The nuclear factor kB (NF-kB) comprises a family of transcription factors involved in the regulation of a wide variety of biological responses. NF-kB plays a well-known function in the regulation of immune responses and inflammation, but growing evidences support a major role in oncogenesis. NF-kB regulates the expression of genes involved in many processes that play a key role in the development and progression of cancer such as proliferation, migration and apoptosis. Aberrant or constitutive NF-kB activation has been detected in many human malignancies. In recent years, numerous studies have focused on elucidating the functional consequences of NF-kB activation as well as its signaling mechanisms. NF-kB has turned out to be an interesting therapeutic target for treatment of cancer.

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We analyzed RASSF1A and NORE1A methylation and BRAF mutation in 89 thyroid tumors, 42 non-neoplastic thyroid tissues and three thyroid tumor cell lines using polymerase chain reaction (PCR), methylation-specific PCR, Western blotting and DNA sequencing in order to study thyroid tumor pathogenesis and progression. RASSF1A promoter methylation was present in all three thyroid cell lines and in 27/78 (35%) of benign and malignant thyroid tumors. We showed for the first time that there was generally good agreement between RASSF1A methylation status and RASSF1A protein expression. We also examined for the first time NORE1A promoter region methylation in thyroid cell lines and primary tumors and showed that two of three thyroid cell lines were methylated in the NORE1A promoter region, while all primary thyroid tumors analyzed (n=51) were unmethylated. BRAF mutation was present in 38% of papillary thyroid carcinomas (PTC), including 20% of PTC with a follicular variant pattern and 67% of the tall cell variant of PTC. Hyalinizing trabecular tumors (n=23), which had nuclear features similar to PTC, did not have BRAF mutations, indicating that the presence of BRAF mutations can help to separate these two tumor types. Phospho-MEK expression was increased in the NPA cell line, which had a BRAF mutation, supporting the importance of the BRAF pathway alterations in PTC pathogenesis. These results indicate that RASSF1A epigenetic changes are an early event in thyroid tumor pathogenesis and progression and that NORE1A methylation is uncommon in primary thyroid tumors. BRAF mutation occurs later in thyroid tumor progression and is restricted mainly to PTC and anaplastic thyroid carcinoma.

The NF-kappaB family of transcription factors regulates a wide variety of cellular processes including cell growth, differentiation, and apoptosis. A tissue microarray was constructed from paraffin wax-embedded blocks from 95 endometrial carcinomas (EC), previously studied for microsatellite instability, as well as for alterations in PTEN, k-RAS and beta-catenin. Immunohistochemical evaluation included members of the NF-kappaB (p50, p65, p52, c-Rel, Rel-B) and the IkappaB (IkappaBalpha, IkappaBbeta, IkappaBepsilon, Bcl-3) families, as well as putative targets of NF-kappaB such as Flip, Bcl-xL, Cyclin D1, and oestrogen and progesterone receptors. Results were correlated with the clinical and pathological data. Nuclear immunostaining for members of the NF-kappaB family was frequent in EC (p50, 20%; p65, 16.5-21.9%; p52, 9.3%; c-Rel, 48.9%; Rel-B, 15.7%); and it correlated with negativity for members of the IkappaB family in some cases. There was a statistically significant association between immunoreaction for p50 and p65 (p = 0.006), suggesting activation of the so-called 'classic form' of NF-kappaB, similar to that described in breast cancer. Bcl-3 nuclear immunostaining was detected in 60.7% of cases. The vast majority of p52-positive tumours showed Bcl-3 nuclear immunoreaction (p = 0.038). Immunostaining for putative targets of NF-kappaB was as follows: Bcl-xL, 76.2% (p = 0.001); Flip 43.0%; Cyclin D1, 64.79%. p65 immunostaining correlated with increased immunoreaction for steroid hormone receptors. No correlation was found between NF-kappaB nuclear pattern and the presence of microsatellite instability, or alterations in PTEN, k-RAS, or beta-catenin. These results suggest that the NF-kappaB and IkappaB families of genes may be important in endometrial carcinogenesis, by controlling apoptosis and cell proliferation.

The tumor suppressor gene PTEN/MMAC1 is located on chromosome 10q23.3. Inactivation of PTEN, either by mutations, deletions, or promoter hypermethylation, has been identified in a wide variety of tumors. Inactivation of the two alleles of PTEN is required, because it is a tumor suppressor gene. Immunohistochemical staining may be an effective screening method to demonstrate the absence of the protein in tumors exhibiting PTEN inactivation. We studied a tissue microarray, constructed from paraffin-embedded blocks of 95 endometrial carcinomas, 38 of them previously evaluated for alterations in PTEN. We also studied cell blocks obtained from one PTEN-defective endometrial cancer cell line, after transfection with either a plasmid encoding wild-type PTEN or the empty vector. The tumor samples were tested with four different anti-PTEN commercial antibodies: a polyclonal antibody, the monoclonal antibody 28H6, the monoclonal antibody 10P03, and the monoclonal antibody 6.H2.1. Results were correlated with the presence of abnormalities in PTEN, as well as with the immunohistochemical expression of phosphorylated AKT. Antibody 28H6 produced a predominant nuclear staining, while the other three antibodies produced a predominant cytoplasmic staining. There was no significant correlation between the results obtained with the four antibodies. The monoclonal antibody 6.H2.1 was the only one that exhibited a correlation with the presence of molecular alterations in PTEN, and a statistically significant association with immunostaining for phosphorylated AKT (r ¼ À0. The tumor suppressor gene PTEN 1 is located on chromosome 10q23. It is somatically mutated or deleted in several types of tumors. 2,3 PTEN encodes a 403 amino-acid dual specificity phosphatase containing a region of homology to tensin and auxilin, which are two cytoskeletal proteins. Among other activities, PTEN antagonizes the PI3K/AKT pathway by dephosphorylating PIP3, resulting in a decreased translocation of AKT to cellular membranes, and subsequent downregulation of AKT phosphorylation and activation. 4,5 The PI3K/AKT pathway plays a key role in the regulation of cellular homeostasis. Activation of cell surface receptors

Proteasome inhibitors are currently used as chemotherapeutic drugs because of their ability to block NF-B, a transcription factor constitutively activated in many different types of human cancer. In the present study, we demonstrate that proteasome inhibitors induce cell death in endometrial carcinoma cell lines and primary explants but, instead of blocking NF-B, they increase its transcriptional activity. Proteasome inhibitors induce phosphorylation of IKK␣/␤, phosphorylation and degradation of IB␣, and phosphorylation of the p65 NF-B subunit on serine 536. Proteasome inhibitor-induced NF-B activity can be blocked by a non-degradable form of IB␣ or dominant negative forms of either IKK␣ or IKK␤. Lentiviral delivery of shRNAs to either IKK␣ or IKK␤ cause blockade of NF-B transcriptional activity and inhibit phosphorylation of p65 on serine 536, but has no effect on IBa degradation. These results suggest a role for p65 phosphorylation in proteasome inhibitor-induced NF-B activation. Accordingly, siRNA knockdown of p65 inhibits proteasome inhibitor-induced NF-B transcriptional activity. Our results demonstrate that proteasome inhibitors, including bortezomib, induce cell death on endometrial carcinoma cells and primary explants. However, they activate NF-B instead of blocking its transcriptional potential. Therefore, the concept that proteasome inhibitors are blockers of NF-B activation should be carefully examined in particular cell types.

The FLICE-inhibitory protein (FLIP) plays a key role in the regulation of apoptosis triggered by death ligands. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in some types of tumor but not in others. To assess the possible role of FLIP in apoptosis resistance in endometrial carcinoma, we performed an immunohistochemical study on a tissue microarray composed of 95 endometrial carcinomas. We found positive signals in 43% of the cases, as well as a significant difference in FLIP expression between stage I and II tumors. Moreover, we observed that endometrial carcinoma cell lines Ishikawa and KLE did not undergo apoptosis after TRAIL treatment.

We present the clinicopathological features of 56 cases of the nested variant of urothelial bladder carcinoma. This is an uncommon variant of bladder cancer, recognized by the current WHO classification of urologic tumors. The nested component represented 100 % of the tumor in 24 cases. The architectural pattern of the tumor varied from solid expansile to infiltrative nests characterized by deceptively bland histologic features resembling von Brunn nests. Typical features of high-grade conventional urothelial carcinoma were present in 32 cases. Most neoplastic cells had nuclei of low to intermediate nuclear grade with occasional nuclear enlargement, most frequently seen in deep areas of tumor. The nested component expressed cytokeratins 7, 20, CAM5.2, and high molecular weight (34ßE12), p63, Ki67, p53, p27, and GATA3. Tumor extension was T1 (n = 9), minimally T2 (n = 10), T2a (n = 1), T2b (n = 4), T3a (n = 8), T3b (n = 13), and T4a (n = 11). On follow-up, 36 of patients died of or were alive with disease from 2 to 80 months (mean 21 months). Four patients died of other causes. Eleven other patients remained disease free. Univariate survival analysis showed no differences for nested carcinoma compared with conventional urothelial carcinoma. As in conventional urothelial carcinoma, in nested carcinoma of the bladder pT category defined different survival groups. In summary, nested variant of urothelial bladder carcinoma is typically associated with advanced stage. In samples of limited volume, it may be misdiagnosed as proliferation of von Brunn nests or other nested-like bladder lesions, delaying definitive therapy.