Biomedical subjects

Alfredo Molinolo

Publications and source records attributed to Alfredo Molinolo.

12 recordsLinked to original sources

PubMed2006

Increased expression of the collagen internalization receptor uPARAP/Endo180 in the stroma of head and neck cancer.

Local growth, invasion, and metastasis of malignancies of the head and neck involve extensive degradation and remodeling of the underlying, collagen-rich connective tissue. Urokinase plasminogen activator receptor-associated protein (uPARAP)/Endo180 is an endocytic receptor recently shown to play a critical role in the uptake and intracellular degradation of collagen by mesenchymal cells. As a step toward determining the putative function of uPARAP/Endo180 in head and neck cancer progression, we used immunohistochemistry to determine the expression of this collagen internalization receptor in 112 human squamous cell carcinomas and 19 normal or tumor-adjacent head and neck tissue samples from the tongue, gingiva, cheek, tonsils, palate, floor of mouth, larynx, maxillary sinus, upper jaw, nasopharynx/nasal cavity, and lymph nodes. Specificity of detection was verified by staining of serial sections with two different monoclonal antibodies against two non-overlapping epitopes on uPARAP/Endo180 and by the use of isotype-matched non-immune antibodies. uPARAP/Endo180 expression was observed in stromal fibroblast-like, vimentin-positive cells. Furthermore, expression of the collagen internalization receptor was increased in tumor stroma compared with tumor-adjacent connective tissue or normal submucosal connective tissue and was most prominent in poorly differentiated tumors. These data suggest that uPARAP/Endo180 participates in the connective tissue destruction during head and neck squamous cell carcinoma progression by mediating cellular uptake and lysosomal degradation of collagen.

Blotting, Western↗

PubMed2006

Natural and synthetic progestins accelerate 7,12-dimethylbenz[a]anthracene-initiated mammary tumors and increase angiogenesis in Sprague-Dawley rats.

PURPOSE: Synthetic progestins are widely used therapeutically; however, there is controversy regarding their proliferative effects. We used a rat 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor model to test the hypothesis that progestins increase angiogenesis and as a result decrease the latency period and increase the multiplicity of mammary tumors. EXPERIMENTAL DESIGN: Medroxyprogesterone acetate (MPA) pellets were implanted 2, 4, or 6 weeks after DMBA exposure; RU-486 was given 3 days before MPA. Experiments were concluded 70 days after DMBA administration. RESULTS: MPA exposure 4 or 6 weeks after DMBA reduced the latency period for appearance of tumors in a dose-dependent manner and increased tumor incidence. Administration of MPA 2 weeks after DMBA administration reduced tumor incidence and was protective. Progesterone did not reduce the latency period but significantly increased tumor incidence. RU-486 delayed the latency period and decreased tumor incidence in animals exposed to MPA at 4 weeks after DMBA treatment, indicating that the progesterone receptor may be partially responsible for transmission of proliferative signals. RU-486 also delayed the latency period but failed to reduce overall tumor incidence when animals were exposed to MPA at 6 weeks after DMBA treatment, indicating that other factors may also control MPA-induced acceleration. Whereas MPA-accelerated tumors were both intraductal and tubular, progesterone-accelerated and/or DMBA-induced tumors were tubular. Progestin treatment increased vascular endothelial growth factor expression within tumors in a ligand- and cell type-dependent manner and increased angiogenesis in correlation with vascular endothelial growth factor expression. No mammary tumors or progesterone receptor were detected in DMBA-treated ovariectomized rats regardless of progestin administration. CONCLUSIONS: We propose that progestins can accelerate the development of mammary tumors and that antiangiogenic agents and/or the use of antiprogestins that can reduce tumor incidence might be a viable therapeutic option for treatment of progestin-accelerated tumors. The model described here is a potentially useful preclinical model for rapidly screening such compounds.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2006

The TSC2/mTOR pathway drives endothelial cell transformation induced by the Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor.

The Kaposi's sarcoma-associated herpesvirus (KSHV), the infectious causative agent of Kaposi's sarcoma (KS), encodes a G protein-coupled receptor (vGPCR) implicated in the initiation of KS. Here we demonstrate that Kaposi's sarcomagenesis involves stimulation of tuberin (TSC2) phosphorylation by vGPCR, promoting the activation of mTOR through both direct and paracrine mechanisms. Pharmacologic inhibition of mTOR with rapamycin prevented vGPCR sarcomagenesis, while overactivation of this pathway was sufficient to render endothelial cells oncogenic. Moreover, mice haploinsufficient for TSC2 are predisposed to vascular sarcomas remarkably similar to KS. Collectively, these results implicate mTOR in KS initiation and suggest that the sarcomagenic potential of KSHV may be a direct consequence of the profound sensitivity of endothelial cells to vGPCR dysregulation of the TSC2/mTOR pathway.

Animals↗

PubMed2006

Involvement of matrix metalloproteinase activity in hormone-induced mammary tumor regression.

Proteolytic activity and remodeling of the extracellular matrix are important players in tumor progression. However, to date the role of the extracellular matrix in tumor regression remains unresolved. To address this, we used a progesterone-dependent in vivo mouse mammary tumor line, C4-HD, which regresses in response to hormone therapy. Within the first 72 hours of treatment, massive apoptosis was accompanied by changes in the staining patterns of laminin and collagens I, III, and IV. We thus hypothesized that an increase in matrix metalloproteinase (MMP) activity could be involved in this process. This indeed was the case as the activities of MMP-2, -9, and -3 increased in regressing tumors, coinciding with the peak of apoptosis. Moreover, cell-cell interactions were disrupted during early hours of regression with E-cadherin levels reduced and fragmentation products detected during regression. Analysis of beta-catenin revealed that although total levels within the tissue did not change, this molecule switched from being involved in cell-cell adhesion in the growing tumor to being expressed in the reactive stroma during regression. Our data provide a novel role for proteolytic activity in tumor regression and question the underlying principle for using MMP inhibitors in cancer treatment.

Animals↗

PubMed2006

Rapid development of salivary gland carcinomas upon conditional expression of K-ras driven by the cytokeratin 5 promoter.

We have used a recently described model in which a ras oncogene is expressed in cytokeratin 5 (K5)-expressing cells on doxycycline administration to explore the effects of this oncogene in salivary glands of adult mice. Inducible expression of a mutated K-ras gene under the control of the K5 promoter led to the development of hyperplastic and dysplastic epithelial lesions and carcinomas, with an incidence of 100% and a minimum latency of a week. All major salivary glands were affected, as well as a set of previously undescribed buccal accessory salivary glands located on the apex of the masseter muscle, close to the oral angle. The tumors appear to arise from the cytokeratin 5-positive basal cell compartment. Myoepithelial cells participated in the hyperplasias but not in carcinomas, because the tumors are negative for smooth muscle actin. Carcinomas did not accumulate immunoreactive p53 but are positive for p63, as assayed by immunohistochemistry using an antibody against the N terminus of DeltaN p63, a splice variant of p63 that can inhibit p53 transcriptional activity. In this study, we provide evidence that the ras oncogene, targeted to a specifically sensitive cell compartment within the salivary glands, can trigger a series of event that are sufficient for full carcinogenesis.

Animals↗

PubMed2006

Delineation of matriptase protein expression by enzymatic gene trapping suggests diverging roles in barrier function, hair formation, and squamous cell carcinogenesis.

The membrane serine protease matriptase is required for epidermal barrier function, hair formation, and thymocyte development in mice, and dysregulated matriptase expression causes epidermal squamous cell carcinoma. To elucidate the specific functions of matriptase in normal and aberrant epidermal differentiation, we used enzymatic gene trapping combined with immunohistochemical, ultrastructural, and barrier function assays to delineate the spatio-temporal expression and function of matriptase in mouse keratinized tissue development, homeostasis, and malignant transformation. In the interfollicular epidermis, matriptase expression was restricted to postmitotic transitional layer keratinocytes undergoing terminal differentiation. Matriptase was also expressed in keratinizing oral epithelium, where it was required for oral barrier function, and in thymic epithelium. In all three tissues, matriptase colocalized with profilaggrin. In staged embryos, the onset of epidermal matriptase expression coincided with that of profilaggrin expression and acquisition of the epidermal barrier. In marked contrast to stratifying keritinized epithelium, matripase expression commenced already in undifferentiated and rapidly proliferating profilaggrin-negative matrix cells and displayed hair growth cycle-dependent expression. Exposure of the epidermis to carcinogens led to the gradual appearance of matriptase in a keratin-5-positive proliferative cell compartment during malignant progression. Combined with previous studies, these data suggest that matriptase has diverging functions in the genesis of stratified keratinized epithelium, hair follicles, and squamous cell carcinoma.

Animals↗

PubMed2005

Antisense oligonucleotides targeting the progesterone receptor inhibit hormone-independent breast cancer growth in mice.

INTRODUCTION: Previous data from our laboratory suggested that progesterone receptors (PRs) are involved in progestin-independent growth of mammary carcinomas. To investigate this possibility further, we studied the effects of PR antisense oligodeoxynucleotides (asPR) on in vivo tumor growth. METHOD: BALB/c mice with subcutaneous 25 mm2 mammary carcinomas expressing estrogen receptor-alpha and PR were either injected intraperitoneally with 1 mg asPR every 24 or 12 hours for 5-10 days, or subcutaneously with RU 486 (6.5 mg/kg body weight) every 24 hours. Control mice received vehicle or scPR. RESULTS: Significant inhibition of tumor growth as well as a significant decrease in bromodeoxyuridine uptake was observed in asPR-treated mice, which correlated with histological signs of regression and increased apoptosis. Mice treated with RU 486 experienced almost complete tumor regression. No differences were detected between vehicle-treated and scPR-treated mice. Anti-progestin-treated and asPR-treated mice were in a continuous estrous/meta-estrous state. Decreased phosphorylated extracellular signal-regulated kinase (ERK)1 and ERK2 levels and estrogen receptor-alpha expression were observed as late events in RU 486-treated and asPR-treated mice with regressing tumors. CONCLUSION: We demonstrate, for the first time, inhibition of tumor growth in vivo using asPR. Our results provide further evidence for a critical and hierarchical role of the PR pathway in mammary carcinomas.

Animals↗

PubMed2005

Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation.

Overexpression of the type II transmembrane serine protease matriptase is a highly consistent feature of human epithelial tumors. Here we show that matriptase possesses a strong oncogenic potential when unopposed by its endogenous inhibitor, HAI-1. Modest orthotopic overexpression of matriptase in the skin of transgenic mice caused spontaneous squamous cell carcinoma and dramatically potentiated carcinogen-induced tumor formation. Matriptase-induced malignant conversion was preceded by progressive interfollicular hyperplasia, dysplasia, follicular transdifferentiation, fibrosis, and dermal inflammation. Furthermore, matriptase induced activation of the pro-tumorigenic PI3K-Akt signaling pathway. This activation was frequently accompanied by H-ras or K-ras mutations in carcinogen-induced tumors, whereas matriptase-induced spontaneous carcinoma formation occurred independently of ras activation. Increasing epidermal HAI-1 expression completely negated the oncogenic effects of matriptase. The data implicate dysregulated matriptase expression in malignant epithelial transformation.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2005

Intermolecular complementation achieves high-specificity tumor targeting by anthrax toxin.

Anthrax toxin protective antigen (PrAg) forms a heptamer in which the binding site for lethal factor (LF) spans two adjacent monomers. This suggested that high cell-type specificity in tumor targeting could be obtained using monomers that generate functional LF-binding sites only through intermolecular complementation. We created PrAg mutants with mutations affecting different LF-binding subsites and containing either urokinase plasminogen activator (uPA) or matrix metalloproteinase (MMP) cleavage sites. Individually, these PrAg mutants had low toxicity as a result of impaired LF binding, but when administered together to uPA- and MMP-expressing tumor cells, they assembled into functional LF-binding heteroheptamers. The mixture of two complementing PrAg variants had greatly reduced toxicity in mice and was highly effective in the treatment of aggressive transplanted tumors of diverse origin. These results show that anthrax toxin, and by implication other multimeric toxins, offer excellent opportunities to introduce multiple-specificity determinants and thereby achieve high therapeutic indices.

Amino Acid Sequence↗

PubMed2005

Isolation of a stromal cell line from an early passage of a mouse mammary tumor line: a model for stromal parenchymal interactions.

We have developed a murine mammary tumor cell line, MC4-L4, and after 15 passages, a spindle-shaped population became evident. The cuboidal cells, MC4-L4E, cloned by limit dilution, proved to be epithelial tumor cells. When inoculated in syngeneic mice, they gave rise to invasive metastatic carcinomas expressing estrogen and progesterone receptors. These tumors regressed after anti-progestin treatment and stopped growing after 17-beta-estradiol administration. In vitro, they were insensitive to medroxyprogesterone acetate (MPA), 17-beta-estradiol, and EGF and were inhibited by TGFbeta1. They expressed mutated p53 and estrogen receptors alpha; progesterone receptors were undetectable. Cells were polyploid and shared the same four common marker chromosomes present in the parental tumor in addition to an exclusive marker. Spindle-shaped cells, MC4-L4F, were selected by differential attachment and detachment and proved to be non-epithelial non-tumorigenic cells. They were cytokeratin negative, showed mesenchymal features by electron microscopy, differentiated to adipocytes when treated with an adipogenic cocktail, were stimulated by TGFbeta1 and EGF, showed a wild-type p53, and did not exhibit the marker chromosomes of the parental tumor. Although they expressed estrogen receptors alpha, they were insensitive to 17-beta-estradiol in proliferation assays. Co-cultures of both cell types had a synergic effect on progesterone receptors expression and on cell proliferation, being the epithelial cells, the most responsive ones, and 17-beta-estradiol increased cell proliferation only in co-cultures. Cytogenetic studies and data on p53 mutations rule out the possibility of an epithelial mesenchymal transition. Their unique characteristics make them an excellent model to be used in studies of epithelial-stromal interactions in the context of hormone responsiveness in hormone related tumors.

Animals↗

PubMed2003

Endothelial infection with KSHV genes in vivo reveals that vGPCR initiates Kaposi's sarcomagenesis and can promote the tumorigenic potential of viral latent genes.

The Kaposi's sarcoma herpesvirus (KSHV) has been identified as the etiologic agent of Kaposi's sarcoma (KS), but initial events leading to KS development remain unclear. Characterization of the KSHV genome reveals the presence of numerous potential oncogenes. To address their contribution to the initiation of the endothelial cell-derived KS tumor, we developed a novel transgenic mouse that enabled endothelial cell-specific infection in vivo using virus expressing candidate KSHV oncogenes. Here we show that transduction of one gene, vGPCR, was sufficient to induce angioproliferative tumors that strikingly resembled human KS. Endothelial cells expressing vGPCR were further able to promote tumor formation by cells expressing KSHV latent genes, suggestive of a cooperative role among viral genes in the promotion of Kaposi's sarcomagenesis.

Animals↗

PubMed2002

p21, p27 and p53 in estrogen and antiprogestin-induced tumor regression of experimental mouse mammary ductal carcinomas.

Metastatic mammary carcinoma tumor lines 59-2-HI and C7-2-HI originated in female BALB/c mice treated with medroxyprogesterone acetate and are maintained by syngeneic transplantation. Both lines express estrogen (ER) and progesterone receptors (PR) and regress completely after estradiol (E(2)) or antiprogestin treatment. The BET tumor line, of similar origin and biological features, regresses only after E(2) treatment. To investigate possible differences between E(2)- and antiprogestin-mediated effects we evaluated the morphological features, mitosis and apoptosis, and the differential expression of cell-cycle inhibitors associated with tumor regression. Treatments started when tumors reached 50-100 mm(2). After 24-96 h, tumors were excised and processed for morphological and immunohistochemical studies. Regression was associated with a significant and early decrease in the number of mitosis and with higher percentages of apoptotic cells. These phenomena were accompanied by an increase in p21 and p27 expression in the E(2) and antiprogestin-responsive lines treated with E(2), RU 38.486 or ZK 98.299 (P < 0.05). In BET tumors treated with E(2), p21 expression remained within basal levels and only p27 increased (P < 0.05). p53 was low in control 59-2-HI and C7-2-HI tumors and increased after treatment (P < 0.05) whereas BET untreated tumors already expressed high levels of p53 and MDM2. Although the immunohistochemical findings were compatible with alterations of p53, SSCP evaluation failed to disclose the presence of mutations, suggesting that the defective expression of p21 is related to an impaired p53 pathway. UV irradiation failed to increase p21 expression in BET, but was able to induce p53 and p21 in 59-2-HI and C7-2-HI tumors. The absence of an increased expression of p21 in E(2)-regressing BET lesions suggests that this protein is not necessary for estrogen-induced regression, but may be essential for antiprogestin action. Our results also suggest that p53/MDM2 alterations may be one of the mechanisms responsible for selected hormone resistance in breast carcinomas.

Animals↗