Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are associated with a poor prognosis. The factors influencing liver metastasis of CRC are poorly characterized, but this information is urgently needed. We have now discovered that small extracellular vesicles (sEVs; exosomes) derived from CRC can be specifically targeted to liver tissue and induce liver macrophage polarization toward an interleukin-6 (IL-6)-secreting proinflammatory phenotype. More importantly, we found that microRNA-21-5p (miR-21) was highly enriched in CRC-derived sEVs and was essential for creating a liver proinflammatory phenotype and liver metastasis of CRC. Silencing either miR-21 in CRC-sEVs or Toll-like receptor 7 (TLR7) in macrophages, to which miR-21 binds, abolished CRC-sEVs’ induction of proinflammatory macrophages. Furthermore, miR-21 expression in plasma-derived sEVs was positively correlated with liver metastasis in CRC patients. Collectively, our data demonstrate a pivotal role of CRC-sEVs in promoting liver metastasis by inducing an inflammatory premetastatic niche through the miR-21–TLR7–IL-6 axis. Thus, sEVs–miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis.

The pro-inflammatory cytokine interleukin-6 (IL-6) in tumor microenvironment has been suggested to promote development and progression of colorectal cancer (CRC). However, the underlying molecular mechanisms remain elusive. In this study, we demonstrate that fos-related antigen-1 (Fra-1) plays a critical role in IL-6 induced CRC aggressiveness and epithelial–mesenchymal transition (EMT). In CRC cell lines, the expression of Fra-1 gene was found significantly upregulated during IL-6-driven EMT process. The Fra-1 induction occurred at transcriptional level in a manner dependent on signal transducer and activator of transcription 3 (STAT3), during which both phosphorylated and acetylated post-translational modifications were required for STAT3 activation to directly bind to the Fra-1 promoter. Importantly, RNA interference-based attenuation of either STAT3 or Fra-1 prevented IL-6-induced EMT, cell migration and invasion, whereas ectopic expression of Fra-1 markedly reversed the STAT3-knockdown effect and enhanced CRC cell aggressiveness by regulating the expression of EMT-promoting factors (ZEB1, Snail, Slug, MMP-2 and MMP-9). Furthermore, Fra-1 levels were positively correlated with the local invasion depth as well as lymph node and liver metastasis in a total of 229 CRC patients. Intense immunohistochemical staining of Fra-1 was observed at the tumor marginal area adjacent to inflammatory cells and in parallel with IL-6 secretion and STAT3 activation in CRC tissues. Together, this study proposes the existence of an aberrant IL-6/STAT3/Fra-1 signaling axis leading to CRC aggressiveness through EMT induction, which suggests novel therapeutic opportunities for the malignant disease.

Colorectal cancer (CRC) has long been known for its tight association with chronic inflammation, thought to play a key role in tumor onset and malignant progression through the modulation of cancer stemness. However, the underlying molecular and cellular mechanisms are still largely elusive. Here we show that the IL-6/STAT3 inflammatory signaling axis induces the deacetylation of FRA1 at the Lys-116 residue located within its DNA-binding domain. The HDAC6 deacetylase underlies this key modification leading to the increase of FRA1 transcriptional activity, the subsequent transactivation of NANOG expression, and the acquisition of stem-like cellular features. As validated in a large (n = 123) CRC cohort, IL-6 secretion was invariably accompanied by increased FRA1 deacetylation at K116 and an overall increase in its protein levels, coincident with malignant progression and poor prognosis. Of note, combined treatment with the conventional cytotoxic drug 5-FU together with Tubastatin A, a HDAC6-specific inhibitor, resulted in a significant in vivo synergistic inhibitory effect on tumor growth through suppression of CRC stemness. Our results reveal a novel transcriptional and posttranslational regulatory cross-talk between inflammation and stemness signaling pathways that underlie self-renewal and maintenance of CRC stem cells and promote their malignant behavior. Combinatorial treatment aimed at the core regulatory mechanisms downstream of IL-6 may offer a novel promising approach for CRC treatment.

The presence of t(4;14)(p16.3;q32.3) in multiple myeloma cells results in dysregulated expression of the fibroblast growth factor receptor 3 (FGFR3). FGFR3 acts as an oncogene to promote multiple myeloma cell proliferation and antiapoptosis. These encourage the clinical development of FGFR3-specific inhibitors. Three short hairpin RNAs (shRNA) targeting different sites of FGFR3 were selected and subsequently transfected into KMS-11, OPM-2, and NCI-H929 human myeloma cell lines, all of which are characterized by t(4;14) and FGFR3 over expression. The combination of these three shRNAs can effectively inhibit FGFR3 expression in all three cell lines. Sequential immunocytochemistry/fluorescence in situ hybridization was employed to validate that the shRNAs specifically inhibited FGFR3 expression in OPM-2 cells. Decreased expression of B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2) and myeloid cell leukemia sequence 1 (MCL1) proteins and increased staining of Annexin V–positive cells showed that inhibition of FGFR3 induces apoptosis. After confirming down-regulation of FGFR3 by real-time PCR, HU-133 plus 2.0 array was employed to compare the gene expression profile of shRNA-treated sample with that of the control. Besides the down-regulation of FGFR3, expression of the antiapoptotic genes CFLAR, BCL2, MCL1, and some members of NF-κB family decreased, whereas expression of the proapoptotic genes CYC, BID, CASP2, and CASP6 increased. Microarray results also revealed changes in genes previously implicated in multiple myeloma pathogenesis (RAS, RAF, IL-6R, and VEGF), as well as others (TLR4, KLF4, and GADD45A) not previously linked to multiple myeloma. Our observations indicate that shRNAs can specifically and effectively inhibit FGFR3 expression. This targeted approach may be worth testing in multiple myeloma patients with t(4;14) and FGFR3 overexpression in the future.

DNA polymerase η (Polη) implements translesion DNA synthesis but has low fidelity in replication. We have previously shown that Polη plays an important role in the genesis of nontargeted mutations at undamaged DNA sites in cells exposed to the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Here, we report that MNNG-induced Polη expression in an interferon regulatory factor 1 (IRF1)-dependent manner in human cells. Mutagenesis analysis showed that four critical residues (Arg-82, Cys-83, Asn-86, and Ser-87) located in the IRF family conserved DNA binding domain-helix α3 were involved in DNA binding and POLH transactivation by IRF1. Furthermore, Polη up-regulation induced by IRF1 was responsible for the increase of mutation frequency in a SupF shuttle plasmid replicated in the MNNG-exposed cells. Interestingly, IRF1 was acetylated by the histone acetyltransferase CBP in these cells. Lys → Arg substitution revealed that Lys-78 of helix α3 was the major acetylation site, and the IRF1-K78R mutation partially inhibited DNA binding and its transcriptional activity. Thus, we propose that IRF1 activation is responsible for MNNG-induced Polη up-regulation, which contributes to mutagenesis and ultimately carcinogenesis in cells.