Cooperative interactions of PTEN deficiency and RAS activation in melanoma metastasis.

Mitogen-activated protein kinase (MAPK) and AKT pathways are frequently co-activated in melanoma through overexpression of receptor tyrosine kinases, mutations in their signaling surrogates, such as RAS and BRAF, or loss of negative regulators such as PTEN. As RAS can be a positive upstream regulator of PI3-K, it has been proposed that the loss of PTEN and the activation of RAS are redundant events in melanoma pathogenesis. Here, in genetically engineered mouse models of cutaneous melanomas, we sought to better understand the genetic interactions between HRAS activation and PTEN inactivation in melanoma genesis and progression in vivo. We showed that HRAS activation cooperates with Pten+/- and Ink4a/Arf-/- to increase melanoma penetrance and promote metastasis. Correspondingly, gain- and loss-of-function studies established that Pten loss increases invasion and migration of melanoma cells and non-transformed melanocytes, and such biological activity correlates with a shift to phosphorylation of AKT2 isoform and E-cadherin down-regulation. Thus, Pten inactivation can drive the genesis and promote the metastatic progression of RAS activated Ink4a/Arf deficient melanomas.

Haploinsufficiency of the mSds3 chromatin regulator promotes chromosomal instability and cancer only upon complete neutralization of p53.

The mSin3 corepressor complex has been linked to diverse cancer signaling pathways through its capacity to regulate target gene expression via chromatin modification. mSds3, a cell essential gene, is a key component of the mSin3 complex serving to maintain its inherent histone deacetylase activity. mSds3 also serves an essential role in the establishment of pericentric heterochromatin, and genetic ablation of mSds3 results in chromosome missegregation. In contrast, mSin3A nullizygous cells show normal chromosome dynamics and cytogenetic profiles. The integral role of mSds3 in controlling chromosome segregation and mSin3-regulated transcriptional networks prompted efforts to determine the neoplastic impact of loss of one copy of mSds3 or mSin3A. In particular, we assessed whether loss of one copy of mSds3, alone or in combination with p53 mutation, results in aneuploidy and promotes a cancer-prone condition in the mouse. We observe that, in a p53 null background, loss of one mSds3 allele results in accelerated tumor onset and increased tumor burden. Notably, these mSds3(+/-) p53(-/-) tumors exhibit a more complex cytogenetic profile characterized by marked aneuploidy and centromeric associations. The presence of even one copy of p53 is sufficient to suppress the accelerated tumorigenesis in mSds3(+/-) mice, consistent with a key role for p53 in monitoring mitotic fidelity. These observations with Sds3 mutant mice contrast with mSin3A(+/-) p53(-/-) mice, which do not show an accelerated or increased tumor incidence relative to mSin3A(+/+)p53(-/-) controls, correlating with the absence of aneuploidy detected upon mSin3A genetic inactivation. This genetic study establishes that the capacity of mSds3 to cooperate with p53 deficiency in cancer predisposition relates to its specific role in chromosome segregation, rather than its central role in maintaining a functional mSin3A complex.

Overlapping functions of the pRb family in the regulation of rRNA synthesis.

The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb(-/-) p130(-/-) fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.

Escape from premature senescence is not sufficient for oncogenic transformation by Ras.

Resistance of primary cells to transformation by oncogenic Ras has been attributed to the induction of replicative growth arrest. This irreversible 'fail-safe mechanism' resembles senescence and requires induction by Ras of p19ARF and p53 (refs 3-5). Mutation of either p19ARF or p53 alleviates Ras-induced senescence and facilitates oncogenic transformation by Ras. Here we report that, whereas Rb and p107 are each dispensable for Ras-induced replicative arrest, simultaneous ablation of both genes disrupts Ras-induced senescence and results in unrestrained proliferation. This occurs despite activation by Ras of the p19ARF /p53 pathway, identifying pRb and p107 as essential mediators of Ras-induced antiproliferative p19ARF/p53 signalling. Unexpectedly, in contrast to p19ARF or p53 deficiency, loss of Rb/p107 function does not result in oncogenic transformation by Ras, as Ras-expressing Rb-/-/p107-/- fibroblasts fail to grow anchorage-independently in vitro and are not tumorigenic in vivo. These results demonstrate that in the absence of both Rb and p107 cells are resistant to p19ARF/p53-dependent protection against Ras-induced proliferation, and uncouple escape from Ras-induced premature senescence from oncogenic transformation.

Ablation of the retinoblastoma gene family deregulates G(1) control causing immortalization and increased cell turnover under growth-restricting conditions.

The retinoblastoma suppressor pRB belongs to the family of so-called pocket proteins, which also includes p107 and p130. These proteins may functionally overlap in cell cycle control and tumor suppression. We have generated an isogenic set of embryonic stem (ES) cell lines carrying single or compound loss-of-function mutations in the Rb gene family, including a cell line completely devoid of all three pocket proteins. None of the knockout combinations affected the growth characteristics of ES cells; however, concomitant ablation of all three pocket proteins strongly impaired their differentiation capacity. For the generated genotypes, primary mouse embryonic fibroblasts (MEFs) also were obtained. While inactivation of Rb alone did not alleviate the senescence response of MEFs, pRB/p107-deficient MEFs, after having adapted to in vitro culturing, continued to proliferate at modest rate. Additional ablation of p130 rendered MEFs completely insensitive to senescence-inducing signals and strongly increased their proliferation rate. Although triple-knockout MEFs retained anchorage dependence, they lacked proper G(1) control and showed increased cell turnover under growth-inhibiting conditions.

p27kip1-independent cell cycle regulation by MYC.

MYC transcription factors are potent stimulators of cell proliferation. It has been suggested that the CDK-inhibitor p27kip1 is a critical G1 phase cell cycle target of c-MYC. We show here that mouse embryo fibroblasts deficient for both p27kip1 and the related p21cip1 are still responsive to stimulation by c-MYC and can be arrested in G1 by a dominant negative mutant of c-MYC. This growth arrest can be overruled by ectopic expression of E2F or adenovirus E1A, but not by a mutant of E1A defective for binding to retinoblastoma family proteins. We show that fibroblasts with a genetic disruption of all three retinoblastoma family members (pRb, p107 and p130) are unresponsive to a dominant negative c-MYC mutant. These data indicate that p27kip1 is not the only rate limiting cell cycle target of c-MYC and suggest that regulation of E2F is also essential for c-MYC's mitogenic activity.

p107 is a suppressor of retinoblastoma development in pRb-deficient mice.

Hemizygosity for the retinoblastoma gene RB in man strongly predisposes to retinoblastoma. In the mouse, however, Rb hemizygosity leaves the retina normal, whereas in Rb-/- chimeras pRb-deficient retinoblasts undergo apoptosis. To test whether concomitant inactivation of the Rb-related gene p107 is required to unleash the oncogenic potential of pRb deficiency in the mouse retina, we inactivated both Rb and p107 by homologous recombination in embryonic stem cells and generated chimeric mice. Retinoblastomas were found in five out of seven adult pRb/p107-deficient chimeras. The retinal tumors showed amacrine cell differentiation, and therefore originated from cells committed to the inner but not the outer nuclear layer. Retinal lesions were already observed at embryonic day 17.5. At this stage, the primitive nuclear layer exhibited severe dysplasia, including rosette-like arrangements, and apoptosis. These findings provide formal proof for the role of loss of Rb in retinoblastoma development in the mouse and the first in vivo evidence that p107 can exert a tumor suppressor function.