Biology and Chemistry Research
The role of TGF-ß signaling in pancreatic stellate cells in pancreatic ductal adenocarcinoma Kelsey Gallant ABSTRACT Pancreatic cancer has a 5-year survival rate of only 6%, and many current therapies are not effective in prolonging survival. Pancreatic cancer contains an abundant fibrotic stroma comprised of extracellular matrix (ECM) proteins like collagen and fibronectin. This dense stroma can promote cancer initiation and metastasis as well as limit the ability of drugs to get to the cancer cells. Therefore, developing therapies that target the fibrotic stroma may inhibit cancer progression and improve the effectiveness of chemotherapy. Pancreatic stellate cells (PSCs) are responsible for making the fibrotic stroma. During pancreatic cancer, PSCs are activated to a myofibroblast state that is characterized by excessive secretion of ECM proteins. Transforming Growth Factor-ß (TGF-ß) signaling activates PSCs to the myofibroblast state. In this study, we analyze the downstream signaling pathways, including the canonical TGF-ß-Smad pathway and the non-canonical TAK1-p38 MAPK and PI3K-Akt pathways, to determine how TGF-ß activates PSCs. We block signaling through these pathways using chemical inhibitors and measure myofibroblast activation of PSCs.
Introduction Pancreatic ductal adenocarcinoma or PDAC is a form of pancreatic cancer that is characterized by a highly malignant phenotype that is resistant to most forms of therapy [1,5,8]. In fact, the incident rate is synonymous with the morality rate [8]. In previous studies, the PDAC tumor has a very aggressive, fibrotic stroma [3]. The PDAC tumor is normally comprised of 80 percent stroma. This stroma is made up of extracellular matrix or ECM proteins such as collagen, growth factors, fibronectin,etc [3]. ECM proteins create a stiff, harsh microenvironment in the stroma that promotes the progression of chronic pancreatitis or pancreatic cancer [3,8]. Additionally the stiffness limits the ability of chemotherapy drugs to reach the cancer cells [3]. The pancreas is an organ that has both exocrine and endocrine features. The function of the endocrine portion is to secrete hormones that regulate carbohydrate metabolism, while the exocrine portion produces enzymes that aid in digestion [1,3,5,8]. The enzyme producing cells are collectively called acinar cells and pump enzymes into the duodenum [6]. One of the several types of acinar cells includes pancreatic stellate cells. Pancreatic stellate cells are myofibroblastlike cells located in the pancreas. The normal function in the cell includes maintaining tissue structure as well as regulating the synthesis and decomposition of proteins within the extracellular matrix (2). Pancreatic stellate cells (PSCs) are unique in their ability to switch from a quiescent to activated state or myofibroblastic state [2,5,6,7]. Normal functioning PSCs stay in the quiescent state until any sort of trauma happens to the pancreas in which they will activate to regulate the proteins in the pancreas and return to the quiescent state when repair has been completed [6]. However, PSCs 12 | 2013-2014 | Volume 3
may activate themselves or sustain activation after repairing and continue proliferation, migration, apoptosis, and synthesis when those functions are unnecessary to the pancreas[5,8]. Previous studies have shown that activated stellate cells promote chronic pancreatitis and pancreatic cancer [2,5,6].When PSCs are activated they secrete the ECM proteins mentioned above which cause the fibrotic stroma of PDAC. Particularly, alpha smooth muscle actin or α-SMA, a cytoskeletan protein, is only secreted when the PSCs are in the myofibroblastic state. This protein is used as a marker for PSC myofibroblastic activity [7]. In addition, growth factors that are secreted from activated PSCs promote cancer progression in neighboring epithelial cells as well as increase density in the fibrotic stroma [2,3]. These growth factors create a crosstalk between the PSCs and cancer cells [8]. This interaction promotes activities such as proliferation, migration, invasion, and apoptosis [3,8]. Transforming Growth Factor Beta (TGF-ß) is the key inducer of stellate cell activation and extracellular matrix secretion [7]. TGF-ß signaling occurs when the TGF-ß ligand binds to its receptors on the cell membrane. The ligand binding causes activationn of downstream signaling pathways. TGF-ß can signal through several downstream pathways including the canonical SMAD pathway [4]. The SMADs are transcription factors. From past studies, it is known that the TGF-ß-p38 MAPK pathway activates more known hepatic stellate cells in the kidney [9]. The TGF-ß activates the p38 MAPK through TRAF6 (TNFΑ Receptor Associated Factor 6) and TAK1 (TGFß Activated Kinase 1) [9]. Since the TGF-ß-p38 MAPK signaling pathway is responsible for the activation of hepatic stellate cells; it is hypothesized that the inhibition of TGF-ß-p38 MAPK and TGF-ß-SMAD signaling will inhibit TGF-ß induced activation of pancreatic stellate
Biology and Chemistry Research cells to the myofibroblastic state. This is important because if the TGF-ß-p38 MAPK signaling pathway is responsible for any activation of PSCs, then by inhibiting parts of the pathway, the activation of the PSCs can be blocked or slowed. If activation is not happening anymore, then the severity of PDAC tumor or chronic pancreatitis can be reduced or obliterated.
1a 1b Figure 1. Normal pancreatic tissue Figure 1a in comparison to one that has been effected with pancreatic ductal adenocarcinoma Figure 1b. Collagen is stained blue. (Shields et al. 2012)1
Figure 4a. TGF-ß-p38MAPK signaling pathway, a noncanonical pathway in TGF-β signaling. We blocked p38 and TAK-1 in the HPSCT and LTC-14 PSCs. (Blobe et al. 2000)3.
Figure 2: Location of pancreatic stellate cells [6]
Figure 3. Features and properties of PSCs in the quiescent versus activated state. α-SMA which was used as a marker for activation is only expressed by activated PSCs [6].
Figure 4b. TGF-ß-Smad signaling pathway, a canonical pathway in TGF-β signaling. We blocked ALK-5 (Receptor 1) in the HPSCT and LTC-14 PSCs (Zhang et al. 2009)2
Shields MA, Dangi-garimella S, Redig AJ, Munshi HG. Biochemical role of the collagen-rich tumour microenvironment in pancreatic cancer progression. Biochem J. 2012;441(2):541-52. 2 Faull C, Prasad R, Griffiths A, et al. TRANSFORMING END OF LIFE CARE THROUGH CLINICAL TEMPLATE DESIGN AND TRAINING. BMJ Support Palliat Care. 2014;4(Suppl_1):A97-A98. 3 Blobe GC, Schiemann WP, Lodish HF. Role of transforming growth factor beta in human disease. N Engl J Med. 2000;342(18):1350-8. 1
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Biology and Chemistry Research Materials and Methodology
to tubes where they were warmed and centrifuged.
The TGF-ß-p38 pathway was studied using two different cell lines: immortalized human pancreatic stellate cells with Tween (HPSCT) and large T immortalized cells (LTC-14). The inhibitors SB203580 p38 MAPK. A selective inhibitor of p38 MAPK. This compound inhibits the activation of MAPKAPK-2 by p38 MAPK and subsequent phosphorylation of HSP27 (9). SB203580 inhibits p38 MAPK catalytic activity by binding to the ATP-binding pocket, but does not inhibit phosphorylation of p38 MAPK by upstream kinases (10). 5Z-7-oxozeanol TAK-.1 Resorcyclic lactone of fungal origin that acts as a potent and selective transforming growth factor-ß-activated kinase 1 (TAK1) mitogen-activated protein kinase kinase kinase (MAPKKK) inhibitor (IC50 = 8 nM). Displays > 33-fold and > 62-fold selectivity over MEKK1 and MEKK4 respectively. Inhibits IL1-induced activation of NF-ȝB (IC50 = 83 nM) and JNK/ p38. Inhibits production of inflammatory mediators, and sensitizes cells to TRAIL- and TNF-Α-induced apoptosis in vitro. SP431542 TGF-ß receptor. ALK-5 (TGF-beta type I receptor). It has also been shown that this compound can replace Sox2 when reprogramming cells to iPS cells. 2-(3-(6-METHYLPYRIDIN-2-YL)-1H-PYRAZOL4-YL)-1,5-NAPHTHYRIDINE. Tissue Culture Each cell line (LTC-14 and HPSCT) was cultured in 8 well dishes with approximately 400,000 cells per well and 2 mL of 10 percent fetal bovine serum (FBS) DMEM media and 1 percent Pen Strep solution. The cells were incubated for 24-48 hours, or until about 85 percent confluency. Serum starvation and inhibitor treatment After both cell lines were serum starved to reduce interference in the enzymatic activity of the cell before inhibitor treatment. The 10 percent FBS was aspirated off the cells, which were rinsed twice with phosphate buffer solution (PBS), and filled with 2mL of 0.5 percent FBS medium in each well. The cells were serum starved for 6 hours. For each cell line there are two groups: the DMSO control and the inhibitor group. The control had 4 wells with 2mL of 0.5 percent FBS with DMSO. The inhibitor group 4 wells had 2mL of 0.5 percent FBS with an inhibitor. Amount of inhibitor used depended on the confluency of the cells at the time of treatment: about 200 liters for normal confluency amount decreased for lower confluencies. The TGFß treatment for both the control and inhibitor groups followed a dose of 0pm , 10pm, 25pm, and 50pm. After 24 hours of treatment the proteins were lysed and harvested with warm 2xSB. The harvested proteins were transferred Preliminary data created by my mentor Rachel Hesler
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Figure 5. Concentration assay of the inhibitors that were used in this experiment. This determines the best concentration of inhibitor to most successfully block signaling (Hesler et. al 2013)4. Western blotting assay The 40 ȝL protein samples were resolved on three gels: two 10 percent polyacrylamide and one 7.5 percent polyacrylamide . The gels ran at 80 V/cm through the stacking portion of the gel and at 120 V/cm after the proteins had gone through the stacking and into the resolving gel. The western blot gel was transferred on nitrocellulose. During the transfer process, the primary and secondary wash was a mixture of tris-buffered saline (TBST), 5 percent milk, and antibody for the corresponding protein: Α-SMA, fibronectin, and ß-actin. The ß-actin and fibronectin western blot used 1ȝL anti-mouse 800 (green) antibody; Α-SMA used 1ȝL anti-mouse 680 (red) and 1ȝL anti-rabbit 800 (green). The western blots were developed and quantitated using a LICOR Odyssey machine. Immunofluorescence assay LTC-14 cells were cultured and plated in an 8 well dish with a coverslip in each well with approximately 400,000 cells per well. There were three groups: a dimethyl sulfoxide (DMSO) control, p38 inhibitorand TAK-1 inhibitor. Each group consisted of 2 samples: a group treated with TGF-ß and one untreated. The samples were fixed with 4 percent formaldehyde in PBS for 15 mins at room temperature and permeabilize with 0.1 percent Triton. The samples were labeled for immunofluorescence with antimouse 680 and DAPI. All values were normalized to their control condition.
Results Western blot assay: LTC-14 cell line with P-p38 The expression of α-SMA was reduced in the samples treated with 0,10,and 25 pm of TGF-ß. However, the well
Biology and Chemistry Research that was treated with 50 pm of TGF-ß had an expression of α-SMA that was greater than the DMSO control. The fibronectin secretion has also clearly showed reduced expression . The ß-actin had a consistent result. Western blot assay: HPSCT cell line with P-p38 This experimental group of samples had a higher expression of α-SMA than the DMSO control. The amount of fibronectin was also higher in the control than the one with the P-p38 inhibitor. The baseline ß-actin was consistent. Western blot assay: LTC-14 cell line with TAK-1 This group had a lower expression of α-SMA than the Figure 6b. control. This could mean that the TAK-1 inhibitor in fact inhibited the activation of the stellate cells. Additionally the expression of fibronectin in the TAK-1 inhibitor cells were lower than the control for 0,10, and 25 pm doses of TGF-ß, however the 50 pm concentration of TGFß sample was higher than the control. Western blot assay: HPSCT cell line with TAK-1 α-SMA had a much lower expression than the DMSO control. However, the dramatically low expression could possibly been a transfer error. There was a clear reduction in the expression of fibronectin in the inhibitor samples in comparison to the DMSO control. Baseline ß-actin was constant. Figure 7a. Western blot assay: LTC-14 cell line with ALK-5 The data from the LTC-14 cell line with the ALK-5 inhibitor was not collected for α-SMA due to a transfer error. The fibronectin results confirm that the ALK-5 inhibitor did reduce the expression of fibronectin in the cell samples. The ß-actin sample was constant. Western blot assay: HPSCT cell line with ALK-5 The ALK-5 inhibitor reduced the expression of α-SMA in the 10pm and 50pm samples of TGFß, while the 0pm and 25pm were much higher than the control. The expression of fibronectin in the samples with the inhibitor was much lower than the DMSO control samples. The ß-actin Figure 7b. baseline was even.
Figure 6a.
Figure 8a.
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Biology and Chemistry Research Discussion
Figure 8b. Figure 6a-8b. Western blotting assays of treated PSCs from the HPSCT and LTC-14 cell lines. Each assay had a DMSO control group and a inhibitor treated group. Within each group had cells that were activated by a different concentration of TGF-β. The cells were quantified for activation by comparing α-SMA and fibronectin activation to the baseline β-actin activation. Immunofluorescence assay In negative DMSO control, untreated PSCs appeared normal. The treated, positive DMSO control showed myofibroblastic stress fibers from the α-SMA. The P-p38 had the most striking results of the three immunofluorescence assays. The sample with TGF-ß almost completely inhibited the activation of the PSCs. The TAK-1 inhibitor did inhibit the some of the activation, but not as much as the P-p38 inhibitor.
The results support my hypothesis that the TGF-β-p38 MAPK pathway that is normally associated with kidney stellate cells is also responsible for the myofibroblastic activation of pancreatic stellate cells which can increase the harsh microenvironment of the fibrotic stroma, chemoresistance in the tumor, and communication with pancreatic cancer cells. The canonical TGF-β-SMAD pathway is also responsible in the signaling of PSCs. This TGFβ-p38 pathway is important in cancer research because, prior to my research it had not been studied for its role in pancreatic cancer. The P-p38 inhibitor was successful in inhibiting the myofibroblastic activation of the PSCs with the concentrations that were use, meaning TGF-β-p38 contributes to cell activation and by inhibiting it, the affects of the PSC activation is decreased or inhibited. Inhibition of TGFB-Smad and TGFB-p38 MAPK signaling may be beneficial to patients with chronic pancreatitis or pancreatic cancer since it can reduce myofibroblast activation of pancreatic stellate cells. This research is significant because, prior to my research, the role of the pathway in pancreatic cancer was previously unexamined. At certain levels of TGF-β activation, the P-p38 inhibitor from the non-canonical pathway was successful in reducing the myofibroblastic activation of the PSCs. The reduction of PSC activation means the reduction in PDAC tumor density making easier to treat. Also this also halts cancerous activity such as proliferation, migration, apoptosis ,and invasion that is normally prevalent during myofibroblastic PSC activation. This means that the TGF-ß-p38 pathway is clearly important in the PSC cell activation .The inhibitors used in this pathway can possibly be used in the treatment of chronic pancreatitis or pancreatic ductal adenocarcinoma in molecular targeted therapy treatment.
Conclusion and Future Directions
Figure 9. Immunofluorescence assay of LTC-14 cells treated with DMSO,p38, and TAK-1 inhibitor. One group was not activated with TGF-β as a control while the other group was treated with 50μL of TGF-β. The bright red staining indicates α-SMA stress fibers that are only seen during myofibroblastic activation. Blue staining indicates the presence of DAPI. 16 | 2013-2014 | Volume 3
The role of TGF-ß signaling in pancreatic stellate cell activation is prevalent and as suggested from the data, the signaling pathway can be inhibited by specific concentrations of kinase inhibitors from the TGF-ß-p38 MAPK pathway and the TGF-ß-SMAD pathway.While there was clear reduction in PSC activation when treated with the inhibitors, that was not the case with every concentration of TGF-β in the Western Blot assay. This could indicate that the inhibitor concentration that was used was too low to completely block the TGF-ß receptor. In the future, in order to get stronger results in Western blot assays, all three inhibitors: P-p38, TAK-1, and ALK-5, will be redone. Additionally, experiments with the inhibitor concentrations can access the best concentrations of the inhibitors that block TGF-ß. As discussed in the introduction. PSC activation can lend to cancerous activity such as apoptosis, migration, invasion ,and proliferation. These would be notable factors to analyze in TGF-ß signaling using a migration or invasion assay.
Biology and Chemistry Research Acknowledgements Special thanks to Rachel Hesler for mentoring me throughout my research project, Dr. Gerard Blobe (Principal Investigator) for hosting me in your lab, Dr. Amy Sheck for sponsoring me, Carolina Livery for providing transportation to and from Duke University, and the Department of Pharmacology and Cancer Biology at Duke University.
References [1] Apte, M. V., Wilson, J. S., Lugea, A., & Pandol, S. J. (2013). A Starring Role for Stellate Cells in the Pancreatic Cancer Microenvironment. Gastroenterology, 144(6), 1210–1219. doi:10.1053/j.gastro.2012.11.037 [2] Bachem, M. G., Schünemann,M., Ramadani,M., Siech, M., Beger, H., Buck, A., … Adler, G. (2005). Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology, 128(4), 907–921. doi:10.1053/j.gastro.2004.12.036 [3] Farrow, B., Albo, D., & Berger, D. H. (2008). The Role of the Tumor Microenvironment in the Progression of Pancreatic Cancer. Journal of Surgical Research, 149(2), 319–328. doi:10.1016/j.jss.2007.12.757 [4] Hanahan, D., &Weinberg, R. A. (2011). Hallmarks of Cancer: The Next Generation. Cell, 144(5), 646–674. doi:10.1016/j.cell.2011.02.013 [5] Mews, P. (2002). Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis. Gut, 50(4), 535–541. doi:10.1136/gut.50.4.535 [6] Omary, M. B., Lugea, A., Lowe, A. W., & Pandol, S. J. (2007). The pancreatic stellate cell: a star on the rise in pancreatic diseases. Journal of Clinical Investigation, 117(1), 50–59. doi:10.1172/JCI30082 [7] Shek, F. W.-T., Benyon, R. C., Walker, F. M., McCrudden, P. R., Pender, S. L. F., Williams, E. J., …Iredale, J. P. (2002). Expression of Transforming Growth Factor-ß1 [Secretion and Turnover in Chronic Pancreatitis. The American Journal of Pathology, 160(5), 1787–1798. doi:10.1016/S0002-9440(10)61125-X [8] Tang, D., Wang, D., Yuan, Z., Xue, X., Zhang, Y., An, Y., …Miao, Y. (2013). Persistent activation of pancreatic stellate cells creates a microenvironment favorable for the malignant behavior of pancreatic ductal adenocarcinoma. International Journal of Cancer, 132(5), 993–1003. doi:10.1002/ijc.27715 [9] Tsukada, S. (2005). SMAD and p38 MAPK Signaling Pathways Independently Regulate 1(I) Collagen Gene Expression in Unstimulated and Transforming Growth Factor- -stimulated Hepatic Stellate Cells. Journal of Biological Chemistry, 280(11), 10055–10064. doi:10.1074/jbc. M409381200
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