2.1 Lung cancer
Lung cancer is one of the leading causes of dead in both male and female worldwide. Cigarette smoke is the main risk factors for lung cancer development, not only active smoking causes cancer but also passive exposure to tobacco can result in the development of lung cancer. Cigarette smoking accounts for about 85% of lung cancer cases worldwide. Cancer risk is dependent on age, smoking duration and intensity. About 15 to 20 % of lung cancer patients minimally or never smoked.
Prolonged exposure to cancer-promoting agents (e.g cigarette smoke, asbestos, radiation, radon, arsenic, chromates, nickel, chloromethyl ethers, polycyclic aromatic hydrocarbons, mustard gas, coke-oven emissions, primitive cooking, heating huts) and multiple genetic mutations are required to develop neoplastic respiratory epithelial cells, which leads to lung cancer development. [1]
2.2 Classification of Lung cancer types
Lung cancer can be divided into two major classifications. It is categorized as small cell lung cancer (15%) or non–small cell lung cancer (85%) [2]. Both types can be further differentiated into four major histological classes: adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma [3]. Small cell carcinomas are aggressive and usually located centrally in the lung, with extensive mediastinal involvement. They are also associated with early stage extra thoracic metastases [4]. Small cell carcinomas are often advanced at the time of diagnosis, which leads to a poor prognosis for the patient, despite its responsiveness to chemotherapeutics.
The three main subtypes of non-small cell lung cancer are adenocarcinomas, Squamous cell carcinomas and large cell carcinomas. Adenocarcinomas are peripheral tumors that metastasize early and are diagnosed in patients with underlying lung disease [3]. Squamous cell carcinomas unlike adenocarcinomas metastasize in later stages of the disease and are mostly located endobronchial.
Large cell carcinomas are associated with cigarette smoking, they grow rapidly and metastasized early. As mentioned earlier, Non-small cell lung cancer (NSCLC) represents 85 % of it. Most patients with advanced NSCLC are treated with chemotherapy regimens that provide an overall survival of less than 1 year. The 5-year survival rate in lung cancer cases did not change in the last 20 years and is less than 20 % [5]. Due to molecular analysis, tumor cells may be distinguished based on various
markers for example, the absence or presence of e.g mutations or oncogenic fusion rearrangements.
One of those markers is the KRAS gene, which KRAS belongs to the RAS family. These genes encode proteins, which are central mediators of growth factor receptor signaling [6]. In about 20-30 % of NSCLC cases a KRAS mutation is existent [7].
2.3 Kirsten-rous avian sarcoma (KRAS)
Of the numerous oncogenes involved in human cancers RAS mutations are the most common ones and is therefore a favorite to target pharmacologically. There are 3 different RAS genes that encode four proteins: HRas, KRAS 4a/b and NRas. In normal cells RAS signaling is crucial to cellular proliferation, cell survival and differentiation. In lung cancer the most commonly mutated gene is the KRAS gene [8]. Mutated KRAS proteins have lost the ability to become inactive due to the fact that the KRAS protein is constitutively bound to guanosine triphosphate (GTP) such that its GTPase activating protein (GAP) cannot bind to the RAS protein. This results in decreased apoptosis, increased cell proliferation, increased angiogenesis and disrupted cellular metabolism [9]. Yun et al. described that cancer cells with a KRAS mutant phenotype, have a consistently up regulated gene expression of the glucose transporter-1 (GLUT1) [10]. Recent studies also described a link between KRAS induced tumorigenesis and its role in metabolic changes. Furthermore it was described that the mRNA expression of Glutathione S- transferase P1 (GSTP1), was elevated in several treatment resistant cancer types. Little is known about the potential role of GSTP1 in the tumorigenesis of NSCLC harboring a KRAS mutant. It is possible that GSTP1 activation has a major impact on cellular regulation in metabolic pathways [11].
2.4 GSTP1
Glutathione S-transferases (GST’s) belong to the family of phase II detoxification enzymes. They catalyze the conjugation of glutathione to endo- or exogenous electrophilic compounds. GST‘s can be divided into 2 super-family members: the membrane bound, microsomal and the cytosolic family.
Cytosolic human GST’s can be derived into six classes: α, µ, ω, π, θ and ζ. GSTπ (in this report referred as GSTP1) and GSTµ have a regulatory role in the mitogen-activated protein kinase (MAPK) pathway. This pathway participates in cellular proliferation and death signals via protein/protein interactions with apoptosis signal-regulating kinase (ASK1) and c-Jun N-terminal kinase (JNK1).
GSTP1 binds to and inhibits Jun-N-terminal kinase (JNK), which is key regulator of cellular
proliferation, differentiation, and apoptosis. Following stimulation by growth factors or cellular stress, JNK gets phosphorylated and activated. Active JNK phosphorylates the transcription factor c-Jun, which then promotes the transcription of genes involved in cell proliferation and differentiation [12].
Most of the GST’s show a high degree of polymorphism. Furthermore dependent on the type of GST, GST’s can have several subunits. Each subunit is approximately 199- 244 amino acids (22 to 29 kD) long. More than a dozen cytosolic GST subunits have been identified. Those subunits contain a catalytic active site, the GSH- binding site (G-site) in the amino- terminal domain, which binds a hydrophobic substrate (‘H-site’) in the carboxy-terminal domain. Expression varies and is dependent on the type of GST. The functional GST enzymes are dimeric, with the exception of GSTα and GSTµ, which can form heterodimers in addition to homodimers. Due to the different subunits isoenzymes can be generated form those subunits. Isoenzymes are named according to their class and composition of present subunits, which is designated with an Arabic number. In case of GSTP1-1 this refers to the heterodimer of the two subunits 1 and 1. GSTπ has in total 2 subunits referred to as subunit 1 and subunit 2 [13]. GSTP1 for example is found mainly in brain, lung, and heart; its expression in liver decreases during embryonic development, becoming very low in non-malignant adult tissue [14].
Recently studies have shown that GST’s act as modulators of signal transduction pathways that control cell proliferation. Their involvement in the development of resistance to anti-cancer drugs has made them attractive research targets. GST isoforms have recently been identified as negative regulators of signal transduction pathways, and thereby protecting cells against apoptosis in stress response, such as Reactive Oxygen Species (ROS). GSTs are well known for its role in phase II drug metabolism, as it catalyzes conjugation of GSH to electrophiles. A variety of human cancers, including of breast, colon, kidney, lung, and ovarian, usually express high levels of GSTP1 compared to normal surrounding tissues. However recently a new role has been described for GSTP, as a catalyst in PSSG reactions [15].
2.5 Protein S-Glutathionylation (PPSG)
Reactive Oxygen Species (ROS) are chemical reactive molecules containing oxygen. These molecules include H2O2, O2-, 1O2, HO-, NO2, RO-, and ROO-. They are formed as a natural byproduct of the normal metabolism of oxygen [16]. Elevated levels of ROS and down regulation of ROS scavengers and antioxidant enzymes are associated with various human diseases including cancer [17]. ROS are normally present in all aerobic cells and are in balance with biochemical antioxidants.
Oxidative stress occurs when the balance is disrupted, because of antioxidants depletion, ROS excess or both. This can result in significant damage to cell structure like e.g. DNA damage (mutagenesis), lipid peroxidation (lipid degradation), amino acid oxidation (protein) and oxidative inactivation of specific enzymes [18]. ROS play an important role in cell signaling and homeostasis.
H2O2 for example is able to control cellular homeostasis and plays a role in de induction of the posttranslational modification of proteins, by selectively oxidizing its cysteine residues, [19,20]. It specifically targets the cysteine’s with low pKa’s, which results in a sulfenic acid intermediate protein.
Protein S-Glutathionylation (PSSG) is the formation of a disulfide bond between the protein cysteine and the glutathione (GSH) (fig. 1). Conjugation of GSH by GST’s such as GSTP1, leads to PSSG of the sulfenic acid intermediate. Due to the conjugation of protein cysteines, GSH changes the size of
the protein by 3 amino acids. This confers a negative charge. This suggest that PSSG can alter the structure and function of the target protein which can than result in an activation or inhibition of this protein [21,22]. Kinase mediated signaling pathways; also play key roles in controlling cell survival, differentiation, apoptosis and stress response. Addressing the complex interactions between GSTP1 and regulatory kinases will help to get a better understanding of the role GSTP plays in tumor pathogenesis and its cancer resistance [23,24].
Figure 1 PKM2 S-glutathionylation: Prior to s-glutathionylation, PKM2 gets oxidized at its cysteine residue, which results in the formation a sulfenic acid (RSOH) derivative form of the protein. Glutathion S Transferase P1 (GSTP1), catalyzes the s-glutathionylation reaction by forming a disulfide bond, between the oxidized cysteine and the thiol residue of the glutathione (GSH).
2.6 TLK-199
Human GSTs are of significant interest because of their high expression levels in tumor tissue and, their possible role in Protein S-Glutathionylation of target proteins. Identifying new inhibitory compounds against human GSTs therefore could utilize new anti cancer drugs [25]. As previous mentioned GSTs exists in homo or heterodimers. Each subunit has its own active site. These active sites consist of a GSH binding site (G-site), which is specific to GSH and the H-site, which binds xenobiotics [26]. At the G-site the substrate enzyme complex (GSTP1-GSH) forms when the hyrdoxyl group of Tyr7, of the GSTP1 enzyme forms a hydrogen bond with the thiol group of the GSH molecule (fig. 2A) [27]. TLK-199 or Ezatiostat hydrochloride is a prodrug and was in phase 2 clinical trial for the treatment of myelodysplastic syndrome. This prodrug has been reported to be a potent inhibitor of GSTP1-1 [28]. The actual working compound is the tripeptide TLK-117
(g-glutamyl(benzyl)cysteinyl-R(-)-phenylglycine). It’s binding affinity to the G-site of the GSTP1-1 enzyme, is greater than that of GSH and GSTP1-1 itself. It’s also highly selective to GSTP1-1. TLK-117 itself interacts and forms a bond with GSTP1. When TLK-199 passes the cell membrane it is hydrolyzed to the phenyl glycine monoethyl ester form TLK-117 [27, 29]. Inside the cell TLK-117 interacts with GSTP1 due to the formation of a hydrogen bond between the Tyr7, of GSTP1 and the thiol group of the TLK-117 molecule (fig. 2 B). This prohibits the binding of GSH to GSTP1, which imaginably inhibits s-glutathionylation of proteins. It also keeps GSTP1 from inhibiting JNK through direct protein-protein Figure 2 Schematic drawing of the residues that interact between the inhibitor/GSH and GSTP1. A) Interaction between the tyrosine of the GSTP1 enzyme and the thiol group of GSH. B) Di-esterfication of the prodrug TLK-199 to the active compound TLK-117 and schematic interaction between GSTP1 and TLK-117.
interactions [26,27,30]. TLK-117 will be used in this project to determine its effect on protein s-Glutathionylation and it’s role in KRASG12D induced tumorigenesis. TLK-117 will be tested in vitro and in vivo.
2.7 PKM2, the Warburg effect and Redox balance
In spite of the presence of an abundant amount of oxygen, cancer cells produce a large amount of lactate, also known as the Warburg effect or aerobic glycolysis. In a rich oxygen environment, normal differentiating cells metabolize glucose to pyruvate by glycolysis. The product is completely oxidized in the mitochondria to CO2, during oxidative phosphorylation. If the oxygen access is limited, cells redirect the produced pyruvate away from the mitochondria and lactate is generated. Cancer cells have the tendency to convert glucose to lactate [31]. It does not matter if oxygen is present or not. This results in minimal ATP production but a high production of waste products such as lactate. Pyruvate kinases are important regulators in the production of lactate [32]. High enzymatic activity results in the production of pyruvate, which enters the TCA cycle. Low enzyme activity of pyruvate kinase, promotes lactate production and therefore promotes the Warburg effect (fig 3). Pyruvate kinases 2 or PKM2 belongs to the pyruvate kinases and is a regulator of anti-oxidative metabolism. It plays an important role in cancer. When the activity of PKM2 is low the substrate phosphoenol pyruvate accumulates. This results in the inhibition of glycolytic enzyme triose phosphate isomerase and leads to activation of the pentose phosphate pathway (PPP). Due to increased activity of the PPP, the cancer cell is protected and promotes the adaptation against oxidative stress and ROS [33]. Anastasiou et.al showed that the activation of the PPP and the associated anti-oxidative activity are essential to the growth of cancer cells. They also established that the oxidation of PKM2 on cysteine (Cys 385), results in a lower enzyme activity of PKM2, in lung cancer cells [34]. This data suggest, that by inducing the Warburg Figure 3 Activation of the pentose
phosphate pathway and its anti-oxidative activity in cancer-cell growth. [20]
effect, cancer cell growth is promoted by activation of the PPP. This maintains the balance of the redox equivalents, providing NADPH an activation the antioxidant defense systems of cancer cells.
2.8 Impact of this project
The precise molecular based mechanisms, where changes in the redox environments of cells promote tumor growth are unknown. This study provides a complete new understanding of the role of GSTP1 as a key regulator of the Warburg effect, due to its ability to catalyze protein S-glutathionylation reactions. Significantly, activation of the PPP provides the cell with GSH [34]. This is required in the catalytic cycle of GSTP1. It provides a feed forward amplification mechanism whereby GSTP1 could induce S- glutathionylation of PKM2, leading to activation of the PPP (fig 4). This leads to a continual increases in GSH required to fuel GSTP1 mediated protein s-glutathionylation (fig. 4).
Thus, this application has the potential to discover a “new” role for an “old” enzyme. Of additional relevance is the potential direct clinical application of this study. TLK-117, a highly selective inhibitor for GSTP1 has been extensively used in patients. Furthermore, TLK-117 is non-toxic and highly suitable for therapy purposes. The information obtained in this study could provide a better understanding of the role of GSTP1 in KRAS induced tumorigenesis, which could lead to the re-purpose of TLK-117 as a new therapeutic for patients with NSCLC, which express high levels of GSTP1 and PSSG.
Figure 4 The role of PSSG in KRAS induced tumorigenesis: (Top) Glucose enters the cell and gets phosphorylated by Hexokinase (HK) to Glucose 6-Phosphate, which initiates glycolysis. Glycolysis converts glucose to pyruvate via a series of intermediate metabolites. The final step of glycolysis, phosphoenolpyruvate gets converted into pyruvate by Pyruvate Dehydrogenase M2 (PKM2). High PKM2 enzymatic activity results in the production of pyruvate which fuels the TCA cycle. Low PKM2 activity results in an accumulation of phosphoenolpyuvate and shunts G6P into the PPP. Consequently NADPH and GSH are produced, which feasibly fuels additional PSSG (feed forward mechanism). This process is possibly mediated by PSSG of PKM2 by GSTP1 (Bottom).