Loss of neurofibromatosis-1 and p19ARF cooperate to induce ... - Nature

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Oncogene (2002) 21, 4978 – 4982 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

Loss of neurofibromatosis-1 and p19ARF cooperate to induce a multiple tumor phenotype Dana King1, Genyan Yang1, Mary Ann Thompson2 and Scott W Hiebert*,1,3 1

Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, TN 37232, USA; 2Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee, TN 37232, USA; 3Vanderbilt-Ingram Center, Vanderbilt University School of Medicine, Nashville, Tennessee, TN 37232, USA

Inactivation of the neurofibromatosis-1 (NF1) gene deregulates RAS and cooperates with mutation or loss of the p53 tumor suppressor to induce tumorigenesis. p19ARF acts upstream of p53 in an oncogene checkpoint to induce apoptosis in response to activated RAS and other factors that stimulate proliferation. Therefore, we bred p19ARF7/7 to NF1+/7 mice to determine if loss of these genes collaborates in tumorigenesis. As expected from the embryonic lethality of NF1 null mice, no mice lacking both p19ARF and NF1 were born. Unexpectedly, the loss of one allele of NF1 did not greatly shorten the time to tumor formation in a p19ARF null background. The tumor types observed were characteristic of p19ARF null animals, not those associated with neurofibromatosis or those observed with NF1+/7/p53+/7 mice. However, seven out of 12 animals developed multiple tumors, some with metastases. This multiple tumor phenotype was not previously observed with p19ARF-null mice and suggests a distinct form of cooperation between the loss of these tumor suppressors. Oncogene (2002) 21, 4978 – 4982. doi:10.1038/sj.onc. 1205632 Keywords: NF1; p19ARF; cell cycle; oncogenesis; sarcoma; lymphoma

Introduction The p16INK4a/p19ARF locus encodes two tumor suppressors with distinct functions. Transcription of p19ARF initiates at an exon 5’ to exon 1 of p16INK4a, and the p19ARF specific first exon is spliced into an alternate reading frame in exon 2, which is shared between both proteins. Consequently, the p19ARF coding region is completely distinct from p16INK4a. In human tumors, the majority of mutations and deletions in this locus affect both genes or specifically target p16INK4a (Sherr and Weber, 2000). However, in mice, the specific deletion of either p16INK4a or p19ARF promotes *Correspondence: SW Hiebert, Department of Biochemistry, Vanderbilt University School of Medicine, PRB 512, 23rd and Pierce, Nashville Tennessee, TN 37232, USA; E-mail: [email protected] Received 12 February 2002; revised 24 April 2002; accepted 29 April 2002

tumorigenesis (Kamijo et al., 1997; Krimpenfort et al., 2001; Sharpless et al., 2001). p16INK4a is a cyclindependent kinase inhibitor that is a key component of the retinoblastoma tumor suppressor pathway. p19ARF acts in an oncogene checkpoint pathway to engage the p53 tumor suppressor in response to various oncogenes including the activation of RAS (Sherr and Weber, 2000). In addition, both p19ARF null and p53 null primary murine embryo fibroblasts fail to undergo replicative crisis and are immortal, and in transgenic mice expressing c-Myc, loss of p19ARF or p53 cooperates with c-Myc in tumorigenesis (Eischen et al., 1999). However, in cell culture models oncogenedependent induction of p53 and apoptosis can be independent of p19ARF (Nip et al., 2001). Type 1 neurofibromatosis is a relatively common genetic disorder characterized by inactivating mutations of neurofibromatosis-1 (NF1) that lead to a predisposition toward benign and malignant tumors whose origins are primarily from neural crest-derived tissues (Zhu and Parada, 2001). Mutation of the p16INK4a/p14ARF locus has been observed in up to 75% of malignant peripheral nerve sheath tumors (MPNSTs), which is associated with inactivation of NF1. Thus, loss of either p16INK4a or p14ARF (or both) cooperates with inactivation of NF1 in tumorigenesis (Zhu and Parada, 2001). NF1 is a negative regulator of RAS activity, acting to accelerate GTP hydrolysis on RAS and its related family members (DeClue et al., 1992). The RAS-GAP activity of NF1 is a key control point for RAS action, as haploinsufficiency of NF1 activates MAPK, presumably by de-regulating RAS (Gutmann et al., 1999; Hiatt et al., 2001; Ingram et al., 2000). For instance, loss of only one NF1 allele is sufficient to partially complement defects in coat color and mast cells in mice containing mutations in the c-Kit receptor tyrosine kinase (W41 mice) that attenuate RAS-dependent signaling (Ingram et al., 2000). Because p19ARF is upstream of p53 in a proposed oncogene checkpoint and the p16INK4a/p14ARF locus is deleted in tumors associated with loss of NF1, we determined whether loss of one allele of NF1 cooperates with complete inactivation of p19ARF by generating NF1+/7/p19ARF7/7 mice. In these animals the time to tumor formation and the tumor types observed were similar to those observed for p19ARF null mice. However, many of these animals displayed

NF1+/7/p19ARF7/7 mice are prone to multiple tumors D King et al

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multiple tumors of distinct origins, some of which were highly metastatic. Thus, loss of one allele of NF1 cooperates with deletion of p19ARF in tumorigenesis. Results and discussion To determine whether loss of p19ARF was equivalent to loss of p53 in response to an indirect activation of the RAS pathway, we bred mice heterozygous for NF1 with p19ARF null mice and followed littermates for up to 18 months for tumor formation. No mice deficient for NF1 or double deficient for NF1 and p19ARF were born, indicating an embryonic lethal phenotype for these mice. This phenotype was not analysed further. Compound heterozygotes and NF1+/7/p19ARF7/7 mice were born at the expected frequencies, although the litter sizes were smaller than expected, perhaps due to the death of the NF1 null embryos, which may have affected the development of other embryos. Twenty eight out of 43 NFI+/7/p19ARF7/7 mice that were prospectively followed for tumor formation died within one year of birth or were sacrificed due to the apparent presence of tumors or other pathologies (Figure 1). Twelve of these mice exhibited spontaneous tumor formation, whereas the cause of death could not be determined for several animals. Two animals were sacrificed due to deformities of the spinal column and one animal displayed developmental defects in the brain. In many animals splenomegaly was present which appeared to be due to extramedullary hematopoiesis, a finding also observed in p19ARF null mice. The median survival time for NF1+/7/p19ARF7/7 mice was 38 weeks as compared to 48 weeks for p19ARF7/7 mice. The average time to tumor formation for NF1+/7/p19ARF7/7 mice was 36 weeks, which is not significantly different from the 38 weeks reported previously for p19ARF7/7 mice (Kamijo et al., 1999).

Nearly half of the mice lacking p19ARF developed sarcomas, often of spindle-cell morphology. Another quarter of these animals developed lymphomas with characteristics of T cells. The remaining tumor types include carcinomas, and neurological tumors (Kamijo et al., 1999). Mice lacking one allele of NF1 are predisposed to neurofibromas, malignant peripheral nerve sheath tumors, pheochromocytoma, astrocytomas, and juvenile myelomonocytic leukemia (Brannan et al., 1994; Jacks et al., 1994; Largaespada et al., 1996; Side et al., 1998). The latency to tumor formation in NF1 heterozygous mice is well over one year and, in general, these tumors have lost the second NF1 allele. The majority of tumors formed in NF1+/7/p19ARF7/7 mice were similar to those observed previously for p19ARF7/7 mice, most developed spindle-cell sarcomas and/or lymphomas with two mice developing carcinomas and one animal a lymphoid leukemia (Table 1, and Figure 2). Similar to p19ARF7/7 mice the majority of these sarcomas displayed a spindle-cell morphology consistent with fibrosarcoma, with one tumor showing evidence of ossification indicative of an osteosarcoma. The lymphomas often involved the spleen, lymph nodes, and liver. A subset of these mice were analysed by paraffin immunohistochemistry, and for these tumors the neoplastic lymphocytes stained strongly with anti-CD45 antibody, but not anti-CD45R (B220, a B-cell marker). A Leder stain for myeloid cells failed to mark these cells. By inference these cells most likely represent a T cell lineage. An example is shown in Figure 2 (compare panels I and J) where CD45+ cells from a T cell leukemia have infiltrated the liver.

Table 1 Tumor development in NF1+/7/p19ARF7/7 and NF1+/7/ p19ARF+/7 mice Case number +/7

Sex

Week of onset

NF1 1 2 3 4 5

/p19 F M F M F

20 27 38 38 55

6 7 8 9 10 11 12

F M M F F F F

29 47 37 49 26 43 25

NF1+/7/p19ARF+/7 1 F 73 2 M 58 3 F 43 4 M 23 5 F 36 6 M 25 Figure 1 Survival plot of NF1+/7, p19ARF7/7, p19ARF+/7, NF1+/7/p19ARF7/7 and NF1+/7/p19ARF+/7 mice. Note that the survival of all mice analysed is shown, which includes those animals with confirmed tumors

7 8 9

Tumor type

ARF7/7

M M F

44 33 63

sarcoma sarcoma+lymphoma sarcoma+leukemia lymphoma sarcoma (mets. to lung)+undefined neoplasm sarcoma lymphoma sarcoma+lymphoma carcinoma sarcoma+lymphoma carcinoma (mets. to liver)+sarcoma sarcoma+lymphoma adenocarcinoma+pheochromocytoma sarcoma pheochromocytoma spindle cell sarcoma lymphoma spindle cell sarcoma+possible malignant peripheral nerve sheath tumor sarcoma+lymphoma sarcoma sarcoma+lymphoma Oncogene


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Figure 2 Morphology of tumors derived from the NF1+/7/ p19ARF7/7 mice that developed multiple tumors (case #5 panels A-F, case #3 panels G-J). Each panel shows an H&E stain of formalin fixed, paraffin embedded sections, except panel J, which shows immunohistochemical staining with anti-CD45, a lymphocyte marker. (a) 106 magnification of a section of the sarcoma found in the left leg, showing tumor cells infiltrating skeletal muscle. (b) 406 magnification of a section of the sarcoma in A. (c) 106 magnification of a portion of the lung showing metastatic sarcoma near a pulmonary vessel and bronchiole. (d) 406 magnification of a portion of the lung showing metastatic sarcoma near a pulmonary vessel. (e) 106 magnification of a section of a poorly differentiated neoplasm (left hand portion of panel) adherent to the stomach wall (right hand portion of the panel). (f) 406 magnification of a section of the poorly differentiated neoplasm near the stomach. (g) 106 magnification of a section of a sarcoma taken from case #3. (h) 406 magnification of a section of a sarcoma taken from case #3. (i) 106 magnification of the liver taken from case #3 showing infiltrating leukemia. (j) 106 magnification of a second section of the liver of case #3 showing CD45 staining of the leukemic cells

The most striking distinction between NF1+/7/ p19ARF7/7 mice and p19ARF7/7 mice was the development of a second independent tumor in seven out of 12 mice (Table 1, Figure 2). This multiple tumor phenotype was not observed in p19ARF7/7 mice unless these animals were mutagenized with dimethylbezanthrene (DMBA) (Kamijo et al., 1999). One of the Oncogene

targets of DMBA treatment is RAS in which DMBA causes oncogenic activation through point mutations (Quintanilla et al., 1986). Thus, the activation of RAS, either by DMBA-induced mutation or loss of NF1 function, may yield a similar phenotype. In addition, two of these mice displayed metastases of a sarcoma or carcinoma and the lymphomas often involved multiple organs. While p19ARF7/7 mice did not develop metastatic tumors, at least one p19ARF7/7 animal treated with DMBA exhibited a metastatic carcinoma (Kamijo et al., 1999). Therefore, our results with NF1+/7/p19ARF7/7 mice more closely resemble the phenotypes observed with DMBA treated p19ARF7/7 mice. Loss of heterozygosity (LOH) is observed in spontaneous tumors arising in NF1+/7 mice and loss of both alleles of NF1 and p53 cooperate in tumorigenesis (Cichowski et al., 1999; Vogel et al., 1999). Therefore, we performed Southern blot analysis on a sampling of tumors taken from NF1+/7/p19ARF7/7 mice using exon 33 as a marker for the retention of the wild-type NF1 allele. Although in some cases EcoRI failed to completely digest the DNA, all but one of the samples contained a similar ratio of wild-type versus targeted allele in control DNA from the tail and DNA extracted from the tumor (Figure 3a, left hand panel denoted with an arrow), indicating that the majority of these tumors retained the wild-type allele of NF1. The one sample that had lost the wild-type NF1 allele was from case 5, which developed metastatic sarcoma and a poorly differentiated neoplasm (Figure 2, compare panels b and f). While this analysis does not rule out point mutations or small deletions that would inactivate NF1, it appears that loss of only one allele of NF1 can cooperate with inactivation of p19ARF in tumorigenesis.

Figure 3 Southern blot analysis of tumor-derived DNA. (a) DNA obtained from the tail (control, C) and tumor (T) derived from p19ARF7/7/NF1+/7 (left hand panel) and p19ARF+/7/ NF1+/7 (right hand panel) mice. M, mutant marker; W, wildtype marker. The case number shown above the pairs of lanes correlates to the case number shown in Table 1. Radiolabeled NF1 exon 33 was used to probe EcoR1 digested genomic DNA. (b) Southern blot analysis of tail and tumor DNA samples from p19ARF+/7/NF1+/7. p19ARF exon 1b was used to probe AflII digested genomic DNA. Arrowheads indicate samples that have lost both alleles of either NF1 or p19ARF. Question marks indicate samples that may have lost both alleles of either NF1 or p19ARF


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While generating the NF1+/7/p19ARF7/7 mice, a large cohort of NF1+/7/p19ARF+/7 mice was generated as controls. The survival curve for these mice was not significantly different from p19ARF or NF1 heterozygotes until around one year at which time the mortality increased (Figure 1). The increase in mortality could be due to an increased propensity for loss of the second p19ARF or NF1 allele. By contrast, to the NF1+/7/p19ARF7/7 mice, the NF1+/7/p19ARF+/7 mice exhibited tumors that were characteristic of loss of either NF1 or p19ARF in that pheochromocytomas were observed in addition to sarcomas and lymphomas (Table 1). This result might suggest LOH at either the NF1 or p19ARF. Southern blot analysis of p19ARF exon 1b or NF1 exon 33, indicated that the wild-type NF1 allele was lost in three tumors (Figure 3a, right hand panel) while p19ARF was deleted in two samples and possibly one other (Figure 3b), which is consistent with LOH of either NF1 or p19ARF contributing to the disease. Only one tumor sample appeared to have retained one allele of both NF1 and p19ARF (Figure 3b, #6). p19ARF mediates an oncogene checkpoint in response to oncogenic RAS, factors that activate RAS (e.g., Abl) or oncogenic transcription factors such as c-Myc (Sherr and Weber, 2000). Expression of c-Myc in p19ARF null mice dramatically shortens the latency for c-Myc induced lymphomagenesis. This was attributed to an attenuated apoptotic response, perhaps due to a failure to induce p53 (Eischen et al., 1999). Expression of oncogenic RAS or c-Myc is sufficient to transform p19ARF-null primary cells (Lin and Lowe, 2001). There is compelling evidence that loss of one allele of NF1 is sufficient to maintain RAS in a hyperactive state (Ingram et al., 2000). However, NF1 haploinsufficiency did not greatly shorten the latency for tumorigenesis (Table 1). The different latency observed in Em-c-Myc transgenic mice and NF1+/7/ p19ARF7/7 mice may be due to the potent effects of c-Myc expression in B-cells or it may reflect qualitative differences in these two model systems. Seven out of the nine NF1+/7/p19ARF+/7 mice analysed exhibited sarcomas and lymphomas (Table 1). While neurofibromatosis type 1 is primarily associated with neurological tumors, NF1 mutation is also associated with juvenile myelomonocytic leukemia. Moreover, the loss of NF1 function stimulates lymphocyte proliferation (Ingram et al., 2000; Side et al., 1997; Zhang et al., 2001). Given that NF1 mutation has not been associated with the sarcomas observed

here, we speculate that the multiple tumor phenotype is due to cooperation between inactivation of NF1 and p19ARF in the hematopoietic compartment allowing the mice to develop lymphomas as well as sarcomas. This is consistent with the loss of the second NF1 allele in two lymphomas (Figure 3a). However, further analysis using tissue specific inducible strategies for gene deletion will be necessary to dissect these effects.

Materials and methods Mouse strains 129sv NF1 heterozygous mice were obtained from Dr Neal Copeland (Brannan et al., 1994 (NCI) and were bred with p19ARF null mice (129svj6C/57BL/6 background) that were purchased from Dr Charles Sherr, St. Jude Children’s Research Hospital (Kamijo et al., 1997). Genotypes were determined by polymerase chain reaction amplification of genomic DNA for NF1 using previously described primers (Brannan et al., 1994) and by Southern blot analysis for p19ARF exon 1b. The mice were examined daily for the presence of tumors or behavioral changes indicative of illness. Analysis and typing of tumors Mouse necropsy was performed by the Vanderbilt necropsy core laboratory. Tumors were either frozen for further analysis or fixed in formalin, embedded in paraffin and the tumor type defined by morphology after hematoxylin and eosin (H&E) staining. For lymphomas and leukemias, the formalin fixed paraffin embedded tissues were further typed using immunohistochemistry with anti-CD45, anti-CD34 (Research Diagnostics), and anti-CD45R (anti-B220, BDPharmingen). In addition, a Leder stain for myeloperoxidase was performed on selected sections. A sampling of tumors from NF1+/7/p19ARF7/7 and NF1+/7/p19ARF+/7 mice were analysed for the loss of the second NF1 allele by Southern blot analysis.

Acknowledgments We thank the members of the Hiebert lab and Wade Clapp for helpful discussions and encouragement, Drs Neal Copeland and Nancy Jenkins for the NF1 heterozygous mice, Yue Hou and Scott Luce for expert technical assistance, and the Vanderbilt-Ingram Cancer Center sequencing facility and mouse necropsy core for support. Special thanks to Kay Washington and Roy Jensen for aid in the pathological analysis of the mice. This work was supported by National Institutes of Health (NIH) grant RO1-CA87549, a Center grant from the National Cancer Institute (CA68485), the Vanderbilt-Ingram Cancer Center.

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