Extracellular Matrix Remodeling Regulates Glucose Metabolism through TXNIP Destabilization

Abstract

The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.

Keywords: GLUT1 trafficking; TXNIP; ZFP36; cell biology; cell migration; extracellular matrix; glucose metabolism; hyaluronidase; mRNA degradation; receptor tyrosine kinase signaling.

Figure 1:. Unbiased analysis identifies ECM engagement as a regulator of glycolytic metabolism.

(A) (Adapted from Hong et al., 2016.) Genes were ranked on the vertical axis by average correlation of expression with glycolytic index in 31 breast cancer cell lines and with FDG maximum standardized uptake value (SUV_max) in 11 patient breast tumors. Tumor transcript levels are presented in order of increasing SUV_max on the horizontal axis.(B) Linear regressions correlating HMMR expression with FDG uptake in patient breast tumors (n=11) and glycolytic index in breast cancer cell lines (n=31). The HMMR probe that generated the highest average correlation between tumors and cell lines is shown.(C) HABP staining (yellow) of LiSa-2 cells after treatment with PBS, 400μg/mL HAase, or 400μg/mL heat-inactivated (HI) HAase (concentrations used throughout study unless otherwise stated). Nuclei were stained with DAPI (blue). Scale bar, 25μm.(D) Glucose uptake and lactate production rates in LiSa-2 cells treated with PBS, HAase, or HI HAase for 24h. (E) HABP staining of LiSa-2 cells treated with 4-MU DMSO 24h prior to fixation. Cells were also treated with PBS or HAase 4h prior to fixation. Scale bar, 50μm.(F) Glucose uptake rates in LiSa-2 cells measured between 24h and 48h after treatment with 4-MU DMSO. (G) Representative PET and CT images of a SCID mouse bearing A549 xenografts on each flank, at baseline and 6h after intratumoral injection of HAase or HI HAase (indicated by red hatched circle). “K” and “B” indicate kidneys and bladder, respectively. (H) Relative tumor FDG signal in each A549 xenografted mouse (n=5 mice), before and after 6h of treatment with HAase and HI HAase. The ratio of mean intensity between the tumor ultimately treated with HAase and that with HI HAase was calculated at baseline and after treatment on a per mouse basis. Ratios for each mouse are displayed. Mouse shown in (G) is highlighted in red. All experiments were biological replicates. Error bars denote SD (n=3, unless otherwise indicated). *p<0.05; **p<0.01; ***p< 0.001.

Figure 2:. Matrix digestion with HAase acutely increases glycolytic metabolism in a broad range of cultured cells.

(A) Glucose uptake and (B) lactate production rates in cultured cells treated with PBS or HAase for 24h. (C) Glucose uptake and lactate production rates in LiSa-2 and MEF cells treated with PBS (24h) or HAase (24h and 5d). (D) Color change of pH-sensitive phenol red indicative acidification of the culture media. Cells of approximately equal confluency were treated with PBS or HAase for times indicated and incubated in equal volumes of fresh media for 24h. Total cell numbers on each plate (×10⁶): 3.31 PBS, 2.92 HAase 24h, 3.25 HAase 5d, 2.94 HAase 10d. (E) Glucose uptake rates in 293T cells treated with PBS (24h) or HAase (24h, 5d). All experiments were biological replicates. Error bars denote SD (n=3). *p<0.05; **p<0.01; ***p< 0.001.

Figure 3:. HAase enriches GLUT1 at the plasma membrane.

(A) Heatmaps showing relative intracellular levels of glycolytic intermediates in LiSa-2 cells treated with PBS or HAase. (B) Immunofluorescence staining of GLUT1 (green) in LiSa-2 cells following 24h treatment with PBS or HAase. Scale bar, 50μm. (C) Immunoblots showing levels of GLUT1, GLUT3, and MCT1 in cytoplasmic (CYTO) and PM fractions of LiSa-2 cells treated with PBS or HAase. (D) ECAR of LiSa-2 cells treated with PBS for 24h or HAase for indicated times (n=12 for each condition). (E) Immunoblots showing levels of GLUT1, GLUT3, and MCT1 in CYTO and PM fractions of LiSa-2 cells treated with PBS for 24h or HAase for indicated times. (F) Transcript levels of SLC2A1-4 in LiSa-2 cells following treatment with PBS or HAase. (G) Immunoblots showing levels of GLUT1 in whole-cell lysates of LiSa-2, 293T, and MDA-686 cells treated with PBS or HAase. (H) Immunoblots showing levels of GLUT1 and GLUT3 in cytoplasmic and PM fractions of 293T and MDA-686 cells treated with PBS or HAase. All experiments were biological replicates. Error bars denote SD (n=3, unless otherwise indicated). **p<0.01; ***p< 0.001.

Figure 4:. TXNIP reduction largely mediates the acute glycolytic response to HAase.

(A) Immunoblots showing levels of TXNIP in 293T, LiSa-2, and MDA-686 cells treated with PBS (4h) or HAase (1h, 2h, 4h). (B) Baseline glucose uptake and lactate production rates in WT and Txnip KO MEFs. (C) Immunoblots showing levels of Glut1 in CYTO and PM fractions of WT and Txnip KO MEFs. (D) Immunoblots showing levels of Txnip in KO MEFs at baseline and in WT MEFs treated with PBS (6h) or HAase for the indicated times. (E) Immunoblots showing levels of Glut1 and Glut3 in CYTO and PM fractions of WT and Txnip KO MEFs treated with PBS (24h) or HAase (6h, 24h). PM Glut1 band intensities were quantified and normalized to Na,K-ATPase; heatmaps represent z-scores from the resulting ratios. (F) Glucose uptake and lactate production rates in WT and Txnip KO MEFs treated with PBS or HAase. Rates for each cell line were normalized to PBS control. All experiments were biological replicates. Error bars denote SD (n=3). *p<0.05; **p<0.01; ***p< 0.001.

Figure 5:. ZFP36 is rapidly induced by HAase and targets TXNIP transcripts for degradation.

(A) Immunoblots showing levels of TXNIP in LiSa-2 cells stably expressing a 3xFLAG-tagged version of the protein. Cells were treated with PBS or HAase. (B) TXNIP transcript levels in 293T, MDA-686, and LiSa-2 cells treated with PBS or HAase. (C) Luciferase activity in lysates of HEK293 cells transfected with a TXNIP 3’ UTR luciferase reporter and treated with HAase (n=6). (D) ZFP36 transcript levels in 293T, MDA-686, and LiSa-2 cells treated with PBS or HAase. (E) Luciferase activity in lysates of HEK293 cells co-transfected with a fixed amount of the TXNIP 3’ UTR luciferase reporter and increasing amounts of a ZFP36 expression vector (M4-ZFP36)—or the empty vector backbone as a control (n=6). (F) Immunoblots showing levels of ZFP36, MYC, and TXNIP in 293T, MDA-686, and LiSa-2 cells treated with a time course of HAase (as indicated), or with PBS (24h). (G) Immunoblots showing levels of ZFP36 and TXNIP in LiSa-2 with stable shRNA knockdown of ZFP36 or expression of a scramble control. Cells were treated with PBS or HAase. (H) Glucose uptake rates in LiSa-2 cells with stable shRNA knockdown of ZFP36 or expression of a scramble control. Cells were treated with PBS or HAase. (I) Immunoblots showing levels of EGFR (Tyr1068 and total), ERK1/2 (Thr202/Tyr204 and total), ZFP36, and TXNIP in MDA-686 cells pretreated for 1h with erlotinib or DMSO control, then with PBS or HAase. (J) Proposed model depicting how ECM remodeling promotes acute upregulation of glycolysis. All experiments were biological replicates. Error bars denote SD (n=3, unless otherwise indicated). *p<0.05; **p<0.01; ***p< 0.001; n.s. - not significant.

Figure 6:. Matrix digestion with HAase accelerates cell migration in a TXNIP- and glucose-dependent fashion.

(A) Scratch assay showing area closed by WT and Txnip KO MEFs after treatment with PBS or HAase for 12h. Representative images are shown in Figure S5D. (B) Immunoblots showing levels of TXNIP in WT and TXNIP KO MDA-MB-231 cells. Lanes were cropped from the same membrane, as indicated by dashed line. (C) Scratch assay showing area closed by WT and TXNIP KO MDA-MB-231 cells after treatment with PBS or HAase. Representative images are shown. (D) Immunoblots showing levels of TXNIP and ZFP36 in LiSa-2 cells expressing tet-TXNIP (inducible overexpression) or an empty vector control. Cells were pretreated with doxycycline (500ng/mL) or vehicle for 12h, then treated with PBS or HAase for 4h. (E) Scratch assay showing area closed by LiSa-2 cells expressing tet-TXNIP or an empty vector control after treatment with PBS or HAase. Cells were pretreated with doxycycline (500ng/mL) or vehicle for 6h. Representative images for tet-TXNIP cells are shown. (F) Scratch assay showing area closed by LiSa-2 cells cultured in 1mM or 25mM glucose after treatment with PBS or HAase. Representative images are shown. All experiments were biological replicates. Error bars denote SD (n=3). *p<0.05; **p<0.01; ***p<0.001; n.s. - not significant.

Figure 7:. ECM remodeling influences Zfp36-Txnip-Glut1 signaling in tissue.

(A) Immunofluorescence staining of hyperplastic murine epidermis 6h after intradermal injection of HAase or HI HAase. For a given marker, representative images following HAase and HI HAase treatment are from the same animal (opposite flanks; n=4 mice). Red boxes indicate informative regions that are expanded below. Scale bar, 100μm. (B) Immunohistochemical staining of an epidermal papilloma (n=5 tumors from 5 mice assessed; 1 shown here). K14 staining distinguishes the epithelial cells within the tissue and demarcates the boundaries of the dermis. Boxes identify comparable architectural reference points with informative staining patterns; they are expanded in a color-coordinated fashion in (C-E) and annotated to demarcate boundaries of tissue structures and to highlight illustrative gradients of signal. Scale bars, 200μm (K14) and 100μm (Ki-67). (C) Region highlighting strong anti-correlation between Txnip and Glut1. (D,E) Regions highlighting strong HABP anti-correlation with cytoplasmic Zfp36 and Ki-67, as well as Glut3 in (E). Concentrated depositions of HA are indicated by arrows. There are two informative gradients in (E), which are highlighted by blue and green arrows in the Ki-67 panel.