Search

SUMMARYCachexia is a debilitating wasting syndrome and highly prevalent comorbidity in cancer patients. It manifests especially with energy and mitochondrial metabolism aberrations that promote tissue wasting. We recently identified nicotinamide adenine dinucleotide (NAD+) loss to associate with muscle mitochondrial dysfunction in cancer hosts. In this study we confirmed that depletion of NAD+ and downregulation of Nrk2, an NAD+ biosynthetic enzyme, are common features of different mouse models and cachectic cancer patients. Testing NAD+ repletion therapy in cachectic mice revealed that NAD+ precursor, vitamin B3 niacin, efficiently corrected tissue NAD+ levels, improved mitochondrial metabolism and ameliorated cancer- and chemotherapy-induced cachexia. To examine NAD+ metabolism in a clinical setting, we showed that the low expression of NRK2 in cancer patients correlated with metabolic abnormalities underscoring the significance of NAD+ in the pathophysiology of human cancer cachexia. Overall, our results propose a novel therapy target, NAD+ metabolism, for cachectic cancer patients.

Rapid tumor growth requires elevated biosynthetic activity, supported by metabolic rewiring occurring both intrinsically in cancer cells and extrinsically in the cancer host. The Warburg effect is one such example, burning glucose to produce a continuous flux of biomass substrates in cancer cells at the cost of energy wasting metabolic cycles in the host to maintain stable glycemia. Amino acid (AA) metabolism is profoundly altered in cancer cells, which use AAs for energy production and for supporting cell proliferation. The peculiarities in cancer AA metabolism allow the identification of specific vulnerabilities as targets of anti-cancer treatments. In the current review, specific approaches targeting AAs in terms of either deprivation or supplementation are discussed. Although based on opposed strategies, both show, in vitro and in vivo, positive effects. Any AA-targeted intervention will inevitably impact the cancer host, who frequently already has cachexia. Cancer cachexia is a wasting syndrome, also due to malnutrition, that compromises the effectiveness of anti-cancer drugs and eventually causes the patient’s death. AA deprivation may exacerbate malnutrition and cachexia, while AA supplementation may improve the nutritional status, counteract cachexia, and predispose the patient to a more effective anti-cancer treatment. Here is provided an attempt to describe the AA-based therapeutic approaches that integrate currently distant points of view on cancer-centered and host-centered research, providing a glimpse of several potential investigations that approach cachexia as a unique cancer disease.

The development of distinct cellular and animal models has allowed the identification and characterization of molecular mechanisms underlying the pathogenesis of cerebral cavernous malformation (CCM) disease. This is a major cerebrovascular disorder of proven genetic origin, affecting 0.5% of the population. Three disease genes have been identified: CCM1/KRIT1, CCM2, and CCM3. These genes encode for proteins implicated in the regulation of major cellular structures and mechanisms, such as cell-cell and cell-matrix adhesion, actin cytoskeleton dynamics, and endothelial-to-mesenchymal transition, suggesting that they may act as pleiotropic regulators of cellular homeostasis. Indeed, accumulated evidence in cellular and animal models demonstrates that emerged pleiotropic functions of CCM proteins are mainly due to their ability to modulate redox-sensitive pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, thus contributing to the preservation of cellular homeostasis and stress defenses. In particular, we demonstrated that KRIT1 loss-of-function affects master regulators of cellular redox homeostasis and responses to oxidative stress, including major redox-sensitive transcriptional factors and antioxidant proteins, and autophagy, suggesting that altered redox signaling and oxidative stress contribute to CCM pathogenesis, and opening novel preventive and therapeutic perspectives.In this chapter, we describe materials and methods for isolation of mouse embryonic fibroblast (MEF) cells from homozygous KRIT1-knockout mouse embryos, and their transduction with a lentiviral vector encoding KRIT1 to generate cellular models of CCM disease that contributed significantly to the identification of pathogenetic mechanisms.

The development of distinct cellular and animal models has allowed the identification and characterization of molecular mechanisms underlying the pathogenesis of cerebral cavernous malformation (CCM) disease. This is a major cerebrovascular disorder of proven genetic origin, affecting 0.5% of the population. Three disease genes have been identified: CCM1/KRIT1, CCM2, and CCM3. These genes encode for proteins implicated in the regulation of major cellular structures and mechanisms, such as cell-cell and cell-matrix adhesion, actin cytoskeleton dynamics, and endothelial-to-mesenchymal transition, suggesting that they may act as pleiotropic regulators of cellular homeostasis. Indeed, accumulated evidence in cellular and animal models demonstrates that emerged pleiotropic functions of CCM proteins are mainly due to their ability to modulate redox-sensitive pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, thus contributing to the preservation of cellular homeostasis and stress defenses. In particular, we demonstrated that KRIT1 loss-of-function affects master regulators of cellular redox homeostasis and responses to oxidative stress, including major redox-sensitive transcriptional factors and antioxidant proteins, and autophagy, suggesting that altered redox signaling and oxidative stress contribute to CCM pathogenesis, and opening novel preventive and therapeutic perspectives.In this chapter, we describe materials and methods for isolation of mouse embryonic fibroblast (MEF) cells from homozygous KRIT1-knockout mouse embryos, and their transduction with a lentiviral vector encoding KRIT1 to generate cellular models of CCM disease that contributed significantly to the identification of pathogenetic mechanisms.

Loss-of-function mutations in the KRIT1 gene are associated with the pathogenesis of cerebral cavernous malformations (CCMs), a major cerebrovascular disease still awaiting therapies. Accumulating evidence demonstrates that KRIT1 plays an important role in major redox-sensitive mechanisms, including transcriptional pathways and autophagy, which play major roles in cellular homeostasis and defense against oxidative stress, raising the possibility that KRIT1 loss has pleiotropic effects on multiple redox-sensitive systems. Using previously established cellular models, we found that KRIT1 loss-of-function affects the glutathione (GSH) redox system, causing a significant decrease in total GSH levels and increase in oxidized glutathione disulfide (GSSG), with a consequent deficit in the GSH/GSSG redox ratio and GSH-mediated antioxidant capacity. Redox proteomic analyses showed that these effects are associated with increased S-glutathionylation of distinct proteins involved in adaptive responses to oxidative stress, including redox-sensitive chaperonins, metabolic enzymes, and cytoskeletal proteins, suggesting a novel molecular signature of KRIT1 loss-of-function. Besides providing further insights into the emerging pleiotropic functions of KRIT1, these findings point definitively to KRIT1 as a major player in redox biology, shedding new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, which may eventually lead to cellular dysfunctions and CCM disease pathogenesis.

Loss-of-function mutations of the gene encoding Krev interaction trapped protein 1 (KRIT1) are associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries and affecting 0.5% of the human population. However, growing evidence demonstrates that KRIT1 is implicated in the modulation of major redox-sensitive signaling pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, suggesting that its loss-of-function mutations may have pathological effects not limited to CCM disease. The aim of this study was to address whether KRIT1 loss-of-function predisposes to the development of pathological conditions associated with enhanced endothelial cell susceptibility to oxidative stress and inflammation, such as arterial endothelial dysfunction (ED) and atherosclerosis. Silencing of KRIT1 in human aortic endothelial cells (HAECs), coronary artery endothelial cells (HCAECs), and umbilical vein endothelial cells (HUVECs) resulted in increased expression of endothelial proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and in enhanced susceptibility to tumor necrosis factor alpha (TNF-&alpha, )-induced apoptosis. These effects were associated with a downregulation of Notch1 activation that could be rescued by antioxidant treatment, suggesting that they are consequent to altered intracellular redox homeostasis induced by KRIT1 loss-of-function. Furthermore, analysis of the aorta of heterozygous KRIT1+/&minus, mice fed a high-fructose diet to induce systemic oxidative stress and inflammation demonstrated a 1.6-fold increased expression of VCAM-1 and an approximately 2-fold enhanced fat accumulation (7.5% vs 3.6%) in atherosclerosis-prone regions, including the aortic arch and aortic root, as compared to corresponding wild-type littermates. In conclusion, we found that KRIT1 deficiency promotes ED, suggesting that, besides CCM, KRIT1 may be implicated in genetic susceptibility to the development of atherosclerotic lesions.

Loss-of-function mutations in the KRIT1 gene are associated with the pathogenesis of cerebral cavernous malformations (CCMs), a major cerebrovascular disease still awaiting therapies. Accumulating evidence demonstrates that KRIT1 plays an important role in major redox-sensitive mechanisms, including transcriptional pathways and autophagy, which play major roles in cellular homeostasis and defense against oxidative stress, raising the possibility that KRIT1 loss has pleiotropic effects on multiple redox-sensitive systems. Using previously established cellular models, we found that KRIT1 loss-of-function affects the glutathione (GSH) redox system, causing a significant decrease in total GSH levels and increase in oxidized glutathione disulfide (GSSG), with a consequent deficit in the GSH/GSSG redox ratio and GSH-mediated antioxidant capacity. Redox proteomic analyses showed that these effects are associated with increased S-glutathionylation of distinct proteins involved in adaptive responses to oxidative stress, including redox-sensitive chaperonins, metabolic enzymes, and cytoskeletal proteins, suggesting a novel molecular signature of KRIT1 loss-of-function. Besides providing further insights into the emerging pleiotropic functions of KRIT1, these findings point definitively to KRIT1 as a major player in redox biology, shedding new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, which may eventually lead to cellular dysfunctions and CCM disease pathogenesis.

Cerebral Cavernous Malformation (CCM) is a major cerebrovascular disease with a prevalence of 0.3–0.5% in the general population and highly variable clinical expressivity. It may cause various symptoms at any age, including recurrent headaches, neurological deficits, seizures, and intracerebral hemorrhage, and has been associated with loss-of-function mutations in three CCM genes, CCM1 (KRIT1), CCM2 and CCM3. However, growing evidence in animal models demonstrates that loss of CCM genes is not sufficient to cause CCM disease, suggesting the contribution of additional triggers occurring locally in the neurovascular microenvironment, including stress factors. Indeed, we found that both cellular and animal models of CCM disease show an enhanced sensitivity to oxidative stress and inflammatory insults due to defects in mechanisms involved in cellular defense against these stressful conditions, including antioxidant responses and autophagy. Furthermore, we provide novel insights into the identification of risk factors and biomarkers associated with CCM disease progression and severity, including the accumulation of major glycation and oxidation products, which may serve as early objective predictors of disease outcome and provide better options for disease prevention and treatment.