Funded Grants

Regulation of PKM2 in glioblastoma Development

Overexpression of EGFR has been reported in many human tumors, including gliomas. Activation of EGFR promotes proliferation, migration, and invasion of tumor cells. Accordingly, targeting EGFR has been intensely pursued. However, the efficacy of clinical treatment of some human cancers with EGFR inhibitors has been less significant than expected because of intrinsic and acquired resistance, which is often due to intrinsic and acquired mutations of EGFR or its downstream genes, such as PTEN, K-Ras, or Raf. The mutations of downstream genes bypass the inhibition of EGFR and drive tumor progression. Thus, the identification of novel key regulators for EGFR-regulated tumorigenesis may provide an alternative approach or combined approaches for treating EGFR-related human cancer.

Tumor cells, unlike their normal counterparts, have elevated rates of glucose uptake and higher lactate production in the presence of oxygen, known as aerobic glycolysis, with reduced mitochondrial oxidative phosphorylation for glucose metabolism. This phenomenon, called the Warburg effect, supports tumor cell growth. Pyruvate kinase regulates the rate-limiting final step of glycolysis, which catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to ADP, yielding one molecule of pyruvate and one molecule of ATP. Four pyruvate kinase isoforms exist in mammals: the L and R isoforms are expressed in liver and red blood cells; the M1 isoform is expressed in most adult tissues; and the M2 isoform is a splice variant of M1 expressed during embryonic development. PKM2 expression is progressively replaced by other isoforms after birth. During tumorigenesis, PKM2 is reexpressed at a high level, which represents a tumor-specific form. PKM2 depletion in lung cancer cells reverses the Warburg effect, as indicated by reduced lactate production and increased oxygen consumption, and profoundly inhibits tumor formation in nude mouse xenografts. These findings point to an essential role of PKM2 expression in tumor growth.

Besides its well-known role in glycolysis, PKM2 has been reported to be involved in other cellular functions, such as being the cytosolic receptor for thyroid hormone. In addition, a neuropeptide hormone, somatostatin, and the structural analogue TT-232 interact with PKM2 and cause PKM2 nuclear translocation and apoptosis of nontransformed cells. In contrast, other evidence indicates that nuclear PKM2 promotes cell proliferation. For example, PKM2 interacts with and enhances Oct-4 transcription factor--mediated transcription, and Oct-4 plays an important role in maintaining the pluripotent state of embryonic stem cells. Furthermore, PKM2 binds directly and selectively to Tyr-phosphorylated peptides, and expression of this phosphotyrosine-binding form of PKM2 is required for rapid growth of cancer cells.

β-catenin, a key component of the Wnt/Wingless signaling pathway, plays a central role in development and cell proliferation and differentiation. In the absence of a Wnt signal, cytoplasmic β-catenin interacts with axin/conductin, GSK-3β, and the adenomatous polyposis coli protein (APC). GSK-3β phosphorylates the N-terminal domain of β-catenin, which leads to β-catenin degradation via the ubiquitin/proteasome pathway. Activation of the Wnt pathway inhibits GSK-3β-dependent phosphorylation of β-catenin. Stabilized and hypophosphorylated β-catenin translocates to the nucleus and interacts with transcription factors of the TCF/LEF-1 family, leading to increased expression of genes, such as CCND1 (coding for cyclin D1) and cmyc. Besides regulation of β-catenin by mutations of components of the Wnt pathway, growth factor receptor activation results in β-catenin transactivation, thereby promoting tumor progression. However, the mechanisms underlying β-catenin-regulated gene transcription in response to growth factor receptor activation remain elusive.

Histones can undergo several different post-translational modifications, including acetylation, phosphorylation, methylation, and ubiquitination. Histone modifications can influence one another, such that one modification is instrumental to generate a different modification for subsequent gene transcription regulation. It is known that phosphorylation of histone H3 Ser or Thr residues can lead to acetylation of adjacent Lys. In response to DNA damage, metabolic stress, growth factor receptor activation, histone H3 is phosphorylated at Ser/Thr residues. However, whether EGFR activation results in β-catenin transactivation-coupled histone H3 phosphorylation and, if so, whether these posttranslational H3 modifications contribute to cell cycle progression and tumorigenesis, remains unclear.

We have shown that EGFR activation results in PKM2 translocation into the nucleus, where it binds to Y333-phosphorylated β-catenin. This protein complex binds to the promoter of the CCND1, leading to the disassociation of HDAC3 from the promoter, cyclin D1 expression, and tumorigenesis. In addition, the levels of nuclear PKM2 expression correlate with those of β-catenin Y333 phosphorylation in human glioblastoma (GBM) specimens, and both correlate with GBM patient prognosis. To further understand the mechanism of PKM2-dependent cyclin D1 expression, we will investigate the mechanims underlying PKM2-dependent HDAC3 removal from histone in PKM2-kinase activity dependent manner. We will further examine PKM2-regulated epigenetic changes and their role in EGFR-promoted gene expression and glioma development. We expect that our proposed research will reveal important mechanisms underlying gene transcription regulation by a nonmetabolic function of PKM2 in EGFR-activated tumor cells. This contribution will be significant, because it could lead to pharmaceutical approaches to interrupt EGFR-induced gene transcription by blocking PKM2 nuclear function, thereby improving the efficacy of treatment for human glioma.