Role of hepatocyte nuclear factor 4 alpha in cell proliferation and gemcitabine resistance in pancreatic adenocarcinoma

Due to the lack of early diagnosis and limited therapy options, pancreatic ductal adenocarcinoma (PDAC) is still among the most lethal tumors and is the fourth leading cause of cancer-related deaths in the United States [1, 2]. Despite enormous progress in the treatment of many other tumor types in recent years, the main treatment for PDAC, other than surgery, is still based on gemcitabine alone or in combination with other chemotherapeutic agents [3]. However, the effect of gemcitabine and other chemotherapeutics remains unsatisfactory, partly due to the intrinsic chemoresistant characteristics of pancreatic cancer cells [4]. Therefore, exploration of potential targets to overcome gemcitabine resistance, which may highlight promising directions for developing novel therapeutic strategies, are urgently needed.

Hepatocyte nuclear factor 4α (HNF4α or NR2A1), is an orphan nuclear receptor that is expressed in pancreas, liver, kidney and intestine [5, 6]. HNF4α has been widely studied as an important regulator of hepatic functions, such as glycometabolism, fatty acid metabolism, and drug metabolism [7, 8]. Although there is abundant evidence that HNF4α plays an important role in hepatic development, its role in the regulation of tumorigenesis and cancer development has been less widely studied. Darsigny et al. reported that HNF4α was highly expressed in colorectal cancer tissue samples and promoted murine tumor development by targeting redox-related genes [9]. Conversely, another study suggested that low HNF4α expression in colorectal cancer reduced the expression of the tumor suppressor gene CDX2 and that the lack of HNF4α function promoted tumor progression [10]. Moreover, a recent study has suggested a role for HNF4α in chemoresistance in gastric cancer, in which they reported that HNF4α may enhance multidrug resistance by regulating cell apoptosis and expression of B-cell lymphoma 2 (Bcl-2) [11]. However, the role of HNF4α in PDAC development has not been reported widely and needs further investigation.

As a highly hydrophilic chemical, gemcitabine requires integral membrane transporters to function as a mediator for drug intake. It has been reported that a membrane transporter protein, human equilibrative nucleoside transporter 1 (hENT1), is responsible for the majority of gemcitabine uptake [12, 13]. Once it is in the cytoplasm, gemcitabine undergoes rate-limiting steps, as the monophosphate is phosphorylated by deoxycytidine kinase (dCK) to generate its active metabolites, gemcitabine diphosphate and gemcitabine triphosphate [14, 15]. The most important mechanism of gemcitabine cytotoxicity is inhibition of DNA synthesis, in which ribonucleotide reductase (RR) plays an important role [16‐18]. First, the diphosphate form of gemcitabine can inactivate RR subunit M1 (RRM1) and RR subunit M2 (RRM2). Additionally, gemcitabine triphosphate can embed into DNA chains, which causes termination of DNA synthesis, resulting in cancer cell apoptosis [19]. In brief, hENT1, dCK and RR are central proteins that contribute to gemcitabine cytotoxicity, and many previous studies have confirmed that their expression correlates with gemcitabine efficacy and patient prognosis [13, 20, 21].

In the present study, we explored the function of HNF4α and its underlying mechanism in PDAC. Our study provided novel findings, including that HNF4α silencing resulted in decreased cell proliferation and increased gemcitabine sensitivity, which was partly due to transcriptional repression of hENT1. Collectively, our present study uncovered novel predictive and treatment targets that show promise for improving overall survival for PDAC.

Considering the significant role of gemcitabine in adjuvant therapy and the treatment of patients with unresectable PDAC, elucidating the underlying mechanisms of gemcitabine resistance could improve treatment response [19, 34]. In the present study, we investigated the role of HNF4α in PDAC. Our results indicated that HNF4α enhances pancreatic cancer cell proliferation and promotes gemcitabine resistance by downregulating the transcription of hENT1. Moreover, HNF4α levels constitute an important prognostic factor for patients with PDAC.

HNF4α is a highly conserved nuclear receptor belonging to the nuclear receptor (NR) superfamily [6]. As the third class of nuclear receptors, orphan receptors are characterized by a lack of endogenous ligands [35]. Despite a well-known role in maintaining cellular homeostasis, NRs have also been found to participate in cancer proliferation and progression [36, 37]. NRs play diverse roles in the oncogenesis and progression of many cancers. Estrogen receptor α and androgen receptor are widely recognized as oncogenes, while other NRs, such as proliferator-activated receptor γ, may have inhibitory functions on cancer proliferation [38‐40]. Moreover, certain NRs may have opposing functions in different cancer types. For example, the orphan nuclear receptor NR4A1 (Nur77) played a role in suppressing hepatocellular carcinoma by inducing a glycolysis to gluconeogenesis switch, whereas it facilitated cancer cell proliferation via the ROS/endoplasmic reticulum stress pathways in pancreatic cancer [41, 42]. NRs also play an important role in regulating drug metabolism in cancer therapy. Holbeck et al. reported that cancer sensitivity to microtubule-disrupting drugs, such as derivatives of vinblastine, colchicines, and Taxol, was increased in cells expressing low levels of NR2F2 based on NR expression profiling, while the high levels of the orphan receptor tailless were correlated with 9α-fluoroprednisolone sensitivity [43].

Considering the significant roles of NRs in cancer development and the high expression of HNF4α in gastrointestinal cancers, such as colorectal carcinoma [43], we investigated whether HNF4α regulated the biological behaviors of pancreatic cancer. We found that abrogation of HNF4α expression inhibited pancreatic cancer cell proliferation and induced cell apoptosis, with increased expression of the cyclin-dependent protein kinase inhibitors p21 and p27. HNF4α has been reported to inhibit hepatocyte proliferation via several proposed mechanisms, including direct inhibition of mitogenic genes, regulation of the c-Myc and cyclin pathways, and an HNF4α-driven miRNA feedback loop [36]. Our results indicated that HNF4α may promote cell proliferation via the cyclin pathways in pancreatic cancer, but further investigation is needed to confirm this hypothesis. Moreover, in vivo experiments are required to support the oncogenic role of HNF4α in PDAC in the future.

The mechanisms of chemoresistance of PDAC may involve the hypoxic tumor microenvironment and deficient vascularization, remodeled metabolism, the capacity for epithelial-mesenchymal transition and altered key signaling pathways, such as the PI3 K/Akt and NF-κB signaling pathways [19, 44‐49]. As a key transporter for the cellular uptake of gemcitabine, the downregulation of hENT1 is one of the recognized mechanisms of gemcitabine resistance [19]. Although the role of hENT1 as a predictive molecule for the response to gemcitabine-based therapy has been confirmed by many studies using adjuvant treatment and studying patients with unresectable tumors, in which higher expression of hENT1 was correlated with better survival, the molecular mechanisms of hENT1 regulation have seldom been described [20, 50‐52]. Pandolfi et al. reported that high levels of glucose could upregulate the formation of hCHOP-C/EBPa, a complex that represses hENT1 expression [53]. A. Hesler et al. found that hENT1 could be negatively regulated by the matricellular protein cysteine-rich angiogenic inducer 61, but the underlying mechanism requires further exploration [54]. Hu et al. proposed the role of FBW7 in the regulation of hENT1 at the protein level, possibly via the inhibition of the lysosome degradation by hENT1 [24].

Our study revealed that hENT1 could be regulated by HNF4α at the transcriptional level. HNF4α mainly binds to a 6-bp repeat (AGGTCA) with a nucleotide spacer called direct repeat 1 (DR1) [55, 56]. Our results showed that HNF4α recognized the consensus binding site located on the promoter region of hENT1 and activated downstream transcription. Moreover, a relatively nonconserved region with abundant CpG dinucleotide could also be recognized by HNF4α, which indicates that HNF4α might participate in the methylation process of hENT1. A previous study demonstrated DNA methylation-independent hENT1 downregulation, which correlated with acquired gemcitabine resistance in cancer cells [57]. Our study supports the need for further investigation of the underlying mechanism of methylation-dependent hENT1 regulation. Moreover, after binding to DNA in the form of a homologous dimer, how HNF4α recruits transcriptional corepressors and accessory proteins to regulate hENT1 is unclear. HNF4α has been reported by previous studies to interact with chromatin modifiers, such as PRMT1 and BMI1 [58, 59], suggesting a potential role of HNF4α in chromatin methylation. Furthermore, in addition to the expression level of hENT1, hENT1 transporter activity is another important factor that affecting the uptake of nucleoside analogs. A study has reported that although the mRNA level of hENT1 in chronic lymphocytic leukemia cells is increased with the presence of interleukin 4 (IL-4), the hENT1-dependent uridine uptake remained unchanged [60]. Another study using a murine model of chronic myelogenous leukemia showed a decreased ENT1 activity reduction following reduced mRNA level of ENT1 [61]. Therefore, the transcription of hENT1 may not perfectly correlated with the uridine uptake activity, dual targeting hENT1 transcription and transporter activity might provide the utmost efficacy in gemcitabine utilization. Thus, a protein interaction screening strategy is needed to determine whether HNF4α is involved in epigenetic regulation or posttranslational modification of hENT1.

In conclusion, we demonstrated that HNF4α functioned as a tumor promoter gene that was upregulated in pancreatic cancer. Moreover, we discovered that higher expression of HNF4α is correlated with poorer prognosis in PDAC patients. HNF4α promotes pancreatic cancer cell proliferation and reduces gemcitabine-induced cell apoptosis. Mechanistically, we found that HNF4α could downregulate hENT1, a membrane transporter of gemcitabine, at the transcriptional level. Taken together, these findings suggest that HNF4α may serve as a potential novel predictive and therapeutic target for pancreatic cancer.