Onno Kranenburg

Cyclin D1 is an essential mediator of apoptotic neuronal cell death.

Many neurons in the developing nervous system undergo programmed cell death, or apoptosis. However, the molecular mechanism underlying this phenomenon is largely unknown. In the present report, we present evidence that the cell cycle regulator cyclin D1 is involved in the regulation of neuronal cell death. During neuronal apoptosis, cyclin D1‐dependent kinase activity is stimulated, due to an increase in cyclin D1 levels. Moreover, artificial elevation of cyclin D1 levels is sufficient to induce apoptosis, even in non‐neural cell types. Cyclin D1‐induced apoptosis, like neuronal apoptosis, can be inhibited by 21 kDa E1B, Bcl2 and pRb, but not by 55 kDa E1B. Most importantly, however, overexpression of the cyclin D‐dependent kinase inhibitor p16INK4 protects neurons from apoptotic cell death, demonstrating that activation of endogenous cyclin D1‐dependent kinases is essential during neuronal apoptosis. These data support a model in which neuronal apoptosis results from an aborted attempt to activate the cell cycle in terminally differentiated neurons.

Tissue-Type Plasminogen Activator Is a Multiligand Cross-β Structure Receptor

Abstract Tissue-type plasminogen activator (tPA) regulates fibrin clot lysis by stimulating the conversion of plasminogen into the active protease plasmin [1]. Fibrin is required for efficient tPA-mediated plasmin generation and thereby stimulates its own proteolysis. Several fibrin regions can bind to tPA [1], but the structural basis for this interaction is unknown. Amyloid β (Aβ) is a peptide aggregate that is associated with neurotoxicity in brains afflicted with Alzheimers disease [2]. Like fibrin, it stimulates tPA-mediated plasmin formation [3–5]. Intermolecular stacking of peptide backbones in β sheet conformation underlies cross-β structure in amyloid peptides [6]. We show here that fibrin-derived peptides adopt cross-β structure and form amyloid fibers. This correlates with tPA binding and stimulation of tPA-mediated plasminogen activation. Prototype amyloid peptides, including Aβ and islet amyloid polypeptide (IAPP) (associated with pancreatic β cell toxicity in type II diabetes [7]), have no sequence similarity to the fibrin peptides but also bind to tPA and can substitute for fibrin in plasminogen activation by tPA. Moreover, the induction of cross-β structure in an otherwise globular protein (endostatin) endows it with tPA-activating potential. Our results classify tPA as a multiligand receptor and show that cross-β structure is the common denominator in tPA binding ligands.

Surgical implantation of an abdominal imaging window for intravital microscopy

High-resolution intravital microscopy through imaging windows has become an indispensable technique for the long-term visualization of dynamic processes in living animals. Easily accessible sites such as the skin, the breast and the skull can be imaged using various different imaging windows; however, long-term imaging studies on cellular processes in abdominal organs are more challenging. These processes include colonization of the liver by metastatic tumor cells and the development of an immune response in the spleen. We have recently developed an abdominal imaging window (AIW) that allows long-term imaging of the liver, the pancreas, the intestine, the kidney and the spleen. Here we describe the detailed protocol for the optimal surgical implantation of the AIW, which takes ∼1 h, and subsequent multiphoton imaging, which takes up to 1 month.

Intravital Microscopy Through an Abdominal Imaging Window Reveals a Pre-Micrometastasis Stage During Liver Metastasis

An abdominal imaging window allows in vivo visualization of dynamic cellular processes, including liver metastasis and islet cell transplantation. Peering Into Cancer Understanding what goes on inside the body, as it is happening, is an ongoing challenge in medical imaging. Conventional imaging methods are only “snapshots,” unable to truly capture biology in action or the progression of disease. In this study by Ritsma and colleagues, an abdominal imaging window (AIW) proves to be the answer, allowing the authors to visualize and quantify metastatic processes in real time, in vivo in mice. The AIW consisted of a titanium ring with a glass coverslip, which could be tightly secured to the abdominal wall of a mouse. This window stayed in place for an average of 5 weeks, which is long enough to visualize many biological phenomena, including single-cell activity in the small intestine, spleen, pancreas, and kidney, as demonstrated by Ritsma et al. Although able to image many organs and cells, the authors chose to focus on tumor cell metastasis—specifically, the metastasis of mouse colorectal cancer C26 cells to the liver. By tracking fluorescently labeled C26 cells over the course of 2 weeks, the authors were able to confirm that the majority of metastatic growth was clonal (that is, from a single founder cell) rather than synergistic. The authors also noticed that the cancer cells had different phenotypic properties at different time points: At day 3, the cells were motile and diffuse in the liver tissue, whereas, at day 5, the cells stopped moving and were densely packed. The authors called this phenotypic shift a “pre-micrometastatic” state, followed by the “micrometastatic state.” Blocking cell migration in the pre-micrometastatic stage with a small-molecule inhibitor reduced cell growth and formation of subsequent micrometastases. Ristma and coauthors have developed a powerful in vivo imaging tool to track biological events in real time. This will hopefully lend insight into many diseases that affect abdominal organs. Although their preliminary findings suggest a new target for pharmacological inhibition of cancer growth and migration, additional preclinical and clinical studies will be needed to follow up this pre-micrometastatic hypothesis and to further confirm its presence in humans. Cell dynamics in subcutaneous and breast tumors can be studied through conventional imaging windows with intravital microscopy. By contrast, visualization of the formation of metastasis has been hampered by the lack of long-term imaging windows for metastasis-prone organs, such as the liver. We developed an abdominal imaging window (AIW) to visualize distinct biological processes in the spleen, kidney, small intestine, pancreas, and liver. The AIW can be used to visualize processes for up to 1 month, as we demonstrate with islet cell transplantation. Furthermore, we have used the AIW to image the single steps of metastasis formation in the liver over the course of 14 days. We observed that single extravasated tumor cells proliferated to form “pre-micrometastases,” in which cells lacked contact with neighboring tumor cells and were active and motile within the confined region of the growing clone. The clones then condensed into micrometastases where cell migration was strongly diminished but proliferation continued. Moreover, the metastatic load was reduced by suppressing tumor cell migration in the pre-micrometastases. We suggest that tumor cell migration within pre-micrometastases is a contributing step that can be targeted therapeutically during liver metastasis formation.

Mouse Models of Colorectal Cancer and Liver Metastases

Colorectal cancer (CRC) is one of the most common malignancies in the western world. Its high mortality rates are particularly related to the occurrence of liver metastases. Many mouse models have been developed to evaluate the various features of CRC in human. Since none of the existing mouse models mimics all the characteristics of human CRC, it is of crucial importance that the optimal model is chosen for each experiment to resolve a specific experimental question. Currently used mouse models for CRC include chemically induced CRC models, genetically engineered mouse models and models in which colon tumors are implanted in recipient mice. Recently, conditional mouse models have been created in which a gene of interest can be (in)activated in a time- and tissue-specific manner. All models have their advantages and limitations. This review highlights the most commonly used mouse models for CRC and its liver metastases, their usefulness and shortcomings, as well as recent improvements, particularly regarding intravital (tumor) imaging.

Comparative Proteomics of Colon Cancer Stem Cells and Differentiated Tumor Cells Identifies BIRC6 as a Potential Therapeutic Target

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that “cell death ” regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.

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

BACKGROUND & AIMSnDeath 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.nnnMETHODSnIsogenic 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.nnnRESULTSnCD95 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.nnnCONCLUSIONSnOncogenic 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 formation.

Differentiated Human Colorectal Cancer Cells Protect Tumor-Initiating Cells From Irinotecan

BACKGROUND & AIMSnStem cells of normal tissues have resistance mechanisms that allow them to survive genotoxic insults. The stem cell-like cells of tumors are defined by their tumor-initiating capacity and may have retained these resistance mechanisms, making them resistant to chemotherapy. We studied the relationship between resistance to the topoisomerase I inhibitor irinotecan and tumor-initiating potential in human colonosphere cultures and in mice with colorectal xenograft tumors.nnnMETHODSnColonosphere cultures were established from human colorectal tumor specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. Stem cell and differentiation markers were analyzed by immunoblotting and fluorescence-activated cell sorting. Clone- and tumor-initiating capacities were assessed by single-cell cloning and in immune-deficient mice. Sensitivity to irinotecan was assessed in vitro and in tumor-bearing mice. The relationship between drug resistance and tumor-initiating capacity was tested by fluorescence-activated cell sorting of colonosphere cells, based on expression of ABCB1 and aldehyde dehydrogenase (ALDH) activity.nnnRESULTSnColonosphere cultures had a high capacity to initiate tumors in mice and were resistant to irinotecan. Inhibition of the drug-efflux pump ABCB1 by PSC-833 allowed irinotecan to eradicate tumor-initiating cells. However, ABCB1 was expressed only by a subpopulation of differentiated tumor cells that did not form clones or tumors. Conversely, tumor-initiating cells were ABCB1-negative and were identified by high ALDH activity. Tumorigenic ALDHhigh/ABCB1negative cells generated nontumorigenic ALDHlow/ABCB1positive daughter cells in vitro and in tumor xenografts. PSC-833 increased the antitumor efficacy of irinotecan in mice.nnnCONCLUSIONSnThe resistance of colorectal tumors to irinotecan requires the cooperative action of tumor-initiating ALDHhigh/ABCB1negative cells and their differentiated, drug-expelling, ALDHlow/ABCB1positive daughter cells.

Recombinant endostatin forms amyloid fibrils that bind and are cytotoxic to murine neuroblastoma cells in vitro

Endostatin is a fragment of collagen XVIII that acts as an endogenous inhibitor of tumor angiogenesis and tumor growth. Anti‐tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study we have investigated the structure and cellular effects of insoluble endostatin. We found that insoluble endostatin shows all the hallmarks of amyloid aggregates. Firstly, it binds Congo red and shows the characteristic apple‐green birefringe when examined under polarized light. Secondly, electron microscopy shows that endostatin forms short unbranched fibrils. Thirdly, X‐ray analysis shows the abundant presence of cross‐β sheets, the tertiary structure that underlies fibrillogenesis. None of these properties was observed when examining soluble endostatin. Soluble endostatin can be triggered to form cross‐β sheets following denaturation, indicating that endostatin is a protein fragment with an inherent propensity to form amyloid deposits. Like β‐amyloid, found in the brains of patients with Alzheimers disease, amyloid endostatin binds to and is toxic to neuronal cells, whereas soluble endostatin has no effect on cell viability. Our results demonstrate a previously unrecognized functional difference between soluble and insoluble endostatin, only the latter acting as a cytotoxic amyloid substance.

Wip1 confers G2 checkpoint recovery competence by counteracting p53-dependent transcriptional repression

Activation of the DNA damage checkpoint causes a cell‐cycle arrest through inhibition of cyclin‐dependent kinases (cdks). To successfully recover from the arrest, a cell should somehow be maintained in its proper cell‐cycle phase. This problem is particularly eminent when a cell arrests in G2, as cdk activity is important to establish a G2 state. Here, we identify the phosphatase Wip1 (PPM1D) as a factor that maintains a cell competent for cell‐cycle re‐entry during an ongoing DNA damage response in G2. We show that Wip1 function is required throughout the arrest, and that Wip1 acts by antagonizing p53‐dependent repression of crucial mitotic inducers, such as Cyclin B and Plk1. Our data show that the primary function of Wip1 is to retain cellular competence to divide, rather than to silence the checkpoint to promote recovery. Our findings uncover Wip1 as a first in class recovery competence gene, and suggest that the principal function of Wip1 in cellular transformation is to retain proliferative capacity in the face of oncogene‐induced stress.