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

Abstract

Since its discovery in mice and humans 19 years ago, the contribution of alternatively spliced Stat3, Stat3β, to the overall functions of Stat3 has been controversial. Tyrosine-phosphorylated (p) Stat3β 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 pStat3α homodimers. Overexpression of one or the other isoform in cell lines demonstrated that Stat3β acted as a dominant-negative of Stat3α 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 Stat3β proteins derived from alternative splicing vs. proteolytic cleavage of Stat3α. We developed monoclonal antibodies that recognize the 7 C-terminal amino acids unique to Stat3β (CT7) and do not cross-react with Stat3α. Immunoblotting studies revealed that levels of Stat3β protein, but not Stat3α, in breast cancer cell lines positively correlated with overall pStat3 levels, suggesting that Stat3β may contribute to constitutive Stat3 activation in this tumor system. The ability to unambiguously discriminate splice alternative Stat3β from proteolytic Stat3β and Stat3α will provide new insights into the contribution of Stat3β vs. Stat3α to oncogenesis, as well as other biological and pathological processes.

Small-molecule targeting of signal transducer and activator of transcription (STAT) 3 to treat non-small cell lung cancer

“…More recent studies demonstrated that increased co-expression of STAT3β relative to STAT3α was shown to increase levels of pSTAT3 and to prolong its phosphorylation (17), presumably through resistance to dephosphorylation of STAT3β homodimers and STAT3α/β heterodimers (17). We developed monoclonal antibodies that recognize the 7 C-terminal amino acids unique to STAT3β (CT7) and were specific for STAT3β (13). Using our STAT3β-specific antibodies, we found that STAT3β protein levels, similar to STAT3α proteins levels, were elevated in 7 of 8 NSCLC cell lines each by 2–6 fold compared to HBEC-3KT (Figure 1B and 1D).…”

“…The blots incubated with primary antibody overnight at 4°C. The primary STAT3β mouse monoclonal antibody was generated in our laboratory against the C-terminal 7 amino acid residues unique to STAT3β (13) and used at a dilution of 1:1000. The membranes were incubated with horseradish-peroxidase (HRP)-conjugated secondary goat anti-mouse antibody (Santa Cruz, catalog# sc2005) at 1:5,000.…”

“…B) Levels of STAT3α, STAT3β and GAPDH were determined by immunoblot in lysates of murine embryonic fibroblast cells (MEF) lines in which the STAT3 gene was deleted followed by transient expression of STAT3α (MEF-STAT3α) or STAT3β (MEF-STAT3β)(13), HBEC3-KT cells, and human NSCLC cell lines (representative gel shown). Levels of STAT3α (C) and STAT3β (D) were quantified by densitometry using Image software.…”

A mix of S and ΔS variants of STAT3 enable survival of activated B-cell-like diffuse large B-cell lymphoma cells in culture

“…In addition to the antibody specific for STAT3α, we used the monoclonal antibody recently developed to detect STAT3β. 27 The result demonstrated that for the most part the expected protein levels were elevated after co-expression of these variants when compared with the EV ( Figure 4a , left panel). However, expression of only the STAT3β variants was associated with a concomitant increase in STAT3Sα.…”

“…After blocking with non-fat milk, the membrane was probed with primary antibodies against proteins of interest. The primary antibodies included anti-STAT3pan (124H6, from Cell Signaling Technology, Danvers, MA, USA), anti-STAT3α (9D8, ab119352, from Abcam, Cambridge, MA, USA), anti-STAT3β, 27 anti-phospho-STAT3 (Tyr-705) (D3A7) (#9145, from Cell Signaling Technology), anti-NFKBIA (L35A5, from Cell Signaling Technology), anti-NFKBIZ (TA502768S, from OriGene Technologies, Rockville, MD, USA), anti-α-tubulin (sc-8035, from Santa Cruz Biotechnology, Dallas, TX, USA), anti-β-actin (#4967, from Cell Signaling Technology) and anti-histone-H3 (Abcam, #ab1791). After blotting with the HRP-conjugated secondary antibody, the membrane was developed using a chemiluminescence HRP substrate kit (Thermo Scientific).…”