Pancreatic ductal adenocarcinoma (PDA) is aggressive, highly metastatic and characterized by a robust desmoplasia. Connexin proteins that form gap junctions have been implicated in tumor suppression for over 30 years. Cx43, the most widely expressed connexin, regulates cell behaviors, including migration and proliferation. Thus, we hypothesized that Cx43 could regulate PDA progression. Phosphorylation of Cx43 by Casein Kinase 1 (CK1) regulates gap junction assembly. We interbred the well-established KrasLSL-G12D/+;p48Cre/+ (KC) mouse model of PDA with homozygous “knock-in” mutant Cx43 mice bearing amino acid substitution at CK1 sites (Cx43CK1A) and found profound and surprising effects on cancer progression. Crossing the Cx43CK1A mouse onto the KC background (termed KC;CxCK1A) led to significant extension of lifespan, from a median of 370 to 486 days (p = 0.03) and a decreased incidence of metastasis (p = 0.045). However, when we examined early stages of disease, we found more rapid onset of tissue remodeling in the KC;CxCK1A mouse followed by divergence to a cystic phenotype. During tumorigenesis, gap junctions are increasingly present in stromal cells of the KC mice but are absent from the KC;Cx43CK1A mice. Tail vein metastasis assays with cells derived from KC or KC;CxCK1A tumors showed that KC;CxCK1A cells could efficiently colonize the lung and downregulate Cx43 expression, arguing that inhibition of metastasis was not occurring at the distal site. Instead, stromal gap junctions, their associated signaling events or other unknown Cx43-dependent events facilitate metastatic capacity in the primary tumor.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Cell Communication and Signaling Open Access 13 January 2023
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
American Cancer Society. Cancer Statistics Center. 2020. https://cancerstatisticscenter.cancer.org.
Aasen T, Mesnil M, Naus CC, Lampe PD, Laird DW. Gap junctions and cancer: communicating for 50 years. Nat Rev Cancer. 2016;16:775–88.
Loewenstein WR. Junctional intercellular communication: the cell-to-cell membrane channel. Physiol Rev. 1981;61:829–913.
Willecke K, Eiberger J, Degen J, Eckardt D, Romualdi A, Guldenagel M, et al. Structural and functional diversity of connexin genes in the mouse and human genome. Biol Chem. 2002;383:725–37.
Saez JC, Berthoud VM, Branes MC, Martinez AD, Beyer EC. Plasma membrane channels formed by connexins: their regulation and functions. Physiol Rev. 2003;83:1359–1400.
Laird DW. Life cycle of connexins in health and disease. Biochem J. 2006;394:527–43.
Solan JL, Lampe PD. Connexin43 phosphorylation: structural changes and biological effects. Biochem J. 2009;419:261–72.
Lastwika KJ, Dunn CA, Solan JL, Lampe PD. Phosphorylation of connexin 43 at MAPK, PKC or CK1 sites each distinctly alter the kinetics of epidermal wound repair. J Cell Sci. 2019;132:jcs234633.
Solan JL, Lampe PD. Specific Cx43 phosphorylation events regulate gap junction turnover in vivo. FEBS Lett. 2014;588:1423–9.
Solan JL, Lampe PD. Kinase programs spatiotemporally regulate gap junction assembly and disassembly: effects on wound repair. Semin Cell Dev Biol. 2016;50:40–48.
Richards TS, Dunn CA, Carter WG, Usui ML, Olerud JE, Lampe PD. Protein kinase C spatially and temporally regulates gap junctional communication during human wound repair via phosphorylation of connexin43 on serine368. J Cell Biol. 2004;167:555–62.
Masamune A, Suzuki N, Kikuta K, Ariga H, Hayashi S, Takikawa T, et al. Connexins regulate cell functions in pancreatic stellate cells. Pancreas. 2013;42:308–16.
Cigliola V, Allagnat F, Berchtold LA, Lamprianou S, Haefliger JA, Meda P. Role of connexins and pannexins in the pancreas. Pancreas. 2015;44:1234–44.
Garcia-Rodriguez L, Perez-Torras S, Carrio M, Cascante A, Garcia-Ribas I, Mazo A, et al. Connexin-26 is a key factor mediating gemcitabine bystander effect. Mol Cancer Ther. 2011;10:505–17.
Kyo N, Yamamoto H, Takeda Y, Ezumi K, Ngan CY, Terayama M, et al. Overexpression of connexin 26 in carcinoma of the pancreas. Oncol Rep. 2008;19:627–31.
Solan JL, Hingorani SR, Lampe PD. Changes in connexin43 expression and localization during pancreatic cancer progression. J Membr Biol. 2012;245:255–62.
Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell. 2003;4:437–50.
Cooper CD, Lampe PD. Casein kinase 1 regulates connexin43 gap junction assembly. J Biol Chem. 2002;277:44962–8.
Lampe PD, Cooper CD, King TJ, Burt JM. Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart. J Cell Sci. 2006;119:3435–42.
Remo BF, Qu J, Volpicelli FM, Giovannone S, Shin D, Lader J, et al. Phosphatase-resistant gap junctions inhibit pathological remodeling and prevent arrhythmias. Circ Res. 2011;108:1459–66.
Solan JL, Marquez-Rosado L, Lampe PD. Cx43 phosphorylation mediated effects on ERK and Akt protect against ischemia reperfusion injury and alter stability of stress-inducible protein NDRG1. J Biol Chem. 2019;294:11762–71.
King TJ, Gurley KE, Prunty J, Shin JL, Kemp CJ, Lampe PD. Deficiency in the gap junction protein connexin32 alters p27Kip1 tumor suppression and MAPK activation in a tissue-specific manner. Oncogene. 2005;24:1718–26.
Trosko JE, Chang CC, Madhukar BV, Klaunig JE. Chemical, oncogene, and growth factor inhibition of gap junctional intercellular communication: an integrative hypothesis of carcinogenesis. Pathobiol. 1990;58:265–78.
Chu GC, Kimmelman AC, Hezel AF, DePinho RA. Stromal biology of pancreatic cancer. J Cell Biochem. 2007;101:887–907.
Maitra A, Fukushima N, Takaori K, Hruban RH. Precursors to invasive pancreatic cancer. Adv Anat Pathol. 2005;12:81–91.
King TJ, Lampe PD. Temporal regulation of connexin phosphorylation in embryonic and adult tissues. Biochim Biophys Acta. 2005;1719:24–35.
Eser S, Schnieke A, Schneider G, Saur D. Oncogenic KRAS signalling in pancreatic cancer. Br J Cancer. 2014;111:817–22.
Mann KM, Ying H, Juan J, Jenkins NA, Copeland NG. KRAS-related proteins in pancreatic cancer. Pharm Ther. 2016;168:29–42.
Cen G, Zhang K, Cao J, Qiu Z. Downregulation of the N-myc downstream regulated gene 1 is related to enhanced proliferation, invasion and migration of pancreatic cancer. Oncol Rep. 2017;37:1189–95.
Angst E, Sibold S, Tiffon C, Weimann R, Gloor B, Candinas D, et al. Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer. Br J Cancer. 2006;95:307–13.
Yen TW, Aardal NP, Bronner MP, Thorning DR, Savard CE, Lee SP, et al. Myofibroblasts are responsible for the desmoplastic reaction surrounding human pancreatic carcinomas. Surgery. 2002;131:129–34.
Sahai E, Astsaturov I, Cukierman E, DeNardo DG, Egeblad M, Evans RM, et al. A framework for advancing our understanding of cancer-associated fibroblasts. Nat Rev Cancer. 2020;20:174–86.
Whittle MC, Hingorani SR. Fibroblasts in pancreatic ductal adenocarcinoma: biological mechanisms and therapeutic targets. Gastroenterology. 2019;156:2085–96.
Lo A, Li CP, Buza EL, Blomberg R, Govindaraju P, Avery D et al. Fibroblast activation protein augments progression and metastasis of pancreatic ductal adenocarcinoma. JCI Insight. 2017;2:e92232.
Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144:646–74.
Vickman RE, Faget DV, Beachy P, Beebe D, Bhowmick NA, Cukierman E, et al. Deconstructing tumor heterogeneity: the stromal perspective. Oncotarget. 2020;11:3621–32.
Helms E, Onate MK, Sherman MH. Fibroblast heterogeneity in the pancreatic tumor microenvironment. Cancer Disco. 2020;10:648–56.
Chen X, Song E. Turning foes to friends: targeting cancer-associated fibroblasts. Nat Rev Drug Disco. 2019;18:99–115.
Ozdemir BC, Pentcheva-Hoang T, Carstens JL, Zheng X, Wu CC, Simpson TR, et al. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell. 2014;25:719–34.
Barrett R, Pure E. Cancer-associated fibroblasts: key determinants of tumor immunity and immunotherapy. Curr Opin Immunol. 2020;64:80–87.
Zhang Z, Chen Y, Zhang T, Guo L, Yang W, Zhang J, et al. Role of myoendothelial gap junctions in the regulation of human coronary artery smooth muscle cell differentiation by laminar shear stress. Cell Physiol Biochem. 2016;39:423–37.
Gomez D, Owens GK. Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc Res. 2012;95:156–64.
Biffi G, Oni TE, Spielman B, Hao Y, Elyada E, Park Y, et al. IL1-induced JAK/STAT signaling Is antagonized by TGFbeta to shape CAF heterogeneity in pancreatic ductal adenocarcinoma. Cancer Disco. 2019;9:282–301.
Mugisho OO, Green CR, Kho DT, Zhang J, Graham ES, Acosta ML, et al. The inflammasome pathway is amplified and perpetuated in an autocrine manner through connexin43 hemichannel mediated ATP release. Biochim Biophys Acta Gen Subj. 2018;1862:385–93.
Daley D, Mani VR, Mohan N, Akkad N, Pandian G, Savadkar S, et al. NLRP3 signaling drives macrophage-induced adaptive immune suppression in pancreatic carcinoma. J Exp Med. 2017;214:1711–24.
Song M, Yu X, Cui X, Zhu G, Zhao G, Chen J, et al. Blockade of connexin 43 hemichannels reduces neointima formation after vascular injury by inhibiting proliferation and phenotypic modulation of smooth muscle cells. Exp Biol Med. 2009;234:1192–1200.
Provenzano PP, Cuevas C, Chang AE, Goel VK, Von Hoff DD, Hingorani SR. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell. 2012;21:418–29.
Salameh A, Dhein S. Effects of mechanical forces and stretch on intercellular gap junction coupling. Biochim Biophys Acta. 2013;1828:147–56.
Best SA, Ding S, Kersbergen A, Dong X, Song JY, Xie Y, et al. Distinct initiating events underpin the immune and metabolic heterogeneity of KRAS-mutant lung adenocarcinoma. Nat Commun. 2019;10:4190.
Kopp JL, von Figura G, Mayes E, Liu FF, Dubois CL, Morris JP. et al. Identification of Sox9-dependent acinar-to-ductal reprogramming as the principal mechanism for initiation of pancreatic ductal adenocarcinoma. Cancer Cell. 2012;22:737–50.
Yang W, Lampe PD, Kensel-Hammes P, Hesson J, Ware CB, Crisa L, et al. Connexin 43 functions as a positive regulator of stem cell differentiation into definitive endoderm and pancreatic progenitors. iScience. 2019;19:450–60.
Mazur PK, Einwachter H, Lee M, Sipos B, Nakhai H, Rad R, et al. Notch2 is required for progression of pancreatic intraepithelial neoplasia and development of pancreatic ductal adenocarcinoma. Proc Natl Acad Sci USA. 2010;107:13438–43.
Schofield HK, Tandon M, Park MJ, Halbrook CJ, Ramakrishnan SK, Kim EC, et al. Pancreatic HIF2alpha stabilization leads to chronic pancreatitis and predisposes to mucinous cystic neoplasm. Cell Mol Gastroenterol Hepatol. 2018;5:169–85.e162.
Izeradjene K, Combs C, Best M, Gopinathan A, Wagner A, Grady WM, et al. Kras(G12D) and Smad4/Dpc4 haploinsufficiency cooperate to induce mucinous cystic neoplasms and invasive adenocarcinoma of the pancreas. Cancer Cell. 2007;11:229–43.
Sin WC, Aftab Q, Bechberger JF, Leung JH, Chen H, Naus CC. Astrocytes promote glioma invasion via the gap junction protein connexin43. Oncogene. 2016;35:1504–16.
Zhang X, Sun Y, Wang Z, Huang Z, Li B, Fu J. Up-regulation of connexin-43 expression in bone marrow mesenchymal stem cells plays a crucial role in adhesion and migration of multiple myeloma cells. Leuk Lymphoma. 2015;56:211–8.
Dvorak HF. Tumors: wounds that do not heal-redux. Cancer Immunol Res. 2015;3:1–11.
Grek CL, Prasad GM, Viswanathan V, Armstrong DG, Gourdie RG, Ghatnekar GS. Topical administration of a connexin43-based peptide augments healing of chronic neuropathic diabetic foot ulcers: a multicenter, randomized trial. Wound Repair Regen. 2015;23:203–12.
Huang GY, Xie LJ, Linask KL, Zhang C, Zhao XQ, Yang Y, et al. Evaluating the role of connexin43 in congenital heart disease: Screening for mutations in patients with outflow tract anomalies and the analysis of knock-in mouse models. J Cardiovasc Dis Res. 2011;2:206–12.
King TJ, Lampe PD. Mice deficient for the gap junction protein Connexin32 exhibit increased radiation-induced tumorigenesis associated with elevated mitogen-activated protein kinase (p44/Erk1, p42/Erk2) activation. Carcinogenesis. 2004;25:669–80.
H & E pathology analysis was performed by Sue Knoblaugh, DVM, Diplomate ACVP. Yutaka Yasui, PhD performed power calculations and aided in the experimental design of the mouse experiments. This work was supported by grants R21CA149554 and GM55632 from the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflict of interest
The authors declare they have no conflict of interest. PDL and JLS receive royalties from the sale of Cx43NT1 antibodies.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Solan, J.L., Hingorani, S.R. & Lampe, P.D. Cx43 phosphorylation sites regulate pancreatic cancer metastasis. Oncogene 40, 1909–1920 (2021). https://doi.org/10.1038/s41388-021-01668-x
This article is cited by
Cell Communication and Signaling (2023)