Matthew P. Fox

Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non-small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation.

Surgical management of pulmonary carcinoid tumors: sublobar resection versus lobectomy.

BACKGROUND Surgical resection of bronchopulmonary carcinoid tumors can be curative and remains the primary treatment modality. There are limited data to delineate the optimal extent of resection for this disease. METHODS A retrospective review of the 3,270 patients diagnosed with typical and atypical carcinoid tumors between 2000 and 2007 in the Surveillance Epidemiology and End Results registry was performed. RESULTS The mean follow-up period was 46 months (range, 1-95 mo). Overall survival (OS) and disease-specific survival at 5 years was 80% and 90%, respectively. The mean OS was slightly better in the lobectomy group compared with those undergoing sublobar resection (86 vs 83 mo; P = .008). After adjusting for age, this finding was no longer present (P = .513). By using multivariate analysis, sublobar resection was noninferior to lobectomy with regard to disease-specific survival and OS (P < .05). CONCLUSIONS Compared with lobectomy, sublobar resection is associated with noninferior survival in patients with typical carcinoid of the lung.

Donors with a history of cocaine use and lung transplant outcomes

crossmatch. Although some of these may have resulted from changes in the crossmatch requirement during listing (ie, initial requirement for traditional crossmatch that was subsequently “relaxed” to a virtual crossmatch), the rationale for specifying the traditional and virtual requirements at listing (93 patients, 12.5% of patients listed with a requirement for prospective crossmatch) is unclear. Because there is no advantage to listing with both requirements simultaneously, such listings may reflect misinterpretation of the requirements by listing centers. Further education about the meaning of prospective crossmatch requirements may be warranted. In summary, we found an increase in the requirement for a prospective crossmatch from 1996 to 2009 due to an increase in use of the virtual crossmatch.

Lymph Node Ratio is a Significant Predictor of Disease Specific Mortality in Patients Undergoing Esophagectomy for Cancer

The seventh edition of the American Joint Committee on Cancer esophageal cancer staging system classifies nodal status by the number of malignant nodes (LNMs) found. This may be confounded by variations in lymphadenectomy and specimen review. The ratio of lymph nodes containing metastases to the total nodes excised (LNR) has been suggested as an alternative. We seek to validate the use of LNR for staging and determine the effect of the total lymph node yield (LNY) on its accuracy. A review of our prospective esophageal database identified 94 patients who underwent esophagectomy for cancer at out institution from 1992 until 2010. Univariate and multivariate analyses were performed. The mean age of our patients was 59.4 years. Transthoracic esophagectomy was performed in all but three instances. The majority of tumors were adenocarcinoma, 76 per cent. Overall survival at 2 and 5 years was 52 and 29 per cent, respectively. LNY correlated with LNM (r = 0.302, P = 0.001) but not LNR (r = 0.012, P = 0.912). Using Kaplan-Meier analysis, LNR had no effect on disease-specific (DS) survival (P = 0.803). However, a Cox proportional hazards regression model showed LNR to be a significant predictor of DS mortality (hazard ratio, 9.47; P = 0.049). The lack of correlation between LNR and LNY suggests that LNR may be a more robust staging method when LNY is low. Furthermore, LNR was found to be a significant predictor of DS mortality when controlling for other factors influencing survival. However, neither a staging system based on LNR nor its efficacy compared with the current system could be determined from these data.

Optimal Therapy in Locally Advanced Esophageal Cancer: a National Cancer Database Analysis

There are over 16,000 new patients diagnosed with esophageal cancer each year in the USA. With the overall 5-year survival of approximately 18%, it is one of the leading causes of cancer deaths. In patients with esophageal cancer, undergoing a complete (R0) resection is the most important predictor of overall survival. In patients fortunate enough to have their cancer diagnosed at an early stage (stages I–IIA), resection alone without neoadjuvant therapy is currently recommended and has proved to be an acceptable therapy with good long-term survival. Unfortunately, patients often present at an advanced stage due to the absence of symptoms during the early stages of the disease. There have been several trials over the past decade designed to determine the optimal treatment for locally advanced esophageal cancer. The MAGIC trial published in 2006 was a randomized control trial comparing survival in patients undergoing neoadjuvant chemotherapy and surgery to surgery alone. This trial demonstrated that patients undergoing neoadjuvant chemotherapy and surgery had improved survival compared to the surgery alone group. Published in 2008, the CALGB 9781 trial, despite closing early due to poor accrual, did demonstrate a survival advantage in patients undergoing chemoradiation followed by surgery compared to surgery alone in patients with locally advanced esophageal cancer. More recently, the CROSS Trial demonstrated a survival benefit in patients undergoing chemotherapy and radiation prior to surgery compared to surgery alone and is arguably the most notable trial to date regarding neoadjuvant chemoradiation for esophageal cancer. Today, most consider the recommended course of treatment in patients with locally advanced esophageal cancer to be neoadjuvant chemotherapy or chemoradiation followed by esophagectomy. The purpose of this study is to determine what proportion of patients with locally advanced esophageal cancer receive the optimal treatment (neoadjuvant chemotherapy ± radiation followed by an R0 resection) and what factors influence whether patients receive this treatment.

Use of drug intoxicated donors for lung transplant: Impact on survival outcomes

The number of increasing deaths due to the opioid epidemic has led to a potential greater supply of organ donors. There is hesitancy to use drug intoxicated donors, and we evaluated their impact on post‐transplant survival.

Abstract 1424: Enhanced pyruvate carboxylation is crucial to non-small cell lung cancer proliferation and anabolism

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Proliferating cancer cells require an active Krebs cycle for generating anabolic precursors, in addition to energy production. Diversion of the Krebs cycle intermediates to meet anabolic demands cannot be sustained without anaplerosis. The two major anaplerotic pathways are the deamidation of glutamine to form glutamate catalyzed by glutaminase (GLS) or glutaminolysis, and the carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase (PC). We determined the expression of PC and GLS proteins in fresh tumor tissue and paired adjacent benign tissue from 86 human NSCLC patients. PC was elevated (median 8-10 fold) in 94% of the tumor tissues, whereas GLS expression did not differ significantly between tumor and paired non tumorous lung tissue. Using 13C6 glucose as tracer and stable isotope-resolved metabolomics (SIRM), we also observed elevated PC activity in vivo in human patients with early stage NSCLC. To examine the importance of PC in the growth and survival of NSCLC, 5 NSCLC cell lines were transduced with a lentiviral vector containing an shRNA against PC. PC knockdown slowed proliferation, induced multinucleation, and reduced colony formation in soft agar. To validate attenuated PC activity and to profile the metabolic effect of PC knockdown, A549 cells were grown in 13C6 glucose or 13C5 glutamine and the incorporation of 13C into various metabolic pathways was measured by NMR and MS by SIRM. We found reduced entry of both glucose and glutamine carbon into the Krebs cycle metabolites, fatty acyl chains of lipids, and nucleotides, suggesting that both energy production and anabolic pathways were hindered and blocking the PC pathway was not compensated by GLS activity. In addition, glutathione biosynthesis and homeostasis were perturbed by PC suppression, leading to compromised anti-oxidation capacity. We further found that PC knockdown in A549 cells reduced their growth rate in mouse xenografts, and recapitulate the metabolic perturbations seen both in cell culture and in human patients. Together, these results suggest that PC is indispensible for the growth and anabolism of NSCLC. This work was funded by 5P20RR018733, 1R01CA118434-01A2, 1P01CA163223-01A1, 1R01ES022191-01, and 3R01CA118434-02S1; and the KLCRP, CTSPGP, and the KY Challenge for Excellence. Citation Format: Katherine E. Sellers, Matthew P. Fox, Michael Bousamra, Jun Yan, Mariia Yuneva, Richard M. Higashi, Andrew N. Lane, Teresa WM Fan. Enhanced pyruvate carboxylation is crucial to non-small cell lung cancer proliferation and anabolism. [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 1424. doi:10.1158/1538-7445.AM2014-1424

Abstract 1271: Human non small cell lung cancer tissue has enhanced expression of pyruvate carboxylase, but not glutaminase

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Altered cellular metabolism in tumors is well established, particularly an increased rate of aerobic glycolysis [1]. However, increased glycolysis alone is insufficient to sustain the enhanced cell growth and anabolic metabolism characteristic of cancer. Another key source of these precursors is the citric acid cycle (CAC). Removal of CAC intermediates for anabolism requires replenishment by other sources; these anaplerotic reactions are vital to cancer development. The two major pathways of anaplerosis are glutaminolyis (deamidation and transamination of glutamine to 2-oxoglutarate) [2] and carboxylation of pyruvate to oxaloacetate (OAA), catalyzed by pyruvate carboxylase (PCB) [2]. We have coupled 13C-labeled glucose tracer with stable isotope resolved metabolomics (SIRM), gene microarray, and western blotting to track the anaplerotic PCB pathway in human lung cancer patients. The resected cancer tissue showed increased flux through the PCB pathway, along with increased transcription of the PCB gene and PCB protein expression compared with the adjacent benign lung tissue [3]. We have extended these findings to 50 paired tissue samples, and have correlated them with histological and clinical data. The protein expression of PCB was on average ten-fold higher in the tumor than in the benign lung tissue (p=0.00001, paired t-test). The normalized protein expression correlated with the fraction of the tumor that was identified as cancer cells, indicating that the increased expression was from the cancer cells rather than other lung cell types. Glutaminase was active in the lung tissue, but its expression was not different from, or lower in the tumors than in the benign lung tissue. These data indicate that in NSCLC, PCB is important in anaplerosis and tumor metabolism. The balance between glycolysis and the choice of different anaplerotic pathways may depend on the tissue types. References 1. Gillies, R.J. and R.A. Gatenby, Adaptive landscapes and emergent phenotypes: why do cancers have high glycolysis? Journal of Bioenergetics and Biomembranes, 2007. 39(3): p. 251-257. 2. Portais, J.C., P. Voisin, M. Merle, and P. Canioni, Glucose and glutamine metabolism in C6 glioma cells studied by carbon 13 NMR. Biochimie, 1996. 78(3): p. 155-164. 3. Fan, T.W., A.N. Lane, R.M. Higashi, M.A. Farag, H. Gao, M. Bousamra, and D.M. Miller, Altered Regulation of Metabolic Pathways in Human Lung Cancer Discerned by 13C Stable Isotope-Resolved Metabolomics (SIRM)) Molecular Cancer 2009. 8 p. 41. 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 1271. doi:10.1158/1538-7445.AM2011-1271