Signal transducer and activator of transcription 3 (STAT3) plays important roles in multiple aspects of cancer aggressiveness including migration, invasion, survival, self-renewal, angiogenesis, and tumor cell immune evasion by regulating the expression of multiple downstream target genes. STAT3 is constitutively activated in many malignant tumors and its activation is associated with high histological grade and advanced cancer stages. Thus, inhibiting STAT3 promises an attracting strategy for treatment of advanced and metastatic cancers. Herein, we identified a STAT3 inhibitor, inS3-54, by targeting the DNA-binding domain of STAT3 using an improved virtual screening strategy. InS3-54 preferentially suppresses proliferation of cancer over non-cancer cells and inhibits migration and invasion of malignant cells. Biochemical analyses show that inS3-54 selectively inhibits STAT3 binding to DNA without affecting the activation and dimerization of STAT3. Furthermore, inS3-54 inhibits expression of STAT3 downstream target genes and STAT3 binding to chromatin in situ. Thus, inS3-54 represents a novel probe for development of specific inhibitors targeting the DNA-binding domain of STAT3 and a potential therapeutic for cancer treatments.

As an alternative to the scanning mechanism of initiation, the direct-internal-initiation mechanism postulates that the translational machinery assembles at the AUG start codon without traversing the entire 5′ untranslated region (5′-UTR) of the mRNA. Although the existence of internal ribosome entry sites (IRESs) in viral mRNAs is considered to be well established, the existence of IRESs in cellular mRNAs has recently been challenged, in part because when testing is carried out using a conventional dicistronic vector, Northern blot analyses might not be sensitive enough to detect low levels of monocistronic transcripts derived via a cryptic promoter or splice site. To address this concern, we created a new promoterless dicistronic vector to test the putative IRES derived from the 5′-UTR of an mRNA that encodes the translation initiation factor eIF4G. Our analysis of this 5′-UTR sequence unexpectedly revealed a strong promoter. The activity of the internal promoter relies on the integrity of a polypyrimidine tract (PPT) sequence that had been identified as an essential component of the IRES. The PPT sequence overlaps with a binding site for transcription factor C/EBPβ. Two other transcription factors, Sp1 and Ets, were also found to bind to and mediate expression from the promoter in the 5′-UTR of eIF4G mRNA. The biological significance of the internal promoter in the eIF4G mRNA might lie in the production of an N-terminally truncated form of the protein. Consistent with the idea that the cryptic promoter we identified underlies the previously reported IRES activity, we found no evidence of IRES function when a dicistronic mRNA containing the eIF4G sequence was translated in vitro or in vivo. Using the promoterless dicistronic vector, we also found promoter activities in the long 5′-UTRs of human Sno and mouse Bad mRNAs although monocistronic transcripts were not detectable on Northern blot analyses. The promoterless dicistronic vector might therefore prove useful in future studies to examine more rigorously the claim that there is IRES activity in cellular mRNAs.