Compound C, a Broad Kinase Inhibitor Alters Metabolic Fingerprinting of Extra Cellular Matrix Detached Cancer Cells

Abstract

Most of the cancer related deaths are caused mainly by metastasis. Therefore, it is highly important to unfold the major mechanisms governing metastasis process in cancer. Throughout the metastatic cascade, cells need the ability to survive without attachment to neighboring cells and the original Extra Cellular Matrix (ECM). Recent reports showed that loss of ECM attachment shifts cancer cell metabolism towards glycolysis mostly through hypoxia. However, AMPK, a master metabolic regulator was also found to be upregulated under ECM detached conditions. Therefore, in this work we aimed to understand the consequences of targeting AMPK and other metabolic kinases by a broad kinase inhibitor namely Compound C in ECM detached cancer cells. Results showed that Compound C impacts glycolysis as evident by increased levels of pyruvate, but reduces its conversion to lactate thereby negatively regulating the Warburg effect. Simultaneously, Compound C induces block at multiple levels in TCA cycle as evident from accumulation of various TCA metabolites. Interestingly Compound C significantly reduces glutamine and reduced glutathione levels, suggesting loss of antioxidant potential of ECM detached cancer cells. Further, we found increased in metabolites associated with nucleotide synthesis, one carbon metabolism and PPP pathway during Compound C treatment of ECM detached cells. Finally, we also found induction in metabolites associated with DNA damage in ECM detached cancer cells during Compound C treatment, suggesting DNA damage regulatory role of metabolic kinases. Overall, our results showed that Compound C represses pyruvate to lactate conversion, reduces antioxidant potential and invokes DNA damage in ECM detached cancer cells. Our data provides a comprehensive metabolic map of ECM detached cancer cells that can be targeted with a broad kinase inhibitor, is Compound C. The data can be used for designing new combinational therapies to eradicate ECM detached cancer cells.

Keywords: AMP-activated protein kinase; compound C; extra cellular matrix detachment; metabolomic analysis; oxidative phosphorylation.

Figure 1

Compound C inhibition reduce cell proliferation and size reduction. (A) Cells are grown in ultra-low attachment 96-well plate and treated with different concentration of Compound C and MTT assay were performed. (B) Cells are grown in ultra-low attachment plate and treated with Compound C, images were acquired, and size were measured using image J software. (C) Cells are grown in ultra-low attachment plat and treated with Compound C; apoptosis assay were performed using Annexin V. * is p-value ≤ 0.05 ** is p-value ≤ 0.01

Figure 2

Metabolomic analysis of ECM detached cancer cells and Compound C treated in ECM detached cancer cells, (A) Metabolites were extracted and run in LTQ-XL linear ion trap LC-MS and its total ion chromatogram with significate features. (B) PCA analysis of total metabolite of HCT 116 and 22RV1 with three different condition. (C) Correlation heat map and expression heat map of differential metabolite expressed between ECM attached, ECM detached, and ECM detached cells treated with Compound C. (D) VIP score based on PCA analysis of top metabolites. (E) Pathway network analysis. (F) Top pathway enriched in metabolome analysis between ECM attached, ECM detached, and ECM detached cells treated with Compound C.

Figure 3

Compound C alter energy enrichment pathway in ECM detachment. (A) Overall energy enrichment pathway with metabolite expression index. (B) Expression of transcript (HCT116) and metabolite involved in glycolysis during ECM detachment and AMPK inhibited ECM detached cells, P-value p < 0.01. (C) LDHA assay and Western blot analysis of protein involved in AMPK activation and glycolysis(AT- ECM attached, DT- ECM detached and CC- Compound C treated).

Figure 4

Compound C modulates TCA cycle. ** is p-value ≤ 0.01.

Figure 5

Compound C alters GSH level. (A) Overall GSH enrichment pathway with metabolite expression index and GSH assay. (B) SOD assay during ECM detachment and AMPK inhibited ECM detached cells, P-value **p < 0.01.

Figure 6

Compound C alter the metabolite involved pentose phosphate pathway. (A) Metabolite involved in PPP pathway. (B) Expression of metabolite involved in PPP during ECM detachment and AMPK inhibited ECM detached cells, P-value p < 0.01. * is p-value ≤ 0.05 ** is p-value ≤ 0.01

Figure 7

Compound C the modulate metabolite expression of one carbon metabolism (A) Metabolite involved in one carbon metabolism pathway. (B) Expression of metabolite involved in One carbon metabolite during ECM detachment and AMPK inhibited ECM detached cells, P-value p < 0.01. ** is p-value ≤ 0.01.

Figure 8

Effect of Compound C on DNA lesion. ** is p-value ≤ 0.01.