Lachnochromonin, a fungal metabolite from Lachnum virgineum, inhibits cell growth and promotes apoptosis in tumor cells through JAK/STAT3 signaling.

Natural compounds that interfere with tumor cell growth have potential to be used as therapeutic agents to treat cancers. Lachnochromonin (p71) is a small molecule isolated from Lachnum virgineum. Here, we reported the effect of p71 on human tumor cells, especially on breast cancer MCF-7 cells. We found that p71 significantly suppresses cell growth and induces apoptosis. The luciferase results demonstrated that p71 specifically attenuates the activation of JAK/STAT3 signaling. Biochemical analysis revealed that p71 blocks the phosphorylation of STAT3 tyrosine 705 and serine 727, resulting in down-regulation of c-Myc and Cyclin D1 expression level. Importantly, p71 inhibited cell growth, colony-formation, and migration through affecting STAT3 activity. These results implied that p71 may be used as a therapeutic agent against breast cancer.

CREPT/RPRD1B promotes tumorigenesis through STAT3-driven gene transcription in a p300-dependent manner.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) has been shown to upregulate gene transcription during tumorigenesis. However, how STAT3 initiates transcription remains to be exploited. This study is to reveal the role of CREPT (cell cycle-related and elevated-expression protein in tumours, or RPRD1B) in promoting STAT3 transcriptional activity. METHODS: BALB/c nude mice, CREPT overexpression or deletion cells were employed for the assay of tumour formation, chromatin immunoprecipitation, assay for transposase-accessible chromatin using sequencing. RESULTS: We demonstrate that CREPT, a recently identified oncoprotein, enhances STAT3 transcriptional activity to promote tumorigenesis. CREPT expression is positively correlated with activation of STAT3 signalling in tumours. Deletion of CREPT led to a decrease, but overexpression of CREPT resulted in an increase, in STAT3-initiated tumour cell proliferation, colony formation and tumour growth. Mechanistically, CREPT interacts with phosphorylated STAT3 (p-STAT3) and facilitates p-STAT3 to recruit p300 to occupy at the promoters of STAT3-targeted genes. Therefore, CREPT and STAT3 coordinately facilitate p300-mediated acetylation of histone 3 (H3K18ac and H3K27ac), further augmenting RNA polymerase II recruitment. Accordingly, depletion of p300 abolished CREPT-enhanced STAT3 transcriptional activity. CONCLUSIONS: We propose that CREPT is a co-activator of STAT3 for recruiting p300. Our study provides an alternative strategy for the therapy of cancers related to STAT3.

Absence of GdX/UBL4A Protects against Inflammatory Diseases by Regulating NF-кB Signaling in Macrophages and Dendritic Cells.

Nuclear factor-kappa B (NF-κB) activation is critical for innate immune responses. However, cellular-intrinsic regulation of NF-κB activity during inflammatory diseases remains incompletely understood. Ubiquitin-like protein 4A (UBL4A, GdX) is a small adaptor protein involved in protein folding, biogenesis and transcription. Yet, whether GdX has a role during innate immune response is largely unknown. Methods: To investigate the involvement of GdX in innate immunity, we challenged GdX-deficient mice with lipopolysaccharides (LPS). To investigate the underlying mechanism, we performed RNA sequencing, real-time PCR, ELISA, luciferase reporter assay, immunoprecipitation and immunoblot analyses, flow cytometry, and structure analyses. To investigate whether GdX functions in inflammatory bowel disease, we generated dendritic cell (DC), macrophage (Mφ), epithelial-cell specific GdX-deficient mice and induced colitis with dextran sulfate sodium. Results: GdX enhances DC and Mφ-mediated innate immune defenses by positively regulating NF-κB signaling. GdX-deficient mice were resistant to LPS-induced endotoxin shock and DSS-induced colitis. DC- or Mφ- specific GdX-deficient mice displayed alleviated mucosal inflammation. The production of pro-inflammatory cytokines by GdX-deficient DCs and Mφ was reduced. Mechanistically, we found that tyrosine-protein phosphatase non-receptor type 2 (PTPN2, TC45) and protein phosphatase 2A (PP2A) form a complex with RelA (p65) to mediate its dephosphorylation whereas GdX interrupts the TC45/PP2A/p65 complex formation and restrict p65 dephosphorylation by trapping TC45. Conclusion: Our study provides a mechanism by which NF-κB signaling is positively regulated by an adaptor protein GdX in DC or Mφ to maintain the innate immune response. Targeting GdX could be a strategy to reduce over-activated immune response in inflammatory diseases.

Dimerization of p15RS mediated by a leucine zipper-like motif is critical for its inhibitory role on Wnt signaling.

We previously demonstrated that p15RS, a newly discovered tumor suppressor, inhibits Wnt/ß-catenin signaling by interrupting the formation of ß-catenin·TCF4 complex. However, it remains unclear how p15RS helps exert such an inhibitory effect on Wnt signaling based on its molecular structure. In this study, we reported that dimerization of p15RS is required for its inhibition on the transcription regulation of Wnt-targeted genes. We found that p15RS forms a dimer through a highly conserved leucine zipper-like motif in the coiled-coil terminus domain. In particular, residues Leu-248 and Leu-255 were identified as being responsible for p15RS dimerization, as mutation of these two leucines into prolines disrupted the homodimer formation of p15RS and weakened its suppression of Wnt signaling. Functional studies further confirmed that mutations of p15RS at these residues results in diminishment of its inhibition on cell proliferation and tumor formation. We therefore concluded that dimerization of p15RS governed by the leucine zipper-like motif is critical for its inhibition of Wnt/ß-catenin signaling and tumorigenesis.

Nuclear termination of STAT3 signaling through SIPAR (STAT3-Interacting Protein As a Repressor)-dependent recruitment of T cell tyrosine phosphatase TC-PTP.

STAT3 is associated with embryo development and survival as well as proliferation and metastasis of tumor cells. In a previous study, we demonstrated that STAT3-Interacting Protein As a Repressor (SIPAR) enhances the dephosphorylation of STAT3 and negatively regulates its activity. However, it remains unclear how SIPAR inhibits phosphorylation of STAT3. Here we demonstrate that SIPAR directly interacts with T cell protein tyrosine phosphatase TC45 and enhances its association with STAT3. This interaction triggers an accelerated dephosphorylation process for STAT3. Furthermore, SIPAR inhibits the transcriptional activity of STAT3 in wild-type MEF cells but not in TC45 null MEF cells. These results suggest that SIPAR terminates the activation of STAT3 through a dephosphorylation process that is dependent upon interaction with TC45 in the nucleus.

p15RS/RPRD1A (p15INK4b-related sequence/regulation of nuclear pre-mRNA domain-containing protein 1A) interacts with HDAC2 in inhibition of the Wnt/ß-catenin signaling pathway.

We previously reported that p15RS (p15INK4b-related sequence), a regulation of nuclear pre-mRNA domain containing protein, inhibited Wnt signaling by interrupting the formation of the ß-catenin·TCF4 complex. However, how p15RS functions as an intrinsic repressor to repress transcription remains unclear. In this study, we show that p15RS, through a specific interaction with HDAC2 (histone deacetylase 2), a deacetylase that regulates gene transcription, maintains histone H3 in a deacetylated state in the promoter region of Wnt-targeted genes where ß-catenin·TCF4 is bound. We observed that histone deacetylase inhibitors impair the ability of p15RS in inhibiting Wnt/ß-catenin signaling. Depletion of HDAC2 markedly disabled p15RS inhibition of Wnt/ß-catenin-mediated transcription. Interestingly, overexpression of p15RS decreases the level of acetylated histone H3 in the c-MYC promoter. Finally, we demonstrate that p15RS significantly enhances the association of HDAC2 and TCF4 and enhances the occupancy of HDAC2 to DNA, resulting in the deacetylation of histone H3 and the failure of ß-catenin interaction. We propose that p15RS acts as an intrinsic transcriptional repressor for Wnt/ß-catenin-mediated gene transcription at least partially through recruiting HDAC2 to occupy the promoter and maintaining deacetylated histone H3.

CREPT/RPRD1B, a recently identified novel protein highly expressed in tumors, enhances the ß-catenin·TCF4 transcriptional activity in response to Wnt signaling.

CREPT (cell cycle-related and expression elevated protein in tumor)/RPRD1B (regulation of nuclear pre-mRNA domain-containing protein 1B), highly expressed during tumorigenesis, was shown to enhance transcription of CCND1 and to promote cell proliferation by interacting with RNA polymerase II. However, which signaling pathway is involved in CREPT-mediated activation of gene transcription remains unclear. In this study, we reveal that CREPT participates in transcription of the Wnt/ß-catenin signaling activated genes through the ß-catenin and the TCF4 complex. Our results demonstrate that CREPT interacts with both ß-catenin and TCF4, and enhances the association of ß-catenin with TCF4, in response to Wnt stimulation. Furthermore, CREPT was shown to occupy at TCF4 binding sites (TBS) of the promoters of Wnt-targeted genes under Wnt stimulation. Interestingly, depletion of CREPT resulted in decreased occupancy of ß-catenin on TBS, and over-expression of CREPT enhances the activity of the ß-catenin·TCF4 complex to initiate transcription of Wnt target genes, which results in up-regulated cell proliferation and invasion. Our study suggests that CREPT acts as an activator to promote transcriptional activity of the ß-catenin·TCF4 complex in response to Wnt signaling.

CHIP/Stub1 functions as a tumor suppressor and represses NF-κB-mediated signaling in colorectal cancer.

The carboxyl terminus of Hsc70-interacting protein (CHIP, also named Stub1), a U-box containing E3 ubiquitin ligase, is involved in degradation of certain oncogenic proteins. Recent studies indicated that CHIP suppresses tumor progression in human cancers by targeting Src-3, hypoxia inducible factor 1α, NF-κB, ErbB2 and c-Myc. Here, we report that CHIP was downregulated, predominantly, in the late stages of human colorectal cancer (CRC), and that the CHIP promoter was hypermethylated in CRC specimens. Overexpression of CHIP in HCT-116 cells resulted in impaired tumor growth in nude mice and decreased abilities of tumor cell migration and invasion. Conversely, depletion of CHIP in HCT-116 cells promoted tumor growth and increased tumor cell migration and invasion. CHIP was further found to negatively regulate NF-κB signaling in HCT-116 cells by promoting ubiquitination and degradation of p65, a subunit of the NF-κB complex. The suppressive effect of CHIP led to decreased expression of NF-κB-targeted oncogenes including Cyclin D1, c-Myc, MMP-2, VEGF and IL-8. We proposed that CHIP inhibits the malignancy of CRC cells, possibly through targeting NF-κB signaling. This study provides functional evidence for CHIP as a potential tumor suppressor in CRC, and CHIP expression may be a marker for stages of CRC.

SIPAR negatively regulates STAT3 signaling and inhibits progression of melanoma.

Persistently activated STAT3 is important for tumorigenesis in a variety of cancers, including melanoma. Although many co-factors in the regulation of STAT3 activity have been identified, it remains unclear how STAT3 phosphorylation is negatively regulated. Here, we report that SIPAR (STAT3-Interacting Protein As a Repressor) inhibits STAT3 activity by accelerating its dephosphorylation. We observed that SIPAR directly interacted with STAT3 upon IL-6 stimulation. Moreover, over-expression of SIPAR reduced, whereas depletion enhanced, the level of phosphorylated STAT3. We further demonstrated that SIPAR inhibited the growth of melanoma cells by decreasing the level of phosphorylated STAT3 and the expression of its target genes. These results suggest that SIPAR, functioning as a new negative regulator, inhibits STAT3 activity by enhancing its dephosphorylation and represses melanoma progression.