Bacterial Genotoxin Accelerates Transient Infection-Driven Murine Colon Tumorigenesis.

Chronic and low-grade inflammation associated with persistent bacterial infections has been linked to colon tumor development; however, the impact of transient and self-limited infections in bacterially driven colon tumorigenesis has remained enigmatic. Here we report that UshA is a novel genotoxin in attaching/effacing (A/E) pathogens, which include the human pathogens enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and their murine equivalent Citrobacter rodentium (CR). UshA harbors direct DNA digestion activity with a catalytic histidine-aspartic acid dyad. Injected via the type III secretion system (T3SS) into host cells, UshA triggers DNA damage and initiates tumorigenic transformation during infections in vitro and in vivo. Moreover, UshA plays an indispensable role in CR infection-accelerated colon tumorigenesis in genetically susceptible Apc MinΔ716/+ mice. Collectively, our results reveal that UshA, functioning as a bacterial T3SS-dependent genotoxin, plays a critical role in prompting transient and noninvasive bacterial infection-accelerated colon tumorigenesis in mice. SIGNIFICANCE: We identified UshA, a novel T3SS-dependent genotoxin in A/E pathogens that possesses direct DNA digestion activity and confers bacterially accelerated colon tumorigenesis in mice. Our results demonstrate that acute and noninvasive infection with A/E pathogens harbors a far-reaching impact on the development of colon cancer.This article is highlighted in the In This Issue feature, p. 1.

Noncoding dsRNA induces retinoic acid synthesis to stimulate hair follicle regeneration via TLR3.

How developmental programs reactivate in regeneration is a fundamental question in biology. We addressed this question through the study of Wound Induced Hair follicle Neogenesis (WIHN), an adult organogenesis model where stem cells regenerate de novo hair follicles following deep wounding. The exact mechanism is uncertain. Here we show that self-noncoding dsRNA activates the anti-viral receptor toll like receptor 3 (TLR3) to induce intrinsic retinoic acid (RA) synthesis in a pattern that predicts new hair follicle formation after wounding in mice. Additionally, in humans, rejuvenation lasers induce gene expression signatures for dsRNA and RA, with measurable increases in intrinsic RA synthesis. These results demonstrate a potent stimulus for RA synthesis by non-coding dsRNA, relevant to their broad functions in development and immunity.

Sam68/KHDRBS1-dependent NF-κB activation confers radioprotection to the colon epithelium in γ-irradiated mice.

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.

Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation.

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.

Ultrasound imaging of splenomegaly as a proxy to monitor colon tumor development in Apc(min716/+) mice.

Animal models of colon cancer are widely used to understand the molecular mechanisms and pathogenesis of the disease. These animal models require a substantial investment of time and traditionally necessitate the killing of the animal to measure the tumor progression. Several in vivo imaging techniques are being used in both human clinics and preclinical studies, albeit at high cost and requiring particular expertise. Here, we report that the progression of splenomegaly coincides with and positively correlates to colon tumor development in Apc(min716/+) mice expressing a mutant gene encoding an adenomatous polyposis coli protein truncated at amino acid 716. Ultrasound image-based spleen size measurement precisely mirrors splenomegaly development in vivo in the tumor-laden Apc(min716/+) mice. Moreover, the spleen dimensions extracted from the ultrasound sonograms are positively correlated with normalized spleen weight and the number and area of colon tumors. Hence, we propose measuring the spleen size in vivo by ultrasound imaging as a novel approach to estimate splenomegaly development and to indirectly monitor colon tumor development in Apc(min716/+) mice. The widespread use of ultrasound machines in the laboratory setting, coupled with the fact that it is a noninvasive method, make it a straightforward and useful tool for monitoring the experimental progress of colon cancer in mice and determining end points without killing animals strictly for diagnostics purposes.

Sam68/KHDRBS1 is critical for colon tumorigenesis by regulating genotoxic stress-induced NF-κB activation.

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.

Caspase-3 cleaved p65 fragment dampens NF-κB-mediated anti-apoptotic transcription by interfering with the p65/RPS3 interaction.

Caspase-3-mediated p65 cleavage is believed to suppress nuclear factor-kappa B (NF-κB)-mediated anti-apoptotic transactivation in cells undergoing apoptosis. However, only a small percentage of p65 is cleaved during apoptosis, not in proportion to the dramatic reduction in NF-κB transactivation. Here we show that the p65(1-97) fragment generated by Caspase-3 cleavage interferes with ribosomal protein S3 (RPS3), an NF-κB "specifier" subunit, and selectively retards the nuclear translocation of RPS3, thus dampening the RPS3/NF-κB-dependent anti-apoptotic gene expression. Our findings reveal a novel cell fate determination mechanism to ensure cells undergo programed cell death through interfering with RPS3/NF-κB-conferred anti-apoptotic transcription by the fragment from partial p65 cleavage by activated Caspase-3.

Metalloprotease NleC suppresses host NF-κB/inflammatory responses by cleaving p65 and interfering with the p65/RPS3 interaction.

Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p65¹â»³8 fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p65¹â»³8 fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a 'specifier' subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.

Sam68 modulates the promoter specificity of NF-κB and mediates expression of CD25 in activated T cells.

CD25, the alpha chain of the interleukin-2 receptor, is expressed in activated T cells and has a significant role in autoimmune disease and tumorigenesis; however, the mechanisms regulating transcription of CD25 remain elusive. Here we identify the Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel non-Rel component in the nuclear factor-kappaB (NF-κB) complex that confers CD25 transcription. Our results demonstrate that Sam68 has an essential role in the induction and maintenance of CD25 in T cells. T-cell receptor engagement triggers translocation of the inhibitor of NF-κB kinase alpha (IKKα) from the cytoplasm to the nucleus, where it phosphorylates Sam68, causing complex formation with NF-κB in the nucleus. These findings reveal the important roles of KH domain-containing components and their spatial interactions with IKKs in determining the binding targets of NF-κB complexes, thus shedding novel insights into the regulatory specificity of NF-κB.

Identification of an N-terminal truncation of the NF-κB p65 subunit that specifically modulates ribosomal protein S3-dependent NF-κB gene expression.

NF-κB is a pleiotrophic transcription factor that plays a prominent regulatory role in various cellular processes. Although previous efforts have focused on its activation, how NF-κB selects specific target genes in response to discrete signals remains puzzling. In addition to the well defined Rel protein components of NF-κB, the ribosomal protein S3 (RPS3) was identified to be an essential component of specific NF-κB complexes. RPS3 synergistically interacts with the NF-κB p65 subunit to achieve optimal binding and transactivation of a subset of NF-κB target genes, thus providing regulatory specificity. Emerging evidence suggests an important role for the RPS3-p65 interaction in context-specific NF-κB gene transcription. The food-borne pathogen Escherichia coli O157:H7 impacts the transcription of a subset of NF-κB target genes encoding proinflammatory cytokines and chemokines in host cells by preventing the nuclear translocation of RPS3, but not p65. The N terminus of p65 is crucial for RPS3 binding. Although several p65 N-terminal fragments are generated by either protease cleavage or alternative mRNA splicing under certain pathophysiological conditions, the role of these fragments in modulating NF-κB signaling, in particular RPS3-dependent selective gene transcription, has not been fully characterized. Here we report that an N-terminal fragment of p65 (amino acids 21-186) can selectively modulate NF-κB gene transcription by competing for RPS3 binding to p65. This 21-186 fragment preferentially localizes in the cytoplasm where it delays stimuli-induced RPS3 nuclear translocation, without affecting the nuclear translocation of p65. Our findings thus uncover a new cytoplasmic function for the N-terminal domain of p65 and provide a novel strategy for selective inhibition of NF-κB gene transcription.