Critical role of the Rb family in myoblast survival and fusion

The tumor suppressor Rb is thought to control cell proliferation, survival and differentiation. We recently showed that differentiating Rb-deficient mouse myoblasts can fuse to form short myotubes that quickly collapse through a mechanism involving autophagy, and that autophagy inhibitors or hypoxia could rescue the defect leading to long, twitching myotubes. Here we determined the contribution of pRb relatives, p107 and p130, to this process. We show that chronic or acute inactivation of Rb plus p107 or p130 increased myoblast cell death and reduced myotube formation relative to Rb loss alone. Treatment with autophagy antagonists or hypoxia extended survival of double-knockout myotubes, which appeared indistinguishable from control fibers. In contrast, triple mutations in Rb, p107 and p130, led to substantial increase in myoblast death and to elongated bi-nuclear myocytes, which seem to derive from nuclear duplication, as opposed to cell fusion. Under hypoxia, some rare, abnormally thin triple knockout myotubes survived and twitched. Thus, mutation of p107 or p130 reduces survival of Rb-deficient myoblasts during differentiation but does not preclude myoblast fusion or necessitate myotube degeneration, whereas combined inactivation of the entire Rb family produces a distinct phenotype, with drastically impaired myoblast fusion and survival.     

Direct and indirect effects of the pRb tumor suppressor on autophagy

Autophagy, an intracellular degradation pathway involved in cell survival or demise, is tightly controlled by complex regulatory mechanisms. A link between the Rb tumor suppressor and autophagy is now emerging. pRb plays a critical role in cell cycle progression and survival as well as the differentiation of certain cell types. Recently, we have reported that during skeletal myogenesis, Rb-deficient myoblasts fuse to form short myotubes that quickly degenerate. Myotube degeneration was associated with increased autophagic flux and inhibition of autophagy rescued the defect leading to long, twitching myotubes. We propose that Rb-loss sensitizes cells to autophagy via direct and indirect mechanisms and we discuss how these might affect cancer progression and response to chemotherapy.     

Direct and indirect effects of the pRb tumor suppressor on autophagy

Autophagy, an intracellular degradation pathway involved in cell survival or demise, is tightly controlled by complex regulatory mechanisms. A link between the Rb tumor suppressor and autophagy is now emerging. pRb plays a critical role in cell cycle progression and survival as well as the differentiation of certain cell types. Recently, we have reported that during skeletal myogenesis, Rb-deficient myoblasts fuse to form short myotubes that quickly degenerate. Myotube degeneration was associated with increased autophagic flux and inhibition of autophagy rescued the defect leading to long, twitching myotubes. We propose that Rb-loss sensitizes cells to autophagy via direct and indirect mechanisms and we discuss how these might affect cancer progression and response to chemotherapy.     

Retinoblastoma protein regulates the crosstalk between autophagy and apoptosis,and favors glioblastoma resistance to etoposide

Glioblastomas (GBMs) are devastating tumors of the central nervous system, with a poor prognosis of 1-year survival. This results from a high resistance of GBM tumor cells to current therapeutic options, including etoposide (VP-16). Understanding resistance mechanisms may thus open new therapeutic avenues. VP-16 is a topoisomerase inhibitor that causes replication fork stalling and, ultimately, the formation of DNA double-strand breaks and apoptotic cell death. Autophagy has been identified as a VP-16 treatment resistance mechanism in tumor cells. Retinoblastoma protein (RB) is a classical tumor suppressor owing to its role in G1/S cell cycle checkpoint, but recent data have shown RB participation in many other cellular functions, including, counterintuitively, negative regulation of apoptosis. As GBMs usually display an amplification of the EGFR signaling involving the RB protein pathway, we questioned whether RB might be involved in mechanisms of resistance of GBM cells to VP-16. We observed that RB silencing increased VP-16-induced DNA double-strand breaks and p53 activation. Moreover, RB knockdown increased VP-16-induced apoptosis in GBM cell lines and cancer stem cells, the latter being now recognized essential to resistance to treatments and recurrence. We also showed that VP-16 treatment induced autophagy, and that RB silencing impaired this process by inhibiting the fusion of autophagosomes with lysosomes. Taken together, our data suggest that RB silencing causes a blockage on the VP-16-induced autophagic flux, which is followed by apoptosis in GBM cell lines and in cancer stem cells. Therefore, we show here, for the first time, that RB represents a molecular link between autophagy and apoptosis, and a resistance marker in GBM, a discovery with potential importance for anticancer treatment.     

Regulation of mitochondrial integrity, autophagy and cell survival by BNIP3

Understanding the role of BNIP3 in the systemic response to hypoxia has been complicated by conflicting results that indicate on the one hand that BNIP3 promotes cell death, and other data, including our own that BNIP3 is not sufficient for cell death, but rather plays a critical role in hypoxia-induced autophagy. This work suggests that rather than promoting death, BNIP3 may actually allow survival either by preventing ATP depletion or by eliminating damaged mitochondria. However, the function of BNIP3 may be subverted under unusual conditions associated with acidosis that arise following extended periods of hypoxia and anaerobic glycolysis. Despite this novel insight into BNIP3 function, much remains to be done in terms of pinning down a molecular activity for BNIP3 that explains both its role in autophagy and how this may be subverted to induce cell death. As a target of the RB tumor suppressor, our work also places BNIP3 at the center of efforts to exploit autophagy to better treat human cancers in which tumor hypoxia is implicated as a progression factor.     

Regulation of Mitochondrial Integrity,Autophagy and Cell Survival by BNIP3

Understanding the role of BNIP3 in the systemic response to hypoxia has been complicated by conflicting results that indicate on the one hand that BNIP3 promotes cell death, and other data, including our own that BNIP3 is not sufficient for cell death, but rather plays a critical role in hypoxia-induced autophagy. This work suggests that rather than promoting death, BNIP3 may actually allow survival either by preventing ATP depletion or by eliminating damaged mitochondria. However, the function of BNIP3 may be subverted under unusual conditions associated with acidosis that arise following extended periods of hypoxia and anaerobic glycolysis. Despite this novel insight into BNIP3 function, much remains to be done in terms of pinning down a molecular activity for BNIP3 that explains both its role in autophagy and how this may be subverted to induce cell death. As a target of the RB tumor suppressor, our work also places BNIP3 at the center of efforts to exploit autophagy to better treat human cancers in which tumor hypoxia is implicated as a progression factor.

Addendum to:

BNIP3 is an RB/E2F Target Gene Required for Hypoxia-Induced Autophagy

K. Tracy, B.C. Dibling, BT. Spike, J. Knabb, P. Schumacker and K.F. Macleod

Mol Cell Biol 2007; In press     

TP53INP1, a tumor suppressor, interacts with LC3 and ATG8-family proteins through the LC3-interacting region (LIR) and promotes autophagy-dependent cell death

TP53INP1 (tumor protein 53-induced nuclear protein 1) is a tumor suppressor, whose expression is downregulated in cancers from different organs. It was described as a p53 target gene involved in cell death, cell-cycle arrest and cellular migration. In this work, we show that TP53INP1 is also able to interact with ATG8-family proteins and to induce autophagy-dependent cell death. In agreement with this finding, we observe that TP53INP1, which is mainly nuclear, relocalizes in autophagosomes during autophagy where it is eventually degraded. TP53INP1-LC3 interaction occurs via a functional LC3-interacting region (LIR). Inactivating mutations of this sequence abolish TP53INP1-LC3 interaction, relocalize TP53INP1 in autophagosomes and decrease TP53INP1 ability to trigger cell death. Interestingly, TP53INP1 binds to ATG8-family proteins with higher affinity than p62, suggesting that it could partially displace p62 from autophagosomes, modifying thereby their composition. Moreover, silencing the expression of autophagy related genes (ATG5 or Beclin-1) or inhibiting caspase activity significantly decreases cell death induced by TP53INP1. These data indicate that cell death observed after TP53INP1-LC3 interaction depends on both autophagy and caspase activity. We conclude that TP53INP1 could act as a tumor suppressor by inducing cell death by caspase-dependent autophagy.     

The 100-kDa Proteolytic Fragment of RB Is Retained Predominantly within the Nuclear Compartment of Apoptotic Cells

The retinoblastoma tumor suppressor protein (RB) has been shown to play a role in regulating the eukaryotic cell cycle, promoting cellular differentiation, and modulating programmed cell death. Although regulation of RB tumor suppressor activity is mediated by reversible phosphorylation, an additional posttranslational modification involves the cleavage of 42 residues from the carboxy terminus of RB during the onset of drug-induced or receptor-mediated apoptosis. We now demonstrate that a recombinant p100cl RB species localizes to the nucleus where it may retain wildtype “pocket” protein binding activity. In addition, using immunocytochemistry, we show that cleavage of the endogenous RB protein occurs in vivo in human cells and that p100cl is predominantly retained within the nuclear compartment of cells during early apoptosis. We also show that the carboxy-terminal cleavage of RB is detected immediately following caspase-3 and PARP cleavage during FAS-mediated apoptosis of MCF10 cells. These findings suggest that this cleavage event may be a component of a downstream cascade during programmed cell death.     

The retinoblastoma protein tumor suppressor is important for appropriate osteoblast differentiation and bone development

Mutation of the retinoblastoma (RB) tumor suppressor gene is strongly linked to osteosarcoma formation. This observation and the documented interaction between the retinoblastoma protein (pRb) and Runx2 suggests that pRb is important in bone development. To assess this hypothesis, we used a conditional knockout strategy to generate pRb-deficient embryos that survive to birth. Analysis of these embryos shows that Rb inactivation causes the abnormal development and impaired ossification of several bones, correlating with an impairment in osteoblast differentiation. We further show that Rb inactivation acts to promote osteoblast differentiation in vitro and, through conditional analysis, establish that this occurs in a cell-intrinsic manner. Although these in vivo and in vitro differentiation phenotypes seem paradoxical, we find that Rb-deficient osteoblasts have an impaired ability to exit the cell cycle both in vivo and in vitro that can explain the observed differentiation defects. Consistent with this observation, we show that the cell cycle and the bone defects in Rb-deficient embryos can be suppressed by deletion of E2f1, a known proliferation inducer that acts downstream of Rb. Thus, we conclude that pRb plays a key role in regulating osteoblast differentiation by mediating the inhibition of E2F and consequently promoting cell cycle exit.     

p53 inhibits autophagy by interacting with the human ortholog of yeast Atg17, RB1CC1/FIP200

The tumor suppressor protein p53 tonically suppresses autophagy when it is present in the cytoplasm. This effect is phylogenetically conserved from mammals to nematodes, and human p53 can inhibit autophagy in yeast, as we show here. Bioinformatic investigations of the p53 interactome in relationship to the autophagy-relevant protein network underscored the possible relevance of a direct molecular interaction between p53 and the mammalian ortholog of the essential yeast autophagy protein Atg17, namely RB1-inducible coiled-coil protein 1 (RB1CC1), also called FAK family kinase-interacting protein of 200 KDa (FIP200). Mutational analyses revealed that a single point mutation in p53 (K382R) abolished its capacity to inhibit autophagy upon transfection into p53-deficient human colon cancer or yeast cells. In conditions in which wild-type p53 co-immunoprecipitated with RB1CC1/FIP200, p53^K382R failed to do so, underscoring the importance of the physical interaction between these proteins for the control of autophagy. In conclusion, p53 regulates autophagy through a direct molecular interaction with RB1CC1/FIP200, a protein that is essential for the very apical step of autophagy initiation.     

DRAM, a p53-induced modulator of autophagy, is critical for apoptosis

Inactivation of cell death is a major step in tumor development, and p53, a tumor suppressor frequently mutated in cancer, is a critical mediator of cell death. While a role for p53 in apoptosis is well established, direct links to other pathways controlling cell death are unknown. Here we describe DRAM (damage-regulated autophagy modulator), a p53 target gene encoding a lysosomal protein that induces macroautophagy, as an effector of p53-mediated death. We show that p53 induces autophagy in a DRAM-dependent manner and, while overexpression of DRAM alone causes minimal cell death, DRAM is essential for p53-mediated apoptosis. Moreover, analysis of DRAM in primary tumors revealed frequent decreased expression often accompanied by retention of wild-type p53. Collectively therefore, these studies not only report a stress-induced regulator of autophagy but also highlight the relationship of DRAM and autophagy to p53 function and damage-induced programmed cell death.     

Autophagy Signaling and the Cogwheels of Cancer

The downregulation of macroautophagy observed in cancer cells is associated with tumor progression. The regulation of macroautophagy by signaling pathways overlaps with the control of cell growth, proliferation, cell survival, and death. Several tumor suppressor genes (PTEN, TSC2 and p53) involved in the mTOR signaling network have been shown to stimulate autophagy. In contrast, the oncoproteins involved in this network have the opposite effect. These findings, together with the discovery that haplo-insufficiency of the tumor suppressor beclin 1 promotes tumorigenesis in various tissues in transgenic mice, give credibility to the idea that autophagy is a tumor suppressor mechanism. The induction of macroautophagy by cancer treatments may also contribute to cell eradication. However, cancer cells sometimes mobilize autophagic capacities in response to various stimuli without a fatal outcome, suggesting that they can also exploit macroautophagy for their own benefit.     

RB-dependent S-phase response to DNA damage

The retinoblastoma tumor suppressor protein (RB) is a potent inhibitor of cell proliferation. RB is expressed throughout the cell cycle, but its antiproliferative activity is neutralized by phosphorylation during the G(1)/S transition. RB plays an essential role in the G(1) arrest induced by a variety of growth inhibitory signals. In this report, RB is shown to also be required for an intra-S-phase response to DNA damage. Treatment with cisplatin, etoposide, or mitomycin C inhibited S-phase progression in Rb(+/+) but not in Rb(-/-) mouse embryo fibroblasts. Dephosphorylation of RB in S-phase cells temporally preceded the inhibition of DNA synthesis. This S-phase dephosphorylation of RB and subsequent inhibition of DNA replication was observed in p21(Cip1)-deficient cells. The induction of the RB-dependent intra-S-phase arrest persisted for days and correlated with a protection against DNA damage-induced cell death. These results demonstrate that RB plays a protective role in response to genotoxic stress by inhibiting cell cycle progression in G(1) and in S phase.     

Autophagy signaling and the cogwheels of cancer

The downregulation of macroautophagy observed in cancer cells is associated with tumor progression. The regulation of macroautophagy by signaling pathways overlaps with the control of cell growth, proliferation, cell survival and death. Several tumor suppressor genes (PTEN, TSC2 and p53) involved in the mTOR signaling network have been shown to stimulate autophagy. In contrast, the oncoproteins involved in this network have the opposite effect. These findings, together with the discovery that haploinsufficiency of the tumor suppressor beclin 1 promotes tumorigenesis in various tissues in transgenic mice, give credibility to the idea that autophagy is a tumor suppressor mechanism. The induction of macroautophagy by cancer treatments may also contribute to cell eradication. However, cancer cells sometimes mobilize autophagic capacities in response to various stimuli without a fatal outcome, suggesting that they can also exploit macroautophagy for their own benefit.     

Distinct patterns of expression of the RB gene family in mouse and human retina

Although RB1 function is disrupted in the majority of human cancers, an undefined cell of developing human retina is uniquely sensitive to cancer induction when the RB1 tumor suppressor gene is lost. Murine retinoblastoma is initiated only when two of the RB family of genes, RB1 and p107 or p130, are inactivated. Although whole embryonic retina shows RB family gene expression by several techniques, when E14 developing retina was depleted of the earliest differentiating cells, ganglion cells, the remaining proliferating murine embryonic retinal progenitor cells clearly did not express RB1 or p130, while the longer splice form of p107 was expressed. Each retinal cell type expressed some member of the RB family at some stage of differentiation. Rod photoreceptors stained for the RB1 protein product, pRB, and p107 in only a brief window of postnatal murine development, with no detectable staining for any of the RB family proteins in adult human and mouse rod photoreceptors. Adult mouse and human Muller glia, ganglion and rare horizontal cells, and adult human, but not adult mouse, cone photoreceptors stained for pRB. The RB gene family is dynamically and variably expressed through retinal development in specific retinal cells.     

Cell-autonomous and non-cell-autonomous functions of the Rb tumor suppressor in developing central nervous system.

The retinoblastoma tumor suppressor (RB) plays an important role in the regulation of cell cycle progression and terminal differentiation of many cell types. Rb(-/-) mouse embryos die at midgestation with defects in cell cycle regulation, control of apoptosis and terminal differentiation. However, chimeric mice composed of wild-type and Rb-deficient cells are viable and show minor abnormalities. To determine the role of Rb in development more precisely, we analyzed chimeric embryos and adults made with marked Rb(-/-) cells. Like their germline Rb(-/-) counterparts, brains of midgestation chimeric embryos exhibited extensive ectopic S-phase entry. In Rb-mutants, this is accompanied by widespread apoptosis. However, in chimeras, the majority of Rb-deficient cells survived and differentiated into neuronal fates. Rescue of Rb(-/-) neurons in the presence of wild-type cells occurred after induction of the p53 pathway and led to accumulation of cells with 4n DNA content. Therefore, the role of Rb during development can be divided into a cell-autonomous function in exit from the cell cycle and a non-cell-autonomous role in the suppression of apoptosis and induction of differentiation.