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Angiostatin, a proteolytic fragment of plasminogen, is a potent angiogenesis inhibitor able to suppress tumor growth and metastasis through inhibition of endothelial cell proliferation and migration. Previously, we showed that angiostatin binds and inhibits F1Fo ATP synthase on the endothelial cell surface and that anti-ATP synthase antibodies reduce endothelial cell proliferation. ATP synthase also occurs on the extracellular surface of a variety of cancer cells, where its function is as yet unknown. Here, we report that ATP synthase is present and active on the tumor cell surface, and angiostatin, or antibody directed against the catalytic β-subunit of ATP synthase, inhibits the activity of the synthase. We show that tumor cell surface ATP synthase is more active at low extracellular pH (pHe). Low pHe is a unique characteristic of the tumor microenvironment. Although the mechanism of action of angiostatin has not been fully elucidated, angiostatin treatment in combination with acidosis decreases the intracellular pH (pHi) of endothelial cells, leading to cell death. We also find that, at low pHe, angiostatin and anti-β-subunit antibody induce intracellular acidification of A549 cells, as well as a direct cytotoxicity that is absent in tumor cells with low levels of extracellular ATP synthase. These results establish angiostatin as an antitumorigenic and antiangiogenic agent through a mechanism implicating tumor cell surface ATP synthase. Furthermore, these data provide evidence that extracellular ATP synthase plays a role in regulating pHi in cells challenged by acidosis. (Cancer Res 2006; 66(2): 875-82)

We have recently reported the identification of kringle 1-5 (K1-5) of plasminogen as a potent and specific inhibitor of angiogenesis and tumor growth. Here, we show that K1-5 bound to endothelial cell surface ATP synthase and triggered caspase-mediated endothelial cell apoptosis. Induction of endothelial apoptosis involved sequential activation of caspases-8, -9, and -3. Administration of neutralizing antibodies directed against the α- and β-subunits of ATP synthase to endothelial cells attenuated activation of these caspases. Furthermore, inhibitors of caspases-3, -8, and -9 also remarkably blocked K1-5-induced endothelial cell apoptosis and antiangiogenic responses. In a mouse tumor model, we show that caspase-3 inhibitors abolished the antitumor activity of K1-5 by protecting the tumor vasculature undergoing apoptosis. These results suggest that the specificity of the antiendothelial effect of K1-5 is attributable, at least in part, to its interaction with the endothelial cell surface ATP synthase and that the caspase-mediated endothelial apoptosis is essential for the angiostatic activity of K1-5. Thus, our findings provide a mechanistic insight with respect to the angiostatic action and signaling pathway of K1-5 and angiostatin.

Circulating autoantibodies against the glucose-regulated protein of 78 kDa (GRP78) are present at high levels in prostate cancer patients and are a biomarker of aggressive tumor behavior. We purified the anti-GRP78 IgGs and examined their effect on 1-LN, PC-3, DU145, and LnCap human prostate cancer cells. We also evaluated its effects on the breast cancer MDA-MB231 and melanoma DM413 cell lines. The anti-GRP78 antibody binds only to cells expressing GRP78 on the surface, to a site also recognized by its physiologic agonist, activated α2-macroglobulin (α2M*). This antibody is completely specific for a peptide, including the primary amino acid sequence CNVKSDKSC, which contains a tertiary structural motif mimicking an epitope in GRP78. Tertiary structual analysis suggested the linear GRP78 primary amino acid sequence LIGRTWNDPSVQQDIKFL (Leu98-Leu115) as the putative binding site, containing the tertiary structual arrangement described above, which was confirmed experimentally. The anti-GRP78 antibodies from prostate cancer patients recognize almost exclusively this epitope. We produced animal antibodies against both these peptides, and they are able to mimic the effects of the human antibody. Our experiments also suggest this epitope as highly immunogenic, thereby explaining the specificity of the immune response against this epitope in GRP78, observed in humans. Using 1-LN cells as a model, we show that anti-GRP78 IgG purified from the sera of these patients mimics the proproliferative effects induced by α2M* via the common receptor, GRP78. Furthermore, increasing concentrations of human anti-GRP78 IgG show a dose-dependent protective effect on apoptosis induced by tumor necrosis factor α. (Cancer Res 2006; 66(23): 11424-31)

Introduction: Western countries have 6 fold higher incidence of prostate cancer (PC). Dietary composition, in addition to other factors, may contribute to this geographic disparity. Cholesterol, the precursor for de novo steroid hormone synthesis, has been hypothesized to promote PC growth via multiple pathways including intratumoral steroidogenesis. While lowering serum cholesterol slows xenograft PC growth, this has not been tested in transgenic models, which allow testing of PC prevention as well as modulating PC growth in an orthotopic position. We determined the effect of cholesterol uptake inhibition, using ezetimibe, on PC growth in Hi-Myc and PTEN transgenic mice. Methods: We randomized 50 c-myc/wt (Hi-Myc) mice and 30 PTENloxP/loxP-Cre+ (PTEN) to one of two diets: high fat high cholesterol (HFHC; 40% fat, 17% protein, 43% carbohydrate, 1.25% cholesterol), or HFHC with ezetimibe (30 mg/kg food) (HFHC+Z). Mice were fed ad libitum starting right after weaning and body weights measured twice weekly. Prostate, liver, spleen, adipose tissue and serum were harvested from Hi-Myc and PTEN mice at age 6 and 4 months, respectively. Serum cholesterol and testosterone (T) and tumor T were measured in Hi-Myc mice and are ongoing in PTEN mice. Results: In Hi-Myc mice, body weights were significantly greater in the HFHC+Z group at sacrifice, relative to the HFHC group (p=0.04), but there were no differences in prostate weight between groups with adjustment for body weight (p=0.84). In PTEN mice, there was no difference in body weights between groups, but prostate weight was significantly lower in the HFHC+Z group, relative to the HFHC group (p=0.046). In Hi-Myc mice, while serum T levels did not vary between groups (p=0.93), tumor T, dihydrotestosterone (DHT) and androstenedione concentrations were significantly reduced in the HFHC+Z group, relative to the HFHC group (p=0.01, p=0.01, p=0.006, respectively). Discussion: Reduction of serum cholesterol levels using cholesterol uptake inhibitor, ezetimibe, significantly reduced prostate weight in PTEN mice consuming a HFHC diet. Furthermore, supplementation of the HFHC diet with ezetimibe significantly reduced tumor concentrations of T, DHT and androstenedione in Hi-Myc mice, although there was no effect on prostate weight. These findings highlight a potentially critical role for cholesterol in PC progression by providing evidence that reduction of hypercholesterolemia may reduce intratumoral de novo steroidogenesis and slow PC growth. This study may provide new insight into the use of cholesterol-lowering drugs for PC prevention and treatment. Citation Format: Emma H. Allott, Elizabeth M. Masko, Alexa Choy, Alexis R. Gaines, Keith R. Solomon, Salvatore V. Pizzo, Stephen J. Freedland. Inhibition of cholesterol uptake with ezetimibe reduces intra-tumoral testosterone levels and slows tumor growth in transgenic mouse models of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3698. doi:10.1158/1538-7445.AM2013-3698

Background: Recently, exciting reports have outlined the existence and function of very thin, membranous conduits between cells that span significant distances, termed tunneling nanotubes (TnTs). TnTs have been observed in neoplastic and normal cells, and may transport soluble proteins, such as P-glycoprotein (MDR1), lipids, and whole organelles, including mitochondria and Golgi vesicles, between cells. Furthermore, TnTs mediate intercellular Ca2+ signals and transmit electrical potentials. However, their functional characterization remains incomplete. Objectives: Our objective is to describe TnT formation and function in melanoma cell lines and primary tumor samples. Methods: We describe the presence and composition of TnTs between cells in the melanoma cell lines SK-Mel-31, DM440, DM443, DM738, and DM598 in vitro with a combination of brightfield and fluorescent microscopy techniques. TnT formation is assessed in response to hypoxia, glucose stress, and low pH. Bulk intercellular transport of mitochondria is quantified via flow cytometry using MitotrackerTM dyes, and GFP-GRP78 fusion proteins are used for studies of protein transport. Additional flow cytometric studies include JC-1 staining to measure mitochondrial membrane potential, annexin V binding to measure apoptosis, and 7-AAD exclusion for live/dead staining. Fluorescent ATP assays are used to measure general metabolic capacity, and confocal microscopy of primary melanoma specimens is performed using melanosome-specific (P100) and dsDNA (H33342) stains. Results All tested cell lines formed TnT-like structures with variable F-actin and tubulin composition. Mitochondria and Golgi vesicles were visible within live and fixed TnTs, which were formed constitutively and were further induced by hypoxia, glucose stress, and low pH. In an actin-dependent process, cells rapidly transferred labeled mitochondria, achieving homogeneity within 24 h as measured by flow cytometry. Transfer of healthy mitochondria from unstressed cells significantly mitigated the acute toxicity of chemotherapeutically damaged cells. GRP78 was also rapidly transferred between cells, and the transfer of GRP78 from healthy donor cells rescued cells damaged by chemotherapeutics as measured by a decreased apoptotic rate. Conclusions: Here, we report for the first time, the characterization of TnTs between melanoma cells. Moreover, melanoma cells acquired primary and secondary chemotherapeutic resistance to alkylating agents melphalan and temozolomide and the BRAFV600E inhibitor, vemurafenib, via bulk intercellular transfer of functional mitochondria and the ER stress protein, GRP78. TnT inhibitors would represent a novel class of chemotherapeutics with implications for invasion, metastasis, and chemotherapeutic resistance. Citation Format: Gustaaf G. de Ridder, Rupa Ray, Salvatore V. Pizzo. Tunneling nanotubes connect melanoma cells and mediate transfer of mitochondria and GRP78. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5248. doi:10.1158/1538-7445.AM2013-5248 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

GRP78 is an important molecular chaperone in the endoplasmic reticulum (ER). However, GRP78 is aberrantly expressed on the surface of melanoma cells. Our objective is to characterize cell-surface GRP78 in melanoma and determine whether immunoconjugates against GRP78 are a viable treatment strategy. We have demonstrated that cleavage of ER GRP78 by Subtilase (SubAB), a GRP78-specific protease, sensitizes melanoma cell lines of different genotypes to melphalan, vemurafenib, and temozolomide. Our data indicate that SubAB cleavage of GRP78 sensitizes melanoma cells to melphalan and vemurafenib. We then coupled the catalytic domain of SubAB, SubA, to GRP78-single chain variable fragments derived from anti-GRP78 mouse monoclonal antibodies. We have previously published that these antibodies target GRP78+ mouse melanomas in a syngeneic mouse melanoma model. These GRP78 scFv-SubA fusion proteins are internalized by cell-surface GRP78 and the SubA moiety cleaves ER GRP78 inside the cell, leading to tumor cell sensitization to chemotherapeutics, and in some instances, tumor cell death. To determine the mechanism by which ER GRP78 cleavage sensitizes melanoma cells to chemotherapeutics, the NH2-terminal 28 kDa and COOH-terminal 50 kDa SubA cleavage products were overexpressed in melanoma cells, and pull-down experiments were performed to identify binding partners. Microarray analyses were also performed on chemotherapy-treated and GRP78 scFv-SubA fusion protein-treated melanoma cells to obtain a broad view of cellular events that occurred subsequent to ER GRP78 cleavage by SubAB or GRP78 scFv-SubA fusion proteins. These fusion proteins will then be tested in mouse melanoma models to determine whether they sensitize cancer cells to chemotherapeutics in vivo. These results of these studies may lead to the development of novel melanoma therapeutics. Citation Format: Rupa Ray, Gustaaf de Ridder, Salvatore V. Pizzo. Development of GRP78 scFv-Subtilase fusion proteins for sensitization of melanoma to chemotherapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1247. doi:10.1158/1538-7445.AM2013-1247

Introduction: Preclinical studies have shown that dietary carbohydrate restriction slows prostate tumor growth in multiple xenograft models in part due to a reduction insulin-like growth factor (IGF) axis signaling. Pomegranate extract (PE) has been shown to slow prostate tumor growth in-vitro and in vivo similarly through this pathway, as well as decrease markers of angiogenesis and inflammation. In this study, we determined the dose-response of PE on prostate tumor growth in the setting of dietary carbohydrate restriction in a slow-growing mouse xenograft model. For this study, we chose a standardized PE (POMx, Pom Wonderful, Los Angeles, CA), at doses which are easily translatable to human equivalents. Materials and Methods: Male athymic nude mice housed 5 per cage (n = 130) were subcutaneously injected with 1 × 10^5 LAPC-4 cells after a 10 day acclimation period. 14 days post-injection, all mice were all placed on an ad-libitum no-carbohydrate ketogenic diet (NCKD: 83% fat, 0% carbohydrate, 17% protein) (day 0). Each cage was randomly assigned to 1 of 4 doses of PE (placebo = 0 mg, low dose = 2.62 mg, intermediate dose = 5.25mg, or high dose = 7.87mg) suspended in 0.2 mL of PBS delivered via oral gavage 5 days per week. These doses are equivalent to 0, 1, 2, or 3 POMx capsules/day consumed by a 70 kg human. Mice will be sacrificed when tumors reach ≥1,000 mm^3. Results: At randomization, there were no differences in median body weight among groups (p = 0.623). Currently, at day 32, overall survival is decreased in the placebo group (hazard ratio 2.26, 95% confidence interval, 0.83 - 6.10, p = 0.109) relative to the low dose PE group with no survival differences among PE groups. Median tumor volumes for the placebo group are 83.3mm^3, compared to 74.5mm^3, 76.3mm^3, and 70.1mm^3 for the low, intermediate, and high PE doses respectively, which are not statistically different (p = 0.98). Conclusions: Preliminary data show a trend toward improved survival with the combination of an NCKD and PE at the selected doses. Currently, tumor volumes are not significantly different, however completed results will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5631. doi:1538-7445.AM2012-5631

Background: Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer (PC), but not the incidence of the disease. This may occur by lowering levels of low-density lipoproteins (LDL). Statins and cholesterol-uptake inhibitors like ezetimibe lower serum LDL and are commonly prescribed for patients with high cholesterol. In this study, we determined the therapeutic role of simvastatin, ezetimibe, or both in vitro using several PC cell lines and in vivo using a LAPC-4 xenograft model. Methods: We treated 4 PC cell lines (LAPC-4, CWR22rv1, PC-3, and DU145) with varying concentrations of simvastatin (0-1000nM) or ezetimibe (0-100μM) for 0-5 days. MTS assays were performed to determine cell proliferation, and the IC50 was calculated using BioDataFit 1.02. For our in vivo study, a total of 90 athymic male nude mice were fed a high-fat, high-cholesterol diet (HFHC; 40% fat, 17% protein, 43% carbohydrates, 1.25% cholesterol), subcutaneously injected with 1 x 10^5 LAPC-4 cells, and randomized 2 weeks later to 1 of 4 treatments: vehicle control, 11mg/kg/day simvastatin (delivered via osmotic pump), 30mg/kg diet ezetimibe (delivered in the food), or simvastatin + ezetimibe. Mice remained on study for 42 days post-randomization, at which point they were harvested and samples analyzed. Results: In vitro, simvastatin directly reduced PC cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no inhibitory effect. In vivo, treatment of low continuous dosing of simvastatin or ezetimibe for 42 days had no effect on tumor growth compared to control. However, relative to control, the combination of simvastatin and ezetimibe accelerated tumor growth (p=0.01). The simvastatin-ezetimibe mice also showed lower fat infiltration in the liver and lower serum cholesterol, but higher tumor cholesterol levels than control. Conclusions: Our results suggest that low continuous dosing of simvastatin or ezetimibe may have no effect on PC growth in a slow-growing LAPC-4 xenograft model, but the combination of simvastatin and ezetimibe may accelerate PC growth, possibly by stimulating cholesterol uptake within the tumor. This novel finding may be important in implementing new treatments for men with slow growing PC and high cholesterol. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3220. doi:1538-7445.AM2012-3220

Introduction and Objectives: Prostate cancer (PC) is affected by cholesterol metabolism and cholesterol-mediating medications. Ezetimibe, a gut cholesterol-uptake inhibitor, inhibits angiogenesis in human PC xenografts when given as preventive therapy. We investigated the effect of ezetimibe on PC growth when given as treatment therapy. Methods: We randomized 125 SCID mice into three initial diets: a high-fat, low-cholesterol diet (HFLC) (40% fat, 43% carbohydrates, 17% protein, 0.21% cholesterol), a high-fat, high-cholesterol diet (HFHC) (40% fat, 43% carbohydrates, 17% protein, 1.25% cholesterol), and a high-fat, high-cholesterol diet supplemented with ezetimibe (30mg/kg) (HFHC+Z). Mice were fed ad libitum. After two weeks, we injected 106 LNCaP cells into the flanks, with 5-10 mice/group serving as non-injected controls. At two weeks post-injection, half the injected and control mice in the HFHC diet group had ezetimibe added to their diet (HFHC+delayed Z). Tumor volumes and body weights were measured twice weekly. Mice were sacrificed once tumors reached 1000mm3. Body weights and tumor volumes were compared across groups using Kruskal-Wallis test. Interim survival outcomes for this on-going study up to 86 days post-injection were evaluated with Cox proportional hazards. Results: At 1 day prior to injection, there were no significant differences in body weights across groups. At post-injection day (PD) 2, HFHC+Z mice with tumors were significantly lighter vs. all other mice (p=0.009). By PD 21, HFHC+delayed Z mice with tumors also became significantly lighter vs. the HFLC and HFHC mice with tumors (p Conclusion: Preliminary evidence from this on-going study demonstrates that ezetimibe is associated with weight loss, but only in mice with PC xenografts. The administration of ezetimibe as preventive vs. treatment therapy does not appear to affect tumor growth or survival. Future studies to explore the interplay between cholesterol metabolism and PC include comparing serum levels of cholesterol, insulin, IGF-1, and IGFBP-3, and tumor markers of proliferation and angiogenesis including akt, pakt, mTOR, VEGF, HIF, CD31 (microvessel density) and ki67 (proliferation). Moreover, we are continuing to follow the mice for any differences in tumor growth or survival. 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 4602. doi:10.1158/1538-7445.AM2011-4602

Introduction: Previous dietary studies indicate carbohydrate intake may influence prostate cancer biology, as LAPC-4 and LNCaP xenograft mice fed a no-carbohydrate ketogenic diet (NCKD; 84% fat-0% carbohydrate-16% protein kcal) had significantly smaller tumors and longer survival times compared to mice fed a Western diet (40% fat-44% carbohydrate-16% protein kcal). The NCKD mice were also found to have higher levels of circulating IGFBP-3 and the lowest levels of insulin, IGF-1, and IGF-1:IGFBP-3 ratio despite consuming more calories than the Western group. As it is nearly impossible for a human to consume and maintain a no-carbohydrate diet similar to that in the previous xenograft studies, we sought to determine whether diets containing 10% or 20% kcal from carbohydrates could slow tumor growth in a similar manner to the NCKD in a xenograft model. METHODS: A total of 150 male SCID mice were injected with LAPC-4 cells and placed on a Western diet (35% fat-49% carbohydrate-16% protein kcal) ad libitum. Two weeks post-injection, all mice were randomized to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate. Ten mice not injected with tumor were fed an ad libitum low-fat diet (12% fat-72% carbohydrate-16% protein kcal) and served as the reference group in a modified-paired feeding protocol for the other three groups. Calorie intake and body weights were measured thrice weekly and tumor volumes twice per week. Mice were sacrificed when tumors reached 1,000mm3. RESULTS: Despite consuming 5-10% extra calories on average, all mice receiving low-carbohydrate diets were significantly lighter than the mice consuming the low-fat diet (p CONCLUSIONS: LAPC-4 xenograft mice fed a low-carbohydrate diet (10-20% carbohydrate kcal) had similar survival to mice consuming a NCKD (0% carbohydrate kcal). Thus, the survival benefit of a NCKD may be achievable with less restrictive low-carbohydrate diets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2816.