Hypoxia-inducible protein 2 is a novel lipid droplet protein and a specific target gene of hypoxia-inducible factor-1.

Hypoxia-inducible protein 2 (HIG2) has been implicated in canonical Wnt signaling, both as target and activator. The potential link between hypoxia and an oncogenic signaling pathway might play a pivotal role in renal clear-cell carcinoma characterized by constitutive activation of hypoxia-inducible factors (HIFs), and hence prompted us to analyze HIG2 regulation and function in detail. HIG2 was up-regulated by hypoxia and HIF inducers in all cell types and mouse organs investigated and abundantly expressed in renal clear-cell carcinomas. Promoter analyses, gel shifts, and siRNA studies revealed that HIG2 is a direct and specific target of HIF-1, but not responsive to HIF-2. Surprisingly, HIG2 was not secreted, and HIG2 overexpression neither stimulated proliferation nor activated Wnt signaling. Instead, we show that HIG2 decorates the hemimembrane of lipid droplets, whose number and size increase on hypoxic inhibition of fatty acid ß-oxidation, and colocalizes with the lipid droplet proteins adipophilin and TIP47. Normoxic overexpression of HIG2 was sufficient to increase neutral lipid deposition in HeLa cells and stimulated cytokine expression. HIG2 could be detected in atherosclerotic arteries and fatty liver disease, suggesting that this ubiquitously inducible HIF-1 target gene may play an important functional role in diseases associated with pathological lipid accumulation.

TIP47 functions in the biogenesis of lipid droplets.

TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.

NF-kappaB-independent down-regulation of XIAP by bortezomib sensitizes HL B cells against cytotoxic drugs.

The proteasome inhibitor bortezomib has been shown to possess promising antitumor activity and significant efficacy against a variety of malignancies. Different studies demonstrated that bortezomib breaks the chemoresistance in different tumor cells basically by altering nuclear factor-kappaB (NF-kappaB) activity. NF-kappaB has been shown to be constitutively active in most primary Hodgkin-Reed-Sternberg (H-RS) cells in lymph node sections and in Hodgkin lymphoma (HL) cell lines and was suggested to be a central molecular switch in apoptosis resistance in HL. Here we report a bimodal effect of bortezomib in HL cells. Whereas high-dose bortezomib induced direct cytotoxicity that correlated with decreased NF-kappaB activity, low-dose bortezomib sensitized HL cells against a variety of cytotoxic drugs without altering NF-kappaB action. Strikingly, bortezomib induced marked XIAP down-regulation at the posttranslational level that was independent of the NF-kappaB status. Similarly, RNA interference (RNAi)-mediated XIAP down-regulation generated susceptibility to cytostatic agents. The results identify XIAP as an NF-kappaB-independent target of bortezomib action that controls the chemoresistant phenotype of HL cells.

XIAP targeting sensitizes Hodgkin lymphoma cells for cytolytic T-cell attack.

The immunosurveillance of Hodgkin lymphoma (HL) by cytotoxic T lymphocytes (CTLs) is insufficient, and the clinical experience with adoptive transfer of CTLs is limited. We have previously reported that defects in mitochondrial apoptotic pathways and elevated XIAP expression confer resistance to different apoptotic stimuli in HL cells. Here, we aimed to develop molecular strategies to overcome the resistance of HL cells against CTL-mediated killing via granzyme B (grzB). In HL cells, grzB-induced mitochondrial release of proapoptotic Smac is blocked, which results in complete abrogation of cytotoxicity mediated by CTLs. Cytosolic expression of recombinant mature Smac enhanced caspase activity induced by grzB and restored the apoptotic response of HL cells. Similarly, down-regulation of XIAP by RNA interference markedly enhanced the susceptibility of HL cells for CTL-mediated cytotoxicity. XIAP gene knockdown sensitized HL cells for killing by antigen-specific CTLs redirected by grafting with a chimeric anti-CD30scFv-CD3zeta immunoreceptor. The results suggest that XIAP targeting by Smac agonists or XIAP-siRNA can be used as a synergistic strategy for cellular immunotherapy of Hodgkin lymphoma.