Inhibition of proprotein convertases activity results in repressed stemness and invasiveness of cancer stem cells in gastric cancer.

BACKGROUND: Gastric cancer (GC), the fourth leading cause of cancer-related death worldwide, with most deaths caused by advanced and metastatic disease, has limited curative options. Here, we revealed the importance of proprotein convertases (PCs) in the malignant and metastatic potential of GC cells through the regulation of the YAP/TAZ/TEAD pathway and epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSC). METHODS: The general PCs inhibitor, decanoyl-RVKR-chloromethyl-ketone (CMK), was used to repress PCs activity in CSCs of various GC cell lines. Their tumorigenic properties, drug resistance, YAP/TAZ/TEAD pathway activity, and invasive properties were then investigated in vitro, and their metastatic properties were explored in a mouse xenograft model. The prognostic value of PCs in GC patients was also explored in molecular databases of GC. RESULTS: Inhibition of PCs activity in CSCs in all GC cell lines reduced tumorsphere formation and growth, drug efflux, EMT phenotype, and invasive properties that are associated with repressed YAP/TAZ/TEAD pathway activity in vitro. In vivo, PCs' inhibition in GC cells reduced their metastatic spread. Molecular analysis of tumors from GC patients has highlighted the prognostic value of PCs. CONCLUSIONS: PCs are overexpressed in GC and associated with poor prognosis. PCs are involved in the malignant and metastatic potential of CSCs via the regulation of EMT, the YAP/TAZ/TEAD oncogenic pathway, and their stemness and invasive properties. Their repression represents a new strategy to target CSCs and impair metastatic spreading in GC.

CD44v3 is a marker of invasive cancer stem cells driving metastasis in gastric carcinoma.

BACKGROUND: Cancer stem cells (CSCs) are at the origin of tumour initiation and progression in gastric adenocarcinoma (GC). However, markers of metastasis-initiating cells remain unidentified in GC. In this study, we characterized CD44 variants expressed in GC and evaluated the tumorigenic and metastatic properties of CD44v3+ cells and their clinical significance in GC patients. METHODS: Using GC cell lines and patient-derived xenografts, we evaluated CD44+ and CD44v3+ GC cells molecular signature and their tumorigenic, chemoresistance, invasive and metastatic properties, and expression in patients-derived tissues. RESULTS: CD44v3+ cells, which represented a subpopulation of CD44+ cells, were detected in advanced preneoplastic lesions and presented CSCs chemoresistance and tumorigenic properties in vitro and in vivo. Molecular and functional analyses revealed two subpopulations of gastric CSCs: CD44v3+ CSCs with an epithelial-mesenchymal transition (EMT)-like signature, and CD44+/v3- CSCs with an epithelial-like signature; both were tumorigenic but CD44v3+ cells showed higher invasive and metastatic properties in vivo. CD44v3+ cells detected in the primary tumours of GC patients were associated with a worse prognosis. CONCLUSION: CD44v3 is a marker of a subpopulation of CSCs with metastatic properties in GC. The identification of metastasis-initiating cells in GC represents a major advance for further development of anti-metastatic therapeutic strategies.

Targeting metastasis-initiating cancer stem cells in gastric cancer with leukaemia inhibitory factor.

Gastric cancer's (GC) bad prognosis is usually associated with metastatic spread. Invasive cancer stem cells (CSC) are considered to be the seed of GC metastasis and not all CSCs are able to initiate metastasis. Targeting these aggressive metastasis-initiating CSC (MIC) is thus vital. Leukaemia inhibitory factor (LIF) is hereby used to target Hippo pathway oncogenic members, found to be induced in GC and associated with CSC features. LIF-treated GC cell lines, patient-derived xenograft (PDX) cells and/or CSC tumourspheres underwent transcriptomics, laser microdissection-associated proteomics, 2D and 3D invasion assays and in vivo xenograft in mice blood circulation. LIFR expression was analysed on tissue microarrays from GC patients and in silico from public databases. LIF-treated cells, especially CSC, presented decreased epithelial to mesenchymal transition (EMT) phenotype and invasion capacity in vitro, and lower metastasis initiation ability in vivo. These effects involved both the Hippo and Jak/Stat pathways. Finally, GC's high LIFR expression was associated with better clinical outcomes in patients. LIF treatment could thus represent a targeted anti-CSC strategy to fight against metastatic GC, and LIFR detection in primary tumours could constitute a potential new prognosis marker in this disease.

Downregulation of Glutamine Synthetase, not glutaminolysis, is responsible for glutamine addiction in Notch1-driven acute lymphoblastic leukemia.

The cellular receptor Notch1 is a central regulator of T-cell development, and as a consequence, Notch1 pathway appears upregulated in > 65% of the cases of T-cell acute lymphoblastic leukemia (T-ALL). However, strategies targeting Notch1 signaling render only modest results in the clinic due to treatment resistance and severe side effects. While many investigations reported the different aspects of tumor cell growth and leukemia progression controlled by Notch1, less is known regarding the modifications of cellular metabolism induced by Notch1 upregulation in T-ALL. Previously, glutaminolysis inhibition has been proposed to synergize with anti-Notch therapies in T-ALL models. In this work, we report that Notch1 upregulation in T-ALL induced a change in the metabolism of the important amino acid glutamine, preventing glutamine synthesis through the downregulation of glutamine synthetase (GS). Downregulation of GS was responsible for glutamine addiction in Notch1-driven T-ALL both in vitro and in vivo. Our results also confirmed an increase in glutaminolysis mediated by Notch1. Increased glutaminolysis resulted in the activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, a central controller of cell growth. However, glutaminolysis did not play any role in Notch1-induced glutamine addiction. Finally, the combined treatment targeting mTORC1 and limiting glutamine availability had a synergistic effect to induce apoptosis and to prevent Notch1-driven leukemia progression. Our results placed glutamine limitation and mTORC1 inhibition as a potential therapy against Notch1-driven leukemia.

mTOR Inhibition via Displacement of Phosphatidic Acid Induces Enhanced Cytotoxicity Specifically in Cancer Cells.

The mTOR is a central regulator of cell growth and is highly activated in cancer cells to allow rapid tumor growth. The use of mTOR inhibitors as anticancer therapy has been approved for some types of tumors, albeit with modest results. We recently reported the synthesis of ICSN3250, a halitulin analogue with enhanced cytotoxicity. We report here that ICSN3250 is a specific mTOR inhibitor that operates through a mechanism distinct from those described for previous mTOR inhibitors. ICSN3250 competed with and displaced phosphatidic acid from the FRB domain in mTOR, thus preventing mTOR activation and leading to cytotoxicity. Docking and molecular dynamics simulations evidenced not only the high conformational plasticity of the FRB domain, but also the specific interactions of both ICSN3250 and phosphatidic acid with the FRB domain in mTOR. Furthermore, ICSN3250 toxicity was shown to act specifically in cancer cells, as noncancer cells showed up to 100-fold less sensitivity to ICSN3250, in contrast to other mTOR inhibitors that did not show selectivity. Thus, our results define ICSN3250 as a new class of mTOR inhibitors that specifically targets cancer cells.Significance: ICSN3250 defines a new class of mTORC1 inhibitors that displaces phosphatidic acid at the FRB domain of mTOR, inducing cell death specifically in cancer cells but not in noncancer cells. Cancer Res; 78(18); 5384-97. ©2018 AACR.

Escaping mTOR inhibition for cancer therapy: Tumor suppressor functions of mTOR.

A master promoter of cell growth, mammalian target of rapamycin (mTOR) is upregulated in a large percentage of cancer cells. Still, targeting mTOR using rapamycin has a limited outcome in patients. Our recent results highlight the additional role of mTOR as a tumor suppressor, explaining these modest results in the clinic.

mTORC1 inhibition in cancer cells protects from glutaminolysis-mediated apoptosis during nutrient limitation.

A master coordinator of cell growth, mTORC1 is activated by different metabolic inputs, particularly the metabolism of glutamine (glutaminolysis), to control a vast range of cellular processes, including autophagy. As a well-recognized tumour promoter, inhibitors of mTORC1 such as rapamycin have been approved as anti-cancer agents, but their overall outcome in patients is rather poor. Here we show that mTORC1 also presents tumour suppressor features in conditions of nutrient restrictions. Thus, the activation of mTORC1 by glutaminolysis during nutritional imbalance inhibits autophagy and induces apoptosis in cancer cells. Importantly, rapamycin treatment reactivates autophagy and prevents the mTORC1-mediated apoptosis. We also observe that the ability of mTORC1 to activate apoptosis is mediated by the adaptor protein p62. Thus, the mTORC1-mediated upregulation of p62 during nutrient imbalance induces the binding of p62 to caspase 8 and the subsequent activation of the caspase pathway. Our data highlight the role of autophagy as a survival mechanism upon rapamycin treatment.

Prolyl hydroxylase domain enzymes and their role in cell signaling and cancer metabolism.

The prolyl hydroxylase domain (PHD) enzymes regulate the stability of the hypoxia-inducible factor (HIF) in response to oxygen availability. During oxygen limitation, the inhibition of PHD permits the stabilization of HIF, allowing the cellular adaptation to hypoxia. This adaptation is especially important for solid tumors, which are often exposed to a hypoxic environment. However, and despite their original role as the oxygen sensors of the cell, PHD are currently known to display HIF-independent and hydroxylase-independent functions in the control of different cellular pathways, including mTOR pathway, NF-kB pathway, apoptosis and cellular metabolism. In this review, we summarize the recent advances in the regulation and functions of PHD in cancer signaling and cell metabolism.