G1 Phase

BackgroundMetformin is a commonly used drug for the treatment of diabetes. Accumulating evidence suggests that it exerts anti-tumor effects in many cancers, including multiple myeloma (MM); however, the underlying molecular mechanisms have not been clearly elucidated.MethodsThe anti-myeloma effects of metformin were evaluated using human MM cell lines (RPMI8226 and U266) in vitro and in vivo NOD-SCID murine xenograft MM model. Cell viability was assessed with CCK8 and cell proliferation was measured by EdU incorporation assay. Cell cycle distribution and apoptosis were examined by flow cytometry. Transmission electron microscopy was used to visualized autophagosomes. Activation of AMPK and inhibition of mTORC1/C2 pathways was assessed by Western blot analysis. RPMI8226 cells and U266 cell lines with AMPK knockdown were generated by transfection with small interfering RNA targeting the AMPK-α1 and α2 subunits using Lipofectamine 2000 reagent.ResultsMetformin effectively inhibited the proliferation of MM cell lines, an effect that was associated with the induction of autophagy and G0/G1 cell cycle arrest, but not apoptosis. Metformin activated AMPK and repressed both mTORC1 and mTORC2 signaling pathways in myeloma cells as well as downstream molecular signaling pathways, such as p-4EBP1 and p-AKT. AMPK activation resulted in direct phosphorylation and activation of tuberous sclerosis complex 2 (TSC2), leading to inhibition of the mammalian target of rapamycin (mTOR). In addition, metformin inhibited myeloma cell growth in an AMPK-dependent manner. The xenograft mouse model further confirmed that metformin inhibited tumor growth by upregulation of AMPK and downregulation of mTOR.ConclusionsMetformin inhibits the proliferation of myeloma cells by inducing autophagy and cell-cycle arrest. Our results suggest that the molecular mechanism involves dual repression of mTORC1 and mTORC2 pathways via AMPK activation. Our study provides a theoretical basis for the development of novel strategies for the treatment of MM using metformin as an already approved and safe drug.

Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ERα) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.

Upon entry into the G1 phase following mitosis, mammalian chromosomal DNA becomes spatially segregated into A and B compartments, which correspond closely to classic euchromatin and heterochromatin, respectively. The functional significance of this spatial segregation, however, has remained unexplored due to the lack of means to manipulate this level of chromosomal organization. Through a genome-wide loss-of-function CRISPR screen, we identify GINS4, a component of the replicative DNA helicase complex, as a factor essential for segregation of A and B compartments during the G1-to-S phase transition. Using GINS4 depletion experiments, we show that proper A/B compartment organization at the time of S-phase entry plays a key role in efficient DNA synthesis. Furthermore, DNA synthesis with attenuated A/B compartments is associated with defects in replication timing regulation. Our findings uncover a previously unrecognized role for GINS4 in regulating nuclear architecture and underscore the biological significance of nuclear compartmentalization in DNA replication control.