PML at Mitochondria-Associated Membranes Is Critical for the Repression of Autophagy and Cancer Development.

The precise molecular mechanisms that coordinate apoptosis and autophagy in cancer remain to be determined. Here, we provide evidence that the tumor suppressor promyelocytic leukemia protein (PML) controls autophagosome formation at mitochondria-associated membranes (MAMs) and, thus, autophagy induction. Our in vitro and in vivo results demonstrate how PML functions as a repressor of autophagy. PML loss promotes tumor development, providing a growth advantage to tumor cells that use autophagy as a cell survival strategy during stress conditions. These findings demonstrate that autophagy inhibition could be paired with a chemotherapeutic agent to develop anticancer strategies for tumors that present PML downregulation.

Hypermethylation-Induced Inactivation of the IRF6 Gene as a Possible Early Event in Progression of Vulvar Squamous Cell Carcinoma Associated With Lichen Sclerosus.

IMPORTANCE: The molecular mechanism leading to the development of vulvar squamous cell carcinoma (VSCC) from vulvar lichen sclerosus (VLS) is unknown. OBJECTIVE: To assess the possible involvement of the IRF6 tumor-suppressor gene in the development of VSCC from VLS. DESIGN: In laboratories at the University of Ferrara, Ferrara, Italy, IRF6 gene expression and promoter methylation were investigated in paraffin-embedded VSCC and adjacent vulvar intraepithelial neoplasia (VIN) and VLS specimens, in cancer-free VLS (cfVLS) specimens, and in healthy skin specimens by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis and by sequencing of PCR-amplified bisulfite-treated DNA. Methylation-induced dysregulation was assessed by expression of p63, the IRF6-transactivator gene. MAIN OUTCOMES AND MEASURES: IRF6 expression, correlation with promoter methylation and p63 expression, and association with development of VSCC from VLS. RESULTS: Specimens from 60 participating women (1 specimen from each) were analyzed for the study (mean [SD] age, 66.3 [12.1] years): 20 paraffin-embedded specimens of VSCC (patient age, 75.3 [8.3] years) with adjacent VLS lesions, in 5 of which VIN preneoplastic tissue was also present (patient age, 64.3 [15.3] years); 20 cfVLS specimens (patient age, 62.1 [11.4] years) obtained from diagnostic biopsies; and 20 normal skin specimens from noncancer surgical patients (patient age, 61.4 [9.1] years). IRF6 messenger RNA was found to be reduced 4.5-, 2.9-, 6.6-, and 2.2-fold in VLS, VIN, VSCC, and cfVLS specimens, respectively, compared with controls, although p63 was still expressed in all specimens. IRF6 promoter was hypermethylated in 9 (45%) of 20 VLS specimens, 1 (20%) of 5 VIN specimens, 16 (80%) of 20 VSCC specimens, 2 (10%) of 20 cfVLS specimens, and 0 normal skin specimens. CONCLUSIONS AND RELEVANCE: IRF6 dysregulation may be involved in the development of VSCC from VLS. Methylation of the IRF6 promoter may be a marker of cancer risk in patients with VLS.

Serologic investigation of undifferentiated nasopharyngeal carcinoma and simian virus 40 infection.

BACKGROUND: The association between undifferentiated nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV) is well established. Nevertheless, available evidence suggests that other cofactors are required for the development of undifferentiated NPC. Several investigations reported simian virus 40 (SV40) footprints in human tumors of different histotypes. METHODS: Serum samples from patients with undifferentiated NPC (n = 64) and healthy subjects (n = 130) were analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) with SV40 synthetic peptides to detect antibodies against viral peptide (VP) capsid proteins VP1, 2, and 3. RESULTS: Immunologic data indicate that in sera from patients with undifferentiated NPC, the prevalence of SV40 antibodies was 25%, whereas in controls it was 16%. This difference is not statistically significant (p > .05). CONCLUSION: A similar prevalence of SV40 antibodies was detected in undifferentiated NPC and healthy subjects. Our serologic data suggest no association between undifferentiated NPC and SV40 infection. This investigation may stimulate further studies aimed at determining the possible contribution of other risk factors in the pathogenesis of undifferentiated NPC.

Gene expression changes in progression of cervical neoplasia revealed by microarray analysis of cervical neoplastic keratinocytes.

To evaluate the gene expression changes involved in neoplastic progression of cervical intraepithelial neoplasia. Using microarray analysis, large-scale gene expression profile was carried out on HPV16-CIN2, HPV16-CIN3, and normal cervical keratinocytes derived from two HPV16-CIN2, two HPV-CIN3 lesions, and two corresponding normal cervical tissues, respectively. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16-CIN2 keratinocytes and in HPV16-CIN2 keratinocytes compared with HPV16-CIN3 keratinocytes; 37 candidate genes with continuously increasing or decreasing expression during CIN progression were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of CIN toward cancer. Gene expression changes occurring during CIN progression were investigated using microarray analysis, for the first time, in CIN2 and CIN3 keratinocytes naturally infected with HPV16. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression.

Impairment of GABA release in the hippocampus at the time of the first spontaneous seizure in the pilocarpine model of temporal lobe epilepsy.

The alterations in GABA release have not yet been systematically measured along the natural course of temporal lobe epilepsy. In this work, we analyzed GABA extracellular concentrations (using in vivo microdialysis under basal and high K(+)-evoked conditions) and loss of two GABA interneuron populations (parvalbumin and somatostatin neurons) in the ventral hippocampus at different time-points after pilocarpine-induced status epilepticus in the rat, i.e. during development and progression of epilepsy. We found that (i) during the latent period between the epileptogenic insult, status epilepticus, and the first spontaneous seizure, basal GABA outflow was reduced to about one third of control values while the number of parvalbumin-positive cells was reduced by about 50% and that of somatostatin-positive cells by about 25%; nonetheless, high K(+) stimulation increased extracellular GABA in a proportionally greater manner during latency than under control conditions; (ii) at the time of the first spontaneous seizure (i.e., when the diagnosis of epilepsy is made in humans) this increased responsiveness to stimulation disappeared, i.e. there was no longer any compensation for GABA cell loss; (iii) thereafter, this dysfunction remained constant until a late phase of the disease. These data suggest that a GABAergic hyper-responsiveness can compensate for GABA cell loss and protect from occurrence of seizures during latency, whereas impaired extracellular GABA levels can favor the occurrence of spontaneous recurrent seizures and the maintenance of an epileptic state.

Polycystin-1 regulates amphiregulin expression through CREB and AP1 signalling: implications in ADPKD cell proliferation.

In autosomal dominant polycystic kidney disease (ADPKD), renal cyst development and enlargement, as well as cell growth, are associated with alterations in several pathways, including cAMP and activator protein 1 (AP1) signalling. However, the precise mechanism by which these molecules stimulate cell proliferation is not yet fully understood. We now show by microarray analysis, luciferase assay, mutagenesis, and chromatin immunoprecipitation that CREB and AP1 contribute to increased expression of the amphiregulin gene, which codifies for an epidermal growth factor-like peptide, in ADPKD cystic cells, thereby promoting their cell growth. Increased amphiregulin (AR) expression was associated with abnormal cell proliferation in both PKD1-depleted and -mutated epithelial cells, as well as primary cystic cell lines isolated from ADPKD kidney tissues. Consistently, normal AR expression and proliferation were re-established in cystic cells by the expression of a mouse full-length PC1. Finally, we show that anti-AR antibodies and inhibitors of AP1 are able to reduce cell proliferation in cystic cells by reducing AR expression and EGFR activity. AR can therefore be considered as one of the key activators of the growth of human ADPKD cystic cells and thus a new potential therapeutic target.

MicroRNA profiling for the identification of cancers with unknown primary tissue-of-origin.

Cancer of unknown primary (CUP) represents a common and important clinical problem. There is evidence that most CUPs are metastases of carcinomas whose primary site cannot be recognized. Driven by the hypothesis that the knowledge of primary cancer could improve patient's prognosis, we investigated microRNA expression profiling as a tool for identifying the tissue of origin of metastases. We assessed microRNA expression from 101 formalin-fixed, paraffin-embedded (FFPE) samples from primary cancers and metastasis samples by using a microarray platform. Forty samples representing ten different cancer types were used for defining a cancer-type-specific microRNA signature, which was used for predicting primary sites of metastatic cancers. A 47-miRNA signature was identified and used to estimate tissue-of-origin probabilities for each sample. Overall, accuracy reached 100% for primary cancers and 78% for metastases in our cohort of samples. When the signature was applied to an independent published dataset of 170 samples, accuracy remained high: correct prediction was found within the first two options in 86% of the metastasis cases (first prediction was correct in 68% of cases). This signature was also applied to predict 16 CUPs. In this group, first predictions exhibited probabilities higher than 90% in most of the cases. These results establish that FFPE samples can be used to reveal the tissue of origin of metastatic cancers by using microRNA expression profiling and suggest that the approach, if applied, could provide strong indications for CUPs, whose correct diagnosis is presently undefined.

Localized delivery of fibroblast growth factor-2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model.

A loss of neurons is observed in the hippocampus of many patients with epilepsies of temporal lobe origin. It has been hypothesized that damage limitation or repair, for example using neurotrophic factors (NTFs), may prevent the transformation of a normal tissue into epileptic (epileptogenesis). Here, we used viral vectors to locally supplement two NTFs, fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when epileptogenic damage was already in place. These vectors were first characterized in vitro, where they increased proliferation of neural progenitors and favored their differentiation into neurons, and they were then tested in a model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in a lesioned hippocampus, FGF-2/BDNF expressing vectors increased neuronogenesis, embanked neuronal damage, and reduced epileptogenesis. It is concluded that reduction of damage reduces epileptogenesis and that supplementing specific NTFs in lesion areas represents a new approach to the therapy of neuronal damage and of its consequences.

Sex hormone receptor levels in laryngeal carcinoma: a comparison between protein and RNA evaluations.

The larynx is a secondary sex organ, and the hormone dependence of laryngeal carcinomas is considered an interesting matter of speculation. However, while tumors of other secondary sex organs, including the prostate, breast, and endometrium, have been recognized as hormone-dependent cancers, the laryngeal carcinomas are still subject to controversy. In this study, samples from 15 laryngeal carcinomas obtained at the time of surgery were assayed for specific estrogen alpha, progesterone, and androgen receptor expression, both at mRNA and protein levels. Detectable levels of specific estrogen and progesterone receptors, 53.3 and 73.3%, respectively, were found in the tumors. This positive detection by immunohistochemical analysis was higher in tumors than in normal mucosa adjacent to the tumor areas and was correlated with the absence of metastatic lymph nodes. No androgen receptor protein was detected in any sample analyzed, even if quantitative RT-PCR revealed high mRNA levels specific for this receptor. A strict correspondence between protein and mRNA hormone receptor levels was not found. This is in agreement with the transcriptional and protein synthesis mechanisms, and it is also compatible with the complex larynx tumorigenesis.

Local in vivo administration of a decoy oligonucleotide targeting NF-kappaB induces apoptosis of osteoclasts after application of orthodontic forces to rat teeth.

In this study, we report the in vivo effects of a decoy oligonucleotide targeting the nuclear factor kappaB (NF-kappaB) on osteoclasts during forced orthodontic tooth movement in rats. Wistar rats were subjected to orthodontic forces, in the absence or presence of treatment with a decoy molecule mimicking a nonsymmetric NF-kappaB binding site (5'-CGC TGG GGA CTT TCC ACG G-3'). TUNEL staining of fragmented DNA revealed that treatment with NF-kappaB decoy but not with scramble double-stranded oligodeoxynucleotides (ODN) induced a high level of osteoclast apoptosis in vivo. Immunohystochemical analysis for death receptor Fas revealed strong positivity only in samples treated with NF-kappaB decoys, demonstrating that osteoclasts are sensitive to death induction via Fas signaling. Induction of apoptosis in osteoclasts could be a strategy for treatment of excessive osteoclast activity in pathologic conditions such as osteoporosis, peri-articular osteolysis, inflammatory arthritis, Paget's syndrome and tumour-associated osteolytic metastases.

PLC-beta2 is highly expressed in breast cancer and is associated with a poor outcome: a study on tissue microarrays.

Despite the identification of many putative biomarkers in breast cancer, a specific pattern of proteins to be used as a prognosticator is not well defined. A growing body of evidence supports the role of phospholipase C (PLC) in the invasion and metastasis of different tumors, including breast cancer. To assess whether the expression of specific PLC isoforms correlates with malignancy-related features of human breast tumors and, hence, could have prognostic significance, an immunohistochemical analysis of PLC-beta2 was performed on tissue microarrays and the relationship between PLC-beta2 expression and biological and clinico-pathological factors was assessed. The analysis of 77 samples of breast tumors with different histotypes revealed that PLC-beta2 is highly expressed in a large majority of the analyzed cancer tissue, particularly ductal and lobular carcinomas, in comparison with normal breast. The expression of PLC-beta2 in primary tumors correlated with size, proliferation index and final grade, while no significant relationship was observed with nodal status or estrogen receptor levels, or with the expression of tumor suppressor p53. Remarkably, high PLC-beta2 levels in primary tumors predict an unfavourable prognosis, suggesting the contribution of this protein to the progression of human mammary carcinomas. Our data indicate that PLC-beta2 expression correlates highly with breast cancer malignancy and suggest that it can be included, as an independent marker, among the prognostic indicators in current use.

Induction of apoptosis of osteoclasts by targeting transcription factors with decoy molecules.

We review the effects of two transcription factor decoy oligonucleotides on apoptosis of human osteoclasts (OCs). The first decoy molecule was designed to inhibit nuclear factor kappa-B (NF-kappaB) binding to target sequence, the second to increase estrogen receptor (ER) alpha expression. We found that both decoy molecules are potent inducers of apoptosis of human OCs, associated with increase of caspase 3 activity and decrease of interleukin 6 expression. In addition, we provide evidence indicating that these oligonucleotides are active in vivo in inducing OCs apoptosis. Because OCs are essential for skeletal development and remodeling throughout the life of animal and man, the approach described is of potential clinical importance.

MicroRNA gene expression deregulation in human breast cancer.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.

Simian virus 40 sequences and expression of the viral large T antigen oncoprotein in human pleomorphic adenomas of parotid glands.

Simian virus 40 (SV40) sequences of the early region coding for the large T antigen (Tag) oncoprotein were investigated in DNA samples from human pleomorphic adenoma (PA) of parotid glands. Specific SV40 sequences were detected, by PCR and filter hybridization with an internal oligoprobe, in 28 of 45 (62%) human PA specimens. None of the DNA samples from 11 normal salivary gland tissues was SV40-positive. DNA sequence analysis, carried out in all PCR amplified products from SV40-positive PA specimens, confirmed the SV40 specificity and indicated that PCR products had a sequence not distinguishable from SV40 DNA wild-type strain 776. SV40 Tag expression was revealed by immunohistochemistry with the specific monoclonal antibody Pab 101 in PA thin sections with a highly sensitive technical approach which retrieved the nuclear viral oncoprotein in 26 out of 28 (93%) samples previously found SV40-positive by PCR. Detection of SV40 sequences and Tag expression in human PA suggests that this oncogenic virus may play a role as a cofactor in the onset and/or progression of this benign neoplasm, or that SV40 DNA could replicate and express the Tag in PA cells.