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Dclk1, a Tumor Stem Cell Marker, Regulates Pro-Survival Signaling And

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Dclk1, a Tumor Stem Cell Marker, Regulates Pro-Survival Signaling And Chandrakesan et al. Molecular Cancer (2017) 16:30 DOI 10.1186/s12943-017-0594-y RESEARCH Open Access Dclk1, a tumor stem cell marker, regulates pro-survival signaling and self-renewal of intestinal tumor cells Parthasarathy Chandrakesan1,2,3*† , Jiannan Yao1,7†, Dongfeng Qu1,2,3, Randal May1,3, Nathaniel Weygant1, Yang Ge1,7, Naushad Ali1,3, Sripathi M. Sureban1,3, Modhi Gude1,4, Kenneth Vega1,5, Eddie Bannerman-Menson8, Lijun Xia6, Michael Bronze1, Guangyu An7* and Courtney W. Houchen1,2,3,8* Abstract Background: More than 80% of intestinal neoplasia is associated with the adenomatous polyposis coli (APC) mutation. Doublecortin-like kinase 1 (Dclk1), a kinase protein, is overexpressed in colorectal cancer and specifically marks tumor stem cells (TSCs) that self-renew and increased the tumor progeny in ApcMin/+ mice. However, the role of Dclk1 expression and its contribution to regulating pro-survival signaling for tumor progression in Apc mutant cancer is poorly understood. Methods: We analyzed DCLK1 and pro-survival signaling gene expression datasets of 329 specimens from TCGA Colon Adenocarcinoma Cancer Data. The network of DCLK1 and pro-survival signaling was analyzed utilizing the GeneMANIA database. We examined the expression levels of Dclk1 and other stem cell-associated markers, pro-survival signaling pathways, cell self-renewal in the isolated intestinal epithelial cells of ApcMin/+mice with high-grade dysplasia and adenocarcinoma. To determine the functional role of Dclk1 for tumor progression, we knocked down Dclk1 and determined the pro-survival signaling pathways and stemness. We used siRNA technology to gene silence pro-survival signaling in colon cancer cells in vitro. We utilized FACS, IHC, western blot, RT-PCR, and clonogenic (self-renewal) assays. Results: We found a correlation between DCLK1 and pro-survival signaling expression. The expression of Dclk1 and stem cell-associated markers Lgr5, Bmi1, and Musashi1 were significantly higher in the intestinal epithelial cells of ApcMin/+mice than in wild-type controls. Intestinal epithelial cells of ApcMin/+mice showed increased expression of pro-survival signaling, pluripotency and self-renewal ability. Furthermore, the enteroids formed from the intestinal Dclk1+ cells of ApcMin/+mice display higher pluripotency and pro-survival signaling. Dclk1 knockdown in ApcMin/+ mice attenuates intestinal adenomas and adenocarcinoma, and decreases pro-survival signaling and self-renewal. Knocking down RELA and NOTCH1 pro-survival signaling and DCLK1 in HT29 and DLD1 colon cancer cells in vitro reduced the tumor cells’ ability to self-renew and survive. (Continued on next page) * Correspondence: [email protected]; [email protected]; [email protected] †Equal contributors 1Division of Digestive Diseases and Nutrition, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA 7Beijing Chao-Yang Hospital Department of Oncology, Capital Medical University, Beijing, China Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 2 of 14 (Continued from previous page) Conclusion: Our results indicate that Dclk1 is essential in advancing intestinal tumorigenesis. Knocking down Dclk1 decreases tumor stemness and progression and is thus predicted to regulate pro-survival signaling and tumor cell pluripotency. This study provides a strong rationale to target Dclk1 as a treatment strategy for colorectal cancer. Keywords: Dclk1, APC mutation, Cancer stem cells, Intestinal epithelial cells, Pro-survival signaling, Self-renewal, Nanoparticles, Colorectal cancer Background that the majority of human malignancies are initiated APC is a tumor suppressor gene that is mutated in pa- and maintained by a distinct population of cells that dis- tients with familial adenomatous polyposis (FAP) and play stem cell properties and self-renewal ability [16]. most sporadic colorectal cancers [1, 2]. The Apc muta- More recently, it has been shown that the development tion dysregulates the Wnt signaling pathway and triggers and progression of colon and pancreatic cancer depend cellular transformation, resulting in the development of upon Dclk1+ cancer stem cells [11, 13–15]. We reported adenomatous polyps [3]. It was suggested that the APC that Dclk1 is overexpressed in many cancers, including gene mutation is required, but is not sufficient, for the colon, pancreas, liver, and esophageal cancer [12, 17–20]. development of cancer in the colon. Since tumorigenesis Previous work from others and us supported the idea that is considered the result of multiple genetic changes, sev- DCLK1 expression is critical for cancer stem cells, cancer eral efforts have made to identify those tumorigenesis- growth, EMT, and metastasis [11, 12, 15, 18, 21–23]. promoting changes. Several genetic changes, including These data provide a basis for Dclk1 as a regulatory factor activation mutations in Ki-RAS/N-RAS, mutations in the for tumor growth and advancement. tumor suppressor TP53, and deletion of a region of Recent studies have indicated that neoplastic cells have chromosome 18 containing SMAD2, SMAD4, and DCC active pro-survival signaling pathways for proliferation, have been identified [4]. Despite improvements in our resistance, self-renewal, and survival [24–26]. Further- understanding of this disease, the molecular events more, progression of cancer, including metastasis and underlying the development and progression of intes- secondary tumor formation of cancer cells with self- tinal tumors are still largely unknown and may be a key renewal ability, is often linked to altered expression of to the development of more effective and novel thera- pro-survival signaling pathways [27]. Understanding the peutic strategies. Therefore, understanding the APC diversity of pro-survival signaling pathways that underlie gene mutation associated changes for intestinal tumori- cancer formation and progression is essential for devel- genesis is important. oping a new generation of effective anticancer drugs for Similar to humans with germline mutations in APC, combinatorial therapeutic strategies. The interdependence Min/+ Apc mice have a heterozygous mutation in the Apc between pro-survival signaling and tumor self-renewal gene, predisposing the mice to intestinal and colon ability with enhanced Dclk1 highlights the collective tumor development. These mice start developing intes- mechanism involved in tumor growth and survival. How- tinal polyps by ~4 weeks of age, with progression to dys- ever, the precise mechanism by which Dclk1 supports plasia at 18–21 weeks; adenocarcinoma is also evident at intestinal tumor progression is poorly understood. With Min/+ Min/+ ~26-34 weeks [5–8]. Eight-to-twelve-week-old Apc the aid of the Apc mouse model, we assessed the con- mice are a good model with which to study the patho- tribution of Dclk1 to intestinal tumorigenesis using small genesis of FAP, while 26-to-34-week-old ApcMin/+ mice interfering RNAs targeting Dclk1 incorporated into develop intestinal high-grade dysplasia and adenocarcin- poly(lactic-co-glycolic acid) nanoparticles (siDclk1-NPs). oma, and are a particularly relevant model for studying We found that Dclk1 is involved in enhancing the pro- tumor progression and developing therapeutic strategies survival signaling pathways and tumor cells’ self-renewal [6, 7]. ApcMin/+ mice develop high-grade dysplasia and ability to facilitate intestinal tumor growth and progression. adenocarcinoma and are a clinically relevant disease model, since a large number of patients diagnosed with Methods advanced colon cancer are elderly and have unresectable TCGA Colon Adenocarcinoma (COAD) Data or widespread disease [9]. The RNA-seq datasets from February 2015 combining Doublecortin-like kinase 1 (Dclk1) is a microtubule- data from 329 patients with colon adenocarcinomas in- associated protein kinase and has been identified as a cluded in the Cancer Genome Atlas (TCGA) dataset tuft cell marker in the small intestine [10]. Dclk1 has were downloaded through the UCSC cancer genome been reported to mark tumor stem cells in the intestine browser (https://www.xenabrowser.net), as previously and pancreas [11–15]. Emerging evidence has confirmed described [28]. Chandrakesan et al. Molecular Cancer (2017) 16:30 Page 3 of 14 Determination of DCLK1-correlated pro-survival signaling anti-CD45, anti-CD31, anti-EpCAM, and anti-Dclk1 in APC mutant COAD antibodies conjugated with the respective fluorochromes APC mutant/APC non-mutant samples and samples for 30 min. The cells were washed and sorted using an − − with high/low DCLK1 expression levels were sorted by Influx-V cell sorter (Cytopeia). CD45 CD31 EpCAM R v3.2. Patients whose DCLK1 expression levels was in +Dclk1+ cells were collected and then subjected to the top 25% or bottom 25% were considered DCLK1- enterosphere
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