Autophagy as a Therapeutic Target in Breast Tumors: The Cancer stem cell perspective.

Breast cancer is a heterogeneous disease, with a subpopulation of tumor cells known as breast cancer stem cells (BCSCs) with self-renewal and differentiation abilities that play a critical role in tumor initiation, progression, and therapy resistance. The tumor microenvironment (TME) is a complex area where diverse cancer cells reside creating a highly interactive environment with secreted factors, and the extracellular matrix. Autophagy, a cellular self-digestion process, influences dynamic cellular processes in the tumor TME integrating diverse signals that regulate tumor development and heterogeneity. Autophagy acts as a double-edged sword in the breast TME, with both tumor-promoting and tumor-suppressing roles. Autophagy promotes breast tumorigenesis by regulating tumor cell survival, migration and invasion, metabolic reprogramming, and epithelial-mesenchymal transition (EMT). BCSCs harness autophagy to maintain stemness properties, evade immune surveillance, and resist therapeutic interventions. Conversely, excessive, or dysregulated autophagy may lead to BCSC differentiation or cell death, offering a potential avenue for therapeutic exploration. The molecular mechanisms that regulate autophagy in BCSCs including the mammalian target of rapamycin (mTOR), AMPK, and Beclin-1 signaling pathways may be potential targets for pharmacological intervention in breast cancer. This review provides a comprehensive overview of the relationship between autophagy and BCSCs, highlighting recent advancements in our understanding of their interplay. We also discuss the current state of autophagy-targeting agents and their preclinical and clinical development in BCSCs.

Autophagy and metabolic aging: Current understanding and future applications.

"Metabolic aging" refers to the gradual decline in cellular metabolic function across various tissues due to defective hormonal signaling, impaired nutrient sensing, mitochondrial dysfunction, replicative stress, and cellular senescence. While this process usually corresponds with chronological aging, the recent increase in metabolic diseases and cancers occurring at younger ages in humans suggests the premature onset of cellular fatigue and metabolic aging. Autophagy, a cellular housekeeping process facilitated by lysosomes, plays a crucial role in maintaining tissue rejuvenation and health. However, various environmental toxins, hormones, lifestyle changes, and nutrient imbalances can disrupt autophagy in humans. In this review, we explore the connection between autophagy and cellular metabolism, its regulation by extrinsic factors and its modulation to prevent the early onset of metabolic aging.

Elevated senescence in the bone marrow mesenchymal stem cells of acquired aplastic anemia patients: A possible implication of DNA damage responses and telomere attrition.

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSC) are an integral part of the BM niche that is essential to maintain hematopoietic homeostasis. In aplastic anemia (AA), a few studies have reported phenotypic defects in the BM-MSC, such as reduced proliferation, imbalanced differentiation, and apoptosis; however, the alterations at the molecular level need to be better characterized. Therefore, the current study aims to identify the causative factors underlying the compromised functions of AA BM-MSC that might eventually be contributing to the AA pathobiology. METHODS: We performed RNA sequencing (RNA-Seq) using the Illumina platform to comprehend the distinction between the transcriptional landscape of AA and control BM-MSC. Further, we validated the alterations observed in senescence by Senescence- associated beta-galactosidase (SA -ß-gal) assay, DNA damage by γH2AX staining, and telomere attrition by relative telomere length assessment and telomerase activity assay. We used qRT-PCR to analyze changes in some of the genes associated with these molecular mechanisms. RESULTS: The transcriptome profiling revealed enrichment of senescence-associated genes and pathways in AA BM-MSC. The senescent phenotype of AA BM-MSC was accompanied by enhanced SA -ß-gal activity and elevated expression of senescence associated genes TP53, PARP1, and CDKN1A. Further, we observed increased γH2AX foci indicating DNA damage, reduced telomere length, and diminished telomerase activity in the AA BM-MSC. CONCLUSION: Our results highlight that AA BM-MSC have a senescent phenotype accompanied by other cellular defects like DNA damage and telomere attrition, which are most likely driving the senescent phenotype of AA BM-MSC thus hampering their hematopoiesis supporting properties as observed in AA.

Targeting autophagy and lipid metabolism in cancer stem cells.

Cancer stem cells (CSCs) are a subset of cancer cells with self-renewal ability and tumor initiating properties. Unlike the other non-stem cancer cells, CSCs resist traditional therapy and remain a major cause of disease relapse. With the recent advances in metabolomics, various studies have demonstrated that CSCs have distinct metabolic properties. Metabolic reprogramming in CSCs contributes to self-renewal and maintenance of stemness. Accumulating evidence suggests that rewiring of energy metabolism is a key player that enables to meet energy demands, maintains stemness, and sustains cancer growth and invasion. CSCs use various mechanisms such as increased glycolysis, redox signaling, and autophagy modulation to overcome nutritional deficiency and sustain cell survival. The alterations in lipid metabolism acquired by the CSCs support biomass production through increased dependence on fatty acid synthesis and ß-oxidation, and contribute to oncogenic signaling pathways. This review summarizes our current understanding of lipid metabolism in CSCs and how pharmacological regulation of autophagy and lipid metabolism influences CSC phenotype. Increased dependence on lipid metabolism appears as an attractive strategy to eliminate CSCs using therapeutic agents that specifically target CSCs based on their modulation of lipid metabolism.

Pharmacological inhibition of CFTR attenuates nonalcoholic steatohepatitis (NASH) progression in mice.

Nonalcoholic steatohepatitis (NASH) is considered a pivotal stage in nonalcoholic fatty liver disease (NAFLD) progression and increases the risk of end-stage liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The etiology of NASH is multifactorial and identifying reliable molecular players has proven difficult. Presently, there are no approved drugs for NASH treatment, which has become a leading cause of liver transplants worldwide. Here, using public human transcriptomic NAFLD dataset, we uncover Cystic fibrosis transmembrane conductance receptor (CFTR) as a differentially expressed gene in the livers of human NASH patients. Similarly, murine Cftr expression was also found to be upregulated in two mouse models of diet-induced NASH. Furthermore, the pharmacological inhibition of CFTR significantly reduced NASH progression in mice and its overexpression aggravated lipotoxicity in human hepatic cells. These results, thus, underscore the involvement of murine Cftr in the pathogenesis of NASH and raise the intriguing possibility of its pharmacological inhibition in human NASH.

ULK1 inhibition attenuates telomerase activity in hepatic cells.

Autophagy and telomere maintenance are two cellular survival processes that show a strong correlation during human ageing and cancer growth, however, their causal relationship remains unclear. In this study, using an unbiased transcriptomics approach, we uncover a novel role of autophagy genes in regulating telomere extension and maintenance pathways. Concomitantly, the pharmacological inhibition of ULK1 (Unc-51 like autophagy activating kinase 1) attenuated human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in HepG2 cells. Furthermore, the suppression of telomerase activity upon ULK1 inhibition was associated with telomere shortening and onset of cellular senescence in HepG2 cells. These results, thus, demonstrate a direct role of autophagy in maintaining cellular longevity via regulation of telomerase activity, which may have implications in the pathophysiology of ageing and cancers.

Lipoic acid blocks autophagic flux and impairs cellular bioenergetics in breast cancer and reduces stemness.

Autophagy inhibition is currently considered a novel therapeutic strategy for cancer treatment. Lipoic acid (LA), a naturally occurring compound found in all prokaryotic and eukaryotic cells, inhibits breast cancer cell growth; however, the effect of LA on autophagy-mediated breast cancer cell death remains unknown. Our study identified that LA blocks autophagic flux by inhibiting autophagosome-lysosome fusion and lysosome activity which increases the accumulation of autophagosomes in MCF-7 and MDA-MB231 cells, leading to cell death of breast cancer cells. Interestingly, autophagic flux blockade limits the recycling of cellular fuels, resulting in insufficient substrates for cellular bioenergetics. Therefore, LA impairs cellular bioenergetics by the inhibition of mitochondrial function and glycolysis. We show that LA-induced ROS generation is responsible for the blockade of autophagic flux and cellular bioenergetics in breast cancer cells. Moreover, LA-mediated blockade of autophagic flux and ROS generation may interfere with the regulation of the BCSCs/progenitor phenotype. Here, we demonstrate that LA inhibits mammosphere formation and subpopulation of BCSCs. Together, these results implicate that LA acts as a prooxidant, potent autophagic flux inhibitor, and causes energetic impairment, which may lead to cell death in breast cancer cells/BCSCs.

ULK1 Signaling in the Liver: Autophagy Dependent and Independent Actions.

Liver is the primary organ for energy metabolism and detoxification in the human body. Not surprisingly, a derangement in liver function leads to several metabolic diseases. Autophagy is a cellular process, which primarily deals with providing molecules for energy production, and maintains cellular health. Autophagy in the liver has been implicated in several hepatic metabolic processes, such as, lipolysis, glycogenolysis, and gluconeogenesis. Autophagy also provides protection against drugs and pathogens. Deregulation of autophagy is associated with the development of non-alcoholic fatty liver disease (NAFLD) acute-liver injury, and cancer. The process of autophagy is synchronized by the action of autophagy family genes or autophagy (Atg) genes that perform key functions at different steps. The uncoordinated-51-like kinases 1 (ULK1) is a proximal kinase member of the Atg family that plays a crucial role in autophagy. Interestingly, ULK1 actions on hepatic cells may also involve some autophagy-independent signaling. In this review, we provide a comprehensive update of ULK1 mediated hepatic action involving lipotoxicity, acute liver injury, cholesterol synthesis, and hepatocellular carcinoma, including both its autophagic and non-autophagic functions.

Multifaceted role of chemokines in solid tumors: From biology to therapy.

Chemokines are small secretory chemotactic cytokines that control the directed migration of immune cells. Chemokines are involved in both anti-and pro-tumorigenic immune responses. Accumulating evidence suggests that the balance between these responses is influenced by several factors such as the stage of tumorigenesis, immune cell activation, recruitment of immune activating or immunosuppressive cells in the tumor microenvironment (TME), and chemokine receptor expression on effector and regulatory target cells. Cancer cells engage in a complex network with their TME components via several factors including growth factors, cytokines and chemokines that are critical for the growth of primary tumor and metastasis. However, chemokines show a multifaceted role in tumor progression including maintenance of stem-like properties, tumor cell proliferation/survival/senescence, angiogenesis, and metastasis. The heterogeneity of solid tumors in primary and metastatic cancers presents a challenge to the development of successful cancer therapy. Despite extensive research on how solid tumors escape immune cell-mediated anti-tumor response, finding an effective therapy for metastatic cancer still remains a challenge. This review discusses the multifarious roles of chemokines in solid tumors including various chemokine signaling pathways such as CXCL8-CXCR1/2, CXCL9, 10, 11-CXCR3, CXCR4-CXCL12, CCL(X)-CCR(X) in primary and metastatic cancers. We further discuss the novel therapeutic approaches that have been developed by major breakthroughs in chemokine research to treat cancer patients by the strategic blockade/activation of these signaling axes alone or in combination with immunotherapies.

Current treatment paradigms and emerging therapies for NAFLD/NASH.

Non-alcoholic fatty liver disease (NAFLD) is one the fastest emerging manifestations of the metabolic syndrome worldwide. Non-alcoholic steatohepatitis (NASH), the progressive form of NAFLD, may culminate into cirrhosis and hepatocellular cancer (HCC) and is presently a leading cause of liver transplant. Although a steady progress is seen in understanding of the disease epidemiology, pathogenesis and identifying therapeutic targets, the slowest advancement is seen in the therapeutic field. Currently, there is no FDA approved therapy for this disease and appropriate therapeutic targets are urgently warranted. In this review we discuss the role of lifestyle intervention, pharmacological agents, surgical approaches, and gut microbiome, with regard to therapy for NASH. In particular, we focus the role of insulin sensitizers, thyroid hormone mimetics, antioxidants, cholesterol lowering drugs, incretins and cytokines as therapeutic targets for NASH. We highlight these targets aiming to optimize the future for NASH therapy.

Autophagic protein ULK1 regulates FOXM1 signalling in human hepatoma cells.

Hepatocellular cancer (HCC) is one of the leading causes of mortality worldwide. Unfortunately, a limited choice of anti-cancer drugs is available for treatment, owing to their minimal efficacy and development of acquired resistance. Autophagy, a cellular survival pathway, often exhibits a pleiotropic role in HCC progression. Studies show increased autophagy in established HCC, promoting the survival of HCC cells in the tumour microenvironment. Therefore, novel anti-autophagy drugs hold promise for preventing HCC progression. Here, using a non-biased transcriptomics analysis in HepG2 cells we demonstrate the existence of an autophagy-FOXM1 nexus regulating growth in HepG2 cells. Additionally, we show that suppression of autophagy by an Unc-51 Like Autophagy Activating Kinase 1(ULK1) inhibitor not only attenuates the expression of FOXM1 and its transcriptional targets, but also has a synergistic effect on the inhibition of HepG2 growth when combined with FOXM1 inhibitors. Thus, the autophagic protein, ULK1, is a promising candidate for preventing HCC progression. Collectively, our results provide new insight into the role of autophagy in HCC growth and are a proof-of concept for combinatorial therapy using ULK1 and FOXM1 inhibitors.

Molecular links between non-alcoholic fatty liver disease and hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) and its advanced complication, non-alcoholic steatohepatitis (NASH), have become leading causes of hepatocellular carcinoma (HCC) worldwide. In this review, we discuss the role of metabolic, gut microbial, immune and endocrine mediators which promote the progression of NAFLD to HCC. In particular, this progression involves multiple hits resulting from lipotoxicity, oxidative stress, inhibition of hepatic autophagy and inflammation. Furthermore, dysbiosis in the gut associated with obesity also promotes HCC via induction of proinflammatory cytokines and Toll like receptor signalling as well as altered bile metabolism. Additionally, compromised T-cell function and impaired hepatic hormonal action promote the development of NASH-associated HCC. Lastly, we discuss the current challenges involved in the diagnosis and treatment of NAFLD/NASH-associated HCC.

Molecular Mechanism of Cancer Susceptibility Associated with Fok1 Single Nucleotide Polymorphism of VDR in Relation to Breast Cancer

Breast cancer is the leading cause of death among women worldwide. It is a multi-factorial disease caused by genetic and environmental factors. Vitamin D has been hypothesized to lower the risk of breast cancer via the nuclear vitamin D receptor (VDR). Genetic variants of these vitamin D metabolizing genes may alter the bioavailability of vitamin D, and hence modulate the risk of breast cancer. Materials and Methods: The distribution of Fok1 VDR gene (rs2228570) polymorphism and its association with breast cancer was analysed in a case­control study based on 125 breast cancer patients and 125 healthy females from North Indian population, using PCR-RFLP. An In silico exploration of the probable mechanism of increased risk of breast cancer was performed to investigate the role of single nucleotide polymorphisms (SNPs) in cancer susceptibility. Results: The Fok1 ff genotype was significantly associated with an increased risk of breast cancer (p=0.001; χ2=13.09; OR=16.909; %95 CI=2.20 - 130.11). In silico analysis indicated that SNPs may lead to a loss in affinity of VDR to calcitriol, and may also cause the impairment of normal interaction of liganded VDR with its heterodimeric partner, the retinoid X receptor (RXR), at protein level, thereby affecting target gene transcription. Conclusion: Breast cancer risk and pathogenesis in females can be influenced by SNPs. SNPs in VDR may cause alterations in the major molecular actions of VDR, namely ligand binding, heterodimerization and transactivation. VDRE binding and co-activator recruitment by VDR appear to be functionally inseparable events that affect vitamin D-elicited gene transcription. This indicates that breast cancer risk and pathogenesis in females may be influenced by SNPs.

PPARß/δ activation of CD300a controls intestinal immunity.

Macrophages are important for maintaining intestinal immune homeostasis. Here, we show that PPARß/δ (peroxisome proliferator-activated receptor ß/δ) directly regulates CD300a in macrophages that express the immunoreceptor tyrosine based-inhibitory motif (ITIM)-containing receptor. In mice lacking CD300a, high-fat diet (HFD) causes chronic intestinal inflammation with low numbers of intestinal lymph capillaries and dramatically expanded mesenteric lymph nodes. As a result, these mice exhibit triglyceride malabsorption and reduced body weight gain on HFD. Peritoneal macrophages from Cd300a-/- mice on HFD are classically M1 activated. Activation of toll-like receptor 4 (TLR4)/MyD88 signaling by lipopolysaccharide (LPS) results in prolonged IL-6 secretion in Cd300a-/- macrophages. Bone marrow transplantation confirmed that the phenotype originates from CD300a deficiency in leucocytes. These results identify CD300a-mediated inhibitory signaling in macrophages as a critical regulator of intestinal immune homeostasis.