Inflammasomes are multimeric protein complexes involved in the processing of IL-1β through Caspase-1 cleavage. NLRP3 is the most widely studied inflammasome, which has been shown to respond to a large number of both endogenous and exogenous stimuli. Although studies have begun to define basic pathways for the activation of inflammasome and have been instrumental in identifying therapeutics for inflammasome related disorders; understanding the inflammasome activation at the molecular level is still incomplete. Recent functional studies indicate that microRNAs (miRs) regulate molecular pathways and can lead to diseased states when hampered or overexpressed. Mechanisms involving the miRNA regulatory network in the activation of inflammasome and IL-1β processing is yet unknown. This report investigates the involvement of miR-133a-1 in the activation of inflammasome (NLRP3) and IL-1β production. miR-133a-1 is known to target the mitochondrial uncoupling protein 2 (UCP2). The role of UCP2 in inflammasome activation has remained elusive. To understand the role of miR-133a-1 in regulating inflammasome activation, we either overexpressed or suppressed miR-133a-1 in differentiated THP1 cells that express the NLRP3 inflammasome. Levels of Caspase-1 and IL-1β were analyzed by Western blot analysis. For the first time, we showed that overexpression of miR-133a-1 increases Caspase-1 p10 and IL-1β p17 cleavage, concurrently suppressing mitochondrial uncoupling protein 2 (UCP2). Surprisingly, our results demonstrated that miR-133A-1 controls inflammasome activation without affecting the basal expression of the individual inflammasome components NLRP3 and ASC or its immediate downstream targets proIL-1β and pro-Caspase-1. To confirm the involvement of UCP2 in the regulation of inflammasome activation, Caspase-1 p10 and IL-1β p17 cleavage in UCP2 of overexpressed and silenced THP1 cells were studied. Suppression of UCP2 by siRNA enhanced the inflammasome activity stimulated by H2O2 and, conversely, overexpression of UCP2 decreased the inflammasome activation. Collectively, these studies suggest that miR-133a-1 suppresses inflammasome activation via the suppression of UCP2.

Incomplete clearance of apoptotic cells and reactive oxygen species (ROS) release are known to trigger inflammasome activation causing severe inflammation in acute lung injury and various metabolic and autoimmune diseases. Moreover, it has been reported that apoptotic cell clearance and ROS-mediated apoptosis critically depend on mitochondrial uncoupling protein-2 (UCP2). However, the relationship between UCP2 and inflammasome activation has not been studied. This report investigates the role of UCP2 in the expression and activation of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in human macrophages. We found that UCP2 overexpression significantly enhanced the expression levels of NLRP3. The NLRP3 expression levels were significantly suppressed in THP1 cells treated with genipin, a UCP2 inhibitor, compared to controls. In addition, genipin altered adenosine triphosphate (ATP)- and hydrogen peroxide (H2O2)-mediated interleukin-1 beta (IL-1β) secretion and significantly suppressed caspase-1 activity in inflammasome-activated human macrophages. Taken together, our results suggest that genipin modulates NLRP3 inflammasome activation and ATP- or H2O2-mediated IL-1β release.

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Hormone therapy with the selective estrogen-receptor modulator tamoxifen (TAM) provides palliative benefit for patients with breast cancer. However, estrogen receptor α (ER hereafter) positive breast tumors with high expression of HER2 receptor tyrosine kinase often develop significant resistance to tamoxifen therapy. Thus, precise understanding of activation of Estrogen (E2)-independent ER regulated gene transcription in tamoxifen-resistant breast tumors could open new therapeutic avenues to target drug-resistance and ameliorate poor prognosis. The non-receptor tyrosine kinase, Ack1 (also known as TNK2) has emerged as a major integrator of signaling from various receptor tyrosine kinases including HER2. Here we demonstrate that heregulin promoted ER activity at Estrogen Response Element (ERE) in the presence of tamoxifen, which was significantly downregulated upon Ack1 knockdown. We observed that Ack1 phosphorylated the ER co-activator, KDM3A, a H3K9 demethylase, at evolutionary conserved tyrosine 1114 in heregulin dependent manner in the presence of tamoxifen. KDM3A demethylates H3 mono- and dimethyl-K9 in vitro and in vivo. Heregulin mediated Ack1 activation resulted in significant decrease in the level of monomethyl-H3K9, while, monomethyl-H3K27 levels remained unchanged. Further, inhibition of Ack1 by small molecule inhibitor AIM-100 or Dasatinib resulted in significant increase in the level of monomethyl-H3K9. Therefore by its ability to regulate the activity of ER co-activator such as KDM3A, activated Ack1 may modulate ER target gene expression in the absence of estrogen and confer TAM-resistance to cancer cells. Thus, Ack1 inhibitors hold the potential as novel therapeutics to treat a subset of tamoxifen resistant breast cancers. Citation Format: Kiran Mahajan, Sayantani Bandyopadhyay, Nupam Mahajan. KDM3A tyrosine phosphorylation by Ack1 promotes tamoxifen-resistance in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2599. doi:10.1158/1538-7445.AM2014-2599

ObjectiveIdiopathic pulmonary fibrosis (IPF) is a progressive scarring disorder characterized by the deposition of extracellular matrix causing impaired gas exchange with excessive proliferation and apoptosis‐resistant state of lung fibroblasts. Therefore, inhibitors of fibroblast proliferation offer considerable therapeutic promise. Diindolylmethane (DIM) is found in cruciferous vegetables and has been known for its anti‐cancer and anti‐proliferative properties. Surprisingly, DIM has never been studied in pulmonary fibrosis. In the current study, we tested the effect of DIM on human lung fibroblast proliferation and apoptosis.MethodsHuman lung fibroblasts were treated with TGF β, a fibroblast proliferation inducer, in the presence or absence of DIM. Proliferation and apoptosis were assessed by using commercially available kits. To further understand the mechanism of DIM, cell lysates were analyzed for gene expression analysis using RT PCR. Protein expressions were determined by western blot analysis.ResultsOur results showed that DIM suppressed TGF β induced proliferation in human lung fibroblasts but not apoptosis. In addition, DIM induced MMP1 expression and suppressed TGF β induced alpha smooth muscle actin and thrombospondin‐2 gene expression. Interestingly, DIM is not influencing ADAM‐TS2 expression (a pro‐fibrotic gene) in human lung fibroblasts.ConclusionsOur findings demonstrated that the natural dietary compound DIM attenuates TGF β mediated pro‐fibrotic effects through regulating of MMP‐1 and thrombospondin‐2 in human lung fibroblasts. These findings suggest that DIM may be a potential therapeutic target for pulmonary fibrosis.