ASO-based PKM Splice-switching Therapy Inhibits Hepatocellular Carcinoma Cell Growth

AbstractThe M2 pyruvate kinase (PKM2) isoform is upregulated in most cancers and plays a crucial role in the Warburg effect, which is characterized by the preference for aerobic glycolysis for energy metabolism. PKM2 is an alternative-splice isoform of the PKM gene, and is a potential therapeutic target. Previously, we developed antisense oligonucleotides (ASOs) that switch PKM splicing from the cancer-associated PKM2 to the PKM1 isoform and induce apoptosis in cultured glioblastoma cells. Here, we explore the potential of ASO-based PKM splice-switching as a targeted therapy for liver cancer. We utilize a lead cEt/DNA ASO, which has a higher potency than MOE modification, to demonstrate that it induces PKM splice-switching and inhibits the growth of cultured hepatocellular-carcinoma (HCC) cells. This PKM isoform switch increases pyruvate-kinase activity and alters glucose metabolism. The lead ASO inhibits tumorigenesis in an orthotopic-xenograft HCC mouse model. Finally, a surrogate mouse-specific ASO induces Pkm splice-switching and inhibits HCC growth, without observable toxicity, in a genetic HCC mouse model.Statement of significanceAntisense oligonucleotides are used to force a change in PKM isoform usage in HCC, reversing the Warburg effect and inhibiting tumorigenesis.

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TSP50 promotes the Warburg effect and hepatocyte proliferation via regulating PKM2 acetylation

Cell Death and Disease ◽

10.1038/s41419-021-03782-w ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Feng Gao ◽

Xiaojun Zhang ◽

Shuyue Wang ◽

Lihua Zheng ◽

Ying Sun ◽

...

Keyword(s):

Pyruvate Kinase ◽

Warburg Effect ◽

Kinase Activity ◽

Aerobic Glycolysis ◽

Tumor Formation ◽

Metabolic Reprogramming ◽

Hepatocyte Proliferation ◽

Pyruvate Kinase Activity ◽

Hcc Cells ◽

The Warburg Effect

AbstractMetabolic reprogramming is a hallmark of malignancy. Testes-specific protease 50 (TSP50), a newly identified oncogene, has been shown to play an important role in tumorigenesis. However, its role in tumor cell metabolism remains unclear. To investigate this issue, LC–MS/MS was employed to identify TSP50-binding proteins and pyruvate kinase M2 isoform (PKM2), a known key enzyme of aerobic glycolysis, was identified as a novel binding partner of TSP50. Further studies suggested that TSP50 promoted aerobic glycolysis in HCC cells by maintaining low pyruvate kinase activity of the PKM2. Mechanistically, TSP50 promoted the Warburg effect by increasing PKM2 K433 acetylation level and PKM2 acetylation site (K433R) mutation remarkably abrogated the TSP50-induced aerobic glycolysis, cell proliferation in vitro and tumor formation in vivo. Our findings indicate that TSP50-mediated low PKM2 pyruvate kinase activity is an important determinant for Warburg effect in HCC cells and provide a mechanistic link between TSP50 and tumor metabolism.

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Differential localization patterns of pyruvate kinase isoforms in murine naïve, formative and primed pluripotent states

10.1101/2020.04.12.036251 ◽

2020 ◽

Author(s):

Joshua G. Dierolf ◽

Andrew J. Watson ◽

Dean H. Betts

Keyword(s):

Stem Cells ◽

Confocal Microscopy ◽

Cell Fate ◽

Pyruvate Kinase ◽

Warburg Effect ◽

Aerobic Glycolysis ◽

Embryonic Stem ◽

Orthogonal Projections ◽

Cellular Expression ◽

The Warburg Effect

AbstractMouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of a pluripotency continuum, referred to as naïve and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naïve cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg effect. Until recently, metabolism has been regarded as a by-product of cell fate; however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg effect. Both isoforms catalyze the last step of glycolysis generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naïve and primed pluripotency is not well understood. The primary objective was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting, flow cytometry and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm but also accumulate in differential subnuclear regions of mESC, mEpiLCs and mEpiSCs as determined by a quantitative, confocal microscopy colocalization employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 shifts during the transition from mESCs, mEpiLCs and mEpiSCs. We have also authenticated a new method of selecting formative pluripotency cells from naïve and primed populations using the cell surface markers SSEA1 and CD24. Finally, we have comprehensively validated the appropriateness and power of the Pearson’s correlation coefficient and Manders’ overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 assists with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naïve, formative and primed pluripotency.

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TRIM35 Interacts with pyruvate kinase isoform M2 to suppress the Warburg effect and tumorigenicity in hepatocellular carcinoma

Oncogene ◽

10.1038/onc.2014.325 ◽

2014 ◽

Vol 34 (30) ◽

pp. 3946-3956 ◽

Cited By ~ 33

Author(s):

Z Chen ◽

Z Wang ◽

W Guo ◽

Z Zhang ◽

F Zhao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Pyruvate Kinase ◽

Warburg Effect ◽

The Warburg Effect

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Valproic acid Suppresses Breast Cancer Cell Growth Through Triggering Pyruvate Kinase M2 Isoform Mediated Warburg Effect

Cell Transplantation ◽

10.1177/09636897211027524 ◽

2021 ◽

Vol 30 ◽

pp. 096368972110275

Author(s):

Zhen Li ◽

Lina Yang ◽

Shuai Zhang ◽

Jiaqi Song ◽

Huanran Sun ◽

...

Keyword(s):

Breast Cancer ◽

Valproic Acid ◽

Cancer Therapy ◽

Pyruvate Kinase ◽

Histone Deacetylases ◽

Warburg Effect ◽

Broad Class ◽

Pyruvate Kinase M2 ◽

Breast Cancer Cell Growth ◽

The Warburg Effect

Energy metabolism programming is a hallmark of cancer, and serves as a potent target of cancer therapy. Valproic acid (VPA), a broad Class I histone deacetylases (HDACs) inhibitor, has been used as a therapeutic agent for cancer. However, the detail mechanism about the potential role of VPA on the Warburg effect in breast cancer remains unclear. In this study, we highlight that VPA significantly attenuates the Warburg effect by decreasing the expression of pyruvate kinase M2 isoform (PKM2), leading to inhibited cell proliferation and reduced colony formation in breast cancer MCF-7 and MDA-MB-231 cells. Mechanistically, Warburg effect suppression triggered by VPA was mediated by inactivation of ERK1/2 phosphorylation through reduced HDAC1 expression, resulting in suppressing breast cancer growth. In summary, we uncover a novel mechanism of VPA in regulating the Warburg effect which is essential for developing the effective approach in breast cancer therapy.

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The Key Role of the WNT/β-Catenin Pathway in Metabolic Reprogramming in Cancers under Normoxic Conditions

Cancers ◽

10.3390/cancers13215557 ◽

2021 ◽

Vol 13 (21) ◽

pp. 5557

Author(s):

Alexandre Vallée ◽

Yves Lecarpentier ◽

Jean-Noël Vallée

Keyword(s):

Tumor Microenvironment ◽

Tumor Growth ◽

Warburg Effect ◽

Target Genes ◽

Glucose Transporter ◽

Aerobic Glycolysis ◽

Lactate Production ◽

Metabolic Reprogramming ◽

Pyruvate Dehydrogenase Kinase ◽

The Warburg Effect

The canonical WNT/β-catenin pathway is upregulated in cancers and plays a major role in proliferation, invasion, apoptosis and angiogenesis. Nuclear β-catenin accumulation is associated with cancer. Hypoxic mechanisms lead to the activation of the hypoxia-inducible factor (HIF)-1α, promoting glycolytic and energetic metabolism and angiogenesis. However, HIF-1α is degraded by the HIF prolyl hydroxylase under normoxia, conditions under which the WNT/β-catenin pathway can activate HIF-1α. This review is therefore focused on the interaction between the upregulated WNT/β-catenin pathway and the metabolic processes underlying cancer mechanisms under normoxic conditions. The WNT pathway stimulates the PI3K/Akt pathway, the STAT3 pathway and the transduction of WNT/β-catenin target genes (such as c-Myc) to activate HIF-1α activity in a hypoxia-independent manner. In cancers, stimulation of the WNT/β-catenin pathway induces many glycolytic enzymes, which in turn induce metabolic reprogramming, known as the Warburg effect or aerobic glycolysis, leading to lactate overproduction. The activation of the Wnt/β-catenin pathway induces gene transactivation via WNT target genes, c-Myc and cyclin D1, or via HIF-1α. This in turn encodes aerobic glycolysis enzymes, including glucose transporter, hexokinase 2, pyruvate kinase M2, pyruvate dehydrogenase kinase 1 and lactate dehydrogenase-A, leading to lactate production. The increase in lactate production is associated with modifications to the tumor microenvironment and tumor growth under normoxic conditions. Moreover, increased lactate production is associated with overexpression of VEGF, a key inducer of angiogenesis. Thus, under normoxic conditions, overstimulation of the WNT/β-catenin pathway leads to modifications of the tumor microenvironment and activation of the Warburg effect, autophagy and glutaminolysis, which in turn participate in tumor growth.

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Proton export drives the Warburg Effect

10.1101/2021.09.20.461019 ◽

2021 ◽

Author(s):

Shonagh Russell ◽

Liping Xu ◽

Yoonseok Kam ◽

Dominique Abrahams ◽

Bryce Ordway ◽

...

Keyword(s):

Warburg Effect ◽

Cancer Metabolism ◽

Aerobic Glycolysis ◽

Lactate Production ◽

Glycolytic Enzymes ◽

Hek293 Cells ◽

Driver Mutations ◽

The Warburg Effect

Aggressive cancers commonly ferment glucose to lactic acid at high rates, even in the presence of oxygen. This is known as aerobic glycolysis, or the “Warburg Effect”. It is widely assumed that this is a consequence of the upregulation of glycolytic enzymes. Oncogenic drivers can increase the expression of most proteins in the glycolytic pathway, including the terminal step of exporting H+ equivalents from the cytoplasm. Proton exporters maintain an alkaline cytoplasmic pH, which can enhance all glycolytic enzyme activities, even in the absence of oncogene-related expression changes. Based on this observation, we hypothesized that increased uptake and fermentative metabolism of glucose could be driven by the expulsion of H+ equivalents from the cell. To test this hypothesis, we stably transfected lowly-glycolytic MCF-7, U2-OS, and glycolytic HEK293 cells to express proton exporting systems: either PMA1 (yeast H+-ATPase) or CAIX (carbonic anhydrase 9). The expression of either exporter in vitro enhanced aerobic glycolysis as measured by glucose consumption, lactate production, and extracellular acidification rate. This resulted in an increased intracellular pH, and metabolomic analyses indicated that this was associated with an increased flux of all glycolytic enzymes upstream of pyruvate kinase. These cells also demonstrated increased migratory and invasive phenotypes in vitro, and these were recapitulated in vivo by more aggressive behavior, whereby the acid-producing cells formed higher grade tumors with higher rates of metastases. Neutralizing tumor acidity with oral buffers reduced the metastatic burden. Therefore, cancer cells with increased H+ export increase intracellular alkalization, even without oncogenic driver mutations, and this is sufficient to alter cancer metabolism towards a Warburg phenotype.

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Cell-Type Specific Metabolic Response of Cancer Cells to Curcumin

International Journal of Molecular Sciences ◽

10.3390/ijms21051661 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1661

Author(s):

Anamarija Mojzeš ◽

Marko Tomljanović ◽

Lidija Milković ◽

Renata Novak Kujundžić ◽

Ana Čipak Gašparović ◽

...

Keyword(s):

Cancer Cells ◽

Cell Lines ◽

Warburg Effect ◽

Aerobic Glycolysis ◽

Intracellular Localization ◽

Lactate Production ◽

Glucose Consumption ◽

Cell Type ◽

Cell Type Specific ◽

The Warburg Effect

In order to support uncontrolled proliferation, cancer cells need to adapt to increased energetic and biosynthetic requirements. One such adjustment is aerobic glycolysis or the Warburg effect. It is characterized by increased glucose uptake and lactate production. Curcumin, a natural compound, has been shown to interact with multiple molecules and signaling pathways in cancer cells, including those relevant for cell metabolism. The effect of curcumin and its solvent, ethanol, was explored on four different cancer cell lines, in which the Warburg effect varied. Vital cellular parameters (proliferation, viability) were measured along with the glucose consumption and lactate production. The transcripts of pyruvate kinase 1 and 2 (PKM1, PKM2), serine hydroxymethyltransferase 2 (SHMT2) and phosphoglycerate dehydrogenase (PHGDH) were quantified with RT-qPCR. The amount and intracellular localization of PKM1, PKM2 and signal transducer and activator of transcription 3 (STAT3) proteins were analyzed by Western blot. The response to ethanol and curcumin seemed to be cell-type specific, with respect to all parameters analyzed. High sensitivity to curcumin was present in the cell lines originating from head and neck squamous cell carcinomas: FaDu, Detroit 562 and, especially, Cal27. Very low sensitivity was observed in the colon adenocarcinoma-originating HT-29 cell line, which retained, after exposure to curcumin, a higher levels of lactate production despite decreased glucose consumption. The effects of ethanol were significant.

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