Clusterin transcript variants expression in thyroid tumor: a potential marker of malignancy?

BACKGROUND: Clusterin (CLU) is a ubiquitous multifunctional factor involved in neoplastic transformation. The CLU transcript variants and protein forms play a crucial role in balancing cells proliferation and death. METHODS: We investigated the regulation of CLU transcript variants expression in an in vivo model system consisting of both neoplastic tissues and fine needle aspiration biopsy (FNAB) samples isolated from patients undergoing thyroidectomy. RESULTS: The immunohistochemical analyses showed an overall CLU up-regulation in papillary carcinoma. A specific CLU2 transcript variant increase was registered using qPCR in papillary carcinomas while CLU1 decreased. In addition, the analysis of CLU transcripts expression level showed an increase of the CLU2 transcript in the TIR 3 patients with histologically confirmed thyroid cancer. CONCLUSIONS: Our results suggest the existence of a specific alteration of CLU2:CLU1 ratio towards CLU2, thus providing the first circumstantial evidence for the potential use of CLU transcript variants as effective biomarkers for a more accurate assessment of the so called "indeterminate" thyroid nodules.

Regulation of the expression of CLU isoforms in endometrial proliferative diseases.

Clusterin (CLU) is a nearly ubiquitous multifunctional protein synthesized in different functionally divergent isoforms, sCLU and nCLU, playing a crucial role by keeping a balance between cell proliferation and death. Studying in vivo CLU expression we found a higher mRNA expression both in neoplastic and hyperplastic tissues in comparison to normal endometria; in particular, by RT-qPCR we demonstrated an increase of the specific sCLU isoform in the neoplastic and hyperplastic endometrial diseases. On the contrary, no CLU increase was detected at the protein level. The CLU gene transcriptional activity was upregulated in the hyperplastic and neoplastic tissues, indicating the existence of a fine post-trans-criptional regulation of CLU expression possibly responsible for the protein decrease in the malignant disease. A specific CLU immunoreactivity was present in all the endometrial glandular cells in comparison to the other cellular compartments where CLU immunoreactivity was lower or absent. In conclusion, our results suggest the existence of a complex regulatory mechanism of CLU gene expression during the progression from normal to malignant cells, possibly contributing to endometrial carcinogenesis. Moreover, the specific alteration of the sCLU:nCLU ratio associated with the pathological stage, suggests a possible usage of CLU as molecular biomarker for the diagnosis/prognosis of endometrial proliferative diseases.

Regulation of TGF-ß1 expression by androgen deprivation therapy of prostate cancer.

In this paper we studied the in vivo neoadjuvant Androgen Deprivation Therapy (ADT) effect on the expression of TGF-ß1 and its receptor Tß-RII. Mechanisms of androgen dependence are critical to understanding prostate cancer progression to androgen independence associated with disease mortality, and TGF-ß is thought to support prostatic apoptosis as its expression coincides with androgen ablation in benign and cancer tissues. Increase of both mRNA and protein level were shown for the first time only in the patients who underwent neoadjuvant ADT for 1-month. This transient increase of TGF-ß expression after androgen ablation suggested cooperation of the pathways in prostate regression. Since no alteration was observed in the gene transcriptional activity, the molecular mechanism of this cooperation, probably act at the post-transcriptional level. TGF-ß1 and Tß-RII specific signals were co-localized within the neoplastic prostate epithelium. Our results suggests that the androgens deprivation by means of ADT for 1-month, involves a shift of the TGF-ß1 mechanism in prostate cancer, suggesting that the TGF-ß1 promotes prostate epithelial cell proliferation and inhibits apoptosis in a autocrine way.

Androgen deprivation therapy affects BCL-2 expression in human prostate cancer.

BCL-2 is an integral protein of the external mitochondrial membrane that inhibits cell apoptotic death. We investigated the effect of androgen deprivation therapy (ADT) on BCL-2 expression in prostate cancer tissues. We studied BCL-2 expression in vivo in prostate cancer tissues obtained from patients who underwent radical prostatectomy after neoadjuvant ADT, by Northern and Western blot analysis, and immunohistochemistry. Moreover, gene transcriptional activity was also measured by nuclear run-on experiments. We demonstrated an increase of BCL-2 mRNA expression in patients who underwent neoadjuvant ADT for 1 month in comparison to patients who had not received any therapy. Moreover, we demonstrated that there were no significant modifications of BCL-2 mRNA levels in patients who underwent neoadjuvant ADT for 3 and 6 months. Furthermore, BCL-2 protein levels in patients who underwent neoadjuvant ADT for 1 month were upregulated in comparison to patients who had not received any therapy. Immunohistochemical analysis showed a strong positivity of prostate cells depending on ADT administration for 1 month. Finally, transcriptional activity was not modified in patients who underwent neoadjuvant ADT, suggesting the absence of hormonal regulation on BCL-2 gene expression at the transcriptional level. Our data show that short-term administration of ADT interferes with BCL-2 expression, suggesting that androgen-mediated mechanisms may act through BCL-2-mediated apoptotic pathways. Moreover, since short-term ADT administration does not interfere with BCL-2 expression at the transcriptional level, the androgen-mediated mechanisms involving BCL-2 pathways, probably act at the post-transcriptional level.

Androgen deprivation therapy regulation of beta1C integrin expression in prostate cancer.

The beta1C integrin is an alternatively spliced variant of the beta1 integrin subfamily that at variance with its wild-type counterpart, i.e., the beta1A integrin, inhibits cell proliferation in prostate cancer cells. We have recently shown that transcriptional, translational and post-translational processes contribute to the selective loss of beta1C integrin during prostate malignant transformation. Here, we investigated whether androgen deprivation therapy (ADT) may affect beta1C mRNA expression in prostate cancer. Neoplastic prostates were obtained from patients undergoing radical prostatectomy who had received neoadjuvant ADT. The beta1C mRNA level was measured by Northern hybridization experiments and compared to normal prostates obtained from patients who underwent radical cystoprostatectomy for bladder cancer. Furthermore, the beta1C integrin gene transcriptional activity was measured by nuclear Run-on assays. We found an increase of beta1C mRNA expression (208+/-11%; p<0.01) in patients who received ADT in comparison to those who did not. Furthermore, we demonstrated an increase of gene transcriptional activity (360+/-10%; p<0.01) possibly partially or completely responsible for the regulation of the beta1C integrin mRNA levels. Short-term administration of ADT seems to interfere with beta1C integrin expression, suggesting the existence of androgen-mediated pathways involving beta1C. Precise characterization of the mechanisms that regulate the expression of this factor in cancer cells will provide further insight into the molecular mechanisms involved in tumor progression and possibly contribute to the identification of molecular targets for the development of new therapeutic strategies.

Hepatocyte 'priming' and increase in transforming growth factor-beta1 mRNA expression are delayed in hypothyroid versus euthyroid rats during liver regeneration.

Hypothyroidism decreases liver weight and delays the compensatory liver growth after partial hepatectomy (PH) as compared with the euthyroid condition. The aim of this study was to investigate, in hypothyroid rats, the mRNA expression of genes modulating these effects, focusing on c-fos and c-myc, hallmarks of hepatocyte 'priming', and on transforming growth factor-beta1 (TGF-beta1) and its receptor, the transforming growth factor-beta1 receptor-type II (TbetaR-II), negative regulators of liver growth. Euthyroid and hypothyroid male Wistar rats underwent 70% PH and total RNA was isolated from frozen liver samples removed at basal state and during regeneration, 0-144 h after surgery. In this study, we show for the first time that, in the basal liver state, hypothyroidism increased TGF-beta1 and TbetaR-II mRNA levels by 45% and 30%, respectively, as compared with the euthyroid condition and, after PH, resulted in a approximately 12-h delay in the activation of c-fos and c-myc mRNA expression. Moreover, the increase in TGF-beta1 mRNA levels, detected 24-48 h after PH in euthyroid rats, was delayed by 72 h in hypothyroid rats, occurring when a concomitant reduction in TbetaR-II was measured. These results suggest that, in hypothyroid rats, at the basal liver level, the increase in mRNA expression of genes that negatively regulate liver growth might be involved in the decrease in liver weight and that, after PH, the delay of hepatocyte 'priming' and coordinated changes in mRNA expression of negative regulators of liver regeneration might be involved in delaying the regenerative process.

Transcriptional regulation of beta1 integrin expression in the physio/pathological states of human endometrial tissues.

beta1C integrin is a spliced variant of the human beta1 integrin family that inhibits cell proliferation, at variance with beta1A that stimulates it. During the transition from normal to neoplastic endometrium, both variants are down-regulated at the mRNA level, but only beta1C at the protein level, suggesting a key role of the regulation of beta1C integrin expression in the pathogenesis of endometrial cancer. In this study we show for the first time that, besides beta1A and beta1C, the beta1B spliced variant is expressed in human endometrium, and is up-regulated in hyperplastic and neoplastic endometria in comparison with normal proliferative endometria. To investigate the mechanisms of regulation of beta1 integrin expression during endometrial cancer progression we compared the transcriptional activity of the beta1 integrin gene in normal and diseased endometria by nuclear run-on analysis and we found it significantly reduced in endometrial adenocarcinoma. On the contrary, hyperplastic endometria showed a 2-fold increase in the beta1 transcription rate that directly correlated with the increase in beta1B, beta1C and beta1A steady-state mRNA levels. Finally, we compared the activity of the distal and proximal promoters of the beta1 gene integrin gene in normal and diseased endometria and we found the activity of the proximal promoter decreased in neoplastic endometria and increased in hyperplastic tissues, whereas the activity of the distal promoter did not change in different endometrial physio/pathological conditions. These findings suggest a complex pattern for regulation of the expression of beta1 integrin variants during endometrial malignant transformation.

Molecular and functional characteristics of erbB2 in normal and cancer breast cells.

The expression pattern of erbB2 and its transmembrane polymorphisms (Ile654Val and Ile655Val) were investigated in a panel of human normal and neoplastic breast cell lines to evaluate whether the expression pattern was affected by changes in the gene structure. At least two peptides of lower molecular mass forms (95 and 68 kDa) than the holoreceptor (185 kDa), comprehensive of the tyrosine kinase domain, were detected in all cells. Both peptides were also phosphorylated, suggesting a functional role in signal transduction. The presence of the polymorphisms found in two cell lines was unrelated to the expression of the lower molecular mass proteins.

Regulation of beta1C and beta1A integrin expression in prostate carcinoma cells.

beta(1C) and beta(1A) integrins are two splice variants of the human beta(1) integrin subfamily that act as an inhibitor and a stimulator of cell proliferation, respectively. In neoplastic prostate epithelium, both these variants are down-regulated at the mRNA level, but only beta(1C) protein levels are reduced. We used an experimental model consisting of PNT1A, a normal immortalized prostate cell line, and LNCaP and PC-3, two prostate carcinoma cell lines, to investigate both the transcription/post-transcription and translation/post-translation processes of beta(1C) and beta(1A). Transcriptional regulation played the key role for the reduction in beta(1C) and beta(1A) mRNA expression in cancer cells, as beta(1C) and beta(1A) mRNA half-lives were comparable in normal and cancer cells. beta(1C) translation rate decreased in cancer cells in agreement with the decrease in mRNA levels, whereas beta(1A) translation rate increased more than 2-fold, despite the reduction in mRNA levels. Both beta(1C) and beta(1A) proteins were degraded more rapidly in cancer than in normal cells, and pulse-chase experiments showed that intermediates and/or rates of beta(1C) and beta(1A) protein maturation differ in cancer versus normal cells. Inhibition of either calpain- or lysosomal-mediated proteolysis increased both beta(1C) and beta(1A) protein levels, the former in normal but not in cancer cells and the latter in both cell types, albeit at a higher extent in cancer than in normal cells. Interestingly, inhibition of the ubiquitin proteolytic pathway increased expression of ubiquitinated beta(1C) protein without affecting beta(1A) protein levels in cancer cells. These results show that transcriptional, translational, and post-translational processes, the last involving the ubiquitin proteolytic pathway, contribute to the selective loss of beta(1C) integrin, a very efficient inhibitor of cell proliferation, in prostate malignant transformation.

beta 1C Integrin expression in human endometrial proliferative diseases.

Integrins are ubiquitous cell adhesion molecules that are involved in maintaining normal tissue morphology and have been implicated in the aggressive behavior of several malignancies. beta 1C integrin is an alternatively spliced variant of the beta 1A integrin subunit that, at variance with beta 1A, inhibits epithelial cell proliferation. beta 1C integrin is expressed in non-proliferative, benign prostatic epithelium and is down-regulated in prostatic adenocarcinoma. In the current study, we examined beta 1C expression at mRNA and protein levels in 18 endometrial adenocarcinoma and in 20 endometrial hyperplastic tissues, using Northern and Western blotting analysis and immunohistochemistry. The pattern of integrin expression was compared to that of the endometrium of 14 normal cycling women. The results of this study document inhibited beta 1C integrin expression in endometrial adenocarcinoma, both at the mRNA and protein levels, at variance with significantly up-regulated beta 1C mRNA expression in endometrial hyperplasia, in comparison with normal proliferative endometria. Our data suggest a key role of the regulation of beta 1C integrin expression in the pathogenesis of endometrial proliferative diseases: beta 1C integrin may act as growth modulator in cancer cells, playing a role in downstream intracellular signaling.

Transcriptional regulation of the beta1C integrin splice variant in human prostate adenocarcinoma.

Members of the integrin family of cell adhesion receptors influence several important aspects of cancer cell behaviour, including motility and invasiveness, cell growth and cell survival. beta1C integrin, an alternatively spliced variant of the human beta1 integrin, has been shown to inhibit cell proliferation. We have previously demonstrated that beta1C integrin mRNA and protein are present in normal prostate and are down-regulated in prostate adenocarcinoma. To explore some of the molecular mechanisms regulating beta1C integrin gene expression, we have analysed the transcriptional activity of the beta1 integrin gene in neoplastic and normal human prostate tissue. Run-on analysis demonstrates that the transcription rate of the beta1 integrin gene is significantly reduced in prostate cancer specimens compared to normal prostate, thus accounting for the reduction in mRNA levels of the beta1 integrin variants. Moreover, the decrease in transcriptional activity of the beta1 integrin gene directly correlates to the reduction of beta1C integrin steady-state mRNA levels (r=0.78).

Differential expression of beta 1c integrin messenger ribonucleic acid and protein levels in human endometrium and decidua during the menstrual cycle and pregnancy.

beta(1C) and beta(1A) integrins are alternatively spliced variants of the human beta(1)-subunit; the former has been shown to inhibit cell proliferation, and the latter to promote it. Although some components of the beta(1) integrin subfamily are expressed in human endometrial and decidual cells during the menstrual cycle and early pregnancy, to date no information is available about the expression of beta(1C) integrin in endometrial and decidual tissues and its possible roles during implantation and pregnancy. To gain further insight on this subject, we have explored beta(1C) integrin expression in endometrial (proliferative, secretory, and atrophic) and decidual (from the first and third trimesters of pregnancy) tissue samples at both gene and protein levels by Northern and Western blotting analyses and by immunohistochemistry. beta(1A) protein levels were also measured in the same tissues as a control. The results of this study demonstrate that both beta(1C)- and beta(1A)-subunits are expressed in the endometrium and decidua. In the former, maximal beta(1C) expression was detected in atrophic endometria, whereas beta(1A) expression levels were increased in secretive and decreased in atrophic endometrial tissues compared with proliferative endometria. In addition, whereas beta(1A) levels were significantly increased in decidual tissues, compared with proliferative endometria, beta(1C) expression was dramatically reduced in the same tissues, thus pointing to selective down-regulation of beta(1C) expression in the decidua. These data suggest that the expression of beta(1C) integrin, a very efficient inhibitor of cell proliferation, may be modulated by the maternal microenvironment and may play a fundamental role in mediating trophoblast outgrowth and migration during pregnancy.