Metabolism-Based Therapeutic Strategies Targeting Cancer Stem Cells

Cancer heterogeneity constitutes the major source of disease progression and therapy failure. Tumors comprise functionally diverse subpopulations, with cancer stem cells (CSCs) as the source of this heterogeneity. Since these cells bear in vivo tumorigenicity and metastatic potential, survive chemotherapy and drive relapse, its elimination may be the only way to achieve long-term survival in patients. Thanks to the great advances in the field over the last few years, we know now that cellular metabolism and stemness are highly intertwined in normal development and cancer. Indeed, CSCs show distinct metabolic features as compared with their more differentiated progenies, though their dominant metabolic phenotype varies across tumor entities, patients and even subclones within a tumor. Following initial works focused on glucose metabolism, current studies have unveiled particularities of CSC metabolism in terms of redox state, lipid metabolism and use of alternative fuels, such as amino acids or ketone bodies. In this review, we describe the different metabolic phenotypes attributed to CSCs with special focus on metabolism-based therapeutic strategies tested in preclinical and clinical settings

Inhibition of mitochondrial dynamics preferentially targets pancreatic cancer cells with enhanced tumorigenic and invasive potential

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumors, partly due to its intrinsic aggressiveness, metastatic potential, and chemoresistance of the contained cancer stem cells (CSCs). Pancreatic CSCs strongly rely on mitochondrial metabolism to maintain their stemness, therefore representing a putative target for their elimination. Since mitochondrial homeostasis de-pends on the tightly controlled balance between fusion and fission processes, namely mitochondrial dynamics, we aim to study this mechanism in the context of stemness. In human PDAC tissues, the mitochondrial fission gene DNM1L (DRP1) was overexpressed and positively correlated with the stemness signature. Moreover, we observe that primary human CSCs display smaller mitochondria and a higher DRP1/MFN2 expression ratio, indicating the activation of the mitochondrial fission. In-terestingly, treatment with the DRP1 inhibitor mDivi-1 induced dose-dependent apoptosis, especially in CD133+ CSCs, due to the accumulation of dysfunctional mitochondria and the subsequent energy crisis in this subpopulation. Mechanistically, mDivi-1 inhibited stemness-related features, such as self-renewal, tumorigenicity, and invasiveness and chemosensitized the cells to the cytotoxic effects of Gemcitabine. In summary, mitochondrial fission is an essential process for pancreatic CSCs and represents an attractive target for designing novel multimodal treatments that will more efficiently eliminate cells with high tumorigenic potentialThis research was funded by the Pancreatic Cancer Research Fund, 2015 Award Round (P.S., C.H.); the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement n 602783 (CAM-PaC) (C.H.), theWorldwide Cancer Research Charity together with Fundación Científica Asociación Española contra el Cáncer (FCAECC) (19-0250) (P.S.); A Fero Foundation grant and a Coordinated grant (GC16173694BARB) from the Fundación Asociación Española Contra el Cáncer (AECC) (B.S.J.); and the Instituto de Salud Carlos III through the Miguel Servet Program (CP16/00121) and Fondo de Investigaciones Sanitarias (PI17/00082) (both co-financed by European funds (FSE: “El FSE invierte en tu futuro” and FEDER: “Una manera de hacer Europa,” respectively) (P.S.

ADAM22/LGI1 complex as a new actionable target for breast cancer brain metastasis

Background: Metastatic breast cancer is a major cause of cancer-related deaths in woman. Brain metastasis is a common and devastating site of relapse for several breast cancer molecular subtypes, including oestrogen receptor-positive disease, with life expectancy of less than a year. While efforts have been devoted to developing therapeutics for extra-cranial metastasis, drug penetration of blood–brain barrier (BBB) remains a major clinical challenge. Defining molecular alterations in breast cancer brain metastasis enables the identification of novel actionable targets.Methods: Global transcriptomic analysis of matched primary and metastatic patient tumours (n = 35 patients, 70 tumour samples) identified a putative new actionable target for advanced breast cancer which was further validated in vivo and in breast cancer patient tumour tissue (n = 843 patients). A peptide mimetic of the target's natural ligand was designed in silico and its efficacy assessed in in vitro, ex vivo and in vivo models of breast cancer metastasis.Results: Bioinformatic analysis of over-represented pathways in metastatic breast cancer identified ADAM22 as a top ranked member of the ECM-related druggable genome specific to brain metastases. ADAM22 was validated as an actionable target in in vitro, ex vivo and in patient tumour tissue (n = 843 patients). A peptide mimetic of the ADAM22 ligand LGI1, LGI1MIM, was designed in silico. The efficacy of LGI1MIM and its ability to penetrate the BBB were assessed in vitro, ex vivo and in brain metastasis BBB 3D biometric biohybrid models, respectively. Treatment with LGI1MIM in vivo inhibited disease progression, in particular the development of brain metastasis.Conclusion: ADAM22 expression in advanced breast cancer supports development of breast cancer brain metastasis. Targeting ADAM22 with a peptide mimetic LGI1MIM represents a new therapeutic option to treat metastatic brain disease

SNPs Associated with Cerebrospinal Fluid Phospho-Tau Levels Influence Rate of Decline in Alzheimer's Disease

Alzheimer's Disease (AD) is a complex and multifactorial disease. While large genome-wide association studies have had some success in identifying novel genetic risk factors for AD, case-control studies are less likely to uncover genetic factors that influence progression of disease. An alternative approach to identifying genetic risk for AD is the use of quantitative traits or endophenotypes. The use of endophenotypes has proven to be an effective strategy, implicating genetic risk factors in several diseases, including anemia, osteoporosis and heart disease. In this study we identify a genetic factor associated with the rate of decline in AD patients and present a methodology for identification of other such factors. We have used an established biomarker for AD, cerebrospinal fluid (CSF) tau phosphorylated at threonine 181 (ptau181) levels as an endophenotype for AD, identifying a SNP, rs1868402, in the gene encoding the regulatory sub-unit of protein phosphatase B, associated with CSF ptau181 levels in two independent CSF series . We show no association of rs1868402 with risk for AD or age at onset, but detected a very significant association with rate of progression of disease that is consistent in two independent series . Our analyses suggest that genetic variants associated with CSF ptau181 levels may have a greater impact on rate of progression, while genetic variants such as APOE4, that are associated with CSF Aβ42 levels influence risk and onset but not the rate of progression. Our results also suggest that drugs that inhibit or decrease tau phosphorylation may slow cognitive decline in individuals with very mild dementia or delay the appearance of memory problems in elderly individuals with low CSF Aβ42 levels. Finally, we believe genome-wide association studies of CSF tau/ptau181 levels should identify novel genetic variants which will likely influence rate of progression of AD

Identification and Validation of Novel Cerebrospinal Fluid Biomarkers for Staging Early Alzheimer's Disease

Ideally, disease modifying therapies for Alzheimer disease (AD) will be applied during the 'preclinical' stage (pathology present with cognition intact) before severe neuronal damage occurs, or upon recognizing very mild cognitive impairment. Developing and judiciously administering such therapies will require biomarker panels to identify early AD pathology, classify disease stage, monitor pathological progression, and predict cognitive decline. To discover such biomarkers, we measured AD-associated changes in the cerebrospinal fluid (CSF) proteome.CSF samples from individuals with mild AD (Clinical Dementia Rating [CDR] 1) (n = 24) and cognitively normal controls (CDR 0) (n = 24) were subjected to two-dimensional difference-in-gel electrophoresis. Within 119 differentially-abundant gel features, mass spectrometry (LC-MS/MS) identified 47 proteins. For validation, eleven proteins were re-evaluated by enzyme-linked immunosorbent assays (ELISA). Six of these assays (NrCAM, YKL-40, chromogranin A, carnosinase I, transthyretin, cystatin C) distinguished CDR 1 and CDR 0 groups and were subsequently applied (with tau, p-tau181 and Aβ42 ELISAs) to a larger independent cohort (n = 292) that included individuals with very mild dementia (CDR 0.5). Receiver-operating characteristic curve analyses using stepwise logistic regression yielded optimal biomarker combinations to distinguish CDR 0 from CDR>0 (tau, YKL-40, NrCAM) and CDR 1 from CDR<1 (tau, chromogranin A, carnosinase I) with areas under the curve of 0.90 (0.85-0.94 95% confidence interval [CI]) and 0.88 (0.81-0.94 CI), respectively.Four novel CSF biomarkers for AD (NrCAM, YKL-40, chromogranin A, carnosinase I) can improve the diagnostic accuracy of Aβ42 and tau. Together, these six markers describe six clinicopathological stages from cognitive normalcy to mild dementia, including stages defined by increased risk of cognitive decline. Such a panel might improve clinical trial efficiency by guiding subject enrollment and monitoring disease progression. Further studies will be required to validate this panel and evaluate its potential for distinguishing AD from other dementing conditions

Glutathione metabolism is essential for self-renewal and chemoresistance of pancreatic cancer stem cells

©The Author(s) 2020.[Background]:Cellular metabolism regulates stemness in health and disease. A reduced redox state is essential for self-renewal of normal and cancer stem cells (CSCs). However, while stem cells rely on glycolysis, different CSCs, including pancreatic CSCs, favor mitochondrial metabolism as their dominant energy-producing pathway. This suggests that powerful antioxidant networks must be in place to detoxify mitochondrial reactive oxygen species (ROS) and maintain stemness in oxidative CSCs. Since glutathione metabolism is critical for normal stem cell function and CSCs from breast, liver and gastric cancer show increased glutathione content, we hypothesized that pancreatic CSCs also rely on this pathway for ROS detoxification.[Aim]:To investigate the role of glutathione metabolism in pancreatic CSCs.[Methods]: Primary pancreatic cancer cells of patient-derived xenografts (PDXs) were cultured in adherent or CSC-enriching sphere conditions to determine the role of glutathione metabolism in stemness. Real-time polymerase chain reaction (PCR) was used to validate RNAseq results involving glutathione metabolism genes in adherent vs spheres, as well as the expression of pluripotency-related genes following treatment. Public TCGA and GTEx RNAseq data from pancreatic cancer vs normal tissue samples were analyzed using the webserver GEPIA2. The glutathione-sensitive fluorescent probe monochlorobimane was used to determine glutathione content by fluorimetry or flow cytometry. Pharmacological inhibitors of glutathione synthesis and recycling [buthionine-sulfoximine (BSO) and 6-Aminonicotinamide (6-AN), respectively] were used to investigate the impact of glutathione depletion on CSC-enriched cultures. Staining with propidium iodide (cell cycle), Annexin-V (apoptosis) and CD133 (CSC content) were determined by flow cytometry. Self-renewal was assessed by sphere formation assay and response to gemcitabine treatment was used as a readout for chemoresistance.[Results]:Analysis of our previously published RNAseq dataset E-MTAB-3808 revealed up-regulation of genes involved in the KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Glutathione Metabolism in CSC-enriched cultures compared to their differentiated counterparts. Consistently, in pancreatic cancer patient samples the expression of most of these up-regulated genes positively correlated with a stemness signature defined by NANOG, KLF4, SOX2 and OCT4 expression (P < 10-5). Moreover, 3 of the upregulated genes (MGST1, GPX8, GCCT) were associated with reduced disease-free survival in patients [Hazard ratio (HR) 2.2-2.5; P = 0.03-0.0054], suggesting a critical role for this pathway in pancreatic cancer progression. CSC-enriched sphere cultures also showed increased expression of different glutathione metabolism-related genes, as well as enhanced glutathione content in its reduced form (GSH). Glutathione depletion with BSO induced cell cycle arrest and apoptosis in spheres, and diminished the expression of stemness genes. Moreover, treatment with either BSO or the glutathione recycling inhibitor 6-AN inhibited self-renewal and the expression of the CSC marker CD133. GSH content in spheres positively correlated with intrinsic resistance to gemcitabine treatment in different PDXs r = 0.96, P = 5.8 × 1011). Additionally, CD133+ cells accumulated GSH in response to gemcitabine, which was abrogated by BSO treatment (P < 0.05). Combined treatment with BSO and gemcitabine-induced apoptosis in CD133+ cells to levels comparable to CD133- cells and significantly diminished self-renewal (P < 0.05), suggesting that chemoresistance of CSCs is partially dependent on GSH metabolism.[Conclusion]: Our data suggest that pancreatic CSCs depend on glutathione metabolism. Pharmacological targeting of this pathway showed that high GSH content is essential to maintain CSC functionality in terms of self-renewal and chemoresistance.ERC Advanced Investigator Grant, No. Pa-CSC 233460; European Community's Seventh Framework Programme, No. 602783; Instituto de Salud Carlos III and European Funds (FSE: “el FSE invierte en tu futuro” and FEDER: “una manera de hacer Europa”) Miguel Servet Fellowship, No. CP16/00121; and Fondo Investigaciones Sanitarias, No. PI17/00082

MYC/PGC-1α balance determines the metabolic phenotype and plasticity of pancreatic cancer stem cells.

The anti-diabetic drug metformin targets pancreatic cancer stem cells (CSCs), but not their differentiated progenies (non-CSCs), which may be related to distinct metabolic phenotypes. Here we conclusively demonstrate that while non-CSCs were highly glycolytic, CSCs were dependent on oxidative metabolism (OXPHOS) with very limited metabolic plasticity. Thus, mitochondrial inhibition, e.g., by metformin, translated into energy crisis and apoptosis. However, resistant CSC clones eventually emerged during treatment with metformin due to their intermediate glycolytic/respiratory phenotype. Mechanistically, suppression of MYC and subsequent increase of PGC-1α were identified as key determinants for the OXPHOS dependency of CSCs, which was abolished in resistant CSC clones. Intriguingly, no resistance was observed for the mitochondrial ROS inducer menadione and resistance could also be prevented/reversed for metformin by genetic/pharmacological inhibition of MYC. Thus, the specific metabolic features of pancreatic CSCs are amendable to therapeutic intervention and could provide the basis for developing more effective therapies to combat this lethal cancer