BRCA1 deficiency enhances the aggressiveness of breast cancer cells expressing HPV16 oncoproteins.

BACKGROUND INFORMATION: The precise etiology of breast cancer is not completely understood, although women with BRCA1 gene mutations have a significantly increased risk of developing the disease. In addition, sporadic breast cancer is frequently associated with decreased BRCA1 gene expression. Growing evidence of Human papillomaviruses (HPVs) infections in breast tumors has raised the possibility of the involvement of HPVs in the pathogenesis of breast cancer. We investigated whether the effects of HPV oncoproteins E6 and E7 were influenced by the expression levels of BRCA1. HPV16E6E7 (prototype or E6D25E/E7N29S Asian variant type) were stably expressed in MDA-MB231 breast cancer cells, wild type for BRCA1, or with BRCA1 knocked down. RESULTS: Expression of HPV16E6E7 oncogenes did not affect BRCA1 levels and the abundance of HPV16E6E7 was not altered by BRCA1 knockdown. BRCA1 levels did not alter HPV16E6E7-dependent degradation of G1-S cell cycle proteins p53 and pRb. However, we found that the expression of G2-M cell cycle protein cyclin B1 enhanced by HPV16E6E7 was impacted by BRCA1 levels. Especially, we found the correlation between BRCA1 and cyclin B1 expression and this was also confirmed in breast cancer samples from a Thai cohort. We further demonstrated that the combination of HPV oncoproteins and low levels of BRCA1 protein appears to enhance proliferation and invasion. Transactivation activities of HPV16E6E7 on genes regulating cell proliferation and invasion (TGF-ß and vimentin) were significantly increased in BRCA1-deficient cells. CONCLUSIONS: Our results indicate that a deficiency of BRCA1 promotes the transactivation activity of HPV16E6E7 leading to increase of cell proliferation and invasion. SIGNIFICANCE: HPV infection appears to have the potential to enhance the aggressiveness of breast cancers, especially those deficient in BRCA1.

Asiatic Acid Inhibits Nasopharyngeal Carcinoma Cell Viability and Migration via Suppressing STAT3 and Claudin-1.

Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Southeast Asia, but effective treatment options remain limited, and chemotherapy has a high resistance rate. Asiatic acid (AA), a triterpenoid found in Centella asiatica, has shown anticancer activity in various cancers. Therefore, this study aims to investigate the anticancer effects and mechanisms of AA in NPC cell lines. The effects of AA on NPC cytotoxicity, apoptosis, and migration were determined in TW-01 and SUNE5-8F NPC cell lines. Western blot analysis was performed to evaluate the protein expression levels affected by AA. The role of AA in proliferation and migration was investigated in STAT3 and claudin-1 knockdown cells. AA inhibited NPC cell viability and migration and induced cell death by increasing cleaved caspase-3 expression. Moreover, AA inhibited STAT3 phosphorylation and reduced claudin-1 expression in NPC cells. Although knockdown of STAT3 or claudin-1 slightly reduced cell viability, it did not enhance the anti-proliferative effect of AA. However, knockdown of STAT3 or claudin-1 increased the anti-migratory effect of AA in NPC cells. These results suggest that AA can be a promising candidate for drug development against NPC.

Comparative Proteomic Analysis of Human Cholangiocarcinoma Cell Lines: S100A2 as a Potential Candidate Protein Inducer of Invasion.

Cholangiocarcinoma (CCA) is a bile duct cancer, commonly found in Asia including Thailand and especially in the northeastern region of Thailand. To identify the proteins involved in carcinogenesis and metastasis of CCA, protein expression profiles of high-invasive KKU-M213 and low-invasive KKU-100 cell lines were compared using a comparative GeLC-MS/MS proteomics analysis. A total of 651 differentially expressed proteins were detected of which 27 protein candidates were identified as having functions involved in cell motility. A total of 22 proteins were significantly upregulated in KKU-M213, whereas 5 proteins were downregulated in KKU-M213. S100A2, a calcium-binding protein in S100 protein family, is upregulated in KKU-M213. S100A2 is implicated in metastasis development in several cancers. The protein expression level of S100A2 was verified by Western blot analysis. Intriguingly, high-invasive KKU-M213 cells showed higher expression of S100A2 than KKU-100 cells, consistent with proteomic data, suggesting that S100A2 may be a key protein involved in the progression of CCA. However, the biological function of S100A2 in cholangiocarcinoma remains to be elucidated. S100A2 might be a potential biomarker as well as a novel therapeutic target in CCA metastasis.

Constitutive suppressor of cytokine signaling 3 expression confers a growth advantage to a human melanoma cell line.

The growth of melanocytes and many early stage melanoma cells can be inhibited by cytokines, whereas late stage melanoma cells have often been reported to be "multi-cytokine-resistant." Here, we analyzed the melanoma cell line 1286, resistant towards the growth-inhibitory effects of interleukin 6 (IL-6), and oncostatin M (OSM), to better understand the mechanisms underlying cytokine resistance. Although the relevant receptors gp130 and OSMR are expressed at the cell surface of these cells, cytokine stimulation hardly led to the activation of Janus kinase 1 and signal transducer and activator of transcription (STAT)3 and STAT1. We found a high-level constitutive expression of suppressors of cytokine signaling 3 (SOCS3) that did not further increase after cytokine treatment. Importantly, upon suppression of SOCS3 by short interfering RNA, cells became susceptible towards OSM and IL-6: they showed an enhanced STAT3 phosphorylation and a dramatically increased STAT1 phosphorylation. Moreover, suppression of SOCS3 rendered 1286 cells sensitive to the antiproliferative action of IL-6 and OSM, but not of IFN-alpha. Interestingly, SOCS3-short interfering RNA treatment also increased the growth-inhibitory effect in cytokine-sensitive WM239 cells expressing SOCS3 in an inducible way. Thus, SOCS3 expression confers a growth advantage to these cell lines. Constitutive SOCS3 mRNA expression, although at lower levels than in 1286 cells, was found in nine additional human melanoma cell lines and in normal human melanocytes, although at the protein level, SOCS3 expression was marginal at best. However, in situ analysis of human melanoma specimens revealed SOCS3 immunoreactivity in 3 out of 10 samples, suggesting that in vivo SOCS3 may possibly play a role in IL-6 resistance in at least a fraction of tumors.

Are STATS arginine-methylated?

Transcription factors of the STAT (signal transducer and activator of transcription) family are important in signal transduction of cytokines. They are subject to post-translational modification by phosphorylation on tyrosine and serine residues. Recent evidence suggested that STATs are methylated on a conserved arginine residue within the N-terminal region. STAT arginine methylation has been described to be important for STAT function and loss of arginine methylation was discussed to be involved in interferon resistance of cancer cells. Here we provide several independent lines of evidence indicating that the issue of arginine methylation of STATs has to be reassessed. First, we show that treatment of melanoma and fibrosarcoma cells with inhibitors used to suppress methylation (N-methyl-2-deoxyadenosine, adenosine, dl-homocysteine) had profound and rapid effects on phosphorylation of STAT1 and STAT3 but also on p38 and Erk signaling cascades which are known to cross-talk with the Jak/STAT pathway. Second, we show that anti-methylarginine antibodies did not precipitate specifically STAT1 or STAT3. Third, we show that mutation of Arg(31) to Lys led to destabilization of STAT1 and STAT3, implicating an important structural role of Arg(31). Finally, purified catalytically active protein arginine methyltransferases (PRMT1, -2, -3, -4, and -6) did not methylate STAT proteins, and cotransfection with PRMT1 did not affect STAT1-controlled reporter gene activity. Taken together, our data suggest the absence of arginine methylation of STAT1 and STAT3.

Janus kinase (Jak) subcellular localization revisited: the exclusive membrane localization of endogenous Janus kinase 1 by cytokine receptor interaction uncovers the Jak.receptor complex to be equivalent to a receptor tyrosine kinase.

The Janus kinases are considered to be cytoplasmic kinases that constitutively associate with the cytoplasmic region of cytokine receptors, and the Janus kinases (Jaks) are crucial for cytokine signal transduction. We investigated Jak1 localization using subcellular fractionation techniques and fluorescence microscopy (immunofluorescence and yellow fluorescent protein-tagged Jaks). In the different experimental approaches we found Jak1 (as well as Jak2 and Tyk2) predominantly located at membranes. In contrast to previous reports we did not observe Jak proteins in significant amounts within the nucleus or in the cytoplasm. The cytoplasmic localization observed for the Jak1 mutant L80A/Y81A, which is unable to associate with cytokine receptors, indicates that Jak1 does not have a strong intrinsic membrane binding potential and that only receptor binding is crucial for the membrane recruitment. Finally we show that Jak1 remains a membrane-localized protein after cytokine stimulation. These data strongly support the hypothesis that cytokine receptor.Janus kinase complexes can be regarded as receptor tyrosine kinases.

Interferon-gamma-mediated growth regulation of melanoma cells: involvement of STAT1-dependent and STAT1-independent signals.

Interferon-gamma, a known inhibitor of tumor cell growth, has been used in several protocols for the treatment of melanoma. We have studied the molecular events underlying interferon-gamma-induced G0/G1 arrest in four metastatic melanoma cell lines with different responsiveness to interferon-gamma. The growth arrest did not result from enhanced expression of cyclin-dependent kinase inhibitors p21 and p27. Instead, it correlated with downregulation of cyclin E and cyclin A and inhibition of their associated kinase activities. We show that interferon-gamma-induced growth inhibition could be abrogated by overexpression of dominant negative STAT1 (signal transducer and activator of transcription 1) in the melanoma cell line A375, suggesting that STAT1 plays a crucial part for the anti-proliferative effect. Erythropoietin stimulation of a chimeric receptor led to a concentration-dependent STAT1 activation and concomitant growth arrest when it contained the STAT recruitment motif Y440 of the interferon-gamma receptor 1. In contrast, dose-response studies for interferon-gamma revealed a discrepancy between levels of STAT1 activation and the extent of growth inhibition; whereas STAT1 was activated by low doses of interferon-gamma (10 U per mL), growth inhibitory effects were only visible with 100-fold higher concentrations. Our results suggest the presence of additional signals emanating from the interferon-gamma receptor, which may counteract the anti-proliferative function of STAT1.

Characterization of methylthioadenosin phosphorylase (MTAP) expression in malignant melanoma.

Homozygous deletions of human chromosomal region 9p21 occur frequently in malignant melanoma and are associated with the loss of the tumor suppressor genes p16(INK4a) and p15(INK4b). In the same chromosomal region the methylthioadenosine phosphorylase (MTAP) gene is localized and therefore may also serve as a tumor suppressor gene. The aim of this study was to analyze MTAP mutations and expression patterns in malignant melanomas. To examine the MTAP gene and expression of MTAP protein we screened 9 human melanoma cell lines and primary human melanocytes by reverse transcriptase-polymerase chain reaction, sequencing, and immunoblotting. Analyzing the melanoma cell lines we found significant down-regulation of MTAP mRNA expression. In only one cell line, HTZ19d, this was due to homozygous deletion of exon 2 to 8 whereas in the other cell lines promoter hypermethylation was detected. MTAP expression was further analyzed in vivo by immunohistochemical staining of 38 tissue samples of benign melanocytic nevi, melanomas, and melanoma metastases. In summary, we demonstrate significant inverse correlation between MTAP protein expression and progression of melanocytic tumors as the amount of MTAP protein staining decreases from benign melanocytic nevi to metastatic melanomas. Our results suggest an important role of MTAP inactivation in the development of melanomas. This finding may be of great clinical significance because recently an association between MTAP activity and interferon sensitivity has been suggested.