A functional screen identifies hDRIL1 as an oncogene that rescues RAS-induced senescence.

Primary fibroblasts respond to activated H-RAS(V12) by undergoing premature arrest, which resembles replicative senescence. This irreversible 'fail-safe mechanism' requires p19(ARF), p53 and the Retinoblastoma (Rb) family: upon their disruption, RAS(V12)-expressing cells fail to undergo senescence and continue to proliferate. Similarly, co-expression of oncogenes such as c-MYC or E1A rescues RAS(V12)-induced senescence. To identify novel genes that allow escape from RAS(V12)-induced senescence, we designed an unbiased, retroviral complementary DNA library screen. We report on the identification of DRIL1, the human orthologue of the mouse Bright and Drosophila dead ringer transcriptional regulators. DRIL1 renders primary murine fibroblasts unresponsive to RAS(V12)-induced anti-proliferative signalling by p19(ARF)/p53/p21(CIP1), as well as by p16(INK4a). In this way, DRIL1 not only rescues RAS(V12)-induced senescence but also causes these fibroblasts to become highly oncogenic. Furthermore, DRIL1 immortalizes mouse fibroblasts, in the presence of high levels of p16(INK4a). Immortalization by DRIL1, whose product binds the pRB-controlled transcription factor E2F1 (ref. 8), is correlated with induction of E2F1 activity. Correspondingly, DRIL1 induces the E2F1 target Cyclin E1, overexpression of which is sufficient to trigger escape from senescence. Thus, DRIL1 disrupts cellular protection against RAS(V12)-induced proliferation downstream of the p19(ARF)/p53 pathway.

An in vivo functional genetic screen reveals a role for the TRK-T3 oncogene in tumor progression.

Over the past decades, much has been learnt about the genes that contribute to oncogenic transformation of primary cells in vitro. However, much less is known about the genes that contribute to the later stages of tumor progression, in which cells of ever increasing malignancy arise through clonal selection in vivo. To search for genes that confer a tumor progression phenotype in vivo, we have used a functional genetic approach. We used adenovirus-transformed mouse embryo fibroblasts, which are tumorigenic in immunodeficient nude mice, but not in immunocompetent mice, due to strong cytotoxic T-cell-mediated immune rejection. We infected these cells in vitro with several high-complexity retroviral cDNA expression libraries and selected rare variants that formed tumors in immunocompetent mice. Using this approach, we identify here the TRK-T3 oncogene as a tumor progression gene. TRK-T3 does not inhibit T-cell reactivity towards the tumor cells. Instead, we find that cells expressing TRK-T3 enhances in vivo growth rate, most likely by stimulating anchorage-independent proliferation in growth factor-limiting conditions. Our data indicate that cDNA expression libraries can be used to identify tumor progression genes in vivo that cannot be readily identified using in vitro cell culture systems.

Protein expression of B-cell lymphoma gene 6 (BCL-6) in invasive breast cancer is associated with cyclin D1 and hypoxia-inducible factor-1alpha (HIF-1alpha).

B-cell lymphoma gene (BCL-6) upregulation contributes to immortalization of mouse embryo fibroblast and primary B cells via upregulation of cyclin D1. As cyclin D1 overexpression is a common phenomenon in different cancers, BCL-6 protein overexpression may not be restricted to lymphomas. In this study, expression of BCL-6 was investigated by immunohistochemistry on paraffin-embedded specimens from 150 breast cancer patients and 10 specimens of normal breast tissue. The results showed BCL-6 overexpression (> or =10% of cells) in 24/150 (16%) breast cancer patients, whereas in normal breast low expression (<1%) of BCL-6 was observed. In linear regression analysis BCL-6 expression was associated with cyclin D1 (r=0.197, P=0.016). Further, in chi2 analyses, BCL-6-positivity was associated with overexpression of p53 (P=0.016), and hypoxia-inducible factor-1alpha (P<0.001). Involvement of BCL-6 in breast carcinogenesis is further underscored by comparative genomic hybridization analysis that showed gains at the BCL-6 locus (3q27) in 14/86 (16%) breast cancer tissues. The cases with amplification in BCL-6 showed an increased (25%) incidence of BCL-6 protein overexpression. Thus, this study is the first to show that BCL-6 oncogene activation plays a role in cancers other than lymphomas.

Expression of hypoxia-inducible factor-1alpha and cell cycle proteins in invasive breast cancer are estrogen receptor related.

BACKGROUND: The transcription factor hypoxia-inducible factor-1 (HIF-1) is a key regulator of the cellular response to hypoxia. Previous studies showed that concentrations of its subunit HIF-1alpha, as a surrogate for HIF-1 activity, are increased during breast carcinogenesis and can independently predict prognosis in breast cancer. During carcinogenesis, the cell cycle is progressively deregulated, and proliferation rate is a strong prognostic factor in breast cancer. In this study we undertook a detailed evaluation of the relationships between HIF-1alpha and cell cycle-associated proteins. METHODS: In a representative estrogen receptor (ER) group of 150 breast cancers, the expression of HIF-1alpha, vascular endothelial growth factor, the ER, HER-2/neu, Ki-67, cyclin A, cyclin D1, p21, p53, and Bcl-2 was investigated by immunohistochemistry. RESULTS: High concentrations (5% or more) of HIF-1alpha were associated with increased proliferation as shown by positive correlations with Ki-67 (P < 0.001) and the late S-G2-phase protein cyclin A (P < 0.001), but not with the G1-phase protein cyclin D1. High HIF-1alpha concentrations were also strongly associated with p53 positivity (P < 0.001) and loss of Bcl-2 expression (P = 0.013). No association was found between p21 and HIF-1alpha (P = 0.105) in the whole group of patients. However, the subgroup of ER-positive cancers was characterized by a strong positive association between HIF-1alpha and p21 (P = 0.023), and HIF-1alpha lacked any relation with proliferation. CONCLUSION: HIF-1alpha overexpression is associated with increased proliferation, which might explain the adverse prognostic impact of increased concentrations of HIF-1alpha in invasive breast cancer. In ER-positive tumors, HIF-1alpha is associated with p21 but not against proliferation. This shows the importance of further functional analysis to unravel the role of HIF-1 in late cell cycle progression, and the link between HIF-1, p21, and ER.

Identification by phage display of single-domain antibody fragments specific for the ODD domain in hypoxia-inducible factor 1alpha.

Hypoxia triggers the transcription of genes responsible for cell survival via the key player transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). Overexpression of this protein has been implicated in cardiovascular disorders, carcinogenesis and cancer progression. For functional and diagnostic studies on the HIF-1alpha protein, we have identified single-domain antibody fragments directed against this protein by using a llama-derived nonimmune phage display library. This library displays the variable domains of the heavy-chain antibody subclass, found in these animals. Phage display selection with six recombinant HIF-1alpha proteins yielded five different antibody fragments. By epitope-mapping, we show that all five antibody fragments bind within the functionally important oxygen-dependent degradation domain of the HIF-1alpha protein. Two of these antibody fragments were engineered into bivalent antibodies that were able to detect human HIF-1alpha by immunohistochemistry, Western blotting and immunoprecipitation, and mouse HIF-1alpha by immunofluorescence and immunoprecipitation. These are the first single-domain antibody fragments that may be used in exploration of HIF-1alpha as a possible therapeutic target through molecular applications.

BRCA1 and p53 protein expression in cultured ovarian surface epithelial cells derived from women with and without a BRCA1 germline mutation.

AIM: Mutations in the BRCA1 and TP53 genes are early genetic events leading to (hereditary) ovarian carcinoma. The human ovarian surface epithelium (OSE) is considered the tissue of origin of at least a subset of these tumours. Therefore, OSE cell cultures derived from women harbouring BRCA1 germline mutations can be a potential model to study hereditary ovarian carcinogenesis. In fact, previous in vitro studies indicate phenotypical differences between OSE from women with and without such germline mutations. Therefore, we have assessed whether differences in the expression of BRCA1 and p53 proteins in cultured OSE cells could contribute to these observations. STUDY DESIGN: Thirty-two OSE cultures derived from women harbouring a BRCA1 mutation (Predisposed OSE [POSE]) and ten cultures from women without a cancer predisposition (Non predisposed OSE [NPOSE]) were grown under standard conditions. Immunocytochemistry was performed to assess the expression of the BRCA1- and p53 proteins. Ki67 immunocytochemical expression was assessed to determine possible differences in cell cycle status between the two groups. In addition, to study whether wild type p53 was expressed, induction of p53 by cis-platinum was assessed by Western blot. RESULTS: On the basis of Ki67 expression, three different groups were analyzed. In the group with all cultures that expressed Ki67 no significant difference was observed in BRCA1 (P = 0.19) and p53 expression (P = 0.09). In the group with moderate to high Ki67 expression no difference in BRCA1 expression (P = 0.50) was observed. However, p53 expression was significantly lower in the case group (P = 0.01). The same observation for p53 was made in the group with only high Ki67 expression (P = 0.02). Furthermore, the expression of both BRCA1 and p53 positively correlates with Ki67 expression. In POSE and NPOSE, p53 was induced by cis-platinum to a similar extent. CONCLUSION: Our study indicates differences in the expression of p53, but not in the expression of BRCA1 between POSE and NPOSE. In addition, our findings do suggest the absence of losses of the wild type BRCA1 and p53 genes in the studied OSE cultures. This indicates that losses in these genes cannot account for observed differences in phenotypical traits between POSE and NPOSE, but that differences in levels of p53 might contribute.

A senescence rescue screen identifies BCL6 as an inhibitor of anti-proliferative p19(ARF)-p53 signaling.

Senescence limits the proliferative capacity of primary cells in culture. We describe here a genetic screen to identify genes that allow bypass of this checkpoint. Using retroviral cDNA expression libraries, we identify BCL6 as a potent inhibitor of senescence. BCL6 is frequently activated in non-Hodgkin's lymphoma, but its mechanism of action has remained unclear. BCL6 efficiently immortalizes primary mouse embryonic fibroblasts and cooperates with RAS in oncogenic transformation. BCL6 overrides the senescence response downstream of p53 through a process that requires induction of cyclin D1 expression, as cyclin D1 knockout fibroblasts are specifically resistant to BCL6 immortalization. We show that BCL6 expression also dramatically extends the replicative lifespan of primary human B cells in culture and induces cyclin D1 expression, indicating that BCL6 has a similar activity in lymphoid cells. Our results suggest that BCL6 contributes to oncogenesis by rendering cells unresponsive to antiproliferative signals from the p19(ARF)-p53 pathway.

Levels of hypoxia-inducible factor-1alpha independently predict prognosis in patients with lymph node negative breast carcinoma.

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays an important role in tumor growth and metastasis by regulating energy metabolism and inducing angiogenesis to survive cellular hypoxia. Increased levels of HIF-1alpha, the O(2)-regulated subunit of HIF-1, were noted during breast carcinogenesis. In this study, the prognostic value of HIF-1alpha expression and its correlation with various clinicopathologic variables in patients with invasive breast carcinoma were investigated. METHODS: Expression levels of HIF-1alpha, HER-2/neu, estrogen receptor, and progesterone receptor were analyzed in 150 patients with early-stage breast carcinoma by immunohistochemistry. HER-2/neu gene amplification was investigated with automated fluorescent in situ hybridization. The mitotic activity index, histologic grade, and tumor type were assessed in hematoxylin and eosinstained specimens. Clinical data included disease-free survival, overall survival, lymph node status, and tumor size. The data were analyzed with two-sided univariate and multivariate tests, with P values < 0.05 considered significant. RESULTS: High levels of HIF-1alpha had an association of borderline significance with decreased overall survival (P = 0.059) and disease-free survival (P = 0.110) that was ascribed completely to the subgroup of women with lymph node negative tumors (n = 81 patients; P = 0.008 and P = 0.004, respectively). HER-2/neu immunoreactivity (P < 0.001) and gene amplification (P < 0.001), vascular endothelial growth factor expression (P = 0.016), and Ki-67 expression (P < 0.001) were correlated strongly with HIF-1alpha positivity, although none of those factors had an independent effect on survival. CONCLUSIONS: Increased levels of HIF-1alpha were associated independently with shortened survival in patients with lymph node negative breast carcinoma. Therefore, the use of immunohistochemical assessment of HIF-1alpha as a new predictor of poor outcome may improve clinical decision-making regarding adjuvant treatment of patients with lymph node negative breast carcinoma.

Cultures of ovarian surface epithelium from women with and without a hereditary predisposition to develop female adnexal carcinoma.

AIM: Conflicting evidence exists on whether in vivo morphological characteristics can distinguish Ovarian Surface Epithelium (OSE) of ovaries obtained from women with and without a predisposition to develop female adnexal (ovarian and fallopian tube) carcinoma. This study aims to detect differences in growth potential and morphology that are maintained or specifically expressed in vitro. STUDY DESIGN: Ovarian surfaces were scraped to retrieve OSE cells from 56 women at hereditary high risk for female adnexal carcinoma, of whom 33 are BRCA1 and four are BRCA2 mutation carriers (Predisposed OSE, POSE) and from 26 women without such risk (Non Predisposed OSE, NPOSE). Number of passages and total cell yield until last passage, as well as morphology was compared between both groups. To confirm morphology, the expression of epithelial, mesothelial, and fibroblast markers was assessed. RESULTS: Both POSE and NPOSE cultures displayed similar growth potential and morphology. The expression of epithelial markers cyto-keratins 7 and 8 was similar between both groups. Only in cultures in which cells did not uniformly exhibit these markers, the percentage of cells expressing these markers was significantly lower at last passage when compared to the initial culture. In these latter cultures, cells that were morphologically indistinguishable from fibroblasts were observed. Mesothelial marker calretinin was expressed in 75% of cells of both POSE and NPOSE cultures and correlates with cyto-keratins 7 and 8 expression. CA 125 expression was equally low in POSE and NPOSE cultures (4.3%). Fibroblast markers FSM and vimentin were expressed in 100% and collagen IV was expressed in 16% of cells in all cultures. CONCLUSION: OSE cells derived from women with a hereditary predisposition to develop female adnexal cancer possess similar in vitro characteristics as OSE from women without this predisposition. On basis of our results, it seems advisable to study only 100% cyto-keratins 7 and 8 positive OSE cultures, since contamination of fibroblasts in some primary OSE cultures cannot be ruled out.