Effect of Combined PD-1 and STAT3 Pathway Blockade Treatment on K-ras Mutant Lung Cancer

https://openworks.mdanderson.org/sumexp21/1175/thumbnail.jp

Substance P Causes Seizures in Neurocysticercosis

Neurocysticercosis (NCC), a helminth infection of the brain, is a major cause of seizures. The mediators responsible for seizures in NCC are unknown, and their management remains controversial. Substance P (SP) is a neuropeptide produced by neurons, endothelial cells and immunocytes. The current studies examined the hypothesis that SP mediates seizures in NCC. We demonstrated by immunostaining that 5 of 5 brain biopsies from NCC patients contained substance P (SP)-positive (+) cells adjacent to but not distant from degenerating worms; no SP+ cells were detected in uninfected brains. In a rodent model of NCC, seizures were induced after intrahippocampal injection of SP alone or after injection of extracts of cysticercosis granuloma obtained from infected wild type (WT), but not from infected SP precursor-deficient mice. Seizure activity correlated with SP levels within WT granuloma extracts and was prevented by intrahippocampal pre-injection of SP receptor antagonist. Furthermore, extracts of granulomas from WT mice caused seizures when injected into the hippocampus of WT mice, but not when injected into SP receptor (NK1R) deficient mice. These findings indicate that SP causes seizures in NCC, and, suggests that seizures in NCC in humans may be prevented and/or treated with SP-receptor antagonists

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, 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

Modulation of STAT3 folding and function by TRiC/CCT chaperonin.

Signal transducer and activator of transcription 3 (Stat3) transduces signals of many peptide hormones from the cell surface to the nucleus and functions as an oncoprotein in many types of cancers, yet little is known about how it achieves its native folded state within the cell. Here we show that Stat3 is a novel substrate of the ring-shaped hetero-oligomeric eukaryotic chaperonin, TRiC/CCT, which contributes to its biosynthesis and activity in vitro and in vivo. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped predominantly to the β-strand rich, DNA-binding domain of Stat3. Notably, enhancing Stat3 binding to TRiC by engineering an additional TRiC-binding domain from the von Hippel-Lindau protein (vTBD), at the N-terminus of Stat3, further increased its affinity for TRiC as well as its function, as determined by Stat3's ability to bind to its phosphotyrosyl-peptide ligand, an interaction critical for Stat3 activation. Thus, Stat3 levels and function are regulated by TRiC and can be modulated by manipulating its interaction with TRiC

Stat3 binds and cross-links to TRiC subunit CCT3 in vitro; CCT3 co-localizes with activated Stat3 within the cell nucleus.

<p>In panel (A), RRLs expressing the indicated ³⁵S-methionine-labeled TRiC subunit were mixed with RRLs expressing an unlabeled Flag-tagged Stat3 construct (Flag-Stat3) or no expression construct (Mock), incubated with DSP, immunoprecipitated with M2 anti-Flag antibody-bound agarose beads, incubated with β-mercaptoethanol, and analyzed by SDS-PAGE and autography. Autoradiographs are shown. In panel (B), transformed MEFs deficient in Stat3 and stably expressing GFP-Stat3α were transiently transfected with mCherry-CCT3 or mCherry-CCT7 and incubated without (<i>−</i>) or with (+) IL-6/sIL-6R (∼250 ng/ml), as indicated (±IL-6), before DAPI staining, fixation, and confocal fluorescent microscopic examination. Photomicrographs shown are representative images filtered to reveal the DAPI signal within the nucleus (column 1), the GFP signal within the cytoplasm and nucleus (column 2), the mCherry signal within the cytoplasm and nucleus (column 3), and the GFP plus mCherry merged signal (column 4). Bar, 10 µM.</p

TRiC knockdown reduces sensitivity of cancer cells to IL-6–mediated Stat3 activation.

<p>CCT2-knockdown (+) or control-knockdown (−) HepG2 cells were immunoblotted for pStat3 and total Stat3 (tStat3) 30 min after incubation in media alone or in media containing IL-6 at the indicated concentrations. Representative immunoblots are shown in the top panel. Densitometry readings of the immunoblot bands were normalized to the IL-6 concentration yielding maximum pStat3 levels (1 ng/ml) in two experiments. The ratios of densitometry values (pStat3/tStat3) for these experiments is shown in the bottom panel; results in CCT2 knockdown cells that are indicated with an asterisk (*) differ from control knockdown cells (<i>p</i><0.05).</p

TRiC binds Stat3 co-translationally and is required for its synthesis in RRLs.

<p>In panel (A), TRiC was immunoprecipitated from RRLs with a combination of antibodies to CCT2 and CCT5 (Anti-TRiC) or with a nonspecific control antibody (Control) following translation of the indicated proteins in the presence of ³⁵S-methionine. Immunoprecipitates were separated by SDS-PAGE and autoradiographed (left panel); the position of the 90-kDa MW marker is indicated. Half of each IP reaction prior to precipitation was run separately on SDS-PAGE and autoradiographed (right panel). In panel (B), RRLs were immunoblotted with CCT1 antibody following TRiC depletion using Protein-A agarose plus CCT1 antibody (TRiC-depleted) or control antibody (Mock-depleted). In panel (C), the indicated protein was translated in mock-depleted or TRiC-depleted RRLs in the presence of ³⁵S-methionine followed by SDS-PAGE and autoradiography. In panel (D), Stat3 was translated in TRiC-depleted RRLs following the addition of purified bovine TRiC in increasing amounts in the presence of ³⁵S-methionine followed by SDS-PAGE and autoradiography. The results shown are representative of three experiments.</p

Formation of TRiC-Stat3 complexes and refolding of guanidine hydrochloride-denatured Stat3 within RRLs containing ATP.

<p>In panel (A), Stat3-containing complexes were detected within RRLs following native-PAGE analysis of <i>in vitro</i> translation and refolding reaction products by Western blot using an anti-Stat3 antibody. Lane 1 shows RRL reaction in the absence of addition of exogenous plasmid. Lane 2 shows RRL reaction in the presence of addition of exogenous plasmid encoding Stat3. Stat3 previously unfolded using 6 M guanidine hydrochloride was added to RRLs without ATP (lane 3) or RRLs containing ATP (lane 4). Arrows indicate the position of Stat3 aggregate, TRiC-Stat3 complex, and native endogenous and refolded Stat3. In panel (B), the blot was stripped and re-probed with anti-CCT1 antibody.</p

Stat3 interacts with TRiC within MEF cells.

<p>In panel (A), lysates of MEF-Stat3^Δ/Δ cells transiently expressing Flag-Stat3α were immunoprecipitated (IP) with a mixture of rabbit antibodies to CCT2 and CCT5 (+) or control rabbit antibody to human IgG (Mock) and Western blotted (WB) with antibodies to Flag tag or CCT1. In panel (B), TRiC lysates of MEF-Stat3^Δ/Δ cells transfected with Flag-tagged Stat3α were prepared in the presence or absence of ATP and MgCl₂, and analyzed by immunoprecipitation and Western blotting. The input is shown to the right.</p