Supplementary Figure 1 from Transcriptional Regulation of Estrogen Receptor-α by p53 in Human Breast Cancer Cells

Figure S1 shows representative GTT and ITT results of the parents used to produce study animals.

Figure S3 shows the FACS gating strategy used to isolate MaSCs.

Diet is a critical environmental factor affecting breast cancer risk, and recent evidence shows that dietary exposures during early development can affect lifetime mammary cancer susceptibility. To elucidate the underlying mechanisms, we used our established crossover feeding mouse model, where exposure to a high-fat and high-sugar (HFHS) diet during defined developmental windows determines mammary tumor incidence and latency in carcinogen-treated mice. Mammary tumor incidence is significantly increased in mice receiving a HFHS post-weaning diet (high-tumor mice, HT) compared to those receiving a HFHS diet during gestation (low-tumor mice, LT). The current study revealed that the mammary stem cell (MaSC) population was significantly increased in mammary glands from HT compared to LT mice. Igf1 expression was increased in mammary stromal cells from HT mice, where it promoted MaSC self-renewal. The increased Igf1 expression was induced by DNA hypomethylation of the Igf1 Pr1 promoter, mediated by a decrease in Dnmt3b levels. Mammary tissues from HT mice also had reduced levels of Igfbp5, leading to increased bioavailability of tissue Igf1. This study provides novel insights into how early dietary exposures program mammary cancer risk, demonstrating that effective dietary intervention can reduce mammary cancer incidence.

The role of stroma is fundamental in the development and behavior of epithelial tumors. In this regard, limited growth of squamous cell carcinomas (SCC) or cell-lines derived from them has been achieved in immunodeficient mice. Moreover, lack of faithful recapitulation of the original human neoplasia complexity is often observed in xenografted tumors. Here, we used tissue engineering techniques to recreate a humanized tumor stroma for SCCs grafted in host mice, by combining CAF (cancer associated fibroblasts)-like cells with a biocompatible scaffold. The stroma was either co-injected with epithelial cell lines derived from aggressive SCC or implanted 15 days before the injection of the tumoral cells, to allow its vascularization and maturation. None of the mice injected with the cell lines without stroma were able to develop a SCC. In contrast, tumors were able to grow when SCC cells were injected into previously established humanized stroma. Histologically, all of the regenerated tumors were moderately differentiated SCC with a well-developed stroma, resembling that found in the original human neoplasm. Persistence of human stromal cells was also confirmed by immunohistochemistry. In summary, we provide a proof of concept that humanized tumor stroma, generated by tissue engineering, can facilitate the development of epithelial tumors in immunodeficient mice.

Psoriasis (PS) and atopic dermatitis (AD) are common inflammatory skin diseases characterized by an imbalance in specific T-cell subsets, resulting in a specific cytokine profile in patients. Obtaining models closely resembling both pathologies along with a relevant clinical impact is crucial for the development of new therapies because of the high prevalence of these diseases. Single-gene mouse models developed until now do not fully reflect the complexity of these disorders, in part not only because of inherent differences between mice and humans but also because of the multifactorial nature of these pathologies. The skin-humanized mouse model developed by our group, based on a tissue engineering approach, has been used to test therapeutic strategies, although this methodology is still technically challenging and not widely available. The skin-humanized mouse models for PS and AD reproduce human skin phenotypes, providing valuable tools for drug development and testing in the preclinical setting. The tissue engineering approach allows the development of personalized medicine, covering the broad genotypic spectrum of these pathologies. This review highlights the main differences between available murine models focusing on the tissue-specific immunity of PS and AD. We discuss their contribution to unravel the complex pathophysiology of these diseases and to translate this knowledge into more accurate therapies.

Psoriasis (PS) and atopic dermatitis (AD) are common inflammatory skin diseases characterized by an imbalance in specific T-cell subsets, resulting in a specific cytokine profile in patients. Obtaining models closely resembling both pathologies along with a relevant clinical impact is crucial for the development of new therapies because of the high prevalence of these diseases. Single-gene mouse models developed until now do not fully reflect the complexity of these disorders, in part not only because of inherent differences between mice and humans but also because of the multifactorial nature of these pathologies. The skin-humanized mouse model developed by our group, based on a tissue engineering approach, has been used to test therapeutic strategies, although this methodology is still technically challenging and not widely available. The skin-humanized mouse models for PS and AD reproduce human skin phenotypes, providing valuable tools for drug development and testing in the preclinical setting. The tissue engineering approach allows the development of personalized medicine, covering the broad genotypic spectrum of these pathologies. This review highlights the main differences between available murine models focusing on the tissue-specific immunity of PS and AD. We discuss their contribution to unravel the complex pathophysiology of these diseases and to translate this knowledge into more accurate therapies.

BackgroundKindler Syndrome (KS) is a rare genodermatosis characterized by skin fragility, skin atrophy, premature aging and poikiloderma. It is caused by mutations in the FERMT1 gene, which encodes kindlin-1, a protein involved in integrin signalling and the formation of focal adhesions. Several reports have shown the presence of non-melanoma skin cancers in KS patients but a systematic study evaluating the risk of these tumors at different ages and their potential outcome has not yet been published. We have here addressed this condition in a retrospective study of 91 adult KS patients, characterizing frequency, metastatic potential and body distribution of squamous cell carcinoma (SCC) in these patients. SCC developed in 13 of the 91 patients.ResultsThe youngest case arose in a 29-year-old patient; however, the cumulative risk of SCC increased to 66.7% in patients over 60years of age. The highly aggressive nature of SCCs in KS was confirmed showing that 53.8% of the patients bearing SCCs develop metastatic disease. Our data also showed there are no specific mutations that correlate directly with the development of SCC; however, the mutational distribution along the gene appears to be different in patients bearing SCC from SCC-free patients. The body distribution of the tumor appearance was also unique and different from other bullous diseases, being concentrated in the hands and around the oral cavity, which are areas of high inflammation in this disease.ConclusionsThis study characterizes SCCs in the largest series of KS patients reported so far, showing the high frequency and aggressiveness of these tumors. It also describes their particular body distribution and their relationship with mutations in the FERMT-1 gene. These data reinforce the need for close monitoring of premalignant or malignant lesions in KS patients.

Female breast cancer (BrCa) is the most common noncutaneous cancer among women in the United States. Human epidemiological studies reveal that a p53 single-nucleotide polymorphism (SNP) at codon 72, encoding proline (P72) or arginine (R72), is associated with differential risk of several cancers, including BrCa. However, the molecular mechanisms by which these variants affect mammary tumorigenesis remain unresolved. To investigate the effects of this polymorphism on susceptibility to mammary cancer, we used a humanized p53 mouse model, homozygous for either P72 or R72. Our studies revealed that R72 mice had a significantly higher mammary tumor incidence and reduced latency in both DMBAinduced and MMTV-Erbb2/Neu mouse mammary tumor models compared to P72 mice. Analyses showed that susceptible mammary glands from E-R72 (R72 x MMTV-Erbb2/Neu) mice developed a senescence-associated secretory phenotype (SASP) with influx of proinflammatory macrophages, ultimately resulting in chronic, protumorigenic inflammation. Mammary tumors arising in E-R72 mice also had an increased influx of tumor-associated macrophages, contributing to angiogenesis and elevated tumor growth rates. These results demonstrate that the p53 R72 variant increased susceptibility to mammary tumorigenesis through chronic inflammation.

Abstract Obesity and alterations in metabolic programming from early diet exposures can affect the propensity to disease in later life. Through dietary manipulation, developing mouse pups were exposed to a hyperinsulinemic, hyperglycemic milieu during three developmental phases: gestation, lactation, and postweaning. Analyses showed that a postweaning high fat/high sugar (HF/HS) diet had the main negative effect on adult body weight, glucose tolerance, and insulin resistance. However, dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis revealed that animals born to a mother fed a HF/HS gestation diet, nursed by a mother on a mildly diet-restricted, low fat/low sugar diet (DR) and weaned onto a HF/HS diet (HF/DR/HF) had the highest mammary tumor incidence, while HF/HF/DR had the lowest tumor incidence. Cox proportional hazards analysis showed that a HF/HS postweaning diet doubled mammary cancer risk, and a HF/HS diet during gestation and postweaning increased risk 5.5 times. Exposure to a HF/HS diet during gestation, when combined with a postweaning DR diet, had a protective effect, reducing mammary tumor risk by 86% (HR = 0.142). Serum adipocytokine analysis revealed significant diet-dependent differences in leptin/adiponectin ratio and IGF-1. Flow cytometry analysis of cells isolated from mammary glands from a high tumor incidence group, DR/HF/HF, showed a significant increase in the size of the mammary stem cell compartment compared with a low tumor group, HF/HF/DR. These results indicate that dietary reprogramming induces an expansion of the mammary stem cell compartment during mammary development, increasing likely carcinogen targets and mammary cancer risk. Cancer Prev Res; 10(10); 553–62. ©2017 AACR. See related editorial by Freedland, p. 551–2.

Epidermolysis bullosa with pyloric atresia ( EB ‐ PA ) is a rare autosomal recessive hereditary disease with a variable prognosis from lethal to very mild. EB ‐ PA is classified into Simplex form ( EBS ‐ PA : OMIM #612138) and Junctional form ( JEB ‐ PA : OMIM #226730), and it is caused by mutations in ITGA 6 , ITGB 4 and PLEC genes. We report the analysis of six patients with EB ‐ PA , including two dizygotic twins. Skin immunofluorescence epitope mapping was performed followed by PCR and direct sequencing of the ITGB 4 gene. Two of the patients presented with non‐lethal EB ‐ PA associated with missense ITGB 4 gene mutations. For the other four, early postnatal demise was associated with complete lack of β 4 integrin due to a variety of ITGB 4 novel mutations (2 large deletions, 1 splice‐site mutation and 3 missense mutations). One of the deletions spanned 278 bp, being one of the largest reported to date for this gene. Remarkably, we also found for the first time a founder effect for one novel mutation in the ITGB 4 gene. We have identified 6 novel mutations in the ITGB 4 gene to be added to the mutation database. Our results reveal genotype–phenotype correlations that contribute to the molecular understanding of this heterogeneous disease, a pivotal issue for prognosis and for the development of novel evidence‐based therapeutic options for EB management.

We report a systematic histopathologic study of papillomas at different times during promotion, correlating the results with those from cytogenetic analysis of the same tumors. Papillomas were induced in SENCAR mice by two- stage carcinogenesis (7,12-dimethylbenz(a)anthracene and phorbol 12-myristate 13-acetate). Individual tumors were ran- domly sampled at different times during promotion, and histopathologic and cytogenetic studies were carried out on every tumor. Early during promotion (10 weeks), most papil- lomas were well-differentiated hyperplastic lesions with mild or no cellular atypia. No tumors showed severe dysplastic changes. By 20 weeks of promotion, a dramatic drop had occurred in the number of lesions with no dysplasia. Most of the tumors presented moderate dysplasia, and some already showed severe dysplastic changes. At later stages (30-40 weeks), most of the papillomas were classified as moderately or severely dysplastic papillomas, and several were considered to be intrapapillomatous carcinomas. This histopathologic eval- uation was supported by nuclear measurements performed on papillomas at different time points. Chromosomal abnormal- ities followed a similar trend. Papillomas seem to start as diploid lesions, but between 10 and 20 weeks of promotion, hyperdiploid cells can be observed in almost every tumor. In some cases the stem line was taken over by aneuploid clones. At 40 weeks of promotion, all papillomas were aneuploid, most of them with hyperdiploid stem lines. A positive correlation was found between the histological and cytogenetic studies, with the most aggressive and atypical tumors being the more aneuploid. These results support the idea that most, if not all, papillomas are truly premalignant lesions in different stages of the poten- tial progression toward malignancy. Chromosomal abnormal- ities might play an important role in the sequence of events leading to malignancy.

This chapter reviews the current use of bioengineered skin with a focus on their clinical and preclinical applications. Skin tissue engineering must take into account the use of three key components: cells, scaffolds, and growth factors. Alternatives of these, depending upon the applicability, are discussed in this chapter. Skin bioengineering is nowadays a reality in clinical practice and different systems of skin equivalents have been successfully used to treat burn patients, chronic ulcers, epidermolysis bullosa patients, and other skin conditions. Bioengineered skin substitutes have been used in a preclinical context, not only to model diseases including genodermatosis, but also to better understand physiological processes. In the near future, the hope of skin tissue engineering is to become a multidisciplinary science where a next generation of biomimic skin substitutes can be developed.

One of the first indications that cancer develops as a multistage process was conceived in the mouse skin carcinogenesis model. The work of Beremblum, Mortram and Boutwell, among others, generated the concept of initiation and promotion (two-stage carcinogenesis) that contributed for many years to a better understanding of cancer development [reviewed by Slaga 1]. This concept is based on studies showing that in mouse skin, it is possible to obtain neoplastic lesions using a subthreshold doses of a mutagenic/carcinogenic agent such as 7,12-dimethylbenz(a)anthracene (DMBA), N-methyl-N-nitrosourea (MNU) or benzo(alpha)pyrene (BP) (initiation stage) followed by a repetitive exposure to a non-carcinogen/non-genotoxic agent such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA), okadaic acid or benzoyl peroxide (promotion stage). Although, this mouse model does not faithfully reproduce the pathology of human skin cancer, it has been very important to the understanding the multistage nature of cancer development and the contribution of both genetic and epigenetic mechanisms in carcinogenesis. It has also been a useful model to test the potential carcinogenicity of different compounds. Until the early 1980s, the initiation was an abstract theoretical concept. The fact that it was caused by mutagens and was irreversible pointed out to a possible mutation or damage at the DNA level, but its true nature remained ignored. However, this changed with the study published by Quintanilla et al. in Nature in 1986 2 (the historical article is freely available in PubMed Central http://www.nature.com/nature/journal/v322/n6074/pdf/322078a0.pdf). These investigators from Allan Balmain laboratory had previously shown that both papillomas and carcinomas had transforming Ha-ras activity 3, 4. Advancing on these results, Quintanilla's et al. 2 showed in a very short but striking paper that carcinogenesis induced by DMBA initiation and TPA promotion was always associated with a specific mutation in the Harvey ras (Ha-ras) gene. Nearly 100% of the tumors produced by these agents presented a mutation in this gene, and even more interestingly, all the mutations occurred in the same position of the gene, a change from CAA to CTA (Gln to Leu) in the 61st codon. However, when another initiation agent N-methyl-N-nitroso-N-nitrosoguanidine (MNNG) was used, none of the tumors presented a detectable mutation in the Ha-ras gene. To rule out a possible effect of the promoter, they used DMBA as the initiation agent followed by Chryserobin, a compound unrelated to TPA, as the tumor promoter. Results with this promoting agent confirmed that tumor initiation with DMBA led to the mutation in the same position of the Ha-ras gene regardless of the promoter agent used. Thus, in that paper, for the first time, the association of DMBA-induced Ha-ras mutation with the initiation event was recognized. Quintanilla and co-workers also presented evidence that amplification of the mutant Ha-ras may occur at later stages of carcinogenesis. In fact, they related the probability of malignant conversion of papillomas to carcinomas with the number of mutated alleles of this gene in the papillomas. In the following years, work from different laboratories confirmed these results and the fact that Ha-ras mutation was actually the initiation event. The direction of the research of the whole field of carcinogenesis changed dramatically with many laboratories focusing in ras-driven molecular alteration in animal models. The genetic revolution had arrived to the field of carcinogenesis. From that point on, experimental cutaneous cancer research moved to the study of genetically modified mouse models targeting specific signal transduction pathways through the use of transgenic technologies 5. In other words, we can say that there is a before and after in the field of mouse skin carcinogenesis as well as in other models of carcinogenesis with Quintanilla's paper. The mouse skin model regained the interest of researchers, and more mechanistic studies were conducted using animal carcinogenesis models. In closing, it is worth mentioning that Dr. Quintanilla, a native Spaniard, went back after a few years in Glasgow to Madrid where he became a successful independent investigator. Furthermore, he turned into a collaborator and more than anything else a good friend of our group. Allan Balmain, the head of the laboratory, moved to the USA to continue a very successful career. This Nature paper, cited 795 times, may not be remembered by the new generations of cancer scientists and is probably seldom referred nowadays. However, there is no doubt in our mind that this paper changed forever a large field of research and made a substantial contribution to understanding the nature of cancer. The manuscript was prepared and written by SGA and CJC, with critical review and comments from FL, MDR and JLJ. The authors have declared no conflicting interests.

Here we describe a spontaneous mutation in the Zdhhc13 (zinc finger, DHHC domain containing 13) gene (also called Hip14l), one of 24 genes encoding palmitoyl acyltransferase (PAT) enzymes in the mouse. This mutation (Zdhhc13luc) was identified as a nonsense base substitution, which results in a premature stop codon that generates a truncated form of the ZDHHC13 protein, representing a potential loss-of-function allele. Homozygous Zdhhc13luc/Zdhhc13luc mice developed generalized hypotrichosis, associated with abnormal hair cycle, epidermal and sebaceous gland hyperplasia, hyperkeratosis, and increased epidermal thickness. Increased keratinocyte proliferation and accelerated transit from basal to more differentiated layers were observed in mutant compared with wild-type (WT) epidermis in untreated skin and after short-term 12-O-tetradecanoyl-phorbol-13-acetate treatment and acute UVB exposure. Interestingly, this epidermal phenotype was associated with constitutive activation of NF-κB (RelA) and increased neutrophil recruitment and elastase activity. Furthermore, tumor multiplicity and malignant progression of papillomas after chemical skin carcinogenesis were significantly higher in mutant mice than WT littermates. To our knowledge, this is the first report of a protective role for PAT in skin carcinogenesis.

Autophagy is a conserved process involved in lysosomal degradation of protein aggregates and damaged organelles. The role of autophagy in cancer is a topic of intense debate, and the underlying mechanism is still not clear. The hypoxia-inducible factor 2α (HIF2α), an oncogenic transcription factor implicated in renal tumorigenesis, is known to be degraded by the ubiquitin–proteasome system (UPS). Here, we report that HIF2α is in part constitutively degraded by autophagy. HIF2α interacts with autophagy–lysosome system components. Inhibition of autophagy increases HIF2α, whereas induction of autophagy decreases HIF2α. The E3 ligase von Hippel-Lindau and autophagy receptor protein p62 are required for autophagic degradation of HIF2α. There is a compensatory interaction between the UPS and autophagy in HIF2α degradation. Autophagy inactivation redirects HIF2α to proteasomal degradation, whereas proteasome inhibition induces autophagy and increases the HIF2α–p62 interaction. Importantly, clear-cell renal cell carcinoma (ccRCC) is frequently associated with monoallelic loss and/or mutation of autophagy-related gene ATG7, and the low expression level of autophagy genes correlates with ccRCC progression. The protein levels of ATG7 and beclin 1 are also reduced in ccRCC tumors. This study indicates that autophagy has an anticancer role in ccRCC tumorigenesis, and suggests that constitutive autophagic degradation of HIF2α is a novel tumor suppression mechanism.

BACKGROUND:Kindler Syndrome (KS) is an autosomal recessive skin disorder characterized by skin blistering, photosensitivity, premature aging, and propensity to skin cancer. In spite of the knowledge underlying cause of this disease involving mutations of FERMT1 (fermitin family member 1), and efforts to characterize genotype-phenotype correlations, the clinical variability of this genodermatosis is still poorly understood. In addition, several pathognomonic features of KS, not related to skin fragility such as aging, inflammation and cancer predisposition have been strongly associated with oxidative stress. Alterations of the cellular redox status have not been previously studied in KS. Here we explored the role of oxidative stress in the pathogenesis of this rare cutaneous disease.METHODS:Patient-derived keratinocytes and their respective controls were cultured and classified according to their different mutations by PCR and western blot, the oxidative stress biomarkers were analyzed by spectrophotometry and qPCR and additionally redox biosensors experiments were also performed. The mitochondrial structure and functionality were analyzed by confocal microscopy and electron microscopy.RESULTS:Patient-derived keratinocytes showed altered levels of several oxidative stress biomarkers including MDA (malondialdehyde), GSSG/GSH ratio (oxidized and reduced glutathione) and GCL (gamma-glutamyl cysteine ligase) subunits. Electron microscopy analysis of both, KS skin biopsies and keratinocytes showed marked morphological mitochondrial abnormalities. Consistently, confocal microscopy studies of mitochondrial fluorescent probes confirmed the mitochondrial derangement. Imbalance of oxidative stress biomarkers together with abnormalities in the mitochondrial network and function are consistent with a pro-oxidant state.CONCLUSIONS:This is the first study to describe mitochondrial dysfunction and oxidative stress involvement in KS.

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human-skin-engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell-based wound therapy consisting of the application of plasma-derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re-epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance.