Keratinocyte senescence acts as a barrier to tumor progression but appears to be lost in late pre-malignancy to yield genetically unstable oral squamous cell carcinomas (GU-OSCC); a subset of OSCC possessing wild-type p53 and are genetically stable (GS-OSCC). In this study, fibroblasts from GU-OSCC were senescent relative to fibroblasts from GS-OSCC, epithelial dysplastic tissues or normal oral mucosa, as demonstrated by increased senescence-associated β-galactosidase (SA β-Gal) activity and overexpression of p16(INK4A). Keratinocytes from GU-OSCC produced high levels of reactive oxygen species (ROS) and this was associated with an increase in the production of transforming growth factor-β1 (TGF-β1) and TGF-β2 in stromal fibroblasts. Treatment of normal fibroblasts with keratinocyte conditioned media (CM) from GU-OSCC, but not GS-OSCC or dysplastic keratinocytes with dysfunctional p53, induced fibroblast senescence. This phenomenon was inhibited by antioxidants and anti-TGF-β antibodies. Fibroblast activation by TGF-β1 preceded cellular senescence and was associated with increased ROS levels; antioxidants inhibited this reaction. Senescent fibroblasts derived from GU-OSCC or normal fibroblasts treated with CM from GU-OSCC or hydrogen peroxide, but not non-senescent fibroblasts derived from GS-OSCC, promoted invasion of keratinocytes in vitro. Epithelial invasion was stimulated by fibroblast activation and amplified further by fibroblast senescence. The data demonstrate that malignant keratinocytes from GU-OSCC, but not their pre-malignant counterparts, produce high levels of ROS, which, in turn, increase TGF-β1 expression and induce fibroblast activation and senescence in a p5-independent manner. Fibroblasts from GU-OSCC were particularly susceptible to oxidative DNA damage because of high levels of ROS production, downregulation of antioxidant genes and upregulation of pro-oxidant genes. The results demonstrate the functional diversity of cancer-associated fibroblasts and show that malignant keratinocytes from GU-OSCC reinforce their malignant behavior by inducing fibroblast activation and senescence through ROS and TGF-β-dependent mechanisms.

Cyclooxygenase-2 and prostaglandin E 2 (PGE 2 ) levels are increased in colorectal cancers and a subset of adenomas. PGE 2 signaling through the EP4 receptor has previously been associated with colorectal tumorigenesis. However, changes in EP4 expression during adenoma to carcinoma progression have not been investigated, neither has whether levels of EP4 influence important markers of malignant potential, such as anchorage-independent growth or the tumors growth response to PGE 2 . We report using immunohistochemistry that in vivo EP4 receptor protein expression was increased in colorectal cancers (100%) as well as adenomas (36%) when compared with normal colonic epithelium. EP4 expression was also higher in colorectal carcinoma compared with adenoma cell lines and increased with in vitro models of tumor progression. Adenoma (PC/AA/C1 and RG/C2) and carcinoma cell lines (HT29) were growth stimulated by PGE 2 up to 0.5 Mmol/L. However, although carcinoma and transformed adenoma (PC/AA/C1SB10C, a transformed derivative of PC/AA/C1) cells remain stimulated by higher doses of PGE 2 (10 Mmol/L), the adenoma cell lines were inhibited. Interestingly, enforced expression of EP4 in the adenoma cell line, RG/ C2, resulted in stimulation of growth by 10 Mmol/L PGE 2 and promoted anchorage-independent growth. Both in vivo and in vitro data from this study suggest that increased EP4 receptor expression is important during colorectal carcinogenesis. We propose that high levels of PGE 2 in a tumor microenvironment would select for cells with increased EP4 expression, and that the EP4 receptor may therefore represent an important target for colorectal cancer prevention and treatment. (Cancer Res 2006; 66(6): 3106-13)

Abstract-Reduced migratory function of circulating angiogenic progenitor cells (CPCs) has been associated with impaired neovascularization in patients with cardiovascular disease (CVD). Previous findings underline the role of the kallikrein-kinin system in angiogenesis. We now demonstrate the involvement of the kinin B2 receptor (B 2 R) in the recruitment of CPCs to sites of ischemia and in their proangiogenic action. In healthy subjects, B 2 R was abundantly present on CD133 ϩ and CD34 ϩ CPCs as well as cultured endothelial progenitor cells (EPCs) derived from blood mononuclear cells (MNCs), whereas kinin B1 receptor expression was barely detectable. In transwell migration assays, bradykinin (BK) exerts a potent chemoattractant activity on CD133 ϩ and CD34 ϩ CPCs and EPCs via a B 2 R/phosphoinositide 3-kinase/eNOS-mediated mechanism. Migration toward BK was able to attract an MNC subpopulation enriched in CPCs with in vitro proangiogenic activity, as assessed by Matrigel assay. CPCs from cardiovascular disease patients showed low B 2 R levels and decreased migratory capacity toward BK. When injected systemically into wild-type mice with unilateral limb ischemia, bone marrow MNCs from syngenic B 2 R-deficient mice resulted in reduced homing of sca-1 ϩ and cKit ϩ flk1 ϩ progenitors to ischemic muscles, impaired reparative neovascularization, and delayed perfusion recovery as compared with wild-type MNCs. Similarly, blockade of the B 2 R by systemic administration of icatibant prevented the beneficial effect of bone marrow MNC transplantation. BK-induced migration represents a novel mechanism mediating homing of circulating angiogenic progenitors. Reduction of BK sensitivity in progenitor cells from cardiovascular disease patients might contribute to impaired neovascularization after ischemic complications.

Objective-We evaluated whether phosphatidylinositol 3-kinase ␥ (PI3K␥) plays a role in reparative neovascularization and endothelial progenitor cell (EPC) function. Methods and Results-Unilateral limb ischemia was induced in mice lacking the PI3K␥ gene (PI3K␥ Ϫ/Ϫ ) or expressing a catalytically inactive mutant (PI3K␥ KD/KD ) and wild-type controls (WT). Capillarization and arteriogenesis were reduced in PI3K␥ Ϫ/Ϫ ischemic muscles resulting in delayed reperfusion compared with WT, whereas reparative neovascularization was preserved in PI3K␥ KD/KD . In PI3K␥ Ϫ/Ϫ muscles, endothelial cell proliferation was reduced, apoptosis was increased, and interstitial space was infiltrated with leukocytes but lacked cKit ϩ progenitor cells that in WT muscles typically surrounded arterioles. PI3K␥ is constitutively expressed by WT EPCs, with expression levels being upregulated by hypoxia. PI3K␥ Ϫ/Ϫ EPCs showed a defect in proliferation, survival, integration into endothelial networks, and migration toward SDF-1. The dysfunctional phenotype was associated with nuclear constraining of FOXO1, reduced Akt and eNOS phosphorylation, and decreased nitric oxide (NO) production. Pretreatment with an NO donor corrected the migratory defect of PI3K␥

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.