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

Malignant pleural mesothelioma and mesothelial hyperplasia: A new molecular tool for the differential diagnosis

Malignant pleural mesothelioma (MPM) is a rare asbestos related cancer, aggressive and unresponsive to therapies. Histological examination of pleural lesions is the gold standard of MPM diagnosis, although it is sometimes hard to discriminate the epithelioid type of MPM from benign mesothelial hyperplasia (MH). This work aims to define a new molecular tool for the differential diagnosis of MPM, using the expression profile of 117 genes deregulated in this tumour. The gene expression analysis was performed by nanoString System on tumour tissues from 36 epithelioid MPM and 17 MH patients, and on 14 mesothelial pleural samples analysed in a blind way. Data analysis included raw nanoString data normalization, unsupervised cluster analysis by Pearson correlation, non-parametric Mann Whitney U-test and molecular classification by the Uncorrelated Shrunken Centroid (USC) Algorithm. The Mann-Whitney U-test found 35 genes upregulated and 31 downregulated in MPM. The unsupervised cluster analysis revealed two clusters, one composed only of MPM and one only of MH samples, thus revealing class-specific gene profiles. The Uncorrelated Shrunken Centroid algorithm identified two classifiers, one including 22 genes and the other 40 genes, able to properly classify all the samples as benign or malignant using gene expression data; both classifiers were also able to correctly determine, in a blind analysis, the diagnostic categories of all the 14 unknown samples. In conclusion we delineated a diagnostic tool combining molecular data (gene expression) and computational analysis (USC algorithm), which can be applied in the clinical practice for the differential diagnosis of MPM

Expression of angiogenesis related factors in glioblastoma and peritumor tissue

Glioblastoma (GBM) is a lethal brain glial tumor characterized by extensive angiogenesis that is mostly triggered by tumor hypoxia. We previous reported that in GBM and in peritumor areas, endothelial cells expressed CD105, which probably marks newly formed vessels with a quite similar morphology. In this study, with the aim of better understanding the involvement of angiogenesis in tumor progression, we analyzed, by immunohistochemistry, the expression of Hypoxia-inducible factor (HIF) 1α and 2α, VEGF and its receptors (VEGFR-1 and -2) in GBM and in peritumor tissue. Twenty two patients were enrolled in this study. Tissue specimens were derived from enhanced lesion (first area) and white matter at a distance ≤1 mm from the tumor edge (second area). Immunoreactivity for all markers was detected not only in the tumor but also in the peritumor tissue and it was present in neoplastic cells, in endothelium and in apparently normal glial cells. HIF1α and 2α expression was mainly confined in the nuclei. VEGF, localized in the cytoplasm, showed diffuse expression with an intense staining in GBM. VEGFR-1 and 2 immupositivity was localized especially to the cell membrane and also to the cytoplasm, as expected. All molecule staining was evident in a heterogeneous manner and there was no significant difference in the expression marker levels between the first and second area also in relation to the presence or absence of tumor cells in the second area. No significant correlation was found between the above molecule expression and survival time. In conclusion, we demonstrated that HIF1α, HIF2α, VEGF and VEGFR-1 and -2 are present in peritumor area, probably reflecting perturbations of oxygenation emanating from the tumor microenvironment. Since, unfortunately, the response to anti-VEGF therapy is transient and the majority of patients eventually relapse, the gain of a deeper knowledge of the above molecule role, in the peritumor tissue, may lead to consider them as the target for new treatment regimens to counteract angiogenesis. Supported by FIRB “Accordi di Programma” 201

Let-7g and miR-21 expression in non-small cell lung cancer: correlation with clinicopathological and molecular features

MicroRNAs (miRNAs) play a key role in cancer pathogenesis and are involved in several human cancers, including non-small cell lung cancer (NSCLC). This study evaluated Let-7g and miR-21 expression by quantitative real-time PCR in 80 NSCLC patients and correlated the results with their main clinicopathological and molecular features. MiR-21 expression was significantly higher in NSCLC tissues compared to non-cancer lung tissues (p<0.0001), while no significant changes in Let-7g expression were observed between the tumor and normal lung tissues. Target prediction analysis led to the identification of 26 miR-21 and 24 Let-7g putative target genes that play important roles in cancer pathogenesis and progression. No significant association was observed between the analysed miRNAs and the main clinicopathological or molecular characteristics of the NSCLC patients, although both miRNAs were downregulated in squamous cell carcinomas compared to adenocarcinomas. Noteworthy, we observed a significant association between low Let-7g expression and metastatic lymph nodes at diagnosis (p=0.046), as well as between high miR-21 expression and K-Ras mutations (p=0.0003). Survival analysis did not show any significant correlation between prognosis and the analysed miRNAs, although the patients with a high Let-7g and miR-21 expression showed a significantly lower short-term progression-free survival (p=0.01 and p=0.0003, respectively) and overall survival (p=0.023 and p=0.0045, respectively). In conclusion, we showed that Let-7g and miR-21 expression was deregulated in NSCLC and we demonstrated a strong relationship between miR-21 overexpression and K-Ras mutations. Our data indicate that Let-7g and miR-21 profiling combined with the determination of K-Ras mutational status may be considered a useful biomarker for a more effective molecular characterization and clinical management of NSCLC patients

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

Relevance of tumour surrounding area in chemoresistance of glioblastoma (GBM)

The mechanisms responsible for resistance to damage in normal cells might determine chemoresistance in both tumor cells and cancer stem cells (CSC). Relapse due to chemoresistant residual disease is a major cause of death in GBM. Increasing body of evidence indicates that not only tumor area (TT), but also tissue surrounding the tumor border (pTT) of GBM contains tumor cells and CSC, which could contribute to the disease progression. Therefore, the need to have a deeper insight in this area through identification of the characteristics that confer chemoresistance. In this study, the expression of molecules involved in chemoresistance was investigated in samples derived from TT and from pTT at <1 cm from the tumor border, in 40 patients with GBM. The expression of O6-methylguanine-DNA methyltransferase (MGMT), a suicidal DNA repair protein; Breast Cancer Resistance Protein (BCRP1), a drug efflux transporter, and A2B5 (c-series gangliosides) has been determined by immunohistochemistry. The percentage of MGMT positive cells was higher (p<0.0001, paired Student’s t test) in pTT (median: 53.5, range: 0.6-92.4) with respect to TT (median: 3.3, range: 0.0-70.7). The same trend was observed in BCRP1 expression (p<0.02; pTT, median: 27.6, range: 1.0-95.6; TT, median: 10.1, range: 0.2-72.1). No difference was found between pTT and TT in A2B5 expression (p=0.69, pTT, median: 29.8, range: 0.0-98.4; TT, median: 26.0, range: 0.0-96.8). Patients were then divided into two groups according to presence (group A) or absence (group B) of tumor cells in pTT. The trend previous observed in MGMT expression was maintained in both groups, while only in group A a statistically significant difference in BCRP1 expression was observed. Our results confirm that the tissue surrounding GBM is not a normal tissue, and that it represents a frontline of tumor invasion, particularly for the presence of some molecules involved in chemoresistance, which could explain the disease recurrence after the conventional treatment of GBM. Experiments about the expression of above mentioned molecules in CSC from pTT and TT are in progress. Supported by FIRB, ACCORDI DI PROGRAMMA 201

Levetiracetam enhances the temozolomide effect on glioblastoma stem cell proliferation and apoptosis

Abstract BACKGROUND: 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. Two CSC populations have been previously identified in GBM, one derived from the GBM area called enhanced lesion (GCSCs) and the other one from the brain area adjacent to the tumor margin (PCSCs) that greatly differ in their growth properties and tumor-initiating ability. To date the most effective chemotherapy to treat GBM is represented by alkylating agents such as temozolomide (TMZ), whose activity can be regulated by histone deacetylases (HDACs) inhibitors through the modulation of O6-methylguanine-DNA methyltransferase (MGMT) expression. Levetiracetam (LEV), a relatively new antiepileptic drug, modulates HDAC levels ultimately silencing MGMT, thus increasing TMZ effectiveness. However, an improvement in the therapeutic efficacy of TMZ is needed. METHODS: Cell proliferation was investigated by BrdU cell proliferation assay and by Western Blot analysis of PCNA expression. Apoptosis was evaluated by Western Blot and Immunofluorescence analysis of the cleaved Caspase-3 expression. MGMT and HDAC4 expression was analyzed by Western Blotting and Immunofluorescence. Statistical analysis was performed using the Student's t test and Mann-Whitney test. RESULTS: Here we evaluated the effect of TMZ on the proliferation rate of the IDH-wildtype GCSCs and PCSCs derived from six patients, in comparison with the effects of other drugs such as etoposide, irinotecan and carboplatin. Our results demonstrated that TMZ was less effective compared to the other agents; hence, we verified the possibility to increase the effect of TMZ by combining it with LEV. Here we show that LEV enhances the effect of TMZ on GCSCs proliferation (being less effective on PCSCs) by decreasing MGMT expression, promoting HDAC4 nuclear translocation and activating apoptotic pathway. CONCLUSIONS: Although further studies are needed to determine the exact mechanism by which LEV makes GBM stem cells more sensitive to TMZ, these results suggest that the clinical therapeutic efficacy of TMZ in GBM might be enhanced by the combined treatment with LEV

Involvement of cancer stem cells in glioblastoma angiogenesis

It is widely accepted that glioblastoma (GBM) develops from cancer stem cells (CSCs), a subset of stem-like cells displaying high resistance to treatment. In fact, despite aggressive therapy, 90% of patients relapse within 2 cm from tumor edge. Our recent findings showed the existence of a CSC type, residing in GBM peritumor tissue (PCSCs), that bears distinct characteristics from CSCs of the tumor mass (GCSCs). It should be considered the possibility that, after surgical resection, PCSCs might represent a reservoir of cells able to recapitulate the tumor. In this setting, characterization of PCSCs appears to be crucial in order to identify novel effective therapeutic targets. Thus, our aim was to investigate GCSCs and PCSCs role in angiogenesis, a key event in both GBM and peritumor tissue, whose vasculature shows features similar to those found in the tumor mass. In particular, we analyzed, by immunocytochemistry (ICC), Western blotting or real-time PCR, the expression of molecules involved in hypoxia and angiogenesis, such as HIF1α, HIF2α, and VEGF along with its receptors (VEGFR1, VEGFR2). ICC has highlighted the presence and the specific localization of these molecules in both GCSCs and PCSCs. The two cell populations showed comparable levels of VEGF. The transcript of VEGFR1 was in general expressed at higher levels in GCSCs than in PCSCs, while VEGFR2 mRNA and protein did not show a unique trend of expression. The expression of VEGF and its receptors in both GCSCs and PCSCs suggests that, besides well-known paracrine loops, autocrine signalings are also involved in tumor angiogenesis. Moreover, the expression of angiogenesis markers in PCSCs suggests these cells to have a direct role in peritumor tissue new vessel formation. In this regard, PCSCs should be considered a promising therapeutic target to counteract the angiogenesis-supported tumor progression