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VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells.

Yoshida A, Shimizu A, Asano H, Kadonosono T, Kondoh SK, Geretti E, Mammoto A, Klagsbrun M, Seo MK - Biol Open (2015)

Bottom Line: DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1.Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor.In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

View Article: PubMed Central - PubMed

Affiliation: Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kyoto 603-8555, Japan.

No MeSH data available.


Related in: MedlinePlus

VEGF-A promoted DJM-1 cell proliferation via NRP1 in an autocrine manner. (A) A western blot shows that DJM-1 cells expressed NRP1, but not NRP2. DJM-1 cells were treated with siRNA (siControl, siNRP1 #1–3, 20 nM each) for immunoblotting the NRP1 protein (arrow indicates NRP1; 130 kDa). Percentages from each blotted protein amount relative to the siControl are indicated below each lane. Actin was immunoblotted to normalize the amounts of NRP1 (upper panel). HUVEC expressed NRP2, whereas DJM-1 cells did not (arrows indicate NRP2; 120–130 kDa, lower panel). (B) DJM-1 cell colony formation assay. Cells treated with 20 nM siControl and siNRP1 #1–3. (C) Colony formation by siVEGF-A- or siNRP1-treated DJM-1 cells. The presence or absence of exogenous VEGF-A (1 µg/ml) was indicated as (+) and (−) respectively. (D) DJM-1 cell colony formation assay in the presence of sNRP (from 20 to 100 ng/ml). (E) The graph shows the effects of VEGF-A family members (1 µg/ml each) in the siVEGF-A-treated DJM-1 cell colony formation assay. These data represent the means±s.d. Percentages from each mean relative to the siControl (B,C,E) or no addition (D) are shown below the graph. ***P<0.001; N.S., not significant.
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BIO010918F3: VEGF-A promoted DJM-1 cell proliferation via NRP1 in an autocrine manner. (A) A western blot shows that DJM-1 cells expressed NRP1, but not NRP2. DJM-1 cells were treated with siRNA (siControl, siNRP1 #1–3, 20 nM each) for immunoblotting the NRP1 protein (arrow indicates NRP1; 130 kDa). Percentages from each blotted protein amount relative to the siControl are indicated below each lane. Actin was immunoblotted to normalize the amounts of NRP1 (upper panel). HUVEC expressed NRP2, whereas DJM-1 cells did not (arrows indicate NRP2; 120–130 kDa, lower panel). (B) DJM-1 cell colony formation assay. Cells treated with 20 nM siControl and siNRP1 #1–3. (C) Colony formation by siVEGF-A- or siNRP1-treated DJM-1 cells. The presence or absence of exogenous VEGF-A (1 µg/ml) was indicated as (+) and (−) respectively. (D) DJM-1 cell colony formation assay in the presence of sNRP (from 20 to 100 ng/ml). (E) The graph shows the effects of VEGF-A family members (1 µg/ml each) in the siVEGF-A-treated DJM-1 cell colony formation assay. These data represent the means±s.d. Percentages from each mean relative to the siControl (B,C,E) or no addition (D) are shown below the graph. ***P<0.001; N.S., not significant.

Mentions: DJM-1 cells express NRP1, but not NRP2. In addition, NRP1 siRNA (siNRP1) #1–3 almost completely abrogated protein expression (siNRP1 #1: 7%, #2: 4%, #3: 3% respectively), inhibiting DJM-1 cell anchorage-independent proliferation from 59 to 94% (Fig. 3A,B). Since siNRP1 #2 was the most effective inhibitor of proliferation, it was used in subsequent experiments. The siNRP1 treatment inhibited the proliferation of DJM-1 cells, similar to siVEGF-A (siControl: 100%, siNRP1: 39%, siVEGF-A: 35%, respectively) (Fig. 3C). The addition of exogenous recombinant VEGF-A did not rescue siNRP1-treated DJM-1 cell proliferation (42%), but did rescue siVEGF-A-treated DJM-1 proliferation (96%) (Fig. 3C). We also assessed the expression of the NRP1 protein by western blotting and VEGF-A by ELISA in other human cancer cell lines: PC3M, prostate cancer and U87MG, glioblastoma. The NRP1 protein (∼130 kDa) was strongly expressed in PC3M and U87MG (supplementary material Fig. S1A). All cell lines expressed NRP1, but did not express VEGFRs. U87MG cells expressed NRP1 and NRP2 (supplementary material Fig. S1A). U87MG cells secreted the highest levels of VEGF-A into conditioned medium, as shown in supplementary material Fig. S1B. The siVEGF-A or siNRP1 treatment inhibited the proliferation of PC3M (siControl: 100%, siVEGF-A: 15%, siNRP1: 23%) and U87MG cells (siControl: 100%, siVEGF-A: 33%, siNRP1: 41%) (supplementary material Fig. S1C). The addition of exogenous VEGF-A rescued the proliferation of siVEGF-A-treated cells (PC3M: 77%, U87MG: 78%). In contrast, the addition of VEGF-A did not recover the proliferation of siNRP1-treated cells (PC3M: 38%, U87MG: 46%), suggesting that NRP1 mediated VEGF-A signaling to induce PC3M and U87MG cell proliferation as in DJM-1 cells (supplementary material Fig. S1C).Fig. 3.


VEGF-A/NRP1 stimulates GIPC1 and Syx complex formation to promote RhoA activation and proliferation in skin cancer cells.

Yoshida A, Shimizu A, Asano H, Kadonosono T, Kondoh SK, Geretti E, Mammoto A, Klagsbrun M, Seo MK - Biol Open (2015)

VEGF-A promoted DJM-1 cell proliferation via NRP1 in an autocrine manner. (A) A western blot shows that DJM-1 cells expressed NRP1, but not NRP2. DJM-1 cells were treated with siRNA (siControl, siNRP1 #1–3, 20 nM each) for immunoblotting the NRP1 protein (arrow indicates NRP1; 130 kDa). Percentages from each blotted protein amount relative to the siControl are indicated below each lane. Actin was immunoblotted to normalize the amounts of NRP1 (upper panel). HUVEC expressed NRP2, whereas DJM-1 cells did not (arrows indicate NRP2; 120–130 kDa, lower panel). (B) DJM-1 cell colony formation assay. Cells treated with 20 nM siControl and siNRP1 #1–3. (C) Colony formation by siVEGF-A- or siNRP1-treated DJM-1 cells. The presence or absence of exogenous VEGF-A (1 µg/ml) was indicated as (+) and (−) respectively. (D) DJM-1 cell colony formation assay in the presence of sNRP (from 20 to 100 ng/ml). (E) The graph shows the effects of VEGF-A family members (1 µg/ml each) in the siVEGF-A-treated DJM-1 cell colony formation assay. These data represent the means±s.d. Percentages from each mean relative to the siControl (B,C,E) or no addition (D) are shown below the graph. ***P<0.001; N.S., not significant.
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BIO010918F3: VEGF-A promoted DJM-1 cell proliferation via NRP1 in an autocrine manner. (A) A western blot shows that DJM-1 cells expressed NRP1, but not NRP2. DJM-1 cells were treated with siRNA (siControl, siNRP1 #1–3, 20 nM each) for immunoblotting the NRP1 protein (arrow indicates NRP1; 130 kDa). Percentages from each blotted protein amount relative to the siControl are indicated below each lane. Actin was immunoblotted to normalize the amounts of NRP1 (upper panel). HUVEC expressed NRP2, whereas DJM-1 cells did not (arrows indicate NRP2; 120–130 kDa, lower panel). (B) DJM-1 cell colony formation assay. Cells treated with 20 nM siControl and siNRP1 #1–3. (C) Colony formation by siVEGF-A- or siNRP1-treated DJM-1 cells. The presence or absence of exogenous VEGF-A (1 µg/ml) was indicated as (+) and (−) respectively. (D) DJM-1 cell colony formation assay in the presence of sNRP (from 20 to 100 ng/ml). (E) The graph shows the effects of VEGF-A family members (1 µg/ml each) in the siVEGF-A-treated DJM-1 cell colony formation assay. These data represent the means±s.d. Percentages from each mean relative to the siControl (B,C,E) or no addition (D) are shown below the graph. ***P<0.001; N.S., not significant.
Mentions: DJM-1 cells express NRP1, but not NRP2. In addition, NRP1 siRNA (siNRP1) #1–3 almost completely abrogated protein expression (siNRP1 #1: 7%, #2: 4%, #3: 3% respectively), inhibiting DJM-1 cell anchorage-independent proliferation from 59 to 94% (Fig. 3A,B). Since siNRP1 #2 was the most effective inhibitor of proliferation, it was used in subsequent experiments. The siNRP1 treatment inhibited the proliferation of DJM-1 cells, similar to siVEGF-A (siControl: 100%, siNRP1: 39%, siVEGF-A: 35%, respectively) (Fig. 3C). The addition of exogenous recombinant VEGF-A did not rescue siNRP1-treated DJM-1 cell proliferation (42%), but did rescue siVEGF-A-treated DJM-1 proliferation (96%) (Fig. 3C). We also assessed the expression of the NRP1 protein by western blotting and VEGF-A by ELISA in other human cancer cell lines: PC3M, prostate cancer and U87MG, glioblastoma. The NRP1 protein (∼130 kDa) was strongly expressed in PC3M and U87MG (supplementary material Fig. S1A). All cell lines expressed NRP1, but did not express VEGFRs. U87MG cells expressed NRP1 and NRP2 (supplementary material Fig. S1A). U87MG cells secreted the highest levels of VEGF-A into conditioned medium, as shown in supplementary material Fig. S1B. The siVEGF-A or siNRP1 treatment inhibited the proliferation of PC3M (siControl: 100%, siVEGF-A: 15%, siNRP1: 23%) and U87MG cells (siControl: 100%, siVEGF-A: 33%, siNRP1: 41%) (supplementary material Fig. S1C). The addition of exogenous VEGF-A rescued the proliferation of siVEGF-A-treated cells (PC3M: 77%, U87MG: 78%). In contrast, the addition of VEGF-A did not recover the proliferation of siNRP1-treated cells (PC3M: 38%, U87MG: 46%), suggesting that NRP1 mediated VEGF-A signaling to induce PC3M and U87MG cell proliferation as in DJM-1 cells (supplementary material Fig. S1C).Fig. 3.

Bottom Line: DJM-1, a human skin cancer cell line, expresses endogenous VEGF-A and NRP1.Furthermore, the inhibition of VEGF-A/NRP1 signaling upregulated p27, a CDK inhibitor.In conclusion, this new signaling pathway of VEGF-A/NRP1 induced cancer cell proliferation by forming a GIPC1/Syx complex that activated RhoA to degrade the p27 protein.

View Article: PubMed Central - PubMed

Affiliation: Division of Engineering (Biotechnology), Graduate School of Engineering, Kyoto Sangyo University, Kyoto 603-8555, Japan.

No MeSH data available.


Related in: MedlinePlus