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1.
J Exp Clin Cancer Res ; 39(1): 2, 2020 Jan 08.
Article in English | MEDLINE | ID: mdl-31910865

ABSTRACT

BACKGROUND: Quiescent/slow cycling cells have been identified in several tumors and correlated with therapy resistance. However, the features of chemoresistant populations and the molecular factors linking quiescence to chemoresistance are largely unknown. METHODS: A population of chemoresistant quiescent/slow cycling cells was isolated through PKH26 staining (which allows to separate cells on the basis of their proliferation rate) from colorectal cancer (CRC) xenografts and subjected to global gene expression and pathway activation analyses. Factors expressed by the quiescent/slow cycling population were analyzed through lentiviral overexpression approaches for their ability to induce a dormant chemoresistant state both in vitro and in mouse xenografts. The correlation between quiescence-associated factors, CRC consensus molecular subtype and cancer prognosis was analyzed in large patient datasets. RESULTS: Untreated colorectal tumors contain a population of quiescent/slow cycling cells with stem cell features (quiescent cancer stem cells, QCSCs) characterized by a predetermined mesenchymal-like chemoresistant phenotype. QCSCs expressed increased levels of ZEB2, a transcription factor involved in stem cell plasticity and epithelial-mesenchymal transition (EMT), and of antiapototic factors pCRAF and pASK1. ZEB2 overexpression upregulated pCRAF/pASK1 levels resulting in increased chemoresistance, enrichment of cells with stemness/EMT traits and proliferative slowdown of tumor xenografts. In parallel, chemotherapy treatment of tumor xenografts induced the prevalence of QCSCs with a stemness/EMT phenotype and activation of the ZEB2/pCRAF/pASK1 axis, resulting in a chemotherapy-unresponsive state. In CRC patients, increased ZEB2 levels correlated with worse relapse-free survival and were strongly associated to the consensus molecular subtype 4 (CMS4) characterized by dismal prognosis, decreased proliferative rates and upregulation of EMT genes. CONCLUSIONS: These results show that chemotherapy-naive tumors contain a cell population characterized by a coordinated program of chemoresistance, quiescence, stemness and EMT. Such population becomes prevalent upon drug treatment and is responsible for chemotherapy resistance, thus representing a key target for more effective therapeutic approaches.


Subject(s)
Colorectal Neoplasms/pathology , Drug Resistance, Neoplasm , Neoplastic Stem Cells/metabolism , Up-Regulation , Zinc Finger E-box Binding Homeobox 2/genetics , Zinc Finger E-box Binding Homeobox 2/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Colorectal Neoplasms/metabolism , Epithelial-Mesenchymal Transition , Female , Fluorouracil/pharmacology , Gene Expression Regulation, Neoplastic , Humans , MAP Kinase Kinase Kinase 5/metabolism , Male , Mice , Middle Aged , Neoplasm Transplantation , Oxaliplatin/pharmacology , Prognosis
2.
Gastroenterology ; 138(7): 2357-67, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20188103

ABSTRACT

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 formation.


Subject(s)
Colorectal Neoplasms/pathology , Liver Neoplasms/secondary , Proto-Oncogene Proteins/physiology , Receptors, TNF-Related Apoptosis-Inducing Ligand/physiology , TNF-Related Apoptosis-Inducing Ligand/physiology , fas Receptor/physiology , ras Proteins/physiology , Animals , Apoptosis , Caspase 8/metabolism , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/physiology , Humans , Lim Kinases/physiology , Mice , Proto-Oncogene Proteins c-raf/physiology , Proto-Oncogene Proteins p21(ras) , rho-Associated Kinases/physiology
3.
Immunobiology ; 207(2): 115-27, 2003.
Article in English | MEDLINE | ID: mdl-12675269

ABSTRACT

Natural Killer (NK) cells can induce apoptosis in target cells in at least four ways: by secretion of granzyme B/perforin (GrB/P) and via the CD95L, TRAIL and TNF-alpha pathways. In this study we examined the pathways used by interleukin-2 activated rat NK (A-NK) cells to induce apoptosis in the rat colon carcinoma cell line CC531s. Co-incubation of A-NK cells with CC531s cells for three hours resulted in 70% apoptosis in the latter. Addition of the GrB/P pathway-inhibitor concanamycin A reduced the number of apoptotic cells to 54%. Blockade of the CD95L, TRAIL and TNF-alpha pathways by specific antibodies hardly had an additional effect. However, co-incubation with transfected MEC cells that expressed CD95L or 2PK3-cells that expressed TRAIL did induce apoptosis in CC531s cells. Furthermore the A-NK cells contained CD95L and TRAIL. However, comparison of non- and permeabilized cells revealed that the majority of TRAIL was present in the cytosol of A-NK cells and was not available for induction of apoptosis. The presence of elevated levels of bcl-2 in CC531 cells reduced the sensitivity towards induction of apoptosis both by A-NK cells as well as the CD95L and TRAIL expressing cell lines. Using the caspase-inhibitors ac-IEPD-CHO, ac-DEVD-CHO and zVAD-fmk, it was shown that inhibition of the effector caspase-3 prevented A-NK cell induced apoptosis in CC531-bcl-2 cells, but not in CC531s cells. In conclusion, A-NK cells kill by secretion of GrB/P and not by the CD95L, TRAIL or TNF pathways albeit both CD95L and TRAIL are produced by the A-NK cells.


Subject(s)
Apoptosis/physiology , Cytotoxicity, Immunologic/physiology , Killer Cells, Natural/physiology , Membrane Glycoproteins/metabolism , Tumor Necrosis Factor-alpha/metabolism , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins , Carcinoma/metabolism , Carcinoma/pathology , Caspase Inhibitors , Caspases/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Enzyme Inhibitors/pharmacology , Fas Ligand Protein , Flow Cytometry , Granzymes , Humans , Immunoblotting , Interleukin-2/metabolism , Male , Mice , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Serine Endopeptidases/metabolism , TNF-Related Apoptosis-Inducing Ligand , Tumor Cells, Cultured
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