Research Article: Subcellular compartmentalization of PKM2 identifies anti-PKM2 therapy response in vitro and in vivo mouse model of human non-small-cell lung cancer

Date Published: May 23, 2019

Publisher: Public Library of Science

Author(s): Akiko Suzuki, Sachin Puri, Pamela Leland, Ankit Puri, Tarsem Moudgil, Bernard A. Fox, Raj K. Puri, Bharat H. Joshi, Aamir Ahmad.

http://doi.org/10.1371/journal.pone.0217131

Abstract

Pyruvate kinase M2 (PKM2) is an alternatively spliced variant, which mediates the conversion of glucose to lactate in cancer cells under normoxic conditions, known as the Warburg effect. Previously, we demonstrated that PKM2 is one of 97 genes that are overexpressed in non-small-cell lung cancer (NSCLC) cell lines. Herein, we demonstrate a novel role of subcellular PKM2 expression as a biomarker of therapeutic response after targeting this gene by shRNA or small molecule inhibitor (SMI) of PKM2 enzyme activity in vitro and in vivo. We examined two established lung cancer cell lines, nine patients derived NSCLC and three normal lung fibroblast cell lines for PKM2 mRNA, protein and enzyme activity by RT-qPCR, immunocytochemistry (ICC), and Western blot analysis. All eleven NSCLC cell lines showed upregulated PKM2 enzymatic activity and protein expression mainly in their cytoplasm. Targeting PKM2 by shRNA or SMI, NSCLC cells showed significantly reduced mRNA, enzyme activity, cell viability, and colony formation, which also downregulated cytosolic PKM2 and upregulated nuclear enzyme activities. Normal lung fibroblast cell lines did not express PKM2, which served as negative controls. PKM2 targeting by SMI slowed tumor growth while gene-silencing significantly reduced growth of human NSCLC xenografts. Tumor sections from responding mice showed >70% reduction in cytoplasmic PKM2 with low or undetectable nuclear staining by immunohistochemistry (IHC). In sharp contrast, non-responding tumors showed a >38% increase in PKM2 nuclear staining with low or undetectable cytoplasmic staining. In conclusion, these results confirmed PKM2 as a target for cancer therapy and an unique function of subcellular PKM2, which may characterize therapeutic response to anti-PKM2 therapy in NSCLC.

Partial Text

Lung cancer is the most common cause of cancer related mortality worldwide, accounting for approximately 1 in 4 cancer deaths [1, 2]. About 85–90% of lung cancers are non-small-cell-lung cancer [3, 4]. For early stage Non-Small-Cell Lung Cancer (NSCLC), surgery is usually the treatment of choice and chemotherapy (sometimes in combination with radiation therapy) may be given as well. Patients with advanced-stage NSCLC are usually treated with chemotherapy, targeted drugs (or a combination of the two), or immunotherapy. Considering the low 5-year survival rate (21%) with currently available therapies, there is a need for improved treatment options [4]. Compared to normal cells, cancer cells display a radical shift in metabolism becoming highly dependent on glucose, which is metabolized through an increased rate of aerobic glycolysis, a metabolic state termed the Warburg effect, which is considered a hallmark of cancer metabolism [5, 6]. Previously, we have demonstrated that human NSCLC cell lines overexpress 97 genes by DNA microarray [7–9]. Among these, pyruvate kinase M2 (PKM2) is highly overexpressed in NSCLC cell lines examined compared to normal lung tissues. PKM2 is an allosteric isoform of pyruvate kinase, which catalyzes the final step in glycolysis and converts phosphoenol-pyruvate (PEP) to pyruvate [10]. PKM2 is shown to divert glycolytic flux into the pentose phosphate pathway associated with attenuated pyruvate kinase activity, thereby meeting the biosynthetic demands for rapid proliferation [10]. Of four isoforms of pyruvate kinase L, R, M1 and M2, proliferating embryonic and tumor cells predominantly express PKM2. In cancer cells, PKM2 can migrate to the nucleus and function as a transcriptional co-factor in response to many extracellular signals such as Epidermal growth factor (EGF) and hypoxia, which activate CYCLIN D1, C-MYC or Hypoxia inducible factor-alpha (HIF-α) [11, 12]. PKM2 is shown to mediate epithelial to mesenchymal transition (EMT), which stimulates PKM2 to migrate to nucleus in cancer cells and acts as a transcription cofactor that in turn inhibits E-cadherin [13]. It is also shown that cytosolic PKM2 is associated with Epidermal growth factor receptor (EGFR) expression and prolongs the protein half-life of EGFR in cancer cells by stabilizing EGFR-Heat shock protein 90 (HSP90) protein complex [14]. PKM2 is reported to act as a protein kinase and exist as a dimer localized in the nucleus promoting cell proliferation, while its tetramer form is an active pyruvate kinase localized in the cytosol [15]. It is also reported that nuclear translocation of PKM2 supports cancer cell survival, which binds to Oct4 promoting expression of cancer stemness-related genes [16].

We have demonstrated overexpression of PKM2 in patient-derived and established NSCLC cell lines. These cell lines not only overexpressed PKM2 protein but also high PKM2 enzyme activity in >55% of the cells with > 2+ immunostaining intensity. The overexpressed PKM2 in NSCLC is inhibitable in a concentration-dependent manner with either a drug or gene silencing by shRNA in vitro. These treatments affected both PKM2 enzyme activity and mRNA transcription, which subsequently led to reduced levels of PKM2 protein, as demonstrated by Western blot analysis. In contrast, three normal lung cell lines, which showed lower enzyme activity and mRNA, did not show such a dramatic response to both types of anti-PKM2 strategies. Further, we observed that PKM2 targeting with SMI or shRNA-PKM2 significantly affected cell viability in all NSCLC cell lines and impaired the ability to form colonies in vitro, suggesting that PKM2 enzyme activity is important for tumor growth in NSCLC cells. The normal lung fibroblast cell lines failed to show such changes after targeting PKM2. Previously published results have shown that PKM2 can interact with mutant EGFR and HSP90 and prolonged the protein half-life of EGFR in lung cancer cells [14]. It is also reported that PKM2 supports tumor growth through regulating the expression of gene that involved in cell proliferation, migration and apoptosis [26]. Our present data have demonstrated a unique role of PKM2 as its enzyme inhibition by either SMI or gene silencing can influence the tumor growth in vitro and in vivo.

 

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http://doi.org/10.1371/journal.pone.0217131

 

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