STAT1 N-terminal domain discriminatively controls type I and type II IFN signaling.

The seven signal transducers of transcription (STATs) are cytokine-inducible modular transcription factors. They transmit the stimulation of cells with type I interferons (IFN-α/IFN-ß) and type II interferon (IFN-É£) into altered gene expression patterns. The N-terminal domain (NTD) of STAT1 is a surface for STAT1/STAT1 homodimer and STAT1/STAT2 heterodimer formation and allows the cooperative DNA binding of STAT1. We investigated whether the STAT1 NTD-mediated dimerization affected the IFN-induced tyrosine phosphorylation of STAT1, its nuclear translocation, STAT1-dependent gene expression, and IFN-dependent antiviral defense. We reconstituted human STAT1-negative and STAT2-negative fibrosarcoma cells with STAT1, NTD-mutated STAT1 (STAT1AA), STAT1 with a mutated DNA-binding domain (DBD), or STAT2. We treated these cells with IFN-α and IFN-É£ to assess differences between IFN-α-induced STAT1 homo- and heterodimers and IFN-É£-induced STAT1 homodimers. Our data demonstrate that IFNs induce the phosphorylation of STAT1 and STAT1AA at Y701 and their nuclear accumulation. We further reveal that STAT1AA can be phosphorylated in response to IFN-α in the absence of STAT2 and that IFN-É£-induced STAT1AA can activate gene expression directly. However, STAT1AA largely fails to bind STAT2 and to activate IFN-α-induced expression of endogenous antiviral STAT1/STAT2 target proteins. Congruent herewith, both an intact STAT1 NTD and STAT2 are indispensable to establish an antiviral state with IFN-α. These data provide new insights into the biological importance of the STAT1 NTD.

PARP-1 protects against colorectal tumor induction, but promotes inflammation-driven colorectal tumor progression.

Colorectal cancer (CRC) is one of the most common tumor entities, which is causally linked to DNA repair defects and inflammatory bowel disease (IBD). Here, we studied the role of the DNA repair protein poly(ADP-ribose) polymerase-1 (PARP-1) in CRC. Tissue microarray analysis revealed PARP-1 overexpression in human CRC, correlating with disease progression. To elucidate its function in CRC, PARP-1 deficient (PARP-1-/-) and wild-type animals (WT) were subjected to azoxymethane (AOM)/ dextran sodium sulfate (DSS)-induced colorectal carcinogenesis. Miniendoscopy showed significantly more tumors in WT than in PARP-1-/- mice. Although the lack of PARP-1 moderately increased DNA damage, both genotypes exhibited comparable levels of AOM-induced autophagy and cell death. Interestingly, miniendoscopy revealed a higher AOM/DSS-triggered intestinal inflammation in WT animals, which was associated with increased levels of innate immune cells and proinflammatory cytokines. Tumors in WT animals were more aggressive, showing higher levels of STAT3 activation and cyclin D1 up-regulation. PARP-1-/- animals were then crossed with O6-methylguanine-DNA methyltransferase (MGMT)-deficient animals hypersensitive to AOM. Intriguingly, PARP-1-/-/MGMT-/- double knockout (DKO) mice developed more, but much smaller tumors than MGMT-/- animals. In contrast to MGMT-deficient mice, DKO animals showed strongly reduced AOM-dependent colonic cell death despite similar O6-methylguanine levels. Studies with PARP-1-/- cells provided evidence for increased alkylation-induced DNA strand break formation when MGMT was inhibited, suggesting a role of PARP-1 in the response to O6-methylguanine adducts. Our findings reveal PARP-1 as a double-edged sword in colorectal carcinogenesis, which suppresses tumor initiation following DNA alkylation in a MGMT-dependent manner, but promotes inflammation-driven tumor progression.

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP.

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs.

The Essential Role of Rac1 Glucosylation in Clostridioides difficile Toxin B-Induced Arrest of G1-S Transition.

Clostridioides difficile infection (CDI) in humans causes pseudomembranous colitis (PMC), which is a severe pathology characterized by a loss of epithelial barrier function and massive colonic inflammation. PMC has been attributed to the action of two large protein toxins, Toxin A (TcdA) and Toxin B (TcdB). TcdA and TcdB mono-O-glucosylate and thereby inactivate a broad spectrum of Rho GTPases and (in the case of TcdA) also some Ras GTPases. Rho/Ras GTPases promote G1-S transition through the activation of components of the ERK, AKT, and WNT signaling pathways. With regard to CDI pathology, TcdB is regarded of being capable of inhibiting colonic stem cell proliferation and colonic regeneration, which is likely causative for PMC. In particular, it is still unclear, the glucosylation of which substrate Rho-GTPase is critical for TcdB-induced arrest of G1-S transition. Exploiting SV40-immortalized mouse embryonic fibroblasts (MEFs) with deleted Rho subtype GTPases, evidence is provided that Rac1 (not Cdc42) positively regulates Cyclin D1, an essential factor of G1-S transition. TcdB-catalyzed Rac1 glucosylation results in Cyclin D1 suppression and arrested G1-S transition in MEFs and in human colonic epithelial cells (HCEC), Remarkably, Rac1-/- MEFs are insensitive to TcdB-induced arrest of G1-S transition, suggesting that TcdB arrests G1-S transition in a Rac1 glucosylation-dependent manner. Human intestinal organoids (HIOs) specifically expressed Cyclin D1 (neither Cyclin D2 nor Cyclin D3), which expression was suppressed upon TcdB treatment. In sum, Cyclin D1 expression in colonic cells seems to be regulated by Rho GTPases (most likely Rac1) and in turn seems to be susceptible to TcdB-induced suppression. With regard to PMC, toxin-catalyzed Rac1 glucosylation and subsequent G1-S arrest of colonic stem cells seems to be causative for decreased repair capacity of the colonic epithelium and delayed epithelial renewal.

Lipoic Acid Synergizes with Antineoplastic Drugs in Colorectal Cancer by Targeting p53 for Proteasomal Degradation.

Lipoic acid (LA) is a redox-active disulphide compound, which functions as a pivotal co-factor for mitochondrial oxidative decarboxylation. LA and chemical derivatives were shown to target mitochondria in cancer cells with altered energy metabolism, thereby inducing cell death. In this study, the impact of LA on the tumor suppressor protein p53 was analyzed in various colorectal cancer (CRC) cell lines, with a focus on the mechanisms driving p53 degradation. First, LA was demonstrated to trigger the depletion of both wildtype and mutant p53 protein in all CRC cells tested without influencing its gene expression and preceded LA-triggered cytotoxicity. Depletion of p53 coincided with a moderate, LA-dependent ROS production, but was not rescued by antioxidant treatment. LA induced the autophagy receptor p62 and differentially modulated autophagosome formation in CRC cells. However, p53 degradation was not mediated via autophagy as shown by chemical inhibition and genetic abrogation of autophagy. LA treatment also stabilized and activated the transcription factor Nrf2 in CRC cells, which was however dispensable for p53 degradation. Mechanistically, p53 was found to be readily ubiquitinylated and degraded by the proteasomal machinery following LA treatment, which did not involve the E3 ubiquitin ligase MDM2. Intriguingly, the combination of LA and anticancer drugs (doxorubicin, 5-fluorouracil) attenuated p53-mediated stabilization of p21 and resulted in synergistic killing in CRC cells in a p53-dependant manner.

AKT2 suppresses pro-survival autophagy triggered by DNA double-strand breaks in colorectal cancer cells.

DNA double-strand breaks (DSBs) are critical DNA lesions, which threaten genome stability and cell survival. DSBs are directly induced by ionizing radiation (IR) and radiomimetic agents, including the cytolethal distending toxin (CDT). This bacterial genotoxin harbors a unique DNase-I-like endonuclease activity. Here we studied the role of DSBs induced by CDT and IR as a trigger of autophagy, which is a cellular degradation process involved in cell homeostasis, genome protection and cancer. The regulatory mechanisms of DSB-induced autophagy were analyzed, focusing on the ATM-p53-mediated DNA damage response and AKT signaling in colorectal cancer cells. We show that treatment of cells with CDT or IR increased the levels of the autophagy marker LC3B-II. Consistently, an enhanced formation of autophagosomes and a decrease of the autophagy substrate p62 were observed. Both CDT and IR concomitantly suppressed mTOR signaling and stimulated the autophagic flux. DSBs were demonstrated as the primary trigger of autophagy using a DNase I-defective CDT mutant, which neither induced DSBs nor autophagy. Genetic abrogation of p53 and inhibition of ATM signaling impaired the autophagic flux as revealed by LC3B-II accumulation and reduced formation of autophagic vesicles. Blocking of DSB-induced apoptotic cell death by the pan-caspase inhibitor Z-VAD stimulated autophagy. In line with this, pharmacological inhibition of autophagy increased cell death, while ATG5 knockdown did not affect cell death after DSB induction. Interestingly, both IR and CDT caused AKT activation, which repressed DSB-triggered autophagy independent of the cellular DNA-PK status. Further knockdown and pharmacological inhibitor experiments provided evidence that the negative autophagy regulation was largely attributable to AKT2. Finally, we show that upregulation of CDT-induced autophagy upon AKT inhibition resulted in lower apoptosis and increased cell viability. Collectively, the findings demonstrate that DSBs trigger pro-survival autophagy in an ATM- and p53-dependent manner, which is curtailed by AKT2 signaling.