Background
Hilar cholangiocarcinoma (HC) is a complex, aggressive disease with an extremely poor prognosis. R0 resection offers the only chance for long-term survival of HC. Even after resection, 5-year survival rates for HC range from 10 to 40 %, and the recurrence rates range from 50 to 70 % [
1]. The most accepted prognostic factors for HC include lymph node metastasis, disease stage, negative margins, and/or neural invasion (NI) [
1]. Tumor markers, specifically, carcinoembryonic antigen and CA19-9, help with diagnosis, treatment, and monitoring the progression of HC [
2]. Additionally, the presence of tumor markers is associated with disease progression and overall survival (OS) [
2‐
4]. However, physicians have successfully applied few of these biomarkers to clinical practice. Therefore, identifying novel biomarkers is of great importance to monitoring progression of, predicting outcome of, and developing effective therapeutic targets for HC.
Cancer cells can switch oxidative phosphorylation to aerobic glycolysis, known as the Warburg effect, to maintain the growth and survival of cancer cells [
5]. Investigators have well established that accelerated aerobic glycolysis is associated with activation of oncogenes or inhibition of tumor suppressors in certain cells [
6]. Three rate-limiting enzymes active in glycolysis—hexokinase (HK), pyruvate kinase (PK), and phosphofructokinase (PFK)—are widely activated in cancer cells [
7]. HK2, which is highly expressed in cancer cells, is thought to be required for tumor initiation and maintenance, and HK2 deletion is therapeutic in mouse models of cancer [
8]. Few studies have demonstrated that HK1 is associated with therapeutic effectiveness against cancer [
9,
10]. Unlike other PK isoforms, PKM2 contributes to tumorigenesis [
11]. Elevated PKM2 expression correlates with poor outcomes of cancer [
12]. In addition, expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), an isoform of PFK2, is elevated in tumors and an important regulator of cancer cell survival [
13]. Additionally, key regulators of aerobic glycolysis are implicated to play roles in predicting outcome and monitoring drug resistance of HC. Therefore, targeting cancer metabolism seems to be a promising therapeutic approach [
14].
Continuous progress in research of the cancer metabolic program has increased our understanding of cancer biology and helped identify novel therapeutic targets and/or predictive biomarkers. However, little is known about the status of the key enzymes HK, PK, and PFK in HC pathogenesis and treatment. However, researchers rarely have explored the therapeutic and prognostic value of glycolytic molecules, including HK, PFKB, and PKM2, regarding HC. Therefore, in the present study, we sought to determine the prognostic value of these three glycolytic enzymes in HC cases and investigated the possible mechanisms underlying PKM2’s promotion of HC growth and NI.
Discussion
In this study, we systematically analyzed the expression profiles of key glycolytic enzymes in patients and mice with HC and found that expression of HK1 and PKM2 was markedly increased and correlated with tumor progression. High PKM2 expression was an independent predictor of recurrence and survival after resection of HC. Additionally, high PKM2 expression correlated directly with NI and NI-related biomarkers, especially SDC2, suggesting a novel molecular basis for the role of PKM2 in HC development and progression.
Aerobic glycolysis is essential for tumorigenesis, and the glycolytic enzymes HK, PFK, and PK are now recognized as potential therapeutic targets for cancer [
14]. Researchers have also linked biological markers of glycolysis (HK2, PFKFB4, PKM2, etc.) with tumor recurrence and OS in certain cancer patients [
12]. Previous studies demonstrated that expression of HK2 but not HK1 was a significant predictor of survival [
16,
17]. Few studies have identified the prognostic value of PFKFB4 regarding tumor recurrence and/or survival [
13]. Other studies demonstrated a significant correlation between PKM2 expression and overall prognosis for cancer [
18,
19]. However, researchers rarely have explored the therapeutic and prognostic value of glycolytic molecules, including HK, PFKB, and PKM2, regarding HC. Therefore, in the present study, we investigated the HK1, PFKB, and PKM2 expression statuses in HC samples and cells and found that HK1 and PKM2 expression but not PFKB expression was higher in tumor than in normal tissue samples. Specifically, HK1 expression was associated with lymph node metastasis and advanced disease stage, whereas PKM2 expression correlated with NI, tumor invasion, lymph node metastasis, and poor or lack of tumor differentiation, suggesting that sustained aerobic glycolysis is a feature of biologically aggressive tumors. Moreover, survival analysis demonstrated that expression of HK1 and PKM2 was associated with recurrence of and prognosis for HC. However, our multivariate analysis demonstrated that expression of PKM2, but not HK1, was an independent predictor of survival of HC. Interestingly, patients with PKM2 expression had shorter times to recurrence and worse outcomes at both early and late stages of HC than did those without PKM2 expression. These findings further confirmed that aerobic glycolysis is essential for development of HC and that PKM2 is a useful biomarker for predicting HC recurrence and outcome.
A variety of risk factors for HC are well established, including chronic inflammation of biliary ducts and cirrhosis [
1]. Also, a previous study identified the proteomic signatures associated with lung cancer and chronic obstructive pulmonary disease and revealed that the relevant signaling between the two diseases was related to inflammatory signaling and gluconeogenesis pathways [
20]. Consistent with these findings, we observed overexpression of inflammatory signaling (ANXA4, ANXA6, etc.) (data not shown) and glycolysis (PKM2) pathways in HC and chronic inflammation of biliary ducts, suggesting that PKM2 expression contributes to tumorigenesis induced by chronic inflammatory diseases for certain cancers. Further analysis revealed that PKM2 expression was gradually upregulated as HC progressed. The predominant expression pattern was increased cytoplasmic PKM2 expression in cancer cells. However, we found nuclear staining for PKM2 at the front edges of invasion margins of primary tumor and metastatic cells. Therefore, nuclear PKM2 expression may be important to HC invasion and metastasis.
NI is highly associated with postoperative recurrence of and poor prognosis for HC [
15]. Researchers have demonstrated that the molecules NGF, neural cell adhesion molecule, MMP, Ach, and transforming growth factor may predict NI of HC
. In the present study, we found that the PKM2 expression rate in HC cases with NI was markedly higher than that in cases without NI, indicating that PKM2 may participate in the NI process. Moreover, PKM2 expression increased along with neural invasiveness of HC, and we observed cytoplasmic PKM2 expression at each step of NI, indicating a relationship between PKM2 expression and HC cells’ ability to adhere to, invade into, and survive in nervous tissue and thus metastasize via the nervous system. The critical role of PKM2 in tumorigenesis in promoting the Warburg effect makes PKM2 an attractive target for cancer therapy. However, PKM2 knockdown has not affected the growth of different types of tumors owing to the fact that slowing glycolysis generates macromolecule precursors, which support cell proliferation [
14]. In our study, we observed modest impairment of survival and proliferation of HC cells after knocking down PKM2 expression. However, the rate of NI of tumors with PKM2 expression was markedly higher than that of NI of tumors without PKM2 expression. Meanwhile, PKM2 was weakly expressed in normal nervous tissue, suggesting an ectopic relationship between PKM2 expression and this tissue. These data suggest that PKM2 expression is positively correlated with NI of HC and is an indicator of NI of and prognosis for HC.
Mechanistically, in HC cells, knockdown of expression of PKM2 decreased the expression of NGF, MMP7, MMP9, and SDC2, molecules potentially related to NI. NGF facilitates cancer cell proliferation by binding to TrkA receptors in the perineurium [
21]. MMP7 and MMP9 are expressed in cancer cells that invade nervous tissue [
15]. SDC2 is a novel PNI-associated gene in pancreatic adenocarcinoma cases, with SDC2 silencing markedly reducing the motility and invasiveness of the cancer cells [
22]. Therefore, PKM2’s mechanisms in regulating NI of HC may be associated with NGF, MMPs, and SDC2. Additionally, silencing of PKM2 leads to downregulation of expression of sodium-coupled neutral amino acid transporter 1 (a potential oncogene) [
23] and upregulation of that of metastasis suppressor 1 [
24], supporting a crucial role for PKM2 in tumor development and progression. However, expression of another candidate tumor suppressor, PCDH9 [
25], was remarkably decreased in HC cells when PKM2 activity was blocked, suggesting an alternative mechanism in PKM2-knockdown-resistant cells.
Expression of the cell surface adhesion receptor SDC2 is increased in a variety of cancers [
26,
27] and correlated with progression and poor prognosis [
28]. Our data demonstrated that SDC2 expression was highly associated with progression and outcome of HC. SDC2 mediates cell adhesion and migration via several signaling pathways, such as syntenin-1, integrin α2, and RACK1 [
29‐
31], and synthesis of SDC2 is enhanced by transforming growth factor-β and melanocortin 1 receptor [
32,
33]. In the present study, we found that knockdown of PKM2 expression decreased SDC2 expression in HC cells. Clinical evidence confirmed a significant correlation between PKM2 and SDC2 expression. Furthermore, PKM2 expression co-localized with SDC2 expression in the majority of HC cells. Thirty-seven HC cases were positive for both PKM2 and SDC2, whereas 26 cases were negative for both of them, accounting for 72 % of all cases. This finding suggested that SDC2 is a novel target of PKM2 and plays an important role in regulation of HC cell migration.
Metabolic targeting has proven to be effective in preventing the development of cancer. Compounds targeting key metabolic steps in glycolysis have inhibited the proliferation, invasion, and metastasis of cancer cells [
14]. Authors recently reported that treatment with metformin, an antidiabetic drug, acts on cell metabolism, inhibiting all energy-consuming processes and cell proliferation [
34,
35]. Evidently, metformin directly inhibits the enzymatic function of HK1 and HK2 in breast cancer cells and markedly reduces tumor glucose consumption and growth [
36]. Our present findings are consistent with those of previous studies in demonstrating the inhibitory effect of treatment with metformin on HC cell proliferation and tumor growth. Furthermore, we demonstrated that metformin decreased PKM2 and SDC2 expression in a dose-dependent manner. These results strongly suggest that inhibition of PKM2 and SDC2 expression contributes to the therapeutic potential of metformin in HC patients.
Competing interests
No potential conflicts of interest were disclosed.
Authors’ contributions
GY, WY, GJ, and YC carried out the cellular and molecular studies, participated in other experiments and drafted the manuscript; GY and YC performed both human and mouse HC pathology studies; TX, AY, DX, WF carried out animal experiments. ES participated in the design of the study and performed the statistical analysis; ZL, and KX conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.