Losses of Both Products of the Cdkn2a/Arf Locus Contribute to Asbestos-Induced Mesothelioma Development and Cooperate to Accelerate Tumorigenesis

The CDKN2A/ARF locus encompasses overlapping tumor suppressor genes p16(INK4A) and p14(ARF), which are frequently co-deleted in human malignant mesothelioma (MM). The importance of p16(INK4A) loss in human cancer is well established, but the relative significance of p14(ARF) loss has been debated. The tumor predisposition of mice singly deficient for either Ink4a or Arf, due to targeting of exons 1 alpha or 1 beta, respectively, supports the idea that both play significant and nonredundant roles in suppressing spontaneous tumors. To further test this notion, we exposed Ink4a(+/-) and Arf(+/-) mice to asbestos, the major cause of MM. Asbestos-treated Ink4a(+/-) and Arf(+/-) mice showed increased incidence and shorter latency of MM relative to wild-type littermates. MMs from Ink4a(+/-) mice exhibited biallelic inactivation of Ink4a, loss of Arf or p53 expression and frequent loss of p15(Ink4b). In contrast, MMs from Arf(+/-) mice exhibited loss of Arf expression, but did not require loss of Ink4a or Ink4b. Mice doubly deficient for Ink4a and Arf, due to deletion of Cdkn2a/Arf exon 2, showed accelerated asbestos-induced MM formation relative to mice deficient for Ink4a or Arf alone, and MMs exhibited biallelic loss of both tumor suppressor genes. The tumor suppressor function of Arf in MM was p53-independent, since MMs with loss of Arf retained functional p53. Collectively, these in vivo data indicate that both CDKN2A/ARF gene products suppress asbestos carcinogenicity. Furthermore, while inactivation of Arf appears to be crucial for MM pathogenesis, the inactivation of both p16(Ink4a) and p19(Arf) cooperate to accelerate asbestos-induced tumorigenesis

VILIP-1 Expression In Vivo Results in Decreased Mouse Skin Keratinocyte Proliferation and Tumor Development

VILIP-1, a member of the neuronal Ca2+ sensor protein family, is able to act as a tumor suppressor in carcinoma cells by inhibiting cell proliferation and migration. In order to study the role of VILIP-1 in skin carcinogenesis we generated transgenic mice overexpressing VILIP-1 in epidermis under the control of the bovine keratin K5 promoter (K5-VILIP-1). We studied the susceptibility of FVB wild type and VILIP-1 transgenic mice to chemically mediated carcinogenesis. After 30 weeks of treatment with a two-stage carcinogenesis protocol, all animals showed numerous skin tumors. Nevertheless, K5-VILIP-1 mice showed decreased squamous cell carcinoma (SCC) multiplicity of ∼49% (p<0.02) with respect to the corresponding SCC multiplicity observed in wild type (WT) mice. In addition, the relative percentage of low-grade cutaneous SCCs grade I (defined by the differentiation pattern according to the Broders grading scale) increased approximately 50% in the K5-VILIP1 mice when compared with SCCs in WT mice. Similar tendency was observed using a complete carcinogenesis protocol for skin carcinogenesis using benzo(a)pyrene (B(a)P). Further studies of tumors and primary epidermal keratinocyte cultures showed that matrix metalloproteinase 9 (MMP-9) levels and cell proliferation decreased in K5-VILIP-1 mice when compared with their wild counterparts. In addition tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was higher in K5-VILIP-1 keratinocytes. These results show that VILIP-1 overexpression decreases the susceptibility to skin carcinogenesis in experimental mouse cancer models, thus supporting its role as a tumor suppressor gene

HnRNPA2 is a Novel Histone Acetyltransferase That Mediates Mitochondrial Stress-Induced Nuclear Gene Expression

Reduced mitochondrial DNA copy number, mitochondrial DNA mutations or disruption of electron transfer chain complexes induce mitochondria-to-nucleus retrograde signaling, which induces global change in nuclear gene expression ultimately contributing to various human pathologies including cancer. Recent studies suggest that these mitochondrial changes cause transcriptional reprogramming of nuclear genes although the mechanism of this cross talk remains unclear. Here, we provide evidence that mitochondria-to-nucleus retrograde signaling regulates chromatin acetylation and alters nuclear gene expression through the heterogeneous ribonucleoprotein A2 (hnRNAP2). These processes are reversed when mitochondrial DNA content is restored to near normal cell levels. We show that the mitochondrial stress-induced transcription coactivator hnRNAP2 acetylates Lys 8 of H4 through an intrinsic histone lysine acetyltransferase (KAT) activity with Arg 48 and Arg 50 of hnRNAP2 being essential for acetyl-CoA binding and acetyltransferase activity. H4K8 acetylation at the mitochondrial stress-responsive promoters by hnRNAP2 is essential for transcriptional activation. We found that the previously described mitochondria-to-nucleus retrograde signaling-mediated transformation of C2C12 cells caused an increased expression of genes involved in various oncogenic processes, which is retarded in hnRNAP2 silenced or hnRNAP2 KAT mutant cells. Taken together, these data show that altered gene expression by mitochondria-to-nucleus retrograde signaling involves a novel hnRNAP2-dependent epigenetic mechanism that may have a role in cancer and other pathologies

Eosinophilic esophagitis-associated epithelial remodeling may limit esophageal carcinogenesis

IntroductionUnder homeostatic conditions, esophageal epithelium displays a proliferation/differentiation gradient that is generated as proliferative basal cells give rise to suprabasal cells then terminally differentiated superficial cells. This proliferation/differentiation gradient is often perturbed in esophageal pathologies. Basal cell hyperplasia may occur in patients with gastroesophageal reflux disease (GERD), a condition in which acid from the stomach enters the esophagus, or eosinophilic esophagitis (EoE), an emerging form of food allergy. While GERD is a primary risk factor for esophageal cancer, epidemiological data suggests that EoE patients do not develop esophageal cancer.MethodsIn order to investigate the impact of EoE and esophageal cancer specifically on the cellular landscape of esophageal epithelium, we perform single cell RNA-sequencing in murine models of EoE and esophageal cancer, specifically esophageal squamous cell carcinoma (ESCC). We further evaluate modules of co-expressed genes in EoE- and ESCC-enriched epithelial cell clusters. Finally, we pair EoE and ESCC murine models to examine the functional relationship between these pathologies.ResultsIn mice with either EoE or ESCC, we find expansion of cell populations as compared to normal esophageal epithelium. In mice with EoE, we detect distinct expansion of 4 suprabasal populations coupled with depletion of 2 basal populations. By contrast, mice with ESCC display unique expansion of 2 basal populations and 1 suprabasal population, as well as depletion of 2 suprabasal populations. Senescence, glucocorticoid receptor signaling, and granulocyte-macrophage colony-stimulating factor pathways are associated with EoE-enriched clusters while pathways associated with cell proliferation and metabolism are identified in ESCC-enriched clusters. Finally, our in vivo data demonstrate that exposure to EoE inflammation limits tumor burden of esophageal carcinogenesis.DiscussionOur findings provide the first functional investigation of the relationship between EoE and esophageal cancer and suggest that esophageal epithelial remodeling events occurring in response to EoE inflammation may limit esophageal carcinogenesis. This investigation may have future implications for leveraging allergic inflammation-associated alterations in epithelial biology to prevent and/or treat esophageal cancer

VILIP-1 Downregulation in Non-Small Cell Lung Carcinomas: Mechanisms and Prediction of Survival

VILIP-1, a member of the neuronal Ca++ sensor protein family, acts as a tumor suppressor gene in an experimental animal model by inhibiting cell proliferation, adhesion and invasiveness of squamous cell carcinoma cells. Western Blot analysis of human tumor cells showed that VILIP-1 expression was undetectable in several types of human tumor cells, including 11 out of 12 non-small cell lung carcinoma (NSCLC) cell lines. The down-regulation of VILIP-1 was due to loss of VILIP-1 mRNA transcripts. Rearrangements, large gene deletions or mutations were not found. Hypermethylation of the VILIP-1 promoter played an important role in gene silencing. In most VILIP-1-silent cells the VILIP-1 promoter was methylated. In vitro methylation of the VILIP-1 promoter reduced its activity in a promoter-reporter assay. Transcriptional activity of endogenous VILIP-1 promoter was recovered by treatment with 5′-aza-2′-deoxycytidine (5′-Aza-dC). Trichostatin A (TSA), a histone deacetylase inhibitor, potently induced VILIP-1 expression, indicating that histone deacetylation is an additional mechanism of VILIP-1 silencing. TSA increased histone H3 and H4 acetylation in the region of the VILIP-1 promoter. Furthermore, statistical analysis of expression and promoter methylation (n = 150 primary NSCLC samples) showed a significant relationship between promoter methylation and protein expression downregulation as well as between survival and decreased or absent VILIP-1 expression in lung cancer tissues (p<0.0001). VILIP-1 expression is silenced by promoter hypermethylation and histone deacetylation in aggressive NSCLC cell lines and primary tumors and its clinical evaluation could have a role as a predictor of short-term survival in lung cancer patients

The putative Tumor Suppressor VILIP-1 Counteracts Epidermal Growth Factor-Induced Epidermal-Mesenchymal Transition in Squamous Carcinoma Cells

Epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1-positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 2′,3′-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner

WWOX protein expression varies among ovarian carcinoma histotypes and correlates with less favorable outcome

BACKGROUND: The putative tumor suppressor WWOX gene spans the common chromosomal fragile site 16D (FRA16D) at chromosome area 16q23.3-24.1. This region is a frequent target for loss of heterozygosity and chromosomal rearrangement in ovarian, breast, hepatocellular, prostate carcinomas and other neoplasias. The goal of these studies was to evaluate WWOX protein expression levels in ovarian carcinomas to determine if they correlated with clinico-pathological parameters, thus providing additional support for WWOX functioning as a tumor suppressor. METHODS: We performed WWOX protein expression analyses by means of immunobloting and immunohistochemistry on normal ovaries and specific human ovarian carcinoma Tissue Microarrays (n = 444). Univariate analysis of clinical-pathological parameters based on WWOX staining was determined by χ(2 )test with Yates' correction. The basic significance level was fixed at p < 0.05. RESULTS: Immunoblotting analysis from normal ovarian samples demonstrated consistently strong WWOX expression while 37% ovarian carcinomas showed reduced or undetectable WWOX protein expression levels. The immunohistochemistry of normal human ovarian tissue sections confirmed strong WWOX expression in ovarian surface epithelial cells and in epithelial inclusion cysts within the cortex. Out of 444 ovarian carcinoma samples analyzed 30% of tumors showed lack of or barely detectable WWOX expression. The remaining ovarian carcinomas (70%) stained moderately to strongly positive for this protein. The two histotypes showing significant loss of WWOX expression were of the Mucinous (70%) and Clear Cell (42%) types. Reduced WWOX expression demonstrated a significant association with clinical Stage IV (FIGO) (p = 0.007), negative Progesterone Receptor (PR) status (p = 0.008) and shorter overall survival (p = 0.03). CONCLUSION: These data indicate that WWOX protein expression is highly variable among ovarian carcinoma histotypes. It was also observed that subsets of ovarian tumors demonstrated loss of WWOX expression and is potentially associated with patient outcome

Study of FoxA Pioneer Factor at Silent Genes Reveals Rfx-Repressed Enhancer at Cdx2 and a Potential Indicator of Esophageal Adenocarcinoma Development

Understanding how silent genes can be competent for activation provides insight into development as well as cellular reprogramming and pathogenesis. We performed genomic location analysis of the pioneer transcription factor FoxA in the adult mouse liver and found that about one-third of the FoxA bound sites are near silent genes, including genes without detectable RNA polymerase II. Virtually all of the FoxA-bound silent sites are within conserved sequences, suggesting possible function. Such sites are enriched in motifs for transcriptional repressors, including for Rfx1 and type II nuclear hormone receptors. We found one such target site at a cryptic “shadow” enhancer 7 kilobases (kb) downstream of the Cdx2 gene, where Rfx1 restricts transcriptional activation by FoxA. The Cdx2 shadow enhancer exhibits a subset of regulatory properties of the upstream Cdx2 promoter region. While Cdx2 is ectopically induced in the early metaplastic condition of Barrett's esophagus, its expression is not necessarily present in progressive Barrett's with dysplasia or adenocarcinoma. By contrast, we find that Rfx1 expression in the esophageal epithelium becomes gradually extinguished during progression to cancer, i.e, expression of Rfx1 decreased markedly in dysplasia and adenocarcinoma. We propose that this decreased expression of Rfx1 could be an indicator of progression from Barrett's esophagus to adenocarcinoma and that similar analyses of other transcription factors bound to silent genes can reveal unanticipated regulatory insights into oncogenic progression and cellular reprogramming