Elsevier

Biophysical Chemistry

Volume 240, September 2018, Pages 107-117
Biophysical Chemistry

Research Article
Antagonistic role of Klotho-derived peptides dynamics in the pancreatic cancer treatment through obstructing WNT-1 and Frizzled binding

https://doi.org/10.1016/j.bpc.2018.07.002Get rights and content

Highlights

  • Expression pattern analysis of human WNTs and Klotho in the pancreatic cancer.

  • Targeting of WNT-1 and Frizzled-CRD association through Klotho-derived peptides.

  • Klotho-derived peptides were scruitinized to evaluate their inhibitory potential against WNT-1 binding region of FZD-1/2- (CRDs).

Abstract

Klotho is an anti-aging protein that is engaged in the suppression of canonical WNT signaling. In this study, we investigated the expression pattern of human WNTs and Klotho in the pancreatic cancer. In the cancerous cells, WNT-1 exhibited much higher expression as compared to other WNTs, while no WNT expression was detected in the normal tissue. In contrast, Klotho expression was significantly low in the cancerous tissue. Based on these observations, we intended to explore Klotho binding to WNT-1 and cystein-rich domains (CRDs) of Frizzled (FZD) homologs through molecular docking and dynamics simulation assays. Interestingly, similar region of WNT-1 was detected in binding with Klotho and CRDs of FZD-1/2. FZD-CRDs were grasped by the association of peripheral hydrophobic residues of WNT-1 U-shaped cavity. Subsequently, WNT-1-bound Klotho-peptides were isolated and reevaluated for their binding abilities against WNT-1 and FZD-CRDs., The conformational readjustements of these complexes were deeply analyzed by calculating the size of WNT-1 U-shaped cavity. In comparison to apo-WNT-1, cavity opening was markedly enhanced (8.2 Å to 15.64 Å, 32.89 Å and 35.11 Å) in WNT-1-a, WNT-1-c and WNT-1-e complexes, respectively. Thus Klotho-derived peptides may facilitate distinct conformational changes in the WNT-1-FZD associated region. As a result, aberrant loss of FZD binding may lead to augment WNT signaling. Overall, current study opens up new avenues in the pancreatic cancer therapeutics through antagonizing WNT-1 by Klotho.

Introduction

Klotho is an anti-aging single-pass transmembrane protein that comprises 1012 residues [1,2] with a large extracellular domain (130 kDa) and a transmembrane domain with a short intracellular domain (10 AA). The extracellular region of Klotho resembles with family 1 glycosidases [3] by containing two homologous domains GS1 and GS2. The soluble or extracellular Klotho is formed by the cleavage of ADAM10 and ADAM17 and may exist either in GS1, GS2 or both domains [4]. The membrane-bound or secreted form of Klotho functions as a humoral factor that is involved in the regulation of growth factor signaling and oxidative stress by binding with ion channels and transporter proteins [5]. It also contributes in phosphate excretion and synthesis of active vitamin D in the kidney tissue [6,7].

Klotho, a WNT antagonist, is released in the serum until 35 years and vanishes rapidly there after resulting in the premature aging phenotype [1,2,8]. WNTs belong to a large protein family that is involved in the regulation of diverse cellular functions such as embryonic development, cell fate speciation, cell polarity and regulates several pathological and physiological processes in mammalian aging [9,10]. The involvement of WNT signaling has been suggested in the pathogenesis of age-associated disorders like breast, colon, pancreatic and hepatic cancer, autoimmune disorders and heart diseases [1,9,[11], [12], [13], [14], [15]]. Klotho binding to WNT-1 and WNT-4 essentially inhibits the downstream WNT signaling, leading to kidney injury disease [16]. Maltare et al. corroborated the binding of Klotho and WNT-7a in the kidney lysates [17]. Essentially, WNT-1 induced luciferase activity has been reduced dose-dependently by Klotho cotransfection [3], while there is no change in the WNT-mediated gene transcription which was detected due to the constitutive overexpression of β-catenin [16]. Based on these observations, it may be worthwhile to address the role of WNT-Klotho paradigm in the context of aging and cancer. WNT-1 and Frizzled-2 (FZD-2) express at low level in the normal pancreata and their concurrent enhanced expression results in pancreatic cancer [18]. In this study, we have investigated the expression pattern of WNTs and Klotho in the pancreatic tissue of a 34 year-old cancerous patient. Due to higher expression of WNT-1, we aim to target its interaction with FZD homologs through computational approaches. Klotho-derived peptides have been scrutinized to evaluate their inhibitory potential against WNT-1 binding regions of FZD-1, FZD-2, FZD-4, FDZ-5, FZD-7, FZD-8 and FZD-10.

Section snippets

Sample collection

Pancreatic cancer biopsies were obtained from Pakistan Institute of Medical Sciences, Islamabad, Pakistan with a written consent of 34-year old patient and stored at −80 °C in National Center for Bioinformatics (NCB), Quaid-i-Azam University, Islamabad, Pakistan.

RNA extraction and RT-PCR analysis

Total RNA was extracted through organic method by using TRIzol (Invitrogen) reagent and treated by DNase (Qiagen). RNA samples were quantified through nanodrop (Thermo Fisher Scientific) and stored at −80 °C for further analysis.

Expression study of Klotho and WNTs

As aberrant behavior of WNT ligands contributes to cancer development and progression, their differential expression was studied in pancreatic cancer tissue. WNT-1, WNT-2b, WNT-7a, WNT-10a and WNT-16 exhibited higher expression in the pancreatic cancer cells as compared to normal tissue (Fig. 1). The expression of WNT-1 was upregulated as compared to other WNTs suggesting that WNT-1 might play a key role in the proliferation of pancreatic cancer. To our surprise, lower level of Klotho

Discussion

Klotho is an antiaging hormone that is secreted in the human serum till 35 years of age and rapidly vanishes thereafter [39]. In Klotho null mice, significant modulation of canonical WNT signaling [3], suggests a fundamental role of WNT signaling in organismal aging and aging-related diseases. Particularly, WNT-1 overexpression results in rheumatoid arthritis and systematic sclerosis [2,40,41]. The secreted Klotho interacts with WNT-1 and functions in blocking WNT-1-mediated β-catenin

Conclusion

In this study, the expression pattern of human WNTs and Klotho was explored in the pancreatic tissue of a 34 year-old cancerous patient. A much lower expression of Klotho and higher expression of WNT ligands, particularly WNT-1 signifies the active involvement of WNT signaling in the pancreatic cancer. Through binding analysis, similar region of WNT-1 (LEU19-VAL365) exhibited interaction with FZD-CRDs and Klotho. These findings led us to investigate structural role of Klotho-derived peptides

Conflict of interest

The authers declare that they have no competing interest.

Acknowledgements

We thank Saima Younis and Sana Zahid for critical reading of manuscript.

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