Joseph Celestino

Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice.

Gliomas are the most common primary malignant brain tumours and are classified into four clinical grades, with the most aggressive tumours being grade 4 astrocytomas (also known as glioblastoma multiforme; GBM). Frequent genetic alterations in GBMs (refs 2–5) result in stimulation of common signal transduction pathways involving Ras, Akt and other proteins. It is not known which of these pathways, if any, are sufficient to induce GBM formation. Here we transfer, in a tissue-specific manner, genes encoding activated forms of Ras and Akt to astrocytes and neural progenitors in mice. We found that although neither activated Ras nor Akt alone is sufficient to induce GBM formation, the combination of activated Ras and Akt induces high-grade gliomas with the histological features of human GBMs. These tumours appear to arise after gene transfer to neural progenitors, but not after transfer to differentiated astrocytes. Increased activity of RAS is found in many human GBMs (ref. 11), and we show here that Akt activity is increased in most of these tumours, implying that combined activation of these two pathways accurately models the biology of this disease.

EphA2 expression is associated with aggressive features in ovarian carcinoma.

Purpose: EphA2 (epithelial cell kinase) is a transmembrane receptor tyrosine kinase that has been implicated in oncogenesis. There are no published data regarding the role of EphA2 in ovarian carcinoma, which is the focus of the present study. Experimental Design: Nontransformed (HIO-180) and ovarian cancer (EG, 222, SKOV3, and A2780-PAR) cell lines were evaluated for EphA2 by Western blot analysis. Five benign ovarian masses, 10 ovarian tumors of low malignant potential, and 79 invasive ovarian carcinomas were also evaluated for EphA2 expression by immunohistochemistry. All samples were scored in a blinded fashion. Univariate and multivariate analyses were used to determine significant associations between EphA2 expression and clinicopathological variables. Results: By Western blot analysis, EG, 222, and SKOV3 cell lines overexpressed EphA2, whereas A2780-PAR and HIO-180 had low to absent EphA2 expression. All of the benign tumors had low or absent EphA2 expression. Among the invasive ovarian carcinomas examined (mean age of patients was 59.2 years), 60 (75.9%) tumors overexpressed EphA2 and the other 19 tumors had negative or minimal EphA2 expression. There was no association of EphA2 overexpression with ascites, likelihood of nodal positivity, pathological subtype, and optimum surgical cytoreduction (residual tumor <1 cm). However, EphA2 overexpression was significantly associated with higher tumor grade (P = 0.02) and advanced stage of disease (P = 0.001). The median survival for patients with tumor EphA2 overexpression was significantly shorter (median, 3.1 years; P = 0.004); the median survival for patients with low or absent EphA2 tumor expression was at least 12 years and has not yet been reached. In multivariate analysis using the Cox proportional hazards model, only volume of residual disease (P < 0.04) and EphA2 overexpression (P < 0.01) were significant and independent predictors of survival. Conclusions: EphA2 overexpression is predictive of aggressive ovarian cancer behavior and may be an important therapeutic target.

A Novel Platform for Detection of CK+ and CK− CTCs

UNLABELLED Metastasis is a complex, multistep process that begins with the epithelial-mesenchymal transition (EMT). Circulating tumor cells (CTC) are believed to have undergone EMT and thus lack or express low levels of epithelial markers commonly used for enrichment and/or detection of such cells. However, most current CTC detection methods target only EpCAM and/or cytokeratin (CK) to enrich epithelial CTCs, resulting in failure to recognize other, perhaps more important, CTC phenotypes that lack expression of these markers. Here, we describe a population of complex aneuploid CTCs that do not express CK or CD45 antigen in patients with breast, ovarian, or colorectal cancer. These cells were not observed in healthy subjects. We show that the primary epithelial tumors were characterized by similar complex aneuploidy, indicating conversion to an EMT phenotype in the captured cells. Collectively, our study provides a new method for highly efficient capture of previously unrecognized populations of CTCs. SIGNIFICANCE Current assays for CTC capture likely miss populations of cells that have undergone EMT. Capture and study of CTCs that have undergone EMT would allow a better understanding of the mechanisms driving metastasis.

Insulin-like growth factor binding protein 2 promotes glioma development and progression

Overexpression of insulin-like growth factor binding protein 2 (IGFBP2) is associated with progression in many types of human cancer. In this study we used a glial-specific transgenic mouse model to examine the active role of IGFBP2 in tumorigenesis and progression. Our studies show that IGFBP2 coexpression results in progression to a higher-grade glioma in platelet-derived growth factor β (PDGFB)-driven tumors. These anaplastic oligodendrogliomas are characterized by increased cellularity, vascular proliferation, small regions of necrosis, increased mitotic activity, and increased activation of the Akt pathway. Combined expression of IGFBP2 or Akt with K-Ras was required to form astrocytomas, indicating that activation of two separate pathways is necessary for gliomagenesis. In ex vivo experiments, blockade of Akt by an inhibitor led to decreased viability of cells coexpressing IGFBP2 versus PDGFB expression alone. Thus, this study provides definitive evidence that IGFBP2 plays a key role in activation of the Akt pathway and collaborates with K-Ras or PDGFB in the development and progression of two major types of glioma.

Astrocytes Give Rise to Oligodendrogliomas and Astrocytomas after Gene Transfer of Polyoma Virus Middle T Antigen in Vivo

The cells of origin for oligodendrogliomas and astrocytomas are not known but are presumed to be oligodendrocyte and astrocyte precursors, respectively. In this paper we report the generation of mixed gliomas from in vivo transformation of glial fibrillary acidic protein (GFAP)-positive cells (differentiated astrocytes) with polyoma virus middle T antigen (MTA). MTA is a powerful oncogene that activates a number of signal transduction pathways, including those proposed to be involved in gliomagenesis, and has been shown to induce tumors in many cell types. We have achieved transfer of MTA expression specifically to GFAP(+) cells in vivo using somatic cell gene transfer, and find resultant formation of anaplastic gliomas with mixed astrocytoma and oligodendroglioma morphological features. We conclude that GFAP- expressing astrocytes, with appropriate signaling abnormalities, can serve as the cell of origin for oligodendrogliomas, astrocytomas, or mixed gliomas.

Enhanced estrogen-induced proliferation in obese rat endometrium

OBJECTIVE We tested the hypothesis that the proliferative estrogen effect on the endometrium is enhanced in obese vs lean animals. STUDY DESIGN Using Zucker fa/fa obese rats and lean control, we examined endometrial cell proliferation and the expression patterns of certain estrogen-regulated proproliferative and antiproliferative genes after short-term treatment with estradiol. RESULTS No significant morphologic/histologic difference was seen between the obese rats and the lean rats. Estrogen-induced proproliferative genes cyclin A and c-Myc messenger RNA expression were significantly higher in the endometrium of obese rats compared with those of the lean control. Expression of the antiproliferative gene p27Kip1 was suppressed by estrogen treatment in both obese and lean rats; however, the decrease was more pronounced in obese rats. Estrogen more strongly induced the antiproliferative genes retinaldehyde dehydrogenases 2 and secreted frizzled-related protein 4 in lean rats but had little or no effect in obese rats. CONCLUSION Enhancement of estrogen-induced endometrial proproliferative gene expression and suppression of antiproliferative gene expression was seen in the endometrium of obese vs lean animals.

Another Surprise from Metformin: Novel Mechanism of Action via K-Ras Influences Endometrial Cancer Response to Therapy

Metformin is an oral biguanide commonly used for the treatment of type II diabetes and has recently been demonstrated to possess antiproliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Using both in vitro and in vivo models, we examined the effects of metformin on endometrial tumors with defined aberrations in the PI3K/PTEN/mTOR and MAPK signaling pathways to understand metformin mechanism of action and identify clinically useful predictors of response to this agent. In vitro assays of proliferation, cytotoxicity, and apoptosis were used to quantify the effects of metformin on endometrial cancer cell lines with mutations in the PI3K/PTEN/mTOR and MAPK signaling pathways. The in vivo effects of oral metformin on tumor progression were further examined using xenograft mouse models of endometrial cancer. K-Ras localization was analyzed by confocal microscopy using GFP-labeled oncogenic K-Ras and by immunoblot following subcellular fractionation. Metformin inhibited cell proliferation, induced apoptosis, and decreased tumor growth in preclinical endometrial cancer models, with the greatest response observed in cells harboring activating mutations in K-Ras. Furthermore, metformin displaces constitutively active K-Ras from the cell membrane, causing uncoupling of the MAPK signaling pathway. These studies provide a rationale for clinical trials using metformin in combination with PI3K-targeted agents for tumors harboring activating K-Ras mutations, and reveal a novel mechanism of action for metformin. Mol Cancer Ther; 12(12); 2847–56. ©2013 AACR.

Primary chemoprevention of endometrial hyperplasia with the peroxisome proliferator-activated receptor gamma agonist rosiglitazone in the PTEN heterozygote murine model.

PTEN mutations have been implicated in the development of endometrial hyperplasia and subsequent cancer. Peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists have demonstrated antineoplastic and chemopreventive effects. The purpose of this study was to evaluate the effects of the PPAR-γ agonist rosiglitazone on both PTEN wild type and PTEN null cell lines and in the PTEN heterozygote(+/−) murine model. Hec-1-A (PTEN wild type) and Ishikawa (PTEN null) cells were treated with rosiglitazone. Thirty-five female PTEN+/− mice were genotyped and placed into one of four groups for treatment for 18 weeks: A) PTEN wild type with 4 mg/kg rosiglitazone, B) PTEN+/− mice with vehicle, C) PTEN+/− mice with 4 mg/kg rosiglitazone, and D) PTEN+/− mice with 8 mg/kg rosiglitazone. Proliferation and apoptosis were measured by bromodeoxyuridine (BrdU) and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragmentation sites assay. Rosiglitazone caused cell growth inhibition in both Hec-1-A and Ishikawa in a dose-dependent manner (P< 0.02 and P< 0.03, respectively). Rosiglitazone also induced apoptosis in both Hec-1-A (P< .001) and Ishikawa (P< .001) cells in a dose-dependent manner. In the murine model, rosiglitazone decreased proliferation of the endometrial hyperplastic lesions (B vs C; 39.7% vs 9.3% and B vs D; 39.7% vs 4.2%; P< 0.0001) and increased apoptosis of glandular endometrial epithelial cells (B vs C; 2.8% vs 22.4%; P< 0.0001 and B vs D; 2.8% vs 30.2%; P= 0.003). PPAR-γ agonist rosiglitazone inhibits proliferation and induces apoptosis in both PTEN intact and PTEN null cancer cell lines and in hyperplastic endometrial lesions in the PTEN+/− murine model.

CGRRF1 as a novel biomarker of tissue response to metformin in the context of obesity

OBJECTIVE Obesity-associated hyperestrogenism and hyperinsulinemia contribute significantly to the pathogenesis of endometrial cancer. We recently demonstrated that metformin, a drug long used for treatment of type 2 diabetes, attenuates both insulin- and estrogen-mediated proliferative signaling in the obese rat endometrium. In this study, we sought to identify tissue biomarkers that may prove clinically useful to predict tissue response for both prevention and therapeutic studies. We identified CGRRF1 (cell growth regulator with ring finger domain 1) as a novel metformin-responsive gene and characterized its possible role in endometrial cancer prevention. METHODS CGRRF1 mRNA expression was evaluated by RT-qPCR in the endometrium of obese and lean rats, and also in normal and malignant human endometrium. CGRRF1 levels were genetically manipulated in endometrial cancer cells, and its effects on proliferation and apoptosis were evaluated by MTT and Western blot. RESULTS CGRRF1 is significantly induced by metformin treatment in the obese rat endometrium. In vitro studies demonstrate that overexpression of CGRRF1 inhibits endometrial cancer cell proliferation. Analysis of human endometrial tumors reveals that CGRRF1 expression is significantly lower in hyperplasia, Grade 1, Grade 2, Grade 3, MMMT, and UPSC endometrial tumors compared to normal human endometrium (p<0.05), suggesting that loss of CGRRF1 is associated with the presence of disease. CONCLUSION CGRRF1 represents a novel, reproducible tissue marker of metformin response in the obese endometrium. Furthermore, our preliminary data suggests that up-regulation of CGRRF1 expression may prove clinically useful in the prevention or treatment of endometrial cancer.

Loss of LKB1 in high-grade endometrial carcinoma

Liver kinase B1 (LKB1) is a serine/threonine kinase that functions as a tumor suppressor and regulates cell polarity, proliferation, and metabolism. Mutations in LKB1 are associated with Peutz‐Jeghers syndrome as well as sporadic cervical and lung cancers. Although LKB1‐null mice develop invasive endometrial cancers, the role and regulation of LKB1 in the pathogenesis of human endometrial cancer are not well defined and are the focus of these studies.