PDAC and Other Lead Causes of Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC), which constitutes 90% of pancreatic cancers, is the fourth leading cause of cancer-related deaths in the world. Unfortunately, it is expected to become the second leading cause of cancer death by 2030, surpassing lung, liver, breast and prostate cancers. This is partly due to increase in incidence of pancreatic cancer and better management options and survival outcomes of other cancers. Currently, available therapeutic options for PDAC are surgery, radiation, chemotherapy, immunotherapy, and use of targeted drugs. Even though, surgery is the cornerstone therapy of PDAC, only 15-20% are candidates for surgery, presenting with localized disease. Majority of the people, around 50% are diagnosed late and present with metastases. In such cases, chemotherapy, either curative or palliative becomes the treatment of choice. The first line therapy of metastatic pancreatic cancers includes Gemcitabine, nab-Paclitaxel and newer combination therapies like FOLFIRINOX and gemcitabine combination therapies. The major challenge in the treatment of pancreatic cancer is its resistance to drug treatments. Several factors such as tumor heterogeneity, stromal density, various inflammatory cells and mediators cause PDAC to develop chemoresistance4. Here, we review the chemotherapy options for PDAC and the various elements involved in chemoresistance to drug treatments.

Gemcitabine

Gemcitabine (dFdC: 2′, 2′difluorodeoxycytidine) is a synthetic pyrimidine, deoxycytidine nucleoside analog. It was approved for treatment of various cancers since 1995. The cytotoxic effects of Gemcitabine are mediated through DNA synthesis inhibition and cell cycle progression blockade at the G1/S-phase. In comparison with other nucleoside analogs, Gemcitabine possess distinct characteristics in terms of mechanism of action, metabolism and pharmacokinetics. The substitution of two hydrogen molecules with fluorine in the deoxycytidine ring and the resulting structural difference between the fluorine molecules in Gemcitabine imparts unique features to the drug such as broad range of disease activity and anti-tumor properties. The intracellular metabolism of Gemcitabine is shown in Figure1. Firstly, Gemcitabine enters into the cells from the blood stream and then it undergoes three-time phosphorylation in order to exert its cytotoxic effects. Chemically, Gemcitabine is hydrophilic in nature and requires various nucleoside transporters such as human concentrative nucleoside transporters (hCNTs) and human equilibrative nucleoside transporters (hENTs, to cross the lipid cell membrane. Therefore, the presence of nucleoside transporters and subsequent intracellular uptake is crucial step in DNA synthesis inhibition and clinical efficacy of the drug. Human cell kinetic studies have demonstrated that the intracellular uptake occurs primarily through hENT1, followed by hENT2, hCNT1 and hCNT3.

Once inside the cell, gemcitabine is activated through a three-step phosphorylation, resulting in the active metabolite, gemcitabine triphosphate (dFdCTP). Because of its structural similarity, dFdCTP competes with deoxycytidine triphosphate (dCTP) in DNA synthesis. Therefore, dFdCTP gets incorporated into DNA, in place of dCTP, during replication. This results in inhibition of chain elongation and masked termination of DNAchains and eventually leads to apoptotic cell death5. Moreover, dFdCDP (difluorodeoxycytidine diphosphate), a metabolite of Gemcitabine, regulates DNA biosynthesis through inhibition of the enzyme ribonucleotide reductase (RR). Nevertheless, the potential of gemcitabine is dependent on the presence of cytosine deaminase (CDA), an intracellular enzyme, that coverts most of the gemcitabine into a less active metabolite, 2′, 2′ -difluorodeoxyuridine (dFdU).

Starting early 1997, gemcitabine has been approved as a first-line medication in the treatment of advanced and metastatic PDCA, primarily in patients with a good patient performance status score. In different individual studies, Gemcitabine had demonstrated superior efficacy compared to its predecessor, 5-Fluorouracil (5-FU). In a comparative phase III study, comprising of 126 people, patients were assigned to treatment with either gemcitabine or 5-FU. An analysis of the results showed that Gemcitabine was more efficient than 5-FU, in terms of clinical benefit proportion (23.8% of gemcitabine-treated patients vs. 4.8% of 5-FU-treated patients), median survival period (5.6 months with Gemcitabine vs. 4.4 months for 5-FU), and one-year survival rate (18% for gemcitabine treatment vs. 2% for 5-FU treatment)11. Besides, gemcitabine showed substantial benefit in improving patients’ disease-related symptoms such as pain, performance status and weight. The clinical efficacy of gemcitabine was reinforced by another phase II/III successive clinical trials, which demonstrated a positive or partial positive response to gemcitabine, in terms of clinical benefit (5.4% to 12%), overall survival (OS) time (5 to 7.2 months), one-year survival (18%) and median survival time (6.2 months). In addition, the studies also demonstrated high safety profile to Gemcitabine, in terms of its lower systemic toxicity compared to 5-FU.

Gemcitabine Combination Therapy

Several combination drugs were tested in clinical trials, some of which showed increased response rate in terms of survival rates compared to gemcitabine alone, but at the cost of increased toxicity and adverse effects. Beginning of 2005, a combination regimen, consisting of cisplatin, epirubicin, fluorouracil, and gemcitabine (PEFG), was tested in a small patient population with advanced PDAC, who were refractory to Gemcitabine alone. PEFG demonstrated superior efficacy in all parameters including overall survival and clinical benefit ratio. However, it resulted in increased incidence of hematological adverse effects such as anemia and thrombocytopenia, compared to gemcitabine. Later, a Phase III trial with a different combination therapy, gemcitabine and erlotinib (EGFR inhibitor) was tested for the treatment of advanced and metastatic PDCA. The combination showed moderate improvement in both median survival rates (23% vs. 17%) and overall survival (6.2 vs. 5.9 months). Based on these results, Food and Drug Administration (FDA) approved gemcitabine/erlotinib combination and it became a preferred option for treatment of advanced, unresectable pancreatic tumors. Other combination drugs such as: capecitabine with oxaliplatin (Cape-Ox) and gemcitabine, docetaxel, and capecitabine (GTX) were proved to be efficient but restricted to good PS patients. However, other combination drugs (eg. gemcitabine and cisplatin/oxaliplatin) showed only moderate or no significant improvement.

FOLFIRINOX

FOLFIRINOX is a combination chemotherapy regimen consisting of FOLinic acid, Fluorouracil, IRINotecan, OXaplatin. It has demonstrated significant clinical benefits over gemcitabine alone, in phase I clinical trials and phase II/III studies, as well. In comparison with Gemcitabine, FOLFIRINOX was found to be superior in all efficacy parameters, including overall survival (11.1 vs. 6.8 months), one-year survival rate (48.4% with FOLFIRINOX vs. 20.6% with Gemcitabine) and progression-free survival (6.4 vs. 3.3 months). Moreover, patients on FOLFIRINOX reported a significant reduction in the deterioration of quality of life, compared to gemcitabine. However, the combination regimen showed increased incidence of adverse effects, such as grade 3 or 4 thrombocytopenia, febrile neutropenia, diarrhea, and grade II alopecia23, compared with gemcitabine. In order to improve the safety profile of the medication, different modifications such as, mFOLFOX-folinic acid, fluorouracil, oxaliplatin- or FOLFIRI-folinic acid, fluorouracil, irinotecan are currently under investigation,. Today, FOLFIRINOX is considered as a first-line treatment option for patients with age D.

Abraxane

Taxanes, such as docetaxel or paclitaxel, have been tested in the treatment of PDAC, but their effectiveness was highly reduced due to poor solubility and subsequent delivery to target organs. This led to the development of nab-paclitaxel. Nab-paclitaxel is a 130-nm, albumin-bound formulation of paclitaxel without any solvent. Nab-paclitaxel particles have a reduced diameter that enhances intracellular paclitaxel delivery, thus increasing antitumor activity. An initial phase I/II trial of Abraxane showed 48% response rate and a median overall survival of 12.2 months. Interestingly, when Abraxane was combined with Gemcitabine, a synergistic response was observed in patients with advanced pancreatic cancer, which was attributed to the improvement in the intra-tumoral delivery of both the drugs by fused albumin. In a phase III trial, with a patient population of 861, the combination treatment showed substantial advantage in all clinical efficacy parameters, including overall survival time (8.5 vs. 6.7 months), progression free survival (5.5 vs. 3.7 months) and one-year survival rates (35% vs. 22%)28, compared to gemcitabine single agent therapy. Conversely, the combination treatment was accompanied by a considerable increase in incidence of adverse events, including grade 3/4 neutropenia, leukopenia, neuropathy and febrile neutropenia. Another positive aspect about Abraxane is that it has demonstrated significant clinical benefits and improvement in quality of life in patients with poor performance status, according to a recent case study.

Chemoresistance

PDAC is notorious for developing chemoresistance that critically impacts the efficacy of gemcitabine. There are two types of chemoresistance: intrinsic and acquired. Intrinsic resistance refers to a condition where chemotherapy is ineffective from the beginning of treatment probably due to patient’s genetic factors, whereas acquired resistance develops only after a certain period of exposure of tumor cells to anticancer drugs, due to genetic or epigenetic alterations in the tumor cells. In acquired resistance, tumor cells might be sensitive to chemotherapy in the beginning of treatment, but continuous treatment eventually makes them refractory to chemotherapy. Pancreatic cancer cells are usually more susceptible to gemcitabine therapy compared to other anticancer agents, however, chemoresistance is observed within weeks of beginning treatment, leading to poor survival. Several mechanisms are involved in controlling both intrinsic and acquired chemoresistance, including enzyme regulation and signaling pathways.

Gemcitabine metabolism pathway related chemoresistance: Gemacitabine chemoresistance ensues due to interplay of several factors in its metabolism pathway including: drug transporters, activating and inactivating enzymes and competitive substrates to active metabolites. Their roles in pancreatic cancer chemoresistance are discussed here.

Nucleoside Transporter (NT) Downregulation

As discussed previously, nucleoside transporters are required for the influx of gemcitabine into the target cells. In patients treated with gemcitabine, a direct relation between the level of expression of nucleoside transporters and survival outcomes has been observed. While patients with low levels of hENT1 and hCNT3 showed a significantly worse survival compared to patients with high levels of NTs, those with lack of NT expression were resistant to gemcitabine cytotoxicity altogether. Therefore, the levels of hENT1 and hCNT3 are considered to be good predictors of gemcitabine response in pancreatic cancer patients. Moreover, in vitro studies have demonstrated an enhanced expression of hENT1 in tumor cells after pretreatment with thymidylate synthase (TS) inhibitors, thus augmenting the cytotoxic effects of gemcitabine.

The association of hCNT1 with gemcitabine cytotoxicity is not yet well established and is of debate. Few studies have demonstrated a decreased expression of hCNT1 in pancreatic tumor cells compared to normal pancreatic cells. Moreover, tumor cells also demonstrated gemcitabine resistance via downregulation of hCNT1 and subsequent decreased transport of gemcitabine through the cell membrane. This indicates the possibility of augmenting gemcitabine cytotoxicity via pharmacological inhibition of hCNT1 degradation38.

Applying this knowledge, many novel mechanisms regulating NT activity have been explained and, drugs targeting the proteins involved in the regulatory pathways, such as, MUC and ErbB2 receptors might boost gemcitabine cytotoxicity. Abnormal expression of Mucin 4 (MUC4), a transmembrane glycoprotein, by the pancreatic cancer is associated with increased invasiveness of the tumor cells, and inversely correlated with prognosis, as well. MUC4 is also associated with inhibition of hCNT1 expression via the NF-κB pathway, eventually resulting in greater resistance to gemcitabine. Moreover, studies have shown that inhibition of MUC4 stimulated increased expression of both hCNT1 and hCNT3, leading to enhanced gemcitabine sensitivity. The transmembrane oncogenic receptor, ErbB2 works similar to MUC4 in inducing resistance to gemcitabine and suppression of ErbB2 leads to upregulation of hCNT1 and hCNT3 expression, ultimately resulting in increased sensitivity to gemcitabine.

Deoxycytidine Kinase (DCK) Downregulation

The main rate-limiting enzyme responsible for intracellular activation and metabolism of gemcitabine is deoxycytidine kinase. There is a direct correlation between dCK activity and degree of gemcitabine sensitivity. Human pancreatic cancer cells display acquired resistance to gemcitabine in an age dependent fashion, through inactivation of dCK. Studies have shown that suppression of dCK resulted in gemcitabine resistance whereas, overexpression of dCK into gemcitabine-resistant cell lines resulted in restored gemcitabine sensitivity,. Studies have shown that the pretreatment levels of dCK and its expression are stable even after development of resistance to gemcitabine. This suggests that determination of dCK levels prior to initiation of gemcitabine therapy may improve patient survival by identifying those who are less likely to respond to the treatment. The mechanism of dCK downregulation and gemcitabine resistance is not yet fully established. The analysis of the coding sequence of dCK in pancreatic cancer cells, and in tumor tissue from patients with disease progression while on gemcitabine treatment, did not identify any mutations. This indicated that genetic alterations or coding polymorphisms of dCK might not be a common mechanism for intrinsic resistance to gemcitabine in pancreatic cancer.

A protein involved in dCK regulation is Hu antigen (HuR), a RNA-binding stress-response protein. Compared to normal pancreatic cells, pancreatic cells treated with gemcitabine showed elevated levels of HuR. High concentration of HuR is associated with increased expression and activity of dCK. When the pancreatic tumor cells are exposed to gemcitabine, it results in cytoplasmic translocation of HuR from the nucleus, where they cause upregulation of dCK, which in turn results in increased sensitivity to gemcitabine. The efficacy of gemcitabine is directly proportional to the levels of HuR antigen and subsequent dCK activity. So, overexpression of HuR elevates dCK, while suppression of HuR reduces dCK activity. In patients receiving adjuvant treatment with gemcitabine, HuR levels are associated with mortality rates, as well. Patients with low cytoplasmic HuR expression reported a seven-fold increased risk of cancer death compared to patients with high HuR levels.

Cytidine Deaminase (CDA) Upregulation

Intracellular gemcitabine gets inactivated primarily through the enzyme cytidine deaminase. It helps in the converting dFdC to dFdU, through a process called deamination (removal of the [-NH2] group from pyrimidine). The level of CDA is inversely proportional to overall survival in pancreatic cancer patients who are on gemcitabine treatment. The higher the concentration of CDA, lesser the overall survival rate for the patients. Several in vitro studies have also demonstrated that upregulation of CDA results in gemcitabine resistance, whereas suppression of CDA reinstates gemcitabine sensitivity. This suggests that regulation of CDA levels in cancer cells ascertains to be a decent mechanism to control gemcitabine resistance. However, further studies are needed to substantiate this assumption.

Ribonucleotide Reductase (RR) Upregulation

Ribonucleotide reductase is an important enzyme in the DNA synthesis pathway. Functionally, the enzyme comprises of two subunits, M1 and M2. While the M1 subunit (RRM1) consists of a binding site responsible for enzyme regulation, M2 subunit (RRM2) is responsible for the enzyme activity. It plays a major role in the conversion of ribonucleotides to dNTPs (deoxynucleotide triphosphate), an essential step for DNA polymerization and repair. So, inhibition of RR causes a drop in the endogenous dNTP pool, thus reducing competition and indirectly facilitating incorporation of dFdCTP into DNA. Therefore, dFdCDP-induced inhibition of RR is the most important mechanism involved in the potentiation of gemcitabine activity. Studies have shown that RRM1 is one of the major factors in intrinsic resistance to gemcitabine. Moreover, there exists an inverse correlation between RRM1 levels and overall survival in patients taking gemcitabine. Higher levels of RRM1 are associated with lower survival rates and vice-versa35,44. Studies of hydroxyurea, a RR inhibitor reported a synergistic effect when prescribed along with gemcitabine. Others have reported the effects of pimasertib on RRM1 levels. Pimasertib, a MEK1/2 inhibitor causes reduced RRM1 protein expression via post-translational modification and also increased gemcitabine sensitivity. This suggests the possibility of combining MEK inhibitors with gemcitabine to improve the cytotoxicity of gemcitabine in pancreatic cancer patients. Studies on M2 subunit reported that its suppression produced enhanced gemcitabine sensitivity and vice-versa.

Thymidylate Synthase (TS) Upregulation

Thymidylate synthase is a folate-dependent enzyme that converts 2′-deoxyuridine-5′-monophosphate (dUMP) into 2′-deoxythymidine-5′-monophosphate (dTMP). dTMP is a vital precursor for DNA synthesis. The deaminated metabolite of gemcitabine, dFdUMP acts as a substrate or inhibitor of TS. Studies have shown that inhibition of TS has augmented gemcitabine sensitivity in various ways including: regulation of intracellular nucleotide pool and activation of hENT1. The benefits of TS inhibition were studied in a randomized phase II study called GEMSAP. In this study, the patients who received a combination of gemcitabine and S-1 (an oral prodrug of 5-FU) exhibited an enhanced overall survival and progression-free survival rates, compared to gemcitabine alone. Few other studies have reported a direct impact of gemcitabine on TS regulation. In gemcitabine-resistant pancreatic cancer cells gemcitabine caused downregulation of TS, whereas in gemcitabine-sensitive pancreatic cancer cells, TS became upregulated. Furthermore, TS expression has an inverse correlation with overall survival and disease free survival rates in pancreatic cancer patients. While, high TS expression produced gemcitabine resistance and shortened patient survival rates, loss of TS expression decreased gemcitabine resistance in the cancer cells.

ATP Binding Cassette (ABC) Transporters

ABC transporters are a family of integral transmembrane proteins, which transport molecules across the plasma and intracellular membranes against their gradient through energy derived from ATP hydrolysis. Structurally, these transporters consist of two hydrophobic transmembrane domains (TMDs) linked to two hydrophilic nucleotide-binding domains (NBDs). TMDs form a pore in the membrane creating a substrate-binding environment, while NBDs are localized in the cytosol,. These proteins export a wide variety of structurally diverse endogenous ligands including amino acids, peptides, vitamins, sugars, hormones, ions, lipids and xenobiotics,,. Normally, ABC transporters are involved in drug absorption, distribution and elimination, determining bioavailability of administered drugs in the gut. Therefore, the expression of these proteins may influence pharmacokinetic characteristics of administered chemotherapeutics.

Studies have demonstrated the association of several proteins in cancer chemoresistance including: p glycoprotein (P-gp)/ABCB1, breast cancer resistance protein (BCRP)/ABCG2, multidrug resistance protein 1 (MRP1)/ABCC1 and other members of ABCC subfamily (e.g., ABCC2, ABCC3). Subfamilies of ABC transporters, ABCC3 and ABCC5 were remarkably overexpressed in pancreatic tumor specimens. ABCC3 transporter mediates the export of bile salts and organic ions where as ABCC5 helps transport of nucleotide analogues. ABCC3 and its expression levels have been associated with survival of patients after tumor resection, suggesting the possibility of its prognostic aspect in PDAC. Interestingly, ABCC5 is involved in the efflux of nucleotide analogues- based drugs, such as 5-FU or gemcitabine and its levels have been directly associated with gemcitabine resistance in pancreatic cancer cell lines. Several clinical trials were initiated targeting inhibition of ABC transporters but unfortunately they failed to achieve success due to reasons such as, increased toxicity in healthy tissues (off-target action) and high doses of the drug in the blood. Therefore, alternative concepts for ABC transporter inhibition are currently being tested including miRNA regulation of ABC transporters and nanoparticle delivery of chemotherapeutics to the targeted cells.

Epithelial-Mesenchymal Transition (EMT)

Epithelial-mesenchymal transition (EMT) is a stage of phenotypic shift of epithelial like cancer cells to an elongated mesenchymal phenotype. This alteration is mediated through a shift in the balance between epithelial (e.g., E-cadherin and Claudin-1) and mesenchymal (e.g., N-cadherin, Snail, Zeb-1 and Twist-1) factors. Gene expression profiling revealed that the resistant cells showed mesenchymal phenotype consistent with EMT. Also, an inverse correlation was observed between E-cadherin and its transcriptional suppressor, Zeb-1. Several factors were documented to induce EMT such as: transforming growth factor-β (TGFβ), epidermal growth factor (EGF), hepatocyte growth factor and Notch pathway.

A study by Wang et al delineated gemcitabine resistant cells with mesenchymal phenotype and reported increased expression of Zeb-1, vimentin, fibronectin and alpha-SMA on these cells. They postulated the possibility of gemcitabine in inducing EMT. In a different study, Güngör et al examined the association of midkine (MK), a heparin binding growth factor in the interplay between Notch, EMT and gemcitabine resistance. They noticed high expression of MK mRNA in pancreatic cancer cell lines that developed gemcitabine resistance. Ma et a l recently showed a positive impact of miR-233 in EMT, invasion, migration and gemcitabine resistance in cancer cells. The association between EMT and chemotherapy resistance has been well established in the literature, but it is not yet fully known how EMT affects cancer cell survival pathways, drug transporter proteins and drug metabolizing enzymes. Nevertheless, it appears beneficial in tempering cancer EMT in combined immunotherapy and molecular-targeted drug strategies to treat pancreatic cancer.

Hypovascular tumor microenvironment and stroma induced chemoresistance: The pancreatic cancer micro environment harbors unique characteristics that directly impact. The current evidence suggests that pancreatic cancer microenvironment has both direct and consequential effects on molecular characteristics of the cancer cells. It is shown to regulate gene expression profile and drug delivery mechanisms, resulting in chemoresistance. The key players in creating hypovascular environment include pancreatic stellate cells, fibroblasts and the cancer stem cells. Immune cells such as macrophages, neutrophils and platelets also infiltrate the tumor microenvironment and play a role in chemoresistance.