Monoclonal Antibodies Specific for STAT3β Reveal Its Contribution to Constitutive STAT3 Phosphorylation in Breast Cancer.

Since its discovery in mice and humans 19 years ago, the contribution of alternatively spliced Stat3, Stat3 beta, to the overall functions of Stat3 has been controversial. Tyrosine-phosphorylated (p) Stat3 beta homodimers are more stable, bind DNA more avidly, are less susceptible to dephosphorylation, and exhibit distinct intracellular dynamics, most notably markedly prolonged nuclear retention, compared to pStat3 alpha homodimers. Overexpression of one or the other isoform in cell lines demonstrated that Stat3 beta acted as a dominant-negative of Stat3 alpha in transformation assays; however, studies with mouse strains deficient in one or the other isoform indicated distinct contributions of Stat3 isoforms to inflammation. Current immunological reagents cannot differentiate Stat3 beta proteins derived from alternative splicing vs. proteolytic cleavage of Stat3 alpha. We developed monoclonal antibodies that recognize the 7 C-terminal amino acids unique to Stat3 beta (CT7) and do not cross-react with Stat3 alpha. Immunoblotting studies revealed that levels of Stat3 beta protein, but not Stat3 alpha, in breast cancer cell lines positively correlated with overall pStat3 levels, suggesting that Stat3 beta may contribute to constitutive Stat3 activation in this tumor system. The ability to unambiguously discriminate splice alternative Stat3 beta from proteolytic Stat3 beta and Stat3 alpha will provide new insights into the contribution of Stat3 beta vs. Stat3 alpha to oncogenesis, as well as other biological and pathological processes.