Ablation of epidermal RXRα in cooperation with activated CDK4 and oncogenic NRAS generates spontaneous and acute neonatal UVB induced malignant metastatic melanomas

Abstract

Background: Understanding the underlying molecular mechanisms involved in the formation of cutaneous malignant melanoma is critical for improved diagnosis and treatment. Keratinocytic nuclear receptor Retinoid X Receptor α (RXRα) has a protective role against melanomagenesis and is involved in the regulation of keratinocyte and melanocyte homeostasis subsequent acute ultraviolet (UV) irradiation.

Methods: We generated a trigenic mouse model system (RXRα ^ep-/- | Tyr-NRAS ^Q61K | CDK4 ^R24C/R24C ) harboring an epidermal knockout of Retinoid X Receptor α (RXRα ^ep-/- ), combined with oncogenic NRAS ^Q61K (constitutively active RAS) and activated CDK4 ^R24C/R24C (constitutively active CDK4). Those mice were subjected to a single neonatal dose of UVB treatment and the role of RXR α was evaluated by characterizing the molecular and cellular changes that took place in the untreated and UVB treated trigenic RXRα ^ep-/- mice compared to the control mice with functional RXRα.

Results: Here we report that the trigenic mice develops spontaneous melanoma and exposure to a single neonatal UVB treatment reduces the tumor latency in those mice compared to control mice with functional RXRα. Melanomas from the trigenic RXRα ^ep-/- mice are substantial in size, show increased proliferation, exhibit increased expression of malignant melanoma markers and exhibit enhanced vascularization. Altered expression of several biomarkers including increased expression of activated AKT, p21 and cyclin D1 and reduced expression of pro-apoptotic marker BAX was observed in the tumor adjacent normal (TAN) skin of acute ultraviolet B treated trigenic RXRα ^ep-/- mice. Interestingly, we observed a significant increase in p21 and Cyclin D1 in the TAN skin of un-irradiated trigenic RXRα ^ep-/- mice, suggesting that those changes might be consequences of loss of functional RXRα in the melanoma microenvironment. Loss of RXRα in the epidermal keratinocytes in combination with oncogenic NRAS ^Q61K and CDK4 ^R24C/R24C mutations in trigenic mice led to significant melanoma invasion into the draining lymph nodes as compared to controls with functional RXRα.

Conclusions: Our study demonstrates the protective role of keratinocytic RxRα in (1) suppressing spontaneous and acute UVB-induced melanoma, and (2) preventing progression of the melanoma to malignancy in the presence of driver mutations like activated CDK4 ^R24C/R24C and oncogenic NRAS ^Q61K .

Keywords: Acute UVB; CDK4R24C/R24C; Keratinocytes; Malignant melanoma; Melanocytes; Microenvironment; NRASQ61K; Retinoid-X-receptor α (RXRα); Spontaneous melanoma; Trigenic.

Fig. 1

Macroscopic and histological characterization of spontaneous and acute UVB induced melanomas from control and RXRα^ep−/−|TyrNRAS ^Q61K|Cdk4^R24C/R24C mice. Mice with epidermal-specific Rxrα ablation in combination with Tyr-NRAS^Q61K and homozygous Cdk4^R24C mutations have (a) spontaneous melanocytic growths; (b) larger melanocytic tumors with some degree of ulceration post single neonatal UVB treatment compared to mice with functional Rxrα. Lesions indicated by arrows. Histological analyses of melanocytic tumors from Tyr-NRAS^Q61K and Cdk4^R24C mice and with functional (left panel) and ablated (right panel) Rxrα. Tyr-NRAS^Q61K /Cdk4^R24C mice with epidermal-specific Rxrα ablation have (c) more pigmented lesions with minimal penetration into epidermal basal layer (inset); (d) greater degree of densely pigmented lesions with enhanced penetration into epidermal basal layer after acute neonatal UVB treatment. E, epidermis; D, dermis; HD, hypodermis. Scale bar =50 μm. e, f Increased epidermal thickness, and (g, h) higher radial growth phase (RGP) and vertical growth phase (VGP) of melanocytic tumors in RXRα^ep−/− |Tyr-NRAS^Q61K |Cdk4^R24C/R24C mice with (e, g) no UVB treatment and (f, h) single UVB treatment relative to their respective controls. Statistical relevance indicated as follows; * = p < 0.05

Fig. 2

Melanomas from RXRα ^ep−/−|TyrNRAS ^Q61K|Cdk4 ^R24C/R24C mice display enhanced proliferation, malignant conversion and angiogenesis relative to their controls. a, b Fluorescent IHC for proliferation marker PCNA (red) and melanocyte marker TYRP1 (green). A trend in overall increase of PCNA+/TYRP1+ cells was observed in lesions from Rxrα^ep−/− mice compared to Rxrα^L2/L2 controls in combination with Tyr-NRAS^Q61K and Cdk4^R24C mutations in no UVB treated mice (a) and single neonatal UVB treated mice (b). c, d Bar-graph represents PCNA + |TYRP2+ melanocytes/ field in no UVB and acute UVB treatment respectively. e, f IHC using antibody cocktail of antibodies directed against malignant melanoma antigens HMB45 and MART-1 (red). Overall, more positive staining was observed in lesions from the triple knockout mice with RXRα ^ep−/− ablation compared to bigenic mice with their respective Rxrα^L2/L2 controls mutations in no UVB treated mice (e) and single neonatal UVB treated mice (f). g, h IHC for tumor angiogenesis marker CD31 (red). Overall, more prominent staining was observed in lesions from trigenic RXRα ^ep−/− mice compared to controls. Loss of epidermal Rxrα results in lesions with multicellular CD31+ blood vessels (g, right panel) in mice with no UVB treatment while acute UVB treated mice resulted in larger multicellular CD31+ blood vessels (h, right panel). a, b, c, d, e, f, g, h White dashed lines, artificially added, indicate epidermal-dermal junction. Blue color corresponds to DAPI staining of the nuclei. E = Epidermis, D = Dermis. Scale bars = 50 μm. Statistical relevance indicated as follows; * = p < 0.05

Fig. 3

Melanomas from trigenic RXRα ^ep−/−|TyrNRAS ^Q61K|Cdk4 ^R24C/R24C mice display reduced apoptosis relative to their controls with functional RXRα. a, b TUNEL assay to label apoptotic cells. Apoptotic cells are indicated by green staining (top panel), blue color corresponds to DAPI staining of the nuclei (middle panel) and merged TUNEL and DAPI cells (lower panel). Overall, reduced TUNEL positive staining was observed in lesions from trigenic Rxrα^ep−/− mice compared to their respective controls in mice with (a) no UVB treatment as well as the (b) acute UVB treated mice. c, d Bar-graph represents TUNEL+ melanocytes/ field in no UVB and acute UVB treatment respectively

Fig. 4

Enhanced metastasis to draining lymph nodes in trigenic RXRα ^ep−/−|Tyr-NRAS ^Q61K|Cdk4 ^R24C/R24C mice relative to Rxrα ^L2/L2 control mice. a, b Excised draining lymph nodes from RXRα^L2/L2/Tyr-NRAS^Q61K/Cdk4^R24C/R24C and RXRα^ep−/−/TyrNRAS ^Q61K/Cdk4^R24C/R24C mice at age (a) 12 months for untreated mice and (b) 6 months for single UVB treated mice. (c, d) Histological analyses of draining lymph nodes from Tyr-NRAS^Q61K/Cdk4^R24C mice with functional and ablated Rxrα in (c) no UVB treatment as well as the (d) acute UVB treated mice. e, f Chromogenic IHC for melanocyte-specific marker TYRP1 (brown). More positive staining overall is observed in RXRα^ep−/−/TyrNRAS ^Q61K/Cdk4^R24C/R24C LNs as opposed to their relative RXRα^L2/L2 control LNs in both the UVB untreated and treated groups. Hematoxylin (purple) was used as a nuclear counterstain. Scale bar =100 μm

Fig. 5

Spontaneous and acute UVB-irradiated skin from trigenic mice exhibit altered expression of regulators of signaling. a Immumnoblotting analyses of non-UV irradiated TAN skin samples showed modest upregulation in pAKT, significant upregulation of p21 and Cyclin D1 and a modest decrease in BAX expression in the Rxrα ^ep−/− mutant mice relative to the controls in the untreated group. b Western blot analysis of single UVB treated groups of control and trigenic Rxrα ^ep−/− mutant mice exhibiting significant upregulation of pAKT, p21 and Cyclin D1 expression and a significant decrease in BAX expression in the trigenic mice relative to the controls. Equal loading was confirmed by β-actin. 2–3 biological replicates for each group are shown for all immunoblots