Cx43 phosphorylation sites regulate pancreatic cancer metastasis

Abstract

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.

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Fig. 1: KC;CxCK1A mice have a longer lifespan and decreased metastatic burden compared to KC mice.
Fig. 2: KC;CxCK1A mice show rapid onset of tumorigenesis and exhibit an MCN phenotype.
Fig. 3: CxCK1A mutation does not inhibit ability of tail vein injected tumor cells to colonize the lung.
Fig. 4: CxCK1A mutation promotes an epithelial phenotype.
Fig. 5: Gap junctions in tumor stroma are absent from KC;CxCK1A tumors.
Fig. 6: Cancer associated fibroblasts increase gap junction expression during tumor progression.

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Acknowledgements

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.

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Correspondence to Paul D. Lampe.

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The authors declare they have no conflict of interest. PDL and JLS receive royalties from the sale of Cx43NT1 antibodies.

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Solan, J.L., Hingorani, S.R. & Lampe, P.D. Cx43 phosphorylation sites regulate pancreatic cancer metastasis. Oncogene (2021). https://doi.org/10.1038/s41388-021-01668-x

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