Stromal localization of inactive CD8+ T cells in metastatic mismatch repair deficient colorectal cancer.

BACKGROUND: The determinants of metastasis in mismatch repair deficiency with high levels of microsatellite instability (MSI-H) in colorectal cancer (CRC) are poorly understood. Here, we hypothesized that distinct immune and stromal microenvironments in primary tumors may discriminate between non-metastatic MSI-H CRC and metastatic MSI-H CRC. METHODS: We profiled 46,727 single cells using high-plex imaging mass cytometry and analyzed both differential cell type abundance, and spatial distribution of fibroblasts and immune cells in primary CRC tumors with or without metastatic capacity. We validated our findings in a second independent cohort using immunohistochemistry. RESULTS: High-plex imaging mass cytometry and hierarchical clustering based on microenvironmental markers separated primary MSI-H CRC tumors with and without metastatic capacity. Primary tumors with metastatic capacity displayed a high stromal content and low influx of CD8+ T cells, which expressed significantly lower levels of markers reflecting proliferation (Ki67) and antigen-experience (CD45RO) compared to CD8+ T cells in non-metastatic tumors. CD8+ T cells showed intra-epithelial localization in non-metastatic tumors, but stromal localization in metastatic tumors, which was validated in a second cohort. CONCLUSION: We conclude that localization of phenotypically distinct CD8+ T cells within stroma may predict metastasis formation in MSI-H CRC.

Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer.

Background: Poor prognosis in colon cancer is associated with a high content of cancer-associated fibroblasts (CAFs) and an immunosuppressive tumor microenvironment. The relationship between these two features is incompletely understood. Here, we aimed to generate a model system for studying the interaction between cancer cells and CAFs and their effect on immune-related cytokines and T cell proliferation. Methods: CAFs were isolated from colon cancer liver metastases and were immortalized to prolong lifespan and improve robustness and reproducibility. Established medium and matrix compositions that support the growth of patient-derived organoids were adapted to also support CAF growth. Changes in growth pattern and cellular re-organization were assessed by confocal microscopy, live cell imaging, and immunofluorescence. Single cell RNA sequencing was used to study CAF/organoid co-culture-induced phenotypic changes in both cell types. Conditioned media were used to quantify the production of immunosuppressive factors and to assess their effect on T cell proliferation. Results: We developed a co-culture system in which colon cancer organoids and CAFs spontaneously organize into superstructures with a high capacity to contract and stiffen the extracellular matrix (ECM). CAF-produced collagen IV provided a basement membrane supporting cancer cell organization into glandular structures, reminiscent of human cancer histology. Single cell RNA sequencing analysis showed that CAFs induced a partial epithelial-to-mesenchymal-transition in a subpopulation of cancer cells, similar to what is observed in the mesenchymal-like consensus molecular subtype 4 (CMS4) colon cancer. CAFs in co-culture were characterized by high expression of ECM components, ECM-remodeling enzymes, glycolysis, hypoxia, and genes involved in immunosuppression. An expression signature derived from CAFs in co-culture identified a subpopulation of glycolytic myofibroblasts specifically residing in CMS1 and CMS4 colon cancer. Medium conditioned by co-cultures contained high levels of the immunosuppressive factors TGFß1, VEGFA and lactate, and potently inhibited T cell proliferation. Conclusion: Co-cultures of organoids and immortalized CAFs recapitulate the histological, biophysical, and immunosuppressive features of aggressive mesenchymal-like human CRC. The model can be used to study the mechanisms of immunosuppression and to test therapeutic strategies targeting the cross-talk between CAFs and cancer cells. It can be further modified to represent distinct colon cancer subtypes and (organ-specific) microenvironments.

Lymphatic Invasion of Plakoglobin-Dependent Tumor Cell Clusters Drives Formation of Polyclonal Lung Metastases in Colon Cancer.

BACKGROUND & AIMS: Patients with colon cancer with liver metastases may be cured with surgery, but the presence of additional lung metastases often precludes curative treatment. Little is known about the processes driving lung metastasis. This study aimed to elucidate the mechanisms governing lung vs liver metastasis formation. METHODS: Patient-derived organoid (PDO) cultures were established from colon tumors with distinct patterns of metastasis. Mouse models recapitulating metastatic organotropism were created by implanting PDOs into the cecum wall. Optical barcoding was applied to trace the origin and clonal composition of liver and lung metastases. RNA sequencing and immunohistochemistry were used to identify candidate determinants of metastatic organotropism. Genetic, pharmacologic, in vitro, and in vivo modeling strategies identified essential steps in lung metastasis formation. Validation was performed by analyzing patient-derived tissues. RESULTS: Cecum transplantation of 3 distinct PDOs yielded models with distinct metastatic organotropism: liver only, lung only, and liver and lung. Liver metastases were seeded by single cells derived from select clones. Lung metastases were seeded by polyclonal clusters of tumor cells entering the lymphatic vasculature with very limited clonal selection. Lung-specific metastasis was associated with high expression of desmosome markers, including plakoglobin. Plakoglobin deletion abrogated tumor cell cluster formation, lymphatic invasion, and lung metastasis formation. Pharmacologic inhibition of lymphangiogenesis attenuated lung metastasis formation. Primary human colon, rectum, esophagus, and stomach tumors with lung metastases had a higher N-stage and more plakoglobin-expressing intra-lymphatic tumor cell clusters than those without lung metastases. CONCLUSIONS: Lung and liver metastasis formation are fundamentally distinct processes with different evolutionary bottlenecks, seeding entities, and anatomic routing. Polyclonal lung metastases originate from plakoglobin-dependent tumor cell clusters entering the lymphatic vasculature at the primary tumor site.

Modeling resistance of colorectal peritoneal metastases to immune checkpoint blockade in humanized mice.

BACKGROUND: The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. METHODS: We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. RESULTS: PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γ signature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. CONCLUSIONS: By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB.

BRAFV600E in colorectal cancer reduces sensitivity to oxidative stress and promotes site-specific metastasis by stimulating glutathione synthesis.

The presence of BRAFV600E in colorectal cancer (CRC) is associated with a higher chance of distant metastasis. Oxidative stress in disseminated tumor cells limits metastatic capacity. To study the relationship between BRAFV600E, sensitivity to oxidative stress, and metastatic capacity in CRC, we use patient-derived organoids (PDOs) and tissue samples. BRAFV600E tumors and PDOs express high levels of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione synthesis. Deletion of GCL in BRAFV600E PDOs strongly reduces their capacity to form distant liver and lung metastases but does not affect peritoneal metastasis outgrowth. Vice versa, the glutathione precursor N-acetyl-cysteine promotes organ-site-specific metastasis in the liver and the lungs but not in the peritoneum. BRAFV600E confers resistance to pharmacologically induced oxidative stress in vitro, which is partially overcome by treatment with the BRAF-inhibitor vemurafenib. We conclude that GCL-driven glutathione synthesis protects BRAFV600E-expressing tumors from oxidative stress during distant metastasis to the liver and the lungs.

KIT promotes tumor stroma formation and counteracts tumor-suppressive TGFß signaling in colorectal cancer.

Expression profiling has identified four consensus molecular subtypes (CMS1-4) in colorectal cancer (CRC). The receptor tyrosine kinase KIT has been associated with the most aggressive subtype, CMS4. However, it is unclear whether, and how, KIT contributes to the aggressive features of CMS4 CRC. Here, we employed genome-editing technologies in patient-derived organoids (PDOs) to study KIT function in CRC in vitro and in vivo. CRISPR-Cas9-mediated deletion of the KIT gene caused a partial mesenchymal-to-epithelial phenotype switch and a strong reduction of intra-tumor stromal content. Vice versa, overexpression of KIT caused a partial epithelial-to-mesenchymal phenotype switch, a strong increase of intra-tumor stromal content, and high expression of TGFß1. Surprisingly, the levels of phosphorylated SMAD2 were significantly lower in KIT-expressing versus KIT-deficient tumor cells. In vitro analyses showed that TGFß signaling in PDOs limits their regenerative capacity. Overexpression of KIT prevented tumor-suppressive TGFß signaling, while KIT deletion sensitized PDOs to TGFß-mediated growth inhibition. Mechanistically, we found that KIT expression caused a strong reduction in the expression of SMAD2, a central mediator of canonical TGFß signaling. We propose that KIT induces a pro-fibrotic tumor microenvironment by stimulating TGFß expression, and protects the tumor cells from tumor-suppressive TGFß signaling by inhibiting SMAD2 expression.

Peritoneal metastases from colorectal cancer belong to Consensus Molecular Subtype 4 and are sensitised to oxaliplatin by inhibiting reducing capacity.

BACKGROUND: Peritoneal metastases (PM) in colorectal cancer (CRC) are associated with therapy resistance and poor survival. Oxaliplatin monotherapy is widely applied in the intraperitoneal treatment of PM, but fails to yield clinical benefit. We aimed to identify the mechanism(s) underlying PM resistance to oxaliplatin and to develop strategies overcoming such resistance. EXPERIMENTAL DESIGN: We generated a biobank consisting of 35 primary tumour regions and 59 paired PM from 12 patients. All samples were analysed by RNA sequencing. We also generated a series of PM-derived organoid (PMDO) cultures and used these to design and test strategies to overcome resistance to oxaliplatin. RESULTS: PM displayed various hallmarks of aggressive CRC biology. The vast majority of PM and paired primary tumours belonged to the Consensus Molecular Subtype 4 (CMS4). PMDO cultures were resistant to oxaliplatin and expressed high levels of glutamate-cysteine ligase (GCLC) causing detoxification of oxaliplatin through glutathione synthesis. Genetic or pharmacological targeting of GCLC sensitised PMDOs to a 1-h exposure to oxaliplatin, through increased platinum-DNA adduct formation. CONCLUSIONS: These results link oxaliplatin resistance of colorectal PM to their CMS4 status and high reducing capacity. Inhibiting the reducing capacity of PM may be an effective strategy to overcome PM resistance to oxaliplatin.

Loss of Neuropilin-2 in Murine Mesenchymal-like Colon Cancer Organoids Causes Mesenchymal-to-Epithelial Transition and an Acquired Dependency on Insulin-Receptor Signaling and Autophagy.

Neuropilin-2 (Nrp2), an important regulator of lymphangiogenesis and lymphatic metastasis, has been associated with progression in colorectal cancer (CRC). However, the tumor cell-intrinsic role of Nrp2 in cancer progression is incompletely understood. To address this question, we employed CRISPR-Cas9 technology to generate Nrp2-knockout organoids derived from murine CRC tumors with a mesenchymal phenotype. Transcriptome profiling and tumor tissue analysis showed that Nrp2 loss resulted in mesenchymal-to-epithelial transition (MET), which was accompanied with restored polarity and tight junction stabilization. Signaling pathway analysis revealed that Nrp2-knockout organoids acquire de novo dependency on insulin receptor (IR) signaling and autophagy as alternative survival mechanisms. Combined inhibition of IR signaling and autophagy prevented the stabilization of cell-cell junctions, reduced metabolic activity, and caused profound cell death in Nrp2-knockout organoids. Collectively, the data demonstrate a key role for Nrp2 in maintaining the aggressive phenotype and survival of tumor-derived CRC organoids. The identified connection between Nrp2, insulin receptor signaling and autophagy may guide the development of novel combination-treatment strategies for aggressive CRC.

Mismatch Repair Status in Patient-Derived Colorectal Cancer Organoids Does Not Affect Intrinsic Tumor Cell Sensitivity to Systemic Therapy.

DNA mismatch repair deficiency (dMMR) in metastatic colorectal cancer (mCRC) is associated with poor survival and a poor response to systemic treatment. However, it is unclear whether dMMR results in a tumor cell-intrinsic state of treatment resistance, or whether alternative mechanisms play a role. To address this, we generated a cohort of MMR-proficient and -deficient Patient-Derived Organoids (PDOs) and tested their response to commonly used drugs in the treatment of mCRC, including 5-fluorouracil (5-FU), oxaliplatin, SN-38, binimetinib, encorafenib, and cetuximab. MMR status did not correlate with the response of PDOs to any of the drugs tested. In contrast, the presence of activating mutations in the KRAS and BRAF oncogenes was significantly associated with resistance to chemotherapy and sensitivity to drugs targeting oncogene-activated pathways. We conclude that mutant KRAS and BRAF impact the intrinsic sensitivity of tumor cells to chemotherapy and targeted therapy. By contrast, tumor cell-extrinsic mechanisms-for instance signals derived from the microenvironment-must underlie the association of MMR status with therapy response. Future drug screens on rationally chosen cohorts of PDOs have great potential in developing tailored therapies for specific CRC subtypes including, but not restricted to, those defined by BRAF/KRAS and MMR status.

Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Biology and Lymphatic Vessel/Tumor Coculture in a 3D Microfluidic Model.

The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in vitro models. Here we present a microfluidic lymphatic vessel model to study lymphangiogenesis and its interaction with colon cancer organoids using a newly developed lymphatic endothelial cell (LEC) line. We generated immortalized human LECs by lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Immortalized LECs showed an increased growth potential, reduced senescence, and elongated lifespan with maintenance of typical LEC morphology and marker expression for over 12 months while remaining nontransformed. Immortalized LECs were introduced in a microfluidic chip, comprising a free-standing extracellular matrix, where they formed a perfusable vessel-like structure against the extracellular matrix. A gradient of lymphangiogenic factors over the extracellular matrix gel induced the formation of luminated sprouts. Adding mouse colon cancer organoids adjacent to the lymphatic vessel resulted in a stable long-lived coculture model in which cancer cell-induced lymphangiogenesis and cancer cell motility can be investigated. Thus, the development of a stable immortalized lymphatic endothelial cell line in a membrane-free, perfused microfluidic chip yields a highly standardized lymphangiogenesis and lymphatic vessel-tumor cell coculture assay.

Distinct and overlapping functions of glutathione peroxidases 1 and 2 in limiting NF-κB-driven inflammation through redox-active mechanisms.

Glutathione peroxidase 2 (GPx2) is one of the five selenoprotein GPxs having a selenocysteine in the active center. GPx2 is strongly expressed in the gastrointestinal epithelium, as is another isoform, GPx1, though with a different localization pattern. Both GPxs are redox-active enzymes that are important for the reduction of hydroperoxides. Studies on GPx2-deficient mice and human HT-29 cells with a stable knockdown (kd) of GPx2 revealed higher basal and IL-1ß-induced expression of NF-κB target genes in vivo and in vitro. The activation of the IKK-IκBα-NF-κB pathway was increased in cultured GPx2 kd cells. Basal signaling was only restored by re-expressing active GPx2 in GPx2 kd cells but not by redox-inactive GPx2. As it is still not clear if the two isoforms GPx1 and GPx2 have different functions, kd cell lines for either GPx1 or GPx2 were studied in parallel. The inhibitory effect of GPx2 on NF-κB signaling and its target gene expression was stronger than that of GPx1, whereas cyclooxygenase (COX)- and lipoxygenase (LOX)-derived lipid mediator levels increased more strongly in GPx1 kd than in GPx2 kd cells. Under unstimulated conditions, the levels of the COX-derived prostaglandins PGE2 and PGD2 were enhanced in GPx2 as well as in GPx1 kd compared to control cells. Specifically, in GPx1 kd cells IL-1ß stimulation led to a dramatic shift of the PGE2/PGD2 ratio towards pro-inflammatory PGE2. Taken together, GPx2 and GPx1 have overlapping functions in controlling inflammatory lipid mediator synthesis and, most probably, exert their anti-inflammatory effects by preventing excessive PGE2 production. In view of the high activity of COX and LOX pathways during inflammatory bowel disease our data therefore provide new insights into the mechanisms of the protective function of GPx1 and GPx2 during colitis as well as inflammation-driven carcinogenesis.

Differential anti-tumour effects of MTH1 inhibitors in patient-derived 3D colorectal cancer cultures.

Reactive oxygen species (ROS) function as second messengers in signal transduction, but high ROS levels can also cause cell death. MTH1 dephosphorylates oxidized nucleotides, thereby preventing their incorporation into DNA and protecting tumour cells from oxidative DNA damage. Inhibitors of MTH1 (TH588 and (S)-crizotinib) were shown to reduce cancer cell viability. However, the MTH1-dependency of the anti-cancer effects of these drugs has recently been questioned. Here, we have assessed anti-tumour effects of TH588 and (S)-crizotinib in patient-derived 3D colorectal cancer cultures. Hypoxia and reoxygenation - conditions that increase intracellular ROS levels - increased sensitivity to (S)-crizotinib, but not to TH588. (S)-crizotinib reduced tyrosine phosphorylation of c-MET and ErbB3 whereas TH588 induced a mitotic cell cycle arrest, which was not affected by adding ROS-modulating compounds. Furthermore, we show that both compounds induced DNA damage that could not be prevented by adding the ROS inhibitor N-acetyl-L-cysteine. Moreover, adding ROS-modulating compounds did not alter the reduction in viability in response to TH588 and (S)-crizotinib. We conclude that TH588 and (S)-crizotinib have very clear and distinct anti-tumour effects in 3D colorectal cancer cultures, but that these effects most likely occur through distinct and ROS-independent mechanisms.

Increased Levels of Oxidative Damage in Liver Metastases Compared with Corresponding Primary Colorectal Tumors: Association with Molecular Subtype and Prior Treatment.

High levels of oxidative stress in disseminated colorectal cancer tumor cells may form a therapeutically exploitable vulnerability. However, it is unclear whether oxidative stress and damage persist in metastases. Therefore, we analyzed markers of oxidative damage in primary colorectal tumors and their corresponding liver metastases. Markers of generic and oxidative DNA damage [phosphorylated histone H2AX (γH2AX) and 8-hydroxy-2'-deoxyguanosine (8-OHdG)] were significantly higher in liver metastases compared with their corresponding primary tumors. Chemotherapy and/or radiotherapy before tumor resection was associated with increased persistent oxidative DNA damage, and this effect was more pronounced in metastases. Immunohistochemistry-based molecular classification into epithelial- and mesenchymal-like molecular subtypes revealed that untreated mesenchymal-like tumors contained lower levels of oxidative DNA damage compared with epithelial-like tumors. Treated mesenchymal-like tumors, but not epithelial-like tumors, showed significantly higher levels of γH2AX and 8-OHdG. Mesenchymal-like tumors expressed significantly lower levels of phosphorylated nuclear factor erythroid 2-related factor 2, a master regulator of the antioxidant response, and nuclear factor erythroid 2-related factor 2-controlled genes. Of interest, a positive 8-OHdG status identified a subgroup of mesenchymal-like metastases with a poor overall survival. An increased capacity to tolerate therapy-induced oxidative damage in mesenchymal-like colorectal cancer may explain, at least in part, the poor responsiveness of these tumors to chemotherapy, which could contribute to the poor survival of this patient subgroup.

Downregulation of DNA repair proteins and increased DNA damage in hypoxic colon cancer cells is a therapeutically exploitable vulnerability.

Surgical removal of colorectal cancer (CRC) liver metastases generates areas of tissue hypoxia. Hypoxia imposes a stem-like phenotype on residual tumor cells and promotes tumor recurrence. Moreover, in primary CRC, gene expression signatures reflecting hypoxia and a stem-like phenotype are highly expressed in the aggressive Consensus Molecular Subtype 4 (CMS4). Therapeutic strategies eliminating hypoxic stem-like cells may limit recurrence following resection of primary tumors or metastases. Here we show that expression of DNA repair genes is strongly suppressed in CMS4 and inversely correlated with hypoxia-inducible factor-1 alpha (HIF1α) and HIF-2α co-expression signatures. Tumors with high expression of HIF signatures and low expression of repair proteins showed the worst survival. In human tumors, expression of the repair proteins RAD51, KU70 and RIF1 was strongly suppressed in hypoxic peri-necrotic tumor areas. Experimentally induced hypoxia in patient derived colonospheres in vitro or in vivo (through vascular clamping) was sufficient to downregulate repair protein expression and caused DNA damage. Hypoxia-induced DNA damage was prevented by expressing the hydroperoxide-scavenging enzyme glutathione peroxidase-2 (GPx2), indicating that reactive oxygen species mediate hypoxia-induced DNA damage. Finally, the hypoxia-activated prodrug Tirapazamine greatly augmented DNA damage and reduced the fraction of stem-like (Aldefluorbright) tumor cells in vitro, and in vivo following vascular clamping. We conclude that decreased expression of DNA repair proteins and increased DNA damage in hypoxic tumor areas may be therapeutically exploited with hypoxia-activated prodrugs, and that such drugs reduce the fraction of Aldefluorbright (stem-like) tumor cells.

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

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 ß-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.

Inhibition of RAF1 kinase activity restores apicobasal polarity and impairs tumour growth in human colorectal cancer.

BACKGROUND AND AIM: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. Novel therapeutics are urgently needed, especially for tumours with activating mutations in KRAS (∼40%). Here we investigated the role of RAF1 in CRC, as a potential, novel target. METHODS: Colonosphere cultures were established from human tumour specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. The role of RAF1 was tested by generating knockdowns (KDs) using three independent shRNA constructs or by using RAF1-kinase inhibitor GW5074. Clone-initiating and tumour-initiating capacities were assessed by single-cell cloning and injecting CRC cells into immune-deficient mice. Expression of tight junction (TJ) proteins, localisation of polarity proteins and activation of MEK-ERK pathway was analysed by western blot, immunohistochemistry and immunofluorescence. RESULTS: KD or pharmacological inhibition of RAF1 significantly decreased clone-forming and tumour-forming capacity of all CRC cultures tested, including KRAS-mutants. This was not due to cytotoxicity but, at least in part, to differentiation of tumour cells into goblet-like cells. Inhibition of RAF1-kinase activity restored apicobasal polarity and the formation of TJs in vitro and in vivo, without reducing MEK-ERK phosphorylation. MEK-inhibition failed to restore polarity and TJs. Moreover, RAF1-impaired tumours were characterised by normalised tissue architecture. CONCLUSIONS: RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS. The effects of RAF1 are kinase-dependent, but MEK-independent. Despite the lack of activating mutations in RAF1, its kinase domain is an attractive therapeutic target for CRC.

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 capacity.

Identification of a pharmacologically tractable Fra-1/ADORA2B axis promoting breast cancer metastasis.

Metastasis confronts clinicians with two major challenges: estimating the patient's risk of metastasis and identifying therapeutic targets. Because they are key signal integrators connecting cellular processes to clinical outcome, we aimed to identify transcriptional nodes regulating cancer cell metastasis. Using rodent xenograft models that we previously developed, we identified the transcription factor Fos-related antigen-1 (Fra-1) as a key coordinator of metastasis. Because Fra-1 often is overexpressed in human metastatic breast cancers and has been shown to control their invasive potential in vitro, we aimed to assess the implication and prognostic significance of the Fra-1-dependent genetic program in breast cancer metastasis and to identify potential Fra-1-dependent therapeutic targets. In several in vivo assays in mice, we demonstrate that stable RNAi depletion of Fra-1 from human breast cancer cells strongly suppresses their ability to metastasize. These results support a clinically important role for Fra-1 and the genetic program it controls. We show that a Fra-1-dependent gene-expression signature accurately predicts recurrence of breast cancer. Furthermore, a synthetic lethal drug screen revealed that antagonists of the adenosine receptor A2B (ADORA2B) are preferentially toxic to breast tumor cells expressing Fra-1. Both RNAi silencing and pharmacologic blockade of ADORA2B inhibited filopodia formation and invasive activity of breast cancer cells and correspondingly reduced tumor outgrowth in the lungs. These data show that Fra-1 activity is causally involved in and is a prognostic indicator of breast cancer metastasis. They suggest that Fra-1 activity predicts responsiveness to inhibition of pharmacologically tractable targets, such as ADORA2B, which may be used for clinical interference of metastatic breast cancer.

Protein phosphatase 2A (B55α) prevents premature activation of forkhead transcription factor FoxM1 by antagonizing cyclin A/cyclin-dependent kinase-mediated phosphorylation.

The forkhead transcription factor FoxM1 controls expression of a large number of genes that are specifically expressed during the G(2) phase of the cell cycle. Throughout most of the cell cycle, FoxM1 activity is restrained by an autoinhibitory mechanism, involving a repressor domain present in the N-terminal part of the protein. Activation of FoxM1 in G(2) is achieved by Cyclin A/Cyclin-dependent kinase (Cdk)-mediated phosphorylation, which alleviates autoinhibition by the N-terminal repressor domain. Here, we show that FoxM1 interacts with B55α, a regulatory subunit of protein phosphatase 2A (PP2A). B55α binds the catalytic subunit of PP2A, and this promotes dephosphorylation and inactivation of FoxM1. Indeed, we find that overexpression of B55α results in decreased FoxM1 activity. Inversely, depletion of B55α results in premature activation of FoxM1. The activation of FoxM1 that is observed upon depletion of B55α is fully dependent on Cyclin A/Cdk-mediated phosphorylation of FoxM1. Taken together, these data demonstrate that B55α acts to antagonize Cyclin A/Cdk-dependent activation of FoxM1, to ensure that FoxM1 activity is restricted to the G(2) phase of the cell cycle.

The death receptor CD95 activates the cofilin pathway to stimulate tumour cell invasion.

The death receptor CD95 promotes apoptosis through well-defined signalling pathways. In colorectal cancer cells, CD95 primarily stimulates migration and invasion through pathways that are incompletely understood. Here, we identify a new CD95-activated tyrosine kinase pathway that is essential for CD95-stimulated tumour cell invasion. We show that CD95 promotes Tyr 783 phosphorylation of phospholipase C-γ1 through the platelet-derived growth factor receptor-ß, resulting in ligand-stimulated phosphatidylinositol (4,5)-bisphosphate (PIP(2)) hydrolysis. PIP(2) hydrolysis liberates the actin-severing protein cofilin from the plasma membrane to initiate cortical actin remodelling. Cofilin activation is required for CD95-stimulated formation of membrane protrusions and increased tumour cell invasion.