Lauren D. Van Wassenhove

Signaling properties of a covalent modification cycle are altered by a downstream target

We used a model system of purified components to explore the effects of a downstream target on the signaling properties of a covalent modification cycle, an example of retroactivity. In the experimental system used, a bifunctional enzyme catalyzed the modification and demodification of its substrate protein, with both activities regulated by a small molecule stimulus. Here we examined how a downstream target for one or both forms of the substrate of the covalent modification cycle affected the steady-state output of the system, the sensitivity of the response to the stimulus, and the concentration of the stimulus required to provide the half-maximal response (S50). When both the modified and unmodified forms of the substrate protein were sequestered by the downstream target, the sensitivity of the response was dramatically decreased, but the S50 was only modestly affected. Conversely, when the downstream target only sequestered the unmodified form of the substrate protein, significant effects were observed on both system sensitivity and S50. Behaviors of the experimental systems were well approximated both by simple models allowing analytical solutions and by a detailed model based on the known interactions and enzymatic activities. Modeling and experimentation indicated that retroactivity may result in subsensitive responses, even if the covalent modification cycle displays significant ultrasensitivity in the absence of retroactivity. Thus, we provide examples of how a downstream target can alter the signaling properties of an upstream signal transduction covalent modification cycle.

RhoC GTPase Is a Potent Regulator of Glutamine Metabolism and N-Acetylaspartate Production in Inflammatory Breast Cancer Cells

Inflammatory breast cancer (IBC) is an extremely lethal cancer that rapidly metastasizes. Although the molecular attributes of IBC have been described, little is known about the underlying metabolic features of the disease. Using a variety of metabolic assays, including 13C tracer experiments, we found that SUM149 cells, the primary in vitro model of IBC, exhibit metabolic abnormalities that distinguish them from other breast cancer cells, including elevated levels of N-acetylaspartate, a metabolite primarily associated with neuronal disorders and gliomas. Here we provide the first evidence of N-acetylaspartate in breast cancer. We also report that the oncogene RhoC, a driver of metastatic potential, modulates glutamine and N-acetylaspartate metabolism in IBC cells in vitro, revealing a novel role for RhoC as a regulator of tumor cell metabolism that extends beyond its well known role in cytoskeletal rearrangement.

Aldehyde dehydrogenase 2 in aplastic anemia, Fanconi anemia and hematopoietic stem cells

Maintenance of the hematopoietic stem cell (HSC) compartment depends on the ability to metabolize exogenously and endogenously generated toxins, and to repair cellular damage caused by such toxins. Reactive aldehydes have been demonstrated to cause specific genotoxic injury, namely DNA interstrand cross-links. Aldehyde dehydrogenase 2 (ALDH2) is a member of a 19 isoenzyme ALDH family with different substrate specificities, subcellular localization, and patterns of expression. ALDH2 is localized in mitochondria and is essential for the metabolism of acetaldehyde, thereby placing it directly downstream of ethanol metabolism. Deficiency in ALDH2 expression and function are caused by a single nucleotide substitution and resulting amino acid change, called ALDH2*2. This genetic polymorphism affects 35-45% of East Asians (about ~560 million people), and causes the well-known Asian flushing syndrome, which results in disulfiram-like reactions after ethanol consumption. Recently, the ALDH2*2 genotype has been found to be associated with marrow failure, with both an increased risk of sporadic aplastic anemia and more rapid progression of Fanconi anemia. This review discusses the unexpected interrelationship between aldehydes, ALDH2 and hematopoietic stem cell biology, and in particular its relationship to Fanconi anemia.

PKC and Breast Cancer

PKC expression is intimately associated with breast cancer initiation, progression, and therapy responsiveness, and these effects are highly isozyme-specific. PKC isozymes play key roles in proliferation and apoptosis of breast cancer cells and exert important modulatory roles in cell cycle progression. A close relationship exists between specific PKC isozymes and estrogen signaling.

Aldehyde dehydrogenase 3A1 activation prevents radiation-induced xerostomia by protecting salivary stem cells from toxic aldehydes

Significance Radiation therapy for head and neck cancer often leads to dry mouth, a debilitating condition that affects speaking, swallowing, and other functions related to quality of life. Since salivary functional recovery after radiation is largely dependent on the number of surviving salivary stem/progenitor cells (SSPCs), we reasoned that protection of SSPCs from injury is critical for mitigating dry mouth. Following radiation, SSPCs accumulate toxic aldehydes that damage DNA, proteins, and lipids, leading to cell death. Here, we identified d-limonene as an activator of aldehyde dehydrogenase 3A1 (ALDH3A1) with a favorable safety profile for clinical use. ALDH3A1 activation decreases aldehyde accumulation in SSPCs, increases sphere-forming ability, reduces apoptosis, and preserves salivary gland structure and function following radiation without reducing the anticancer effects. Xerostomia (dry mouth) is the most common side effect of radiation therapy in patients with head and neck cancer and causes difficulty speaking and swallowing. Since aldehyde dehydrogenase 3A1 (ALDH3A1) is highly expressed in mouse salivary stem/progenitor cells (SSPCs), we sought to determine the role of ALDH3A1 in SSPCs using genetic loss-of-function and pharmacologic gain-of-function studies. Using DarkZone dye to measure intracellular aldehydes, we observed higher aldehyde accumulation in irradiated Aldh3a1−/− adult murine salisphere cells and in situ in whole murine embryonic salivary glands enriched in SSPCs compared with wild-type glands. To identify a safe ALDH3A1 activator for potential clinical testing, we screened a traditional Chinese medicine library and isolated d-limonene, commonly used as a food-flavoring agent, as a single constituent activator. ALDH3A1 activation by d-limonene significantly reduced aldehyde accumulation in SSPCs and whole embryonic glands, increased sphere-forming ability, decreased apoptosis, and improved submandibular gland structure and function in vivo after radiation. A phase 0 study in patients with salivary gland tumors showed effective delivery of d-limonene into human salivary glands following daily oral dosing. Given its safety and bioavailability, d-limonene may be a good clinical candidate for mitigating xerostomia in patients with head and neck cancer receiving radiation therapy.

Abstract 5239: Unraveling the complex regulatory relationship between PI3K signaling and metabolic transformation in breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cancer cells exhibit a metabolic phenotype characterized by high rates of glucose uptake and lactate production, known as the Warburg effect. While the Warburg effect and normal proliferative metabolism appear similar, important molecular differences exist. We hypothesize that molecular and metabolic drivers of the Warburg effect can be modulated to impede cancer proliferation without substantial effects on normal tissue growth. Intracellular networks exhibit a variety of emergent non-linear behaviors and, as a result, the use of experimental intuition alone will not be enough to identify these drivers. Using a combination of experimental and theoretical methods, we developed a model of breast cancer progression that includes metabolism and the phosphatidylinositol-3 kinase (PI3K) signaling pathway, an important regulator of carbon metabolism. A key component of our model is a detailed logic network of molecular interactions associated with PI3K signaling as well as regulatory connections to central carbon metabolism, including the ATP/AMP ratio, GLUT receptor activation, hexokinase activation, and changes in the catalytic activity of pyruvate kinase. To validate our model, a series of phospho Western blot analyses were performed using a normal-like breast cell line and a diverse set of breast cancer cell lines exposed to PI3K pathway inhibitors. From these data, a series of predictive network models were constructed representing distinct stages of breast cancer progression. We also generated detailed metabolic flux maps for each cell line using metabolic flux analysis (MFA), a method that relies on carbon-13 tracers, mass-spectrometry, and measurements of extracellular flux to infer intracellular flux. In agreement with recent studies, we found an increase in the reductive carboxylation of glutamine derived alpha-ketoglutarate in cells constitutively adapted to hypoxia. We also identified a potentially important metabolic vulnerability in aggressive breast cancers. Moreover, we found important PI3K network differences at the RNA and protein levels, some of which were isoform specific. Together our data indicate that very different system-level properties are associated with distinct stages of breast cancer progression and metabolic transformation. Our model is suitable for performing in silico molecular perturbations to predict a normal as well as tumor level response to a targeted therapy or combination of therapies. Our approach also serves as a prototype for the use of systems biology methods in personalized medicine where molecular and metabolic data collected from a patients biopsied tumor is input into a predictive model designed to develop a strategic treatment plan for the patient. The use of predictive models to integrate data from an individual patient will have a profound impact on cancer care decisions and patient outcomes in the future. Citation Format: Michelle L. Wynn, Megan Egbert, Lauren D. Van Wassenhove, Zhi Fen Wu, Firas Midani, Charles Evans, Charles F. Burant, Santiago Schnell, Sofia D. Merajver. Unraveling the complex regulatory relationship between PI3K signaling and metabolic transformation in breast 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 5239. doi:10.1158/1538-7445.AM2013-5239

Abstract 1695: Quantification of mitochondria in MCF-10A, MDA-MB-231, and SUM149 cells to understand potential defects in oxidative phosphorylation in cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Cancer cells are known to exhibit altered metabolism. The Warburg effect, whereby cancer cells preferentially use glycolysis instead of oxidative phosphorylation to produce ATP, plays an important role in the tumorigenic capacity of breast cancer. Previously, we found that SUM149 (inflammatory breast cancer) and MDA-MB-231 (invasive breast cancer) cells have decreased oxidative phosphorylation. Since mitochondria are the powerhouses of oxidative phosphorylation, examining their concentrations, phenotypes, and the distribution of their key enzymes can provide crucial information. These data can be used to further clarify whether the observed metabolic discrepancies stem from abundance differences or from other sources, such as defective enzymes and/or intermediate participation in alternate metabolic pathways. The purpose of our research was to elucidate if differential mitochondria concentrations accounted for the observed marked decrease in oxidative phosphorylation in cancer cells. Specifically, we focused on quantifying the number of mitochondria in two aggressive cancer cell lines, MDA-MB-231 and SUM149, and a normal-like breast cell line, MCF-10A. In order to measure the number of mitochondria in the cancerous and noncancerous cells, a commercially available antibody conjugated to green fluorescent protein (GFP) and directed against the E1-alpha subunit of pyruvate dehydrogenase was used in each cell line. The resulting cells were imaged under high-resolution confocal microscopy and their relative fluorescence assessed. The average intensity of GFP in each cell was calculated and used as an indicator for the amount of mitochondria in each cell line. Additionally, these results were compared to transmission electron microscopy (TEM) images of each of these cell lines to validate the immunofluorescent quantifications. Our results showed that there is no significant difference among the number of mitochondria in MDA-MB-231, SUM149, and MCF-10A cells. Additionally, the distribution of mitochondria among all three cell lines was relatively uniform, with a slightly greater abundance of mitochondria around the nuclei. These findings are surprising given that the published research on the Warburg effect, as well as our own prior observations on these three specific cell lines, suggested that cancer cells may have different numbers of mitochondria, although the previous work did not offer definitive proof of this interpretation. Therefore, these results contradict that hypothesis, and direct further studies to identify qualitative functional defects within mitochondria such as mutated or silenced mitochondrial genes, and to explore alternate pathways that breast cancer cells use to derive energy. Our investigation highlights the importance of taking a multi-facetted approach to studying cancer cell metabolism. Citation Format: Sepideh Ashrafzadeh, Lauren D. Van Wassenhove, Sofia D. Merajver. Quantification of mitochondria in MCF-10A, MDA-MB-231, and SUM149 cells to understand potential defects in oxidative phosphorylation in 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 1695. doi:10.1158/1538-7445.AM2013-1695

Abstract 5164: Using metabolomic flux to uncover new targets for the modulation of breast cancer metastasis

Inflammatory breast cancer (IBC) is the most deadly breast cancer because of its ability to rapidly metastasize, often before the primary lesion is detected. To better understand what drives this highly aggressive form of cancer, we studied central carbon metabolism in IBC. The Warburg effect, which is characterized by high rates of glucose uptake and glycolysis even under aerobic conditions, occurs in most cancer cells and plays an important role in the tumorigenic capacity of breast cancer. Specifically, we examined metabolic changes that occur when breast cancer cells switch from a proliferating phenotype to a highly motile phenotype. In primary tumor formation rapid proliferation is crucial, while in metastasis motility to new sites is critical. We hypothesize that alterations in the regulation of metabolic pathways leads to the direction of energy from proliferation to motility, and that this change is important in the progression of breast cancer towards metastases. Understanding if a coincident metabolic shift occurs when malignant cells switch from a proliferative to a more aggressive motile form may help identify new therapeutics for highly aggressive breast cancers. For our studies, we are using “normal-like” MCF-10A breast cells, which proliferate quickly and move slower than cancer cells, and two highly metastatic breast cancer cell lines (MDA-MB-231 and the IBC-derived line SUM 149), which have the ability to move quickly and proliferate relatively slowly. Using these cell lines as a model, we have examined metabolic changes as cancer progresses. We are conducting targeted metabolomic studies to determine relative concentrations of metabolites in the glycolysis, tricarboxylic acid (TCA) cycle, and mevalonate pathway. In addition, we are using 13 C-labeled pyruvate and glucose to measure the transient and steady state flux through these metabolic pathways. Our preliminary results indicate interesting and unexpected changes in both the transient and steady state metabolic fluxes across the different cell lines. In addition, the metabolic flux through the TCA cycle is very active in the metastatic cancer cells despite an increase in glycolysis. In agreement with flux studies of other cancer cell lines, our results suggest that the cancer cells are likely using intermediates from the TCA cycle to generate nucleotides and fatty acids needed for replication. The results from this work, once validated, can be used to identify new therapeutic metabolic targets to modulate breast cancer metastasis. 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 5164. doi:1538-7445.AM2012-5164

Abstract 1397: The effects of mevalonate pathway inhibitors on the motility and invasion of aggressive breast cancers

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The mevalonate pathway is a metabolic pathway responsible for synthesizing prenyl (both farnesyl and geranylgeranyl) groups and activating small GTPase proteins through transfer of these groups by covalent modification. These GTPase proteins include RhoA, RhoC, Rac and Cdc42, which regulate the actin cytoskeleton during cellular migration and are over-expressed in many cancers. This project explores the effects of three mevalonate pathway inhibitors–zoledronic acid, atorvastatin and geranylgeranyl transferase inhibitor (GGTI)–on the single-cell motility, collective cell migration and invasion of an aggressive breast cancer line (MDA-MB-231) and an inflammatory breast cancer derived (IBC) line (SUM149). We are using MCF10A immortalized breast cells as our “normal-like” control. We hypothesize that these cancers rely on this pathway for cellular motility, invasion and the structure of the actin cytoskeleton more than normal epithelial cells. We observed significant decreases in total quantities of Rac, RhoC and Cdc42 and a striking increase in RhoA upon treatment with each separate inhibitor, observed by western blot. The effect is much more potent with atorvastatin and GGTI, resulting in an almost complete loss of lamellipodia and filopodia. We assayed for invasion through 3D culture transwell chambers, individual cell motility using a bead motility assay and live-cell imaging, and collective cell migration using a wound healing assay. In general, all three drugs were highly effective at inhibiting both types of cell migration and invasion in MDA-MB-231 cells. While all drugs inhibited the collective cell migration of SUM149 cells, only GGTI additionally inhibited invasion and single cell migration. This result suggests that an approach targeting geranylgeranylation may be more effective for inhibition of IBC cells. Typically IBC tumors use collective cell migration mechanisms to metastasize, whereas MDA-MB-231 tumors metastasize through single-cell migration. Our results show that aggressive breast cancers heavily rely on the mevalonate pathway for cellular motility in comparison to normal epithelial cells, and that inhibitors of this pathway have significant potential to inhibit the metastatic properties of these two tumor types, given their mechanism of tissue invasion. We are continuing to study collective cell movement by assaying for cell-cell adhesion molecules upon treatment with our three inhibitors. In addition, we are studying the effects of these inhibitors on metastasis in vivo using xenograft mouse models and Her2Neu transgenic mouse models. 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 1397. doi:10.1158/1538-7445.AM2011-1397

Abstract 37: Alterations in the metabolism of highly motile cells: Implications for cancer metastasis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC It has been known for some time that cancer cells have aberrant metabolism. Our lab observed that highly proliferative cells are not very motile, and likewise, highly motile cells do not proliferate rapidly. Thus, we hypothesized that cells direct their metabolism toward rapidly dividing or toward moving, but not both processes. This may be important in the progression of cancer from primary tumor to end-stage metastasis. By understanding how the metabolism and signaling pathways change in these very different situations, it may be possible to discover new drug targets to prevent cancers from progressing. Traditionally, studies of cellular metabolism and proliferation have been measured in fixed cells. In order to truly understand a living system, quantitative measurements in live cells are necessary. To this end, we utilized the Fluorescent Ubiquitination-based Cell Cycle Indicator or FUCCI, developed by Sakaue-Sawano et al. This system involves two vectors, one of which encodes Geminin fused to a green fluorescent probe, and the other which encodes Cdt1 fused to a red fluorescent probe. The levels of these proteins are regulated inversely during the cell cycle, with Geminin at its highest level during S, G2, and M phase, while Cdt1 is highest during G1. Therefore, when our stable transfectants are dividing (in S, G2, or M phases), their nuclei fluoresce green, while in the G1 phase of the cell cycle, their nuclei fluoresce red. Utilizing this system in our aggressive breast cancer cell lines enabled us to measure proliferation and metabolism in live cells. This also allowed us to directly test our hypothesis that actively motile cells are not proliferating (red), and proliferating cells (green) are not actively motile. By treating with different anti-proliferative and metabolic agents, we measured what percentage of cells cease dividing, and what percentage continued to grow. We used flow cytometry to separate these different populations and to determine genetic differences that may make them more or less resistant to these drugs. We also examined known genes involved in both metabolism and motility. To do this, we measured protein and mRNA expression of several proteins in the glycolysis, lipid metabolism, and cholesterol synthesis pathways, as well as the small motility-related GTPases. From our findings, a preliminary model of the interaction between metabolism and metastasis is emerging. The proteins likely involved in metastasis are small GTPases. These proteins are modified by prenylation, allowing them to localize to the cell membrane and direct motility. Our results suggest that MDA-MB-231 cells may have a slower rate of glycolysis, but display increased cholesterol synthesis to prenylate proteins, while SUM 149 cells have an increased rate of glycolysis. This may help explain the differences in the phenotypes of these cancers, and may allow for the prediction of future drug targets to prevent cancer metastasis. 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 37.