B7-1 drives TGF-β stimulated pancreatic carcinoma cell migration and expression of EMT target genes

B7-1 proteins are routinely expressed on the surface of antigen presenting cells (APC) and within the innate immune system. They function to establish a biologically optimal and dynamic balance between immune activation and inhibition or self-tolerance. Interactions between B7-1 and its receptors, which include CD28, CTLA4 and PD-L1, contribute to both stimulatory as well as inhibitory or homeostatic regulation. In the current study, we investigated whether the tumor-promoting actions of transforming growth factor beta (TGF-β) disrupted this equilibrium in pancreatic cancer to promote malignant progression and an enhanced means to evade immune detection. The data show that B7-1 is (i) upregulated following treatment of pancreatic carcinoma cells with TGF-β; (ii) induced by TGF-β via both Smad2/3-dependent and independent pathways; (iii) required for pancreatic tumor cell in vitro migration/invasion; and (iv) necessary for TGF-β regulated epithelial-mesenchymal transition (EMT) through induction of Snail family members. Results from the proposed studies provide valuable insights into mechanisms whereby TGF-β regulates both the innate immune response and intrinsic properties of pancreatic tumor growth.


Introduction
The American Cancer Society has reported that approximately 44,330 people will die from pancreatic cancer in Cancer Facts & Figures 2018. Although this reflects the 4th leading cause of cancer-associated deaths in the United States, unfortunately, recent advances in diagnostics, staging, and therapy have not resulted in significant improvements in long-term survival. A major component impacting pancreatic cancer's mortality is its ability to maintain an immunosuppressive microenvironment characterized by poor T cell penetration [1]. Furthermore, the known ability of pancreatic cancer antigens to generate relatively weak immune responses coupled with the expression of multidrug-resistant genes and growth factors such as transforming growth factor-β (TGF-β), tumor-necrosis factor-α (TNFα), and vascular endothelial

Scratch assay
For scratch assays, 3 X 10 5 cells in 10% FBS/DMEM were seeded into 6 well plates. The next day, following disruption of the monolayer with a sterile 200 μl pipet tip the cultures were washed and incubated ± TGF-β for 24 hours at 37˚C in 0.1% FBS/DMEM. Images were obtained with an EVOS XL Core Imaging System at 0 and 24 hr.

Migration assay
For migration assays, 1 X 10 5 cells in 0.1% FBS/DME were seeded in the upper chamber of 8 μm pore size transwell plates (Costar, Cambridge, MA). Following 24 hr incubation at 37˚C, any remaining cells in the upper chamber were removed with a cotton swab, and cells which migrated to the lower chamber were fixed and stained using a Differential Quik Stain Kit according to the manufacturer's instructions (Polysciences, Inc., PA, USA). Each experiment was performed in duplicate and cell counting was done in 3 randomly selected fields.

Flow cytometry
2.5 X 10 5 cells were seeded into 6 well plates and following overnight incubation the medium was replaced with fresh 0.1% FBS/DMEM at 37˚C for 18 hr. Cultures were then placed in fresh 0.1% FBS/DMEM for 24 hr ± TGF-β, trypsinized, washed, and then 1 X 10 5 cells stained with B7-1-Alexa 596 (BD Pharmingen San Jose, CA) for 30 min at room temperature. After MACS buffer (Miltenyi Biotec, San Diego, CA) washing and 2% paraformaldehyde fixation, FACScan flow cytometer was performed by gating 1 X 10 4 living cells.

Statistical analysis
All data are presented as mean ± SEM. Two-tailed Student's t-test or ANOVA was used to evaluate statistical significance using GraphPad Prism 5 software (GraphPad Software, Inc). P < 0.05 was considered statistically significant.

B7-1 (CD80) is highly expressed in human pancreatic cancer and regulated by TGF-β in human pancreatic cancer cells
Several studies have reported that the immune checkpoint protein B7-1 is more highly expressed in human pancreatic tumors than normal pancreatic tissue [17,18]. This was further documented in Fig 1 where we not only show increased B7-1 staining in pancreatic cancer tissue ( Fig 1A), but that expression colocalizes with the epithelial marker CK19 (Fig 1B). CK-19 is mainly expressed in ductal epithelial (bile and pancreatic duct, renal collecting ducts) and gastrointestinal epithelia [31,32]. In the pancreas, CK-19 expression is usually observed in the exocrine ducts, but not in the exocrine acinar and endocrine islet cells [31]. Tumor progression/metastasis reflects a highly regulated interaction between both intrinsic and extrinsic factors [33]. Since TGF-β has a critical role in numerous homeostatic as well as pathological processes [34,35], we investigated the inter-relation of the growth-promoting/ migratory role of TGF-β with the expression of B7-1 in pancreatic cancer cells. As shown in Fig 2A, TGF-β induced the expression of B7-1, but not B7-2, in PANC-1 and PANC04.03 cells. Kinetic analysis showed increased B7-1 protein and gene expression significantly increased by TGF-β beginning within 6-12 hr and maintained for at least 24 hr (Fig 2B and 2C). Since B7-1 is a surface glycoprotein [36], we confirmed cell surface B7-1 expression following exposure to TGF-β using FACS analysis ( Fig 2D). Last, to further document that the observed increase in B7-1 protein and mRNA was specific to TGF-β receptor activation, treatment of PANC04.03 cells with the type I TGF-β receptor (TβRI) kinase inhibitor SB431542 was shown to abrogate the response (Fig 2E).

Loss of B7-1 prevents TGF-β mediated pancreatic tumor cell migration and invasion
Immune checkpoint proteins have been reported to provide tumors with both a growth and survival advantage [37,38]. In that TGF-β is similarly known to promote tumor progression and invasion [39], we next investigated whether there was any causal relationship between the induction of B7-1 (Fig 2) and TGF-β's protumorigenic activity. To address this question, siRNA was used to knockdown B7-1 protein/mRNA (Fig 3A). While the protein level of B7-1 in the absence of TGF-β was essentially the same in the siCon and siB7-1 cultures, this simply reflects the baseline value as the actual fold level of B7-1 mRNA following siB7-1 treatment was 0.47 relative to siCon. Most importantly, while B7-1 mRNA in siCon treated cells increased 2.7 fold with TGF-β, treatment with siB7-1 prevented any increase by TGF-β (i.e., went from 0.47 to 0.5 in siB7-1 cultures -/+ TGF-β, respectively). This was similarly seen in the Western analysis where siB7-1 abrogated the stimulatory effect of TGF-β on B7-1 protein and human pancreatic tumor tissue (n = 2) was obtained from US Biomax (Rockville, MD, USA). Immunofluorescence confocal microscopy was performed on 3 slides from each of the two Normal and Pancreatic Cancer tissue specimens for B7-1 (red) and nuclear DAPI (blue). Expression of B7-1 is quantitated in the right panel. Magnification X 40. n = 6, ��� P<0.001. (B) Immunofluorescent co-staining on the indicated pancreatic tissue for the epithelial marker cytokeratin19 (green), B7-1 (red) and DAPI nuclear counterstain (blue). Merge shows the co-localization of B7-1 with CK-19 (yellow) and is quantitated in the right panel. Magnification X 40. n = 3, ��� P<0.001.

B7-1 regulates TGF-β induced EMT in PANC04.03 cells
Primary in vitro evidence reflecting the pro-tumorigenic actions of TGF-β is the epithelial-tomesenchymal transition (EMT) where the epithelial phenotype associated with cell-cell contact and apical-basal polarity is lost [40][41][42]. Since this process has been reported to be regulated by several transcription factors including paralogs of the Snail, Twist, and/or ZEB families [43][44][45], we assessed the effect of TGF-β on their expression in PANC04.03 cells by RT-qPCR. As shown in Fig 4A, while TGF-β treatment had no significant impact on the expression of Twist1/2 or ZEB1/2, Snail1 and Snail2 were induced 4-6 fold. Given that Snail transcription factors are known to regulate the expression of various mesenchymal markers associated with tumor invasion, metastasis, and cell motility [46][47][48], we determined, first, whether there was an analogous relation between the induction of Snail1/2 and profibrotic targets connected with TGF-β mediated EMT ( Fig 4B); and most importantly, second, if B7-1 was required for the response (Fig 4C-4E). As shown in Fig 4B, coincident with the increase in Snail protein, treatment of PANC04.03 cells with TGF-β induced expression of the mesenchymal markers N-cadherin and Vimentin. Of particular note, not only was the increase in Snail mRNA and protein by TGF-β dependent upon B7-1 (Fig 4C and 4D), but mesenchymal targets associated with the mesenchymal transition were similarly diminished in the absence of B7-1 (Fig 4D and  4E). Thus, B7-1 has a fundamental role in promoting both the biologic and mechanistic aspects of the TGF-β driven EMT in PANC04.03 cells.

B7-1 induction by TGF-β is dependent upon both Smad-dependent and -independent pathways
The preceding data show that B7-1 is highly expressed in pancreatic cancer (Fig 1), induced by TGF-β in two pancreatic cancer cell lines (Fig 2), and required for the induction of EMT in PANC04.03 cells (Figs 3 and 4). Since TGF-β signals via both Smad-dependent [49][50][51] as well as Smad-independent pathways (i.e., Erk, SAPK/JNK, mTOR, PI3K, and p38 MAPK) [27, 52, 53], we utilized both a genetic as well as pharmacologic approach to determine the operative pathway(s) regulating B7-1 induction. PANC04.03 cultures were first treated with siRNA targeting Smad2/3 or NT control and the effect on B7-1 protein and mRNA induction by TGF-β determined. As shown in Fig 5A and 5B, knockdown of Smad2 and/or Smad3 essentially abrogated the ability of TGF-β to induce B7-1. In that TGF-β is known to regulate target gene expression via multiple mechanisms [53][54][55][56][57], we assessed the role of the PI3K and MAPK pathways as they reflect the most upstream non-Smad activities coupled to the activated TGFβ receptor complex [27]. By using pharmacologic inhibitors, it was observed that while inhibition of the MAPK pathway with the MEK inhibitor U0126 had no negative impact on B7-1 expression by TGF-β, inhibition of PI3K and/or its downstream mediators Akt and mTOR abrogated the response (Fig 5C). A model depicting these findings is provided in Fig 5D.

Discussion
Pancreatic cancer is the fourth leading cause of cancer-related deaths among both men and women with a 5 year survival rate in the USA of around 7-8% [58]. The high number of pancreatic cancer-related deaths is reflective of a number of elements including delayed diagnosis, natural resistance to chemotherapy or radiation therapy, and the relatively weak immune responses elicited by pancreatic antigens [59]. This latter finding is likely due to a combination of factors including the gain and/or loss of various oncogenes or tumor suppressor genes as well as the expression of checkpoint proteins such as B7-1 capable of dampening the immune response [60].
B7-1 is a transmembrane protein normally expressed on antigen presenting cells capable of providing either a co-stimulatory or co-inhibitory signal depending upon whether it interacts with its counter-receptor CD28 or CTLA4, respectively [61,62]. The expression has been reported to portend a poor prognosis for patients with pancreatic cancer [63] and the combination of B7-1 and B7-H1 has been suggested as a diagnostic factor [18]. As such, determining the manner(s) by which B7-1 is regulated and its role in pancreatic cancer progression is critical to developing effective immunotherapeutic approaches for this deadly disease as recently shown for other solid tumors such as lung, melanoma, head and renal cancers [64][65][66][67]. To that end, as TGF-β is a known mediator of tumor progression/EMT and immune regulation [68], we addressed the following general questions: first, does TGF-β regulate B7-1 expression in pancreatic cancer cell lines; and if so, second, would B7-1 have a critical role in TGF-β stimulated cell migration/invasion and the induction of genes critical to EMT development?
In this study, we demonstrate that B7-1 protein is upregulated in human pancreatic cancer and TGF-β stimulates B7-1 expression in pancreatic cancer cell lines via both Smad-dependent as well as -independent pathways (Figs 1, 2 and 5). While B7-1 was induced by TGF-β in human PANC-1 and PANC-0403 as well as murine mKPC cells (Figs 2A and 5A), this was not observed in human BXPC-3 or MIA PaCa-2 cultures (not shown). This is the first time, to our knowledge, that B7-1 expression is described being regulated by TGF-β signaling and further document both the various responses of transformation to TGF-β and the importance of personalized approaches.
TGF-β is a known growth suppressor of pancreatic epithelial cells [20,21]. While it is currently unknown if B7-1 has any role in this process, as TGF-β ligands are commonly overexpressed in pancreatic cancer and can promote epithelial to mesenchymal transition (EMT) and invasion [69,70], we next investigated whether B7-1 was required for the induction of TGF-β target/regulatory genes and biological phenotypes associated with pancreatic cell EMT. To initially address that question the effect of B7-1 knockdown on TGF-β-stimulated cell migration and transwell invasion were assessed in PANC04.03 cells. Somewhat surprisingly, in the absence of B7-1 both responses were significantly inhibited (Fig 3). Since these activities are associated with the TGF-β regulated EMT, it was subsequently determined whether this was coincident with an inability to express EMT-inducing transcription factors such as Snail1/ 2, Twist1/2, and/or Zeb1/2 [71]. While Snail1/2 mRNA and protein were significantly increased by TGF-β, there was no appreciable impact on Twist1/2 or Zeb1/2 mRNA expression (Fig 4). Moreover, in the absence of B7-1 the TGF-β induction of Snail was diminished RT-qPCR expression of B7-1 and Snail, Twist, and ZEB following 24 hr stimulation of PANC04.03 cells in the absence (Con) or presence of 5 ng/ml TGF-β. ��� P<0.001 and n = 3. (B) Cells were treated with (+) or without (-) TGF-β (5 ng/ml) for 24 hr and total cell lysates subjected to Western blot analysis for expression of the indicated proteins. (C) PANC04-03 cultures were transfected with non-targeting (siCon) or B7-1 siRNA as in Fig 3A and processed for RT-qPCR subsequent to 24 hr vehicle (-; 4 mM HCl, 0.1% BSA) or TGF-β (+; 5 ng/ml) treatment. �� P<0.01, ��� P<0.001 and n = 3. (D and E) siCon or siB7-1 PANC04.03 cells were stimulated ± TGF-β as in (C) and assessed by Western blotting (D) or RT-qPCR (E) for the indicated targets. �� P<0.01, ��� P<0.001 and n = 3 for E.
https://doi.org/10.1371/journal.pone.0222083.g004 B7-1 checkpoint regulation by TGF-β in pancreatic cancer with a concomitant loss in EMT marker proteins (i.e., N-cadherin and Vimentin) as well as fibronectin and collagen α1 mRNA (Fig 4). As B7-1 is believed critical to the genesis and progression of tumor disorders, these findings (i) further document the various pathways and mechanisms by which TGF-β can regulate cell proliferation; and, most importantly, (ii) are consistent with the possibility that B7-1 inhibition might reflect a novel and effective means to impact both intrinsic and extrinsic aspects of pancreatic cancer.