Publications by authors named "Annalisa Lorenzato"

19 Publications

  • Page 1 of 1

Extracellular Vesicles in Human Preterm Colostrum Inhibit Infection by Human Cytomegalovirus In Vitro.

Microorganisms 2020 Jul 21;8(7). Epub 2020 Jul 21.

Laboratory of Molecular Virology, Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy.

Breast milk is a complex biofluid that nourishes infants, supports their growth and protects them from diseases. However, at the same time, breastfeeding is a transmission route for human cytomegalovirus (HCMV), with preterm infants being at a great risk of congenital disease. The discrepancy between high HCMV transmission rates and the few reported cases of infants with severe clinical illness is likely due to the protective effect of breast milk. The aim of this study was to investigate the anti-HCMV activity of human preterm colostrum and clarify the role of colostrum-derived extracellular vesicles (EVs). Preterm colostrum samples were collected and the EVs were purified and characterized. The in vitro anti-HCMV activity of both colostrum and EVs was tested against HCMV, and the viral replication step inhibited by colostrum-purified EVs was examined. We investigated the putative role EV surface proteins play in impairing HCMV infection using shaving experiments and proteomic analysis. The obtained results confirmed the antiviral action of colostrum against HCMV and demonstrated a remarkable antiviral activity of colostrum-derived EVs. Furthermore, we demonstrated that EVs impair the attachment of HCMV to cells, with EV surface proteins playing a role in mediating this action. These findings contribute to clarifying the mechanisms that underlie the protective role of human colostrum against HCMV infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/microorganisms8071087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7409124PMC
July 2020

EGFR Blockade Reverts Resistance to KRAS Inhibition in Colorectal Cancer.

Cancer Discov 2020 Aug 19;10(8):1129-1139. Epub 2020 May 19.

Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, New York.

Most patients with -mutant non-small cell lung cancer (NSCLC) experience clinical benefit from selective KRAS inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRAS inhibitors in colorectal cancer, we examined the effects of AMG510 in colorectal cancer cell lines. Unlike NSCLC cell lines, colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRAS inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRAS blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRAS inhibitors. The combinatorial targeting of EGFR and KRAS is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with colorectal cancer. SIGNIFICANCE: The efficacy of KRAS inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRAS inhibition in colorectal cancer. EGFR and KRAS should be concomitantly inhibited to overcome resistance to KRAS blockade in colorectal tumors...
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-20-0187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416460PMC
August 2020

Vitamin C Restricts the Emergence of Acquired Resistance to EGFR-Targeted Therapies in Colorectal Cancer.

Cancers (Basel) 2020 Mar 14;12(3). Epub 2020 Mar 14.

Candiolo Cancer Institute, FPO-IRCCS, Candiolo 10060 (TO), Italy.

The long-term efficacy of the Epidermal Growth Factor Receptor (EGFR)-targeted antibody cetuximab in advanced colorectal cancer (CRC) patients is limited by the emergence of drug-resistant (persister) cells. Recent studies in other cancer types have shown that cells surviving initial treatment with targeted agents are often vulnerable to alterations in cell metabolism including oxidative stress. Vitamin C (VitC) is an antioxidant agent which can paradoxically trigger oxidative stress at pharmacological dose. Here we tested the hypothesis that VitC in combination with cetuximab could restrain the emergence of secondary resistance to EGFR blockade in CRC wild-type models. We found that addition of VitC to cetuximab impairs the emergence of drug persisters, limits the growth of CRC organoids, and significantly delays acquired resistance in CRC patient-derived xenografts. Mechanistically, proteomic and metabolic flux analysis shows that cetuximab blunts carbohydrate metabolism by blocking glucose uptake and glycolysis, beyond promoting slow but progressive ROS production. In parallel, VitC disrupts iron homeostasis and further increases ROS levels ultimately leading to ferroptosis. Combination of VitC and cetuximab orchestrates a synthetic lethal metabolic cell death program triggered by ATP depletion and oxidative stress, which effectively limits the emergence of acquired resistance to anti-EGFR antibodies. Considering that high-dose VitC is known to be safe in cancer patients, our findings might have clinical impact on CRC patients treated with anti-EGFR therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12030685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140052PMC
March 2020

High-dose vitamin C enhances cancer immunotherapy.

Sci Transl Med 2020 02;12(532)

Department of Oncology, University of Torino, 10060 Candiolo (TO), Italy.

Vitamin C (VitC) is known to directly impair cancer cell growth in preclinical models, but there is little clinical evidence on its antitumoral efficacy. In addition, whether and how VitC modulates anticancer immune responses is mostly unknown. Here, we show that a fully competent immune system is required to maximize the antiproliferative effect of VitC in breast, colorectal, melanoma, and pancreatic murine tumors. High-dose VitC modulates infiltration of the tumor microenvironment by cells of the immune system and delays cancer growth in a T cell-dependent manner. VitC not only enhances the cytotoxic activity of adoptively transferred CD8 T cells but also cooperates with immune checkpoint therapy (ICT) in several cancer types. Combination of VitC and ICT can be curative in models of mismatch repair-deficient tumors with high mutational burden. This work provides a rationale for clinical trials combining ICT with high doses of VitC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aay8707DOI Listing
February 2020

A Subset of Colorectal Cancers with Cross-Sensitivity to Olaparib and Oxaliplatin.

Clin Cancer Res 2020 03 12;26(6):1372-1384. Epub 2019 Dec 12.

Candiolo Cancer Institute, FPO - IRCCS, Candiolo, Torino, Italy.

Purpose: Defects in the homologous recombination (HR) repair pathway are of clinical interest due to sensitivity of HR-deficient cells to PARP inhibitors. We were interested in defining PARP vulnerability in patients with metastatic colorectal cancer (mCRC) carrying and mutations who display poor prognosis, have limited therapeutic options, and represent an unmet clinical need.

Experimental Design: We tested colorectal cancer cell lines, patient-derived organoids (PDO), and patient-derived xenografts (PDX) enriched for and mutations for sensitivity to the PARP inhibitor olaparib, and the chemotherapeutic agents oxaliplatin and 5-fluorouracil (5-FU). Genomic profiles and DNA repair proficiency of colorectal cancer models were compared with pharmacologic response.

Results: Thirteen of 99 (around 13%) colorectal cancer cell lines were highly sensitive to clinically active concentrations of olaparib and displayed functional deficiency in HR. Response to PARP blockade was positively correlated with sensitivity to oxaliplatin in colorectal cancer cell lines as well as patient-derived organoids. Treatment of PDXs with olaparib impaired tumor growth and maintenance therapy with PARP blockade after initial oxaliplatin response delayed disease progression in mice.

Conclusions: These results indicate that a colorectal cancer subset characterized by poor prognosis and limited therapeutic options is vulnerable to PARP inhibition and suggest that PDO-based drug-screening assays can be used to identify patients with colorectal cancer likely to benefit from olaparib. As patients with mCRC almost invariably receive therapies based on oxaliplatin, "maintenance" treatment with PARP inhibitors warrants further clinical investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-19-2409DOI Listing
March 2020

Evolving neoantigen profiles in colorectal cancers with DNA repair defects.

Genome Med 2019 06 28;11(1):42. Epub 2019 Jun 28.

Candiolo Cancer Institute, FPO-IRCCS, 10060, Candiolo (TO), Italy.

Background: Neoantigens that arise as a consequence of tumor-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumor types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time.

Methods: We performed whole exome sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines, in vitro and in vivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures, and predicted neoantigens.

Results: The majority of CRC models showed remarkably stable mutational and neoantigen profiles; however, those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly evolving CRC.

Conclusions: These results indicate that CRCs carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13073-019-0654-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599263PMC
June 2019

Radiologic and Genomic Evolution of Individual Metastases during HER2 Blockade in Colorectal Cancer.

Cancer Cell 2018 07;34(1):148-162.e7

Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan 20162, Italy.

Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2018.06.004DOI Listing
July 2018

Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies.

Nat Commun 2018 06 12;9(1):2287. Epub 2018 Jun 12.

Candiolo Cancer Institute-FPO, IRCCS, 10060, Candiolo, Turin, Italy.

Attempts at eradicating metastatic cancers with targeted therapies are limited by the emergence of resistant subclones bearing heterogeneous (epi)genetic changes. We used colorectal cancer (CRC) to test the hypothesis that interfering with an ancestral oncogenic event shared by all the malignant cells (such as WNT pathway alterations) could override heterogeneous mechanisms of acquired drug resistance. Here, we report that in CRC-resistant cell populations, phylogenetic analysis uncovers a complex subclonal architecture, indicating parallel evolution of multiple independent cellular lineages. Functional and pharmacological modulation of WNT signalling induces cell death in CRC preclinical models from patients that relapsed during the treatment, regardless of the drug type or resistance mechanisms. Concomitant blockade of WNT and MAPK signalling restrains the emergence of drug-resistant clones. Reliance upon the WNT-APC pathway is preserved throughout the branched genomic drift associated with emergence of treatment relapse, thus offering the possibility of a common therapeutic strategy to overcome secondary drug resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-04506-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997733PMC
June 2018

PARP1 expression drives the synergistic antitumor activity of trabectedin and PARP1 inhibitors in sarcoma preclinical models.

Mol Cancer 2017 04 28;16(1):86. Epub 2017 Apr 28.

Sarcoma Unit, Medical Oncology, Candiolo Cancer Institute - FPO, IRCCS, Candiolo, Torino, Italy.

Background: Enhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death.

Methods: We investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role.

Results: Trabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing.

Conclusions: PARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12943-017-0652-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410089PMC
April 2017

The integrin-linked kinase-associated phosphatase (ILKAP) is a regulatory hub of ovarian cancer cell susceptibility to platinum drugs.

Eur J Cancer 2016 06 13;60:59-68. Epub 2016 Apr 13.

Department of Oncology, University of Torino School of Medicine, Turin, Italy; Candiolo Cancer Institute, Fondazione del Piemonte per l'Oncologia (FPO), Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS), SP 142, Km. 3.95, 10060 Candiolo, Italy. Electronic address:

Background: Platinum drugs are the most powerful chemotherapeutic agents in the treatment of ovarian cancer. We demonstrated previously that unexpectedly ovarian cancer cells are sensitised to cisplatin (CDDP) by the hepatocyte growth factor (HGF), usually considered an anti-apoptotic factor.

Methods: We used quantitative polymerase chain reaction and Western blot analysis to evaluate gene and protein expression, immunofluorescence to evaluate protein localisation and functional assays to measure cell viability and apoptosis.

Results: In ovarian cancer cells, CDDP induced the phosphorylation, i.e. the activation, of the p90RSK. Surprisingly, a 48-h-long cell pre-treatment with HGF reverted this activation. HGF pre-treatment also resulted in the increased expression of the integrin-linked kinase (ILK)-associated phosphatase (ILKAP) that dephosphorylated the p90RSK. Conversely, CDDP down-modulated ILKAP expression. This impaired CDDP efficacy, as ILKAP silencing protected cells from CDDP-induced death. In line, the biochemical inhibition of the p90RSK or the combined silencing of the most expressed RSK isoforms, namely RSK1 and RSK2, increased the efficacy of CDDP. However, p90RSK inhibition was not sufficient to revert cell protection from death after ILKAP suppression, because of the simultaneous increased activity of the anti-apoptotic kinases ILK and ILK substrate AKT, which were both dephosphorylated, i.e. negatively regulated, by ILKAP. Only the combined inhibition of p90RSK and ILK reverted the effect of ILKAP suppression.

Conclusions: As RSKs, ILK and AKT are vital kinases for ovarian cancer onset and progression, data suggest that ILKAP is a regulatory hub of ovarian cancer cell survival by controlling the activation of these kinases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ejca.2016.02.022DOI Listing
June 2016

Peritoneal and hematogenous metastases of ovarian cancer cells are both controlled by the p90RSK through a self-reinforcing cell autonomous mechanism.

Oncotarget 2016 Jan;7(1):712-28

Department of Oncology, University of Torino School of Medicine, Turin, Italy.

The molecular mechanisms orchestrating peritoneal and hematogenous metastases of ovarian cancer cells are assumed to be distinct. We studied the p90RSK family of serine/threonine kinases that lie downstream the RAS-ERK/MAPK pathway and modulate a variety of cellular processes including cell proliferation, survival, motility and invasiveness. We found the RSK1 and RSK2 isoforms expressed in a number of human ovarian cancer cell lines, where they played redundant roles in sustaining in vitro motility and invasiveness. In vivo, silencing of both RSK1 and RSK2 almost abrogated short-term and long-term metastatic engraftment of ovarian cancer cells in the peritoneum. In addition, RSK1/RSK2 silenced cells failed to colonize the lungs after intravenous injection and to form hematogenous metastasis from subcutaneous xenografts. RSK1/RSK2 suppression resulted in lessened ovarian cancer cell spreading on endogenous fibronectin (FN). Mechanistically, RSK1/RSK2 knockdown diminished FN transcription, α5β1 integrin activation and TGF-β1 translation. Reduced endogenous FN deposition and TGF-β1 secretion depended on the lack of activating phosphorylation of the transcription/translation factor YB-1 by p90RSK. Altogether data show how p90RSK activates a self-reinforcing cell autonomous pro-adhesive circuit necessary for metastatic seeding of ovarian cancer cells. Thus, p90RSK inhibitors might hinder both the hematogenous and the peritoneal metastatic spread of human ovarian cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.6412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808028PMC
January 2016

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

Exp Cell Res 2013 Oct 13;319(17):2627-36. Epub 2013 Aug 13.

Department of Oncology, University of Torino School of Medicine, Torino, Italy; Institute for Cancer Research at Candiolo, Candiolo, Torino, Italy.

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexcr.2013.07.030DOI Listing
October 2013

Cells lacking the fumarase tumor suppressor are protected from apoptosis through a hypoxia-inducible factor-independent, AMPK-dependent mechanism.

Mol Cell Biol 2012 Aug 29;32(15):3081-94. Epub 2012 May 29.

Department of Oncological Sciences, University of Torino, School of Medicine, Turin, Italy.

Loss-of-function mutations of the tumor suppressor gene encoding fumarase (FH) occur in individuals with hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). We found that loss of FH activity conferred protection from apoptosis in normal human renal cells and fibroblasts. In FH-defective cells, both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α accumulated, but they were not required for apoptosis protection. Conversely, AMP-activated protein kinase (AMPK) was activated and required, as evidenced by the finding that FH inactivation failed to protect AMPK-null mouse embryo fibroblasts (MEFs) and AMPK-depleted human renal cells. Activated AMPK was detected in renal cysts, which occur in mice with kidney-targeted deletion of Fh1 and in kidney cancers of HLRCC patients. In Fh1-null MEFs, AMPK activation was sustained by fumarate accumulation and not by defective energy metabolism. Addition of fumarate and succinate to kidney cells led to extracellular signal-regulated kinase 1/2 (ERK1/2) and AMPK activation, probably through a receptor-mediated mechanism. These findings reveal a new mechanism of tumorigenesis due to FH loss and an unexpected pro-oncogenic role for AMPK that is important in considering AMPK reactivation as a therapeutic strategy against cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.06160-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3434507PMC
August 2012

The cellular apoptosis susceptibility CAS/CSE1L gene protects ovarian cancer cells from death by suppressing RASSF1C.

FASEB J 2012 Jun 2;26(6):2446-56. Epub 2012 Mar 2.

Department of Oncological Sciences, University of Torino School of Medicine, Turin, Italy.

The cellular apoptosis susceptibility gene CAS/CSE1L is overexpressed in cancer, although it was originally identified as a gene that renders cells vulnerable to apoptotic stimuli. CAS/CSE1L has roles in the nucleocytoplasmic recycling of importin-α and in the regulation of gene expression, cell migration, and secretion. We identified CAS/CSE1L as a survival factor for ovarian cancer cells in vitro and in vivo. In 3/3 ovarian cancer cell lines, CAS/CSE1L was down-modulated by the unorthodox proapoptotic signaling of the MET receptor. CAS/CSE1L knockdown with RNA interference committed the ovarian cancer cells to death, but not immortalized normal cells and breast and colon cancer cells. In 70 and 95% of these latter cells, respectively, CAS/CSE1L was localized in the cytoplasm, while it accumulated in the nucleus in >90% of ovarian cancer cells. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells. In the nucleus, CAS/CSE1L regulated the expression of the proapoptotic Ras-association domain family 1 gene products RASSF1C and RASSF1A, which mediated death signals evoked by depletion of CAS/CSE1L. Our data show that CAS/CSE1L protects ovarian cancer cells from death through transcriptional suppression of a proapoptotic gene and suggest that the localization of CAS/CSE1L dictates its function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.11-195982DOI Listing
June 2012

Fumarase tumor suppressor gene and MET oncogene cooperate in upholding transformation and tumorigenesis.

FASEB J 2010 Aug 30;24(8):2680-8. Epub 2010 Mar 30.

Department of Oncological Sciences, University of Torino School of Medicine, Turin, Italy.

Loss of the fumarate hydratase (FH) tumor suppressor gene results in the development of benign tumors that rarely, but regrettably, progress to very aggressive cancers. Using mouse embryo fibroblasts (MEFs) to model transformation, we found that fh knockdown results in increased expression of the met oncogene-encoded tyrosine kinase receptor through hypoxia-inducible factor (hif) stabilization. MET-increased expression was alone able to stabilize hif, thus establishing a feed forward loop that might enforce tumor progression. The fh-defective MEFs showed increased motility and protection from apoptosis. Motility, but not survival, relied on hif-1alpha and was greatly enhanced by MET ligand hepatocyte growth factor. Met cooperated with a weakly oncogenic ras in making MEFs transformed and tumorigenic, as shown by in vitro and in vivo assays. Loss of fh was not equally effective by itself but enhanced the transformed and tumorigenic phenotype induced by ras and MET. Consistently, the rescue of fumarase expression abrogated the motogenic and transformed phenotype of fh-defective MEFs. In conclusion, the data suggest that the progression of tumors where FH is lost might be boosted by activation of the MET oncogene, which is able to drive cell-autonomous tumor progression and is a strong candidate for targeted therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.09-146928DOI Listing
August 2010

A cancer-predisposing "hot spot" mutation of the fumarase gene creates a dominant negative protein.

Int J Cancer 2008 Feb;122(4):947-51

Laboratory of Cancer Genetics, Institute for Cancer Research and Treatment (IRCC), University of Turin Medical School, SP142, KM 395, 10060 Candiolo, Turin, Italy.

The Fumarase (Fumarate Hydratase, FH) is a tumor suppressor gene whose germline heterozygous mutations predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). The FH gene encodes an enzyme of the Krebs cycle, functioning as a homotetramer and catalyzing the hydration of fumarate to malate. Among the numerous FH mutations reported so far, the R190H missense mutation is the most frequent in HLRCC patients. Here we show the functional analyses of the R190H, in comparison to the better characterized E319Q mutation. We first expressed wild-type and mutated proteins in FH deficient human skin fibroblasts, using lentiviral vectors. The wild-type transgene was able to restore the FH enzymatic activity in cells, while the R190H- and E319Q-FH were not. More interestingly, when the same transgenes were expressed in normal, FH-proficient cells, only the R190H-FH reduced the endogenous FH enzymatic activity. By enforcing the expression of equal amount of wild-type and R190H-FH in the same cell, we showed that the mutated FH protein directly inhibited enzymatic activity by nearly abrogating the FH homotetramer formation. These data demonstrate the dominant negative effect of the R190H missense mutation in the FH gene and suggest that the FH tumor-suppressing activity might be impaired in cells carrying a heterozygous mutation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ijc.23209DOI Listing
February 2008

Mitochondrial succinate is instrumental for HIF1alpha nuclear translocation in SDHA-mutant fibroblasts under normoxic conditions.

Hum Mol Genet 2005 Nov 29;14(21):3263-9. Epub 2005 Sep 29.

INSERM U676, Hôpital Robert Debré, Paris, France

The genes encoding succinate dehydrogenase (SDH) subunits B, C and D, act as tumour suppressors in neuro-endocrine tissues. Tumour formation has been associated with succinate accumulation. In paraganglioma cells, two forms of SDHA (type I, II) were found which might preclude significant succinate accumulation in the case of a mutation in either form. In fibroblasts only SDHA type I is found. In these cells, SDHA type I mutation leads to SDH deficiency, succinate accumulation and hypoxia-inducible factor 1alpha(HIF1alpha) nuclear translocation. HIF1alpha nuclear translocation was not observed in ATPase-deficient fibroblasts with increased superoxide production and was found to be independent of cellular iron availability in SDHA-mutant cells. This suggests that neither superoxides nor iron were causative of HIF1alpha nuclear translocation. Conversely, alpha-ketoglutarate (alpha-KG) inhibits this nuclear translocation. Therefore, the pseudo-hypoxia pathway in SDH-deficient cells depends on the HIF1alphaprolyl hydroxylase product/substrate (succinate/alpha-KG) equilibrium. In SDH deficiency, organic acids thus appear instrumental in the HIF1alpha-dependent cascade suggesting a direct link between SDH and tumourigenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddi359DOI Listing
November 2005

The RON and MET oncogenes are co-expressed in human ovarian carcinomas and cooperate in activating invasiveness.

Exp Cell Res 2003 Aug;288(2):382-9

Laboratory of Cancer Genetics of the Institute for Cancer Research and Treatment, University of Torino School of Medicine, SP 142, Km 3.95, 10060 Candiolo (Torino), Italy.

RON is a member of the receptor tyrosine kinase gene family that includes the MET oncogene, whose germline mutations have been causally related to human tumorigenesis. In vitro, RON and MET receptors cross-talk, synergize in intracellular signaling, and cooperate in inducing morphogenic responses. Here we show that the RON and MET oncogenes were expressed in 55% and 56% of human ovarian carcinomas, respectively, and were significantly coexpressed in 42% (P < 0.001). In ovarian carcinoma samples and cell lines we did not find mutations in RON and MET gene kinase domain, nor coexpression of RON and MET receptor ligands (MSP and HGF, respectively). We show that motility and invasiveness of ovarian cancer cells coexpressing MET and RON receptors were elicited by HGF and, to a lesser extent, by MSP. More interestingly, invasion of both reconstituted basement membrane and collagen gel was greatly enhanced by the simultaneous addition of the two ligands. These data suggest that coexpression of the MET and RON receptors confer a selective advantage to ovarian cancer cells and might promote ovarian cancer progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0014-4827(03)00250-7DOI Listing
August 2003

Novel somatic mutations of the MET oncogene in human carcinoma metastases activating cell motility and invasion.

Cancer Res 2002 Dec;62(23):7025-30

Laboratory of Cancer Genetics, Institute for Cancer Research and Treatment, Torino, Italy.

Several gene mutations responsible for human cancer initiation have been discovered, whereas only a few have been identified in association with the progression to metastasis. In this study, we screened a large panel of human sporadic cancers, metastases, and tumor cell lines for mutations in the tyrosine kinase domain of the MET receptor, crucially involved in invasive cell growth and motility during embryogenesis. MET activating mutations have been described previously in hereditary papillary renal cell carcinoma and in a few sporadic tumors. Summarizing results of this and our previous studies, we did not detect mutations in the MET kinase domain from 153 sporadic human cancers and 25 cancer cell lines, whereas we found somatic MET mutations in 10 of 46 lymph nodal and 2 of 14 pulmonary metastases. We identified four MET mutations in metastases. Two were known as MET germ-line mutations (H1112R and Y1248C), which predispose to hereditary renal cell carcinoma. One of the two novel mutations (N1118Y) changed an asparagine in the region of the glycine-rich ATP binding site, which is highly conserved in all of the kinases. The other (Y1253D) changed a critical tyrosine, known to regulate MET kinase activity, to a negatively charged residue. The MET receptors carrying either the N1118Y or the Y1253D mutation were constitutively active and conferred a motile-invasive phenotype on transduced carcinoma cells. The latter phenotype was additionally stimulated by the MET receptor ligand scatter factor/hepatocyte growth factor. These data suggest that MET might be one of the long sought oncogenes controlling progression of primary cancers to metastasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
December 2002