Abstract 2380: Induction of gap junctional chemotherapy bystander effect in breast cancer cells through regulation of the protein kinase A pathway
Authors: Pradeep Prarthana , Urban Alexander E. , Jones Jennifer C. , Bodenstine Thomas M. 
Published: August 2018

Gap junctional intercellular communication (GJIC) mediates the regulated transfer of cellular metabolites between cells and represents an integral component of homeostasis in epithelial cells. The effects of dysregulation of GJIC in cancer has proven to be complex and context dependent, however, a lack of GJIC between cancer cells and a causative role in cancer progression have been demonstrated. In the context of treatment, delivery of therapeutics is often hindered by a lack of vascularization in hypoxic solid tumors. Transfer of small molecular weight therapies between cells through gap junctions, a process known as bystander effect, represents a potential mechanism for transfer of therapies within a tumor in the absence of a blood supply. Previous evidence has shown that upregulation of the Protein Kinase A (PKA) pathway can restore GJIC in cancer cells by promoting the assembly of gap junction channels. As a proof-of-principle experiment, we hypothesized that stimulation of the PKA pathway in breast cancer cells could facilitate the intercellular transfer and subsequent cellular effects of chemotherapy. Using the MDA-MB-231 and T47D breast cancer cell lines which exhibit low GJIC, we utilized multiple PKA activators (8-Br-cAMP, forskolin, LY294002) and the PKA inhibitor H89 to regulate the activity of PKA. Pathway activation was confirmed by western blot analysis of phosphorylated CREB at serine 133 and immunofluorescent localization of PKA catalytic subunits. A corresponding increase in GJIC was assessed by following the transfer of the fluorescent gap-junction permeable dye calcein from labeled donor cells to non-labeled acceptor cells. Results were recorded by live-cell fluorescence microscopy and quantified using flow cytometry. The Hs578T breast cancer cell line which exhibits high levels of GJIC was used as a positive control in these experiments. To assess the effects of chemotherapeutic spread, cells were treated with doxorubicin and subsequently co-cultured with non-treated cells. Transfer of doxorubicin was traced by its autofluorescent properties and induction of apoptosis in doxorubicin treated and untreated cells was assessed using annexin V staining by flow cytometry. Transfer of doxorubicin to non-treated cells did not occur under control conditions. Following co-culture in the presence of PKA activators, transfer of doxorubicin into non-treated cells was evident and resulted in the induction of apoptosis. These results demonstrate that upregulation of GJIC by mediating changes to PKA promote the spread of chemotherapeutic effects and indicate a possible role for modulation of PKA in breast cancer cells to improve the transfer of chemotherapeutics.

Citation Format: Prarthana Pradeep, Alexander E. Urban, Jennifer C. Jones, Thomas M. Bodenstine. Induction of gap junctional chemotherapy bystander effect in breast cancer cells through regulation of the protein kinase A pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2380.