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

Abstract

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 13 C 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. Inflammatory breast cancer (IBC)3 is a highly aggressive cancer that affords the lowest 5-year survival rate of any breast cancer diagnosis, with a large proportion of patients exhibiting nodal metastases at diagnosis (1). IBC disproportionally affects both younger women and African American women and is typically characterized by rapid and specific changes in skin appearance, such as peau d'orange, erythema, skin thickening, and rapid enlargement of the affected breast (2). Instead of forming solid masses, IBC cells tend to form abundant tumor emboli that have the capacity to rapidly invade the lymphatic system and spread to distant sites. Although the molecular attributes of IBC are well described and are suggestive of constitutive adaptation to hypoxia (3), little is known about the underlying metabolic features of the disease.Metabolic reprogramming is increasingly recognized as a fundamental hallmark of cancer, and efforts to identify clinically viable drugs that target cancer metabolism are underway (4). It is well established that many tumor cells consume glucose and produce lactate at significantly higher rates than the surrounding tissue, even when sufficient oxygen is present, via a phenomenon known as aerobic glycolysis or the Warburg effect (5). It was originally hypothesized that cancer cells utilize aerobic glycolysis because of mitochondrial respiratory dysfunction. However, even though a number of mitochondrial defects in tumor cells have been reported, evidence suggests that most tumor cells have functional tricarboxylic acid (TCA) cycles and electron transport chains (6, 7).In addition to increased glucose uptake, many cancer cells also consume glutamine at higher rates than normal cells, both in culture and in vivo (8, 9). The Rho GTPase family of proteins has recently been linked to the regulation of glutaminase activity (10). The oncogene RhoC, a GTPase involved in cytoskeletal rearrangement and cell motility, is a driver of the metastatic phenotype exhibited by IBC (11,12). RhoC expression increases with tumor stage, is a robust predictor of breast cancer aggressiveness and surv...

Integration of high-content fluorescence imaging into the metabolic flux assay reveals insights into mitochondrial properties and functions

“…We then examined how the Rho-like family of Rho-GTPases impact mitochondrial fragmentation, based on their known role in regulating cytoskeletal dynamics. And, more specifically, our previous work found that inhibition of RhoC impairs the metabolic properties of inflammatory breast cancer cells 33 . Application of the MitoTracker strategy downstream of the metabolic flux assay revealed that RhoC deletion significantly increases the number of fragmented mitochondria in inflammatory breast cancer cells, which we hypothesize contributes to the depleted OCR levels ( Fig.…”

“…The Rho-like family of Rho-GTPases have been well characterized for their participation in the progression of breast cancer 32,34–36 and the alteration of metabolic phenotype 33 . Therefore, we next applied our imaging and analysis strategy to explore the potential for fragmented mitochondria in breast cancer cells that lack either the Rho-like family of Rho-GTPases RhoA or RhoC, via CRISPR/Cas9 knockout (WT, RhoA KO, RhoC KO) ( Supplementary Fig.…”

“…Multiple normalization methodologies have been used with varying degrees of acceptance by the research community. Examples include normalization to post-assay protein harvest or post-assay cell counting, normalization to pre-assay cell counting 18 , or normalization via one of a variety of chemical colorimetric or fluorometric readouts (e.g. MTT, ATPGlo, WST-1).…”

The importance of accurately correcting for the natural abundance of stable isotopes

“…The workflow of the experimental approach is outlined in Figure 7 and the metabolic network is illustrated in Figure 6. The network includes reactions involved in glycolysis and the tricarboxylic acid (TCA) cycle in breast cancer cell lines (Wynn et al, 2016). The input fluxes are glucose and glutamine, and the output fluxes are alanine, alpha-ketoglutarate, aspartate, glutamine, lactate, and oxaloacetate.…”

Effects of RhoA and RhoC upon the sensitivity of prostate cancer cells to glutamine deprivation

“…RhoA and RhoC are highly homologous GTPases (approximately 92% amino acid identity) that have each been implicated in glutamine metabolism [5][6][7] . RhoA has been better characterized than RhoC, however several studies have demonstrated that they present distinct functions in cancer.…”