<i>MIR506</i> induces autophagy-related cell death in pancreatic cancer cells by targeting the STAT3 pathway

<p>Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive and lethal cancer. The role of autophagy in the pathobiology of PDAC is intricate, with opposing functions manifested in different cellular contexts. <i>MIR506</i> functions as a tumor suppressor in many cancer types through the regulation of multiple pathways. In this study, we hypothesized that <i>MIR506</i> exerted a tumor suppression function in PDAC by inducing autophagy-related cell death. Our results provided evidence that downregulation of <i>MIR506</i> expression was associated with disease progression in human PDAC. <i>MIR506</i> triggered autophagic flux in PDAC cells, which led to autophagy-related cell death through direct targeting of the <i>STAT3</i> (signal transducer and activator of transcription 3)-<i>BCL2-BECN1</i> axis. Silencing and inhibiting STAT3 recapitulated the effects of <i>MIR506</i>, whereas forced expression of <i>STAT3</i> abrogated the effects of <i>MIR506</i>. We propose that the apoptosis-inhibitory protein BCL2, which also inhibits induction of autophagy by blocking BECN1, was inhibited by <i>MIR506</i> through targeting <i>STAT3</i>, thus augmenting BECN1 and promoting autophagy-related cell death. Silencing <i>BECN1</i> and overexpression of <i>BCL2</i> abrogated the effects of <i>MIR506</i>. These findings expand the known mechanisms of <i>MIR506</i>-mediated tumor suppression to activation of autophagy-related cell death and suggest a strategy for using <i>MIR506</i> as an anti-STAT3 approach to PDAC treatment.</p

Knocking down of p16 enabled the HPNE/K-ras cells to overcome senescence.

<p>(A) Representative fields of senescence-associated β-galactosidase (SA-β-gal) activity in HPNE cell lines. (B) Quantification of SA-β-gal staining in HPNE cells, data are showed as percentage of positive cells **<i>P</i><0.01 versus the corresponding control groups. (C) The expression of cyclin-dependent inhibitors – p15, p21 and p27 was analyzed by Western blot in HPNE cell lines. β-Actin was used as loading control.</p

Molecular analysis of HPNE cell lines offers insight into malignant transformation in human pancreatic cancer.

<p>(A) Activation of the Akt signaling pathway by K-ras in HPNE cell lines as detected by Western blot analysis. (B) Activation of the MAPK signaling pathway by K-ras in HPNE cell lines. The expression of total and phosphorylated-Erk and p38 was detected by Western blot analysis. β-Actin was used as loading control. (C) Expression of p16 was decreased after siRNA depletion of HBP1, as detected by real-time PCR. (D) Relative quantification of SA-β-gal staining cells after siRNA depletion of HBP1 in HPNE/K-ras cells (E) Expression of EGF and TGFα was increased in HPNE/Kras cells as detected by real-time PCR. (F) Expression of TGFα was increased in HPNE-iKras cells as detected by real-time PCR. (G) The proposed model for transformation of HPNE cell line. hTERT enabled cell to acquire replicating immortality; oncogenic K-ras rendered HPNE cells able to sustain proliferative signaling, and thus increased cell proliferation and cell growth; and inactivation of p16 by shRNA silencing enabled HPNE cells to evade growth suppressors, disrupt the senescence checkpoint, and ultimately induce transformation of HPNE cells.</p

Activation of K-ras and silencing of p16 in HPNE cells increased cell proliferation and growth.

<p>(A) The cell growth curve of HPNE cell lines as detected by using a cell counter. (B) Cell cycle analysis of HPNE cell lines. The percentage of cells in each phase of the cell cycle is shown. (C) Anchorage-independent cell growth of HPNE cell lines in soft agar assay. A representative field of soft agar for each cell line is shown. (D) Cells were incubated with gemcitabine for 72 h, and cell viability was measured by MTT assay. (E) The expression of cell cycle proteins cyclins and c-Myc was increased by mutant K-ras in HPNE cell lines, as determined by Western blot analysis. β-Actin was used as loading control. (F) Relative mRNA levels of E2F target genes in HPNE/K-ras and HPNE/K-ras/p16sh cells. All data are presented as mean ± SD (n = 3 independent experiments). **<i>P</i><0.01 versus the corresponding control groups.</p

Activation of K-ras in HPNE cells increased cell invasion.

<p>(A) The quantification of stained HPNE cell lines per field in a transwell-matrigel penetration assay. Representative micrographs for each cell line are shown. Data are presented as mean ± SD (n = 3 independent experiments). **<i>P</i><0.01 versus the HPNE control cells. (B) The expression of EMT markers cytokeratin-19, E-cadherin, vimentin, and N-cadherin was detected in HPNE cell lines by Western blot analysis. (C) The expression of invasion-related proteins MMP2 and uPA in HPNE cell lines was detected by Western blot analysis. β-Actin was used as loading control.</p

HPNE cells with activation of K-ras and inactivation of p16 induced tumorigenesis <i>in vivo</i>.

<p>(A) <i>In vivo</i> bioluminescence imaging of tumor growth by HPNE, HPNE/K-ras and HPNE/K-ras/p16shRNA cells at 8 weeks' after injection in NOD/SCID mice is shown. (B) The rates of tumor formation and metastasis in NOD/SCID mice. (C) Representative micrographs showing the histology of the orthotopic tumors formed by HPNE/K-ras/p16shRNA cells as revealed by H&E staining: (i) undifferentiated ductal carcinoma with sarcomatoid features, (ii) necrosis, (iii) liver metastasis, and (iv) spleen metastasis. (D) Immunohistochemical analysis of the expression of HER-2 and EGFR in tumors formed <i>in vivo</i> by the HPNE/K-ras/p16shRNA cells compared with those in human pancreatic cancer and human normal pancreatic duct. Scale bar: 100 µm.</p

SMAD4 Regulates Cell Motility through Transcription of N-Cadherin in Human Pancreatic Ductal Epithelium

<div><p>Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT). The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β) superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs). Here we describe a mechanism for control of <i>CDH2</i>, the gene encoding N-cadherin, through the canonical TGFβ–SMAD4 pathway. We first identified four previously undescribed SBEs within the <i>CDH2</i> promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at −3790 bp to −3795 bp within the promoter region of <i>CDH2</i> was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after <i>CDH2</i> knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.</p></div

Pearson’s correlation analysis between MAP4K5 and CDH1 mRNA expression measured by RNA sequencing analysis in 112 pancreatic ductal adenocarcinoma samples from The Cancer Genome Atlas (TCGA) data portal.

<p>Pearson’s correlation analysis between MAP4K5 and CDH1 mRNA expression measured by RNA sequencing analysis in 112 pancreatic ductal adenocarcinoma samples from The Cancer Genome Atlas (TCGA) data portal.</p

Prognostic and Functional Significance of MAP4K5 in Pancreatic Cancer - Fig 4

<p>A. Immunoblotting for MAP4K5 and E-cadherin expression in HPDE cells, immortalized human pancreatic ductal cells, and pancreatic cancer cell lines. The 293T cells transfected with MAP4K5 cDNA construct saved as a positive control for immunoblotting. B. Pearson’s correlation analysis between the MAP4K5 mRNA and CDH1 mRNA expression by RNA-sequencing analysis in 11 pancreatic cancer cell lines.</p

Expression of MAP4K5 in non-neoplastic pancreas and pancreatic ductal adenocarcinoma samples.

<p>Representative micrographs show the expression of MAP4K5 in normal pancreatic tissue (A) and chronic pancreatitis (B). The benign pancreatic ductal cells in normal pancreas and chronic pancreatitis show strong cytoplasmic staining for MAP4K5. The adjacent pancreatic acinar cells and pancreatic islet cells are either negative or have very low expression of MAP4K5. C & D, Representative micrographs show the loss of MAP4K5 expression in two different pancreatic ductal adenocarcinoma samples. Strong cytoplasmic staining for MAP4K5 in benign pancreatic ductal cells in the left upper corner in D served as internal positive control. Original magnifications: 200X.</p