Daniela Spano

Molecular networks that regulate cancer metastasis

Tumor metastases are responsible for approximately 90% of all cancer-related deaths. Although many patients can be cured, in the US and UK, cancer still causes 730,000 deaths every year, and it is second only to cardiovascular disease as a cause of death. The functional roles of many critical players involved in metastasis have been delineated in great detail in recent years, due to the draft of the human genome and to many associated discoveries. Here, we address several genetic events and critical factors that define the metastatic phenotype acquired during tumorigenesis. This involves molecular networks that promote local cancer-cell invasion, single-cell invasion, formation of the metastatic microenvironment of primary tumors, intravasation, lymphogenic metastasis, extravasation, and metastatic outgrowth. Altogether, these functional networks of molecules contribute to the development of a selective environment that promotes the seeding and malignant progression of tumorigenic cells in distant organs. We include here candidate target proteins and signaling pathways that are now under clinical investigation. Although many of these trials are still ongoing, they provide the basis for the development of new aspects in the treatment of metastatic cancers, which involves inhibition of these proteins and their molecular networks.

Mutations affecting the secretory COPII coat component SEC23B cause congenital dyserythropoietic anemia type II

Congenital dyserythropoietic anemias (CDAs) are phenotypically and genotypically heterogeneous diseases. CDA type II (CDAII) is the most frequent CDA. It is characterized by ineffective erythropoiesis and by the presence of bi- and multinucleated erythroblasts in bone marrow, with nuclei of equal size and DNA content, suggesting a cytokinesis disturbance. Other features of the peripheral red blood cells are protein and lipid dysglycosylation and endoplasmic reticulum double-membrane remnants. Development of other hematopoietic lineages is normal. Individuals with CDAII show progressive splenomegaly, gallstones and iron overload potentially with liver cirrhosis or cardiac failure. Here we show that the gene encoding the secretory COPII component SEC23B is mutated in CDAII. Short hairpin RNA (shRNA)-mediated suppression of SEC23B expression recapitulates the cytokinesis defect. Knockdown of zebrafish sec23b also leads to aberrant erythrocyte development. Our results provide in vivo evidence for SEC23B selectivity in erythroid differentiation and show that SEC23A and SEC23B, although highly related paralogous secretory COPII components, are nonredundant in erythrocyte maturation.

Suppressor of cytokine signaling 3 (SOCS3) expression and hepatitis C virus-related chronic hepatitis: Insulin resistance and response to antiviral therapy.

The response to antiviral therapy is lower in hepatitis C virus (HCV) patients with genotype 1 than in those with genotype 2. Overexpression of the suppressor of cytokine signaling 3 (SOCS3) gene in liver tissue is associated with a poorer treatment outcome in patients with chronic hepatitis C viral genotype 1. Also, insulin resistance has been implicated in nonresponse to an anti‐HCV treatment. To understand why HCV genotype 1 patients respond differently, we investigated SOCS3 gene expression, metabolic syndrome (MS), and the response to therapy in a cohort of patients with HCV‐related hepatitis. A total of 198 patients (108 with genotype 1 and 90 with genotype 2) treated with pegylated interferon plus ribavirin were consecutively enrolled in the study. We measured SOCS3 expression in Epstein‐Barr virus–transformed lymphoblastoid cell lines derived from peripheral lymphocytes of a subset of 130 patients. MS was more frequent in genotype 1 patients than in genotype 2 patients (P < 0.01). Nonresponders (P < 0.01), MS (P < 0.001), and genotype 1 (P < 0.001) were significantly related to SOCS3 overexpression. However, SOCS3 levels were higher in nonresponders also, regardless of the genotype (P < 0.01). In a univariate analysis, the genotype (P < 0.001), age (P < 0.001), SOCS3 (P < 0.001), and MS (P < 0.001) were significantly related to the response to therapy. However, in a multivariate analysis, SOCS3 was the only independent predictor of the response (odds ratio = 6.7; P < 0.005). Conclusion: We speculate that SOCS3 expression per se may influence the response to antiviral therapy and that the genotype 1b virus might induce its up‐regulation. This may account for the different responses to therapy between genotype 1–infected and genotype 2–infected patients. (HEPATOLOGY 2007.)

Tumor microenvironment: a main actor in the metastasis process

Over recent decades, various studies have argued that the metastatic tissue microenvironment is fully controlled by the intrinsic properties of the cancer cells (growth, motility and invasion, angiogenesis, extracellular matrix remodeling, immune escape) and additional cells types. Overall, the extrinsic factors and determinants mediate the contribution of the host microenvironment to metastasis formation. The tumor microenvironment carries out these functions by secretion of molecules that can influence and modulate its phenotype, making these complex interactions the basis for support for the progression of a cancer. Here, we undertake a summary of the “state of the art” of the functions and actions of these cells, as the main actors in the promotion of the formation of the microenvironment of the metastatic niche, and the associated network of interactions. The unraveling of the relationships between tumorigenic cells and their microenvironment represents an important issue for the development of new therapeutic agents that can fight both initiation and recurrence of cancer.

Targeting monocyte chemotactic protein-1 synthesis with bindarit induces tumor regression in prostate and breast cancer animal models

Prostate and breast cancer are major causes of death worldwide, mainly due to patient relapse upon disease recurrence through formation of metastases. Chemokines are small proteins with crucial roles in the immune system, and their regulation is finely tuned in early inflammatory responses. They are key molecules during inflammatory processes, and many studies are focusing on their regulatory functions in tumor growth and angiogenesis during metastatic cell seeding and spreading. Bindarit is an anti-inflammatory indazolic derivative that can inhibit the synthesis of MCP-1/CCL2, with a potential inhibitory function in tumor progression and metastasis formation. We show here that in vitro, bindarit can modulate cancer-cell proliferation and migration, mainly through negative regulation of TGF-β and AKT signaling, and it can impair the NF-κB signaling pathway through enhancing the expression of the NF-κB inhibitor IkB-α. In vivo administration of bindarit results in impaired metastatic disease in prostate cancer xenograft mice (PC-3M-Luc2 cells injected intra-cardially) and impairment of local tumorigenesis in syngeneic Balb/c mice injected under the mammary gland with murine breast cancer cells (4T1-Luc cells). In addition, bindarit treatment significantly decreases the infiltration of tumor-associated macrophages and myeloid-derived suppressor cells in 4T1-Luc primary tumors. Overall, our data indicate that bindarit is a good candidate for new therapies against prostate and breast tumorigenesis, with an action through impairment of inflammatory cell responses during formation of the tumor–stroma niche microenvironment.

Galectin-1 and its involvement in hepatocellular carcinoma aggressiveness.

Hepatocellular carcinoma is one of the most common cancers worldwide. Despite several efforts to elucidate hepatocellular carcinoma molecular pathogenesis, it is still not fully understood. To acquire further insights into the molecular mechanisms of hepatocellular carcinoma, we performed a systematic functional genomic approach on human HuH-7 and JHH-6 cells. The subsequent analysis of the differentially expressed genes in human specimens revealed a molecular signature of 11 genes from which we selected the LGALS1 gene, which was overexpressed in hepatocellular carcinoma. The expression analysis in humans of Galectin-1 (Gal-1), the protein encoded by LGALS1, showed a Gal-1 preferential accumulation in the stromal tissue around hepatocellular carcinoma tumors. Moreover, a significant association between increased expression of Gal-1 in hepatocellular carcinoma and the presence of metastasis was observed. Interestingly, Gal-1 overexpression resulted in an increase of cell migration and invasion. In conclusion, this study provides a portfolio of targets useful for future investigations into molecular marker-discovery studies on a large number of patients and functional assays. In addition, our data provide evidence that Gal-1 plays a role in hepatocellular carcinoma cell migration and invasion, and we suggest that further studies should be conducted to fully establish the role of Gal-1 in hepatocellular carcinoma pathogenesis and evaluate Gal-1 as a potential molecular therapeutic target.

The Ras Homolog Rhes Affects Dopamine D1 and D2 Receptor-Mediated Behavior in Mice

Dopamine activates five different receptor subtypes and a complex array of intracellular signaling pathways. Rhes is a striatally expressed guanidine triphosphate-binding protein involved in dopamine signaling. Here we have used mutant mice to test whether Rhes (Ras homolog enriched in striatum) is involved in D1 and D2 dopamine receptor-mediated behaviors. Rhes was not necessary for the expression of normal D1/D2 receptor synergism, as measured by apomorphine-induced stereotypy. The stereotypic responses to D1/D2 costimulation and to D2 stimulation alone were significantly increased in mice lacking Rhes, but D1 receptor-mediated grooming was reduced in these mice. These results suggest that Rhes is normally inhibitory to behaviors induced by D1/D2 receptor costimulation and by D2 receptor stimulation alone. Rhes, however, seems to facilitate the D1-specific behavior of grooming.

Attenuation of Rhes Activity Significantly Delays the Appearance of Behavioral Symptoms in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) is a neuropsychiatric disorder characterized by choreiform movement of the limbs, cognitive disability, psychosis and dementia. It is invariably associated with an abnormally long CAG expansion within the IT15 gene on human chromosome 4. Although the mutant huntingtin protein is ubiquitously expressed in HD patients, cellular degeneration occurs predominantly in neurons within the corpus striatum and cerebral cortex. The Ras homolog Rhes is expressed very selectively in the precise brain areas affected by HD. Recent in vitro work suggests that Rhes may be a co-factor with mutant huntingtin in cell death. The objective of the present study was to examine whether the inhibition of Rhes would attenuate or delay the symptoms of HD in vivo. We used a transgenic mouse model of HD crossed with Rhes knockout mice to show that the behavioral symptoms of HD are regulated by Rhes. HD+/Rhes−/− mice showed significantly delayed expression of HD-like symptoms in this in vivo model. Drugs that block or inhibit the actions of Rhes may be useful as the first treatments for HD.

Effects of the Ras homolog Rhes on Akt/protein kinase B and glycogen synthase kinase 3 phosphorylation in striatum

G protein-coupled receptors (GPCR) signal not only through heterotrimeric G proteins, but also through alternate pathways. Thus, dopamine D2 receptors in the striatum signal through Gαi/o and also by promoting formation of a multi-protein complex containing β-arrestin2, protein phosphatase 2A (PP2A), and Akt in order to dephosphorylate Akt. Lithium, on the other hand, disrupts this complex to increase Akt phosphorylation. Rhes is a striatally enriched GTP-binding protein that has been shown to inhibit dopamine receptor-mediated behavior and signaling through heterotrimeric G proteins. Therefore, our objective was to test whether Rhes similarly affects signaling through the Akt/GSK3 pathway in the striatum. Rhes(-/-) mice showed basally increased Akt and GSK3β phosphorylation relative to rhes(+/+) mice that was not further enhanced by lithium treatment. Furthermore, they responded to the D1/D2 agonist apomorphine with increased Akt and GSK3 phosphorylation. Co-immunoprecipitation experiments revealed that apomorphine treatment recruits PP 2A-C to Akt in both rhes(+/+) and rhes(-/-) mice. Lithium did not disrupt their interaction in rhes(-/-) mice as there was little basal interaction. Rhes co-immunoprecipitated with β-arrestins, suggesting that it is integral to the multi-protein complex. Thus, Rhes is necessary for Akt dephosphorylation by the striatal multi-protein complex, and in its absence, a lithium-treated phenotype results.

SOCS3 and IRS-1 gene expression differs between genotype 1 and genotype 2 hepatitis C virus-infected HepG2 cells

Abstract Background: The poor response to antiviral treatment of hepatitis C virus (HCV)-infected patients with genotype 1b has been associated with a higher prevalence of metabolic syndrome. However, the molecular link between these clinical entities is not clear. The goal of this study was to clarify the role of genotype 1b and 2 in the genetic expression of suppressor of cytokine signaling 3 (SOCS3) and insulin receptor substrate 1 (IRS-1). Methods: We infected human hepatocellular carcinoma cell line (HepG2) cells with human HCV genotype 1b or 2 and measured the gene and protein expression of SOCS3 at various times. We also evaluated impairment in the insulin pathway by analysis of IRS-1 and phospho-AKT. For the control, we used HepG2 cell cultures treated with non-infectious serum. We also demonstrated the occurrence of HCV infection by the detection of both positive and negative strands in the cells and culture medium. To test infection of the HepG2 cells, we performed quantitative real-time polymerase chain reaction (qRT-PCR) of viral load at different time points. We analyzed the viral genotype in the pellet and supernatant. Results: At each time point, we found positive and negative strands in the infected cells, while in the medium we found positive, but no negative strands. We also detected the presence of the correct genotype in the medium. Two weeks following infection when the viral load was higher, we tested genotype 1b and 2 infected cells. SOCS3 gene expression was significantly higher in genotype 1b-infected cells (median 2.56; mean 2.82±0.59) compared with genotype 2 (median 1.34; mean 1.46±0.31) (p=0.04) and control cells (median 1.09; mean 1.02±0.11, p=0.02). There was no difference between cells exposed to genotype 2 and control cells. Conversely, IRS-1 was significantly lower in genotype 1b-infected cells (median 15.97; mean 15.45±0.67) compared with genotype 2-infected cells (median 16.45; mean 16.44±0.01, p=0.04). Statistically significant differences were seen when comparing the pAKT/AKT ratio in genotype 1b-infected cells (0.19±0.034) and not genotype 1b-infected (genotype 2-infected and non-infected) cells (0.253±0.004, p=0.03). This inverse regulation is compatible with interactions between the molecular expression of SOCS3, IRS-1 and phospho-AKT mediated by the genotype 1b virus. Conclusions: Up-regulation of the SOCS3 gene might be one of the mechanisms governing non-response to therapy and expression of insulin resistance mediated via a direct mechanism at this level of genotype 1b HCV. Clin Chem Lab Med 2009;47:1217–25.