Autophagy deactivation is associated with severe prostatic inflammation in patients with lower urinary tract symptoms and benign prostatic hyperplasia.

Autophagy is a conserved evolutionary process that allows cells to maintain macromolecular synthesis and energy homeostasis during starvation and stressful conditions. We prospectively evaluated the relationship between autophagy and prostatic inflammation in a series of transurethral prostatic resection samples. Inflammatory infiltrates were defined according to the standardized classification of chronic prostatitis of the National Institute of Health. The inflammatory score (IS score) was calculated. High IS score was defined as ≥7. Each sample was stained for anti-LC3B and for anti-P62/SQSTM1 and scored. High p62 or LC3B percentage was defined as >25%, whereas low was defined as <25% of cells with dots. We analyzed 94 specimens. Overall, 18/94 (19%) showed no sign of prostatic inflammation, whereas 76/94 (81%) presented inflammatory infiltrates. Inflammation was mild in 61/76 (80%), moderate/severe in 15/76 (20%). Patients with high p62 percentage were 62/94 (66%) while 32 (34%) showed low p62 percentage. Patients with high LC3B percentage were 37/94 (39%) while 57(61%) showed low LC3B percentage. Overall 42/94 (44%) patients presented a high p62 percentage and concomitant a low LC3B percentage. IS score was significantly higher in patients with a with high p62 percentage (median IS 7 (6/8) vs 5 (3/7); p= 0.04) and in patients with a low LC3B percentage (median IS 7 (6/8) vs 5 (3/7); p= 0.004) when compared to patients with a low p62 percentage or a high LC3B percentage respectively. On multivariate analysis, p62 (OR: 10.1, 95%CI: 2.6-38.6; p= 0,001) and LC3B expression (OR: 0.319; 95%CI: 0.112-0.907; p= 0.032) were independent predictors of a high IS. Here we present the first evidence of autophagy deregulation in prostatic inflammation. These results raise many questions about the mechanisms mediating the autophagy dysfunction and the links to prostatic inflammation that need to be addressed.

Antiproliferative Effects of 1α-OH-vitD3 in Malignant Melanoma: Potential Therapeutic implications.

Early detection and surgery represent the mainstay of treatment for superficial melanoma, but for high risk lesions (Breslow's thickness >0.75 mm) an effective adjuvant therapy is lacking. Vitamin D insufficiency plays a relevant role in cancer biology. The biological effects of 1α hydroxycholecalciferol on experimental melanoma models were investigated. 105 melanoma patients were checked for 25-hydroxycholecalciferol (circulating vitamin D) serum levels. Human derived melanoma cell lines and in vivo xenografts were used for studying 1α-hydroxycholecalciferol-mediated biological effects on cell proliferation and tumor growth. 99 out of 105 (94%) melanoma patients had insufficient 25-hydroxycholecalciferol serum levels. Interestingly among the six with vitamin D in the normal range, five had a diagnosis of in situ/microinvasive melanoma. Treatment with 1α-hydroxycholecalciferol induced antiproliferative effects on melanoma cells in vitro and in vivo, modulating the expression of cell cycle key regulatory molecules. Cell cycle arrest in G1 or G2 phase was invariably observed in vitamin D treated melanoma cells. The antiproliferative activity induced by 1α-hydroxycholecalciferol in experimental melanoma models, together with the discovery of insufficient 25-hydroxycholecalciferol serum levels in melanoma patients, provide the rationale for using vitamin D in melanoma adjuvant therapy, alone or in association with other therapeutic options.

The loss of the p53 activator HIPK2 is responsible for galectin-3 overexpression in well differentiated thyroid carcinomas.

BACKGROUND: Galectin-3 (Gal-3) is an anti-apoptotic molecule involved in thyroid cells transformation. It is specifically overexpressed in thyroid tumour cells and is currently used as a preoperative diagnostic marker of thyroid malignancy. Gal-3 expression is downregulated by wt-p53 at the transcriptional level. In well-differentiated thyroid carcinomas (WDTCs) there is an unexplained paradoxical concomitant expression of Gal-3 and wt-p53. HIPK2 is a co-regulator of different transcription factors, and modulates basic cellular processes mainly through the activation of wt-p53. Since we demonstrated that HIPK2 is involved in p53-mediated Gal-3 downregulation, we asked whether HIPK2 deficiency might be responsible for such paradoxical Gal-3 overexpression in WDTC. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed HIPK2 protein and mRNA levels, as well as loss of heterozygosity (LOH) at the HIPK2 locus (7q32-34), in thyroid tissue samples. HIPK2 protein levels were high in all follicular hyperplasias (FHs) analyzed. Conversely, HIPK2 was undetectable in 91.7% of papillary thyroid carcinomas (PTCs) and in 60.0% of follicular thyroid carcinomas (FTCs). HIPK2 mRNA levels were upregulated in FH compared to normal thyroid tissue (NTT), while PTC showed mean HIPK2 mRNA levels lower than FH and, in 61.5% of cases, also lower than NTT. We found LOH at HIPK-2 gene locus in 37.5% of PTCs, 14.3% of FTCs and 18.2% of follicular adenomas. To causally link these data with Gal-3 upregulation, we performed in vitro experiments, using the PTC-derived K1 cells, in which HIPK2 expression was manipulated by RNA interference (RNAi) or plasmid-mediated overexpression. HIPK2 RNAi was associated with Gal-3 upregulation, while HIPK2 overexpression with Gal-3 downregulation. CONCLUSIONS/SIGNIFICANCE: Our results indicate that HIPK2 expression and function are impaired in WDTCs, in particular in PTCs, and that this event explains Gal-3 overexpression typically observed in these types of tumours. Therefore, HIPK2 can be considered as a new tumour suppressor gene for thyroid cancers.

COX-2 is induced by HGF stimulation in Met-positive thyroid papillary carcinoma cells and is involved in tumour invasiveness.

Thyroid papillary carcinoma (TPC) cells express high levels of cytoplasmic cyclo-oxygenase 2 protein. Analysis of microdissected samples of the tumour and of the paired normal thyroid tissue confirmed that mRNA transcripts for cyclo-oxygenase 2 (COX-2) were significantly more numerous in the tumour (7.6 +/- 13-fold; p = 0.01). High levels of COX-2 mRNA were not associated with age, sex, tumour size or lymph node metastasis. COX-2 was not homogeneously expressed throughout the tumour, but was significantly higher at the tumour invasion front. Hepatocyte growth factor (HGF) can up-regulate the expression of COX-2 mRNA. A marked increase in COX-2 mRNA levels was observed in 8/8 primary TPC cultures after HGF stimulation (6.3 +/- 6-fold) and in two papillary carcinoma cell lines (TPC-1 and NPA). Specific involvement of the high-affinity HGF receptor (Met protein) was suggested by the observation that PHA-665752, an inhibitor of the catalytic activity of c-Met kinase, caused a 54% reduction of the hepatocyte growth factor-induced COX-2 up-regulation. The possibility that HGF-Met interactions also had a causative role in the up-regulation of COX-2 in vivo was investigated in 30 tumour samples, where it was found that there was a statistically significant correlation (p = 0.001, r = 0.85) in the levels of expression of MET and COX-2 RNAs. The biological role of COX-2 in TPC cells was investigated by treating the TPC cell lines with the specific COX-2 inhibitor NS-398. It was found that NS-398 treatment significantly reduced the migration (50-75%) and invasiveness (47-92%) of tumour cells, but did not alter cell proliferation. Our data suggest that the increased expression of Met protein in TPC cells has a role in up-regulating the expression of COX-2, which in turn contributes to the invasive capacity of TPC cells.

Gal-3 is stimulated by gain-of-function p53 mutations and modulates chemoresistance in anaplastic thyroid carcinomas.

Galectin-3 (Gal-3) is an anti-apoptotic molecule of the beta-galactoside-binding lectin family. Gal-3 is down-regulated by wt-p53 and this repression is required for p53-induced apoptosis. Since poorly differentiated thyroid carcinomas (PDTCs) and anaplastic thyroid carcinomas (ATCs) frequently harbour p53 mutations, we asked whether Gal-3 expression and activity could be influenced by such mutations in these tumours. We found a positive correlation between Gal-3 expression and p53 mutation in human thyroids and in thyroid carcinoma cell lines (TCCLs) harbouring different p53 mutations. Gal-3 was over-expressed in most ATCs and TCCLs, especially those with the most frequently detected p53 mutation (p53(R273H)). Over-expression of p53(R273H) in two p53-null cells (SAOS-2 and SW-1736) as well as in two wt-p53-carrying TCCLs (TPC-1 and K1), stimulated Gal-3 expression, while interference with p53(R273H) endogenous expression in ARO cells down-regulated Gal-3 expression. Conversely, over-expression of wt-p53 in ARO cells restored the inhibitory effect on Gal-3 expression. ARO cells are highly resistant to apoptosis and express both p53 and Gal-3, which are increased upon cisplatin treatment. Interference with Gal-3 expression in these cells stimulated their chemosensitivity. In conclusion, gain-of-function p53 mutant acquires the de novo ability to stimulate Gal-3 expression and to increase chemoresistance in ATCs.