Parthasarathy Chandrakesan

DCLK1 Regulates Pluripotency and Angiogenic Factors via microRNA-Dependent Mechanisms in Pancreatic Cancer

Stem cell pluripotency, angiogenesis and epithelial-mesenchymal transition (EMT) have been shown to be significantly upregulated in pancreatic ductal adenocarcinoma (PDAC) and many other aggressive cancers. The dysregulation of these processes is believed to play key roles in tumor initiation, progression, and metastasis, and is contributory to PDAC being the fourth leading cause of cancer-related deaths in the US. The tumor suppressor miRNA miR-145 downregulates critical pluripotency factors and oncogenes and results in repressed metastatic potential in PDAC. Additionally, the miR-200 family regulates several angiogenic factors which have been linked to metastasis in many solid tumors. We have previously demonstrated that downregulation of DCLK1 can upregulate critical miRNAs in both in vitro and in vivo cancer models and results in downregulation of c-MYC, KRAS, NOTCH1 and EMT-related transcription factors. A recent report has also shown that Dclk1 can distinguish between normal and tumor stem cells in Apc min/+ mice and that ablation of Dclk1+ cells resulted in regression of intestinal polyps without affecting homeostasis. Here we demonstrate that the knockdown of DCLK1 using poly(lactide-co-glycolide)-encapsulated-DCLK1-siRNA results in AsPC1 tumor growth arrest. Examination of xenograft tumors revealed, (a) increased miR-145 which results in decreased pluripotency maintenance factors OCT4, SOX2, NANOG, KLF4 as well as KRAS and RREB1; (b) increased let-7a which results in decreased pluripotency factor LIN28B; and (c) increased miR-200 which results in decreased VEGFR1, VEGFR2 and EMT-related transcription factors ZEB1, ZEB2, SNAIL and SLUG. Specificity of DCLK1 post-transcriptional regulation of the downstream targets of miR-145, miR-200 and let-7a was accomplished utilizing a luciferase-based reporter assay. We conclude that DCLK1 plays a significant master regulatory role in pancreatic tumorigenesis through the regulation of multiple tumor suppressor miRNAs and their downstream pro-tumorigenic pathways. This novel concept of targeting DCLK1 alone has several advantages over targeting single pathway or miRNA-based therapies for PDAC.

XMD8-92 inhibits pancreatic tumor xenograft growth via a DCLK1-dependent mechanism

XMD8-92 is a kinase inhibitor with anti-cancer activity against lung and cervical cancers, but its effect on pancreatic ductal adenocarcinoma (PDAC) remains unknown. Doublecortin-like kinase1 (DCLK1) is upregulated in various cancers including PDAC. In this study, we showed that XMD8-92 inhibits AsPC-1 cancer cell proliferation and tumor xenograft growth. XMD8-92 treated tumors demonstrated significant downregulation of DCLK1 and several of its downstream targets (including c-MYC, KRAS, NOTCH1, ZEB1, ZEB2, SNAIL, SLUG, OCT4, SOX2, NANOG, KLF4, LIN28, VEGFR1, and VEGFR2) via upregulation of tumor suppressor miRNAs let-7a, miR-144, miR-200a-c, and miR-143/145; it did not however affect BMK1 downstream genes p21 and p53. These data taken together suggest that XMD8-92 treatment results in inhibition of DCLK1 and downstream oncogenic pathways (EMT, pluripotency, angiogenesis and anti-apoptotic), and is a promising chemotherapeutic agent against PDAC.

Novel Changes in NF-κB Activity during Progression and Regression Phases of Hyperplasia: ROLE OF MEK, ERK, AND p38*

Utilizing the Citrobacter rodentium-induced transmissible murine colonic hyperplasia (TMCH) model, we measured hyperplasia and NF-κB activation during progression (days 6 and 12 post-infection) and regression (days 20–34 post-infection) phases of TMCH. NF-κB activity increased at progression in conjunction with bacterial attachment and translocation to the colonic crypts and decreased 40% by day 20. NF-κB activity at days 27 and 34, however, remained 2–3-fold higher than uninfected control. Expression of the downstream target gene CXCL-1/KC in the crypts correlated with NF-κB activation kinetics. Phosphorylation of cellular IκBα kinase (IKK)α/β (Ser176/180) was elevated during progression and regression of TMCH. Phosphorylation (Ser32/36) and degradation of IκBα, however, contributed to NF-κB activation only from days 6 to 20 but not at later time points. Phosphorylation of MEK1/2 (Ser217/221), ERK1/2 (Thr202/Tyr204), and p38 (Thr180/Tyr182) paralleled IKKα/β kinetics at days 6 and 12 without declining with regressing hyperplasia. siRNAs to MEK, ERK, and p38 significantly blocked NF-κB activity in vitro, whereas MEK1/2-inhibitor (PD98059) also blocked increases in MEK1/2, ERK1/2, and IKKα/β thereby inhibiting NF-κB activity in vivo. Cellular and nuclear levels of Ser536-phosphorylated (p65536) and Lys310-acetylated p65 subunit accompanied functional NF-κB activation during TMCH. RSK-1 phosphorylation at Thr359/Ser363 in cellular/nuclear extracts and co-immunoprecipitation with cellular p65-NF-κB overlapped with p65536 kinetics. Dietary pectin (6%) blocked NF-κB activity by blocking increases in p65 abundance and nuclear translocation thereby down-regulating CXCL-1/KC expression in the crypts. Thus, NF-κB activation persisted despite the lack of bacterial attachment to colonic mucosa beyond peak hyperplasia. The MEK/ERK/p38 pathway therefore seems to modulate sustained activation of NF-κB in colonic crypts in response to C. rodentium infection.

Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1

BackgroundDoublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous in vitro and in vivo studies have demonstrated the therapeutic effects of inhibiting DCLK1 with small interfering RNA (siRNA) as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Therefore, we assessed the effects of inhibiting DCLK1 kinase activity using the novel small molecule kinase inhibitor, LRRK2-IN-1, which demonstrates significant affinity for DCLK1.ResultsHere we report that LRRK2-IN-1 demonstrates potent anti-cancer activity including inhibition of cancer cell proliferation, migration, and invasion as well as induction of apoptosis and cell cycle arrest. Additionally we found that it regulates stemness, epithelial-mesenchymal transition, and oncogenic targets on the molecular level. Moreover, we show that LRRK2-IN-1 suppresses DCLK1 kinase activity and downstream DCLK1 effector c-MYC, and demonstrate that DCLK1 kinase activity is a significant factor in resistance to LRRK2-IN-1.ConclusionsGiven DCLK1’s tumor stem cell marker status, a strong understanding of its biological role and interactions in gastrointestinal tumors may lead to discoveries that improve patient outcomes. The results of this study suggest that small molecule inhibitors of DCLK1 kinase should be further investigated as they may hold promise as anti-tumor stem cell drugs.

Critical Roles of Notch and Wnt/β-Catenin Pathways in the Regulation of Hyperplasia and/or Colitis in Response to Bacterial Infection

ABSTRACT Notch and Wnt/β-catenin signals play essential roles in intestinal development and homeostasis. Citrobacter rodentium induces transmissible murine colonic hyperplasia (TMCH) and various degrees of inflammation, depending upon the genetic background. We aimed at delineating the role of the Notch and Wnt/β-catenin pathways in the regulation of colonic crypt hyperplasia and/or colitis following C. rodentium infection. During TMCH, relative levels of the Notch intracellular domain (NICD) increased significantly, along with increases in Jagged-1 and Hes-1 coinciding with the progression and regression phases of hyperplasia. Blocking of Notch signaling with dibenzazepine (DBZ) for 5 days before the onset of hyperplasia also blocked Wnt/β-catenin signaling. Targeting the Notch pathway for 5 days after the onset of hyperplasia failed to inhibit Wnt/β-catenin-regulated crypt hyperplasia. Chronic DBZ administration for 10 days blocked both Notch and Wnt signaling, disrupted the intestinal barrier, and induced colitis. Core-3−/− mice, which are defective in mucin secretion and are susceptible to experimental triggers of colitis, also exhibited significant colitis in response to C. rodentium plus DBZ. Chronic DBZ administration in these mice did not result in depletion of the putative stem cell marker doublecortin-like kinase-1 (DCLK1) in the crypts. Dietary bael (Aegle marmelos) extract (4%) and curcumin (4%) restored signaling via the Notch and Wnt/β-catenin pathways, thereby promoting crypt regeneration, and also replenished the mucus layer, leading to amelioration of C. rodentium- and DBZ-induced colitis in NIH:Swiss mice. Thus, the balancing act between cell proliferation and mucus production to restore barrier integrity seems to depend upon the interplay between the Wnt/β-catenin and Notch pathways in the TMCH model.

Brief Report: Dclk1 Deletion in Tuft Cells Results in Impaired Epithelial Repair After Radiation Injury

The role of Dclk1+ tuft cells in the replacement of intestinal epithelia and reestablishing the epithelial barrier after severe genotoxic insult is completely unknown. Successful restoration requires precise coordination between the cells within each crypt subunit. While the mechanisms that control this response remain largely uncertain, the radiation model remains an exceptional surrogate for stem cell‐associated crypt loss. Following the creation of Dclk1‐intestinal‐epithelial‐deficient Villin‐Cre;Dclk1flox/flox mice, widespread gene expression changes were detected in isolated intestinal epithelia during homeostasis. While the number of surviving crypts was unaffected, Villin‐Cre;Dclk1flox/flox mice failed to maintain tight junctions and died at approximately 5 days, where Dclk1flox/flox mice lived until day 10 following radiation injury. These findings suggest that Dclk1 plays a functional role critical in the epithelial restorative response. Stem Cells 2014;32:822–827

Distinct Compartmentalization of NF-κB Activity in Crypt and Crypt-Denuded Lamina Propria Precedes and Accompanies Hyperplasia and/or Colitis following Bacterial Infection

ABSTRACT Citrobacter rodentium induces transmissible murine colonic hyperplasia (TMCH) and variable degrees of inflammation and necrosis depending upon the genetic background. Utilizing C. rodentium-induced TMCH in C3H/HeNHsd inbred mice, we observed significant crypt hyperplasia on days 3 and 7 preceding active colitis. NF-κB activity in the crypt-denuded lamina propria (CLP) increased within 24 h postinfection, followed by its activation in the crypts at day 3, which peaked by day 7. Increases in interleukin-α1 (IL-1α), IL-12(p40), and macrophage inflammatory protein 1α (MIP-1α) paralleled NF-κB activation, while increases in IL-1α/β, IL-6/IL-12(p40)/granulocyte colony-stimulating factor (G-CSF)/keratinocyte-derived chemokine (KC)/monocyte chemotactic protein 1 (MCP-1), and MIP-1α followed NF-κB activation leading to significant recruitment of neutrophils to the colonic mucosa and increased colonic myeloperoxidase (MPO) activity. Phosphorylation of the crypt cellular and nuclear p65 subunit at serines 276 and 536 led to functional NF-κB activation that facilitated expression of its downstream target, CXCL-1/KC, during TMCH. Distinct compartmentalization of phosphorylated extracellular signal-regulated kinase 1 and 2 ([ERK1/2] Thr180/Tyr182) and p38 (Thr202/Tyr204) in the CLP preceded increases in the crypts. Inhibition of ERK1/2 and p38 suppressed NF-κB activity in both crypts and the CLP. Dietary administration of 6% pectin or 4% curcumin in C. rodentium-infected mice also inhibited NF-κB activity and blocked CD3, F4/80, IL-1α/β, G-CSF/MCP-1/KC, and MPO activity in the CLP while not affecting NF-κB activity in the crypts. Thus, distinct compartmentalization of NF-κB activity in the crypts and the CLP regulates crypt hyperplasia and/or colitis, and dietary intervention may be a novel strategy to modulate NF-κB-dependent protective immunity to facilitate crypt regeneration following C. rodentium-induced pathogenesis.

Doublecortin-like kinase 1 is elevated serologically in pancreatic ductal adenocarcinoma and widely expressed on circulating tumor cells.

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.

Dclk1 + small intestinal epithelial tuft cells display the hallmarks of quiescence and self-renewal

To date, no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. In order to elucidate the potential function of Dclk1+ cells, we FACS-sorted Dclk1+ cells from mouse small intestinal epithelium using transgenic mice expressing YFP under the control of the Dclk1 promoter (Dclk1-CreER;Rosa26-YFP). Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFP− cells. Dclk1+ population display ~6 fold enrichment for the putative quiescent stem cell marker Bmi1. We observed significantly greater expression of pluripotency genes, pro-survival genes, and quiescence markers in the Dclk1+ population. A significant increase in self-renewal capability (14-fold) was observed in in vitro isolated Dclk1+ cells. The unique genetic report presented in this manuscript suggests that Dclk1+ cells may maintain quiescence, pluripotency, and metabolic activity for survival/longevity. Functionally, these reserve characteristics manifest in vitro, with Dclk1+ cells exhibiting greater ability to self-renew. These findings indicate that quiescent stem-like functionality is a feature of Dclk1-expressing tuft cells.

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.