Biomedical subjects

Massimo Santoro

Publications and source records attributed to Massimo Santoro.

At least 55 records · Page 3Linked to original sources

PubMed2003

Suppression of HMGA2 protein synthesis could be a tool for the therapy of well differentiated liposarcomas overexpressing HMGA2.

Atypical lipomatous tumors (ALTs)/well-differentiated liposarcomas represent a distinctive subset of mesenchymal neoplasms featuring mature adipocytic proliferation. These tumors are characterized cytogenetically by the presence of supernumerary ring and/or long marker chromosomes that contain several copies of the chromosomal region 12q13-15, in which the HMGA2 gene is located. Deregulation of the HMGA2 gene is a common molecular alteration implicated in the development of a variety of benign tumors, such as lipomas, uterine leiomyomas, and pulmonary chondroid hamartomas. In this study, we observed HMGA2 overexpression in 7 of 12 ALT primary cell cultures examined. Subsequently, we generated an adenovirus containing the HMGA2 gene in the antisense orientation (Ad-A2as) to study the effect of HMGA2 protein suppression in ALT cells. The infection of six ALT cells, three of which were positive for HMGA2 expression, resulted in growth inhibition coupled with a significant increase in apoptosis. In addition, the growth of the ALT cells negative for HMGA2 expression was not affected by the infection with either the Ad-A2as or the control virus. On the basis of these findings, the targeting of the HMGA2 protein expression may represent a promising approach for treating the well-differentiated liposarcomas resistant to conventional therapies.

Adenoviruses, Human↗

PubMed2003

A truncated HMGA1 gene induces proliferation of the 3T3-L1 pre-adipocytic cells: a model of human lipomas.

The high mobility group A (HMGA) proteins are non-histone chromosomal proteins implicated in the organization of chromatin structure and in the assembly of protein complexes on the promoters of several inducible genes. Rearrangements of HMGA1 and HMGA2 genes, consequent to chromosomal translocation, have been frequently detected in human benign tumours of mesenchymal origin including lipomas. We have demonstrated previously that 3T3-L1 adipocytic differentiation is associated with increased HMGA1 protein levels, and that the block of HMGA1 synthesis dramatically increases the growth rate of 3T3-L1 cells and suppresses adipocytic differentiation. Here we have examined the role of a truncated HMGA1 gene in adipocytic cell growth. We have found that expression of the truncated Hmga1 gene (Hmga1/T) dramatically increases 3T3-L1 cell growth without blocking adipocytic differentiation. The Hmga1/T 3T3-L1 cells had higher E2F activity than the wild-type cells, and a deregulated cell cycle. In fact, the Hmga1/T cells had a reduced G0/G1 fraction, and a greater number of cells in S-phase. However, consistent with the benign nature of tumours associated with HMGA1 rearrangements, the Hmga1/T 3T3-L1 cells did not acquire the malignant phenotype. These results suggest a critical role played by HMGA1 rearrangements in the generation of human lipomas.

3T3-L1 Cells↗

PubMed2003

Tyrosine kinase oncoprotein, RET/PTC3, induces the secretion of myeloid growth and chemotactic factors.

Differentiated thyroid carcinomas are the most frequent endocrine neoplasms, but account for few cancer-related deaths. Although the indolent growth of these cancers correlates well with longevity, the biological basis for this good prognosis is not known. In contrast, two of the most frequent autoimmune diseases involve the thyroid suggesting a high propensity for this organ to invoke destructive immunity. Unfortunately, the mechanism linking malignancy and autoimmunity is not clear, although the expression of the oncogenic fusion protein RET/PTC3 (RP3) in both of these disorders may provide a clue. Interestingly, the signaling caused by activated RET kinase involves overlapping pathways and some common to the inflammatory response. Accordingly, we analyzed the function of RP3 and a mutant RP3 molecule to induce proinflammatory pathways in thyroid epithelial cells. Indeed, we find that RP3 alone causes increases in nuclear NF-kappaB activity and secretion of MCP-1 and GM-CSF. Finally, transfer of RP3-expressing thyrocytes into mice in vivo attracted dense macrophage infiltrates, which lead to rapid thyroid cell death. Further, cytokine synthesis and inflammation was largely abrogated by mutation of RP3 Tyr588; an important protein-binding site for downstream signaling. Together, these studies implicate oncogene-induced cytokine-signaling pathways in a new mechanism linking inflammation with cancer.

Animals↗

PubMed2003

Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.

We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.

3T3 Cells↗

PubMed2003

Conditional expression of RET/PTC induces a weak oncogenic drive in thyroid PCCL3 cells and inhibits thyrotropin action at multiple levels.

Chromosomal rearrangements linking the promoter(s) and N-terminal domain of unrelated gene(s) to the C terminus of RET result in constitutively activated chimeric forms of the receptor in thyroid cells (RET/PTC). RET/PTC rearrangements are thought to be tumor-initiating events; however, the early biological consequences of RET/PTC activation are unknown. To explore this, we generated clonal lines derived from well-differentiated rat thyroid PCCL3 cells with doxycycline-inducible expression of either RET/PTC1 or RET/PTC3. As previously shown in other cell types, RET/PTC1 and RET/PTC3 oligomerized and displayed constitutive tyrosine kinase activity. Neither RET/PTC1 nor RET/PTC3 conferred cells with the ability to grow in the absence of TSH, likely because of concomitant stimulation of both DNA synthesis and apoptosis, resulting in no net growth in the cell population. Effects of RET/PTC on DNA synthesis and apoptosis did not require direct interaction of the oncoprotein with either Shc or phospholipase Cgamma. Acute expression of the oncoprotein decreased TSH-mediated growth stimulation due to interference of TSH signaling by RET/PTC at multiple levels. Taken together, these data indicate that RET/PTC is a weak tumor-initiating event and that TSH action is disrupted by this oncoprotein at several points, and also predict that secondary genetic or epigenetic changes are required for clonal expansion.

Adenylyl Cyclases↗

PubMed2003

Cloning and molecular characterization of a novel gene strongly induced by the adenovirus E1A gene in rat thyroid cells.

Expression of the adenovirus E1A gene in the rat thyroid differentiated cell line PC Cl 3 induces thyrotropin-independent cell growth and impairs differentiation. However, the malignant phenotype is achieved only when the PC E1A cells are infected with other murine retroviruses carrying the v-abl, v-raf or polyoma middle-T genes. To determine through which genes E1A affects thyroid cells, we differentially screened PC Cl 3 and PC E1A cells. Here we report a new gene, named CL2, that is upregulated in PC E1A cells. The CL2 transcript is 4.4 kb long and encodes a 949 amino-acid protein. Conceptual translation of the open reading frame showed one product with a signal peptide, multiple nuclear localization signals and three newly described domains. Furthermore, in vivo, this protein was located juxtanuclear, which is suggestive of Golgian localization, and also in cytoplasm and nucleus/nucleolus. Finally, CL2 gene expression was drastically downregulated in human thyroid neoplastic cell lines and tissues.

Adenovirus E1A Proteins↗

PubMed2003

An adenovirus carrying the rat protein tyrosine phosphatase eta suppresses the growth of human thyroid carcinoma cell lines in vitro and in vivo.

We demonstrated previously that rat tyrosine phosphatase r-PTPeta expression was suppressed in rat and human thyroid neoplastic cells, and that restoration of r-PTPeta expression reverted the malignant phenotype. To investigate the potential of this gene for cancer therapy, we generated an adenovirus carrying the r-PTPeta cDNA (Ad-r-PTPeta). This virus infected human thyroid carcinoma cells and overexpressed the r-PTPeta protein. Overexpression of r-PTPeta significantly inhibited the growth of four thyroid carcinoma cell lines. Cell growth inhibition was associated with down-regulation of extracellular signal-regulated kinase 1/2 activity, with increased levels of the cell-cycle inhibitor p27(kip1) protein and with dephosphorylation of PLCgamma1, a substrate of DEP-1, the human homologue of r-PTPeta. Finally, the growth of xenograft tumors induced in athymic mice by anaplastic thyroid carcinoma ARO cells transduced with the Ad-r-PTPeta virus was drastically reduced. These data suggest that gene therapy based on restoration of PTPeta function has potential in the treatment of human thyroid malignant neoplasias.

Adenocarcinoma, Follicular↗

PubMed2003

Ras-mediated apoptosis of PC CL 3 rat thyroid cells induced by RET/PTC oncogenes.

RET gene rearrangements, which generate chimeric RET/PTC oncogenes, are early events in the evolution of thyroid papillary carcinomas. Expression of RET/PTC oncogenes promotes neoplastic transformation of cultured thyroid cells and of thyroid glands in transgenic mice. Notwithstanding these oncogenic effects, we have found that the expression of two RET/PTC oncogenes (H4-RET and RFG-RET) induces apoptosis of rat thyroid PC CL 3 cells. Promotion of thyroid cell death depends on the kinase activity of RET/PTC and on the phosphorylation of a tyrosine residue (tyrosine 1062) that maps in the carboxy-terminus of the RET protein. Tyrosine 1062 is essential for RET/PTC-mediated activation of the Ras/ERK pathway. Inhibition of Ras/ERK by a dominant negative Ras or by the MEKI inhibitor, PD98059, obstructed RET/PTC-mediated apoptosis. We also show that signals transmitted by tyrosine 1062 mediate proapoptotic events like Bcl-2 down regulation and Bax upregulation, and that adoptive overexpression of Bcl-2 overcomes RET/PTC-induced apoptosis. Thus, gene rearrangements that generate RET/PTC oncogenes subvert RET function by converting it into a chronically active kinase that is constitutively phosphorylated on tyrosine 1062. In turn, Y1062 phosphorylation transmits not only mitogenic but also proapoptotic signals to thyroid cells.

Animals↗

PubMed2003

Antineoplastic ribonucleases selectively kill thyroid carcinoma cells via caspase-mediated induction of apoptosis.

Bovine seminal ribonuclease (BS-RNase), a natural dimeric homolog of bovine pancreatic RNase (RNase A), and HHP2-RNase, an engineered dimeric form of human pancreatic RNase (HP-RNase), are endowed with powerful antitumor effects. Here we show that BS- and HHP2-RNases, but not monomeric RNase A, induce apoptosis of human thyroid carcinoma cell lines. RNase-induced apoptosis was associated with activation of initiation caspase-8 and -9. This was followed by activation of executioner caspase-3, leading to the proteolytic cleavage of poly(ADP-ribose) polymerase. The caspase inhibitor Z-Val-Ala-Asp-(OMe)-fluoromethylketone protected thyroid cancer cells from BS-RNase-induced apoptosis. RNase-triggered apoptosis and caspase activation were accompanied by reduced phosphorylation of Akt/protein kinase B (PKB), a serine-threonine kinase that when phosphorylated is able to deliver survival signals to cancer cells. BS-RNase antitumor effects in nude mice were accompanied by caspase activation and apoptosis. Because of the high selectivity of apoptotic effects for malignant cells, BS- and HHP2-RNase are promising tools for the treatment of aggressive thyroid cancer.

Amino Acid Chloromethyl Ketones↗

PubMed2003

Efficient inhibition of RET/papillary thyroid carcinoma oncogenic kinases by 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2).

Inappropriate activation of the RET receptor tyrosine kinase causes development of papillary and medullary thyroid cancer. We have previously shown that pyrazolopyrimidine is a potent inhibitor of the RET kinase. Here, we show that 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) (PP2), another pyrazolopyrimidine, blocks the enzymatic activity of the isolated RET kinase and RET/PTC1 oncoprotein at IC(50) in the nanomolar range. PP2 blocked in vivo phosphorylation and signaling of the RET/PTC1 oncoprotein. PP2 prevented serum-independent growth of RET/PTC1-transformed NIH3T3 fibroblasts and of TPC1 and FB2, two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, PP2 blocked invasion of type I collagen matrix by TPC1 cells. Thus, pyrazolopirimidines hold promise for the treatment of human cancers sustaining oncogenic activation of RET.

3T3 Cells↗

PubMed2003

BRAF mutations in thyroid tumors are restricted to papillary carcinomas and anaplastic or poorly differentiated carcinomas arising from papillary carcinomas.

Activating point mutations of the BRAF gene have been recently reported in papillary thyroid carcinomas. In this study, we analyzed 320 thyroid tumors and six anaplastic carcinoma cell lines and detected BRAF mutations in 45 (38%) papillary carcinomas, two (13%) poorly-differentiated carcinomas, three (10%) anaplastic carcinomas, and five (83%) thyroid anaplastic carcinoma cell lines but not in follicular, Hürthle cell, and medullary carcinomas, follicular and Hürthle cell adenomas, or benign hyperplastic nodules. All mutations involved a T-->A transversion at nucleotide 1796. In papillary carcinomas, BRAF mutations were associated with older age, classic papillary carcinoma or tall cell variant histology, extrathyroidal extension, and more frequent presentation at stages III and IV. All BRAF-positive poorly differentiated and anaplastic carcinomas contained areas of preexisting papillary carcinoma, and mutation was present in both the well-differentiated and dedifferentiated components. These data indicate that BRAF mutations are restricted to papillary carcinomas and poorly differentiated and anaplastic carcinomas arising from papillary carcinomas. They are associated with distinct phenotypical and biological properties of papillary carcinomas and may participate in progression to poorly differentiated and anaplastic carcinomas.

Adult↗

PubMed2003

HMGA2 locus rearrangement in a case of acute lymphoblastic leukemia.

We report the first case of acute lymphoblastic leukemia associated with a t(9;12)(p22;q14) translocation within the HMGA2 locus. The breakpoint lies in the 5' region. Fluorescence in situ hybridization with a BAC clone covering the 5' region produced three signals versus two signals with a BAC clone covering the 3' region including intron 3 of HMGA2, which harbors most of the breakpoints described in benign mesenchymal tumors. HMGA2 locus rearrangement was associated with overexpression of an HMGA2 mRNA that lacked a carboxy-terminal tail. These results suggest that the HMGA2 gene rearrangement may contribute to the malignancy in leukemias harboring a 12q translocation.

Child↗

PubMed2002

ZD6474, an orally available inhibitor of KDR tyrosine kinase activity, efficiently blocks oncogenic RET kinases.

RET/papillary thyroid carcinoma (PTC) oncogenes, generated by recombination of the tyrosine kinase-encoding domain of RET with different heterologous genes, are prevalent in papillary carcinomas of the thyroid. Point mutations of RET cause multiple endocrine neoplasia type 2 (MEN2) familial cancer syndrome and are found in sporadic medullary thyroid carcinomas. Here, we show that ZD6474, a low molecular weight tyrosine kinase inhibitor, blocks the enzymatic activity of RET-derived oncoproteins at a one-half maximal inhibitory concentration of 100 nM. ZD6474 blocked in vivo phosphorylation and signaling of the RET/PTC3 and RET/MEN2B oncoproteins and of an epidermal growth factor (EGF)-activated EGF-receptor/RET chimeric receptor. RET/PTC3-transformed cells-treated ZD6474 lost proliferative autonomy and showed morphological reversion. ZD6474 prevented the growth of two human PTC cell lines that carry spontaneous RET/PTC1 rearrangements. Finally, it blocked anchorage-independent growth of RET/PTC3-transformed NIH3T3 fibroblasts and the formation of tumors after injection of NIH-RET/PTC3 cells into nude mice. Thus, targeting RET oncogenes with ZD6474 might offer a potential treatment strategy for carcinomas sustaining oncogenic activation of RET.

3T3 Cells↗

PubMed2002

Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27(Kip1) by PKB/Akt-mediated phosphorylation in breast cancer.

The cyclin-dependent kinase inhibitor p27(kip1) is a putative tumor suppressor for human cancer. The mechanism underlying p27(kip1) deregulation in human cancer is, however, poorly understood. We demonstrate that the serine/threonine kinase Akt regulates cell proliferation in breast cancer cells by preventing p27(kip1)-mediated growth arrest. Threonine 157 (T157), which maps within the nuclear localization signal of p27(kip1), is a predicted Akt-phosphorylation site. Akt-induced T157 phosphorylation causes retention of p27(kip1) in the cytoplasm, precluding p27(kip1)-induced G1 arrest. Conversely, the p27(kip1)-T157A mutant accumulates in cell nuclei and Akt does not affect p27(kip1)-T157A-mediated cell cycle arrest. Lastly, T157-phosphorylated p27(kip1) accumulates in the cytoplasm of primary human breast cancer cells coincident with Akt activation. Thus, cytoplasmic relocalization of p27(kip1), secondary to Akt-mediated phosphorylation, is a novel mechanism whereby the growth inhibitory properties of p27(kip1) are functionally inactivated and the proliferation of breast cancer cells is sustained.

Breast Neoplasms↗

PubMed2002

Generation and characterization of novel monoclonal antibodies to the Ret receptor tyrosine kinase.

Ret is a tyrosine kinase receptor involved in several human diseases germ-line mutations are responsible for multiple endocrine neoplasia type 2 syndromes while somatic mutations of Ret are found in sporadic medullary thyroid carcinomas. In the present work, we describe the generation and characterization of a panel of novel monoclonal antibodies to Ret obtained by immunizing mice with a Ret-FC fusion protein. Fifty-five independent monoclonal antibodies recognize Ret-FC by enzyme linked immunosorbent assay but not a non-related FC fusion protein. Twenty antibodies further characterized recognize Ret expressing cells by flow cytometry. Finally, immunoprecipitation analysis showed that these antibodies recognize Ret mature glycosylated and immature forms. Thus, these monoclonal antibodies could be used as diagnostic tools to detect Ret expression, as well as therapeutic tools to downmodulate Ret or to deliver cytotoxic drugs to malignancies that overexpress Ret as neuroblastomas, medullary and papillary thyroid carcinomas, seminomas, and leukemia.

Animals↗

PubMed2002

Overexpression of the HMGA2 gene in transgenic mice leads to the onset of pituitary adenomas.

Overexpression of the HMGA2 gene is a common feature of neoplastic cells both in experimental and human models. Intragenic and extragenic HMGA2 rearrangements responsible for HMGA2 gene overexpression have been frequently detected in human benign tumours of mesenchymal origin. To better understand the role of HMGA2 overexpression in human tumorigenesis, we have generated transgenic mice carrying the HMGA2 gene under the transcriptional control of the cytomegalovirus promoter. High expression of the transgene was demonstrated in all the mouse tissues analysed, whereas no expression of the endogenous HMGA2 gene was detected in the same tissues from wild-type mice. In this study, two independent lines of transgenic mice have been generated. By 6 months of age, 85% of female animals of both transgenic lines developed pituitary adenomas secreting prolactin and growth hormone. The transgenic males developed the same phenotype with a lower penetrance (40%) and a longer latency period (about 18 months). Therefore, these data demonstrate that the overexpression of HMGA2 leads to the onset of mixed growth hormone/prolactin cell pituitary adenomas. These transgenic mice may represent an important tool for the study of this kind of neoplasia.

Adenoma↗

PubMed2002

Thyroid cell transformation requires the expression of the HMGA1 proteins.

Elevated expression of HMGA1 and HMGA2 proteins is correlated with a highly malignant phenotype in several human tumors. We previously demonstrated that the block of HMGA2 protein synthesis prevented rat thyroid cell transformation by murine retroviruses. Suppression of HMGA2 synthesis was associated with lack of induction of HMGA1 proteins suggesting that both HMGA1 and HMGA2 play a role in the process of neoplastic transformation. To determine the role of the HMGA1 gene in thyroid cell transformation, we blocked HMGA1 protein synthesis by an antisense methodology. Here we report that transfection of an HMGA1 cDNA antisense construct into a normal rat thyroid cell line (FRTL-5 Cl2), followed by infection with Kirsten murine sarcoma virus (KiMSV), generated a transformed cell line that expresses high levels of the v-ras-Ki oncogene and that does not require thyroid-stimulating hormones for growth. However, this cell line does not show the malignant phenotype, i.e., it neither grows in soft agar nor induces tumors after injection in athymic mice. Moreover, the lack of the neoplastic phenotype in the virus-infected thyroid cells carrying the HMGA1 antisense construct correlates with the absence of induction of AP-1 transcriptional activity.

Animals↗

PubMed2002

Glial cell line-derived neurotrophic factor induces proliferative inhibition of NT2/D1 cells through RET-mediated up-regulation of the cyclin-dependent kinase inhibitor p27(kip1).

Growth factors of the glial cell line-derived neurotrophic factor (GDNF) family control the differentiation of neuronal cells of the central and peripheral nervous systems. Intracellular signalling of these growth factors is, at least in part, mediated by activation of the RET receptor tyrosine kinase. Here, we demonstrate that GDNF triggering inhibits the proliferation of the embryonal carcinoma cell line NT2/D1. This anti-proliferative effect is accompanied by down-regulation of the SSEA-3 antigen, a marker typical of undifferentiated NT2/D1 cells. We show that these effects are mediated by activation of RET signalling. The block of RET by a kinase-deficient dominant negative mutant impairs GDNF-dependent growth inhibition, whereas the adoptive expression of a constitutively active RET, the RET-MEN2A oncogene, promotes effects similar to those exerted by GDNF. We show that RET signalling increases the expression of the cyclin-dependent kinase inhibitor p27(kip1) in NT2/D1 cells. Both DNA synthesis inhibition and SSEA-3 down-regulation are prevented if p27(kip1) expression is blocked by an antisense construct, which demonstrates that RET-triggered effects are mediated by p27(kip1).

Animals↗