little was known pertaining to Prx1 expression in CaP except that Prx1 is elevated in metastatic CaP cells. Characterization of Prx1 expression in CaP found that Prx1 is elevated in clinical CaP tissues and the preclinical TRAMP model. Reduction of Prx1 in subcutaneous models of CaP led to reduced tumor growth that could be rescued by restoration of Prx1 levels or by injection of cells in an angiogenic rich matrigel matrix. Immunohistochemical and immunofluorescence analysis of tumors with reduced Prx1 expression found reduced mature vasculature and vessel numbers. Magnetic resonance imaging of tumors found reduced vascular functionality when Prx1 was reduced. Furthermore, Prx1 activation of TLR4 in vivo resulted in vessel recruitment, measured by hemoglobin continent in a collagen plug; and in vitro Prx1 increases endothelial cell proliferation, migration, and differentiation. Dramatic changes in vasculature led us to examine angiogenic factor expression in s.c. tumors through a vascular microarray; Prx1 expression was positively correlated with numerous angiogenic factors including vascular endothelial growth factor (VEGF), which is a mitigating factor in CaP and regulated by TLR4. VEGF is a major promoter of both vascular networking and permeability; VEGF can directly mediate proliferative, migratory, and maturation effects on endothelial cells, the major components of the vessels. Restoration of VEGF in tumors with reduced Prx1 expression partially restored tumor growth. ELISA and molecular analysis of VEGF expression and secretion confirmed that Prx1 regulates VEGF expression in vitro and in vivo in both tumor and host cells. Further examination by quantitative PCR and luciferase assays yielded that Prx1 stimulates VEGF mRNA and promoter activation in a TLR4 dependent manner. In the literature, LPS stimulates VEGF expression through NF-κB dependent control of HIF-1α transcription and therein HIF-1 activation of the VEGF promoter. We hypothesized that Prx1/TLR4 interaction controlled VEGF expression through NF-κB dependent control of HIF-1 activity . The thesis work here demonstrates that Prx1 activates NF-κB in a TLR4 dependent manner to increase HIF-1 activity and NF-κB interaction with the HIF-1α promoter, measured by chromatin immunoprecipitation. Transcriptional increases of HIF-1α due to Prx1 stimulation translate to increased HIF-1α mRNA and protein expression. Similarly, Prx1/TLR4 interaction drives HIF-1 activation of a luciferase reporter and increases the interaction of HIF-1 with the VEGF promoter. Mutation of the hypoxic regulatory element (HRE) that HIF-1 binds to in the VEGF promoter abrogated VEGF promoter activation due to Prx1 stimulation. Cumulatively, this body of work supports that Prx1 interacts with TLR4 to promote NF-κB dependent control of HIF-1α transcription and activation of VEGF to mediate CaP vasculature and CaP tumor growth. This work is the first evidence that Prx1 interacts with TLR4 and controls NF-κB and HIF-1 in normoxic conditions. Also, demonstrated here is that Prx1 is elevated in clinical and preclinical models of CaP and the elevation of Prx1 promotes CaP vasculature and tumor growth. This work gives rise to a therapeutic target that has the potential to more effectively combat CaP because of its potential inhibitory effects on the multiple facets of tumor biology: including the vascular network, immune system and oxidative microenvironment.