Chiara Puggioni

BRAF and NRAS Mutations are Heterogeneous and Not Mutually Exclusive in Nodular Melanoma

Inhibitors of RAF inhibit the MAPK pathway that plays an important role in the development and progression of those melanoma carrying the V600E BRAF mutation, but there’s a subset of such patients who do not respond to the therapy. Various mechanisms of drug resistance have been proposed which include the clonal heterogeneity of the tumor. We have studied a population of nodular melanoma to investigate the intratumor and intertumor heterogeneity by Laser Capture Microdissection (LCM) analysis. Our results showed that BRAF and NRAS mutations were detected in 47% and 33% of nodular melanoma, respectively, and that there is a discrepancy in mutational pattern of tumoral sample because in the 36% of patients a different mutation, in at least 1 area of the tumor, was found by LCM analysis, giving evidence of the presence of different clonal cells populations. Moreover, we found that mutations in BRAF and NRAS are not mutually exclusive because they were simultaneously present in the same tumor specimens and we observed that when the 2 different mutations were present one is a high-frequency mutation and the other is a low-frequency mutation. This was more evident in lymphonodal metastasis that resulted from wild type to mutational analysis, but showed different mutations following LCM analysis. Therefore, we believed that, when primary tumoral sample results negative to mutational analysis, if it is possible, metastases should be investigated to verify the presence of mutations. Generally, it should be searched for other mutations, in addition to BRAF V600E, so as to better understand the mechanism of drug resistance.

Ezrin silencing remodulates the expression of Phosphoinositide-specific Phospholipase C enzymes in human osteosarcoma cell lines

Ezrin, a protein belonging to the Ezrin, radixin and moesin (ERM) family, was engaged in the metastatic spread of osteosarcoma. The Protein 4.1, Ezrin, radixin, moesin (FERM) domain of Ezrin binds the membrane Phosphatydil inositol (4,5) bisphosphate (PIP2), a crucial molecule belonging to the Phosphoinositide (PI) signal transduction pathway. The cytoskeleton cross-linker function of Ezrin largely depends on membrane PIP2 levels, and thus upon the activity of related enzymes belonging to the PI-specific phospholipase C (PI-PLC) family. Based on the role of Ezrin in tumour progression and metastasis, we silenced the expression of Vil2 (OMIM *123900), the gene which codifies for Ezrin, in cultured human osteosarcoma 143B and Hs888 cell lines. After Ezrin silencing, the growth rate of both cell lines was significantly reduced and morphogical changes were observed. We also observed moderate variations both of selected PI-PLC enzymes within the cell and of expression of the corresponding PLC genes. In 143B cell line the transcription of PLCB1 decreased, of PLCG2 increased and of PLCE differed in a time-dependent manner. In Hs888, the expression of PLCB1 and of PLCD4 significantly increased, of PLCE moderately increased in a time dependent manner; the expression of PLCG2 was up-regulated. These observations indicate that Ezrin silencing affects the transcription of selected PLC genes, suggesting that Ezrin might influence the expression regulation of PI-PLC enzymes.

Expression of Phosphoinositide-specific phospholipase C enzymes in human osteosarcoma cell lines

The definition of the number and nature of signal transduction pathways networking in the pathogenesis of osteosarcoma raised great interest. Intracellular calcium ions are important second messengers implicated in the control of cell death. The calcium concentration is regulated by signal transduction pathways, including the Phosphoinositides (PI) signaling. Phosphatydil inositol (4,5) bisphosphate (PIP2) is critical for many cellular activities. The levels of PIP2 are regulated by means of Phosphoinositide-specific Phospholipase C (PI-PLC) family of enzymes. We delineated the panel of expression of PI-PLC enzymes in four human osteosarcoma cell lines. In MG-63 cell line, PI-PLC β1, β2, β3, β4, γ1, γ2, δ1, δ3 and ε resulted expressed. In 143B cell line, PI-PLC β1, β2, β3, β4, γ1, γ2, δ1, δ3 and ε were expressed. In SaOS-2 cell line, PI-PLC β1, β3, β4, γ1, γ2, δ1, δ3, ε and η1. In Hs888 cell line, PI-PLC β1, β3, β4, γ1, δ1, δ3, δ4, ε and η1 the administration of U-73122 to cultures briefly modifies the levels of PI-PLC transcripts. The obtained complete expression panel of PI-PLC isoforms will be a useful tool for further functional studies about the role of the PI signal transduction pathway in osteosarcoma.

Neuropeptide Y reduces the expression of PLCB2, PLCD1 and selected PLC genes in cultured human endothelial cells

Endothelial cells (EC) are the first elements exposed to mediators circulating in the bloodstream, and react to stimulation with finely tuned responses mediated by different signal transduction pathways, leading the endothelium to adapt. Neuropeptide Y (NPY), the most abundant peptide in heart and brain, is mainly involved in the neuroendocrine regulation of the stress response. The regulatory roles of NPY depend on many factors, including its enzymatic processing, receptor subtypes and related signal transduction systems, including the phosphoinositide (PI) pathway and related phospholipase C (PI-PLC) family of enzymes. The panel of expression of PI-PLC enzymes differs comparing quiescent versus differently stimulated human EC. Growing evidences indicate that the regulation of the expression of PLC genes, which codify for PI-PLC enzymes, might act as an additional mechanism of control of the PI signal transduction pathway. NPY was described to potentiate the activation of PI-PLC enzymes in different cell types, including EC. In the present experiments, we stimulated human umbilical vein EC using different doses of NPY in order to investigate a possible role upon the expression PLC genes. NPY reduced the overall transcription of PLC genes, excepting for PLCE. The most significant effects were observed for PLCB2 and PLCD1, both isoforms recruited by means of G-proteins and G-protein-coupled receptors. NPY behavior was comparable with other PI-PLC interacting molecules that, beside the stimulation of phospholipase activity, also affect the upcoming enzymes’ production acting upon gene expression. That might represent a mode to regulate the activity of PI-PLC enzymes after activation.

Expression of phosphoinositide-specific phospholipase C enzymes in human skin fibroblasts

Fibroblasts are involved in a number of functions regulated by different signal transduction pathways, including the phosphoinositide (PI) signaling system and related converting enzymes, such as phosphoinositide-specific phospholipase C (PI-PLC). The PI-PLC family comprises crucial effector enzymes in the PI signal transduction pathway. Once activated, PI-PLC cleaves an important membrane PI, the phosphatidylinositol (4,5) bisphosphate into inositol trisphosphate and diacylglycerol—both are crucial molecules in the transduction of signals. The activity of selected PI-PLC enzymes was reported in fibroblasts, although the complete panel of expression was not available. Each cell type expresses a group of selected PI-PLC isoforms, and knowledge of the panel of expression is a necessary and preliminary tool to address further studies. In the present study, we delineated the expression panel of PI-PLC enzymes in human skin fibroblasts. PI-PLC β1, PI-PLC β3, PI-PLC β4, PI-PLC γ1, PI-PLC γ2, PI-PLC δ1, PI-PLC δ3, PI-PLC δ4, and PI-PLC ϵ were expressed. PI-PLC β1 was weakly expressed, PI-PLC δ4 was inconstantly expressed, and PI-PLC γ2 was weakly expressed.

Whole-exome analysis in osteosarcoma to identify a personalized therapy

Osteosarcoma is the most common pediatric primary non-hematopoietic bone tumor. Survival of these young patients is related to the response to chemotherapy and development of metastases. Despite many advances in cancer research, chemotherapy regimens for osteosarcoma are still based on non-selective cytotoxic drugs. It is essential to investigate new specific molecular therapies for osteosarcoma to increase the survival rate of these patients. We performed exomic sequence analyses of 8 diagnostic biopsies of patients with conventional high grade osteosarcoma to advance our understanding of their genetic underpinnings and to correlate the genetic alteration with the clinical and pathological features of each patient to identify a personalized therapy.We identified 18,275 somatic variations in 8,247 genes and we found three mutated genes in 7/8 (87%) samples (KIF1B, NEB and KMT2C). KMT2C showed the highest number of variations; it is an important component of a histone H3 lysine 4 methyltransferase complex and it is one of the histone modifiers previously implicated in carcinogenesis, never studied in osteosarcoma. Moreover, we found a group of 15 genes that showed variations only in patients that did not respond to therapy and developed metastasis and some of these genes are involved in carcinogenesis and tumor progression in other tumors.These data could offer the opportunity to get a key molecular target to identify possible new strategies for early diagnosis and new therapeutic approaches for osteosarcoma and to provide a tailored treatment for each patient based on their genetic profile.Osteosarcoma is the most common pediatric primary non-hematopoietic bone tumor. Survival of these young patients is related to the response to chemotherapy and development of metastases. Despite many advances in cancer research, chemotherapy regimens for osteosarcoma are still based on non-selective cytotoxic drugs. It is essential to investigate new specific molecular therapies for osteosarcoma to increase the survival rate of these patients. We performed exomic sequence analyses of 8 diagnostic biopsies of patients with conventional high grade osteosarcoma to advance our understanding of their genetic underpinnings and to correlate the genetic alteration with the clinical and pathological features of each patient to identify a personalized therapy. We identified 18,275 somatic variations in 8,247 genes and we found three mutated genes in 7/8 (87%) samples (KIF1B, NEB and KMT2C). KMT2C showed the highest number of variations; it is an important component of a histone H3 lysine 4 methyltransferase complex and it is one of the histone modifiers previously implicated in carcinogenesis, never studied in osteosarcoma. Moreover, we found a group of 15 genes that showed variations only in patients that did not respond to therapy and developed metastasis and some of these genes are involved in carcinogenesis and tumor progression in other tumors. These data could offer the opportunity to get a key molecular target to identify possible new strategies for early diagnosis and new therapeutic approaches for osteosarcoma and to provide a tailored treatment for each patient based on their genetic profile.

Correction: The nuclear-cytoplasmic trafficking of a chromatin-modifying and remodelling protein (KMT2C), in osteosarcoma

[This corrects the article DOI: 10.18632/oncotarget.25755.].

The nuclear-cytoplasmic trafficking of a chromatin-modifyng and remodelling protein (KMT2C), in osteosarcoma

Osteosarcoma is the most common paediatric primary non-hematopoietic bone tumor; the survival is related to the response to chemotherapy and development of metastases. KMT2C is a chromatin-modifying and remodelling protein and its expression has never been studied in osteosarcoma. The aim of this study was to understand the role of KMT2C in the osteosarcoma carcinogenesis and metastatic progression to identify a new molecular target and to provide new therapeutic approach. We performed the immunohistochemical and gene expression analysis of KMT2C in 32 samples of patients with diagnosis of osteosarcoma with known clinic-pathological data and we analysed the expression of genes involved in the metastatic pathway in four osteosarcoma cell lines by blocking the KMT2C expression using siRNA. We found a nuclear-cytoplamic trafficking of KMT2C and the cytoplasmic localization was higher than the nuclear localization (p < 0.0001). Moreover, the percentage of cells with cytoplasmic positivity increased from low grade primary tissue to metastatic tissues. The cytoplasmic localization of KMT2C could lead to a change in its function supporting osteosarcoma carcinogenesis and progression. Our hypothesis is that KMT2C could affect the enhancer activity of genes influencing the invasive properties and metastatic potential of osteosarcoma.