Phosphorylation serves as a crucial cellular signaling mechanism that governs protein activity, function, and interactions, impacting various physiological processes. In the context of tumor development, perturbed cellular metabolism stands out as a significant hallmark. Cancer cells adeptly reprogram their metabolism to adapt to dynamic environments, fueling their rapid proliferation and survival. Consequently, deciphering the regulatory intricacies of tumor cell metabolism holds the key to unraveling cancer's underlying mechanisms and devising innovative therapeutic approaches.
The intersection of phosphorylation and metabolism has garnered heightened attention. With its widespread reach as an intracellular signaling mode, phosphorylation exerts influence over protein functions and interactions, thereby modulating the orchestration of cellular metabolic pathways. The phosphorylation status of proteins can wield direct or indirect impact on diverse metabolic routes, encompassing glycolysis, lipid metabolism, and amino acid metabolism. As a consequence, these influences reverberate through processes like cellular energy equilibrium, biosynthesis, and oxidative stress management.
Anaplastic Lymphoma Kinase (ALK), a tyrosine kinase, displays aberrant activation across various cancers, implicated in tumor cell proliferation, survival, and invasion. Recent investigations propose an extended role for ALK, encompassing tumor metabolic reprogramming in tandem with its established involvement in cell proliferation signaling pathways. By modulating phosphorylation, ALK potentially oversees the function of several metabolic enzymes, thereby affecting pivotal metabolic pathways including glucose utilization, lipid synthesis, and amino acid metabolism.
Metabolomics technology can comprehensively detect the composition and changes of intracellular metabolites, thus revealing changes in the metabolic state of cells. Combining phosphoproteomics data and metabolomics analysis allows researchers to deeply explore the regulatory mechanisms of ALK in tumor metabolic reprogramming. This comprehensive approach helps identify potential metabolic pathways, enzymes, and related signaling pathways, providing new insights into the role of ALK in tumor metabolism.
Case. Phosphorylation + Metabolomics Unveiling the Regulatory Role and Mechanisms of ALK in Tumor Metabolic Reprogramming
Anaplastic Large Cell Lymphoma (ALCL) is a subtype of peripheral T-cell lymphoma that is relatively rare but represents a significant portion of childhood lymphomas. A notable portion of pediatric ALCL cases are characterized by a specific genetic abnormality involving the fusion of two genes, ALK and NPM, resulting in the creation of a constitutively active enzyme called NPM-ALK. This enzyme is a tyrosine kinase that drives the development of ALCL.
Metabolic reprogramming, particularly the "Warburg effect," is a hallmark of cancer cells, where they prioritize glycolysis and lactate production for energy and biomass generation, even in the presence of oxygen. This metabolic shift is important for cell proliferation.
To better understand the signaling pathways involved in ALK-driven oncogenesis, researchers conducted phosphoproteomic studies to identify proteins and pathways influenced by ALK. This led to further investigations using mass spectrometry-based metabolomics to characterize the metabolic changes driven by ALK. The integrated analysis of these studies revealed a novel metabolic switch induced by ALK.
Phosphorylation Modification Analysis
As a key cancer-related gene, ALK has been studied quite a bit, therefore, in order to systematically resolve the action network of ALK and to uncover new mechanisms of action, and considering that ALK itself is a kinase, the authors examined untreated versus ALK kinase inhibitor-treated lymphoma cells using quantitative phosphorylation modifier group analysis. A total of 671 proteins showed changes in phosphorylation (569 down-regulated and 102 up-regulated), of which 49 were significantly changed (43 down-regulated and 6 up-regulated). These altered phosphorylated proteins constitute a direct or indirect regulatory network for ALK.
GO and KEGG analysis further revealed that these proteins that changed with ALK were enriched for many metabolic processes, especially nucleotide metabolism, macromolecule metabolism, biosynthesis, and pathways such as glycolysis, pentose phosphate, and the TCA cycle.
Metabolomics Analysis
Since proteins altered by phosphorylation modifications are mostly associated with metabolic pathways, is the role of ALK closely related to metabolic regulation? To clarify this query, the authors performed quantitative metabolomic analysis on untreated versus ALK inhibitor-treated cells. Unsurprisingly, the content of nucleotide metabolites, glycolysis, pentose phosphate, and TCA cycling pathway metabolites were all altered with ALK inhibition. It suggests that ALK indeed plays an important role in metabolic regulation.
Joint Analysis of Omics Data using KEGG Pathway Mapping:
For those familiar with omics analysis, it's widely recognized that KEGG provides a comprehensive overview of numerous metabolic pathways, elucidating the direct relationships between metabolic processes and their corresponding participating genes/proteins. Consequently, in order to systematically elucidate how ALK orchestrates metabolic processes through phosphorylation, the authors conducted a combined analysis of phosphoproteomics and metabolomics data within the context of KEGG metabolic pathways.
By mapping both sets of data onto the KEGG pathways, a clear visualization emerges, showcasing the proteins that directly influence metabolic regulation, and whether alterations in their activity lead to corresponding changes in metabolite levels. This integrated approach provides a tangible insight into the altered proteins that are intricately involved in the orchestration of metabolic adaptations.
To further substantiate these findings, the authors verified by metabolic flow analysis that ALK activation triggers a change in the direction of metabolic flow from energy production to substance synthesis.
Uncovering Key Molecules through Integrative Omics Data for Subsequent In-depth Investigations:
Leveraging the wealth of integrative omics data and employing meticulous metabolomics and metabolic flux experiments, critical nodes within key metabolic pathways can be identified, serving as focal points for subsequent in-depth exploration. From the aforementioned omics data, the authors identified the protein kinase - PKM2, wherein both its protein modification levels and its regulation of pyruvate metabolism exhibited corresponding changes. As a result, PKM2 emerged as a plausible pivotal molecule in the context of ALK's role.
Initial validation and investigation:
(1) Western blot (WB) experiments were conducted to corroborate that ALK indeed influences PKM2 modification levels.
(2) Co-immunoprecipitation (Co-IP) experiments were employed to ascertain that ALK can directly interact with PKM2, regulating PKM2's metabolic enzyme activity through phosphorylation of PKM2 Y105.
Further Functional Validation of Selected Key Protein
(1) Cell-Level Verification of PKM2 Activity: Substantiating that the activation of PKM2 (via TEPP-46 and NCGC-527 PKM2 activators) induces alterations in cellular metabolism and restrains cell proliferation.
(2) Cellular-Level Phenotypic Validation of PKM2: Treatment with PKM2 activators and mutation at PKM2 Y105 site both lead to the inhibition of growth in ALK1 ALCL cells at the cellular level, demonstrating a corresponding suppression of tumor growth in vivo in tumor-bearing animals.
Reference
- McDonnell, Scott RP, et al. "Integrated phosphoproteomic and metabolomic profiling reveals NPM-ALK–mediated phosphorylation of PKM2 and metabolic reprogramming in anaplastic large cell lymphoma." Blood, The Journal of the American Society of Hematology 122.6 (2013): 958-968.
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