Carcinogen treatment in mouse selectively expressing activated N-RasQ61K in melanocytes recapitulates metastatic cutaneous melanoma development

Authors


  • Correction added after online publication January 2012: The fifth author Samuel Gehrke was added after the original Early View publication of this paper.

Emmanuel Contassot, e-mail: emmanuel.contassot@usz.ch

Summary

The incidence of melanoma has significantly increased, and a better understanding of its pathogenesis and development of new therapeutic strategies are urgently needed. Here, we describe a murine model of metastatic cutaneous melanoma using C57BL/6 mice expressing a mutated human N-Ras gene under the control of a tyrosinase promoter (TyrRas). These mice were topically exposed to 7,12- dimethylbenzanthracene (DMBA) for brief exposure periods. Cutaneous melanoma developed at the site of exposure on average by 19 weeks of age and in 80% of mice. Importantly, as in humans, melanoma development was associated with subsequent metastasis to tumor-draining lymph nodes. Critically, such metastatic behavior is transplantable, as intradermal inoculation of melanoma cells from TyrRas-DMBA mice into non-transgenic mice led to the growth of melanoma and, again, metastasis to skin-draining lymph nodes. This metastatic melanoma model closely mimics human pathology and should be a useful tool for studying melanoma pathogenesis and developing new therapies.

Significance

Mouse melanoma models that mirror the course and pathology of human disease are of great interest for both basic research and preclinical testing of new therapeutic approaches. Herein, we describe experiments where C57BL/6 mice expressing oncogenic N-Ras under the control of a tyrosinase promoter were topically exposed to 7,12-dimethylbenzanthracene (DMBA). These mice developed cutaneous melanoma and lymph node metastases. Importantly, we show for the first time that cell suspensions, isolated from either primary tumors or lymph node metastases, that are implanted subcutaneously to non-transgenic recipients also develop into cutaneous tumors that metastasize to the lymph nodes.

Current evidence suggests that human melanoma arises as a consequence of cumulative mutations in genes governing melanocyte growth, differentiation, and death (Chin, 2003; Chudnovsky et al., 2005). These include mutations that activate Ras and members of its effector cascade. Notably, mutations in the N-Ras gene at codon 61 are a frequent event in primary melanomas (Alsina et al., 2003; Kumar et al., 2003) and metastatic lesions (Gorden et al., 2003). Progress in developing therapeutic approaches for the therapy of melanoma is dependent on the availability of mouse models as surrogates of the human disease. Most currently used murine models of melanoma make use of long-term in vitro propagated melanoma cell lines implanted subcutaneously. These models do not optimally reflect human melanoma in that they do not occur spontaneously, and their growth results in encapsulated tumors with no junctional or epidermal activity. Murine models using oncogenic SV40 T antigen targeted to melanocytes (Bradl et al., 1991; Mintz and Silvers, 1993) (Penna et al., 1998) have been produced, but these mice essentially develop ocular melanoma and only rarely develop cutaneous melanomas and metastatic disease. Furthermore, a decrease or loss of pigmentation and tyrosinase activity was frequently reported (Orlow et al., 1995, 1998). More recently, models using melanocyte-targeted expression of activated Ras have been developed. A model that has been widely studied is based on the expression of activated Ras in melanocytes of mice expressing little or no p16 (Ackermann et al., 2005; Chin et al., 1997). Although these transgenic models are more reminiscent of human melanoma, some do not metastasize. Herein, we characterize an autochthonous model of metastatic melanoma that develops in the skin of transgenic C57BL/6 mice selectively expressing a mutated N-Ras in melanocytes after a short post-natal period of cutaneous exposure to the carcinogen DMBA. TyrRas transgenic mice have been previously described (Ackermann et al., 2005). Briefly, both the 6.1-kb promoter sequence and the 3.6-kb distal control region of the mouse tyrosinase gene were used to restrict expression of a mutant human NRAS gene (NRASQ61K) to the melanocytic lineage. DMBA in 50 μl acetone (0.5 mg/ml) was applied once a week on a defined 1 cm2 surface of the back skin of mice, during five consecutive weeks, starting at 3 weeks of age. Macroscopically visible tumors appeared with a median latency of 18 weeks, and 47 of 59 DMBA-treated TyrRas mice (80%) had macroscopically identifiable cutaneous tumors by week 26 (Figure 1A,B). None of the untreated TyrRas mice developed macroscopic tumors during this time frame nor did DMBA-treated wild-type mice (not shown). Tumor growth was exponential, and in 10 of 12 cases (83%), a tumor volume of 1 cm3 was achieved within 5 to 10 weeks after the first appearance with a doubling time of 8.5 days (Figure 1C). These observations indicate that large homogenous groups of tumor-bearing TyrRas mice can be generated. Hyperpigmented nodules compatible with melanocyte-derived tumors (Figure 1B) were observed on TyrRas mouse skin exposed to DMBA. During the 30-week follow-up period, no morphological changes of the skin were observed outside of the treated areas. Histological analyses revealed that in addition to the normal distribution of melanocytes found around hair follicles, pigmented cells were also diffusely distributed in the reticular and papillary dermis of tumor-free TyrRas mice (Figure 2A). Generalized vertical growth, complete invasion of the dermis, asymmetry and invasion beyond the fat layer were observed in DMBA-induced tumors (Figure 2A, right panel). Analysis of DMBA-treated TyrRas mice systematically revealed pigmentation of axillary and inguinal skin-draining lymph nodes. Importantly, tumor-draining lymph nodes were enlarged, suggestive of lymph node metastasis (Figure 2B, left panel). ‘Small’ lymph nodes included large areas of focally pigmented cells within the sinus without disruption of normal lymph node architecture (Figure 2B, right panel). In contrast, enlarged skin-draining lymph nodes exhibited abundant infiltration by large pigmented cells that were identical in morphology to those observed in cutaneous tumors and were associated with disruption of normal lymph node architecture. Importantly, mRNA specific for melanoma-associated antigens (MAA), including TRP-1, TRP-2, Pmel-17, Melan-A (MART-1), and tyrosinase, was detected in tumor samples confirming a melanocytic origin for the tumors (Figure 2C). These observations strongly suggest that DMBA-induced melanoma cells are able to migrate to and proliferate within draining lymph tissues, a characteristic of the metastatic process. To confirm the tumorigenic and metastatic potential of melanoma cells, we further analyzed their capacity to grow subcutaneously after intradermal injection into healthy Rag-2−/− mice. Tumors from DMBA-treated TyrRas mice and tumor-invaded skin-draining lymph nodes were dissociated mechanically, and 1 × 105 pigmented cells were injected intradermally in immunodeficient Rag-2−/−C57BL/10 or WT mice as a suspension in 100 μl of HBSS. Twelve to 16 weeks after intradermal inoculation of cutaneous or lymph node-invading melanoma cells, large cutaneous tumors were observed in eight of eight sex-matched Rag-2−/−C57BL/10 recipients. Critically, metastases to the lymph nodes were identified in all of these mice (Figure 3). To follow this up, melanoma-invaded lymph nodes of Rag-2−/−C57BL/10 implanted mice were dilacerated, and cell suspensions were intradermally injected into syngeneic recipients. In three of three implanted mice, cutaneous melanoma developed and metastases to the lymph nodes were again observed. Identical results have been obtained in five of five WT C57BL/6 mice. Histological analyses of invaded lymph nodes confirmed the presence of atypical highly pigmented cells, and notably, no difference have been observed between Rag-2−/−C57BL/10 and WT C57BL/6 recipients (Figure 3C). Such serial transplantations of TyrRas melanoma cells from either primary lesions or invaded lymph nodes demonstrate the high metastatic potential for this model. Such a property could be of interest for the study of melanoma stem cells and/or the emerging switching theory in an appropriate model. Transgenic models using melanocyte-targeted expression of the HRAS oncogene on a p16 (INK4aΔ2/3)-deficient background have been reported (see (Beermann et al., 1999) and (Tietze and Chin, 2000) for review). In the model described by Chin and coworkers, mice develop cutaneous as well as ocular melanoma, with a high penetrance and after a relatively short latency. However, melanoma metastases were never observed in these mice (Chin et al., 1997, 1999). More recently, another model of melanoma in melanocyte-targeted N-Ras-p16 (INK4a)-deficient mice was reported (Ackermann et al., 2005). In this model, mice developed multiple highly invasive pigmented primary cutaneous melanomas with a penetrance greater than 90%. The observation that our mouse model is both reminiscent of metastatic behavior in human melanoma and expresses classical MAA in primary and metastatic lesions strongly suggests that it also constitutes a valuable tool for the in vivo study of spontaneous antitumor immune responses and the development of immunotherapeutic approaches. Preclinical investigations for new drugs such as demethylating agents, small-molecule kinase inhibitors, and specific oligonucleotides are often performed in vitro and in animal models which are relatively poorly representative of the human disease. Our model, with its short latency period, predictable and visible exponential tumor growth, and its high metastatic potential will be of use in the selection and testing the preclinical therapeutic candidates to identify those most likely to succeed in human clinical trials.

Figure 1.

 Tumor development and growth characteristics in TyrRas-7,12- dimethylbenzanthracene (DMBA) mice. (A) Kaplan–Meier representation of DMBA-induced tumor occurrence in TyrRas transgenic mice (full line: DMBA-treated Tyr-Ras mice n = 59; dashed line: non-treated Tyr-Ras mice n = 10). (B) Representative tumor on the back of a 20-week-old TyrRas-DMBA mouse. (C) Individual tumor growth in DMBA-treated TyrRas mice (left panel). Day 0 corresponds to the day when individual tumors became palpable. Mice were sacrificed when tumors reached a diameter of 1 cm2, according to animal experimentation guidelines. Modeling of exponential tumor growth during the first 5 weeks (right panel, no euthanized animal at this time) revealed the following equation: Y = 0.02259*exp(0.5672*X). The calculated tumor-doubling time was 1.22 weeks.

Figure 2.

 Characterization of TyrRas-7,12- dimethylbenzanthracene (DMBA) tumors as melanoma. (A) Histological representation of skin treated or not with DMBA. In non-treated skin, pigmented cells accumulated around pilli follicles and were also present in the dermis (left panel). Histology of DMBA-induced tumor revealed a proliferation of pigmented cells tightly attached to the epidermis and the invasion of the deeper tissues (right panel). (B) Histological analysis of a skin-draining lymph node from a non-DMBA exposed TyrRas mouse illustrating a conserved lymph node architecture (top panel) and a representative tumor-draining lymph node (bottom panel). Note that pigmented cells invaded the draining lymph node only in mice bearing a DMBA-induced tumor. The proliferation of pigmented cells caused a notable disruption of the lymph node architecture. (C) RT-PCR analysis of RNA from TyrRas skin and TyrRas-DMBA tumors for Tyrosinase, Trp-1, Trp-2, Melan-A and Pmel-17 (gp100) mRNA. 30-cycle PCR cDNA-amplification products from two representative mice are presented.

Figure 3.

 TyrRas-7,12- dimethylbenzanthracene (DMBA) tumors can be transplanted and retain their ability to metastasize. (A) Intradermal injection of 1 × 105 cells from a dilacerated primary cutaneous tumor of TyrRas-DMBA mice into immunodeficient Rag-2−/−C57BL/10 mice resulted in the development of a new tumor (white arrowhead) and subsequent metastasis to the draining inguinal lymph node (white arrow). Identical observations were made in C57BL/6 WT mice. (B) Individual tumor growth in Rag-2−/−C57BL/10 mice having received a cell suspension from DMBA-treated TyrRas mice (n = 8, black squares), in Rag-2−/−C57BL/10 mice having received a cell suspension from an invaded lymph node of a tumor-bearing Rag-2−/−C57BL/10 mouse (black circles, n = 3), and in WT C57BL/6 mice having received a cell suspension from an invaded lymph node of a tumor-bearing Rag-2−/−C57BL/10 mouse (white circles, n = 3). Mice were sacrificed when tumors reached a diameter of 1 cm2, according to animal experimentation guidelines. At that time, all the animals exhibited invaded inguinal lymph nodes. (C) Histological analysis of a tumor-invaded lymph node from a Rag-2−/−C57BL/10 (week 20, left panel) and a C57BL/6 WT mouse (week 25, right panel) having received a cell suspension from an invaded lymph node of tumor-bearing syngeneic mice.

Acknowledgements

We thank Dr. Keith Hoek for critical reading. This work was supported by grants from the Swiss National Science Foundation, the Association for International Cancer Research and the Swiss Cancer league (Oncosuisse).

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