Abstract Introduction: Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide. Chronic infections with hepatitis B or C viruses are the leading cause of HCC. The cancer stem cell (CSC) marker, doublecortin-like kinase (DCLK1), is believed to be a key factor for the development of multiple cancer types including HCC. However, its impact on signaling pathways that promote hepatocarcinogenesis is largely unknown. We have shown DCLK1's enhanced expression in HCC and its stimulatory role in hepatitis C virus (HCV) replication. Our published reports further suggest that DCLK1 positively affects tumor-related miRNAs and transcription factors that promote epithelial-mesenchymal transition (EMT). These observations prompted us to investigate DCLK1 signaling module in virus-induced liver pathogenesis and HCC. Methods: Genome-wide transcriptome analysis was carried out in total RNAs isolated from DCLK1-overexpressing and control hepatoma cells. One hundred five clinical cases representing various stages of liver diseases in patients with chronic hepatitis B or C were analyzed for an array of markers including DCLK1 by immunohistochemical staining and Western blot. Huh7 hepatoma cells were transplanted into the flanks of athymic nude mice to generate HCC-like tumor xenografts. The tumors were analyzed for proteins, mRNAs and miRNAs or treated with siRNA against DCLK1 and scrambled siRNA to monitor tumor growth arrest. Results: We found that the expression of 19 genes (14 upregulated, 5 downregulated) was specifically affected by DCLK1 overexpression in hepatoma cell lines. Among these, the level of kinase-suppressor of Ras 1 (KSR1) scaffold protein of Ras-MAPK pathway was increased significantly when recombinant DCLK1 was co-expressed with HCV replicon. Examination of liver tissues derived from patients with chronic hepatitis B/C and HCC suggests that a pro-inflammatory S100A9 protein tends to correlate with DCLK1 overexpression in cirrhosis with or without HCC. Both DCLK1 and S100A9 proteins were mainly localized in the regenerative nodules, fibrotic septa, mesenchymal cells, endothelium and areas with lymphocytes aggregates. Analysis of tumor xenografts developed by hepatoma cells suggest that overexpression of DCLK1 is accompanied by high-level expression of S100A9 and c-Myc as well as activation of NFκB. Conversely, siRNA-led inhibition of DCLK1 causes decrease in tumor volume considerably in a xenograft model. Conclusions: DCLK1 overexpression appears to be intimately related to the activation of pro-inflammatory and MAPK signaling pathways during the development of virus-induced pre-neoplastic conditions and initiation of tumors in liver. Thus, targeting DCLK1 at early stage of liver diseases may prevent virus-induced cirrhosis and HCC. Citation Format: Naushad Ali, Parthasarathy Chandrakesan, Mark Huycke, Sanam Husain, Allison F. Gillaspy, Randal May, William L. Berry, Sripathi Sureban, Dongfeng Qu, Nathaniel Weygant, Michael S. Bronze, Danny N. Dhanasekaran, Courtney W. Houchen. Overexpression of a cancer stem cell marker doublecortin-like kinase (DCLK1) leads to activation of inflammatory cascade during development of virus-induced hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3171. doi:10.1158/1538-7445.AM2014-3171
Abstract Gastrointestinal cancers, including gastric cancer (GC) and colorectal cancer (CRC), continue to be devastating diseases despite advances in screening, early detection, and removal of pre-neoplastic and early cancerous lesions. Even with advancements in targeted chemotherapy and immunotherapies, advanced and metastatic diseases continue to be responsible for the high mortality associated with GC and CRC. Newer, safer, and more effective therapies to prevent, inhibit, or reverse advanced disease progression are urgently needed. Doublecortin-like kinase 1 (DCLK1) is a microtubule associated protein kinase that has been reported to drive tumor initiation, progression, metastasis, and drug resistance in many solid tumor cancers including GC and CRC, where it is upregulated in patient tumors and associated with poor overall survival. An anti-DCLK1 monoclonal antibody has demonstrated potential therapeutic activity against CRC. In this study, we established patient-derived organoids (PDOs) to evaluate the effects of inhibiting DCLK1 kinase activity (DCLK1-IN-1) or targeting DCLK1 with a highly specific CAR-T cells on the treated PDOs. Two GC PDOs were treated with various concentrations of DCLK1 kinase inhibitor DCLK1-IN-1 (0-32 μM) for 72 hrs, viability of the PDOs were determined, histochemistry and immunohistochemistry staining were used to image the live cells and DCLK1 expression. For CRC PDOs, five CRC PDOs were cultured in the presence or absence of PBMC, treated with Mock CAR-T cells or DCLK1 CAR-T cells (three doses) for 4 days. Tumor size, tumor total area, and viability of these PDOs were determined. Here, we demonstrate that treating GC PDOs with DCLK1 kinase inhibitor resulted in a significant dose-dependent inhibition in PDO numbers in both PDOs (two-way ANOVA P<0.0001), a decrease of cell viability, revealing IC50 values of 7.566 and 9.958 μM respectively, and a near total loss of GC PDO viability at 10 μM using CellTiterGlo assay. In CRC PDOs, two of five CRC PDOs showed a significant decrease in total tumor size, tumor areas, and an increase in cell death measured by the DRAQ7 method. Compared to Mock CAR-T cells, treatment with DCLK1 CAR-T cells resulted in a dose dependent decrease of tumor size and total tumor area, with more than a 50% reduction at the 50000 cells dose (p<0.05). Treatment with DCLK1 CAR-T cells also resulted in a dose dependent increase in cell death, with approximately 20% cell death without PBMC, and 50% cell death with PBMC at the 50000 cells dose (p<0.05). These results suggest that targeting DCLK1 represents a potential novel therapeutic strategy for GI cancers, especially in combination with FDA approved drugs: 5-fluorouracil (5-FU) or Gefitinib. Citation Format: Dongfeng Qu, Jinsen Shi, Zhiyun Cao, Eddie BAnnerman-Menson, Nathaniel Weygant, Courtney Houchen. DCLK1 is a novel therapeutic target for gastric and colorectal cancers as demonstrated by patient-derived organoids assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB219.
Doublecortin-like kinase 1 (DCLK1) is a putative pancreatic stem cell marker and is upregulated in pancreatic cancer, colorectal cancer, and many other solid tumors. It marks tumor stem cells in mouse models of intestinal neoplasia. Here we sought to determine whether DCLK1 protein can be detected in the bloodstream and if its levels in archived serum samples could be quantitatively assessed in pancreatic cancer patients. DCLK1 specific ELISA, western blotting, and immunohistochemical analyses were used to determine expression levels in the serum and staining intensity in archived tumor tissues of pancreatic ductal adenocarcinoma (PDAC) patients and in pancreatic cancer mouse models. DCLK1 levels in the serum were elevated in early stages of PDAC (stages I and II) compared to healthy volunteers (normal controls). No differences were observed between stages III/IV and normal controls. In resected surgical tissues, DCLK1 expression intensity in the stromal cells was significantly higher than that observed in tumor epithelial cells. Circulating tumor cells were isolated from KPCY mice and approximately 52% of these cells were positive for Dclk1 staining. Dclk1 levels in the serum of KPC mice were also elevated. We have previously demonstrated that DCLK1 plays a potential role in regulating epithelial mesenchymal transition (EMT). Given the increasingly recognized role of EMT derived stem cells in cancer progression and metastasis, we hypothesize that DCLK1 may contribute to the metastatic process. Taken together, our results suggest that DCLK1 serum levels and DCLK1 positive circulating tumor cells should be further assessed for their potential diagnostic and prognostic significance.
Abstract Crypt epithelial survival and regeneration after injury require highly coordinated complex interplay between resident stem cells and diverse cell types. The function of Dclk1 expressing tuft cells regulating intestinal epithelial DNA damage response for cell survival/self-renewal after radiation-induced injury is unclear. Intestinal epithelial cells (IECs) were isolated and purified and utilized for experimental analysis. We found that small intestinal crypts of Villin Cre ;Dclk1 f/f mice were hypoplastic and more apoptotic 24 h post-total body irradiation, a time when stem cell survival is p53-independent. Injury-induced ATM mediated DNA damage response, pro-survival genes, stem cell markers, and self-renewal ability for survival and restitution were reduced in the isolated intestinal epithelial cells. An even greater reduction in these signaling pathways was observed 3.5 days post-TBI, when peak crypt regeneration occurs. We found that interaction with Dclk1 is critical for ATM and COX2 activation in response to injury. We determined that Dclk1 expressing tuft cells regulate the whole intestinal epithelial cells following injury through paracrine mechanism. These findings suggest that intestinal tuft cells play an important role in regulating the ATM mediated DNA damage response, for epithelial cell survival/self-renewal via a Dclk1 dependent mechanism, and these processes are indispensable for restitution and function after severe radiation-induced injury.
Abstract Introduction: Lung cancers possess a rare population of cells that have the capacity to self-renew, differentiation and ultimately promote tumorigenesis. These cells are often resistance to conventional therapies and are responsible for tumor relapse and metastatic spread, which contribute to the overall mortality observed in non-small cell lung cancer (NSCLC). These cells are termed tumor stem cells (TSCs) and as such are highly desirable targets for novel therapeutic strategies. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is dysregulated in many solid tumor cancers including lung cancer. Increased expression has been demonstrated to correlate with poor survival in several solid tumor cancers. Thus these data taken together provide the rational for targeting DCLK1 expressing cells in NSCLC. Methods: Small interfering RNA (siRNA) against DCLK1 was transfected into NSCLC cell lines (A549 - wt-p53; H1299 –homozygous deletion of p53) of highly drug resistant (A549) and metastatic (H1299). Cell proliferation, colony formation, self-renewal, cell migration and invasion and drug resistance assays were performed to assess the tumor stem cell characteristics. A549 cells were treated with siDCLK1 and cisplatin in order to assess the role of DCLK1 to reverse the drug-resistance. A549 cells were utilized to generate tumor xenograft in order to assess the role of DCLK1 knockdown on NSCLC tumorigenesis in vivo. The data were generated utilizing experimental protocols for clonogenic culture, immunohistochemistry, RT-qPCR and Western blotting. Results: Silencing DCLK1 via RNA interference decreased the colony formation (p<0.001), and self-renewal ability (p<0.0001) of the NSCLC cell lines in vitro. Knocking down DCLK1 reduced (p<0.001) NSCLC cells migration and invasion in vitro. Furthermore, DCLK1 knockdown decreased the expression of EMT associated factors (Slug, Snail, Twist, Zeb1/2, Vimentin) and pluripotency factors. Initial cisplatin treatment results in the isolation of cisplatin resistant A549 cells. Retreatment of cisplatin resistant A549 cells results in greater resistance and survival ability. However, combination therapy of these resistance cells with siDCLK1 and cisplatin overcame cisplatin-induced resistance, resulting in greater cell death compared to cisplatin or siDCLK1 alone treatments. Nanoparticle encapsulated siDCLK1 treatment in xenograft tumors resulted in tumor growth arrest in vivo. Conclusions: NSCLC cells highly express DCLK1 display enhanced self-renewal, increased migration and invasion. Combination therapy of siDCLKL1 with cisplatin overcame cisplatin induced drug resistance. Targeting DCLK1 reduced tumor xenograft in vivo. Taken together these data suggest that DCLK1 inhibition reduces TSC related properties, overcome drug resistance and inhibits tumor growth, thus making DCLK1 targeted therapy an attractive tool for combating NSCLC. Citation Format: Parthasarathy Chandrakesan, Dongfeng Qu, Randal May, Nathaniel Weygant, William Berry, Naushad Ali, Sripathi Sureban, Eddie Bannerman-Menson, Michael Bronze, Courtney Houchen. DCLK1 a novel therapeutic target in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4147. doi:10.1158/1538-7445.AM2017-4147
OBJECTIVES: Esophageal cancer (EC) is a significant cause of cancer death with 5-year survival of 10%–15% and males more frequently affected. Genetic evaluation for loci highlighting risk has been performed, but survival data are limited. The Cancer Genome Atlas (TCGA) data sets allow for potential prognostic marker assessment in large patient cohorts. The study aimed to use the TCGA EC data set to assess whether survival varies by sex and explore genetic alterations that may explain variation observed. METHODS: TCGA clinical/RNA-seq data sets (n = 185, 158 males/27 females) were downloaded from the cancer genome browser. Data analysis/figure preparation was performed in R and GraphPad Prism 7. Survival analysis was performed using the survival package. Text mining of PubMed was performed using the tm, RISmed, and wordcloud packages. Pathway analysis was performed using the Reactome database. RESULTS: In EC, male sex/high tumor grade reduced overall survival (hazard ratio = 2.27 [0.99–5.24] for M vs F and 2.49 [0.89–6.92] for low vs high grade, respectively) and recurrence-free survival (hazard ratio = 4.09 [0.98–17.03] for M vs F and 3.36 [0.81–14.01] for low vs high grade, respectively). To investigate the genetic basis for sex-based survival differences in EC, corresponding gene expression data were analyzed. Sixty-nine genes were dysregulated at the P < 0.01 level by the Wilcox test, 33% were X-chromosome genes, and 7% were Y-chromosome genes. DISCUSSION: Female sex potentially confers an EC survival advantage. Importantly, we demonstrate a genetic/epigenetic basis for these survival differences that are independent of lifestyle-associated risk factors overrepresented in males. Further research may lead to novel concepts in treating/measuring EC aggressiveness by sex.
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