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Cancer Cell International

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Open Access 01-12-2022 | Research

Establishment of anti-DKK3 peptide for the cancer control in head and neck squamous cell carcinoma (HNSCC)

Authors: Naoki Katase, Shin-ichiro Nishimatsu, Akira Yamauchi, Shinji Okano, Shuichi Fujita

Published in: Cancer Cell International | Issue 1/2022

Abstract

Background

Head and neck squamous cell carcinoma (HNSCC) is the most common malignant tumor of the head and neck. We identified cancer-specific genes in HNSCC and focused on DKK3 expression. DKK3 gene codes two isoforms of proteins (secreted and non-secreted) with two distinct cysteine rich domains (CRDs). It is reported that DKK3 functions as a negative regulator of oncogenic Wnt signaling and, is therefore, considered to be a tumor suppressor gene. However, our series of studies have demonstrated that DKK3 expression is specifically high in HNSCC tissues and cells, and that DKK3 might determine the malignant potentials of HNSCC cells via the activation of Akt. Further analyses strongly suggested that both secreted DKK3 and non-secreted DKK3 could activate Akt signaling in discrete ways, and consequently exert tumor promoting effects. We hypothesized that DKK3 might be a specific druggable target, and it is necessary to establish a DKK3 inhibitor that can inhibit both secreted and non-secreted isoforms of DKK3.

Methods

Using inverse polymerase chain reaction, we generated mutant expression plasmids that express DKK3 without CRD1, CRD2, or both CRD1 and CRD2 (DKK3ΔC1, DKK3ΔC2, and DKK3ΔC1ΔC2, respectively). These plasmids were then transfected into HNSCC-derived cells to determine the domain responsible for DKK3-mediated Akt activation. We designed antisense peptides using the MIMETEC program, targeting DKK3-specific amino acid sequences within CRD1 and CRD2. The structural models for peptides and DKK3 were generated using Raptor X, and then a docking simulation was performed using CluPro2. Afterward, the best set of the peptides was applied into HNSCC-derived cells, and the effects on Akt phosphorylation, cellular proliferation, invasion, and migration were assessed. We also investigated the therapeutic effects of the peptides in the xenograft models.

Results

Transfection of mutant expression plasmids and subsequent functional analyses revealed that it is necessary to delete both CRD1 and CRD2 to inhibit Akt activation and inhibition of proliferation, migration, and invasion. The inhibitory peptides for CRD1 and CRD2 of DKK3 significantly reduced the phosphorylation of Akt, and consequently suppressed cellular proliferation, migration, invasion and in vivo tumor growth at very low doses.

Conclusions

This inhibitory peptide represents a promising new therapeutic strategy for HNSCC treatment.

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Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR. Head and neck squamous cell carcinoma. Nat Rev Dis Primers. 2020;6(1):92.PubMedPubMedCentralCrossRef

Solomon B, Young RJ, Rischin D. Head and neck squamous cell carcinoma: genomics and emerging biomarkers for immunomodulatory cancer treatments. Semin Cancer Biol. 2018;52(Pt 2):228–40.PubMedCrossRef

Kobayashi K, Hisamatsu K, Suzui N, Hara A, Tomita H, Miyazaki T. A review of hpv-related head and neck cancer. J Clin Med. 2018;7(9):241.PubMedCentralCrossRef

Leemans CR, Snijders PJF, Brakenhoff RH. The molecular landscape of head and neck cancer. Nat Rev Cancer. 2018;18(5):269–82.PubMedCrossRef

The Cancer Genome Atlas Network. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature. 2015;517(7536):576–82.CrossRef

Katase N, Gunduz M, Beder L, Gunduz E, Lefeuvre M, Hatipoglu OF, Borkosky SS, Tamamura R, Tominaga S, Yamanaka N, Shimizu K, Nagai N, Nagatsuka H. Deletion at Dickkopf (dkk)-3 locus (11p15.2) is related with lower lymph node metastasis and better prognosis in head and neck squamous cell carcinomas. Oncol Res. 2008;17(6):273–82.PubMedCrossRef

Katase N, Nagano K, Fujita S. DKK3 expression and function in head and neck squamous cell carcinoma and other cancers. J Oral Biosci. 2020;62(1):9–15.PubMedCrossRef

Leonard JL, Leonard DM, Wolfe SA, Liu J, Rivera J, Yang M, Leonard RT, Johnson JPS, Kumar P, Liebmann KL, Tutto AA, Mou Z, Simin KJ. The Dkk3 gene encodes a vital intracellular regulator of cell proliferation. PLoS ONE. 2017;12(7):e0181724.PubMedPubMedCentralCrossRef

Lee EJ, Nguyen QTT, Lee M. Dickkopf-3 in human malignant tumours: a clinical viewpoint. Anticancer Res. 2020;40(11):5969–79.PubMedCrossRef

10.

Fujii M, Katase N, Lefeuvre M, Gunduz M, Buery RR, Tamamura R, Tsujigiwa H, Nagatsuka H. Dickkopf (Dkk)-3 and β-catenin expressions increased in the transition from normal oral mucosal to oral squamous cell carcinoma. J Mol Histol. 2011;42(6):499–504.PubMedCrossRef

11.

Katase N, Lefeuvre M, Gunduz M, Gunduz E, Beder LB, Grenman R, Fujii M, Tamamura R, Tsujigiwa H, Nagatsuka H. Absence of Dickkopf (Dkk)-3 protein expression is correlated with longer disease-free survival and lower incidence of metastasis in head and neck squamous cell carcinoma. Oncol Lett. 2012;3(2):273–80.PubMedCrossRef

12.

Katase N, Lefeuvre M, Tsujigiwa H, Fujii M, Ito S, Tamamura R, Buery RR, Gunduz M, Nagatsuka H. Knockdown of Dkk-3 decreases cancer cell migration and invasion independently of the Wnt pathways in oral squamous cell carcinoma-derived cells. Oncol Rep. 2013;29(4):1349–55.PubMedCrossRef

13.

Katase N, Kudo K, Ogawa K, Sakamoto Y, Nishimatsu SI, Yamauchi A, Fujita S. DKK3/CKAP4 axis is associated with advanced stage and poorer prognosis in oral cancer. Oral Dis. 2022. https://doi.org/10.1111/odi.14277.CrossRefPubMed

14.

Katase N, Nishimatsu SI, Yamauchi A, Yamamura M, Terada K, Itadani M, Okada N, Hassan NMM, Nagatsuka H, Ikeda T, Nohno T, Fujita S. DKK3 overexpression increases the malignant properties of head and neck squamous cell carcinoma cells. Oncol Res. 2018;26(1):45–58.PubMedPubMedCentralCrossRef

15.

Katase N, Nishimatsu SI, Yamauchi A, Yamamura M, Fujita S. DKK3 knockdown confers negative effects on the malignant potency of head and neck squamous cell carcinoma cells via the PI3K/Akt and MAPK signaling pathways. Int J Oncol. 2019;54(3):1021–32.PubMed

16.

Baranyi L, Campbell W, Ohshima K, Fujimoto S, Boros M, Okada H. The antisense homology box: a new motif within proteins that encodes biologically active peptides. Nat Med. 1995;1(9):894–901.PubMedCrossRef

17.

Campbell W, Kleiman L, Barany L, Li Z, Khorchid A, Fujita E, Okada N, Okada H. A novel genetic algorithm for designing mimetic peptides that interfere with the function of a target molecule. Microbiol Immunol. 2002;46(3):211–5.PubMedCrossRef

18.

Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE. The Protein Data Bank. Nucleic Acids Res. 2000;28(1):235–42.PubMedPubMedCentralCrossRef

19.

Poorebrahim M, Sadeghi S, Rahimi H, Karimipoor M, Azadmanesh K, Mazlomi MA, Teimoori-Toolabi L. Rational design of DKK3 structure-based small peptides as antagonists of Wnt signaling pathway and in silico evaluation of their efficiency. PLoS ONE. 2017;12(2):e0172217.PubMedPubMedCentralCrossRef

20.

Fujii Y, Hoshino T, Kumon H. Molecular simulation analysis of the structure complex of C2 domains of DKK family members and beta-propeller domains of LRP5/6: explaining why DKK3 does not bind to LRP5/6. Acta Med Okayama. 2014;68(2):63–78.PubMed

21.

Martí-Renom MA, Stuart AC, Fiser A, Sánchez R, Melo F, Sali A. Comparative protein structure modeling of genes and genomes. Annu Rev Biophys Biomol Struct. 2000;29:291–325.PubMedCrossRef

22.

Källberg M, Wang H, Wang S, Peng J, Wang Z, Lu H, Xu J. Template-based protein structure modeling using the RaptorX web server. Nat Protoc. 2012;7(8):1511–22.PubMedPubMedCentralCrossRef

23.

Xu J. Distance-based protein folding powered by deep learning. Proc Natl Acad Sci USA. 2019;116(34):16856–65.PubMedPubMedCentralCrossRef

24.

Wang S, Sun S, Li Z, Zhang R, Xu J. Accurate de novo prediction of protein contact map by ultra-deep learning model. PLoS Comput Biol. 2017;13(1):e1005324.PubMedPubMedCentralCrossRef

25.

Xu J, Wang S. Analysis of distance-based protein structure prediction by deep learning in CASP13. Proteins. 2019;87(12):1069–81.PubMedCrossRef

26.

Wang S, Li Z, Yu Y, Xu J. Folding membrane proteins by deep transfer learning. Cell Syst. 2017;5(3):202–11.PubMedPubMedCentralCrossRef

27.

Wang S, Sun S, Xu J. Analysis of deep learning methods for blind protein contact prediction in CASP12. Proteins. 2018. https://doi.org/10.1002/prot.25377.CrossRefPubMed

28.

Futaki S, Suzuki T, Ohashi W, Yagami T, Tanaka S, Ueda K, Sugiura Y. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J Biol Chem. 2001;8:5836–40.CrossRef

29.

Takeuchi T, Futaki S. Current understanding of direct translocation of arginine-rich cell-penetrating peptides and its internalization mechanisms. Chem Pharm Bull (Tokyo). 2016. https://doi.org/10.1248/cpb.c16-00505.CrossRef

30.

Rothbard JB, Garlington S, Lin Q, Kirschberg T, Kreider E, McGrane PL, Wender PA, Khavari PA. Conjugation of arginine oligomers to cyclosporin a facilitates topical delivery and inhibition of inflammation. Nat Med. 2000;6(11):1253–7.PubMedCrossRef

31.

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem. 2004;25(13):1605–12.PubMedCrossRef

32.

Desta IT, Porter KA, Xia B, Kozakov D, Vajda S. Performance and its limits in rigid body protein–protein docking. Structure. 2020;28(9):1071–81.PubMedPubMedCentralCrossRef

33.

Vajda S, Yueh C, Beglov D, Bohnuud T, Mottarella SE, Xia B, Hall DR, Kozakov D. New additions to the ClusPro server motivated by CAPRI. Proteins. 2017;85(3):435–44.PubMedPubMedCentralCrossRef

34.

Kozakov D, Hall DR, Xia B, Porter KA, Padhorny D, Yueh C, Beglov D, Vajda S. The ClusPro web server for protein–protein docking. Nat Protoc. 2017;12(2):255–78.PubMedPubMedCentralCrossRef

35.

Kozakov D, Beglov D, Bohnuud T, Mottarella S, Xia B, Hall DR, Vajda S. How good is automated protein docking? Proteins. 2013;81(12):2159–66.PubMedPubMedCentralCrossRef

36.

Deshmukh A, Rao KN, Arora RD, Nagarkar NM, Singh A, Shetty OS. Molecular insights into oral malignancy. Indian J Surg Oncol. 2022;13(2):267–80.PubMedCrossRef

37.

Niehrs C. Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene. 2006;25(57):7469–81.PubMedCrossRef

38.

Veeck J, Dahl E. Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3. Biochim Biophys Acta. 2012;1825(1):18–28.PubMed

39.

Tsuji T, Miyazaki M, Sakaguchi M, Inoue Y, Namba M. A REIC gene shows down-regulation in human immortalized cells and human tumor-derived cell lines. Biochem Biophys Res Commun. 2000;268(1):20–4.PubMedCrossRef

40.

Kobayashi K, Ouchida M, Tsuji T, Hanafusa H, Miyazaki M, Namba M, Shimizu N, Shimizu K. Reduced expression of the REIC/Dkk-3 gene by promoter-hypermethylation in human tumor cells. Gene. 2002;282(1–2):151–8.PubMedCrossRef

41.

Hamzehzadeh L, Caraglia M, Atkin SL, Sahebkar A. Dickkopf homolog 3 (DKK3): a candidate for detection and treatment of cancers? J Cell Physiol. 2018;233(6):4595–605.PubMedCrossRef

42.

Kumon H, Ariyoshi Y, Sasaki K, Sadahira T, Araki M, Ebara S, Yanai H, Watanabe M, Nasu Y. Adenovirus vector carrying REIC/DKK-3 gene: neoadjuvant intraprostatic injection for high-risk localized prostate cancer undergoing radical prostatectomy. Cancer Gene Ther. 2016;23(11):400–9.PubMedPubMedCentralCrossRef

43.

Oyama A, Shiraha H, Uchida D, Iwamuro M, Kato H, Takaki A, Ikeda F, Onishi H, Yasunaka T, Takeuchi Y, Wada N, Iwasaki Y, Sakata M, Okada H, Kumon H. A Phase I/Ib trial of Ad-REIC in liver cancer: study protocol. Future Oncol. 2019;31:3547–54.CrossRef

44.

Westin SN, Fellman B, Sun CC, Broaddus RR, Woodall ML, Pal N, Urbauer DL, Ramondetta LM, Schmeler KM, Soliman PT, Fleming ND, Burzawa JK, Nick AM, Milbourne AM, Yuan Y, Lu KH, Bodurka DC, Coleman RL, Yates MS. Prospective phase II trial of levonorgestrel intrauterine device: nonsurgical approach for complex atypical hyperplasia and early-stage endometrial cancer. Am J Obstet Gynecol. 2021;224(2):191.e1-191.e15.CrossRef

45.

Zenzmaier C, Hermann M, Hengster P, Berger P. Dickkopf-3 maintains the PANC-1 human pancreatic tumor cells in a dedifferentiated state. Int J Oncol. 2012;40(1):40–6.PubMed

46.

Zhou L, Husted H, Moore T, Lu M, Deng D, Liu Y, Ramachandran V, Arumugam T, Niehrs C, Wang H, Chiao P, Ling J, Curran MA, Maitra A, Hung MC, Lee JE, Logsdon CD, Hwang RF. Suppression of stromal-derived Dickkopf-3 (DKK3) inhibits tumor progression and prolongs survival in pancreatic ductal adenocarcinoma. Sci Transl Med. 2018. https://doi.org/10.1126/scitranslmed.aat3487.CrossRefPubMedPubMedCentral

47.

Kajiwara C, Fumoto K, Kimura H, Nojima S, Asano K, Odagiri K, Yamasaki M, Hikita H, Takehara T, Doki Y, Morii E, Kikuchi A. p63-dependent Dickkopf3 expression promotes esophageal cancer cell proliferation via CKAP4. Cancer Res. 2018;78(21):6107–20.PubMedCrossRef

48.

Wang Z, Lin L, Thomas DG, Nadal E, Chang AC, Beer DG, Lin J. The role of Dickkopf-3 overexpression in esophageal adenocarcinoma. J Thorac Cardiovasc Surg. 2015;150(2):377-385.e2.PubMedPubMedCentralCrossRef

49.

Kikuchi A, Matsumoto S, Sada R. Dickkopf signaling, beyond Wnt-mediated biology. Semin Cell Dev Biol. 2022;125:55–65.PubMedCrossRef

50.

Krupnik VE, Sharp JD, Jiang C, Robison K, Chickering TW, Amaravadi L, Brown DE, Guyot D, Mays G, Leiby K, Chang B, Duong T, Goodearl AD, Gearing DP, Sokol SY, McCarthy SA. Functional and structural diversity of the human Dickkopf gene family. Gene. 1999;238(2):301–13.PubMedCrossRef

51.

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–9.PubMedPubMedCentralCrossRef

52.

Patel S, Barkell AM, Gupta D, Strong SL, Bruton S, Muskett FW, Addis PW, Renshaw PS, Slocombe PM, Doyle C, Clargo A, Taylor RJ, Prosser CE, Henry AJ, Robinson MK, Waters LC, Holdsworth G, Carr MD. Structural and functional analysis of Dickkopf 4 (Dkk4): new insights into Dkk evolution and regulation of Wnt signaling by Dkk and Kremen proteins. J Biol Chem. 2018;293(31):12149–66.PubMedPubMedCentralCrossRef

53.

Zebisch M, Jackson VA, Zhao Y, Jones EY. Structure of the dual-mode wnt regulator Kremen1 and insight into ternary complex formation with LRP6 and Dickkopf. Structure. 2016;24(9):1599–605.PubMedPubMedCentralCrossRef

54.

Kano J, Wang H, Zhang H, Noguchi M. Roles of DKK3 in cellular adhesion, motility, and invasion through extracellular interaction with TGFBI. FEBS J. 2022. https://doi.org/10.1111/febs.16529.CrossRefPubMed

55.

Lee EJ, Jo M, Rho SB, Park K, Yoo YN, Park J, Chae M, Zhang W, Lee JH. Dkk3, downregulated in cervical cancer, functions as a negative regulator of beta-catenin. Int J Cancer. 2009;124(2):287–97.PubMedCrossRef

56.

Kinoshita R, Watanabe M, Huang P, Li SA, Sakaguchi M, Kumon H, Futami J. The cysteine-rich core domain of REIC/Dkk-3 is critical for its effect on monocyte differentiation and tumor regression. Oncol Rep. 2015;33(6):2908–14.PubMedCrossRef

57.

Marqus S, Pirogova E, Piva TJ. Evaluation of the use of therapeutic peptides for cancer treatment. J Biomed Sci. 2017;24(1):21.PubMedPubMedCentralCrossRef

58.

Štambuk N, Konjevoda P, Turčić P, Šošić H, Aralica G, Babić D, Seiwerth S, Kaštelan Ž, Kujundžić RN, Wardega P, Žutelija JB, Gračanin AG, Gabričević M. Targeting tumor markers with antisense peptides: an example of human prostate specific antigen. Int J Mol Sci. 2019;20(9):2090.PubMedCentralCrossRef

59.

Centuori SM, Bauman JE. c-Met signaling as a therapeutic target in head and neck cancer. Cancer J. 2022;28(5):346–53.PubMedCrossRef

60.

Khatoon E, Hegde M, Kumar A, Daimary UD, Sethi G, Bishyaee A, Kunnumakkara AB. The multifaceted role of STAT3 pathway and its implication as a potential therapeutic target in oral cancer. Arch Pharm Res. 2022;45(8):507–34.PubMedCrossRef

Title: Establishment of anti-DKK3 peptide for the cancer control in head and neck squamous cell carcinoma (HNSCC)
Authors: Naoki Katase
Shin-ichiro Nishimatsu
Akira Yamauchi
Shinji Okano
Shuichi Fujita
Publication date: 01-12-2022
Publisher: BioMed Central
Published in: Cancer Cell International / Issue 1/2022
Electronic ISSN: 1475-2867
DOI: https://doi.org/10.1186/s12935-022-02783-9

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