13C-pyruvate imaging reveals alterations in glycolysis that precede c-Myc-induced tumor formation and regression

Abstract

Tumor cells have an altered metabolic phenotype characterized by increased glycolysis and diminished oxidative phosphorylation. Despite the suspected importance of glycolysis in tumorigenesis, the temporal relationship between oncogene signaling, in vivo tumor formation, and glycolytic pathway activity is poorly understood. Moreover, how glycolytic pathways are altered as tumors regress remains unknown. Here, we use a switchable model of Myc-driven liver cancer, along with hyperpolarized (13)C-pyruvate magnetic resonance spectroscopic imaging (MRSI) to visualize glycolysis in de novo tumor formation and regression. LDHA abundance and activity in tumors is tightly correlated to in vivo pyruvate conversion to lactate and is rapidly inhibited as tumors begin to regress, as are numerous glycolysis pathway genes. Conversion of pyruvate to alanine predominates in precancerous tissues prior to observable morphologic or histological changes. These results demonstrate that metabolic changes precede tumor formation and regression and are directly linked to the activity of a single oncogene.

Figure 1. Switchable MYC expression in Primary Liver Tumor Formation and Early Regression

A. Gross tissues, corresponding histology and MYC staining at different stages of tumor formation and early regression. No apparent histologic differences were noted between primary tumors and early-regression sections. MYC expression (indicated with arrowheads) is heterogeneous in pre-tumor tissue, is significantly increased in tumors, and is absent following 72 hrs of doxy treatment. B. Quantitative PCR and C. western blots show negligible MYC expression in early stage pre-tumor liver compared to elevated expression in established tumors. MYC mRNA and protein expression is switched off when doxy is administered for 3 days. C. The liver tumor marker, AFP is highly expressed in tumor tissue and persists once MYC is switched off. D. Comparison of staining for the Ki67 proliferation marker and E. TUNEL assay of liver samples at different stages of tumor development and regression. F. Graph depicting percentage of Ki67 positive cells. G. Graph depicting percentage of TUNEL positive cells. Error Bars represent S.E.M. * p < 0.004, N.S. = not significant.

Figure 2. Changes in Gene Expression During MYC-Driven Tumor Development and Regression

GSEA analysis of 120 previously reported MYC-regulated genes. Shown are subsets of the 120 genes that are most abundantly enriched or down-regulated when comparing A. normal liver versus tumor tissues or B. tumor tissues versus early regression samples. See also Table S1.

Figure 3. Hyperpolarized ¹³C metabolic Imaging of Liver Tumor Formation and Regression

A. Diagram showing the conversion of ¹³C-pyruvate to lactate or alanine. B. Color overlay maps from spectroscopic grids show clear differences in metabolic profiles between liver tumors and other tissues. C. Hyperpolarized imaging reveals changes in metabolic profile during MYC-driven tumor formation. High pyruvate is detected in normal liver. Elevated alanine is associated with pre-tumor liver with modest MYC induction but no apparent phenotypic changes. A substantial increase in lactate is seen in developed tumor masses. D. A significant and reproducible pattern of high alanine in pre-tumor liver compared to controls (p = 0.003) and tumor (p = 0.0002) is observed. E. Heterogeneous elevation of alanine may correspond to areas of eventual tumor development. Color overlays generated from spectral grids are shown for a single animal over the course of disease progression after oncogene activation. The first column shows a healthy liver that has uniformly low alanine and lactate levels. The middle column shows a change in metabolic profile one week later, with substantially elevated alanine signal now observed. The alanine overlay highlights heterogeneity in the liver (bright red regions corresponding to highest signal intensity). The final column shows the liver 9 weeks after MYC activation, now consisting of a heterogeneous collection of distinct tumor nodules and regions of non-tumor. The lactate overlay shows the tumor nodules as the regions with the highest lactate signal intensity. Color intensities were normalized across the different time points according to metabolite. F. Imaging early tumor regression before and 72 hours after MYC inhibition. A dramatic decrease in lactate is detected in voxels at similar locations prior and after doxy treatment. Spectra for the indicated voxel and corresponding anatomic imaging is shown. G. Repeated studies demonstrated a significant increase in lactate during tumor formation compared to controls (p < 0.0001) and decrease upon early regression (p < 0.0002). Lactate and alanine are reported as normalized to total carbon (the sum of lactate, alanine, and pyruvate). Error Bars represent S.E.M. See also Figure S1.

Figure 4. Expression of Key Regulators of the Alanine and Glycolysis Biosynthetic Pathways are Altered at Different Stages of MYC-driven Liver Tumor Development

A. Gpt1 mRNA expression significantly increases during early stage pre-tumors and decreases in fully developed liver tumors. B. GSEA analysis of alanine-pathway genes reveals up-regulation of many genes in the pathway in pre-tumor samples compared to normal liver (p < 0.004). Enriched genes are shown. C. GSEA analysis of alanine-pathway genes comparing pre-tumor and tumor samples shows down-regulation of genes in this pathway (p < 0.005). Genes enriched in pre-tumors are shown. Alanine-pathway genes described in the text are indicated with an asterix. D. Ldha mRNA and E. protein expression show elevated Ldha expression only in primary tumors. F. GSEA analysis of glycolysis genes showing two-way comparisons of glycolytic pathway gene expression at different states of tumor formation and regression. Key glycolysis regulators, Ldha and Pkm2 are indicated with an asterix. Error Bars represent S.E.M. See also Figure S2, Table S2.

Figure 5. ALT and LDH Enzyme Activities Correlate with the Corresponding Metabolic Imaging Biomarkers

A. Graph of ALT activity versus alanine/tCar levels across various treatment conditions demonstrates a correlation between imaging and enzyme activity (p = 0.05). B. Graph of LDH activity versus lactate/tCar levels across various treatment conditions demonstrates a correlation between imaging and enzyme activity (p < 0.0005).

Figure 6. Expression of Glutamine and TCA Cycle Pathway Genes as Tumors Form and Regress

A. GSEA analyses of a subset of glutamine metabolism genes show an increase in expression as tumors develop and decrease in expression as they regress. Key regulators discussed in the text are indicated with an asterix. B. GSEA analysis of TCA cycle genes. Key regulators Pdha1 and Pdk1 are indicated with an asterix. See also Table S3.

Figure 7. Changes in Metabolism Correlate with Stages of MYC-Driven Liver Tumor Development and Regression

Schema summarizing the Lactate/tot Carbon, LDHA Vmax, Alanine/tot Carbon, ALT activity levels and metabolic gene expression changes in the different stages of MYC-induced liver tumor progression and regression.