Anita Sjölander

Wnt-5a Protein Expression in Primary Dukes B Colon Cancers Identifies a Subgroup of Patients with Good Prognosis

Oncogenic Wnt/beta-catenin signaling occurs in a majority of colorectal cancers. In contrast, very little is known about the role of the nontransforming Wnt protein family member Wnt-5a in those tumors. In the most common of the three colon cancer stages, Dukes B or lymph node-negative, the outcome is the hardest to predict. We searched for a predictive marker in this group and observed loss of or reduced Wnt-5a expression in 50% of Dukes B tumors. Such Wnt-5a negativity was a strong predictor of adverse outcome, with a relative risk of death of 3.007 (95% confidence interval, 1.336-6.769; P = 0.008) after 5 years in Wnt-5a-negative patients. Furthermore, the median survival time after diagnosis was 109.1 months for patients with Wnt-5a-positive primary tumors but only 58 months for those with Wnt-5a-negative primary tumors. To find a possible biological explanation for these results, we studied the invasive and poorly differentiated human colon cancer cell line, SW480, which does not express Wnt-5a protein and the Wnt-5a-expressing and moderately differentiated Caco2 colon cancer cell line. We found that the addition of recombinant/purified Wnt-5a significantly reduced the migratory capacity of SW480 cells. By comparison, equivalent treatment did not significantly alter migration in the Wnt-5a-expressing Caco2 colon cancer cell line. These findings indicate that the expression of Wnt-5a in primary Dukes B colon cancer tissue constitutes a good prognostic marker for longer survival, which can be explained by the ability of Wnt-5a to impair tumor cell migration and thus reduce invasiveness and metastasis.

The pro-inflammatory mediator leukotriene D4 induces phosphatidylinositol 3-kinase and Rac-dependent migration of intestinal epithelial cells.

Inflammatory bowel diseases are associated with increased risk of developing colon cancer. A possible role of the pro-inflammatory leukotriene D4 (LTD4) in this process has been implicated by the findings that LTD4 can signal increased proliferation and survival, both hallmarks of a cancer cell, in non-transformed intestinal epithelial cells. Here we make the novel finding that LTD4 can also signal increased motility in these cells. In parallel, we found that LTD4 induced a simultaneous transient 10-fold increase in Rac but not Cdc42 activity. These data were also supported by the ability of LTD4 to activate the Rac GDP/GTP exchange factor Vav2. Further, LTD4 triggered a 3-fold transient increase in phosphatidylinositol 3-kinase (PI3K) phosphorylation, a possible upstream activator of the Vav2/Rac signaling pathway. The activation of Rac was blocked by the PI3K inhibitors LY294002 and wortmannin and by transfection of a kinase-negative mutant of PI3K or a dominant-negative form of Vav2. Furthermore, Rac was found to co-localize with actin in LTD4-generated membrane ruffles that were formed by a PI3K-dependent mechanism. In accordance, the inhibition of the PI3K and Rac signaling pathway also blocked the LTD4-induced migration of the intestinal cells. The present data reveal that an inflammatory mediator such as LTD4 cannot only increase proliferation and survival of non-transformed intestinal epithelial cells but also, via a PI3K/Rac signaling pathway, trigger a motile response in such cells. These data demonstrate the capacity of inflammatory mediators to participate in the process by which inflammatory bowel conditions increase the risk for colon cancer development.

Low expression of CysLT1R and high expression of CysLT2R mediate good prognosis in colorectal cancer

Colorectal cancer is the third most common cancer type in the Western world. In search of new treatment possibilities, the inflammation mediators, know as cysteinyl leukotrienes (CysLTs), have been shown to regulate intestinal epithelial cell survival and proliferation via the CysLT(1)R, and cell differentiation via the CysLT(2)R. These results prompted us to investigate the significance of CysLT(1)R and CysLT(2)R expression in colorectal cancer tissue for patient survival. The CysLT(1)R, CysLT(2)R, beta-catenin and Bcl-xL protein expression levels were evaluated by immunohistochemistry in a tissue microarray of 329 colorectal patients. We found that high nuclear expression of CysLT(1)R is associated with a poor prognosis, whereas high nuclear expression of CysLT(2)R is associated with a good prognosis. We also observed that patients with colorectal tumours characterised by high CysLT(1)R but low CysLT(2)R nuclear expression had the lowest survival expectancy, whereas patients with colorectal tumours characterised by low CysLT(1)R but high CysLT(2)R nuclear expression had the best survival expectancy. Interestingly, beta-catenin as a single prognostic marker did not exhibit any prognostic value. However, in patients with tumours characterised by a high CysLT(1)R nuclear expression, an elevated beta-catenin nuclear expression had a significantly prognostic value. In conclusion these data indicate that nuclear expressions of CysLTRs are potential prognostic indicators of colorectal cancer.

The inflammatory mediator leukotriene D4 induces β-catenin signaling and its association with antiapoptotic Bcl-2 in intestinal epithelial cells

Increased levels of the inflammatory mediator leukotriene D4 (LTD4) are present at sites of inflammatory bowel disease, and such areas also exhibit an increased risk for subsequent cancer development. It is known that LTD4 affects the expression of many proteins that influence survival and proliferation of intestinal epithelial cells. We demonstrate here that after LTD4 exposure, β-catenin translocates to the nucleus where it signals activation of the TCF/LEF family of transcription factors. These events are mediated via a phosphatidylinositol 3-kinase-dependent phosphorylation of the inhibitory Ser-9 residue of glycogen synthase kinase 3β. We also show that in the presence of LTD4, free β-catenin translocates to the mitochondria where it associates with the cell survival protein Bcl-2. We hypothesize that LTD4 may enhance cell survival via activation of β-catenin signaling, in particular, by promoting the association of β-catenin with Bcl-2 in the mitochondria. Similar to Wnt-1 signaling, LTD4 signals an increased level of free β-catenin and elevated TCF/LEF promotor activity. This work in intestinal epithelial cells further lends credence to the idea that inflammatory signaling pathways are intrinsically linked with potential oncogenic signals involved in cell survival and apoptosis.

Crosstalk between colon cancer cells and macrophages via inflammatory mediators and CD47 promotes tumour cell migration.

Tumour-associated macrophages (TAMs) of the M2 phenotype are present in the stroma of many tumours and are frequently associated with the progression of several types of cancer. We investigated the role of M2 macrophages in colon cancer progression and found that human colon cancer tissue had elevated numbers of CD68(+) (macrophage marker) cells and CD206(+) (M2 macrophage marker) cells and increased CD47 expression. To explore potential interplay between colon cancer cells and M2 macrophages, we differentiated the monocyte cell line THP-1 into M1 and M2 macrophages (CD206(high) and Th2 cytokine-secreting cells), respectively. M2 macrophages migrated faster than M1 macrophages towards SW480-conditioned medium. Similarly, M2 macrophage-conditioned medium induced SW480 cell migration and CD47 expression. Factors released by macrophages were involved in this induction. In addition, SW480 cells migrated faster when co-cultured with M2 macrophages. Inhibition of CD47 with blocking antibodies or siRNA significantly reduced the migration of SW480 cells in the presence of M2 macrophages. This effect was further decreased via blocking antibodies against the CD47 ligand signal-regulatory protein α (SIRPα). Additionally, cancer cells also secreted significant levels of IL-10, thereby promoting M2 macrophage differentiation. These findings indicate that a TAM-enriched tumour microenvironment promotes colon cancer cell migration and metastasis.

Endogenous production of leukotriene D(4) mediates autocrine survival and proliferation via CysLT(1) receptor signalling in intestinal epithelial cells.

The cysteinyl leukotriene1 (CysLT1) receptor (CysLT1R) enhances survival and proliferation of intestinal cells via distinct pathways. Here, we have demonstrated that there is significant endogenous production of CysLTs from both non-tumour- and tumour-derived intestinal epithelial cells. Treatment of two non-tumour cell lines, Int 407 and IEC-6, with CysLT1R antagonists led to shrinkage and detachment of cells, confirmed as apoptotic cell death, and a dose-dependent reduction in proliferation. However, in the tumour intestinal cell lines Caco-2, SW480, HCT-116 and HT-29, treatment with CysLT1R antagonists significantly reduced proliferation, but had no effect on apoptosis. A unique characteristic of intestinal cancer cells is the presence of nuclear CysLT1Rs, which are inaccessible to receptor antagonists. In these cells, inhibition of the endogenous production of CysLTs indirectly, by 5-lipoxygenase inhibition, impaired CysLT1R signalling throughout the cell, and resulted in apoptosis of the tumour cells. These data reveal the existence of constitutive CysLT1R signalling that mediates both survival and proliferation in intestinal cells. Importantly, we propose that tumour-derived intestinal cells are resistant to CysLT1R antagonist-induced apoptosis, a phenomena that could be explained by nuclear CysLT1R signalling.

The Role of Leukotriene Receptor Signaling in Inflammation and Cancer

Leukotrienes (LTs) and prostaglandins (PGs) are metabolites of arachidonic acid that play major roles in various inflammatory conditions. The release of these mediators, by cells recruited to or present at the site of inflammation, modulate/influence the magnitude of the inflammatory response. A better understanding of eicosanoids and how their receptors trigger intracellular signaling during inflammatory conditions is helping to elucidate the well-known connection between chronic inflammatory disease and neoplastic transformation. In the current review, we summarize the role of LTs and PGs in chronic inflammation and, in particular, we focus on recent insights into the role of CysLT1 receptor signaling pathway. In addition, we delineate how continuous CysLT1 receptor activation and signaling can increase cell survival and proliferation as important early steps toward oncogenicity.

An increased expression of cysteinyl leukotriene 2 receptor in colorectal adenocarcinomas correlates with high differentiation

Increased levels of inflammatory mediators such as cysteinyl leukotrienes (CysLT) have been found in and around tumors. These data, along with our previous observation that the G-protein-coupled receptor CysLT(1)R, which signals survival and proliferation, is up-regulated in colon cancer, suggest an important role for CysLT(1)R in tumor development. The objective of this study was to examine the expression and function of the low-affinity CysLT2 receptor (CysLT2R) in colon cancer. We found lower expression levels of CysLT2R compared with CysLT(1)R in cancer cell lines as well as clinical tumor material. Interestingly, CysLT2R, like CysLT(1)R, was found to be one of few G-protein-coupled receptors that are located both at the plasma membrane and the nuclear membrane. No effect of CysLT2R signaling on cell proliferation was observed, nor was there a correlation between CysLT2R and different proliferation markers such as Ki-67 and cyclooxygenase-2 in the tumor material. Instead, we found that activation of this receptor in colon cancer cells led to cellular differentiation similar to the effects of butyrate treatment. In accordance with this finding, we found that reduced expression of CysLT2R in colon cancer was associated with poor prognosis. We report the novel finding that CysLT2R signaling leads to terminal differentiation of colon carcinoma cells and growth inhibition, and that its expression is relatively high in less malignant forms of colon cancer. These data suggest that the balance between these two receptors is important for tumor progression and disease outcome.

Leukotriene D4-induced adhesion of Caco-2 cells is mediated by prostaglandin E2 and upregulation of α2β1-integrin

Cell-cell and extracellular matrix adhesions play important roles in the progression of cancer. We investigated the involvement of the inflammatory mediator leukotriene D4 (LTD4) in the regulation of cell-matrix adhesion of colon cancer (Caco-2) cells. We observed that LTD4 acted via its CysLT1 receptor in these cells to induce increased adhesion to collagen I. LTD4 also enhanced the activation and expression of α2β1-integrins on the cell surface, which we found to be responsible for mediating the increased adhesion to collagen I. LTD4 simultaneously augmented expression of the prostaglandin-generating enzyme cyclooxygenase-2 (COX-2) and increased prostaglandin E2 (PGE2) production in Caco-2 cells. The adhesive capacity of the Caco-2 cells was reduced by specific inhibition of COX-2 and was subsequently restored by PGE2, but not by LTD4. A selective PGE2 receptor antagonist abolished the increased adhesion and the augmented α2β1-integrin expression induced by both PGE2 and LTD4. Summarizing, the inflammatory mediator LTD4 regulates the adhesive properties and migration of the Caco-2 cell line by upregulating COX-2 and stimulating PGE2-induced expression of α2β1-integrins. This suggests that inflammatory mediators such as LTD4 can be involved in the dissemination and survival of colon cancer cells.

The inflammatory mediator leukotriene D4 induces subcellular β-catenin translocation and migration of colon cancer cells.

The abnormal activation of the Wnt/β-catenin pathway frequently occurs in colorectal cancer. The nuclear translocation of β-catenin activates the transcription of target genes that promote cell proliferation, survival, and invasion. The pro-inflammatory mediator leukotriene D4 (LTD4) exerts its effects through the CysLT1 receptor. We previously reported an upregulation of CysLT1R in patients with colon cancer, suggesting the importance of leukotrienes in colon cancer. The aim of this study was to investigate the impact of LTD4 on Wnt/β-catenin signaling and its effects on proliferation and migration of colon cancer cells. LTD4 stimulation led to an increase in β-catenin expression, β-catenin nuclear translocation and the subsequent transcription of MYC and CCND1. Furthermore, LTD4 significantly reduced the expression of E-cadherin and β-catenin at the plasma membrane and increased the migration and proliferation of HCT116 colon cancer cells. The effects of LTD4 can be blocked by the inhibition of CysLT1R. Furthermore, LTD4 induced the inhibition of glycogen synthase kinase 3 (GSK)-3β activity, indicating a crosstalk between the G-protein-coupled receptor CysLT1 and the Wnt/β-catenin pathway. In conclusion, LTD4, which can be secreted from macrophages and leukocytes in the tumor microenvironment, induces β-catenin translocation and the activation of β-catenin target genes, resulting in the increased proliferation and migration of colon cancer cells.