Transcription analysis in the MeLiM swine model identifies RACK1 as a potential marker of malignancy for human melanocytic proliferation

Abstract

Background: Metastatic melanoma is a severe disease. Few experimental animal models of metastatic melanoma exist. MeLiM minipigs exhibit spontaneous melanoma. Cutaneous and metastatic lesions are histologically similar to human's. However, most of them eventually spontaneously regress. Our purpose was to investigate whether the MeLiM model could reveal markers of malignancy in human melanocytic proliferations.

Results: We compared the serial analysis of gene expression (SAGE) between normal pig skin melanocytes and melanoma cells from an early pulmonary metastasis of MeLiM minipigs. Tag identification revealed 55 regulated genes, including GNB2L1 which was found upregulated in the melanoma library. In situ hybridisation confirmed GNB2L1 overexpression in MeLiM melanocytic lesions. GNB2L1 encodes the adaptor protein RACK1, recently shown to influence melanoma cell lines tumorigenicity. We studied the expression of RACK1 by immunofluorescence and confocal microscopy in tissues specimens of normal skin, in cutaneous and metastatic melanoma developped in MeLiM minipigs and in human patients. In pig and human samples, the results were similar. RACK1 protein was not detected in normal epidermal melanocytes. By contrast, RACK1 signal was highly increased in the cytoplasm of all melanocytic cells of superficial spreading melanoma, recurrent dermal lesions and metastatic melanoma. RACK1 partially colocalised with activated PKCalphabeta. In pig metastases, additional nuclear RACK1 did not associate to BDNF expression. In human nevi, the RACK1 signal was low.

Conclusion: RACK1 overexpression detected in situ in human melanoma specimens characterized cutaneous and metastatic melanoma raising the possibility that RACK1 can be a potential marker of malignancy in human melanoma. The MeLiM strain provides a relevant model for exploring mechanisms of melanocytic malignant transformation in humans. This study may contribute to a better understanding of melanoma pathophysiology and to progress in diagnosis.

Figure 1

Expression of RACK1 mRNA in pig tissues. (A-B) In situ hybridisation autoradiography of RACK1 antisense (A) and sense (B) probes on depigmented sections. For each probe, normal (N) tissues are displayed on the left (skin, lung, liver and lymph node) and tumoral (T) tissues on the right (cutaneous melanoma, metastatic melanoma from lung, liver and lymph node). Note the intense signal on the tumors compared to the healthy or non-compromised tissues, with the antisense probe, except for lymph node. (C) Darkfield photomicrograph taken from a MeLiM melanoma lung metastasis hybridised with the RACK1 antisense probe. (D) Consecutive section stained with hematoxylin and eosin. The pigmented area in the tumor matches the region which exhibits silver grains on (C). Bar = 1 cm for A and B and 100 μm for C and D.

Figure 2

Identification of melanoma cells from MeLiM by MITF. Melanocytes were visualized as brown nuclear granules, by immunohistochemistry with MITF antibody (A-C) and without the primary antibody (D-F). (A, D) Normal skin. (B, E) Cutaneous melanoma. (C, F) Melanoma metastasis in a lung. a, alveolae; be, bronchiolar epithelium; d, dermis; e, epidermis; hf, hair follicle. Arrows point to normal melanocytes (black) and melanoma cells (white). Bar = 100 μm.

Figure 3

RACK1 expression in normal pig epidermis. (A, C, D) Confocal microscopy analysis of RACK1 protein (green fluorescence), and double labelling for either MITF (A, C) or DCT (D) (red fluorescence) on pig skin. Normal epidermis were from control Meishan minipig (A, B), and MeLiM (C, D). (B) Transmission photograph corresponding to (A). (C) Three dimensional 'orthogonal' slice projection analysis is included: the large central panel shows a single optical slice through which an x axis (green line) and a y axis (red line) were defined for sliced z-axis reconstruction. The corresponding results for the x, z slice (top) and the y, z slice (right) are shown. The blue line represents the position of the central panel image in the z stack. Nuclear counterstaining is shown in blue. Note the RACK1 cytosolic spotty signal on keratinocytes and its absence in the melanocyte indicated by the white dashed line. Dotted lines indicate epidermis-dermis boundaries. e, epidermis; d, dermis. Bar = 5 μm.

Figure 4

Cellular distribution of RACK1 in MeLiM melanoma at different progression stages. Confocal microscopy analysis of RACK1 (green fluorescence), and double labelling for MITF (red fluorescence). (A) Cutaneous melanoma. (B-D) Melanoma metastasis in a lymph node (B), lung (C) and heart (D). Three dimensional 'orthogonal' slice projection analyses are presented as in Figure 3. Nuclei are shown in blue. (A1) Transmission photograph corresponding to (A2). (A3) Zoom on (A2) inset. White arrowheads in (A1-A3) point at a dermal cutaneous melanoma cell positive for MITF and analysed by orthogonal projection. Note the comparable RACK1 cytosolic signal on dermal melanoma cells and epidermal keratinocytes. High levels of RACK1 are seen in cutaneous and metastatic melanoma cells with perinuclear localization. Furthermore, in metastases, RACK1 is seen within the nuclei, as indicated by yellow arrowheads on the optical slice and the orthogonal projections. Dotted lines in (A1) and (A2) indicate epidermis-dermis boundaries. e, epidermis; d, dermis. Bar = 5 μm.

Figure 5

Cellular distribution of RACK1 in human cutaneous melanocytic proliferations. Confocal microscopy analysis of double labelling of RACK1 protein (green fluorescence), with MITF (red fluorescence). (A) Control human skin: an MITF-positive melanocyte is localised to the basal membrane. (B-D) Nevi: lentiginous proliferation in B, junctional nest of melanocytes in C, with an additional dermal component in D. (E, F) Cutaneous melanoma samples. Basal and suprabasal keratinocytes display a strong cytoplasmic RACK1 signal. RACK1 is almost not detected in normal melanocytes (A). This also holds true in hyperproliferative lesions of nevi (B-D). In some nevi, RACK1 heterogeneous expression is recognized in melanocytic cells (C). By contrast, in cutaneous melanoma, all MITF+ cells displayed a strong RACK1 signal (big arrow in E and F). Sections of skin are from 6 different patients. Arrowheads point to melanocytes where RACK1 is not detected. Arrows indicate melanocytes expressing cytoplasmic RACK1. Nuclear counterstaining is shown in blue in B. Dotted line indicates epidermis-dermis boundary. e, epidermis; d, dermis. Bar = 10 μm.

Figure 6

RACK1 in human melanoma metastasis. Confocal microscopy analysis of double labelling of RACK1 protein (green fluorescence) and MITF (red fluorescence). (A, B) Melanoma metastasis in lymph node. (C, D) Melanoma metastasis in liver. High levels of RACK1 are seen in the cytoplasm of all metastatic human melanocytes. Sections are from 4 different patients. Nuclear counterstaining is shown in blue in D. Bar = 10 μm.

Figure 7

Cellular distribution of activated PKC in human nevi and melanoma. (A, B) Confocal microscopy analysis of double labelling of phospho-PKCαβ protein (green fluorescence, biotin amplified signal) and MITF (red fluorescence). (C, D) Confocal microscopy analysis of double labelling of RACK1 protein (green fluorescence, biotin amplified signal), with phospho-PKCαβ (red fluorescence). (A, C) Nevi. (B, D) Melanoma metastasis in lymph node. High levels of activated PKC are seen on MITF-positive melanoma cells in metastasis compared to MITF-positive melanocytes in nevus (arrowheads in B and A, respectively). Dermal melanocytes in nevus display low cytoplasmic RACK1 and nuclear phospho-PKC signals (arrow in C). Abundant signals for RACK1 and activated PKCαβ partially colocalise (arrow in D). Nuclear counterstaining is shown in blue. Dotted line indicates epidermis-dermis boundary. e, epidermis; d, dermis. Bar = 10 μm.