Volume 125, Issue 3, Pages 891-905 (September 2003) Activated signal transducer and activator of transcription 3 (STAT3) supports the malignant phenotype of human pancreatic cancer  Arne Scholz, Sandra Heinze, Katharina M Detjen, Michael Peters, Martina Welzel, Peter Hauff, Michael Schirner, Bertram Wiedenmann, Stefan Rosewicz  Gastroenterology  Volume 125, Issue 3, Pages 891-905 (September 2003) DOI: 10.1016/S0016-5085(03)01064-3

Figure 1 Expression of active, tyrosine-phosphorylated STAT3 in human pancreatic cancer. Sections of paraffin-embedded pancreatic tissue were immunostained with a p(tyr)STAT3 antibody by using the alkaline phosphatase/anti-alkaline phosphatase detection method. Representative examples of immunohistochemical staining are shown for (A) normal pancreas, (B) chronic pancreatitis, and (C-F) pancreatic carcinoma. (A, B, and C [left side]) Ductal epithelia of nontransformed pancreas are predominantly negative. In malignant transformed ductal structures of pancreatic carcinoma, (C [right side] and D) strong nuclear immunostaining was observed as well as (E) in perineural tumor infiltration and (F) lymph node metastasis. Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)

Figure 2 Expression and activation of STAT3 in human pancreatic carcinoma cell lines. Whole cell lysates of 7 exocrine human pancreatic cancer cell lines and a human neuroendocrine cell line (LCC-18) were analyzed by immunoblotting. Equal amounts of protein (26 μg) were subjected to SDS-PAGE and were immunoblotted by using an antibody against STAT3. The blot was stripped and sequentially incubated with antibodies against the tyrosine- and serine-phosphorylated form of STAT3, respectively. Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)

Figure 3 Autocrine EGF-R activation stimulates STAT3 activity of human pancreatic cancer cells. (A) Effect of tyrphostin AG1517 (10 μmol/L, 48 hours) on STAT3 expression and phosphorylation as well as on the expression of regulatory SOCS-1 and SOCS-3 molecules. (B) Time course of AG1517-induced effects on STAT3 and SOCS. CAPAN-1 cells were cultured overnight before stimulation with 10 μmol/L AG1517 for the indicated periods. Representative immunoblots from 2 experiments that yielded similar results are shown. (C) Effects of EGF-R inhibition on STAT3 DNA binding. Nuclear extracts from CAPAN-1 cells were incubated with a [γ-32P]adenosine triphosphate-labeled STAT3 consensus oligonucleotide (wt) and electrophoretic mobility shift assays were conducted (lanes 1–6). For competition and supershift experiments, extracts were preincubated with a 100-fold molar excess of either unlabeled consensus (wt) or mutated (mut, deficient in STAT3 DNA binding) oligonucleotide or 2 μg of antibodies to STAT1 or STAT3. For stimulation with IL-6 (lanes 7 and 8), cells were serum starved for 4 hours and incubated with 100 ng/mL of IL-6 in serum-free medium for 15 minutes before nuclear extracts were prepared. Treatment with AG1517 (lanes 9–18) and treatment with the anti-EGF-R antibody (10 μg/mL) (lanes 19 and 20) were performed in the presence of medium. Concentrations are in μmol/L. Co, control; V, vehicle (dimethyl sulfoxide). Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)

Figure 4 Functional inhibition of STAT3 in CAPAN-1 cells by stable transfection with dominant-negative STAT3 constructs. Two HA-tagged dominant-negative constructs and empty vector controls were stably transfected in CAPAN-1 cells. (A) Expression of dominant-negative STAT3 in CAPAN-1 cells. Whole cell lysates of dominant-negative STAT3 transfectants (D4, F2, and F5) in CAPAN-1 (D16, D19, and F8 in BxPc-3 cells), wild-type (WT), and vector-transfected (V) controls with equal amounts of protein (20 μg) were subjected to SDS-PAGE and immunoblotted with an antibody against the HA tag. Blots were stripped and reprobed with STAT3-, p(tyr)STAT3-, and p(ser)STAT3-specific antibodies. For loading control, expression of proliferating nuclear antigen (PCNA) was evaluated. (B) DNA-binding activity of dominant-negative STAT3 clones from CAPAN-1 cells (lanes 5–7; clones D4, F2, and F5) and BxPC-3 cells (lanes 10–12; clones D16, D19, and F8) was compared with wild-type (WT) and vector-transfected controls (V) (lanes 1–4, 8, and 9) by electrophoretic mobility shift assay. Ten micrograms of nuclear extracts from serum starved (4 hours) cells was incubated with a [γ-32P]adenosine triphosphate-labeled STAT3 consensus oligonucleotide (cons). Nuclear extracts from CAPAN-1 were incubated in parallel with a [γ-32P]adenosine triphosphate-labeled SP1 consensus oligonucleotide and SP1 DNA binding was examined (lanes 13–17). Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)

Figure 5 Growth inhibition of CAPAN-1 and BxPC-3 cells by dominant-negative STAT3. (A and B) For anchorage-dependent growth, 105 (CAPAN-1) or 104 (BxPC-3) cells per clone were seeded. Viable cells were counted manually with a hemocytometer at the indicated times (CAPAN-1) or after 96 hours (BxPC-3), respectively. (C and D) Anchorage-independent growth was estimated by colony formation in soft agar. Cell agar suspension of 103 cells per clone were seeded. Colonies (>20 cells) were scored 10 days after plating. (A-D) Each experiment was performed in triplicate. Data represent mean ± SEM of 3 independent experiments. ∗Significantly different from wild-type (WT) and vector-transfected controls (V) (P < 0.05). Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)

Figure 6 Activated STAT3 promotes tumor growth in vivo. (A) Ten mice were injected with 1.5 × 106 CAPAN-1 cells of vector-transfected control (V) and dominant-negative STAT3 clones (D4, F2, F5). Tumor volume was measured twice weekly by calculating the product of the largest diameter and its perpendicular. Data represent mean ± SEM. ∗Significantly different from vector controls (P < 0.05) at indicated times. (B) Immunodetection of dominant-negative STAT3 expression in tumor tissue of mice. A total of 200 mg frozen tissue of the xenotransplanted tumor at day 21 was lysed and homogenized. A total of 50 μg protein was subjected to SDS-PAGE and incubated with the antibody against HA tag. Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)

Figure 7 Effect of dominant-negative STAT3 on cell cycle progression. Vector (control) and dominant-negative STAT3-transfected CAPAN-1 cells (clone F2) were synchronized in G0/G1 phase by serum starvation for 24 hours. Following stimulation with medium/15% FCS, cells were prepared for flow cytometry analysis after 0, 6, 12, 18, and 24 hours. Fluorescence-activated cell sorter analysis was performed based on DNA content of 104 cells. (A) A representative analysis of a time-course experiment is shown. (B) Statistical analysis of cell cycle distribution. Data represent the mean ± SEM of 5 independent experiments. ∗Significantly different compared with vector-transfected controls (P < 0.05). Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)

Figure 8 Inactivation of STAT3 inhibits CDK-2 activity in CAPAN-1 cells. (A and B) Vector-transfected controls (V) and dominant-negative STAT3-transfected F2 cells were synchronized in G0/G1 phase by serum starvation for 24 hours. Cells were then released and whole cell lysates prepared at the indicated times. Cell cycle progression was controlled by simultaneous flow cytometry analysis (see Figure 7). Expression levels of cell cycle components were analyzed by immunoblotting using 50 μg of protein lysate per lane and the respective specific antibodies. Representative results of 3–4 independent experiments are shown. For histone H1 and GST-Rb kinase assays, lysates were immunoprecipitated with CDK-2- or CDK-4-specific antibodies, respectively. After the kinase reaction was terminated, the whole reaction volume was subjected to SDS-PAGE and kinase activity determined by autoradiography of the dried gels. Each kinase experiment was independently repeated at least 3 times. Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)

Figure 9 Inhibition of activated STAT3 increases the p21WAF1 content of CDK-2 complexes. (A) In analogy to the experimental protocol previously outlined, CDK-2 immunoprecipitation of vector control (V) and F2 cells was performed and sequentially immunoblotted with antibodies against p21WAF1 and CDK-2. (B) Up-regulation of p21WAF1 expression in vivo. Tumor tissue lysates derived from nude mice xenotransplanted with dominant-negative STAT3 (D4, F2, F5) and vector-transfected CAPAN-1 cells were subjected to immunoblotting using p21WAF1 antibody. (C) Effect of dominant-negative STAT3 on expression levels of putative STAT3 target genes investigated in CAPAN-1 cells by immunoblot analysis of whole cell lysates using specific antibodies as indicated. Proliferating cell nuclear antigen content of CDK-2 complexes was not altered, suggesting that p21WAF1 regulation was specific. Arrows indicate the molecular sizes of the detected bands. Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)

Figure 10 Effect of tyrphostin AG490 on CAPAN-1 cells. (A) Inhibition of JAK2 tyrosine phosphorylation. Cells were incubated with AG490 or vehicle (dimethyl sulfoxide) for 24 hours. A total of 2200 μg of whole cell lysate was immunoprecipitated with a JAK2 antibody and immunoblotted using an antibody against p-tyrosine. To control for equal amounts of immunoprecipitates, blots were stripped and reprobed with JAK2 antibody. (B) Dose-dependent inhibition of anchorage-dependent growth. Triplicates of 104 cells were incubated with the indicated concentrations of AG490. After 96 hours, viable cells were counted manually with a hemocytometer. Data represent mean ± SEM of 3 independent experiments. ∗Significantly different from vehicle-treated control (P < 0.05). (C) Cell cycle distribution. After synchronization as previously outlined, cells were exposed to 100 μmol/L AG490 and submitted to flow cytometry analysis at the indicated times. A representative fluorescence-activated cell sorter analysis of 4 independent experiments, all yielding similar results, is shown. (D) Down-regulation of STAT3 activity and up-regulation of p21WAF1 expression. CAPAN-1 cells were treated with the indicated concentrations of tyrphostin for 24 hours. Whole cell lysates (40 μg) were analyzed by immunoblotting using specific antibodies as indicated. Proliferating cell nuclear antigen remained unchanged, suggesting that induction of p21WAF1 was not due to unspecific changes in protein synthesis. Representative blots of 4 independent experiments are shown. Gastroenterology 2003 125, 891-905DOI: (10.1016/S0016-5085(03)01064-3)