Melanoma-associated cancer-testis antigen 16 (CT16) regulates the expression of apoptotic and antiapoptotic genes and promotes cell survival.

Cancer-testis (CT) antigens are predominantly expressed in testis or placenta, but absent in most adult tissues. During malignant transformation CT genes are often activated. CT antigen 16 (CT16, PAGE5) is frequently expressed in advanced melanoma but its biological function has been unknown. To examine the role of CT16 in cell survival we knocked it down in A2058 melanoma cells using specific siRNAs and exposed the cells to cancer drug cisplatin known to induce apoptosis. As a result, cell survival was markedly decreased. To study the effects of CT16 on cell survival in more detail, the cellular gene expression profiles were investigated after CT16 silencing in CT16 positive A2058 melanoma cells, as well as after CT16 overexpression in CT16 negative WM-266-4 melanoma cells. Among the 11 genes both upregulated by CT16 silencing and downregulated by CT16 overexpression or vice versa, 4 genes were potentially apoptotic or antiapoptotic genes. CT16 was recognized as a positive regulator of antiapoptotic metallothionein 2A and interleukin 8 genes, whereas it inhibited the expression of apoptosis inducing dickkopf 1 (DKK1) gene. In addition CT16 enhanced the expression of fatty acid binding protein 7, a known promoter of melanoma progression. The effect of CT16 on DKK1 expression was p53 independent. Furthermore, CT16 did not regulate apoptotic genes via DNA methylation. In twenty melanoma metastasis tissue samples average DKK1 mRNA level was shown to be significantly (p<0.05) lower in high CT16 expressing tumors (n = 3) when compared to the tumors with low CT16 expression (n = 17). Thus, our results indicate that CT16 promotes the survival of melanoma cells and is therefore a potential target for future drug development.

Detection of melanoma-derived cancer-testis antigen CT16 in patient sera by a novel immunoassay.

Cancer-testis antigens (CTAs) are expressed mainly in various cancer tissues and in testis or placenta. Because of their restricted expression pattern, the CTAs can be potentially used for vaccine development and diagnostic applications. CTA CT16 has been found to be expressed in lung and renal cancers as well as in melanomas. Detection of CT16 protein directly from patient serum could facilitate monitoring of tumor growth and response to therapy in CT16-positive patients. A highly sensitive time-resolved fluorescence-based immunoassay measuring CTA CT16 in serum was developed. Generally, CTAs have not been measured directly from body fluids. CT16 level was detectable in 14 of 23 (61%) patients with metastatic melanoma, whereas none of the nine healthy volunteers collected by us had measurable CT16 level. For an unknown reason, 1 of 20 commercial control serum samples gave a positive result. The Wilcoxon-Mann-Whitney exact test showed statistically significant difference when patients with metastatic melanoma were compared to our control group (p = 0.006) or to the commercial set (p < 0.001). Four melanoma patients had exceptionally high serum CT16 level. CT16 did not correlate either with S100B, a recognized marker of progressing melanoma, or with unspecific serum marker lactate dehydrogenase. Elevation of CT16 titers preceded or followed the clinical diagnosis of disease progression in four patients with metastatic melanoma. As a conclusion, our results show that CT16 protein can be measured directly from patient serum, and the developed assay has a potential for clinical use.

Calpain 1 and 2 are required for RNA replication of echovirus 1.

Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.

Increased gene expression levels of collagen receptor integrins are associated with decreased survival parameters in patients with advanced melanoma.

Expression of collagen receptor integrins alpha1beta1 and alpha2beta1 has been associated with progression and metastatic potential of malignant melanoma. Integrin alpha2beta1 was originally characterized as a melanoma progression antigen. We have used real-time quantitative PCR to study the mRNA expression levels of three collagen receptor integrin chains, that is alpha1, alpha2 and alpha11 in metastases from 26 patients with melanoma. Interestingly, we find that survival after initiation of chemoimmunotherapy was significantly decreased in all patients whose tumours expressed high mRNA levels of alpha1 integrin, alpha2 integrin or alpha11 integrin when compared with lower tumour expression levels (P<0.05, log rank test). Moreover, those patients with high mRNA levels of all studied integrins had a significantly shorter survival from the appearance of the first metastasis than the patients with low levels of integrins (P<0.05). Furthermore, a high mRNA expression level of integrin alpha2 was found to be associated with poorer overall survival. High alpha2 mRNA levels (n=6) were associated with median survival of 35 months and low alpha2 mRNA levels (n=20), with median survival of 53 months (P=0.033). We conclude that collagen receptor integrins are important in the progression and prognosis of metastatic melanoma, and their measurements might be used as predictive markers when assessing disease progression.

Regulation of prostate cell collagen receptors by malignant transformation.

Cell adhesion receptors, including the integrin-type collagen receptors (alpha1beta1, alpha2beta1, alpha10beta1 and alpha11beta1) participate in cancer progression and invasion. Quantitative RT-PCR indicated that all 4 receptors are abundantly expressed in sarcoma-derived cell lines, whereas most carcinoma-derived cells express alpha1beta1 and alpha2beta1 only. This was surprising because alpha11beta1 has been connected previously to the progression of lung adenocarcinomas. To test the hypothesis that alpha11 expression may not persist in cultured cancer cells we analyzed fresh tissue samples of 104 total prostatectomies, keeping in mind that prostate cancer cell lines showed negligible alpha11 mRNA levels. In prostate alpha2 expression was significantly lower in poorly differentiated carcinomas when compared to benign lesions (p = 0.0331). In immunohistochemistry the protein levels of alpha2 integrin decreased significantly (p = 0.0001) and the protein levels of alpha11 subunit increased significantly (p = 0.029) with the increasing grade of carcinoma. Thus alpha11beta1 may replace alpha2beta1 during tumor progression. Our observations support the idea that alpha11beta1 may be expressed in tumors but the corresponding cell lines may lose the expression of this integrin. Previous studies have shown that in cell culture androgen receptor (AR) controls alpha2beta1 expression. We measured AR mRNA levels and the number of AR positive nuclei in the prostate samples and the results showed a significant correlation between alpha2beta1 and AR. Androgen receptors may control the mechanisms regulating integrin expression in prostate.