Notch receptor/ligand diversity: contribution to colorectal cancer stem cell heterogeneity.

Cancer cell heterogeneity is a key contributor to therapeutic failure and post-treatment recurrence. Targeting cell subpopulations responsible for chemoresistance and recurrence seems to be an attractive approach to improve treatment outcome in cancer patients. However, this remains challenging due to the complexity and incomplete characterization of tumor cell subpopulations. The heterogeneity of cells exhibiting stemness-related features, such as self-renewal and chemoresistance, fuels this complexity. Notch signaling is a known regulator of cancer stem cell (CSC) features in colorectal cancer (CRC), though the effects of its heterogenous signaling on CRC cell stemness are only just emerging. In this review, we discuss how Notch ligand-receptor specificity contributes to regulating stemness, self-renewal, chemoresistance and cancer stem cells heterogeneity in CRC.

Notch3 regulates Mybl2 via HeyL to limit proliferation and tumor initiation in breast cancer.

Notch signaling is a conserved signaling pathway that participates in many aspects of mammary gland development and homeostasis, and has extensively been associated with breast tumorigenesis. Here, to unravel the as yet debated role of Notch3 in breast cancer development, we investigated its expression in human breast cancer samples and effects of its loss in mice. Notch3 expression was very weak in breast cancer cells and was associated with good patient prognosis. Interestingly, its expression was very strong in stromal cells of these patients, though this had no prognostic value. Mechanistically, we demonstrated that Notch3 prevents tumor initiation via HeyL-mediated inhibition of Mybl2, an important regulator of cell cycle. In the mammary glands of Notch3-deficient mice, we observed accelerated tumor initiation and proliferation in a MMTV-Neu model. Notch3-null tumors were enriched in Mybl2 mRNA signature and protein expression. Hence, our study reinforces the anti-tumoral role of Notch3 in breast tumorigenesis.

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network.

Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

Targeting netrin-3 in small cell lung cancer and neuroblastoma.

The navigation cue netrin-1 is well-documented for its key role in cancer development and represents a promising therapeutic target currently under clinical investigation. Phase 1 and 2 clinical trials are ongoing with NP137, a humanized monoclonal antibody against netrin-1. Interestingly, the epitope recognized by NP137 in netrin-1 shares 90% homology with its counterpart in netrin-3, the closest member to netrin-1 in humans, for which little is known in the field of cancer. Here, we unveiled that netrin-3 appears to be expressed specifically in human neuroblastoma (NB) and small cell lung cancer (SCLC), two subtypes of neuroectodermal/neuroendocrine lineages. Netrin-3 and netrin-1 expression are mutually exclusive, and the former is driven by the MYCN oncogene in NB, and the ASCL-1 or NeuroD1 transcription factors in SCLC. Netrin-3 expression is correlated with disease stage, aggressiveness, and overall survival in NB. Mechanistically, we confirmed the high affinity of netrin-3 for netrin-1 receptors and we demonstrated that netrin-3 genetic silencing or interference using NP137, delayed tumor engraftment, and reduced tumor growth in animal models. Altogether, these data support the targeting of netrin-3 in NB and SCLC.

Shaping of the Tumor Microenvironment by Notch Signaling.

The tumor microenvironment (TME) has become a major concern of cancer research both from a basic and a therapeutic point of view. Understanding the effect of a signaling pathway-and thus the effect of its targeting-in every aspect of the microenvironment is a prerequisite to predict and analyze the effect of a therapy. The Notch signaling pathway is involved in every component of the TME as well as in the interaction between the different parts of the TME. This review aims at describing how Notch signaling is impacting the TME and the consequences this may have when modulating Notch signaling in a therapeutic perspective.

Notch Signaling in the Tumor Microenvironment.

The Notch signaling pathway regulates many aspects of cancer biology. Most attention has been given to its role in the transformed cell. However, it is now clear that cancer progression and metastasis depend on the bidirectional interactions between cancer cells and their environment, forming the tumor microenvironment (TME). These interactions are mediated and constantly evolve through paracrine and juxtacrine signaling. In this review, we discuss how Notch signaling takes an important part in regulating the crosstalk between the different compartments of the TME. We also address the consequences of the Notch-TME involvement from a therapeutic perspective.

Non-canonical NOTCH3 signalling limits tumour angiogenesis.

Notch signalling is a causal determinant of cancer and efforts have been made to develop targeted therapies to inhibit the so-called canonical pathway. Here we describe an unexpected pro-apoptotic role of Notch3 in regulating tumour angiogenesis independently of the Notch canonical pathway. The Notch3 ligand Jagged-1 is upregulated in a fraction of human cancer and our data support the view that Jagged-1, produced by cancer cells, is inhibiting the apoptosis induced by the aberrant Notch3 expression in tumour vasculature. We thus present Notch3 as a dependence receptor inducing endothelial cell death while this pro-apoptotic activity is blocked by Jagged-1. Along this line, using Notch3 mutant mice, we demonstrate that tumour growth and angiogenesis are increased when Notch3 is silenced in the stroma. Consequently, we show that the well-documented anti-tumour effect mediated by γ-secretase inhibition is at least in part dependent on the apoptosis triggered by Notch3 in endothelial cells.

Regulation by miR181 family of the dependence receptor CDON tumor suppressive activity in neuroblastoma.

BACKGROUND: The Sonic Hedgehog (SHH) signaling pathway plays an important role in neural crest cell fate during embryonic development and has been implicated in the progression of multiple cancers that include neuroblastoma, a neural crest cell-derived disease. While most of the SHH signaling is mediated by the well-described canonical pathway leading to the activation of Smoothened and Gli, it has recently been shown that cell-adhesion molecule-related/downregulated by oncogenes (CDON) serves as a receptor for SHH and contributes to SHH-induced signaling. CDON has also been recently described as a dependence receptor, triggering apoptosis in the absence of SHH. This CDON proapoptotic activity has been suggested to constrain tumor progression. METHODS: CDON expression was analyzed by quantitative-reverse transcription-polymerase chain reaction in a panel of 226 neuroblastoma patients and associated with stages, overall survival, and expression of miR181 family members using Kaplan Meier and Pearson correlation methods. Cell death assays were performed in neuroblastoma cell lines and tumor growth was investigated in the chick chorioallantoic model. All statistical tests were two-sided. RESULTS: CDON expression was inversely associated with neuroblastoma aggressiveness (P < .001). Moreover, re-expression of CDON in neuroblastoma cell lines was associated with apoptosis in vitro and tumor growth inhibition in vivo. We show that CDON expression is regulated by the miR181 miRNA family, whose expression is directly associated with neuroblastoma aggressiveness (survival: high miR181-b 73.2% vs low miR181-b 54.6%; P = .03). CONCLUSIONS: Together, these data support the view that CDON acts as a tumor suppressor in neuroblastomas, and that CDON is tightly regulated by miRNAs.

Dishevelled limits Notch signalling through inhibition of CSL.

Notch and Wnt are highly conserved signalling pathways that are used repeatedly throughout animal development to generate a diverse array of cell types. However, they often have opposing effects on cell-fate decisions with each pathway promoting an alternate outcome. Commonly, a cell receiving both signals exhibits only Wnt pathway activity. This suggests that Wnt inhibits Notch activity to promote a Wnt-ON/Notch-OFF output; but what might underpin this Notch regulation is not understood. Here, we show that Wnt acts via Dishevelled to inhibit Notch signalling, and that this crosstalk regulates cell-fate specification in vivo during Xenopus development. Mechanistically, Dishevelled binds and directly inhibits CSL transcription factors downstream of Notch receptors, reducing their activity. Furthermore, our data suggest that this crosstalk mechanism is conserved between vertebrate and invertebrate homologues. Thus, we identify a dual function for Dishevelled as an inhibitor of Notch signalling and an activator of the Wnt pathway that sharpens the distinction between opposing Wnt and Notch responses, allowing for robust cell-fate decisions.

Is there more to Wnt signalling in breast cancer than stabilisation of beta-catenin?

Increased Wnt signalling has been implicated in the aetiology of many different human cancers, including breast cancers. In most cases, Wnt signalling is thought to drive tumourigenesis through the stabilisation of cytosolic beta-catenin and the subsequent changes in the expression of T-cell factor (TCF)-dependent genes. However, this is not necessarily the only mechanism, as Wnt proteins can signal through a number of different intracellular signalling pathways. The ongoing work from Nancy Hynes' laboratory continues to highlight this latter possibility.

Notch activation induces Akt signaling via an autocrine loop to prevent apoptosis in breast epithelial cells.

The Notch pathway is aberrantly activated in a wide range of cancers, including breast carcinoma, and is required to maintain the transformed phenotype of many of these tumors. Notch signaling contributes to the transformed phenotype, in part, by preventing apoptosis in response to many different stimuli. However, it is unclear how Notch activation can lead to a general suppression of apoptosis. We show here that Notch signaling induced an autocrine signaling loop that activates Akt in breast epithelial cells. This activation of Akt was necessary for Notch-induced protection against apoptosis in the nontransformed breast epithelial cell line MCF10A. Moreover, inhibiting Notch signaling in breast cancer cells induced a decrease in Akt activity and an increase in sensitivity to apoptosis. Finally, the inhibition of ASK1 by Akt was responsible for the protection from apoptosis induced by DNA damage, as it prevented c-Jun NH(2)-terminal kinase-mediated phosphorylation and activation of p53.

Cisplatin-induced apoptosis involves membrane fluidification via inhibition of NHE1 in human colon cancer cells.

We have previously shown that cisplatin triggers an early acid sphingomyelinase (aSMase)-dependent ceramide generation concomitantly with an increase in membrane fluidity and induces apoptosis in HT29 cells. The present study further explores the role and origin of membrane fluidification in cisplatin-induced apoptosis. The rapid increase in membrane fluidity following cisplatin treatment was inhibited by membrane-stabilizing agents such as cholesterol or monosialoganglioside-1. In HT29 cells, these compounds prevented the early aggregation of Fas death receptor and of membrane lipid rafts on cell surface and significantly inhibited cisplatin-induced apoptosis without altering drug intracellular uptake or cisplatin DNA adducts formation. Early after cisplatin treatment, Na+/H+ membrane exchanger-1 (NHE1) was inhibited leading to intracellular acidification, aSMase was activated, and ceramide was detected at the cell membrane. Treatment of HT29 cells with Staphylococcus aureus sphingomyelinase increased membrane fluidity. Moreover, pretreatment with cariporide, a specific inhibitor of NHE1, inhibited cisplatin-induced intracellular acidification, aSMase activation, ceramide membrane generation, membrane fluidification, and apoptosis. Finally, NHE1-expressing PS120 cells were more sensitive to cisplatin than NHE1-deficient PS120 cells. Altogether, these findings suggest that the apoptotic pathway triggered by cisplatin involves a very early NHE1-dependent intracellular acidification leading to aSMase activation and increase in membrane fluidity. These events are independent of cisplatin-induced DNA adducts formation. The membrane exchanger NHE1 may be another potential target of cisplatin, increasing cell sensitivity to this compound.

TRAIL induces receptor-interacting protein 1-dependent and caspase-dependent necrosis-like cell death under acidic extracellular conditions.

Tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is a potential anticancer agent that induces apoptosis in cancer cells but not in most normal cells. How tumor physiology, particularly acidic extracellular pH (pH(e)), would modify sensitivity of cancer cells to TRAIL-induced cell death is not known. We have previously shown that cancer cells, resistant to TRAIL-induced apoptosis at physiologic pH(e) (7.4), could be sensitized to TRAIL at acidic pH(e) (6.5). However, at this acidic pH(e), cell death was necrotic. We show here that, in spite of a necrosis-like cell death morphology, caspases are activated and are necessary for TRAIL-induced cell death at acidic pH(e) in HT29 human colon cancer cells. Furthermore, we observed that, whereas receptor-interacting protein (RIP) was cleaved following TRAIL treatment at physiologic pH(e) (7.4), it was not cleaved following TRAIL treatment at acidic pH(e) (6.5). Moreover, RIP degradation by geldanamycin or decrease expression of RIP by small RNA interference transfection inhibited TRAIL-induced necrosis at acidic pH(e), showing that RIP was necessary for this necrotic cell death pathway. We also show that RIP kinase activity was essential for this cell death pathway. Altogether, we show that, under acidic pH(e) conditions, TRAIL induces a necrosis-like cell death pathway that depends both on caspases and RIP kinase activity. Thus, our data suggest for the first time that RIP-dependent necrosis might be a major death pathway in TRAIL-based therapy in solid tumors with acidic pH(e).

Cytotoxicity of TRAIL/anticancer drug combinations in human normal cells.

TRAIL (TNF-alpha-Related Apoptosis-Inducing Ligand) is a promising anticancer agent. In fact, it induces apoptosis in cancer cells and not in most normal cells. Nevertheless, certain cancer cells are resistant to TRAIL-induced apoptosis and this could limit TRAIL's efficiency in cancer therapy. To overcome TRAIL resistance, a combination of TRAIL with chemotherapy could be used in cancer treatment. However, sensitivity of human normal cells to such combinations is not well known. We showed in this study that TRAIL/cisplatin, in contrast to TRAIL/5-fluorouracil, was toxic toward human primary hepatocytes and resting lymphocytes. Furthermore, both combinations are toxic toward PHA-IL2-activated lymphocytes. In contrast, freshly isolated neutrophils are resistant to TRAIL in combination or not with anticancer drugs.

Maintenance of embryonic auxin distribution for apical-basal patterning by PIN-FORMED-dependent auxin transport in Arabidopsis.

Molecular mechanisms of pattern formation in the plant embryo are not well understood. Recent molecular and cellular studies, in conjunction with earlier microsurgical, physiological, and genetic work, are now starting to define the outlines of a model where gradients of the signaling molecule auxin play a central role in embryo patterning. It is relatively clear how these gradients are established and interpreted, but how they are maintained is still unresolved. Here, we have studied the contributions of auxin biosynthesis, conjugation, and transport pathways to the maintenance of embryonic auxin gradients. Auxin homeostasis in the embryo was manipulated by region-specific conditional expression of indoleacetic acid-tryptophan monooxygenase or indoleacetic acid-lysine synthetase, bacterial enzymes for auxin biosynthesis or conjugation. Neither manipulation of auxin biosynthesis nor of auxin conjugation interfered with auxin gradients and patterning in the embryo. This result suggests a compensatory mechanism for buffering auxin gradients in the embryo. Chemical and genetic inhibition revealed that auxin transport activity, in particular that of the PIN-FORMED1 (PIN1) and PIN4 proteins, is a major factor in the maintenance of these gradients.

Role of early plasma membrane events in chemotherapy-induced cell death.

Most current anticancer therapies induce tumor cell death through the induction of apoptosis. However, the pathways leading to cell death are not always understood. For example, for several DNA-damaging agents the specific biochemical lesions (DNA damage) have been associated with the induction of apoptosis. However, several of these DNA-damaging agents (cisplatin, 1-beta-arabinofuranosylcytosine, daunorubicin or doxorubicin) as well as other antitumor agents, such as edelfosine or resveratrol, have been recently shown to induce apoptosis via signaling through plasma membrane lipid rafts involving the death receptor pathway. In this review we focus on the role of early plasma membrane events in chemotherapy-induced cell death. Special attention is given to changes in plasma membrane fluidity, activation of the acid sphingomyelinase and the Fas death pathway in response to chemotherapy as well as their possible interrelationships.

Role of intracellular glutathione in cell sensitivity to the apoptosis induced by tumor necrosis factor {alpha}-related apoptosis-inducing ligand/anticancer drug combinations.

PURPOSE: We have recently shown that combination of tumor necrosis factor alpha-related apoptosis-inducing ligand (TRAIL) with anticancer drugs induced an apoptotic cell death pathway involving both caspases and mitochondria. The present work further explores the role of intracellular reduced glutathione (GSH) level in cell sensitivity to this cell death pathway. EXPERIMENTAL DESIGN: Intracellular GSH level was measured by high-performance liquid chromatography. Cell death was detected by immunofluorescence after Hoechst 33342/propidium iodide staining. Reactive oxygen species production was evaluated by flow cytometry after dihydroethidium probe labeling. Western blot analysis was done to study stress-activated protein kinase/c-jun NH(2)-terminal kinase (SAPK/JNK) phosphorylation. The Student's t test was used to determine significance of the results. Three to six experiments were done. RESULTS: GSH depletion enhanced apoptosis induced by TRAIL/cisplatin (CDDP) or TRAIL/5-fluorouracil (5-FU) combinations in both human HT29 colon carcinoma and HepG2 hepatocarcinoma cells, whereas it enhanced cytotoxicity induced only by TRAIL/CDDP in human primary hepatocytes. Our results further suggested that GSH depletion enhanced SAPK/JNK phosphorylation upon TRAIL/5-FU exposure and likely reduced the detoxification mechanisms of CDDP in HT29 cells. Resistance of Bcl-2-expressing HT29 and HepG2 cells to combined treatment was not overcome by GSH depletion, thus indicating that Bcl-2-mediated antiapoptotic effect occurs independently of intracellular GSH level. CONCLUSION: GSH depletion could be useful to increase the therapeutic efficacy of cancer treatment by TRAIL/anticancer drug combinations. Furthermore, TRAIL/5-FU combination might be a potential anticancer treatment of human tumors, being ineffective on human primary hepatocytes and thus could be of interest in clinical cancer treatment. Nevertheless, Bcl-2 expression remains an important resistance factor.

TRAIL (TNF-related apoptosis-inducing ligand) induces necrosis-like cell death in tumor cells at acidic extracellular pH.

How tumor microenvironment, more specifically low extracellular pH (6.5), alters cell response to TNF-related apoptosis-inducing ligand (TRAIL)-based cancer therapy has yet to be determined. The aim of the current work was to test the effect of acidic extracellular pH on TRAIL-induced cell death in human HT29 colon carcinoma and HepG2 hepatocarcinoma cell lines as well as in human primary hepatocytes. We found an increase in TRAIL sensitivity at low extracellular pH, which is partially inhibited by Bcl-2 expression in HT29 cells. At low extracellular pH, TRAIL induced a new form of cell death, sharing necrotic and apoptotic features in tumor cells. By contrast, human primary hepatocytes were resistant to TRAIL-induced cell death even at acidic extracellular pH.