A STAT3-decoy oligonucleotide induces cell death in a human colorectal carcinoma cell line by blocking nuclear transfer of STAT3 and STAT3-bound NF-κB

Abstract

BackgroundThe transcription factor STAT3 (signal transducer and activator of transcription 3) is frequently activated in tumor cells. Activated STAT3 forms homodimers, or heterodimers with other TFs such as NF-κB, which becomes activated. Cytoplasmic STAT3 dimers are activated by tyrosine phosphorylation; they interact with importins via a nuclear localization signal (NLS) one of which is located within the DNA-binding domain formed by the dimer. In the nucleus, STAT3 regulates target gene expression by binding a consensus sequence within the promoter. STAT3-specific decoy oligonucleotides (STAT3-decoy ODN) that contain this consensus sequence inhibit the transcriptional activity of STAT3, leading to cell death; however, their mechanism of action is unclear.ResultsThe mechanism of action of a STAT3-decoy ODN was analyzed in the colon carcinoma cell line SW 480. These cells' dependence on activated STAT3 was verified by showing that cell death is induced by STAT3-specific siRNAs or Stattic. STAT3-decoy ODN was shown to bind activated STAT3 within the cytoplasm, and to prevent its translocation to the nucleus, as well as that of STAT3-associated NF-κB, but it did not prevent the nuclear transfer of STAT3 with mutations in its DNA-binding domain. The complex formed by STAT3 and the STAT3-decoy ODN did not associate with importin, while STAT3 alone was found to co-immunoprecipitate with importin. Leptomycin B and vanadate both trap STAT3 in the nucleus. They were found here to oppose the cytoplasmic trapping of STAT3 by the STAT3-decoy ODN. Control decoys consisting of either a mutated STAT3-decoy ODN or a NF-κB-specific decoy ODN had no effect on STAT3 nuclear translocation. Finally, blockage of STAT3 nuclear transfer correlated with the induction of SW 480 cell death.ConclusionsThe inhibition of STAT3 by a STAT3-decoy ODN, leading to cell death, involves the entrapment of activated STAT3 dimers in the cytoplasm. A mechanism is suggested whereby this entrapment is due to STAT3-decoy ODN's inhibition of active STAT3/importin interaction. These observations point to the high potential of STAT3-decoy ODN as a reagent and to STAT3 nucleo-cytoplasmic shuttling in tumor cells as a potential target for effective anti-cancer compounds.

Systemic Administration of a Cyclic Signal Transducer and Activator of Transcription 3 (STAT3) Decoy Oligonucleotide Inhibits Tumor Growth without Inducing Toxicological Effects

“…Preclinical studies with the linear formulation of the STAT3 decoy have shown significant antitumor effects in vivo in HNSCC as well as in several other cancers (11,13,23,24). Moreover, a phase 0 clinical trial of the linear STAT3 decoy in HNSCC patients demonstrated pharmacodynamics effects of the decoy on STAT3 target gene expression in tumor tissues (17).…”

“…In addition, comparable antitumor effects indicate a lack of dose response at the two dose levels investigated. STAT3 decoy inhibited proliferation and STAT3-mediated gene expression in head and neck squamous cell carcinoma (HNSCC) (10), as well as other cancers (11)(12)(13). Several natural products are also under development to target STAT3, including curcumin, resveratrol and cucurbitacin, among others, although these agents lack specificity (5).…”

Serum-resistant CpG-STAT3 decoy for targeting survival and immune checkpoint signaling in acute myeloid leukemia

“…46 The CpG-assisted delivery of STAT3dODN expands the therapeutic potential of decoy strategy, which has been limited to certain types of solid tumors, mainly head and neck cancers. 28,47,48 CpG-STAT3dODN overcomes obstacles in oligonucleotide delivery to hematopoietic cells, which are challenging targets for lipid-and polymer-based approaches. 49 We do not exclude that the antitumor effect of CpGSTAT3dODN is partly derived from concomitant blocking of STAT3 together with STAT1 which reportedly have overlapping tumor-promoting roles in leukemia and certain other tumors.…”

“…[26][27][28] To verify whether a CpGSTAT3dODN conjugate exerts similar activity, we tested binding of STAT3 to its radiolabeled target DNA sequences using EMSA. As shown in Figure 2A, CpG-STAT3dODN abrogated interleukin-6 (IL-6)-and IL-10-induced STAT3 DNA binding in mouse splenocytes and also reduced constitutive STAT3 activity in KG1a AML cells.…”

A sensible approach to targeting STAT3-mediated transcription

“…Additionally, the modified STAT3 ASO did not inhibit STAT1 or STAT5 underscoring the specificity of the ASO. This is a key finding since another group of STAT3 inhibitors, the STAT3 decoy oligonucleotides, have been shown to inhibit both STAT3 and STAT1 (10,11). STAT1 plays an important role in negatively regulating cell growth (12,13); therefore, cross-inhibition of STAT1 by STAT3 decoys may diminish the antitumor effects of STAT3 blockade.…”