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1.
Osteoporos Int ; 29(4): 993-997, 2018 04.
Article in English | MEDLINE | ID: mdl-29380000

ABSTRACT

Tumor-induced osteomalacia (TIO) is a rare paraneoplastic condition in which phosphaturic mesenchymal tumors (PMTs) secrete high levels of fibroblast growth factor 23 (FGF23) into the circulation. This results in renal phosphate wasting, hypophosphatemia, muscle weakness, bone pain, and pathological fractures. Recent studies suggest that fibronectin-fibroblast growth factor receptor 1 (FN1-FGFR1) translocations may be a driver of tumorigenesis. We present a patient with TIO who also exhibited clinical findings suggestive of Cowden syndrome (CS), a rare autosomal dominant disorder characterized by numerous benign hamartomas, as well as an increased risk for multiple malignancies, such as thyroid cancer. While CS is a clinical diagnosis, most, but not all, harbor a mutation in the tumor suppressor gene PTEN. Genetic testing revealed a somatic FN1-FGFR1 translocation in the FGF23-producing tumor causing TIO; however, a germline PTEN mutation was not identified. To our knowledge, this is the first reported case of concurrent TIO and CS.


Subject(s)
Hamartoma Syndrome, Multiple/complications , Neoplasms, Connective Tissue/etiology , Paraneoplastic Syndromes/etiology , Fibroblast Growth Factor-23 , Fibroblast Growth Factors/biosynthesis , Hamartoma Syndrome, Multiple/pathology , Hamartoma Syndrome, Multiple/surgery , Humans , Male , Middle Aged , Mutation , Neoplasms, Connective Tissue/metabolism , Osteomalacia , PTEN Phosphohydrolase/genetics
2.
Oncogene ; 35(29): 3771-80, 2016 07 21.
Article in English | MEDLINE | ID: mdl-26616858

ABSTRACT

G proteins and their cognate G protein-coupled receptors (GPCRs) function as critical signal transduction molecules that regulate cell survival, proliferation, motility and differentiation. The aberrant expression and/or function of these molecules have been linked to the growth, progression and metastasis of various cancers. As such, the analysis of mutations in the genes encoding GPCRs, G proteins and their downstream targets provides important clues regarding how these signaling cascades contribute to malignancy. Recent genome-wide sequencing efforts have unveiled the presence of frequent mutations in GNA13, the gene encoding the G protein Gα13, in Burkitt's lymphoma and diffuse large B-cell lymphoma (DLBCL). We found that mutations in the downstream target of Gα13, RhoA, are also present in Burkitt's lymphoma and DLBCL. By multiple complementary approaches, we now show that that these cancer-specific GNA13 and RHOA mutations are inhibitory in nature, and that the expression of wild-type Gα13 in B-cell lymphoma cells with mutant GNA13 has limited impact in vitro but results in a remarkable growth inhibition in vivo. Thus, although Gα13 and RhoA activity has previously been linked to cellular transformation and metastatic potential of epithelial cancers, our findings support a tumor suppressive role for Gα13 and RhoA in Burkitt's lymphoma and DLBCL.


Subject(s)
Burkitt Lymphoma/genetics , GTP-Binding Protein alpha Subunits, G12-G13/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Mutation , rhoA GTP-Binding Protein/genetics , Animals , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Blotting, Western , Burkitt Lymphoma/pathology , Cell Line, Tumor , DNA Mutational Analysis , Dogs , GTP-Binding Protein alpha Subunits, G12-G13/metabolism , HEK293 Cells , Humans , Lymphoma, Large B-Cell, Diffuse/pathology , Madin Darby Canine Kidney Cells , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Microscopy, Confocal , Signal Transduction/genetics , Transplantation, Heterologous , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , rhoA GTP-Binding Protein/metabolism
3.
Oncogene ; 33(18): 2405-12, 2014 May 01.
Article in English | MEDLINE | ID: mdl-23708663

ABSTRACT

Dysregulation of the PI3K/Akt/mTOR pathway is one of the most frequent events in human cancer. However, the clinical benefits of PI3K/Akt/mTOR inhibitors have not yet achieved their predicted potential in many of the most prevalent human cancers. Of interest, treatment of Kaposi's sarcoma (KS) patients with rapamycin provided the first evidence of the antineoplastic activity of mTOR inhibitors in humans, becoming the standard of care for KS arising in renal transplant patients. Thus, the study of KS may provide a unique opportunity to dissect the contribution of specific mTOR downstream targets to cancer development. The KS-associated herpesvirus (KSHV) is the etiological agent for KS, and the KSHV-encoded oncogene viral-G protein-coupled receptor (vGPCR) promotes the potent activation of the PI3K-Akt-mTOR pathway by both direct and paracrine mechanisms. We focused on a direct target of mTOR, EIF4EBP1/2/3 (4EBP), which inhibits the translation of eukaryotic initiation factor 4E (eiF4E)-bound mRNAs. 4EBP phosphorylation by mTOR relieves its inhibitory activity, hence resulting in increased eiF4E-dependent mRNA translation. We developed a paracrine transformation model, recapitulating the cellular composition of KS lesions, in which vGPCR-expressing cells promote the rapid proliferation of endothelial cells, thus expressing KSHV-latent genes by the release of growth factors. Using this model, we show here that the accumulation of dephosphorylated 4EBP in response to rapamycin or by the expression of an mTOR-insensitive mutant of 4EBP1 is sufficient to disrupt the eiF4E function downstream of mTOR to a similar extent than the mTOR catalytic inhibitor Torin2 and to halt KS development. We also provide evidence that eiF4E contributes to paracrine neoplastic, signaling through the release of pro-angiogenic factors that are acting on endothelial cells, expressing KSHV-latent genes. These findings may provide a preclinical platform and the rationale for the development of novel mTOR, inhibiting agents that may selectively disrupt the mTOR-4EBP interaction for the treatment of KS and other tumor lesions, exhibiting hyperactive mTOR pathway function.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/metabolism , Cell Transformation, Viral , Eukaryotic Initiation Factors/metabolism , Herpesvirus 8, Human , Phosphoproteins/metabolism , Receptors, Chemokine/metabolism , Sarcoma, Kaposi/pathology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Antibiotics, Antineoplastic/pharmacology , Cell Cycle Proteins , Humans , Oncogenes , Paracrine Communication , Receptors, Chemokine/genetics , Sarcoma, Kaposi/genetics , Sarcoma, Kaposi/virology , Sirolimus/pharmacology
4.
Oncogene ; 31(28): 3322-32, 2012 Jul 12.
Article in English | MEDLINE | ID: mdl-22037217

ABSTRACT

The molecular mechanisms that contribute to the initiation and progression of head and neck squamous cell carcinoma (HNSCC) have not been completely delineated. Our observations indicate that defects in the transforming growth factor-ß and PI3K/Akt signaling pathways are common in human HNSCCs. Conditional activation of the PI3K/Akt pathway due to Pten deletion in the mouse head and neck epithelia gives rise to hyperproliferation, but only a few lesions progress to HNSCC. However, Pten-deficient mice developed full-penetrance HNSCC in combination with type I TGF-ß receptor (Tgfbr1) deletion. Molecular analysis revealed enhanced cell proliferation, decreased apoptosis, and increased expression of CCND1 in the basal layer of the head and neck epithelia, as well as in the tumors of Tgfbr1/Pten double conditional knockout (2cKO) mice. Furthermore, neoplastic transformation involves senescence evasion, and is associated with an increased number of putative cancer stem cells. In addition, the nuclear factor-κB pathway activation, myeloid-derived suppressor cell infiltration, angiogenesis and immune suppression in the tumor microenvironment, all of which are characteristics of human HNSCCs, contribute significantly to head and neck carcinogenesis in 2cKO mice. These tumors display pathology and multiple molecular alterations resembling human HNSCCs. This suggests that the Tgfbr1/Pten 2cKO mouse model is suitable for preclinical intervention, and that it has significant implications in the development of diagnostic cancer biomarkers and effective strategies for prevention and treatment of HNSCCs.


Subject(s)
Carcinoma, Squamous Cell/pathology , Cellular Senescence , Gene Knockout Techniques , Head and Neck Neoplasms/pathology , PTEN Phosphohydrolase/metabolism , Signal Transduction , Transforming Growth Factor beta1/metabolism , Animals , Apoptosis/genetics , Carcinoma, Squamous Cell/complications , Carcinoma, Squamous Cell/genetics , Cell Count , Cell Line, Tumor , Cell Proliferation , Cellular Senescence/genetics , Epithelial Cells/metabolism , Epithelial Cells/pathology , Head and Neck Neoplasms/complications , Head and Neck Neoplasms/genetics , Humans , Inflammation/complications , Mice , Myeloid Cells/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , PTEN Phosphohydrolase/deficiency , PTEN Phosphohydrolase/genetics , Penetrance , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/genetics , Transforming Growth Factor beta1/genetics
5.
Oncogene ; 31(32): 3679-95, 2012 Aug 09.
Article in English | MEDLINE | ID: mdl-22139080

ABSTRACT

Colitis-associated colorectal cancers are an etiologically distinct subgroup of colon cancers that occur in individuals suffering from inflammatory bowel disease and arise as a consequence of persistent exposure of hyperproliferative epithelial stem cells to an inflammatory microenvironment. An intrinsic defect in the intestinal epithelial barrier has been proposed to be one of several factors that contribute to the inappropriate immune response to the commensal microbiota that underlies inflammatory bowel disease. Matriptase is a membrane-anchored serine protease encoded by Suppression of Tumorigenicity-14 (ST14) that strengthens the intestinal epithelial barrier by promoting tight junction formation. Here, we show that intestinal epithelial-specific ablation of St14 in mice causes formation of colon adenocarcinoma with very early onset and high penetrance. Neoplastic progression is preceded by a chronic inflammation of the colon that resembles human inflammatory bowel disease and is promoted by the commensal microbiota. This study demonstrates that inflammation-associated colon carcinogenesis can be initiated and promoted solely by an intrinsic intestinal permeability barrier perturbation, establishes St14 as a critical tumor-suppressor gene in the mouse gastrointestinal tract and adds matriptase to the expanding list of pericellular proteases with tumor-suppressive functions.


Subject(s)
Adenocarcinoma/enzymology , Colitis/enzymology , Colonic Neoplasms/enzymology , Genes, Tumor Suppressor , Serine Endopeptidases/genetics , Adenocarcinoma/pathology , Adenoma/enzymology , Animals , Anti-Bacterial Agents/pharmacology , Basement Membrane/enzymology , Basement Membrane/metabolism , Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Colitis/microbiology , Colitis/pathology , Colon/pathology , Colonic Neoplasms/pathology , Epithelium/enzymology , Epithelium/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Inflammatory Bowel Diseases/enzymology , Inflammatory Bowel Diseases/microbiology , Inflammatory Bowel Diseases/pathology , Intestinal Absorption , Membrane Proteins , Metagenome/drug effects , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Neoplasm Invasiveness , Precancerous Conditions , Serine Endopeptidases/metabolism , Signal Transduction , beta Catenin/metabolism
6.
Oncogene ; 30(17): 2003-16, 2011 Apr 28.
Article in English | MEDLINE | ID: mdl-21217780

ABSTRACT

The progression and negative outcome of a variety of human carcinomas are intimately associated with aberrant activity of the c-Met oncogene. The underlying cause of this dysregulation, however, remains a subject of discussion, as the majority of cancer patients do not present with activating mutations in c-Met receptor itself. In this study, we show that the oncogenic protease matriptase is ubiquitously co-expressed with the c-Met in human squamous cell carcinomas and amplifies migratory and proliferative responses of primary epithelial cells to the cognate ligand for c-Met, pro-hepatocyte growth factor/scatter factor (proHGF/SF), through c-Met and Gab1 signaling. Furthermore, the selective genetic ablation of c-Met from matriptase-expressing keratinocytes completely negates the oncogenic potential of matriptase. In addition, matriptase-dependent carcinoma formation could be blocked by the pharmacological inhibition of the Akt-mammalian target of Rapamycin (mTor) pathway. Our data identify matriptase as an initiator of c-Met-Akt-mTor-dependent signaling axis in tumors and reveal mTor activation as an essential component of matriptase/c-Met-induced carcinogenesis. The study provides a specific example of how epithelial transformation can be promoted by epigenetic acquisition of the capacity to convert a widely available paracrine growth factor precursor to its signaling competent state.


Subject(s)
Carcinoma, Squamous Cell/pathology , Cell Transformation, Neoplastic , Epithelial Cells/enzymology , Epithelial Cells/pathology , Head and Neck Neoplasms/pathology , Proto-Oncogene Proteins c-met/metabolism , Serine Endopeptidases/metabolism , Animals , Carcinoma, Squamous Cell/enzymology , Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/enzymology , Head and Neck Neoplasms/genetics , Hepatocyte Growth Factor/metabolism , Humans , Keratinocytes/enzymology , Keratinocytes/metabolism , Keratinocytes/pathology , Mice , Protein Precursors/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-met/deficiency , Proto-Oncogene Proteins c-met/genetics , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Up-Regulation
7.
Oncogene ; 26(35): 5078-85, 2007 Aug 02.
Article in English | MEDLINE | ID: mdl-17334398

ABSTRACT

Epithelial stem cells in the bulge region within the hair follicle maintain the cyclic hair growth, but whether these stem cells also contribute to the epidermal renewal remains unclear. Here, we observed that the conditional deletion of the Rac1 gene in the mouse skin, including the potential follicular and epidermal stem cell compartments, results in alopecia owing to defective hair development. Surprisingly, mice lacking the expression of this Rho GTPase do not display major alterations in the interfollicular skin. Furthermore, Rac1 excision from primary epithelial keratinocytes results in the inability to reconstitute hair follicles and sebaceous glands when grafted onto mice, but epithelial cells lacking Rac1 can nonetheless form a healthy epidermis. Together, these findings support the emerging view that the epidermis and the hair follicles are maintained by different epithelial stem cells, and provide evidence that the requirement for Rac1 function can distinguish these distinct stem cells populations.


Subject(s)
Epidermis/physiology , Hair Follicle/cytology , Keratinocytes/physiology , Neuropeptides/physiology , Regeneration , Stem Cells/physiology , rac GTP-Binding Proteins/physiology , Animals , Cell Movement/genetics , Epidermal Cells , Epidermis/enzymology , Epithelial Cells/enzymology , Epithelial Cells/physiology , Gene Deletion , Hair Follicle/abnormalities , Hair Follicle/growth & development , Keratinocytes/enzymology , Mice , Mice, Mutant Strains , Neuropeptides/antagonists & inhibitors , Neuropeptides/genetics , Regeneration/genetics , Stem Cells/enzymology , rac GTP-Binding Proteins/antagonists & inhibitors , rac GTP-Binding Proteins/genetics , rac1 GTP-Binding Protein
8.
Oncogene ; 26(11): 1546-56, 2007 Mar 08.
Article in English | MEDLINE | ID: mdl-16983341

ABSTRACT

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor that forms inhibitor complexes with several trypsin-like serine proteases and is required for mouse placental development and embryo survival. Here we show that the essential function of HAI-1 in placentation and all other embryonic processes is to restrict the activity of the type II transmembrane serine protease, matriptase. Enzymatic gene trapping of matriptase combined with HAI-1 immunohistochemistry revealed that matriptase is co-expressed with HAI-1 in both extraembryonic and embryonic tissues. As early as embryonic day 8.5, matriptase and HAI-1 were expressed in a population of chorionic trophoblasts. Ablation of HAI-1 disrupted the epithelial integrity of this cell population, causing disorganized laminin deposition and altered expression of E-cadherin and beta-catenin. This led to a complete loss of undifferentiated chorionic trophoblasts after embryonic day 9.5 and prevented the formation of the placental labyrinth. Genetic ablation of matriptase activity in HAI-1-deficient embryos, however, restored the integrity of chorionic trophoblasts and enabled placental labyrinth formation and development to term. Furthermore, matriptase/HAI-1 double-deficient mice were phenotypically indistinguishable from matriptase single-deficient littermates.


Subject(s)
Membrane Glycoproteins/physiology , Placentation , Serine Endopeptidases/drug effects , Animals , Base Sequence , DNA Primers , Female , Humans , Immunohistochemistry , Mice , Proteinase Inhibitory Proteins, Secretory
9.
J Oral Pathol Med ; 35(4): 212-7, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16519768

ABSTRACT

BACKGROUND: The fibroblastic growth factor (FGF)-2 has been shown to induce angiogenesis in several tumor types. To date, the activity of FGF during the development of oral pre-cancerous lesions has not been analyzed. We herein evaluated the role of FGF-2 in the pre-cancerous and cancerous lesions in the hamster cheek pouch oral cancer model. METHODS: Expression of FGF-2 and its receptors FGFR-2 and FGFR-3 was assessed by immunohistochemistry at different stages of the carcinogenesis protocol. Activity of FGF-2 isoforms was analyzed by Western blots. RESULTS: Increase and abnormal localization of FGF-2 expression was evident in cancerized epithelium before it was possible to detect morphologic alterations. The changes in FGF-2 are concomitant with the evolution of subepithelial fibrosis. Immunolabeling of carcinomas was faint or completely negative. Increases of FGF-2 activity are mainly due to the increase in the 18 kDa isoform. Receptors 2 and 3 of FGF are present in epithelium, fibroblasts, and vascular endothelia of control samples and in all stages of malignant transformation. CONCLUSIONS: Our results would suggest a role for FGF-2 in the epithelium-connective interactions and a deregulation of its expression in the early stages of oral cancerization. In pre-cancerous tissue FGF-2 would play a central role in the development of fibrosis and a more collateral role in the induction of angiogenesis. The data would indicate its involvement in the process via the 18 kDa isoform.


Subject(s)
Fibroblast Growth Factor 2/genetics , Fibrosis/genetics , Mouth Neoplasms/genetics , Precancerous Conditions/genetics , Animals , Blotting, Western , Cheek , Cricetinae , Fibroblast Growth Factor 2/biosynthesis , Fibrosis/metabolism , Gene Expression , Immunohistochemistry , Mesocricetus , Mouth Mucosa/metabolism , Mouth Mucosa/pathology , Mouth Neoplasms/blood supply , Mouth Neoplasms/metabolism , Neovascularization, Pathologic/genetics , Precancerous Conditions/blood supply , Precancerous Conditions/metabolism , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Receptor, Fibroblast Growth Factor, Type 2/biosynthesis , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Fibroblast Growth Factor, Type 3/biosynthesis , Receptor, Fibroblast Growth Factor, Type 3/genetics
10.
Immunology ; 104(1): 80-6, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11576224

ABSTRACT

In the present study we examine the effects of medroxyprogesterone acetate (MPA) on the specific antibody secretion to T-dependent antigens. Our results show that the in vivo administration of MPA to mice, 7 or 90 days before immunization with sheep red blood cells (SRBC), significantly enhanced both, primary and secondary antibody responses, without affecting delayed-type hypersensitivity (DTH). These effects could be counteracted by the anti-progestin onapristone or ZK 98299 (ZK) suggesting that MPA interacted with progesterone (PRG) receptors to increase B-cell response. To better understand the mechanisms involved in MPA activity we carried out cultures of splenocytes, bone marrow cells or lymph node cells from immunized mice in the presence of MPA, and evaluated the amount of antibody release to supernatants. We found that low doses of MPA (10(-9) M and 10(-10) M) significantly enhanced the in vitro production of specific immunoglobulin G (IgG) antibodies, an effect that appears to involve the interaction of the progestin with PRG receptors, as judged by the inhibition of MPA effects with ZK (10(-8) M) or RU486 (10(-9) M). These receptors were detected by flow cytometry analysis in a proportion of T lymphocytes. Because MPA did not increase the number of immunoglobulin-secreting cells, our findings suggest that MPA enhanced the capacity of individual cells to produce specific immunoglobulin.


Subject(s)
Immunoglobulins/biosynthesis , Medroxyprogesterone Acetate/immunology , Progesterone Congeners/immunology , Animals , Cell Culture Techniques , Dose-Response Relationship, Immunologic , Erythrocytes/immunology , Female , Hypersensitivity, Delayed/immunology , Immunologic Memory , Lymph Nodes/immunology , Mice , Mice, Inbred BALB C , Ovalbumin/immunology , Receptors, Progesterone/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
11.
Cancer Res ; 61(1): 293-302, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11196177

ABSTRACT

We have developed an experimental model of mammary carcinogenesis in which the administration of medroxyprogesterone acetate (MPA) to female BALB/c mice induces progestin-dependent ductal metastatic mammary tumors with high levels of estrogen receptor (ER) and progesterone receptor (PR). Through selective transplants in untreated mice, we have obtained progestin-independent variants, still expressing high levels of ER and PR. Primary cultures of the MPA-induced carcinomas C4-HD and C7-HI were set up, and after 3-4 months, several different cell lines were obtained. Four of these, MC4-L1, MC4-L2, MC4-L3, and MC4-L5 were established from C4-HD and a fifth, MC7-L1, from C7-HI. All cells were of epithelial origin, as demonstrated by electron microscopy and by immunocytochemical identification of cytokeratin and cadherin. In vitro MC4-L1, MC4-L3, and MC4-L5 showed a typical epithelial morphology; when transplanted in vivo, they originated metastatic carcinomas with different degrees of differentiation. MC4-L2 and MC7-L1 deviated from the standard epithelial picture; they disclosed a spindle-shaped morphology in vitro and in vivo gave rise to a biphasic spindle cell/tubular carcinoma and an anaplastic carcinoma, respectively; both lines gave rise to metastases. This differential morphology correlated with a higher degree of aggressiveness, as compared with MC4-L1, MC4-L3, and MC4-L5. ERs and PRs were detected by binding, immunocytochemistry, and Western blot. In vitro, MC4-L2 and MC7-L1 were stimulated by MPA (nM to microM) and 17beta-estradiol (nM and 10 nM); no significant stimulation was observed in MC4-L1, MC4-L3, and MC4-L5 under the same experimental conditions. In vivo, MPA significantly stimulated tumor growth in all epithelioid lines but not in MC4-L2 and MC7-L1. A progestin-dependent growth pattern was confirmed for MC4-L1, MC4-L3, and MC4-L5 in successive transplants, whereas MC4-L2 and MC7-L1 behaved as progestin independent. This is the first description of mouse mammary carcinoma cell lines expressing ER and PR. The different in vitro hormone responses as compared with in vivo and the differential effects of 17beta-estradiol in the parental tumors and in cell lines render these lines useful tools for the in vitro and in vivo study of hormone regulation of tumor growth and metastases.


Subject(s)
Carcinoma, Ductal, Breast/pathology , Mammary Neoplasms, Experimental/pathology , Neoplasms, Hormone-Dependent/pathology , Tumor Cells, Cultured , Animals , Carcinoma, Ductal, Breast/metabolism , Cell Differentiation/physiology , Cell Division/drug effects , Cell Division/physiology , Disease Models, Animal , Estradiol/pharmacology , Female , Immunohistochemistry , Mammary Neoplasms, Experimental/metabolism , Medroxyprogesterone Acetate/pharmacology , Mice , Mice, Inbred BALB C , Microscopy, Electron , Neoplasm Transplantation , Neoplasms, Hormone-Dependent/metabolism , Receptor, ErbB-2/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism
12.
Leuk Res ; 25(2): 165-7, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11166832

ABSTRACT

In a previous paper we reported the occurrence of a high incidence of lymphomas in N-methyl-N-nitrosourea (MNU)-treated mice, in the course of an experiment of combined chemical-hormonal carcinogenesis in mammary gland, in which we used medroxyprogesterone acetate (MPA) and MNU in different treatment protocols. In this report we have analyzed the action of MPA in the leukemogenic effects of MNU, by specifically selecting for the analysis experimental groups in which only few mammary carcinomas had developed. A high incidence of lymphomas (65%, median latency: 176 days) was registered in MNU-treated mice, and the administration of MPA was associated with a significant reduction in the incidence of lymphomas (P<0.001) in all protocols.


Subject(s)
Lymphoma/prevention & control , Medroxyprogesterone Acetate/pharmacology , Methylnitrosourea/toxicity , Animals , Female , Immunohistochemistry , Incidence , Lymphoma/chemically induced , Mice , Mice, Inbred BALB C
13.
Breast Cancer Res Treat ; 54(2): 93-9, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10424399

ABSTRACT

From mouse mammary progestin-dependent (PD) adenocarcinomas induced with medroxyprogesterone acetate (MPA) we developed several in vivo lines that are maintained by subcutaneous syngeneic passages in MPA-treated mice and express estrogen (ER) and progesterone receptors (PR). Although most lines remained PD, with time some progestin-independent (PI) variants arose. Both PD and PI tumors regress with estrogen treatment although estrogen-resistant variants may also arise. The object of this paper was to investigate the reversibility of estrogen-resistance and its possible relation with hormone receptor down-regulation. Tumor regression was induced in a progestin-independent tumor line (BET) by treatment with a 5 mg 17beta-estradiol (E2) silastic pellet. One of the tumors started to grow disclosing an estrogen-resistant pattern of growth. This tumor line (BET-R) was transplanted into E2-treated and untreated animals (n = 4-6), selecting for the next passage tumors growing in treated animals. Seven new sublines were obtained at different passages by selecting for the next passage the tumors that had grown in untreated mice (BET-Ra-BET-Rg), until no tumors grew in E2-treated mice. ER and PR were measured by a ligand-binding, dextran-coated charcoal method using a single saturating point. From the seven sublines initiated, the first four proved to be reversible after 3-6 generation transplantation and the last three did not revert. A difference in PR expression between BET and BET-R (p < 0.05) was registered, but it did not correlate with the specific hormone behavior since two reverted lines had a pattern similar to that of BET and the other two were similar to BET-R. The expression of PR was higher in E2-treated mice (p < 0.05) and highly variable in the parental line. This led us to study the expression of PR at different stages of the estrous cycle. Higher levels of PR were observed in proestrous, estrous, and metestrous (p < 0.05) than in diestrous, and undetectable levels were found in ovariectomized mice. No differences in ER expression were detected during the estrous cycle. It can be concluded that under certain experimental conditions, estrogen-resistance is a reversible phenomenon. The experimental manipulation of hormone resistance may help develop strategies to modify the response to anti-hormones in humans.


Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Agents, Hormonal/therapeutic use , Estradiol/therapeutic use , Mammary Neoplasms, Experimental/drug therapy , Adenocarcinoma/chemically induced , Adenocarcinoma/pathology , Animals , Cell Division , Drug Implants , Drug Resistance, Neoplasm , Female , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/pathology , Medroxyprogesterone Acetate , Mice , Mice, Inbred BALB C , Progestins/toxicity , Receptors, Estrogen/drug effects , Receptors, Estrogen/metabolism , Receptors, Progesterone/drug effects , Receptors, Progesterone/metabolism
14.
J Steroid Biochem Mol Biol ; 68(1-2): 11-21, 1999 Jan.
Article in English | MEDLINE | ID: mdl-10215033

ABSTRACT

We have developed a model of hormonal carcinogenesis in BALB/c female mice, in which MPA induced ductal mammary adenocarcinomas, expressing high levels of estrogen and progesterone receptors (ER and PR). A series of tumor lines, retaining both PR and ER expression, were obtained from selected tumors, which are maintained by syngeneic passages. In this model progesterone behaves as the growth-stimulating hormone (progesterone-dependent or PD tumors), whereas estrogens induce tumor regression. Through selective treatments we were able to derive a series of progesterone-independent (PI) variants. These lines do not require progesterone treatment to grow in ovariectomized female BALB/c mice, but retain, however, the expression of ER and PR. The aim of this paper is to investigate a possible regulatory role of the progesterone receptor (PR) on PI tumor growth. ER and PR were detected by immunocytochemistry in all lines studied. They were also characterized using biochemical assays and Scatchard plots. No differences in Kd of PR or ER were detected in PI variants. AR or GR were not detected in tumor samples using biochemical assays. Estradiol (5 mg silastic pellet) induced complete tumor regression in all tumors tested. We also evaluated the effects of different antiprogestins on tumor growth. Onapristone (10 mg/kg/day) and mifepristone (4.5 mg/kg/day) were able to induce complete tumor regression. The antiandrogen flutamide (5 mg silastic pellet) had no effect on tumor growth in agreement with the lack of androgen receptors. We used an in vitro approach to corroborate that the antiprogestin-induced inhibition was not attributable to an intrinsic effect. Cultures of a selected PI line were treated with PR antisense oligodeoxynucleotides (ASPR) to inhibit in vitro cell proliferation. A significant decrease of 3H-thymidine uptake was observed in cells of a PI line growing in the presence of 2.5% charcoalized fetal calf serum and 0.8-20 microg/ml ASPR. It can be concluded that the PR pathway is an essential path in the growth stimulation of PI tumors.


Subject(s)
Adenocarcinoma/metabolism , Mammary Neoplasms, Experimental/metabolism , Receptors, Progesterone/metabolism , Adenocarcinoma/chemically induced , Adenocarcinoma/drug therapy , Androgens/metabolism , Animals , Binding Sites , Estradiol/pharmacology , Female , Flutamide/pharmacology , Glucocorticoids/metabolism , Gonanes/pharmacology , Hormone Antagonists/pharmacology , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/drug therapy , Medroxyprogesterone Acetate/toxicity , Mice , Mice, Inbred BALB C , Mifepristone/pharmacology , Neoplasms, Hormone-Dependent/chemically induced , Neoplasms, Hormone-Dependent/drug therapy , Neoplasms, Hormone-Dependent/metabolism , Oligonucleotides, Antisense/pharmacology , Ovariectomy , Receptors, Estrogen/metabolism , Receptors, Progesterone/antagonists & inhibitors , Receptors, Progesterone/genetics , Thymidine/metabolism , Tumor Cells, Cultured
15.
J Steroid Biochem Mol Biol ; 70(4-6): 133-42, 1999.
Article in English | MEDLINE | ID: mdl-10622401

ABSTRACT

Primary cultures of the medroxyprogesterone acetate-induced mouse mammary tumor line C4-HD are stimulated by medroxyprogesterone acetate (MPA) or progesterone. Serum obtained from ovariectomized, MPA-treated animals (OVX-MPA) exerts a stimulatory effect that is significantly higher than that induced by serum obtained from OVX mice with the exogenous addition of MPA, suggesting the involvement of MPA-induced serum factors potentiating the proliferative effect of MPA. The object of this paper is to further explore the stimulatory effect of mouse serum and to investigate the role of aFGF and bFGF on cell proliferation. The role of PR as possible mediators was tested using two different antiprogestins and antisense oligodeoxynucleotides of PR A isoform. Serum was obtained from OVX untreated or MPA-treated mice and was charcoalized and/or heat-inactivated. The effect of MPA or mifepristone at 10 nM concentrations was tested. Charcoalization and heat inactivation exerted a stimulatory effect (P<0.01) when OVX-serum was used. This effect was potentiated by MPA. Charcoalized OVX-MPA serum induced a significant inhibition of cell proliferation that was restored by the exogenous addition of MPA or by heat inactivation. Mifepristone induced an inhibition of 3H-thymidine uptake when OVX-MPA serum was used. These results suggest that serum factors activated by different manipulations may replace the stimulatory effect of MPA. When charcoalized fetal calf serum (chFCS) was used, a higher proliferative activity was obtained using higher serum concentrations. Mifepristone and onapristone 10 nM also inhibited this effect. aFGF and bFGF 100 ng/ml were both able to stimulate 3H-thymidine uptake. MPA exerted an additive effect. Mifepristone 10 nM inhibited bFGF and MPA+bFGF induced cell proliferation. Antisense oligodeoxynucleotides of PR (ASPR) were used to further confirm the participation of PR in the proliferative pathway of these cells. They inhibited serum and bFGF-induced cell proliferation in a specific dose-dependent manner. Our results suggest that PR play a central role in proliferation and suggest the existence of a cross-talk between steroid and growth factor signaling pathways.


Subject(s)
Cell Division/drug effects , Growth Substances/pharmacology , Mammary Neoplasms, Experimental/pathology , Medroxyprogesterone Acetate/pharmacology , Progesterone/pharmacology , Animals , Blood , Culture Media , DNA Replication/drug effects , Epidermal Growth Factor/pharmacology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Female , Fibroblast Growth Factor 1/pharmacology , Fibroblast Growth Factor 2/pharmacology , Mammary Neoplasms, Experimental/chemically induced , Mice , Mice, Inbred BALB C , Mifepristone/pharmacology , Oligodeoxyribonucleotides, Antisense/pharmacology , Ovariectomy , Receptors, Progesterone/genetics , Thymidine/metabolism , Tumor Cells, Cultured
16.
Cancer Lett ; 126(1): 49-57, 1998 Apr 10.
Article in English | MEDLINE | ID: mdl-9563648

ABSTRACT

In previous papers we have demonstrated that sialoadenectomy inhibited MPA-induced mammary tumorigenesis in BALB/c mice. To further explore the role of EGF in this experimental model, we evaluated its effects on mammary glands of sialoadenectomized (sialox) MPA-treated female mice and on tumor growth. MPA-treated sialox mice were injected s.c. (n = 3) or not (n = 6) with 5 microg EGF every 36 h for 45 days; MPA-treated sham-operated mice were used as controls (n = 6). Mammary glands from sialox MPA-treated mice are considerably less developed as compared with sham-operated animals. The exogenous administration of EGF restores the usual MPA-induced growth pattern of the glands, thus confirming a role for EGF either in mediating or cooperating with MPA in inducing the mammary architectural changes observed in MPA-treated mice. On the other hand, primary cultures of progestin-dependent (PD) ductal mammary adenocarcinoma in vivo tumor lines and of lobular progestin-independent (PI) tumor lines were used to evaluate the effect of EGF on tumor growth. In vitro EGF was found to stimulate cell proliferation of lobular PI tumor cells and of fibroblastic cells from both types of tumors at concentrations higher than 0.1-0.5 ng/ml and in the presence of 1-5% of charcoal-stripped fetal calf serum. Conversely, no proliferative effects were observed in ductal PD cells under the same experimental conditions, regardless of the presence of 10 nM MPA. It can be concluded that although EGF plays an important role in MPA-induced mammary carcinogenesis, it is not necessary in PD tumor growth.


Subject(s)
Adenocarcinoma/chemically induced , Carcinogens , Epidermal Growth Factor/physiology , Mammary Glands, Animal/pathology , Mammary Neoplasms, Experimental/chemically induced , Medroxyprogesterone Acetate , Animals , Cell Division/drug effects , Female , Hyperplasia/chemically induced , Mice , Mice, Inbred BALB C , Salivary Glands/physiology , Tumor Cells, Cultured
17.
Carcinogenesis ; 19(3): 529-31, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9525291

ABSTRACT

The promoter effect of medroxyprogesterone acetate (MPA) on mammary carcinogenesis in female BALB/c mice was investigated using methylnitrosourea (MNU) as initiator. Nine out of 43 animals developed mammary carcinomas in the group treated with MNU (50 mg/kg) and MPA (administration of 40 mg every 3 months) starting 1 week after MNU administration. No tumors appeared in controls receiving only MNU or MPA during the time course of the experiment (9 months). The tumors were lobular adenocarcinomas showing different degrees of squamous differentiation with low or undetectable estrogen and progesterone receptors, and expressing epidermal growth factor receptors. These results support the hypothesis that MPA promotes the growth of MNU induced lesions.


Subject(s)
Adenocarcinoma/chemically induced , Carcinogens/toxicity , Mammary Neoplasms, Experimental/chemically induced , Medroxyprogesterone Acetate/toxicity , Methylnitrosourea/toxicity , Animals , Cocarcinogenesis , Female , Mice , Mice, Inbred BALB C
18.
J Steroid Biochem Mol Biol ; 67(4): 305-17, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9883987

ABSTRACT

The role of the insulin-like growth factors (IGFs) system was investigated in hormone-dependent (HD) and -independent (HI) in vivo lines of the medroxyprogesterone acetate (MPA)-induced mammary tumor model in Balb/c mice. IGF-II protein and message showed a three- to four-fold increase in HD lines growing in MPA-treated mice, as compared with HD tumors growing in untreated mice. Progression to a hormone-independent phenotype in all these lines was accompanied by a high constitutive expression of IGF-II. Similar IGF-I mRNA levels were detected in HD and HI lines. Both IGF-I and -II messages arose from the malignant epithelial cells, as shown by in situ hybridization studies. A significant decrease in Man-6P/type II IGF-R content was detected in HD tumors growing in MPA-treated mice as compared with HD lines growing in untreated mice. On the other hand, in HI tumors, notwithstanding high IGF-II synthesis, the levels of Man-6P/type II IGF-R remain high. Competitive inhibition and affinity labeling studies showed an almost exclusive binding of IGF-II to Man-6P/type II IGF-R on tumor membranes. The involvement of IGFs in the growth of epithelial primary cultures of the C4-HD line was evaluated. Exogenous IGF-I potentiated MPA stimulatory effect at concentrations of 50-100 ng/ml. Treatment of C4-HD cells with antisense oligodeoxynucleotides (ASODNs) to type I IGF-R and to IGF-II RNA resulted in a dose-dependent inhibition of MPA-mediated cell proliferation. The inhibition caused by IGF-II ASODNs could not be overcome by the addition of IGF-II up to 150 ng/ml. ASODNs to type I IGF-R at 40 microg/ml reduced by 75% the number of type I IGF-R; ASODNs to IGF-II at 1 microM decreased by 83% the levels of IGF-II protein. Our results provide support for the involvement of IGF-I and -II in MPA-induced mammary tumor growth by autocrine pathways.


Subject(s)
Insulin-Like Growth Factor II/physiology , Insulin-Like Growth Factor I/physiology , Mammary Neoplasms, Experimental/pathology , Medroxyprogesterone Acetate/pharmacology , Receptor, IGF Type 1/physiology , Receptor, IGF Type 2/physiology , Adenocarcinoma/pathology , Animals , Base Sequence , Cell Division/drug effects , Cell Division/physiology , Female , In Situ Hybridization , Mice , Mice, Inbred BALB C , Oligonucleotides, Antisense/pharmacology , Tumor Cells, Cultured
19.
Medicina (B Aires) ; 57 Suppl 2: 55-69, 1997.
Article in Spanish | MEDLINE | ID: mdl-9567344

ABSTRACT

We have developed an experimental model in which the administration of progestins induces mammary tumors in female virgin BALB/c mice. In this paper we review the morphological and biological features of progestin-induced tumors, such as estrogen receptor (ER) and progesterone receptor (PR) patterns of expression, hormone dependence and epidermal growth factor receptors (EGF-R) we also examine our data concerning the systemic effects of medroxyprogesterone acetate (MPA) as regards its stimulating EGF synthesis in salivary glands and its subsequent increase in serum. This growth factor seems to play an important role in the induction of mammary tumors. Direct MPA proliferative effects mediated by PR were demonstrated using primary cultures of progestin-dependent (PD) mammary tumors. Antiprogestins inhibited cell growth beyond control values, suggesting that PR are involved in cell proliferation even in the absence of the ligand. Progesterone-independent (PI) tumors expressing high levels of PR and ER are also inhibited by estrogen or antiprogestin treatment, suggesting that PR are involved in the control of autonomous tumor growth. Estrogen-resistant variants may be selected which may revert to an estrogen-sensitive phenotype after several transplants in untreated mice. The similarities between the tumors obtained with this model and human breast cancer as regards morphological features, evolution and the regulation of growth control converts this model into a useful tool to explore the mechanisms related with acquisition of hormone independence and autonomous tumor growth.


Subject(s)
Adenocarcinoma/pathology , ErbB Receptors/analysis , Mammary Neoplasms, Experimental/pathology , Neoplasms, Hormone-Dependent/pathology , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis , Adenocarcinoma/chemically induced , Animals , Child , Disease Progression , Female , Humans , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/chemically induced , Medroxyprogesterone Acetate , Mice , Mice, Inbred BALB C
20.
Breast Cancer Res Treat ; 35(2): 173-86, 1995 Aug.
Article in English | MEDLINE | ID: mdl-7647339

ABSTRACT

The effect of progesterone (Pg), medroxyprogesterone acetate (MPA), estradiol (E2), dihydrotestosterone (DHT) and dexamethasone (DEXA) was studied on the in vitro growth rate of a progestin-dependent (PD), estrogen-sensitive mammary tumor line originated in an MPA-treated BALB/c mouse (C4-HD), and on its estrogen-resistant variant (C4-HDR). The specificity of hormone action was further investigated using the anti-hormones RU-486 and hydroxyflutamide (FLU). Cell growth was evaluated in epithelial and fibroblast-enriched cultures using 3H-thymidine and/or autoradiography and immunocytochemistry. The results indicate that cell growth is directly stimulated by MPA and Pg at concentrations ranging from 10(-11) to 10(-7) M. RU486 prevented MPA-induced stimulation in concentrations 10 to 100 fold lower than those of MPA. When used alone, it inhibited cell proliferation only in concentrations higher than 10(-11) M. At nM concentrations, neither DEXA nor DHT stimulated 3H-thymidine uptake except DEXA at 100 nM. MPA-induced stimulation was not reverted by micromolar concentrations of FLU. As for E2 (10(-7)-10(-9) M) it prevented MPA stimulation only in cultures of estrogen-sensitive tumors. Progesterone receptors (PR) (475 +/- 115 fmoles/10(5) cells, n = 5) and estrogen receptors (ER) (ND-115 fmoles/10(5) cells, n = 5) were detected only in epithelial-enriched cultures. Serum from 7 day-MPA-treated mice induced a significant increase of 3H-thymidine uptake; an increase was also obtained with serum from untreated ovariectomized animals to which 1 nM-100 nM concentrations of MPA had been added. The stimulatory effect of the exogenous MPA was much lower than that of the serum obtained from MPA-treated animals. It is concluded that MPA stimulates cell growth of primary cultures of MPA-induced PD tumors via PR. The results provide support for a direct effect of MPA which may be mediated or potentiated by serum factors.


Subject(s)
Adenocarcinoma/pathology , Mammary Neoplasms, Experimental/pathology , Medroxyprogesterone Acetate/pharmacology , Adenocarcinoma/blood , Adenocarcinoma/chemically induced , Androgen Antagonists/pharmacology , Animals , Blood Physiological Phenomena , Cell Division/drug effects , Dexamethasone/pharmacology , Dihydrotestosterone/pharmacology , Estradiol/pharmacology , Female , Flutamide/analogs & derivatives , Flutamide/pharmacology , Mammary Neoplasms, Experimental/blood , Mice , Mice, Inbred BALB C , Mifepristone/pharmacology , Ovariectomy , Progesterone/pharmacology , Tumor Cells, Cultured
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