Elda Perlino

Regulation of mRNA and Protein Levels of β1 Integrin Variants in Human Prostate Carcinoma

Alterations of integrin expression levels in cancer cells correlate with changes in invasiveness, tumor progression, and metastatic potential. The beta1C integrin, an alternatively spliced form of the human beta1 integrin, has been shown to inhibit prostate cell proliferation. Furthermore, beta1C protein levels were found to be abundant in normal prostate glandular epithelium and down-regulated in prostatic adenocarcinoma. To gain further insights into the molecular mechanisms underlying abnormal cancer cell proliferation, we have studied beta1C and beta1 integrin expression at both mRNA and protein levels by Northern and immunoblotting analysis using freshly isolated neoplastic and normal human prostate tissue specimens. Steady-state mRNA levels were evaluated in 38 specimens: 33 prostatic adenocarcinomas exhibiting different Gleasons grade and five normal tissue specimens that did not show any histological manifestation of benign prostatic hypertrophy. Our results demonstrate that beta1C mRNA is expressed in normal prostate and is significantly down-regulated in neoplastic prostate specimens. In addition, using a probe that hybridizes with all beta1 variants, mRNA levels of beta1 are found reduced in neoplastic versus normal prostate tissues. We demonstrate that beta1C mRNA down-regulation does not correlate with either tumor grade or differentiation according to Gleasons grade and TNM system evaluation, and that beta1C mRNA levels are not affected by hormonal therapy. In parallel, beta1C protein levels were analyzed. As expected, beta1C is found to be expressed in normal prostate and dramatically reduced in neoplastic prostate tissues; in contrast, using an antibody to beta1 that recognizes all beta1 variants, the levels of beta1 are comparable in normal and neoplastic prostate, thus indicating a selective down-regulation of the beta1C protein in prostate carcinoma. These results demonstrate for the first time that beta1C and beta1 mRNA expression is down-regulated in prostate carcinoma, whereas only beta1C protein levels are reduced. Our data highlight a selective pressure to reduce the expression levels of beta1C, a very efficient inhibitor of cell proliferation, in prostate malignant transformation.

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

β1C and β1A integrins are two splice variants of the human β1 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 β1C 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 β1C and β1A. Transcriptional regulation played the key role for the reduction in β1C and β1A mRNA expression in cancer cells, as β1C and β1A mRNA half-lives were comparable in normal and cancer cells. β1C translation rate decreased in cancer cells in agreement with the decrease in mRNA levels, whereas β1A translation rate increased more than 2-fold, despite the reduction in mRNA levels. Both β1C and β1A proteins were degraded more rapidly in cancer than in normal cells, and pulse-chase experiments showed that intermediates and/or rates of β1C and β1A protein maturation differ in cancer versus normal cells. Inhibition of either calpain- or lysosomal-mediated proteolysis increased both β1C and β1A 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 β1C protein without affecting β1A 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 β1C integrin, a very efficient inhibitor of cell proliferation, in prostate malignant transformation.

Down-regulated expression of transforming growth factor beta 1 mRNA in endometrial carcinoma

Transforming growth factor beta1 (TGF-beta1) is a potent modulator of cell proliferation in vitro, and recent studies have demonstrated its overexpression in several different tumours; nevertheless, the molecular mechanisms of TGF-beta1 action on cell growth and differentiation have not been fully elucidated. To clarify the role of TGF-beta and its receptor in human endometrial proliferation and differentiation, TGF-beta1 expression at both the mRNA and protein levels has been evaluated by using Northern blotting and immunohistochemistry, in both normal (atrophic, proliferative and secretory) and neoplastic (adenocarcinoma) endometrial samples. This study demonstrates that TGF-beta1 mRNA expression is dramatically reduced in endometrial carcinomas with respect to non-neoplastic tissues, whereas the immunohistochemical expression of TGF-beta1 is enhanced in the epithelial component of endometrial carcinomas compared with non-neoplastic tissues. These data suggest that TGF-beta1 acts as a paracrine regulator of endometrial cell proliferation and that it may contribute to the carcinogenic mechanisms of endometrial carcinoma.

Insulin-like growth factor-I expression in normal and diseased endometrium

While the role of steroid hormones in the regulation of endometrial proliferation and differentiation is well established, the effects of growth factors and their receptors in normal and neoplastic endometrium remain a matter of debate. Previous studies have documented the positive effects of insulin‐like growth factor‐I (IGF‐I) on epithelial cell proliferation and the active production of this growth factor in endometrial tissues. In view of decreased expression of transforming growth factor‐β1 (TGF‐β1), an antagonist of IGF‐I, in endometrial carcinoma, we investigated the expression of IGF‐I, at both the mRNA and protein levels, and the immunoreactivity for type I IGF‐I receptor in 30 formalin‐fixed, paraffin‐embedded tissue samples of normal and neoplastic endometrium, in order to possibly clarify the role of IGF‐I in endometrial proliferation and differentiation. Our results demonstrate a reduced expression of IGF‐I mRNA in endometrial carcinomas compared with non‐neoplastic tissues, despite equivalent immunohistochemical expression of IGF‐I and IGF‐I receptor. Our data suggest that IGF‐I and its corresponding receptor may not be directly involved in endometrial cancer cell proliferation and differentiation in vivo, though other components of the IGF‐I system (e.g., IGF binding proteins) may affect endometrial malignant transformation and tumor progression. Int. J. Cancer 80:188–193, 1999.

The IGF-I axis in thyroid carcinoma.

Insulin like-growth factor I (IGF-I) has been involved in the pathogenesis of a variety of human neoplasia due to the mitogenic and anti-apoptotic properties of its cognate receptor. In human thyroid carcinomas, we have previously documented an increased immunoreactivity of both IGF-I and the IGF-I receptor (IGF-I R) associated with up regulation of IGF-I mRNA . Immunoreactivity of IGF-I and cognate receptor positively correlated with tumor diameter and wide intrathyroidal extension but not with patients gender and age or with the stage of the tumors and the occurrence of limph node metastases. Most experimental studies indicate that the effects of IGF-I on target cells are regulated in a complex fashion and depend on the simultaneous occurrence of IGF-IR and the binding proteins.

Regulation of TGF-β1 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-β1 and its receptor Tβ-RII. Mechanisms of androgen dependence are critical to understanding prostate cancer progression to androgen independence associated with disease mortality, and TGF-β 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-β 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-β1 and Tβ-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-β1 mechanism in prostate cancer, suggesting that the TGF-β1 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.

β1C 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.

TGF-beta 1 and IGF-1 expression in atrophic post-menopausal endometrium.

OBJECTIVES Endometrial cells may synthetize cytokines and growth factors which may modulate some of the molecular mechanisms of endometrial proliferation and differentiation. PATIENTS AND METHODS We investigated the role of transforming growth factor beta-1 (TGF-beta 1), insulin-like growth factor-1 (IGF-1) and relative receptors in five tissue samples from atrophic post-menopausal endometria. The control group was represented by proliferative and secretory endometria from 10 healthy, normally-menstratued women. TGF-beta 1 and IGF-1 m-RNA expression was evaluated by Northern hybridization analysis, while TGF-beta 1 and IGF-1 receptors distribution was studied by immunohistochemistry. RESULTS In atrophic endometria Northern hybridization analysis showed a significant decrease of IGF-1 expression, and an increase of TGF-beta 1 expression compared to proliferative and secretory endometria. By immunohistochemistry it was demonstrated that TGF-beta 1 and IGF-1 receptors were both localized in cell cytoplasm, mainly in the stromal compartment. CONCLUSIONS The results of our study would suggest a possible role of IGF-1 and TGF-beta 1 in maintaining the quiescent differentiative state of atrophic post-menopausal endometrium. The persistence of IGF-1 and TGF-beta 1 receptors in epithelial compartment could play a key role in proliferative response of atrophic endometrium to exogenous hormone replacement therapy (HRT) or endogenous intervening high estrogens levels.

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.