RESULTS

Pituitary tumorigenesis in Men1+/− mice depends on Cdk4
Previous studies demonstrated that Men1+/− mice develop tumors in the anterior pituitary with a latency of 9-13 months 3, 4. Cdk4−/− mice exhibit hypoplasia of the anterior pituitary and pancreatic islets during postnatal periods, while embryonic development of these endocrine tissues occurs normally 21-23. To determine whether CDK4 activity is required for tumorigenesis initiated by the loss of menin, Men1+/−; Cdk4−/− mice were generated by crossbreeding and characterized in comparison with Men1+/−; Cdk4+/+ (Cdk wild-type) littermates. Mice were euthanized at 15 months of age for pathological examinations. None of 12 wild-type (Men1+/+) mice generated from the breeding displayed pituitary tumors, while 45% of Men1+/− mice (n=31) showed pituitary tumors (Fig. 1A, B). It is noteworthy that no Men1+/− males exhibited macroscopic pituitary tumors at 15 months of age (n=11), whereas 14 (70%) of 20 Men1+/− female mice had developed pituitary tumors. This gender-specific effect on pituitary tumorigenesis is consistent with a previous study, which showed 79% of Men1+/− mice that developed pituitary tumors were females 4. Strikingly, none of 30 Men1+/−; Cdk4−/− mice (11 males and 19 females) displayed pituitary tumors. These observations indicate that pituitary tumorigenesis induced by menin deficiency depends on CDK4 activity. Furthermore, the pituitaries from the entire cohort of Men1+/−; Cdk4−/− mice remained hypoplastic, in a manner similar to the single Cdk4−/− mice throughout their 15-month lifespan (Fig. 1A). Our previous study demonstrated that CDK4 is an essential driving force for estrogen- or GHRH-induced proliferation in murine pituicytes 23. Thus, these data indicate that CDK4 is required for both physiological and tumorigenic control of cell cycle progression in this particular endocrine tissue.
To determine whether the requirement for CDK4 in menin-associated tumorigenesis is unique among the G1-regulatory CDKs or there is functional overlap, we next examined the impact of Cdk2-deficiency on tumorigenesis of Men1+/− mice, by generating Men1+/−; Cdk2−/− and Men1+/−; Cdk2+/+ (Cdk wild-type) littermates. In sharp contrast to Men1+/−; Cdk4−/− mice, 51% of examined Men1+/−; Cdk2−/− mice (n=29) showed macroscopic pituitary tumors comparable to those in Men1+/−; Cdk wild-type mice (Fig. 1A, 1B). Interestingly, we observed tumors in 4 (24%) of 17 Men1+/−; Cdk2−/− male mice and 11 (92%) of 12 Men1+/−; Cdk2−/− female mice. This may imply that Cdk2 deficiency modestly promoted pituitary tumorigenesis under the conditions of the study, although examinations of a larger cohort will be necessary to be conclusive. Regardless, these data indicate that the absence of CDK2 can be compensated, and has no impact on restraining the process of pituitary tumorigenesis induced by menin deficiency.

Pancreatic islet tumorigenesis in Men1+/− mice depends on Cdk4
Previous studies reported that 30-50% of Men1+/− mice exhibited tumors in the endocrine pancreas, mostly insulinomas, during 8-18 months of age, and almost 80% of Men1+/− mice developed islet adenomas or carcinomas by 18-26 months 3-5. To determine the effects of Cdk4 or Cdk2 deficiency on islet tumorigenesis, we examined pancreatic tissues from 15-month-old Men1+/−; Cdk4−/− mice, Men1+/−; Cdk2−/− mice and control littermates with Men1+/+ and/or Cdk wild-type genotypes. Histological analyses showed that 62% of Men1+/−; Cdk wild-type mice (n=29) had islet tumors (Fig. 2A, C). Of the samples examined, islet adenomas were found in 12 (67%) of 18 Men1+/−; Cdk wild-type females and 6 (55%) of 11 Men1+/−; Cdk wild-type males. Essentially all islet tumors displayed insulin immunoreactivity (Fig. 2B), indicating these were insulinomas. No glucagonomas were observed in the examined groups of mice. Spontaneous islet tumors could not be found in Men1+/+ mice regardless of the Cdk4 or Cdk2 genotypes. In sharp contrast to Men1+/−; Cdk wild-type mice, none of 30 Men1+/−; Cdk4−/− mice (19 females and 11 males examined) had islet tumors or showed evidence of dysplasia. Indeed, pancreatic islets of Men1+/−; Cdk4−/− mice were markedly hypoplastic, essentially identical in appearance to the hypoplasia in the islets of Men1+/+; Cdk4−/− mice. On the other hand, 10 (77%) of 13 Men1+/−; Cdk2−/− females and 12 (57%) of 21 Men1+/−; Cdk2−/− males exhibited islet tumors, again demonstrating the lack of restraining effect of Cdk2 deficiency on both normal growth of the islets and neoplastic transformation..
We then examined proliferation of islet cells using immunohistochemistry with the Ki67 proliferation marker (Fig. 3A, B). In normal islets of wild-type mice, approximately 0.2% of endocrine cells stained positive for Ki67 immunoreactivity (Fig. 3B, Group 1), which is consistent with previous reports 24, 25. In Men1+/−; Cdk wild-type mice, even islets that appeared normal in size showed higher percentages (0.7%) of Ki67-positive endocrine cells (Group 2). In hyperplastic or dysplastic Men1+/− islets, about 1.5% of cells showed Ki67 immunoreactivity (Group 3), and 6.5% of cells in islet adenomas were Ki67-positive (Group 4). In Men1+/−; Cdk4−/− islets and Men1+/+; Cdk4−/− islets, Ki67-positive endocrine cells were virtually undetectable (Group 5), indicating that the absence of CDK4 severely impaired proliferation of islet cells in adult mice. In Men1+/−; Cdk2−/− mice, 0.5%, 2.2% and 6.0% of cells were Ki67-positive in normal, hyperplastic/dysplastic and adenomatous islets, respectively (Groups 6-8). Thus, increased proliferation is correlated with tumorigenic changes in Men1+/− islets, regardless of the Cdk2 genotype. Previous studies showed that adult murine islets express relatively high levels of CDK4 protein 22, consistent with its role in β cell proliferation. Our immunohistochemical analysis not only confirmed readily detectable expression of CDK4 in wild-type and Cdk2−/−islets, but also demonstrated robust CDK4 expression in islet tumors of Men1+/−; Cdk wild-type and Men1+/−; Cdk2−/− mice (Fig. 3C). CDK4 expression was not detected in Cdk4−/− islets, confirming the specificity of the immunoreactivity. These data indicate that Cdk4 is required for islet tumorigenesis initiated by hemizygous loss of Men1, while Cdk2 function can be compensated in this process.

LOH of the Men1 locus in pituitary tumors
It is well established that endocrine tumors that arise in MEN1+/− humans and Men1+/− mice exhibit LOH 3, 4, which is consistent with the classical “two-hit” model of tumorigenesis operative with inactivation of tumor suppressor genes 26. In order to determine whether Cdk2 or Cdk4 deficiency had a differential impact on Men1 LOH, we examined whether LOH had occurred at the Men1 locus in pituitary tissues and tumors from Men1+/− mice with Cdk4−/−, Cdk2−/− and Cdk-wild type backgrounds. Genomic PCR analyses unambiguously indicated that pituicytes in Men1+/−; Cdk wild-type mice and Men1+/−; Cdk2−/− mice underwent LOH, i.e., the loss of the wild-type Men1 allele in genomic DNA, while livers from those mice as non-tumorigenic control tissues retained both wild-type and mutant alleles (Fig. 4, compare lanes 4, 8 to lanes 3, 7). In contrast, pituitary tissues from Men1+/−; Cdk4−/− mice showed no sign of LOH (Fig. 4, lane 6). These results suggest that Cdk4 deficiency impedes early tumorigenic changes in Men1+/− pituicytes that normally occur in prior to the complete loss of menin expression. In contrast, Cdk2 deficiency does not alter LOH that occurs as part of the multi-step tumorigenic process.

CDK4 depletion inhibits cell cycle progression of insulinoma cells with suppressed Rb phosphorylation
To obtain insight into the mechanism underlying the requirement for CDK4 in neuroendocrine tumorigenesis, we examined INS-1 insulinoma cells, which express very low levels of menin and have been widely used for functional studies by forced expression of wild-type and mutant menin proteins 27, 28. At 24 hrs after transfection with siRNA against CDK2 or CDK4, cells were treated with medium containing a low level of glucose (0.1 mM) and incubated for 48 more hours to suppress proliferation. Cell cycle progression was then stimulated by switching to medium with a high glucose concentration (11 mM). Immunoblotting (Fig. 5A) demonstrated that both CDK2 and CDK4 siRNAs successfully downregulated the expression of the target proteins. The stimulation of cells with 11 mM glucose resulted in marked upregulation of Cyclin D1 and substantial increases in Cyclin D3 expression, for which the knockdown of Cdk2 or Cdk4 had minimal impact. The expression levels of Cyclin E and CDK6 did not significantly alter during glucose starvation and stimulation. It was noted that CDK2 knockdown resulted in modest increases in CDK4 levels (2nd panel, C vs. K2 at 0, 6 and 24h). Immunoblots for total RB showed accumulation of the slower migrating hyperphosphorylated forms of RB after the switch to 11 mM glucose, and CDK4 knockdown clearly inhibited the hyperphosphorylation of RB. In contrast, CDK2 knockdown resulted in slightly accelerated kinetics of RB phosphorylation, compared with control cells. Immunoblots using phospho-specific RB antibodies showed that phosphorylation of RB at Ser780 was delayed in cells with CDK4 knockdown (8th panel, K4-6 h vs. C-6 h and K4-24 h vs. C-24 h), whereas phosphorylation at Ser807/811 was minimally affected by CDK2 or CDK4 siRNA. We did not observe major effects of CDK2 or CDK4 knockdown in phosphorylation of p130 at Ser952. Consistently with the suppression of hyperphosphorylated forms of RB, CDK4-depleted cells exhibited a substantial delay in glucose-mediated cell cycle stimulation relative to control or CDK2 depleted cells, as shown as partially inhibited G1-S transition in flow cytometric analysis using DNA staining with propidium iodide and immunodetection of incorporated bromodeoxyuridine (BrdU)(Fig. 5B). Using another insulinoma cell line, Min6 cells, we confirmed that siRNA-mediated silencing of CDK4 significantly decreased Ser780 phosphorylation of RB, as well as hyperphosphorylated forms of the protein (Fig. 5C). Thus, these data using insulinoma cells demonstrate that CDK4 depletion inhibits site-specific RB phosphorylation and cell cycle stimulation in this cell type, whereas CDK2 depletion has minimal effect on these molecular processes.