Doublecortin like Kinase-1 is Overexpressed in Breast Cancer Tissues and Correlated with Epithelial-mesenchymal Transition Markers

Rasha S. Abo El Alaa¹, Samia A. Ebeid¹, Nadia A. Abd El Moneim², Sanaa A. El-Benhawy^3, and Ahmed S. Ahmed⁴

¹Applied Medical Chemistry Department, Medical Research Institute, Alexandria University, Alexandria, Egypt

²Cancer Management and Research Department, Medical Research Institute, Alexandria University, Alexandria, Egypt

³Radiation Sciences Department, Medical Research Institute, Alexandria University, Alexandria, Egypt

⁴Experimental and Clinical Surgery Department, Medical Research Institute, Alexandria University, Alexandria, Egypt

Cite this paper:
Rasha S. Abo El Alaa, Samia A. Ebeid, Nadia A. Abd El Moneim, Sanaa A. El-Benhawy and Ahmed S. Ahmed. Doublecortin like Kinase-1 is Overexpressed in Breast Cancer Tissues and Correlated with Epithelial-mesenchymal Transition Markers. Journal of Cancer Research and Treatment. 2019; 7(1):1-9. doi: 10.12691/jcrt-7-1-1

Abstract

Background: Much of the current literature support, the idea that epithelial to mesenchymal transition (EMT) is the key mechanism by which tumor cells gain invasive and metastatic ability, as EMT enables separation of individual cells from the primary tumor mass as well as promote migration. After undergoing EMT, thereby enabling access to hematogenous or lymphatic routes of dissemination, tumor cells can extravasate into secondary organs and establish micro-metastases. Objective: The main target of this work was to study the relation between expression of Doublecortin-like kinase-1 (DCLK-1) and epithelial to mesenchymal transition markers (E-cadherin, vimentin and transforming growth factor-beta (TGF-β)) in breast cancer patients and assess its role in cancer prognosis. Materials and Methods: This study included 60 breast cancer patients and 40 healthy females as control group. Tumor tissues and adjacent normal breast tissues were collected from patients with breast cancer. A single venous blood sample was collected concurrently from breast cancer patients (Before surgery) and from the control group. Tissue expression of DCLK-1, E-cadherin and vimentin were evaluated in breast cancer tissues and normal breast tissues by real-time reverse transcription polymerase chain reaction (RT-PCR). Serum level of TGF-β was assayed by enzyme linked-immunossorbant assay. Results: According to the results of the present study DCLK-1 is highly expressed in breast cancer tissues compared to normal breast tissues. Overexpression of DCLK-1 was significantly correlated with higher tissues Vimentin expression, increased serum TGF-β and lower tissues E-cadherin expression. Kaplan-Meier survival curves for breast cancer patients revealed that, patients with elevated tissue DCLK-1 and Vimentin expression and higher serum TGF-β were significantly associated with poor prognosis in primary breast cancer patients. However, patients with lower tissue E-Cadherin expression had shorter disease free survival time than patients with higher levels. Conclusion: DCLK-1 was significantly increased in breast cancer tissues in comparison to normal breast tissues and its overexpression was significantly correlated with EMT markers and poor prognosis in breast cancer patients. Further prospective studies using greater numbers of patients are required to confirm our findings.

Keywords:
breast cancer DCLK-1 EMT markers

This work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]	Hanahan D, Weberg RA. The hallmarks of cancer. Cell 2000; 100: 57-70.
[2]	Nik-Zainal S, Alexandrov LB, Wedge DC, Van Loo P, Greenman CD, Raine K, et al. Mutational processes molding the genomes of 21 breast cancers. Cell 2012; 149: 979-93.
[3]	Nakanishi Yn, Seno H, Fukuoka A, Ueo T, Yamaga Y, Maruno T, et al. Dclk1 distinguishes between tumor and normal stem cells in the intestine. Nat Genet 2013; 45: 98-103.
[4]	Bailey JM, Alsina J, Rasheed ZA, McAllister FM, Fu YY, Plentz R, et al. DCLK1 marks a morphologically distinct subpopulation of cells with stem cell properties in preinvasive pancreatic cancer. Gastroenterology 2014; 146: 245-56.
[5]	Weygant N, Qu D, Berry WL, May R, Chandrakesan P, Owen DB, et al. Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1. Mol Cancer 2014.
[6]	Weygant N, Qu D, May R, Tierney RM, Berry WL, Zhao L, et al. DCLK1 is a broadly dysregulated target against epithelial-mesenchymal transition, focal adhesion, and stemness in clear cell renal carcinoma. Oncotarget 2015; 6: 2193-205.
[7]	Whorton J, Sureban SM, May R, Qu D, Lightfoot SA, Madhoun M, et al. DCLK1 is detectable in plasma of patients with Barrett's esophagus and esophageal adenocarcinoma. Dig Dis Sci 2015; 60: 509-13.
[8]	Chandrakesan P, Weygant N, May R, Qu D, Chinthalapally HR, Ali N, et al. DCLK1 facilitates intestinal tumor growth via enhancing pluripotency and epithelial mesenchymal transition. Oncotarget 2014; 5: 9269-80.
[9]	Choi E, Petersen CP, Lapierre LA, Williams JA, Weis VG, Nam KT, et al. Dynamic Expansion of Gastric Mucosal Doublecortin-Like Kinase 1 - Expressing Cells in Response to Parietal Cell Loss Is Regulated by Gastrin. Am J Pathol 2015; 185: 2219-31.
[10]	Chandrakesan P, Panneerselvam J, Qu D, Weygant N, May R, Bronze MS, et al. Regulatory Roles of Dclk1 in Epithelial Mesenchymal Transition and Cancer Stem Cells. J Carcinog Mutagen 2016.
[11]	Kalluri R, Weinberg RA. The basics of epithelial-mesenchymal transition. J Clin Invest 2009; 119:1420-8.
[12]	Kuang J, Li L, Guo L, Su Y, Wang Y, Xu Y, et al. RNF8 promotes epithelial mesenchymal transition of breast cancer cells. J Exp Clin Cancer Res 2016.
[13]	Wu Y, Sarkissyan M, Vadgama JV. Epithelial-Mesenchymal Transition and Breast Cancer. J Clin Med 2016.
[14]	Heldin C, Moustakas A. Role of Smads in TGFβ signaling. Cell Tissue Res 2012; 347: 21-36.
[15]	Wang D, Lu P, Zhang H, Luo M, Zhang X, Wei X, et al. Oct-4 and Nanog promote the epithelial-mesenchymal transition of breast cancer stem cells and are associated with poor prognosis in breast cancer patients. Oncotarget 2014; 5: 10803-15.
[16]	Punglia RS, Morrow M, Winer EP, Harris JR. Local therapy and survival in breast cancer. N Engl J Med 2007; 356: 2399-405.
[17]	Lee JM, Dedhar S, Kalluri R, Thompson EW. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J Cell Biol 2006; 172: 973-81.
[18]	Sureban SM, Madhoun MF, May R, Qu D, Ali N, Fazili J, et al. Plasma DCLK1 is a marker of hepatocellular carcinoma (HCC): Targeting DCLK1 prevents HCC tumor xenograft growth via a microRNA-dependent mechanism. Oncotarget 2015; 6: 37200-15.
[19]	Wenhua Shi, Fangwei Li, Shaojun Li, Jian Wang, Qingting Wang, Xin Yan, et al. Increased DCLK1 correlates with the malignant status and poor outcome in malignant tumors: a meta-analysis. Oncotarget 2017; 8: 100545-57.
[20]	Koizumi H, Higginbotham H, Poon T, Tanaka T, Brinkman BC, Gleeson JG. Doublecortin maintains bipolar shape and nuclear translocation during migration in the adult forebrain. Nat Neurosci 2006; 9: 779-86.
[21]	May R, Sureban SM, Lightfoot SA, Hoskins AB, Brackett DJ, Postier RG, et al. Identification of a novel putative pancreatic stem/progenitor cell marker DCAMKL-1 in normal mouse pancreas. Am J Physiol Gastrointest Liver Physiol 2010; 299u: G303-10.
[22]	Westphalen CB, Takemoto Y, Tanaka T, Macchini M, Jiang Z, Renz BW, et al. Dclk1 Defines Quiescent Pancreatic Progenitors that Promote Injury-Induced Regeneration and Tumorigenesis. Cell stem cell 2016; 18: 441-55.
[23]	Sureban SM, May R, Ramalingam S, Subramaniam D, Natarajan G, Anant S, et al. Selective blockade of DCAMKL-1 results in tumor growth arrest by a Let-7a MicroRNA-dependent mechanism. Gastroenterology 2009; 137: 649-59, 59. e1-2.
[24]	Ito H, Tanaka S, Akiyama Y, Shimada S, Adikrisna R, Matsumura S, et al. Dominant Expression of DCLK1 in Human Pancreatic Cancer Stem Cells Accelerates Tumor Invasion and Metastasis. PLoS One 2016; 11: e0146564.
[25]	Sureban SM, May R, Qu D, Weygant N, Chandrakesan P, Ali N, et al. DCLK1 regulates pluripotency and angiogenic factors via microRNA-dependent mechanisms in pancreatic cancer. PLoS One 2013; 8: e73940.
[26]	Weygant N, Qu D, Berry WL, May R, Chandrakesan P, Owen DB, et al. Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1. Mol Cancer 2014; 13: 103.
[27]	Chandrakesan P, Weygant N, May R, Qu D, Chinthalapally HR, Sureban SM, Ali N, Lightfoot SA, Umar S, Houchen CW. DCLK1 facilitates intestinal tumor growth via enhancing pluripotency and epithelial mesenchymal transition. Oncotarget. 2014; 5: 9269-80.
[28]	Li J, Wang Y, Ge J, Li W, Yin L, Zhao Z, et al. Doublecortin-Like Kinase 1 (DCLK1) Regulates B Cell-Specific Moloney Murine Leukemia Virus Insertion Site 1 (Bmi-1) and is Associated with Metastasis and Prognosis in Pancreatic Cancer. Cell Physiol Biochem 2018; 51: 262-77.
[29]	Gao T, Wang M, Xu L, Wen T, Liu J, An G. DCLK1 is up-regulated and associated with metastasis and prognosis in colorectal cancer. J Cancer Res Clin Oncol 2016; 142: 2131-40.
[30]	Shi W, Li F, Li S, Wang J, Wang Q, Yan X, et al. Increased DCLK1 correlates with the malignant status and poor outcome in malignant tumors: a meta-analysis. Oncotarget 2017; 8: 100545-57.
[31]	Jiang WG, Mansel RE. E-cadherin complex and its abnormalities in human breast cancer. Surg Oncol 2000; 9: 151-71.
[32]	Takeichi M. Cadherins in cancer: implications for invasion and metastasis. Curr Opin Cell Biol 1993; 5: 806-11.
[33]	Wang Y, Zhou BP. Epithelial-mesenchymal Transition-A Hallmark of Breast Cancer Metastasis. Cancer Hallm 2013; 1: 38-49.
[34]	Suciu C, Cimpean AM, Muresan AM, Izvernariu D, Raica M. E-cadherin expression in invasive breast cancer. Rom J Morphol Embryol 2008; 49: 517-23.
[35]	Duprey P, Paulin D. What can be learned from intermediate filament gene regulation in the mouse embryo. Int J Dev Biol 1995; 39: 443-57.
[36]	Calaf GM, Balajee AS, Montalvo-Villagra MT, Leon M, Daniela NM, Alvarez RG, et al. Vimentin and Notch as biomarkers for breast cancer progression. Oncol Lett 2014; 7: 721-7.
[37]	Whipple RA, Balzer EM, Cho EH, Matrone MA, Yoon JR, Martin SS. Vimentin filaments support extension of tubulin-based microtentacles in detached breast tumor cells. Cancer Res 2008; 68: 5678-88.
[38]	Hendrix MJ, Seftor EA, Seftor RE, Trevor KT. Experimental co-expression of vimentin and keratin intermediate filaments in human breast cancer cells results in phenotypic interconversion and increased invasive behavior. Am J Pathol 1997; 150: 483-95.
[39]	Panis C, Herrera AC, Victorino VJ, ARANOME AM, Cecchini R. Screening of circulating TGF-β levels and its clinicopathological significance in human breast cancer. Anticancer Res 2013; 33: 737-42.
[40]	Ciftci R, Tas F, Yasasever CT, Aksit E, Karabulut S, Sen F, et al. High serum transforming growth factor beta 1 (TGFB1) level predicts better survival in breast cancer. Tumor Biol 2014; 35: 6941-8.
[41]	Nacif M, Talaat R, Shaker O. Transforming growth factor-β1 (TGF-β1) gene expression and protein level in blood as prognostic and diagnostic tools in cancer patients. Euro J Biotechnol Biosci 2014; 2: 01-7.
[42]	Desruisseau S, Palmari J, Giusti C, Romain S, Martin PM, Berthois Y. Determination of TGFβ1 protein level in human primary breast cancers and its relationship with survival. Br J Cancer 2006; 94: 239-46.
[43]	Liesheng L, Lei Y, Zhenshun S, Lijun Z, Donglei Z, Zhen Y. Comparison Study of E-cadherin expression in primary breast cancer and its corresponding metastatic lymph node. J Cytol Histol 2014; 5: 1.
[44]	Niknami Z, Eslamifar A, Emamirazavi A, Ebrahimi A, Shirkoohi R. The association of vimentin and fibronectin gene expression with epithelial-mesenchymal transition and tumor malignancy in colorectal carcinoma. Excli j 2017; 16: 1009-17.
[45]	Rakshith V, Harendra Kumar HL, Bhaskaran A, Suresh TN. Expression levels of e-cadherin and vimentin in breast cancer. Int J Curr Res 2017; 9: 56505-9.
[46]	Duffy MJ, Shering S, Sherry F, McDermott E, O'Higgins N. CA 15-3: a prognostic marker in breast cancer. Int J Biol Markers 2000; 15: 330-3.
[47]	Li Z, Yin S, Zhang L, Liu W, Chen B. Prognostic value of reduced E-cadherin expression in breast cancer: a meta-analysis. Oncotarget 2017; 8: 16445-55.
[48]	Hemalatha A, Suresh TN, Kumar ML. Expression of vimentin in breast carcinoma, its correlation with Ki67 and other histopathological parameters. Indian J Cancer 2013; 50: 189-94.