Therapeutic potential of Wnt/β-catenin/TCF4 signaling pathway downregulators against gallbladder cancer

Abstract

Gallbladder cancer (GBC) is the most common malignancy of the biliary tract, ranked sixth among gastrointestinal cancers, and is associated with a poor prognosis. According to GLOBOCAN 2018 data, globally about 219420 GBC new cases and 165087 deaths were estimated to occur in 2018. India is among the highest incidence area for GBC similar to the Latin American countries like Chile, Bolivia, and Columbia. In India, GBC is most prevalent in the north, east, northeast, and central part of India. Cancer incidences and mortality are rapidly growing worldwide because of exposure to carcinogenic chemicals, pathogenic microorganism, dietary patterns and lifestyle factors. The development of GBC occurs over a span of 5–15 years, from chronic inflammation to tissue alterations, including metaplasia, dysplasia, carcinoma in situ, and invasive cancer. The early diagnosis of GBC is not possible due to the lack of specific biomarkers. The mean survival period for patients with advanced GBC is 6 months. However, if the disease is diagnosed in stage IV, the 5-year survival rate is less than 5%. There is certainly an unmet need to understand the pathophysiology of gallbladder cancer and identify novel targets to manage both the early and advanced disease states. Wnt/β-catenin signaling pathway dysregulation has been involved in cancer initiation, cancer metastasis, and cancer stem cells development. Aberrant Wnt/β catenin signaling is an early progression event in 90% of colorectal cancers and hepatocellular carcinomas. Overactivation of β-catenin/TCF4 signaling by accumulated β-catenin in the nucleus has been shown to play a crucial role in the development of colorectal cancer and hepatocellular carcinoma. TCF4 expression is potentially xxi involved in various tumors, including colorectal cancer and hepatocellular carcinoma, and in the progression of drug resistance. A great body of literature indicates that deregulation of Wnt/β-catenin signaling in GBC leads to increased nuclear localization of β-catenin. Gallbladder is an accessory organ of the gastrointestinal tract, GBC may share similar pathophysiological characteristics of CRC and HCC. As activation of β-catenin/TCF4 signaling primarily depends on nuclear β-catenin, we hypothesized that TCF4 may have a critical role in the progression of GBC. The aim for the experiment 1 was to investigate the expression patterns of TCF4 in gallbladder cancer tissue samples by reverse transcription polymerase chain reaction (RTPCR) and immunohistochemistry (IHC) and attempted to correlate its expression with different clinicopathological parameters. From our study, we found relative mRNA expression levels of β-catenin and TCF4 in GBC tissues were significantly (P < 0.05) higher than in CC samples. TCF4 protein expression was observed in 81.82% (27/33) GBC cases. Specifically, among GBC samples, 21.21% (7/33) was graded as strongly positive, 60.61% (20/33) graded as moderately positive, whereas 18.18% (6/33) graded as negative. All 12 CC samples graded as negative. Overall, TCF4 expression in GBC tissues was statistically significant over CC samples (P < 0.05). Moreover, we observed that TCF4 expression was significantly higher (P < 0.05) in high tumor grades than low grade, higher (P < 0.05) in stage 2 and stage 3 than stage 1. The present study suggests that TCF4 may exert an oncogenic role in the progression of GBC, and may serve as a new potential candidate biomarker for tumor progression, and it might be a potential therapeutic target against gallbladder cancer. Moreover, we hypothesized that downregulation of Wnt/β-catenin/TCF4 signaling or selective β-catenin/TCF4 protein-protein interaction xxii inhibitors could be a potential therapeutic agent against gallbladder cancer which might also be helpful in overcoming the prevalence of drug resistance. Adjuvant chemotherapy in GBC is very limited, and the drugs that are commonly recommended include gemcitabine, capecitabine, fluorouracil (5-FU), cisplatin, and oxaliplatin. With various chemotherapeutic agents response rates reported are 0-36% of cases, which confer limited benefit and are also ineffective at eliminating self-renewing cancer stem cells. Therefore, there is certainly an urgent need to design and develop more efficacious drug therapies for the treatment of such cancers patients. Knowledge about the molecular pathophysiology and oncogenic mechanisms underlying the development and progression of cancer has led to the discovery and development of new class of targeted therapeutics for the treatment of cancer. Many researchers indicates that aberrant Wnt/β‐catenin signaling pathway by accumulated nuclear β catenin plays a crucial role in the development and progression of gastrointestinal cancers including colon cancer (CC), hepatocellular carcinoma (HCC) and gallbladder cancer (GBC). Over-activation of the Wnt/β-catenin signaling pathway results in increased nuclear localization of β-catenin from the cytoplasm and thereby binds to T cell factor/Lymphoid enhancer factor (TCF/LEF) and transcriptional co-activators (BCL9, Pygo, CBP/p300), which then activate the transcription of Wnt target genes. Wnt target genes such as cyclin D1, c-myc, c-Jun, survivin, CD44, PPARδ, VEGF, uPAR, Met, fra-1, Msl1, MMPs, and endothelin-1 are known to play an essential roles in several aspects of tumor development such as cell growth, proliferation, migration, angiogenesis, invasion, transformation, survival, epithelial to mesenchymal transition, and development of cancer stem cells. Interaction of nuclear β-catenin with T cell factor 4 (TCF4) (also named as Transcription factor 4 or transcription factor 7‐like 2) is a key step for the activation of Wnt target genes in response to upstream signals of the Wnt/β- xxiii catenin pathway. So far, no effective anti-cancer drug targeting the β-catenin/TCF4 signaling pathway has been approved by the Food and Drug Administration (FDA). Hence, targeting downstream events by selective β-catenin/TCF4 protein–protein interaction inhibitors or by down regulation of Wnt/β-catenin/TCF4 signaling could be a potential therapeutic strategy against such cancers. Gefitinib, lapatinib, erlotinib, afatinib, and vandetanib are some examples bearing quinazoline scaffold used clinically as anti-cancer drugs. In the recent decade, many researchers reported that small molecules with 4-amino substituent quinazoline core have significant anti-cancer activities and also inhibiting the β-catenin/TCF4 signaling pathway. The aim for the experiment 2 was to design and develop some novel 4,7- disubstituted 8-methoxyquinazoline derivatives and assessing these compounds for β catenin/TCF4 protein–protein interaction inhibition and to evaluate the cytotoxic potential of these derivatives against constitutively activated β-catenin/TCF4 signaling pathway cancer cells (HCT116 and HepG2 cells) using the sulforhodamine B assay. The most potent compound was selected for further evaluation for anti-carcinogenic activities and for ascertaining the underlying mechanism involved. Cell morphology, Hoechst 33342 and Annexin V/PI staining were used to detect apoptosis, while inhibition of cell migration was assessed by in vitro wound healing assay against HCT116 and HepG2 cells. Effect on β-catenin/TCF mediated transcriptional activity was assessed by TOPFlash/FOPFlash assay, TCF4 and β-catenin protein expression by immunocytofluorescence, and Wnt target genes (like c-MYC and Cyclin D1) mRNA levels by RTPCR against HCT116 cells. In vitro cytotoxic potential was evaluated against primary human gallbladder cancer cells. Molecular docking studies revealed that these compounds showed interactions with the active site residues on β-catenin and thus capable of hindering the TCF4 binding, thereby disrupting β-catenin/TCF4 xxiv interactions. These novel quinazoline derivatives were identified as potential cytotoxic agents against constitutively activated β-catenin/TCF4 signaling cancer cells (HCT116 and HepG2) when treated for 48 hours with IC50 values ranged from 5.64 ± 0.68 to 23.18 ± 0.45 μM and were found to be comparable in potency with imatinib mesylate (IC50 value = 12.98 ± 0.21 μM, 16.69 ± 0.38 μM against HCT116 and HepG2 cells, respectively), a widely used clinical anti-cancer drug for the treating gastrointestinal cancers. Compound [N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-8-methoxy-7-(3-(4- methylpiperazin-1-yl)propoxy)quinazolin-4-amine], the most potent compound among the series, induced apoptosis and inhibited cell migration at IC50 concentration when treated for 48 hours against HCT116 and HepG2 cells (based on cells morphological changes, Hoechst 33342 and Annexin V/PI staining and in vitro wound healing assay). Mechanistic studies indicated that compound [N-(3-chloro-4-((3- fluorobenzyl)oxy)phenyl)-8-methoxy-7-(3-(4-methylpiperazin-1- yl)propoxy)quinazolin-4-amine] when treated for 24 hours at IC50 concentration significantly downregulated the β-catenin/TCF4 mediated transcriptional activity (reduced TOP-luciferase activity), downregulated β-catenin and TCF4 protein expression (based on immunocytofluorescence), downregulated mRNA levels of c MYC and Cyclin D1 (Wnt target genes) against HCT116 cells (based on RTPCR). Compound [N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-8-methoxy-7-(3-(4- methylpiperazin-1-yl)propoxy)quinazolin-4-amine] when treated for 48 hours was found to be more potent (IC50 8.50 ± 1.44 μM) than imatinib mesylate (IC50 15.56 ± 1.26 μM) against primary human GBC cells and induced morphological changes like cell shrinkage and membrane blebbing at IC50 concentration, suggesting its promising therapeutic potential against gallbladder cancer. All these observations indicated that compound [N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-8-methoxy-7-(3-(4- xxv methylpiperazin-1-yl)propoxy)quinazolin-4-amine] downregulated Wnt/β catenin/TCF4 signaling pathway and this could be one of the mechanisms by which it exerts its anticancer activity. Idelalisib, raltitrexed and nolatrexed are some examples having quinazolinone scaffold used clinically as anti-cancer drugs. In the recent decade, many researchers from both industry and academia reported that small molecules with quinazolin-4(3H)- one core have significant anti-cancer activities and also inhibiting the TopFlash luciferase assay activity (downregulating the Wnt/β-catenin/TCF4 signaling pathway). In our continuing study aimed (experiment 3) at the discovery and development of novel and more potent anti-cancer drug candidates, we thought of designing some novel 2,7-disubstituted 6-methoxyquinazolin-4(3H)-one derivatives and assessing these compounds for β-catenin/TCF4 protein–protein interaction inhibition and for anti cancer activities. Observations based on the molecular docking studies indicated that these compounds showed interactions with the residues of β-catenin on the active site and therefore capable of hindering the TCF4 binding, thereby disrupting the β catenin/TCF4 protein-protein interactions. The cytotoxic potencies of these novel 2,7- disubstituted 6-methoxyquinazolin-4(3H)-one derivatives when treated for 48 hours against HCT116 and HepG2 cells were found to be comparable (IC50 values ranged from 3.89 ± 0.49 to 21.81 ± 0.77 μM) to those of imatinib mesylate (IC50 value = 12.73 ± 0.40 μM, 16.91 ± 0.55 μM against HCT116 and HepG2 cells, respectively). Compound [2-benzhydryl-6-methoxy-7-(3-(4-methylpiperazin-1- yl)propoxy)quinazolin-4(3H)-one], the most potent compound, induced apoptosis against HCT116 and HepG2 cells at IC50 concentration when treated for 48 hours (based on cells morphological changes, Hoechst 33342 and Annexin V/PI staining). Mechanistic studies revealed that when treated for 24 hours at IC50 concentration xxvi significantly downregulated the β-catenin/TCF4 transcriptional activity (reduced TOP luciferase activity), downregulated β-catenin and TCF4 protein expression against HCT116 cells (based on immunocytofluorescence), downregulated the expression of c Myc and cyclin D1 (Wnt target genes) against HCT116 cells (based on RTPCR). Compound [2-benzhydryl-6-methoxy-7-(3-(4-methylpiperazin-1- yl)propoxy)quinazolin-4(3H)-one] when treated for 48 hours was found to be more potent (IC50 7.26 ± 1.06 μM) than imatinib mesylate (IC50 15.27 ± 0.91 μM) against primary human GBC cells and induced morphological changes like cell shrinkage and membrane blebbing at IC50 concentration, suggesting its promising therapeutic potential against gallbladder cancer. All these observations indicated that compound [2- benzhydryl-6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4(3H)-one] downregulated Wnt/β-catenin/TCF4 signaling pathway and this could be one of the mechanisms by which it exerts its anticancer activity. These findings suggest that these derivatives have the potential to be developed as a new class of drug candidate molecules though needs more preclinical experiments to verify their practicabilities. Compound [N-(3-chloro-4-((3-fluorobenzyl)oxy)phenyl)-8- methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4-amine] and compound [2- benzhydryl-6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinazolin-4(3H)-one] represents a promising lead molecule and chemical probe as anticancer agent against colon, hepatocellular and gallbladder cancer targeting Wnt/β-catenin/TCF4 signaling pathway.