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1.
Int J Mol Sci ; 23(10)2022 May 21.
Article in English | MEDLINE | ID: mdl-35628590

ABSTRACT

The MET oncogene encodes a tyrosine kinase (TK) receptor. Its activation protects cells from death but also stimulates DNA damage response by triggering excess replicative stress. Transcriptomic classification of cancer cell lines based on MET expression showed that response to the PARP inhibitor (PARPi) olaparib is poorer in MET overexpressing cell lines. Accordingly, a high MET expressing lung carcinoma cell line was sensitized to PARPi by MET TK inhibition. This was not linked solely to MET overexpression: other MET overexpressing cell lines were biochemically but not functionally responsive to combined inhibition. Moreover, exogenously induced MET overexpression was unable to induce resistance to PARPi. The MET overexpressing cell line, responsive to the combined PARP and MET inhibition, carried a heterozygous mutation of the ATM gene and showed an attenuated response of ATM to PARPi. Among the downstream targets of ATM activation, NuMA was phosphorylated only in response to the combined PARP and MET inhibition. Given the role played by NuMA in mitosis, data show that the latter is affected by MET and PARP inhibition in cells with haploinsufficient ATM. This is important as ATM heterozygous mutation is frequently found in human cancer and in lung carcinomas in particular.


Subject(s)
Antineoplastic Agents , Lung Neoplasms , Antineoplastic Agents/pharmacology , Ataxia Telangiectasia Mutated Proteins/metabolism , Haploinsufficiency , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
2.
NPJ Syst Biol Appl ; 7(1): 1, 2021 01 05.
Article in English | MEDLINE | ID: mdl-33402683

ABSTRACT

Single-cell RNA sequencing (scRNAseq) is an essential tool to investigate cellular heterogeneity. Thus, it would be of great interest being able to disclose biological information belonging to cell subpopulations, which can be defined by clustering analysis of scRNAseq data. In this manuscript, we report a tool that we developed for the functional mining of single cell clusters based on Sparsely-Connected Autoencoder (SCA). This tool allows uncovering hidden features associated with scRNAseq data. We implemented two new metrics, QCC (Quality Control of Cluster) and QCM (Quality Control of Model), which allow quantifying the ability of SCA to reconstruct valuable cell clusters and to evaluate the quality of the neural network achievements, respectively. Our data indicate that SCA encoded space, derived by different experimentally validated data (TF targets, miRNA targets, Kinase targets, and cancer-related immune signatures), can be used to grasp single cell cluster-specific functional features. In our implementation, SCA efficacy comes from its ability to reconstruct only specific clusters, thus indicating only those clusters where the SCA encoding space is a key element for cells aggregation. SCA analysis is implemented as module in rCASC framework and it is supported by a GUI to simplify it usage for biologists and medical personnel.


Subject(s)
Data Mining/methods , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Algorithms , Base Sequence/genetics , Cluster Analysis , Humans , Neural Networks, Computer , Software , Systems Biology/methods , Exome Sequencing/methods
3.
Cancers (Basel) ; 12(12)2020 Dec 15.
Article in English | MEDLINE | ID: mdl-33333972

ABSTRACT

For many decades, basic and preclinical cancer research has been based on the use of established, commercially available cell lines, originally derived from patients' samples but adapted to grow indefinitely in artificial culture conditions, and on xenograft models developed by injection of these cells in immunocompromised animals [...].

4.
Cancers (Basel) ; 12(12)2020 Dec 08.
Article in English | MEDLINE | ID: mdl-33302341

ABSTRACT

Ovarian cancer is still the most lethal gynecologic malignancy with a median five-year survival of 48%, including the less malignant and early diagnosed cases [...].

5.
Cells ; 9(2)2020 02 14.
Article in English | MEDLINE | ID: mdl-32075097

ABSTRACT

Identifying cancer drivers and actionable mutations is critical for precision oncology. In epithelial ovarian cancer (EOC) the majority of mutations lack biological or clinical validation. We fully characterized 43 lines of Patient-Derived Xenografts (PDXs) and performed copy number analysis and whole exome sequencing of 12 lines derived from naïve, high grade EOCs. Pyrosequencing allowed quantifying mutations in the source tumours. Drug response was assayed on PDX Derived Tumour Cells (PDTCs) and in vivo on PDXs. We identified a PIK3R1W624R variant in PDXs from a high grade serous EOC. Allele frequencies of PIK3R1W624R in all the passaged PDXs and in samples of the source tumour suggested that it was truncal and thus possibly a driver mutation. After inconclusive results in silico analyses, PDTCs and PDXs allowed the showing actionability of PIK3R1W624R and addiction of PIK3R1W624R carrying cells to inhibitors of the PI3K/AKT/mTOR pathway. It is noteworthy that PIK3R1 encodes the p85α regulatory subunit of PI3K, that is very rarely mutated in EOC. The PIK3R1W624R mutation is located in the cSH2 domain of the p85α that has never been involved in oncogenesis. These data show that patient-derived models are irreplaceable in their role of unveiling unpredicted driver and actionable variants in advanced ovarian cancer.


Subject(s)
Carcinoma, Ovarian Epithelial/genetics , Class Ia Phosphatidylinositol 3-Kinase/genetics , Cystadenocarcinoma, Serous/genetics , Ovarian Neoplasms/genetics , Animals , Carcinoma, Ovarian Epithelial/drug therapy , Carcinoma, Ovarian Epithelial/enzymology , Cell Line, Tumor , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Cystadenocarcinoma, Serous/drug therapy , Cystadenocarcinoma, Serous/enzymology , Female , Heterografts , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Targeted Therapy , Mutation , Neoplasm Grading , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/enzymology , Random Allocation
6.
J Ovarian Res ; 12(1): 17, 2019 Feb 13.
Article in English | MEDLINE | ID: mdl-30760286

ABSTRACT

OBJECTIVE: Relapsed epithelial ovarian cancer (EOC) is frequently treated with pegylated liposomal doxorubicin (PLD). Unfortunately, most patients do not benefit from treatment. Prediction of response is crucial to optimize PLD use and avoid unnecessary toxicities. We aimed at assessing the value of topoisomerase II alpha (TOP2A) expression as predictive marker of response to PLD-based therapy in patients with relapsed EOCs. METHODS: We retrospectively analyzed Formalin Fixed Paraffin Embedded (FFPE) tissues from 101 patients with platinum resistant (PR) or partially platinum-sensitive (PPS) EOCs treated with PLD-based chemotherapy beyond second line in three referral cancer centers between January 2010 and June 2018. TOP2A expression was measured by immunohistochemistry (IHC): images of each sample were acquired by optical microscope and analyzed by using automatic counter software. Correlation between TOP2A expression and response to PLD was assessed. Since no cut-off for positivity has been validated yet, we dichotomized TOP2A expression based on a cut-off of 18% (mean value in this study). RESULTS: TOP2A expression beyond cut-off was not prognostic for primary platinum-free interval in our series (p = 0.77) neither for optimal cytoreduction (p = 0.9). TOP2A > 18% was associated with a longer time to progression (TTP) following PLD-treatment, although not statistically significant (p = 0.394). No difference was observed between PR and PPS patients' groups (p = 0.445 and p = 0.185, respectively). Not unexpectedly, patients with TOP2A expression > 18% treated with PLD monotherapy achieved a longer TTP compared with PLD-doublet therapy (p = 0.05). CONCLUSIONS: Our data suggest that TOP2A status might predict activity of PLD in patients with PR/PPS EOCs.


Subject(s)
Antibiotics, Antineoplastic/therapeutic use , Carcinoma, Ovarian Epithelial/drug therapy , DNA Topoisomerases, Type II/metabolism , Doxorubicin/analogs & derivatives , Neoplasm Recurrence, Local , Ovarian Neoplasms/drug therapy , Antineoplastic Combined Chemotherapy Protocols , Biomarkers, Tumor/metabolism , Carcinoma, Ovarian Epithelial/metabolism , Carcinoma, Ovarian Epithelial/pathology , DNA Topoisomerases, Type II/genetics , Doxorubicin/therapeutic use , Drug Resistance, Neoplasm , Female , Humans , Kaplan-Meier Estimate , Middle Aged , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Platinum/pharmacology , Polyethylene Glycols/therapeutic use , Retrospective Studies
7.
Oncotarget ; 7(26): 40073-40084, 2016 Jun 28.
Article in English | MEDLINE | ID: mdl-27223077

ABSTRACT

Inhibition of the mechanistic target of rapamycin (mTOR) is a promising treatment strategy for several cancer types. Rapamycin derivatives such as everolimus are allosteric mTOR inhibitors acting through interaction with the intracellular immunophilin FKBP12, a prolyl isomerase with different cellular functions. Although mTOR inhibitors have significantly improved survival of different cancer patients, resistance and lack of predictive factors of response remain unsolved issues. To elucidate the mechanisms of resistance to everolimus, we evaluated Met activation in everolimus-sensitive/resistant human cancer cells, in vitro and in vivo. Biochemical and computational analyses were performed. Everolimus-resistant cells were xenografted into mice (10/group) and studied for their response to everolimus and Met inhibitors. The statistical significance of the in vitro results was evaluated by Student's t test.Everolimus reduced Met phosphorylation in everolimus-sensitive cells. This event was mediated by the formation of a Met-FKBP12 complex, which in turn is disrupted by everolimus. Aberrant Met activation in everolimus-resistant cells and overexpression of wild-type/mutant Met caused everolimus resistance. Pharmacological inhibition and RNA silencing of Met are effective in condition of everolimus resistance (P<0.01). In mice xenografted with everolimus-resistant cells, the combination of everolimus with the Met inhibitor PHA665752 reduced tumor growth and induced a statistically significant survival advantage (combination vs control P=0.0005).FKBP12 binding is required for full Met activation and everolimus can inhibit Met. Persistent Met activation might sustain everolimus resistance. These results identify a novel everolimus mechanism of action and suggest the development of clinical strategies based on Met inhibitors in everolimus-resistant cancers.


Subject(s)
Drug Resistance, Neoplasm , Everolimus/pharmacology , Gene Expression Regulation, Neoplastic , Receptor Protein-Tyrosine Kinases/metabolism , TOR Serine-Threonine Kinases/metabolism , Allosteric Site , Animals , Cell Line, Tumor , Female , HCT116 Cells , Humans , Mice , Mice, Nude , Neoplasm Transplantation , Phosphorylation , RNA Interference
8.
Eur J Cancer ; 60: 59-68, 2016 06.
Article in English | MEDLINE | ID: mdl-27065457

ABSTRACT

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.


Subject(s)
Hepatocyte Growth Factor/physiology , Ovarian Neoplasms/enzymology , Protein Serine-Threonine Kinases/physiology , Antineoplastic Agents/therapeutic use , Apoptosis/drug effects , Cisplatin/therapeutic use , Female , Hepatocyte Growth Factor/metabolism , Humans , Ovarian Neoplasms/drug therapy , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Tumor Cells, Cultured
9.
Oncotarget ; 7(18): 26181-91, 2016 May 03.
Article in English | MEDLINE | ID: mdl-27027433

ABSTRACT

Platinum-based chemotherapy is the recommended first-line treatment for high-grade serous (HGS) epithelial ovarian cancer (EOC). However, most patients relapse because of platinum refractory/resistant disease. We aimed at assessing whether other drugs, commonly used to treat relapsed HGS-EOC and poorly active in this clinical setting, might be more effective against chemotherapy-naïve cancers. We collected couples of HGS-EOC samples from the same patients before and after neo-adjuvant platinum-based chemotherapy. Samples were propagated as Patient Derived Xenografts (PDXs) in immunocompromised mice ("xenopatients"). Xenopatients were treated in parallel with carboplatin, gemcitabine, pegylated liposomal doxorubicin (PLD) and trabectedin. PDXs derived from a naïve HSG-EOC showed responsiveness to carboplatin, trabectedin and gemcitabine. The PDXs propagated from a tumor mass of the same patient, grown after carboplatin therapy, did no longer respond to trabectedin and gemcitabine and showed heterogeneous response to carboplatin. In line, the patient experienced clinically platinum-sensitivity first and then discordant responses of different tumor sites to platinum re-challenge. Loss of PDX responsiveness to drugs was associated with 4-fold increase of NR2F2 gene expression. PDXs from another naïve tumor showed complete response to PLD, which was lost in the PDXs derived from a mass grown in the same patient after platinum-based chemotherapy. This patient showed platinum refractoriness and responded poorly to PLD as second-line treatment. PDX response to PLD was associated with high expression of TOP2A protein. PDXs demonstrated that chemotherapy-naïve HGS-EOC might display susceptibility to agents not used commonly as first line treatment. Data suggest the importance of personalizing also chemotherapy.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Ovarian Neoplasms/drug therapy , Precision Medicine , Aged , Animals , Apoptosis , Carboplatin/administration & dosage , Cell Proliferation , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Dioxoles/administration & dosage , Doxorubicin/administration & dosage , Doxorubicin/analogs & derivatives , Female , Humans , Mice , Middle Aged , Neoadjuvant Therapy , Ovarian Neoplasms/pathology , Polyethylene Glycols/administration & dosage , Tetrahydroisoquinolines/administration & dosage , Trabectedin , Tumor Cells, Cultured , Xenograft Model Antitumor Assays , Gemcitabine
10.
Oncotarget ; 7(1): 712-28, 2016 Jan 05.
Article in English | MEDLINE | ID: mdl-26625210

ABSTRACT

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, α5ß1 integrin activation and TGF-ß1 translation. Reduced endogenous FN deposition and TGF-ß1 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.


Subject(s)
Lung Neoplasms/genetics , Ovarian Neoplasms/genetics , Peritoneal Neoplasms/genetics , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Animals , Blotting, Western , Cell Adhesion/genetics , Cell Line, Tumor , Cell Movement/genetics , Female , Fibronectins/genetics , Fibronectins/metabolism , Gene Expression Regulation, Neoplastic , HeLa Cells , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , Mice, Inbred NOD , Mice, SCID , Microscopy, Confocal , Neoplasm Invasiveness , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Peritoneal Neoplasms/metabolism , Peritoneal Neoplasms/secondary , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism , Transplantation, Heterologous
11.
J Proteome Res ; 13(11): 4970-82, 2014 Nov 07.
Article in English | MEDLINE | ID: mdl-25099161

ABSTRACT

Platinum-based chemotherapy is widely used to treat various cancers, but many patients ultimately relapse due to drug resistance. We employed phosphoproteomic analysis and functional assays of the response of SK-OV-3 ovarian cancer cells to cisplatin as a strategy to identify kinases as candidate druggable targets to sensitize cells to platinum. A SILAC-based approach combined with TiO2-based phosphopeptide enrichment allowed the direct identification of ERK1/2, p90RSK, and ERBB2 as kinases whose phosphorylation is regulated by cisplatin. Bioinformatic analysis revealed enrichment in linear phosphorylation motifs predicted to be targets of p38MAPK, CDK2, and PIM2. All three PIM kinases were found expressed in a panel of 10 ovarian cancer cell lines, with the oncogenic PIM2 being the most commonly induced by cisplatin. Targeting PIM2 kinase by either biochemical inhibitors or RNA interference impaired cell growth, decreased cisplatin-triggered BAD phosphorylation, and sensitized ovarian cancer cells to drug-induced apoptosis. Overexpression of PIM2 triggered anchorage-independent growth and resulted in increased BAD phosphorylation and cell resistance to DNA damaging agents. The data show that the PIM2 kinase plays a role in the response of ovarian cancer cells to platinum drugs and suggest that PIM inhibitors may find clinical application as an adjunct to platinum-based therapies.


Subject(s)
Cisplatin/pharmacology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Protein Serine-Threonine Kinases/metabolism , Proteomics/methods , Proto-Oncogene Proteins/metabolism , Amino Acid Motifs , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor/drug effects , Cell Proliferation/drug effects , Female , Humans , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Tandem Mass Spectrometry/methods , bcl-Associated Death Protein/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
12.
Oncotarget ; 5(15): 5992-6002, 2014 Aug 15.
Article in English | MEDLINE | ID: mdl-25115388

ABSTRACT

CDT2/L2DTL/RAMP is one of the substrate receptors of the Cullin Ring Ubiquitin Ligase 4 that targets for ubiquitin mediated degradation a number of substrates, such as CDT1, p21 and CHK1, involved in the regulation of cell cycle and survival. Here we show that CDT2 depletion was alone able to induce the apoptotic death in 12/12 human cancer cell lines from different tissues, regardless of the mutation profile and CDT2 expression level. Cell death was associated to rereplication and to loss of CDT1 degradation. Conversely, CDT2 depletion did not affect non-transformed human cells, such as immortalized kidney, lung and breast cell lines, and primary cultures of endothelial cells and osteoblasts. The ectopic over-expression of an activated oncogene, such as the mutation-activated RAS or the amplified MET in non-transformed immortalized breast cell lines and primary human osteoblasts, respectively, made cells transformed in vitro, tumorigenic in vivo, and susceptible to CDT2 loss. The widespread effect of CDT2 depletion in different cancer cells suggests that CDT2 is not in a synthetic lethal interaction to a single specific pathway. CDT2 likely is a non-oncogene to which transformed cells become addicted because of their enhanced cellular stress, such as replicative stress and DNA damage.


Subject(s)
Neoplasms/enzymology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Cell Line, Tumor , DNA Replication , DNA, Neoplasm/biosynthesis , DNA, Neoplasm/genetics , Female , Human Umbilical Vein Endothelial Cells , Humans , Neoplasms/genetics , Neoplasms/pathology , Phenotype
13.
FASEB J ; 28(9): 4055-67, 2014 Sep.
Article in English | MEDLINE | ID: mdl-24903273

ABSTRACT

The tyrosine kinase encoded by the MET oncogene is activated by gene mutation or amplification in tumors, which in most instances maintain addiction, i.e., dependency, to MET activation. This makes MET an attractive candidate for targeted therapies. Here we show that, in 3/3 MET-addicted human gastric cancer cell lines, MET kinase inhibition resulted in a 3- to 4-fold increased expression of the antiapoptotic small heat-shock protein of 27 kDa (HSP27, HSPB1). HSP27 increase depended on the inhibition of the MEK/ERK pathway and on heat-shock factor 1 (HSF1) and hypoxia-inducible factor-1α (HIF-1α) regulation. Importantly, HSP27-silenced MET-addicted cells underwent 2- and 3-fold more apoptosis following MET inhibition in vitro and in vivo, respectively. Likewise, in human cancer cells susceptible to epidermal growth factor receptor (EGFR) inhibition, EGFR inhibitors induced HSP27 expression and were strengthened by HSP27 suppression. In control cell lines that were not affected by drugs targeting MET or EGFR, these drugs did not induce HSP27 increase. Therefore, in cancer therapies targeting the MET pathway, the induction of HSP27 might limit the efficacy of anti-MET agents. As HSP27 increase also impairs the effectiveness of EGFR inhibitors and is known to protect cells from chemotherapeutics, the induction of HSP27 by targeted agents might strongly affect the success of combination treatments.


Subject(s)
Gene Expression Regulation, Neoplastic/drug effects , HSP27 Heat-Shock Proteins/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Stomach Neoplasms/drug therapy , Animals , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Blotting, Western , Cell Cycle/drug effects , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/drug effects , HSP27 Heat-Shock Proteins/antagonists & inhibitors , HSP27 Heat-Shock Proteins/genetics , Heat-Shock Proteins , Humans , Immunoenzyme Techniques , Mice , Molecular Chaperones , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Stomach Neoplasms/enzymology , Stomach Neoplasms/pathology , Tumor Cells, Cultured , Up-Regulation , Xenograft Model Antitumor Assays
14.
Int J Cancer ; 134(6): 1289-99, 2014 Mar 15.
Article in English | MEDLINE | ID: mdl-23996744

ABSTRACT

The hepatocyte growth factor (HGF) also known as scatter factor activates cancer cell invasion and metastasis. We show that in ovarian cancer cells HGF induced the phosphorylation of the small heat shock protein of 27 kDa (HSP27) by activating the p38MAPK. HSP27 is increased in many cancers at advanced stage including ovarian cancer and associated with cancer resistance to therapy and poor patients' survival. The phosphorylation of HSP27 regulates both its chaperone activity and its control of cytoskeletal stability. We show that HSP27 was necessary for the remodeling of actin filaments induced by HGF and that motility in vitro depended on the p38MAPK-MK2 axis. In vivo, HSP27 silencing impaired the ability of the highly metastatic, HGF-secreting ovarian cancer cells to give rise to spontaneous metastases. This was due to defective motility across the vessel wall and reduced growth. Indeed, HSP27 silencing impaired the ability of circulating ovarian cancer cells to home to the lungs and to form experimental hematogenous metastases and the capability of cancer cells to grow as subcutaneous xenografts. Moreover, HSP27 suppression resulted in the sensitization of xenografts to low doses of the chemotherapeutic paclitaxel, likely because HSP27 protected microtubules from bundling caused by the drug. Altogether, these data show that the HSP27 is required for the proinvasive and prometastatic activity of HGF and suggest that HSP27 might be not only a marker of progression of ovarian cancer, but also a suitable target for therapy.


Subject(s)
Cell Movement , Drug Resistance, Neoplasm , HSP27 Heat-Shock Proteins/metabolism , Hepatocyte Growth Factor/pharmacology , Lung Neoplasms/secondary , Ovarian Neoplasms/pathology , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis , Blotting, Western , Cell Proliferation , Female , Fluorescent Antibody Technique , HSP27 Heat-Shock Proteins/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Invasiveness , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Paclitaxel/pharmacology , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
15.
Exp Cell Res ; 319(17): 2627-36, 2013 Oct 15.
Article in English | MEDLINE | ID: mdl-23948303

ABSTRACT

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.


Subject(s)
Cell Nucleus/metabolism , Cellular Apoptosis Susceptibility Protein/metabolism , Ovarian Neoplasms/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Transcriptional Activation , Active Transport, Cell Nucleus , Cell Line, Tumor , Cell Movement , Cellular Apoptosis Susceptibility Protein/genetics , Cytoplasm/metabolism , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Nuclear Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Phosphorylation , Transcription, Genetic
16.
BMC Vet Res ; 9: 80, 2013 Apr 15.
Article in English | MEDLINE | ID: mdl-23587222

ABSTRACT

BACKGROUND: Triple negative breast cancer (TNBC) in humans is defined by the absence of oestrogen receptor (ER), progesterone receptor (PR) and HER2 overexpression. Mammalian target of rapamycin (mTOR) is overexpressed in TNBC and it represents a potential target for the treatment of this aggressive tumour. Feline mammary carcinoma (FMC) is considered to be a model for hormone-independent human breast cancer. This study investigated mTOR and p-mTOR expression in FMC in relation to triple negative (TN) phenotype. RESULTS: The expression of mTOR, p-mTOR, ERα, PR and HER2 was evaluated in 58 FMCs by immunohistochemistry and in six FMC cell lines by Western blot analysis. 53.5% of FMC analyzed were ER, PR, HER2 negative (TN-FMC) while 56.9% and 55.2% of cases expressed mTOR and p-mTOR respectively. In this study we found that m-TOR and p-mTOR were more frequently detected in TN-FMC and in HER2 negative samples. CONCLUSIONS: In this study, we demonstrate that there is also a FMC subset defined as TN FMC, which is characterised by a statistically significant association with m-TOR and p-mTOR expression as demonstrated in human breast cancer.


Subject(s)
Cat Diseases/metabolism , Mammary Neoplasms, Animal/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Blotting, Western/veterinary , Cat Diseases/pathology , Cats , Cell Line, Tumor , Disease Models, Animal , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation, Neoplastic , Mammary Glands, Animal/metabolism , Mammary Glands, Animal/pathology , Mammary Neoplasms, Animal/pathology , Phenotype , Receptor, ErbB-2/metabolism , Receptors, Progesterone/metabolism
17.
Eur J Cancer ; 49(4): 964-73, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23079474

ABSTRACT

BACKGROUND: The Interferon Regulatory Factors (IRFs) are transcription factors involved in immune responses and oncogenesis and most of them are classified as tumour suppressors. The expression and activation of IRF(s) are stimulated by several cytokines and by DNA damage. Here we examine the role of the IRF-1 in the response of ovarian cancer cells to the front-line chemotherapeutic drug cisplatin (CDDP). METHODS: We evaluated the transcriptional response of three ovarian cancer cell lines to CDDP both under control conditions and after IRF-1 silencing using expression microarrays. The role played by IRF-1 in the response of these cells to CDDP was evaluated after silencing and overexpressing IRF-1. We studied cell cycle progression, colony forming ability in monolayer culture and semisolid medium, and apoptosis in the response to the drug. RESULTS: The treatment of ovarian cancer cells with CDDP boosted the expression and the nuclear translocation of IRF-1, which in turn modulated the expression of putative IRF-1 target genes. Accordingly, IRF-1 silencing re-orchestrated the expression profiles of CDDP-treated cells. In agreement with its role as a tumour suppressor, overexpressing IRF-1 suppressed the transformed phenotype of ovarian cancer cells. Nevertheless, IRF-1 silencing sensitized cells to the apoptotic death induced by CDDP. Over-expression was associated with cell G1 arrest and p21 induction irrespective of p53 proficiency, while IRF-1 silencing reduced the induction of p21 by CDDP. CONCLUSIONS: These data demonstrate that IRF-1 is up-regulated by CDDP in ovarian cancer cells and might limit the cell response to CDDP, likely by inhibiting cell proliferation. Data suggest that IRF-1 induction might interfere with the effectiveness of combination therapy with platinum drugs and cytokines.


Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/genetics , Cell Transformation, Neoplastic/genetics , Cisplatin/pharmacology , Drug Resistance, Neoplasm , Interferon Regulatory Factor-1/metabolism , Ovarian Neoplasms/genetics , Apoptosis/drug effects , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Female , Gene Expression Profiling , Genes, Tumor Suppressor , Humans , Interferon Regulatory Factor-1/antagonists & inhibitors , Interferon Regulatory Factor-1/genetics , Oligonucleotide Array Sequence Analysis , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Up-Regulation
18.
BMC Res Notes ; 5: 517, 2012 Sep 23.
Article in English | MEDLINE | ID: mdl-22999213

ABSTRACT

BACKGROUND: Hepatocyte Growth Factor (HGF) enhances cytotoxicity of paclitaxel (PTX) and cisplatin (CDDP) in human ovarian cancer cells. Because of potential pitfalls of HGF exogenous administration, we investigated whether HGF serum concentration might be alternatively raised in vivo by administering low molecular weight heparin (LMWH). METHODS: The main HGF pharmacokinetic parameters were evaluated following acute and chronic LMWH treatment. First, women, operated on for gynaecological tumors, were treated with a single dose of calcium nadroparin and studied for 12 hours. Next, women operated on for benign or malignant gynaecological tumors were treated daily with calcic nadroparin for one month. Subsequently, the biological activity of the measured HGF serum levels was tested in assays of ovarian cancer cell sensitization to drugs. RESULTS: In the short-term treated group, median HGF AUCss, Cmax and Caverage were about four-fold that of the control group, whereas Cmin was three-fold. In the patients treated chronically median HGF serum levels rose about six-fold in the first week, and decreased but remained significantly higher after one month. The pharmacokinetic of nadroparin-dependent HGF increase were similar in the two groups. The HGF concentrations measured after both acute and chronic treatment were found to be effective in sensitising ovarian cancer cells to chemotherapeutics. CONCLUSIONS: This study raises the possibility of using LMWH to increase HGF serum concentration and to take advantage of its biological activities. In particular, nadroparin might be used as a chemo-potentiating agent in epithelial cell ovarian carcinoma through its action on HGF serum concentration. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT01523652.


Subject(s)
Genital Neoplasms, Female/drug therapy , Heparin, Low-Molecular-Weight/therapeutic use , Hepatocyte Growth Factor/blood , Ovarian Neoplasms/drug therapy , Adult , Aged , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Line, Tumor , Cisplatin/pharmacology , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Genital Neoplasms, Female/blood , Genital Neoplasms, Female/pathology , Hepatocyte Growth Factor/pharmacology , Humans , Middle Aged , Nadroparin/therapeutic use , Ovarian Neoplasms/blood , Ovarian Neoplasms/pathology , Prospective Studies , Time Factors
19.
Mol Cell Biol ; 32(15): 3081-94, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22645311

ABSTRACT

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.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Fumarate Hydratase/genetics , Fumarates/metabolism , AMP-Activated Protein Kinases/deficiency , AMP-Activated Protein Kinases/genetics , Animals , Apoptosis , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line , Fumarate Hydratase/deficiency , Fumarate Hydratase/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Kidney Neoplasms/genetics , Leiomyomatosis/genetics , Mice , Mitogen-Activated Protein Kinase 1/biosynthesis , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/biosynthesis , Mitogen-Activated Protein Kinase 3/metabolism , Neoplastic Syndromes, Hereditary/genetics , RNA Interference , RNA, Small Interfering , Reactive Oxygen Species/analysis , Signal Transduction , Skin Neoplasms , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Uterine Neoplasms
20.
FASEB J ; 26(6): 2446-56, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22389439

ABSTRACT

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.


Subject(s)
Cellular Apoptosis Susceptibility Protein/genetics , Ovarian Neoplasms/genetics , Tumor Suppressor Proteins/drug effects , Apoptosis/drug effects , Cell Nucleus/metabolism , Cisplatin/pharmacology , Cytoplasm/metabolism , Female , Gene Expression Regulation, Neoplastic , Hepatocyte Growth Factor/pharmacology , Humans , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Tumor Suppressor Proteins/biosynthesis , Up-Regulation
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