Archives of Dermatological Research

, Volume 304, Issue 10, pp 839–845

Role of translocator protein in melanoma growth and progression

Authors

    • Department of PathophysiologyKrasnoyarsk State Medical University
    • Department of Biochemistry and Molecular and Cellular BiologyGeorgetown University Medical Center
  • Maria Aksenenko
    • Department of PathophysiologyKrasnoyarsk State Medical University
  • Vassilios Papadopoulos
    • Department of Biochemistry and Molecular and Cellular BiologyGeorgetown University Medical Center
    • The Research Institute of the McGill University Health Center and Departments of Medicine, Biochemistry, Pharmacology and TherapeuticsMcGill University
Short Communication

DOI: 10.1007/s00403-012-1294-5

Cite this article as:
Ruksha, T., Aksenenko, M. & Papadopoulos, V. Arch Dermatol Res (2012) 304: 839. doi:10.1007/s00403-012-1294-5

Abstract

The 18 kDa translocator protein (TSPO) is a primarily mitochondrial protein that participates in steroid biosynthesis, cell proliferation, differentiation, apoptosis, and the regulation of mitochondrial function in general. TSPO has been implicated in carcinogenesis via its ability to transport cholesterol into mitochondria to meet the increased energy needs of tumor cells. The purpose of this study was to investigate TSPO involvement in melanoma pathogenesis. TSPO expression in melanoma and melanocytic nevi was analyzed by immunohistochemistry and real-time PCR, and TSPO levels were correlated to the invasiveness of the tumor. The number of TSPO-positive melanoma samples increased with tumor progression irrespective of age or gender of patients. Similar findings were obtained while examining TSPO expression levels in relation to the Clark invasion stage of the tumor. Indeed, the immunohistochemical index was elevated in invasive tumors characterized as Clark level V compared to those characterized as levels I and II. Besides, the elevation of immunohistochemical index was accompanied with a shift of homogeneous cytoplasmic subcellular expression pattern of the protein to nuclear and perinuclear. Taken together, these results suggest TSPO participation in melanoma growth and progression.

Keywords

Cutaneous melanomaMelanocytic nevusTSPO

Introduction

Translocator protein (TSPO) is an 18 kDa drug- and cholesterol-binding protein that localizes to the outer mitochondrial membrane. TSPO was discovered in 1977 by Braestrup and Squires [4, 28] as peripheral binding site for benzodiazepines and was thus, originally named peripheral-type benzodiazepine receptor. Later, TSPO expression was detected in most organs and cell types with an abundance in steroidogenesis-related tissues [5]. Such prominent distribution and the highly conserved sequence across species [12, 36] suggested fundamental functions regulated by this protein. Indeed, TSPO was shown to be involved in the regulation of steroid biosynthesis, mitochondrial respiration, mitochondrial permeability transition pore opening, cell proliferation, and apoptosis [3].

The participation of TSPO in the regulation of cell proliferation and apoptosis spurred the investigation of the role of TSPO in carcinogenesis. TSPO expression was found to be elevated in high-grade astrocytomas [25], glioblastomas [8], and breast carcinomas [15, 17, 18]. Increased levels of TSPO were also observed in colon carcinoma [22], prostatic intraepithelial neoplasia, primary prostate cancer [11], and oral squamous cell carcinoma [17, 27]. Although the exact mechanism of TSPO involvement in cancer development and progression is unclear, the most plausible explanation implies TSPO function as a cholesterol carrier that enhances transport of cholesterol to intracellular organelles for membrane biogenesis, in order to adapt to the requirements for elevated tumor cell growth and division [35].

Skin functions as a barrier against extrinsic pathogen factors, including UV radiation [30], which is considered to be a major contributor to the high incidence of skin cancer [34]. Cutaneous melanoma is the most aggressive type of skin malignancy and is associated with poor prognosis for disseminated forms [16]. Approximately 50–60 % of melanomas harbor B-RAF mutations that lead to pathological activation of mitogen-activated protein kinase (MAPK) pathway [1]. The most frequent B-RAF mutation is a substitution of Glu for Val at amino acid 600 (V600E, [9]). Although B-RAF V600E has been detected in melanocytic nevi, this mutation is considered as one of the key elements in melanoma development [29]. MAPK is a part of kinase signaling network that regulates fundamental biological processes, such as cell proliferation, growth, differentiation, and apoptosis [21]. MAPK inhibitors are under intensive study as possible therapeutic agents for melanoma [19].

In skin, TSPO was described for the first time in 1999 [32]. TSPO expression was detected in the dermis around blood vessels, in fibroblasts, in eccrine sweat ducts, and in hair follicles. The presence of TSPO was prominent in the granular and spinous layers of the epidermis. TSPO was also identified in Langerhans cells. In Mrl/Lpr mice, which develop an inflammatory pathology similar to lupus erythematodes, treatment with TSPO ligands prevented acanthosis, hyperkeratosis, and dermal infiltrate formation [6]. TSPO has been shown to participate in UV-mediated reactions: TSPO-transfected Jurkat cells were found to be more resistant to apoptosis and showed delayed caspase-3 activation early after UV exposure [31]. Furthermore, following UVA exposure, TSPO forms stable homopolymers with different molecular mass complexes ranging from 36 to 130 kDa in the presence of an abundance of reactive oxygen species (ROS) [13]. Spectroscopic studies revealed that, formation of polymers with TSPO occurs via covalent linkage of tyrosines [10]. TSPO polymeric forms are prevalent in breast cancer cell lines [18].

Recent studies showed that MAPK regulates TSPO gene expression and MAPK inhibitors control TSPO promoter activity [2]. Taken together, these data suggest that TSPO can regulate melanoma development and progression.

Materials and methods

Skin biopsies and immunohistochemistry (IHC)

Melanoma (n = 77) and benign melanocytic nevi (n = 13) specimens were collected from patients with primary melanocytic lesions at the Krasnoyarsk State Oncological Dispensary in 2004–2009. The study was approved by the Local Ethics Committee of Krasnoyarsk State Medical University. Skin biopsies from melanoma patients were divided into four groups in accordance with the Clark levels of invasion (I–II, III, IV, and V). For immunohistochemical evaluation, tissue samples were fixed in a 10 % neutral buffered formalin, dehydrated in an alcohol series, and embedded in paraffin. The 3–4 μm sections were preincubated with Peroxidase Block (NovoLink Polymer Detection System, Leica Microsystems, Newcastle, UK) for 5 min and Protein Block (NovoLink Polymer Detection System, Leica Microsystems, Newcastle, UK) for 5 min to block non-specific background staining. Then the slides were incubated with anti-TSPO antibodies [23] for 1 h, which were used at a dilution of 1:200. Negative controls were made by performing the same immunohistochemistry protocol without application of anti-TSPO antibodies. Washes were performed in 1× phosphate-buffered saline (PBS) obtained from PBS tablets (Sigma®, St Louis, MO, USA) dissolved in 200 mL deionized water that yields 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4. After washing in PBS, the sections were incubated with Post primary Block for 30 min followed by NovoLink Polymer (NovoLink Polymer Detection System). Thereafter, diaminobenzidine substrate chromogen solution obtained by adding 50 μl DAB Chromogen to 1 mL of NovoLink DAB Substrate Buffer (NovoLink Polymer Detection System) was applied to the sections for 5 min. The sections were counterstained with hematoxylin and mounted.

The slides were viewed with an Olympus BX-41 microscope with a magnification of 600×. The number of TSPO-positive cells was determined per 100 cancer or nevus cells. In positive samples, the staining patterns were further classified as strong (+++), moderate (++), or weak (+) using an immunohistochemistry (IHC) index score calculated as the number of (+++) cells × 3 + number of (++) cells × 2 + number of (+) cells × 1 [24]. Digital images were recorded with an Infinity camera (Lumenera Corporation, Canada) and analyzed using Infinity Capture Software, Infinity Analyze Software (V.4.6.0).

Real-time PCR

Skin biopsies from melanoma, melanocytic nevi patients and healthy voluntaries were incubated in RNAlater™ (Qiagen, USA) with further processing for RNA isolation with the Ribo-Zol B kit (Amplisense, Russia). Reverse transcription reactions were performed to obtain complementary DNA for 30 min at 37 °C using the Reverta reverse transcription kit (Amplisense, Russia). The mRNA expression of TSPO was analyzed by the StepOne Real-Time PCR system (Applied Biosystems, USA) using TaqMan® Gene Expression Master Mix (Applied Biosystems, USA). Human TSPO was amplified using the probe set Hs00559362 TaqMan® Gene Expression assays (Applied Biosystems, USA). The readout for each reaction was the cycle threshold (Ct), which corresponds to the cycle number in which the fluorescent signal became higher than a pre-defined threshold. Relative gene expression levels were calculated by normalization to β-actin expression (Applied Biosystems, USA).

Statistics

Statistical analyses were carried out by the Student’s unpaired t test, Mann–Whitney test and analysis of variance. Mean values of the IHC index score were presented with SD. p values lower 0.05 were considered to be statistically significant.

Results

TSPO expression was detected in 87.5 % of the melanoma skin biopsies and 100 % of melanocytic nevi (Table 1; Fig. 1a–d). In unaffected epidermis the TSPO+ cells showed an homogenous brown color in the cytoplasm indicative of the intracellular localization of the protein, with prevalence in the spinous and granular layers of epidermis (Fig. 1a, b). TSPO+ cells were also found in sweat and sebaceous gland cells, in hair follicle cells, where preferential cytoplasmic localization was also observed (Fig. 1d). In benign nevi TSPO+ cells were present in epidermis and dermal nests of melanocytes (Fig. 1e, f). TSPO expression level revealed by IHC index score was 120.8. In melanoma slides, TSPO-positive cells were found in tumor nodes and epidermal keratinocytes and also in appendages of skin. TSPO-positive tumor samples exhibited an homogeneous pattern of TSPO expression throughout the tumor node. In melanoma samples intensively immunopositive for TSPO, redistribution in the subcellular localization was observed with the shift from cytosolic to perinuclear and nuclear (Fig. 2a–c). No differences in TSPO expression levels between males and females were found (Table 2). No age dependence of TSPO expression levels was established. The IHC index score in patients with Clark levels I-II tumor invasion was 44.4, and this score was significantly lower than that from the Clark level V melanomas, where the IHC index was 116.7. For tumors with Clark invasion levels III and IV, TSPO expression IHC scores were 93.2 and 77, respectively, although the values did not present statistical differences as compared to Clark I and II tumor invasion levels. Melanoma IHC score indexes of I–II and IV levels of invasion were significantly lower in comparison to melanocytic nevi IHC score index (Fig. 3a). Comparison of all the melanocytic nevi and melanoma cases revealed diminished TSPO levels in melanoma samples (Fig. 3b). The mRNA levels of TSPO derived from melanoma tissues were not significantly different as compared to normal skin samples but decreased in benign nevi (Fig. 3c).
Table 1

TSPO immunoreactivity in melanoma and melanocytic nevi: dependence of Clark invasion level in melanoma cases

Clark invasion level

Number of cases

TSPO-positive cases (%)

I–II

20

70

III

21

90.5

IV

19

89.5

V

17

100

Melanocytic nevi

13

100

https://static-content.springer.com/image/art%3A10.1007%2Fs00403-012-1294-5/MediaObjects/403_2012_1294_Fig1_HTML.jpg
Fig. 1

TSPO expression in skin biopsies studied by immunohistochemistry: a Expression of TSPO in epidermis and dermal melanoma cells, ×100. b Increased immunoreactivity of TSPO in spinous and granular layers of epidermis, ×400. c Example of TSPO-negative melanoma cells, ×400. d TSPO-positive cells found in sweat glands in primary melanoma, ×100. e Immunohistochemical staining with antibodies to TSPO in melanocytic nevi, ×100. f The same slide, ×400

https://static-content.springer.com/image/art%3A10.1007%2Fs00403-012-1294-5/MediaObjects/403_2012_1294_Fig2_HTML.jpg
Fig. 2

Melanoma cells immunostained for TSPO with different levels of TSPO expression. a Strong (with nuclear and perinuclear subcellular localization of TSPO), ×400. b Moderate expression of TSPO, ×400. c Weak immunostaining with antibodies to TSPO, ×400. d Immunostaining for TSPO in malignant melanoma. e Negative control (the same sample without anti-TSPO antibodies), ×100. f Immunostaining for TSPO in malignant melanoma. g Negative control (the same sample without anti-TSPO antibodies), ×400

Table 2

Demographics and immunohistochemical findings of TSPO expression in melanoma biopsies

 

Median age

Gender

≤50

>51

Male

Female

TSPO level (immunohistochemistry index)

75.7 ± 47.1

73.0 ± 31.8

58.2 ± 22.4

48.8 ± 18.1

https://static-content.springer.com/image/art%3A10.1007%2Fs00403-012-1294-5/MediaObjects/403_2012_1294_Fig3_HTML.gif
Fig. 3

a Comparative analysis of TSPO expression in normal melanocytes in benign nevi and malignant melanoma cells: TSPO expression elevation corresponds to increasing tumor invasion in the derma. Results are presented as the mean ± SD, *p < 0.05. b Mean values of IHC score for TSPO expression in melanocytic nevi and melanoma cells, *p < 0.05. cTSPO mRNA expression in normal skin, melanocytic nevi and melanoma relative to β-actin. Results are the mean of four experiments, *p < 0.05

Discussion

TSPO expression was decreased in malignant melanocytes in comparison to normal melanocytes. This observation is in accordance with the previous data demonstrating the reduction of TSPO expression levels in both basal cell carcinoma and squamous cell carcinoma [17, 26]. The mRNA TSPO levels were significantly lower in nevus samples as opposed to normal skin biopsies but no differences in TSPO mRNA expression levels between normal and melanoma skin were found. These results could be explained by the absence of TSPO expression alterations in the epidermis or other dermal components, such as endothelial cells and fibroblasts. On the other hand, it is in accordance with the data on the decreased TSPO levels in skin tumors [17, 26].

In melanoma skin biopsies, TSPO levels were significantly elevated in Clark invasion level V tumors as opposed to tumors of levels I and II. In addition, the number of TSPO-positive biopsies increased as the Clark invasion level elevated. Clark invasion level and Breslow thickness are well established markers for prognosis of disease as well as for the invasiveness of the tumor [20]. Our data are in agreement with the previous results that showed elevated TSPO levels in tumor cells with an aggressive phenotype [18]. Our findings also point to the putative role of TSPO in cancer cell development as a carrier of cholesterol to biological membranes for membrane biogenesis. Melanoma mitotic index rises as the tumor becomes thicker, and this result may stem from the increased requirement for cholesterol for cell division [33]. We demonstrated that although TSPO levels are diminished in melanoma cells, melanoma progression is associated with a rise in TSPO levels. Furthermore, the TSPO expression patterns in melanoma do not depend on the gender or age of patients. In tumors with high IHC score indices, the perinuclear and nuclear localization of a protein was earlier revealed in highly aggressive breast cancer cell lines and related to TSPO-mediated transport of cholesterol to the nuclei for membranogenesis of dividing cells [18]. This observation supports the hypothesis of TSPO involvement in carcinogenesis via regulation of tumor cell metabolic atypism.

Recent studies demonstrated that the MAPK (Raf-1-ERK1/2) signal transduction pathway modulates TSPO promoter activity and TSPO expression levels, and this pathway is under the control of protein kinase C epsilon [3]. MAPK signal transduction pathway dysregulation and pathological activation are considered crucial events in melanoma onset and evolution [14]. Interestingly, activation of MAPK signal transduction pathway, mediated by protein kinase C epsilon and C delta, takes place after UVB irradiation [7]. Taken together, these data suggest that UVB-induced TSPO expression in melanoma cells may be under the control of MAPK and support the hypothesis that altered MAPK activity in skin cancer cells drives TSPO expression in a manner parallel to increased levels of tumor invasion. Whether TSPO is at the origin or is a consequence of increased cell growth remains to be determined.

Acknowledgments

This study was supported by grants from Boehringer Ingelheim Funds for Biomedical Research (to T.R.) and National Institutes of Health, USA, and Canadian Institutes of Health Research (to V.P.).

Conflict of interest

The authors declare no conflicts of interest.

Copyright information

© Springer-Verlag Berlin Heidelberg 2012