Joshua Matthew Rhett

Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1

Cx43 gap junctions (GJs) are integral to the function of the mammalian heart. It is shown that ZO-1 dynamically regulates the transition between Cx43 connexons and GJ intercellular channels, determining the balance of connexon-mediated cell permeability to GJ communication. Importantly, a novel domain proximal to GJs is identified—the perinexus.

The perinexus: A new feature of Cx43 gap junction organization

The intercellular propagation of action potential is a necessary prerequisite of cardiac function. It is widely held that this is achieved by direct coupling between myocytes mediated by gap junctions – aggregates of cell-to-cell connexon channels. Connexons in the plasma membrane of cardiomyocytes not docked within gap junction aggregates are thought tightly regulated, though there is evidence that so-called connexon hemichannels may open in certain pathological conditions. It is presently not known whether undocked connexons are concentrated in specialized domains of membrane. Recently, we reported that interaction between the MAGUK scaffolding protein Zonula Occludens-1 (ZO-1) and the gap junction protein connexin 43 (Cx43) is concentrated in a region of the plasma membrane surrounding the gap junction plaque called the perinexus. It was found that ZO-1-Cx43 interaction governs a balance between undocked connexons in the perinexus and connexons docked in functional intercellular channels in the gap junction. In ongoing work it has been determined that the perinexus of cardiomyocyte gap junctions likely does contain high concentrations of undocked connexons composed of Cx43. This connexon-enriched zone of membrane appears to be a specialized nidus for integration of channel, junctional, and signal transduction molecules. Further insight into the function of the perinexus could provide new therapeutic avenues for the treatment of arrhythmia and other cardiac diseases.

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.

Targeting connexin 43 with α–connexin carboxyl-terminal (ACT1) peptide enhances the activity of the targeted inhibitors, tamoxifen and lapatinib, in breast cancer: clinical implication for ACT1

BackgroundTreatment failure is a critical issue in breast cancer and identifying useful interventions that optimize current cancer therapies remains a critical unmet need. Expression and functional studies have identified connexins (Cxs), a family of gap junction proteins, as potential tumor suppressors. Studies suggest that Cx43 has a role in breast cancer cell proliferation, differentiation, and migration. Although pan-gap junction drugs are available, the lack of specificity of these agents increases the opportunity for off target effects. Consequently, a therapeutic agent that specifically modulates Cx43 would be beneficial and has not been tested in breast cancer. In this study, we now test an agent that specifically targets Cx43, called ACT1, in breast cancer.MethodsWe evaluated whether direct modulation of Cx43 using a Cx43-directed therapeutic peptide, called ACT1, enhances Cx43 gap junctional activity in breast cancer cells, impairs breast cancer cell proliferation or survival, and enhances the activity of the targeted inhibitors tamoxifen and lapatinib.ResultsOur results show that therapeutic modulation of Cx43 by ACT1 maintains Cx43 at gap junction sites between cell-cell membrane borders of breast cancer cells and augments gap junction activity in functional assays. The increase in Cx43 gap junctional activity achieved by ACT1 treatment impairs proliferation or survival of breast cancer cells but ACT1 has no effect on non-transformed MCF10A cells. Furthermore, treating ER+ breast cancer cells with a combination of ACT1 and tamoxifen or HER2+ breast cancer cells with ACT1 and lapatinib augments the activity of these targeted inhibitors.ConclusionsBased on our findings, we conclude that modulation of Cx43 activity in breast cancer can be effectively achieved with the agent ACT1 to sustain Cx43-mediated gap junctional activity resulting in impaired malignant progression and enhanced activity of lapatinib and tamoxifen, implicating ACT1 as part of a combination regimen in breast cancer.

The Potential for Connexin Hemichannels to Drive Breast Cancer Progression through Regulation of the Inflammatory Response

Over the past few decades, connexin hemichannels have become recognized as major players in modulating the inflammatory response. Chronic inflammation is documented to promote tumorigenesis and is a critical component of tumor progression. Furthermore, inflammation is strongly linked to angiogenesis, immunotolerance, invasiveness, metastasis, and resistance in breast cancers. In this review, the literature on the role of connexin hemichannels in inflammation is summarized, and the potential role for hemichannel-mediated inflammation in driving breast cancer progression is discussed. Lastly, the potential for connexin-based therapeutics to modulate the inflammatory component of the tumor microenvironment as an avenue for the treatment of breast cancer is also discussed.

Abstract 4358: Targeting connexin 43 with α-connexin carboxyl-terminal (ACT1) peptide in breast cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Expression and functional studies have identified connexins (Cxs), a family of gap junction proteins, as potential tumor suppressors. Studies suggest that connexin 43 (Cx43) has a role in breast cancer cell proliferation, differentiation, and migration. Although pan-gap junction drugs are available, the lack of specificity of these agents increases the opportunity for off target effects. Consequently, a therapeutic agent that specifically modulates Cx43 would be beneficial and has not been tested in breast cancer. In this study, we now test a Cx43 specific agent, called α-connexin carboxyl-terminal (ACT1), in breast cancer. Our studies demonstrate that modulation of Cx43 activity in breast cancer can be effectively achieved with the agent ACT1 to sustain Cx43-mediated gap junctional activity resulting in impaired malignant progression. Furthermore, using ER+ and HER2+ breast cancer cell models, we show that ACT1 treatment enhances the activity of lapatinib and tamoxifen. Citation Format: Christina Grek, Joshua Matthew Rhett, Melissa Abt, Jaclynn Bruce, Gautam Ghatnekar, Elizabeth S. Yeh. Targeting connexin 43 with α-connexin carboxyl-terminal (ACT1) peptide in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4358. doi:10.1158/1538-7445.AM2015-4358