HGF modulates actin cytoskeleton remodeling and contraction in testicular myoid cells

The presence of the HGF/Met system in the testicular myoid cells was first discovered by our group. However, the physiological role of this pathway remains poorly understood. We previously reported that HGF increases uPA secretion and TGF-β activation in cultured tubular fragments and that HGF is maximally expressed at Stages VII–VIII of the seminiferous epithelium cycle, when myoid cell contraction occurs. It is well known that the HGF/Met pathway is involved in cytoskeletal remodeling; moreover, the interaction of uPA with its receptor, uPAR, as well as the activation of TGF-β have been reported to be related to the actin cytoskeleton contractility of smooth muscle cells. Herein, we report that HGF induces actin cytoskeleton remodeling in vitro in isolated myoid cells and myoid cell contraction in cultured seminiferous tubules. To better understand these phenomena, we evaluated: (1) the regulation of the uPA machinery in isolated myoid cells after HGF administration; and (2) the effect of uPA or Met inhibition on HGF-treated tubular fragments. Because uPA activates latent TGF-β, the secretion of this factor was also evaluated. We found that both uPA and TGF-β activation increase after HGF administration. In testicular tubular fragments, HGF-induced TGF-β activation and myoid cell contraction are abrogated by uPA or Met inhibitor administratio

R-spondin 1/Dickkopf-1/beta-catenin machinery is involved in testicular embryonic angiogenesis

Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today's most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors' observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis

Correction: C-MET receptor as potential biomarker and target molecule for malignant testicular germ cell tumors

This article has been corrected: The correct author name is given below: Mariano Bizzarri

c-MET proto-oncogene in malignant testicular germ cell tumours

THE THESIS EXPLAINED The biological issue: Testicular Germ Cell Tumours (TGCTs), seminomas and non-seminomas, represent the most frequent malignancy in Caucasian males (20–40 years). Even if diagnosed in an early stage, resulting in good prognosis, a small percentage of cases progress resulting in death in young men. It is commonly accepted that these cancers arise from a disturbed testicular embryonic niche, that leads to the block of gonocyte differentiation. The subsequent development of seminomas and non-seminomas is due to the combination of genetic, epigenetic and microenvironment-based alterations (genvironment). Hepatocyte Growth Factor (HGF) is available in the testicular microenvironment, together with its receptor c-MET, from early embryonic development to adult stage. Moreover, c-MET is a well-known proto-oncogene involved in the onset and progression of various human cancers. Herein, we have investigated the expression and availability of HGF and c-MET in TCam-2, NCCIT and NT2D1 cells, that are Type II GCT representative cell lines, and the effect of c-MET activation/repression in the regulation of cancer biological processes. Moreover, we evaluated the immunoreactivity of c-MET in a cohort of patients affected by TGCTs with the aim to correlate in vitro study with clinical information. Results: Our results demonstrate that TCam-2 (seminoma cell line) and NT2D1 (embryonal carcinoma cell line) show multiple copies of c-MET gene, whereas NCCIT (mixed seminoma/non-seminoma cell line) show a normal gene copy number. They differentially express c-MET receptor, but they do not secrete HGF. Moreover, we found that NT2D1 cells, which have the higher availability of the protein, increase their proliferation, polarized migration and invasion in response to HGF administration. NCCIT respond to HGF stimulation only partially, whereas TCam-2 cells do not respond to HGF at least for the investigated parameters. Moreover, preliminary data show that HGF stimulation is able to modulate, in NT2D1 cells, the expression of several miRNAs, probably involved in cancer progression, but these results need further investigations. Interestingly, the immunohistochemical study of c-MET distribution pattern in TGCT histological samples confirm its presence both in seminoma and non-seminoma lesions with different scores. Noteworthy, we found a higher c-MET immunoreactivity in non-seminoma tumours, in particular in all the most differentiated cancer specimens. Conclusions: On the basis of the results herein reported, this study could shed light on the molecular pathways underlying TGCT onset and progression, and opens new fields of research on the use of c-MET receptor as a potential biomarker for testicular germ cell tumours, since this protein is differentially expressed and distributed in TGCT different histotypes. Moreover, a deeper investigation of HGF/c-MET system in this pathology could allow better prediction of clinical outcome and/or identification of patients at low/high risk of progression

HGF/c-Met pathway in seminoma and non-seminoma cell lines: role in cell invasion.

HGF/c-Met pathway in seminoma and non-seminoma cell lines: role in cell invasion

Microgravity promotes gap junctional contact formation in TCam-2 seminoma cells grown in low adhesion as embryoid bodies.

The study of how mechanical forces may influence cell behavior is a relevant challenge of nowadays that may allow us to define the relationship between mechanics and biochemistry. Literature data reported that microgravity alters cell behavior through cytoskeleton and/or junction modifications. It has been already reported, by our group, that TCam-2 seminoma cells are sensible to microgravity when cultured in adhesion on plastic or glass dishes (Ferranti et al., 2014, Biomed. Res. Int.). However, TCam-2 cells are able to grow also when cultured in low adhesion conditions, forming floating embryoid bodies (EB). This study has the aim to verify if microgravity can affect TCam-2 cell behavior even in absence of cell-substrate adhesion. We investigated the morphological, cytoskeletal and junctional features of TCam-2 cell EB cultured for 24 hours in control and simulated microgravity conditions. Simulated microgravity was obtained using the Random Positioning Machine (RPM; Dutch Space, Netherlands). We observed by TEM, that junctional capability of TCam-2 EB, changes in microgravity condition, being RPM cultured EB involved in gap junctional contacts that do not form in control condition. Noteworthy, gap junction loss is related to seminoma neoplastic progression, and seminoma samples are characterized by an aberrant connexin-43 (CX-43) localization in the Golgi apparatus (Roger et al., 2004, J Pathol; Brehm et al., 2006, Neoplasia). Interestingly, we observed that simulated microgravity-exposed samples show a strong CX-43 membranous staining, whereas, consistent to literature data, a CX-43 low positivity is observable only in the cytoplasm of control cells. In addition, we found that microgravity also affects TCam-2 microtubule organization, strongly indicating that microtubule-dependent membrane trafficking changes in RPM cultured cells. All together these data indicate that TCam-2 cell are sensitive to gravity force changes even in absence of cell-substrate adhesion

IFN-stimulated gene expression is independent of the IFNL4 genotype in chronic HIV-1 infection

This study aimed to evaluate the association between the IFNL4 rs368234815 (ΔG/TT) dinucleotide polymorphism and the IFN response during chronic HIV-1 infection. We carried out genotyping analysis and measured the expression of IFN-stimulated genes (ISGs) (myxovirus resistance protein A [MxA], ISG15, ISG56, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like [APOBEC] 3F and APOBEC3G) on peripheral blood mononuclear cells collected from naïve and HAART-treated HIV-1-infected patients. There were no statistically significant differences in endogenous ISGs mRNA levels among HIV-1-positive patients bearing different IFNL4 genotypes, suggesting that ISG expression is independent of the IFNL4 genotype in HIV-1 infection

c-MET receptor: a potential biomarker for Testicular Germ Cell Tumours.

Testicular Germ Cell Tumours (TGCTs) are a heterogeneous family of neoplasms classified into three groups: type I (infants), type II (adolescents and young adults) and type III (elderly) [1]. The incidence of type II or malignant TGCTs (seminomas and non-seminomas) is drastically increased. They originate from a common precursor, the Germ Cell Neoplasia In Situ, which likely arises during embryogenesis due to an alteration of the testicular niche that blocks gonocyte differentiation [2]. They show similarities to embryogenesis, including the formation of both embryonic as well as extra-embryonic lineages, and present clinically often with metastases. Hepatocyte growth factor (HGF) exerts multiple biological responses (proliferation, migration, morphogenesis, differentiation) through c-MET receptor [3]. We demonstrated that HGF/c-MET signalling contributes to the maintenance of testicular microenvironment both pre-natally and post-natally. However, c-MET is altered in many human cancers, making it a suitable molecular target for therapies. My research is focused on the role of HGF/c-MET system in type II TGCT onset and/or progression to metastatic disease and treatment resistance. We have already evaluated its expression in TGCT derived cell lines and its contribution to proliferation, migration and invasion. With this communication, I present the c-MET immunoreactivity performed in a cohort of histological samples from all major variants of type II TGCTs, using Tissue Microarrays evaluation after immunohistochemical staining with a validated antibody. Our scoring results show a clearly different c-MET distribution and staining intensity between the analysed TGCT histotypes, allowing us to speculate that c-MET receptor could be used as a novel biomarker for these tumours