A systematic search for downstream mediators of tumor suppressor function of p53 reveals a major role of BTG2 in suppression of Ras-induced transformation

Abstract

Factors that mediate p53 tumor suppressor activity remain largely unknown. In this study we describe a systematic approach to identify downstream mediators of tumor suppressor function of p53, consisting of global gene expression profiling, focused short hairpin RNA (shRNA) library creation, and functional selection of genetic elements cooperating with oncogenic Ras in cell transformation. This approach is based on our finding that repression of gene expression is a major event, occurring in response to p53 inactivation during transformation and immortalization of primary cells. Functional analysis of the subset of genes universally down-regulated in the cells that lacked functional p53 revealed BTG2 as a major downstream effector of p53-dependent proliferation arrest of mouse and human fibroblasts transduced with oncogenic Ras. shRNA-mediated knockdown of BTG2 cooperates with oncogenic Ras to transform primary mouse fibroblasts containing wild-type transcriptionally active p53. Repression of BTG2 results in up-regulation of cyclins D1 and E1 and phosphorylation of Rb and, in cooperation with other oncogenic elements, induces neoplastic transformation of primary human fibroblasts. BTG2 expression was found to be significantly reduced in a large proportion of human kidney and breast carcinomas, suggesting that BTG2 is a tumor suppressor that links p53 and Rb pathways in human tumorigenesis.

Figure 1.

General outline of the study. See the explanation in the text.

Figure 2.

Subcloning and selection of cell cultures with properties of complete neoplastic transformation. (A) Cell morphology at day 14 after MEFs were transduced with indicated constructs and randomly subcloned into cell subcultures for further analysis of their properties. (B) Western blots of cellular lysates that indicate expression levels of GSE56, oncogenic Ras and its downstream target cyclin D1 in described cell cultures. (C) Doubling time and growth factor dependence of MEFs infected with GSE56 (clones 56SN1, 56SN5), GSE56-IRES-Ras (clones 56R1, 56R2, 56R4, 56R7) and with EGFP. (D) Growth of indicated MEF cell subcultures in the absence of anchorage support. (E) Ability of indicated cell subcultures to form tumors in vivo. Cells (5 × 10⁶) were injected into athymic nu/nu mice. Twenty-four days later mice were sacrificed and tumors were weighed.

Figure 3.

Microarray profiling of transformation mediated by suppression of p53 and overexpression of H-Ras^v12. (A) Hierarchical clustering of expression measurements from 1327 genes in MEFs with down-regulated p53 (56SN1, 56SN5) and in MEFs with down-regulated p53 that overexpress H-RAS^v12 (56R1, 56R2 56R4 56R7) and that show a consistent pattern of up- or down-regulation compared with primary MEFs. Each row represents a genetically modified MEF cell line and each column represents expression of a single gene across MEF specimens. Red indicates increased gene expression and blue indicates decreased gene expression relative to the median expression level in a primary MEF culture. The right panel shows the proportion of up- and down-regulated genes that exhibit the same pattern of regulation in hierarchically close MEF species. (B) Venn diagram of the number of genes whose suppression was common and unique between p53 dependent immortalization and transformation of MEFs. Down-regulated genes that exhibit the same pattern of regulation in hierarchically close transformed MEFs were intersected with down-regulated genes from immortalized MEFs to identify the targets whose inhibition was mediated only by repression of p53, as opposed to the genes that required additional activity of oncogenic Ras in order to be repressed.

Figure 4.

Comparison of behavior of the genes that become repressed after p53 knockdown in the presence or absence of oncogenic Ras among three types of cell cultures. (A) Relative expression of the genes that were commonly down-regulated in both immortalized and transformed cells. Values of expression are calculated relative to average gene expression level (dotted line) determined for all genes represented in the array. (B) Northern blot analysis of indicated genes that were down-regulated in MEFs in response to p53 suppression and independent of oncogenic Ras overexpression. (C) Relative expression of the genes that were down-regulated exclusively in transformed cells. Values of expression are calculated relative to average gene expression level (dotted line) determined for all genes represented in the array. (D) Northern blot analysis of indicated genes whose down-regulation was only specific for transformed MEFs where p53 inhibition was accompanied by overexpression of H-Ras^v12.

Figure 5.

shRNA-mediated knockdown of BTG2 or p53 allows Ras-mediated transformation of primary MEFs. (A) Primary MEFs were infected with a mixture as well as with individual shRNAs against candidate tumor suppressor genes; all cultures were then superinfected with LRasSN virus-containing supernatants and then seeded at 5 × 10⁵ cells per 100-mm plate. Infected populations were grown in the presence of 0.4 mg/mL of G418. Morphologically transformed colonies appearing 2 wk later were photographed, stained, and counted. (B) Real-time PCR analysis of BTG2 mRNA levels in cells expressing shRNAs for BTG2, p53, and GFP. (C) Semi-quantitative RT-PCR analysis of p53 mRNA levels in cells expressing shRNA for p53, BTG2, and GFP. (D) MEFs transformed by a combination of oncogenic Ras and shRNAs for the indicated genes were resuspended in normal culture medium containing 1.4% methyl cellulose and seeded at 10⁴ cells per well, and in duplicates into six-well plates coated with 1.2% low-melting-point agarose. Cultures were grown under standard tissue culture conditions, cells were fed twice weekly, and the number of macroscopically visible foci was scored after 3 wk. (E) The ability to form tumors in vivo was assessed by injecting 4 × 10⁶ transformed cells under the skin of both flanks of athymic nu/nu mice; growth of tumors was analyzed 18 d later. (F) Levels of BTG2 mRNA in MEFs expressing GSE56 + HRas, GSE56, or EGFP used for microarray analysis determined by real-time PCR. (G) p53 retains its transactivation ability in the cell with down-regulated BTG2 (results of detection of the activity of p53-responsive lacZ after induction with the indicated DNA-damaging drugs). (H) Western blot analysis of p21 expression in MEFs transduced with constructs expressing shRNAs against the indicated genes with or without oncogenic H-RAS^v12. β-Actin is used as loading control. (J) Semi-quantitative RT-PCR analysis of ARF mRNA levels in MEFs expressing shRNA against p53, BTG2 and GFP, with or without oncogenic H-RAS^v12.

Figure 6.

Inhibition of BTG2 rescues human diploid embryonic lung fibroblasts, IMR90, from oncogene-mediated growth arrest. (A) IMR-90 cells infected with shRNAs against BTG2, p53, and GFP were superinfected with lentivirus-expressing H-Ras^v12 + Neo^r cassette and selected in G418-containing media for 14 d, after which surviving resistant cells were fixed and stained with Methylene Blue. (B) Resistant IMR-90 cells expressing H-Ras^v12 + Neo^r and shRNAs for the indicated genes were stained for senescence-associated endogenous β-galactosidase activity and photographed. Results were calculated as the percentage of visible cells that had positive staining (at least five different fields were counted for each plate). (C) BTG2 expression is down-regulated in IMR-90 cells that express shRNA for BTG2 or p53 as measured by real-time PCR.

Figure 7.

BTG2 cooperates with p53 to prevent transformation of human IMR90 cells. (A) Primary IMR90 cells were sequentially infected to express a combination of oncogenic elements that are required but not sufficient for tumorigenic transformation (TERT/st/GSE56/Ras), and then super infected with shRNA lentiviral constructs against the indicated genes. Cells were grown in methyl cellulose and the number of macroscopically visible foci was scored after 3 wk. (B) Western blot analysis of genetically transduced IMR90 cells for the expression of the indicated proteins. (C) A model in which BTG2 integrates p53 and other signaling networks to control cell division. Cell proliferation and, ultimately, formation of malignant tumors largely depends on pRb phosphorylation status, which controls the activity of the E2F family of transcription factors necessary for induction of the genes that regulate DNA synthesis. Rb phosphorylation status is, in turn, mediated by CDKs bound to activating cyclins. Oncogenic and other stress signals that might lead to the formation of a malignant phenotype activate BTG2 through p53-dependent and p53-independent mechanisms leading to reduced expression of D- and E-type cyclins, hypophosphorylation of Rb, and the inhibition of cell growth.

Figure 8.

BTG2 expression analysis in human cancers using BD Clontech Cancer Profiling Array I, consisting of genetically matched cDNA pairs derived from tumor and adjacent normal tissue samples.