PHLPP Regulates Hexokinase 2-Dependent Glucose Metabolism in Colon Cancer Cells

Increased glucose metabolism is considered as one of the most important metabolic alterations adapted by cancer cells in order to generate energy as well as high levels of glycolytic intermediates to support rapid proliferation. PH domain leucine-rich repeat protein phosphatase (PHLPP) belongs to a novel family of Ser/Thr protein phosphatases that function as tumor suppressors in various types of human cancer. Here we determined the role of PHLPP in regulating glucose metabolism in colon cancer cells. Knockdown of PHLPP increased the rate of glucose consumption and lactate production, whereas overexpression of PHLPP had the opposite effect. Bioenergetic analysis using Seahorse Extracelluar Flux Analyzer revealed that silencing PHLPP expression induced a glycolytic shift in colon cancer cells. Mechanistically, we found that PHLPP formed a complex with Akt and hexokinase 2 (HK2) in the mitochondrial fraction of colon cancer cells and knockdown of PHLPP enhanced Akt-mediated phosphorylation and mitochondrial localization of HK2. Depletion of HK2 expression or treating cells with Akt and HK2 inhibitors reversed PHLPP loss-induced increase in glycolysis. Furthermore, PHLPP knockdown cells became addicted to glucose as a major energy source in that glucose starvation significantly decreased cancer cell survival. As HK2 is the key enzyme that determines the direction and magnitude of glucose flux, our study identified PHLPP as a novel regulator of glucose metabolism by controlling HK2 activity in colon cancer cells

Temperature Induces Significant Changes in Both Glycolytic Reserve and Mitochondrial Spare Respiratory Capacity in Colorectal Cancer Cell Lines

Thermotherapy, as a method of treating cancer, has recently attracted considerable attention from basic and clinical investigators. A number of studies and clinical trials have shown that thermotherapy can be successfully used as a therapeutic approach for various cancers. However, the effects of temperature on cancer bioenergetics have not been studied in detail with a real time, in a microplate, label-free detection approach. This study investigate how changes in temperature affect the bioenergetics characteristics (mitochondrial function and glycolysis) of three colorectal cancer (CRC) cell lines utilizing the Seahorse XF96 technology. Experiments were performed at 32°C, 37°C and 42°C using assay medium conditions and equipment settings adjusted to produce equal oxygen and pH levels ubiquitously at the beginning of all experiments. The results suggest that temperature significantly changes multiple components of glycolytic and mitochondrial function of all cell lines tested. Under hypothermia conditions (32°C), the extracellular acidification rates (ECAR) of CRC cells were significantly lower compared to the same basal ECAR levels measured at 37°C. Mitochondrial stress test for SW480 cells at 37°C vs 42°C demonstrated increased proton leak while all other OCR components remained unchanged (similar results were detected also for the patient-derived xenograft cells Pt.93). Interestingly, the FCCP dose response at 37°C vs 42°C show significant shifts in profiles, suggesting that single dose FCCP experiments might not be sufficient to characterize the mitochondrial metabolic potential when comparing groups, conditions or treatments. These findings provide valuable insights for the metabolic and bioenergetic changes of CRC cells under hypo- and hyperthermia conditions that could potentially lead to development of better targeted and personalized strategies for patients undergoing combined thermotherapy with chemotherapy

Adipocytes Activate Mitochondrial Fatty Acid Oxidation and Autophagy to Promote Tumor Growth in Colon Cancer

Obesity has been associated with increased incidence and mortality of a wide variety of human cancers including colorectal cancer. However, the molecular mechanism by which adipocytes regulate the metabolism of colon cancer cells remains elusive. In this study, we showed that adipocytes isolated from adipose tissues of colon cancer patients have an important role in modulating cellular metabolism to support tumor growth and survival. Abundant adipocytes were found in close association with invasive tumor cells in colon cancer patients. Co-culture of adipocytes with colon cancer cells led to a transfer of free fatty acids that released from the adipocytes to the cancer cells. Uptake of fatty acids allowed the cancer cells to survive nutrient deprivation conditions by upregulating mitochondrial fatty acid β-oxidation. Mechanistically, co-culture of adipocytes or treating cells with fatty acids induced autophagy in colon cancer cells as a result of AMPK activation. Inhibition of autophagy attenuated the ability of cancer cells to utilize fatty acids and blocked the growth-promoting effect of adipocytes. In addition, we found that adipocytes stimulated the expression of genes associated with cancer stem cells and downregulated genes associated with intestinal epithelial cell differentiation in primary colon cancer cells and mouse tumor organoids. Importantly, the presence of adipocytes promoted the growth of xenograft tumors in vivo. Taken together, our results show that adipocytes in the tumor microenvironment serve as an energy provider and a metabolic regulator to promote the growth and survival of colon cancer cells

Increased Expression of Fatty Acid Synthase Provides a Survival Advantage to Colorectal Cancer Cells via Upregulation of Cellular Respiration

Fatty acid synthase (FASN), a lipogenic enzyme, is upregulated in colorectal cancer (CRC). Increased de novo lipid synthesis is thought to be a metabolic adaptation of cancer cells that promotes survival and metastasis; however, the mechanisms for this phenomenon are not fully understood. We show that FASN plays a role in regulation of energy homeostasis by enhancing cellular respiration in CRC. We demonstrate that endogenously synthesized lipids fuel fatty acid oxidation, particularly during metabolic stress, and maintain energy homeostasis. Increased FASN expression is associated with a decrease in activation of energy-sensing pathways and accumulation of lipid droplets in CRC cells and orthotopic CRCs. Immunohistochemical evaluation demonstrated increased expression of FASN and p62, a marker of autophagy inhibition, in primary CRCs and liver metastases compared to matched normal colonic mucosa. Our findings indicate that overexpression of FASN plays a crucial role in maintaining energy homeostasis in CRC via increased oxidation of endogenously synthesized lipids. Importantly, activation of fatty acid oxidation and consequent downregulation of stress-response signaling pathways may be key adaptation mechanisms that mediate the effects of FASN on cancer cell survival and metastasis, providing a strong rationale for targeting this pathway in advanced CRC

Redox Proteomic Identification of HNE-Bound Mitochondrial Proteins in Cardiac Tissues Reveals a Systemic Effect on Energy Metabolism After Doxorubicin Treatment

Doxorubicin (DOX), one of the most effective anticancer drugs, is known to generate progressive cardiac damage, which is due, in part, to DOX-induced reactive oxygen species (ROS). The elevated ROS often induce oxidative protein modifications that result in alteration of protein functions. This study demonstrates that the level of proteins adducted by 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, is significantly increased in mouse heart mitochondria after DOX treatment. A redox proteomics method involving two-dimensional electrophoresis followed by mass spectrometry and investigation of protein databases identified several HNE-modified mitochondrial proteins, which were verified by HNE-specific immunoprecipitation in cardiac mitochondria from the DOX-treated mice. The majority of the identified proteins are related to mitochondrial energy metabolism. These include proteins in the citric acid cycle and electron transport chain. The enzymatic activities of the HNE-adducted proteins were significantly reduced in DOX-treated mice. Consistent with the decline in the function of the HNE-adducted proteins, the respiratory function of cardiac mitochondria as determined by oxygen consumption rate was also significantly reduced after DOX treatment. Treatment with Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, an SOD mimic, averted the doxorubicin-induced mitochondrial dysfunctions as well as the HNE–protein adductions. Together, the results demonstrate that free radical-mediated alteration of energy metabolism is an important mechanism mediating DOX-induced cardiac injury, suggesting that metabolic intervention may represent a novel approach to preventing cardiac injury after chemotherapy