Hiromi I. Wettersten

Epoxy metabolites of docosahexaenoic acid (DHA) inhibit angiogenesis, tumor growth, and metastasis

Epidemiological and preclinical evidence supports that omega-3 dietary fatty acids (fish oil) reduce the risks of macular degeneration and cancers, but the mechanisms by which these omega-3 lipids inhibit angiogenesis and tumorigenesis are poorly understood. Here we show that epoxydocosapentaenoic acids (EDPs), which are lipid mediators produced by cytochrome P450 epoxygenases from omega-3 fatty acid docosahexaenoic acid, inhibit VEGF- and fibroblast growth factor 2-induced angiogenesis in vivo, and suppress endothelial cell migration and protease production in vitro via a VEGF receptor 2-dependent mechanism. When EDPs (0.05 mg⋅kg−1⋅d−1) are coadministered with a low-dose soluble epoxide hydrolase inhibitor, EDPs are stabilized in circulation, causing ∼70% inhibition of primary tumor growth and metastasis. Contrary to the effects of EDPs, the corresponding metabolites derived from omega-6 arachidonic acid, epoxyeicosatrienoic acids, increase angiogenesis and tumor progression. These results designate epoxyeicosatrienoic acids and EDPs as unique endogenous mediators of an angiogenic switch to regulate tumorigenesis and implicate a unique mechanistic linkage between omega-3 and omega-6 fatty acids and cancers.

Grade-Dependent Metabolic Reprogramming in Kidney Cancer Revealed by Combined Proteomics and Metabolomics Analysis

Kidney cancer [or renal cell carcinoma (RCC)] is known as the internists tumor because it has protean systemic manifestations, suggesting that it utilizes complex, nonphysiologic metabolic pathways. Given the increasing incidence of this cancer and its lack of effective therapeutic targets, we undertook an extensive analysis of human RCC tissue employing combined grade-dependent proteomics and metabolomics analysis to determine how metabolic reprogramming occurring in this disease allows it to escape available therapeutic approaches. After validation experiments in RCC cell lines that were wild-type or mutant for the Von Hippel-Lindau tumor suppressor, in characterizing higher-grade tumors, we found that the Warburg effect is relatively more prominent at the expense of the tricarboxylic acid cycle and oxidative metabolism in general. Further, we found that the glutamine metabolism pathway acts to inhibit reactive oxygen species, as evidenced by an upregulated glutathione pathway, whereas the β-oxidation pathway is inhibited, leading to increased fatty acylcarnitines. In support of findings from previous urine metabolomics analyses, we also documented tryptophan catabolism associated with immune suppression, which was highly represented in RCC compared with other metabolic pathways. Together, our results offer a rationale to evaluate novel antimetabolic treatment strategies being developed in other disease settings as therapeutic strategies in RCC.

Dual inhibition of cyclooxygenase-2 and soluble epoxide hydrolase synergistically suppresses primary tumor growth and metastasis

Significance Our study suggests that cyclooxygenase (COX)-2 and soluble epoxide hydrolase (sEH) pathways have potent synergistic antiangiogenic and anticancer activity. Dual pharmacological inhibition of COX-2 and sEH pathways may be useful in treating cancer with minimal toxicity associated with COX-2 inhibition. Prostaglandins derived from the cyclooxygenase (COX) pathway and epoxyeicosatrienoic acids (EETs) from the cytochrome P450/soluble epoxide hydrolase (sEH) pathway are important eicosanoids that regulate angiogenesis and tumorigenesis. COX-2 inhibitors, which block the formation of prostaglandins, suppress tumor growth, whereas sEH inhibitors, which increase endogenous EETs, stimulate primary tumor growth and metastasis. However, the functional interactions of these two pathways in cancer are unknown. Using pharmacological inhibitors as probes, we show here that dual inhibition of COX-2 and sEH synergistically inhibits primary tumor growth and metastasis by suppressing tumor angiogenesis. COX-2/sEH dual pharmacological inhibitors also potently suppress primary tumor growth and metastasis by inhibiting tumor angiogenesis via selective inhibition of endothelial cell proliferation. These results demonstrate a critical interaction of these two lipid metabolism pathways on tumorigenesis and suggest dual inhibition of COX-2 and sEH as a potential therapeutic strategy for cancer therapy.

Mealtime, Temporal, and Daily Variability of the Human Urinary and Plasma Metabolomes in a Tightly Controlled Environment

While metabolomics has tremendous potential for diagnostic biomarker and therapeutic target discovery, its utility may be diminished by the variability that occurs due to environmental exposures including diet and the influences of the human circadian rhythm. For successful translation of metabolomics findings into the clinical setting, it is necessary to exhaustively define the sources of metabolome variation. To address these issues and to measure the variability of urinary and plasma metabolomes throughout the day, we have undertaken a comprehensive inpatient study in which we have performed non-targeted metabolomics analysis of blood and urine in 26 volunteers (13 healthy subjects with no known disease and 13 healthy subjects with autosomal dominant polycystic kidney disease not taking medication). These individuals were evaluated in a clinical research facility on two separate occasions, over three days, while on a standardized, weight-based diet. Subjects provided pre- and post-prandial blood and urine samples at the same time of day, and all samples were analyzed by “fast lane” LC-MS-based global metabolomics. The largest source of variability in blood and urine metabolomes was attributable to technical issues such as sample preparation and analysis, and less variability was due to biological variables, meals, and time of day. Higher metabolome variability was observed after the morning as compared to the evening meal, yet day-to-day variability was minimal and urine metabolome variability was greater than that of blood. Thus we suggest that blood and urine are suitable biofluids for metabolomics studies, though nontargeted mass spectrometry alone may not offer sufficient precision to reveal subtle changes in the metabolome. Additional targeted analyses may be needed to support the data from nontargeted mass spectrometric analyses. In light of these findings, future metabolomics studies should consider these sources of variability to allow for appropriate metabolomics testing and reliable clinical translation of metabolomics data.

Inhibition of PPARα Induces Cell Cycle Arrest and Apoptosis, and Synergizes with Glycolysis Inhibition in Kidney Cancer Cells

Renal cell carcinoma (RCC) is the sixth most common cancer in the US. While RCC is highly metastatic, there are few therapeutics options available for patients with metastatic RCC, and progression-free survival of patients even with the newest targeted therapeutics is only up to two years. Thus, novel therapeutic targets for this disease are desperately needed. Based on our previous metabolomics studies showing alteration of peroxisome proliferator-activated receptor α (PPARα) related events in both RCC patient and xenograft mice materials, this pathway was further examined in the current study in the setting of RCC. PPARα is a nuclear receptor protein that functions as a transcription factor for genes including those encoding enzymes involved in energy metabolism; while PPARα has been reported to regulate tumor growth in several cancers, it has not been evaluated in RCC. A specific PPARα antagonist, GW6471, induced both apoptosis and cell cycle arrest at G0/G1 in VHL(+) and VHL(−) RCC cell lines (786-O and Caki-1) associated with attenuation of the cell cycle regulatory proteins c-Myc, Cyclin D1, and CDK4; this data was confirmed as specific to PPARα antagonism by siRNA methods. Interestingly, when glycolysis was blocked by several methods, the cytotoxicity of GW6471 was synergistically increased, suggesting a switch to fatty acid oxidation from glycolysis and providing an entirely novel therapeutic approach for RCC.

Synergistic tumor suppression by combined inhibition of telomerase and CDKN1A

Significance Over 90% of cancer cells express telomerase, which is required for their survival. However, telomerase inhibitors alone have so far failed to provide any significant clinical benefit. Therefore, identifying and targeting genes that can enhance the effects of telomerase inhibitors will greatly benefit a large population of cancer patients. We find that simultaneous inhibition of p21 and telomerase synergistically suppresses tumor growth. We also show that our approach is useful for treating p53 mutant cancers, when used with therapies that restore the function of mutant p53. We anticipate that simultaneous targeting of p21 and telomerase will overcome the current limitation of single-agent telomerase therapeutics and provide an effective method to treat cancers that rely on telomerase activity for survival. Tumor suppressor p53 plays an important role in mediating growth inhibition upon telomere dysfunction. Here, we show that loss of the p53 target gene cyclin-dependent kinase inhibitor 1A (CDKN1A, also known as p21WAF1/CIP1) increases apoptosis induction following telomerase inhibition in a variety of cancer cell lines and mouse xenografts. This effect is highly specific to p21, as loss of other checkpoint proteins and CDK inhibitors did not affect apoptosis. In telomerase, inhibited cell loss of p21 leads to E2F1- and p53-mediated transcriptional activation of p53-upregulated modulator of apoptosis, resulting in increased apoptosis. Combined genetic or pharmacological inhibition of telomerase and p21 synergistically suppresses tumor growth. Furthermore, we demonstrate that simultaneous inhibition of telomerase and p21 also suppresses growth of tumors containing mutant p53 following pharmacological restoration of p53 activity. Collectively, our results establish that inactivation of p21 leads to increased apoptosis upon telomerase inhibition and thus identify a genetic vulnerability that can be exploited to treat many human cancers containing either wild-type or mutant p53.

Metabolic reprogramming in clear cell renal cell carcinoma

Research in many cancers has uncovered changes in metabolic pathways that control tumour energetics and biosynthesis, so-called metabolic reprogramming. Studies in clear cell renal cell carcinoma (ccRCC) have been particularly revealing, leading to the concept that ccRCC is a metabolic disease. ccRCC is generally accompanied by reprogramming of glucose and fatty acid metabolism and of the tricarboxylic acid cycle. Metabolism of tryptophan, arginine and glutamine is also reprogrammed in many ccRCCs, and these changes provide opportunities for new therapeutic strategies, biomarkers and imaging modalities. In particular, metabolic reprogramming facilitates the identification of novel and repurposed drugs that could potentially be used to treat ccRCC, which when metastatic has currently limited long-term treatment options. Further research and dissemination of these concepts to nephrologists and oncologists will lead to clinical trials of therapeutics specifically targeted to tumour metabolism, rather than generally toxic to all proliferating cells. Such novel agents are highly likely to be more effective and to have far fewer adverse effects than existing drugs.

Potential biofluid markers and treatment targets for renal cell carcinoma

Renal cell carcinoma (RCC) is the 13th most common cancer in the world and one of the few cancers for which incidence is increasing. This disease is generally asymptomatic at an early stage and is highly metastatic. Frequently discovered by physicians in the process of working up other diseases such as acute kidney injury, RCC is often discovered in an advanced form and many patients have metastases at the time of diagnosis. Given that life expectancy with currently approved therapies for metastatic RCC is approximately 1–2 years, biomarkers for RCC that will enable early detection are urgently needed. Although it is unlikely that highly sensitive and specific biomarkers will be identified in the near future that are useful for screening the general population, a noninvasive marker or set of markers could soon be used in general medicine, nephrology, and urology clinics to screen patients at increased risk of RCC. In addition to the ongoing need for RCC biomarkers, the frequent resistance reported with currently available targeted therapies makes the identification of new therapeutic targets similarly important. Many promising leads for new targeted therapies have come to light; some of these therapies are in clinical trials and others are still being evaluated in the laboratory.

Small-molecule inhibitors of p21 as novel therapeutics for chemotherapy-resistant kidney cancer

“In the search for novel approaches for the treatment of resistant cancers, small molecules targeting the cyclin dependent kinase inhibitors have shown considerable promise”

A novel p21 attenuator which is structurally related to sorafenib

p21 is a member of the cyclin kinase inhibitor family of proteins and plays pivotal roles in cellular proliferation as well as in the regulation of apoptosis, and thus has diverse functions in diseases as varied as cancer and atherosclerosis. In light of its pleiotropic effects and potential clinical relevance, new methods of attenuation of p21 protein levels by selective inhibitors are therefore powerful tools to probe malignant, infectious and other diseases. Here we introduce a novel p21 attenuator, UC2288, which possesses consistent and relatively selective activity for p21. UC2288 was synthesized based on the chemical model of sorafenib, a multikinase inhibitor that also attenuates p21, but unlike sorafenib, UC2288 did not inhibit Raf kinases or alter p-ERK protein levels. UC2288 decreased p21 mRNA expression independently of p53, and attenuated p21 protein levels with minimal effect on p21 protein stability. In addition, UC2288 inhibits cell growth in the kidney cancer cell lines (GI50 = approximately 10 µM) as well as multiple other cancer cell lines. Thus, this novel p21 inhibitor will be indispensable for exploring the function of p21, and upon further study may be translatable to the clinic.