Expression profile of the amino acid transporters SLC7A5, SLC7A7, SLC7A8 and the enzyme TDO2 in basal cell carcinoma E Tina1, S Prosén2, S Lennholm3, G Gasparyan3, M Lindberg2,3, A Göthlin Eremo1 1. Department of Clinical Research Laboratory, Faculty of Medicine and Health, Örebro University, Örebro, Sweden 2. Department of Dermatology, Örebro University hospital, Örebro, Sweden 3. School of Medical Sciences, Faculty of Medicine and Health, Örebro university, Örebro, Sweden British Journal of Dermatology. DOI: 10.111/bjd.16905

Introduction What’s already known? The incidence of basal cell carcinoma is increasing and consequently also the costs for care. The transport and metabolism of amino acids are often altered in tumours although the knowledge whether this applies to basal cell carcinomas is limited. Illustration of basal cell carcinoma with incipient signs of tissue disruption. Invasive growth and metastasis is rarely observed.

Objective To study expression profiles in basal cell carcinomas compared to individually matched non-tumour skin, with focus on finding differences associated to tumour metabolism. The Hallmarks of Cancer. Originally published in Cell, 144, Hanahan D & Weinberg RA, Hallmarks of Cancer: The Next Generation, 646-674, © Elsevier, 2011

Methods Tissues from each patient (n=14); 4 mm punch biopsy taken centrally in basal call carcinoma 4 mm punch biopsy taken from gluteal skin Paraffin embedded tumour tissues routinely taken for pathological examination. Paraffin embedded control skin from healthy donors (n=2). RNA extraction using RNeasy Microarray Tissue Mini Kit (Qiagen). RNA concentration and quality assessed by NanoDrop Spectrophotometer ND-1000 (Thermo Fisher Scientific) and 2100 Bioanalyzer Instrument (Agilent Technologies). Snap frozen and stored at -80°C

Methods Microarray qPCR IHC Comparison of gene expression between BCC (n=4) and non-tumour skin (n=4) and selection of differentially expressed genes for further analysis. Validation of gene expressions SLC7A5, SLC7A7, SLC7A8 and TDO2 in patients’ (n=14) matched tissues Investigation of the proteins SLC7A5, SLC7A7, SLC7A8 and TDO2 localisation and level of expression in tumours Gene expression; SurePrint G3 microarray v3 8x60k (Agilent technologies) and TaqMan® Gene expression assays (Applied Biosystems). Protein expression; Automated slide stainer IntelliPATH FLX™ and MACH 1 Universal HRP-Polymer Detection system (Biocare Medical).

Results 1 Analysis of microarray data resulted in 1225 differently expressed genes (DEGs) between non-matched tissues. Tumour upregulation of SLC7A5, SLC7A7 and SLC7A8 as well as TDO2 among the findings qPCR-analysis of matched tissues confirmed upregulation of SLC7A5, SLC7A8 and TDO2 (Figure 1).

Results 2 Figure 1. SLC7A5 protein expression in BCC by IHC (immunochemitstry). Epidermis with normal appearance adjacent to the tumor, weak IHC staining (x20). B-C) Strong SLC7A5 staining in tumour cells (x20). D) The inner mass of tumour cells with membrane staining and negative palisading cells (x40).

Results 3 Figure 2. SLC7A7 protein expression in BCC by IHC. Epidermis with normal appearance adjacent to the tumor, weak IHC staining in basal cell layer and strong staining in the stratum granulosum cells (x20). Epidermis above the tumor mass shows strong SLC7A7 staining in stratum granulosum cells similar to normal cells (x20). Cells in stratum granulosum with SLC7A7 protein expression in membrane and cytoplasms (x40). Tumor cells absent from SLC7A7 protein expression (x40).

Results 4 Figure 3. SLC7A8 expression in BCC. Epidermis with normal appearance adjacent to the tumor, none to weak IHC staining (x20). Epidermis directly above the tumor mass shows none to weak staining. The tumor cells seem to express SLC7A8 heterogeneously as they display various degrees of staining intensity (x20). Some tissues had areas of tumor cells with strong membrane and cytoplasmic staining (x20). Staining of SLC7A8 in membranes of tumor cells (x40). Figure 4. TDO2 expression in BCC. Epidermis with normal appearance adjacent to the tumor show weak IHC staining (x20) Epidermis directly above the tumor mass shows none to weak staining. Stroma surrounding the masses of tumor cells shows immunoreactivity (x20). Immune cells with cytoplasmic TDO2-staining (x40). Staining of TDO2 in tumor cells (x40).

Discussion 1 SLC7A5 upregulation is implicated in other cancer types and inhibitory drugs are being explored. The present study implies that SLC7A5 is candidate target for further evaluation in BCC. Surprisingly, SLC7A7 protein expression was exclusively found in stratum granulosum and most likely not involved in BCC carcinogenesis.

Discussion 2 SLC7A8 implication in cancer is less known however present study could show upregulation in BCC. TDO2 was expressed by immune cells as well as in stroma, possibly contributing to tumour phenotype.

Conclusions What does this study add? Alterations of amino acid transporters SLC7A5 and SLC7A8 and the cytosolic enzyme TDO2 is suggested in basal cell carcinoma and possibly potential targets for treatment. SLC7A7 (transporter of e.g. lysine) is expressed in stratum granulosum of normal epidermis and may be involved in the cornification process.

The research team Sophie Lennholm (MSc, MD) Magnus Lindberg (Professor, MD) Gajana Gasparyan (BSc) Elisabet Tina (PhD, Head of Department) Sara Prosén (MSc, MD) Anna Göthlin Eremo (PhD)

Call for correspondence Why not join the debate on this article through our correspondence section? Rapid responses should not exceed 350 words, four references and one figure Further details can be found here