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TCGA data and patient-derived orthotopic xenografts highlight pancreatic cancer-associated angiogenesis.

Gore J, Craven KE, Wilson JL, Cote GA, Cheng M, Nguyen HV, Cramer HM, Sherman S, Korc M - Oncotarget (2015)

Bottom Line: Inhibition of the type I TGF-β receptor with SB505124 does not alter endothelial activation in vitro, but decreases pro-angiogenic gene expression and suppresses angiogenesis in vivo.Conversely, STAT3 silencing or JAK1-2 inhibition with ruxolitinib blocks CM-enhanced EC proliferation.Thus, targeting JAK1-2 with ruxolitinib blocks a final pathway that is common to multiple pro-angiogenic factors, suppresses EC-mediated PCC proliferation, and may be useful in PDACs with a strong pro-angiogenic signature.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

ABSTRACT
Pancreatic ductal adenocarcinomas (PDACs) overexpress pro-angiogenic factors but are not viewed as vascular. Using data from The Cancer Genome Atlas we demonstrate that a subset of PDACs exhibits a strong pro-angiogenic signature that includes 37 genes, such as HDAC9, that are overexpressed in PDAC arising in KRC mice, which express mutated Kras and lack RB. Moreover, patient-derived orthotopic xenografts can exhibit tumor angiogenesis, whereas conditioned media (CM) from KRC-derived pancreatic cancer cells (PCCs) enhance endothelial cell (EC) growth and migration, and activate canonical TGF-β signaling and STAT3. Inhibition of the type I TGF-β receptor with SB505124 does not alter endothelial activation in vitro, but decreases pro-angiogenic gene expression and suppresses angiogenesis in vivo. Conversely, STAT3 silencing or JAK1-2 inhibition with ruxolitinib blocks CM-enhanced EC proliferation. STAT3 disruption also suppresses endothelial HDAC9 and blocks CM-induced HDAC9 expression, whereas HDAC9 re-expression restores CM-enhanced endothelial proliferation. Moreover, ruxolitinib blocks mitogenic EC/PCC cross-talk, and suppresses endothelial p-STAT3 and HDAC9, and PDAC progression and angiogenesis in vivo, while markedly prolonging survival of KRC mice. Thus, targeting JAK1-2 with ruxolitinib blocks a final pathway that is common to multiple pro-angiogenic factors, suppresses EC-mediated PCC proliferation, and may be useful in PDACs with a strong pro-angiogenic signature.

No MeSH data available.


Related in: MedlinePlus

TβRI inhibition does not block endothelial activation but suppresses angiogenic gene expression in PCCs(A) Compared with control media (closed bars), conditioned media (CM) from three KRC cell lines (purple, green or blue bars) significantly enhance EC proliferation (top) and migration (bottom). (B) GSEA shows that genes up-regulated in KRC tumors correlate with genes up-regulated by TGF-β (FWER < 0.001). (C) Canonical TGF-β signaling pathways are active in KRC tumors as evidenced by the abundance of nuclear p-Smad2 (top) and p-Smad3 (bottom). (D) Vehicle-treated tumors (top) display abundant CD31 (left) and CD34 (right) immunoreactivity, both of which are markedly attenuated in SB505124-treated tumors. Shown in (C–D) are representative images from 3 mice per group. Scale bars, 50 μm. (E) CM from KRC cells markedly increase p-Smad2 and p-Smad3 levels in ECs, which is blocked by SB505124 [2 μM]. Shown are representative immunoblots from three independent experiments. (F) SB505124 [2 μM] does not prevent KRC CM from enhancing EC proliferation (top) or migration (bottom). Data in (A, F–H) are mean ± SEM. **P < 0.01; *P < 0.05. (G) SB505124 (2 μM] significantly attenuates the levels of the indicated mRNAs in KRC PCCs. (H) ELISA shows the levels of the indicated cytokines in KRC CM in the absence (open bars) or presence (closed bars) of TGF-β1. Data are mean ± SD from two different cell lines.
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Figure 3: TβRI inhibition does not block endothelial activation but suppresses angiogenic gene expression in PCCs(A) Compared with control media (closed bars), conditioned media (CM) from three KRC cell lines (purple, green or blue bars) significantly enhance EC proliferation (top) and migration (bottom). (B) GSEA shows that genes up-regulated in KRC tumors correlate with genes up-regulated by TGF-β (FWER < 0.001). (C) Canonical TGF-β signaling pathways are active in KRC tumors as evidenced by the abundance of nuclear p-Smad2 (top) and p-Smad3 (bottom). (D) Vehicle-treated tumors (top) display abundant CD31 (left) and CD34 (right) immunoreactivity, both of which are markedly attenuated in SB505124-treated tumors. Shown in (C–D) are representative images from 3 mice per group. Scale bars, 50 μm. (E) CM from KRC cells markedly increase p-Smad2 and p-Smad3 levels in ECs, which is blocked by SB505124 [2 μM]. Shown are representative immunoblots from three independent experiments. (F) SB505124 [2 μM] does not prevent KRC CM from enhancing EC proliferation (top) or migration (bottom). Data in (A, F–H) are mean ± SEM. **P < 0.01; *P < 0.05. (G) SB505124 (2 μM] significantly attenuates the levels of the indicated mRNAs in KRC PCCs. (H) ELISA shows the levels of the indicated cytokines in KRC CM in the absence (open bars) or presence (closed bars) of TGF-β1. Data are mean ± SD from two different cell lines.

Mentions: We next assessed the ability of conditioned media (CM) from KRC cells to enhance the proliferation and migration of murine SVEC4–10 ECs that are commonly used to study angiogenesis pathways in vitro [35]. CM from three KRC-derived PCCs markedly enhanced EC proliferation and migration (Figure 3A), suggesting that KRC cells may secrete factors that promote EC proliferation in vivo. To identify mechanisms through which KRC cell-derived factors could promote angiogenesis in vivo, we conducted an Ingenuity Pathway Analysis (IPA) of our KRC tumor array data. Based on the overall gene expression profile of KRC tumors, IPA identified TGF-β as the top regulator of gene expression (P = 1.91 × 10−16). GSEA confirmed these findings, and indicated that genes up-regulated in KRC tumors correlated strongly with genes up-regulated by TGF-β (Figure 3B). Moreover, KRC mPDACs (Figure 3C) and human PDACs [32] are often associated with nuclear p-Smad2 and p-Smad3, indicating that canonical TGF-β signaling pathways are active in both settings, and raising the possibility that PDAC-derived TGF-βs could promote angiogenesis [36].


TCGA data and patient-derived orthotopic xenografts highlight pancreatic cancer-associated angiogenesis.

Gore J, Craven KE, Wilson JL, Cote GA, Cheng M, Nguyen HV, Cramer HM, Sherman S, Korc M - Oncotarget (2015)

TβRI inhibition does not block endothelial activation but suppresses angiogenic gene expression in PCCs(A) Compared with control media (closed bars), conditioned media (CM) from three KRC cell lines (purple, green or blue bars) significantly enhance EC proliferation (top) and migration (bottom). (B) GSEA shows that genes up-regulated in KRC tumors correlate with genes up-regulated by TGF-β (FWER < 0.001). (C) Canonical TGF-β signaling pathways are active in KRC tumors as evidenced by the abundance of nuclear p-Smad2 (top) and p-Smad3 (bottom). (D) Vehicle-treated tumors (top) display abundant CD31 (left) and CD34 (right) immunoreactivity, both of which are markedly attenuated in SB505124-treated tumors. Shown in (C–D) are representative images from 3 mice per group. Scale bars, 50 μm. (E) CM from KRC cells markedly increase p-Smad2 and p-Smad3 levels in ECs, which is blocked by SB505124 [2 μM]. Shown are representative immunoblots from three independent experiments. (F) SB505124 [2 μM] does not prevent KRC CM from enhancing EC proliferation (top) or migration (bottom). Data in (A, F–H) are mean ± SEM. **P < 0.01; *P < 0.05. (G) SB505124 (2 μM] significantly attenuates the levels of the indicated mRNAs in KRC PCCs. (H) ELISA shows the levels of the indicated cytokines in KRC CM in the absence (open bars) or presence (closed bars) of TGF-β1. Data are mean ± SD from two different cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4480696&req=5

Figure 3: TβRI inhibition does not block endothelial activation but suppresses angiogenic gene expression in PCCs(A) Compared with control media (closed bars), conditioned media (CM) from three KRC cell lines (purple, green or blue bars) significantly enhance EC proliferation (top) and migration (bottom). (B) GSEA shows that genes up-regulated in KRC tumors correlate with genes up-regulated by TGF-β (FWER < 0.001). (C) Canonical TGF-β signaling pathways are active in KRC tumors as evidenced by the abundance of nuclear p-Smad2 (top) and p-Smad3 (bottom). (D) Vehicle-treated tumors (top) display abundant CD31 (left) and CD34 (right) immunoreactivity, both of which are markedly attenuated in SB505124-treated tumors. Shown in (C–D) are representative images from 3 mice per group. Scale bars, 50 μm. (E) CM from KRC cells markedly increase p-Smad2 and p-Smad3 levels in ECs, which is blocked by SB505124 [2 μM]. Shown are representative immunoblots from three independent experiments. (F) SB505124 [2 μM] does not prevent KRC CM from enhancing EC proliferation (top) or migration (bottom). Data in (A, F–H) are mean ± SEM. **P < 0.01; *P < 0.05. (G) SB505124 (2 μM] significantly attenuates the levels of the indicated mRNAs in KRC PCCs. (H) ELISA shows the levels of the indicated cytokines in KRC CM in the absence (open bars) or presence (closed bars) of TGF-β1. Data are mean ± SD from two different cell lines.
Mentions: We next assessed the ability of conditioned media (CM) from KRC cells to enhance the proliferation and migration of murine SVEC4–10 ECs that are commonly used to study angiogenesis pathways in vitro [35]. CM from three KRC-derived PCCs markedly enhanced EC proliferation and migration (Figure 3A), suggesting that KRC cells may secrete factors that promote EC proliferation in vivo. To identify mechanisms through which KRC cell-derived factors could promote angiogenesis in vivo, we conducted an Ingenuity Pathway Analysis (IPA) of our KRC tumor array data. Based on the overall gene expression profile of KRC tumors, IPA identified TGF-β as the top regulator of gene expression (P = 1.91 × 10−16). GSEA confirmed these findings, and indicated that genes up-regulated in KRC tumors correlated strongly with genes up-regulated by TGF-β (Figure 3B). Moreover, KRC mPDACs (Figure 3C) and human PDACs [32] are often associated with nuclear p-Smad2 and p-Smad3, indicating that canonical TGF-β signaling pathways are active in both settings, and raising the possibility that PDAC-derived TGF-βs could promote angiogenesis [36].

Bottom Line: Inhibition of the type I TGF-β receptor with SB505124 does not alter endothelial activation in vitro, but decreases pro-angiogenic gene expression and suppresses angiogenesis in vivo.Conversely, STAT3 silencing or JAK1-2 inhibition with ruxolitinib blocks CM-enhanced EC proliferation.Thus, targeting JAK1-2 with ruxolitinib blocks a final pathway that is common to multiple pro-angiogenic factors, suppresses EC-mediated PCC proliferation, and may be useful in PDACs with a strong pro-angiogenic signature.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA.

ABSTRACT
Pancreatic ductal adenocarcinomas (PDACs) overexpress pro-angiogenic factors but are not viewed as vascular. Using data from The Cancer Genome Atlas we demonstrate that a subset of PDACs exhibits a strong pro-angiogenic signature that includes 37 genes, such as HDAC9, that are overexpressed in PDAC arising in KRC mice, which express mutated Kras and lack RB. Moreover, patient-derived orthotopic xenografts can exhibit tumor angiogenesis, whereas conditioned media (CM) from KRC-derived pancreatic cancer cells (PCCs) enhance endothelial cell (EC) growth and migration, and activate canonical TGF-β signaling and STAT3. Inhibition of the type I TGF-β receptor with SB505124 does not alter endothelial activation in vitro, but decreases pro-angiogenic gene expression and suppresses angiogenesis in vivo. Conversely, STAT3 silencing or JAK1-2 inhibition with ruxolitinib blocks CM-enhanced EC proliferation. STAT3 disruption also suppresses endothelial HDAC9 and blocks CM-induced HDAC9 expression, whereas HDAC9 re-expression restores CM-enhanced endothelial proliferation. Moreover, ruxolitinib blocks mitogenic EC/PCC cross-talk, and suppresses endothelial p-STAT3 and HDAC9, and PDAC progression and angiogenesis in vivo, while markedly prolonging survival of KRC mice. Thus, targeting JAK1-2 with ruxolitinib blocks a final pathway that is common to multiple pro-angiogenic factors, suppresses EC-mediated PCC proliferation, and may be useful in PDACs with a strong pro-angiogenic signature.

No MeSH data available.


Related in: MedlinePlus