4.2. Agents Interacting with HuR Inducing Chemoresistance
PIM1 represents a hypoxia-inducing, pro-oncogenic, serine-threonine kinase that only recently turned out to be a key regulator of hypoxia-induced chemotherapy resistance in PDAs. The molecular mechanism underlying its overexpression in pancreatic carcinomas is based on the presence of cis-acting AREs in the PIM1’s mRNA 3′ untranslated region, which mediates an interaction with HuR in a tumor hypoxia context 
. More specifically, HuR, in response to hypoxic stress, translocates from the nucleus to the cytoplasm of PDA cells and stabilizes PIM1 mRNA transcript, causing PIM1 protein overexpression. The HuR-mediated PIM1 protein overexpression prevents cancerous cells from hypoxia through phosphorylation and inactivation of BAD (Bcl-2-associated death promoter) and activation of MEK 1/2 (mitogen activated protein kinase kinase) 
. Selective inhibition of HuR by MS-444 blocks its homodimerization and its cytoplasmic translocation, therefore rendering the PDA cells susceptible to oxaliplatin and 5-FU 
. These results elucidate the role of HuR and its prosurvival properties in PDA and provide evidence that its selective inhibition and disruption of PIM1 regulation could be the key to interrupting this chemotherapeutic resistance mechanism.
IDH1 is a NADPH (nicotinamide adenine dinucleotide phosphate)-generating enzyme that has been demonstrated to be posttranscriptionally stabilized by HuR; via this posttranscriptional regulation, HuR manages an anti-ROS (reactive oxygen species) defense system. It is well known that HuR protects PDA cells not just from hypoxia but also from nutrient-derived stress 
. A study conducted by Zarei et al. showed that, under nutrient-deprived conditions, PDA cells were less sensitive to gemcitabine in PDA xenografts in hypoglycemic mice, compared with the hyperglycemic mice 
. Similar results were observed in a retrospective study of patients with elevated serum glucose levels treated with gemcitabine, as they revealed an improved OS (overall survival). Furthermore, Zarei et al. 
identified an enhanced antioxidant defense as a driver of chemoresistance. More precisely, ROS levels were increased in vitro either by nutrient deprivation or gemcitabine treatment, but withdrawing nutrients from PDA cells before gemcitabine treatment enhanced this effect 
. However, HuR expression reduced ROS levels under low glucose, whereas HuR silencing augmented ROS levels. Investigation via CRISPR and RNAi (RNA interference) of the factor responsible for maintaining survival of PDA cancer cells under nutrient-deprived conditions revealed HuR to be the implicated agent. Importantly, studies in HuR-null PDA cell lines demonstrated IDH1 as the single HuR-regulated antioxidant enzyme 
. These findings support the notion that selective inhibition of HuR could break the HuR-IDH1 regulatory axis and serve as a promising therapeutic target.
Lal et al. 
demonstrated that stressing PDA cancer cells with DNA damaging anti-cancer agents (carboplatin, cisplatin, oxaliplatin, mitomycin C and PARP-inhibitors) resulted in HuR’s translocation from the nucleus to the cytoplasm 
. What is even more interesting, is that HuR knockdown in PDA cells resulted in their sensitization to the above agents, while HuR’s overexpression led to resistance. HuR was implicated with DNA-damaging anti-cancer agents by the acute upregulation of WEE1. Actually, WEE1, a mitotic inhibitor kinase, participates in the regulation of the DNA damage repair pathway, and its therapeutic inhibition along with chemotherapy is currently under clinical trials investigation for cancer treatment 
. Furthermore, Lal et al. demonstrated the role of WEE1 as a HuR target, both in vitro and in vivo, by revealing the direct binding of HuR to WEE1 mRNA and that HuR small interfering RNA (siRNA) knockdown and/or overexpression affects the WEE1 protein levels, especially following DNA damage. HuR stimulation of WEE1 subsequently leads to an increase in the γH2AX levels, causes Cdk1 phosphorylation and facilitates cell cycle arrest at the G2/M transition. Additionally, they demonstrated a novel acute checkpoint mechanism that involves WEE1 and by which cells can block and potentially withstand any sudden DNA damage insult experienced 
. Taking these parameters into consideration, targeting the HuR-WEE1 interactions could be a promising novel approach towards patients receiving chemotherapy and, thereby, enhancing their therapy’s outcomes. Generally, therapies focusing on translocating targets, such as HuR, and its targeting mRNAs, such as WEE1, could turn out to be more efficient therapeutic strategies than the targeting of PDA cells’ genetic alterations 
GPRC5A (G protein-coupled receptor class C group 5 member A) is a protein coding gene that demonstrates a dual behavior—acting as an oncogene in certain cancers and as a tumor suppressor in other cancers 
. Zhou et al. 
attempted to establish the impact of GPRC5A overexpression in PDA cell lines and provided an association between its overexpression and HuR’s role in pancreatic cancer. After exhibiting that GPRC5A mRNA levels hold the second highest average expression among different cancer types in pancreatic cancer, they examined and compared its expression levels in normal pancreatic tissues, primary PDAs and metastatic tumors. As a result, GPRC5A was shown to be overexpressed in primary and metastatic PDA tumors, and even more in the metastatic sites 
. This was further validated through immunohistochemical analysis. In an attempt to analyze the posttranscriptional regulation of GPRC5A, the authors performed luciferase assays, demonstrating that HuR binds to at least one site in the 3′-UTR of GPRC5A. More specifically, after the cellular stress caused by gemcitabine treatment, HuR translocates to the cytoplasm where it binds with the GPRC5A mRNA and induces a monotonous increase in GPRC5A protein levels for at least 18 h. After this, HuR and GPRC5A mRNA association returns to background levels and its posttranscriptional control decreases 
. Additionally, GPRC5A levels were increased after treatment both with 5-FU and oxaliplatin suggesting that other factors could also be involved in regulating GPRC5A expression in response to chemical stressors. These interactions of GPRC5A with HuR, gemcitabine and the other chemotherapeutic agents imply a potential pro-oncogenic role for this gene; therefore, targeting of these interactions could augment the death rate of pancreatic cancer cells post-chemotherapy treatment.
TRAIL (TNF-related apoptosis-inducing ligand) is a type II transmembrane protein harvesting an important role in cancer onset, progression and apoptosis 
. TRAIL directly induces apoptosis by engaging cell surface death receptors (DR) DR4 and DR5, constituting a possible molecular target in cancer therapeutics. It has been previously demonstrated that HuR and DR5 expression share an inverse relation in vitro and in PDA patient tissues. Additionally, HuR is capable of binding to DR5 mRNA and suppressing its protein expression, leading to a decrease in apoptosis 
. Nevertheless, due to the fact that DR4, and not DR5, has been proven to be a more potent trigger for TRAIL-induced apoptosis in PDA cells 
, Romeo et al. studied the effects of HuR levels and their correlation with DR4 expression levels and TRAIL resistance in PDA 
. In order to obtain information regarding the association of DR4 and DR5 expression with PDA cell sensitivity to soluble (s)TRAIL treatment, the authors compared the IC50
for every cell line from the sTRAIL killing curve, revealing a strong direct correlation among DR4 cell surface expression and TRAIL sensitivity, compared with the association observed among DR5 expression and TRAIL sensitivity 
. They further demonstrated that HuR not only translocates to the cytoplasm in response to sTRAIL treatment but also that HuR has the ability to posttranscriptionally bind DR4 mRNA through the 3′-UTR. Additionally, they utilized specific siRNA to silence HuR in PDA cell lines in the presence of sTRAIL, which resulted in an increase in DR4 cell surface protein expression, suggesting that HuR plays a role in the downregulation of TRAIL-induced DR4 mRNA expression 
. Therefore, strategies focused on decreasing HuR cytoplasmic concentration in PDA patients could enhance the efficacy of certain treatment regimens, such as TRAIL.
Poly (ADP-ribose) polymerase (PARP) is a family of proteins involved in a number of cellular processes, such as DNA repair, genomic stability and programmed cell death 
. Although PARP inhibitors (PARPi) initially showed promising results, it turned out that most tumors would develop drug resistance 
. Chand et al. 
demonstrated that the antitumor response to PARPi in PDA is largely controlled by the HuR-dependent stabilization of poly (ADP-ribose) glycohydrolase (PARG) 
. More specifically, they attempted to assess the role of HuR in PARPi response in PDA cell lines via HuR’s knockout. As a result, CRISPR knockout of HuR ensued a 20-fold increase in sensitivity to several PARPis (olaparib and veliparib) 
. Furthermore, they demonstrated that PARPis induced a cytoplasmic HuR translocation, which, however, could be blocked using a small molecule inhibitor, MS-444, that would prevent HuR dimerization. Additionally, HuR inhibition with MS-444 resulted in a significant decrease in PARG expression and an associated accumulation of total polyADP-ribosylation (PARylation) 
. Taking these data together, inhibition of HuR inhibits PARG overexpression and function and could possibly be utilized to enhance the efficacy of PARPi.
Considering, the aforementioned, properties of HuR and the effects that it is capable of exerting both in tumor progression and in therapy induction, it automatically raises the probability of becoming a potential drug target. Inhibiting HuR could assist in overcoming the major issue of chemoresistance that PDA cancer patients encounter, because to date, no matter of the protocol used, PDA entails one of the most dismal prognoses among cancers.