Background
Endometrial cancer (EC) is the most common gynecological cancer in developed countries, and its prevalence is rapidly increasing in Japan [
1,
2]. Although many efforts have been made to develop targeted therapy and immunotherapy, the prognosis of patients with advanced-stage disease remains poor [
3]. The mechanisms by which genetic and epigenetic alterations contribute to EC progression and influence the response to cancer therapies are incompletely understood.
Epithelial-to-mesenchymal transition (EMT) is a multistep process defined by the loss of epithelial phenotypes, and the gain of mesenchymal and cancer stem cell (CSC)-like characteristics [
4,
5]. EMT can be triggered by various genetic and epigenetic signaling pathways in tumor cells [
4]. Cancer cells frequently exhibit enhanced glycolysis and lactate production even in the presence of abundant oxygen, known as the Warburg effect or aerobic glycolysis [
6]. Hexokinase 2 (HK2) is a glycolytic enzyme that catalyzes the first committed step in glucose metabolism, and its expression is markedly induced in cancer cells by multiple mechanisms [
6]. Systemic HK2 deletion showed therapeutic effects in mice bearing lung tumors without adverse physiological consequences [
7]. There is increasing evidence demonstrating an association between EMT execution and the reprogramming of glucose metabolism [
8]. For instance, Snail serves as a positive regulator of EMT and glucose metabolism in gastric cancer [
9], and HK2 was reported to increase glycolytic activity and ovarian cancer cell invasiveness through upregulation of the EMT activator, focal adhesion kinase (FAK) [
10,
11], indicating that the overlapping mechanisms exist to affect both EMT and aerobic glycolysis.
Dysregulation of non-coding RNAs, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), was known to be critical to cancer metastasis and metabolism [
12‐
14]. LncRNAs mediate many important cancer phenotypes through their cross-talk with other macromolecules including DNA, RNA, and protein [
15]. Although lncRNA DLEU2 is implicated in the tumorigenesis and progression of several malignancies [
16,
17], the functional role and the mechanisms underlying its function in EMT and glycolysis in EC are still unknown.
In this study, we show that HK2 confers an oncogenic function in promoting EMT and glycolysis in EC cells. DLEU2 employs two distinct mechanisms to induce EMT and sustain glycolysis through HK2. First, DLEU2 competitively binds with miR-455 to induce HK2 expression. Second, DLEU2 interacts with EZH2 to silence a direct inhibitor of HK2, miR-181a.
Discussion
Despite a better understanding of EC genetic and epigenetic changes in recent years, the development of metastatic dissemination and drug resistance remain the major challenges for treating this cancer. Previous evidence has indicated that EMT-guided invasive growth and enhanced aerobic glycolysis are closely connected with each other, and serve as two main manifestations of tumor progression [
8‐
11]. Thus, unraveling the precise mechanisms that underlie both EMT and glycolysis would facilitate a deeper understanding of the biological complexity of cancer metastasis and may lead to more effective and selective cancer treatments.
Here, we describe a previously unappreciated regulatory mechanism whereby DLEU2, HK2, and FAK form a functional axis that plays a causal role in EMT and glycolysis, representing an advance in the field of genetic and epigenetic regulation of these features of EC cells. Importantly, we demonstrate that DLEU2 regulates the HK2/FAK/ERK1/2 signaling via competitively binding with miR-455 and by interacting with EZH2 to silence miR-181a expression (Fig.
8d).
DLEU2, HK2 and FAK are selectively overexpressed in EC cells but not in normal cells, act as key EMT/glycolysis-stimulating factors in EC, and are inversely correlated with patient prognosis. We also utilized EC cell models to illustrate that inhibition of DLEU2 and HK2 reduces cell migration, invasion, sphere formation and glycolysis, while enhancing chemotherapy sensitivities. These findings raise the possibility that targeting the DLEU2-driven HK2 signaling axis may provide a therapeutic strategy for EC patients with highly aggressive and glycolytic tumors.
Numerous studies have reported that aberrant HK2 overexpression stimulates cancer development, metastasis, and therapeutic resistance to chemotherapy and radiation [
33‐
35]. HK2 is highly expressed in a wide range of human cancers [
6] and is associated with poor outcomes of patients with diverse cancers [
36]. Of note, forced overexpression of HK2 induces cancer cell invasiveness, EMT, and cancer stemness [
37,
38]. Our present study has provided a better understanding of how enhanced HK2 expression confers aggressive tumor progression and poor prognosis in EC patients through the maintenance of mesenchymal state and increased glycolytic activity.
FAK promotes cell survival, growth, angiogenesis, migration, invasion, EMT, and CSC-like characteristics, thus controlling cancer initiation, progression, metastasis, recurrence, and drug resistance [
20]. We found that the expression of FAK was significantly higher in EC tissues than in normal tissues. This was consistent with a previous study showing that FAK protein expression was increased in endometrial hyperplasia and EC tissues compared with normal endometrium, and FAK overexpression in EC correlates with higher FIGO grade [
39]. Furthermore, our data suggest that the oncogenic function of HK2 in EC is due to its ability to activate FAK-dependent signaling, however, the precise mechanism by which this HK2/FAK axis works in EC remains largely elusive.
Previous evidence has indicated that FAK exerts its oncogenic functions through the modulation of multiple downstream signaling cascades, such as ERK1/2, PI3K/AKT, and JNK pathways [
40]. Consistent with these findings, our results support that HK2 drives EMT and glycolysis through activation of FAK and its downstream ERK1/2 signaling. However, other researchers have found that FAK might trigger PI3K/AKT (but not ERK1/2) signaling to affect melanoma cell invasion and metastasis [
41]. Importantly, in EC cells, treatment with the selective FAK inhibitor abolished estrogen-induced cell migration, and pharmacological inhibition of PI3K/AKT and ERK1/2 pathways prevented the phosphorylation of FAK [
42], implying an interesting possibility whereby FAK might induce the activation of PI3K/AKT and/or ERK1/2 pathways, and then high PI3K/AKT and/or ERK1/2 activity feeds back to initiate FAK, thus establishing a positive feedback loop to mediate HK2-induced EMT and glycolysis in EC cells. Further studies are thus required to elucidate the detailed molecular regulatory circuitry between them, and to expand our understanding of the complexity of HK2-dependent gene networks contributing to the metastatic and metabolic phenotypes of EC cells.
MiRNAs are key regulators of the human transcriptome and dysregulation of miRNAs has a critical role in cancer metastasis and metabolism [
12,
13]. Reduced expression of miR-455 and its tumor suppressor function has been reported in colon [
43], pancreatic [
44], lung [
45], and prostate [
46] cancers. MiR-455 inhibits the EMT process in pancreatic cancer [
44] and hepatocellular carcinoma [
47] by modulating the Wnt/β-catenin and STK17B/AKT/GSK-3β/Snail signaling, respectively. Here, we demonstrated a new function of miR-455 in suppressing EMT and glycolysis of EC cells via repressing the expression of HK2. Thus, these previous results, together with our findings, collectively suggest that miR-455 could suppress multiple oncogenic pathways, making it a novel therapeutic target for EC and other cancers. Future studies will reveal how miR-455 functions and loss of miR-455 expression occurs in human tumors.
The list of lncRNAs involved in tumor progression is expanding rapidly. Although a few lncRNAs have been implicated in EMT and cancer metabolism regulation [
15,
48], the functions and underlying mechanisms of most lncRNAs in EC remain poorly understood. Previous studies on DLEU2 have mainly focused on its oncogenic role in controlling tumor cell proliferation, migration, and invasion [
16,
17]. In this study, we found that the overexpression of DLEU2 could regulate HK2/FAK/ERK1/2 signaling through binding miR-455 and an EZH2/miR-181a axis-mediated mechanism, resulting in the gain of EMT and glycolytic properties in EC cells. Additional studies will be needed to clarify the functional contribution of DLEU2 in cancer biology, the precise mechanisms downstream of DLEU2, and how its expression is regulated in tumor cells.
Identifying potential biomarkers to detect EC in the early stage would contribute to improved survival rates. Although prior studies using genomic and proteomic technologies have provided molecular insights into the pathogenesis and biology of EC, but have yet to yield reliable biomarkers to impact the early diagnosis of this disease [
49]. Overexpression of EZH2 has been previously discovered in pre-cancerous lesions of the endometrium and EC tissues, but not in normal endometrium samples [
50]. On the basis of GEO EC datasets, we observed that EZH2 (data not shown), HK2, FAK, and DLEU2 exhibited significantly increased expression in stage I EC tissues compared to normal tissues (Figs.
1,
3,
5). Consistently, in the TCGA dataset, we verified their overexpression or amplification in EC tissues (Fig.
8). These results demonstrate that these molecules might be valuable biomarkers for the early diagnosis of EC.
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