Nuclear EGFR-PKM2 axis induces cancer stem cell-like characteristics in irradiation-resistant cells

Highlights

• The EGFR and PKM2 complex facilitates radioresistance in an EGFR dependent manner.

• Depletion of EGFR diminishes stem-like properties through reducing PKM2 binding to stemness gene promoters.

• Targeting the nuclear intact complex of EGFR and PKM2 overcomes radioresistence in cancer.

Abstract

Radiation therapy has become an important tool in the treatment of cancer patients, but most patients relapse within 5 years. Relapse is due to the presence of cancer stem cells (CSCs), but the molecular mechanism of radioresistance in CSCs remains largely elusive. Here, we found that irradiation-resistant (IR) cells exhibited increased stem cell-like properties together with elevated anchorage-independent growth and metastasis ability. EGFR not only leads to increased acquisition of endometrial cancer stem cell markers in radioresistant sublines but is critical for the cancer stem-cell phenotype and tumorigenicity. Moreover, PKM2 functions as an interacting partner of EGFR, which induces the EMT phenotype and stem cell-like properties in IR cells. Finally, we found that the regulatory function of the EGFR-PKM2 axis is dependent on nuclear EGFR. In sum, our study indicated that EGFR and PKM2 directly interact and bind with each other to regulate the transcription of stemness-related genes and promote the stem-like phenotype, thus promoting invasion and metastasis.

Introduction

Radiation therapy is one of the major tools of cancer treatment and is broadly used for a variety of malignant tumors. Within tumors, a very small subpopulation of cells is considered to possess the ability to self-renew and differentiate, leading to radioresistance, metastasis and recurrent disease. Such cells have been designated “cancer stem cells” (CSCs) [1,2] or “stem-like cancer cells” (SLCCs) [2]. Subsequently, CSCs have been discovered and isolated from many types of cancer [[3], [4], [5], [6], [7], [8], [9]]. Treatments targeting CSCs may substantially improve therapy in cancer patients. Hence, recent studies have increasingly focused on the identification of CSC-specific markers, such as CD44, CD133, ABCG2 (CD338), and ALDH1, and the embryonic stem cell transcription factors Sox2, Oct4 and Nanog [[10], [11], [12]]. However, tumor cells with CSC-like phenotypes or markers have been found to be radiation resistant, and the molecular mechanisms of the radioresistance of CSCs are poorly understood [13].

EGFR is a receptor tyrosine kinase that belongs to the ErbB family, which includes ErbB1, ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) [14]. Importantly, EGFR and its downstream signaling pathway play important roles in the control of cell proliferation, survival, epithelial-to-mesenchymal transition (EMT), migration and differentiation in a wide variety of human cancers [15]. Over-expression or elevated activity of EGFR is associated with unfavorable prognosis and resistance against chemotherapy and radiation therapy [16]. In addition, EGFR signaling has been implicated in the functions of CSCs in various cancers and is known to increase tumor invasiveness. There is a growing body of evidence that activation of the EGFR pathway mediates the regulation of multiple CSC-related genes, including Sox2 [17,18], CD44 [19], CD133 [20], Oct4 [21], and ALDH1 [22], although the underlying molecular mechanisms are not clear.

PKM2 is a rate-limiting enzyme, which catalyzes the final step in glycolysis by converting phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) to pyruvate and adenosine-triphosphate (ATP) [23]. It has been demonstrated to play a critical role in tumor metabolism and be involved in the Warburg effect, conferring cancer cells with the glycolytic phenotype to generate the energy required for cellular processes [[24], [25], [26]]. Consistently, enhanced PKM2 expression has been found in multiple human cancers, suggesting that PKM2 is a potential diagnostic biomarker and therapeutic target for tumors.

Beyond its metabolic function, PKM2 has recently received much attention for its noncanonical roles in tumorigenesis. The PKM2 dimer is also well known as a nuclear protein kinase that regulates gene transcription induced by Oct4, STAT3, β-catenin, FGFR1 or HIF-1α [24]. In addition, nuclear translocation of PKM2 may occur in response to EGFR activation. For instance, cytosolic PKM2 can directly interact with and stabilize mutant EGFR protein in lung cancer cells [27]. Activation of EGFR can also induce the translocation of PKM2 to the nucleus, where PKM2 promotes the transcriptional activity of β-catenin by binding to c-Src-phosphorylated β-catenin, resulting in transcriptional activation of cyclin D1 and Myc [28].

The aim of the present study is to investigate whether activation of the EGFR-PKM2 axis in the nucleus contributes to radioresistance and induces CSC-like properties in irradiation-resistant cells. We also examined whether the EGFR-PKM2 axis affects colony formation, tumor sphere formation, tumorigenicity and expression of stem cell-like genes in irradiation-resistant cells. Finally, we propose a new concept for targeting EGFR-PKM2 to overcome radioresistance.