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1.
EMBO Mol Med ; 12(12): e11889, 2020 12 07.
Article in English | MEDLINE | ID: mdl-33151035

ABSTRACT

An open debate in antiangiogenic therapies is about their consequence on tumor invasiveness and metastasis, which is undoubtedly relevant for patients currently treated with antiangiogenics, such as renal cell carcinoma patients. To address, this we developed an extensive series of 27 patient biopsy-derived orthotopic xenograft models (Ren-PDOX) that represent inter-patient heterogeneity. In specific tumors, antiangiogenics produced increased invasiveness and metastatic dissemination, while in others aggressiveness remained unchanged. Mechanistically, species-discriminative RNA sequencing identified a tumor cell-specific differential expression profile associated with tumor progression and aggressivity in TCGA RCC patients. Gene filtering using an invasion-annotated patient series pinpointed two candidate genes, of which ALDH1A3 differentiated the pro-invasive subtype of Ren-PDOXs. Validation in an independent series of 15 antiangiogenic-treated patients confirmed that pre-treatment ALDH1A3 can significantly discriminate patients with pro-aggressive response upon treatment. Overall, results confirm that effects of antiangiogenic drugs on tumor invasion and metastasis are heterogeneous and may profoundly affect the natural progression of tumors and promote malignancy. Furthermore, we identify a specific molecular biomarker that could be used to select patients that better benefit from treatment.


Subject(s)
Angiogenesis Inhibitors/adverse effects , Carcinoma, Renal Cell/pathology , Kidney Neoplasms/pathology , Models, Biological , Neoplasm Invasiveness/pathology , Neoplasm Metastasis/pathology , Animals , Cell Line, Tumor , Humans , Male , Mice , Mice, Nude , Precision Medicine , Xenograft Model Antitumor Assays
2.
Sci Rep ; 10(1): 13987, 2020 08 19.
Article in English | MEDLINE | ID: mdl-32814805

ABSTRACT

Classification of variants in the BRCA1 and BRCA2 genes has a major impact on the clinical management of subjects at high risk for breast and ovarian cancer. The identification of a pathogenic variant allows for early detection/prevention strategies in healthy carriers as well as targeted treatments in patients affected by BRCA-associated tumors. The BRCA2 c.9227G>T p.(Gly3076Val) variant recurs in families from Northeast Italy and is rarely reported in international databases. This variant substitutes the evolutionary invariant glycine 3076 with a valine in the DNA binding domain of the BRCA2 protein, thus suggesting a high probability of pathogenicity. We analysed clinical and genealogic data of carriers from 15 breast/ovarian cancer families in whom no other pathogenic variants were detected. The variant was shown to co-segregate with breast and ovarian cancer in the most informative families. Combined segregation data led to a likelihood ratio of 81,527:1 of pathogenicity vs. neutrality. We conclude that c.9227G>T is a BRCA2 pathogenic variant that recurs in Northeast Italy. It can now be safely used for the predictive testing of healthy family members to guide preventive surgery and/or early tumor detection strategies, as well as for PARP inhibitors treatments in patients with BRCA2-associated tumors.


Subject(s)
BRCA2 Protein/genetics , Breast Neoplasms/genetics , Genetic Predisposition to Disease/genetics , Ovarian Neoplasms/genetics , Polymorphism, Single Nucleotide , Adult , Aged , Aged, 80 and over , BRCA1 Protein/genetics , Breast Neoplasms/diagnosis , Family Health , Female , Genetic Testing , Humans , Male , Middle Aged , Ovarian Neoplasms/diagnosis , Pedigree
3.
Carcinogenesis ; 36(1): 115-21, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25355291

ABSTRACT

Activation of the NOTCH pathway occurs commonly in T acute lymphoblastic leukemia (T-ALL) mainly due to mutations in NOTCH1 or alterations in FBW7 and is involved in the regulation of cell proliferation and survival. Since mutations hit different domains of the receptor, they are predicted to heterogeneously perturb ligand-induced NOTCH1 activity. Moreover, T-ALL cells also co-express NOTCH3 receptors which could be triggered by different ligands. In this study, we aimed to investigate the role of DLL4 in the regulation of NOTCH signaling in T-ALL cells in the context of different types of NOTCH1 mutation or wild-type NOTCH receptor, as well as the effects of DLL4 neutralization on T-ALL engraftment in mice. We found that NOTCH signaling can be stimulated in T-ALL cells in vitro by either human or murine DLL4 with heterogeneous effects, according to NOTCH1/FBW7 mutation status, and that these effects can be blocked by antibodies neutralizing DLL4, NOTCH1 or NOTCH2/3. In vivo, DLL4 is expressed in the spleen and the bone marrow (BM) of NOD/SCID mice bearing T-ALL xenografts as well as the BM of T-ALL patients. Importantly, DLL4 blockade impaired growth of T-ALL cells in NOD/SCID mice and increased leukemia cell apoptosis. These results show that DLL4 is an important component of the tumor microenvironment which contributes to the early steps of T-ALL cell growth.


Subject(s)
Intercellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptors, Notch/metabolism , Adaptor Proteins, Signal Transducing , Animals , Apoptosis , Blotting, Western , Calcium-Binding Proteins , Cell Proliferation , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression Regulation, Neoplastic , Humans , Immunoenzyme Techniques , Intercellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptors, Notch/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
4.
Cancer Discov ; 4(1): 31-41, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24356098

ABSTRACT

Tumor growth requires induction of an angiogenic program, and targeting of this program with antiangiogenic drugs shows an impact on tumor progression. However, although they are effective at reducing angiogenesis, these therapies have not produced widespread or enduring clinical benefit, which openly exposes their limitations. Here, we describe the current limitations of these therapies, including the known mechanisms and current controversies. Further, we present some of the recent approaches to predict these limitations and strategies to overcome them. With the development of meaningful predictive biomarkers and effective treatments that impede these limitations, longer and more robust efficacies will be achieved for a wider population of patients.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Neoplasms/drug therapy , Neovascularization, Pathologic/drug therapy , Animals , Biomarkers, Tumor/metabolism , Drug Resistance, Neoplasm/drug effects , Drug Therapy, Combination , Humans , Neoplasms/metabolism , Neovascularization, Pathologic/metabolism
5.
Hepatology ; 58(3): 1042-53, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23505219

ABSTRACT

UNLABELLED: Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)ß. PDGF-D, a PDGFRß agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRß blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration. CONCLUSION: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRß and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach.


Subject(s)
Bile Duct Neoplasms/physiopathology , Bile Ducts, Intrahepatic , Cell Movement/physiology , Cholangiocarcinoma/physiopathology , Fibroblasts/pathology , Lymphokines/physiology , Platelet-Derived Growth Factor/physiology , rho GTP-Binding Proteins/physiology , Animals , Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Bile Duct Neoplasms/pathology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Cholangiocarcinoma/pathology , Epithelial-Mesenchymal Transition/physiology , Heterografts , Humans , Imatinib Mesylate , In Vitro Techniques , Male , Mice , Mice, SCID , Piperazines/pharmacology , Pyrimidines/pharmacology , Signal Transduction/physiology
7.
Hepatology ; 54(3): 890-9, 2011 Sep 02.
Article in English | MEDLINE | ID: mdl-21618579

ABSTRACT

UNLABELLED: Cholangiocarcinoma (CCA) carries a severe prognosis because of its strong invasiveness and early metastasization. In several patients, otherwise eligible for surgical resection, micrometastasis are already present at the time of surgery. The mechanisms responsible for CCA invasiveness are unclear. S100A4, a member of the S100 family of small Ca(2+)-binding proteins, is expressed in mesenchymal cells, regulates cell motility in several cell types, and is expressed in some epithelial cancers. Thus, we aimed to study the role of S100A4 in CCA invasiveness and metastasization. The expression of S100A4 was studied by immunohistochemistry in 93 human liver samples of CCA patients undergoing surgical resection and correlated with metastases development (67 cases) and patient survival following surgery using log rank tests and multivariate analysis. S100A4 expression was studied in EGI-1 and TFK-1, human CCA cell lines with and without nuclear S100A4 expression, respectively. Metastatic properties of CCA cells were assessed by xenotransplantation in severe combined immunodeficiency (SCID) mice after transduction with lentiviral vectors encoding firefly luciferase gene. Proliferation, motility (wound healing), invasiveness (Boyden chamber), and metalloproteinases (MMPs) secretion were studied in CCA cells, with or without lentiviral silencing of S100A4. Nuclear expression of S100A4 by neoplastic ducts was a strong predictor of metastasization and reduced survival after resection (P < 0.01). EGI-1 CCA cells showed stronger metastatic properties than TFK-1 when xenotransplanted in SCID mice. S100A4-silenced EGI-1 cells showed significantly reduced motility, invasiveness, and MMP-9 secretion in vitro, without changes in cell proliferation. CONCLUSION: Nuclear S100A4 identifies a subset of CCA patients with a poor prognosis after surgical resection. Nuclear expression of S100A4 increases CCA cells invasiveness and metastasization, indicating that S100A4 may also represent a potential therapeutic target.


Subject(s)
Bile Duct Neoplasms/pathology , Bile Ducts, Intrahepatic , Cell Nucleus/chemistry , Cholangiocarcinoma/pathology , S100 Proteins/physiology , Aged , Animals , Apoptosis , Bile Duct Neoplasms/chemistry , Bile Duct Neoplasms/surgery , Cell Movement , Cell Proliferation , Cholangiocarcinoma/chemistry , Cholangiocarcinoma/surgery , Female , Humans , Male , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mice , Middle Aged , Neoplasm Invasiveness , Neoplasm Metastasis , Prognosis , S100 Calcium-Binding Protein A4 , S100 Proteins/analysis
8.
J Pathol ; 224(4): 448-60, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21598247

ABSTRACT

Increased Notch1 activity has been observed in intestinal tumours, partially accomplished by ß-catenin-mediated up-regulation of the Notch ligand Jagged-1. Whether further mechanisms of Notch activation exist and other Notch receptors might be involved is unclear. Microarray data indicated that Notch3 transcript levels are significantly up-regulated in primary and metastatic CRC samples compared to normal mucosa. Moreover, Notch3 protein was expressed at strong/moderate levels by 19.7% of 158 CRC samples analysed, and at weak levels by 51.2% of the samples. Intrigued by these findings, we sought to investigate whether Notch3 modulates oncogenic features of CRC cells. By exploiting xenografts of CRC cells with different tumourigenic properties in mice, we found that the aggressive phenotype was associated with altered expression of components of the Notch pathway, including Notch3, Delta-like 4 (DLL4), and Jagged-1 ligands. Stimulation with immobilized recombinant DLL4 or transduction with DLL4-expressing vectors dramatically increased Notch3 expression in CRC cells, associated with accelerated tumour growth. Forced expression of an active form of Notch3 mirrored the effects of DLL4 stimulation and increased tumour formation. Conversely, attenuation of Notch3 levels by shRNA resulted in perturbation of the cell cycle followed by reduction in cell proliferation, clonogenic capacity, and inhibition of tumour growth. Altogether, these findings indicate that Notch3 can modulate the tumourigenic properties of CRC cells and contributes to sustained Notch activity in DLL4-expressing tumours.


Subject(s)
Colorectal Neoplasms/pathology , Receptors, Notch/physiology , Adaptor Proteins, Signal Transducing , Animals , Calcium-Binding Proteins/metabolism , Cell Transformation, Neoplastic/metabolism , Colorectal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic/physiology , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Jagged-1 Protein , Male , Membrane Proteins/metabolism , Mice , Mice, Nude , Mice, SCID , Neoplasm Proteins/metabolism , Neoplasm Proteins/physiology , Neoplasm Transplantation , Receptor, Notch3 , Receptors, Notch/metabolism , Serrate-Jagged Proteins , Signal Transduction/physiology , Transplantation, Heterologous , Tumor Cells, Cultured , Up-Regulation
9.
Cancer Res ; 71(12): 4214-25, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21546569

ABSTRACT

VEGF antagonists are now widely used cancer therapeutics, but predictive biomarkers of response or toxicity remain unavailable. In this study, we analyzed the effects of anti-VEGF therapy on tumor metabolism and therapeutic response by using an integrated set of imaging techniques, including bioluminescence metabolic imaging, 18-fluorodeoxyglucose positron emission tomography, and MRI imaging and spectroscopy. Our results revealed that anti-VEGF therapy caused a dramatic depletion of glucose and an exhaustion of ATP levels in tumors, although glucose uptake was maintained. These metabolic changes selectively accompanied the presence of large necrotic areas and partial tumor regression in highly glycolytic tumors. In addition, we found that the central metabolic protein kinase AMP-activated protein kinase (AMPK)-a cellular sensor of ATP levels that supports cell viability in response to energy stress-was activated by anti-VEGF therapy in experimental tumors. AMPK-α2 attenuation increased glucose consumption, tumor cell sensitivity to glucose starvation, and tumor necrosis following anti-VEGF therapy. Taken together, our findings reveal functional links between the Warburg effect and the AMPK pathway with therapeutic responses to VEGF neutralization in tumor xenograft models.


Subject(s)
Adenylate Kinase/physiology , Glycolysis , Neoplasms, Experimental/drug therapy , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Cell Line, Tumor , Fluorodeoxyglucose F18/pharmacokinetics , Humans , Magnetic Resonance Imaging , Magnetic Resonance Spectroscopy , Mice , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Phenotype
10.
Stem Cells Dev ; 20(4): 709-19, 2011 Apr.
Article in English | MEDLINE | ID: mdl-20695752

ABSTRACT

Bone marrow mesenchymal stromal cells (BM-MSCs) may survive and proliferate in the presence of cycling neoplastic cells. Exogenously administered MSCs are actively incorporated in the tumor as stromal fibroblasts, thus competing with the local mesenchymal cell precursors. For this reason, MSCs have been suggested as a suitable carrier for gene therapy strategies, as they can be genetically engineered with genes encoding for biologically active molecules that can inhibit tumor cell proliferation and enhance the antitumor immune response. We used BM-MSCs engineered with the murine interferon-alpha (IFN-α) gene (BM-MSCs/IFN-α) to assess in a mouse plasmacytoma model the efficacy of this approach toward neoplastic plasma cells. We found that IFN-α can be efficiently produced and delivered inside the tumor microenvironment. Subcutaneous multiple administration of BM-MSCs/IFN-α significantly hampered the tumor growth in vivo and prolonged the overall survival of mice. The antitumor effect was associated with enhanced apoptosis of tumor cells, reduction in microvessel density, and ischemic necrosis. By contrast, intravenous administration of BM-MSCs/IFN-α did not significantly modify the survival of mice, mainly as a consequence of an excessive entrapment of injected cells in the pulmonary vessels. In conclusion, BM-MSCs/IFN-α are effective in inhibiting neoplastic plasma cell growth; however, systemic administration of engineered MSCs needs to be improved to make this approach potentially suitable for the treatment of multiple myeloma.


Subject(s)
Interferon-alpha/metabolism , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Plasmacytoma/therapy , Animals , Apoptosis , Bone Marrow Cells/cytology , Cell Line, Tumor , Cell Survival , Coculture Techniques , Genetic Therapy , Interferon-alpha/genetics , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Neovascularization, Pathologic/therapy , Plasma Cells/pathology , Plasmacytoma/blood supply , Plasmacytoma/pathology , Recombinant Proteins/metabolism , Thy-1 Antigens/metabolism , Transplantation, Heterologous , Tumor Burden
11.
Cancer Lett ; 288(1): 1-9, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-19523754

ABSTRACT

New studies indicate that the side population (SP) and cancer stem cells (CSC) drive and maintain many types of human malignancies. SP and CSC appear to be highly resistant to chemo- and radio-therapy and this knowledge is now reshaping our therapeutic approach to cancer. Several studies have pioneered the possibility of specifically targeting CSC and SP cells by exploiting pathways involved in drug resistance, or forcing these cells to proliferate and differentiate thus converting them into a target of conventional therapies. Moreover, certain cytokines - such as IFN-alpha - appear to modulate SP and stem cell functions, and this associates with remarkable therapeutic activity in animal models. These recent findings underscore the need of a more comprehensive view of the interactions between cytokines and key regulatory pathways in SP and CSC.


Subject(s)
Antineoplastic Agents/therapeutic use , Immunotherapy , Neoplasms/therapy , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/radiation effects , Animals , Cell Differentiation , Cell Proliferation , Cytokines/metabolism , Cytokines/therapeutic use , Drug Resistance, Neoplasm , Humans , Neoplasms/immunology , Neoplasms/pathology , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/pathology , Radiation Tolerance , Signal Transduction
12.
Cancer Res ; 69(4): 1314-23, 2009 Feb 15.
Article in English | MEDLINE | ID: mdl-19208840

ABSTRACT

The Notch ligand Dll4 has a recognized role during both physiologic and tumor angiogenesis, as it contributes to regulate Notch activity in endothelial cells (EC). The effects of Dll4 on Notch signaling in tumor cells expressing Notch receptors remain, however, largely unknown. Here, we report that escape of human T-cell acute lymphoblastic leukemia (T-ALL) cells or colorectal cancer cells from dormancy is associated with Dll4 expression in the tumor microenvironment and increased Notch3 signaling in tumor cells. Dll4 was expressed at early time points during the angiogenic process, and its expression preceded perfusion of the newly established vessels. Treatment of EC with angiogenic factors induced Dll4 expression and increased Notch3 activation in cocultured T-ALL cells. Neutralization of Dll4 greatly reduced EC-mediated activation of Notch 3 signaling in T-ALL cells and blocked tumorigenesis. Moreover, silencing Notch3 by RNA interference had marked antiproliferative and proapoptotic effects on T-ALL cells in vitro and reduced tumorigenicity in vivo. Our results elucidate a novel mechanism by which a direct interplay between endothelial and tumor cells promotes survival and triggers tumor growth.


Subject(s)
Cell Communication/physiology , Colorectal Neoplasms/physiopathology , Endothelial Cells/physiology , Intercellular Signaling Peptides and Proteins/physiology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/physiopathology , Receptors, Notch/physiology , Adaptor Proteins, Signal Transducing , Animals , Calcium-Binding Proteins , Cell Line, Tumor , Cell Survival , Coculture Techniques , Colorectal Neoplasms/pathology , Female , Gene Expression Regulation, Neoplastic , Gene Silencing , Genes, Reporter , Humans , Intercellular Signaling Peptides and Proteins/genetics , Jurkat Cells , Mice , Mice, Inbred NOD , Mice, SCID , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , RNA, Messenger/genetics , RNA, Neoplasm/genetics , Receptor, Notch3 , Receptors, Notch/genetics , Reverse Transcriptase Polymerase Chain Reaction
13.
Carcinogenesis ; 30(5): 851-60, 2009 May.
Article in English | MEDLINE | ID: mdl-19237608

ABSTRACT

Interferon (IFN)-alpha is a cytokine with marked therapeutic activity in transplantable tumor models, that is in part due to angiogenesis inhibition. Aim of this study was to investigate the effects of IFN-alpha during the early phases of tumor development in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. To provide sustained IFN-alpha production, TRAMP mice were injected intraperitoneally with lentiviral vectors. IFN-alpha administration resulted in rapid and protracted upregulation of IFN-alpha-regulated genes associated with antiangiogenic and antiproliferative functions in the prostate of TRAMP mice, including guanylate-binding protein 1 (GBP-1), IFI204 and CXCL10-11. These transcriptional changes were accompanied by effects on the tumor vasculature, including significant reduction of intraductal microvessel density and increased pericyte coverage, and marked reduction of tumor cell proliferation, without induction of tumor necrosis. Intriguingly, GBP-1 and myxovirus resistance A, two IFN-regulated proteins, were found expressed in approximately 40% of human prostate cancer samples analyzed, suggesting expression of endogenous IFN-alpha. Overall, these findings demonstrate that IFN-alpha is able to counteract the angiogenic switch and impairs tumor cell proliferation in preinvasive lesions. Since the angiogenic switch also marks progression of human prostatic cancer, these results highlight the potential of angiogenesis inhibitors for the development of chemoprevention strategies in high-risk individuals.


Subject(s)
Interferon-alpha/physiology , Neovascularization, Pathologic/physiopathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Cell Division , GTP-Binding Proteins/genetics , Humans , Male , Myxovirus Resistance Proteins , Neovascularization, Pathologic/genetics , Prostatectomy , Prostatic Neoplasms/surgery , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic
14.
Am J Pathol ; 173(4): 1186-201, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18772337

ABSTRACT

Hypoxia and the acquisition of a glycolytic phenotype are intrinsic features of the tumor microenvironment. The hypoxia inducible factor-1alpha (HIF-1alpha) pathway is activated under hypoxic conditions and orchestrates a complex transcriptional program that enhances cell survival. Although the consequences of HIF-1alpha inactivation in cancer cells have been widely investigated, only a few studies have addressed the role of HIF-1alpha in the survival of cancer cells endowed with different glycolytic capacities. In this study, we investigated this aspect in ovarian cancer cells. Hypoxia-induced toxicity was increased in highly glycolytic cells compared with poorly glycolytic cells; it was also associated with a sharp decrease in intracellular ATP levels and was prevented by glucose supplementation. Stable HIF-1alpha silencing enhanced hypoxia-induced cell death in vitro due to a lack of cell cycle arrest. Tumors bearing attenuated HIF-1alpha levels had similar growth rates and vascularization as did controls, but tumors showed higher proliferation levels and increased necrosis. Moreover, tumors formed by HIF-1alpha deficient cells had higher levels of lactate and lower ATP concentrations than controls as shown by metabolic imaging. The findings that such metabolic properties can affect the survival of cancer cells under hypoxic conditions and that these properties contribute to the determination of the consequences of HIF-1alpha inactivation could have important implications on the understanding of the effects of anti-angiogenic and HIF-1alpha-targeting drugs in cancer.


Subject(s)
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Animals , Cell Death , Cell Hypoxia , Cell Line, Tumor , Cell Proliferation , Female , Gene Silencing , Humans , Lentivirus , Mice , Mice, SCID , Phenotype , RNA, Small Interfering/metabolism
15.
Cancer Res ; 68(14): 5658-68, 2008 Jul 15.
Article in English | MEDLINE | ID: mdl-18632618

ABSTRACT

The side population (SP), recently identified in several normal tissues and in a variety of tumors based on its ability to extrude some fluorescent dyes, may comprise cells endowed with stem cell features. In this study, we investigated the presence of SP in epithelial ovarian cancer and found it in 9 of 27 primary tumor samples analyzed, as well as in 4 of 6 cultures from xenotransplants. SP cells from one xenograft bearing a large SP fraction were characterized in detail. SP cells had higher proliferation rates, were much less apoptotic compared with non-SP cells, and generated tumors more rapidly than non-SP cells. We also investigated the effects of IFN-alpha, a cytokine that has widely been used to treat solid tumors, on epithelial ovarian cancer cells and observed that IFN-alpha exerted marked antiproliferative and proapoptotic effects on primary cultures containing high numbers of SP cells. In vitro, IFN-alpha treatment invariably caused a dramatic reduction in SP size in tumor cell lines of different origins; moreover, IFN-alpha treatment of purified SP cells was associated with a distinctive change in their transcriptional profile. Gene therapy with human IFN-alpha resulted in regression of established tumors bearing a large SP fraction, which was not observed when tumors bearing low SP levels were treated. These findings could have relevant clinical implications because they imply that tumors bearing large SP numbers, albeit rare, could be sensitive to IFN-alpha treatment.


Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation, Neoplastic , Interferon-alpha/metabolism , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Animals , Apoptosis , Cell Line, Tumor , Cell Proliferation , Female , Humans , Mice , Mice, SCID , Neoplasm Transplantation , Stem Cells/cytology , Transcription, Genetic
16.
Mol Aspects Med ; 28(1): 59-86, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17306360

ABSTRACT

Delivery of cytokine genes at the tumor site in pre-clinical models has been shown to recruit host inflammatory cells followed by inhibition of tumor growth. This local effect is often accompanied by systemic protection mediated by the immune system, mainly by CD8(+) T and NK cells. On this basis, cytokine gene-transduced tumor cells have widely been used as vaccines in clinical trials, which have shown good safety profiles and some local responses but substantial lack of systemic efficacy. Are these findings the end of the story? Possibly not, if major improvements will be attained in the coming years. These should be directed at the level of gene selection and delivery, in order to identify the optimal cytokine and achieve efficient and durable cytokine expression, and at the level of improving immune stimulation, i.e. by co-administration of co-stimulatory molecules including B7 and CD40, or boosting the expression of tumor antigens or MHC class I molecules. Interestingly, some of the cytokines which have shown encouraging anti-tumor activity, including IFNs, IL-4, IL-12 and TNF-alpha, are endowed with anti-angiogenic or vasculotoxic effects, which may significantly contribute to local tumor control. Therapeutic exploitation of this property may result in the design of novel approaches which, by maximizing immune-stimulating and anti-angiogenic effects, could possibly lead to starvation of established tumors in patients.


Subject(s)
Allergy and Immunology , Cytokines/genetics , Genetic Therapy , Neoplasms/immunology , Neoplasms/therapy , Neovascularization, Pathologic/therapy , Cytokines/metabolism , Humans , Neoplasms/genetics , Neoplasms/metabolism , Neovascularization, Pathologic/metabolism
17.
Eur J Cancer ; 42(10): 1475-83, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16759848

ABSTRACT

The BRCA1 gene is responsible for a high number of hereditary breast and ovarian cancers that cluster in families with a strong genetic predisposition. Despite intense investigation, the accumulating findings on BRCA1 biological functions have not yet been translated into specific therapeutic approaches, also due to the lack of suitable experimental models. The purpose of this study was to establish and characterize cell cultures and xenografts from patients with BRCA1 -/- ovarian cancers. We derived two ovarian cancer cell lines, termed PD-OVCA1 and PD-OVCA2, both from patients previously treated with chemotherapy, that propagate in SCID mice as well as in vitro for a limited number of passages. Both cell lines expressed cytokeratins and the CA125 tumour marker. A detailed molecular characterization highlighted both constitutive and somatic genetic events that abrogate BRCA1 gene function. Both cell lines were shown to lose the wild type BRCA1 allele; intriguingly, these deletions were apparently accompanied by gain of one or more copies of the mutant alleles. Finally, a genomic profile of major chromosomal aberrations was obtained by the Multiplex Ligation-dependent Probe Amplification (MLPA) technique, which disclosed chromosomal imbalances targeting specific genes in each cell line. The PD-OVCA1 and PD-OVCA2 ovarian cancer cell lines will provide a valuable tool for new experimental models for the study of BRCA1-associated tumour biology.


Subject(s)
Genes, BRCA1 , Ovarian Neoplasms/pathology , Tumor Cells, Cultured/pathology , Aged , Animals , CA-125 Antigen/metabolism , Cell Culture Techniques/methods , Female , Genome , Genotype , Humans , Mice , Ovarian Neoplasms/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transplantation, Heterologous/methods , Tumor Cells, Cultured/metabolism
18.
Hum Gene Ther ; 16(8): 957-70, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16076254

ABSTRACT

Ovarian cancer represents a suitable disease for gene therapy because of the containment of neoplastic cells in the peritoneal cavity even at advanced tumor stages. The aim of this study was to investigate whether intraperitoneal administration of a lentiviral vector encoding murine interferon-alpha (LV-IFN) could have therapeutic activity in a transplantable ovarian cancer model. Multiple injections of low amounts of LV-IFN into severe combined immunodeficiency (SCID) mice bearing IGROV-1 or OC316 ovarian cancer cells elicited remarkable antitumor activity, leading to prolongation of survival in the majority of animals. A definitive cure was obtained in animals bearing PD-OVA#1 tumors, generated by injecting tumor cells isolated from the ascitic fluid of a patient into SCID mice. Interferon-alpha levels were detected in the peritoneal fluids but not in the serum of treated mice, indicating that production of the cytokine is mainly local, by both tumor and normal cells of the host. Antitumor effects were associated with a remarkable decrease in the formation of hemorrhagic ascites, an increase in ischemic tumor necrosis, and a reduction in microvessel density. In conclusion, our findings show that intracavitary IFN-alpha gene therapy, using a lentiviral vector, provides strong antitumor effects in murine models of ovarian cancer and reinforces the evidence that angiogenesis inhibition is a promising strategy for the treatment of localized tumors.


Subject(s)
Antineoplastic Agents/therapeutic use , Gene Transfer Techniques , Genetic Therapy/methods , Interferon-alpha/genetics , Interferon-alpha/therapeutic use , Neovascularization, Pathologic , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Disease Models, Animal , Female , Genetic Vectors , Humans , Infusions, Parenteral , Interferon-alpha/administration & dosage , Interferon-alpha/pharmacokinetics , Lentivirus/genetics , Mice , Mice, SCID , Ovarian Neoplasms/veterinary , Survival
19.
J Clin Invest ; 113(11): 1651-9, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15173892

ABSTRACT

The IL-12Rbeta2 gene is expressed in human mature B cell subsets but not in transformed B cell lines. Silencing of this gene may be advantageous to neoplastic B cells. Our objective was to investigate the mechanism(s) and the functional consequence(s) of IL-12Rbeta2 gene silencing in primary B cell tumors and transformed B cell lines. Purified tumor cells from 41 patients with different chronic B cell lymphoproliferative disorders, representing the counterparts of the major mature human B cell subsets, tested negative for IL-12Rbeta2 gene expression. Hypermethylation of a CpG island in the noncoding exon 1 was associated with silencing of this gene in malignant B cells. Treatment with the DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine restored IL-12Rbeta2 mRNA expression in primary neoplastic B cells that underwent apoptosis following exposure to human recombinant IL-12 (hrIL-12). hrIL-12 inhibited proliferation and increased the apoptotic rate of IL-12Rbeta2-transfected B cell lines in vitro. Finally, hrIL-12 strongly reduced the tumorigenicity of IL-12Rbeta2-transfected Burkitt lymphoma RAJI cells in SCID-NOD mice through antiproliferative and proapoptotic effects, coupled with neoangiogenesis inhibition related to human IFN-gamma-independent induction of hMig/CXCL9. The IL-12Rbeta2 gene acts as tumor suppressor in chronic B cell malignancies, and IL-12 exerts direct antitumor effects on IL-12Rbeta2-expressing neoplastic B cells.


Subject(s)
Azacitidine/analogs & derivatives , Genes, Tumor Suppressor/physiology , Leukemia, B-Cell/genetics , Receptors, Interleukin/genetics , Animals , Azacitidine/pharmacology , B-Lymphocytes/metabolism , Chronic Disease , DNA Modification Methylases/antagonists & inhibitors , Decitabine , Humans , Interleukin-12/metabolism , Leukemia, B-Cell/metabolism , Lymphoproliferative Disorders/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Palatine Tonsil/cytology , Palatine Tonsil/metabolism , Receptors, Interleukin/metabolism , Receptors, Interleukin-12
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