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COX-2 Alters the Metabolic Secretome in Triple Negative Human Breast Cancer Xenografts
Santosh Kumar Bharti1, Paul T Winnard Jr.1, Yelena Mironchik1, Louis Dore-Savard2, Balaji Kirshnamachary1, and Zaver M Bhujwalla1,3

1Division of Cancer Imaging Research, Department of Radiology, Johns Hopkins University, School of Medicine, Baltimore, MD, United States, 2McGill University Health Centre and RI-MUHC, Montreal, QC, Canada, 3Department of Oncology, Johns Hopkins University, School of Medicine, Baltimore, MD, United States

Synopsis

Tumor interstitial fluid (TIF) contains the tumor secretome and forms a critical component of the tumor microenvironment. Cyclooxygenase-2 (COX2) mediates the inflammatory response of cells and is upregulated in cancers. In cancers, COX-2 expression has been related to increased invasion and metastasis. Here, for the first time, using 1H MR spectroscopy we characterized changes in the metabolic patterns of TIF in tumors derived from triple negative SUM-149 human breast cancer cells with COX-2 overexpressed. COX-2 overexpression significantly altered several fundamental metabolic pathways. These data provide new insights into the role of COX-2 in tumor aggressiveness, and identify new metabolic targets.

Introduction

Cyclooxygenase-2 (COX-2) is an active mediator of the inflammatory response of cells and plays an important role in the development, progression, invasion, and metastasis of several cancers including breast cancer [1-2]. Our ongoing studies of the tumor microenvironment (TME) of have focused on understanding and targeting COX-2 in triple negative breast cancer [3-4], the most lethal form of breast cancer. Tumor interstitial fluid (TIF), the milieu that contains the tumor secretome, is one of the least examined aspects of the TME because of the difficulty in sampling this fluid from tumors. Recently we adapted a previous published method [5] to sample TIF in human tumor xenografts in SCID mice [5]. Here, for the first time, we have sampled TIF from COX-2 overexpressing triple negative SUM-149 human breast cancer xenografts and empty vector SUM-149 xenografts. The metabolic profile of TIF from both tumor types was characterized using 1H magnetic resonance spectroscopy (1H MRS). These data represent the first ever characterization of the COX-2 metabolic secretome and reveal new insights into the role of COX-2 in altering the TME.

Methods

The cloning, construction of a lentivirus vector expressing COX-2 gene, and the establishment of SUM-149 cells stably overexpressing COX-2 (SUM-COX-2) were reported by us previously [4]. Two million SUM-COX-2 or empty vector control cells (SUM-EV) were injected subcutaneously in female SCID mice to obtain tumor pieces for TIF chamber transplantation. A home-built TIF collection chamber [6] was inserted subcutaneously in female SCID mice with 4-6 1-2 mm tumor pieces packed around the chamber; the incision was closed with suture clips. Within 4-6 weeks the growing tumor encapsulated the chamber allowing the collection of TIF. Once tumors were ~ 400 mm3, TIF was carefully removed from the chamber in terminally anesthetized mice. TIF samples that were not clear fluid were discarded, resulting in 4 samples from SUM-COX-2 tumors and 4 samples from SUM-EV tumors. Each chamber yielded ~50mL of TIF that was diluted to a total volume of 500μL with D2O for high-resolution 1H MRS performed on an Avance III 750 MHz Bruker Spectrometer equipped with a 5 mm probe. 1H MR spectra with water suppression were acquired using a one-dimensional Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence with the following parameters: spectral width of 15495.8 Hz, time domain data points of 64K, effective 90° flip angle, relaxation delay 10s, acquisition time of 2.1s, 64 scans with 8 dummy scans, a receiver gain of 1030, and echo time 25ms. All spectra were processed using line broadening for exponential window function of 0.3 Hz prior to Fourier transformation, manually phased, and automatically baseline corrected using TOPSPIN 2.1. All surgical procedures and animal handling were performed in accordance with protocols approved by the Johns Hopkins University Institutional Animal Care and Use Committee, and conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH. Immunoblot analysis of COX-2 expression was performed as previously described by us [4].

Results and Discussion

SUM-COX-2 tumors showed consistently higher COX-2 expression compared to SUM-EV tumors (Figure 1). Representative spectra from SUM-COX-2 and SUM-EV tumors in Figure 2 demonstrate the profound differences in metabolites with COX-2 overexpression. These data are summarized for significant metabolic differences in the heat map in Figure 3. COX-2 overexpression resulted in a significant increase of lactate, glutamate, acetate, and succinate, and a significant decrease of glucose, glutamine, citrate, formate, and lipids; pyruvate tended to decrease. These data are displayed as quantitative bar graphs in Figure 4. The changes in lactate and lipids are consistent with our earlier observations where COX-2 downregulation in triple negative MDMB-231 human breast cancer cells resulted in a significant decrease of lactate and an increase of lipids and lipid droplets in intact perfused cells [7]. Here COX-2 overexpression increased glycolysis. Depletion of pyruvate observed here in COX-2 overexpressing cells would limit production of acetyl-CoA and consequently citrate to also diminish fatty acid synthesis/lipids. Increased succinate parallels the increase in glutamate/a-ketoglutarate, the upstream intermediate to succinate in the tricarboxylic acid cycle (TCA), and indicates an increased utilization/depletion of glutamine to supplement the TCA. Importantly, accumulation of succinate inhibits HIF-1a prolyl hydroxylase that stabilizes HIF-1a driven cancer promoting metabolic pathways such as enhanced glycolysis and increased ROS [8]. Moreover, increased succinate also, through the succinate/succinate dehydrogenase reaction, provides necessary electrons to the electron transport chain upstream of the COX-2 reaction [9]. The depletion of formate in COX-2 overexpressing cells indicates a disruption of several vital cellular metabolic pathways [9]. These data provide new insights into the role of COX-2 in the metabolic secretome and tumor metabolism, and identify metabolic pathways as potential targets for reducing the effects of COX-2 expression in cancer.

Acknowledgements

This work was supported by NIH R35CA209960, R01CA82337 and NIH P30CA06973.

References

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Figures

Figure 1: Immunoblot of COX-2 expression in tumors derived from SUM-COX-2 (n=6) and SUM-EV (n=6) cells. GAPDH was used as a loading control. Higher COX-2 expression is consistently observed in the SUM-COX-2 tumors.

Figure 2: Representative 1H MR spectra of tumor interstitial fluid obtained from SUM-COX-2 (red) and SUM-EV (green) tumors.

Figure 3: Heat map representing significant differences in metabolites between TIF obtained from SUM-EV (n=4) and SUM-COX-2 (n=4) tumors. (* represents P<0.05, two tailed t-test; pyruvate P<0.05 with one-tailed t-test)

Figure 4: Quantitative estimation of metabolites in TIF obtained from SUM-EV (n=4) and SUM- COX-2 (n=4) tumors. Values represent mean±SE. Statistical significance was determined using an unpaired two-tailed student-t test; * represents P<0.05: pyruvate P<0.05 with one-tailed t-test.

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)
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