The role of caveolin-1 in the regulation of angiogenesis

Caveolin-1 (cav-1) is the principal structural component of caveolae which functions as scaffolding protein for the integration of a variety of signaling pathways. In this study, we show that siRNA-induced cav-1 down regulation in human endothelial cells (EC) increased cell size and provoked cell cycle arrest at G1/S phase transition. In addition, silencing of cav-1 reduced matrix metalloproteinases (MMPs) activity which, in turn, affected cell migration and VEGF-induced tube formation of EC in vitro. These data indicate that proper expression of cav-1 is required for maintaining typical functions of EC such as proliferation and the formation of new blood vessels. In addition, we observed a marked increase of cell size, after cav-1 silencing, which might indicate the involvement of this scaffolding protein in the way by which cells perceive changes in their microenvironment. In conclusion, this study proposes cav- 1 as an interesting target molecule for studying cellular mechanisms which occur in physiological as well as pathological conditions such as senescence and tumorigenesis

Expression of Reelin in cancer stem cells isolated from human glioblastoma

Reelin is a large secreted extracellular matrix glycoprotein which contributes to positioning, migration and laminar organization of several central nervous system structures during neurodevelopment. Recent studies reported the expression of Reelin and its intracellular adapter protein DAB1 in neuroblastoma, where it appears to be involved in cell motility and invasiveness. Interestingly, our data obtained by immunolocalization analysis show the expression of Reelin in both tumor and peritumoral area of glioblastoma (GBM). It is known that many solid tumors may originate from cancer stem cells (CSC) which are usually resistant to common therapies and might be involved in tumor progression. Therefore, we evaluated the expression of Reelin in CSC neurospheres isolated from both tumor (GCSC) and peritumoral area (PCSC) of GBM. Immunocytochemistry analysis showed the expression of Reelin in both cell types, suggesting that this protein may contribute to neurosphere tridimensional organization and possibly to cell migration during tumor progression. This is the first evidence of Reelin expression in human GBM which might indicate a pivotal role of this protein in the regulation of tumor development. Our data may open potential avenues for GBM treatment by targeting Reelin signaling activity

Levetiracetam enhances the Temozolomide effect on glioblastoma stem cell proliferation

Glioblastoma Multiforme (GBM) is a highly aggressive brain tumor in which cancer cells with stem cell-like features, called Cancer Stem Cells (CSCs), were identified. CSCs show a high capacity to resist to standard therapies, finally leading to a poor prognosis. Thus, the development of efficient strategies targeting these cells are urgently needed. We have previously demonstrated the presence of two CSC populations in GBM, one derived from the GBM area called enhanced lesion (GCSC) and the other one from the brain area adjacent to the tumor margin (PCSC), that greatly differ in their growth properties and tumor-initiating ability. Tumor recurrence occurs in tissue neighboring GBM suggesting a growing relevance for this area in translational research. To date the most effective chemotherapies to treat GBM are alkylating agents such as temozolomide (TMZ). Epigenetic mechanisms are increasingly recognized as a major factor contributing to pathogenesis of cancer including glioblastoma. Histone deacetylase (HDAC) inhibitors can interfere with TMZ activity by modulating methylguanine methyltransferase (MGMT) expression, resulting in increased TMZ efficacy. Levetiracetam (LEV), an antiepileptic drug, is known to modulate the transcription of HDAC, ultimately silencing MGMT.Since TMZ is the chemotherapeutic agent most widely used in newly diagnosed adult glioblastoma patients, we evaluated its effects on the proliferation rate of both GCSC and PCSC deriving from five patients, in comparison with the effects of other drugs such as etoposide, irinotecan and car-boplatin. Our results demonstrated that TMZ was the less efficient agent, hence we verified the pos-sibility to increase the effect of TMZ by combining it with LEV. Here we show that LEV signifi-cantly enhances the inhibitory effect of TMZ on the proliferation of the GCSC deriving from four patients and of the PCSC deriving from two patients. This effect seems to be mediated by HDAC6 since its expression is up-regulated in the TMZ resistant cells and correlates with MGMT expression. Taken together our results suggest that GCSC and PCSC differ in their ability to respond to the combined chemotherapeutic treatment we used and that the manipulation of HDAC6 expression might be a potential strategy for treating glioblastoma and overcoming resistance to TMZ

Effects of conditioned medium from human amniotic mesenchymal tissue cell cultures on prostate cancer cells

It has been recently demonstrated that human amniotic mesenchymal tissue cells (hAMTC) derived from term placenta inhibit lymphocyte proliferation and significantly reduce the growth of haemopoietic and non haemopoietic cancer cell lines (HeLa and Saos cells) in vitro (1). The aim of our study was to evaluate the effects of hAMTC-conditioned medium (CM) on two human prostate cancer cells lines: LNCaP, androgen responsive and well differentiated, and PC-3, androgen unresponsive and less differentiated. Cells were grown in their standard culture conditions in the absence or in the presence of various concentrations (0.001–50%) of hAMTC-CM or their own exhausted medium. Cell numbers were determined by using a haemocytometer, after three days. Moreover, E- and N-cadherin expression was evaluated in PC-3 cells cultured in medium with 0.01, 1 or 25% hAMTC-CM by Immunocytochemistry and Western blot analysis. Our findings indicate that hAMTC-CM reduces the growth of both PC-3 and LNCaP cells. The effect is more pronounced in PC-3 cells in which inhibition is about 25% vs control (p<0.001) at a very low concentration (0.001%) and reaches the maximum (about 55% vs control, p<0.001) with the highest concentration used (50%). In LNCaP cells only the highest concentration of hAMTC-CM (50%) inhibits cell proliferation (about 40% vs control, p<0.001). Interestingly, growth of LNCaP cells is reduced by their own exhausted medium, while proliferation of PC-3 cells is not affected by their spent medium. Both E- and N-cadherin expression have been detected at the membrane level in untreated PC-3 cells and the localization does not change in hAMTC-CM-treated cells. Preliminary data obtained by Western blot analysis seem to indicate an increase in both E- and N-cadherin levels. Our findings show that hAMTC-CM reduces prostate cancer cell proliferation in relationship to their androgen sensitivity and modifies the expression levels of adhesion molecules. Experiments are in progress to determine the mechanisms which underlie the observed effects and assess if hAMTC-CM can determine any variation in the differentiation status of prostate cancer cells

Role of Stearoyl-CoA Desaturase 1 and 5 in breast cancer cell migration and survival

We previously reported that a major component of breast tumor stroma, the “cancer-associated fibroblasts” (CAFs), induced epithelial-mesenchymal transition and an increase in cell membrane fluidity as well as in migration speed and directness in poorly (MCF-7) and highly invasive (MDA-MB-231) breast cancer cells. We next investigated the mechanisms responsible for the CAF-promoted tumor cell migration demonstrating the crucial role of Stearoyl-CoA desaturase 1 (SCD1), one of the main enzyme regulating membrane fluidity. We found SCD1 to be upregulated in tumor cells co-cultured with CAFs and that its inhibition (pharmacological or siRNA-based) impaired both intrinsic and CAF-driven tumor cell migration. In the present study, we deepen the understanding of the mechanisms involved in the SCD1-based modulation of tumor cell migration, as well as the possible role of the other human SCD isoform, SCD5. Thus, in the two above mentioned cell lines we studied whether the inhibitory effect produced on cell migration by SCD1 depletion was due to the deficiency of oleic acid (OA), the main SCD1 enzymatic product. By a wound healing assay, we found that the addition of OA nullified the inhibitory effects produced on tumor cell migration by the SCD1 inhibition in both the cell lines while SCD5 appeared not to be involved in the regulation of their motility but it was upregulated in MCF-7 cells co-cultured with CAFs. Because of the high number of detached MCF-7 cells silenced for SCD5, we investigated the role of the desaturase on tumor cell survival and an induction of necrosis was found. Consistently with the promotion of tumor cell migration, CAFs have also been found to induce the activated form of the hepatocyte growth factor receptor, p-MET, in the two cell lines. These results provide further insights in understanding the role of SCD1 in both intrinsic and CAF-stimulated mammary tumor cell migration. Moreover, our data seem to suggest the ability of CAFs to promote the maintenance of tumor cell survival by the induction of SCD5 levels

B cell depletion in diffuse progressive systemic sclerosis: safety, skin score modification and IL-6 modulation in an up to thirty-six months follow-up open-label trial

INTRODUCTION: An over-expression of CD19 has been shown in B cells of systemic sclerosis (SSc) and B cells are thought to contribute to the induction of skin fibrosis in the tight skin mouse model. The aim was to define the outcome on safety and the change in skin score after rituximab therapy in SSc patients and to correlate the clinical characteristics with the levels of interleukin (IL)-6 and with the immune cell infiltrate detected by immunohistochemistry. METHODS: Nine patients with SSc with mean age 40.9 +/- 11.1 years were treated with anti-CD20, 1 g at time 0 and after 14 days. Skin biopsy was performed at baseline and during the follow-up. B-cell activating factor (BAFF) and IL-6 levels were also determined at the follow-up times. RESULTS: After 6 months patients presented a median decrease of the skin score of 43.3% (range 21.1-64.0%), and a decrease in disease activity index and disease severity index. IL-6 levels decreased permanently during the follow up. After treatment, a complete depletion of peripheral blood B cells was observed in all but 2 patients. Only 3 patients presented CD20 positive cells in the biopsy of the involved skin at baseline. CONCLUSIONS: Anti-CD20 treatment has been well tolerated and SSc patients experienced an improvement of the skin score and of clinical symptoms. The clear fall in IL-6 levels could contribute to the skin fibrosis improvement, while the presence of B cells in the skin seems to be irrelevant with respect to the outcome after B cell depletion

Epithelium-stroma reciprocal influence in breast cancer. Focus on plasma membrane features related to cell migratory/invasive ability

Interactions occurring between malignant cells and stromal microenvironment greatly influence progression of breast cancer. In a previous study, we co-cultured mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB- 231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or breast tumor stroma (cancer-associated fibroblasts, CAFs). In this system, we demonstrated the influence exerted in particular by CAFs on malignant cells, leading to the acquisition of a more aggressive/invasive phenotype (i.e. reduced adhesion, epithelial-mesenchymal transition, enhanced plasma membrane fluidity and migration velocity/directness). In the present study, we evaluated the reciprocal effect of breast tumor cells and fibroblasts in co-culture on the expression of the main enzyme regulating the fatty acids membrane composition, Stearoyl-CoA desaturase 1 (SCD1). Abnormally high levels of SCD1 have been reported in different cancers and transformed cells and the enzyme has been recently raised to the role of key regulator of cell growth, programmed cell death and carcinogenesis. In our experience, Western blot analysis demonstrated a strong increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells, resulting from their interaction with CAFs and, at a lesser extent, with NFs. High levels of SCD1 were also observed in both NFs and CAFs when co-cultured with MCF-7 cells. MDA-MB-231 cells more slightly up-regulated the enzyme expression in NFs or even induced a certain inhibition in CAFs. The fibroblast-triggered up-regulation of SCD1 in cancer cells could reasonably be considered the molecular event underlying the increase of membrane fluidity, previously observed in tumor cells co-cultured with NFs and, notably, with CAFs. This change might downstream promote the previously described increase in tumor cell motility

Multifaceted Functional Role of Semaphorins in Glioblastoma

Glioblastoma (GBM) is the most malignant tumor type affecting the adult central nervous system. Despite advances in therapy, the prognosis for patients with GBM remains poor, with a median survival of about 15 months. To date, few treatment options are available and recent trials based on the molecular targeting of some of the GBM hallmark pathways (e.g., angiogenesis) have not produced any significant improvement in overall survival. The urgent need to develop more efficacious targeted therapies has led to a better molecular characterization of GBM, revealing an emerging role of semaphorins in GBM progression. Semphorins are a wide group of membrane-bound and secreted proteins, originally identified as axon guidance cues, signaling through their receptors, neuropilins, and plexins. A number of semaphorin signals involved in the control of axonal growth and navigation during development have been found to furthermore participate in crosstalk with different dysfunctional GBM pathways, controlling tumor cell proliferation, migration, and invasion, as well as tumor angiogenesis or immune response. In this review, we summarize the regulatory activities mediated by semaphorins and their receptors on the oncogenic pathways implicated in GBM growth and invasive/metastatic progression

PDGFRα depletion attenuates glioblastoma stem cells features by modulation of STAT3, RB1 and multiple oncogenic signals.

Platelet derived growth factor receptors (PDGFRs) play an important role in tumor pathogenesis, and they are frequently overexpressed in glioblastoma (GBM). Earlier we have shown a higher protein expression of PDGFR isoforms (α and β) in peritumoral-tissue derived cancer stem cells (p-CSC) than in tumor core (c-CSC) of several GBM affected patients. In the current study, in order to assess the activity of PDGFRα/PDGF-AA signaling axis, we performed time course experiments to monitor the effects of exogenous PDGF-AA on the expression of downstream target genes in c-CSC vs p-CSC. Interestingly, in p-CSC we detected the upregulation of Y705-phosphorylated Stat3, concurrent with a decrement of Rb1 protein in its active state, within minutes of PDGF-AA addition. This finding prompted us to elucidate the role of PDGFRα in self-renewal, invasion and differentiation in p-CSC by using short hairpin RNA depletion of PDGFRα expression. Notably, in PDGFRα-depleted cells, protein analysis revealed attenuation of stemness-related and glial markers expression, alongside early activation of the neuronal marker MAP2a/b that correlated with the induction of tumor suppressor Rb1. The in vitro reduction of the invasive capacity of PDGFRα-depleted CSC as compared to parental cells correlated with the downmodulation of markers of epithelial-mesenchymal transition phenotype and angiogenesis. Surprisingly, we observed the induction of anti-apoptotic proteins and compensatory oncogenic signals such as EDN1, EDNRB, PRKCB1, PDGF-C and PDGF-D. To conclude, we hypothesize that the newly discovered PDGFRα/Stat3/Rb1 regulatory axis might represent a potential therapeutic target for GBM treatment.BRC, Q

Breast cancer cells and fibroblasts in co-culture: reciprocal influences on cell adhesion, membrane fluidity and migration

The growing role of the reciprocal interaction between epithelial and stromal cells in the development and progression of breast cancer has been recognized. In particular, the migratory/invasive behaviour of tumor cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of the tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive, poorly invasive and low metastasizing (MCF-7) or ER-negative, highly invasive and metastatic (MDA-MB-231) breast cancer cells with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to respectively induce or inhibit the epithelial adhesion molecule, E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells, originally negative for N-cad. Two-photon microscopy imaging of cells labeled with Laurdan, a membrane fluorescent probe, was used to investigate fluidity changes in plasma membranes of all the cell types in monolayer cultures. Tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while tumor cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the interaction of mammary cancer cells with NFs or CAFs determined a definite increment in tumor cell migration velocity, even with a marked enhancement of the migration directionality induced by CAFs. Our results demonstrate a reciprocal influence of mammary cancer and stromal cells on various adhesiveness/invasiveness features. In particular, an overall pro-tumoral/-invasive effect of CAFs on both well- and poorly differentiated mammary cancer cells was exteriorized by reduction of cell adhesion, induction of membrane fluidity, and migration velocity and directionality, along with a promotion of epithelial-mesenchymal transition