Abstract
Shank-associated RH domain-interacting protein (SHARPIN) is a cytosolic protein that plays a key role in activation of nuclear factor κ-light-chain enhancer of activated B cells and regulation of inflammation. Furthermore, SHARPIN controls integrin-dependent cell adhesion and migration in several normal and malignant cell types, and loss of SHARPIN correlates with increased integrin activity in mice. Arginyl-glycyl-aspartic acid (RGD), a cell adhesion tripeptide motif, is an integrin recognition sequence that facilitates PET imaging of integrin upregulation during tumor angiogenesis. We hypothesized that increased integrin activity due to loss of SHARPIN protein would affect the uptake of α(v)β(3)-selective cyclic, dimeric peptide (68)Ga-DOTA-E[c(RGDfK)](2), where E[c(RGDfk)](2) = glutamic acid-[cyclo(arginyl-glycyl-aspartic acid-D-phenylalanine-lysine)], both in several tissue types and in the tumor microenvironment. To test this hypothesis, we used RGD-based in vivo PET imaging to evaluate wild-type (wt) and SHARPIN-deficient mice (Sharpin(cpdm) , where cpdm = chronic proliferative dermatitis in mice) with and without melanoma tumor allografts. Methods:Sharpin(cpdm) mice with spontaneous null mutation in the Sharpin gene and their wt littermates with or without B16-F10-luc melanoma tumors were studied by in vivo imaging and ex vivo measurements with cyclic-RGD peptide (68)Ga-DOTA-E[c(RGDfK)](2) After the last (68)Ga-DOTA-E[c(RGDfK)](2) peptide PET/CT, tumors were cut into cryosections for autoradiography, histology, and immunohistochemistry. Results: The ex vivo uptake of (68)Ga-DOTA-E[c(RGDfK)](2) in the mouse skin and tumor was significantly higher in Sharpin(cpdm) mice than in wt mice. B16-F10-luc tumors were detected 4 d after inoculation, without differences in volume or blood flow between the mouse strains. PET imaging with (68)Ga-DOTA-E[c(RGDfK)](2) peptide at day 10 after inoculation revealed significantly higher uptake in the tumors transplanted into Sharpin(cpdm) mice than in wt mice. Furthermore, tumor vascularization was increased in the Sharpin(cpdm) mice. Conclusion:Sharpin(cpdm) mice demonstrated increased integrin activity and vascularization in B16-F10-luc melanoma tumors, as demonstrated by RGD-based in vivo PET imaging. These data indicate that SHARPIN, a protein previously associated with increased cancer growth and metastasis, may also have important regulatory roles in controlling the tumor microenvironment.