• CD95 ligand induces senescence in mismatch repair-deficient human colon cancer via chronic caspase-mediated induction of DNA damage.

CD95 ligand induces senescence in mismatch repair-deficient human colon cancer via chronic caspase-mediated induction of DNA damage.

Cell death & disease (2017-03-17)
Danielle A Raats, Nicola Frenkel, Susanne J van Schelven, Inne HMBorel Rinkes, Jamila Laoukili, Onno Kranenburg

CD95 is best known for its ability to induce apoptosis via a well-characterized pathway involving caspase-mediated proteolytic events. However, in apoptosis-resistant cell lines of diverse cancer types stimulation of CD95 primarily has pro-tumorigenic effects that affect many of the hallmarks of cancer. For instance, in colon cancer cells with a mutant KRAS gene CD95 primarily promotes invasion and metastasis. In the current study, we further investigated the context dependency of the consequences of CD95 activation in colon cancer. We used a series of patient-derived three-dimensional colon cancer cultures and studied their response to stimulation with CD95 ligand (CD95L). CD95L had a strong inhibitory effect on the clone-forming capacity of five out of nine cultures. In line with previous work, these cultures all had a wild-type KRAS gene and expressed high levels of CD95. Furthermore, the most sensitive cultures were characterized by microsatellite instability (MSI) and deficient mismatch repair. The reduced clonogenic growth of MSI-type colonospheres resulting from chronic CD95 stimulation was only partly due to apoptosis as many tumor cells survived treatment, yet were unable to regenerate clones. CD95 stimulation caused an irreversible cell cycle arrest, which was associated with cytokine secretion, similar to the senescence-associated secretory phenotype (SASP), and expression of senescence-associated β-galactosidase. In human colon cancer cohorts, CD95 expression was strongly correlated with the recently identified consensus molecular subtype 1 (CMS1), which mainly consists of MSI-high tumors, and with two independent SASP signatures. Mechanistically, CD95-induced senescence was caused by chronic DNA damage via caspase-activated DNAse resulting in p53 activation and p21 expression, with a minor contribution of the SASP. We conclude that induction of senescence is a hitherto unrecognized consequence of high CD95 expression, which appears to be most relevant for CMS1.

Product Number
Product Description

5-Carboxyfluorescein diacetate, ~95% (HPLC)
Trypsin-EDTA solution, 10 ×, sterile-filtered, BioReagent, suitable for cell culture, 5.0 g porcine trypsin and 2 g EDTA, 4Na per liter of 0.9% sodium chloride
Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture
Ribonuclease A from bovine pancreas, Type II-A, ≥60% (SDS-PAGE), >= 60 Kunitz units/mg protein
5(6)-Carboxyfluorescein diacetate N-succinimidyl ester, BioReagent, suitable for fluorescence, ≥90% (HPLC)
Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301, clone JBW301, Upstate®, from mouse
Anti-Fas Antibody (human, activating), clone CH11, clone CH11, Upstate®, from mouse
Human IgG, Fc Fragment, Plasma