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. 2011 Mar 15;71(6):2328-38.
doi: 10.1158/0008-5472.CAN-10-2738. Epub 2011 Feb 1.

DCAMKL-1 regulates epithelial-mesenchymal transition in human pancreatic cells through a miR-200a-dependent mechanism

Sripathi M Sureban  1 , Randal May, Stan A Lightfoot, Aimee B Hoskins, Megan Lerner, Daniel J Brackett, Russell G Postier, Rama Ramanujam, Altaf Mohammed, Chinthalapally V Rao, James H Wyche, Shrikant Anant, Courtney W Houchen
Affiliations

DCAMKL-1 regulates epithelial-mesenchymal transition in human pancreatic cells through a miR-200a-dependent mechanism

Sripathi M Sureban et al. Cancer Res. .

Abstract

Pancreatic cancer is an exceptionally aggressive disease in great need of more effective therapeutic options. Epithelial-mesenchymal transition (EMT) plays a key role in cancer invasion and metastasis, and there is a gain of stem cell properties during EMT. Here we report increased expression of the putative pancreatic stem cell marker DCAMKL-1 in an established KRAS transgenic mouse model of pancreatic cancer and in human pancreatic adenocarcinoma. Colocalization of DCAMKL-1 with vimentin, a marker of mesenchymal lineage, along with 14-3-3 σ was observed within premalignant PanIN lesions that arise in the mouse model. siRNA-mediated knockdown of DCAMKL-1 in human pancreatic cancer cells induced microRNA miR-200a, an EMT inhibitor, along with downregulation of EMT-associated transcription factors ZEB1, ZEB2, Snail, Slug, and Twist. Furthermore, DCAMKL-1 knockdown resulted in downregulation of c-Myc and KRAS through a let-7a microRNA-dependent mechanism, and downregulation of Notch-1 through a miR-144 microRNA-dependent mechanism. These findings illustrate direct regulatory links between DCAMKL-1, microRNAs, and EMT in pancreatic cancer. Moreover, they demonstrate a functional role for DCAMKL-1 in pancreatic cancer. Together, our results rationalize DCAMKL-1 as a therapeutic target for eradicating pancreatic cancers.

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Conflict of interest statement

Authors have no conflict of interest

Figures

Figure 1
Figure 1. DCAMKL-1 expression in P48Cre-LSL-KRASG12D pancreatic cancer mouse model
Pancreatic tissues from 5-month-old WT littermate (400X) (A) and from 5-month-old (100X) (B) P48Cre-LSL-KRASG12D mouse were immunostained for DCAMKL-1. (C) A magnified portion of the image (B) demonstrating cells positive for DCAMKL-1 in the pancreatic duct (400X). (D) A magnified portion of the image (B) demonstrating cells positive for DCAMKL-1 in the islets (400X). Brown colored cells (arrows) indicate cells positive for DCAMKL-1. These data demonstrate an increased expression of DCAMKL-1 correlated with progressive neoplastic changes. (E) PanIN lesions of the 5-month-old P48Cre-LSL-KRASG12D mouse expressed DCAMKL-1 (brown) and 14-3-3 σ (purple). Cells positive for DCAMKL-1 and nuclear 14-3-3 σ are indicated by arrows (400X). (F and G) Areas of co-localization in figure 5E (arrows) are shown as magnified images.
Figure 2
Figure 2. DCAMKL-1 and 14-3-3 σ expression in human pancreatic adenocarcinoma
(A) DCAMKL-1 expression (brown) in histologically normal appearing tissue from human pancreatic cancer resection specimen (top left) (200X). DCAMKL-1 in neoplastic pancreatic islet tissue (top right) (200X). DCAMKL-1 in ductal epithelial cells (bottom left) (400X). Intervening stromal elements demonstrate fibrillar DCAMKL-1 immunoreactivity (bottom right) (200X). Representative cells are indicated by arrows. (B) 14-3-3 σ (purple) and DCAMKL-1 (brown) at the islet periphery in normal appearing human pancreatic tissue (left) (100X). Representative cell demonstrating the cytoplasmic expression of 14-3-3 σ in magnified portion of the left image (right – arrow) (400X). (C) 14-3-3 σ (purple) and DCAMKL-1 (brown) expression in human pancreatic adenocarcinoma (left) (100X). In a magnified portion of the left image, nuclear localized 14-3-3 σ (purple) co-localized with cytoplasmic DCAMKL-1 (brown) (right – arrowhead) (400X). Fibrillar DCAMKL-1 staining in the intervening stroma (arrows). (D) DCAMKL-1 (brown) expression in ductal epithelium of a PanIN type lesion in human pancreatic adenocarcinoma (left – arrow) (400X). Intense cytoplasmic and nuclear staining of 14-3-3 σ (purple) and cytoplasmic DCAMKL-1 (brown) in a PanIN lesion (right – arrow) (400X). Insets in the images on the right in the panel B, C and D are magnified images.
Figure 3
Figure 3. DCAMKL-1 and vimentin expression in human pancreatic adenocarcinoma
(A) DCAMKL-1 expressing cell in a PanIN type lesion (left – arrow). Vimentin expressing cell in the ductal epithelium of a PanIN type lesion (right – arrow). (400X). (B) DCAMKL-1 (red) and vimentin (green) in a PanIN lesion. Co-localization demonstrated in merged image (arrows) and nuclei are stained blue with Hoechst dye (400X). (C) DCAMKL-1 (red) and vimentin (green) in stromal compartment of human pancreatic adenocarcinoma. Co-localization demonstrated in merged image and nuclei are stained blue with Hoechst dye (100X). (D) A magnified portion of bottom right of 4C demonstrating immunolocalization of DCAMKL-1 (red) and vimentin (green) indicated by arrows.
Figure 4
Figure 4. Knockdown of DCAMKL-1 inhibits EMT
(A) DCAMKL-1 specific siRNA (siDCAMKL-1) decreases DCAMKL-1 mRNA expression (left panel), DCAMKL-1 protein expression (middle panel) and increases expression of pri-miR-200a (right panel) compared to scrambled siRNA (siSCR)-treated or Control untreated AsPC-1 human pancreatic cancer cells. (B) AsPC-1-siDCAMKL-1 cancer cells demonstrated decreased expression ZEB1 (left panel), ZEB2 (middle panel) and rescues/upregulates E-cadherin (right panel). (C) DCAMKL-1 (red) and Snail (green) in human pancreatic adenocarcinoma. Co-localization demonstrated in merged image and nuclei are stained blue with Hoechst dye (100X). (D) DCAMKL-1 (red) and Slug (green) in human pancreatic adenocarcinoma. Co-localization demonstrated in merged image and nuclei are stained blue with Hoechst dye (100X). (E) siRNA-mediated knockdown of DCAMKL-1 decreases Snail (left panel), Slug (middle panel) and Twist (right panel) mRNA expression in AsPC-1 cancer cells. Insets in the images on the right in the panel C and D are magnified images. For panels A, B and E values given as mean ± SEM, and asterisks denote statistically significant differences (*p<0.01) compared with control.
Figure 5
Figure 5. DCAMKL-1 regulates oncogenes c-Myc and KRAS via let-7a miRNA
(A) siRNA-mediated knockdown of DCAMKL-1 results in upregulation of pri-miR-let-7a. (B) Knockdown of DCAMKL-1 decreases luciferase activity (luciferase units) following transfection with plasmid encoding luciferase containing let-7a binding site in AsPC-1 cells. (C) A decreased expression of c-Myc mRNA (left panel) and protein (right panel) was observed in AsPC-1 cells following the knockdown of DCAMKL-1. (D) AsPC-1-siDCAMKL-1 cells demonstrated a decrease in KRAS mRNA. (E) Knockdown of DCAMKL-1 decreases luciferase activity (luciferase units) following transfection with plasmid encoding luciferase containing binding sites for let-7 family members (similar to KRAS 3’ UTR) in AsPC-1 cells. Values represented as mean ± SEM, and asterisks denote statistically significant differences (*p<0.01) compared with control.
Figure 6
Figure 6. Knockdown of DCAMKL-1 downregulates Notch-1 via miR-144
(A) siRNA-mediated knockdown of DCAMKL-1 decreases Notch-1 mRNA in AsPC-1 cells. (B) A putative binding site for miR-144 at 189th base pair position on Notch-1 3’ UTR (source: www.microrna.org). (C) AsPC-1-siDCAMKL-1 cells demonstrate increased expression of pri-miR-144. (D) Knockdown of DCAMKL-1 decreases luciferase activity (luciferase units) following transfection with plasmid encoding luciferase containing miR-144 binding site in AsPC-1 cells. Values represented as mean ± SEM, and asterisks denote statistically significant differences (*p<0.01) compared with control.
Figure 7
Figure 7. Selective blockade of DCAMKL-1 results in inhibition of EMT and tumorigenesis in CSCs of pancreatic cancer
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