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Mammary Epithelial Cell Differentiation Is Regulated by Programmed Autophagy and SIM2s
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Normal physiological or developmental processes, including invasion, proliferation, metabolic reprogramming, and apoptotic escape, are often hijacked in malignant states to drive disease occurrence or progression. As malignant states often involve a complicated combination of initiating factors, these processes are best studied in their native environment during tissue development. Metabolic reprogramming is a hallmark of tumor cells; however, the underlying cause remains unknown. Therefore, we investigated metabolic reprogramming in the mammary gland between gestation and lactation when a dramatic metabolic transition occurs under the influence of local and hormonal signals. To do so, we utilized an in vitro model of mouse mammary epithelial cell differentiation in combination with mouse models to interrogate changes in mitochondrial homeostasis, including fission, fusion, biogenesis, and mitophagy (targeted macroautophagy of mitochondria). Through transmission electron microscopy and real-time fluorescent time course studies, we found that mitochondria underwent mitophagy in response to differentiation cues in vitro. Importantly, full differentiation was impaired if autophagy was inhibited pharmacologically or genetically, via knockdown of Atg5 or Atg7. Furthermore, differentiation was completely abrogated with knockdown of the mitophagy factor, parkin (Prkn). To address the upstream mechanism, we evaluated mitophagy and mitochondrial function in two in-house mouse models that have development phenotypes: MMTV-Sim2s mice, which demonstrate precocious differentiation, and Sim2fl/fl conditional knockout mice, which we demonstrate here to have reduced lactation function. Sim2s is the short splice variant of single-minded 2s, a master regulator of central midline development in drosophila as well as a tumor suppressor in breast cancer. Interestingly, we found that over-expression of Sim2s enhanced mitophagy and prolonged lactation capability, whereas loss of Sim2s impaired lactation performance. To address the mechanism of these effects, we evaluated the localization of SIM2s. Surprisingly, SIM2s localized to mitochondria and interacted with LC3B, which associates with the phagophore membrane. Mutation of two putative LC3B interacting region motifs in Sim2s abrogated the differentiation enhancement of Sim2s overexpression. Together, these data suggest that SIM2s is an integral link in the programmed mitophagy that occurs during mammary epithelial cell differentiation. Future studies will contribute to our understanding of the tumor suppressive role of SIM2s in breast cancer as well as to how the regulatory biology of mitochondria contributes to development. We expect continuation of this work to advance therapeutic approaches to combatting mitochondrial dysfunction and resulting disease states.
Elswood, Jessica Lee (2020). Mammary Epithelial Cell Differentiation Is Regulated by Programmed Autophagy and SIM2s. Doctoral dissertation, Texas A&M University. Available electronically from