Circulating miR-378 in plasma: a reliable, haemolysis-independent biomarker for colorectal cancer.

BACKGROUND: Plasma circulating tumour-specific microRNAs (miRNAs) are promising biomarkers of tumour presence and recurrence, especially for diseases whose best chance of successful treatment requires early diagnosis and timely surgery of an already malignant but not yet invasive tumour, such as colorectal cancer (CRC). METHODS: Expression levels of miRNAs previously found to be differently expressed in tumour vs normal colon tissues were investigated by quantitative real-time PCR in plasma from CRC patients and from healthy donors and confirmed in independent case control series. The validated miRNAs were also measured after surgery. Analyses were repeated on the subsets of haemolysis-free samples. RESULTS: We identified four miRNAs differently expressed between the compared groups, two (miR-21 and miR-378) of which were validated. miR-378 expression decreased in non-relapsed patients 4-6 months after surgery and miR-378 ability to discriminate CRC patients from healthy individuals was not influenced by haemolysis levels of plasma samples. CONCLUSION: The miRNA analysis on plasma samples represents a useful non-invasive tool to assess CRC presence as well as tumour-free status at follow-up. Plasma levels of miR-378 could be used to discriminate CRC patients from healthy individuals, irrespective of the level of haemoglobin of plasma samples.

The psychological impact of breast and ovarian cancer preventive options in BRCA1 and BRCA2 mutation carriers.

This study was performed to describe the impact of preventive options on the psychological condition of BRCA1/BRCA2 carriers. A sample of 52 cancer-affected (C-A) and 27 cancer-unaffected (C-UN) women were enrolled after gene test disclosure (T0). Psychological evaluations were performed at T0 and 15 months later (T1). The surgical options were more likely to be chosen in C-A women (62%), although a consistent proportion of C-UN women (30%) also opt for these preventive measures. At the baseline, both samples had average anxiety and depression scores below the cut-off value, restrained average cancer worry scores and a risk perception consistent with the risk percentage provided during genetic counselling. The longitudinal results indicated no clinically meaningful variations in the anxiety and depression scores in either of the two samples. Statistically significant reductions in cancer-risk perception emerged in women who chose surgery in both C-A and C-UN women. In BRCA1/BRCA2 mutation carriers, surveillance does not influence their initial psychological condition, whereas prophylactic surgery has a significant impact in reducing the perceived risk and worry about getting sick. C-A and C-UN women have to be considered as two separate populations of BRCA mutation carriers requiring personalized approaches to risk management.

A major susceptibility locus to murine lung carcinogenesis maps on chromosome 6.

Lung tumours represent a major cause of death in humans, and although smoking represents the main pathogenetic factor, inheritance also plays a part. However, the identification of possible predisposing genetic factors is difficult, because of their low penetrance. We took advantage of murine strains that are genetically susceptible or resistant to lung tumour development, to map murine genes associated with susceptibility to lung carcinogenesis. An F2 population of urethan-treated A/J x C3H/He mice was scored with 83 genetic markers. A chromosome 6 distal region, spanning mice was scored with 83 genetic markers. A chromosome 6 distal region, spanning 35 centiMorgans, contained a major lung tumour susceptibility locus. No other chromosomal region was significantly associated with lung tumour development.

Genetic mapping of a pulmonary adenoma resistance (Par1) in mouse.

Lung cancer, a major cause of death in the Western world, has a poor prognosis. So far, therapeutic strategies have had only a limited effect. Lung cancer risk is strongly associated with cigarette smoking and lung cancer pedigrees are rare. However, a possible polygenic nature of inherited predisposition to this cancer has been envisaged. Mouse inbred strains with inherited predisposition and resistance to lung cancer provide an important tool for the dissection of the genetics of this complex disease. The A/J strain carries the pulmonary adenoma susceptibility 1 (Pas1) locus and develops many lung tumours. We have mapped the M. spretus-derived locus that strongly resists the lung tumorigenesis in Pas1/+ mice. This locus, pulmonary adenoma resistance 1 (Par1) maps to mouse chromosome 11, near the Rara locus, with a lod score of 5.3. In Pas1/+ mice Par1 accounts for 23% of the phenotypic variance and 10 fold reduction in total tumour volume. These results provide evidence for a major resistance locus affecting the expression of an inherited predisposition to lung cancer.

Oncogenic and ligand-dependent activation of KIT/PDGFRA in surgical samples of imatinib-treated gastrointestinal stromal tumours (GISTs).

As the range of receptor tyrosine kinase (RTK) inhibitors widens, a detailed understanding of the activating mechanisms of KIT/platelet-derived growth factor receptor (PDGFR)A and the related downstream pathways involved in the development and maintenance of GISTs is becoming increasingly important. We analysed areas with different histological response ratios in surgical specimens taken from imatinib-treated and untreated GIST patients in order to investigate KIT and PDGFRA expression/activation, the presence of their cognate ligands and the activation of downstream signalling, by means of biochemistry, immunohistochemistry and flow cytometry. All of the cases showed KIT and PDGFRA co-expression. In addition to the oncogenic activation of mutated receptors, activation of wild-type KIT and wild-type PDGFRA, sustained by heterodimerization and an autocrine-paracrine loop, was demonstrated by the presence of their specific ligands, stem cell factor (SCF) and PDGFA. To confirm RTK activation further, all of the samples (including those with the highest regression ratios) were investigated for downstream effectors, and all proved to have activated downstream signalling. The results show that after the mutated receptors are switched off, heterologous wild-type receptors become important in imatinib-treated GISTs as a means of maintaining signalling activation. Taken together, our findings suggest that drugs targeting wild-type receptors should be tested in imatinib-treated GIST patients.

Gene expression profiling integrated into network modelling reveals heterogeneity in the mechanisms of BRCA1 tumorigenesis.

BACKGROUND: Gene expression profiling has distinguished sporadic breast tumour classes with genetic and clinical differences. Less is known about the molecular classification of familial breast tumours, which are generally considered to be less heterogeneous. Here, we describe molecular signatures that define BRCA1 subclasses depending on the expression of the gene encoding for oestrogen receptor, ESR1. METHODS: For this purpose, we have used the Oncochip v2, a cancer-related cDNA microarray to analyze 14 BRCA1-associated breast tumours. RESULTS: Signatures were found to be molecularly associated with different biological processes and transcriptional regulatory programs. The signature of ESR1-positive tumours was mainly linked to cell proliferation and regulated by ER, whereas the signature of ESR1-negative tumours was mainly linked to the immune response and possibly regulated by transcription factors of the REL/NFkappaB family. These signatures were then verified in an independent series of familial and sporadic breast tumours, which revealed a possible prognostic value for each subclass. Over-expression of immune response genes seems to be a common feature of ER-negative sporadic and familial breast cancer and may be associated with good prognosis. Interestingly, the ESR1-negative tumours were substratified into two groups presenting slight differences in the magnitude of the expression of immune response transcripts and REL/NFkappaB transcription factors, which could be dependent on the type of BRCA1 germline mutation. CONCLUSION: This study reveals the molecular complexity of BRCA1 breast tumours, which are found to display similarities to sporadic tumours, and suggests possible prognostic implications.

PI3KCA/PTEN deregulation contributes to impaired responses to cetuximab in metastatic colorectal cancer patients.

BACKGROUND: It has been reported that KRAS mutations (and to a lesser extent KRAS mutations with the BRAF V600E mutation) negatively affect response to anti-epidermal growth factor receptor (EGFR) mAbs in metastatic colorectal cancer (mCRC) patients, while the biological impact of the EGFR pathway represented by PI3K/PTEN/AKT on anti-EGFR treatment is still not clear. PATIENTS AND METHODS: We analysed formalin-fixed samples from a cohort of 32 mCRC patients treated with cetuximab by means of EGFR immunohistochemistry, EGFR and PTEN FISH analysis, and KRAS, BRAF, PI3KCA, and PTEN genomic sequencing. RESULTS: Ten (31%) of 32 patients showed a partial response to cetuximab and 22 (69%) did not [nonresponder (NR)]. EGFR immunophenotype and FISH-based gene status did not predict an anti-EGFR mAb response, whereas KRAS mutations (24%) and PI3K pathway activation, by means of PI3KCA mutations (13%) or PTEN mutation (10%)/loss (13%), were significantly restricted to, respectively, 41% and 37% of NRs. CONCLUSION: These findings suggested that KRAS mutations and PI3KCA/PTEN deregulation significantly correlate with resistance to cetuximab. In line with this, patients carrying KRAS mutations or with activated PI3K profiles can benefit from targeted treatments only by switching off molecules belonging to the downstream signalling of activated EGFR, such as mammalian target of rapamycin.

Proteomics study of medullary thyroid carcinomas expressing RET germ-line mutations: identification of new signaling elements.

Proteomics may help to elucidate differential signaling networks underlying the effects of compounds and to identify new therapeutic targets. Using a proteomic-multiplexed analysis of the phosphotyrosine signaling together with antibody-based validation techniques, we identified several candidate molecules for RET (rearranged during transfection) tyrosine kinase receptor carrying mutations responsible for the multiple endocrine neoplasia type 2A and 2B (MEN2A and MEN2B) syndromes in two human medullary thyroid carcinoma (MTC) cell lines, TT and MZ-CRC-1, which express the RET-MEN2A and RET-MEN2B oncoproteins, respectively. Signaling elements downstream of these oncoproteins were identified after treating cells with the indolinone tyrosine kinase inhibitor RPI-1 to knock down RET phosphorylation activity. We detected 23 and 18 affinity-purified phosphotyrosine proteins in untreated TT and MZ-CRC-1 cells, respectively, most of which were shared and sensitive to RPI-1 treatment. However, our data clearly point to specific signaling features of the RET-MEN2A and RET-MEN2B oncogenic pathways. Moreover, the detection of high-level expression of minimally phosphorylated epidermal growth factor receptor (EGFR) in both TT and MZ-CRC-1 cells, together with our data on the effects of EGF stimulation on the proteomic profiles and the response to Gefitinib treatment, suggest the relevance of EGFR signaling in these cell lines, especially since analysis of 14 archival MTC specimens revealed EGFR mRNA expression in all samples. Together, our data suggest that RET/EGFR multi-target inhibitors might be beneficial for therapy of MTC.

Malignant activation of a K-ras oncogene in lung carcinoma but not in normal tissue of the same patient.

A single genetic alteration, a guanine-to-cytosine transversion, is responsible for the acquisition of malignant properties by K-ras genes of two human tumor cell lines established from carcinomas of the bladder (A1698) and lung (A2182). As a consequence, arginine instead of the normal glycine is incorporated into the K-ras-coded p21 proteins at amino acid position 12. This mutation creates a restriction enzyme polymorphism that can be used to screen human cells for transforming K-ras genes. This approach was used to identify the mutational event responsible for the malignant activation of a K-ras oncogene in a squamous cell lung carcinoma of a 66-year-old man; this point mutation was not present in either the normal bronchial or parenchymal tissue or in the blood lymphocytes. Hence, malignant activation of a ras oncogene appears to be specifically associated with the development of a human neoplasm.

Detection of novel mRNA splice variants of human ING4 tumor suppressor gene.

Inhibitor of growth (ING)4, member of a gene family encoding potential tumor suppressors, is implicated as a repressor of angiogenesis and tumor growth and suppresses loss of contact inhibition in vitro. Here, we report that ING4 undergoes alternative splicing. Expression analysis identified novel ING4 spliced variant mRNAs encoding proteins devoid of different portions. The ING4 variants were detected in both normal and tumor tissues. The existence of ING4 variants was confirmed by several approaches, including reverse transcriptase-polymerase chain reaction, real-time PCR and in silico experiments. To investigate the functional consequences of alternative splicing the ING4 variant cDNAs were expressed in mammalian cells. Our studies indicated that (i) the ING4 variants do not differ from wild-type in their nuclear localization, interaction with p53 and association to HBO1 complex; and (ii) the ING4-DeltaEx6A variant, devoid of the C-terminal portion, loses the capability to inhibit NF-kappaB. On the whole our data suggest that alternative splicing could modulate the activity of ING4 tumor suppressor protein.

Analysis of gene expression identifies PLAB as a mediator of the apoptotic activity of fenretinide in human ovarian cancer cells.

Fenretinide (4-HPR) is a synthetic retinoid with antitumor activity, which induces apoptosis in cancer cell lines of different histotypes. To identify genes contributing to its apoptotic activity in ovarian cancer cells, we monitored, by cDNA arrays, gene expression changes after 4-HPR exposure in A2780, a human ovarian carcinoma cell line sensitive to the retinoid. Among the differentially expressed transcripts, PLAcental Bone morphogenetic protein (PLAB), a proapoptotic gene, was the most highly induced. In a panel of ovarian carcinoma cell lines with different 4-HPR sensitivities, PLAB upregulation was associated with cellular response to 4-HPR, its overexpression increased basal apoptosis and its silencing by small interfering RNA decreased the ability of 4-HPR to induce apoptosis. PLAB induction by 4-HPR was p53- and EGR-1 independent and was regulated, at least in part, by increased stability of PLAB mRNA. PLAB up-modulation by 4-HPR also occurred in vivo: in ascitic cells collected from patients with ovarian cancer before and after 4-HPR treatment, PLAB was upmodulated in 2/4 patients. Our results in certain ovarian cancer cell lines indicate a role for PLAB as a mediator of 4-HPR-induced apoptosis. The correlation of increased PLAB in vivo with antitumor activity remains to be established.

SH2B1beta adaptor is a key enhancer of RET tyrosine kinase signaling.

The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.

Quantitative and qualitative characterization of plasma DNA identifies primary and recurrent colorectal cancer.

Because plasma DNA may be a useful tool for cancer detection, we screened primary tumors and related multiple plasma samples at the time of surgery and during the follow-up period for plasma DNA level as well as for K-Ras mutations and p16INK4a promoter hypermethylation in colorectal cancer patients. At the time of surgery, DNA levels were higher in tumor patients than in healthy donors, and K-Ras and p16INK4a alterations were detected in 7 and 11 cancers respectively, and in all related plasma samples. During the follow-up, plasma DNA levels decrease progressively but rapidly increased when a relapse occurred, whereas K-Ras and p16INK4a alterations were detected only in relapsed patients. Therefore, combined quantitative and qualitative analyses of plasma DNA confirm the presence of colorectal cancer, define disease-free status and indicate the presence of relapse.

Functional analyses and molecular modeling of two c-Kit mutations responsible for imatinib secondary resistance in GIST patients.

Imatinib-acquired resistance related to the presence of secondary point mutations has become a frequent event in gastrointestinal stromal tumors. Here, transient transfection experiments with plasmids carrying two different KIT-acquired point mutations were performed along with immunoprecipitation of total protein extracts, derived from imatinib-treated and untreated cells. The molecular mechanics/Poisson Boltzmann surface area computational techniques were applied to study the interactions of the wild-type and mutated receptors with imatinib at the molecular level. Biochemical analyses showed KIT phosphorylation in cells transfected with vectors carrying the specific mutant genes. Imatinib treatment demonstrated that T670I was insensitive to the drug at all the applied concentrations, whereas V654A was inhibited by 6 microM of imatinib. The modeling of the mutated receptors revealed that both substitutions affect imatinib-binding site, but to a different extent: T670I substantially modifies the binding pocket, whereas V654A induces only relatively confined structural changes. We demonstrated that T670I and V654A cause indeed imatinib-acquired resistance and that the former is more resistant to imatinib than the latter. The application of molecular simulations allowed us to quantify the interactions between the mutated receptors and imatinib, and to propose a molecular rationale for this type of drug resistance.

Gene p53 mutations are restricted to poorly differentiated and undifferentiated carcinomas of the thyroid gland.

The p53 gene was analyzed in tumor specimens obtained from 52 patients with various types of carcinoma of the thyroid gland by a combined molecular and immunocytochemical approach. The histologic types included 37 well-differentiated papillary and follicular carcinomas, 8 poorly differentiated, and 7 undifferentiated carcinomas. The p53 gene was shown to be unaffected in all differentiated tumors by single-strand conformation polymorphism analysis. However, in two out of eight (25%) of poorly differentiated carcinomas and five out of seven (71%) undifferentiated carcinomas, p53 mutations were identified and subsequently characterized by DNA sequencing. One undifferentiated carcinoma displayed two areas with varying degrees of differentiation. The comparative analysis of the p53 gene, in both the more and the less differentiated area of this tumor, clearly showed that the p53 mutation was confined to the latter component of the tumor specimen. These results indicate that mutations of the p53 gene are associated with the most aggressive histologic types of thyroid tumors, such as the undifferentiated carcinoma and, to a certain extent, the poorly differentiated carcinoma, and that the alterations of this gene represent a late genetic event in human thyroid carcinogenesis.

Ret oncogene activation in human thyroid neoplasms is restricted to the papillary cancer subtype.

We have recently reported the activation of a new oncogene in human papillary thyroid carcinomas. This oncogene, papillary thyroid carcinoma (PTC), is a novel rearranged version of the ret tyrosine-kinase protooncogene. Thyroid neoplasms include a broad spectrum of malignant tumors, ranging from well-differentiated tumors to undifferentiated anaplastic carcinomas. To determine the frequency of ret oncogene activation, we analyzed 286 cases of human thyroid tumors of diverse histologic types. We found the presence of an activated form of the ret oncogene in 33 (19%) of 177 papillary carcinomas. By contrast, none of the other 109 thyroid tumors, which included 37 follicular, 15 anaplastic, and 18 medullary carcinomas, and 34 benign lesions, showed ret activation.

RET oncoproteins induce tyrosine phosphorylation changes of proteins involved in RNA metabolism.

We report the identification of proteins induced in response to RET/PTC2, an oncogene implicated in thyroid cancers. Anti-phosphotyrosine antibody affinity resin was used to purify Tyr(P)-containing and interacting proteins from 293T and NIH3T3 cells which were transfected with kinase active or inactive RET/PTC and RETMEN2 oncogenes. Proteins were separated by one-dimensional SDS-PAGE, extracted by in-gel digestion, and identified by MALDI-TOF peptide mass fingerprinting. The expression and tyrosine phosphorylation of Sam68, a protein implicated in mRNA nucleocytoplasmic translocation and splicing, were further examined in RET-transfected cells and thyroid tumors. Of relevance, cells transfected with RETMEN2B examined for anti-phosphotyrosine bound proteins, showed other proteins implicated in splicing: DEAD-box p68 RNA helicase, SYNCRIP, and hnRNP K. Western blotting analysis suggested that these proteins are singularly tyrosine phosphorylated in RETMEN2B-transfected cells, and that they constitutively bind with Sam68. The study concludes that regulation of splicing factors is likely to be important in RET-mediated thyroid carcinogenesis.

The DNA rearrangement that generates the TRK-T3 oncogene involves a novel gene on chromosome 3 whose product has a potential coiled-coil domain.

Oncogenic rearrangements of the NTRK1 gene (also designated TRKA), encoding one of the receptors for the nerve growth factor, are frequently detected in thyroid carcinomas. Such rearrangements fuse the NTRK1 tyrosine kinase domain to 5'-end sequences belonging to different genes. In previously reported studies we have demonstrated that NTRK1 oncogenic activation involves two genes, TPM3 and TPR, both localized similarly to the receptor tyrosine kinase, on the q arm of chromosome 1. Here we report the characterization of a novel NTRK1-derived thyroid oncogene, named TRK-T3. A cDNA clone, capable of transforming activity, was isolated from a transformant cell line. Sequence analysis revealed that TRK-T3 contains 1,412 nucleotides of NTRK1 preceded by 598 nucleotides belonging to a novel gene that we have named TFG (TRK-fused gene). The TRK-T3 amino acid sequence displays, within the TFG region, a coiled-coil motif that could endow the oncoprotein with the capability to form complexes. The TRK-T3 oncogene encodes a 68-kDa cytoplasmic protein reacting with NTRK1-specific antibodies. By sedimentation gradient experiments the TRK-T3 oncoprotein was shown to form, in vivo, multimeric complexes, most likely trimers or tetramers. The TFG gene is ubiquitously expressed and is located on chromosome 3. The breakpoint producing the TRK-T3 oncogene occurs within exons of both the TFG gene and the NTRK1 gene and produces a chimeric exon that undergoes alternative splicing. Molecular analysis of the NTRK1 rearranged fragments indicated that the chromosomal rearrangement is reciprocal and balanced and involves loss of a few nucleotides of germ line sequences.

The full oncogenic activity of Ret/ptc2 depends on tyrosine 539, a docking site for phospholipase Cgamma.

RET/PTC oncogenes, generated by chromosomal rearrangements in papillary thyroid carcinomas, are constitutively activated versions of proto-RET, a gene coding for a receptor-type tyrosine kinase (TK) whose ligand is still unknown. RET/PTCs encode fusion proteins in which proto-RET TK and C-terminal domains are fused to different donor genes. The respective Ret/ptc oncoproteins display constitutive TK activity and tyrosine phosphorylation. We found that Ret/ptcs associate with and phosphorylate the SH2-containing transducer phospholipase Cgamma (PLCgamma). Two putative PLCgamma docking sites, Tyr-505 and Tyr-539, have been identified on Ret/ptc2 by competition experiments using phosphorylated peptides modelled on Ret sequence. Transfection experiments and biochemical analysis using Tyr-->Phe mutants of Ret/ptc2 allowed us to rule out Tyr-505 and to identify Tyr-539 as a functional PLCgamma docking site in vivo. Moreover, kinetic measurements showed that Tyr-539 is able to mediate high-affinity interaction with PLCgamma. Mutation of Tyr-539 resulted in a drastically reduced oncogenic activity of Ret/ptc2 on NIH 3T3 cells (75 to 90% reduction) both in vitro and in vivo, which correlates with impaired ability of Ret/ptc2 to activate PLCgamma. In conclusion, this paper demonstrates that Tyr-539 of Ret/ptc2 (Tyr-761 on the proto-RET product) is an essential docking site for the full transforming potential of the oncogene. In addition, the present data identify PLCgamma as a downstream effector of Ret/ptcs and suggest that this transducing molecule could play a crucial role in neoplastic signalling triggered by Ret/ptc oncoproteins.