CD95 signaling in colorectal cancer

CD95 and its ligand (CD95L) are widely expressed in colorectal tumors, but their role in shaping tumor behavior is unclear. CD95 activation on tumor cells can lead to apoptosis, while CD95L attracts neutrophils, suggesting a function in tumor suppression. However, CD95 can also promote tumorigenesis, at least in part by activating non-apoptotic signaling pathways that stimulate tumor cell proliferation, invasion and survival. In addition, CD95 signaling in stromal cells and tumor-infiltrating inflammatory cells has to be taken into account when addressing the function of CD95 and its ligand in colorectal tumor biology. We present a model in which the tumor-suppressing and tumor-promoting activities of CD95/CD95L together determine colorectal tumor behavior. We also discuss how these multiple activities are changing our view of CD95 and CD95L as potential therapeutic targets in the treatment of colorectal cancer. We conclude that locking CD95 in apoptosis-mode may be a more promising anti-cancer strategy than simply inhibiting or stimulating CD9

Posterior Malleolar Fracture Patterns

To characterize posterior malleolar fracture morphology using Cole fracture mapping and to study reliability of quantification of 3-dimensional computed tomography (CT)-modeling for posterior malleolar fractures with respect to quantification of fragment size (in cubic millimeter) and true articular involvement (in square millimeter). CT scans of a consecutive series of 45 patients with an ankle fracture involving the posterior malleolus were reconstructed to calculate (1) fracture maps, (2) fragment volume, (3) articular surface of the posterior malleolar fragment, (4) articular surface of intact tibia, and (5) articular surface of the medial malleolus by 3 independent observers. Three-dimensional animation of this technique is shown on www.traumaplatform.org. Fracture mapping revealed (1) a continuous spectrum of posterolateral oriented fracture lines and (2) fragments with posterolateral to posteromedial oriented fracture lines extending into the medial malleolus. Reliability of measurements of the volume and articular surface of posterior malleolar fracture fragments was defined as almost perfect according to the categorical system of Landis (interclass coefficient, range, 0.978-1.000). Mapping of posterior malleolar fractures revealed a continuous spectrum of Haraguchi III to I fractures and identified Haraguchi type II as a separate pattern. Quantification of 3-dimensional CT-modeling is reliable to assess fracture characteristics of posterior malleolar fracture fragments. Morphology might be more important than posterior malleolar fracture size alone for clinical decision makin

Mice lacking functional CD95-ligand display reduced proliferation of the intestinal epithelium without gross homeostatic alterations

Homeostasis of the continuously self-renewing intestinal tract involves cell proliferation, migration, differentiation along the crypt-villus-axis and shedding of cells into the gut lumen. CD95-ligand (FAS-ligand, CD95L) is a cytokine that is known for its capacity to induce apoptosis by binding its cognate receptor, CD95 (Fas). More recently, it was discovered that CD95L can also induce other cellular responses, such as proliferation, differentiation and cell migration. CD95L is highly expressed in Paneth cells of the small intestine which are in close contact with intestinal stem cells. This suggests a potential role for CD95L in controlling stem cell function and, possibly, intestinal homeostasis. We analyzed the intestines of mice deficient for functional CD95L (gld) for potential alterations in the diversity of stem-cell-lineages and parameters of intestinal homeostasis. Stem cell diversity was assessed by analyzing methylation patterns of the non-transcribed mMYOD gene. Proliferation was analyzed by BrdU labeling and differentiation was assessed by immunohistochemistry. Of all parameters analyzed, only epithelial cell proliferation was significantly reduced in the small intestines of gld-mice, but not in their colons which lack CD95L expression. We conclude that CD95L has a proliferation-stimulating role during normal turnover of the small intestine, but has a marginal effect on overall intestinal homeostasis

CD95 is a key mediator of invasion and accelerated outgrowth of mouse colorectal liver metastases following radiofrequency ablation

Recently, we have shown that micro-metastases, in the hypoxic transition zone surrounding lesions generated by radiofrequency ablation (RFA), display strongly accelerated outgrowth. CD95 is best known for its ability to induce apoptosis but can also promote tumorigenesis in apoptosis-resistant tumor cells. Therefore, we tested whether CD95 signaling plays a role in accelerated outgrowth of colorectal liver metastases following RFA. Hypoxia-induced invasion was assessed in three-dimensional EGFP-expressing C26 tumor cell cultures by confocal microscopy. CD95 localization was tested by immunofluorescence. Invasion and outgrowth of liver metastases following RFA were analyzed by post-mortem confocal microscopy and by morphometric assessment of tumor load. Neutralization of CD95L was performed by using antibody MFL4. CD95 was suppressed by lentiviral RNA interference. The role of host CD95L was assessed using gld mice. Micro-metastases in the hypoxic transition zone following RFA displayed a highly invasive phenotype and increased expression of CD95 and CD95L. Hypoxia-induced tumor cell invasion in vitro increased the expression of CD95 and CD95L and induced translocation of CD95 to the invasive front. In vitro invasion, metastasis invasion, and accelerated tumor growth in the transition zone were strongly suppressed by neutralizing CD95L or by suppressing tumor cell CD95. In contrast, metastasis invasion and outgrowth were unaffected in gld mice. Hypoxia causes autocrine activation of CD95 on colorectal tumor cells, thereby promoting local invasion and accelerated metastasis outgrowth in the hypoxic transition zone following RFA. Further pre-clinical work is needed to assess the role of CD95L neutralization, either alone or in combination with chemotherapy, in limiting aggressive recurrence of liver metastases following RF

PDGFRB Promotes Liver Metastasis Formation of Mesenchymal-Like Colorectal Tumor Cells

AbstractIn epithelial tumors, the platelet-derived growth factor receptor B (PDGFRB) is mainly expressed by stromal cells of mesenchymal origin. Tumor cells may also acquire PDGFRB expression following epithelial-to-mesenchymal transition (EMT), which occurs during metastasis formation. Little is known about PDGFRB signaling in colorectal tumor cells. We studied the relationship between PDGFRB expression, EMT, and metastasis in human colorectal cancer (CRC) cohorts by analysis of gene expression profiles. PDGFRB expression in primary CRC was correlated with short disease-free and overall survival. PDGFRB was co-expressed with genes involved in platelet activation, transforming growth factor beta (TGFB) signaling, and EMT in three CRC cohorts. PDGFRB was expressed in mesenchymal-like tumor cell lines in vitro and stimulated invasion and liver metastasis formation in mice. Platelets, a major source of PDGF, preferentially bound to tumor cells in a non-activated state. Platelet activation caused robust PDGFRB tyrosine phosphorylation on tumor cells in vitro and in liver sinusoids in vivo. Platelets also release TGFB, which is a potent inducer of EMT. Inhibition of TGFB signaling in tumor cells caused partial reversion of the mesenchymal phenotype and strongly reduced PDGFRB expression and PDGF-stimulated tumor cell invasion. These results suggest that PDGFRB may contribute to the aggressive phenotype of colorectal tumors with mesenchymal properties, most likely downstream of platelet activation and TGFB signaling

GPx2 suppression of H2O2 stress links the formation of differentiated tumor mass to metastatic capacity in colorectal cancer

Colorectal tumorigenesis is accompanied by the generation of oxidative stress, but how this controls tumor development is poorly understood. Here, we studied how the H2O2-reducing enzyme glutathione peroxidase 2 (GPx2) regulates H2O2 stress and differentiation in patient-derived "colonosphere" cultures. GPx2 silencing caused accumulation of radical oxygen species, sensitization to H2O2-induced apoptosis, and strongly reduced clone- and metastasis-forming capacity. Neutralization of radical oxygen species restored clonogenic capacity. Surprisingly, GPx2-suppressed cells also lacked differentiation potential and formed slow-growing undifferentiated tumors. GPx2 overexpression stimulated multilineage differentiation, proliferation, and tumor growth without reducing the tumor-initiating capacity. Finally, GPx2 expression was inversely correlated with H2O2-stress signatures in human colon tumor cohorts, but positively correlated with differentiation and proliferation. Moreover, high GPx2 expression was associated with early tumor recurrence, particularly in the recently identified aggressive subtype of human colon cancer. We conclude that H2O2 neutralization by GPx2 is essential for maintaining clonogenic and metastatic capacity, but also for the generation of differentiated proliferating tumor mass. The results reveal an unexpected redox-controlled link between tumor mass formation and metastatic capacit

Oncogenic K-Ras Turns Death Receptors Into Metastasis-Promoting Receptors in Human and Mouse Colorectal Cancer Cells

BACKGROUND & AIMS: Death receptors expressed on tumor cells can prevent metastasis formation by inducing apoptosis, but they also can promote migration and invasion. The determinants of death receptor signaling output are poorly defined. Here we investigated the role of oncogenic K-Ras in determining death receptor function and metastatic potential. METHODS: Isogenic human and mouse colorectal cancer cell lines differing only in the presence or absence of the K-Ras oncogene were tested in apoptosis and invasion assays using CD95 ligand and tumor necrois factor-related apoptosis-inducing ligand (TRAIL) as stimuli. Metastatic potential was assessed by intrasplenic injections of green fluorescent protein-or luciferase-expressing tumor cells, followed by intravital fluorescence microscopy or bioluminescence imaging, and confocal microscopy and immunohistochemistry. Ras-effector pathway control of CD95 output was assessed by an RNA-interference and inhibitor-based approach. RESULTS: CD95 ligand and TRAIL stimulated invasion of colorectal tumor cells and liver metastases in a K-Ras-dependent fashion. Loss of mutant K-Ras switched CD95 and TRAIL receptors back into apoptosis mode and abrogated metastatic potential. Raf1 was essential for the switch in CD95 function, for tumor cell survival in the liver, and for K-Ras-driven formation of liver metastases. K-Ras and Raf1 suppressed Rho kinase (ROCK)/LIM kinase-mediated phosphorylation of the actin-severing protein cofilin. Overexpression of ROCK or LIM kinase allowed CD95L to induce apoptosis in K-Ras-proficient cells and prevented metastasis formation, whereas their suppression protected K-Ras-deficient cells against apoptosis. CONCLUSIONS: Oncogenic K-Ras and its effector Raf1 convert death receptors into invasion-inducing receptors by suppressing the ROCK/ LIM kinase pathway, and this is essential for K-Ras/Raf1-driven metastasis formatio