Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. CD133, a transmembrane glycoprotein, is an important cell surface marker for both stem cells and cancer stem cells in various tissues including liver. CD133 expression has been recently linked to poor prognosis in HCC patients. CD1331 liver cancer cells are characterized by resistance to chemotherapy, self-renewal, multilineage potential, increased colony formation, and in vivo cancer initiation at limited dilution. Recent studies demonstrate that CD133 expression is regulated by DNA methylation. In this study, we explored the role of transforming growth factor b (TGFb), a multifunctional cytokine that plays a critical role in chronic liver injury, in the regulation of CD133 expression. TGFb1 is capable of up-regulating CD133 expression specifically within the Huh7 HCC cell line in a time-and dose-dependent manner. Most important, TGFb1-induced CD1331 Huh7 cells demonstrate increased tumor initiation in vivo. Forced expression of inhibitory Smads, including Smad6 and Smad7, attenuated TGFb1-induced CD133 expression. Within CD1332 Huh7 cells, TGFb1 stimulation inhibited the expression of DNA methyltransferases (DNMT) 1 and DNMT3b, which are critical in the maintenance of regional DNA methylation, and global DNMT activity in CD1332 Huh7 cells was inhibited by TGFb1. DNMT3b inhibition by TGFb1 was partially rescued with overexpression of inhibitory Smads. Lastly, TGFb1 treatment led to significant demethylation in CD133 promoter-1 in CD1332 Huh7 cells. Conclusion: TGFb1 is able to regulate CD133 expression through inhibition of DNMT1 and DNMT3b expression and subsequent demethylation of promoter-1. TGFb1-induced CD1331 Huh7 cells are tumorigenic. The mechanism by which TGFb induces CD133 expression is partially dependent on the Smads pathway. (HEPATOLOGY 2010;51:1635-1644 We have recently demonstrated that CD133 reliably identifies liver cancer stem cells (CSCs) in two independent murine models of chronic injury. [10][11][12] In the human HCC cell line Huh7, CD133þ cells demonstrated many stem cell-like properties including colony formation, self-renewal and differentiation ability, as well as a greater ability to initiate tumors in vivo compared to CD133À cells. 3Despite the high volume of recent publications related to CD133, little is known about the regulation

Background c-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF), plays a critical role in cancer growth, invasion and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c-Met pathway has been proposed for the treatment of multiple cancers, the effect of c-Met inhibition in HCC remains unclear. Human HCC cell lines, Huh7, Hep3B, MHCC97-L and MHCC97-H, were utilized in this study to investigate the effect of c-Met inhibition using the small molecule, selective c-Met tyrosine kinase inhibitor PHA665752. Results MHCC97-L and MHCC97-H cells demonstrate a mesenchymal phenotype with decreased expression of E-cadherin and increased expression of c-Met, Fibronectin and Zeb2 compared to Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c-Met and downstream PI3K/Akt and MAPK/Erk pathways, inhibited cell proliferation and induced apoptosis in c-Met positive MHCC97-L and MHCC97-H cells. In xenograft models, administration of PHA665752 significantly inhibited c-Met positive MHCC97-L and MHCC97-H tumor growth, and PHA665752 treated tumors demonstrated marked reduction of both c-Met phosphorylation and cell proliferation. c-Met negative Huh7 and Hep3B cells were not affected by c-Met inhibitor treatment in vitro or in vivo. In addition, c-Met positive MHCC97-L and MHCC97-H cells demonstrated cancer stem cell-like characteristics, such as resistance to chemotherapy, tumor sphere-formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere-formation and inhibited CD44 expression. Conclusion c-Met represents a potential target of personalized treatment for HCC with an active HGF/c-Met pathway.

Monocyte/macrophage recruitment correlates strongly with the progression of diabetic nephropathy. Tumor necrosis factor-alpha (TNF-α) is produced by monocytes/macrophages but the direct role of TNF-α and/or macrophage-derived TNF-α in the progression of diabetic nephropathy remains unclear. Here we tested whether inhibition of TNF-α confers kidney protection in diabetic nephropathy via a macrophage-derived TNF-α dependent pathway. Compared to vehicle-treated mice, blockade of TNF-α with a murine anti-TNF-α antibody conferred kidney protection in Ins2Akita mice as indicated by reductions in albuminuria, plasma creatinine, histopathologic changes, kidney macrophage recruitment and plasma inflammatory cytokine levels at 18 weeks of age. To assess the direct role of macrophage-derived TNF-α in diabetic nephropathy, we generated macrophage specific TNF-α deficient mice (CD11bCre/TNF-αFlox/Flox). Conditional ablation of TNF-α in macrophages significantly reduced albuminuria, the increase in plasma creatinine and BUN, histopathologic changes and kidney macrophage recruitment compared to diabetic TNF-αFlox/Flox control mice after 12 weeks of streptozotocin-induced diabetes. Thus, production of TNF-α by macrophages plays a major role in diabetic renal injury. Hence, blocking TNF-α could be a novel therapeutic approach for treatment of diabetic nephropathy.

Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN), yet their direct role is not clear. We hypothesized that macrophages contribute to direct podocyte injury and/or an abnormal podocyte niche leading to DN. Experiments were conducted in CD11b-DTR mice treated with diphtheria toxin (DT) to deplete macrophages after streptozotocin-induced diabetes. Additional experiments were conducted in bone marrow chimeric (CD11b-DTR→ C57BL6/J) mice. Diabetes was associated with an increase in the M1-to-M2 ratio by 6 wk after the induction of diabetes. Macrophage depletion in diabetic CD11b-DTR mice significantly attenuated albuminuria, kidney macrophage recruitment, and glomerular histological changes and preserved kidney nephrin and podocin expression compared with diabetic CD11b-DTR mice treated with mutant DT. These data were confirmed in chimeric mice indicating a direct role of bone marrow-derived macrophages in DN. In vitro, podocytes grown in high-glucose media significantly increased macrophage migration compared with podocytes grown in normal glucose media. In addition, classically activated M1 macrophages, but not M2 macrophages, induced podocyte permeability. These findings provide evidence showing that macrophages directly contribute to kidney injury in DN, perhaps by altering podocyte integrity through the proinflammatory M1 subset of macrophages. Attenuating the deleterious effects of macrophages on podocytes could provide a new therapeutic approach to the treatment of DN.