Gadd45b Is a Pro-Survival Factor Associated with Stress-Resistant Tumors
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Oncogene (2008) 27, 1429–1438 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Gadd45b is a pro-survival factor associated with stress-resistant tumors A Engelmann1, D Speidel1, GW Bornkamm2, W Deppert1 and C Stocking1 1Heinrich-Pette-Institut, Hamburg, Germany and 2GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Institut fu¨r Klinische Molekularbiologie und Tumorgenetik, Munich, Germany Tumors that acquire resistance against death stimuli recognized. It is now widely accepted that most cancer constitute a severe problem in the context of cancer therapies work by inducing the intrinsic apoptotic path- therapy. To determine genetic alterations that favor the way; however other mechanisms may also be involved, development of stress-resistant tumors in vivo, we took including activation of senescence, necrosis, mitotic advantage of polyclonal tumors generated after retro- catastrophe or autophagic cell death (Johnstone et al., viral infection of newborn Ek-MYC mice, in which the 2002; Okada and Mak, 2004). Altered expression or retroviral integration acts as a mutagen to enhance tumor mutation of genes encoding key proteins involved in any progression. Tumor cells were cultivated ex vivo and of these processes can provide cancer cells with both an exposed to c-irradiation prior to their transplantation into intrinsic survival advantage and inherent resistance to syngenic recipients, thereby providing a strong selective therapeutic-induced stress. pressure for pro-survival mutations. Secondary tumors To identify novel genes and thus mechanisms by developing from stress-resistant tumor stem cells were which tumor cells escape stress response, we have deve- analysed for retroviral integration sites to reveal candi- loped an approach in which tumor growth is accelerated date genes whose dysregulation confer survival. In addi- by retroviral insertional mutagenesis, and subclones tion to the gene encoding the antiapoptotic Bcl-xL protein, that have acquired stress resistance are selected. This we identified the gadd45b locus to be a novel common approach takes advantage of the fact that retroviral integration site in these tumors, leading to enhanced infection of transgenic mice expressing the human MYC expression. In accord with a thus far undocumented role of oncogene under control of the Igl enhancers, mimicking Gadd45b in tumorigenesis, we showed that NIH3T3 cells human Burkitt’s lymphoma (Kovalchuk et al., 2000), overexpressing Gadd45b form tumors in NOD/SCID mice. leads to the rapid development of polyclonal tumors. Interestingly and differently to other known ‘classical’ Retrovirus infection accelerates tumor progression by antiapoptotic factors, high Gadd45b levels did not protect integration in the vicinity of oncogenes and/or tumor against MYC-, UV- or c-irradiation-induced apoptosis, but suppressor genes and their subsequent activation or conferred a strong and specific survival advantage to serum inactivation, conferring a selective advantage and withdrawal. subsequent outgrowth of the affected cell (Mikkers Oncogene (2008) 27, 1429–1438; doi:10.1038/sj.onc.1210772; and Berns, 2003). In the approach developed here, the published online 24 September 2007 tumor cells are subsequently subjected to stress factors (ex vivo cultivation and g-radiation) prior to transplan- Keywords: B-cell lymphoma; MYC; p53; Bcl-xL; retroviral tation into recipient mice, thus providing a strong insertional mutagenesis; stress-resistance selective pressure for tumor cells with integrations that confer pro-survival mutations. The strength of this approach was demonstrated by identifying the known antiapoptotic factor Bcl-xL, but also identifying Introduction Gadd45b as a pro-survival factor in this system. Recent studies using gadd45b knockout mice have suggested Despite considerable progress over the past fifty years an antiapoptotic function for Gadd45b (Gupta et al., in the treatment of human malignancies, intrinsic 2005). Extending these studies, we provide evidence that and acquired resistance to chemotherapeutic agents normal levels of Gadd45b may be critical to prevent and radiation remains a severe problem (Bernier et al., apoptosis, but increased levels of Gadd45b contribute 2004; Chabner and Roberts, 2005). Our understanding to tumorigenesis and tumor resistance by a mechanism of mechanisms leading to therapeutic resistance has distinct to classical apoptosis. been expanded considerably during this time, and the crucial role of stress resistance pathways is increasingly Results Correspondence: Dr C Stocking, Molecular Pathology, Heinrich- Mouse model for the identification of tumor-related Pette-Institut, PO Box 201652, D-20206 Hamburg, Germany. E-mail: [email protected] pro-survival genes Received 27 March 2007; revised 11 July 2007; accepted 6 August 2007; To define genetic alterations that contribute to published online 24 September 2007 development of stress-resistant hematopoietic tumors Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1430 in vivo, we extended the method of retroviral insertional show that infection of newborn El-MYC mice with mutagenesis in a mouse model for leukemia by adding Moloney-murine leukemia virus (Mo-MuLV) enhanced an additional selection step. In the first step, we could tumor progression and decreased the median survival Oncogene Pro-survival function of Gadd45b in tumorigenicity A Engelmann et al 1431 time of the mice from 100 to 49 days (Figure 1a). Similarly to uninfected controls, the mice succumbed to an aggressive B-cell (B220 þ IgM þ ) or pro-B-cell (B220 þ IgMÀ) neoplasia, characterized by lymphoma and hepatosplenomegaly, as well as high leukocyte counts in the peripheral blood (mean 114 Â 106 cells mlÀ1, n ¼ 44). The accelerated tumor progression can be attributed to additional oncogenic mutations caused by retrovirus integration. To specifically select for tumor subclones in which mutations imparted a pro-survival advantage to stress stimuli, we explanted the tumors, cultivated the tumor cells ex vivo and exposed them to 7 Gy g-radiation, prior to their transplantation into syngenic recipient animals. Such treatments combine several stress stimuli and eliminate the vast majority of tumor cells by apoptosis as indicated by fragmentation of DNA and the presence of active cleaved caspase-3 (Figures 1b and c). Although irradiation clearly provided the major death stimulus, ex vivo cultivation alone was sufficient to provoke a measurable apoptotic response in some tumor cells within a short time, indicating the sensitivity of primary tumor cells to non-genotoxic environmental changes. In concordance with the massive cell death upon irradiation, tumor-initiating cells giving rise to second- ary tumors were detected at a frequency four orders of Figure 2 Secondary tumors arising from irradiated tumors have magnitude lower than that of non-irradiated controls, acquired mutations conferring radiation resistance. (a) Schematic representation of experimental design to test if tumors selected as determined by serial dilution (Figure 1d). Southern after radiation are less sensitive to stress signals. (b) Survival curves blot analysis performed with a Mo-MuLV env probe of mice transplanted with 5 Â 105 secondary tumor cells that were confirmed that the stress treatment induced a strong either irradiated (lightning bolt and solid lines) or untreated selection for specific clones; the viral integration pattern (dashed line). Two sets of secondary tumors were tested: tumors changed significantly from a predominantly diffuse generated from irradiated primary tumors (gray lines) or non- irradiated primary tumors (black lines). Results depicted are a picture in the parental primary tumor (P), indicative of compilation of five experiments with independent primary tumors, a highly polyclonal tumor, to a more distinct band for which three to four mice were used for each experimental pattern in the different, primarily monoclonal, second- condition (total of 68 mice). ary tumors (S1–S9) (Figure 1e). Of note, some bands not prominently appearing in the parental tumor could resistance (secondary tumors derived from irradiated be detected in several of the different independent primary cells), tumor growth was significantly faster and secondary tumors, with specific differences between the with a higher penetrance (95 versus 50%) than that of sets of non-irradiated and irradiated secondary tumors. irradiated tumor cells derived from secondary tumors This finding confirms that both treatments, ex vivo culti- from non-irradiated primary cells (Figure 2b). These vation alone and ex vivo cultivation plus g-irradiation, results demonstrate that tumor cells had been selected provide a distinct selection pressure, selecting for that were significantly less sensitive to irradiation different tumor cell clones. treatment. To determine if spontaneous mutations A second round of radiation and re-transplantation of within the Trp53 gene were responsible for the acquired both irradiated and non-irradiated secondary tumors stress resistance, sequence analysis was performed. Only was performed to verify the selection for stress-resistant 25% of the tumors carried point mutations in the p53 tumors (Figure 2a). Although irradiation still delayed coding region, in agreement with the low incidence of the in vivo growth of tumors cells selected for stress p53 inactivation in Mo-MuLV-induced murine and Figure 1 Mouse model to identify pro-survival genes. (a)