The Function of V-ATPases in Cancer.

The vacuolar ATPases (V-ATPases) are a family of proton pumps that couple ATP hydrolysis to proton transport into intracellular compartments and across the plasma membrane. They function in a wide array of normal cellular processes, including membrane traffic, protein processing and degradation, and the coupled transport of small molecules, as well as such physiological processes as urinary acidification and bone resorption. The V-ATPases have also been implicated in a number of disease processes, including viral infection, renal disease, and bone resorption defects. This review is focused on the growing evidence for the important role of V-ATPases in cancer. This includes functions in cellular signaling (particularly Wnt, Notch, and mTOR signaling), cancer cell survival in the highly acidic environment of tumors, aiding the development of drug resistance, as well as crucial roles in tumor cell invasion, migration, and metastasis. Of greatest excitement is evidence that at least some tumors express isoforms of V-ATPase subunits whose disruption is not lethal, leading to the possibility of developing anti-cancer therapeutics that selectively target V-ATPases that function in cancer cells.

Recent Insights into the Structure, Regulation, and Function of the V-ATPases.

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and are also present at the plasma membrane. They function in such processes as membrane traffic, protein degradation, virus and toxin entry, bone resorption, pH homeostasis, and tumor cell invasion. V-ATPases are large multisubunit complexes, composed of an ATP-hydrolytic domain (V1) and a proton translocation domain (V0), and operate by a rotary mechanism. This review focuses on recent insights into their structure and mechanism, the mechanisms that regulate V-ATPase activity (particularly regulated assembly and trafficking), and the role of V-ATPases in processes such as cell signaling and cancer. These developments have highlighted the potential of V-ATPases as a therapeutic target in a variety of human diseases.

Regulation of V-ATPase assembly and function of V-ATPases in tumor cell invasiveness.

V-ATPases are ATP-driven proton pumps that function within both intracellular compartments and the plasma membrane in a wide array of normal physiological and pathophysiological processes. V-ATPases are composed of a peripheral V(1) domain that hydrolyzes ATP and an integral V(0) domain that transports protons. Regulated assembly of the V-ATPase represents an important mechanism of regulating V-ATPase activity in response to a number of environmental cues. Our laboratory has demonstrated that glucose-dependent assembly of the V-ATPase complex in yeast is controlled by the Ras/cAMP/PKA pathway. By contrast, increased assembly of the V-ATPase during dendritic cell maturation involves the PI-3 kinase and mTORC1 pathways. Recently, we have shown that amino acids regulate V-ATPase assembly in mammalian cells, possibly as a means to maintain adequate levels of amino acids upon nutrient starvation. V-ATPases have also been implicated in cancer cell survival and invasion. V-ATPases are targeted to different cellular membranes by isoforms of subunit a, with a3 targeting V-ATPases to the plasma membrane of osteoclasts. We have shown that highly invasive human breast cancer cell lines express higher levels of the a3 isoform than poorly invasive lines and that knockdown of a3 reduces both expression of V-ATPases at the plasma membrane and in vitro invasion of breast tumor cells. Moreover, overexpression of a3 in a non-invasive breast epithelial line increases both plasma membrane V-ATPases and in vitro invasion. Finally, specific ablation of plasma membrane V-ATPases in highly invasive human breast cancer cells using either an antibody or small molecule approach inhibits both in vitro invasion and migration. These results suggest that plasma membrane and a3-containing V-ATPases represent a novel and important target in the development of therapeutics to limit breast cancer metastasis. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Activity of plasma membrane V-ATPases is critical for the invasion of MDA-MB231 breast cancer cells.

The vacuolar (H(+))-ATPases (V-ATPases) are a family of ATP-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. Previous studies have implicated V-ATPases in cancer cell invasion. It has been proposed that V-ATPases participate in invasion by localizing to the plasma membrane and causing acidification of the extracellular space. To test this hypothesis, we utilized two separate approaches to specifically inhibit plasma membrane V-ATPases. First, we stably transfected highly invasive MDA-MB231 cells with a V5-tagged construct of the membrane-embedded c subunit of the V-ATPase, allowing for extracellular expression of the V5 epitope. We evaluated the effect of addition of a monoclonal antibody directed against the V5 epitope on both V-ATPase-mediated proton translocation across the plasma membrane and invasion using an in vitro Matrigel assay. The addition of anti-V5 antibody resulted in acidification of the cytosol and a decrease in V-ATPase-dependent proton flux across the plasma membrane in transfected but not control (untransfected) cells. These results demonstrate that the anti-V5 antibody inhibits activity of plasma membrane V-ATPases in transfected cells. Addition of the anti-V5 antibody also inhibited in vitro invasion of transfected (but not untransfected) cells. Second, we utilized a biotin-conjugated form of the specific V-ATPase inhibitor bafilomycin. When bound to streptavidin, this compound cannot cross the plasma membrane. Addition of this compound to MDA-MB231 cells also inhibited in vitro invasion. These studies suggest that plasma membrane V-ATPases play an important role in invasion of breast cancer cells.

Structural analysis of the N-terminal domain of subunit a of the yeast vacuolar ATPase (V-ATPase) using accessibility of single cysteine substitutions to chemical modification.

The vacuolar ATPase (V-ATPase) is a multisubunit complex that carries out ATP-driven proton transport. It is composed of a peripheral V1 domain that hydrolyzes ATP and an integral V0 domain that translocates protons. Subunit a is a 100-kDa integral membrane protein (part of V0) that possesses an N-terminal cytoplasmic domain and a C-terminal hydrophobic domain. Although the C-terminal domain functions in proton transport, the N-terminal domain is critical for intracellular targeting and regulation of V-ATPase assembly. Despite its importance, there is currently no high resolution structure for subunit a of the V-ATPase. Recently, the crystal structure of the N-terminal domain of the related subunit I from the archaebacterium Meiothermus ruber was reported. We have used homology modeling to construct a model of the N-terminal domain of Vph1p, one of two isoforms of subunit a expressed in yeast. To test this model, unique cysteine residues were introduced into a Cys-less form of Vph1p and their accessibility to modification by the sulfhydryl reagent 3-(N-maleimido-propionyl) biocytin (MPB) was determined. In addition, accessibility of introduced cysteine residues to MPB modification was compared in the V1V0 complex and the free V0 domain to identify residues protected from modification by the presence of V1. The results provide an experimental test of the proposed model and have identified regions of the N-terminal domain of subunit a that likely serve as interfacial contact sites with the peripheral V1 domain. The possible significance of these results for in vivo regulation of V-ATPase assembly is discussed.

Prevalence of Lynch syndrome in women with mismatch repair-deficient ovarian cancer.

BACKGROUND: There are limited data on the prevalence of Lynch syndrome (LS) in women with primary ovarian cancer with mismatch repair deficiency (MMR-D) by immunohistochemistry (IHC). MATERIALS AND METHODS: Three hundred and eight cases of primary ovarian, fallopian, and peritoneal cancer between January 2012 and December 2019 were evaluated for MMR-D by IHC. The incidence of LS in this cohort was evaluated. RESULTS: MMR-D by IHC was identified in 16 of 308 (5.2%) (95% CI: 3.2%-8.3%) primary ovarian-related cancers. Most cases with MMR-D were endometrioid (n = 11, 68.7%); (95% CI: 44.2%-86.1%). MSH2/MSH6 protein loss was detected in eight cases (50.0%); (95% CI: 28.0%-72.0%) and MLH1/PMS2 protein loss was detected in four cases (25.0%); (95% CI: 9.7%-50.0%). MSH6 protein loss was detected in two cases (12.5%); (95% CI: 2.2%-37.3%) and PMS2 protein loss was detected in two cases (12.5%); (95% CI: 2.2%-37.3%). All four cases with MLH1/PMS2 protein loss had MLH1 promotor hypermethylation. All 12 women with ovarian cancer suggestive of LS underwent germline testing and 8 (66.6%); (95% CI: 38.8%-86.5%) were confirmed to have LS. CONCLUSIONS: Most ovarian cancers with somatic MMR-D were confirmed to have LS in this cohort. Germline testing for LS in addition to BRCA1/2 for all women with an epithelial ovarian cancer would be efficient and would approach 100% sensitivity for identifying Lynch syndrome. Utilization of a multigene panel should also be considered, given the additional non-Lynch germline mutation identified in this cohort.

Positive Attitudes and Therapeutic Misconception Around Hypothetical Clinical Trial Participation in the Huntington's Disease Community.

BACKGROUND: New therapies that could modify the disease course of Huntington's disease (HD) are entering clinical trials. However, conceptions about clinical research from the HD community are unknown. This knowledge could help inform patient-clinician discussions surrounding clinical trial participation. OBJECTIVE: The purpose of this study was to assess clinical trial attitudes and understanding in the HD community. METHODS: We developed a survey incorporating two measures of trial understanding and attitudes and the impact of therapeutic route of administration on hypothetical trial participation. The survey was distributed via emails, flyers, and social media through HD-related organizations. RESULTS: There were 73 responses. Individuals self-reported as clinically diagnosed with HD, gene positive but asymptomatic, or primary caregivers. Respondents viewed clinical trials positively and generally viewed trials as safe. Individuals with prior HD-related research experience were less likely to have negative expectations about trials than those without research experience (p = 0.002), and women had higher information needs than men (p = 0.001). Individuals with HD were more likely than the other groups to experience therapeutic misconception (p = 0.002). All respondents were able to appraise risks and benefits of research but exhibited optimism about trial outcomes. Willingness to participate was highest when the route of administration was minimally invasive. CONCLUSIONS: While the HD community views clinical trials positively, patients with HD are at high risk for therapeutic misconception and all groups are optimistic about trial outcomes. Limitations of this study include a small sample that may be inclined to view research positively given past trial participation and interest in participating in HD surveys. However, the findings from this study can be used to strengthen informed consent during HD clinical trial recruitment.

Regulation of V-ATPase activity.

V-ATPases are ATP-driven proton pumps present in both intracellular and cell surface membranes of eukaryotes that function in many normal and disease processes. V-ATPases are large, multi-subunit complexes composed of a peripheral domain (V1) that hydrolyzes ATP and a membrane integral domain (V0) that translocates protons. Because of the diversity of their functions, V-ATPase activity is controlled by a number of mechanisms. Regulated assembly of the V1 and V0 domains rapidly modulates V-ATPase activity in response to a variety of cues, including nutrient availability, growth factor stimulation and cellular differentiation. Considerable information has recently emerged concerning the cellular signaling pathways controlling regulated assembly. Acid secretion by epithelial cells in the kidney and epididymus is controlled by regulated trafficking of V-ATPases to the cell surface. Isoforms of subunit a of the V0 domain both control trafficking of V-ATPases to distinct cellular membranes and confer properties to the resultant complexes that help account for differences in pH between cellular compartments. Finally, differential expression of genes encoding V-ATPases subunits occurs in a number of contexts, including cancer.

Public- and Private-Sector Contributions to the Research and Development of the Most Transformational Drugs in the Past 25 Years: From Theory to Therapy.

BACKGROUND: With available funding from the public sector decreasing while medical needs and scientific complexity increase, private-sector collaborations with academia and government have become increasingly key in furthering medical innovation. Nonetheless, some skeptics diminish the contribution of the private sector to the discovery and development of truly innovative drugs on the one hand, while on the other hand they assert that research and development (R&D) of new medicines could and should be exclusively within control (at least financially) of the government. This begs the question, How much government funding would be needed to replace industry new drug R&D spending? METHODS: We address the respective roles of the private and public sectors in drug development by examining a diverse array of evidentiary materials on the history of 19 individual drugs, 6 drug classes, and 1 drug combination identified as the most transformative drugs in health care over the past 25 years by a survey of over 200 physicians. RESULTS: Only 4 of the individual drugs appear to have been almost completely researched and developed by one sector. One sector or the other, however, did dominate particular phases of the R&D continuum. For example, 54% of basic science milestones were achieved predominantly by the public sector and 27% by the private sector. For discovery milestones, it was 15% by the public sector and 58% by the private sector. The private sector was also dominant in achieving the major milestones for both the production and drug development phases (81% and 73% of the drugs reviewed, respectively). For 19% to 27% of the case histories for the various categories, dominance of one sector versus the other could not be determined. On the question of replacing industry's spending on the R&D of medicines, we estimate quite conservatively that the amount that would have to be spent by government would be nearly double the budget of the National Institutes of Health just to maintain the flow of the most innovative drug approvals and would have to increase nearly 2.5 times that level to maintain the development of all new drugs. CONCLUSIONS: Our analysis indicates that industry's contributions to the R&D of innovative drugs go beyond development and marketing and include basic and applied science, discovery technologies, and manufacturing protocols, and that without private investment in the applied sciences there would be no return on public investment in basic science.

The a3 isoform of subunit a of the vacuolar ATPase localizes to the plasma membrane of invasive breast tumor cells and is overexpressed in human breast cancer.

The vacuolar (H+)-ATPases (V-ATPases) are a family of ATP-driven proton pumps that acidify intracellular compartments and transport protons across the plasma membrane. Previous work has demonstrated that plasma membrane V-ATPases are important for breast cancer invasion in vitro and that the V-ATPase subunit a isoform a3 is upregulated in and critical for MDA-MB231 and MCF10CA1a breast cancer cell invasion. It has been proposed that subunit a3 is present on the plasma membrane of invasive breast cancer cells and is overexpressed in human breast cancer. To test this, we used an a3-specific antibody to assess localization in breast cancer cells. Subunit a3 localizes to the leading edge of migrating breast cancer cells, but not the plasma membrane of normal breast epithelial cells. Furthermore, invasive breast cancer cells express a3 throughout all intracellular compartments tested, including endosomes, the Golgi, and lysosomes. Moreover, subunit a3 knockdown in MB231 breast cancer cells reduces in vitro migration. This reduction is not enhanced upon addition of a V-ATPase inhibitor, suggesting that a3-containing V-ATPases are critical for breast cancer migration. Finally, we have tested a3 expression in human breast cancer tissue and mRNA prepared from normal and cancerous breast tissue. a3 mRNA was upregulated 2.5-47 fold in all breast tumor cDNA samples tested relative to normal tissue, with expression generally correlated to cancer stage. Furthermore, a3 protein expression was increased in invasive breast cancer tissue relative to noninvasive cancer and normal breast tissue. These studies suggest that subunit a3 plays an important role in invasive human breast cancer.

Pharmacological validation of Trypanosoma brucei phosphodiesterases B1 and B2 as druggable targets for African sleeping sickness.

Neglected tropical disease drug discovery requires application of pragmatic and efficient methods for development of new therapeutic agents. In this report, we describe our target repurposing efforts for the essential phosphodiesterase (PDE) enzymes TbrPDEB1 and TbrPDEB2 of Trypanosoma brucei , the causative agent for human African trypanosomiasis (HAT). We describe protein expression and purification, assay development, and benchmark screening of a collection of 20 established human PDE inhibitors. We disclose that the human PDE4 inhibitor piclamilast, and some of its analogues, show modest inhibition of TbrPDEB1 and B2 and quickly kill the bloodstream form of the subspecies T. brucei brucei . We also report the development of a homology model of TbrPDEB1 that is useful for understanding the compound-enzyme interactions and for comparing the parasitic and human enzymes. Our profiling and early medicinal chemistry results strongly suggest that human PDE4 chemotypes represent a better starting point for optimization of TbrPDEB inhibitors than those that target any other human PDEs.