Biological/clinical parameters (patient age, PSA levels, Gleason score) and glucose and fructose levels in benign and PCa patients.

AbstractClinical localization of primary tumors and sites of metastasis by PET is based on the enhanced cellular uptake of 2-deoxy-2-[18F]-fluoro-D-glucose (FDG). In prostate cancer, however, PET-FDG imaging has shown limited clinical applicability, suggesting that prostate cancer cells may utilize hexoses other than glucose, such as fructose, as the preferred energy source. Our previous studies suggested that prostate cancer cells overexpress fructose transporters, but not glucose transporters, compared with benign cells. Here, we focused on validating the functional expression of fructose transporters and determining whether fructose can modulate the biology of prostate cancer cells in vitro and in vivo. Fructose transporters, Glut5 and Glut9, were significantly upregulated in clinical specimens of prostate cancer when compared with their benign counterparts. Fructose levels in the serum of patients with prostate cancer were significantly higher than healthy subjects. Functional expression of fructose transporters was confirmed in prostate cancer cell lines. A detailed kinetic characterization indicated that Glut5 represents the main functional contributor in mediating fructose transport in prostate cancer cells. Fructose stimulated proliferation and invasion of prostate cancer cells in vitro. In addition, dietary fructose increased the growth of prostate cancer cell line–derived xenograft tumors and promoted prostate cancer cell proliferation in patient-derived xenografts. Gene set enrichment analysis confirmed that fructose stimulation enriched for proliferation-related pathways in prostate cancer cells. These results demonstrate that fructose promotes prostate cancer cell growth and aggressiveness in vitro and in vivo and may represent an alternative energy source for prostate cancer cells.Significance:This study identifies increased expression of fructose transporters in prostate cancer and demonstrates a role for fructose as a key metabolic substrate supporting prostate cancer cells, revealing potential therapeutic targets and biomarkers.

Activation of glucose transporter-1 (Glut-1) gene expression is a molecular feature of cancer cells that increases glucose uptake and metabolism. Increased glucose uptake is the basis for the clinical localization of primary tumors using positron emission tomography (PET) and 2-deoxy-2-[18F]-fluoro-D-glucose (FDG) as a radiotracer. However, previous studies have demonstrated that a considerable number of cancers, which include prostate cancer (CaP), express low to undetectable levels of Glut-1 and that FDG-PET has limited clinical applicability in CaP. This observation could be explained by a low metabolic activity of CaP cells that may be overcome using different hexoses, such as fructose, as the preferred energy source. However, these hypotheses have not been examined critically in CaP. This review article summarizes what is currently known about transport and metabolism of hexoses, and more specifically fructose, in CaP and provides experimental evidences indicating that CaP cells may have increased capacity to transport and metabolize fructose in vitro and in vivo. Moreover, this review highlights recent findings that allow better understanding of how metabolism of fructose may regulate cancer cell proliferation and how fructose uptake and metabolism, through the de novo lipogenesis pathway, may provide new opportunities for CaP early diagnosis, staging, and treatment.

Introduction PET-scanning can detect primary tumours relaying on their high glucose uptake ability through overexpression of the glucose transporter Glut-1. However, this method has shown limited clinical applicability for prostate cancer (PCa) diagnosis, suggesting that PCa cells do not use glucose as its primary source of energy. Preliminary data from our laboratory has shown overexpression of the fructose transporter Glut-5 in PCa cell lines and in clinical specimens of PCa, which suggest that fructose could play an important role in PCa biology. Material and methods We analysed the effect of fructose on aggressiveness and metabolic reprogramming in benign prostate epithelial and PCa cell lines. Cells were incubated with glucose or fructose in the media for 24, 48, and 72 hour and then we evaluated: 1) the proliferative rate of benign and PCa cells, 2) the invasion and migration capacity of PCa cells, and 3) the mRNA levels of the enzymes involved in glycolysis, pentose phosphate and de novo lipogenesis pathways using real-time PCR in PCa cells. The effect of fructose on tumour growth was analysed by a PC3 cell line xenograft in immunosuppressed NSG mice. 15% fructose was added to the drinking water for 8 weeks. Tumour weight and the expression of glycolytic enzymes by qPCR was evaluated with respect to the control (water without additives). Results and discussions PCa cells incubated with fructose or glucose, showed similar proliferative rate, invasion and migration capacities. However, fructose evokes a different expression profile of the enzymes involve in glycolysis, pentose phosphate and de novo lipogenesis pathways. Fructose promotes tumour growth of PC3 cells in the NSG mice. Conclusion Our data suggest that fructose could play an important role in PCa pathophysiology, promoting proliferation and migration of PCa cells in vitro , and increasing tumour growth in vivo thanks to a reprogram in their metabolism that allows PCa cells to use fructose as effectively as glucose.

Introduction The clinical localization of primary cancers and sites of metastasis by positron emission tomography (PET) is based on the enhanced cellular uptake of 2-deoxy-2-[ 18 F]-fluoro-d-glucose (FDG). In prostate cancer (CaP), however, FDG-PET imaging has shown limited clinical applicability. This striking difference suggests that CaP cells utilise hexoses other than glucose, such as fructose, as the principal energy source. The purpose of this study was to determine whether or not fructose is a/the principal source of energy for CaP cells. Material and methods We determined the glucose and fructose concentration in serum from benign and CaP patients, the protein expression of Glut1, Glut5 and Glut9 and mRNA expression of Glut1, Glut5, Glut7, Glut9 and Glut11 in clinical specimens of benign and malignant prostate tissue using immunostaining and qRT-PCR. Moreover, mRNA and protein expression for the glucose transporters was analysed in vitro in benign (RWPE-1) and malignant (LNCaP, LNCaP-C4-2, DU-145, and PC3) CaP cell lines using qRT-PCR and western blot. Fructose and glucose uptake was measured in vitro in prostate cell lines using radiolabelled d-[U- 14 C]-fructose or 2-[1,2–3 hour]-deoxy-d-[ 3 H]-glucose, respectively. The effect of fructose and glucose on mitochondrial metabolism was analysed in prostate cell lines using seahorse analysis and the effect of fructose on tumour growth was analysed in vivo using a PC3 cell line xenograft model using immunosuppressed NSG mice. Results and discussions Fructose concentration was elevated in CaP patients compared to benign and no difference in glucose levels was observed. The expression of the fructose transporters, Glut5 and Glut9, was increased in CaP cell lines and in human CaP tissues compared to benign cell lines and benign prostate tissues, respectively. Glut1 expression, however, did not differ between benign and malignant human prostate cells. Transport assays demonstrated that CaP cell lines have a higher capacity to transport fructose compared to benign cell lines. However, glucose uptake was not altered between benign and malignant human prostate cell lines. ATP levels, basal and maximal respiration in bening and CaP cells were similar in the presence of fructose or glucose. Lastly, fructose was able to increase the growth rate, size and weight of the tumours in the NSG mice compared to controls (without fructose). Conclusion Our results indicate that fructose may represent an alternative energy source for CaP cells and may promote CaP tumour growth of CaP cells in vivo .

Abstract The elevated level of glucose uptake and metabolism in cancers is the basis for the clinical localization of primary cancers and sites of metastasis by positron emission tomography (PET scanning), based on the enhanced cellular uptake of 2-deoxy-2-[18F]-fluoro-D-glucose (FDG). In prostate cancer (CaP), however, FDG-PET imaging has shown limited clinical applicability. This striking difference suggests that CaP cells utilize hexoses other than glucose, such as fructose, as the principal energy source. The purpose of this study was to determine whether or not fructose is a/the principal source of energy for CaP cells. mRNA and protein expression for the glucose transporter Glut-1 and fructose transporters Glut-2, Glut-5, Glut-7, Glut-9 and Glut-11 was analyzed in benign (PWR-1E, RWPE-1) and malignant (LNCaP, vCaP, LNCaP-C4-2, DU-145, and PC-3) human prostate cell lines using qRT-PCR and western blot, respectively. In addition, Glut(s) protein expression was analyzed on a tissue microarray containing 200 formalin-fixed paraffin-embedded benign and malignant human prostate tissues using immunohistochemistry. Fructose and glucose uptake was measured in vitro in benign and malignant human prostate cell lines using radiolabelled D-[U-14C]-fructose or 2-[1,2-3H]-deoxy-D-[3H]-glucose, respectively. Lastly, the effect of fructose or glucose on the levels of ATP, mitochondrial metabolism, and expression of the enzymes hexokinase-2 (HK2), type-C fructokinase (KHK-C), pyruvate kinase M2 (PKM2) and type-A lactate dehydrogenase (LDH-A) was analyzed in benign and malignant human prostate cell lines using chemiluminescence, seahorse, and qRT-PCR analyses, respectively. Our results indicated that expression of the fructose transporters, Glut-5 and Glut-9, was increased in CaP cell lines and in human CaP tissues compared to benign cell lines and benign prostate tissues, respectively. Glut-1 expression, however, did not differ between benign and malignant human prostate cells. Transport assays demonstrated that CaP cell lines have a higher capacity to transport fructose compared to benign cell lines. However, glucose uptake was not altered between benign and malignant human prostate cell lines. ATP levels in CaP cells were similar in the presence of fructose or glucose. Fructose, but not glucose, significantly altered mRNA expression of HK2, KHK-C, PKM2, and LDH-A in malignant human prostate cells. Taken together, our results suggest that fructose may represent an alternative energy source and may reprogram hexose metabolism in CaP cells. Citation Format: Daniela Carreño, Nestor Corro, Marcia Arredondo, Carmen Navarro, Verónica Torres, Viviana Montecinos, Paula Sotomayor, Francisco Nualart, Julio Cesar Cárdenas, Alejandro S. Godoy. Role of fructose in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 448. doi:10.1158/1538-7445.AM2017-448

Abstract Prostate cancer (CaP) is the most commonly diagnosed cancer and the second leading cause of cancer deaths among males in the United States. Androgen deprivation therapy (ADT) is the standard treatment for advanced or metastatic CaP. However, during ADT, CaP progresses from an androgen-sensitive (AS-CaP) to a more aggressive, and eventually lethal, castration-resistant (CRPC) phenotype. There is evidence to suggest that the prostate tumor mass is under tight control of endothelial microvasculature due to an increase in angiogenesis by tumor cells. Nevertheless, now there is evidence to support that this influence is not one-directional and that the endothelial cells secrete a large number of active substances (angiocrine factors), which may directly or indirectly influence tumor growth and progression. However, the direct impacts of the endothelium on prostate tumor progression or the molecular mechanisms that are involved in this communication remain unclear. Here we investigated the potential influence of endothelium-derived paracrine factors on prostate cancer biology and the role of connexins in these interactions, since connexins play a major role in cell-cell communication and form a bidirectional signaling pathway to assemble gap junctions and alter cell behaviors. We measured the effect of conditioned media (CM) obtained from a primary culture of human endothelial cells isolated from umbilical vein (HUVEC) on viability, proliferation, migration and invasion of CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) and in the metastatic potential by in vivo assays using co-injection of CaP cell with HUVEC or injection of CaP cells pre-incubated with CM from HUVEC in a zebrafish embryo model. Finally, we studied the expression and the role of connexins on this stimulation using pharmacological (GJIC inhibitors) approaches. All together, our results showed that CM from endothelial cell induces an increases in the viability and proliferation in all CaP cell lines (LNCaP, LNCaP-C4-2 and PC3) but only increases migration of the CRPC cell lines (LNCaP-C4-2 and PC3). We also observed in our in vivo model that endothelial cells either through cell-cell interaction or by paracrine communication increases the metastatic ability of the CaP cells. Moreover, the increase in viability and migration of CaP cells observed with CM from endothelial cells was blocked using inhibitors of gap junctions. Real-time PCR analyses detected an up-regulation of Cx43 mRNA after exposition to CM from endothelial cells. Our data suggest that angiocrine communication between endothelial cells and CaP cells increases proliferation and migration of more aggressive CaP cells which could be important for the acquisition of the aggressive phenotype of the disease, and this interaction could be mediated by Cx43. Delineation of such critical players may culminate in identifying therapeutic targets or biomarkers to counteract CaP, especially advanced CaP. Citation Format: Verónica Torres-Estay, Patricia Fuenzalida, Catalina Ascencio, Carla Cembrano, Daniela Carreño, Néstor Corro, Viviana Montecinos, Gareth Owen, Xavier Figueroa, Julio Amigo, Juan Carlos Saéz, Alejandro Godoy. Paracrine effect of the endothelium on prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2017-2969