[Abstract unavailable

Metabolic reprogramming is a hallmark of cancer cells and contributes to their adaption within the tumour microenvironment and resistance to anticancer therapies. Recently, glycogen metabolism has become a recognised feature of cancer cells since it is upregulated in many tumour types, suggesting that it is an important aspect of cancer cell pathophysiology. Here, we provide an overview of glycogen metabolism and its regulation, with a focus on its role in metabolic reprogramming of cancer cells under stress conditions such as hypoxia, glucose deprivation and anticancer treatment. The various methods to detect glycogen in tumours in vivo as well as pharmacological modulators of glycogen metabolism are also reviewed. Finally, we discuss the therapeutic value of targeting glycogen metabolism as a strategy for combinational approaches in cancer treatment.

Autophagy is a self-degradation mechanism by which cells recycle their own cytoplasmic constituents and dispose of excess or defective organelles after starvation and oxygen deprivation. An antibody to the microtubule-associated protein 1 light chain 3 (LC3A), recognizing both the soluble (LC3A-I) and the membrane-bound form (LC3A-II) of the protein, was used to detect autophagic activity in 102 breast carcinomas. Three distinct patterns were recognized: (1) diffuse cytoplasmic, (2) cytoplasmic/juxta-nuclear, and (3) "stone-like" pattern -dense , rounded , amorphous structures, 5 m on average, typically enclosed within cytoplasmic vacuoles. The diffuse cytoplasmic pattern showed a direct association with estrogen and progesterone receptor expression. The juxta-nuclear pattern indicated a similar association with hormone receptors, an inverse association with tumor size, and a favorable prognosis. By contrast, an increased number of stonelike structures, probably representing an excessive autophagic response, was related to high-grade tumors and a less favorable outcome. Interestingly, 60 additional epithelial tumors of nonbreast origin disclosed identical autophagic patterns , and so did MDA231 breast cancer xenografts and HCT116 colon tumor spheroids (also analyzed by electron microscopy). Moreover, MCF-7 human breast cancer cell lines confirmed induction of LC3A by anoxia and Thapsigargin. It is concluded that autophagy can be readily recognized in breast carcinomas by light microscopy, after immunohistochemical staining with LC3A, but the signifi- Cancer cells survive the adverse conditions of the extracellular milieu (i.e., hypoxia, nutrient deprivation, and reduced growth factors) through angiogenesis and anaerobic glycolysis.1 Yet rapidly proliferating malignant neoplasms, having high metabolic demands, are not sufficiently supplied by these processes.2-6 An alternative metabolic pathway for providing energy, when both oxygen and glucose are depleted, is autophagy-a self-degradation mechanism by which cells recycle their own cytoplasmic constituents and dispose of excess or defective organelles resulting in protein and ATP synthesis.7-9 However, autophagy when prolonged can also cause cell death, but how the outcome is determined and the relationship of autophagy to clinical outcome is, at present, poorly understood. Angiogenesis and anaerobic glycolysis (i.e., the shifting from oxidative phosphorylation to anaerobic metabolism) have both been studied extensively in relation to malignant disease, including breast cancer. 10 -15 The activation of antiapoptotic pathways, as a complementary cell surviving mechanism, has also been considered in a number of studies. 16,17 By contrast, the phenomenon of autophagy only recently received attention for its biological significance in human malignancies. It is now known, for example, that under suboptimal microenvironmental conditions, cytoplasmic constituents are first entrapped by a membrane sac, called the isolation membrane, which subsequently closes to form dou...

Exosomes are secreted extracellular vesicles carrying diverse molecular cargos, which can modulate recipient cell behaviour. They are thought to derive from intraluminal vesicles formed in late endosomal multivesicular bodies ( MVB s). An alternate exosome formation mechanism, which is conserved from fly to human, is described here, with exosomes carrying unique cargos, including the GTP ase Rab11, generated in Rab11‐positive recycling endosomal MVB s. Release of Rab11‐positive exosomes from cancer cells is increased relative to late endosomal exosomes by reducing growth regulatory Akt/mechanistic Target of Rapamycin Complex 1 ( mTORC 1) signalling or depleting the key metabolic substrate glutamine, which diverts membrane flux through recycling endosomes. Vesicles produced under these conditions promote tumour cell proliferation and turnover and modulate blood vessel networks in xenograft mouse models in vivo . Their growth‐promoting activity, which is also observed in vitro , is Rab11a‐dependent, involves ERK ‐ MAPK ‐signalling and is inhibited by antibodies against amphiregulin, an EGFR ligand concentrated on these vesicles. Therefore, glutamine depletion or mTORC 1 inhibition stimulates release from Rab11a compartments of exosomes with pro‐tumorigenic functions, which we propose promote stress‐induced tumour adaptation.

LC3s (MAP1-LC3A, B and C) are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

Autophagy or Type II programmed cell death (PCD) is a major intracellular pathway for the degradation and recycling of proteins, ribosomes and entire organelles. The role of this pathway in the antitumor effect of radiotherapy and in radiation toxicity is obscure. A complicated machinery of genes and proteins is involved in the regulation of autophagy as a response to a variety of stress factors including hypoxia, nutrient deprivation, cytotoxic agents and radiotherapy. Continuously accumulating data suggest that autophagic response of cancer cells to radiotherapy is a major pathway which, in contrast to apoptosis that leads to death, may lead to either death or cellular survival. A variety of agents have been recognized that induce or block autophagy, directly interfering with the cytotoxic effect of radiotherapy. Simultaneous targeting of autophagy and apoptosis during radiotherapy seems to further augment the antitumor effect. Radiobiology research should focus on the differential effect of fractionation on the induction of autophagy in different tumors and on the manipulation of this with autophagy triggering agents. Whether manipulation of this pathway in normal tissues may be used to confer cytoprotection also deserves thorough investigation. Moreover, the role of pretreatment autophagic indices in tumor cells in predicting radiotherapy and chemotherapy outcome should be examined in translational studies.

SummaryLate-phase clinical trials investigating metformin as a cancer therapy are underway. However, there remains controversy as to the mode of action of metformin in tumors at clinical doses. We conducted a clinical study integrating measurement of markers of systemic metabolism, dynamic FDG-PET-CT, transcriptomics, and metabolomics at paired time points to profile the bioactivity of metformin in primary breast cancer. We show metformin reduces the levels of mitochondrial metabolites, activates multiple mitochondrial metabolic pathways, and increases 18-FDG flux in tumors. Two tumor groups are identified with distinct metabolic responses, an OXPHOS transcriptional response (OTR) group for which there is an increase in OXPHOS gene transcription and an FDG response group with increased 18-FDG uptake. Increase in proliferation, as measured by a validated proliferation signature, suggested that patients in the OTR group were resistant to metformin treatment. We conclude that mitochondrial response to metformin in primary breast cancer may define anti-tumor effect.

Tumor hypoxia is associated with poor patient outcomes in estrogen receptor-α–positive (ERα+) breast cancer. Hypoxia is known to affect tumor growth by reprogramming metabolism and regulating amino acid (AA) uptake. Here, we show that the glutamine transporter, SNAT2, is the AA transporter most frequently induced by hypoxia in breast cancer, and is regulated by hypoxia both in vitro and in vivo in xenografts. SNAT2 induction in MCF7 cells was also regulated by ERα, but it became predominantly a hypoxia-inducible factor 1α (HIF-1α)–dependent gene under hypoxia. Relevant to this, binding sites for both HIF-1α and ERα overlap in SNAT2’s cis-regulatory elements. In addition, the down-regulation of SNAT2 by the ER antagonist fulvestrant was reverted in hypoxia. Overexpression of SNAT2 in vitro to recapitulate the levels induced by hypoxia caused enhanced growth, particularly after ERα inhibition, in hypoxia, or when glutamine levels were low. SNAT2 up-regulation in vivo caused complete resistance to antiestrogen and, partially, anti-VEGF therapies. Finally, high SNAT2 expression levels correlated with hypoxia profiles and worse outcome in patients given antiestrogen therapies. Our findings show a switch in the regulation of SNAT2 between ERα and HIF-1α, leading to endocrine resistance in hypoxia. Development of drugs targeting SNAT2 may be of value for a subset of hormone-resistant breast cancer.