Connexins in cancer: bridging the gap to the clinic

Gap junctions comprise arrays of intercellular channels formed by connexin proteins and provide for the direct communication between adjacent cells. This type of intercellular communication permits the coordination of cellular activities and plays key roles in the control of cell growth and differentiation and in the maintenance of tissue homoeostasis. After more than 50 years, deciphering the links among connexins, gap junctions and cancer, researchers are now beginning to translate this knowledge to the clinic. The emergence of new strategies for connexin targeting, combined with an improved understanding of the molecular bases underlying the dysregulation of connexins during cancer development, offers novel opportunities for clinical applications. However, different connexin isoforms have diverse channel-dependent and -independent functions that are tissue and stage specific. This can elicit both pro- and anti-tumorigenic effects that engender significant challenges in the path towards personalised medicine. Here, we review the current understanding of the role of connexins and gap junctions in cancer, with particular focus on the recent progress made in determining their prognostic and therapeutic potential

Genome Wide CRISPR/Cas9 Screen Identifies the Coagulation Factor IX (F9) as a Regulator of Senescence

[Abstract] During this last decade, the development of prosenescence therapies has become an attractive strategy as cellular senescence acts as a barrier against tumour progression. In this context, CDK4/6 inhibitors induce senescence and reduce tumour growth in breast cancer patients. However, even though cancer cells are arrested after CDK4/6 inhibitor treatment, genes regulating senescence in this context are still unknown limiting their antitumour activity. Here, using a functional genome-wide CRISPR/Cas9 genetic screen we found several genes that participate in the proliferation arrest induced by CDK4/6 inhibitors. We find that downregulation of the coagulation factor IX (F9) using sgRNA and shRNA prevents the cell cycle arrest and senescent-like phenotype induced in MCF7 breast tumour cells upon Palbociclib treatment. These results were confirmed using another breast cancer cell line, T47D, and with an alternative CDK4/6 inhibitor, Abemaciclib, and further tested in a panel of 22 cancer cells. While F9 knockout prevents the induction of senescence, treatment with a recombinant F9 protein was sufficient to induce a cell cycle arrest and senescence-like state in MCF7 tumour cells. Besides, endogenous F9 is upregulated in different human primary cells cultures undergoing senescence. Importantly, bioinformatics analysis of cancer datasets suggest a role for F9 in human tumours. Altogether, these data collectively propose key genes involved in CDK4/6 inhibitor response that will be useful to design new therapeutic strategies in personalised medicine in order to increase their efficiency, stratify patients and avoid drug resistance.This paper was funded by the BBSRC (BB/P000223/1), the MRC (MR/K501372/1), The Royal Society (RG170399) and Barts Charity (MGU0497 and G-002158) grants to A.O. M.M. was funded by PI19/00145, IN607B2020/12 and 858014. J.F.L. is funded by Xunta de Galicia (ED481B 2017/117). M.B. was funded by MRC (MR/K501372/1). T.P.M. was funded by a QMUL PhD programme and T.D.N.’s lab is currently funded by a Barts Charity project grant (MGU0534). P.C.F. is currently funded by GAIN (IN606C 2021/006) Xunta de GaliciaReino Unido. Biotechnology and Biological Sciences Research Council; BB/P000223/1Reino Unido. Medical Research Council; MR/K501372/1Reino Unido. Royal Society; RG170399Barts Charity (Londres); MGU0497Barts Charity (Londres); G-002158Xunta de Galicia; IN607B2020/12Xunta de Galicia; ED481B 2017/117Barts Charity (Londres); MGU0534Xunta de Galicia; IN606C 2021/00

Targeting of chondrocyte plasticity via connexin43 modulation attenuates cellular senescence and fosters a pro-regenerative environment in osteoarthritis

Osteoarthritis (OA), a chronic disease characterized by articular cartilage degeneration, is a leading cause of disability and pain worldwide. In OA, chondrocytes in cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favouring disease progression. Similar to other wound-healing disorders, chondrocytes from OA patients show a chronic increase in the gap junction channel protein connexin43 (Cx43), which regulates signal transduction through the exchange of elements or recruitment/release of signalling factors. Although immature or stem-like cells are present in cartilage from OA patients, their origin and role in disease progression are unknown. In this study, we found that Cx43 acts as a positive regulator of chondrocyte-mesenchymal transition. Overactive Cx43 largely maintains the immature phenotype by increasing nuclear translocation of Twist-1 and tissue remodelling and proinflammatory agents, such as MMPs and IL-1β, which in turn cause cellular senescence through upregulation of p53, p16INK4a and NF-κB, contributing to the senescence-associated secretory phenotype (SASP). Downregulation of either Cx43 by CRISPR/Cas9 or Cx43-mediated gap junctional intercellular communication (GJIC) by carbenoxolone treatment triggered rediferentiation of osteoarthritic chondrocytes into a more differentiated state, associated with decreased synthesis of MMPs and proinflammatory factors, and reduced senescence. We have identified causal Cx43- sensitive circuit in chondrocytes that regulates dedifferentiation, redifferentiation and senescence. We propose that chondrocytes undergo chondrocyte-mesenchymal transition where increased Cx43-mediated GJIC during OA facilitates Twist-1 nuclear translocation as a novel mechanism involved in OA progression. These findings support the use of Cx43 as an appropriate therapeutic target to halt OA progression and to promote cartilage regeneration.This work was supported in part through funding from the Spanish Society for Rheumatology (SER; FER 2013) and Spanish Foundation for Research on Bone and Mineral Metabolism (FEIOMM), grant PRECIPITA-2015-000139 from the FECYT-Ministry of Economy and Competitiveness (to M.D.M.), grant PI16/00035 from the Health Institute ‘Carlos III’ (ISCIII, Spain), the European Regional Development Fund, ‘A way of making Europe’ from the European Union (to M.D.M.) and a grant from Xunta de Galicia IN607B 2017/21 (to M.D.M.) and pre-doctoral fellowship to M.V.-E. T.A. acknowledges support from Instituto de Salud Carlos III grants PI16/00772 and CPII16/00042, co-financed by the European Regional Development Fund (ERDF)S

Extracellular Vesicles Enriched in Connexin 43 Promote a Senescent Phenotype in Bone and Synovial Cells Contributing to Osteoarthritis Progression

[Abstract] The accumulation of senescent cells is a key characteristic of aging, leading to the progression of age-related diseases such as osteoarthritis (OA). Previous data from our laboratory has demonstrated that high levels of the transmembrane protein connexin 43 (Cx43) are associated with a senescent phenotype in chondrocytes from osteoarthritic cartilage. OA has been reclassified as a musculoskeletal disease characterized by the breakdown of the articular cartilage affecting the whole joint, subchondral bone, synovium, ligaments, tendons and muscles. However, the mechanisms that contribute to the spread of pathogenic factors throughout the joint tissues are still unknown. Here, we show for the first time that small extracellular vesicles (sEVs) released by human OA-derived chondrocytes contain high levels of Cx43 and induce a senescent phenotype in targeted chondrocytes, synovial and bone cells contributing to the formation of an inflammatory and degenerative joint environment by the secretion of senescence-associated secretory associated phenotype (SASP) molecules, including IL-1ß and IL-6 and MMPs. The enrichment of Cx43 changes the protein profile and activity of the secreted sEVs. Our results indicate a dual role for sEVs containing Cx43 inducing senescence and activating cellular plasticity in target cells mediated by NF-kß and the extracellular signal-regulated kinase 1/2 (ERK1/2), inducing epithelial-to-mesenchymal transition (EMT) signalling programme and contributing to the loss of the fully differentiated phenotype. Our results demonstrated that Cx43-sEVs released by OA-derived chondrocytes spread senescence, inflammation and reprogramming factors involved in wound healing failure to neighbouring tissues, contributing to the progression of the disease among cartilage, synovium, and bone and probably from one joint to another. These results highlight the importance for future studies to consider sEVs positive for Cx43 as a new biomarker of disease progression and new target to treat OA.This work was supported in part through funding from Health Institute ‘Carlos III’ (ISCIII, Spain), the European Regional Development Fund, ‘A way of making Europe’ from the European Union (to MDM): grant PI19/00145; a grant from the Joint Transnational Call for Proposals for “European Innovative Research & Technological Development Projects in Nanomedicine” EURONANOMED III (AC21_2/00026) (to MDM); a grant from Xunta de Galicia (IN607B2020/12) (to MDM) and from H2020, Future and Emerging Technologies (grant 858014 “PANACHE”) to MDM. MV-E was funded with a predoctoral (ED481A-2015/188) and post-doctoral fellowship (IN606B-2019/004) from Xunta de Galicia. AG-C was funded with a predoctoral fellowship (FIS20/00310) from ISCIII. PC-F was funded with a post-doctoral fellowship and a grant from Xunta de Galicia (INB606B 2017/014 and IN606C 2021/006). We thank members of the CellCOM group for helpful technical suggestion, María Dolores Álvarez Alvariño (CHUS) for generously collecting tissue samples in the operating room after surgery and Arantxa Tabernero (INCYL, University of Salamanca) for kindly providing the human Cx43 plasmid used in this studyXunta de Galicia; IN607B2020/12Xunta de Galicia; ED481A-2015/188Xunta de Galicia; IN606B-2019/004Xunta de Galicia; INB606B 2017/014Xunta de Galicia; IN606C 2021/00

Emerging functions and clinical prospects of connexins and pannexins in melanoma

© 2020 The Author(s) Cellular communication through gap junctions and hemichannels formed by connexins and through channels made by pannexins allows for metabolic cooperation and control of cellular activity and signalling. These channel proteins have been described to be tumour suppressors that regulate features such as cell death, proliferation and differentiation. However, they display cancer type-dependent and stage-dependent functions and may facilitate tumour progression through junctional and non-junctional pathways. The accumulated knowledge and emerging strategies to target connexins and pannexins are providing novel clinical opportunities for the treatment of cancer. Here, we provide an updated overview of the role of connexins and pannexins in malignant melanoma. We discuss how targeting of these channel proteins may be used to potentiate antitumour effects in therapeutic settings, including through improved immune-mediated tumour elimination

Structure-Based Design and Synthesis of Stapled 10Panx1 Analogues for Use in Cardiovascular Inflammatory Diseases

Following a rational design, a series of macrocyclic ("stapled") peptidomimetics of10Panx1, the most established peptide inhibitor of Pannexin1 (Panx1) channels, were developed and synthesized. Two macrocyclic analoguesSBL-PX1-42andSBL-PX1-44outperformed the linear native peptide. Duringin vitroadenosine triphosphate (ATP) release and Yo-Pro-1 uptake assays in a Panx1-expressing tumor cell line, both compounds were revealed to be promising bidirectional inhibitors of Panx1 channel function, able to induce a two-fold inhibition, as compared to the native10Panx1 sequence. The introduction of triazole-based cross-links within the peptide backbones increased helical content and enhancedin vitroproteolytic stability in human plasma (>30-fold longer half-lives, compared to10Panx1). In adhesion assays, a "double-stapled" peptide,SBL-PX1-206inhibited ATP release from endothelial cells, thereby efficiently reducing THP-1 monocyte adhesion to a TNF-α-activated endothelial monolayer and making it a promising candidate for futurein vivoinvestigations in animal models of cardiovascular inflammatory disease