Malignant features of minipig melanomas prior to spontaneous regression.

In MeLiM minipigs, melanomas develop around birth, can metastasize, and have histopathologic characteristics similar to humans. Interestingly, MeLiM melanomas eventually regress. This favorable outcome raises the question of their malignancy, which we investigated. We clinically followed tens of tumors from onset to first signs of regression. Transcriptome analysis revealed an enrichment of all cancer hallmarks in melanomas, although no activating or suppressing somatic mutation were found in common driver genes. Analysis of tumor cell genomes revealed high mutation rates without UV signature. Canonical proliferative, survival and angiogenic pathways were detected in MeLiM tumor cells all along progression stages. Functionally, we show that MeLiM melanoma cells are capable to grow in immunocompromised mice, with serial passages and for a longer time than in MeLiM pigs. Pigs set in place an immune response during progression with dense infiltration by myeloid cells while melanoma cells are deficient in B2M expression. To conclude, our data on MeLiM melanomas reveal several malignancy characteristics. The combination of these features with the successful spontaneous regression of these tumors make it an outstanding model to study an efficient anti-tumor immune response.

Deciphering the immune reaction leading to spontaneous melanoma regression: initial role of MHCII+ CD163- macrophages.

The human cutaneous metastatic melanoma is the deadliest skin cancer. Partial, or less frequently complete spontaneous regressions could be observed, mainly mediated by T cells. Nevertheless, the underlying mechanisms are not fully unraveled. We investigated the first events of the immune response related to cancer regression in Melanoma-bearing Libechov Minipigs (MeLiM), a unique swine model of cutaneous melanoma that regresses spontaneously. Using a multiparameter flow cytometry strategy and integrating new clinical and histological criteria of the regression, we show that T cells and B cells are present only in the late stages, arguing against their role in the initial destruction of malignant cells. NK cells infiltrate the tumors before T cells and therefore might be involved in the induction of the regression process. Myeloid cells represent the main immune population within the tumor microenvironment regardless of the regression stage. Among those, MHCII+ CD163- macrophages that differ phenotypically and functionally compared to other tumor-associated macrophages, increase in number together with the first signs of regression suggesting their crucial contribution to initiating the regression process. Our study supports the importance of macrophage reprogramming in humans to improve current immunotherapy for metastatic melanoma.

Loss of CD24 promotes radiation­ and chemo­resistance by inducing stemness properties associated with a hybrid E/M state in breast cancer cells.

Cancer stem cells (CSCs) serve an essential role in failure of conventional antitumor therapy. In breast cancer, CD24­/low/CD44+ phenotype and high aldehyde dehydrogenase activity are associated with CSC subtypes. Furthermore, CD24­/low/CD44+ pattern is also characteristic of mesenchymal cells generated by epithelial­mesenchymal transition (EMT). CD24 is a surface marker expressed in numerous types of tumor, however, its biological functions and role in cancer progression and treatment resistance remain poorly documented. Loss of CD24 expression in breast cancer cells is associated with radiation resistance and control of oxidative stress. Reactive oxygen species (ROS) mediate the effects of anticancer drugs as well as ionizing radiation; therefore, the present study investigated if CD24 mediates radiation­ and chemo­resistance of breast cancer cells. Using a HMLE breast cancer cell model, CD24 expression has been artificially modulated and it was observed that loss of CD24 expression induced stemness properties associated with acquisition of a hybrid E/M phenotype. CD24­/low cells were more radiation­ and chemo­resistant than CD24+ cells. The resistance was associated with lower levels of ROS; CD24 controlled ROS levels via regulation of mitochondrial function independently of antioxidant activity. Together, these results suggested a key role of CD24 in de­differentiation of breast cancer cells and promoting acquisition of therapeutic resistance properties.

Radiation Enhancer Effect of Platinum Nanoparticles in Breast Cancer Cell Lines: In Vitro and In Silico Analyses.

High-Z metallic nanoparticles (NPs) are new players in the therapeutic arsenal against cancer, especially radioresistant cells. Indeed, the presence of these NPs inside malignant cells is believed to enhance the effect of ionizing radiation by locally increasing the dose deposition. In this context, the potential of platinum nanoparticles (PtNPs) as radiosensitizers was investigated in two breast cancer cell lines, T47D and MDA-MB-231, showing a different radiation sensitivity. PtNPs were internalized in the two cell lines and localized in lysosomes and multivesicular bodies. Analyses of cell responses in terms of clonogenicity, survival, mortality, cell-cycle distribution, oxidative stress, and DNA double-strand breaks did not reveal any significant enhancement effect when cells were pre-exposed to PtNPs before being irradiated, as compared to radiation alone. This result is different from that reported in a previous study performed, under the same conditions, on cervical cancer HeLa cells. This shows that the efficacy of radio-enhancement is strongly cell-type-dependent. Simulation of the early stage ionization processes, taking into account the irradiation characteristics and realistic physical parameters in the biological sample, indicated that PtNPs could weakly increase the dose deposition (by 3%) in the immediate vicinity of the nanoparticles. Some features that are potentially responsible for the biological effect could not be taken into account in the simulation. Thus, chemical and biological effects could explain this discrepancy. For instance, we showed that, in these breast cancer cell lines, PtNPs exhibited ambivalent redox properties, with an antioxidant potential which could counteract the radio-enhancement effect. This work shows that the efficacy of PtNPs for enhancing radiation effects is strongly cell-dependent and that no effect is observed in the case of the breast cancer cell lines T47D and MDA-MB-231. Thus, more extensive experiments using other relevant biological models are needed in order to evaluate such combined strategies, since several clinical trials have already demonstrated the success of combining nanoagents with radiotherapy in the treatment of a range of tumor types.

The Composition of Circulating Leukocytes Varies With Age and Melanoma Onset in the MeLiM Pig Biomedical Model.

Immunological research in pigs benefits from many improvements with a direct impact on the veterinary control of pig husbandry and on biomedical models. We compiled the available knowledge to develop gating strategies to monitor simultaneously all blood immune cell types by multicolor flow cytometry in Melanoblastoma-bearing Libechov Minipigs (MeLiM). The MeLiM pig spontaneously develops cutaneous melanomas that regress few months later. We monitored lymphoid and myeloid cell subsets in 3 to 21 weeks old pigs. Interestingly, neutrophils, type III monocytes (CD163+ CD14+ MHC II-) and CD4- CD8α- T cells are less abundant in oldest animals in contrast to eosinophils, type II monocytes (CD163- CD14low MHC II+), B cells, γδ T cells, CD4+ CD8α+ and CD4- CD8α+ T cells. Melanoma occurrence led to changes in the blood cell composition. Higher proportions of NK cells, CD4+ and CD4+ CD8α+ T cells, and CD21- B cells among B cells are found in young melanoma-bearing piglets, consistent with the immune-mediated spontaneous regression in the MeLiM model.

Survey of allele specific expression in bovine muscle.

Allelic imbalance is a common phenomenon in mammals that plays an important role in gene regulation. An Allele Specific Expression (ASE) approach can be used to detect variants with a cis-regulatory effect on gene expression. In cattle, this type of study has only been done once in Holstein. In our study we performed a genome-wide analysis of ASE in 19 Limousine muscle samples. We identified 5,658 ASE SNPs (Single Nucleotide Polymorphisms showing allele specific expression) in 13% of genes with detectable expression in the Longissimus thoraci muscle. Interestingly we found allelic imbalance in AOX1, PALLD and CAST genes. We also found 2,107 ASE SNPs located within genomic regions associated with meat or carcass traits. In order to identify causative cis-regulatory variants explaining ASE we searched for SNPs altering binding sites of transcription factors or microRNAs. We identified one SNP in the 3'UTR region of PRNP that could be a causal regulatory variant modifying binding sites of several miRNAs. We showed that ASE is frequent within our muscle samples. Our data could be used to elucidate the molecular mechanisms underlying gene expression imbalance.

New susceptibility loci for cutaneous melanoma risk and progression revealed using a porcine model.

Despite major advances, it is estimated that a large part of melanoma predisposing genes remains to be discovered. Animal models of spontaneous diseases are valuable tools and experimental crosses can be used to identify and fine-map new susceptibility loci associated with melanoma. We performed a Genome-Wide Association Study (GWAS) of melanoma occurrence and progression (clinical ulceration and presence of metastasis) in a porcine model of spontaneous melanoma, the MeLiM pig. Five loci on chromosomes 2, 5, 7, 8 and 16 showed genome-wide significant associations (p < 5 × 10-6) with either one of these phenotypes. Suggestive associations (p < 5 × 10-5) were also found at 16 additional loci. Moreover, comparison of the porcine results to those reported by human melanoma GWAS indicated shared association signals notably at CDKAL1 and TERT loci but also nearby CCND1, FTO, PLA2G6 and TMEM38B-RAD23B loci. Extensive search of the literature revealed a potential key role of genes at the identified porcine loci in tumor invasion (DST, PLEKHA5, CBY1, LIMK2 and ETV5) and immune response modulation (ETV5, HERC3 and DICER1) of the progression phenotypes. These biological processes are consistent with the clinico-pathological features of MeLiM tumors and can open new routes for future melanoma research in humans.

Impact of a CD4 gene haplotype on the immune response in minipigs.

The cluster of differentiation 4 (CD4) molecule functions as a co-receptor for MHC class II binding to TCR in T helper cells. A CD4 epitope deficiency was identified in the swine MeLiM (melanoblastoma-bearing Libechov minipig) strain, a model for spontaneous cutaneous melanoma development and regression. Extensive sequencing revealed a high genetic variability of CD4 and the existence of several haplotypes segregating in MeLiM. Forty polymorphisms were identified in the coding sequence, out of which 20 correspond to non-synonymous variants and 10 are located in the 3'UTR of CD4 transcripts. One of the haplotypes segregating in the MeLiM explained the epitope deficiency observed. An association analysis between CD4 genotype and several phenotypes related to tumor regression was performed in 267 animals. An association was evidenced between a MeLiM alternative CD4 haplotype and skin and eye depigmentation, as well as the extent of hair depigmentation. Also, seric IgG concentration was shown to be higher in pigs carrying the alternative haplotype at the homozygous state. In conclusion, the genetic variability of the CD4 gene is associated with immune response-related phenotypes in MeLiM minipigs.

The MeLiM Minipig: An Original Spontaneous Model to Explore Cutaneous Melanoma Genetic Basis.

Melanoma is the deadliest skin cancer and is a major public health concern with a growing incidence worldwide. As for other complex diseases, animal models are needed in order to better understand the mechanisms leading to pathology, identify potential biomarkers to be used in the clinics, and eventually molecular targets for therapeutic solutions. Cutaneous melanoma, arising from skin melanocytes, is mainly caused by environmental factors such as UV radiation; however a significant genetic component participates in the etiology of the disease. The pig is a recognized model for spontaneous development of melanoma with features similar to the human ones, followed by a complete regression and a vitiligo-like depigmentation. Three different pig models (MeLiM, Sinclair, and MMS-Troll) have been maintained through the last decades, and different genetic studies have evidenced a complex inheritance of the disease. As in humans, pigmentation seems to play a prominent role, notably through MC1R and MITF signaling. Conversely, cell cycle genes as CDKN2A and CDK4 have been excluded as predisposing for melanoma in MeLiM. So far, only sparse studies have focused on somatic changes occurring during oncogenesis, and have revealed major cytological changes and a potential dysfunction of the telomere maintenance system. Finally, the spontaneous tumor progression and regression occurring in these models could shed light on the interplay between endogenous retroviruses, melanomagenesis, and adaptive immune response.

Transcriptome of melanoma cells from two mouse models, Tyr:NRasQ61K and Tyr:Rack1-HA, Tyr:NRasQ61K.

The transcriptome sequencing of melanoma cells from two mouse models differing in the expression level of the scaffold protein Receptor for activated C kinase (RACK1) are presented. Primary melanoma cells were harvested from Tyr:NRasQ61K; Pax3GFP/+ mice, with or without the Tyr:Rack1-HA transgene. Cells were cultured and infected with scramble shRNA or Rack1-targeting shRNA, on technical triplicates of viral infection. Libraries were prepared by selecting polyadenylated mRNAs and RNA Sequencing (RNASeq) was performed. Samples are described in the SRA portal (SRP096162) and FASTQ files have been deposited in Sequence Read Archive (accession numbers: SRR5150106 to SRR5150117). The interpretation of these data is presented in the following research article: "RACK1 cooperates with NRASQ61K to promote melanoma in vivo" (Campagne et al., 2017, doi: 10.1016/j.cellsig.2017.03.015) [1].

Transcription variants of SLA-7, a swine non classical MHC class I gene.

In pig, very little information is available on the non classical class I (Ib) genes of the Major Histocompatibility Complex (MHC) i.e. SLA-6, -7 and -8. Our aim was to focus on the transcription pattern of the SLA-7 gene. RT-PCR experiments were carried out with SLA-7 specific primers targeting either the full coding sequence (CDS) from exon 1 to the 3 prime untranslated region (3UTR) or a partial CDS from exon 4 to the 3UTR. We show that the SLA-7 gene expresses a full length transcript not yet identified that refines annotation of the gene with eight exons instead of seven as initially described from the existing RefSeq RNA. These two RNAs encode molecules that differ in cytoplasmic tail length. In this study, another SLA-7 transcript variant was characterized, which encodes a protein with a shorter alpha 3 domain, as a consequence of a splicing site within exon 4. Surprisingly, a cryptic non canonical GA-AG splicing site is used to generate this transcript variant. An additional SLA-7 variant was also identified in the 3UTR with a splicing site occurring 31 nucleotides downstream to the stop codon. In conclusion, the pig SLA-7 MHC class Ib gene presents a complex transcription pattern with two transcripts encoding various molecules and transcripts that do not alter the CDS and may be subject to post-transcriptional regulation.

Genetic and functional evaluation of MITF as a candidate gene for cutaneous melanoma predisposition in pigs.

Cutaneous melanoma arises from transformed melanocytes and is caused mainly by environmental effects such as ultraviolet radiation and to a lesser extent by predisposing genetic variants. Only a few susceptibility genes for cutaneous melanoma have been identified so far in human; therefore, animal models represent a valuable alternative for genetic studies of this disease. In a previous quantitative trait locus (QTL) study, several susceptibility regions were identified in a swine biomedical model, the MeLiM (Melanoblastoma-bearing Libechov minipig) pigs. This article details the fine-mapping of a QTL located on SSC13 (Sus scrofa chromosome 13) through an increase in marker density. New microsatellites were used to confirm the results of the first analysis, and MITF (microphthalmia-associated transcription factor) was selected as a candidate gene for melanoma development. A single-marker association analysis was performed with single-nucleotide polymorphisms (SNPs) spread over the locus, but it did not reveal a significant association with diverse melanoma-related traits. In parallel, MITF alternative transcripts were characterized and their expression was investigated in different porcine tissues. The obtained results showed a complex transcriptional regulation concordant with the one present in other mammals. Notably, the ratio between MITF+ and MITF- isoforms in melanoma samples followed the same pattern as in human tumors, which highlights the adequacy of the MeLiM pig as a model for human melanoma. In conclusion, although MITF does not seem to be the causal gene of the QTL initially observed, we do not exclude a prominent role of its transcription and function in the outbreak and evolution of the tumors observed in pigs.

Microarray analysis of differential gene expression in the liver of lean and fat chickens.

Excessive adiposity has become a major drawback in meat-type chicken production. However, few studies were conducted to analyze the liver expression of genes involved in pathways and mechanisms leading to adiposity. A previous study performed by differential display on RNAs extracted from chicken livers from lean and fat lines allowed us to isolate cDNA products of genes with putative differential expression. In this study, a cDNA microarray resource was developed from these products together with cDNAs from genes involved in or related to lipid metabolism. This resource was used to analyze gene expression in the liver from lean and fat chickens. Some genes were found with a difference in expression between lean and fat animals and/or correlated to adipose tissue weight. Cytochrome P450 2C45, thought to play a role in biotransformation of steroids and poly-unsaturated fatty acids, was more expressed in lean chickens whereas fatty acid synthase, stearoyl-CoA desaturase, sterol response element binding factor 1 and hepatocyte nuclear factor 4, respectively involved in lipogenesis and its regulation, were more expressed in fat chickens. These results indicate that mechanisms involved in the expression and regulation of lipogenic genes could play a key role in fatness ontogenesis in chickens from lean and fat lines.

Differential expression and genetic variation of hepatic messenger RNAs from genetically lean and fat chickens.

Although excessive adiposity has become a major drawback in meat type chicken production, few of the genes involved in this process have been characterized so far. In order to identify putative genes involved in adiposity, we performed differential display analysis of RNAs extracted from the liver of divergently selected lean and fat chickens. Twenty-six differential products were selected and purified by single strand conformation polymorphism gel electrophoresis before sequencing and Northern blot analyses. An orthologous sequence of a mammalian cytochrome P450 2C subfamily member was proven to be differentially expressed in the liver of lean and fat chickens and could play an important role in the regulation of adiposity. In mammals, these genes are involved in detoxification of xenobiotics and metabolism of some important biological compounds. Four other genes were found differentially expressed to a lower extent. Some unidentified products were shown to be lean or fat specific, with sequence polymorphism and liver specific expression, strongly suggesting that the related gene could be directly involved in adiposity. Our data indicate that differential display can evidence genes with differential expression and with sequence polymorphism, making this strategy more accurate for differential analysis of messenger RNAs.