Giuliana Cassinelli

Inhibition of transforming activity of the ret/ptc1 oncoprotein by a 2- indolinone derivative

ret‐derived oncogenes are frequently and specifically expressed in thyroid tumors. In contrast to the ret receptor, ret oncoproteins are characterized by ligand‐independent tyrosine‐kinase activity and tyrosine phosphorylation. In this study, novel synthetic arylidene 2‐indolinone compounds were evaluated as inhibitors of the ret/ptc1 tyrosine kinase. Four compounds inhibited ret/ptc1 activity in immunokinase assay (IC50 27 − 42 μM) including one (1,3‐dihydro‐5,6‐dimethoxy‐3‐[(4‐hydroxyphenyl) methylene)‐2H‐indol‐2‐one) (Cpd 1) that selectively inhibited the anchorage‐independent growth of NIH3T3 transformants expressing the ret/ptc1 gene (NIH3T3ptc1 cells). Following exposure to Cpd 1, the transformed phenotype of NIH3T3ptc1 cells was reverted, within 24 hr, to a normal fibroblast‐like morphology in adherent‐cell culture. In these cells, the constitutive tyrosine phosphorylation of ret/ptc1, of the transducing adaptor protein shc and of a series of co‐immunoprecipitated peptides became much reduced, as demonstrated by immunoprecipitation/Western‐blot analyses. Data presented provide additional evidence that ret/ptc1 is directly implicated in malignant transformation, and demonstrate the ability of Cpd 1 to interfere in the signal transduction pathway constitutively activated by the ret/ptc1 oncoprotein. These results confirm the interest of the arylidene 2‐indolinone class of tyrosine‐kinase inhibitors as tools for the study of ret signaling and the control of cell proliferation in ret‐ and ret/ptcs‐associated diseases. Int. J. Cancer 85:384–390, 2000. ©2000 Wiley‐Liss, Inc.

A role for loss of p53 function in sensitivity of ovarian carcinoma cells to taxanes

Loss of p53 function has been linked to increased responsiveness to taxane treatment of ovarian carcinoma in clinical studies. We recently reported that the acquisition of cisplatin resistance in an ovarian carcinoma cell line (IGROV‐1) was associated with mutation of p53 and collateral sensitivity to paclitaxel. The increased sensitivity to paclitaxel of the cisplatin‐resistant subline appeared to be pharmacologically relevant since it was reflected in an in vivo sensitization to taxanes. To investigate the cellular and molecular basis of this phenomenon, we performed a comparative study of cellular response to taxanes (paclitaxel and the novel analog IDN 5109) in the parental cell line, containing wild‐type p53 and its cisplatin‐resistant p53 mutant subline (IGROV‐1/Pt1). IDN 5109 was included in this study because of its higher potency and efficacy compared with paclitaxel on both tumor systems. The pattern of cellular response of the two ovarian cell lines was different. In IGROV‐1 cells, apoptosis was an early event consequent to a transient mitotic arrest. The cell death of IGROV‐1/Pt1 cells was a somewhat slow and delayed event, following mitotic arrest and appearance of hyperploid cells. The increased cytotoxic effect of IDN 5109, compared with paclitaxel, was associated with more marked p34cdc2 dephosphorylation in IGROV‐1 cells and higher Bcl‐2 phosphorylation in IGROV‐1/Pt1 cells after 24 hr of treatment. In each cell line, these biochemical events were not correlated with parallel levels of mitotic cells. Attempts to reintroduce wild‐type p53 in IGROV‐1/Pt1 were unsuccessful. However, in other p53‐deficient cells (osteosarcoma SAOS), taxane treatment was associated with hyperploid progression and the introduction of wild‐type p53 resulted in a reduced sensivity. Although our approach does not allow definitive conclusions, these results suggest that loss of p53‐dependent post‐mitotic checkpoint results in a different time‐course of taxane‐induced cell death following DNA reduplication. These events, more evident after exposure to the potent analog IDN 5109, support the notion that the enhanced sensitivity of p53 mutant cells is closely related to the different mode of cell death.

Targeting RET for thyroid cancer therapy

The limited efficacy of conventional treatments in progressive thyroid carcinomas indicates the need for new therapeutic options. Activating mutations of the receptor tyrosine kinase-encoding RET gene have been identified as driving oncogenic events in subsets of papillary (PTC) and medullary (MTC) thyroid carcinomas suggesting the interest of targeted therapy. The role of RET oncogenes and the encoded constitutively active oncoproteins as potential targets has been investigated by different strategies including gene therapy and pharmacological approaches, but targeted treatment for RET-driven cancers is not clinically available in current therapy. Small molecule tyrosine kinase inhibitors, including sorafenib, sunitinib, motesanib and vandetanib, which have already shown efficacy against other neoplastic diseases, are being evaluated in clinical trials for treatment of thyroid carcinomas. Most of them, also described as Ret inhibitors, are multi-kinase inhibitors with antiangiogenic activity related to inhibition of receptor tyrosine kinases, such as the vascular endothelial growth factor receptors. Preclinical evidence supports the relevance of Ret oncoproteins as therapeutic targets for a subset of thyroid neoplastic diseases and, although targeting the original causal genetic change may not be sufficient to control the disease efficiently, the available knowledge outlines therapeutic opportunities for exploiting Ret inhibition.

Induction of apoptosis and stress response in ovarian carcinoma cell lines treated with ST1926, an atypical retinoid

AbstractTo understand the molecular mechanisms mediating apoptosis induction by a novel atypical retinoid, ST1926, the cellular response to drug treatment was investigated in IGROV-1 ovarian carcinoma cells carrying wild-type p53 and a cisplatin-resistant p53 mutant subline (IGROV-1/Pt1). Despite a similar extent of drug-induced DNA strand breaks, the level of apoptosis was substantially higher in p53 wild-type cells. p53 activation and early upregulation of p53-target genes were consistent with p53-dependent apoptosis in IGROV-1 cells. Stress-activated protein kinases were activated in both cell lines in response to ST1926. This event and activation of AP-1 were more pronounced in IGROV-1/Pt1 cells, in which the modulation of DNA repair-associated genes suggests an increased ability to repair DNA damage. Inhibition of JNK or p38 stimulated ST1926-induced apoptosis only in IGROV-1 cells, whereas inhibition of ERKs enhanced apoptosis in both the cell lines. Such a pattern of cellular response and modulation of genes implicated in DNA damage response supports that the genotoxic stress is a critical event mediating drug-induced apoptosis. The results are consistent with apoptosis induction through p53-dependent and -independent pathways, regulated by MAP kinases, which likely play a protective role.

Targeting the Akt Kinase to Modulate Survival, Invasiveness and Drug Resistance of Cancer Cells

The deregulation of oncogenic signaling pathways which provide survival advantages to tumor cells is mediated by multiple cellular networks. Among them, the PI3K-Akt-mTOR axis, in particular the serine/threonine kinase Akt, is recognized as a key player. The kinase is hyperactivated due to a variety of mechanisms including loss of PTEN, mutations in the PI3K catalytic subunit, receptor tyrosine kinase and Ras activation. Indeed, inappropriate activation of the Akt kinase is a common event in human tumors and Akt appears to be a critical player in cell survival that may also account for the therapeutic resistance and the invasive phenotype of tumors. Inhibition of Akt signalling results in apoptosis and growth inhibition of tumour cells with elevated Akt activity. A functional role in drug resistance is supported by evidence that tumor cells with acquired resistance to antitumor agents may display increased Akt activation and that treatment with molecularly targeted agents can activate feed-back loops involving Akt. This serine/threonine kinase may therefore represent an amenable target for modulation of sensitivity to compounds with different molecular features due to its pleiotropic role in cell survival. Different types of Akt inhibitors [i.e., ATP mimetics and pleckstrin-homology (PH) domain binders] have been generated and some of them have reached the clinical setting. The present review focuses on the i) mechanisms implicating Akt in increased survival and invasive potential of tumor cells of different tumor types and ii) on the development of Akt inhibitors as modulators of drug resistance.

Decreased Drug Accumulation and Increased Tolerance to DNA Damage in Tumor Cells with a Low Level of Cisplatin Resistance

In an attempt to examine the cellular changes associated with cisplatin resistance, we selected a cisplatin-resistant (A43 1/Pt) human cervix squamous cell carcinoma cell line following continuous in vitro drug exposure. The resistant subline was characterized by a 2.5-fold degree of resistance. In particular, we investigated the expression of cellular defence systems and other cellular factors probably involved in dealing with cisplatin-induced DNA damage. Resistant cells exhibited decreased platinum accumulation and reduced levels of DNA-bound platinum and interstrand cross-link frequency after short-term drug exposure. Analysis of the effect of cisplatin on cell cycle progression revealed a cisplatin-induced G2M arrest in sensitive and resistant cells. Interestingly, a slowdown in S-phase transit was found in A431/Pt cells. A comparison of the ability of sensitive and resistant cells to repair drug-induced DNA damage suggested that resistant cells were able to tolerate higher levels of cisplatin-induced DNA damage than their parental counterparts. Analysis of the expression of proteins involved in DNA mismatch repair showed a decreased level of MSH2 in resistant cells. Since MSH2 seems to be involved in recognition of drug-induced DNA damage, this change may account for the increased tolerance to DNA damage observed in the resistant subline. In conclusion, the involvement of accumulation defects and the increased tolerance to cisplatin-induced DNA damage in these cisplatin-resistant cells support the notion that multiple changes contribute to confer a low level of cisplatin resistance.

Inhibition of c-Met and prevention of spontaneous metastatic spreading by the 2-indolinone RPI-1

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. [Mol Cancer Ther 2006;5(9):2388–97]

Antitumor efficacy of the heparanase inhibitor SST0001 alone and in combination with antiangiogenic agents in the treatment of human pediatric sarcoma models

The activity of heparanase is responsible for heparan sulfate cleavage, thus resulting in the release of heparan sulfate-bound growth factors. Since heparanase activity is upregulated in several tumor types and is implicated in the malignant behavior, the enzyme is regarded as a promising target for antitumor therapy. Based on previous evidence that the heparanase inhibitor SST0001, a non-anticoagulant N-acetylated glycol split heparin, is effective against an Ewings sarcoma model, the present study was performed to extend the preclinical evaluation of SST0001 to a panel of pediatric sarcoma models, representative of various tumor histotypes (soft tissue and bone sarcomas) and to further elucidate its mode of action. SST0001 treatment downregulated several angiogenic factors in the conditioned media of sarcoma cells, inhibited the pro-invasive effect of heparin-binding factors (VEGF, bFGF, HGF, PDGF), and abrogated PDGF receptor tyrosine phosphorylation. Subcutaneous administration of SST0001 was very effective, resulting in a significant growth inhibition (range, 64-95%) of all tested tumor xenografts. The efficacy of SST0001 was enhanced in combination with antiangiogenic agents (bevacizumab, sunitinib) as documented by the high rate of complete response. The synergistic effect of SST0001 in combination with antiangiogenic agents is consistent with the heparanase mode of action and with the relevant role of heparin-binding proangiogenic/growth factors in the malignant behavior of sarcoma cells.

Pre-clinical and clinical significance of heparanase in Ewing’s sarcoma

Heparanase is an endoglycosidase that specifically cleaves heparan sulphate side chains of heparan sulphate proteoglycans, activity that is strongly implicated in cell migration and invasion associated with tumour metastasis, angiogenesis and inflammation. Heparanase up‐regulation was documented in an increasing number of human carcinomas, correlating with reduced post‐operative survival rate and enhanced tumour angiogenesis. Expression and significance of heparanase in human sarcomas has not been so far reported. Here, we applied the Ewing’s sarcoma cell line TC71 and demonstrated a potent inhibition of cell invasion in vitro and tumour xenograft growth in vivo upon treatment with a specific inhibitor of heparanase enzymatic activity (compound SST0001, non‐anticoagulant N‐acetylated, glycol split heparin). Next, we examined heparanase expression and cellular localization by immunostaining of a cohort of 69 patients diagnosed with Ewing’s sarcoma. Heparanase staining was noted in all patients. Notably, heparanase staining intensity correlated with increased tumour size (P = 0.04) and with patients’ age (P = 0.03), two prognostic factors associated with a worse outcome. Our study indicates that heparanase expression is induced in Ewing’s sarcoma and associates with poor prognosis. Moreover, it encourages the inclusion of heparanase inhibitors (i.e. SST0001) in newly developed therapeutic modalities directed against Ewing’s sarcoma and likely other malignancies.

Dasatinib reduces FAK phosphorylation increasing the effects of RPI-1 inhibition in a RET/PTC1- expressing cell line

BackgroundTPC-1 is a papillary thyroid carcinoma (PTC)-derived cell line that spontaneously expresses the oncogene RET/PTC1. TPC-1 treated with the RET/PTC1 inhibitor RPI-1 displayed a cytostatic and reversible inhibition of cell proliferation and a strong activation of focal adhesion kinase (FAK). As dasatinib inhibition of Src results in reduction of FAK activation, we evaluated the effects of TPC-1 treatment with dasatinib in combination with RPI-1.ResultsDasatinib (100 nM) strongly reduced TPC-1 proliferation and induced marked changes in TPC-1 morphology. Cells appeared smaller and more contracted, with decreased cell spreading, due to the inhibition of phosphorylation of important cytoskeletal proteins (p130CAS, Crk, and paxillin) by dasatinib. The combination of RPI-1 with dasatinib demonstrated enhanced effects on cell proliferation (more than 80% reduction) and on the phosphotyrosine protein profile. In particular, RPI-1 reduced the phosphorylation of RET, MET, DCDB2, CTND1, and PLCγ, while dasatinib acted on the phosphorylation of EGFR, EPHA2, and DOK1. Moreover, dasatinib completely abrogated the phosphorylation of FAK at all tyrosine sites (Y576, Y577, Y861, Y925) with the exception of the autoactivation site (Y397). Notably, the pharmacological treatments induced an overexpression of integrin β1 (ITB1) that was correlated with a mild enhancement in phosphorylation of ERK1/2 and STAT3, known for their roles in prevention of apoptosis and in increase of proliferation and survival. A reduction in Akt, p38 and JNK1/2 activation was observed.ConclusionsAll data demonstrate that the combination of the two drugs effectively reduced cell proliferation (by more than 80%), significantly decreased Tyr phosphorylation of almost all phosphorylable proteins, and altered the morphology of the cells, supporting high cytostatic effects. Following the combined treatment, cell survival pathways appeared to be mediated by STAT3 and ERK activities resulting from integrin clustering and FAK autophosphorylation. EphA2 may also contribute, at least in part, to integrin and FAK activation. In conclusion, these data implicate ITB1 and EphA2 as promising therapeutic targets in PTC.