Breast cancers that disseminate to bone marrow acquire aggressive phenotypes through CX43-related tumor-stroma tunnels
Saptarshi Sinha, Brennan W. Callow, Alex P. Farfel, Suchismita Roy, Siyi Chen, Maria Masotti, Shrila Rajendran, Johanna M. Buschhaus, Celia R. Espinoza, Kathryn E. Luker, Pradipta Ghosh, Gary D. Luker
Saptarshi Sinha, Brennan W. Callow, Alex P. Farfel, Suchismita Roy, Siyi Chen, Maria Masotti, Shrila Rajendran, Johanna M. Buschhaus, Celia R. Espinoza, Kathryn E. Luker, Pradipta Ghosh, Gary D. Luker
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Research Article Oncology

Breast cancers that disseminate to bone marrow acquire aggressive phenotypes through CX43-related tumor-stroma tunnels

Abstract

Estrogen receptor-positive (ER+) breast cancer commonly disseminates to bone marrow, where interactions with mesenchymal stromal cells (MSCs) shape disease trajectory. We modeled these interactions with tumor-MSC co-cultures and used an integrated transcriptome-proteome-network-analyses workflow to identify a comprehensive catalog of contact-induced changes. Conditioned media from MSCs failed to recapitulate genes and proteins, some borrowed and others tumor-intrinsic, induced in cancer cells by direct contact. Protein-protein interaction networks revealed the rich connectome between “borrowed” and “intrinsic” components. Bioinformatics prioritized one of the borrowed components, CCDC88A/GIV, a multi-modular metastasis-related protein that has recently been implicated in driving a hallmark of cancer, growth signaling autonomy. MSCs transferred GIV protein to ER+ breast cancer cells (that lack GIV) through tunnelling nanotubes via connexin (Cx)43-facilitated intercellular transport. Reinstating GIV alone in GIV-negative breast cancer cells reproduced approximately 20% of both the borrowed and the intrinsic gene induction patterns from contact co-cultures; conferred resistance to anti-estrogen drugs; and enhanced tumor dissemination. Findings provide a multiomic insight into MSC→tumor cell intercellular transport and validate how transport of one such candidate, GIV, from the haves (MSCs) to have-nots (ER+ breast cancer) orchestrates aggressive disease states.

Authors

Saptarshi Sinha, Brennan W. Callow, Alex P. Farfel, Suchismita Roy, Siyi Chen, Maria Masotti, Shrila Rajendran, Johanna M. Buschhaus, Celia R. Espinoza, Kathryn E. Luker, Pradipta Ghosh, Gary D. Luker

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Figure 7

Cx43 and GIV interact and are co-transported through intercellular communication sites.

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Cx43 and GIV interact and are co-transported through intercellular commu...
(A) Equal aliquots of lysates of MCF7 cells in monoculture or recovered after co-culture with HS5 cells were analyzed for Cx43 and actin (as a loading control) by immunoblotting (IB). (B) MCF7 cells in monoculture or in co-culture with HS5 cells were treated with the indicated concentrations of carbenoxolone or fulvestrant prior to lysis. Equal aliquots of lysates were analyzed for GIV, Cx43 or actin (the latter as a loading control) by IB. See Supplemental Figure 5 for similar studies on HS5 monocultures. (C) Electron micrographs of immunogold stained MCF7↔HS5 contact co-cultures. Red arrowhead denotes 6 nm gold particles (GIV); white arrow denotes 12 nm gold particles (Cx43). Scale bar: 1000 nm (top left), 100 nm (bottom left, middle and right panels), 200 nm (right panel insets). (D) Schematic of constructs used in pull-down assays with recombinant GST-tagged GIV N- or C-terminal fragments or GST alone (negative control) proteins immobilized on glutathione beads and lysates of Cos7 cells transiently expressing GFP-Cx43. (E) Bound Cx43 was visualized by IB (top). Equal loading of GST proteins was confirmed by ponceau S staining (bottom). (F) Bar graphs display the relative binding of Cx43 to various GST proteins. Data are represented as mean ± SEM (n = 3); P value determined by 1-way ANOVA with Tukey’s HSD test for post hoc pairwise comparisons.