Characterization of Epithelial-Mesenchymal and Neuroendocrine Differentiation States in Pancreatic and Small Cell Ovarian Tumor Cells and Their Modulation by TGF-β1 and BMP-7

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis, due in part to early invasion and metastasis, which in turn involves epithelial-mesenchymal transition (EMT) of the cancer cells. Prompted by the discovery that two PDAC cell lines of the quasi-mesenchymal subtype (PANC-1, MIA PaCa-2) exhibit neuroendocrine differentiation (NED), we asked whether NED is associated with EMT. Using real-time PCR and immunoblotting, we initially verified endogenous expressions of various NED markers, i.e., chromogranin A (CHGA), synaptophysin (SYP), somatostatin receptor 2 (SSTR2), and SSTR5 in PANC-1 and MIA PaCa-2 cells. By means of immunohistochemistry, the expressions of CHGA, SYP, SSTR2, and the EMT markers cytokeratin 7 (CK7) and vimentin could be allocated to the neoplastic ductal epithelial cells of pancreatic ducts in surgically resected tissues from patients with PDAC. In HPDE6c7 normal pancreatic duct epithelial cells and in epithelial subtype BxPC-3 PDAC cells, the expression of CHGA, SYP, and neuron-specific enolase 2 (NSE) was either undetectable or much lower than in PANC-1 and MIA PaCa-2 cells. Parental cultures of PANC-1 cells exhibit EM plasticity (EMP) and harbor clonal subpopulations with both M- and E-phenotypes. Of note, M-type clones were found to display more pronounced NED than E-type clones. Inducing EMT in parental cultures of PANC-1 cells by treatment with transforming growth factor-β1 (TGF-β1) repressed epithelial genes and co-induced mesenchymal and NED genes, except for SSTR5. Surprisingly, treatment with bone morphogenetic protein (BMP)-7 differentially affected gene expressions in PANC-1, MIA PaCa-2, BxPC-3, and HPDE cells. It synergized with TGF-β1 in the induction of vimentin, SNAIL, SSTR2, and NSE but antagonized it in the regulation of CHGA and SSTR5. Phospho-immunoblotting in M- and E-type PANC-1 clones revealed that both TGF-β1 and, surprisingly, also BMP-7 activated SMAD2 and SMAD3 and that in M- but not E-type clones BMP-7 was able to dramatically enhance the activation of SMAD3. From these data, we conclude that in EMT of PDAC cells mesenchymal and NED markers are co-regulated, and that mesenchymal-epithelial transition (MET) is associated with a loss of both the mesenchymal and NED phenotypes. Analyzing NED in another tumor type, small cell carcinoma of the ovary hypercalcemic type (SCCOHT), revealed that two model cell lines of this disease (SCCOHT-1, BIN-67) do express CDH1, SNAI1, VIM, CHGA, SYP, ENO2, and SSTR2, but that in contrast to BMP-7, none of these genes was transcriptionally regulated by TGF-β1. Likewise, in BIN-67 cells, BMP-7 was able to reduce proliferation, while in SCCOHT-1 cells this occurred only upon combined treatment with TGF-β and BMP-7. We conclude that in PDAC-derived tumor cells, NED is closely linked to EMT and TGF-β signaling, which may have implications for the therapeutic use of TGF-β inhibitors in PDAC management.

Keywords: TGF-β; epithelial–mesenchymal transition; mesenchymal–epithelial transition; neuroendocrine differentiation; pancreatic ductal adenocarcinoma.

Figure 1

Relative expression of NED markers in PDAC-derived cell lines, PANC-1, MIA PaCa-2, and BxPC-3, and in the normal pancreatic duct epithelial cell line, HPDE. The panNET-derived cell lines, BON and NT-3, were employed here as controls. Cells were lysed at different times during continuous culture and subjected to RNA isolation and analyzed by qPCR. Expression levels for the indicated genes are displayed relative to those in PANC-1 cells, set arbitrarily at 1.0. Data represent the means ± SD of 3–6 independent preparations. Please note the extra scales for BON and NT-3 cells in the SSTR2, 5 and CHGA graphs, which indicate the orders of magnitude higher expression. Data shown are the mean ± SD of at least three independent experiments. The asterisks (∗) denote significant differences (two-tailed unpaired Student’s t-test). The asterisks above BON and NT-3 cells indicate a significant difference relative to PANC-1 cells.

Figure 2

NED markers are enriched in PANC-1 single-cell-derived clones with an M-phenotype over those with an E-phenotype as indicated in parentheses in (B). (A) Seven individual clones previously grouped according to their EMT state (three E-type and four M-type clones) [6] were subjected to qPCR analysis for the indicated NED markers. Data represent the ratio of mean values between M and E-type clones for each marker. At 1.0, expression is equally high in M and E-type clones (indicated by the stippled line). (B) Immunoblot analysis of SYP in the same clones analyzed in (A). For quantitative analysis, the two closely spaced bands (indicated by arrows on the right-hand side) from two clones each (harvested at different times during continuous culture) were densitometrically scanned using the program NIH image. Hence, data represent the means ± SD of four bands per individual clone. Significant differences between the various clones are marked by asterisks (∗).

Figure 3

IHC of the indicated NED and EMT markers in human PDAC specimens, primary tumors and a liver metastasis. CHGA, SSTR2, and SYP are primarily, and CK7 exclusively, expressed in ductal epithelial tumor cells lining the pancreatic ducts, while VIM is present in tumor cells lining the pancreatic ducts (arrows) as well as in stromal fibroblasts (considered as an internal control). Representative images are shown. For negative controls, see Supplementary Figure S3. The scalebar represents 100 µm.

Figure 4

Effect of treatment with TGF-β1 or BMP-7 on EMT and NED markers in parental PANC-1 cells. Cells were treated with TGF-β1 (5 ng/mL), BMP-7 (200 ng/mL), or vehicle (Ctrl), either singly or in combination, for 24 h and subsequently subjected to qPCR (A) or immunoblot (B) analysis of the indicated EMT- and NED-associated genes. (C) As in (A), only NED-associated markers were detected. (D) Five single-cell-derived clones were treated with TGF-β1 (5 ng/mL) for 24 h and subjected to qPCR analysis for the indicated markers. The graphs below the blots in (B) display the results of densitometry-based quantification of band intensities. Data shown (mean ± SD of three parallel wells) are each from a representative experiment out of at least three experiments performed in total. The asterisks (∗) denote significant differences (two-tailed unpaired Student’s t-test).

Figure 5

Effect of treatment with TGF-β1 or BMP-7 on EMT and NED markers in MIA PaCa-2 cells. Cells were treated with 5 ng/mL of TGF-β1, 200 ng/mL of BMP-7, or vehicle (Ctrl) either singly, or with both growth factors simultaneously, for 24 h and subjected to qPCR (A) or immunoblot (B) analysis of the indicated EMT- and NED-associated markers. The graphs below the blots in (B) display the results of densitometric band quantification. Data shown (mean ± SD of three parallel wells) are from a representative experiment out of at least three experiments performed in total. The asterisks (∗) denote significant differences (two-tailed unpaired Student’s t-test).

Figure 6

Effect of treatment with TGF-β1 or BMP-7 on the activation of SMAD3 and SMAD2 in PANC-1 clones P1C3 (M-type) and P3D2 (E-type). Cells (3 wells each) were treated for 1 h with either vehicle (Control, C), TGF-β1 (T, 10 ng/mL), BMP-7 (B, 200 ng/mL), either singly or in combination (TGF-β1+BMP-7, T+B), lysed, and subjected to phospho-immunoblotting of SMAD3 (upper panels) and SMAD2 (lower panels). The graphs below the blots display the results of densitometric signal quantification. Data shown (mean ± SD of three parallel wells) are from a representative experiment out of three experiments performed in total. The asterisks (∗) denote significant differences (two-tailed unpaired Student’s t-test).

Figure 7

Expression of EMT and NED markers in response to MET induction via TDC-IIT. Parental PANC-1 (upper graphs) and MIA PaCa-2 (lower graphs) cells were subjected to TDC-IIT or control culture (Ctrl) for 72 h followed by lysis and qPCR analysis of markers of EMT (A) or NED (B). The data represent the mean ± SD of quadruplicate wells of a representative assay out of at least three assays performed in total. The asterisks (∗) denote significant differences relative to Ctrl.

Figure 8

Expression of EMT and NED markers in response to TGF-β1 and BMP-7 in SCCOHT. SCCOHT-1 cells were treated with TGF-β1 (10 ng/mL) or BMP-7 (200 ng/mL) followed by lysis and qPCR analysis of markers of EMT (A) or NED (B). The graphs represent the mean ± SD of quadruplicate wells of a representative assay out of at least three independent assays performed in total. The asterisks (∗) denote a significant difference.

Figure 9

Impact of single and combined treatment with TGF-β1 and BMP-7 on functional activities in parental PANC-1, BIN-67, and SCCOHT-1 cells. (A) Migratory activity of parental PANC-1 cells as determined by RTCA using xCELLigence technology. PANC-1 cells received either vehicle (C) or were exposed to TGF-β1 (T, 10 ng/mL), BMP-7 (B, 200 ng/mL), or a combination of both growth factors (T+B) and ran on an xCELLigence device with CIM-plates-16 for a total of 7:45 h. Data were recorded by RTCA software and represent the mean ± SD of three parallel wells. A significant difference between the red and the green curves was first noted at the 3:25 h time point (indicated by the asterisk) and all later time points. (B) BIN-67 and SCCOHT-1 cells were treated for 24 h with either vehicle (C), TGF-β1 (T, 10 ng/mL), BMP-7 (B, 200 ng/mL), or a combination of both growth factors (T+B), after which cells were detached and counted. The graphs represent the mean ± SD of six independent assays (n = 6). The asterisks (∗) denote significant differences relative to C set arbitrarily to 1.00 (two-tailed unpaired Student’s t-test).