Scott D. Cramer

Lipid Catabolism via CPT1 as a Therapeutic Target for Prostate Cancer

Prostate cancer is the most commonly diagnosed malignancy among Western men and accounts for the second leading cause of cancer-related deaths. Prostate cancer tends to grow slowly and recent studies suggest that it relies on lipid fuel more than on aerobic glycolysis. However, the biochemical mechanisms governing the relationships between lipid synthesis, lipid utilization, and cancer growth remain unknown. To address the role of lipid metabolism in prostate cancer, we have used etomoxir and orlistat, clinically safe drugs that block lipid oxidation and lipid synthesis/lipolysis, respectively. Etomoxir is an irreversible inhibitor of the carnitine palmitoyltransferase (CPT1) enzyme that decreases β oxidation in the mitochondria. Combinatorial treatments using etomoxir and orlistat resulted in synergistic decreased viability in LNCaP, VCaP, and patient-derived benign and prostate cancer cells. These effects were associated with decreased androgen receptor expression, decreased mTOR signaling, and increased caspase-3 activation. Knockdown of CPT1A enzyme in LNCaP cells resulted in decreased palmitate oxidation but increased sensitivity to etomoxir, with inactivation of AKT kinase and activation of caspase-3. Systemic treatment with etomoxir in nude mice resulted in decreased xenograft growth over 21 days, underscoring the therapeutic potential of blocking lipid catabolism to decrease prostate cancer tumor growth. Mol Cancer Ther; 13(10); 2361–71. ©2014 AACR.

The Autophagy Machinery Controls Cell Death Switching between Apoptosis and Necroptosis

Although autophagy controls cell death and survival, underlying mechanisms are poorly understood, and it is unknown whether autophagy affects only whether or not cells die or also controls other aspects of programmed cell death. MAP3K7 is a tumor suppressor gene associated with poor disease-free survival in prostate cancer. Here, we report that Map3k7 deletion in mouse prostate cells sensitizes to cell death by TRAIL (TNF-related apoptosis-inducing ligand). Surprisingly, this death occurs primarily through necroptosis, not apoptosis, due to assembly of the necrosome in association with the autophagy machinery, mediated by p62/SQSTM1 recruitment of RIPK1. The mechanism of cell death switches to apoptosis if p62-dependent recruitment of the necrosome to the autophagy machinery is blocked. These data show that the autophagy machinery can control the mechanism of programmed cell death by serving as a scaffold rather than by degrading cargo.

Inhibition of Lipid Oxidation Increases Glucose Metabolism and Enhances 2-Deoxy-2-[ 18 F]Fluoro- d -Glucose Uptake in Prostate Cancer Mouse Xenografts

PurposeProstate cancer (PCa) is the second most common cause of cancer-related death among men in the United States. Due to the lipid-driven metabolic phenotype of PCa, imaging with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG) is suboptimal, since tumors tend to have low avidity for glucose.ProceduresWe have used the fat oxidation inhibitor etomoxir (2-[6-(4-chlorophenoxy)-hexyl]oxirane-2-carboxylate) that targets carnitine-palmitoyl-transferase-1 (CPT-1) to increase glucose uptake in PCa cell lines. Small hairpin RNA specific for CPT1A was used to confirm the glycolytic switch induced by etomoxir in vitro. Systemic etomoxir treatment was used to enhance [18F]FDG-positron emission tomography ([18F]FDG-PET) imaging in PCa xenograft mouse models in 24 h.ResultsPCa cells significantly oxidize more of circulating fatty acids than benign cells via CPT-1 enzyme, and blocking this lipid oxidation resulted in activation of the Warburg effect and enhanced [18F]FDG signal in PCa mouse models.ConclusionsInhibition of lipid oxidation plays a major role in elevating glucose metabolism of PCa cells, with potential for imaging enhancement that could also be extended to other cancers.

Antenatal endotoxin disrupts lung vitamin D receptor and 25-hydroxyvitamin D 1α-hydroxylase expression in the developing rat.

Vitamin D [vit D; 1,25-(OH)2D] treatment improves survival and lung alveolar and vascular growth in an experimental model of bronchopulmonary dysplasia (BPD) after antenatal exposure to endotoxin (ETX). However, little is known about lung-specific 1,25-(OH)2D3 regulation during development, especially regarding maturational changes in lung-specific expression of the vitamin D receptor (VDR), 1α-hydroxylase (1α-OHase), and CYP24A1 during late gestation and the effects of antenatal ETX exposure on 1,25-(OH)2D3 metabolism in the lung. We hypothesized that vit D regulatory proteins undergo maturation regulation in the late fetal and early neonatal lung and that prenatal exposure to ETX impairs lung growth partly through abnormal endogenous vit D metabolism. Normal fetal rat lungs were harvested between embryonic day 15 and postnatal day 14. Lung homogenates were assayed for VDR, 1α-OHase, and CYP24A1 protein contents by Western blot analysis. Fetal rats were injected on embryonic day 20 with intra-amniotic ETX, ETX + 1,25-(OH)2D3, or saline and delivered 2 days later. Pulmonary artery endothelial cells (PAECs) from fetal sheep were assessed for VDR, 1α-OHase, and CYP24A1 expression after treatment with 25-(OH)D3, 1,25-(OH)2D3, ETX, ETX + 25-(OH)D3, or ETX + 1,25-(OH)2D3. We found that lung VDR, 1α-OHase, and CYP2741 protein expression dramatically increase immediately before birth (P < 0.01 vs. early fetal values). Antenatal ETX increases CYP24A1 expression (P < 0.05) and decreases VDR and 1α-OHase expression at birth (P < 0.001), but these changes are prevented with concurrent vit D treatment (P < 0.001). ETX-induced reduction of fetal PAEC growth and tube formation and lung 1α-OHase expression are prevented by vit D treatment (P < 0.001). We conclude that lung VDR, 1α-OHase, and CYP24A1 protein content markedly increase before birth and that antenatal ETX disrupts lung vit D metabolism through downregulation of VDR and increased vit D catabolic enzyme expression, including changes in developing endothelium. We speculate that endogenous vitamin D metabolism modulates normal fetal lung development and that prenatal disruption of vit D signaling may contribute to impaired postnatal lung growth at least partly through altered angiogenic signaling.

Autophagy RIPs into cell death.

Programmed cell death in recent years has extended to include many other cell death pathways besides apoptosis. The finding of these new cell death pathways is proving to have critical roles in how we evaluate cell death in cancer. One such cell death pathway is necroptosis, a type of programmed necrosis. Necroptosis mediates plasma membrane permeablization through phosphorylation of MLKL (mixed lineage kinase domain-like protein) by formation of the necrosome that contains RIPK3 (receptor interacting protein 3) in complex with RIPK1, FADD, and caspase-8. Death through necroptosis, but not apoptosis, promotes anti-tumor immune responses. A role for necroptosis in cancer is further strengthened by the fact that RIPK3 expression is often silenced in cancers making them unable to undergo necroptosis. This implies that necroptosis may be selected against during tumor evolution so that tumors can avoid adaptive anti-tumor immunity. Moreover, apoptosis has been shown to release growth stimulating signals which could lead to more effective tumor repopulation. Thus, apoptosis may be a less effective form of tumor cell killing than necroptosis because it is not as good at activating anti-tumor immunity, but is capable of promoting more rapid tumor repopulation. Based on these findings, it is critical to understand the underlying mechanisms that determine how a cell dies and specifically whether death occurs by apoptosis or necroptosis. A recent paper from our group provides new insights into this question. In our recent article we show that mouse prostate cells that had lost Map3k7, a gene that is lost in 30–40% of human prostate cancers and associated with aggressive disease, were hypersensitive to TNF and TRAIL (TNFa-related apoptosisinducing ligand)-induced cell death. Despite the well-established mechanism of TRAIL-induced cell death being by apoptosis, Map3k7-null cells preferentially died through necroptosis (Fig. 1). Moreover, the cell death mechanism could switch to apoptosis when necroptosis was prevented by direct inhibition of the necrosome. Establishing that cells lacking Map3k7 die preferentially by necroptosis in response to TRAIL could have significant immunogenic outcomes for patients whose tumors show loss of this gene. Consistent with this, and suggesting that tumors evolve ways to suppress these mechanisms, we also found that prostate cancer patients whose tumors had lost MAP3K7 also tended to have deletions of the receptors that are activated by TRAIL. Previously, we reported TRAIL-induced apoptosis can be regulated by autophagy, therefore we further investigated the role of autophagy in necroptosis in the Map3k7-null cells. As expected, inhibition of late stage autophagy enhanced cell death in response to TRAIL. However, inhibition of early/mid-stage autophagy by both genetic (Atg5, Atg7, and Beclin-1) and pharmacological inhibition (Wortmannin) prevented cell death. We hypothesized that components of the autophagy machinery, not the turnover of cellular components, were mediating cell death by functioning as a scaffold for necrosome complex formation and activation. We confirmed our hypothesis by performing dual proximity ligation assays, co-immunoprecipitations, and immuno-gold transmission electron microscopy showing localization and activation of the necrosome complex on the autophagosome. Interestingly, binding of p62/SQSTM1 to RIPK1 was shown to localize the necrosome to the autophagosome, and its loss was sufficient to switch cell death from necroptosis to apoptosis. These data led us to conclude that the mechanism that determines the cell death outcome is through p62-dependent recruitment of the necrosome components to the autophagy machinery. Our studies provide insights into how autophagy regulates necroptosis and presents a mechanism by which controlled switching between necroptosis and apoptosis can be achieved. More importantly, our studies raise a number of significant issues in regards to cancer treatment, specifically in tumors lacking Map3k7. Firstly, the use of autophagy inhibitors in the clinic could have very different outcomes based on which point of the pathway is inhibited with inhibitors that block early steps in the autophagy pathway having completely different effects than inhibitors that block later steps. Secondly, our work adds to accumulating evidence that the most important question for cancer therapy might not be just whether or not cancer cells are killed, but how they are killed. And, if adaptive immunity is activated through necroptosis, finding ways to kill cancer cells

Abstract PR11: The molecular implications of lifestyle associated metabolites (AGEs) to prostate cancer disparity

Poor diet, low income, obesity, and a lack of exercise are established lifestyle factors that are known to increase cancer burden and are often more prevalent in African American communities. As our understanding of tumor biology advances, it is becoming increasingly clear that these inter-related lifestyle factors have distinct molecular consequences on the biologic make up of tumors, altering cell signaling events and gene expression profiles to contribute to cancer disparity outcomes such as its earlier development or its progression to more aggressive disease. Advanced glycation end-products (AGEs), are reactive metabolites produced endogenously as a consequence of glucose uptake during glycolysis. AGEs accumulate in tissues and organs as we grow older to promote multiple chronic disease phenotypes. AGE pathogenic effects are mediated through modification of protein function, genetic fidelity, stress responses and cellular signaling pathways. Critically, cancer disparity factors such as a sedentary lifestyle, obesity and an unhealthy diet are external influences that also contribute to the AGE accumulation pool in the body. The investigators studies support the concept that AGE metabolites represent a biological consequence of the socioeconomic and environmental factors that promote cancer health disparity. This research group examined circulating and tumor AGE levels in clinical specimens of prostate cancer and identified a race specific, tumor-dependent pattern of accumulation. The aberrant activation and recruitment of immune cells is a major pathogenic consequence of AGE accumulation and a series of studies have highlighted the tumor associated immune response as a critical pathway contributing to cancer disparity. Using patient derived primary tumor cells, the investigators found that AGEs released into the extracellular matrix can recapitulate the tumor associated immune response observed in race specific prostate tumor tissues. Activated immune cells show a similar metabolic profile as a glycolytic tumor cell with a shift towards increased glucose metabolism and aerobic glycolysis (i.e. the Warburg effect). Evidence suggests that abnormal glucose uptake may occur earlier in African American cancer patients with aggressive disease. Further preliminary studies indicate that AGE treatment of prostate cancer cells can alter how cancer cells metabolize glucose to promote an aggressive phenotype. Based on associations between active metabolism, lifestyle, and race, increases in AGE accumulation may represent a novel biologic mechanism contributing to cancer disparity and may represent a new paradigm to explaining the increased cancer incidence and mortality figures observed within health disparity populations. Given the potential benefits of lifestyle changes and the potential biological role of AGEs in promoting cancer, opportunities exist for collaborations impacting basic, translational, epidemiological and cancer prevention initiatives. Citation Format: Danzell Smith, Dion Foster, Laura Spruill, Lourdes Nogueira, Bradley Krisanits, Scott Cramer, Marvella Ford, Stephen Savage, Thomas Keane, Victoria Findlay, David Turner. The molecular implications of lifestyle associated metabolites (AGEs) to prostate cancer disparity. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr PR11.

Abstract 445: Coordinate loss of CHD1 and MAP3K7 promotes aggressive prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The genomic complexity of prostate cancer contributes to disease progression, resistance to therapy, and development of metastases. The genes CHD1 and MAP3K7 are signficantly co-deleted in 10-15% of primary tumors and combined loss of CHD1 and MAP3K7 expression is associated with poor disease-free survival. We recently validated MAP3K7 as a prostate tumor suppressor, but CHD1s role in prostate tumorigenesis has not been evaluated. In this study, we used a mouse prostate epithelial stem cell (PrP/SC) model and a human prostate cancer cell line (LNCaP) model to investigate how loss of CHD1 alone or in combination with MAP3K7 suppression affects prostate tumorigenesis. Suppression of Map3k7 increased proliferation of PrP/SCs. Chd1 suppression decreased growth of wildtype PrP/SCs but not PrP/SCs already deficient in Map3k7 expression. Similarly, suppression of CHD1 in LNCaP cells decreased proliferation when MAP3K7 expression was present, but not when MAP3K7 was suppressed. These data suggest that loss of CHD1 alone is deleterious to growth, but that MAP3K7 suppression can compensate for this growth inhibition. Chd1 and Map3k7-deficient PrP/SCs were then evaluated in vivo using tissue recombination. When Chd1-deficient PrP/SCs were recombined with fetal rat urogenital mesenchyme (rUGM) and implanted under the renal capsules of immunocompromised mice, grafts contained mostly benign glandular tissue with focal areas of mild hyperplasia/prostatic intraepithelial neoplasia (PIN). A few nuclear abnormalities were observed. Map3k7-deficient recombinants generated a mixture of benign tissue, PIN, and adenocarcinoma (consistent with our previous findings). The nuclear morphology of these grafts was unremarkable. Strikingly, dual Chd1-Map3k7 knockdown recombinants displayed massive glandular atypia with glands completely filled with AR-negative and p63-negative cells. These cells had large nuclei, prominent nucleoli, and abundant cytoplasm. Tumorigenicity of CHD1 and/or MAP3K7-deficient LNCaP cells was also assessed by subcutaneous implantation of cells into immunocompromised mice. Mice implanted with CHD1-deficient xenografts formed smaller tumors and had increased survival relative to mice injected with control LNCaP cells (p=0.0083). MAP3K7-deficient xenografts were similar in size and in survival rate compared to control LNCAP cells. Dual CHD1-MAP3K7 deficient xenografts, in contrast, showed increased tumor growth and decreased survival relative to control (p=0.0242), MAP3K7 alone (p=0.0462) and CHD1 alone (p=0.0002). Collectively, these data suggest that loss of CHD1 alone is anti-tumorigenic but that combined loss of CHD1 and MAP3K7 drives aggressive prostate cancer development. Citation Format: Lindsey Ulkus, Leah Rider, Cera Nieto, Lina Romero, Haitao Chen, Massimo Loda, Wennuan Liu, Jianfeng Xu, Scott Cramer. Coordinate loss of CHD1 and MAP3K7 promotes aggressive prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 445. doi:10.1158/1538-7445.AM2014-445

Abstract 107: Lipid metabolism inhibitors enhance glycolysis and FDG-PET imaging of prostate cancer tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Positron Emission Tomography (PET) with18F-deoxyglucose (FDG) has gained a major role in the clinical setting for detection, staging and assessment of treatment response for a number of epithelial cancers. However, staging of primary or metastatic prostate cancer with 18F-FDG-PET is suboptimal, likely due to the low glucose uptake and enhanced lipid metabolism characteristic of primary prostate tumors. The exact mechanisms of aerobic glycolysis (Warburg effect) and lipid metabolism in prostate cancer (PCa) cells remain unknown. However, 18F-FDG-PET imaging of metastatic PCa might be enhanced by altering the intrinsic energy metabolism of involved sites. Experimental Procedures: In vitro radioactive studies were carried out to confirm the specificity of etomoxir for blocking lipid oxidation and enhancing glucose uptake at 24 hours in prostate cancer cell lines. For in vivo studies, three mouse models of PCa were used: Male nude mice with subcutaneous xenograts or orthotopic injections and TRAMP mice that develop prostate cancer with age. All mice were treated with a single dose of the lipid oxidation inhibitor etomoxir (20 mg/kg) or vehicle (saline) for 24 hours. A basal FDG-PET scan was performed before the drug treatment, followed by a second FDG-PET scan after 24 hours. Western blot analysis was used to validate the molecular mechanisms of increased FDG uptake. Results: The clinically safe drug etomoxir blocks fat oxidation within 4 hours and increases glucose uptake in cultured PCa cells. Subcutaneous xenografts showed a significant increase in normalized FDG uptake (NUV) after a single dose of etomoxir compared to basal NUV (1.4 fold change, p =0.03). This enhancing effect was not seen in mice treated with saline (vehicle). Mice with orthotopic xenografts and 20-week old TRAMP mice showed a 2.7-fold (p=0.03) and 1.6-fold (p=0.07) increase in FDG uptake, respectively, over basal. Protein examination of the excised subcutaneous tumors showed increased hexokinase-II content and activated mTOR and p70-S6 Kinase proteins in the etomoxir-treated tumors when compared to saline-treated tumors. Conclusion: 18F-FDG is a commercially available tracer that is widely used for standard-of-care and research-based oncologic imaging. Blocking lipid oxidation in PCa tumors has the potential to improve diagnostic imaging, since 18F-FDG that accumulates inside the cell can enhance FDG-PET imaging of prostate cancer tumors to detectable and quantifiable levels. Citation Format: Isabel R. Schlaepfer, Colton T. Pac, Natalie J. Serkova, Gagan Deep, Rajesh Agarwal, Scott D. Cramer, Robert H. Eckel, L. Michael Glode. Lipid metabolism inhibitors enhance glycolysis and FDG-PET imaging of prostate cancer tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 107. doi:10.1158/1538-7445.AM2014-107

Abstract A86: Silibinin inhibits prostate cancer and stromal cells interaction through targeting TGFβ and α-smooth muscle actin

The tumor microenvironment is now established as an integral and essential component of carcinogenesis playing a critical role in early tumor development. Therefore, targeting the interaction between a growing cancerous lesion and its microenvironment is considered an important translational cancer preventive strategy. Fibroblasts are a key cellular component of the prostate cancer (PCA) microenvironment as they have the capacity to remodel that microenvironment, promoting tumor growth, angiogenesis, invasiveness, and metastasis. Earlier studies have shown that the cancer preventive agent silibinin has broad spectrum efficacy against PCA but its effect on the PCA-fibroblast interaction remains unknown. In the present study we developed cell culture models to study the molecular interaction between PCA and fibroblasts and the effect of silibinin therein. We treated human PCA PC3 cells with DMSO or silibinin (30-60 μM dose) and collected conditioned media labeled as CCM (control conditioned media) or SBCM (silibinin treated conditioned media) respectively. Human prostate fibroblasts (PrSC) were exposed to CCM or SBCM (volume normalized with respective cell number) and we analyzed cell growth, morphology, invasiveness and molecular alterations. It was revealed that CCM exposure resulted in PrSC attaining an elongated morphology, increased invasiveness, and enhanced α-SMA (alpha-smooth muscle actin) and vimentin expression, overall transforming into a ‘myofibroblast’ phenotype permissive to prostate cancer growth and progression. In contrast, PrSC exposed to SBCM failed to manifest a myofibroblast phenotype lacking an elongated morphology, exhibiting decreased invasiveness and reduced α-SMA and vimentin expression. Analyses of CCM and SBCM revealed that the inhibitory effect of silibinin could be the result of decreasing levels of TGFβ1 secreted by PCA cells. These results are currently being validated in other PCA cells (Du145, C4-2B). To investigate the effect of silibinin directly on the fibroblasts component of PCA, we employed both PrSC treated with CCM as well as cancer-associated fibroblasts (CAFs) obtained from PCA patients. Silibinin (30-90 μM dose) exposure inhibited the invasiveness of and α-SMA expression in both CCM-treated PrSC as well as CAFs. Ongoing studies also suggest that silibinin targets CAF-induced EMT (epithelial-to-mesenchymal transition) and invasiveness of human PCA LNCaP cells. These results collectively show that silibinin could target PCA, PrSC, and CAFs as well as their cancer promoting interactions and suggest a role for silibinin in PCA prevention and intervention. Citation Format: Harold J. Ting, Gagan Deep, Chapla Agarwal, Scott D. Cramer, Lina M. Romero, Rajesh Agarwal. Silibinin inhibits prostate cancer and stromal cells interaction through targeting TGFβ and α-smooth muscle actin. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A86.