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Activated Hgf-Met Signaling Cooperates with Oncogenic BRAF to Drive Primary Cutaneous Melanomas and Angiotropic Lung Metastases in Mice
Journal Pre-proof Activated Hgf-Met signaling cooperates with oncogenic Braf to drive primary cutaneous melanomas and angiotropic lung metastases in mice Andreas Dominik Braun, Miriam Mengoni, Susanne Bonifatius, Thomas Tüting, Evelyn Gaffal PII:
S0022-202X(20)30019-1
DOI:
https://doi.org/10.1016/j.jid.2019.12.020
Reference:
JID 2262
To appear in:
The Journal of Investigative Dermatology
Received Date: 30 September 2019 Revised Date:
17 December 2019
Accepted Date: 18 December 2019
Please cite this article as: Braun AD, Mengoni M, Bonifatius S, Tüting T, Gaffal E, Activated Hgf-Met signaling cooperates with oncogenic Braf to drive primary cutaneous melanomas and angiotropic lung metastases in mice, The Journal of Investigative Dermatology (2020), doi: https://doi.org/10.1016/ j.jid.2019.12.020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
Activated Hgf-Met signaling cooperates with oncogenic Braf to drive primary cutaneous melanomas and angiotropic lung metastases in mice
Andreas Dominik Braun1, Miriam Mengoni1, Susanne Bonifatius1, Thomas Tüting1, Evelyn Gaffal1*
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Laboratory for Experimental Dermatology, Department of Dermatology, University
Hospital Magdeburg, 39120 Magdeburg, Germany
*
Corresponding Author
Evelyn Gaffal, MD Laboratory for Experimental Dermatology Department of Dermatology University Hospital Magdeburg Leipziger Straße 44 39120 Magdeburg Germany Tel: 0391-67-21249 E-Mail: [email protected]
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ABSTRACT Oncogenic mutations in the Braf-kinase gene represent the most frequent genomic driver in acquired melanocytic nevi and in cutaneous melanomas. It is currently thought that oncogeneinduced senescence and cell cycle arrest limit the ability of oncogenic Braf to promote melanocyte proliferation in benign nevi. The molecular and cellular mechanisms that allow an oncogenic Braf mutation to fully transform melanocytes into invasively growing melanoma cells that are able to metastasize systemically are only partially understood. Here we show in a genetic mouse model that constitutively enhanced Hgf-Met signaling cooperates with oncogenic Braf to drive tumor development and metastatic spread. Activation of oncogenic Braf in mice with transgenic Hgf overexpression and an oncogenic Cdk4 germline mutation accelerated and increased the development of primary cutaneous melanomas. Primary melanomas showed considerable phenotypic heterogeneity with frequent signs of dedifferentiation. Braf activation in Hgf-Cdk4 mice also increased the number of lung metastases. Intriguingly, melanoma cells showed a pronounced angiotropic growth pattern both at the invasive front in primary tumors and in metastatic lesions of the lung. Taken together, our work supports the notion that activated Hgf-Met signaling and oncogenic Braf can cooperate in melanoma pathogenesis.
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INTRODUCTION Genomic analyses have identified somatic point mutations in the kinase domain of the Braf gene as the most frequent oncogenic driver in both benign melanocytic nevi as well as cutaneous melanoma (Akbani et al. 2015; Pollock et al. 2003; Poynter et al. 2006). In nevi, Braf-driven MAP-kinase signaling is thought to activate cellular safety mechanisms that halt aberrant proliferation in a process termed oncogene-induced senescence. On a molecular level this is thought to be initiated by the tumor suppressor p16/Ink4a (Gray-Schopfer et al. 2006; Michaloglou et al. 2005). However, many Braf-driven nevi also arise in mice and men with germline mutations in the p16/Ink4a gene that abrogate its function, indicating that additional mechanisms limit Braf-driven melanomagenesis. Furthermore, clinical studies suggest that Braf mutations could be associated with an increased likelihood of hepatic or cerebral metastasis (Adler et al. 2017; Maxwell et al. 2017). The underlying mechanisms that allow the oncogenic Braf mutation to fully transform melanocytes into invasively growing melanoma cells that are able to metastasize systemically are incompletely understood.
Genetically engineered mouse models unequivocally demonstrated that oncogenic Braf mutations can cause the development of nevi and melanoma (Damsky and Bosenberg 2017; Pérez-Guijarro et al. 2017; Walker et al. 2011). Initial work revealed that the conditional activation of a BrafV600E mutation in the melanocyte lineage of mice is fatal during embryonal development or shortly after birth (Dhomen et al. 2010). To circumvent this problem, models for tamoxifen-inducible melanocyte-specific activation of the mutant BrafV600E kinase using CreERT2 have been generated (Dankort et al. 2009; Dankort et al. 2007; Dhomen et al. 2009; Mercer et al. 2005). In our previous work we established the Braf-Cdk4 mouse model by combining an inducible BrafV600E mutation using CreERT2 specifically expressed in melanocytes with an oncogenic Cdk4R24C germline mutation that abrogates p16 binding and 3
p16-dependent oncogene-induced senescence and cell cycle arrest (Dhomen et al. 2009; Hölzel et al. 2016; Rane et al. 1999; Yajima et al. 2006). In this model we observed many melanocytic nevi but only occasionally primary melanomas. Intriguingly, these primary melanomas showed considerable morphologic heterogeneity with both highly pigmented as well as amelanotic melanoma cell phenotypes. However, Braf-Cdk4 mice did not develop distant metastases similar to many other Braf-driven mouse models for melanoma (Goel et al. 2009; Hooijkaas et al. 2012).
Overexpression of the hepatocyte growth factor (Hgf) can promote the development of primary cutaneous melanomas and of systemic metastases in mice (Noonan et al. 2001; Otsuka et al. 1998). Melanoma development and metastatic progression is increased in mice that overexpress Hgf and carry the oncogenic Cdk4R24C germline mutation (Tormo et al. 2006a). Inflammatory responses in the microenvironment of incipient Hgf-driven melanomas caused by sun-burning doses of UV irradiation further accelerate melanomagenesis and promote the development of lung metastasis (Bald et al. 2014; Gaffal et al. 2011; Zaidi et al. 2011). Here we experimentally investigated the hypothesis that constitutively enhanced HgfMet signaling as a result of broad transgenic Hgf overexpression promotes the development and metastatic progression of melanocytic neoplasms driven by oncogenic Braf. For this, we introduced the Mt::Hgf transgene heterozygously into Braf-Cdk4 mice that carry one allele of a LoxP flanked oncogenic V600E mutation in the Braf gene, one allele with a Tyr::CreERT2 transgene that allows for conditional activation of oncogenic Braf in melanocytes, and a homozygous R24C germline mutation in the Cdk4 gene. The resulting Hgf-Braf-Cdk4 mice enabled us to investigate whether enhanced Hgf-Met signaling cooperates with the activation of oncogenic Braf to increase the incidence and growth of primary cutaneous melanomas, to affect their phenotype and to enhance their ability to grow invasively and to metastasize.
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RESULTS All Hgf-Braf-Cdk4 mice spontaneously develop nodular, rapidly growing melanoma In the beginning Hgf-Cdk4 and Braf-Cdk4 mice were crossed in order to establish breeding pairs between male Hgf-Braf-Cdk4 and female Braf-Cdk4 mice (Figure 1a). Genetic analyses of the offspring from this breeding revealed 8 genotypes in their expected frequencies. These genotypes can be combined to four different phenotypes, one of which represents the desired Hgf-Braf-Cdk4 mice (Figure S1). Unexpectedly, spontaneous development of cutaneous melanomas without tamoxifen treatment was more frequent in Hgf-Braf-Cdk4 mice when compared to Hgf-Cdk4 mice. All Hgf-Braf-Cdk4 mice showed rapidly growing, nodular melanomas within the first year of life (Figure 1b, Hgf-Braf-Cdk4 11/11 mice with tumor, Hgf-Cdk4 15/25 mice with tumor). Accordingly, the median survival of Hgf-Braf-Cdk4 mice was significantly shorter than that of Hgf-Cdk4 mice (235 vs. 296 days). As expected, BrafCdk4 mice did not develop spontaneous melanomas (Figure 1c).
Spontaneous recombination of the mutant Braf-allele in Hgf-Braf-Cdk4 mice is associated with amelanotic, dedifferentiated melanoma phenotypes Hgf-Braf-Cdk4 mice not only developed pigmented melanomas but also melanomas with an amelanotic appearance in the same animals (Figure 2a). Because we previously observed similar amelanotic cutaneous melanoma phenotypes only in Braf-Cdk4 mice but not in HgfCdk4 mice, we suspected that the conditional mutant Braf allele was spontaneously activated. Indeed, PCR based genotyping revealed a spontaneous recombination of the BrafLSL-V600E allele without any contact to tamoxifen (Figure S2). Interestingly, the activated Braf allele was found in all amelanotic melanomas but only in a fraction of pigmented melanomas (Figure 2b). 5
Amelanotic HBC-melanomas show an immune-cell enriched phenotype Histomorphologic examinations of spontaneous Hgf-Braf-Cdk4 melanomas showed that pigmented cutaneous melanomas resemble those arising in Hgf-Cdk4 mice with highly pigmented epitheloid cells arranged in nests throughout the lesion (Figure 3, left panel, and Figure S3). Immunohistochemical stainings were strongly positive for the melanocytic marker gp100 and only very few interspersed immune cells expressing CD45. In contrast, amelanotic cutaneous melanomas showed a heterogeneous picture with alternating hypocellular areas with a myxoid stroma and hypercellular areas with spindle-shaped melanomas cells reminiscent of malignant peripheral nerve sheath tumors (Figure 3, right panel). Accordingly, tumor cells did not express gp100 but were strongly positive for Ngfr, a marker for neural crest-derived precursor cells, and showed a considerable infiltrate of CD45+ immune cells. This infiltrate consisted mainly of myeloid immune cells as identifiable in Giemsa stained sections with only few CD3+ T cells (Figure S4). We previously observed a similar tumor architecture in amelanotic melanomas arising in Braf-Cdk4 mice (Hölzel et al. 2016).
Tamoxifen-induced activation of the mutated Braf-allele promotes melanoma development in Hgf-Braf-Cdk4 mice Next, we treated 4-6 week old Hgf-Braf-Cdk4 mice with 4-hydroxytamoxifen epicutaneously on three consecutive days to induce cre-mediated recombination of the mutated Braf allele and assessed the impact on melanoma development (Figure 4a). Additional cohorts of mice were treated with the model carcinogen DMBA and with a combination of 4hydroxytamoxifen and DMBA to induce melanoma formation. We observed a marked acceleration of melanoma development in all three treatment groups. Mice receiving 4hydroxytamoxifen developed numerous differently sized tumors, primarily located in the
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treated skin areas, i.e. the back skin, which showed a hyperpigmented phenotype (Figure 4b). Additionally, these groups of mice frequently showed pronounced symptoms of tumor burden prior to the endpoint tumor size of 100 mm2, reducing the median survival (Figure 4c). Mice treated with the combination of 4-hydroxytamoxifen and DMBA showed the shortest median survival of 65 days. This was significantly lower when compared with mice receiving 4hydroxytamoxifen alone (p=0.0009) or DMBA alone (p=0.007).
Melanomas in Hgf-Braf-Cdk4 mice treated with tamoxifen also show pigmented and amelanotic phenotypes In Hgf-Braf-Cdk4 mice treated with 4-hydroxytamoxifen we again observed both pigmented and amelanotic melanomas with the same morphologic heterogeneity seen in spontaneous melanomas of untreated Hgf-Braf-Cdk4 mice (Figure 5a). Simultaneous application of DMBA shifted the melanoma phenotype towards pigmented tumors (Figure 5b). Genotyping of tumors revealed a recombination of the mutant Braf-allele in all melanomas from 4hydroxytamoxifen and amelanotic 4-hydroxytamoxifen +DMBA treated mice and none of the DMBA treated mice (Figure S5). From these observations we concluded that the activation of the mutant Braf kinase promotes the growth of both pigmented, immune cell-poor as well as unpigmented, dedifferentiated, immune cell rich melanomas in Hgf-Braf-Cdk4 mice.
Primary Hgf-Braf-Cdk4 melanomas spontaneously metastasize to the lungs and show an angiotropic growth pattern Next, we investigated the appearance of melanoma metastases in Hgf-Braf-Cdk4 mice carrying primary, spontaneous melanomas in the skin. We were able to detect pulmonary metastasis in 3 of 11 mice (27%) both macroscopically and microscopically (Figure 6a and b). All metastases showed a differentiated, pigmented phenotype. Amelanotic metastasis were
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not observed. The targeted activation of the mutant Braf allele further increased the frequency of distant metastasis, with all 6 of 6 mice developing pulmonary metastasis. Interestingly, these mice showed numerous metastases disseminated throughout the lungs, resembling a “miliary” spread. The combination of tamoxifen-induced Braf activation with DMBA treatment did not increase the number of pulmonary metastasis. Histological examinations of lung tissues from Hgf-Braf-Cdk4 mice revealed a close association of melanoma cells with endothelial cells with pulmonary blood vessels being ensheathed by tumor cells on the abluminal side, a phenomenon known as “angiotropism” (Figure 6c).
DISCUSSION Mice carrying only a conditionally activatable mutated Braf allele develop nevi but only very rarely melanomas (Dankort et al. 2009). Additional genetic alterations that abrogate the function of key tumor suppressor genes such as the deletion of the Cdnk2a gene encoding p16/Ink4a or the PTEN gene develop more nevi and melanomas (Damsky et al. 2015; Dankort et al. 2009; Goel et al. 2009; Viros et al. 2014). The data presented here demonstrate that activation of oncogenic Braf in mice efficiently cooperates with transgenic overexpression of Hgf and readily transforms melanocytes into invasively growing melanoma cells. This leads to an increased incidence of primary cutaneous melanomas in Hgf-Cdk4 mice and a decreased latency of tumor onset. Importantly, activation of oncogenic Braf in Hgf-Cdk4 mice also increased the propensity of melanoma cells to metastasize to the lungs. Hepatocyte growth factor was initially discovered as a growth factor enabling liver regeneration after hepatic injury (Leffert et al. 1979; Nakamura et al. 1989). Subsequently, it was shown that binding of Hgf to its receptor c-Met promotes cell migration and tumor metastasis (Nakamura et al. 1987; Weidner et al. 1991). In human melanoma, Hgf and its receptor Met are also abundantly expressed especially in distant metastases, suggesting a 8
clinically relevant role for Hgf-Met signaling in systemic dissemination (Moore et al. 2008; Natali et al. 1993).
Hgf-Met signaling might impact on the melanoma cell phenotype through the Wnt-signaling pathway, which has previously been shown to promote metastatic dissemination in another Braf-driven melanoma model. In this model, conditional genetic stabilization of β-catenin enhances metastasis in Braf-mutant, Pten-deficient mice (Damsky et al. 2011). Similarly, activation of Wnt-signaling in melanocytes via deletion of Smad4 increases metastatic dissemination in Nras mutated, Ink4a deficient mice (Tuncer et al. 2019). As the transgenic Hgf overexpression is broadly expressed, increased Hgf-Met signaling could also have microenvironmental effects in addition to its potential cell-intrinsic roles for transformation. Hgf-Met signaling is well known to promote immunosuppression (Benkhoucha et al. 2010; Glodde et al. 2017; Okunishi et al. 2005) and can cause microenvironmental resistance to signal transduction inhibitors (Straussman et al. 2012). We further speculate that increased Hgf-Met signaling can reshape the microenvironment at metastatic sites such as the lungs to create a favorable niche for the outgrowth of metastases, an assumption that will be tested in future experiments.
We previously observed that UV irradiation of Hgf-Cdk4 mice promoted melanoma cells to grow invasively along the abluminal surfaces of endothelial cells, a process termed angiotropism (Bald et al. 2014). This angiotropic growth pattern was also found in Hgf-BrafCdk4 melanomas both at the invasive front in primary tumors and in metastatic lesions of the lung. Similar correlations between angiotropism and distant metastasis are described in human melanoma. It has been hypothesized that melanoma cells metastasize through “extravascular migration” along vessel surfaces (Barnhill et al. 2002; Barnhill and Lugassy
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2004). Whether melanoma cells actually migrate on endothelial cells or only expand along the perivascular niche and gain increased access to the blood stream will also have to be further addressed in future studies.
In our experiments we observed spontaneous melanoma formation in Hgf-Braf-Cdk4 mice without any exposure to tamoxifen. A similar “leakiness” of CreERT2 driven recombination systems has been described also for a mouse melanoma model combining Braf mutation with loss of PTEN (Hooijkaas et al. 2012) and is well known in the field. Upon tamoxifen treatment, the conditionally expressed mutant Braf allele is simultaneously introduced as an oncogenic driver in a large number of melanocytes. Not unexpected, this resulted in the development of numerous nevi and some melanomas, suggesting a polyclonal origin of these lesions. In contrast, spontaneous recombination of the mutated Braf allele without tamoxifen might represent a clonal event. From an experimental standpoint this could be a desirable feature as it would more closely portray our evolutionary understanding of tumor pathogenesis in which initially single cells acquire malignant potential and subsequently give rise to a genetically and phenotypically heterogeneous cancer (Merlo et al. 2006). Further analyses are required to elucidate the clonal dynamics in genetically engineered mouse melanoma models in more detail.
Primary melanomas in untreated Hgf-Braf-Cdk4 mice showed considerable morphologic heterogeneity with both pigmented, highly differentiated, immune cell poor phenotypes as well as amelanotic, dedifferentiated, immune cell enriched phenotypes. Surprisingly, all amelanotic spontaneous melanomas showed a recombination of the conditional mutant Braf allele while a similar recombination could only be detected in a few pigmented melanomas. In fact, amelanotic melanomas appeared only after activation of the mutant Braf allele
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suggesting that oncogenic Braf-driven signaling can promote cellular dedifferentiation. However, activation of mutant Braf in amelanotic progenitor cells with subsequent hierarchical differentiation cannot be fully excluded. Dynamic dedifferentiation has been observed in another mouse melanoma model where conditional activation of mutated Braf was combined with loss of Pten. Here the activation of the Braf mutation locally on the tail skin initiated the clonal expansion of interfollicular melanocytes which subsequently acquired a dedifferentiated phenotype and progressed towards invasively growing melanoma (Köhler et al. 2017). Recent reports support the concept that melanoma cells can show progressive stages of dedifferentiation during tumor progression both in mice and men (Tsoi et al. 2018). A similar phenotype switch has been observed during signal transduction inhibitor therapy, where melanoma cells shifted towards an intermediary, “starved-like” phenotype before either undergoing dedifferentiation or becoming fully differentiated with both phenotypes conferring resistance to the signal transduction inhibitor (Rambow et al. 2018). This dynamic phenotypic plasticity is thought to be due to aberrant reactivation of embryonic developmental programs with melanoma cells acquiring stem-cell like phenotypes and properties. This has been shown to enable invasive growth and metastatic dissemination (Dongre and Weinberg 2019; Hoek et al. 2008; Verfaillie et al. 2015).
Taken together, we established the Hgf-Braf-Cdk4 mouse melanoma model and observed that activated Hgf-Met signaling cooperates with oncogenic Braf to drive primary cutaneous melanomas and systemic metastases in the lungs. In this experimental system oncogenic Braf also promotes melanoma cell dedifferentiation. A unique feature of the model is the emergence of angiotropic growth patterns during melanoma cell invasion and metastasis. The Hgf-Braf-Cdk4 mouse model represents an attractive experimental system to further
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investigate mechanisms underlying the process of malignant transformation of melanocytes and the progression towards metastatic disease.
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MATERIALS AND METHODS Mice Hgf-Cdk4 and Braf-Cdk4 mice were bred and genotyped as previously described (Mercer et al. 2005; Rane et al. 1999; Takayama et al. 1996; Yajima et al. 2006). Hgf-Braf-Cdk4 mice were generated by breeding male Hgf-Cdk4 mice with female Braf-Cdk4 mice. Breeding and experiments were conducted in individually ventilated cages. Mice were regularly screened for pathogens. All experiments were performed in compliance with federal and international guidelines for animal experiments and with the approval of the responsible authorities (Landesverwaltungsamt Saxony-Anhalt, Germany).
Induction of primary melanomas with DMBA and 4OHT All experiments were performed in 6-8 week old mice. Cohorts of age and sex matched mice were randomly assigned to experimental groups. Primary melanoma development was induced by single dose application of 100 nmol of the carcinogen DMBA (dissolved in 100 µl acetone) on the shaved back skin as previously described (Landsberg et al. 2012). Induction of Cre-mediated recombination and activation of the mutant Braf-allele in melanocytes was triggered by epicutaneously application of 4-hydroxy-Tamoxifen (4OHT, 33 µg dissolved in 100 µl acetone ≈ 853 µmol) on three consecutive days. Tumor area was measured twice weekly and calculated by the formula area in mm2 = length x width. Mice were sacrificed when tumors exceeded 100 mm2 tumor area. Cohort sizes of experimental groups are stated in the corresponding figure legends.
Histomorphologic sample preparation and immunohistochemical analyses Melanomas were fixed in zinc-solution for 24 h (BD Pharmingen) and dehydrated, embedded in paraffin, sectioned and stained with haematoxylin and eosin or giemsa according to 13
standard protocols. Highly pigmented samples were bleached with H2O2 prior to staining (3% H2O2 and 0,5% KOH for 20 minutes at 37°C, then 1% acetic acid for 20 seconds). Immunohistochemical stainings were conducted with polyclonal rabbit anti-mouse gp100 (NBP1-69571, Novus Biologicals), monoclonal rat anti-mouse CD45 (Catalog Nr. 550539, BD Biosciences), polyclonal, biotinylated goat anti-mouse Ngfr (AF1157, R&D Systems), monoclonal rat anti-human CD3 (Catalog Nr. MCA1477, Bio-Rad), secondary antibody polyclonal goat anti-rabbit IgG (Catalog Nr. 111-067-003, dianova) and polyclonal goat antirat IgG (Catalog Nr. 112-067-003, dianova) as well as the ZytoChem Fast Red AP LSAB kit. Stained sections were imaged with an Axio Imager A1 light microscope (Zeiss) and digitized with a NanoZoomer SQ digital slide scanner (Hamamatsu).
Statistical analyses Survival probabilities were calculated with a Kaplan-Meier estimator and significance tested by logrank test. Post-hoc correction for multiple testing was performed by Bonferronicorrection. Significance of spontaneous Braf-activation between pigmented and amelanotic tumors was calculated with Fishers exact test. Frequency of pulmonary metastasis was tested for significance with Kruskal-Wallis test and post-hoc Benjamini-Hochberg correction of the false discovery rate. p values less than 0.05 were considered significant. All calculations were performed in the R computing environment.
DATA AVAILABILITY STATEMENT No datasets were generated or analyzed during the current study.
CONFLICT OF INTEREST The authors declare no conflict of interest.
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ACKNOWLEDGEMENTS We thank J. Herz for expert assistance with the histological procedures. A.D.B. was supported by a scholarship from the Else Kröner-Forschungskolleg Magdeburg. E.G. was supported by the SFB854. T.T. was funded in part by grants from the DFG (TU 90/8-1 and A27 in the SFB854).
CRediT STATEMENT Conceptualization, data curation, formal analysis, writing of manuscript: E.G., T.T., A.D.B. and M.M.; Experiments: E.G., A.D.B. and S.B.; Funding Acquisition: T.T.
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Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM, et al. High frequency of BRAF mutations in nevi. Nat. Genet. 2003;33(1):19–20 Poynter J, Elder J, Fullen D, Nair R, Soengas M, Johnson T, et al. BRAF and NRAS mutations in melanoma and melanocytic nevi. Melanoma Research. 2006;16(4):267–73 Rambow F, Rogiers A, Marin-Bejar O, Aibar S, Femel J, Dewaele M, et al. Toward Minimal Residual Disease-Directed Therapy in Melanoma. Cell. 2018;174(4):843-855.e19 Rane SG, Dubus P, Mettus RV, Galbreath EJ, Boden G, Reddy EP, et al. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in β-islet cell hyperplasia. Nature Genetics. 1999;22(1):44–52 Straussman R, Morikawa T, Shee K, Barzily-Rokni M, Qian ZR, Du J, et al. Tumour microenvironment elicits innate resistance to RAF inhibitors through HGF secretion. Nature. 2012;487(7408):500–4 Takayama H, La Rochelle WJ, Anver M, Bockman DE, Merlino G. Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development. Proceedings of the National Academy of Sciences. 1996;93(12):5866–71 Tormo D, Ferrer A, Bosch P, Gaffal E, Basner-Tschakarjan E, Wenzel J, et al. Therapeutic Efficacy of Antigen-Specific Vaccination and Toll-Like Receptor Stimulation against Established Transplanted and Autochthonous Melanoma in Mice. Cancer Research. 2006;66(10):5427–35 Tsoi J, Robert L, Paraiso K, Galvan C, Sheu KM, Lay J, et al. Multi-stage Differentiation Defines Melanoma Subtypes with Differential Vulnerability to Drug-Induced Iron-Dependent Oxidative Stress. Cancer Cell. 2018; Available from: http://linkinghub.elsevier.com/retrieve/pii/S1535610818301223 Tuncer E, Calçada RR, Zingg D, Varum S, Cheng P, Freiberger SN, et al. SMAD signaling promotes melanoma metastasis independently of phenotype switching. J Clin Invest. 2019;129(7):2702–16 Verfaillie A, Imrichova H, Atak ZK, Dewaele M, Rambow F, Hulselmans G, et al. Decoding the regulatory landscape of melanoma reveals TEADS as regulators of the invasive cell state. Nature Communications. 2015;6(1) Available from: http://www.nature.com/articles/ncomms7683 Viros A, Sanchez-Laorden B, Pedersen M, Furney SJ, Rae J, Hogan K, et al. Ultraviolet radiation accelerates BRAF-driven melanomagenesis by targeting TP53. Nature. 2014;511(7510):478–82 Walker GJ, Soyer HP, Terzian T, Box NF. Modelling melanoma in mice. Pigment Cell & Melanoma Research. 2011;24(6):1158–76 Weidner KM, Arakaki N, Hartmann G, Vandekerckhove J, Weingart S, Rieder H, et al. Evidence for the identity of human scatter factor and human hepatocyte growth factor. PNAS. 1991;88(16):7001–5
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Yajima I, Belloir E, Bourgeois Y, Kumasaka M, Delmas V, Larue L. Spatiotemporal gene control by the Cre‐ERT2 system in melanocytes. genesis. 2006;44:34–43 Zaidi MR, Davis S, Noonan FP, Graff-Cherry C, Hawley TS, Walker RL, et al. Interferon-γ links ultraviolet radiation to melanomagenesis in mice. Nature. 2011;469(7331):548–53
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FIGURE LEGENDS Figure 1: Hgf-Braf-Cdk4 mice spontaneously develop rapidly growing, nodular melanoma with complete penetrance. a. Breeding strategy for the generation of Hgf-Braf-Cdk4 mice. b. Representative macroscopic pictures of spontaneously arising nodular cutaneous melanomas of Hgf-Braf-Cdk4 and Hgf-Cdk4 mice. c. Kaplan-Meyer graphs showing the survival of Hgf-Braf-Cdk4 (HBC), Hgf-Cdk4 (HC) as well as Braf-Cdk4 and Cdk4 (BC/C) control mice (**p<0.01, logrank test).
Figure 2: Spontaneous recombination of the mutant Braf-allele drives amelanotic melanomas in HgfBraf-Cdk4 mice a. Table indicating the number of both pigmented and amelanotic melanomas in a cohort of 15 Hgf-Cdk4 and 11 Hgf-Braf-Cdk4 mice (mean±SD). b. Analysis of Braf-allele-status in pigmented and amelanotic Hgf-Braf-Cdk4 mice (***p<0.005, Fishers exact test).
Figure 3: Amelanotic Hgf-Braf-Cdk4 melanomas show an immune-cell rich phenotype Representative histopathologic images of pigmented and amelanotic Hgf-Braf-Cdk4 melanomas. Similar results were seen in 5 pigmented and 5 amelanotic tumors. Magnification and staining are shown in individual pictures.
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Figure 4: Activation of the mutant Braf allele and epicutaneous treatment with DMBA accelerate melanomagenesis. a. Experimental setup for the treatment of Hgf-Braf-Cdk4 mice with 4-OHT and/or DMBA. b. Representative macroscopic pictures of Hgf-Braf-Cdk4 mice left untreated or treated with 4OHT, DMBA and 4OHT+DMBA at the indicated age. c. Mean survival of Hgf-Braf-Cdk4 mice in the different cohorts (*p<0.05, ***p<0.005, logrank test with Bonferroni-correction).
Figure 5: Activation of the mutant Braf allele promotes the appearance of amelanotic, immune cell rich Hgf-Braf-Cdk4 melanomas. a. Representative macroscopic and microscopic images of melanomas from Hgf-Braf-Cdk4 mice treated and stained as indicated. Similar results were found in 5 pigmented and 5 amelanotic tumors. b. Incidence of pigmented and amelanotic phenotypes in cohorts of Hgf-Braf-Cdk4 mice treated with 4OHT, DMBA or 4OHT+DMBA (mean ± SD).
Figure 6: Figure 6: Activation of the mutant Braf allele drives the development of angiotropic lung metastases a. Representative macroscopic and microscopic images of lungs from Hgf-Braf-Cdk4 mice treated and stained as indicated. b. Percentages of Hgf-Cdk4 (n=25), Braf-Cdk4 (spontaneous n=10, 4OHT n=6) and HgfBraf-Cdk4 mice (spontaneous n=11, 4OHT n=6, DMBA n=9, 4OHT+DMBA n=11) with
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pulmonary metastases in the different cohorts treated as indicated (*p<0.05, ***p<0.005, Kruskal-Wallis-test with post-hoc Benjamini-Hochberg correction of the false discovery rate). c. Representative macroscopic and microscopic images of angiotropism in Hgf-Braf-Cdk4 primary tumors and lung metastases, Magnification and staining as indicated. Similar results were found in 5 mice.
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Supplementary Figure 1: Supplementary Figure 1: Genotype and phenotype distribution of 60 offspring from breeding between male C57BL/6 Mt::Hgf+/o- BRAFLSL-V600E/wt-Cdk4R24C/R24C-Tyr::CreERT2 +/o and female C57BL/6 BRAFLSL-V600E/wt-Cdk4R24C/R24C-Tyr::CreERT2 +/o mice. Supplementary Figure 2: Supplementary Figure 2: Recombination of the Braf-allele in Hgf-Braf-Cdk4 mice Schematic representation and photograph of a Braf locus genotyping-PCR with Primers A+B and A+C before (a.) and after (b.) recombination. Sample 1: Hgf-Cdk4 skin, Sample 2: HgfBraf-Cdk4 skin, Sample 3: Hgf-Braf-Cdk4 amelanotic melanoma Supplementary Figure 3: Representative histopathology of Hgf-Cdk4 melanomas. Magnification and staining are shown in individual images. Supplementary Figure 4: Characterization of the immune infiltrate in amelanotic Hgf-Braf-Cdk4 melanomas. Magnification and staining are shown in individual images. Supplementary Figure 5: Braf-Status of melanomas from Hgf-Braf-Cdk4 mice treated with 4OHT, DMBA or both.
S0022-202X(20)30019-1
DOI:
https://doi.org/10.1016/j.jid.2019.12.020
Reference:
JID 2262
To appear in:
The Journal of Investigative Dermatology
Received Date: 30 September 2019 Revised Date:
17 December 2019
Accepted Date: 18 December 2019
Please cite this article as: Braun AD, Mengoni M, Bonifatius S, Tüting T, Gaffal E, Activated Hgf-Met signaling cooperates with oncogenic Braf to drive primary cutaneous melanomas and angiotropic lung metastases in mice, The Journal of Investigative Dermatology (2020), doi: https://doi.org/10.1016/ j.jid.2019.12.020. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 The Authors. Published by Elsevier, Inc. on behalf of the Society for Investigative Dermatology.
Activated Hgf-Met signaling cooperates with oncogenic Braf to drive primary cutaneous melanomas and angiotropic lung metastases in mice
Andreas Dominik Braun1, Miriam Mengoni1, Susanne Bonifatius1, Thomas Tüting1, Evelyn Gaffal1*
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Laboratory for Experimental Dermatology, Department of Dermatology, University
Hospital Magdeburg, 39120 Magdeburg, Germany
*
Corresponding Author
Evelyn Gaffal, MD Laboratory for Experimental Dermatology Department of Dermatology University Hospital Magdeburg Leipziger Straße 44 39120 Magdeburg Germany Tel: 0391-67-21249 E-Mail: [email protected]
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ABSTRACT Oncogenic mutations in the Braf-kinase gene represent the most frequent genomic driver in acquired melanocytic nevi and in cutaneous melanomas. It is currently thought that oncogeneinduced senescence and cell cycle arrest limit the ability of oncogenic Braf to promote melanocyte proliferation in benign nevi. The molecular and cellular mechanisms that allow an oncogenic Braf mutation to fully transform melanocytes into invasively growing melanoma cells that are able to metastasize systemically are only partially understood. Here we show in a genetic mouse model that constitutively enhanced Hgf-Met signaling cooperates with oncogenic Braf to drive tumor development and metastatic spread. Activation of oncogenic Braf in mice with transgenic Hgf overexpression and an oncogenic Cdk4 germline mutation accelerated and increased the development of primary cutaneous melanomas. Primary melanomas showed considerable phenotypic heterogeneity with frequent signs of dedifferentiation. Braf activation in Hgf-Cdk4 mice also increased the number of lung metastases. Intriguingly, melanoma cells showed a pronounced angiotropic growth pattern both at the invasive front in primary tumors and in metastatic lesions of the lung. Taken together, our work supports the notion that activated Hgf-Met signaling and oncogenic Braf can cooperate in melanoma pathogenesis.
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INTRODUCTION Genomic analyses have identified somatic point mutations in the kinase domain of the Braf gene as the most frequent oncogenic driver in both benign melanocytic nevi as well as cutaneous melanoma (Akbani et al. 2015; Pollock et al. 2003; Poynter et al. 2006). In nevi, Braf-driven MAP-kinase signaling is thought to activate cellular safety mechanisms that halt aberrant proliferation in a process termed oncogene-induced senescence. On a molecular level this is thought to be initiated by the tumor suppressor p16/Ink4a (Gray-Schopfer et al. 2006; Michaloglou et al. 2005). However, many Braf-driven nevi also arise in mice and men with germline mutations in the p16/Ink4a gene that abrogate its function, indicating that additional mechanisms limit Braf-driven melanomagenesis. Furthermore, clinical studies suggest that Braf mutations could be associated with an increased likelihood of hepatic or cerebral metastasis (Adler et al. 2017; Maxwell et al. 2017). The underlying mechanisms that allow the oncogenic Braf mutation to fully transform melanocytes into invasively growing melanoma cells that are able to metastasize systemically are incompletely understood.
Genetically engineered mouse models unequivocally demonstrated that oncogenic Braf mutations can cause the development of nevi and melanoma (Damsky and Bosenberg 2017; Pérez-Guijarro et al. 2017; Walker et al. 2011). Initial work revealed that the conditional activation of a BrafV600E mutation in the melanocyte lineage of mice is fatal during embryonal development or shortly after birth (Dhomen et al. 2010). To circumvent this problem, models for tamoxifen-inducible melanocyte-specific activation of the mutant BrafV600E kinase using CreERT2 have been generated (Dankort et al. 2009; Dankort et al. 2007; Dhomen et al. 2009; Mercer et al. 2005). In our previous work we established the Braf-Cdk4 mouse model by combining an inducible BrafV600E mutation using CreERT2 specifically expressed in melanocytes with an oncogenic Cdk4R24C germline mutation that abrogates p16 binding and 3
p16-dependent oncogene-induced senescence and cell cycle arrest (Dhomen et al. 2009; Hölzel et al. 2016; Rane et al. 1999; Yajima et al. 2006). In this model we observed many melanocytic nevi but only occasionally primary melanomas. Intriguingly, these primary melanomas showed considerable morphologic heterogeneity with both highly pigmented as well as amelanotic melanoma cell phenotypes. However, Braf-Cdk4 mice did not develop distant metastases similar to many other Braf-driven mouse models for melanoma (Goel et al. 2009; Hooijkaas et al. 2012).
Overexpression of the hepatocyte growth factor (Hgf) can promote the development of primary cutaneous melanomas and of systemic metastases in mice (Noonan et al. 2001; Otsuka et al. 1998). Melanoma development and metastatic progression is increased in mice that overexpress Hgf and carry the oncogenic Cdk4R24C germline mutation (Tormo et al. 2006a). Inflammatory responses in the microenvironment of incipient Hgf-driven melanomas caused by sun-burning doses of UV irradiation further accelerate melanomagenesis and promote the development of lung metastasis (Bald et al. 2014; Gaffal et al. 2011; Zaidi et al. 2011). Here we experimentally investigated the hypothesis that constitutively enhanced HgfMet signaling as a result of broad transgenic Hgf overexpression promotes the development and metastatic progression of melanocytic neoplasms driven by oncogenic Braf. For this, we introduced the Mt::Hgf transgene heterozygously into Braf-Cdk4 mice that carry one allele of a LoxP flanked oncogenic V600E mutation in the Braf gene, one allele with a Tyr::CreERT2 transgene that allows for conditional activation of oncogenic Braf in melanocytes, and a homozygous R24C germline mutation in the Cdk4 gene. The resulting Hgf-Braf-Cdk4 mice enabled us to investigate whether enhanced Hgf-Met signaling cooperates with the activation of oncogenic Braf to increase the incidence and growth of primary cutaneous melanomas, to affect their phenotype and to enhance their ability to grow invasively and to metastasize.
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RESULTS All Hgf-Braf-Cdk4 mice spontaneously develop nodular, rapidly growing melanoma In the beginning Hgf-Cdk4 and Braf-Cdk4 mice were crossed in order to establish breeding pairs between male Hgf-Braf-Cdk4 and female Braf-Cdk4 mice (Figure 1a). Genetic analyses of the offspring from this breeding revealed 8 genotypes in their expected frequencies. These genotypes can be combined to four different phenotypes, one of which represents the desired Hgf-Braf-Cdk4 mice (Figure S1). Unexpectedly, spontaneous development of cutaneous melanomas without tamoxifen treatment was more frequent in Hgf-Braf-Cdk4 mice when compared to Hgf-Cdk4 mice. All Hgf-Braf-Cdk4 mice showed rapidly growing, nodular melanomas within the first year of life (Figure 1b, Hgf-Braf-Cdk4 11/11 mice with tumor, Hgf-Cdk4 15/25 mice with tumor). Accordingly, the median survival of Hgf-Braf-Cdk4 mice was significantly shorter than that of Hgf-Cdk4 mice (235 vs. 296 days). As expected, BrafCdk4 mice did not develop spontaneous melanomas (Figure 1c).
Spontaneous recombination of the mutant Braf-allele in Hgf-Braf-Cdk4 mice is associated with amelanotic, dedifferentiated melanoma phenotypes Hgf-Braf-Cdk4 mice not only developed pigmented melanomas but also melanomas with an amelanotic appearance in the same animals (Figure 2a). Because we previously observed similar amelanotic cutaneous melanoma phenotypes only in Braf-Cdk4 mice but not in HgfCdk4 mice, we suspected that the conditional mutant Braf allele was spontaneously activated. Indeed, PCR based genotyping revealed a spontaneous recombination of the BrafLSL-V600E allele without any contact to tamoxifen (Figure S2). Interestingly, the activated Braf allele was found in all amelanotic melanomas but only in a fraction of pigmented melanomas (Figure 2b). 5
Amelanotic HBC-melanomas show an immune-cell enriched phenotype Histomorphologic examinations of spontaneous Hgf-Braf-Cdk4 melanomas showed that pigmented cutaneous melanomas resemble those arising in Hgf-Cdk4 mice with highly pigmented epitheloid cells arranged in nests throughout the lesion (Figure 3, left panel, and Figure S3). Immunohistochemical stainings were strongly positive for the melanocytic marker gp100 and only very few interspersed immune cells expressing CD45. In contrast, amelanotic cutaneous melanomas showed a heterogeneous picture with alternating hypocellular areas with a myxoid stroma and hypercellular areas with spindle-shaped melanomas cells reminiscent of malignant peripheral nerve sheath tumors (Figure 3, right panel). Accordingly, tumor cells did not express gp100 but were strongly positive for Ngfr, a marker for neural crest-derived precursor cells, and showed a considerable infiltrate of CD45+ immune cells. This infiltrate consisted mainly of myeloid immune cells as identifiable in Giemsa stained sections with only few CD3+ T cells (Figure S4). We previously observed a similar tumor architecture in amelanotic melanomas arising in Braf-Cdk4 mice (Hölzel et al. 2016).
Tamoxifen-induced activation of the mutated Braf-allele promotes melanoma development in Hgf-Braf-Cdk4 mice Next, we treated 4-6 week old Hgf-Braf-Cdk4 mice with 4-hydroxytamoxifen epicutaneously on three consecutive days to induce cre-mediated recombination of the mutated Braf allele and assessed the impact on melanoma development (Figure 4a). Additional cohorts of mice were treated with the model carcinogen DMBA and with a combination of 4hydroxytamoxifen and DMBA to induce melanoma formation. We observed a marked acceleration of melanoma development in all three treatment groups. Mice receiving 4hydroxytamoxifen developed numerous differently sized tumors, primarily located in the
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treated skin areas, i.e. the back skin, which showed a hyperpigmented phenotype (Figure 4b). Additionally, these groups of mice frequently showed pronounced symptoms of tumor burden prior to the endpoint tumor size of 100 mm2, reducing the median survival (Figure 4c). Mice treated with the combination of 4-hydroxytamoxifen and DMBA showed the shortest median survival of 65 days. This was significantly lower when compared with mice receiving 4hydroxytamoxifen alone (p=0.0009) or DMBA alone (p=0.007).
Melanomas in Hgf-Braf-Cdk4 mice treated with tamoxifen also show pigmented and amelanotic phenotypes In Hgf-Braf-Cdk4 mice treated with 4-hydroxytamoxifen we again observed both pigmented and amelanotic melanomas with the same morphologic heterogeneity seen in spontaneous melanomas of untreated Hgf-Braf-Cdk4 mice (Figure 5a). Simultaneous application of DMBA shifted the melanoma phenotype towards pigmented tumors (Figure 5b). Genotyping of tumors revealed a recombination of the mutant Braf-allele in all melanomas from 4hydroxytamoxifen and amelanotic 4-hydroxytamoxifen +DMBA treated mice and none of the DMBA treated mice (Figure S5). From these observations we concluded that the activation of the mutant Braf kinase promotes the growth of both pigmented, immune cell-poor as well as unpigmented, dedifferentiated, immune cell rich melanomas in Hgf-Braf-Cdk4 mice.
Primary Hgf-Braf-Cdk4 melanomas spontaneously metastasize to the lungs and show an angiotropic growth pattern Next, we investigated the appearance of melanoma metastases in Hgf-Braf-Cdk4 mice carrying primary, spontaneous melanomas in the skin. We were able to detect pulmonary metastasis in 3 of 11 mice (27%) both macroscopically and microscopically (Figure 6a and b). All metastases showed a differentiated, pigmented phenotype. Amelanotic metastasis were
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not observed. The targeted activation of the mutant Braf allele further increased the frequency of distant metastasis, with all 6 of 6 mice developing pulmonary metastasis. Interestingly, these mice showed numerous metastases disseminated throughout the lungs, resembling a “miliary” spread. The combination of tamoxifen-induced Braf activation with DMBA treatment did not increase the number of pulmonary metastasis. Histological examinations of lung tissues from Hgf-Braf-Cdk4 mice revealed a close association of melanoma cells with endothelial cells with pulmonary blood vessels being ensheathed by tumor cells on the abluminal side, a phenomenon known as “angiotropism” (Figure 6c).
DISCUSSION Mice carrying only a conditionally activatable mutated Braf allele develop nevi but only very rarely melanomas (Dankort et al. 2009). Additional genetic alterations that abrogate the function of key tumor suppressor genes such as the deletion of the Cdnk2a gene encoding p16/Ink4a or the PTEN gene develop more nevi and melanomas (Damsky et al. 2015; Dankort et al. 2009; Goel et al. 2009; Viros et al. 2014). The data presented here demonstrate that activation of oncogenic Braf in mice efficiently cooperates with transgenic overexpression of Hgf and readily transforms melanocytes into invasively growing melanoma cells. This leads to an increased incidence of primary cutaneous melanomas in Hgf-Cdk4 mice and a decreased latency of tumor onset. Importantly, activation of oncogenic Braf in Hgf-Cdk4 mice also increased the propensity of melanoma cells to metastasize to the lungs. Hepatocyte growth factor was initially discovered as a growth factor enabling liver regeneration after hepatic injury (Leffert et al. 1979; Nakamura et al. 1989). Subsequently, it was shown that binding of Hgf to its receptor c-Met promotes cell migration and tumor metastasis (Nakamura et al. 1987; Weidner et al. 1991). In human melanoma, Hgf and its receptor Met are also abundantly expressed especially in distant metastases, suggesting a 8
clinically relevant role for Hgf-Met signaling in systemic dissemination (Moore et al. 2008; Natali et al. 1993).
Hgf-Met signaling might impact on the melanoma cell phenotype through the Wnt-signaling pathway, which has previously been shown to promote metastatic dissemination in another Braf-driven melanoma model. In this model, conditional genetic stabilization of β-catenin enhances metastasis in Braf-mutant, Pten-deficient mice (Damsky et al. 2011). Similarly, activation of Wnt-signaling in melanocytes via deletion of Smad4 increases metastatic dissemination in Nras mutated, Ink4a deficient mice (Tuncer et al. 2019). As the transgenic Hgf overexpression is broadly expressed, increased Hgf-Met signaling could also have microenvironmental effects in addition to its potential cell-intrinsic roles for transformation. Hgf-Met signaling is well known to promote immunosuppression (Benkhoucha et al. 2010; Glodde et al. 2017; Okunishi et al. 2005) and can cause microenvironmental resistance to signal transduction inhibitors (Straussman et al. 2012). We further speculate that increased Hgf-Met signaling can reshape the microenvironment at metastatic sites such as the lungs to create a favorable niche for the outgrowth of metastases, an assumption that will be tested in future experiments.
We previously observed that UV irradiation of Hgf-Cdk4 mice promoted melanoma cells to grow invasively along the abluminal surfaces of endothelial cells, a process termed angiotropism (Bald et al. 2014). This angiotropic growth pattern was also found in Hgf-BrafCdk4 melanomas both at the invasive front in primary tumors and in metastatic lesions of the lung. Similar correlations between angiotropism and distant metastasis are described in human melanoma. It has been hypothesized that melanoma cells metastasize through “extravascular migration” along vessel surfaces (Barnhill et al. 2002; Barnhill and Lugassy
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2004). Whether melanoma cells actually migrate on endothelial cells or only expand along the perivascular niche and gain increased access to the blood stream will also have to be further addressed in future studies.
In our experiments we observed spontaneous melanoma formation in Hgf-Braf-Cdk4 mice without any exposure to tamoxifen. A similar “leakiness” of CreERT2 driven recombination systems has been described also for a mouse melanoma model combining Braf mutation with loss of PTEN (Hooijkaas et al. 2012) and is well known in the field. Upon tamoxifen treatment, the conditionally expressed mutant Braf allele is simultaneously introduced as an oncogenic driver in a large number of melanocytes. Not unexpected, this resulted in the development of numerous nevi and some melanomas, suggesting a polyclonal origin of these lesions. In contrast, spontaneous recombination of the mutated Braf allele without tamoxifen might represent a clonal event. From an experimental standpoint this could be a desirable feature as it would more closely portray our evolutionary understanding of tumor pathogenesis in which initially single cells acquire malignant potential and subsequently give rise to a genetically and phenotypically heterogeneous cancer (Merlo et al. 2006). Further analyses are required to elucidate the clonal dynamics in genetically engineered mouse melanoma models in more detail.
Primary melanomas in untreated Hgf-Braf-Cdk4 mice showed considerable morphologic heterogeneity with both pigmented, highly differentiated, immune cell poor phenotypes as well as amelanotic, dedifferentiated, immune cell enriched phenotypes. Surprisingly, all amelanotic spontaneous melanomas showed a recombination of the conditional mutant Braf allele while a similar recombination could only be detected in a few pigmented melanomas. In fact, amelanotic melanomas appeared only after activation of the mutant Braf allele
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suggesting that oncogenic Braf-driven signaling can promote cellular dedifferentiation. However, activation of mutant Braf in amelanotic progenitor cells with subsequent hierarchical differentiation cannot be fully excluded. Dynamic dedifferentiation has been observed in another mouse melanoma model where conditional activation of mutated Braf was combined with loss of Pten. Here the activation of the Braf mutation locally on the tail skin initiated the clonal expansion of interfollicular melanocytes which subsequently acquired a dedifferentiated phenotype and progressed towards invasively growing melanoma (Köhler et al. 2017). Recent reports support the concept that melanoma cells can show progressive stages of dedifferentiation during tumor progression both in mice and men (Tsoi et al. 2018). A similar phenotype switch has been observed during signal transduction inhibitor therapy, where melanoma cells shifted towards an intermediary, “starved-like” phenotype before either undergoing dedifferentiation or becoming fully differentiated with both phenotypes conferring resistance to the signal transduction inhibitor (Rambow et al. 2018). This dynamic phenotypic plasticity is thought to be due to aberrant reactivation of embryonic developmental programs with melanoma cells acquiring stem-cell like phenotypes and properties. This has been shown to enable invasive growth and metastatic dissemination (Dongre and Weinberg 2019; Hoek et al. 2008; Verfaillie et al. 2015).
Taken together, we established the Hgf-Braf-Cdk4 mouse melanoma model and observed that activated Hgf-Met signaling cooperates with oncogenic Braf to drive primary cutaneous melanomas and systemic metastases in the lungs. In this experimental system oncogenic Braf also promotes melanoma cell dedifferentiation. A unique feature of the model is the emergence of angiotropic growth patterns during melanoma cell invasion and metastasis. The Hgf-Braf-Cdk4 mouse model represents an attractive experimental system to further
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investigate mechanisms underlying the process of malignant transformation of melanocytes and the progression towards metastatic disease.
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MATERIALS AND METHODS Mice Hgf-Cdk4 and Braf-Cdk4 mice were bred and genotyped as previously described (Mercer et al. 2005; Rane et al. 1999; Takayama et al. 1996; Yajima et al. 2006). Hgf-Braf-Cdk4 mice were generated by breeding male Hgf-Cdk4 mice with female Braf-Cdk4 mice. Breeding and experiments were conducted in individually ventilated cages. Mice were regularly screened for pathogens. All experiments were performed in compliance with federal and international guidelines for animal experiments and with the approval of the responsible authorities (Landesverwaltungsamt Saxony-Anhalt, Germany).
Induction of primary melanomas with DMBA and 4OHT All experiments were performed in 6-8 week old mice. Cohorts of age and sex matched mice were randomly assigned to experimental groups. Primary melanoma development was induced by single dose application of 100 nmol of the carcinogen DMBA (dissolved in 100 µl acetone) on the shaved back skin as previously described (Landsberg et al. 2012). Induction of Cre-mediated recombination and activation of the mutant Braf-allele in melanocytes was triggered by epicutaneously application of 4-hydroxy-Tamoxifen (4OHT, 33 µg dissolved in 100 µl acetone ≈ 853 µmol) on three consecutive days. Tumor area was measured twice weekly and calculated by the formula area in mm2 = length x width. Mice were sacrificed when tumors exceeded 100 mm2 tumor area. Cohort sizes of experimental groups are stated in the corresponding figure legends.
Histomorphologic sample preparation and immunohistochemical analyses Melanomas were fixed in zinc-solution for 24 h (BD Pharmingen) and dehydrated, embedded in paraffin, sectioned and stained with haematoxylin and eosin or giemsa according to 13
standard protocols. Highly pigmented samples were bleached with H2O2 prior to staining (3% H2O2 and 0,5% KOH for 20 minutes at 37°C, then 1% acetic acid for 20 seconds). Immunohistochemical stainings were conducted with polyclonal rabbit anti-mouse gp100 (NBP1-69571, Novus Biologicals), monoclonal rat anti-mouse CD45 (Catalog Nr. 550539, BD Biosciences), polyclonal, biotinylated goat anti-mouse Ngfr (AF1157, R&D Systems), monoclonal rat anti-human CD3 (Catalog Nr. MCA1477, Bio-Rad), secondary antibody polyclonal goat anti-rabbit IgG (Catalog Nr. 111-067-003, dianova) and polyclonal goat antirat IgG (Catalog Nr. 112-067-003, dianova) as well as the ZytoChem Fast Red AP LSAB kit. Stained sections were imaged with an Axio Imager A1 light microscope (Zeiss) and digitized with a NanoZoomer SQ digital slide scanner (Hamamatsu).
Statistical analyses Survival probabilities were calculated with a Kaplan-Meier estimator and significance tested by logrank test. Post-hoc correction for multiple testing was performed by Bonferronicorrection. Significance of spontaneous Braf-activation between pigmented and amelanotic tumors was calculated with Fishers exact test. Frequency of pulmonary metastasis was tested for significance with Kruskal-Wallis test and post-hoc Benjamini-Hochberg correction of the false discovery rate. p values less than 0.05 were considered significant. All calculations were performed in the R computing environment.
DATA AVAILABILITY STATEMENT No datasets were generated or analyzed during the current study.
CONFLICT OF INTEREST The authors declare no conflict of interest.
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ACKNOWLEDGEMENTS We thank J. Herz for expert assistance with the histological procedures. A.D.B. was supported by a scholarship from the Else Kröner-Forschungskolleg Magdeburg. E.G. was supported by the SFB854. T.T. was funded in part by grants from the DFG (TU 90/8-1 and A27 in the SFB854).
CRediT STATEMENT Conceptualization, data curation, formal analysis, writing of manuscript: E.G., T.T., A.D.B. and M.M.; Experiments: E.G., A.D.B. and S.B.; Funding Acquisition: T.T.
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FIGURE LEGENDS Figure 1: Hgf-Braf-Cdk4 mice spontaneously develop rapidly growing, nodular melanoma with complete penetrance. a. Breeding strategy for the generation of Hgf-Braf-Cdk4 mice. b. Representative macroscopic pictures of spontaneously arising nodular cutaneous melanomas of Hgf-Braf-Cdk4 and Hgf-Cdk4 mice. c. Kaplan-Meyer graphs showing the survival of Hgf-Braf-Cdk4 (HBC), Hgf-Cdk4 (HC) as well as Braf-Cdk4 and Cdk4 (BC/C) control mice (**p<0.01, logrank test).
Figure 2: Spontaneous recombination of the mutant Braf-allele drives amelanotic melanomas in HgfBraf-Cdk4 mice a. Table indicating the number of both pigmented and amelanotic melanomas in a cohort of 15 Hgf-Cdk4 and 11 Hgf-Braf-Cdk4 mice (mean±SD). b. Analysis of Braf-allele-status in pigmented and amelanotic Hgf-Braf-Cdk4 mice (***p<0.005, Fishers exact test).
Figure 3: Amelanotic Hgf-Braf-Cdk4 melanomas show an immune-cell rich phenotype Representative histopathologic images of pigmented and amelanotic Hgf-Braf-Cdk4 melanomas. Similar results were seen in 5 pigmented and 5 amelanotic tumors. Magnification and staining are shown in individual pictures.
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Figure 4: Activation of the mutant Braf allele and epicutaneous treatment with DMBA accelerate melanomagenesis. a. Experimental setup for the treatment of Hgf-Braf-Cdk4 mice with 4-OHT and/or DMBA. b. Representative macroscopic pictures of Hgf-Braf-Cdk4 mice left untreated or treated with 4OHT, DMBA and 4OHT+DMBA at the indicated age. c. Mean survival of Hgf-Braf-Cdk4 mice in the different cohorts (*p<0.05, ***p<0.005, logrank test with Bonferroni-correction).
Figure 5: Activation of the mutant Braf allele promotes the appearance of amelanotic, immune cell rich Hgf-Braf-Cdk4 melanomas. a. Representative macroscopic and microscopic images of melanomas from Hgf-Braf-Cdk4 mice treated and stained as indicated. Similar results were found in 5 pigmented and 5 amelanotic tumors. b. Incidence of pigmented and amelanotic phenotypes in cohorts of Hgf-Braf-Cdk4 mice treated with 4OHT, DMBA or 4OHT+DMBA (mean ± SD).
Figure 6: Figure 6: Activation of the mutant Braf allele drives the development of angiotropic lung metastases a. Representative macroscopic and microscopic images of lungs from Hgf-Braf-Cdk4 mice treated and stained as indicated. b. Percentages of Hgf-Cdk4 (n=25), Braf-Cdk4 (spontaneous n=10, 4OHT n=6) and HgfBraf-Cdk4 mice (spontaneous n=11, 4OHT n=6, DMBA n=9, 4OHT+DMBA n=11) with
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pulmonary metastases in the different cohorts treated as indicated (*p<0.05, ***p<0.005, Kruskal-Wallis-test with post-hoc Benjamini-Hochberg correction of the false discovery rate). c. Representative macroscopic and microscopic images of angiotropism in Hgf-Braf-Cdk4 primary tumors and lung metastases, Magnification and staining as indicated. Similar results were found in 5 mice.
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Supplementary Figure 1: Supplementary Figure 1: Genotype and phenotype distribution of 60 offspring from breeding between male C57BL/6 Mt::Hgf+/o- BRAFLSL-V600E/wt-Cdk4R24C/R24C-Tyr::CreERT2 +/o and female C57BL/6 BRAFLSL-V600E/wt-Cdk4R24C/R24C-Tyr::CreERT2 +/o mice. Supplementary Figure 2: Supplementary Figure 2: Recombination of the Braf-allele in Hgf-Braf-Cdk4 mice Schematic representation and photograph of a Braf locus genotyping-PCR with Primers A+B and A+C before (a.) and after (b.) recombination. Sample 1: Hgf-Cdk4 skin, Sample 2: HgfBraf-Cdk4 skin, Sample 3: Hgf-Braf-Cdk4 amelanotic melanoma Supplementary Figure 3: Representative histopathology of Hgf-Cdk4 melanomas. Magnification and staining are shown in individual images. Supplementary Figure 4: Characterization of the immune infiltrate in amelanotic Hgf-Braf-Cdk4 melanomas. Magnification and staining are shown in individual images. Supplementary Figure 5: Braf-Status of melanomas from Hgf-Braf-Cdk4 mice treated with 4OHT, DMBA or both.