Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity.

Colon carcinoma is one of the leading causes of death from cancer and is characterized by a heterogenic pool of cells with distinct differentiation patterns. Recently, it was reported that a population of undifferentiated cells from a primary tumor, so-called cancer stem cells (CSC), can reconstitute the original tumor on xenotransplantation. Here, we show that spheroid cultures of these colon CSCs contain expression of CD133, CD166, CD44, CD29, CD24, Lgr5, and nuclear beta-catenin, which have all been suggested to mark the (cancer) stem cell population. More importantly, by using these spheroid cultures or freshly isolated tumor cells from multiple colon carcinomas, we now provide compelling evidence to indicate that the capacity to propagate a tumor with all differentiated progeny resides in a single CSC. Single-cell-cloned CSCs can form an adenocarcinoma on xenotransplantation but do not generate the stroma within these tumors. Moreover, they can self-renew and are capable of multilineage differentiation. Further analysis indicated that the lineage decision is dictated by phosphoinositide 3-kinase (PI3K) signaling in CSCs. These data support the hypothesis that tumor hierarchy can be traced back to a single CSC that contains multilineage differentiation capacity, and provides clues to the regulation of differentiation in colon cancers in vivo.

Cancer stem cells--old concepts, new insights.

Cancer has long been viewed as an exclusively genetic disorder. The model of carcinogenesis, postulated by Nowell and Vogelstein, describes the formation of a tumor by the sequential accumulation of mutations in oncogenes and tumor suppressor genes. In this model, tumors are thought to consist of a heterogeneous population of cells that continue to acquire new mutations, resulting in a highly dynamic process, with clones that out compete others due to increased proliferative or survival capacity. However, novel insights in cancer stem cell research suggest another layer of complexity in the process of malignant transformation and preservation. It has been reported that only a small fraction of the cancer cells in a malignancy have the capacity to propagate the tumor upon transplantation into immuno-compromised mice. Those cells are termed 'cancer stem cells' (CSC) and can be selected based on the expression of cell surface markers associated with immature cell types. In this review, we will critically discuss these novel insights in CSC-related research. Where possible we integrate these results within the genetic model of cancer and illustrate that the CSC model can be considered an extension of the classic genetic model rather than a contradictory theory. Finally, we discuss some of the most controversial issues in this field.

Specific resistance upon lentiviral TRAIL transfer by intracellular retention of TRAIL receptors.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in many transformed cells, suggesting TRAIL as an ideal candidate for cancer gene therapy. A main obstacle in cancer therapy is intrinsic or acquired therapy resistance of malignant cells. To study induction of resistance against TRAIL, we generated lentiviral vectors allowing efficient TRAIL expression and apoptosis induction in a variety of human cancer cell lines. Within days upon TRAIL overexpression, cells became resistant towards TRAIL, but not to CD95 ligation or DNA damage by cisplatin. Cell surface expression of TRAIL receptors 1 and 2 was completely abrogated in resistant cells due to intracellular retention of the receptors by TRAIL. SiRNA directed against TRAIL resensitized the resistant cells by restoring cell surface expression of TRAIL receptors. These findings represent a novel resistance mechanism towards TRAIL, specifically caused by TRAIL overexpression, and question the use of TRAIL expression in tumor-cell targeting gene therapy.

Apoptosis mediated by lentiviral TRAIL transfer involves transduction-dependent and -independent effects.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, which selectively induces apoptosis in many transformed cells without apparent toxic side effects in normal tissue. We recently described the construction and characterization of a lentiviral vector for expression of TRAIL. In this report, we evaluate its suitability for therapeutic application. In vitro, we observed specific induction of apoptosis upon transduction in human lung cancer cells. Cell death was partially dependent on successful integration and TRAIL expression by the vectors, but was to some extent mediated by protein carryover, as we found TRAIL protein associated with virus particles. Transduction of subcutaneously growing lung tumors on nude mice with lentiviral TRAIL mediated a transient suppression of tumor growth. Analysis of tumor sections revealed that transduction efficiency of lentiviral control vector but not of lentiviral TRAIL vector was high. This was because of the direct cytotoxic activity of recombinant TRAIL present in viral particles, which prevented efficient tumor transduction. These data therefore suggest that enveloped viral vectors constitutively expressing TRAIL are well suited for ex vivo applications, such as the transduction of tumor-homing cells, but may have a lower effect when used directly for the transduction of tumor cells in vivo.

Enhanced caspase-8 recruitment to and activation at the DISC is critical for sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by chemotherapeutic drugs.

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumour activity upon systemic administration in mice without showing the deleterious side effects observed with other apoptosis-inducing members of the TNF family such as TNF and CD95L. TRAIL may, thus, have great potential in the treatment of human cancer. However, about 60% of tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of tumour resistance to TRAIL, we investigated hepatocellular carcinoma (HCC) cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs, for example, 5-fluorouracil (5-FU), rendered the TRAIL-resistant HCC cell lines sensitive to TRAIL-induced apoptosis. Analysis of the TRAIL death-inducing signalling complex (DISC) revealed upregulation of TRAIL-R2. Caspase-8 recruitment to and its activation at the DISC were substantially increased after 5-FU sensitisation, while FADD recruitment remained essentially unchanged. 5-FU pretreatment downregulated cellular FLICE-inhibitory protein (cFLIP) and specific cFLIP downregulation by small interfering RNA was sufficient to sensitise TRAIL-resistant HCC cell lines for TRAIL-induced apoptosis. Thus, a potential mechanism for TRAIL sensitisation by 5-FU is the increased effectiveness of caspase-8 recruitment to and activation at the DISC facilitated by the downregulation of cFLIP and the consequent shift in the ratio of caspase-8 to cFLIP at the DISC.

CD95 promotes metastatic spread via Sck in pancreatic ductal adenocarcinoma.

Cancer stem cells (CSCs) have been implicated in the initiation and maintenance of tumour growth as well as metastasis. Recent reports link stemness to epithelial-mesenchymal transition (EMT) in cancer. However, there is still little knowledge about the molecular markers of those events. In silico analysis of RNA profiles of 36 pancreatic ductal adenocarcinomas (PDAC) reveals an association of the expression of CD95 with EMT and stemness that was validated in CSCs isolated from PDAC surgical specimens. CD95 expression was also higher in metastatic pancreatic cells than in primary PDAC. Pharmacological inhibition of CD95 activity reduced PDAC growth and metastasis in CSC-derived xenografts and in a murine syngeneic model. On the mechanistic level, Sck was identified as a novel molecule indispensable for CD95's induction of cell cycle progression. This study uncovers CD95 as a marker of EMT and stemness in PDAC. It also addresses the molecular mechanism by which CD95 drives tumour growth and opens tantalizing therapeutic possibilities in PDAC.

Therapy-resistant tumor microvascular endothelial cells contribute to treatment failure in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a devastating disease with high mortality and poor prognosis. Cancer stem cells (CSCs) have recently been defined as a fraction of tumor cells highly resistant to therapy and subsequently considered to be responsible for tumor recurrence. These cells have been characterized in GBM and suggested to reside in and be supported by the tumor microvascular niche. Here we evaluated the response of tumor microvascular endothelial cells (tMVECs) to radio- and chemotherapy, and analyzed how this affects their interaction with CSCs. Our data demonstrate that tMVECs exhibit extreme resistance to both therapies, with the main response to irradiation being senescence. Importantly, senescent tMVECs can be detected in human GBM samples as well as in mice upon irradiation. Even though permanently arrested, they are still viable and able to support CSC growth with the same efficacy as non-senescent tMVECs. Intriguingly, GBM CSCs themselves are capable of differentiating into cells with similar features as tMVECs that subsequently undergo senescence when exposed to radiation. This indicates that endothelial-like cells are therapy resistant and, more importantly, support expansion of GBM cells.

Suppression of cFLIP is sufficient to sensitize human melanoma cells to TRAIL- and CD95L-mediated apoptosis.

Death ligands such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and certain forms of CD95L are attractive therapeutic options for metastatic melanoma. Since knowledge about the regulation of death receptor sensitivity in melanoma is sparse, we have analysed these signaling pathways in detail. The loss of CD95 or TRAIL-R1, but not of TRAIL-R2, surface expression correlated with apoptosis sensitivity in a panel of melanoma cell lines. In contrast, the expression of proteins of the apical apoptosis signaling cascade (FADD, initiator caspases-8 and cFLIP) did not predict apoptosis sensitivity. Since both TRAIL-R1 and -R2 transmit apoptotic signals, we asked whether cFLIP, highly expressed in several of the cell lines tested, is sufficient to maintain resistance to TRAIL-R2-mediated apoptosis. Downregulation of cFLIP in TRAIL-R2-positive, TRAIL-resistant IGR cells dramatically increased TRAIL sensitivity. Conversely ectopic expression of cFLIP in TRAIL-sensitive, TRAIL-R2-expressing RPM-EP melanoma cells inhibited TRAIL- and CD95L-mediated cell death. Thus, modulation of cFLIP is sufficient to sensitize TRAIL-R2-expressing cells for TRAIL. Taken together, albeit expressing all proteins necessary for death receptor-mediated apoptosis, TRAIL-R1 negative melanoma cells cannot undergo TRAIL- or CD95L-induced apoptosis due to expression of cFLIP. Hence, cFLIP represents an attractive therapeutic target for melanoma treatment, especially in combination with TRAIL receptor agonists.

FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2.

Apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO-2L) has been shown to exert important functions during various immunological processes. The involvement of the death adaptor proteins FADD/MORT1, TRADD, and RIP and the apoptosis-initiating caspases-8 and -10 in death signaling by the two death-inducing TRAIL receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are controversial. Analysis of the native TRAIL death-inducing signaling complex (DISC) revealed ligand-dependent recruitment of FADD/MORT1 and caspase-8. Differential precipitation of ligand-stimulated TRAIL receptors demonstrated that FADD/MORT1 and caspase-8 were recruited to TRAIL-R1 and TRAIL-R2 independently of each other. FADD/MORT1- and caspase-8-deficient Jurkat cells expressing only TRAIL-R2 were resistant to TRAIL-induced apoptosis. Thus, FADD/MORT1 and caspase-8 are essential for apoptosis induction via TRAIL-R2.