Fibroblast activation and abnormal extracellular matrix remodelling as common hallmarks in three cancer-prone genodermatoses.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three cancer-prone genodermatoses whose causal genetic mutations cannot fully explain, on their own, the array of associated phenotypic manifestations. Recent evidence highlights the role of the stromal microenvironment in the pathology of these disorders. OBJECTIVES: To investigate, by means of comparative gene expression analysis, the role played by dermal fibroblasts in the pathogenesis of RDEB, KS and XPC. METHODS: We conducted RNA-Seq analysis, which included a thorough examination of the differentially expressed genes, a functional enrichment analysis and a description of affected signalling circuits. Transcriptomic data were validated at the protein level in cell cultures, serum samples and skin biopsies. RESULTS: Interdisease comparisons against control fibroblasts revealed a unifying signature of 186 differentially expressed genes and four signalling pathways in the three genodermatoses. Remarkably, some of the uncovered expression changes suggest a synthetic fibroblast phenotype characterized by the aberrant expression of extracellular matrix (ECM) proteins. Western blot and immunofluorescence in situ analyses validated the RNA-Seq data. In addition, enzyme-linked immunosorbent assay revealed increased circulating levels of periostin in patients with RDEB. CONCLUSIONS: Our results suggest that the different causal genetic defects converge into common changes in gene expression, possibly due to injury-sensitive events. These, in turn, trigger a cascade of reactions involving abnormal ECM deposition and underexpression of antioxidant enzymes. The elucidated expression signature provides new potential biomarkers and common therapeutic targets in RDEB, XPC and KS. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three genodermatoses with high predisposition to cancer development. Although their causal genetic mutations mainly affect epithelia, the dermal microenvironment likely contributes to the physiopathology of these disorders. What does this study add? We disclose a large overlapping transcription profile between XPC, KS and RDEB fibroblasts that points towards an activated phenotype with high matrix-synthetic capacity. This common signature seems to be independent of the primary causal deficiency, but reflects an underlying derangement of the extracellular matrix via transforming growth factor-ß signalling activation and oxidative state imbalance. What is the translational message? This study broadens the current knowledge about the pathology of these diseases and highlights new targets and biomarkers for effective therapeutic intervention. It is suggested that high levels of circulating periostin could represent a potential biomarker in RDEB.

Autophagy mediates HIF2α degradation and suppresses renal tumorigenesis.

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.

Transgenic expression of E2F3a causes DNA damage leading to ATM-dependent apoptosis.

Many early stage human tumors display markers of a DNA-damage response (DDR), including ataxia telangiectasia mutated (ATM) kinase activation. This suggests that DNA damage accumulates during the process of carcinogenesis and that the ATM-dependent response to this damage may function to suppress cancer progression. The E2F3a transcription factor plays an important role in regulating cell proliferation and is amplified in a subset of human cancers. Similar to human premalignant lesions, we find activated ATM and other markers of the DDR in the hyperplastic epidermis of transgenic mice expressing E2F3a through a keratin 5 (K5) promoter. Primary keratinocytes from K5 E2F3a transgenic mice contain increased levels of DNA breaks compared to wild-type cells. E2F3a overexpression also induced DNA damage in primary human fibroblasts that was inhibited by blocking DNA replication. The absence of ATM impaired apoptosis induced by E2F3a and treating K5 E2F3a transgenic mice with caffeine, an inhibitor of ATM and Rad3-related (ATR), promoted skin tumor development. These findings demonstrate that the deregulated expression of E2F3a causes DNA damage under physiological conditions and indicate that the ATM-dependent response to this damage is important for the induction of apoptosis and tumor suppression.

Cyclin D2 and cyclin D3 play opposite roles in mouse skin carcinogenesis.

D-type cyclins are components of the cell-cycle engine that link cell signaling pathways and passage throughout G1 phase. We previously described the effects of overexpression cyclin D1, D2 or D3 in mouse epidermis and tumor development. We now asked whether cyclin D2 and/or cyclin D3 play a relevant role in ras-dependent tumorigenesis. Here, we described the effect of cyclin D3 and cyclin D2 overexpression in mouse skin tumor development. Notably, overexpression of cyclin D3 results in reduced tumor development and malignant progression to squamous cell carcinomas (SCC). Biochemical analysis of keratinocytes shows that overexpression of cyclin D3 results in strong reduction of cyclin D2 and its associated kinase activity. Furthermore, we found that reinstatement of cyclin D2 level in the cyclin D3/cyclin D2 bigenic mice results in a complete reversion of the inhibitory action of cyclin D3. Supporting these results, ablation of cyclin D2 results in reduced tumorigenesis and malignant progression. On the other hand, overexpression of cyclin D2 results in an increased number of papillomas and malignant progression. We conclude that cyclin D3 and cyclin D2 play opposite roles in mouse skin tumor development and that the suppressive activity of cyclin D3 is associated with cyclin D2 downregulation.

A variant of pulmonary alveolar microlithiasis in nackt mice.

Four adult mutant nackt mice, which demonstrate alopecia and CD4+ T-cell deficiency, and two outbred SENCAR mice (sentinels) were presented for routine health surveillance. Lesions were not observed at necropsy. Microscopically, all four nackt mice demonstrated multiple concretions that were 30-100 microm in diameter, irregularly rounded to oval, nonbirefringent, and amphophilic to basophilic. Many of the concretions appeared attached to or within the alveolar walls of all lung lobes. Approximately half of the concretions had irregular fracture lines. All concretions were periodic acid-Schiff positive, and Von Kassa staining revealed diffuse calcification. None of the concretions were associated with inflammatory cell infiltrates, and metaplastic ossification was not evident. A diagnosis of pulmonary alveolar microlithiasis, a rare disease in both humans and animals, was made based on the size and location of the concretions and the lack of an inflammatory response. This is the first report of a laboratory mouse demonstrating pulmonary alveolar microlithiasis.

Regulation of the differentiation-related gene Drg-1 during mouse skin carcinogenesis.

Differentiation-related gene-1 (Drg-1) has been identified as a gene whose expression is increased in several processes related to differentiation, but its function is currently unknown. In this report, we show that Drg-1 was expressed in keratinocytes, this expression being rapidly increased as a result of induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) or the presence of an activating form of Ha-ras. Induction by TPA occurred both in cultured cell lines and primary keratinocytes as well as in mouse skin after a single TPA application. Overexpression of Drg-1 was also observed in TPA-induced hyperplastic skin. In agreement, mouse skin papillomas and carcinomas also overexpressed Drg-1. In addition, Drg-1 was induced when keratinocytes were forced to differentiate by calcium switch or serum starvation. Analysis of the expression of Drg-1 during the keratinocyte cell cycle demonstrated relatively high levels of Drg-1 mRNA in G(0), which increased in early G(1) and decreased afterwards in late G(1)/S. In situ analysis showed an accumulation of Drg-1 in the suprabasal layers of the skin, as well as in the more differentiated areas of mouse skin papillomas. These results suggest that, in addition to being upregulated during keratinocyte differentiation, the Drg-1 gene might have a complex function in skin tumorigenesis.

Myc lacks E2F1's ability to suppress skin carcinogenesis.

Myc and E2F1 can each stimulate proliferation, induce apoptosis, and contribute to oncogenic transformation. However, only E2F1 has been shown to have a tumor suppressive activity under some conditions. To examine the potential of Myc to suppress tumorigenesis under one of the conditions in which E2F1 functions to suppress tumorigenesis, transgenic mice expressing Myc under the control of a keratin 5 (K5) promoter were generated. Like K5 E2F1 transgenic mice, K5 Myc transgenic mice have hyperplastic and hyperproliferative epidermis and develop spontaneous tumors in the skin and oral epithelium. In addition, K5 Myc and K5 E2F1 transgenic mice both display aberrant, p53-dependent apoptosis in the epidermis. It has been demonstrated that deregulated expression of E2F1 in the epidermis of transgenic mice inhibits tumorigenesis in a two-stage skin carcinogenesis assay. In sharp contrast to those results, deregulated expression of Myc in the epidermis of transgenic mice resulted in an enhanced response to two-stage skin carcinogenesis. We conclude that while Myc and E2F1 have similar proliferative, apoptotic and oncogenic properties in mouse epidermis, Myc lacks E2F1's tumor suppressive property. This suggests that E2F1's unique ability to inhibit skin carcinogenesis is not simply a consequence of promoting p53-dependent apoptosis.

Transgenic expression of cyclin-dependent kinase 4 results in epidermal hyperplasia, hypertrophy, and severe dermal fibrosis.

In a previous report we have described the effects of expression of D-type cyclins in epithelial tissues of transgenic mice. To study the involvement of the D-type cyclin partner cyclin-dependent kinase 4 (CDK4) in epithelial growth and differentiation, transgenic mice were generated carrying the CDK4 gene under the control of a keratin 5 promoter. As expected, transgenic mice showed expression of CDK4 in the epidermal basal-cell layer. Epidermal proliferation increased dramatically and basal cell hyperplasia and hypertrophy were observed. The hyperproliferative phenotype of these transgenic mice was independent of D-type cyclin expression because no overexpression of these proteins was detected. CDK4 and CDK2 kinase activities increased in transgenic animals and were associated with elevated binding of p27(Kip1) to CDK4. Expression of CDK4 in the epidermis results in an increased spinous layer compared with normal epidermis, and a mild hyperkeratosis in the cornified layer. In addition to epidermal changes, severe dermal fibrosis was observed and part of the subcutaneous adipose tissue was replaced by connective tissue. Also, abnormal expression of keratin 6 associated with the hyperproliferative phenotype was observed in transgenic epidermis. This model provides in vivo evidence for the role of CDK4 as a mediator of proliferation in epithelial cells independent of D-type cyclin expression.

The CD4 T cell-deficient mouse mutation nackt (nkt) involves a deletion in the cathepsin L (CtsI) gene.

We recently reported a novel autosomal recessive mouse mutation designated nackt (nkt). Homozygous mutant mice have diffuse alopecia and a marked reduction in the proportion of CD4+ T cells in the thymus and peripheral lymphoid tissues. Here we show that the CD4 T-cell deficiency is due to a defect in the thymic microenvironment rather than the hematopoietic compartment. Furthermore, we identified the molecular basis of the mutant phenotype by demonstrating that the nkt mutation represents a 118-bp deletion of the cathepsin L (Ctsl) gene which is required for degradation of the invariant chain, a critical chaperone for major histocompatibility complex class II molecules. This finding explains the similarities in skin and immune defects observed in nkt/nkt and Ctsl -/- mice. The data reported here provide further in vivo evidence that the lysosomal cysteine protease cathepsin L plays a critical role in CD4+ T-cell selection in the thymus.

PCR-based microsatellite analysis for differentiation and genetic monitoring of nine inbred SENCAR mouse strains.

Sixteen DNA microsatellites or simple sequence length polymorphisms (SSLPs), generated by polymerase chain reaction (PCR) were selected for use in the genetic quality control of the nine inbred SENCAR strains currently available. The SENCAR strains constitute a powerful tool for mechanistic studies of multi-stage skin carcinogenesis, as well as for studies to understand the underlying genetic basis of resistance to tumour promotion and progression. SSLP analysis is a fast and economical way for detecting genetic contamination (unexpected outcrosses) among these closely-related albino strains, where standard immunological and biochemical markers have been shown to be insufficient.

Mutational and functional analyses reveal that ST7 is a highly conserved tumor-suppressor gene on human chromosome 7q31.

Loss of heterozygosity (LOH) of markers on human chromosome 7q31 is frequently encountered in a variety of human neoplasias, indicating the presence of a tumor-suppressor gene (TSG). By a combination of microcell-fusion and deletion-mapping studies, we previously established that this TSG resides within a critical region flanked by the genetic markers D7S522 and D7S677. Using a positional cloning strategy and aided by the availability of near-complete sequence of this genomic interval, we have identified a TSG within 7q31, named ST7 (for suppression of tumorigenicity 7; this same gene was recently reported in another context and called RAY1). ST7 is ubiquitously expressed in human tissues. Analysis of a series of cell lines derived from breast tumors and primary colon carcinomas revealed the presence of mutations in ST7. Introduction of the ST7 cDNA into the prostate-cancer-derived cell line PC3 had no effect on the in vitro proliferation of the cells, but abrogated their in vivo tumorigenicity. Our data indicate that ST7 is a TSG within chromosome 7q31 and may have an important role in the development of some types of human cancer.

Cyclin D2 overexpression in transgenic mice induces thymic and epidermal hyperplasia whereas cyclin D3 expression results only in epidermal hyperplasia.

In a previous report, we described the effects of cyclin D1 expression in epithelial tissues of transgenic mice. To study the involvement of D-type cyclins (D1, D2, and D3) in epithelial growth and differentiation and their putative role as oncogenes in skin, transgenic mice were developed which carry cyclin D2 or D3 genes driven by a keratin 5 promoter. As expected, both transgenic lines showed expression of these proteins in most of the squamous tissues analyzed. Epidermal proliferation increased in transgenic animals and basal cell hyperplasia was observed. All of the animals also had a minor thickening of the epidermis. The pattern of expression of keratin 1 and keratin 5 indicated that epidermal differentiation was not affected. Transgenic K5D2 mice developed mild thymic hyperplasia that reversed at 4 months of age. On the other hand, high expression of cyclin D3 in the thymus did not produce hyperplasia. This model provides in vivo evidence of the action of cyclin D2 and cyclin D3 as mediators of proliferation in squamous epithelial cells. A direct comparison among the three D-type cyclin transgenic mice suggests that cyclin D1 and cyclin D2 have similar roles in epithelial thymus cells. However, overexpression of each D-type cyclin produces a distinct phenotype in thymic epithelial cells.

Microsatellite DNA variants between the inbred SENCAR mouse strains.

The two-stage model, initiation with 7,12-dimethylbenz[a]anthracene and promotion with 12-O-tetradecanoylphorbol-13-acetate, of mouse skin carcinogenesis has been the protocol of choice to study the genetic susceptibility to carcinogens, the outbred SENCAR mouse being the most widely used skin tumor-sensitive animal model. Squamous cell carcinomas (SCCs) develop from many of the papillomas, making these mice a useful model for epithelial tumorigenesis and for the progression to malignant tumors. Nine different inbred strains derived from outbred SENCAR mice have been recently reported. Interestingly, these strains display different sensitivities to two-stage carcinogenesis, and, in particular, some of them show a dissociation between susceptibility to papilloma development and the malignant conversion of these into SCC. However, the utility of these SENCAR strains for genetic mapping is limited by the lack of information regarding DNA variant alleles among them. Therefore, we analyzed the nine inbred strains with microsatellite markers distributed along the 20 chromosomes and in this article report the variant alleles found. The information presented is likely to be helpful for linkage analysis and marker-assisted development of congenic strains between SENCAR-derived inbred strains.

Allelotype analysis of chemically induced squamous cell carcinomas in F(1) hybrids of two inbred mouse strains with different susceptibility to tumor progression.

Loss of heterozygosity (LOH) at specific chromosomal loci is generally considered indirect evidence for the presence of putative suppressor genes. Allelotyping of tumors using polymorphic markers distributed throughout the entire genome allows the analysis of specific allelic losses. In the field of chemical carcinogenesis, the outbred SENCAR mouse has been commonly used to analyze the multistage nature of skin tumor development. In the study reported here we generated F(1) hybrids between two inbred strains (SENCARB/Pt and SSIN/Sprd) derived from the SENCAR stock that differ in their susceptibility to tumor progression. We typed 24 7, 12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate-induced squamous cell carcinomas for LOH using 56 microsatellite markers distributed among all autosomal chromosomes. The highest percentage of LOH, 78%, was found on chromosome 7, but there was no preferential loss of one particular allele, indicating that the putative suppressor genes found in this area are not involved in genetic susceptibility. High levels of LOH were also found on chromosomes 16 (39%), 6 (29%), 4 (25%), 9 (25%), 14 (22%), 10 (20%) and 19 (20%), but with no preferential loss of the alleles of one strain. The chromosomal regions with LOH on mouse chromosomes 4, 6, 7, 9, 10, 14, 16 and 19 correspond to regions in the human genome where LOH has been reported and have been suggested to harbor tumor suppressor genes.

Definitive functional evidence for a tumor suppressor gene on human chromosome 7q31.1 neighboring the Fra7G site.

We have previously shown that loss of heterozygosity (LOH) on human chromosome (hchr) 7 at q31.1 is common in a variety of tumors of epithelial origin. Frequent LOH of a specific chromosomal marker is indicative of a closely linked tumor suppressor gene (TSG). However, recent reports have also indicated that such a high frequency of LOH could be due to the presence in this region of the second most common aphidicolin-inducible fragile site in the human genome (Fra7G). To address this controversy, we introduced single copies of hchr7 or hchr12 into a highly aggressive human prostate carcinoma cell line (PC3) by microcell-mediated transfer. The tumorigenicity of six clones of PC3/hchr7 hybrids and three clones of PCRhchr12 hybrids, obtained in four separate fusion experiments, were studied in BALB/c nude mice. All but one of the PC3/hchr7 hybrids increased tumor latency by at least twofold, whereas none of the PC3/hchr12 hybrids delayed tumor onset. No differences in the in vitro growth rate were observed among any of the cell lines assayed (parental and hybrids) suggesting that the observed tumor suppression was due to factors other than cell cycle regulation. Deletion mapping of the PC3/hchr7 tumors obtained after reversion to the malignant phenotype revealed a common region of loss centred around 7q31.1, supporting the TSG hypothesis. The smallest commonly deleted region was approximately 1.5 Mb in size and flanked by the markers D7S486 and D7S655.

Development and initial characterization of several new inbred strains of SENCAR mice for studies of multistage skin carcinogenesis.

The development and initial characterization of five new inbred strains of SENCAR mice are described in this paper. Ten randomly selected pairs of outbred SENCAR mice were mated and offspring from each separately maintained parental line were sib mated at each successive generation to result in inbred strains. Due to poor reproductive performance only five of the original 10 lines were bred to homogeneity. Initial characterization of the five remaining lines (referred to as SL2/sprd, SL5/sprd, SL7/sprd, SL8/sprd and SLl0/sprd) at F12 for their responsiveness to a two-stage carcinogenesis protocol (10 nmol 7,12-dimethylbenz[a]anthracene and 0.25 microg 12-O-tetradecanoylphorbol-13 acetate) revealed three groups of responders in terms of the number of papillomas per mouse: SL2/sprd and SL8/sprd > SL7/sprd and SL10/sprd >> SL5/sprd. The papilloma responses in SL2/sprd and SL8/sprd were very similar to SENCAR B/Pt compared at the same doses. Papillomas induced on SL2/sprd had the highest propensity to progress to squamous cell carcinomas, similar to that observed in outbred SENCAR and SENCAR B/Pt mice. More detailed comparison of the responsiveness of SL2/sprd and SL5/sprd at Fl5 showed that these two inbred strains differed in their sensitivity to TPA-induced epidermal hyperplasia and that the dose of TPA required to produce a tumor response in SL5/sprd in comparison with that in SL2/sprd was 4-20 times higher. Overall, the availability of the different inbred SENCAR strains will greatly aid mechanistic studies of multistage skin carcinogenesis as well as studies to understand the underlying genetic basis of resistance to tumor promotion and progression in this model system.

Transgenic expression of cyclin D1 in thymic epithelial precursors promotes epithelial and T cell development.

We previously reported that precursors within the keratin (K) 8+5+ thymic epithelial cell (TEC) subset generate the major cortical K8+5- TEC population in a process dependent on T lineage commitment. This report demonstrates that expression of a cyclin D1 transgene in K8+5+ TECs expands this subset and promotes TEC and thymocyte development. Cyclin D1 transgene expression is not sufficient to induce TEC differentiation in the absence of T lineage-committed thymocytes because TECs from both hCD3epsilon transgenic and hCD3epsilon/cyclin D1 double transgenic mice remain blocked at the K8+5+ maturation stage. However, enforced cyclin D1 expression does expand the developmental window during which K8+5+ cells can differentiate in response to normal hemopoietic precursors. Thus, enhancement of thymic function may be achieved by manipulating the growth and/or survival of TEC precursors within the K8+5+ subset.

Deregulated expression of cyclin D1 overrides antimitogenic signals.

Several types of epithelial neoplasms exhibit high expression of transforming growth factor beta1 (TGFbeta-1), indicating that they have acquired tolerance to this normally growth inhibitory cytokine. Since cyclin D1 is expressed at high levels in murine skin tumors coincident with high levels of TGFbeta-1 expression, we hypothesized that cyclin D1 may override TGFbeta-1 induced growth arrest. We observed that in primary murine keratinocytes treated with TGFbeta-1, cyclin D1 is quickly suppressed at both the mRNA and protein level. Since changes in other cell cycle proteins occur at a later time during TGFbeta-1 treatment, the early suppression of cyclin D1 suggests that this gene is a critical target for TGFbeta-1 growth suppression. Using primary keratinocytes from transgenic mice that overexpress cyclin D1 (K5-D1 mice), we observed partial resistance to TGFbeta-1 growth inhibition. This resistance involves changes in the cyclin/cdk/inhibitor complexes rather than differences in expression of the TGFbeta receptors or signaling. Comparison of cdk associated kinase activity between wild-type and K5-D1 cells shows differential regulation. We conclude that deregulated cyclin D1 and subsequent alterations in cell cycle machinery provides keratinocytes the ability to at least partially override growth inhibitory signals.

Persistence of p53 mutations and resistance of keratinocytes to apoptosis are associated with the increased susceptibility of mice lacking the XPC gene to UV carcinogenesis.

Like xeroderma pigmentosum (XP) patients, transgenic mice lacking nucleotide excision repair (NER) genes such as XPA and XPC are extremely susceptible to ultraviolet (UV)-induced skin cancer. Because the p53 gene is an important target for UV carcinogenesis and because the p53 protein modulates NER, we investigated the consequences of NER deficiency on UV-induced p53 mutations in XPC-/- mouse skin tumors. Thirty-eight (76%) of 50 UV-induced XPC-/- skin tumor analysed displayed C-->T or CC-->TT transitions at dipyrimidine sites on the untranscribed strand of the p53 gene. A major hot spot for p53 mutation occurred at codon 270, which is also a hot spot in UV-induced skin tumors from NER-proficient C3H and SKH-hr 1 mice. Interestingly, codon 270 mutations were induced in both XPC-/- and +/+ mouse skin after 1 week of UV irradiation, but the mutations persisted only in XPC-/- mouse skin after 3 - 4 weeks of chronic UV. The persistence of UV-induced p53 mutations in XPC-/- mouse skin was associated with decreased apoptosis and increased proliferation of keratinocytes, suggesting that these events may contribute to the accelerated development of UV-induced skin tumors in XPC-/- mice.