Marzia Toscano

Beclin 1 and LC3 autophagic gene expression in cutaneous melanocytic lesions.

Beclin 1 and LC3 autophagic genes are altered in several human cancer types. This study was designed to assess the expression of Beclin 1 and LC3 in cutaneous melanocytic lesions, in which they have not yet been investigated. In melanoma, we correlated their expression with conventional histopathologic prognostic factors. In 149 lesions, including benign nevi, dysplastic nevi, radial growth phase melanomas, vertical growth phase melanomas, and melanoma metastases, proteins were evaluated by immunohistochemistry, and, in representative cases of benign nevi, vertical growth phase melanomas and melanoma metastases were evaluated by Western blotting. In most lesions, messenger RNA level was also assessed by real-time reverse transcriptase polymerase chain reaction. Both genes were expressed in all the investigated conditions. Beclin 1 cytoplasmic protein and messenger RNA, as well as LC3 messenger RNA, significantly decreased with tumor progression (P < .05). The percentage of cases with high cytoplasmic expression of beclin 1 from 100% in benign nevi declined to 86.4% in dysplastic nevi, 54.5% in radial growth phase melanomas, 54.3% in vertical growth phase melanomas, and 26.7% in melanoma metastases. The lowest expression of LC3 II protein was observed in melanoma metastases (53.3% of cases) (P < .05); LC3 II protein overexpression was, however, found in several nonbenign lesions, with the highest percentage (45.5%) in radial growth phase melanomas. LC3 II protein expression was inversely correlated to thickness, ulceration, and mitotic rate. In a multivariate analysis, messenger RNAs for both genes discriminated between nonmalignant (benign and dysplastic nevi) and malignant (radial, vertical growth phase melanomas, and melanoma metastases) lesions. Our results, therefore, indicate that beclin 1 and LC3 II autophagic gene expression is altered also in melanocytic neoplasms.

Cigarette smoke affects keratinocytes SRB1 expression and localization via H2O2 production and HNE protein adducts formation.

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H2O2) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H2O2, induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake.

Different involvement of autophagy in human malignant glioma cell lines undergoing irradiation and temozolomide combined treatments.

Glioblastoma (GB) has a poor prognosis, despite current multimodality treatment. Beside surgical resection, adjuvant ionizing radiation (IR) combined with Temozolomide (TMZ) drug administration is the standard therapy for GB. This currently combined radio‐chemotherapy treatment resulted in glial tumor cell death induction, whose main molecular death pathways are still not completely deciphered. In this study, the autophagy process was investigated, and in vitro modulated, in two different GB cell lines, T98G and U373MG (known to differ in their radiosensitivity), after IR or combined IR/TMZ treatments. T98G cells showed a high radiosensitivity (especially at low and intermediate doses), associated with autophagy activation, assessed by Beclin‐1 and Atg‐5 expression increase, LC3‐I to LC3‐II conversion and LC3B‐GFP accumulation in autophagosomes of irradiated cells; differently, U373MG cells resulted less radiosensitive. Autophagy inhibition, using siRNA against BECN1 or ATG‐7 genes, totally prevented decrease in viability after both IR and IR/TMZ treatments in the radiosensitive T98G cells, confirming the autophagy involvement in the cytotoxicity of these cells after the current GB treatment, contrary to U373MG cells. However, rapamycin‐mediated autophagy, that further radiosensitized T98G, was able to promote radiosensitivty also in U373MG cells, suggesting a role of autophagy process in enhancing radiosensitivity. Taken together, these results might enforce the concept that autophagy‐associated cell death might constitute a possible adjuvant therapeutic strategy to enhance the conventional GB treatment. J. Cell. Biochem. 113: 2308–2318, 2012.

Cisplatin-Induced Apoptosis Inhibits Autophagy, Which Acts as a Pro-Survival Mechanism in Human Melanoma Cells

The interplay between a non-lethal autophagic response and apoptotic cell death is still a matter of debate in cancer cell biology. In the present study performed on human melanoma cells, we investigate the role of basal or stimulated autophagy in cisplatin-induced cytotoxicity, as well as the contribution of cisplatin-induced activation of caspases 3/7 and conventional calpains. The results show that, while down-regulating Beclin-1, Atg14 and LC3-II, cisplatin treatment inhibits the basal autophagic response, impairing a physiological pro-survival response. Consistently, exogenously stimulated autophagy, obtained with trehalose or calpains inhibitors (MDL-28170 and calpeptin), protects from cisplatin-induced apoptosis, and such a protection is reverted by inhibiting autophagy with 3-methyladenine or ATG5 silencing. In addition, during trehalose-stimulated autophagy, the cisplatin-induced activation of calpains is abrogated, suggesting the existence of a feedback loop between the autophagic process and calpains. On the whole, our results demonstrate that in human melanoma cells autophagy may function as a beneficial stress response, hindered by cisplatin-induced death mechanisms. In a therapeutic perspective, these findings suggest that the efficacy of cisplatin-based polychemotherapies for melanoma could be potentiated by inhibitors of autophagy.

Combined EGFR and autophagy modulation impairs cell migration and enhances radiosensitivity in human glioblastoma cells

Glioblastoma (GBM) remains the most aggressive and lethal brain tumor due to its molecular heterogeneity and high motility and invasion capabilities of its cells, resulting in high resistance to current standard treatments (surgery, followed by ionizing radiation combined with Temozolomide chemotherapy administration). Locus amplification, gene overexpression, and genetic mutations of epidermal growth factor receptor (EGFR) are hallmarks of GBM that can ectopically activate downstream signaling oncogenic cascades such as PI3K/Akt/mTOR pathway. Importantly, alteration of this pathway, involved also in the regulation of autophagy process, can improve radioresistance in GBM cells, thus promoting the aggressive phenotype of this tumor. In this work, the endogenous EGFR expression profile and autophagy were modulated to increase radiosensitivity behavior of human T98G and U373MG GBM cells. Our results primarily indicated that EGFR interfering induced radiosensitivity according to a decrease of the clonogenic capability of the investigated cells, and an effective reduction of the in vitro migratory features. Moreover, EGFR interfering resulted in an increase of Temozolomide (TMZ) cytotoxicity in T98G TMZ‐resistant cells. In order to elucidate the involvement of the autophagy process as pro‐death or pro‐survival role in cells subjected to EGFR interfering, the key autophagic gene ATG7 was silenced, thereby producing a transient block of the autophagy process. This autophagy inhibition rescued clonogenic capability of irradiated and EGFR‐silenced T98G cells, suggesting a pro‐death autophagy contribution. To further confirm the functional interplay between EGFR and autophagy pathways, Rapamycin‐mediated autophagy induction during EGFR modulation promoted further impairment of irradiated cells, in terms of clonogenic and migration capabilities. Taken together, these results might suggest a novel combined EGFR‐autophagy modulation strategy, to overcome intrinsic GBM radioresistance, thus improving the efficacy of standard treatments. J. Cell. Physiol. 229: 1863–1873, 2014.

Usefulness of CDX2 in the diagnosis of extramammary Paget disease associated with malignancies of intestinal type.

1 Del Castillo LF, Garcia C, Schoendorff C et al. Spontaneous apparent clinical resolution with histologic persistence of a case of extramammary Paget’s disease: response to topical 5-fluorouracil. Cutis 2000; 65:331–3. 2 Imakado S, Abe M, Okuno T et al. Two cases of genital Paget’s disease with bilateral axillary involvement: mutability of axillary lesions. Arch Dermatol 1991; 127:1243. 3 Archer CB, Louback JB, MacDonald DM. Spontaneous regression of perianal extramammary Paget’s disease after partial surgical excision. Arch Dermatol 1987; 123:379–82. 4 Brown RS, McCormack M, Lankester KJ, Spittle MF. Spontaneous apparent clinical resolution with histologic persistence of a case of extramammary Paget’s disease: response to topical 5-fluorouracil. Cutis 2000; 66:454–5.

The combined EGFR protein expression analysis refines the prognostic value of the MGMT promoter methylation status in glioblastoma

BACKGROUND/AIMS To investigate the combined prognostic value of the EGFR expression level and the MGMT promoter methylation status in Glioblastoma (GB). METHODS We assessed the EGFR protein expression level by immune-histochemical (IHC) evaluation and the MGMT promoter methylation status by Polymerase Chain Reaction (PCR) in 169 patients affected by GB. We assessed the prognostic significance of combined MGMT methylation status and EGFR expression level in terms of Overall Survival (OS) with univariate and multivariate analysis, and validated this finding using an external data set of GB patient. RESULTS Clustering survival analysis for the methylation status of MGMT (methMGMT/unmethMGMT) and EGFR expression (High EGFR: H-EGFR; Low EGFR: L-EGFR), identified three different prognostic groups (p=0.001), as follows. Patients with unmethMGMT/H-EGFR had the shortest survival time (median OS: 5 months) and patients co-expressing methMGMT/L-EGFR had the best prognosis (median OS: 35 months), as compared to the other two sub-groups (methMGMT/H-EGFR; unmethMGMT/L-EGFR), which had respectively median OSs of 11 and 12 months. The combined MGMT methylation and EGFR amplification status analysis showed a similar prognostic impact in an independent series, which we used for validation (p=0.001). CONCLUSIONS The EGFR expression evaluation refines the prognostic value of MGMT methylation status in GBs.

Combined Epidermal Growth Factor Receptor and Beclin1 Autophagic Protein Expression Analysis Identifies Different Clinical Presentations, Responses to Chemo- and Radiotherapy, and Prognosis in Glioblastoma

Dysregulated EGFR in glioblastoma may inactivate the key autophagy protein Beclin1. Each of high EGFR and low Beclin1 protein expression, independently, has been associated with tumor progression and poor prognosis. High (H) compared to low (L) expression of EGFR and Beclin1 is here correlated with main clinical data in 117 patients after chemo- and radiotherapy. H-EGFR correlated with low Karnofsky performance and worse neurological performance status, higher incidence of synchronous multifocality, poor radiological evidence of response, shorter progression disease-free (PDFS), and overall survival (OS). H-Beclin1 cases showed better Karnofsky performance status, higher incidence of objective response, longer PDFS, and OS. A mutual strengthening effect emerges in correlative power of stratified L-EGFR and H-Beclin1 expression with incidence of radiological response after treatment, unifocal disease, and better prognosis, thus identifying an even longer OS group (30 months median OS compared to 18 months in L-EGFR, 15 months in H-Beclin1, and 11 months in all GBs) (P = 0.0001). Combined L-EGFR + H-Beclin1 expression may represent a biomarker in identifying relatively favorable clinical presentations and prognosis, thus envisaging possible EGFR/Beclin1-targeted therapies.

Correlative study of squash smear cytology with histopathology in a rare case of anaplastic giant cell ependymoma of the pineal

Anaplastic giant cell ependymoma (AGCE) is a very rare neoplasm. Its cytological features, helpful for the intraoperative diagnosis, have been reported only once. AGCE is characterized by giant cells with intranuclear inclusions, besides other findings, observable in ependymal neoplasms, such as intracytoplasmic vacuoles, epithelial and glial features of the tumor cells and ependymal pseudorosettes. These findings can be detected also in intraoperative squash smear. Herein we describe a pineal AGCE, highlighting the cytological and histological correlations and underlining some useful diagnostic clues of this unusual entity.

Cell Death Pathways, with Special Regard to Ionizing Radiation and Temozolomide

Tumor cell death is the final goal of both radio- and chemotherapy. For several decades, apoptosis has been considered as the principal type of programmed cell death (PCD) in mammalian tissues. Resistance to apoptosis was closely linked to tumorigenesis, and has taken a central position in cell death research. However, in the last years it has become evident that radiotherapy as well as other therapeutic agents, including temozolomide (TMZ) are able to elicit non-apoptotic pathways of PCD, which are not always mutually exclusive, as thought in the past.In this chapter we will address this topic, focusing on apoptosis and autophagy-related cell death in cancer, their induction by IR and TMZ, and implication in glioblastoma (GB) treatment.We will review currently known mechanisms of both IR and TMZ in inducing cell death, the possibility to modulate PCD as well as other approaches which will open new perspectives for improving the efficacy of therapy in GB.