Tumor Stem Cell Marker Research Articles

Abstract 3884: A tumor stem cell marker DCLK1 promotes hepatocellular carcinoma by regulating β-catenin, EMT and clonogenic properties of hepatocytes

Abstract Introduction: Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related deaths worldwide with a 5-year survival rate of <15%. Chronic viral hepatitis, NASH, and cirrhosis are major risk factors for the HCC development. Thus, HCC represents a major public health issue worldwide. Currently, sorafenib is the only FDA-approved drug used for HCC treatment. Unfortunately, it increases patient's survival by only 3 months. Tumor stem cells (TSCs) are likely responsible for resistance to chemotherapy, relapse, and metastasis. We have demonstrated that a TSC marker, doublecortin-like kinase (DCLK1), is induced during hepatic injury, cirrhosis and HCC and its overexpression in HCC patient’s liver significantly reduces survival. Therefore, understanding of the mechanism(s) hepatic TSCs regulation is important for the development of effective therapy against HCC. Methods: Hepatoma Huh7 cells and primary human hepatocytes were infected with lentiviruses expressing recombinant RFP-DCLK1 fusion protein. The RFP-DCLK1-positive cells were isolated using FACS and subjected to immunofluorescence confocal microscopy, clonogenic assay, Western blot and xenograft in mice. Results: DCLK1-overexpressing human hepatocytes formed spheroids but not the control cells. DCLK1 overexpression induced accumulation of active β-catenin in the perinuclear and nuclear regions in Huh7 cells with concomitant increase in c-Myc. The DCLK1-negative cells lacked active β-catenin and total β-catenin was observed only in their cell membranes. Hepatic β-catenin mRNA levels were unaffected during various stages of HCC. Analysis of HCC patients’ liver tissues (n= 369) suggested that elevated DCLK1 mRNAs expression significantly correlates with increased levels of MMP2, vimentin, Zeb2, Twist1 and VEGF-C which are involved in epithelial-to-mesenchymal transition (EMT) and tumor invasion. Huh7-derived tumors also exhibited high level expression of these factors. Conclusions: These observations suggest that DCLK1 induces tumor/cancer stem cell-like properties and EMT in liver cells. DCLK1 appears to enhance active β-catenin levels and its cellular localization to the nucleus. Thus, the DCLK1 represents an important target for HCC therapy. Citation Format: Naushad Ali, Charles B. Nguyen, Parthasarathy Chandrakesan, Randal May, Danny Dhanasekaran, Michael S. Bronze, Courtney W. Houchen. A tumor stem cell marker DCLK1 promotes hepatocellular carcinoma by regulating β-catenin, EMT and clonogenic properties of hepatocytes [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 3884. doi:10.1158/1538-7445.AM2017-3884

Synergistic effect of IFN-γ on breast cancer targeted therapy

Abstract ErbB2 is amplified in ~30% of breast cancer patients, and its amplification is associated with poor prognosis and worse survival outcome. We examined the synergistic effect of interferon-γ (IFN-γ) with anti-erbB2/neu mAb on erbB2-positive cancer model. IFN-γ showed marginal effects on its own, indicating that immune therapies mediated by this cytokine alone are unlikely to be beneficial. However, we discovered that treatment of the tumors with anti-erbB2/neu mAb concomitant with IFN-γ led to dramatic inhibition of in vivo tumor growth in the syngeneic tumor model and the preventional MMTV-neu transgenic mouse tumor model. We noted an increase of M1 macrophage accumulation in the tumor tissues as well as diminished myeloid derived suppressor cells, and increased CD8+ T cell cytotoxicity against tumor cells in the IFN-γ plus anti-erbB2/neu mAb treated mice. The tumor cells treated with both mAb and IFN-γ underwent changes in tumor stem cell marker ALDH-1, indicating a loss of stem cell-like properties, while mAb treatment alone did not accomplish this phenotypic change. We found that IFN-γ treatment significantly increase tumor PD-L1 expression, so we included anti-PD-1 or PD-L1 antibody with this combination therapy. Interestingly, anti-PD-L1 antibody further reduced tumor growth, while anti-PD-1 did not affect very much. These results indicate that the treatment of IFN-γ can improve targeted therapy, and further induce PD-L1 expression on tumor cells, creating a new target on the tumor cells for anti-PD-L1 therapy.

Doublecortin and CaM kinase-like-1 expression in pathological stage I non-small cell lung cancer

Doublecortin and CaM kinase-like-1 (DCLK1) regulates microtubule polymerization in migrating neurons. Recently, DCLK1 has been reported to act as an intestinal tumor stem cell marker and has been shown to be expressed in cancer cells and in the stroma of breast, colon, pancreatic, and prostate cancers. Here, we studied DCLK1 expression in non-small cell lung cancer (NSCLC) by immunohistochemistry in association with clinicopathological factors and patient prognosis. DCLK1 expression was analyzed by immunohistochemical staining of surgical specimens from 232 patients with pathological stage I NSCLC, including 187 adenocarcinomas. Relationships between the expression status of DCLK1 and clinicopathological factors were examined. The impact of DCLK1 expression status and other clinicopathological factors on survival was evaluated by univariate and multivariate analyses. Thirty-three (14.2%) of 232 patients had DCLK1-positive cancer cells. DCLK1 was also expressed in the tumor stroma in most of the specimens and was significantly associated with DCLK1 expression in cancer cells. DCLK1-positive cancer cells were more common in non-adenocarcinoma tissues (44.4%) than in adenocarcinoma tissues (7.0%). Moreover, positive DCLK1 expression in cancer cells and stromal cells was correlated with a worse prognosis. Histological analyses revealed that the presence of DCLK1-positive cancer cells was an independent risk factor for poor prognosis in adenocarcinoma, but was not significantly associated with prognosis in non-adenocarcinoma. DCLK1 expression was observed in tumor cells in patients with pathological stage I NSCLC and was correlated with adverse prognosis, especially in patients with adenocarcinoma. DCLK1 may be a potential new therapeutic target.

High expression of SALL4 and fascin, and loss of E-cadherin expression in undifferentiated/dedifferentiated carcinomas of the endometrium

Undifferentiated/dedifferentiated endometrial carcinomas (UCE/DCEs) of the endometrium are rare tumors with poor prognosis. There are few clinicopathologic studies with detailed immunohistochemical analysis regarding UCE/DCEs.We evaluated the diagnostic value of a selected tumor stem-cell marker and epithelial-mesenchymal transition (EMT) markers, in addition to previously studied markers in identifying UCE/DCEs from other types of high-grade endometrial carcinomas.Eleven cases of UCE/DCEs with complete clinical follow-up that were diagnosed between 2006 and 2015 were included in the study. For immunohistochemical comparison, 11 clinically matched cases for each type of other high-grade endometrial carcinomas (high-grade endometrioid (F3-EC), serous [SC], and clear cell carcinoma [CCC]) were used as a control group. An immunohistochemical analysis including fascin, SALL4, E-cadherin, and β-catenin, in addition to epithelial and neuroendocrine markers was performed in each case.The majority of UCE/DCEs displayed diffuse expression of fascin (81.9%) and loss of E-cadherin expression (54.5%). SALL4 expression was detected in 36.3% of the UCE/DCE cases. SALL4 expression was significantly more frequent in UCE/DCEs than all other high-grade carcinomas (P < 0.001). Loss of E-cadherin and fascin expression was significantly more frequent in UCE/DCEs than high-grade endometrioid and clear cell adenocarcinomas (P = 0.012, 0.014 and P = 0.01, 0.003, respectively).We suggest that loss of E-cadherin expression together with fascin and SALL4 immunopositivity in addition to morphologic features have an impact in differential diagnosis of UCE/DCEs from other high-grade endometrial carcinomas.

Role of cancer stem-cell marker doublecortin-like kinase 1 in head and neck squamous cell carcinoma

BackgroundSo far, no data is available on the role of the tumor stem cell marker doublecortin-like kinase 1 (DCLK1) in head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to evaluate DCLK1 expression in HNSCC patients that underwent surgery and postoperative radiotherapy, and to assess its potential as a therapeutic target in vitro. MethodsWe immunohistochemically stained for DCLK1 in 127 sections of HNSCC samples obtained during surgery of HNSCC patients and correlated the expression to patients’ overall- and disease-free survival, as well as human papilloma virus (HPV) status. Additionally, we compared our survival data with data obtained from The Cancer Genome Atlas (TCGA). The effects of the DCLK1 inhibitor LRRK-2-in-1 on HNSCC cell lines alone and in combination with irradiation. ResultsExpression of DCLK1 in 127 patients was associated with poor survival. In particular, DCLK1 expression had a significant impact on survival of oropharyngeal carcinoma patients. Specifically, DCLK1+/HPV− patients had the worst prognosis after simultaneous assessment of DCLK1 and HPV status in comparison to the other three possible DCLK1/HPV constellations. Higher levels of DCLK1 mRNA were also associated with poor clinical outcome. Inhibition of DCLK1 in our HNSCC cell lines led to growth arrest and induction of apoptosis. The combination of DCLK1 inhibition with irradiation had a synergistic effect. ConclusionFirstly, DCLK1 is a prognostic biomarker for shortened survival. Secondly, through inhibition of DCLK1, it may serve as a therapeutic target as well.

MicroRNA-424 inhibits cell migration, invasion, and epithelial mesenchymal transition by downregulating doublecortin-like kinase 1 in ovarian clear cell carcinoma

Doublecortin-like kinase 1 (DCLK1) is overexpressed in many cancers and acts as a tumor stem cell marker. Here, we investigated the role of DCLK1 and microRNA-424 (miR-424) in ovarian clear cell carcinoma (OCCC), a histopathologically distinct subtype of epithelial ovarian cancer associated with poor prognosis and chemotherapy resistance. Analysis of samples from 30 OCCC patients showed that DCLK1 was upregulated and miR-424 was downregulated in tumors compared with adjacent non-tumor tissues. DCLK1 overexpression promoted OCCC cell proliferation, migration, and invasion, whereas DCLK1 knockdown reduced cell viability and invasion and induced growth arrest in vitro and in vivo. Dual-luciferase reporter assays revealed that miR-424 directly targets DCLK1 and downregulates its expression. Transfection of ES-2 cells with miR-424 mimics downregulated DCLK1 and suppressed the effects of DCLK1 overexpression on upregulating matrix metalloprotease-9 and promoting epithelial-mesenchymal transition (EMT). Taken together, these data demonstrate that miR-424 has the capacity to suppress cell invasion and EMT in OCCC by downregulating DCLK1, suggesting potential therapeutic targets and strategies for the treatment of this disease.

Dclk1, a tumor stem cell marker, regulates pro-survival signaling and self-renewal of intestinal tumor cells

BackgroundMore 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.MethodsWe 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.ResultsWe 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.ConclusionOur 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.

Abstract 2493: Knocking down the expression of DCLK1 reduces mammary tumor formation, tumor cell self-renewal and metastasis: DCLK1 a novel therapeutic target in breast cancer

Abstract Introduction: Breast cancer is a heterogeneous disease, and is comprised of a rare population of cells that display stem cell-like properties with high metastatic potential and relative resistance to conventional therapies. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is overexpressed in many cancers including breast cancer. In this study, we targeted DCLK1 as a novel therapeutic strategy in breast cancer cells to reduce cancer cell self-renewal, tumorigenesis and metastasis. Methods: Anti-DCLK1-siRNA was transfected into breast cancer cell lines (MCF7 [luminal] and MDAMB231 [basal-triple negative breast cancer cell line]). For analysis of circulating tumor cells, MDAMB231/MCF7 cells were stably transfected with RFP (red fluorescence protein). Tumor cell colony formation, mammosphere formation (self-renewal), cell migration and invasion were assessed to evaluate the effect of DCLK1 knockdown and or DCLK1 overexpression on breast cancer cells self-renewal and metastatic potential. Xenograft model was used to identify the role of DCLK1 on breast cancer cell growth and circulating tumor cells in vivo. The data were generated utilizing experimental protocols for clonogenic culture, circulating tumor cells, immunohistochemistry, RT-qPCR and Western blotting. Results: Expression of DCLK1 was ∼36% higher in human breast cancer tissues than in normal breast tissue. Silencing DCLK1 via RNA interference decreased the colony formation (∼70%), and self-renewal ability (∼85%), of the breast cancer cell lines in vitro. In vitro migration and invasion assays demonstrated that knocking down DCLK1 led to less (40-80%) breast cancer cells migration and invasion. Furthermore, expression of EMT associated factors (Slug, Snail, Twist, Zeb-1, Zeb-2 and Vimentin) and pluripotency factors (c-Myc, Sox2, Nanog and Oct-4) were markedly reduced between 30 and 50%, after silencing DCLK1. Inhibition of DCLK1 in xenograft tumors resulted in reduced tumor growth (∼81%) and CTCs (∼95%) in vivo. However, overexpression of DCLK1 in the non-metastatic MCF7 breast cancer cell line increased its in vitro migration and invasion potency and pluripotency along with in vivo tumor formation and progression. Conclusions: DCLK1 expression is associated with breast cancer cell self-renewal, metastasis and tumorigenesis. Targeting DCLK1 reduced breast cancer cell self-renewal, CTCs, metastasis and tumor growth. DCLK1 inhibition may represent a novel targeted therapeutic approach to reduce the morbidity and mortality associated with breast cancer. Citation Format: PARTHASARATHY CHANDRAKESAN, Nathaniel Weygant, Dongfeng Qu, William Berry, Randal May, Naushad Ali, Sripathi Sureban, Eddie Bannerman-Menson, Michael Schlosser, Courtney Houchen. Knocking down the expression of DCLK1 reduces mammary tumor formation, tumor cell self-renewal and metastasis: DCLK1 a novel therapeutic target in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2493.

Novel MET/TIE2/VEGFR2 inhibitor altiratinib inhibits tumor growth and invasiveness in bevacizumab-resistant glioblastoma mouse models.

Glioblastoma highly expresses the proto-oncogene MET in the setting of resistance to bevacizumab. MET engagement by hepatocyte growth factor (HGF) results in receptor dimerization and autophosphorylation mediating tumor growth, invasion, and metastasis. Evasive revascularization and the recruitment of TIE2-expressing macrophages (TEMs) are also triggered by anti-VEGF therapy. We investigated the activity of altiratinib (a novel balanced inhibitor of MET/TIE2/VEGFR2) against human glioblastoma stem cell lines in vitro and in vivo using xenograft mouse models. The biological activity of altiratinib was assessed in vitro by testing the expression of HGF-stimulated MET phosphorylation as well as cell viability after altiratinib treatment. Tumor volume, stem cell and mesenchymal marker levels, microvessel density, and TIE2-expressing monocyte infiltration were evaluated in vivo following treatment with a control, bevacizumab alone, bevacizumab combined with altiratinib, or altiratinib alone. In vitro, HGF-stimulated MET phosphorylation was completely suppressed by altiratinib in GSC17 and GSC267, and altiratinib markedly inhibited cell viability in several glioblastoma stem cell lines. More importantly, in multiple xenograft mouse models, altiratinib combined with bevacizumab dramatically reduced tumor volume, invasiveness, mesenchymal marker expression, microvessel density, and TIE2-expressing monocyte infiltration compared with bevacizumab alone. Furthermore, in the GSC17 xenograft model, altiratinib combined with bevacizumab significantly prolonged survival compared with bevacizumab alone. Together, these data suggest that altiratinib may suppress tumor growth, invasiveness, angiogenesis, and myeloid cell infiltration in glioblastoma. Thus, altiratinib administered alone or in combination with bevacizumab may overcome resistance to bevacizumab and prolong survival in patients with glioblastoma.

Association between ALDH1+/CD133+ stem-like cells and tumor angiogenesis in invasive ductal breast carcinoma.

The growth and metastasis of tumors is dependent on angiogenesis; however, the association between tumor stem cells (TSCs) and tumor angiogenesis remains to be elucidated. The present study aimed to investigate the expression of the TSC markers aldehyde dehydrogenase 1 (ALDH1) and cluster of differentiation 133 (CD133) in invasive ductal breast carcinoma, and identify their correlation with tumor angiogenesis. Stem-like cells from the breast tissue of 120 patients, who were diagnosed with invasive ductal breast carcinoma at The First Affiliated Hospital of Zhengzhou University (Zhengzhou, Henan, China) between January 2009 and December 2010, were collected by surgical resection and analyzed using immunohistochemical double staining. The expression of the vascular markers CD34, CD105 and vascular endothelial growth factor (VEGF) were determined using single staining. Overall, 25.83% (31/120) of the specimens contained a large number of ALDH1+/CD133+ stem-like cells (ALDH1+/CD133+ tumor). ALDH1+/CD133+ expression is associated with microvessel density, VEGF-positive rate and estrogen receptor expression (P<0.05); however, ALDH1+/CD133+ expression was not associated with age, tumor diameter, lymph node metastasis, histological classification, progesterone receptor expression or human epidermal growth factor receptor 2 expression (P>0.05). The ALDH1+/CD133+ tumor phenotype and expression of VEGF were identified to be correlated in the present study (P=0.020). The present study revealed a close association between breast cancer TSC markers, including ALDH1 and CD133, and tumor angiogenesis. The results of the present study may provide a novel target and treatment strategy for future studies investigating tumor growth and metastasis.

Expression and clinical significance of Oct-4 and E-cad in non-small-cell lung cancer.

Non-small-lung cancer (NSCLC) is a common malignant tumor and is a leading cause of cancer mortality. Tumor stem cells are associated with tumor pathogenesis and development as well as invastion and metastasis. In the present study, the expression and correlation of tumor stem cell markers, octamer-binding transcription factor 4 (Oct-4) and E-cadherin (E-cad) in NSCLC and normal lung tissue were investigated. Additionally, the molecular mechanisms of invasion and metastasis of NSCLC were assessed. The expression of Oct-4 and E-cad was examined in 65 pathologically diagnosed cases of NSCLC using immunohistochemistry. The correlation between Oct-4 and E-cad, as well as the association with pathological grade and clinical staging were also analyzed. Fifteen cases of normal lung tissues served as the control. The positive expression of Oct-4 and abnormal expression of E-cad was higher in the NSCLC tissue compared to the normal lung tissue, and increased as NSCLC malignancy increased. The differences in each grade each stage were statistically significant (P<0.05). A correlation was identified between the abnormal expression of Oct-4 and E-cad (P<0.05, coefficient of contingency C=0.439). In conclusion, the expression of Oct-4 promoted the epithelial-mesenchymal transition in lung cancer.

Colorectal cancer: role of commensal bacteria and bystander effects

For years the human microbiota has been implicated in the etiology of colorectal cancer (CRC). However, identifying the molecular mechanisms for how aneuploidy and chromosomal instability (CIN) arise in sporadic and colitis-associated CRC has been difficult. In this Addendum we review recent work from our laboratory that explore mechanisms by which intestinal commensals polarize colon macrophages to an M1 phenotype to generate a bystander effect (BSE) that leads to mutations, spindle malfunction, cell cycle arrest, tetraploidy, and aneuploidy in epithelial cells. BSE represents the application of a phenomenon initially described in the radiation biology field. The result of commensal-driven BSE on colon epithelial cells is aneuploidy, chromosomal instability (CIN), expression of stem cell and tumor stem cell markers and, ultimately, malignant transformation. Our findings provide a conceptual framework for integrating the microbiota with aging, cyclooxygenase (COX)-2, and inflammation as risk factors for CRC.

Plasma DCLK1 is a marker of hepatocellular carcinoma (HCC): Targeting DCLK1 prevents HCC tumor xenograft growth via a microRNA-dependent mechanism.

Tumor stem cell marker Doublecortin-like kinase1 (DCLK1) is upregulated in several solid tumors. The role of DCLK1 in hepatocellular carcinoma (HCC) is unclear. We immunostained tissues from human livers with HCC, cirrhosis controls (CC), and non-cirrhosis controls (NCC) for DCLK1. Western blot and ELISA analyses for DCLK1 were performed with stored plasma samples. We observed increased immunoreactive DCLK1 in epithelia and stroma in HCC and CCs compared with NCCs, and observed a marked increase in plasma DCLK1 from patients with HCC compared with CC and NCC. Analysis of the Cancer Genome Atlas' HCC dataset revealed that DCLK1 is overexpressed in HCC tumors relative to adjacent normal tissues. High DCLK1-expressing cells had more epithelial-mesenchymal transition (EMT). Various tumor suppressor miRNAs were also downregulated in HCC tumors. We evaluated the effects of DCLK1 knockdown on Huh7.5-derived tumor xenograft growth. This was associated with growth arrest and a marked downregulation of cMYC, and EMT transcription factors ZEB1, ZEB2, SNAIL, and SLUG via let-7a and miR-200 miRNA-dependent mechanisms. Furthermore, upregulation of miR-143/145, a corresponding decrease in pluripotency factors OCT4, NANOG, KLF4, and LIN28, and a reduction of let-7a, miR-143/145, and miR-200-specific luciferase activity was observed. These findings suggest that the detection of elevated plasma DCLK1 may provide a cost-effective, less invasive tool for confirmation of clinical signs of cirrhosis, and a potential companion diagnostic marker for patients with cirrhosis and HCC. Our results support evaluating DCLK1 as a biomarker for detection and as a therapeutic target for eradicating HCC.

Dietary Pectin Increases Intestinal Crypt Stem Cell Survival following Radiation Injury.

Gastrointestinal (GI) mucosal damage is a devastating adverse effect of radiation therapy. We have recently reported that expression of Dclk1, a Tuft cell and tumor stem cell (TSC) marker, 24h after high dose total-body gamma-IR (TBI) can be used as a surrogate marker for crypt survival. Dietary pectin has been demonstrated to possess chemopreventive properties, whereas its radioprotective property has not been studied. The aim of this study was to determine the effects of dietary pectin on ionizing radiation (IR)-induced intestinal stem cell (ISC) deletion, crypt and overall survival following lethal TBI. C57BL/6 mice received a 6% pectin diet and 0.5% pectin drinking water (pre-IR mice received pectin one week before TBI until death; post-IR mice received pectin after TBI until death). Animals were exposed to TBI (14 Gy) and euthanized at 24 and 84h post-IR to assess ISC deletion and crypt survival respectively. Animals were also subjected to overall survival studies following TBI. In pre-IR treatment group, we observed a three-fold increase in ISC/crypt survival, a two-fold increase in Dclk1+ stem cells, increased overall survival (median 10d vs. 7d), and increased expression of Dclk1, Msi1, Lgr5, Bmi1, and Notch1 (in small intestine) post-TBI in pectin treated mice compared to controls. We also observed increased survival of mice treated with pectin (post-IR) compared to controls. Dietary pectin is a radioprotective agent; prevents IR-induced deletion of potential reserve ISCs; facilitates crypt regeneration; and ultimately promotes overall survival. Given the anti-cancer activity of pectin, our data support a potential role for dietary pectin as an agent that can be administered to patients receiving radiation therapy to protect against radiation-induces mucositis.

Abstract 4220: Silencing DCLK1 prevents breast cancer cell self-renewal, epithelial mesenchymal transition, circulating tumor cells and metastasis

Abstract Introduction: Solid tumor cancers including breast cancer possess a rare population of cells that display stem cell-like properties with high metastatic potential and relative resistance to conventional therapies. These features render these cells as highly desirable albeit elusive therapeutic targets. Doublecortin-like Kinase 1 (DCLK1) has recently been identified as a tumor stem cell marker in the intestine and pancreas and is overexpressed in many cancers including breast cancer. In this study, we establish the scientific rationale for targeting DCLK1 to inhibit breast cancer proliferation, tumor stem cell growth, self-renewal, EMT, circulating tumor cells (CTCa) and metastasis. Methods: Small interfering RNA (siRNA) against DCLK1 was transfected into breast cancer cell lines (MCF7 and MDAMB231 (triple negative breast cancer cell line)). For analyzing circulating tumor cells MDAMB231 cells are stably transfected with RFP (red fluorescence protein. Cell proliferation, colony formation, mammosphere formation assay (self-renewal), cell migration and invasion assays were assessed to evaluate the effect of DCLK1 knockdown on breast cancer cells self-renewal and metastatic potential. Isograft model was used to identify the role of DCLK1 knockdown on breast cancer growth and circulating tumor cells in vivo. The assays were analyzed utilizing experimental protocols for cell culture, clonogenic culture, circulating tumor cells by gradient centrifugation, immunohistochemistry, real-time PCR and Western blotting. Results: Expression of DCLK1 was ∼36% higher in human breast cancer tissues than in normal tissue. DCLK1 siRNA inhibited the expression of DCLK1 mRNA and protein in breast cancer cells. Silencing DCLK1 decreased the proliferation (∼40%), colony formation (∼70%), and self-renewal (∼85%), of the breast cancer cell lines in vitro. In vitro migration and invasion assays demonstrated that knocking down DCLK1 led to significantly less (40-80%) breast cancer cells migrated and invaded. Further, expression of EMT associated factors (Slug, Snail, Twist, Zeb-1, Zeb-2 and Vimentin) and pluripotency factors (c-Myc, Sox2, Nanog and Oct-4) were significantly (p&amp;lt;0.05) reduced between 30 and 50%, after silencing DCLK1. Inhibition of DCLK1 in isograft models resulted in reduced tumor growth (∼81%) and CTCs (∼98%) in vivo. Conclusions: DCLK1 expression is associated with breast cancer cell survival, self-renewal, metastasis and tumorigenesis. Silencing DCLK1 reduced breast cancer cell self-renewal, CTCs, metastasis, tumor growth and survival. DCLK1 inhibition may represent a novel targeted therapeutic approach to reduce the morbidity and mortality associated with breast cancer. Citation Format: PARTHASARATHY CHANDRAKESAN, Nathaniel Weygant, Vivian Taylor, William Berry, Randal May, Dongfeng Qu, Meghna Singh, Sripathi Sureban, Naushad Ali, Michael Bronze, Courtney Houchen. Silencing DCLK1 prevents breast cancer cell self-renewal, epithelial mesenchymal transition, circulating tumor cells and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4220. doi:10.1158/1538-7445.AM2015-4220

Abstract 2239: The tumor stem cell marker doublecortin-like kinase (DCLK1) activates inflammatory and carcinogenic signals in hepatocellular carcinoma

Abstract Introduction: Hepatocellular Carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide. Chronic viral hepatitis, non-alcoholic steatohepatitis and cirrhosis are considered to be major risk factors for the development of HCC. The risk of HCC increases significantly in patients with chronic hepatitis C virus (HCV) infection and metabolic diseases. Thus, HCC represents a wide-ranging public health issue worldwide. Our recent studies have revealed a positive correlation between expression of the tumor stem cell (TSC) marker, doublecortin-like kinase (DCLK1) and cancer growth in liver, colon and pancreas. However, the molecular mechanism by which its overexpression impacts key signaling pathways during hepatocarcinogenesis is largely unknown. We have previously shown a dynamic relationship between active HCV replication and expression of a few TSC markers including DCLK1. Our published reports further suggest that DCLK1 positively affects the viral replication and transcription factors that promote epithelial-mesenchymal transition. These observations prompted us to investigate a possible DCLK1-controlled signaling network in HCC. Methods: We used transcriptome analysis of DCLK1-overexpressing and HCV replicon co-expressing cell lines, RNA interference, Huh7 hepatoma cells-derived tumor xenografts, patient's liver tissues and normal human hepatocytes-derived hepatospheroids to investigate DCLK1-regulated signaling pathways in the liver. Immunohistochemical staining, real-time PCR and Western blot were used to validate our observations. The siRNAs and shRNAs against DCLK1 were used to monitor inhibition of cell migration and tumor growth. Results: Although normal human liver lacks detectable DCLK1, extensive DCLK1 expression was observed in lymphoid aggregates, epithelial and stromal cells, lymphocytes, and bile ducts in HCV-positive patients with cirrhosis and HCC. The DCLK1 expression correlated with pro-inflammatory S100A9 levels and activation of NFκB. Normal human hepatocytes-derived spheroids acquired DCLK1 expression, and differentiated into stem/progenitor-like and neuron-like cells upon stimulation with serum. Silencing of DCLK1 inhibited S100A9 expression and hepatoma cell migration. Based on transcriptome data and validation at protein levels, we found a novel DCLK1-controlled feed-forward-like signaling network that potentially drives inflammation and tumorigenesis in the liver. DCLK1 was also found to regulate SMARCA2 (hBRM) protein that participates in the formation of tumor-associated SW1/SNF1 chromatin remodeling complexes. Conclusions: The TCS marker, DCLK1, appears to be a master regulator of inflammatory, oncogenic and chromatic remodeling networks. It is possible to target this signal regulator for preventing hepatic inflammation and liver neoplasia. Citation Format: Naushad Ali, Parthasarathy Chandrakesan, Charles B. Nguyen, Sanam Husain, Allison F. Gillaspy, Mark Huycke, William L. Berry, Randal May, Dongfeng Qu, Nathaniel Weygant, Sripathi M. Sureban, Danny N. Dhanasekaran, Courtney W. Houchen. The tumor stem cell marker doublecortin-like kinase (DCLK1) activates inflammatory and carcinogenic signals in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2239. doi:10.1158/1538-7445.AM2015-2239

Abstract 362: Molecular characterization of circulating tumor cells allows stratifying for prognosis in breast cancer patients undergoing neoadjuvant therapy

Abstract Background: Primary systemic neoadjuvant therapy in breast cancer (BC) is considered to convert inoperable to operable primary tumors and is undertaken to improve surgical options. However, patients might have metastatic relapse despite achieving a pathologic complete remission (pCR). Here we characterized CTCs of BC patients before and after primary systemic neoadjuvant therapy by molecular profiling including HER2, the hormonal receptors estrogen (ER) and progesterone (PR), the tumor stemcell marker ALDH1 as well as markers involved in epithelial mesenchymal transition (EMT) to a) correlate these findings with response to therapy, progression free survival (PFS) and overall survival (OS), b) identify insufficiently treated patients for possible secondary treatment options. Materials and Methods: Two x 5 ml blood samples from BC patients were collected before (n = 142) and after (n = 132) primary systemic therapy. CTCs were analyzed using the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1 and HER-2 transcripts. Expression of the ER and PR receptor was assessed in an additional RT-PCR. Furthermore the recovered c-DNA was additionally multiplex tested for three EMT markers (Twist1, Akt2, PI3Kα) using the AdnaTest EMT and separately for ALDH1 applying the AdnaTest StemCell. The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100. Results: Before therapy, CTCs were detected in 36/142 (25%) of the patients expressing EpCAM (17%), MUC-1 (37%), HER-2 (79%), ER (23%) and PR (6%), respectively. After therapy, 13/132 (10%) of the patients were still positive for CTCs expressing EpCAM and HER2 (each 54%), MUC-1 (43%), ER (17%) and PR (0%), respectively. In addition, 49% of the patients were positive for at least one of the EMT markers before and 14% after therapy, respectively. For ALDH1, the values were 18% and 12%, respectively. CTCs detected after therapy significantly correlated with therapy response whereas no significant associations were found for all CTC-subgroups with regard to PFS and OS. Interestingly, when CTCs were stratified for ER-pos and ER-neg CTCs, ER-neg CTCs before therapy were significantly correlated with reduced PFS (p = 0.03) and OS (p = 0.05) while ER-neg CTCs in EMT before therapy were associated with reduced PFS (p = 0.05). Conclusion: CTCs found before treatment, displaying either ER-neg or EMT characteristics, might serve as an indicator for therapy resistant tumor cell populations. On the other hand, patients with ER-pos CTCs before therapy had the best prognosis. In conclusion, these findings indicate that the identification of the molecular CTC-subtype appears mandatory to differentiate such populations that are correlated with poor prognosis but also such CTC that indicate good responsiveness as well as long term benefit of systemic therapeutic regimens. Citation Format: Lisa König, Oliver Hoffmann, Rainer Kimmig, Sabine Kasimir-Bauer. Molecular characterization of circulating tumor cells allows stratifying for prognosis in breast cancer patients undergoing neoadjuvant therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 362. doi:10.1158/1538-7445.AM2015-362

A Study on the Mechanism of Low-Expressed Cancer Stem Cell Marker Lgr5 in Inhibition of the Proliferation and Invasion of Colorectal Carcinoma.

The present study intends to explore the influence of Lgr5 as a marker of tumor stem cells after siRNA interference on the proliferation and invasion of colorectal carcinoma (CRC) and its mechanism. The tissue samples were taken for biopsy from 32 cases of patients and 32 cases of normal subjects by colonoscopy. Real-time quantitative PCR was used to detect the differential expression of Lgr5. After siRNA interference of Lgr5 in CRC cell line CT-26 cells, RT-PCR method was used to detect the mRNA expression level of Lgr5 after interference of CT-26 cells. CCK8 method was used to observe the influence of Lgr5 interference on the proliferation, colony formation, and invasion of CT-26 cells. RT-PCR and Western blot were used to detect the E-cadherin mRNA and protein levels in CT-26 cells. Lgr5 expression level in CRC tissue was significantly higher than that in the corresponding para-carcinoma tissue and the control group, and the differences were statistically significant (P<0.05). Lgr5 mRNA expression level in tissue with lymph node metastasis was significantly higher than that in the tissue without lymph node metastasis, and the difference was statistically significant (P<0.05). Compared with the control group, CT-26 cell proliferation, colony formation, and migration capability after Lgr5 siRNA transfection were all significantly reduced, and the differences were statistically significant (P<0.05). CT-26 cells after Lgr5 interference were found with significantly reduced E-cadherin mRNA and protein levels. Lgr5 facilitates the cell proliferation, colony formation, and migration of colorectal carcinoma, which may be closely related to the expression level of E-cadherin.

Inflammatory and oncogenic roles of a tumor stem cell marker doublecortin-like kinase (DCLK1) in virus-induced chronic liver diseases.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality worldwide. We previously showed that a tumor/cancer stem cell (CSC) marker, doublecortin-like kinase (DCLK1) positively regulates hepatitis C virus (HCV) replication, and promotes tumor growth in colon and pancreas. Here, we employed transcriptome analysis, RNA interference, tumor xenografts, patient's liver tissues and hepatospheroids to investigate DCLK1-regulated inflammation and tumorigenesis in the liver. Our studies unveiled novel DCLK1-controlled feed-forward signaling cascades involving calprotectin subunit S100A9 and NFκB activation as a driver of inflammation. Validation of transcriptome data suggests that DCLK1 co-expression with HCV induces BRM/SMARCA2 of SW1/SNF1 chromatin remodeling complexes. Frequently observed lymphoid aggregates including hepatic epithelial and stromal cells of internodular septa extensively express DCLK1 and S100A9. The DCLK1 overexpression also correlates with increased levels of S100A9, c-Myc, and BRM levels in HCV/HBV-positive patients with cirrhosis and HCC. DCLK1 silencing inhibits S100A9 expression and hepatoma cell migration. Normal human hepatocytes (NHH)-derived spheroids exhibit CSC properties. These results provide new insights into the molecular mechanism of the hepatitis B/C-virus induced liver inflammation and tumorigenesis via DCLK1-controlled networks. Thus, DCLK1 appears to be a novel therapeutic target for the treatment of inflammatory diseases and HCC.

Su2003 Nrip2, a Novel Regulator of the Non-Canonical Wnt Pathway, Promotes the Self-Renewal of Colorectal Cancer Initiating Cells

As normal tissues contain normal tissue stem cells (NSCs), tumors are thought to contain tumor stem cells (TSCs) which perform self-renewal and generate their progenies. TSC can be a therapeutic target; however, cancer therapies targeting TSCs are limited, because most of the TSC markers are shared by NSCs. Therefore, it is important to identify TSC-specific markers. Previously, we reported that doublecortin-like kinase 1 (Dclk1)-positive epithelial cells do not produce any progenies in the normal small intestine but supply progeny tumor cells in the tumors of Apc mice. Selective ablation of Dclk1-positive TSCs collapses the intestinal tumors of Apc mice. Therefore, Dclk1 appears to be a specific marker of TSCs in the mouse intestinal tumors. In the present study, we performed microarray analysis in order to investigate the characteristics of Dclk1-positive TSCs in the tumors of Apc mice. Dclk1-positive tumor cells had higher expression levels of cyclooxygenase 1 (Cox1), leukotriene C4 synthase (Ltc4s), and arachidonate 5-lipoxygenase activating protein (Alox5ap) than Dclk1-negative cells in the tumors of Apc mice, suggesting that Dclk1positive cells in the tumors of Apc mice had higher expression levels of the genes which were related to prostaglandin and leukotriene metabolism. As well, microarray analysis revealed that gustducin alpha and gamma subunits, transient receptor potential cation channel, subfamily M, member 5 (TRPM5), and phospholipase C beta 2 were more highly expressed in Dclk1-positive tumor cells. Thus, Dclk1-positive tumor cells seemed to possess the characteristic of Tuft cells. Furthermore, Dclk1-positive tumor cells had higher expression level of Ras-related C3 botulinum toxin substrate 2 (Rac2) than Dclk1-negative tumor cells in Apc mouse intestine. In order to elucidate the role of Rac2 in Dclk1-positive TSCs, we generated tumor spheroids from Apc mouse tumors, and administrated EHT1864, Rac inhibitor to the spheroids. Following 5-day treatment with EHT1864, the growth of the tumor spheroids was significantly suppressed. Importantly, on day1, EHT1864 treatment significantly reduced the proportion of Dclk1-positive cells in the tumor spheroids. These results suggested that Rac2 played some roles for the maintenance of the intestinal tumor spheroids. In conclusion, this study may provide new insights into the maintenance mechanisms of Dclk1-positive TSC in the intestinal tumors, and give us hints to develop novel therapeutic strategies targeting TSCs against colorectal cancers.