-
Valentina Nicolini,
Giuliana Cassinelli, Giuditta Cuccuru,
Italia Bongarzone,
Giovanna Petrangolini,
Monica Tortoreto,
Piera Mondellini,
Patrizia Casalini,
Enrica Favini,
Nadia Zaffaroni,
Franco Zunino,
Cinzia Lanzi
[show abstract]
[hide abstract]
ABSTRACT: Emerging evidence suggests that Ret oncoproteins expressed in medullary thyroid cancer (MTC) might evade the pro-apoptotic function of the dependence receptor proto-Ret by directly impacting the apoptosis machinery. Identification of the molecular determinants of the interplay between Ret signaling and apoptosis might provide a relevant contribution to the optimization of Ret-targeted therapies. Here, we describe the cross-talk between Ret-M918T oncogenic mutant responsible for type 2B multiple endocrine syndrome (MEN2B), and components of death receptor-mediated extrinsic apoptosis pathway. In the human MEN2B-type MTC cell line MZ-CRC-1 expressing Ret-M918T, Ret was found associated with Fap-1, known as inhibitor of the CD95 death receptor trafficking to the cell membrane, and with procaspase-8, the initiator pro-form caspase in the extrinsic apoptosis pathway. Cell treatment with the anti-tumor Ret kinase inhibitor RPI-1 inhibited tyrosine phosphorylation of procaspase-8, likely inducing its local activation, followed by downregulation of both Ret and Fap-1, and translocation of CD95 into lipid rafts. According to the resulting increase of CD95 cell surface expression, the CD95 agonist antibody CH11 enhanced RPI-1-induced cell growth inhibition and apoptosis. RET RNA interference downregulated Fap-1 protein in MZ-CRC-1 cells, whereas exogenous RET-M918T upregulated Fap-1 in HEK293 cells. Overall, these data indicate that the Ret oncoprotein exerts opposing controls on Fap-1 and CD95, increasing Fap-1 expression and decreasing CD95 cell surface expression. The functional interplay of the Ret mutant with the extrinsic apoptosis pathway provides a mechanism possibly contributing to MTC malignant phenotype and a rational basis for novel therapeutic strategies combining Ret inhibitors and CD95 agonists.
Biochemical pharmacology 10/2011; 82(7):778-88. · 4.25 Impact Factor
-
Giuliana Cassinelli,
Domenica Ronchetti,
Diletta Laccabue,
Michela Mattioli, Giuditta Cuccuru,
Enrica Favini,
Valentina Nicolini,
Angela Greco,
Antonino Neri,
Franco Zunino,
Cinzia Lanzi
[show abstract]
[hide abstract]
ABSTRACT: The identification of proliferation/survival pathways constitutively activated by genetic alterations in multiple myeloma (MM), or sustained by the bone marrow (BM) microenvironment, provides novel opportunities for the development of targeted therapies. The deregulated function of protein tyrosine kinases plays a critical role in driving MM malignant phenotype. We investigated the effects of the multi-target tyrosine kinase inhibitor RPI-1 in a panel of human MM cell lines, including t(4;14) positive cell lines expressing the TK receptor FGF-R3. Cells harboring FGF-R3 activating mutations (KMS11 and OPM2) displayed the highest sensitivity to RPI-1 antiproliferative effect. The stimulating effect of the aFGF ligand was abrogated in cells harboring a non-constitutively active receptor. Drug treatment inhibited activation and expression of the FGF-R3(Y373C) mutant as well as aFGF-dependent signaling involving AKT and ERKs. Inhibition of JAK2, an additional RPI-1 target, resulted in STAT3 inactivation. Blockade of these proliferation/survival pathways was associated with caspase-dependent apoptosis. Moreover, drug treatment abrogated proliferative and pro-invasive stimuli provided by conditioned medium from mesenchymal stromal cells. Gene expression profile of KMS11 cells showed 22 upregulated and 52 downregulated genes upon RPI-1 treatment, with an early modulation of genes implicated in MM pathobiology such as SAT-1, MYC, MIP-1alpha/beta, FGF-R3, and the growth factor receptor B-cell maturation antigen (BCMA). Thus, concomitant blockade of FGF-R3 and JAK2 results in inhibition of several MM-promoting pathways, including BCMA-regulated signaling, and downregulation of disease-associated proteins. These data may have therapeutic implications in the design of treatment strategies resulting in the concomitant inhibition of FGF-R3 and JAK2 signaling pathways in t(4;14) MM.
Biochemical pharmacology 07/2009; 78(9):1139-47. · 4.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A novel series of 3-arylureidobenzylidene-indolin-2-ones was synthesized and their inhibitory activity against Ret tyrosine kinase investigated in comparison with the Ret inhibitor RPI-1 as a reference compound for this series. A few compounds were able to revert the RETC634R oncogene-transformed morphologic phenotype of NIH3T3(MEN2A) cells and showed a selective antiproliferative activity against these cells as compared to parental NIH3T3 cells or NIH3T3 cells transformed with a non-tyrosine kinase oncogene (NIH3T3(H-RAS)). Inhibition of Ret enzyme activity by effective derivatives was confirmed in a kinase assay. Structure-activity relationship indicated a favourable activity for 5,6-dimethoxyindolinone derivatives with H, OH, or OMe in the para position of the distal aryl ring.
Bioorganic & Medicinal Chemistry Letters 08/2007; 17(14):3962-8. · 2.55 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The proto-oncogene c-myc is involved in multiple cell pathways with opposite effects on cell outcome of death or proliferation. It has been proposed that these different roles depend on the sequestration of c-Myc protein in cellular compartments and/or its phosphorylation. We speculated that subcellular localization of c-Myc protein and of its phosphorylated form (P-c-Myc) could have a role in the different response to paclitaxel (PTX) in two prostate carcinoma cell lines, PC3 and DU145, which undergo either multinucleation or c-myc-dependent apoptosis, respectively. c-myc is amplified only in PC3, but a similar extent of c-Myc phosphorylation was observed in both cell lines after PTX treatment. We found that PTX-induced upregulation of c-myc in DU145 cells, not occurring in PC3 cells, cannot be ascribed to a different protein localization, and that a comparable c-Myc and P-c-Myc nuclear translocation occurs in both cell lines after drug treatment. Thus, subcellular localization of c-Myc and P-c-Myc is not crucial in determining the mode of cell death in these prostate carcinoma cell lines.
Annals of the New York Academy of Sciences 01/2007; 1095:175-81. · 3.15 Impact Factor
-
Giuliana Cassinelli,
Cinzia Lanzi,
Giovanna Petrangolini,
Monica Tortoreto,
Graziella Pratesi, Giuditta Cuccuru,
Diletta Laccabue,
Rosanna Supino,
Sara Belluco,
Enrica Favini,
Anna Poletti,
Franco Zunino
[show abstract]
[hide abstract]
ABSTRACT: Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. We investigated the biochemical and biological effects of the tyrosine kinase inhibitor RPI-1 on the human lung cancer cell lines H460 and N592, which express constitutively active Met. RPI-1-treated cells showed down-regulation of Met activation and expression, inhibition of HGF/Met-dependent downstream signaling involving AKT, signal transducers and activators of transcription 3 and paxillin, as well as a reduced expression of the proangiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. Cell growth in soft agar of H460 cells was strongly reduced in the presence of the drug. Furthermore, RPI-1 inhibited both spontaneous and HGF-induced motility/invasiveness of both H460 and human endothelial cells. Targeting of Met signaling by alternative methods (Met small interfering RNA and anti-phosphorylated Met antibody intracellular transfer) produced comparable biochemical and biological effects. Using the spontaneously metastasizing lung carcinoma xenograft H460, daily oral treatment with well-tolerated doses of RPI-1 produced a significant reduction of spontaneous lung metastases (-75%; P < 0.001, compared with control mice). In addition, a significant inhibition of angiogenesis in primary s.c. tumors of treated mice was observed, possibly contributing to limit the development of metastases. The results provide preclinical evidence in support of Met targeting pharmacologic approach as a new option for the control of tumor metastatic dissemination.
Molecular Cancer Therapeutics 10/2006; 5(9):2388-97. · 5.23 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Recent evidence indicates that the success of molecular targeted therapies may depend on the identification of drug targets which are essential for the survival of subsets of tumors. RET oncogenes that have been implicated in the development of thyroid carcinomas are emerging as potential therapeutic targets. In the present study, we investigated the efficacy and the cellular bases of antitumor activity of the indolinone Ret tyrosine kinase inhibitor RPI-1 against large established s.c. TT tumor xenograft, a human medullary thyroid carcinoma (MTC) harboring oncogenic MEN-2A-type RET mutation. Oral treatment with RPI-1 caused growth arrest or regression in 81% treated tumors. Following treatment suspension, tumor inhibition was maintained (51%, P<0.05, 100 days) and cures were achieved in 2/11 mice. In treated tumors, Ret was tyrosine dephosphorylated. Moreover, compared to control tumors, a significant increase in apoptotic cells (210%, P<0.0001), loss of cellularity (47%, P<0.0001) and reduction of microvessel density (36%, P<0.0005) were detected. In vivo effects of RPI-1 were reflected in activation of BAD, cleavage of caspases, apoptotic DNA fragmentation and inhibition of VEGF production observed in in vitro RPI-1-treated TT cells. These findings thus indicate that RPI-1 antitumor effect on the MTC was characterized by apoptosis induction and angiogenesis inhibition. The results, consistent with a dependence on RET oncogene activation for maintenance and survival of MEN2A-type MTC, provide further preclinical rationale for a pharmacological RET-targeted intervention in thyroid cancer.
Biochemical Pharmacology 08/2006; 72(4):405-14. · 4.70 Impact Factor
-
Giuditta Cuccuru,
Cinzia Lanzi,
Giuliana Cassinelli,
Graziella Pratesi,
Monica Tortoreto,
Giovanna Petrangolini,
Ettore Seregni,
Antonia Martinetti,
Diletta Laccabue,
Chiara Zanchi,
Franco Zunino
[show abstract]
[hide abstract]
ABSTRACT: The RET proto-oncogene encodes a receptor tyrosine kinase. RET oncogenes arise through sporadic and inherited gene mutations and are involved in the etiopathogenesis of medullary thyroid carcinoma, a cancer that responds poorly to conventional chemotherapy. Medullary thyroid carcinoma is a component of multiple endocrine neoplasia type 2 or MEN2 syndromes.
We investigated the cellular effects of RPI-1, a novel 2-indolinone Ret tyrosine kinase inhibitor on cells that express RET C634 oncogenic mutants common in the MEN2A syndrome: NIH3T3 fibroblasts transfected with RET(C634R) and human medullary thyroid carcinoma TT cells that express endogenous RET(C634W). RPI-1 antiproliferative activity was determined by cell proliferation and anchorage-independent growth assays. Expression and phosphorylation of Ret and of proteins involved in downstream signaling pathways were examined by immunoblotting. Antitumor activity of oral RPI-1 treatment was tested by using two dosing levels in nude mice bearing subcutaneous TT xenograft tumors. All statistical tests were two-sided.
The RPI-1 IC50 value for cell proliferation was 3.6 microM (95% confidence interval [CI] = 1.8 to 5.4 microM) in NIH3T3 cells expressing the Ret mutant compared with 16 microM (95% CI = 12.3 to 19.7 microM) in non-transfected NIH3T3 cells, and that for colony formation in soft agar was 2.4 microM (95% CI = 0.8 to 4.0 microM) and 26 microM (95% CI = 17 to 35 microM) in RET mutant-transfected and H-RAS-transfected NIH3T3 cells, respectively. In NIH3T3 cells expressing the Ret mutant, Ret protein and tyrosine phosphorylation were undetectable after 24 hours of RPI-1 treatment. In TT cells, RPI-1 inhibited proliferation, Ret tyrosine phosphorylation, Ret protein expression, and the activation of PLCgamma, ERKs and AKT. In mice, oral daily RPI-1 treatment inhibited the tumor growth of TT xenografts by 81% (P<.001 versus control mice) and reduced the plasma levels of the specific biomarker calcitonin (P =.01 versus control mice). Twenty-five percent of RPI-1-treated mice were tumor-free.
Ret oncoproteins represent exploitable targets for therapeutic intervention in MEN2A-associated medullary thyroid carcinoma. The antitumor efficacy and oral bioavailability of RPI-1 support its therapeutic potential.
CancerSpectrum Knowledge Environment 08/2004; 96(13):1006-14. · 14.07 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Taxane-based therapies appear to have a significant efficacy in clinical trials on hormone-refractory prostate carcinoma. In the present study, we investigated the cellular response of androgen-independent prostate carcinoma cell lines to the novel taxane IDN 5109 (BAY 59-8862) and evaluated its antitumor activity. In previous preclinical studies, this new paclitaxel (PTX) analogue was characterized by high tolerability and antitumor efficacy, ability to overcome multidrug resistance, and activity by oral administration. Upon treatment, DU145 and PC3 prostate carcinoma cell lines underwent a transient mitotic arrest. This was followed by G1 arrest and rapid occurrence of apoptosis in DU145 cells, whereas in PC3 cells, which are defective for the postmitotic checkpoint, a slow cell death was preceded by DNA endoreduplication. At the biochemical level, such events were associated with tubulin polymerization, activation of the mitosis-promoting factor, and phosphorylation of Bcl-X(L)/Bcl-2/Raf-1. In addition, IDN 5109 shared with PTX the ability to down-regulate the expression of the two potent angiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. These findings indicated that IDN 5109 affected the same pathways involved in the cellular response to PTX and suggested that an antiangiogenic effect mediated by inhibition of paracrine stimulation of endothelial cells might contribute to the antitumor effect of both drugs. In in vivo experiments, the new taxane displayed a superior and more persistent effect compared with PTX against DU145 tumor xenografts. Such an effect was associated with pronounced reduction of the tumor microvessel density, superior to that achieved by PTX. These results support a potential therapeutic advantage of IDN 5109 over PTX against hormone-refractory prostate carcinoma.
Clinical Cancer Research 09/2002; 8(8):2647-54. · 7.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: BACKGROUND
Defects in the cell cycle machinery of prostate cancer cells might impair the efficiency of cell cycle checkpoints and affect the cell response to chemotherapeutic drugs. We examined the relationship between the status of microtubule damage-activated checkpoints and the response of hormone-refractory prostate cancer cells to paclitaxel.METHODS
The two cell lines DU145 and PC3 harboring defects at proteins involved in the regulation of checkpoints activated by microtubule damage were examined for cell sensitivity, apoptotic response, and efficiency of checkpoints in response to paclitaxel.RESULTSIn spite of a comparable sensitivity to the antiproliferative effects of paclitaxel, DU145 and PC3 cells exhibited different cell cycle control at checkpoints activated by microtubule damage. A transient mitotic arrest was induced by the taxane in both cell lines. However, PC3 cells underwent a rapid mitotic slippage and displayed a defective postmitotic checkpoint as evidenced by the appearance of polyploid cells. In this cell line, paclitaxel-induced cell death was a slow and delayed event, occurring also after S-phase re-entry. The mitotic checkpoint appeared to be more stringent in DU145 cells compared to PC3 cells. Moreover, despite the expression of mutated proteins involved in the prevention of DNA endoreduplication (p53, pRb, and p16INK4A), these cells did not progress into the cell cycle but efficiently underwent apoptosis by 24 hr. Such a response of DU145 cells was associated with phosphorylation of the p21WAF1 protein.CONCLUSIONS
These observations evidence that activation of checkpoints following microtubule damage in prostate cancer may be regulated through complex mechanisms possibly involving p21WAF1. Our findings support that the status of cell cycle checkpoints might affect the modality of cell death. However, the relevance of the mode of cell death for the sensitivity to taxanes remains to be determined. Prostate 48:254–264, 2001. © 2001 Wiley-Liss, Inc.
The Prostate 08/2001; 48(4):254 - 264. · 3.48 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ret oncoproteins expressed in thyroid carcinomas represent possible targets for therapeutic intervention. Oncogenic activation of the receptor tyrosine kinase encoding RET gene occurs typically by gene rearrangement in papillary thyroid carcinomas (PTC) and by missense mutation in medullary thyroid carcinomas (MTC). These genetic alterations lead to the expression of deregulated products characterized by ligand-independent activation of the intrinsic tyrosine kinase of Ret. Such features suggest the possibility of using specific tyrosine kinase inhibitors to block the Ret oncoproteins signaling. The present report summarizes the cellular effects of the arylidene 2-indolinone Ret inhibitor RPI-1 (formerly Cpd1) on the human PTC cell line TPC-1 which spontaneously harbors the RET/PTC1 oncogene. The results provide evidence that RPI-1 is able to inhibit cell growth and to interfere with Ret/ptc1-driven signaling. These findings support a role for Ret oncoproteins as therapeutic targets and the pharmacological interest of RPI-1 as a candidate drug for preclinical evaluation on thyroid tumors expressing RET oncogenes.
Tumori 89(5):520-2. · 0.86 Impact Factor
