Tumor-associated macrophages (TAMs) constitute major infiltrates of solid tumors and express a marker profile that characterizes alternatively activated macrophages (MФs). TAMs accumulate in hypoxic tumor regions, express high amounts of hypoxia-inducible factor-1 (HIF-1) and contribute to tumor angiogenesis and invasiveness. However, the precise role of HIF-1 on MФ infiltration and phenotype alterations remains poorly defined. Therefore, we cocultured wild type (wt) versus HIF-1α(-/-) MФs with tumor spheroids. Both, wt and HIF-1α(-/-) MФs, infiltrated hypoxic regions of tumor spheroids at equal rates and got alternatively activated. Interestingly, significantly higher amounts of HIF-1α(-/-) MФs expressed the TAM markers CD206 and stabilin-1 compared with wt phagocytes. Stimulation of infiltrated TAMs with lipopolysaccharide (LPS)/interferon-γ revealed a reduced expression of the pro-inflammatory markers interleukin (IL)-6, tumor necrosis factor-α and inducible nitric oxide synthase in HIF-1α(-/-) MФs. Furthermore, HIF-1α(-/-) MФs were less cytotoxic toward tumor cells. Although infiltration of MФs increased the invasive potential of tumor spheroids independently of HIF-1, the ability to stimulate differentiation of stem cells toward CD31-positive cells was triggered by wt but not by HIF-1α(-/-) MФs. Our data suggest that HIF-1α-deficient MФs develop a more prominent TAM marker profile accompanied by reduced cytotoxicity, whereas HIF-1 seems indispensable for the angiogenesis-promoting properties of TAMs.

Hypoxia-inducible factors (HIFs) provoke adaptation to hypoxic stress occurring in rapidly growing tumor tissues. Therefore, overexpression of HIF-1 or HIF-2 is a common feature in hepatocellular carcinoma but their specific function is still controversially discussed. To analyze HIF function in hypoxia-induced cell death we created a stable knockdown of HIF-1a and HIF-2a in HepG2 cells and generated tumor spheroids as an in vitro hepatocellular carcinoma model. Knockdown of HIF-1a enhanced expression of HIF-2a and vice versa. Unexpectedly, knockdown of HIF-1a or HIF-2a increased cell viability as well as spheroid size and decreased caspase-3 activity. Antiapoptotic Bcl-X L expression increased in both knockdown spheroids, whereas proapoptotic Bax was only reduced in HIF-1a-knockdown cells. Furthermore, an HIF-2a-knockdown significantly increased Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) expression in an HIF-1a-dependent manner. Concomitantly, electron microscopy revealed a substantial increase in autophagosomal structures in HIF-2a-knockdown spheroids and mito-/lysotracker costaining confirmed lysosomal activity of these autophagosomes. Blocking autophagosome maturation using 3-methyladenine restored cell death in HIF-2a-knockdown clones comparable to wildtype cells. Conclusion: An HIF-1a-knockdown increases HIF-2a expression and shifts the balance of Bcl-2 family members toward survival. The knockdown of HIF-2a raises autophagic activity and attenuates apoptosis by enhancing HIF-1a expression. Our data indicate that enhanced expression of one HIF-isoform causes a survival advantage in hepatocellular carcinoma development. (HEPATOLOGY 2010;51:2183-2192

A challenging task of the immune system is to fight cancer cells. However, a variety of human cancers educate immune cells to become tumor supportive. This is exemplified for tumor-associated macrophages (TAMs), which are polarized towards an antiinflammatory and cancer promoting phenotype. Mechanistic explanations, how cancer cells influence the macrophage phenotype are urgently needed to address potential anti-cancer strategies along this line. One potential immune modulating compound, sphingosine-1-phosphate (S1P), was recently highlighted in both tumor growth and immune modulation. Using a xenograft model in nude mice, we demonstrate a supportive role of sphingosine kinase 2 (SphK2), one of the S1P-producing enzymes for tumor progression. The growth of SphK2-deficient MCF-7 breast tumor xenografts was markedly delayed when compared with controls. Infiltration of macrophages in SphK2-deficient and control tumors was comparable. However, TAMs from SphK2-deficient tumors displayed a pronounced anti-tumor phenotype, showing an increased expression of pro-inflammatory markers/mediators such as NO, TNF-a, IL-12 and MHCII and a low expression of anti-inflammatory IL-10 and CD206. These data suggest a role for S1P, generated by SphK2, in early tumor development by affecting macrophage polarization. ' 2009 UICC

IntroductionTumor-associated macrophages (TAMs) infiltrate tumors, which often predicts a poor survival prognosis. TAMs apparently support tumor development by improving vascularization and cell survival rather than behaving tumoricidally (for review, see Lewis and Pollard 1 ).Features characterizing TAMs are often elicited by alternative activation, which polarizes cells toward regulatory macrophages, a subgroup of the so-called M2 phenotype. They share anti-inflammatory characteristic, for example, reduced interleukin (IL)-12 production 2,3 compared with classical stimulation and M1 polarization. 2 Unfortunately, it is only poorly understood which factors influence the TAM phenotype profile. The phenotype alteration occurs in the tumor microenvironment, with the likely contribution of tumor-derived molecules such as IL-4, IL-10, transforming growth factor (TGF)-␤, prostaglandin E 2 (PGE 2 ), chemokines, or tumor hypoxia. 1,2 Interestingly, apoptotic cells (AC) also release anti-inflammatory cytokines such as TGF-␤ and IL-10 4,5 and reduce tumor-directed macrophage cytotoxicity. 6 It became apparent that phagocytosis of AC by macrophages is an immunoregulatory process that shapes the regulatory macrophage phenotype. [7][8][9][10] Macrophages cocultured in vitro with AC or exposed to the supernatant of AC express a pattern of markers that overlaps with those established for TAMs. [11][12][13] Furthermore, sphingosine-1-phosphate (S1P) provoked M2 macrophage polarization, 13,14 and S1P is released from AC. 13,15 S1P activates distinct G proteincoupled receptors, 16 elicits a variety of immune cell responses, 17 and promotes tumor angiogenesis. 18,19 Hypoxia-inducible factor (HIF)-1 is known for its importance in tumorigenesis, and controls angiogenesis, proliferation, metabolism, metastasis, and differentiation. 20 HIF-1, composed of a HIF-1␣ and HIF-1␤ subunit, senses and coordinates adaptation to low oxygen availability. Under normoxia (21% O 2 ), HIF-1␤ is constitutively expressed, whereas HIF-1␣ is hydroxylated by prolyl hydroxylases, polyubiquitinated by an E3-ubiquitin ligase complex containing the von Hippel Lindau protein, and concomitantly degraded (for review, see Semenza 21 ). Because hypoxia mainly affects HIF-1␣ protein stability, HIF-1␣ mRNA expression regulation only recently attracted some interest. In macrophages, lipopolysaccharide (LPS) induced nuclear factor (NF)-B binding to the HIF-1␣ promoter and increased its mRNA expression. 22 Nuclear factor of activated T cells (NFAT)c1 increased the HIF-␣ mRNA content in mast cells after stimulation with ionomycin, 23 and signal transducer and activator of transcription (STAT)3 up-regulated HIF-1␣ mRNA in human tumor cells, 24 substantiating the importance of transcriptional control mechanisms governing HIF-1 appearance/activity.We provide evidence that HIF-1␣ under normoxia is transcriptionally activated via NFAT in response to apoptotic cell-derived S1P and TGF-␤. As verified in cells from HIF-1␣ conditional knockout mice, activation of HIF-1 in pola...

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1α (HIF1α) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent manner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1α protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1α protein half-life from ~58 min to 38 min under hypoxic conditions. The reduced HIF1α protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1α. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1α. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1α protein accumulation and signaling.