Glycolytic Regulation of Intestinal Stem Cell Self-Renewal and Differentiation.

BACKGROUND AND AIMS: The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation, how glycolysis contributes to intestinal epithelial homeostasis remains to be defined. METHODS: Small intestines were harvested from mice with specific hexokinase 2 (HK2) deletion in the intestinal epithelium or LGR5+ stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 mitogen-activated protein kinase, the transcription factor atonal homolog 1, and intestinal cell differentiation markers lysozyme, mucin 2, and chromogranin A were determined by Western blot, quantitative real-time reverse transcription polymerase chain reaction, or immunofluorescence, and immunohistochemistry staining. RESULTS: HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout or inhibition of glycolysis resulted in increased numbers of Paneth, goblet, and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically, HK2 knockout resulted in activation of p38 mitogen-activated protein kinase and increased expression of ATOH1; inhibition of p38 mitogen-activated protein kinase signaling attenuated the phenotypes induced by HK2 knockout in intestinal organoids. HK2 knockout significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 knockout. CONCLUSIONS: Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 mitogen-activated protein kinase/atonal homolog 1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function.

Regulation of SIRT2 by Wnt/ß-catenin signaling pathway in colorectal cancer cells.

Activation of the Wnt/ß-catenin pathway is one of the hallmarks of colorectal cancer (CRC). Sirtuin 2 (SIRT2) protein has been shown to inhibit CRC proliferation. Previously, we reported that SIRT2 plays an important role in the maintenance of normal intestinal cell homeostasis. Here, we show that SIRT2 is a direct target gene of Wnt/ß-catenin signaling in CRC cells. Inhibition or knockdown of Wnt/ß-catenin increased SIRT2 promoter activity and mRNA and protein expression, whereas activation of Wnt/ß-catenin decreased SIRT2 promoter activity and expression. ß-Catenin was recruited to the promoter of SIRT2 and transcriptionally regulated SIRT2 expression. Wnt/ß-catenin inhibition increased mitochondrial oxidative phosphorylation (OXPHOS) and CRC cell differentiation. Moreover, inhibition of OXPHOS attenuated the differentiation of CRC cells induced by Wnt/ß-catenin inhibition. In contrast, inhibition or knockdown of SIRT2 decreased, while overexpression of SIRT2 increased, OXPHOS activity and differentiation in CRC cells. Consistently, inhibition or knockdown or SIRT2 attenuated the differentiation induced by Wnt/ß-catenin inhibition. These results demonstrate that SIRT2 is a novel target gene of the Wnt/ß-catenin signaling and contributes to the differentiation of CRC cells.

mTOR Inhibitor Everolimus in Regulatory T Cell Expansion for Clinical Application in Transplantation.

BACKGROUND: Experimental and preclinical evidence suggest that adoptive transfer of regulatory T (Treg) cells could be an appropriate therapeutic strategy to induce tolerance and improve graft survival in transplanted patients. The University of Kentucky Transplant Service Line is developing a novel phase I/II clinical trial with ex vivo expanded autologous Treg cells as an adoptive cellular therapy in renal transplant recipients who are using everolimus (EVR)-based immunosuppressive regimen. METHODS: The aim of this study was to determine the mechanisms of action and efficacy of EVR for the development of functionally competent Treg cell-based adoptive immunotherapy in transplantation to integrate a common EVR-based regimen in vivo (in the patient) and ex vivo (in the expansion of autologous Treg cells). CD25 Treg cells were selected from leukapheresis product with a GMP-compliant cell separation system and placed in 5-day (short) or 21-day (long) culture with EVR or rapamycin (RAPA). Multi-parametric flow cytometry analyses were used to monitor the expansion rates, phenotype, autophagic flux, and suppressor function of the cells. phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway profiles of treated cells were analyzed by Western blot and cell bioenergetic parameters by extracellular flux analysis. RESULTS: EVR-treated cells showed temporary slower growth, lower metabolic rates, and reduced phosphorylation of protein kinase B compared with RAPA-treated cells. In spite of these differences, the expansion rates, phenotype, and suppressor function of long-term Treg cells in culture with EVR were similar to those with RAPA. CONCLUSIONS: Our results support the feasibility of EVR to expand functionally competent Treg cells for their clinical use.

Investigating the Role of Mitochondrial Respiratory Dysfunction during Hexavalent Chromium-Induced Lung Carcinogenesis.

Hexavalent chromium [Cr(VI)] is a lung carcinogen and its complete mechanism of action remains to be investigated. Metabolic reprogramming of key energy metabolism pathways (e.g., increased anaerobic glycolysis in the presence of oxygen or "Warburg effect", dysregulated mitochondrial function, and lipogenesis) are important to cancer cell and tumor survival and growth. In our current understanding of Cr(VI)-induced carcinogenesis, the role for metabolic reprogramming remains unclear. In this study, we treated human lung epithelial cells (BEAS-2B) with Cr(VI) for 6 months and obtained malignantly transformed cells from an isolated colony grown in soft agar. We also used Cr(VI)-transformed cells from two other human lung cell lines (BEP2D and WTHBF-6 cells). Overall, we found that all the Cr(VI)-transformed cells had no changes in their mitochondrial respiratory functions (measured by the Seahorse Analyzer) compared with passaged-matched control cells. Using a xenograft tumor growth model, we generated tumors from these transformed cells in Nude mice. Using cells obtained from the xenograft tumor tissues, we observed that these cells had decreased maximal mitochondrial respiration, spare respiratory capacity, and coupling efficiency. These results provide evidence that, although mitochondrial dysfunction does not occur during Cr(VI)-induced transformation of lung cells, it does occur during tumor development.

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, microplate based, label-free detection approach. This study investigates 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.