Postdoctoral fellowship, Johns Hopkins University
Cells use signaling pathways to detect and respond to harmful conditions by switching on genes that keep the cell healthy. One important pathway is the nuclear factor kappa B (NF-κB) signaling pathway, which is activated by many stimuli. These stimuli may come from infections from outside the cell or may originate inside the cell, as seen for DNA damage caused by irradiation, chemicals or rapid DNA replication in cancer cells. Most of a cell’s DNA is located in the cell nucleus. However, NF-κB proteins are normally located outside the nucleus, in the cell’s cytoplasm. Damage to DNA triggers a signal from the nucleus to the cytoplasm. This signal activates the NF-κB proteins, which move into the nucleus and turn on genes that help the cell to recover from the damage. These genes include those that prevent the cell from self-destructing. In one step of the NF-κB activation process, chain-like molecules called polymers are made from a compound called poly(ADP-ribose), or PAR for short. However, few other details are known about how the damaged DNA in the nucleus signals to the cytoplasm. A protein called Sam68, which is found in the cell nucleus, has been linked to DNA damage signaling. We now present evidence that suggests that if mouse cells lack Sam68, they do not produce PAR polymers in response to DNA damage. In addition, these cells could not trigger the PAR-dependent signaling cascade that is essential for activating NF-κB and for turning on the protective genes. Consequently, cells that lacked Sam68 were extremely sensitive to agents that cause DNA damage, such as chemicals and irradiation. The NF-κB pathway is regulated incorrectly in some cancers, but is also activated by DNA damage caused by cancer treatments. Therefore, we also explored the role of Sam68 in cancer. Reducing the levels of Sam68 made human colon cancer cells more likely to self-destruct when they were exposed to DNA-damaging agents. Furthermore, removing Sam68 from mice that spontaneously grow colon cancer caused their tumors to develop more slowly than mice that retained Sam68 in their cells. Overall, Our findings suggest that Sam68 regulates the signal from the nucleus to the cytoplasm that activates NF-κB proteins in response to DNA damage. Sam68 also appears to be important for helping colon cancer cells grow and survive.
Abstract: Nuclear factor kappa B (NF-κB)-mediated transcription is an important mediator for cellular responses to DNA damage. Genotoxic agents trigger a 'nuclear-to-cytoplasmic' NF-κB activation signaling pathway; however, the early nuclear signaling cascade linking DNA damage and NF-κB activation is poorly understood. Here we report that Src-associated-substrate-during-mitosis-of-68kDa/KH domain containing, RNA binding, signal transduction associated 1 (Sam68/KHDRBS1) is a key NF-κB regulator in genotoxic stress-initiated signaling pathway. Sam68 deficiency abolishes DNA damage-stimulated polymers of ADP-ribose (PAR) production and the PAR-dependent NF-κB transactivation of anti-apoptotic genes. Sam68 deleted cells are hypersensitive to genotoxicity caused by DNA damaging agents. Upregulated Sam68 coincides with elevated PAR production and NF-κB-mediated anti-apoptotic transcription in human and mouse colon cancer. Knockdown of Sam68 sensitizes human colon cancer cells to genotoxic stress-induced apoptosis and genetic deletion of Sam68 dampens colon tumor burden in mice. Together our data reveal a novel function of Sam68 in the genotoxic stress-initiated nuclear signaling, which is crucial for colon tumorigenesis.
Pub.: 28 Jul '16, Pinned: 29 Jun '17
Abstract: The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production.
Pub.: 17 Sep '16, Pinned: 29 Jun '17
Abstract: Previously we reported that Src-associated-substrate-during-mitosis-of-68kDa (Sam68/KHDRBS1) is pivotal for DNA damage-stimulated NF-κB transactivation of anti-apoptotic genes (Fu et al., 2016). Here we show that Sam68 is critical for genotoxic stress-induced NF-κB activation in the γ-irradiated colon and animal and that Sam68-dependent NF-κB activation provides radioprotection to colon epithelium in vivo. Sam68 deletion diminishes γ-irradiation-triggered PAR synthesis and NF-κB activation in colon epithelial cells (CECs), thus hampering the expression of anti-apoptotic molecules in situ and facilitating CECs to undergo apoptosis in mice post whole-body γ-irradiation (WBIR). Sam68 knockout mice suffer more severe damage in the colon and succumb more rapidly from acute radiotoxicity than the control mice following WBIR. Our results underscore the critical role of Sam68 in orchestrating genotoxic stress-initiated NF-κB activation signaling in the colon tissue and whole animal and reveal the pathophysiological relevance of Sam68-dependent NF-κB activation in colonic cell survival and recovery from extrinsic DNA damage.
Pub.: 21 Dec '16, Pinned: 29 Jun '17