PMC:4135037 / 17065-24170
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4135037","sourcedb":"PMC","sourceid":"4135037","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4135037","text":"DISCUSSION\nThe present study demonstrated that Cdk4 disruption completely inhibited pituitary and islet tumorigenesis in Men1+/− mice. This is in contrast with the finding that Cdk2 disruption did not affect pituitary and islet tumorigenesis. Cdk4 deficiency markedly inhibited islet proliferation in both wild-type and Men1+/− mice, while Cdk2 deficiency showed no appreciable effects. LOH at the Men1 locus was observed in Cdk-wild type or Cdk2-null pituitary tumors, whereas no sign of LOH was detected in Cdk4-null pituitaries. These observations suggest that CDK4 plays a unique and essential role in islet and pituitary tumorigenesis initiated by the loss of menin.\nMEN1 tumors in humans are restricted to endocrine tissues such as the pituitary, pancreatic islet and parathyroid, although the tumor suppressor menin is ubiquitously expressed. The mechanism of the tissue-specific tumor suppression is yet to be elucidated. Our data indicating the unique requirement for CDK4 suggest that CDK4 activation is essential for a pre-LOH stage of the oncogenic process in the neuroendocrine cell types. While parathyroid tumorigenesis was not examined in the present study, complete inhibition of pituitary and islet tumor development in the Cdk4-null background indicates tight linkage between menin loss and CDK4 activation. Activating mutations of the human CDK4 gene in pituitary, islet or other neuroendocrine tumors have not been reported, whereas activating germline mutations of CDK4 have been described in melanoma-prone families 29-31. These mutations in melanomas affect the Arg24 residue and render the mutant protein resistant to INK4-mediated inhibition. For MEN1 tumorigenesis, downregulation of CDK inhibitors seems to be involved in CDK4 activation. In particular, p18 and p27 have been proposed to play important roles. Menin together with its partner MLL has been shown to control histone methylation at the p18 (CDKN2C) and p27 (CDKN1B) loci for transactivation 32, 33. The levels of p18 and p27 mRNAs in pancreatic islets of Men1+/− mice decline within 28 weeks of age 33. Intriguingly, Cdkn1b−/− mice develop pituitary tumors spontaneously 9, and Cdkn1b−/−; Cdkn2c−/− mice exhibit tumorigenesis with the tissue spectrum overlapping with that of the MEN1 syndrome, including the endocrine pancreas, parathyroid, thyroid, adrenal and pituitary glands 13. A gene expression profiling study, which analyzed pituitary and pancreatic tumors from Men1+/− mice, confirmed p18 downregulation 34. Thus, it is conceivable that downregulation of the CDK inhibitors accounts substantially for ectopic CDK4 activation in pre-oncogenic menin-deficient neuroendocrine cells.\nThe requirement for CDK4 in tumorigenesis of Men1+/− mice expands upon its physiologic role previously demonstrated in the pituitary and pancreatic islets in Cdk4−/− mice 14, 15. It is important that this phenotype is restricted to adult mice, while embryonic development of these organs is completely intact 18-20. Regarding murine pancreatic islets, it is possible that CDK4 activity is essential for proliferation of differentiated β cells, which is thought to be a major mechanism for the adaptive regulation of islet mass in response to homeostatic requirement for insulin secretion 35. A similar scenario might apply to the regulation of adult pituicyte proliferation. A recent study proposed that downregulation of menin promotes β cell proliferation during pregnancy 36. Thus, menin could be a limiting factor for not only tumorigenesis but also physiological proliferation in adult endocrine tissues, presumably via CDK4 inhibition. Pancreatic islets and pituitaries in Cdkn2c−/−; Cdk4−/− double mutant mice are as hypoplastic as Cdk4−/− mice 37, indicating a linear functional relationship between p18 and CDK4. While CDK6 is another target of p18 and this kinase has also been implicated in the control of human islet proliferation 38, genetic evidence suggests that murine islet cells depend solely on CDK4 for proliferation and tumorigenesis. Moreover, CDK4 expression was robust in islet tumors of Men1+/− mice, suggesting that upregulation of CDK4 protein may cooperate with downregulation of the CDK inhibitors in Men1+/− neuroendocrine cells. This notion is consistent with a recent report that a number of human pancreatic neuroendocrine tumors expressed high levels of CDK4 39. We hypothesize that CDK4 activation not only accelerates proliferation but also generates a selective pressure for cells with LOH at the Men1 locus. To further delineate the role of CDK4 in pituitary tumorigenesis triggered by the loss of menin, we recently generated mice with pituitary-specific disruption of MEN1, using Cre transgenic mice under the growth hormone promoter (rGHp-Cretg+) crossed with mice with a floxed Men1 allele (Men1lox/lox). While rGHp-Cretg+; Men1 lox/lox; Cdk4+/+ mice displayed large pituitary tumors by 7 months of age with complete penetrance, no tumor was detected in rGHp-Cretg+; Men lox/lox; Cdk4−/− mice autopsied at 9 months (Gillam et al., unpublished observations). Since the GH promoter becomes active during late embryogenesis, homozygous loss of menin in this model occurs earlier than any detectable impact of Cdk4 deficiency on pituitary proliferation or differentiation. Thus, the abrogation of tumorigenesis observed in Men1+/−; Cdk4−/− mice results from the requirement for CDK4 in neuroendocrine tumor initiation, and is unlikely to reflect other functional defects in hypoplastic pituitary or islets in Cdk4−/− mice before LOH occurs in the Men1 locus.\nUnlike Cdk4 deficiency, Cdk2 deficiency does not affect the homeostasis and function of the neuroendocrine tissues, and does not inhibit menin-associated tumorigenesis. These results are congruent with our previous observation that Cdk2 deficiency does not prevent p27-deficient mice from developing pituitary tumors 40. In sharp contrast, Cdk2 deficiency and Cdk4 deficiency are both inhibitory on HER2/neu- induced murine mammary tumorigenesis 41, 42. The embryonic lethality of Cdk4−/−; Cdk2−/− double mutant mice 43 implies that these two kinases have overlapping function in embryonic tissues as well as in most non-endocrine organs. CDK4 abundantly expressed in islet cells of Men1+/−; Cdk2−/− mice may be sufficient to compensate for the absence of CDK2 during tumorigenesis. It is thought that CDK4 and CDK2 collaborate in phosphorylating the critical substrate RB, sharing different phosphorylation sites on the protein 44. In the present study using the islet cell lines, CDK4 depletion inhibited glucose-stimulated hyperphosphorylation of RB, including Ser780 phosphorylation, and G1-S progression, whereas CDK2 depletion had no effect. Collectively, these data underscore the significance of the CDK4-RB pathway in the control of neuroendocrine cell proliferation. Further investigations are necessary to understand how multiple CDK complexes control phosphorylation of RB at the 16 CDK-consensus sites 44 in neuroendocrine-specific manners, and whether non-RB substrates of CDK4 are involved in MEN1 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