Purpose: Breast cancer is the most frequent tumor in women, afflicting African American (AA) females to a greater degree than Caucasians (CAs). Recently, we and others have found that Kaiso expression is elevated in AA patients relative to CA patients, and its expression correlates with tumor recurrence and metastasis. Exosomes are considered important modulators of cellular behavior through their cellular communication by transferring mRNA, microRNAs, proteins among cells. Here, we study the role of Kaiso on the biological function of breast cancer exosomes.
Experimental procedures: Exosomes were isolated by ultracentrifugation method and characterized by antibody array. We performed exosome internalization assay, cell proliferation assay and migration assay. Cellular metabolic activity was measured by Seahorse Analyzer. Exosome proteomics was performed by LC-MS/MS and analyzed quantitatively by Panther and DAVID bioinformatics resources.
Results: Isolated exosomes were effectively internalized by MCF7 cells. MCF7 cells treated with exosomes of Kaiso-knock-downed MDA-MB-231 cells (sh-Kaiso) showed a decreased proliferation as compared to exosomes of Kaiso-scrambled MDA-MB-231 cells (sh-Scr). We further observed that treatment of MCF7 cells with sh-Kaiso exosomes decreased cell migration when compared to sh-Scr. To determine the proteins responsible for this observation, we performed exosome proteomics profiling. Exosomes released from sh-Kaiso compared to sh-Scr cells showed differential enrichment of protein expression. In sh-Kaiso exosomes, 36 proteins were down-regulated and most of these proteins are involved in cell invasion and metastasis; whereas 172 proteins were up-regulated in sh-Kaiso of which most of them are involved in protein folding, protein complex assembly, biogenesis and repair. In proteomics data, lactate dehydrogenases (LDH) A&B which are metabolically important proteins and are involved in the critical step of inter-conversion of lactate to pyruvate, expressed differentially between sh-SCR and sh-kaiso. Furthermore, using Seahorse Analyzer we determined enhanced oxygen consumption rate, an indicator of oxidative phosphorylation and elevated extracellular acidification rate, an indicator of enhanced metabolism; in sh-SCR exosome treated MCF7 cells as compared to sh-Kaiso exosome treated MCF7 cells.
Conclusions: Our findings demonstrate that Kaiso plays an important role in the content of exosome cargo, which in turn has an effect promoting cell growth, migration and metabolism of breast cancer cells. We suggest that Kaiso has defined role in limiting important cellular information in breast cancer exosome cargo.
Citation Format: Md Shakir U. Ahmed, Shweta Triphati, William E. Grizzle, Honghe Wang, Clayton C. Yates. Kaiso influences the exosome profile required to induce cell proliferation, migration and metabolism in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5414. doi:10.1158/1538-7445.AM2017-5414