Inactivation of Cancer-Associated-Fibroblasts Disrupts Oncogenic Signaling in Pancreatic Cancer Cells and Promotes Its Regression

Abstract

Resident fibroblasts that contact tumor epithelial cells (TEC) can become irreversibly activated as cancer-associated-fibroblasts (CAF) that stimulate oncogenic signaling in TEC. In this study, we evaluated the cross-talk between CAF and TEC isolated from tumors generated in a mouse model of KRAS/mut p53-induced pancreatic cancer (KPC mice). Transcriptomic profiling conducted after treatment with the anticancer compound Minnelide revealed deregulation of the TGFβ signaling pathway in CAF, resulting in an apparent reversal of their activated state to a quiescent, nonproliferative state. TEC exposed to media conditioned by drug-treated CAFs exhibited a decrease in oncogenic signaling, as manifested by downregulation of the transcription factor Sp1. This inhibition was rescued by treating TEC with TGFβ. Given promising early clinical studies with Minnelide, our findings suggest that approaches to inactivate CAF and prevent tumor-stroma cross-talk may offer a viable strategy to treat pancreatic cancer.Significance: In an established mouse model of pancreatic cancer, administration of the promising experimental drug Minnelide was found to actively deplete reactive stromal fibroblasts and to trigger tumor regression, with implications for stromal-based strategies to attack this disease. Cancer Res; 78(5); 1321-33. ©2018 AACR.

Figure 1. Triptolide/Minnelide inhibited CAF mediated invasion, tumor development and desmoplasia of TECs in pancreatic cancer cells and animal model

Co-culture of TEC (mouse derived KPC, Panc-1 or human derived S2VP10) with CAF cells (mouse derived CAF-1, CAF-5 or human derived SC00A5) resulted in increased invasion in a boyden chamber, this was decreased upon treatment with 25nM triptolide of CAF cells, TECs or both. (A–C). Co-injection of CAFs with tumor cells increased tumor growth (D) in orthotopic PDAC mouse model, but Minnelide treatment suppressed tumor-promoting roles of CAFs in vivo as can be seen by the end of study tumor weight. Analysis of tumors by HE staining, sirius red staining and BrdU staining showed that CAFs induced increased collagen synthesis, proliferation in TECs, however, this was decreased upon treatment with Minnelide. Minnelide also induced apoptosis as seen by cleaved PARP staining and TUNEL staining (E). Though CAFs promoted metastasis, treatment with Minnelide resulted in decreased metastatic lesions as can be observed by the metastatic index (F). In summary, Minnelide suppressed CAFs mediated collagen deposition, cell proliferation, metastasis and αSMA and induced apoptosis in orthotopic PDAC model. (*, P<0.05; **, P<0.01).

Figure 2. Transcriptome analysis of triptolide treated CAFs

Differentially expressed genes in CAFs before and after triptolide treatment were shown in heatmap (A). Principal component analysis showed three distinct groups of all samples that correlate to different treatments (B). Pathways that are significantly deregulated in 24h triptolide treatment samples were shown as bar plot (C). Visualization of TGF-β pathway in response to triptolide treatment in CAFs. Green label means down-regulated genes and red-label mean up-regulated genes (D). GSEA indicated obvious suppressive role of triptolide on TGF-β pathway down-stream genes’ expression (E). Heatmap of TGF-β pathway down-stream genes’ expression before and after triptolide treatment. Red means high-expression and blue means low-expression (F).

Figure 3. Triptolide downregulated TGFβ pathway in the CAFs

Activated pancreatic stellate cells (PSCs) produced more active as well as total TGF-β compared to the TECs in culture supernatant (A) as well as in the cell lysate (B) as seen by the ratio of the active: total TGF-β. Treatment with 25nM triptolide (sub-lethal) decreased mRNA expression (C) as well as active: total TGF-β in hPSC supernatant (D) as well as cell lysate (E). mRNA expression of several genes in the TGF-β pathway were downregulated by triptolide treatment of activated PSC. This was rescued by addition of recombinant TGF-β (F). Triptolide treatment also downregulated the activity of SMAD2 and SMAD3 proteins as seen by decreased phosphorylation in western blot. This was rescued by addition of recombinant TGF-β (G). * signifies P<0.05 in control vs. triptolide treatment; # signifies P<0.05 in triptolide treated vs. TGF–β+ triptolide–treated samples.

Figure 4. Downregulation of TGF-β pathway in CAFs by triptolide reverts them from activated to inactivated state

Treatment of TEC (KPC001) with conditioned media from CAF7 treated with 25nM triptolide decreased proliferation rate of KPC001 as measured by ECIS (A), resulted in Vitamin A accumulation as observed by change of fluorescence at 338nM (B) and lipid droplet accumulation by Oil Red staining (C). Further, analysis a-SMA expression by flow cytometry showed a decrease in the intensity of staining indicating an inactivation of CAF cells in the presence of triptolide (D). * signifies P<0.05 in control vs. triptolide treatment; # signifies P<0.05 in triptolide treated vs. TGF-β+ triptolide–treated samples.

Figure 5. Treatment with triptolide decreased ECM secretion by CAFs

Treatment with 25 nM triptolide decreased the secretion of fibronectin (Figure 5A), periostin (Figure 5B), collagen (Figure 5C), hyaluronic acid (Figure 5D), MMP2 (Figure 5E), and MMP9 (Figure 5F). Further, treatment with TGF-β reverted this decreased ECM production (Figure 5G, H). * signifies P<0.05 in control vs. triptolide treatment; # signifies P<0.05 in triptolide treated vs. TGF-β+ triptolide–treated samples.

Figure 6. Downregulation of TGFβ in CAFs inhibits oncogenic signaling in TECs

Treatment of TEC (MIA-PACA2 or S2VP10) with conditioned media from activated PSC treated with 25nM triptolide decreased SMAD transcriptional activity as seen by dual luciferase assay (A). This resulted in decreased Sp1 expression in the TECs (B). Further, DNA binding ability of Sp1 was inhibited (C). Expression of genes downstream of Sp1 like HSP70, HSF1 and components of NF-kB pathway (RelA and NFKB1) were also downregulated. These were rescued upon addition of recombinant TGF-β(D). * signifies P<0.05 in control vs. triptolide treatment; # signifies P<0.05 in triptolide treated vs. TGF-β+ triptolide–treated samples.

Figure 7. Schematic Figure illustration of how Minnelide mediated inactivation of CAFs affects oncogenic signaling in TECs

Minnelide inactivates the CAF cells leading to decreased production of TGFβ, which in turn downregulates pro-oncogenic signaling in the tumor epithelial cells.