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.

Targeting metabolism for cancer treatment and prevention: metformin, an old drug with multi-faceted effects.

Understanding the complexity of cancer and of the underlying regulatory networks provides a new paradigm that tackles cancer development and treatment through a system biology approach, contemporarily acting on various intersecting pathways. Cancer cell metabolism is an old pathogenetic issue that has recently gained new interest as target for therapeutic approaches. More than 70 years ago, Warburg discovered that malignant cells generally have altered metabolism with high rates of glucose uptake and increased glycolysis, even under aerobic condition. Observational studies have provided evidence that impaired metabolism, obesity, hyperglycemia and hyperinsulinemia may have a role in cancer development, progression and prognosis, and actually diabetic and obese patients have increased cancer risk. On the other hand, caloric restriction has been shown to prolong life span and reduce cancer incidence in several animal models, having an impact on different metabolic pathways. Metformin, an antidiabetic drug widely used for over 40 years, mimics caloric restriction acting on cell metabolism at multiple levels, reducing all energy-consuming processes in the cells, including cell proliferation. By overviewing molecular mechanisms of action, epidemiological evidences, experimental data in tumor models and early clinical study results, this review provides information supporting the promising use of metformin in cancer prevention and treatment.

TIMP3 regulates migration, invasion and in vivo tumorigenicity of thyroid tumor cells.

Papillary thyroid carcinoma (PTC) arises from the thyroid follicular epithelium and represents the most frequent thyroid malignancy. PTC is associated with gene rearrangements generating RET/PTC and TRK oncogenes, and to the BRAFV600E activating point mutation. A role of tumor-suppressor genes in the pathogenesis of PTC has not been assessed yet. The tissue inhibitor of metalloproteinase-3 (TIMP3) gene, encoding a metalloproteinases inhibitor and capable of inhibiting growth, angiogenesis, invasion and metastasis of several cancers, was found to be silenced by promoter methylation in a consistent fraction of PTCs, in association with tumor aggressiveness and BRAFV600E mutation, thus suggesting an oncosuppressor role. To explore this possibility, in this study we performed gene expression and functional studies. Analysis of gene expression data produced in our laboratory as well as meta-analysis of publicly available data sets confirmed the downregulation of TIMP3 gene expression in PTC with respect to normal thyroid. The functional consequences of TIMP3 downregulation were investigated in the PTC-derived NIM1 cell line, in which the expression of TIMP3 is silenced. Restoration of TIMP3 expression by exposure to soluble TIMP3 protein or by complementary DNA transfection had no effect on the growth rate of NIM1 cells. Instead, it affected the adhesive, migratory and invasive capabilities of NIM1 cells by modulating several proteins involved in these processes. A striking effect was observed in vivo, as TIMP3 reduced the tumorigenicity of NIM1 cells by repressing angiogenesis and macrophage infiltration. Our data indicate that the loss of TIMP3 expression exerts a functional role in the pathogenesis of PTC.

IGFBP7: an oncosuppressor gene in thyroid carcinogenesis.

Insulin-like growth factor-binding protein 7 (IGFBP7) is a secreted protein involved in several cellular processes, including proliferation, senescence and apoptosis. Loss of IGFBP7 expression is a critical step in the development of human tumors, including melanoma and colon cancer. By microarray gene expression studies, we have detected downregulation of IGFBP7 gene expression in follicular and papillary thyroid tumors in comparison with normal thyroid tissue. Evaluation of publicly available PTC microarray gene expression data sets confirmed, in a consistent fraction of tumors, the downregulation of IGFBP7 transcript levels. The functional consequence of IGFBP7 downregulation was addressed in the PTC-derived NIM1 cell line in which IGFBP7 expression is repressed by promoter hypermethylation. Exposure to soluble IGFBP7 protein or restoration of IGFBP7 expression by complementary DNA transfection reduced growth rate, migration, anchorage-independent growth and tumorigenicity of NIM1 cells. We show that the effects of IGFBP7 are related to apoptosis. Our data suggest that loss of IGFBP7 expression has a functional role in thyroid carcinogenesis, and it may represent a possible basis for therapeutic strategies.

Rearrangements of NTRK1 gene in papillary thyroid carcinoma.

TRK oncogenes are observed in a consistent fraction of papillary thyroid carcinoma (PTC); they arise from the fusion of the 3' terminal sequences of the NTRK1/NGF receptor gene with 5' terminal sequences of various activating genes, such as TPM3, TPR and TFG. TRK oncoproteins display constitutive tyrosine-kinase activity, leading to in vitro and in vivo transformation. In this review studies performed during the last 20 years will be summarized. The following topics will be illustrated: (a) frequency of TRK oncogenes and correlation with radiation and tumor histopathological features; (b) molecular mechanisms underlying NTRK1 oncogenic rearrangements; (c) molecular and biochemical characterization of TRK oncoproteins, and their mechanism of action; (d) role of activating sequences in the activation of TRK oncoproteins.

Molecular pathology of differentiated thyroid cancer.

Thyroid cancer is the most common endocrine malignancy; it accounts for approximately 1% of all new case of cancer each year, and its incidence has increased significantly over the last few decades. The majority of thyroid tumors originate from follicular epithelial cells. Among them, papillary (PTC) and follicular carcinomas (FTC) represent the most common forms of differentiated thyroid cancer and account for approximately 80% and 15% of all cases, respectively. Specific genetic lesions are associated to each thyroid tumor histotype: BRAF mutations and RET/PTC and TRK oncogenes have been detected in PTC, whereas FTC is characterized by PAX8/PPARgamma rearrangements and RAS mutations. In this review we summarize studies on the molecular biology of the differentiated thyroid tumors, with particular interest in the associated genetic lesions and their role in thyroid carcinogenesis. We also report recent findings on gene expression and miRNA profiles of PTC and FTC.

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.

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.

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.

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.

Breast cancer metastases are molecularly distinct from their primary tumors.

Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether 'phenotypic' disease progression is really associated with 'molecular' progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.

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.

Analysis of potential receptor tyrosine kinase targets in intimal and mural sarcomas.

Primary sarcomas of the great vessels are very rare neoplasms and only a few cases have been reported. They are divided into the two broad categories of intimal or luminal and mural sarcomas. We analysed eight advanced high-grade sarcomas originating from major vessels (seven intimal and one mural sarcoma) by means of immunohistochemistry and FISH analysis for PDGFRA, PDGFRB, EGFR and KIT receptor tyrosine kinases (RTKs), together with immunoprecipitation/western blotting, sequencing of the corresponding genes, and the search for cognate ligands. The intimal sarcomas showed a wide spectrum of morphologies and immunophenotypes, whereas the mural sarcoma had common leiomyosarcomatous features. Regardless of their category, all of the cases had a PDGFRA-deregulated cytogenetic profile mainly consisting of an amplification cluster; five were also polysomic for PDGFRB, whereas three showed disomy. Six cases had a deregulated EGFR gene, and c-Kit gene status was similar to that of PDGFRA. In one case, biochemical analysis revealed the presence of activated and highly expressed PDGFRA, PDGFRB and EGFR, whereas KIT was expressed at reference level. Sequencing of the corresponding genes revealed no activating mutations in any of the analysed receptors. The cognate ligands were detected in all cases. In predictive terms, the evidence of gene amplification/high polysomy of several RTKs, together with PDGFRA, PDGFRB and EGFR expression and phosphorylation, suggests that these tumours may be sensitive to RTK-inhibiting treatments.

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.