This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bai, F.
Right arrow Articles by Xiong, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bai, F.
Right arrow Articles by Xiong, Y.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, February 2007, p. 1495-1504, Vol. 27, No. 4
0270-7306/07/$08.00+0     doi:10.1128/MCB.01764-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

p18Ink4c, but Not p27Kip1, Collaborates with Men1 To Suppress Neuroendocrine Organ Tumors{triangledown}

Feng Bai,{dagger} Xin-Hai Pei,{dagger} Toru Nishikawa,{ddagger} Matthew D. Smith, and Yue Xiong*

Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295

Received 18 September 2006/ Returned for modification 22 October 2006/ Accepted 25 November 2006


arrow
ABSTRACT
 
Mutant mice lacking both cyclin-dependent kinase (CDK) inhibitors p18Ink4c and p27Kip1 develop a tumor spectrum reminiscent of human multiple endocrine neoplasia (MEN) syndromes. To determine how p18 and p27 genetically interact with Men1, the tumor suppressor gene mutated in familial MEN1, we characterized p18-Men1 and p27-Men1 double mutant mice. Compared with their corresponding single mutant littermates, the p18–/–; Men1+/– mice develop tumors at an accelerated rate and with an increased incidence in the pituitary, thyroid, parathyroid, and pancreas. In the pituitary and pancreatic islets, phosphorylation of the retinoblastoma (Rb) protein at both CDK2 and CDK4/6 sites was increased in p18–/– and Men1+/– cells and was further increased in p18–/–; Men1+/– cells. The remaining wild-type Men1 allele was lost in most tumors from Men1+/– mice but was retained in most tumors from p18–/–; Men1+/– mice. Combined mutations of p27–/– and Men1+/–, in contrast, did not exhibit noticeable synergistic stimulation of Rb kinase activity, cell proliferation, and tumor growth. These results demonstrate that functional collaboration exists between p18 and Men1 and suggest that menin may regulate additional factor(s) that interact with p18 and p27 differently.


arrow
INTRODUCTION
 
Retinoblastoma family proteins, Rb, p107, and p130, collaboratively regulate G1 cell cycle progression and maintain cells at quiescent G0 state (34). When present in hypophosphorylated states, these proteins bind to E2F transcription factors and repress the transcription of various E2F target genes, thereby preventing S phase entry. Mitogen-induced expression of D-type cyclins activates cyclin D-dependent CDK4 and CDK6 kinases, leading to phosphorylation and functional inactivation of Rb proteins. Conversely, binding with an INK4 protein or depriving cyclin D synthesis inhibits CDK4/6, retaining Rb proteins in their growth-suppressive states and preventing the G1-to-S transition. Functional inactivation of this pathway, consisting of INK4-cyclin D/CDK4/6-Rb-E2F, is a common event for the development of most types of cancer (29).

A long-standing puzzle from the studies of mice is that, despite the fundamental function of the Rb pathway in cell cycle control and frequent mutations in many different types of human cancers, mice with mutations impairing the Rb pathway develop tumors predominantly in neuroendocrine organs. Characteristic intermediate lobe pituitary tumors develop in mice heterozygous for Rb (12, 17), chimeric for Rb–/– (19, 25, 35), and deficient for CDK inhibitors p18Ink4c (8, 9, 16) and p27Kip1 (7, 15, 24). Furthermore, mice with compounded mutations in both Rb1 and p53 genes develop tumors with high penetrance in the pituitary, pancreatic islets, and thyroid (5, 10, 35). More strikingly, mice carrying a INK4-insensitive mutation (R24C) in CDK4 or mice carrying simultaneous deletions of both the CDK inhibitors p18Ink4c and p27Kip1 develop tumors in multiple endocrine organs, including the pituitary, thyroid, parathyroid, adrenal glands, pancreas, and testis (9, 30), a tumor spectrum that is congruous with the human multiple endocrine neoplasia (MEN) syndrome. Many major tumor types which sustain frequent mutations targeting the genes on the Rb pathway in humans, such as glioblastoma and cancer of the breast, lung, skin, bone, head, and neck, are rarely observed in mice with mutations targeting the Rb pathway.

Endocrine glands include seven so-called classical tissues or organs (pituitary, thyroid, parathyroid, pancreatic islets, adrenals, testes, and ovaries), as well as various other tissue systems (e.g., lung and gastrointestinal carcinoid) that are not usually considered endocrine glands but that contain cells that produce and secrete hormones, cytokines, and secondary messengers (31). Clinical diagnoses of hereditary MEN syndromes include the multiplicity of tumors in a given endocrine organ and the multiplicity of endocrine organs developing tumors in an individual. The recognition of Mendelian inheritance within MEN syndromes has led efforts over the past decade to identify causal genes by linkage analysis and positional cloning, including the type 1 MEN gene, Men1, which encodes a nuclear transcriptional regulator protein, known as menin (4). Despite the widespread expression of menin in nearly all tissues during embryonal development and in adults (32), loss of function of the Men1 gene, perplexingly, causes a unique and restricted pattern of endocrine tumors in both humans and mice. Germ line mutations in Men1 predispose humans to endocrine tumor development, predominantly in the anterior lobe of pituitary, parathyroid, and pancreatic islet cells (21). Likewise, mice heterozygous for Men1 developed multiple endocrine tumors with a spectrum very similar to human Men1 kindred (2, 6). Both the biochemical function of menin and the cellular pathway that Men1 regulates are currently under investigation. In particular, how Men1 may genetically interact with other genes involved in tumor suppression has not been examined.

Development of multiple neuroendocrine tumors in p18-p27 double mutant mice led us to determine how Men1 genetically interacts with p18 and p27. These genetic, histological, and pathological studies revealed that p18, but not p27, genetically collaborates with Men1 to suppress the development of multiple types of endocrine tumors.


arrow
MATERIALS AND METHODS
 
Mouse strains. The generation of p18 and Men1 mutant mice and conditions for genotyping were described previously (6, 8), and both p18 and Men1 mutant mice have been backcrossed into, and maintained on, a C57BL/6 background. Mice deficient for p18 were bred with mice heterozygous for Men1 to create doubly heterozygous mice. Mice heterozygous for both p18 and Men1 were then intercrossed to generate all of the genotypes analyzed in this study. Animals were genotyped by PCR and monitored as described previously. Cohorts were housed and analyzed in a common setting, and littermate controls were used for all experiments as indicated below. All mice were sacrificed and subjected to complete necropsy as age-matched controls. The mice were mainly divided into five groups based on different time points (3 months, 6 to 9 months, 9 to 12 months, 12 to 16 months, and 16 to 22 months of age) (Table 1). Most groups of mice younger than 1 year of age had at least three mice for each genotype, except for a few groups from wild-type and p27–/– mice, some of which contained two mice.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Spontaneous tumor formation and incidence in p18–/–, p27–/–, Men1+/–, p18–/–, Men1+/–, and p27–/–; Men1+/– micea

Histopathology and IHC. Tissues of most organs were removed, fixed in 10% neutral buffered formalin, and examined histologically by two pathologists after hematoxylin-eosin staining. Lesions were photographed, and additional sections were taken for immunohistochemical analyses. Immunohistochemistry (IHC) was performed as described previously (1). To measure proliferating and mitotic cells, sections were blocked with normal goat serum in phosphate-buffered saline and incubated with either a polyclonal antibody against mitosis-specific phosphorylated histone H3 (5 µg ml–1; Upstate Biotechnology) or a polyclonal antibody against Ki67 (NCL-Ki67 at a dilution of 1:1,000; Novocastra Laboratories) for 1 h and with biotin-conjugated secondary antibody (Vector Laboratories) for 30 min. Other primary antibodies used were as follows: to insulin (Dako Cytomation), to phospho-Rb(Ser608) (Cell Signaling), to phospho-Rb(Thr821) (Biosource), and to menin (Bethyl). Immunocomplexes were detected using the Vectastain ABC alkaline phosphatase kit according to the manufacturer's instructions (Vector Laboratories) or using rhodamine-conjugated secondary antibody. For apoptosis assays, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays were carried out using an in situ ApopTag kit (Chemicon) according to the manufacturer's protocol.

LCM and loss-of-heterozygosity (LOH) analysis. Laser capture microdissection (LCM) was used to obtain pure cell populations of selected areas from formalin-fixed, paraffin-embedded tissue sections. Ten-micrometer sections were deparaffinized and lightly stained with hematoxylin. Using a PixCell lle Laser Capture Microdissection system (Arcturus, Mountain View, CA), the lesions that were clearly separated from normal tissues were isolated from the slides. Particular care was taken to avoid contamination by surrounding tissue. DNA isolation from the microdissected tissue samples was performed as described before (1).

Pancreatic islet size determination. Pancreatic islet size (number of cells/islet section) was measured as previously reported (20). Briefly, islet size was determined in at least three cut sections from matched pancreatic regions of three animals per genotype at each stage of development. Sections were more than 500 µm apart from each other to avoid the overestimation of large islets in this analysis. For pancreatic islet number counting, the whole pancreas was dissected together with the surrounding spleen and stomach tissue to preserve the shape of the pancreas during subsequent procedures of fixation and paraffin embedding. Dissected pancreases were flatly embedded, with similar orientations and maximized surface areas showing. Islet number per section was determined from age-matched pancreases of three animals per genotype and averaged from at least six sections (two each from the top, the center, and the bottom of the pancreas).


arrow
RESULTS
 
p18 collaborates with Men1 in tumor suppression. Men1–/– mice die in utero (between embryonic day 11.5 to 13.5), and Men1+/– mice develop endocrine tumors later in their life with a spectrum similar to those in MEN1 patients (2, 6). While conditional alleles have been generated to allow tissue-specific homozygous deletion of Men1, we performed our initial studies in Men1+/– mice to gain an unbiased assessment of the functional in vivo interaction between Men1 with p18 and p27 in diverse tissues. To generate mice of several genotypes, Men1+/– mice were crossed with p18–/– or p27–/– mice. The double-heterozygote p18+/–-Men1+/– and p27+/–-Men1+/– mice were mated to generate mice with the following genotypes: p18–/–; Men1+/–, p18+/–; Men1+/–, p18+/+; Men1+/–, p18–/–; Men1+/+, p27–/–; Men1+/–, p27+/–; Men1+/–, p27+/+; Men1+/–, and p27–/–; Men1+/+. Genotype analysis of more than 250 offspring did not identify any p18–/– or p27–/–; Men1–/– double null mice, nor were there any viable p18–/–; Men1–/– or p27–/–; Men1–/– embryos beyond embryonic day 15.5 (data not shown). These results indicate that homozygous deletion of p18 or p27 did not rescue the embryonic lethality caused by Men1 loss. p18–/–; Men1+/–, p27–/–; Men1+/–, and Men1+/– mice developed normally and did not show significant phenotypic abnormalities at a young age (<3 months) but subsequently developed various hyperplastic and tumor phenotypes.

Consistent with previous reporting, histological and pathological examination revealed that specific lesions developed predominantly in multiple endocrine organs of p18, p27, and Men1 single mutant mice, including the pituitary, thyroid, parathyroid, pancreas, adrenal, and testis. Compared to these single mutant mice, tumor phenotypes in nearly all these endocrine organs were accelerated in double mutant p18–/–; Men1+/– mice but not in p27–/–; Men1+/– mice (Table 1 and Fig. 1) (see also Fig. 2 to 4).


Figure 1
View larger version (50K):
[in this window]
[in a new window]

  		FIG. 1. p18, not p27, collaborates with Men1 to suppress pituitary tumors. Age-matched pituitary glands from different genotypes of mice were microscopically examined after hematoxylin-eosin staining. Anterior lobe (A), intermediate lobe (I), neurohypophysis (N), and tumor (T) are indicated. Note the tumors in both intermediate and anterior lobes of p18–/–-Men1+/– pituitary.


Figure 2
View larger version (52K):
[in this window]
[in a new window]

  		FIG. 2. p18, not p27, collaborates with Men1 to suppress testis, thyroid, and parathyroid tumors. (A) Age-matched testis glands from different genotypes of mice were microscopically examined after hematoxylin-eosin staining. Leydig cell hyperplasia (H) and Leydig cell tumor (T) are indicated. (B) hematoxylin-eosin staining of thyroid (Th) and parathyroid (Pth) from different genotypes of mice at 12 months of age. Hyperplasia (H) and tumor (T) are indicated. Frame e shows a parathyroid tumor, and frame f shows a thyroid tumor from the same p18–/–-Men1+/– mouse (frame d is the same area at lower magnification).


Figure 4
View larger version (91K):
[in this window]
[in a new window]

  		FIG. 4. Combined p18 and Men1 mutations increased cell proliferation and apoptosis. Sections from pituitary glands and pancreases from different genotypes of mice at 9 to 12 months of age were examined for cell proliferation by immunostaining with an antibody recognizing phosphorylated histone H3 (A and C) and for apoptotic cell death by TUNEL assay (B and D). At least three mice for each genotype were examined. Pancreatic islets in the top row (A) are magnified in the respective insets. Blue, cells positive for PH3; brown, cells positive in TUNEL assay. Positive cells for PH3 and TUNEL were counted in 10 randomly chosen fields. The P value is less than 0.05 for p18–/–; Men1+/– versus either p18–/– or Men1+/– samples and larger than 0.05 for p27–/–; Men1+/– versus either p27–/– or Men1+/–.

p18, not p27, collaborates with Men1 to suppress both intermediate and anterior lobe pituitary tumors. Before 1 year of age, 2 of 13 (15%) p18–/– mice developed pituitary tumors in the intermediate lobe. Thereafter, four of six (67%) developed intermediate lobe pituitary tumors. At similar stages, the incidence of intermediate lobe tumor was increased to 8 of 20 (40%) and 5 of 5 (100%) in p18–/–; Men1+/– mice (Table 1). Although there was statistically no significant difference in tumor incidence between these two mutant mice, intermediate lobe tumors were detected in p18–/–; Men1+/– mice at as early as 3 months of age (40%; n = 5) while no such tumor was observed in p18–/– mice at a similar age. In addition, pathologically more aggressive features (see below) were frequently found in p18–/–; Men1+/– tumors than in p18–/– tumors (Fig. 1). These results reveal a previously unrecognized function of Men1 in the intermediate lobe of the pituitary.

Anterior lobe pituitary tumors were not found in the p18–/– mice up to 22 months of age. In Men1 heterozygotes, only 1 of 18 (6%) developed pituitary tumors in the anterior lobe at 1 year of age, a frequency that increased to 3 of 7 (43%) in mice older than 1 year. Anterior lobe pituitary tumors were found in 20% of p18–/–; Men1+/– mice (n = 20) prior to 1 year and 80% (n = 5) thereafter. In these animals, we also noted the formation of adenomas and adenocarcinomas in both anterior and intermediate lobes. Although developed at similar incidences, the anterior lobe pituitary tumors in the p18–/–; Men1+/– mice exhibited aggressive features, including hemorrhaging, necrosis, and invasion into adjacent tissues (Fig. 1), that were not noted in either p18–/– or Men1+/– mice. This result indicates that p18, in addition to suppressing intermediate lobe pituitary tumors, also plays a role in suppressing anterior lobe pituitary tumors. By contrast, the frequency of pituitary tumors did not differ significantly between p27–/–; Men1+/– and p27–/– single mutant mice (n = 12) (data not shown). Collectively, these results demonstrate a functional collaboration between Men1 and p18 but not between Men1 and p27 in suppression of pituitary tumors.

p18, not p27, collaborates with Men1 to suppress testis Leydig cell tumors. Before 1 year of age, no testis Leydig cell tumors were found in any mutant mice lacking p18 or p27 or in Men1 heterozygotes. Although half of the p18 null mice (after 1 year) developed evident hyperplasia in the testis (Fig. 2A), no Leydig cell tumors were found in p18–/– mice, even in mice 2 years of age (Table 1 and Fig. 2A). When the mice aged beyond 1 year, two of nine (22%) Men1+/– mice developed Leydig cell adenoma in the testis and three of six p18–/–; Men1+/– male mice (50%) developed Leydig tumors (one adenoma and two adenocarcinomas; both adenocarcinomas metastasized to the lung). No Leydig cell hyperplasia or tumors were detected in p27–/– mice, even in mice 2 years of age (Table 1), nor was there any evidence that p27 loss accelerates the Leydig tumor phenotype in Men1+/– mice (Fig. 2A). These results also provide evidence supporting a functional collaboration between p18, but not p27, and Men1 in suppressing Leydig cell tumors in the testis.

p18, not p27, collaborates with Men1 to suppress thyroid and parathyroid tumors. Neither p18–/–, p27–/–, nor Men1+/– mice developed a significant tumor phenotype in the thyroid up to 22 months of age. In the p18–/–; Men1+/–mice, 4 of 5 (80%) developed thyroid adenoma by the age of 22 months, while only 1 of 16 (6%) p27–/–; Men1+/– mice developed thyroid tumors at 12 months of age (Table 1 and Fig. 2B). Ninety percent of MEN1 patients over 40 years old have parathyroid tumors, making this the most common tumor of this syndrome in humans (21). In Men1+/– mice, 3 of 12 (25%) developed detectable parathyroid tumors by the age of 22 months. p18 loss alone did not result in parathyroid tumors but increased the incidence of parathyroid adenomas from 25% in Men1+/– mice to 80% in p18–/–; Men1+/– mice (n = 5). All parathyroid and thyroid adenomas developed concurrently in p18–/–; Men1+/– mice (Fig. 2B). The underlying basis for this concurrent parathyroid and thyroid tumor formation in p18–/–; Men1+/– mice is not clear. As for pituitary tumor development, p27 deficiency alone did not result in parathyroid tumors, nor did it increase the incidence of parathyroid tumors in Men1+/– background mice (Table 1).

p18, not p27, collaborates with Men1 to suppress pancreatic islet insulinomas. Both p18–/– and Men1+/– mice developed an islet ß-cell tumor phenotype in the pancreas before 1 year of age. (2, 6, 26) (Table 1). Islet tumors developed in 15% (n = 13) of p18–/– and 22% (n = 18) of Men1+/– mice between 9 to 12 months of age; the incidence was increased to 45% (n = 20) in p18–/–; Men1+/– mice, and the onset time was shortened to as early as 3 months of age (Table 1 and Fig. 2). After 12 months of age, islet tumors were found in all p18–/–; Men1+/– mice (n = 5), compared to 33% (n = 6) of p18–/– mice and 43% (n = 7) of Men1+/– mice (Table 1). Despite their hyperplastic or dysplastic appearance, islet tumors were highly immunoreactive for insulin (Fig. 3A), consistent with the ß-cell origin of these tumors. Detailed analysis revealed that tumor masses were comparably enlarged in p18–/–; Men1+/– pancreases. Two of five insulinomas that developed in older (12 to 22 months) p18–/–; Men1+/– mice had features of multifocal islet carcinomas (Fig. 3A), while no carcinomas were found in mice of other genotypes. In contrast to these effects from p18 mutation, loss of p27 alone did not result in islet hyperplasia (7, 15, 24) (Table 1), nor did p27 deficiency have any additional effects on islet hyperplasia and tumor frequency (Table 1 and Fig. 3A).


Figure 3
View larger version (42K):
[in this window]
[in a new window]

  		FIG. 3. p18, not p27, collaborates with Men1 to suppress insulinoma development and to regulate pancreatic ß islet cell proliferation. (A) Results of hematoxylin-eosin staining of pancreatic sections obtained from different genotypes of mice at 12 months of age are shown (top). The islet cell carcinoma shown in frame f has been magnified in the inset. Mitotic figures (black arrows) and tumor cells invading a blood vessel (yellow arrow) are indicated. Pancreatic sections from 12-month-old mice of the indicated genotypes were stained with an antibody to insulin (bottom). (B) The number of pancreatic islets per section was determined as described in Materials and Methods from 12-month-old mice of the indicated genotypes. (C) A total of 250 islets from wild-type and mutant pancreases from mice at 1 year of age were counted. Islets are categorized into three groups, small (containing less than 40 cells), medium (containing 41 to 400 cells), and large (containing more than 400 cells), and percentages of each group are plotted.

p18, not p27, collaborates with Men1 to regulate islet cell proliferation and size. To further examine how Men1 differentially interacts with p18 and p27 in regulating pancreatic islet ß cells, we determined both the number and size of islets in mice of different genotypes. The number of islets was increased on the average of 43 ± 17 per section in p18–/– and 46 ± 18 in Men1+/– to 70 ± 14 in p18–/–; Men1+/– mice. Although p27 deletion slightly increased the number of islets from 28 ± 7 in wild-type mice to 34 ± 6, p27–/–; Men1+/– mice actually had a decrease in the number of islets to 24 ± 5 (Fig. 3B). The basis for this decrease is not clear at present.

There is an evident increase in the size of islets in p18–/–; Men1+/– mice. There could be two different cellular mechanisms accounting for the increase in islet size: an increase in the size and/or the cell number of islet ß cells. Microscopic examination did not show any visible difference in cell size. We therefore counted the cell numbers in 250 randomly chosen islets from mice of six different genotypes and divided them into three groups: small (less than 40 cells per islet), medium (40 to 400 cells), and large (more than 400 cells) islets. The percentage of medium- and large-sized islets increased from 25% ± 8% and 21% ± 11% in p18–/– mice and 22% ± 6% and 26% ± 8% in Men1+/– mice to 26% ± 2% and 40% ± 6% in p18–/–; Men1+/– mice, respectively (Fig. 3C). By contrast with Men1+/– mice, the p27–/–-Men1+/– mice, while having a slight increase in medium islets (30% ± 2%), had a substantially decreased number of large islets (5% ± 1%). Altogether, these data suggest that p18, but not p27, functionally collaborates with Men1 to control ß islet cell number and to suppress insulinoma development in the pancreas.

Increased proliferation and apoptosis in pituitary and pancreatic tumors of p18–/–; Men1+/– mice. To determine the cellular basis for the functional collaboration of p18 and Men1, we examined two cellular processes that are often deregulated during tumor growth—cell proliferation and apoptosis. Mitotic index, as determined by the immunostaining of anti-phospho-histone H3 (PH3), was low at baseline in the wild-type pancreases and pituitaries but increased in all three single mutant mice (Fig. 4A). Consistent with pathological analysis, there were further substantial increases of the mitotic index in both p18–/–; Men1+/– pancreases and pituitaries but not in p27–/–; Men1+/– pancreases and pituitaries.

The increase in cell proliferation following loss of p18, p27, and Men1 correlated with an increased level of apoptosis in the pituitary and pancreas, as assessed by TUNEL assay. A higher number of TUNEL-positive cells was detected in p18–/–; Men1+/– pancreases and pituitaries than in either p18–/– or Men1+/– mice (Fig. 4B). There was no detectable increase in apoptotic cells in either p27–/–-Men1+/– pancreases or pituitaries.

p18 deficiency protects the loss of wild-type Men1 allele. MEN1 patients typically inherit loss-of-function mutations in the Men1 gene, and tumors arise after loss of the remaining wild-type allele, which appears to fit the classical definition of a "two-hit" tumor suppressor. Development of tumors from Men1 heterozygotes offered an opportunity to address the issue of Men1 haploinsufficiency in tumor suppression. We took two approaches to this end: IHC of Men1 and LOH analysis of the remaining Men1 wild-type alleles. As determined by IHC, menin expression was lost in most pancreatic tumors (five of seven) and all pituitary (three of three) tumors that we examined (Fig. 5A and C), indicating that the remaining wild-type Men1 allele was lost in most tumor cells. In contrast, menin protein expression was retained in most pancreatic (9 of 10) and pituitary (5 of 6) tumors developed in p18–/–; Men1+/– mice, suggesting that loss of the wild-type Men1 allele in p18–/–; Men1+/– mice is protected by p18 loss.


Figure 5
View larger version (46K):
[in this window]
[in a new window]

  		FIG. 5. p18 deficiency protects loss of the wild-type Men1 allele. (A) Sections of pancreatic tumors and pituitary tumors derived from Men1+/– and p18–/–; Men1+/– mice were immunostained with an antibody against menin. Note the strong menin staining in the tumors from p18–/–; Men1+/– mice and very faint (or negative) menin staining in the tumors from Men1+/– mice. Frames a and b, pancreatic tumors at a magnification of x100 (inset, x400); frame c, pituitary anterior lobe tumor from Men1+/– mouse at a magnification of x100 (inset, x400); frame d, pituitary intermediate lobe and anterior lobe tumors from p18–/– Men1+/– mouse at a magnification of x100 (inset x400). (B) DNA extracted from the microdissected tumors of different genotypes was amplified by PCR to detect wild-type (wt) and mutant (mt) alleles of Men1 and p18, respectively. Lane 1, tail DNA; lanes 2 and 3, pituitary tumors; lanes 4 to 8, pancreatic tumors. (C) Summary of loss of menin expression by IHC and of wild-type Men1 allele by PCR analysis for tumors from the pancreas and pituitary. The results are shown as the number of menin-negative staining tumors or absence of wild-type Men1 allele divided by the number of total samples examined.

To further confirm this, we conducted LCM and genomic PCR. DNA was extracted from the tumor tissues under the microscope, and PCR-based LOH analysis was performed. Four of five pancreatic islet tumors and three of three pituitary tumors developed in Men1+/– mice exhibited loss of the wild-type Men1 allele and retention of the null allele, whereas all six pancreatic tumors and three of four pituitary tumors derived from p18–/–; Men1+/– mice retained the wild-type Men1 allele (Fig. 5B and C). Together, these results demonstrate that complete loss of Men1 function conveys a strong growth advantage for tumor development to the Men1+/– cells but not the p18–/–; Men1+/– cells, and they provide additional evidence for the notion that p18 and Men1 regulate two separate pathways that collaboratively suppress tumorigenesis.

Phosphorylation of Rb protein at CDK2 and CDK4/6 sites was increased in p18–/–; Men1+/– but not in p27–/–-Men1+/– cells. To search for the underlying biochemical basis for the different genetic interactions between p18, p27, and Men1, we examined the kinase activity of CDK2 and CDK4/6, three major targets of p18 and p27, in normal and tumor cells of different genotypes. Taking advantage of the availability of several well-characterized antibodies specifically recognizing Rb proteins phosphorylated by CDK at different sites and their suitability for immunostaining of embedded tissue samples, we directly examined two representative organs, the pituitary and pancreatic islet, where p18 but not p27 exhibited an evident functional interaction with Men1. Two well-characterized phosphorylation sites in the Rb protein, S608 phosphorylated by CDK4 and CDK6 and T821 phosphorylated by CDK2 (14, 37), were examined. A visible increase of Rb phosphorylations at both CDK2 site T821 and CDK4/6 site S608 were detected in pituitaries of p18–/–, p27–/–, and Men1+/– mice (Fig. 6A and B). In pancreatic islets, p18–/–, p27–/–, and Men1+/– mutations all evidently increased S608 phosphorylation of Rb, but an increase of T821 phosphorylation was less pronounced in all three mutants. Combined mutation of p18–/– and Men1+/– substantially increased phosphorylation of Rb at both T821 and S608 sites in both the pituitary and pancreatic islet. Combined mutations of p27–/– and Men1+/–, on the other hand, caused no significant change of T821 phosphorylation in either tissue type but a surprisingly evident decrease of S608 phosphorylation in both pituitary and islet (Fig. 6). Together, these results are consistent with the pathological analyses showing that cell proliferation and tumor growth were synergistically stimulated in p18–/–; Men1+/– tissues but were either not enhanced (in pituitary) or actually alleviated (in islet) in p27–/–; Men1+/– tissues.


Figure 6
View larger version (98K):
[in this window]
[in a new window]

  		FIG. 6. Combined p18 and Men1, but not p27 and Men1, mutations stimulate pRB phosphorylation at CDK sites. Sections of normal and tumorigenic pituitary and pancreatic islets of different genotypes of mice at 12 months of age were examined for pRb protein phosphorylation at two CDK sites, S608 phosphorylated by CDK4 and CDK6 and T821 phosphorylated by CDK2. Counterstaining is blue, and positive staining is brown.


arrow
DISCUSSION
 
In this paper, we provide evidence that p18 functionally collaborates with Men1 to suppress development of tumors in five endocrine organs, including the pituitary, testis, thyroid, parathyroid, and pancreatic islets. We previously reported that loss of p18 resulted in spontaneous development of hyperplastic tissues and/or tumors in the pituitary (8), and loss of both p18 and p27 resulted in spontaneous development of hyperplastic tissues and/or tumors in the pituitary, adrenals, thyroid, parathyroid, testis, and pancreas (9). These phenotypes are reminiscent of human MEN syndrome, implicating p18 (and p27) as an MEN gene. One puzzling difference between the clinical manifestation of human MEN syndromes and mouse phenotypes is that while MEN patients develop pituitary tumors in the anterior lobe, nearly all pituitary tumors developed in p18–/–; p27–/– mice were from the intermediate lobe. Demonstration of a p18 function and its functional collaboration with Men1 in suppressing anterior lobe pituitary tumor growth (Fig. 1) provide further support for a role of p18 in suppression of MEN.

In all five endocrine organs, p18–/–; Men1+/– mice developed tumors at an earlier age and with increased incidence; these tumors are more aggressive than those found in either p18–/– or Men1+/– mice, further supporting p18 as a critical regulator of tumor progression in endocrine neoplasms. Mutations in Men1 have been frequently found in familial MEN1 patients and in sporadic neuroendocrine tumors. Mutations in the p18 gene, however, have rarely been detected in human cancers, raising the question of whether p18 may play different roles in endocrine tumor suppression between human and mouse. Two lines of evidence, however, argue against this possibility and support a function of p18 in suppressing human tumors. p18 is haploinsufficient for tumor suppression in mice (1), suggesting that a complete inactivation of both alleles by classical two-hit mutational event criteria is not necessary for tumor development. Consistent with this p18 haploinsufficiency, loss or reduced expression of p18 has been observed in several types of human cancers, including Hodgkin lymphomas (27), parathyroid tumors (3), hepatocellular carcinomas (23), and medulloblastomas (33).

One surprising conclusion derived from this study is that Men1 genetically interacts with p18 and p27 differently; while an increased tumorigenesis is seen in five different organs in p18–/–; Men1+/– mice, no evidence was obtained to support a similar functional collaboration between Men1 and p27 in suppressing endocrine tumors. Men1 encodes a 76-kDa nuclear protein (4, 18) and associates with mixed lineage leukemia family proteins of a histone methyltransferase (HMTase) complex (11, 36). Genome-wide analysis of genomic occupancy revealed that menin can potentially regulate the expression of a large number of target genes, possibly by recruiting HMTase (28). A direct binding by menin and increase of histone H3 lysine 4 methylation have been demonstrated for five genes, including HOX cluster genes HOXA9, c6, and c8 (11, 36) and, notably, both p18 and p27 (13, 22, 28). Loss of menin is conversely associated with the reduction of both p18 and p27 gene expression (13, 22).

Why, then, are the genetic results that p18 but not p27 interacts with Men1 inconsistent with the biochemical studies illustrating that both genes are targets regulated by menin? Loss of one allele of Men1 reduced the expression of both p18 and p27 in pancreatic islet cells as mice aged, without causing any appreciable change to the four other CDK inhibitor genes examined (p15, p19, p21, and p16 were not detectably expressed) (13), suggesting that, at least in pancreatic islet cells, a differential compensation between INK4 and CIP/KIP proteins is unlikely to have contributed to the different tumor phenotypes developed in p18–/–; Men1+/– and p27–/–; Men1+/– mice. The mRNA and protein levels of both p18 and p27 were reduced to an undetectably low level in the islets of Men1+/– mice after 28 weeks (13), arguing against the possibility that a differential effect of Men1 heterozygosity on p18 and p27 transcription is the explanation for the functional collaboration of Men1 with p18 but not p27. Given the potentially large number of genes regulated by menin-associated HMTases, we speculate that another menin target gene, whose level is affected by Men1 heterozygosity, differentially interacts with p18 and p27. One possible scenario is that Men1 activity may negatively regulate a factor(s) that functions in stimulating CDK activity and that an increased expression of this factor in Men1 mutant cells may antagonistically inhibit the increase of CDK activity caused by p27 loss.


arrow
ACKNOWLEDGMENTS
 
We thank Francis Collins and Judy Crabtree for providing Men1 mutant mice, Jerrold M. Ward and Virginia Godfrey for helping with histological examination, and Xianxin Hua for providing the menin antibody. We also thank Seung Kim, Matthew Meyerson, and Ned Sharpless for reading the manuscript.

F.B. is supported in part by a U.S. Department of Defense Career Postdoctoral fellowship. This study was supported by NIH grant CA68377 to Y.X.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Lineberger Comprehensive Cancer Center, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7295. Phone: (919) 962-2142. Fax: (919) 966-8799. E-mail: yxiong{at}email.unc.edu. Back

{triangledown} Published ahead of print on 4 December 2006. Back

{dagger} F.B. and X.-H.P. contributed equally to this work. Back

{ddagger} Present address: Division of Breast and Endocrine Surgery, St. Marianna University School of Medicine, Kawasaki, Japan. Back


arrow
REFERENCES
 
    1
  1. Bai, F., X. H. Pei, V. L. Godfrey, and Y. Xiong. 2003. Haploinsufficiency of p18INK4c sensitizes mice to carcinogen-induced tumorigenesis. Mol. Cell. Biol. 23:1269-1277.[Abstract/Free Full Text]
    2. 2
  2. Bertolino, P., W. M. Tong, D. Galendo, Z. Q. Wang, and C. X. Zhang. 2003. Heterozygous Men1 mutant mice develop a range of endocrine tumors mimicking multiple endocrine neoplasia type 1. Mol. Endocrinol. 17:1880-1892.[Abstract/Free Full Text]
    3. 3
  3. Buchwald, P. C., G. Akerstrom, and G. Westin. 2004. Reduced p18INK4c, p21CIP1/WAF1 and p27KIP1 mRNA levels in tumours of primary and secondary hyperparathyroidism. Clin. Endocrinol. 60:389-393.[CrossRef][Medline]
    4. 4
  4. Chandrasekharappa, S., S. C. Guru, P. Manickam, S. Olufemi, F. S. Collins, M. R. Emmert-Buck, L. V. Debelenko, Z. Zhuang, I. A. Lubensky, L. A. Liotta, J. S. Crabtree, Y. Wang, B. A. Roe, J. Weisemann, M. S. Boguski, S. K. Agawal, M. B. Kester, Y. S. Kim, C. Heppner, Q. Dong, A. M. Spiegel, A. L. Burns, and S. J. Marx. 1997. Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 276:404-407.[Abstract/Free Full Text]
    5. 5
  5. Coxon, A. B., J. M. Ward, J. Geradts, G. A. Otterson, M. Zajac-Kaye, and F. J. Kaye. 1998. RET cooperates with RB/p53 inactivation in a somatic multi-step model for murine thyroid cancer. Oncogene 17:1625-1628.[CrossRef][Medline]
    6. 6
  6. Crabtree, J. S., P. C. Scacheri, J. M. Ward, L. Garrett-Beal, M. R. Emmert-Buck, K. A. Edgemon, D. Lorang, S. K. Libutti, S. C. Chandrasekharappa, S. J. Marx, A. M. Spiegel, and F. S. Collins. 2001. A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors. Proc. Natl. Acad. Sci. USA 98:1118-1123.[Abstract/Free Full Text]
    7. 7
  7. Fero, M. L., M. Rivkin, M. Tasch, P. Porter, C. E. Carow, E. Firpo, K. Polyak, L.-H. Tsai, V. Broudy, R. M. Perlmutter, K. Kaushansky, and J. M. Roberts. 1996. A syndrome of multiorgan hyperplasia with features of gigantism, tumorigenesis, and female sterility in p27Kip1-deficient mice. Cell 85:733-744.[CrossRef][Medline]
    8. 8
  8. Franklin, D. S., V. L. Godfrey, H. Lee, G. I. Kovalev, R. Schoonhoven, S. Chen-Kiang, L. Su, and Y. Xiong. 1998. CDK inhibitors p18INK4c and p27KIP1 mediate two separate pathways to collaboratively suppress pituitary tumorigenesis. Genes Dev. 12:2899-2911.[Abstract/Free Full Text]
    9. 9
  9. Franklin, D. S., V. L. Godfrey, D. A. O'Brien, C. Deng, and Y. Xiong. 2000. Functional collaboration between different cyclin-dependent kinase inhibitors suppresses tumor growth with distinct tissue specificity. Mol. Cell. Biol. 20:6147-6158.[Abstract/Free Full Text]
    10. 10
  10. Harvey, M., H. Vogel, E. Y.-H. P. Lee, A. Bradley, and L. A. Donehower. 1995. Mice deficient in both p53 and Rb develop tumors primarily of endocrine origin. Oncogene 55:1146-1151.
    11. 11
  11. Hughes, C. M., O. Rozenblatt-Rosen, T. A. Milne, T. D. Copeland, S. S. Levine, J. C. Lee, D. N. Hayes, K. S. Shanmugam, A. Bhattacharjee, C. A. Biondi, G. F. Kay, N. K. Hayward, J. L. Hess, and M. Meyerson. 2004. Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus. Mol. Cell 13:587-597.[CrossRef][Medline]
    12. 12
  12. Jacks, T., A. Fazeli, E. M. Schmitt, R. T. Bronson, M. A. Goodell, and R. A. Weinberg. 1992. Effects of an Rb mutation in the mouse. Nature 359:295-300.[CrossRef][Medline]
    13. 13
  13. Karnik, S. K., C. M. Hughes, X. Gu, O. Rozenblatt-Rosen, G. W. McLean, Y. Xiong, M. Meyerson, and S. K. Kim. 2005. Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27Kip1 and p18INK4c. Proc. Natl. Acad. Sci. USA 102:14659-14664.[Abstract/Free Full Text]
    14. 14
  14. Kitagawa, M., H. Higashi, H. K. Jung, I. Suzuki-Takahashi, M. Ikeda, K. Tamai, J. Kato, K. Segawa, E. Yoshida, S. Nishimura, and Y. Taya. 1996. The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2. EMBO J. 15:7060-7069.[Medline]
    15. 15
  15. Kiyokawa, H., R. D. Kineman, K. O. Manova-Todorova, V. C. Soares, E. S. Hoffman, M. Ono, D. Khanam, A. C. Hayday, L. A. Frohman, and A. Koff. 1996. Enhanced growth of mice lacking the cyclin-dependent kinase inhibitor function of p27Kip1. Cell 85:721-732.[CrossRef][Medline]
    16. 16
  16. Latres, E., M. Malumbres, R. Sotillo, J. Martin, S. Ortega, J. Martin-Caballero, J. M. Flores, C. Cordon-Cardo, and M. Barbacid. 2000. Limited overlapping roles of p15INK4b and p18INK4c cell cycle inhibitors in proliferation and tumorigenesis. EMBO J. 19:3496-3506.[CrossRef][Medline]
    17. 17
  17. Lee, Y.-H. P., C.-Y. Chang, N. Hu, Y.-C. J. Wang, C.-C. Lai, K. Herrup, W.-H. Lee, and A. Bradley. 1992. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 359:288-294.[CrossRef][Medline]
    18. 18
  18. Lemmens, I., W. J. Van de Ven, K. Kas, C. X. Zhang, S. Giraud, V. Wautot, N. Buisson, K. De Witte, J. Salandre, G. Lenoir, M. Pugeat, A. Calender, F. Parente, D. Quincey, P. Gaudray, M. J. De Wit, C. J. Lips, J. W. Hoppener, S. Khodaei, A. L. Grant, G. Weber, S. Kytola, B. T. Teh, F. Farnebo, R. V. Thakker, et al. 1997. Identification of the multiple endocrine neoplasia type 1 (MEN1) gene. Hum. Mol. Genet. 6:1177-1183.[Abstract/Free Full Text]
    19. 19
  19. Maandag, E. C., M. Van Der Valk, M. Vlaar, C. Feltkamp, J. O'Brien, M. Van Roon, N. Van Der Lugt, A. Berns, and H. Te Riele. 1994. Developmental rescue of an embryonic-lethal mutation in the retinoblastoma gene in chimeric mice. EMBO J. 13:4260-4268.[Medline]
    20. 20
  20. Martin, J., S. L. Hunt, P. Dubus, R. Sotillo, F. Nehme-Pelluard, M. A. Magnuson, A. F. Parlow, M. Malumbres, S. Ortega, and M. Barbacid. 2003. Genetic rescue of Cdk4 null mice restores pancreatic beta-cell proliferation but not homeostatic cell number. Oncogene 22:5261-5269.[CrossRef][Medline]
    21. 21
  21. Marx, S. J. 1998. Multiple endocrine neoplasia type 1, p. 489-506. In B. Vogelstein and K. W. Kinzler (ed.), The genetic basis of human cancer. McGraw-Hill, New York, NY.
    22. 22
  22. Milne, T. A., C. M. Hughes, R. Lloyd, Z. Yang, O. Rozenblatt-Rosen, Y. Dou, R. W. Schnepp, C. Krankel, V. A. Livolsi, D. Gibbs, X. Hua, R. G. Roeder, M. Meyerson, and J. L. Hess. 2005. Menin and MLL cooperatively regulate expression of cyclin-dependent kinase inhibitors. Proc. Natl. Acad. Sci. USA 102:749-754.[Abstract/Free Full Text]
    23. 23
  23. Morishita, A., T. Masaki, H. Yoshiji, S. Nakai, T. Ogi, Y. Miyauchi, S. Yoshida, T. Funaki, N. Uchida, Y. Kita, F. Funakoshi, H. Usuki, S. Okada, K. Izuishi, S. Watanabe, K. Kurokohchi, and S. Kuriyama. 2004. Reduced expression of cell cycle regulator p18INK4C in human hepatocellular carcinoma. Hepatology 40:677-686.[CrossRef][Medline]
    24. 24
  24. Nakayama, K., N. Ishida, M. Shirane, A. Inomata, T. Inoue, N. Shishido, I. Horii, D. Y. Loh, and K.-i. Nakayama. 1996. Mice lacking p27Kip1 display increased body size, multiple organ hyperplasia, retinal dysplasia, and pituitary tumors. Cell 85:707-720.[CrossRef][Medline]
    25. 25
  25. Nikitin, A. Y., M. I. Juarez-Perez, S. Li, L. Huang, and W. H. Lee. 1999. RB-mediated suppression of spontaneous multiple neuroendocrine neoplasia and lung metastases in Rb+/– mice. Proc. Natl. Acad. Sci. USA 96:3916-3921.[Abstract/Free Full Text]
    26. 26
  26. Pei, X. H., F. Bai, T. Tsutsui, H. Kiyokawa, and Y. Xiong. 2004. Genetic evidence for functional dependency of p18Ink4c on Cdk4. Mol. Cell. Biol. 24:6653-6664.[Abstract/Free Full Text]
    27. 27
  27. Sanchez-Aguilera, A., J. Delgado, F. I. Camacho, M. Sanchez-Beato, L. Sanchez, C. Montalban, M. F. Fresno, C. Martin, M. A. Piris, and J. F. Garcia. 2004. Silencing of the p18INK4c gene by promoter hypermethylation in Reed-Sternberg cells in Hodgkin lymphomas. Blood 103:2351-2357.[Abstract/Free Full Text]
    28. 28
  28. Scacheri, P. C., S. Davis, D. T. Odom, G. E. Crawford, S. Perkins, M. J. Halawi, S. K. Agarwal, S. J. Marx, A. M. Spiegel, P. S. Meltzer, and F. S. Collins. 2006. Genome-wide analysis of menin binding provides insights into MEN1 tumorigenesis. PLOS Genet. 2:e51.[CrossRef][Medline]
    29. 29
  29. Sherr, C. J. 1996. Cancer cell cycle. Science 274:1672-1677.[Abstract/Free Full Text]
    30. 30
  30. Sotillo, R., P. Dubus, J. Martin, E. de La Cueva, S. Ortega, M. Malumbres, and M. Barbacid. 2001. Wide spectrum of tumors in knock-in mice carrying a Cdk4 protein insensitive to INK4 inhibitors. EMBO J. 20:6637-6647.[CrossRef][Medline]
    31. 31
  31. Spiegel, A. M. 2004. Focus on hereditary endocrine neoplasia. Cancer Cell 6:327-332.[CrossRef][Medline]
    32. 32
  32. Stewart, C., F. Parente, F. Piehl, F. Farnebo, D. Quincey, G. Silins, L. Bergman, G. F. Carle, I. Lemmens, S. Grimmond, C. Z. Xian, S. Khodei, B. T. Teh, J. Lagercrantz, P. Siggers, A. Calender, V. V. de Vem, K. Kas, G. Weber, N. Hayward, P. Gaudray, and C. Larsson. 1998. Characterization of the mouse Men1 gene and its expression during development. Oncogene 17:2485-2493.[CrossRef][Medline]
    33. 33
  33. Uziel, T., F. Zindy, S. Xie, Y. Lee, A. Forget, S. Magdaleno, J. E. Rehg, C. Calabrese, D. Solecki, C. G. Eberhart, S. E. Sherr, S. Plimmer, S. C. Clifford, M. E. Hatten, P. J. McKinnon, R. J. Gilbertson, T. Curran, C. J. Sherr, and M. F. Roussel. 2005. The tumor suppressors Ink4c and p53 collaborate independently with Patched to suppress medulloblastoma formation. Genes Dev. 19:2656-2667.[Abstract/Free Full Text]
    34. 34
  34. Weinberg, R. A. 1995. The retinoblastoma protein and cell cycle control. Cell 81:323-330.[CrossRef][Medline]
    35. 35
  35. Williams, B. O., L. Remington, D. M. Albert, S. Mukai, R. T. Bronson, and T. Jacks. 1994. Cooperative tumorigenic effects of germline mitations in Rb and p53. Nat. Genet. 7:480-484.[CrossRef][Medline]
    36. 36
  36. Yokoyama, A., Z. Wang, J. Wysocka, M. Sanyal, D. J. Aufiero, I. Kitabayashi, W. Herr, and M. L. Cleary. 2004. Leukemia proto-oncoprotein MLL forms a SET1-like histone methyltransferase complex with menin to regulate Hox gene expression. Mol. Cell. Biol. 24:5639-5649.[Abstract/Free Full Text]
    37. 37
  37. Zarkowska, T., S. U, E. Harlow, and S. Mittnacht. 1997. Monoclonal antibodies specific for underphosphorylated retinoblastoma protein identify a cell cycle regulated phosphorylation site targeted by CDKs. Oncogene 14:249-254.[CrossRef][Medline]

Molecular and Cellular Biology, February 2007, p. 1495-1504, Vol. 27, No. 4
0270-7306/07/$08.00+0     doi:10.1128/MCB.01764-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Gracanin, A., Dreijerink, K. M. A., van der Luijt, R. B., Lips, C. J. M., Hoppener, J. W. M. (2009). Tissue Selectivity in Multiple Endocrine Neoplasia Type 1-Associated Tumorigenesis. Cancer Res. 69: 6371-6374 [Abstract] [Full Text]  
  • Shen, H.-C. J., He, M., Powell, A., Adem, A., Lorang, D., Heller, C., Grover, A. C., Ylaya, K., Hewitt, S. M., Marx, S. J., Spiegel, A. M., Libutti, S. K. (2009). Recapitulation of Pancreatic Neuroendocrine Tumors in Human Multiple Endocrine Neoplasia Type I Syndrome via Pdx1-Directed Inactivation of Men1. Cancer Res. 69: 1858-1866 [Abstract] [Full Text]  
  • Quereda, V., Malumbres, M. (2009). Cell cycle control of pituitary development and disease. J Mol Endocrinol 42: 75-86 [Abstract] [Full Text]  
  • van Veelen, W., van Gasteren, C. J.R., Acton, D. S., Franklin, D. S., Berger, R., Lips, C. J.M., Hoppener, J. W.M. (2008). Synergistic Effect of Oncogenic RET and Loss of p18 on Medullary Thyroid Carcinoma Development. Cancer Res. 68: 1329-1337 [Abstract] [Full Text]  
  • Matoso, A., Zhou, Z., Hayama, R., Flesken-Nikitin, A., Nikitin, A. Yu. (2008). Cell lineage-specific interactions between Men1 and Rb in neuroendocrine neoplasia. Carcinogenesis 29: 620-628 [Abstract] [Full Text]  
  • Pei, X.-H., Bai, F., Smith, M. D., Xiong, Y. (2007). p18Ink4c Collaborates with Men1 to Constrain Lung Stem Cell Expansion and Suppress Non-Small-Cell Lung Cancers. Cancer Res. 67: 3162-3170 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bai, F.
Right arrow Articles by Xiong, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bai, F.
Right arrow Articles by Xiong, Y.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»