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Regulation of V-APTase Activity and Its Role in Breast Cancer Metastasis.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Regulation of V-APTase Activity and Its Role in Breast Cancer Metastasis./
作者:	McGuire, Christina Miriam.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2019,
面頁冊數:	195 p.
附註:	Source: Dissertations Abstracts International, Volume: 80-12, Section: B.
Contained By:	Dissertations Abstracts International80-12B.
標題:	Molecular biology. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=13805580
ISBN:	9781392197448

Regulation of V-APTase Activity and Its Role in Breast Cancer Metastasis.
McGuire, Christina Miriam.

Regulation of V-APTase Activity and Its Role in Breast Cancer Metastasis. - Ann Arbor : ProQuest Dissertations & Theses, 2019 - 195 p.

Source: Dissertations Abstracts International, Volume: 80-12, Section: B.

Thesis (Ph.D.)--Sackler School of Graduate Biomedical Sciences (Tufts University), 2019.

This item must not be sold to any third party vendors.

The vacuolar H+ ATPase (V-ATPase) is an ATP driven proton pump made up of fourteen different subunits that are arranged into two domains, the peripheral V1 domain and the integral V0 domain. Present in both intracellular membranes and at the plasma membrane of specialized cell types, V-ATPase activity is required for numerous basic cellular processes. Dysregulation of V-ATPase activity has been implicated in various diseases including cancer, renal tubular acidosis, and osteopetrosis. One of the major ways pump activity is controlled, is through the reversible assembly of its two domains. The process of reversible assembly is conserved across eukaryotes ranging from yeast to mammals, though the signaling pathways controlling it are not fully understood. Here, we identify glucose starvation as a novel regulator of V-ATPase assembly in mammalian cells. Acute glucose starvation induced a rapid and reversible increase in both assembly and activity of the V-ATPase.Because the V-ATPase is required for the activation of AMP Kinase (AMPK), a critical cellular energy sensor that is also activated upon glucose starvation, we hypothesized that V-ATPase assembly regulated AMPK activation. To test this, we compared the time course of AMPK activation and V-ATPase activity upon glucose starvation. We observed that activation of AMPK preceded an increase in pump activity, suggesting that regulated assembly was not contributing to AMPK activation during glucose starvation. Moreover, a pharmacological AMPK inhibitor prevented the starvation-induced increase in V-ATPase activity and assembly. These results suggested that increased assembly and activity of the V-ATPase during glucose starvation are due to AMPK signaling. Surprisingly, after performing a genetic knockout of AMPK we still observed an increase in pump activity after glucose starvation, indicating that our AMPK inhibitor was targeting a molecule other than AMPK. Using additional pharmacological inhibitors of important signaling pathways within the cell we found that the PI3K/Akt pathway, which has previously been implicated in controlling V-ATPase assembly in mammalian cells, plays a role in the starvation-induced increase in V-ATPase assembly and activity.In addition to functioning in cellular homeostasis, V-ATPase activity also promotes cancer cell survival and metastasis. Previous work in our lab identified plasma membrane V-ATPases as key players in breast cancer cell invasiveness. The two subunit-a isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, where expression of a3 promotes plasma membrane pump expression, and thereby invasiveness, of human invasive breast cancer cell lines. We sought to analyze the role of each of the subunit a-isoforms in the invasive, 4T1-12B mouse breast cancer cell line. Based on work in human breast cancer cells, we hypothesized that a3 would be the dominant a-isoform that also promoted the invasive phenotype of these cells. Surprisingly, we found that a4 was the dominantly expressed a-isoform in these cells. Genetic knockout of each of the four a-isoforms using CRISPR/Cas9 revealed that the a4 isoform promoted plasma membrane expression of the V-ATPase in 4T1-12B cells, and knockout of this isoform, but not isoforms a1-a3, reduced cell migration and invasion. These findings suggest that while different a-isoforms may upregulate plasma membrane V-ATPase localization, cell surface pumps promote an invasive phenotype.This work identifies glucose starvation as a novel regulator of V-ATPase assembly during glucose starvation in mammalian cells and attempts to understand the mechanism through which V-ATPases contribute to AMPK activation. Furthermore, it identifies subunit a4 as the dominant a-isoform in 4T1-12B cells, that also localizes the V-ATPase to the cell surface to promote invasiveness. Together, this provides novel insights into both the mechanisms of cellular homeostasis and cancer cell metastasis.

ISBN: 9781392197448Subjects--Topical Terms:

517296
Molecular biology.

Regulation of V-APTase Activity and Its Role in Breast Cancer Metastasis.
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300 $a 195 p.
500 $a Source: Dissertations Abstracts International, Volume: 80-12, Section: B.
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506 $a This item must not be sold to any third party vendors.
520 $a The vacuolar H+ ATPase (V-ATPase) is an ATP driven proton pump made up of fourteen different subunits that are arranged into two domains, the peripheral V1 domain and the integral V0 domain. Present in both intracellular membranes and at the plasma membrane of specialized cell types, V-ATPase activity is required for numerous basic cellular processes. Dysregulation of V-ATPase activity has been implicated in various diseases including cancer, renal tubular acidosis, and osteopetrosis. One of the major ways pump activity is controlled, is through the reversible assembly of its two domains. The process of reversible assembly is conserved across eukaryotes ranging from yeast to mammals, though the signaling pathways controlling it are not fully understood. Here, we identify glucose starvation as a novel regulator of V-ATPase assembly in mammalian cells. Acute glucose starvation induced a rapid and reversible increase in both assembly and activity of the V-ATPase.Because the V-ATPase is required for the activation of AMP Kinase (AMPK), a critical cellular energy sensor that is also activated upon glucose starvation, we hypothesized that V-ATPase assembly regulated AMPK activation. To test this, we compared the time course of AMPK activation and V-ATPase activity upon glucose starvation. We observed that activation of AMPK preceded an increase in pump activity, suggesting that regulated assembly was not contributing to AMPK activation during glucose starvation. Moreover, a pharmacological AMPK inhibitor prevented the starvation-induced increase in V-ATPase activity and assembly. These results suggested that increased assembly and activity of the V-ATPase during glucose starvation are due to AMPK signaling. Surprisingly, after performing a genetic knockout of AMPK we still observed an increase in pump activity after glucose starvation, indicating that our AMPK inhibitor was targeting a molecule other than AMPK. Using additional pharmacological inhibitors of important signaling pathways within the cell we found that the PI3K/Akt pathway, which has previously been implicated in controlling V-ATPase assembly in mammalian cells, plays a role in the starvation-induced increase in V-ATPase assembly and activity.In addition to functioning in cellular homeostasis, V-ATPase activity also promotes cancer cell survival and metastasis. Previous work in our lab identified plasma membrane V-ATPases as key players in breast cancer cell invasiveness. The two subunit-a isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, where expression of a3 promotes plasma membrane pump expression, and thereby invasiveness, of human invasive breast cancer cell lines. We sought to analyze the role of each of the subunit a-isoforms in the invasive, 4T1-12B mouse breast cancer cell line. Based on work in human breast cancer cells, we hypothesized that a3 would be the dominant a-isoform that also promoted the invasive phenotype of these cells. Surprisingly, we found that a4 was the dominantly expressed a-isoform in these cells. Genetic knockout of each of the four a-isoforms using CRISPR/Cas9 revealed that the a4 isoform promoted plasma membrane expression of the V-ATPase in 4T1-12B cells, and knockout of this isoform, but not isoforms a1-a3, reduced cell migration and invasion. These findings suggest that while different a-isoforms may upregulate plasma membrane V-ATPase localization, cell surface pumps promote an invasive phenotype.This work identifies glucose starvation as a novel regulator of V-ATPase assembly during glucose starvation in mammalian cells and attempts to understand the mechanism through which V-ATPases contribute to AMPK activation. Furthermore, it identifies subunit a4 as the dominant a-isoform in 4T1-12B cells, that also localizes the V-ATPase to the cell surface to promote invasiveness. Together, this provides novel insights into both the mechanisms of cellular homeostasis and cancer cell metastasis.
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