Susan Frost | University of Florida (original) (raw)

Papers by Susan Frost

I would like to express my gratitude toward Drs. Bobbi Langkamp-Henken and Robert McMahon for the... more I would like to express my gratitude toward Drs. Bobbi Langkamp-Henken and Robert McMahon for their continuous guidance, advice, and support throughout the course of my Ph.D. work. My great appreciation also goes to my committee, Drs. Robert Cousins, Jesse Gregory III, and Susan Frost, for constantly providing valuable comments and suggestions during my Ph.D. experience. I would also like to express my gratitude towards my wife, Darci, and my parents, Pete and Sharon Lewis, for their support and undying faith that this would eventually be finished. Finally, I would like to thank Sara Rathman for all her help and especially her reliability in the lab and Amy Mackey for her support and enthusiasm in answering my many questions.

Journal of Biological Chemistry, May 1, 2011

Carbonic anhydrase IX (CAIX) is a membrane-bound, tumorrelated enzyme whose expression is often c... more Carbonic anhydrase IX (CAIX) is a membrane-bound, tumorrelated enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification of the tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using 18 O exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO 2 species at 25 mM and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells.

PLOS ONE

Hypoxia is a common feature of most solid tumors, one that favors tumor progression and limits tr... more Hypoxia is a common feature of most solid tumors, one that favors tumor progression and limits treatment effectiveness. Targeting hypoxia has long been a goal in cancer therapy, by identifying factors that reverse or ameliorate the effects of hypoxia on cancer cells. We, and others, have shown that β-caryophyllene (BCP) exhibits anti-proliferative properties in cancer cells. We have further shown that non-cytotoxic concentrations of BCP affect cholesterol and lipid biosynthesis in hypoxic hBrC cells at both transcriptional and translational levels. This led us to hypothesize that BCP may reverse the hypoxic phenotype in hBrC cells. To test this, we determined the effect of BCP on hypoxic sensitive pathways, including oxygen consumption, glycolysis, oxidative stress, cholesterol and fatty acid biosynthesis, and ERK activation. While each of these studies revealed new information on the regulation by hypoxia and BCP, only the lipidomic studies showed reversal of hypoxic-dependent effe...

Analytical Biochemistry, Aug 1, 2010

Current research into the function of carbonic anhydrases in cell physiology emphasizes the role ... more Current research into the function of carbonic anhydrases in cell physiology emphasizes the role of membrane-bound carbonic anhydrases, such as carbonic anhydrase IX that has been identified in malignant tumors and is associated with extracellular acidification as a response to hypoxia. We present here a mass spectrometric method to determine the extent to which total carbonic anhydrase activity is due to extracellular carbonic anhydrase in whole cell preparations. The method is based on the biphasic rate of depletion of 18 O from CO 2 measured by membrane inlet mass spectrometry. The slopes of the biphasic depletion are a sensitive measure of the presence of carbonic anhydrase outside and inside of the cells. This property is demonstrated here using suspensions of human red cells in which external carbonic anhydrase was added to the suspending solution. It is also applied to breast and prostate cancer cells which both express exofacial carbonic anhydrase IX. Inhibition of external carbonic anhydrase is achieved by use of a membrane impermeant inhibitor that was synthesized for this purpose, p-aminomethylbenzenesulfonamide attached to a polyethyleneglycol polymer.

Biochimica et biophysica acta. Molecular cell research, 2011

Carbonic anhydrase IX (CAIX) is a zinc metalloenzyme that catalyzes the reversible hydration of C... more Carbonic anhydrase IX (CAIX) is a zinc metalloenzyme that catalyzes the reversible hydration of CO 2. CAIX is overexpressed in many types of cancer, including breast cancer, but is most frequently absent in corresponding normal tissues. CAIX expression is strongly induced by hypoxia and is significantly associated with tumor grade and poor survival. Herein, we show that hypoxia induces a significant increase in CAIX protein in MDA-MB-231 breast cancer cells. Using a unique mass spectrophotometric assay, we demonstrate that CAIX activity in plasma membranes isolated from MDA-MB-231 is correlated with CAIX content. We also show that CAIX exists predominantly as a dimeric, high-mannose N-linked glycoprotein. While there is some evidence that the dimeric form resides specifically in lipid rafts, our data do not support this hypothesis. EGF, alone, did not affect the distribution of CAIX into lipid rafts. However, acute EGF treatment in the context of hypoxia increased the amount of CAIX in lipid rafts by about 5-fold. EGF did not stimulate tyrosine phosphorylation of CAIX, although EGFR and downstream signaling pathways were activated by EGF. Interestingly, hypoxia activated Akt independent of EGF action. Together, these data demonstrate that the active form of CAIX in the MDA-MB-231 breast cancer cell line is dimeric but that neither lipid raft localization nor phosphorylation are likely required for its dimerization or activity.

PLOS ONE, Nov 19, 2018

Carbonic anhydrases (CAs) have been linked to tumor progression, particularly membranebound CA is... more Carbonic anhydrases (CAs) have been linked to tumor progression, particularly membranebound CA isoform IX (CA IX). The role of CA IX in the context of breast cancer is to regulate the pH of the tumor microenvironment. In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureidosubstituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The ability of these sulfonamides to block CA IX activity in breast cancer cells is less effective than their ability to block activity of the recombinant protein (by one to two orders of magnitude depending on the inhibitor). The same is true for CA XII activity but now they are two to three orders of magnitude less effective. Thus, there is significantly greater specificity for CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this again occurs at two orders of magnitude above the K i values for inhibition of CA IX and CA XII activity in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells where CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth potentiation.

The FASEB Journal, Apr 1, 2012

Cell migration and invasion were determined using trans-well chambers. Panel A. UFH-001 ... more Cell migration and invasion were determined using trans-well chambers. Panel A. UFH-001 cells (empty vector and CRISPR-CAIX knockdown cells from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199476#pone.0199476.g005" target="_blank">Fig 5</a>) were plated in the upper transwell chambers and allowed to migrate or invade across the membrane for 24 h (upper images) or 48 h (lower images), respectively. Tabulation of results is shown to the right (p < 0.05). Panel B. T47D cells (empty vector or CAXII knockdown cells from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199476#pone.0199476.g004" target="_blank">Fig 4</a>) were plated in the upper transwell chambers and allowed to migrate or invade across the membrane for 24 h (upper images) or 48 h (lower images), respectively. Tabulation of results is shown to the right.</p

18O exchange activity was used to measure CA activity in empty ve... more 18O exchange activity was used to measure CA activity in empty vector controls and knockdown cells (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199476#pone.0199476.g004" target="_blank">Fig 4</a>) exposed to normoxic or hypoxic conditions. Panel A. UFH-001 cells. Panel B. T47D cells.</p

PLOS ONE, Jul 2, 2018

Carbonic anhydrase IX (CAIX) and XII (CAXII) are transmembrane proteins that are associated with ... more Carbonic anhydrase IX (CAIX) and XII (CAXII) are transmembrane proteins that are associated with cancer progression. We have previously described the catalytic properties of CAIX in MDA-MB-231 breast cancer cells, a line of cells that were derived from a patient with triple negative breast cancer. We chose this line because CAIX expression in breast cancer is a marker of hypoxia and a prognosticator for reduced survival. However, CAXII expression is associated with better survival statistics than those patients with low CAXII expression. Yet CAIX and CAXII have similar catalytic activities. Here we compare the potential roles of CAIX and CAXII in the context of TNBC and estrogen receptor (ER)-positive breast cancer. In tumor graft models, we show that CAIX and CAXII exhibit distinct expression patterns and non-overlapping. We find the same pattern across a panel of TNBC and luminal breast cancer cell lines. This affords an opportunity to compare directly CAIX and CAXII function. Our data suggest that CAIX expression is associated with growth potentiation in the tumor graft model and in a TNBC line using knockdown strategies and blocking activity with an impermeant sulfonamide inhibitor, N-3500. CAXII was not associated with growth potentiation. The catalytic activities of both CAIX and CAXII were sensitive to inhibition by N-3500 and activated at low pH. However, pH titration of activity in membrane ghosts revealed significant differences in the catalytic efficiency and pKa values. These features provide evidence that CAIX is a more efficient enzyme than CAXII at low pH and that CAIX shifts the equilibrium between CO 2 and bicarbonate in favor of CO 2 production by consuming protons. This suggests that in the acidic microenvironment of tumors, CAIX plays a role in stabilizing pH at a value that favors cancer cell survival.

The FASEB Journal, Apr 1, 2015

Carbonic anhydrase IX (CAIX) has emerged as a potential target for triple negative breast cancer ... more Carbonic anhydrase IX (CAIX) has emerged as a potential target for triple negative breast cancer (TNBC). Our goal is to utilize the tumor graft model to demonstrate the efficacy of carbonic anhydra...

CA IX knockout in UFH-001 cells was accomplished using Crispr/Cas9 technology. CA XII kn... more CA IX knockout in UFH-001 cells was accomplished using Crispr/Cas9 technology. CA XII knockdown in T47D cells was performed using shRNAi lentiviral strategies. Cells were grown similarly to those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0207417#pone.0207417.g003" target="_blank">Fig 3</a>. Panel A. CA activity was measured in 1 x 106 UFH-001 cells (EV controls, CA IX KO, or cells treated with the pegylated sulfonamide, N-3500). Panel B. CA activity was measured in 5 x 105 T47D cells (EV controls, CA XII KO, or cells treated with N-3500. Data represent duplicate experiments.</p

Panel A. Protein expression in a normal immortalized basal type breast cell line (MCF 10... more Panel A. Protein expression in a normal immortalized basal type breast cell line (MCF 10A) and a triple negative breast cancer (TNBC) cell line (UFH-001), using western blot analysis (under normoxic (N) or hypoxic conditions (H) for 16 h, respectively). Panel B. Immunoblots for CAs II, IX and XII protein expression in MCF 10A and the luminal ER positive breast cancer cell line (T47D). Panels C and D. UFH-001 and T47D cells were grown for 3 days at which point they were exposed to normoxia or 16 h of hypoxia and the cells assayed for CA IX and XII activity, respectively, using the MIMS assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0207417#pone.0207417.s003" target="_blank">S3A and S3B Fig</a> for a description of a pictorial representation of the MIMS assay). First order rate constants for CA activity in UFH-001 cells (Panel A) and T47D cells (Panel B), as described in the methods, are shown. These data represent three independent experiments and are reported as the mean ± SEM.</p

Panel A. Ribbon diagram of monomeric CA isoform II (gray). Panel C. Ribbon diagram of th... more Panel A. Ribbon diagram of monomeric CA isoform II (gray). Panel C. Ribbon diagram of the catalytic domains of dimeric CA isoform IX (cyan). Panel E. Ribbon diagram of the catalytic domains of dimeric CA isoform XII (wheat). Surface representation of compounds U-CH3 (pink), U-F (green) and U-NO2 (yellow) in complex with monomers of CA II (Panel B), the CA IX-mimic (Panel D), and modeled into the active site of CA XII (Panel F). Catalytic zinc (magenta sphere), hydrophilic (blue) and hydrophobic (orange) residues are as shown. Red double-headed arrows indicate isoform specificity relative to residue 131 (labeled in white). These arrows also show flexibility in tail conformations seen in CA II and CA IX but not in CA XII. Previously published Ki values of each compound bound to purified CA II, CA IX and CA XII are noted next to the inhibitor name [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0207417#pone.0207417.ref067" target="_blank">67</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0207417#pone.0207417.ref068" target="_blank">68</a>]. Figures were designed using PyMol.</p

The lipids of rat milk, the contents of 910-dayold rat stomach and intestine, lymph, plasma, and ... more The lipids of rat milk, the contents of 910-dayold rat stomach and intestine, lymph, plasma, and liver were quantitated and their fatty acids were analyzed. Rat milk consists of 97% triacylglycerols, of which 35% of the fatty acids are of medium chain length (C8-C12). However, stomach triacylglycerols show a 25% reduction in medium chain fatty acids, which indicates preferential hydrolysis of medium chain fatty acids in the stomach. The intestinal lumen free fatty acid composition shows decreased medium chain fatty acids compared to long chain fatty acids, indicating preferential absorption of the former. Lymph was shown to contain a significant amount ( -22%) of medium chain fatty acids. The decreased content of medium chain fatty acids in vena cava blood compared to portal blood, and also the lower concentration of medium chain fatty acids in liver compared to blood, indicates preferential use of these fatty acids by the liver. -FernandoWarnakulasuriya, G. J. P., J . E. Staggers, ...

PloS one, 2018

Public reporting burden for this collection of information is estimated to average 1 hour per res... more Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number.

Subcellular Biochemistry, 2014

Cancer Research, 2011

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Carbonic anhydrase IX (C... more Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme the expression of which is often considered a marker for hypoxia, an indicator of poor prognosis, and associated with acidification of the tumor microenvironment. Many studies have shown that CAIX expression is induced by hypoxia exposing its catalytic domain to the interstitial milieu. Several recent studies have suggested that hypoxic conditions may also permit activation of CAIX, perhaps by causing a conformational change which exposes the catalytic pocket. Our goal was to assess the effect of anoxic conditions on CAIX activity in MDA-MB-231 cells, previously exposed to hypoxia which increases CAIX expression in the absence of other membrane-bound carbonic anhydrase (CA) family members. We have taken advantage of membrane inlet mass spectrometry (MIMS) to directly analyze CA activity in intact cells by measuring the 18O exchange between CO2 and H2O. This method distinguishes between intracellular and extracellular CA activity. MDA-MB-231 cells were exposed to 1% oxygen for 16 hours after which they were isolated under normoxic or anoxic conditions. CA activity was then measured, again under normoxic or anoxic conditions. These data show biphasic depletion of 18O from CO2 under both normoxic and anoxic assay conditions. The first phase (which occurs over the first 20-40 seconds) represents a rapid diffusion of CO2 into cells where it is exposed to intracellular CAII. A catalytic cycle depletes 18O followed by efflux of CO2 from the cell. There was no difference in this phase between cells prepared and assayed under normoxic or anoxic conditions. The second phase (from 200-500 sec) is dominated by the hydration-dehydration reaction of CO2/HCO3− catalyzed by exofacial CA activity (CAIX). In cells exposed to anoxia, the slope of the second phase was greater than that observed with cells exposed to normoxic conditions indicating elevated CAIX activity. While this provided evidence that oxygen limitation might influence CAIX activity, the Ki values for two impermeant CA inhibitors, Cpd 5C and a polymeric sulfonamide, did not differ between anoxic and normoxic cells. We conclude from these data that the catalytic site of CAIX is exposed and functional under both anoxic or normoxic conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2056. doi:10.1158/1538-7445.AM2011-2056