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// Silvia Vidali 1 , Sepideh Aminzadeh-Gohari 1 , Rene Gunther Feichtinger 1 , Renaud Vatrinet 2 , Andreas Koller 1 , Felix Locker 1 , Tricia Rutherford 3 , Maura O’Donnell 3 , Andrea Stoger-Kleiber 3 , Bridget Lambert 3 , Thomas Klaus Felder 4 , Wolfgang Sperl 5 and Barbara Kofler 1 1 Laura Bassi Centre of Expertise-THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria 2 Department of Pharmacy and Biotechnology (FABIT), University of Bologna, Bologna, Italy 3 Clinical Nutrition, Vitaflo International Ltd, Liverpool, UK 4 Department of Laboratory Medicine, Paracelsus Medical University, Salzburg, Austria 5 Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria Correspondence to: Barbara Kofler, email: b.kofler@salk.at Keywords: renal cell carcinoma, mitochondria, ketogenic diet, metabolism, Warburg effect Received: March 10, 2017 Accepted: June 27, 2017 Published: July 17, 2017 ABSTRACT The ketogenic diet (KD), a high-fat low-carbohydrate diet, has shown some efficacy in the treatment of certain types of tumors such as brain tumors and neuroblastoma. These tumors are characterized by the Warburg effect. Because renal cell carcinoma (RCC) presents similar energetic features as neuroblastoma, KD might also be effective in the treatment of RCC. To test this, we established xenografts with RCC 786-O cells in CD-1 nu/nu mice and then randomized them to a control diet or to KDs with different triglyceride contents. Although the KDs tended to reduce tumor growth, mouse survival was dramatically reduced due to massive weight loss. A possible explanation comes from observations of human RCC patients, who often experience secondary non-metastatic hepatic dysfunction due to secretion of high levels of inflammatory cytokines by the RCCs. Measurement of the mRNA levels of tumor necrosis factor alpha (TNFα) and interleukin-6 revealed high expression in the RCC xenografts compared to the original 786-O cells. The expression of TNFα, interleukin-6 and C-reactive protein were all increased in the livers of tumor-bearing mice, and KD significantly boosted their expression. KDs did not cause weight loss or liver inflammation in healthy mice, suggesting that KDs are per se safe, but might be contraindicated in the treatment of RCC patients presenting with Stauffer’s syndrome, because they potentially worsen the associated hepatic dysfunction.

// Raphael Johannes Morscher 1, 4 , Sepideh Aminzadeh-Gohari 1 , Cornelia Hauser-Kronberger 2 , Rene Gunther Feichtinger 1 , Wolfgang Sperl 3 , Barbara Kofler 1 1 Laura Bassi Centre of Expertise-THERAPEP, Department of Pediatrics, Paracelsus Medical University, 5020 Salzburg, Austria 2 Department of Pathology, Paracelsus Medical University, 5020 Salzburg, Austria 3 Department of Pediatrics, Paracelsus Medical University, 5020 Salzburg, Austria 4 Division of Medical Genetics, Medical University Innsbruck, 6020 Innsbruck, Austria Correspondence to: Raphael Johannes Morscher, e-mail: raphael.morscher@pmu.ac.at Keywords: neuroblastoma, ketogenic diet, glucose, metronomic cyclophosphamide, anti-angiogenic Received: September 15, 2015 Accepted: February 05, 2016 Published: March 05, 2016 ABSTRACT Background: MYCN -amplification in high-grade Neuroblastoma (NB) tumors correlates with increased vascularization and therapy resistance. This study combines an anti-angiogenic approach with targeting NB metabolism for treatment. Methods and Results: Metronomic cyclophosphamide (MCP) monotherapy significantly inhibited NB growth and prolonged host survival. Growth inhibition was more pronounced in MYCN -amplified xenografts. Immunohistochemical evaluation of this subtype showed significant decrease in blood vessel density and intratumoral hemorrhage accompanied by blood vessel maturation and perivascular fibrosis. Up-regulation of VEGFA was not sufficient to compensate for the effects of the MCP regimen. Reduced Bcl-2 expression and increased caspase-3 cleavage were evident. In contrast non MYCN -amplified tumors developed resistance, which was accompanied by Bcl-2-up-regulation. Combining MCP with a ketogenic diet and/or calorie-restriction significantly enhanced the anti-tumor effect. Calorie-restricted ketogenic diet in combination with MCP resulted in tumor regression in all cases. Conclusions: Our data show efficacy of combining an anti-angiogenic cyclophosphamide dosing regimen with dietary intervention in a preclinical NB model. These findings might open a new front in NB treatment.

Neuroblastoma (NB) is a pediatric malignancy characterized by a marked reduction in aerobic energy metabolism. Recent preclinical data indicate that targeting this metabolic phenotype by a ketogenic diet (KD), especially in combination with calorie restriction, slows tumor growth and enhances metronomic cyclophosphamide (CP) therapy of NB xenografts. Because calorie restriction would be contraindicated in most cancer patients, the aim of the present study was to optimize the KD such that the tumors are sensitized to CP without the need of calorie restriction. In a NB xenograft model, metronomic CP was combined with KDs of different triglyceride compositions and fed to CD1-nu mice ad libitum. Metronomic CP in combination with a KD containing 8-carbon medium-chain triglycerides exerted a robust anti-tumor effect, suppressing growth and causing a significant reduction of tumor blood-vessel density and intratumoral hemorrhage, accompanied by activation of AMP-activated protein kinase in NB cells. Furthermore, the KDs caused a significant reduction in the serum levels of essential amino acids, but increased those of serine, glutamine and glycine. Our data suggest that targeting energy metabolism by a modified KD may be considered as part of a multimodal treatment regimen to improve the efficacy of classic anti-NB therapy.

The glycolytic enzyme pyruvate kinase (PK) is required for cancer development, and has been implicated in the metabolic transition from oxidative to fermentative metabolism, the Warburg effect. However, the global metabolic response that follows changes in PK activity is not yet fully understood. Using shotgun proteomics, we identified 31 yeast proteins that were regulated in a PK-dependent manner. Selective reaction monitoring confirmed that their expression was dependent on PK isoform, level and activity. Most of the PK targets were amino acid metabolizing enzymes or factors of protein translation, indicating that PK plays a global regulatory role in biosynthethic amino acid metabolism. Indeed, we found strongly altered amino acid profiles when PK levels were changed. Low PK levels increased the cellular glutamine and glutamate concentrations, but decreased the levels of seven amino acids including serine and histidine. To test for evolutionary conservation of this PK function, we quantified orthologues of the identified PK targets in thyroid follicular adenoma, a tumor characterized by high PK levels and low respiratory activity. Aminopeptidase AAP-1 and serine hydroxymethyltransferase SHMT1 both showed PKM2- concentration dependence, and were upregulated in the tumor. Thus, PK expression levels and activity were important for maintaining cellular amino acid homeostasis. Mediating between energy production, ROS clearance and amino acid biosynthesis, PK thus plays a central regulatory role in the metabolism of proliferating cells.