Diet and microbiome derived indole derivatives are known to activate the ligand induced transcription factor, the Aryl hydrocarbon Receptor (AhR). While the current understanding of AhR biology has ...confirmed its role in mucosal lymphocytes, its function in intestinal antigen presenting cells (APCs) is poorly understood. Here, we report that Cre-mediated deletion of AhR in CD11c-expressing cells in C57/BL6 mice is associated with altered intestinal epithelial morphogenesis in vivo. Moreover, when co-cultured with AhR-deficient DCs ex vivo, intestinal organoids showed reduced SRY (sex determining region Y)-box 9 and increased Mucin 2 expression, which correlates with reduced Paneth cells and increased goblet cell differentiation, similar to the data obtained in vivo. Further, characterization of intestinal APC subsets, devoid of AhR, revealed an expression pattern associated with aberrant intrinsic Wnt pathway regulation. At a functional level, the loss of AhR in APCs resulted in a dysfunctional epithelial barrier, associated with a more aggressive chemically induced colitis compared to wild type animals. Our results are consistent with a model whereby the AhR signalling pathway may participate in the regulation of innate immunity through intestinal epithelium development and mucosal immunity.
The tumour suppressor ARF (alternative reading frame), which is mutated or silenced in various tumours, has a crucial role in tumour surveillance to suppress unwarranted cell growth and ...proliferation. ARF has also been linked to the DNA‐damage‐induced response of p53 because of its ability to inhibit murine double minute 2 (MDM2). Here, however, we provide genetic evidence for a role of ARF in nucleotide excision repair (NER) that is independent of p53. Cells lacking ARF are deficient in NER. Expression of ARF restores the repair activity, which coincides with increased expression of the damaged‐DNA recognition protein xeroderma pigmentosum, complementation group C (XPC). We provide evidence that, by disrupting the interaction between E2F transcription factor 4 (E2F4) and DRTF polypeptide 1 (DP1), ARF reduces the interaction of the E2F4–p130 repressor complex with the promoter of XPC to ensure high‐level expression of XPC. Together, our results point to an important ‘care‐taker’‐type tumour‐suppression function for ARF in NER through the increased expression of XPC.
Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these ...processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.
The E3 ubiquitin ligase Mule is often overexpressed in human colorectal cancers, but its role in gut tumorigenesis is unknown. Here, we show in vivo that Mule controls murine intestinal stem and ...progenitor cell proliferation by modulating Wnt signaling via c-Myc. Mule also regulates protein levels of the receptor tyrosine kinase EphB3 by targeting it for proteasomal and lysosomal degradation. In the intestine, EphB/ephrinB interactions position cells along the crypt-villus axis and compartmentalize incipient colorectal tumors. Our study thus unveils an important new avenue by which Mule acts as an intestinal tumor suppressor by regulation of the intestinal stem cell niche.
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•Mule regulates murine intestinal stem and progenitor cell proliferation•Mule modulates the Wnt pathway via c-Myc•Mule targets EphB3 to the proteasome and lysosome•Proper protein levels of EphB3 are required to maintain the EphB/ephrinB gradient
Dominguez-Brauer et al. show that the ubiquitin ligase Mule fine-tunes the Wnt pathway via c-Myc to control unwarranted proliferation and stem cell expansion. Mule also regulates the levels of EphB3 to maintain the EphB/ephrinB gradient, which is responsible for cell positioning along the crypt-villus axis.
The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G1/S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we ...examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G2/M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G2/M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1.
The cell cycle is an evolutionarily conserved process necessary for mammalian cell growth and development. Because cell-cycle aberrations are a hallmark of cancer, this process has been the target of ...anti-cancer therapeutics for decades. However, despite numerous clinical trials, cell-cycle-targeting agents have generally failed in the clinic. This review briefly examines past cell-cycle-targeted therapeutics and outlines how experience with these agents has provided valuable insight to refine and improve anti-mitotic strategies. An overview of emerging anti-mitotic approaches with promising pre-clinical results is provided, and the concept of exploiting the genomic instability of tumor cells through therapeutic inhibition of mitotic checkpoints is discussed. We believe this strategy has a high likelihood of success given its potential to enhance therapeutic index by targeting tumor-specific vulnerabilities. This reasoning stimulated our development of novel inhibitors targeting the critical regulators of genomic stability and the mitotic checkpoint: AURKA, PLK4, and Mps1/TTK.
Dominguez-Brauer et al. review the therapeutic potential of exploiting the genomic instability of tumor cells by inhibiting mitotic checkpoints as a strategy to selectively target tumor-specific vulnerabilities.
Gain-of-function mutations in isocitrate dehydrogenase 1 (IDH1) are key drivers of hematopoietic malignancies. Although these mutations are most commonly associated with myeloid diseases, they also ...occur in malignancies of the T-cell lineage. To investigate their role in these diseases and provide tractable disease models for further investigation, we analyzed the T-cell compartment in a conditional knock-in (KI) mouse model of mutant Idh1. We observed the development of a spontaneous T-cell acute lymphoblastic leukemia (T-ALL) in these animals. The disease was transplantable and maintained expression of mutant IDH1. Whole-exome sequencing revealed the presence of a spontaneous activating mutation in Notch1, one of the most common mutations in human T-ALL, suggesting Idh1 mutations may have the capacity to cooperate with Notch1 to drive T-ALL. To further investigate the Idh1 mutation as an oncogenic driver in the T-cell lineage,we crossed Idh1-KI mice with conditional Trp53 null mice, a well-characterized model of T-cell malignancy, and found that T-cell lymphomagenesis was accelerated in mice bearing both mutations. Because both IDH1 and p53 are known to affect cellular metabolism, we compared the requirements for glucose and glutamine in cells derived from these tumors and found that cells bearing the Idh1 mutation have an increased dependence on both glucose and glutamine. These data suggest that mutant IDH1 contributes to malignancy in the T-cell lineage and may alter the metabolic profile of malignant T cells.
ARF is a vital tumor suppressor and its loss contributes significantly to cancer. The frequency in which ARF is mutated, deleted or silenced is second to the loss of p53. The most documented and ...widely accepted activity of ARF is mediated through its activation of the p53 transcriptional program by inhibiting MDM2 function. However, several lines of evidence have surfaced demonstrating that ARF possesses p53-independent functions. One of these p53 independent functions is ARF's regulation of the E2F family. The E2F/DP transcription factor is critical for cell cycle progression. The balance between activator and repressor E2Fs regulates the expression of E2F target genes and thus cell proliferation as well as other cellular functions such as checkpoint, chromosome assembly and repair. Through its ability to bind directly to DP1, ARF can cause dissociation of both activator and repressor E2Fs. While the regulation of the activator E2Fs is related to cell cycle arrest, there is evidence that the regulation of the repressors, E2F4 and E2F5, is significant in maintaining genomic stability.
Protein tyrosine kinase 6 (PTK6), also called breast tumor kinase (BRK), is expressed in epithelial cells of various tissues including the prostate. Previously it was shown that PTK6 is localized to ...epithelial cell nuclei in normal prostate, but becomes cytoplasmic in human prostate tumors. PTK6 is also primarily cytoplasmic in the PC3 prostate adenocarcinoma cell line. Sequencing revealed expression of wild type full-length PTK6 transcripts in addition to an alternative transcript lacking exon 2 in PC3 cells. The alternative transcript encodes a 134 amino acid protein, referred to here as ALT-PTK6, which shares the first 77 amino acid residues including the SH3 domain with full length PTK6. RT-PCR was used to show that ALT-PTK6 is coexpressed with full length PTK6 in established human prostate and colon cell lines, as well as in primary cell lines derived from human prostate tissue and tumors. Although interaction between full-length PTK6 and ALT-PTK6 was not detected, ALT-PTK6 associates with the known PTK6 substrates Sam68 and β-catenin in GST pull-down assays. Coexpression of PTK6 and ALT-PTK6 led to suppression of PTK6 activity and reduced association of PTK6 with tyrosine phosphorylated proteins. While ALT-PTK6 alone did not influence β-catenin/TCF transcriptional activity in a luciferase reporter assay, it enhanced PTK6-mediated inhibition of β-catenin/TCF transcription by promoting PTK6 nuclear functions. Ectopic expression of ALT-PTK6 led to reduced expression of the β-catenin/TCF targets Cyclin D1 and c-Myc in PC3 cells. Expression of tetracycline-inducible ALT-PTK6 blocked the proliferation and colony formation of PC3 cells. Our findings suggest that ALT-PTK6 is able to negatively regulate growth and modulate PTK6 activity, protein-protein associations and/or subcellular localization. Fully understanding functions of ALT-PTK6 and its impact on PTK6 signaling will be critical for development of therapeutic strategies that target PTK6 in cancer.
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