Metabolism In Colon Cancer Cells Research Articles

P53 Orchestrates Cancer Metabolism: Unveiling Strategies to Reverse the Warburg Effect

Cancer cells exhibit significant alterations in their metabolism, characterised by a reduction in oxidative phosphorylation (OXPHOS) and an increased reliance on glycolysis, even in the presence of oxygen. This metabolic shift, known as the Warburg effect, is pivotal in fuelling cancer’s uncontrolled growth, invasion, and therapeutic resistance. While dysregulation of many genes contributes to this metabolic shift, the tumour suppressor gene p53 emerges as a master player. Yet, the molecular mechanisms remain elusive. This study introduces a comprehensive mathematical model, integrating essential p53 targets, offering insights into how p53 orchestrates its targets to redirect cancer metabolism towards an OXPHOS-dominant state. Simulation outcomes align closely with experimental data comparing glucose metabolism in colon cancer cells with wild-type and mutated p53. Additionally, our findings reveal the dynamic capability of elevated p53 activation to fully reverse the Warburg effect, highlighting the significance of its activity levels not just in triggering apoptosis (programmed cell death) post-chemotherapy but also in modifying the metabolic pathways implicated in treatment resistance. In scenarios of p53 mutations, our analysis suggests targeting glycolysis-instigating signalling pathways as an alternative strategy, whereas targeting solely synthesis of cytochrome c oxidase 2 (SCO2) does support mitochondrial respiration but may not effectively suppress the glycolysis pathway, potentially boosting the energy production and cancer cell viability.

Multi-omics Analysis of the Role of PHGDH in Colon Cancer.

Objectives: Serine metabolism is essential for tumor cells. Endogenous serine arises from de novo synthesis pathways. As the rate-limiting enzyme of this pathway, PHGDH is highly expressed in a variety of tumors including colon cancer. Therefore, targeted inhibition of PHGDH is an important strategy for anti-tumor therapy research. However, the specific gene expression and metabolic pathways regulated by PHGDH in colon cancer are still unclear. Our study was aimed to clarified the role of PHGDH in serine metabolism in colon cancer to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer. Methods: In this study, we analyzed the gene expression and metabolic remodeling process of colon cancer cells (SW620) after targeted inhibition of PHGDH by gene transcriptomics and metabolomics. LC-MS analysis was performed in 293T cells to PHGDH gene transcription and protein post-translational modification under depriving exogenous serine. Results: We found that amino acid transporters, amino acid metabolism, lipid synthesis related pathways compensation and other processes are involved in the response process after PHGDH inhibition. And ATF4 mediated the transcriptional expression of PHGDH under exogenous serine deficiency conditions. While LC-MS analysis of post-translational modification revealed that PHGDH produced changes in acetylation sites after serine deprivation that the K289 site was lost, and a new acetylation site K21was produced. Conclusion: Our study performed transcriptomic and metabolomic analysis by inhibiting PHGDH, thus clarifying the role of PHGDH in gene transcription and metabolism in colon cancer cells. The mechanism of high PHGDH expression in colon cancer cells and the acetylation modification that occurs in PHGDH protein were also clarified by serine deprivation. In our study, the role of PHGDH in serine metabolism in colon cancer was clarified by multi-omics analysis to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer.

Metabolic Effects of Cedar Tar on Colon Cancer Cell Line (HCT-116): A Follow-up Study

Objective: Colon cancer (CC) is considered to be the most common cancers seen worldwide. Surgery and chemotherapy are the leading methods used in treatment. In recent years, the therapeutic properties of natural products have been investigated and used in drug development. Cedrus libani plant that normally grows in Syria and Turkey, some studies have shown that the plant’s extract (cedar tar) has anti-microbial and anti-cancer properties on colon cancer. Recently, metabolic changes have been identified as the hallmark of cancer, and various metabolic drugs are being investigated in clinical trials. This study was carried out to investigate the effects of cedar tar on the amino acid metabolism of colon cancer cells. Materials and Methods: Colon cancer cells (HCT-116) were homogenized in cold PBS medium after incubation with 30 µg/mL dose of cedar tar for 24 hours in appropriate medium. The intracellular free amino acid profile in the samples was analyzed by LC-MS/MS method. Statistical analysis of the obtained data was done with SPSS and MetaboAnalyst 5.0 program. Results: According to the statistical analysis results, significant positive correlations were found between β-alanine, anserine amino acids and apoptotic effects of cedar tar between the untreated control and cedar tar-treated groups, while a negative correlation was found with O-Phosphorylethanolamine amino acid. Conclusion: By increasing the levels of β-alanine and anserine amino acids in colon cancer cells, cedar tar reduces both the metastatic character of the cells and inhibits tumor formation; It caused cell proliferation and energy deprivation by reducing the level of O-Phosphorylethanolamine amino acid, which is defined as a pro-tumorigenic metabolite. Cedar tar both triggered apoptosis and changed amino acid metabolism in colon cancer cells. It has been determined that cedar tar has the potential to be used as a natural product in the treatment of colon cancer.

Curcumin Synergizes with Cisplatin to Inhibit Colon Cancer through Targeting the MicroRNA-137-Glutaminase Axis.

Colorectal cancer (CRC) is one of the most lethal and prevalent malignancies world-wide. Currently, surgery, radiotherapy and chemotherapy are clinically applied as common approaches for CRC patients. Cisplatin is one of the most frequently used chemotherapy drugs for diverse cancers. Although chemotherapeutic strategies have improved the prognosis and survival of cancer patients, development of cisplatin resistance has led to cancer recurrence. Curcumin, isolated from turmeric, has been used as an effective anti-cancer agent. However, the molecular mechanisms for curcumin-mediated cisplatin sensitivity of CRC have not been elucidated. This study aimed to investigate the effects of curcumin treatment on cisplatin-resistant CRC cells. Expression levels of miRNAs and mRNAs were determined by qRT-PCR. Protein expression levels were detected by Western blotting. Cell responses to curcumin treatments were evaluated by MTT assay, Clonogenic assay and Annexin V apoptosis assay. The glutamine metabolism of colon cancer cells was assessed by glutamine uptake and glutaminase (GLS) activity. The binding of miR-137 on 3' UTR of GLS was validated by Western blotting and luciferase assay. Results demonstrated that curcumin significantly synergized with cisplatin (combination index <1) to suppress proliferation of colon cancer cells compared with curcumin or cisplatin alone. Moreover, from the established cisplatin-resistant cell line (HT-29), glutamine metabolism was remarkedly elevated in cisplatin-resistant CRC cells that displayed a glutamine addictive phenotype. Furthermore, curcumin treatments attenuated glutamine metabolism in colon cancer cells. Under low glutamine supply, colon cancer cells showed less sensitivity to curcumin. Using a microRNA (miRNA) microArray assay, miR-137, a tumor suppressor in colon cancer, was significantly induced by curcumin treatments in CRC cells. Bioinformatics analysis and a luciferase assay illustrated miR-137 directly targeted the 3' UTR of GLS mRNA. Rescue experiments demonstrated that miR-137-induced cisplatin sensitization was through targeting of GLS. Finally, curcumin treatment overcame cisplatin resistance through miR-137-mediated glutamine inhibition. Collectively, these results indicate that curcumin could be clinically applied as an anti-chemoresistance approach against CRC by modulating miR-137-inhibited glutamine metabolism.

Abstract 2400: PINK1 is a novel regulator of mitochondria and iron metabolism in colon cancer

Abstract Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover and cellular health. In particular, PINK and Parkin mediate one of the canonical mitophagy pathways. The mechanisms behind PINK1-Parkin mediated mitophagy is well established, yet its role in cancer remains equivocal. Based on differential gene expression analyses on colon cancer patients, higher PINK1 expressions are correlated with worsened survival. To confirm increased mitophagy in colon cancer cells, we assessed lysosomal proteome under nutrient stress and observed an enrichment of mitochondrial peptides. Then, we generated inducible shRNA knockdown (KD) models for PINK1 in a panel of colon cancer cell lines. Preliminary data suggest loss of PINK1 hinders colon cancer cell growth and induces cell death. We showed that PINK1 KD hinders mitophagy assessed by decreased phospho-ubiquitin, a marker of PINK1 activity. We observed high levels of basal mitophagy in colon cancer cells, and PINK1 KD can efficiently inhibit mitophagy using a fluorescent mitophagy reporter. Interestingly, we demonstrated PINK KD disrupts mitochondria dynamics by decreasing MFN2 protein level, suggesting mitochondrial fusion could be inhibited. In summary, we demonstrated colon cancer cells have high levels of mitophagy and PINK1 KD decreases mitophagy and disrupts mitochondria network. Mitochondria are important organelles that integrates cellular metabolic pathways. With PINK1's role as an overall mitochondrial health sensor, we hypothesized disrupting PINK1 could affect mitochondria metabolism in colon cancer cells. We demonstrated alterations in mitochondrial metabolites as assessed by metabolomics, NAD/NADH imbalance, membrane hyperpolarization, and decreased oxygen consumption rate. It is known that TCA cycle and electron transport chain require iron sulfur clusters (ISC) to maintain proper function. Although the exact quantification of mitochondrial iron fraction is elusive, some studies suggested that mitochondria could harbor up to 20% of cellular iron. These evidences suggest mitochondria play pivotal roles in cellular iron homeostasis. Upon PINK1 KD, we saw an accumulation of cytosolic and mitochondrial iron storage protein, ferritin. Interestingly, we also observed a decrease in ISC-containing ETC proteins upon PINK1 KD. We believe PINK1 could have a non-canonical function in regulating mitochondria iron and play a profound role in regulating cellular iron balance. Overall, these results indicate that PINK1 is essential for maintaining mitochondria metabolism and survival in colon cancers. Here, we showcased that PINK has a unique role in regulating mitochondrial metabolism. In conclusion, the growth and mitochondrial defects we observed from PINK1 KD cells suggest a potential oncogenic role of PINK1 in colorectal cancer that can be targeted for future therapeutic purposes. Citation Format: Brandon Chen, Samantha Devenport, Costas Lyssiotis, Yatrik Shah. PINK1 is a novel regulator of mitochondria and iron metabolism in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2400.

Inhibition of APE1-endonuclease activity affects cell metabolism in colon cancer cells via a p53-dependent pathway

The pathogenesis of colorectal cancer (CRC) involves different mechanisms, such as genomic and microsatellite instabilities. Recently, a contribution of the base excision repair (BER) pathway in CRC pathology has been emerged. In this context, the involvement of APE1 in the BER pathway and in the transcriptional regulation of genes implicated in tumor progression strongly correlates with chemoresistance in CRC and in more aggressive cancers. In addition, the APE1 interactome is emerging as an important player in tumor progression, as demonstrated by its interaction with Nucleophosmin (NPM1). For these reasons, APE1 is becoming a promising target in cancer therapy and a powerful prognostic and predictive factor in several cancer types. Thus, specific APE1 inhibitors have been developed targeting: i) the endonuclease activity; ii) the redox function and iii) the APE1-NPM1 interaction. Furthermore, mutated p53 is a common feature of advanced CRC. The relationship between APE1 inhibition and p53 is still completely unknown. Here, we demonstrated that the inhibition of the endonuclease activity of APE1 triggers p53-mediated effects on cell metabolism in HCT-116 colon cancer cell line. In particular, the inhibition of the endonuclease activity, but not of the redox function or of the interaction with NPM1, promotes p53 activation in parallel to sensitization of p53-expressing HCT-116 cell line to genotoxic treatment. Moreover, the endonuclease inhibitor affects mitochondrial activity in a p53-dependent manner. Finally, we demonstrated that 3D organoids derived from CRC patients are susceptible to APE1-endonuclease inhibition in a p53-status correlated manner, recapitulating data obtained with HCT-116 isogenic cell lines. These findings suggest the importance of further studies aimed at testing the possibility to target the endonuclease activity of APE1 in CRC.

N-Acetylcysteine Serves as Substrate of 3-Mercaptopyruvate Sulfurtransferase and Stimulates Sulfide Metabolism in Colon Cancer Cells.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST.

A noncoding RNA LINC00504 interacts with c-Myc to regulate tumor metabolism in colon cancer.

Accumulating evidence has shown a critical role of long-non-coding RNAs (lncRNAs) during multiple tumor progression. However, the potential functions of LINC00504 in colon cancer as well as its mechanisms remain obscure. By lncRNA profiling, we identified LINC00504 as a novel oncogenic lncRNA in colon cancer. The lncRNA LINC00504 was markedly upregulated in colon cancer cell lines and specimens. LINC00504 increases viability and migration of colon cells in vitro. Furthermore, LINC00504 also enhances colon cancer xenograft tumors in vivo. We noted that LINC00504 regulates metabolism at a transcriptional level which influences multiple metabolic pathways, such as glucose metabolism, pentose phosphate pathway, and tricarboxylic acid cycle. Mechanistic study showed that LINC00504 could interact with c-Myc to promote chromatin recruitment of c-Myc and enhance its transactivation activity. Collectively, our results showed that LINC00504 serves as an important transcriptional regulator for c-Myc in colon cancer cells. LINC00504 can reprogram central metabolism in colon cancer cells implying that LINC00504 may serve as a potential target for therapeutic intervention.

SRSF3, a Splicer of the PKM Gene, Regulates Cell Growth and Maintenance of Cancer-Specific Energy Metabolism in Colon Cancer Cells.

Serine and arginine rich splicing factor 3 (SRSF3), an SR-rich family protein, has an oncogenic function in various kinds of cancer. However, the detailed mechanism of the function had not been previously clarified. Here, we showed that the SRSF3 splicer regulated the expression profile of the pyruvate kinase, which is one of the rate-limiting enzymes in glycolysis. Most cancer cells express pyruvate kinase muscle 2 (PKM2) dominantly to maintain a glycolysis-dominant energy metabolism. Overexpression of SRSF3, as well as that of another splicer, polypyrimidine tract binding protein 1 (PTBP1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), in clinical cancer samples supported the notion that these proteins decreased the Pyruvate kinase muscle 1 (PKM1)/PKM2 ratio, which positively contributed to a glycolysis-dominant metabolism. The silencing of SRSF3 in human colon cancer cells induced a marked growth inhibition in both in vitro and in vivo experiments and caused an increase in the PKM1/PKM2 ratio, thus resulting in a metabolic shift from glycolysis to oxidative phosphorylation. At the same time, the silenced cells were induced to undergo autophagy. SRSF3 contributed to PKM mRNA splicing by co-operating with PTBP1 and hnRNPA1, which was validated by the results of RNP immunoprecipitation (RIP) and immunoprecipitation (IP) experiments. These findings altogether indicated that SRSF3 as a PKM splicer played a positive role in cancer-specific energy metabolism.

Novel Polyphenols That Inhibit Colon Cancer Cell Growth Affecting Cancer Cell Metabolism

New series of polyphenols with a hydrophilic galloyl-based head and a hydrophobic N-acyl tail, linked through a serinol moiety, have been synthesized and tested against colon cancer cell growth. Our structure activity relationship studies revealed that galloyl moieties are essential for growth inhibition. Moreover, the length of the N-acyl chain is crucial for the activity. Introduction of a (Z) double bond in the acyl chain increased the anticancer properties. Our findings demonstrate that 16, the most potent compound within this series, has inhibitory effects on colon cancer cell growth and metabolism (glycolysis and mitochondrial respiration) at the same time that it activates 5'AMP-activated kinase (AMPK) and induces apoptotic cell death. Based on these results, we propose that 16 might reprogram colon cancer cell metabolism through AMPK activation. This might lead to alterations on cancer cell bioenergy compromising cancer cell viability. Importantly, these antiproliferative and proapoptotic effects are selective for cancer cells. Accordingly, these results indicate that 16, with an unsaturated C18 chain, might be a useful prototype for the development of novel colon cancer cell growth inhibitors affecting cell metabolism.

P-381 - H2S metabolism in colon cancer cells: Effect of hypoxia

Hydrogen sulfide (H2S) plays key roles in human (patho)physiology. Synthesized endogenously by known enzymatic systems, H2S is mainly metabolized through a mitochondrial sulfide-oxidizing pathway that comprises sulfide:quinone oxidoreductase (SQR) and a few other enzymes. H2S degradation through this pathway is coupled to electron injection into the respiratory chain and, thus, to stimulation of ATP synthesis. In cancer cells, H2S was reported to be synthesized at high levels and to stimulate energy metabolism and cell proliferation. Under hypoxic conditions, commonly found in the microenvironment of solid tumours, H2S is known to be more stable and to be overproduced with pro-survival effects. Here, H2S catabolism was investigated in colon cancer model cells grown under either normoxic (20% O2) or hypoxic (1% O2) conditions, comparing their maximal ability to dispose H2S at the level of the mitochondrion by high resolution respirometry. Intriguingly, cell exposure to hypoxic conditions was found to have effects on H2S metabolism, both in functional terms and at the level of protein expression. The potential implications of these findings will be discussed.

MicroRNA-661 modulates redox and metabolic homeostasis in colon cancer.

Cancer cell survival and metastasis are dependent on metabolic reprogramming that is capable of increasing resistance to oxidative and energetic stress. Targeting these two processes can be crucial for cancer progression. Herein, we describe the role of microRNA‐661 (miR661) as epigenetic regulator of colon cancer (CC) cell metabolism. MicroR661 induces a global increase in reactive oxygen species, specifically in mitochondrial superoxide anions, which appears to be mediated by decreased carbohydrate metabolism and pentose phosphate pathway, and by a higher dependency on mitochondrial respiration. MicroR661 overexpression in non‐metastatic human CC cells induces an epithelial‐to‐mesenchymal transition phenotype, and a reduced tolerance to metabolic stress. This seems to be a general effect of miR661 in CC, since metastatic CC cell metabolism is also compromised upon miR661 overexpression. We propose hexose‐6‐phosphate dehydrogenase and pyruvate kinase M2 as two key players related to the observed metabolic reprogramming. Finally, the clinical relevance of miR661 expression levels in stage‐II and III CC patients is discussed. In conclusion, we propose miR661 as a potential modulator of redox and metabolic homeostasis in CC.

Oroxylin A suppresses the development and growth of colorectal cancer through reprogram of HIF1\u03b1-modulated fatty acid metabolism

The occurrence and progress of colon cancer are closely associated with obesity. Therefore, the lipid metabolism, especially fatty acid metabolism, is a significant section of energy homeostasis in colon cancer cells, and it affects many important cellular processes. Oroxylin A is one of the main bioactive flavonoids of Scutellariae radix, which has a strong anticancer effect but low toxicity to normal tissue. In previous studies, we have proved that oroxylin A reprogrammes metabolism of cancer cells by inhibiting glycolysis. Here, we further investigated the metabolism-modulating effects of oroxylin A on the fatty acid metabolism in colon cancer cells under hypoxia. We found that HIF1α upregulated adipophilin, fatty acid synthase and sterol regulatory element-binding protein 1, and downregulated carnitine palmitoyltransferase 1 (CPT1), resulting in the promoted lipid uptake and transport, increased de novo fatty acid synthesis and suppressed fatty acid oxidation. Oroxylin A inactivated HIF1α and reprogrammed fatty acid metabolism of HCT116 cells, decreasing intracellular fatty acid level and enhancing fatty acid oxidation. Furthermore, the rapid decrease of fatty acid level caused by oroxylin A inhibited the nuclear translocation of β-cantenin and inactivated the Wnt pathway, arousing cell cycle arrest in G2/M phase. In vivo studies demonstrated that high-fat diet increased the incidence of colon cancer and accelerated tumor development. Importantly, besides the growth inhibitory effects on colon cancer xenograft, oroxylin A prevented carcinogenesis and delayed progress of primary colon cancer as well. Our studies enriched the metabolic regulatory mechanism of oroxylin A, and suggested that oroxylin A was a potent candidate for the treatment and prevention of colorectal cancer.

PHLPP regulates hexokinase 2-dependent glucose metabolism in colon cancer cells

Increased glucose metabolism is considered as one of the most important metabolic alterations adapted by cancer cells in order to generate energy as well as high levels of glycolytic intermediates to support rapid proliferation. PH domain leucine-rich repeat protein phosphatase (PHLPP) belongs to a novel family of Ser/Thr protein phosphatases that function as tumor suppressors in various types of human cancer. Here we determined the role of PHLPP in regulating glucose metabolism in colon cancer cells. Knockdown of PHLPP increased the rate of glucose consumption and lactate production, whereas overexpression of PHLPP had the opposite effect. Bioenergetic analysis using Seahorse Extracelluar Flux Analyzer revealed that silencing PHLPP expression induced a glycolytic shift in colon cancer cells. Mechanistically, we found that PHLPP formed a complex with Akt and hexokinase 2 (HK2) in the mitochondrial fraction of colon cancer cells and knockdown of PHLPP enhanced Akt-mediated phosphorylation and mitochondrial localization of HK2. Depletion of HK2 expression or treating cells with Akt and HK2 inhibitors reversed PHLPP loss-induced increase in glycolysis. Furthermore, PHLPP knockdown cells became addicted to glucose as a major energy source in that glucose starvation significantly decreased cancer cell survival. As HK2 is the key enzyme that determines the direction and magnitude of glucose flux, our study identified PHLPP as a novel regulator of glucose metabolism by controlling HK2 activity in colon cancer cells.

Cross-Talk between Alternatively Spliced UGT1A Isoforms and Colon Cancer Cell Metabolism.

Alternative splicing at the human glucuronosyltransferase 1 gene locus (UGT1) produces alternate isoforms UGT1A_i2s that control glucuronidation activity through protein-protein interactions. Here, we hypothesized that UGT1A_i2s function as a complex protein network connecting other metabolic pathways with an influence on cancer cell metabolism. This is based on a pathway enrichment analysis of proteomic data that identified several high-confidence candidate interaction proteins of UGT1A_i2 proteins in human tissues-namely, the rate-limiting enzyme of glycolysis pyruvate kinase (PKM), which plays a critical role in cancer cell metabolism and tumor growth. The partnership of UGT1A_i2 and PKM2 was confirmed by coimmunoprecipitation in the HT115 colon cancer cells and was supported by a partial colocalization of these two proteins. In support of a functional role for this partnership, depletion of UGT1A_i2 proteins in HT115 cells enforced the Warburg effect, with a higher glycolytic rate at the expense of mitochondrial respiration, and led to lactate accumulation. Untargeted metabolomics further revealed a significantly altered cellular content of 58 metabolites, including many intermediates derived from the glycolysis and tricarboxylic acid cycle pathways. These metabolic changes were associated with a greater migration potential. The potential relevance of our observations is supported by the down-regulation of UGT1A_i2 mRNA in colon tumors compared with normal tissues. Alternate UGT1A variants may thus be part of the expanding compendium of metabolic pathways involved in cancer biology directly contributing to the oncogenic phenotype of colon cancer cells. Findings uncover new aspects of UGT functions diverging from their transferase activity.

Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells.

AKT is a central protein in many cellular pathways such as cell survival, proliferation, glucose uptake, metabolism, angiogenesis, as well as radiation and drug response. The three isoforms of AKT (AKT1, AKT2 and AKT3) are proposed to have different physiological functions, properties and expression patterns in a cell type-dependent manner. As of yet, not much is known about the influence of the different AKT isoforms in the genome and their effects in the metabolism of colorectal cancer cells. In the present study, DLD-1 isogenic AKT1, AKT2 and AKT1/2 knockout colon cancer cell lines were used as a model system in conjunction with the parental cell line in order to further elucidate the differences between the AKT isoforms and how they are involved in various cellular pathways. This was done using genome wide expression analyses, metabolic profiling and cell migration assays. In conclusion, downregulation of genes in the cell adhesion, extracellular matrix and Notch-pathways and upregulation of apoptosis and metastasis inhibitory genes in the p53-pathway, confirm that the knockout of both AKT1 and AKT2 will attenuate metastasis and tumor cell growth. This was verified with a reduction in migration rate in the AKT1 KO and AKT2 KO and most explicitly in the AKT1/2 KO. Furthermore, the knockout of AKT1, AKT2 or both, resulted in a reduction in lactate and alanine, suggesting that the metabolism of carbohydrates and glutathione was impaired. This was further verified in gene expression analyses, showing downregulation of genes involved in glucose metabolism. Additionally, both AKT1 KO and AKT2 KO demonstrated an impaired fatty acid metabolism. However, genes were upregulated in the Wnt and cell proliferation pathways, which could oppose this effect. AKT inhibition should therefore be combined with other effectors to attain the best effect.

Abstract 1012: Systems analysis of colon cancer cell metabolism rewired by p53 and KRAS mutations

Abstract Introduction. Somatic mutations in proto-oncogenes and tumour-suppressor genes contribute to rewire the already deregulated metabolic network in cancer cells, resulting in uncontrolled proliferation and oncogenesis. In this study, we set out to establish a dynamic mathematical model of bioenergetics and to exploit it to explore the multifaceted cross-talk between bioenergetics, somatic gene mutations in KRAS and p53, and cell proliferation and survival. Model Development. We have developed an ordinary differential equations-based model of central carbon metabolism in cancer cells which includes glycolysis, pentose phosphate pathway, citric acid cycle and respiratory chain, based on our previous work and published models. The model describes how nutrients (glucose, glutamine, lactate, pyruvate, serine and glycine) from the extracellular micro-environment affect bioenergetics parameters. The resulting model predictions are linked to cell proliferation via a heuristic function. Enzymatic activities regulated by p53 and KRAS mutations were obtained by mining publically available datasets and their regulation by the mutational status was modelled by adapting the corresponding kinetic parameters. To estimate the kinetic parameters, model simulation outputs were fitted to a portfolio of experimental data both generated de novo in house and gathered from the literature in HCT-116 colon cancer cells. Experiments were performed on parental HCT-116 (p53 competent; harbouring a KRAS mutation on exon 2 of codon G13) and three derived mutant cell lines covering all four combinations of p53 and KRAS mutational status to isolate their relative and joint effect on bioenergetics signatures. HCT-116-derived cell lines included: p53 proficient cells with the KRAS allelic mutation silenced by homologous recombination in the presence or absence of p53 knockout by lentiviral shRNA. Results. The model was calibrated against ATP concentrations measured via single-cell microscopy (ATeam probe and TMRM dye) following pharmacological inhibition of respiratory chain complexes (rotenone, sodium azide and oligomycin) as a function of nutrients availability (glucose, lactate, pyruvate). Modelling results revealed that p53 and KRAS mutations drive a shift in metabolic signatures and L-lactate emerged as a pivotal metabolite to stratify the different phenotypes. Systems analysis revealed that in KRAS mutated cells p53 deficiency leads to an increase in glucose uptake and flux through the pentose phosphate pathway and a decrease in lactate production. Indeed, p53 deficient HCT-116 cells showed a decrease in extracellular lactate with respect to p53 proficient cells in validation experiments. Conclusions. The computational model developed can be used to benchmark mechanistic hypotheses by which tumour suppressors and/or oncogenic mutations rewire metabolism and to identify putative targets for therapeutic intervention. Citation Format: Manuela Salvucci, Robert O’Byrne, Natalia Niewidok, Séan Kilbride, Caoimhín G. Concannon, Heiko Düssmann, Heinrich H. Huber, Jochen HM Prehn. Systems analysis of colon cancer cell metabolism rewired by p53 and KRAS mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1012.

Rosemary polyphenols induce unfolded protein response and changes in cholesterol metabolism in colon cancer cells

Several studies have demonstrated that rosemary polyphenols exert changes in the lipid metabolism in adipose and hepatic cells. In this work, the effects of a polyphenol-enriched supercritical rosemary extract (SC-RE) and carnosic acid (CA) on the transcriptome and cholesterol metabolism in HT-29 colon cancer cells were examined using a Foodomics approach. Targeted metabolomics analysis indicated that the SC-RE treatment induced cholesterol accumulation after 24 h. Transcriptomic analysis suggested that most of the changes induced by the SC-RE and CA were orchestrated by unfolded protein response (UPR) and triggered by endoplasmic reticulum stress. Results suggested up-regulation of VLDLR gene as the principal contributor to the observed cholesterol accumulation in SC-RE-treated cells. In addition, the SC-RE attenuated the activity of E2F transcription factor, down-regulating several genes involved in G1–S transition of the cell cycle.

Abstract 2431: Downregulation of PHLPP promotes glycolysis in colon cancer cells

Abstract PHLPP belongs to a novel family of Ser/Thr protein phosphatases. Our previous studies have shown that PHLPP can antagonize multiple signaling pathways by negatively regulating signaling molecules such as Akt, S6K, and PKC. Functionally, PHLPP serves as a tumor suppressor to inhibit cell proliferation, promote apoptosis and increase therapeutic sensitivity in colon cancer cells. Here, we further examined the role of PHLPP in regulating metabolic pathways in colon cancer. Control and PHLPP knockdown cells colon cancer cells, as well as wild-type and Phlpp knockout MEF cells, were subjected to bioenergetic phenotype analysis using Seahorse XF96 analyzer. The mitochondrial respiration measurements revealed that decreased PHLPP expression resulted in an increase in oxygen consumption rate (OCR) suggesting enhanced cellular respiration. In addition, PHLPP depletion significantly stimulated glycolysis as indicated by increased extracellular acidification rate (ECAR). Consistently, the rates of glucose consumption and lactate production were significantly increased in PHLPP knockdown cells. Furthermore, the numbers of apoptotic cells were increased in PHLPP knockdown cells compared to control cells when cultured in glucose-free medium, suggesting that loss of PHLPP expression render cells more sensitive to glucose deprivation as they became relied on glycolysis. To begin determining the underlying molecular mechanism, we found that the activity of both Akt and S6K was largely induced in PHLPP-knockdown cancer cells and PHLPP-knockout MEF cells. Since both kinases are known to play important roles in re-programming metabolic networks to favor tumor growth, it is likely that PHLPP inhibits glycolysis by negatively regulating Akt- and S6K-mediated metabolic effects. Taken together, our studies identified PHLPP as a novel regulator of glucose metabolism in colon cancer cells. Downregulation of PHLPP may promote tumorigenesis by providing metabolic advantages in colon cancer Citation Format: Yang-an Wen, Xiaopeng Xiong, Tianyan Gao. Downregulation of PHLPP promotes glycolysis in colon cancer cells. [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 2431. doi:10.1158/1538-7445.AM2014-2431

PKM2 depletion induces the compensation of glutaminolysis through β-catenin/c-Myc pathway in tumor cells

The metabolic activity in cancer cells primarily rely on aerobic glycolysis. Besides glycolysis, some tumor cells also exhibit excessive addition to glutamine, which constitutes an advantage for tumor growth. M2-type pyruvate kinase (PKM2) plays a pivotal role in sustaining aerobic glycolysis, pentose phosphate pathway and serine synthesis pathway. However, the participation of PKM2 in glutaminolysis is little to be known. Here we demonstrated that PKM2 depletion could provoke glutamine metabolism by enhancing the β-catenin signaling pathway and consequently promoting its downstream c-Myc-mediated glutamine metabolism in colon cancer cells. Treatment with 2-deoxy-d-glucose (2-DG), a glycolytic inhibitor, got consistent results with the above. In addition, the dimeric form of PKM2, which lacks the pyruvate kinase activities, plays a critical role in regulating β-catenin. Moreover, we found that overexpression of PKM2 negatively regulated β-catenin through miR-200a. These insights supply evidence that glutaminolysis plays a compensatory role for cell survival upon glucose metabolism impaired.