The aim of this study was to examine the role of GSTP1 in KRAS induced tumorigenesis and its involvement in cancer metabolism. Here we demonstrated that GSTP1 protein levels are differentially expressed between several human lung cancer cell lines. Interestingly there appears to be an association between the levels of GSTP1 expression and the sensitivity to TLK-117, a GSTP1 inhibitor. In cells with a higher abundance of GSTP1, TLK-117 reduced the lactate production and migratory capacity, in contrast, cells with low GSTP1 expression were not sensitive to TLK-117 treatment. Conflicting results where found by Zhang et al., which described that genetic ablation of GSTP1, resulted in an increase in myeloproliferation and migration [36]. Similarly it has been observed that TLK199 stimulated cytokine-induced myeloproliferation in wild type mice but not in GSTπ–/– mice [37]. A possible explanation is that GSTP1 has distinctive roles in specific cell types. Our findings suggest that GSTP1 inhibition and the use of TLK-117 could be applicable in reducing cell migration, cancer progression and the formation of metastases. GSTP is a member of a larger family of Glutathione S-transferases, and is well known for its role in phase II drug metabolism, as it catalyzes conjugation of GSH to electrophiles. GSTP is overexpressed in a variety of cancers [38,39], where it has been associated with resistance to chemotherapeutic drugs [40]. GSTP1 can also act as a modulator of signal transduction and has recently been discovered to catalyze protein s-glutathionylation (PSSG) reactions. Our data suggest that TLK-117 inhibits overall s-glutathionylation in cells with high GSTP1 expression and more specifically TLK inhibits PKM2 glutathionylation in A549 cells. Since PKM2 is an important regulator of the Warburg effect, the Pentose Phosphate Pathway and PKM2 appears to be a potential target for GSTP1 induced PSSG, the findings in this study highlight the possible role and contribution of GSTP1 and PKM2, in glycolysis and involvement in human cancers [26,27,34]. In addition to its role as a catalyst of PSSG, GSTP1 also acts as a ligandin [41,42]. GSTP1 can bind to a number of proteins to alter their function. For example, GSTP1 can bind to c-Jun-N-terminal kinase (JNK) and the adaptor protein, TRAF2 to inhibit their activation [12]. This can be an additional potential mechanism to elucidate the effect observed with TLK-117 but was beyond the scope of this study.
Lung cancer is a disease that encompasses many different cell types, and is thus impossible to entirely recapitulate in cell culture models. In order to elucidate a connection between these two
processes, we will need to utilize the whole mouse as a biological system to investigate if genetic manipulation is a possible form of lung cancer treatment. There are several ways to induce cancer and specific NSCLC in experimental animals. Cho et al. described this inducible mouse model where they established that mice carrying KRAS mutations were highly susceptible to early onset lung cancer.
They established that bronchiolar Club cells are the origin of cells and tumorigenesis for KRASG12D -induced neoplasia in lungs. [43] Correspondingly in approximately 20-30 % KRAS is frequently mutated in human tumors of the lung [44,45,46,47]. To further investigate the relevance of GSTP1 in KRAS induced NSCLC we bred a triple transgenic mouse that expresses the KRASG12G oncogene. In comparison to a xenograft model, in which human lung cancer cell lines have been subcutaneously implanted in immunodeficient mice, we utilized an inducible model of KRAS driven tumorigenesis [48].
Food containing doxycycline, led to a controlled activation of the oncogene KRASG12D, inducing tumor formation originated from CCSP positive lung epithelial cells. An immense advantage of this model is that it can recapitulate spontaneous cancer formation due to a mutation and activation of an oncogene. We investigated KRAS induced tumor formation by evaluating physiological differences of lung sections that were obtained from the CCSP-rtTA/tetO-Cre/LSL-KRASG12D. After 6 weeks, KRASG12D mice revealed the development of epithelial hyperplasia and tumor formation along the left lung lobe. After evaluating protein, mRNA expression and lactate production, we confirmed that KRASG12D expressing mice produced an excessive amount of lactate compared to KRASWt mice.
Protein expression of PKM2, PD, G6PD, HKII and PFKFB3 was increased as well as mRNA expression of PKM2, HKII, HKIII, Glut2 and PFKFB3. All these are biomarkers for cancer and are known to be elevated in human cancers [49,50,51,52,53,54]. These findings exposes the CCSP-rtTA/tetO-Cre/LSL-KRASG12D mouse model as a suitable preclinical model for intervention studies that make it possible to assess therapy responses and evaluate new therapeutic agents.
In the next part of this study we determined whether pharmacological inhibition of GSTP1 attenuated KRAS induced tumorigenesis in vivo. To examine this, we used adenovirus expressing Cre recombinase (AdCre), which induces tumor formation by activating the KRASG12D in adeno-infected cell. KRASG12D expressing mice and wild type mice were exposed to TLK-117, a selective GSTP1 inhibitor [55]. Preliminary data suggested that TLK-117 attenuated tumor growth and cancer progression in mutant KRASG12D induced mice. In addition, as shown in previous studies, epithelial hyperplasia was observed in AdCre induced KRASG12D mice, and TLK treatment appeared to
attenuate epithelial hyperplasia. Unfortunately we found no differences in GST activity in the lungs of KRASG12D mice treated with TLK-117 compared to mice treated with a vehicle control. Since this model shows a higher tumor burden at 12 weeks we will conduct future studies with a greater number of animals and a prolonged time course. This will allow a better understanding of the impact of TLK-117 on reducing tumorigenesis.
As described before the most common mutation in NSCLC is the KRAS gene mutation, which is believed to play a key role in NSCLC [56]. As previously mentioned GSTP1 expression is increased in several forms of cancers including lung cancer. Another goal of this study was to evaluate GSTP1 expression and overall s-glutathionylation in human NSCLC tumors. For this purpose, we obtained normal and cancerous tissue from 106 patients diagnosed with NSCLC. After estimating GSTP1 expression an overall s-glutathionylation in cancerous and normal tissue in these patients, we found that 41% of these patients expressed an excessive amount of GSTP1 in their tumor tissue.
Interestingly, overall s-glutathionylation was correspondingly elevated in tumor tissue of patients that expressed large amounts of the GSTP1 protein. We are in the process of correlating overall s-glutathionylation and GSTP1 expression to KRAS mutation status in these patients.
In conclusion, this study suggests that GSTP1 plays a role in KRAS induced tumorigenesis. The presented data demonstrate that GSTP1 controls lactate production and protein expression of glycolysis-associated proteins in NSCLC cells. Further, pharmacological inhibition of GSTP1 attenuates migration in GSTP1 high expressing NSCLC cells. This study further describes a possible role of GSTP1 in overall protein s-glutathionylation and more specifically s-glutathionylation of PKM2.
Excitingly, preliminary data suggest that pharmacological inhibition of GSTP1 attenuates tumor burden in a mouse model of KRASG12D induced tumor formation. Finally this study reveals GSTP1 as a relevant therapeutic target in NSCLC patients sine about 40% of all investigated tumors were found positive for GSTP1. Furthermore, if GSTP1 over expression in human lung tumors correlates with an existent KRAS mutation, GSTP1 could be introduced as a new biomarker for KRAS positive tumors.
These findings demonstrate that TLK-117 could be a potential new therapeutic for the treatment of NSCLC especially when a KRAS mutation is present.