MITO END-3: efficacy of avelumab immunotherapy according to molecular profiling in first-line endometrial cancer therapy.

BACKGROUND: Immunotherapy combined with chemotherapy significantly improves progression-free survival (PFS) compared to first-line chemotherapy alone in advanced endometrial cancer (EC), with a much larger effect size in microsatellite instability-high (MSI-H) cases. New biomarkers might help to select patients who may have benefit among those with a microsatellite-stable (MSS) tumor. PATIENTS AND METHODS: In a pre-planned translational analysis of the MITO END-3 trial, we assessed the significance of genomic abnormalities in patients randomized to standard carboplatin/paclitaxel without or with avelumab. RESULTS: Out of 125 randomized patients, 109 had samples eligible for next-generation sequencing analysis, and 102 had MSI tested. According to The Cancer Genome Atlas (TCGA), there were 29 cases with MSI-H, 26 with MSS TP53 wild type (wt), 47 with MSS TP53 mutated (mut), and 1 case with POLE mutation. Four mutated genes were present in >30% of cases: TP53, PIK3CA, ARID1A, and PTEN. Eleven patients (10%) had a BRCA1/2 mutation (five in MSI-H and six in MSS). High tumor mutational burden (≥10 muts/Mb) was observed in all MSI-H patients, in 4 out of 47 MSS/TP53 mut, and no case in the MSS/TP53 wt category. The effect of avelumab on PFS significantly varied according to TCGA categories, being favorable in MSI-H and worst in MSS/TP53 mut (P interaction = 0.003); a similar non-significant trend was seen in survival analysis. ARID1A and PTEN also showed a statistically significant interaction with treatment effect, which was better in the presence of the mutation (ARID1A P interaction = 0.01; PTEN P interaction = 0.002). CONCLUSION: The MITO END-3 trial results suggest that TP53 mutation is associated with a poor effect of avelumab, while mutations of PTEN and ARID1A are related to a positive effect of the drug in patients with advanced EC.

Alternative academic approaches for testing homologous recombination deficiency in ovarian cancer in the MITO16A/MaNGO-OV2 trial.

BACKGROUND: The detection of homologous recombination deficiency (HRD) can identify patients who are more responsive to platinum and poly ADP ribose polymerase inhibitors (PARPi). MyChoice CDx (Myriad) is the most used HRD test in ovarian cancer (OC). However, some limitations of commercial tests exist, because of the high rate of inconclusive results, costs, and the impossibility of evaluating functional resistance mechanisms. PATIENTS AND METHODS: Two academic genomic tests and a functional assay, the RAD51 foci, were evaluated to detect HRD. One hundred patients with high-grade OC enrolled in the MITO16A/MaNGO-OV2 trial and treated with first-line therapy with carboplatin, paclitaxel, and bevacizumab were analyzed. RESULTS: The failure rate of the two genomic assays was 2%. The sensitivity in detecting HRD when compared with Myriad was 98.1% and 90.6%, respectively. The agreement rate with Myriad was 0.92 and 0.87, with a Cohen's κ coefficient corresponding to 0.84 and 0.74, respectively. For the RAD51 foci assay, the failure rate was 30%. When the test was successful, discordant results for deficient and proficient tumors were observed, and additional HRD patients were identified compared to Myriad; sensitivity was 82.9%, agreement rate was 0.65, and Cohen's κ coefficient was 0.18. The HRD detected by genomic assays and residual tumor at primary surgery and stage was correlated with progression-free survival at multivariate analysis. CONCLUSIONS: Results suggest the feasibility of academic tests for assessing HRD status that show robust concordance with Myriad and correlation with clinical outcome. The contribution of the functional information related to the RAD51 foci test to the genomic data needs further investigation.

IFN-γ increases susceptibility to influenza A infection through suppression of group II innate lymphoid cells.

Increased levels of interferon-γ (IFN-γ) are routinely observed in the respiratory tract following influenza virus infection, yet its potential role remains unclear. We now demonstrate that influenza-induced IFN-γ restricts protective innate lymphoid cell group II (ILC2) function in the lung following challenge with the pandemic H1N1 A/CA/04/2009 (CA04) influenza virus. Specifically, IFN-γ deficiency resulted in enhanced ILC2 activity, characterized by increased production of interleukin (IL)-5 and amphiregulin, and improved tissue integrity, yet no change in ILC2 numbers, viral load or clearance. We further found that IFN-γ-deficient mice, as well as wild-type animals treated with neutralizing anti-IFN-γ antibody, exhibited decreased susceptibility to lethal infection with H1N1 CA04 influenza virus, and moreover that survival was dependent on the presence of IL-5. The beneficial effects of IFN-γ neutralization were not observed in ILC2-deficient animals. These data support the novel concept that IFN-γ can have a detrimental role in the pathogenesis of influenza through a restriction in ILC2 activity. Thus, regulation of ILC2 activity is a potential target for post-infection therapy of influenza.

Trabectedin in Ovarian Cancer: is it now a Standard of Care?

In patients with recurrent ovarian cancer, the choice of second-line therapy is complex. Several factors have to be considered, such as platinum-free interval (PFI), residual toxicity from the previous treatments, BRCA1/2 gene mutation status. Trebectedin is a minor groove DNA binder derived from a marine organism that has shown efficacy in different settings in ovarian cancer therapy. It has been approved in the treatment of partially platinum sensitive (PPS) (PFI between 6 and 12 months) relapsed ovarian cancer according to the statistically significant progression-free survival (7.3 versus 5.8 months) and overall survival (22.2 versus 18.9 months) benefit compared with single-agent pegylated liposomal doxorubicin (PLD) in the OVA 301 phase III trial. This drug has been shown to prolong the time to first subsequent treatment and improve the efficacy of further platinum-based chemotherapy. The role of trabectedin/PLD followed by platinum combination compared with the reverse sequence in PPS is actually in evaluation in the INOVATYON phase III study, which will clarify the best sequence to be adopted in this setting. Trabectedin has been shown to be active in patient carriers of BRCA mutations, probably for its mechanism of action directly affecting DNA and it is actually tested as a single agent in some phase III trials in BRCA mutated and BRCAness ovarian cancer patients. Trabectedin is also active on the immune system. There is, therefore, the rational for new trials of a combination with immune checkpoint inhibitors.

Overexpression of the HIP gene coding for a heparin/heparan sulfate-binding protein in human thyroid carcinomas.

A subtractive library screening was performed to identify changes in gene expression that occur during the process of neoplastic transformation of thyroid cells. A cDNA library was constructed from a human thyroid papillary carcinoma cell line (NPA) subtracted with cDNAs from normal thyroid cells (HTC 2). The differential screening of this library lead to the isolation of 39 cDNA clones; six of them showed homology with a recently isolated gene, named HIP, that codes for a protein belonging to a novel class of heparin/heparan sulfate-binding proteins. Northern blot analysis revealed HIP gene overexpression in all of the human thyroid carcinoma cell lines analyzed, as compared to the HTC 2 cells. HIP expression was particularly abundant in the anaplastic carcinoma-derived cell lines. The analysis of surgically removed thyroid tumors showed overexpression of HIP gene in all of the carcinomas, independent of the histotype, although the largest increase in HIP expression was observed in the undifferentiated forms. In contrast, none of the benign adenomas or normal thyroid tissues showed HIP overexpression. To establish the role of HIP overexpression in cell transformation, the NPA cell line was transfected with an eukaryotic expression vector carrying the HIP gene in the antisense orientation. Stable transfectants expressed reduced HIP mRNA levels and showed morphological changes, such as becoming spindle-shaped and growing scattered. The growth rate of the antisense clones was greatly reduced compared to the NPA cells transfected with the backbone vector. Taken together, these results indicate that HIP gene overexpression is associated with thyroid carcinogenesis and strongly suggest its involvement in thyroid cell growth regulation.

Production of transgenic mice expressing the Ki-ras oncogene under the control of a thyroglobulin promoter.

Transgenic mice have been generated bearing three fusion genes consisting of: (a) a 900-base pair rat thyroglobulin promoter followed by a gene coding for a chloramphenicol acetyl transferase activity; (b) the same promoter followed by the complementary DNA of the human activated Ki-ras oncogene; (c) a 2000-base pair rat thyroglobulin promoter followed by the complementary DNA of the human activated Ki-ras. We have shown that the 900-base pair rat thyroglobulin promoter is able to direct the expression of the reporter gene specifically in the thyroid gland of transgenic mice. The mice bearing the two Ki-ras constructs, which express the transgene in thyroid glands, show thyroid abnormalities, although at very low incidence. These lesions appear after a long latency and with a benign aspect, thus suggest that, in agreement with literature data on naturally occurring human thyroid tumors, the action of an activated ras gene is not sufficient to attain a complete malignant conversion of thyroid glands in vivo. However, ras expression in thyroid follicular cells represents a favorable ground for tumor development, as shown by the fact that goitrogen stimulation experiments increase the occurrence of tumors.

Thymosin beta-10 gene overexpression correlated with the highly malignant neoplastic phenotype of transformed thyroid cells in vivo and in vitro.

A subtractive thyroid cDNA library was constructed from two human thyroid carcinoma cell lines originating from an anaplastic carcinoma and a papillary thyroid carcinoma. The library was used to identify genes correlated with the progression to a highly malignant phenotype. The thymosin beta-10 gene was isolated and found to be expressed at much higher levels in the anaplastic cell line than in the papillary cells. The thymosin beta-10 gene was overexpressed in five carcinoma cell lines compared with normal thyroid tissue and normal thyroid primary culture cells. The highest expression occurred in the most malignant cell lines. Thymosin beta-10 gene expression was also increased in surgically removed human thyroid carcinomas and was highest in the anaplastic carcinomas. Thymosin beta-10 gene expression was correlated with the degree of the malignant phenotype also in rat thyroid cells transfected with cellular and viral oncogenes of different tumorigenicity. These results show that thymosin beta-10 overexpression is a general event of thyroid cell neoplastic transformation and suggest that the gene is involved in the progression of thyroid carcinogenesis. Finally, the thymosin beta-10 gene was located on chromosome 2q37 by fluorescence in situ hybridization analysis.

Activated RET/PTC oncogene elicits immediate early and delayed response genes in PC12 cells.

The expression of the receptor-like tyrosine kinase RET is associated with tumors, tissues or cell lines of neural crest origin. In addition RET products (Ret) are involved in determining cell fate during the differentiation of the enteric nervous system and during renal organogenesis. However, as yet, no direct evidence exists to indicate that the Ret kinase activity might interfere in a specific way with cellular differentiation, or proliferation, of a neural crest derived cell line. By using two constitutively activated forms of RET (RET/PTC1 and RET/PTC3) in transient transfection experiments, we have obtained evidence that active RET could reprogramme the gene expression pattern in the rat pheochromocytoma PC12 cell line. Transcription driven by gene promoters, such as NGFI-A and vgf, which belong, respectively, to primary and delayed response genes to nerve growth factor (NGF), and by the neuron-specific enolase (NSE) promoter, is rapidly induced by the expression of activated RET oncogenes. This induction is not elicited in other non neural derived cell types tested. We also demonstrate that endogenous ras activity is required for RET induction of these neural markers. Finally, in the RET/PTC transfected PC12 cells, NGF is unable to induce further their transcription. This suggests that RET/PTC could share an intracellular signalling pathway with the NGF-receptor.

Upregulation of the angiogenic factors PlGF, VEGF and their receptors (Flt-1, Flk-1/KDR) by TSH in cultured thyrocytes and in the thyroid gland of thiouracil-fed rats suggest a TSH-dependent paracrine mechanism for goiter hypervascularization.

Placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) represent two closely related angiogenic growth factors active as homodimers or heterodimers. Since goiters of the thyroid gland are extremely hypervascular, we investigated the expression of PlGF, VEGF and their receptors, Flt-1 and Flk-1/KDR, in a small panel of human goiters from patients with Graves's disease, in an animal model of thyroid goitrogenesis and in in vitro cultured thyroid cells. Here we report that the mRNA expression of PlGF, VEGF and their receptors is markedly enhanced in biopsies of goiters resected from Graves's patients. In vivo studies demonstrated that in the thyroid gland of thiouracil-fed rats, increased mRNA and protein expression of PIGF, VEGF, Flt-1 and Flk-1/KDR occurred subsequent to the rise in the serum thyroid stimulating hormone (TSH) levels and in parallel with thyroid capillary proliferation. In vitro studies confirmed the existence of such TSH-dependent paracrine communication between thyroid epithelial cells and endothelium since the conditioned medium collected from TSH-stimulated thyrocytes acquired mitogenic activity for human umbilical vein endothelial (HUVE) cells. Altogether, these data suggest that PlGF and VEGF, released by thyrocytes in response to the chronic activation of the TSH receptor pathway, may act through a paracrine mechanism on thyroid endothelium.

Neovascularization in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not the placenta-derived growth factor.

Neoangiogenesis is a prerequisite for tumor growth and metastasis. In germ cell cancer patients with the disease limited to the testicle (stage A), tumor-associated neovascularization is predictive of metastatic disease (stage B). To investigate the molecular mechanisms underlying neovascularization in human germ cell tumors (GCTs), we analysed the expression of two angiogenic growth factors, vascular endothelial growth factor (VEGF) and placenta growth factor (P1GF), and of their receptors (FLT-1) and Flk-1/KDR) in a panel of testicular tumors. In this study we show a marked increase in VEGF expression in 36/44 (81.8%) primary testicular-derived GCTs, as compared to normal testis, that significantly correlates with a high density of intratumor microvessels (r = 0.72461, P < 0.001; n = 24). As determined by RT - PCR and/or Western blot, the predominant VEGF isoforms expressed in GCTs are the VEGF121 and VEGF165, which are more efficiently secreted by the cells, and thus more active in eliciting angiogenesis. Conversely, in the case of PIGF, only a weak correlation with the vascular density of tumors is observed (r = 0.26599, P < 0.05; n = 24). Northern blot analysis also revealed significant up-regulation of VEGF/ PIGF receptors in highly vascularized germ cell tumors, compared to normal testes. These findings suggest that VEGF may act in a paracrine manner to induce neovascularization, oedema extravasation and cyst formation in human germ cell tumors. The correlation between VEGF expression and the vascular density of tumors, suggest that the evaluation of VEGF expression may be of help in predicting patients at risk for metastatic diseases. Finally, we demonstrate that VEGF up-regulation may occur at the RNA level since no gene amplification is observed; conversely, in in vitro models such as the embryonal stem cell line NTERA-2 and the choricarcinoma JEG-3 cell line, VEGF (but not PIGF) mRNA expression is regulated by hypoxic stress.

The tyrosine phosphatase Shp-2 mediates intracellular signaling initiated by Ret mutants.

The Src homology 2-containing tyrosine phosphatase, Shp-2, is a crucial enzyme that mediates intracellular signaling and is implicated in cell proliferation and differentiation. Here we investigated the involvement of the Shp-2 tyrosine phosphatase in determining the downstream signaling pathways initiated by the Ret oncogene, carrying either the cysteine 634 to tyrosine or the methionine 918 to threonine substitutions. These mutations convert the receptor tyrosine kinase, Ret, into a dominant transforming protein and induce constitutive activation of its intrinsic tyrosine kinase activity leading to congenital and sporadic cancers in neuroendocrine organs. Using the PC12, rat pheochromocytoma cell line, as model system, we show that Shp-2 mediates immediate-early gene expression if induced by either of the mutant alleles. Furthermore, we show that Shp-2 activity is required for RetM918T-induced Akt activation. The results indicate that Shp-2 is a downstream mediator of the mutated receptors RetC634Y and RetM918T, thus suggesting that it may act as a limiting factor in Ret-associated endocrine tumors, in the neoplastic syndromes multiple endocrine neoplasia types 2A and 2B.

Human dbl proto-oncogene in 85 kb of xq26, and determination of the transcription initiation site.

The dbl oncogene is generated by substitution of the 5' portion of its normal counterpart with an unrelated human sequence. To analyze the genomic structure and transcriptional regulation of the dbl proto-oncogene, we have isolated human genomic clones containing the entire human proto-dbl gene, localized in Xq26. Restriction mapping of a 600kb YAC clone (yWXD311) placed proto-dbl about 50kb telomeric to the coagulation Factor IX gene. The genomic DNA fragment containing the 5' end of proto-dbl was subcloned into plasmid vectors and the nucleotide sequences of exon 1, the flanking intronic region and genomic DNA 5' of the first codon were determined. Sequence analysis of 85119bp from the region revealed the genomic structure of proto-dbl. It contains 25 exons coding for a 4.7kb transcript including large 5'- and 3'- (1218bp and 701bp, respectively) untranslated regions (UTRs). RNase protection and primer extension assays on RNA from medullary thyroid carcinoma (TT) cells, which normally express dbl, revealed a transcription start site 1218bp upstream of the ATG of the first exon. A 1.6kb genomic 5' of the translation start sites drives the expression of a CAT-reporter in transient transfections in the TT cell line, though lacking TATA or CAAT boxes.

Low frequency of p53 mutations in human thyroid tumours; p53 and Ras mutation in two out of fifty-six thyroid tumours.

OBJECTIVE: p53 is a well-known nuclear phosphoprotein encoded by a suppressor gene know to be mutated in various kinds of human tumours. A relationship between p53 gene mutation and tumour progression seems to be a common feature of several neoplasias. DESIGN: In order to investigate the role of p53 mutations in human thyroid tumours, DNA samples derived from fifty-six neoplastic tissues, ranging from benign adenomas to undifferentiated carcinomas, were examined for the presence of p53 gene mutations. METHODS: The analysis has been conducted using polymerase chain reaction (PCR) amplification of the exons 5-9 of the p53 gene followed by single strand conformation polymorphism (SSCP) and sequence analyses. RESULTS: One anaplastic carcinoma and one papillary carcinoma showed p53 gene mutations in exons 5 and 8, respectively. A cell line established from the papillary carcinoma showed the same mutation present in the original tumour. Both p53 mutations were heterozygous. The p53 positive samples were analysed for other genetic alterations frequently detected in human thyroid carcinomas (mutations of the RET, TRK, and ras oncogenes): both p53-mutated samples proved to be mutated at level of codon 13 of the c-Ki-ras gene. CONCLUSIONS: Our data confirm that p53 gene alterations are rare in well-differentiated thyroid tumours, that they are an important requirement for the establishment in culture of human thyroid carcinoma cell lines, and that they can be associated with other genetic alterations, namely ras mutations, in the malignant progression of thyroid tumours.

Regulation of thymosin beta10 expression by TSH and other mitogenic signals in the thyroid gland and in cultured thyrocytes.

OBJECTIVE: To investigate the expression of thymosin beta10 - a small conserved acidic protein involved in the inhibition of actin polymerization - in human and experimental thyroid goiters as well as the regulation exerted by TSH on thymosin beta10 expression in thyroid follicular cells both in vivo and in vitro. DESIGN: To this aim, we have used 5 bioptic specimens from patients affected by thyroid goiter, a well known experimental model of thyroid goitrogenesis (rat fed with the drug propylthiouracil) and a cultured rat thyroid cell line (PC Cl 3 cells) as a model system. RESULTS: We report that the mRNA expression of thymosin beta10 is markedly enhanced in human goiters compared with normal thyroid. In vivo results showed that the steady-state level of thymosin beta10 mRNA is up-regulated in the thyroid gland of propylthiouracil-fed rats in parallel with follicular cell proliferation: iodide administration to goitrous rats, which induced a marked involution of thyroid hyperplasia, reduced the mRNA level of thymosin beta10. Finally, in vitro studies showed that in cultured rat thyrocytes, the expression of thymosin beta10 mRNA is induced in a time- and dose-dependent manner by the activation of pathways which are mitogenic for thyroid cells (i.e. the protein kinase (PK) A and PKC pathways). CONCLUSION: Taken together, the findings reported here demonstrate that thymosin beta10 expression is regulated by extracellular signals that stimulate growth of thyroid cells both in vitro and in vivo, and suggest a role for this protein in thyroid diseases characterized by proliferation of follicular cells.

Transgenic mice carrying the human KRAS oncogene under the control of a thyroglobulin promoter: KRAS expression in thyroids analyzed by in situ hybridization.

We have previously generated transgenic mice bearing a molecular construct obtained by fusing the rat thyroglobulin promoter with the human Kirsten ras oncogene (KRAS). These mice showed thyroid abnormalities, although at very low incidence and after a long latency period. A six-month thyrotropin (TSH) stimulation of thyroid glands, followed by a two-month suspension, induced a significant increase in the number of lesions in transgenic mice as compared with a nontransgenic control group. Our goal was to follow the progression and the reversion of the tumorigenesis process in relationship with the levels of expression of the KRAS in this experimental model. In situ hybridization was used to detect expression of KRAS mRNA in sections of thyroids of the various groups of mice. A positive hybridization was observed in follicular cells of TSH-stimulated transgenic mice, whereas no expression could be appreciated in control nontransgenic mice. A positive signal was also observed in thyroid glands excised from transgenic mice after the 2-month suspension of treatment; however, the number of expressing cells was decreased compared with transgenic mice killed immediately after 6 months of a goitrogen regimen. Finally, every cell in the single thyroid carcinoma observed after the two-month suspension was positive for the transgene mRNA. This study further strengthens the role of the expression of mutated KRAS in the early stages of thyroid follicular cell transformation and indicates that when the expression of the mutated KRAS becomes independent of exogenous TSH stimulation, this event coincides with a further progression towards tumorigenesis.

Synthesis and biological activity of pseudopeptides inhibitors of Ras farnesyl transferase containing unconventional amino acids.

A study was performed on the structure-activity relationships of a series of phenol derivatives, CVFM analogs, derived from the two most active compounds of a first series (1A and 1B) of inhibitors of Ras farnesyl transferase (FTase) that we have recently described. We report the synthesis and the activity of a second series of compounds in which the phenylalanine residue was replaced by unconventional aromatic and non-aromatic amino acids, with varying electronic, lipophilic, steric and conformational properties. The compounds showed to be significantly less active than reference compounds against FT, with the only exception of derivative 3A (IC50 = 3 microM), which is slightly more active than 1A but not 1B. Subsequently we tested the effects of compounds 1A, 1B and 3A, 3B on the anchorage-dependent growth of two epithelial cell lines of rats, FRTL-5 and the same line v-Ha-ras transformed. Compound 3A derived from lead compound 1A, showed an appreciable selectivity against transformed cells. In contrast, compounds derived from derivative 1B had only a modest cellular activity.

Mutated human Kirsten ras, driven by a thyroglobulin promoter, induces a growth advantage and partially dedifferentiates rat thyroid epithelial cells in vitro.

We have earlier shown that expression of the human activated Ki-ras, directed by the rat thyroglobulin (TG) promoter in the thyroid gland of transgenic mice, is able to induce thyroid benign tumors, albeit at low incidence. A likely explantation of our results is that the low levels of exogenous Ki-ras transcripts are not sufficient to induce multifocal tumors in the thyroid gland. We have performed experiments to analyze the effects of a similar construct in vitro upon thyroid-cell proliferation and differentiation. Transfection of FRTL-5 rat thyroid cells with the human Ki-rasval12 fused to the rat TG promoter is rapidly followed by reduced expression of the differentiation markers thyroglobulin, thyroperoxydase and thyrotropin receptor, but not by fully malignant cell transformation. The data reported support the hypothesis that Ki-ras mRNA levels are critical to the process of complete neoplastic transformation of thyroid epithelial differentiated cells in vitro.

Isolation of a SIR-like gene, SIR-T8, that is overexpressed in thyroid carcinoma cell lines and tissues.

We used subtractive library screening to identify the changes that occur in gene expression during thyroid cell neoplastic transformation. Complementary DNA from normal thyroid cells (HTC 2) was subtracted from a complementary DNA library constructed from a human thyroid papillary carcinoma cell line. The library was screened for genes upregulated in human thyroid papillary carcinoma cell line cells, and several cDNA clones were isolated. One of these clones has a sirtuin core and high homology with the human silent information regulator protein family. This clone, designated "SIR-T8", was overexpressed in human thyroid carcinoma cell lines and tissues, but not in adenomas. The human SIR-T8 protein has a molecular weight of 39 kDa and is primarily located in the cytoplasm under the nuclear membrane. The SIR-T8 gene is located on chromosome 17q25-1.

Signaling through Ras is essential for ret oncogene-induced cell differentiation in PC12 cells.

Specific germline mutations of the receptor tyrosine kinase, Ret, predispose to multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma. The mechanisms by which different Ret isoforms (Ret-2A and Ret-2B) cause distinct neoplastic diseases remain largely unknown. On the other hand, forced expression of these mutated versions of Ret induces the rat pheochromocytoma cell line, PC12, to differentiate. Here we used an inducible vector encoding a dominant-negative Ras (Ras p21(N17)) to investigate the contributions of the Ras pathway to the phenotype induced in PC12 cells by the expression of either Ret-2A or Ret-2B mutants. We show that the Ret-induced molecular and morphological changes are both mediated by Ras-dependent pathways. However, even though inhibition of Ras activity was sufficient to revert Ret-induced differentiation, the kinetics of morphological reversion of the Ret-2B- was more rapid than the Ret-2A-transfected cells. Further, we show that in Ret-transfected cells the suc1-associated neurotrophic factor-induced tyrosine phosphorylation target, SNT, is chronically phosphorylated in tyrosine residues, and associates with the Sos substrate. These results indicate the activation of the Ras cascade as an essential pathway triggered by the chronic active Ret mutants in PC12 cells. Moreover, our data indicate SNT as a substrate for both Ret mutants, which might mediate the activation of this cascade.