Prognostic implications of markers of the metabolic phenotype in human cutaneous melanoma

1. Prognostic implications of markers of the metabolic phenotype in human cutaneous melanoma L. Nájera, M. Alonso-Juarranz, M. Garrido, C. Ballestín, L. Moya, M. Martínez-Díaz, R. Carrillo, A. Juarranz, F. Rojo, J.M. Cuezvaand J.L.Rodríguez-Peralto Hospital Universitario Puerta de Hierro, Fundación Jiménez Díaz, Hospital Universitario 12 de Octubre, Hospital Universitario Ramón y Cajal, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma. Madrid, Spain. British Journal of Dermatology. DOI: 10.111/bjd.17513

2. Professor José L. Rodríguez-Peralto Head of DermopathologyUnit 12 Octubre Hospital Professor José Manuel Cuezva Centro de Biología Molecular Severo Ochoa Universidad Autónoma de Madrid

3. Introduction What’s already known? • Normal differentiated cells depend primarily on mitochondrial oxidative phosphorylation (OXPHOS) to generate the energy. However proliferating and cancer cells activate aerobic glycolysis, a phenomenon known as the Warburg effect. • Reprogramming of energy metabolism to enhanced aerobic glycolysis has been defined as a hallmark of cancer.

4. Objective • To investigate the role of the mitochondrial proteins, β-subunit of the H+-ATP synthase (β-F1-ATPase), and heat-shock protein 60 (HSP60), and the glycolytic markers, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pyruvate kinase M2 (PKM2), as well as the bioenergetic cellular (BEC) index, in melanoma progression.

5. Methods Patients and samples • A training cohort of 63 nevi, 55 primary melanomas and 35 metastases were evaluated; and 108 primary melanoma and 30 metastases were used for validation. • Specimens from formalin-fixed paraffin-embedded (FFPE) blocks were retrospectively selected (1998-2000) from two different Spanish institutions.

6. Methods Cell lines and Cell proliferation, Spheroid formation and Migration assays • Melanoma cell lines with a gradient of aggressiveness were used: melanocytes (MN), radial (WM35) and vertical growth phase (WM278), and poorly (C8161-PA, PA) and highly aggressive melanoma with metastatic ability (C8161-HA, HA). Xenografts of melanoma cells in nude mice • Tumourigenicity of cells was assessed in hairless athymic nude mice (BALB/cByJ-Hfh11nu). Tumours were stained with H/E or subjected to immunohistochemistry.

7. Methods Immunofluorescence, immunohistochemistry and Western blot • Highly specific and validated mouse monoclonal for β-F1-ATPase, HSP60, PK and GAPDH antibodies were used. RNA extraction, quantitative RT-PCR • Total RNA was isolated and retrotranscribed to cDNA. Relative mRNA levels were calculated using the comparative CT method and RNA 18S as reference. Statistics • Statistical analysis was carried out with SPSS.

8. Results A B C Morphology of the melanoma cells. Ability of PA and HA cellstoformspheroids. Abilityfo induce tumors in miceby PA and HA cells Differentialexpression of thestudiedproteins in the melanoma cells. Variations in the BEC indexaccordingtotheaggresiveness of thecells. D E

9. Results C Figure A and B: Overexpression of HSP60, GAPDH and PKM2 was detected in melanoma human samples compared with nevi, showing a gradient of increased expression from radial growth phase to metastatic melanoma. Figure C: The BEC index was also significantly reduced in melanoma samples and correlated with worse overall and disease-free survival

10. Results Table: The multivariate Cox analysis showed that BEC index (HR, 0.64; CI95%, 0.4-1.2) is an independent predictor for overall survival. Figure: Overall survival and expression of studied markers in the training (A) and validation (B) cohort.

11. Discussion • Alterations in cancer cell metabolism are a hallmark of cancer. • This work demonstrates that, in the malignant transformation, melanomas suffer a profound shift towards an enhanced expression of proteins of glycolysis (GADPH and PKM2) and mitochondria (HSP60).

12. Discussion • These alterations are not present in normal melanocytes, or in benign melanocytic lesions, whereas a significant overexpression is observed in radial and vertical growth phase cells (PA, WM35 and WM278) and human melanoma tumours, reaching maximum expression in metastatic cells (HA) and corresponding human lesions.

13. Discussion • This alteration strongly suggests that progression of melanoma involves dysregulationin the pathways that adjust the supply of precursor for biosynthetic purposes (glycolysis) and the energy (OXPHOS) to support proliferation. • Similar findings have been described in different tumour types (breast, gastric, lung, kidney and colorectal cancer)showing a gradient of expression levels of these proteins from normal to malignant cells.

14. ConclusionsWhat does this study add? • Melanoma progression yields an enhanced expression of aerobic glycolysis and OXPHOS proteins. • The mitochondrial and glycolytic proteins are dysregulated and BEC index reduced in the malignant transformation of melanocytes. • A BEC index reduction confers an advantage for progression and dissemination in melanoma cells and correlates with tumour aggressiveness and worse overall and disease-free survival in melanoma patients.

15. Research team Laura Nájera Miguel Alonso-Juarranz Marta Martínez Ángeles Juarranz Federico Rojo

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