Inhibition of oncogenic transformation by mammalian Lin-9, a pRB-associated protein

Abstract

Genetic studies in Caenorhabditis elegans identified lin-9 to function together with the retinoblastoma homologue lin-35 in vulva differentiation. We have now identified a human homologue of Lin-9 (hLin-9) and provide evidence about its function in the mammalian pRB pathway. hLin-9 binds to pRB and cooperates with pRB in flat cell formation in Saos-2 cells. In addition, hLin-9 synergized with pRB and Cbfal to transactivate an osteoblast-specific reporter gene. In contrast, hLin-9 was not involved in pRB-mediated inhibition of cell cycle progression or repression of E2F-dependent transactivation. Consistent with these data, hLin-9 was able to associate with partially penetrant pRB mutants that do not bind to E2F, but retain the ability to activate transcription and to promote differentiation. hLin-9 can also inhibit oncogenic transformation, dependent on the presence of a functional pRB protein. RNAi-mediated knockdown of Lin-9 can substitute for the loss of pRB in transformation of human primary fibroblasts. These data suggest that hLin-9 has tumor-suppressing activities and that the ability of hLin-9 to inhibit transformation is mediated through its association with pRB.

Mammalian Mip/LIN-9 interacts with either the p107, p130/E2F4 repressor complex or B-Myb in a cell cycle-phase-dependent context distinct from the Drosophila dREAM complex

“…We should point out that to our surprise, we have not been able to detect an association between Mip/LIN-9 and pRB as previously reported by Gagrica et al (2004), although identical cell lines (that is HeLa cells) and a variety of anti-pRB antibodies were used. This is illustrated in Figure 1f, in which the anti-Mip/LIN-9 mAbdoes not co-precipitate pRB, although this pocket protein is easily detected in immunoprecipitates with the anti-pRB antibody or by an antibody directed against a known partner of pRB such as E2F3 (lanes 4 and 5).…”

“…Mip/LIN-9 interacts with mammalian homologs of the Drosophila dREAM complex: B-Myb, p107, p130 and E2F4 Previous reports indicated that in mammalian cells, Mip/LIN-9 interacts with B-Myb (Osterloh et al, 2007;Pilkinton et al, 2007) and it is pulled down with the pocket proteins (Gagrica et al, 2004;Korenjak et al, 2004;Osterloh et al, 2007). Yet, the existence of a dREAM complex and its possible role in cell cycle regulation in mammalian cells has not been addressed.…”

“…It has been recently shown that expression of B-Myb is regulated by Mip/LIN-9 and that both genes function together in the induction of S phase and mitotic genes (Osterloh et al, 2007;Pilkinton et al, 2007). This requirement for cell cycle progression was partially unexpected since Gagrica et al (2004) first described that although mammalian Mip/LIN-9 was a collaborator of pRB in the regulation of transformation, it had no effect on cell cycle progression. Interestingly, it was independently reported that mammalian Mip/LIN-9, first named as BARA/LIN-9 (Sandoval et al, 2006), was inhibited by cyclin D/CDK4 activity as its mutation rescued the CDK4 null phenotype including the cell cycle re-entry defects and the expression of E2F-regulated genes (Sandoval et al, 2006).…”

“…Interestingly, although interactions between pocket proteins, Mip/LIN-9 and B-Myb were also described in mammalian cells (Gagrica et al, 2004;Osterloh et al, 2007;Pilkinton et al, 2007), the existence of an equivalent of the dREAM complex is uncertain. Moreover, some findings would argue against such a complex.…”

Loss of LIN9, a member of the DREAM complex, cooperates with SV40 large T antigen to induce genomic instability and anchorage-independent growth

“…Early-passage (p2) Lin9 fl/f CreER T2 MEFs were infected with retroviral expression constructs encoding SV40 LT, LT K1 or LTD434-44 as described (Cotsiki et al, 2004;Gagrica et al, 2004). Cells were selected with 400 mg/ml neomycin for 5 days.…”