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Laboratory of Experimental Biology and Physiology  

Lab. Limonta

 

Responsable
Prof. Patrizia Limonta  Associate Professor patrizia.limonta@unimi.it

Team
Dr. Roberta Manuela Moretti  Assistant Professor  roberta.moretti@unimi.it
Dr. Marina Montagnani Marelli Assistant Professor marina.marellimontagnani@unimi.it
Dr. Monica Marzagalli PhD student monica.marzagalli@unimi.it

 

Research interests

The main field of research of the laboratory is the clarification of the molecular/cellular mechanisms of the neoplastic process. In particular, the aim of the research performed is to investigate the expression of  hormone receptors and their role in the molecular mechanisms controlling tumor growth and progression. The ultimate goal of the research is to identify new biological markers and, therefore, molecular targets for the development of npvel cancer therapeutic strategies. 

Role of GnRH and its receptors in the control of prostate cancer growth and progression
Prostate cancer is the second leading cause of cancer death in Western countries. Early-stage prostate cancer is often androgen-dependent, the treatment of choice is represented by agonistic analogs of Gonadotropin-Releasing Hormone (GnRH) which cause a blockage of the pituitary-gonadal axis and, therefore, the production of testosterone. However, after an initial phase of regression, the tumor starts growing with characteristics of androgen-independence. Treatment options for androgen-independent prostate cancer are still limited.

Studies carried out in our laboratory have shown that GnRH receptors are expressed not only in pituitary cells , but also in prostate cancer cells , both at the mRNA and at the protein level. Activation of these receptors, obtained by using GnRH agonists, significantly reduces cell proliferation, both in vitro and in vivo (in nude mice xenografted with human prostate cancer cells), as well as the migratory and invasive behavior of prostate cancer cells, and this action is mediated by a reduction of the expression and / or activity of molecules involved in the metastatic behavior of cancer cells (integrins and matrix metalloproteases). GnRH agonists act by interfering with the mitogenic and pro-metastatic action of growth factors such as EGF and IGF-I, blocking the signal transduction pathways associated with their tyrosine kinase receptors. Since these compounds act on prostate cancer cells both androgen-dependent and androgen-independent (castration resistant), the results obtained from these studies suggest that they may exert a direct antitumor effects  also on prostate cancer in its advanced stages. Finally, recent data suggest that GnRH agonists are able to sensitize and re-sensitize chemotherapy-resistant cells to the proapototic activity of chemotherapeutic drugs. 

Role of GnRH and its receptor in the control of melanoma growth and progression
Cutaneous melanoma is a cancer characterized by a rapid growth and early acquisition of high metastatic properties. Treatment options for melanoma are still very limited. Studies conducted by the laboratory have shown that GnRH receptors are expressed on melanoma cells (derived from patients at different stages of disease progression), a tumor not classically related to the reproductive system. The activation of these receptors significantly reduces both proliferation that the metastatic behavior of cancer cells. This effect is accompanied by a reduction of α3 integrin subunit expression levels and of the activity of matrix metalloproteinases-2. Gene expression analysis experiments (microarray studies performed using Affymetrix Human Genome U133 Plus 2.0 Array) have shown that the activation of the GnRH receptors significantly reduces the expression of the pro-angiogenic factor VEGF. These observations were also confirmed by Real Time-PCR and ELISA assay of VEGF secreted by the cells. Moreover, the GnRH receptor is also expressed in endothelial cells of human umbilical vein (HUVEC) and its activation counteracts the pro-angiogenic action of VEGF. Thus, the activation of the GnRH receptors reduces not only the growth and the metastatic behavior of melanoma, but also its proangiogenic properties.

Taken together, these results indicate that GnRH receptors can be considered as a new molecular target for the development of novel therapies for the treatment of melanoma.

Role of two different isoforms of clusterin in the process of prostate tumorigenesis
Clusterin is a heterodimeric glycoprotein, highly glycosylated, which is expressed in several tissues where it is involved in the control of different biological functions. In addition to this isoform, named sCLU as it is secreted by cells, a shorter form has been identified, which lacks the signal peptide and, therefore, it does not enter the endoplasmic reticulum and is addressed to the nucleus (nCLU, nuclear clusterin). nCLU would result from alternative splicing or alternative translation of clusterin RNA. Research conducted in the laboratory, by means of overexpression experiments, have shown that nCLU isoform is responsible for a significant proapoptotic and antimetastatic activity, both in non tumoral epithelial prostatic cells and in tumoral cells. On the contrary, sCLU is proapoptotic and antimetastatic in normal cells but not in tumor cells. To understand the causes of the different role of the two isoforms of CLU, it is speculated that the protein can undergo a different pattern of expression in normal cells or tumor cells. In the Laboratory it has been demonstrated that, after overexpression of the full-length form of CLU in normal cells, both sCLU and nCLU are formed, while in tumor cells only sCLU is formed. This indicates that cancer cells loose the mechanisms that lead to the formation of proapoptotic isoform of the protein. These results demonstrate that the pattern of production of the two clusterin isoforms may play a crucial role in the tumorigenesis of the prostate.

Molecular basis for the development of novel chemopreventive and therapeutic strategies in melanoma: role of estrogen receptor beta and tocotrienols
As already pointed out, based on the increased incidence of melanoma, the poor prognosis and the poor results obtained with current drugs therapies (interferon-α, dacarbazine, IL-2, etc.), it clearly emerges that the identification of new molecular markers for the improvement of the diagnosis and the development of novel therapeutic strategies, more effective and less toxic, is imperative.

Different experimental observations indicate that the estrogen receptor β (ERb ) is associated with antitumor activity, on the basis of clinical reports demonstrating that ERβ, but not ERa, is the main estrogen receptor expressed in melanocytic lesions and its expression decreases significantly during tumor progression. Furthermore, it is known that the nutraceuticals can exert a specific antitumor effect not only on the basis of their antioxidant properties, but also on the basis of their ability to interfere with the molecular processes underlying the development of tumors. Preliminary data obtained in the laboratory indicate that ERb is expressed in human melanoma cells and its activation reduces cell proliferation. Moreover, tocotrienols, derivatives of vitamin E, are able to exert an antiproliferative effect on melanoma cells, through the activation of ERβ. Studies are at present ongoing to elucidate the molecular mechanisms of the anticancer action of ERβ in melanoma and its interaction with tocotrienols. The purpose of this field of research is to identify novel therapeutic and/or chemopreventive strategies for this tumor which is still incurable.

 

Techniques

Cell  cultures
Methods for assessment of cell growth, motility and invasiveness: cell count, MTT, Boyden's chamber, invasion and adhesion assays
Clonogenic assay
Methods for assessment of angiogenic behavior: endothelial tube formation
Gel electrophoresis: Western blot, immunoprecipitation and zymography
Immunofluorescence
RT-PCR and real time PCR
Recombinant DNA techniques
Gene silencing (RNAi)

 

Selected publications

1. Limonta P, Montagnani Marelli M, Mai S, Motta M, Martini L, Moretti RM (2012) GnRH receptors in cancer: from cell biology to novel targeted therapeutic strategies. Endocr Rev33:784-811.

2. Moretti RM, Mai S, Montagnani Marelli M, Rizzi F, Bettuzzi S, Limonta P (2011) Molecular mechanisms of the antimetastatic activity of nucelar clusterin in prostate cancer cells. Int J Oncol39:225-34.

3. Moretti RM, Mai S, Montagnani Marelli M, Bani MR, Ghilardi C, Giavazzi R, Taylor DM, Martini PG, Limonta P (2010) Dual targeting of tumor and endothelial cells by gonadotropin- releasing hormone agonists to reduce melanoma angiogenesis. Endocrinology151:4643-53.

4. M. Montagnani Marelli M, R.M. Moretti  RM, S. Mai S, J. Januszkiewicz-Caulier J, M. Motta M, P. Limonta P (2009) Type I gonadotropin-releasing hormone  receptor mediates the  antiproliferative effects of GnRH-II on prostate cancer cells. J Clin Endocrinol Metab94:1761-67.

5. Moretti RM, Montagnani Marelli M, Mai S, Cariboni A, Scaltriti M, Bettuzzi S, Limonta P (2007) Clusterin isoforms differentially affect growth and motility of prostate cells: possible implications in prostate tumorigenesis. Cancer Res67:10325-33.

6. Montagnani Marelli M,  Moretti RM, Junuszkiewicz-Caulier J,  Motta M, Limonta P (2006) Gonadotropin.-Releasing Hormone (GnRH) receptors in tumors: a new rationale for the therapeutic application of GnRH analogs in cancer patients. Current Cancer Drug Target 6:257-269.

7. Montagnani Marelli M, Moretti RM,  Procacci P, Motta M, Limonta P (2006) Insulin-like growth factor-I promotes migration in human androgen-independent prostate cancer cells via the alphavbeta3 integrin and PI3/Akt signaling. Int J Oncol28:723-30.

8. Limonta P, Moretti RM, Montagnani Marelli M, Motta M (2003) The biology of gonadotropin hormone- releasing hormone: role in the control of tumor growth and progression in humans. Front Neuroendocrinol   24:279-95.

9. Moretti RM, Montagnani Marelli M, van Groeninghen JC, Motta M, Limonta P (2003) Inhibitory activity of luteinizing hormone-releasing hormone on tumor growth and progression. Endocr-Relat Cancer10:161-167.

10. Moretti RM, Montagnani Marelli M, Groeninghen JC, Limonta P (2002) Locally expressed luteinizing hormone-releasing hormone (LHRH) receptors mediate the oncostatic and antimetastatic activity of LHRH agonists on melanoma cells. J Clin Endocrinol Metab87:3791-97.

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