Stabilization but No Functional Influence of HIF-1α Expression in the Intestinal Epithelium during Salmonella Typhimurium Infection.

Hypoxia-inducible transcription factor 1 (HIF-1) has been shown to enhance microbial killing and ameliorate the course of bacterial infections. While the impact of HIF-1 on inflammatory diseases of the gut has been studied intensively, its function in bacterial infections of the gastrointestinal tract remains largely elusive. With the help of a publicly available gene expression data set, we inferred significant activation of HIF-1 after oral infection of mice with Salmonella enterica serovar Typhimurium. Immunohistochemistry and Western blot analyses confirmed marked HIF-1α protein stabilization, especially in the intestinal epithelium. This prompted us to analyze conditional Hif1a-deficient mice to examine cell type-specific functions of HIF-1 in this model. Our results demonstrate enhanced noncanonical induction of HIF-1 activity upon Salmonella infection in the intestinal epithelium as well as in macrophages. Surprisingly, Hif1a deletion in intestinal epithelial cells did not impact inflammatory gene expression, bacterial spread, or disease outcomes. In contrast, Hif1a deletion in myeloid cells enhanced intestinal Cxcl2 expression and reduced the cecal Salmonella load. In vitro, HIF-1α-deficient macrophages showed overall impaired transcription of mRNA encoding proinflammatory factors; however, the intracellular survival of Salmonella was not impacted by HIF-1α deficiency.

Deletion of mTOR in liver epithelial cells enhances hepatic metastasis of colon cancer.

Activation of the mechanistic target of rapamycin (mTOR) pathway is frequently found in cancer, but mTOR inhibitors have thus far failed to demonstrate significant antiproliferative efficacy in the majority of cancer types. Besides cancer cell-intrinsic resistance mechanisms, it is conceivable that mTOR inhibitors impact on non-malignant host cells in a manner that ultimately supports resistance of cancer cells. Against this background, we sought to analyze the functional consequences of mTOR inhibition in hepatocytes for the growth of metastatic colon cancer. To this end, we established liver epithelial cell (LEC)-specific knockout (KO) of mTOR (mTORLEC ) mice. We used these mice to characterize the growth of colorectal liver metastases with or without partial hepatectomy to model different clinical settings. Although the LEC-specific loss of mTOR remained without effect on metastasis growth in intact liver, partial liver resection resulted in the formation of larger metastases in mTORLEC mice compared with wildtype controls. This was accompanied by significantly enhanced inflammatory activity in LEC-specific mTOR KO livers after partial liver resection. Analysis of NF-ĸB target gene expression and immunohistochemistry of p65 displayed a significant activation of NF-ĸB in mTORLEC mice, suggesting a functional importance of this pathway for the observed inflammatory phenotype. Taken together, we show an unexpected acceleration of liver metastases upon deletion of mTOR in LECs. Our results support the notion that non-malignant host cells can contribute to resistance against mTOR inhibitors and encourage testing whether anti-inflammatory drugs are able to improve the efficacy of mTOR inhibitors for cancer therapy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Macrophages protect against loss of adipose tissue during cancer cachexia.

BACKGROUND: Cancer cachexia represents a central obstacle in medical oncology as it is associated with poor therapy response and reduced overall survival. Systemic inflammation is considered to be a key driver of cancer cachexia; however, clinical studies with anti-inflammatory drugs failed to show distinct cachexia-inhibiting effects. To address this contradiction, we investigated the functional importance of innate immune cells for hepatocellular carcinoma (HCC)-associated cachexia. METHODS: A transgenic HCC mouse model was intercrossed with mice harbouring a defect in myeloid cell-mediated inflammation. Body composition of mice was analysed via nuclear magnetic resonance spectroscopy and microcomputed tomography. Quantitative PCR was used to determine adipose tissue browning and polarization of adipose tissue macrophages. The activation state of distinct areas of the hypothalamus was analysed via immunofluorescence. Multispectral immunofluorescence imaging and immunoblot were applied to characterize sympathetic neurons and macrophages in visceral adipose tissue. Quantification of pro-inflammatory cytokines in mouse serum was performed with a multiplex immunoassay. Visceral adipose tissue of HCC patients was quantified via the L3 index of computed tomography scans obtained during routine clinical care. RESULTS: We identified robust cachexia in the HCC mouse model as evidenced by a marked loss of visceral fat and lean mass. Computed tomography-based analyses demonstrated that a subgroup of human HCC patients displays reduced visceral fat mass, complementing the murine data. While the myeloid cell-mediated inflammation defect resulted in reduced expression of pro-inflammatory cytokines in the serum of HCC-bearing mice, this unexpectedly did not translate into diminished but rather enhanced cachexia-associated fat loss. Defective myeloid cell-mediated inflammation was associated with decreased macrophage abundance in visceral adipose tissue, suggesting a role for local macrophages in the regulation of cancer-induced fat loss. CONCLUSIONS: Myeloid cell-mediated inflammation displays a rather unexpected beneficial function in a murine HCC model. These results demonstrate that immune cells are capable of protecting the host against cancer-induced tissue wasting, adding a further layer of complexity to the pathogenesis of cachexia and providing a potential explanation for the contradictory results of clinical studies with anti-inflammatory drugs.

Non-canonical HIF-1 stabilization contributes to intestinal tumorigenesis.

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1's role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of Wnt/ß-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts (TAF) and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with HIF-1α, arguing for an importance of hypoxia-independent, i.e., non-canonical, HIF-1 stabilization for intestinal tumorigenesis that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1α stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.

CSN5/JAB1 suppresses the WNT inhibitor DKK1 in colorectal cancer cells.

The COP9 signalosome (CSN) is a multi-protein complex that is highly conserved in eukaryotes. Due to its regulatory impact on processes such as cell cycle, DNA damage response and apoptosis, the CSN is essential for mammalian cells. One of the best-studied functions of the CSN is the deNEDDylation of cullin-RING ligases (CRLs) via its catalytically active subunit CSN5/JAB1, thereby triggering the degradation of various target proteins. CSN5 was found to be overexpressed in many human cancer entities, including colon adenocarcinoma. Overactivation of WNT signaling is known as a key step in colon cancer development. Recently, we found that depletion of CSN5 in colorectal cancer (CRC) cells affects WNT signaling by downregulation of ß-catenin. To investigate changes in gene expression associated with the CSN5 knockdown, we performed a microarray using cDNA from the CRC cell line SW480. We found the WNT ligand WNT6 and the WNT inhibitors DKK1 and DKK4 differentially regulated in CSN5 knockdown cells. DKK1 expression and DKK1 protein levels depended on CSN5 in different CRC cell lines. In addition, DKK1 secretion was increased following CSN5 knockdown, affecting WNT signaling in SW480 cells. Consequently, blocking of secreted DKK1 in cell-conditioned media abolished ß-catenin downregulation in SW480 cells, while treatment with recombinant DKK1 mimicked the CSN5 knockdown effect. Furthermore, knockdown of DKK1 was able to rescue the proliferative deficiency of CSN5 knockdown cells. We conclude that downregulation of WNT signaling in colorectal cancer cells resulting from CSN5 knockdown is mediated, at least in part, by elevated DKK1 secretion. Moreover, experiments with the NEDDylation inhibitor MLN-4924 indicated that DKK1 expression is regulated by a so far unidentified repressor, the stability of which could be controlled by a CSN-regulated CRL.

The ß-catenin E3 ubiquitin ligase SIAH-1 is regulated by CSN5/JAB1 in CRC cells.

COP9 signalosome subunit 5 (CSN5) plays a decisive role in cellular processes such as cell cycle regulation and apoptosis via promoting protein degradation, gene transcription, and nuclear export. CSN5 regulates cullin-RING-E3 ligase (CRL) activity through its deNEDDylase function. It is overexpressed in several tumor entities, but its role in colorectal cancer (CRC) is poorly understood. Wnt/ß-catenin signaling is aberrant in most CRC cells, resulting in increased levels of oncogenic ß-catenin and thus tumor progression. Under physiological conditions, ß-catenin levels are tightly regulated by continuous proteasomal degradation. We recently showed that knockdown of CSN5 in model and CRC cells results in decreased (phospho)-ß-catenin levels. Reduced ß-catenin levels were associated with an attenuated proliferation rate of different CRC cell types after CSN5 knockdown. The canonical Wnt pathway involves degradation of ß-catenin by a ß-TrCP1-containing E3 ligase, but is mostly non-functional in CRC cells. We thus hypothesized that alternative ß-catenin degradation mediated by SIAH-1 (seven in absentia homolog-1), is responsible for the effect of CSN5 on ß-catenin signaling in CRC cells. We found that SIAH-1 plays an essential role in ß-catenin degradation in HCT116 CRC cells and that CSN5 affects ß-catenin target gene expression in these cells. Of note, CSN5 affected SIAH-1 mRNA and SIAH-1 protein levels. Moreover, ß-catenin and SIAH-1 form protein complexes with CSN5 in HCT116 cells. Lastly, we demonstrate that CSN5 promotes SIAH-1 degradation in HCT116 and SW480 cells and that this is associated with its deNEDDylase activity. In conclusion, we have identified a CSN5/ß-catenin/SIAH-1 interaction network that might control ß-catenin degradation in CRC cells.

Role of CSN5/JAB1 in Wnt/ß-catenin activation in colorectal cancer cells.

CSN5/JAB1 is a critical subunit of the COP9 signalosome (CSN) and is overexpressed in many human cancers, but little is known about the role of CSN5 in colorectal cancer (CRC). To explore the functional role of CSN5 in colorectal tumorigenesis, we applied siRNA technology to silence CSN5 in HeLa, SW480, HCT116, HT29, and CaCo2 cells. CSN5 knock-down led to reduced ß-catenin and phospho-bcatenin levels and this was paralleled by reduced CRC cell proliferation and reduced apoptosis rates, whereas the short-term ß-catenin protein stability was enhanced by CSN5 knock-down in SW480 cells. Together, these data implicate the CSN in the pathogenesis of CRC via regulation of the Wnt/ß-catenin pathway