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Background: Patients with neuroendocrine tumors (NETs) may have metastatic disease and unknown primary site. NETs commonly arise from the bronchopulmonary (BP) and gastrointestinal (GI) tract. The largest subgroups of well-differentiated BP-NETs are typical carcinoids (TCs). The homeodomain transcription factor NKX2.2 regulates development of gut serotonin cells and is a marker of GI-NETs. Previous work on a limited number of samples suggested that BP-TCs do not express NKX2.2. We hypothesized that lack of NKX2.2 expression in BP-TCs might be useful to distinguish BP- from GI-NETs, and evaluated NKX2.2 expression in a larger number of BP-TCs. Methods: Archived formalin-fixed, paraffin-embedded tissues were obtained from 13 previously undescribed patients with BP-TCs. Expression of NKX2.2, serotonin, and the NE marker chromogranin A (CgA) were assessed by immunohistochemistry. Results: CgA expression was robust in all 13 BP-TCs, confirming the NE phenotype. Serotonin expression was less frequent (9/13; 69%). Two patients with BP-TCs in which serotonin expression was absent exhibited Cushing''s syndrome due to ectopic ACTH production. NKX2.2 expression was not observed in any of the 13 tumors. Conclusions: Bronchopulmonary TCs uniformly express CgA but not NKX2.2. Because most of these tumors express serotonin, our findings suggest that NKX2.2 may not be required for serotonin production by BP-TCs. We conclude that the presence or absence of NKX2.2 expression may assist in the determination of the primary tumor site in patients with NET metastases of unknown origin. NET metastases that are CgA-positive/NKX2.2-negative would suggest a BP primary, whereas those that are CgA-positive/NKX2.2-positive would suggest a GI primary. [Copyright &y& Elsevier]

Abstract: Wnt inhibitory factor-1 (WIF-1) is a secreted protein that antagonizes Wnt signaling. We recently demonstrated the importance of aberrant activation of the Wnt signaling pathway in various cancers including malignant pleural mesothelioma. In this study, we revealed downregulated WIF-1 expression in cell lines and primary tissue when compared to normal mesothelial cell lines and adjacent pleura, respectively. We observed hypermethylation in four of four mesothelioma cell lines, but not in two normal mesothelial cell lines. In primary tissue samples, we observed methylation in three paired tumor specimens compared to their adjacent normal pleura and methylation in eight of nine unpaired tumor tissue samples. Taken together, our studies suggest that WIF-1 silencing due to its promoter hypermethylation is an important mechanism underlying the constitutively activated Wnt signaling in mesothelioma. New therapies toward inhibition of the Wnt pathway through WIF-1 might be promising for the future treatment of malignant mesothelioma. [Copyright &y& Elsevier]

Abstract: Aberrant activation of Wingless-type (Wnt) signaling pathway plays a critical role in oncogenesis of various human cancers. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist of Wnt signaling and acts through direct binding to Wnt in the extracelllular space. Recently, we reported Wnt signaling in various human malignancies. In addition, we identified in lung cancer that WIF-1 is silenced due to promoter hypermethylation. In this study, we found constitutive activation of Wnt signaling and WIF-1 silencing in nasopharyngeal carcinoma (NPC) cell lines. Furthermore, by utilizing methylation-specific PCR and sequence analysis, we demonstrated that frequent hypermethylation of the WIF-1 promoter correlates with WIF-1 silencing in NPC cell lines. Our results indicate that aberrant Wnt signaling is a common event in NPC carcinogenesis linked with WIF-1 silencing in at least cell lines. Strategies targeting these molecules should be potentially promising in treating NPC. [Copyright &y& Elsevier]

Dickkopf-1 (Dkk-1) is a secreted protein that acts as a potent inhibitor of the Wnt signal transduction pathway. It is thought that the antagonistic effect of Dkk-1 is specific to the canonical (Wnt/β-catenin) pathway. In this study, we demonstrate that restoration of Dkk-1 expression suppresses cell growth and induces apoptotic cell death in β-catenin-deficient mesothelioma cell lines H28 and MS-1. Furthermore, we found that a small-molecule inhibitor of JNK inhibited the apoptosis induced by Dkk-1 overexpression in these cells. Together, our data suggest that Dkk-1 may be able to antagonize Wnt signaling and exert its tumor suppressive effects through β-catenin-independent non-canonical pathways (i.e., the Wnt/JNK pathway). [Copyright &y& Elsevier]

Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist of Wnt signaling and functions by directly binding to Wnt ligands in the extracellular space. Here we report the identification of the 5′ promoter region (∼1.5kb) of the human WIF-1 gene. Functional analysis of this region shows that a whole fragment displays high basal promoter activity in different cell lines, while the truncated forms do not, indicating that integrity of the WIF-1 promoter region may be important for WIF-1 activity. Moreover, we found that the expression level of β-catenin in cancer cell lines correlates with the WIF-1 promoter activity, suggesting that the WIF-1 promoter may be regulated by the Wnt/β-catenin pathway and may function in a negative feedback manner. Our results also suggest that a methylated CpG island, which we observed recently in human lung cancer, lies within the functional WIF-1 promoter region and therefore bears the importance of the methylation-status of this CpG island as an important key in Wnt activation in human cancer. [Copyright &y& Elsevier]

SOCS-3 is a member of a newly discovered protein family that inhibits LIF-activated Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling in a negative auto-regulatory manner. In this study, we have cloned and characterized the promoter region of the human SOCS-3 gene. This region is ∼1.1 kbp in length and consists of two putative STAT-binding elements, a G-rich element, and a putative TATA box. These elements are highly conserved in both murine and rat SOCS-3 promoters. Functional analysis of this region shows that the whole fragment (∼1.1 kbp) has high basal promoter activity and is responsive to growth factors. We also found that the wild type SOCS-3 promoter construct has significantly greater activity in non-small-cell lung cancer cell lines than in normal cells in accordance with STAT3 disregulation in these cells. Cloning of the human SOCS-3 promoter should help uncover mechanisms of regulation of the JAK-STAT pathway in human cancer. [Copyright &y& Elsevier]