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Cordycepin (3'-deoxyadenosine) suppressed HMGA2, Twist1 and ZEB1-dependent melanoma invasion and metastasis by targeting miR-33b.

Zhang P, Huang C, Fu C, Tian Y, Hu Y, Wang B, Strasner A, Song Y, Song E - Oncotarget (2015)

Bottom Line: Cordycepin, the active component of Cordyceps spp., has been identified to have anti-metastatic effect on tumor progression and thus possesses pharmacological and therapeutic potentials.Cordycepin-mediated miR-33b expression was dependent on LXR-RXR heterodimer activation. miR-33b directly binds to HMGA2, Twist1 and ZEB1 3'-UTR to suppress their expression.In spontaneous metastasis models, cordycepin suppressed tumor metastasis without altering primary tumor growth.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.

ABSTRACT
Malignant melanoma, the most deadly form of skin cancer, has a high propensity for metastatic spread and is notoriously chemotherapy-resistant. Cordycepin, the active component of Cordyceps spp., has been identified to have anti-metastatic effect on tumor progression and thus possesses pharmacological and therapeutic potentials. However, the mechanisms of anti-metastatic effects of cordycepin at cellular levels remain elusive. We analyzed the effect of cordycepin on human melanoma miRNA expression profiles by miRNAarray and found that miR-33b was upregulated in highly-metastatic melanoma cell lines following cordycepin exposure. Cordycepin-mediated miR-33b expression was dependent on LXR-RXR heterodimer activation. miR-33b directly binds to HMGA2, Twist1 and ZEB1 3'-UTR to suppress their expression. The negative correlations between miR-33b levels and HMGA2, Twist1 or ZEB1 expression were detected in 72 patient melanoma tissue samples. By targeting HMGA2 and Twist1, miR-33b attenuated melanoma migration and invasiveness upon cordycepin exposure. miR-33b knockdown or ZEB1 overexpression reverted cordycepin-mediated mesenchymal-epithelial transition (MET), triggering the expression of N-cadherin. In spontaneous metastasis models, cordycepin suppressed tumor metastasis without altering primary tumor growth. We showed for the first time that targeting miRNA by cordycepin indicates a new mechanism of cordycepin-induced suppression of tumor metastasis and miR-33b/HMGA2/Twist1/ZEB1 axis plays critical roles in regulating melanoma dissemination.

No MeSH data available.


Related in: MedlinePlus

Cordycepin suppresses melanoma migration through miR-33b(A–C) sh-miR-33b reverted cordycepin-mediated suppression of Lu1205 melanoma migration capacities as measured by wound healing assay. (A) Representative images photographed at 0 and 24 hr post-wounding were shown at magnification of 20X. (B) Increasing cordycepin concentration reduced Lu1205 and A375 cell motility. (C) sh-miR-33b but not sh-miR-200b, sh-miR-200c, sh-miR-205 and sh-miR-211 reverted cordycepin-mediated suppression of Lu1205 and A375 motility. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with control. †p < 0.05 compared with sh-NT. (D) siLXRβ/RXRα cordycepin-mediated suppression of Lu1205 and A375 motility. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with control. (E) sh-miR-33b reverted cordycepin-mediated suppression of phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA in Lu1205 and A375 cells. Cells were untransfected or transfected with sh-NT or sh-miR-33b for 24 hr before being treated or untreated with 200 μg/ml cordycepin for 24 hr. Cells were lysed and subjected to Western blotting analysis of phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA. Total FAK, Src, MLC and RhoA were used as loading controls. (F) HMGA2 and Twist1 overexpression reverted cordycepin-mediated suppression of Lu1205 migration as measured by wound healing assay. Lu1205 monolayer were transfected with either mock, HMGA2 or Twist1 construct before being treated with 200 μg/ml cordycepin for 24 hr. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with cordycepin + mock. (G) Cordycepin inhibited Lu1205 melanoma stress fiber and focal adhesion formation via miR-33b. Lu1205 cells were transfected with a variety of miRNA mimic, sh-miRNA, and/or overexpressing constructs before being treated or untreated with cordycepin. Lu1205 cells were stained with rhodamine-phalloidin and paxillin antibody (Geen = paxillin, red = F-actin). Bar = 10 μm. (H–I) Quantification of the average number and size (μm2) of paxillin-containing focal adhesions in Lu1205 cells using ImageJ software.12 cells were analyzed per condition in each experiment. Data are expressed as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 compared with ctrl + scramble.
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Figure 4: Cordycepin suppresses melanoma migration through miR-33b(A–C) sh-miR-33b reverted cordycepin-mediated suppression of Lu1205 melanoma migration capacities as measured by wound healing assay. (A) Representative images photographed at 0 and 24 hr post-wounding were shown at magnification of 20X. (B) Increasing cordycepin concentration reduced Lu1205 and A375 cell motility. (C) sh-miR-33b but not sh-miR-200b, sh-miR-200c, sh-miR-205 and sh-miR-211 reverted cordycepin-mediated suppression of Lu1205 and A375 motility. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with control. †p < 0.05 compared with sh-NT. (D) siLXRβ/RXRα cordycepin-mediated suppression of Lu1205 and A375 motility. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with control. (E) sh-miR-33b reverted cordycepin-mediated suppression of phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA in Lu1205 and A375 cells. Cells were untransfected or transfected with sh-NT or sh-miR-33b for 24 hr before being treated or untreated with 200 μg/ml cordycepin for 24 hr. Cells were lysed and subjected to Western blotting analysis of phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA. Total FAK, Src, MLC and RhoA were used as loading controls. (F) HMGA2 and Twist1 overexpression reverted cordycepin-mediated suppression of Lu1205 migration as measured by wound healing assay. Lu1205 monolayer were transfected with either mock, HMGA2 or Twist1 construct before being treated with 200 μg/ml cordycepin for 24 hr. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with cordycepin + mock. (G) Cordycepin inhibited Lu1205 melanoma stress fiber and focal adhesion formation via miR-33b. Lu1205 cells were transfected with a variety of miRNA mimic, sh-miRNA, and/or overexpressing constructs before being treated or untreated with cordycepin. Lu1205 cells were stained with rhodamine-phalloidin and paxillin antibody (Geen = paxillin, red = F-actin). Bar = 10 μm. (H–I) Quantification of the average number and size (μm2) of paxillin-containing focal adhesions in Lu1205 cells using ImageJ software.12 cells were analyzed per condition in each experiment. Data are expressed as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 compared with ctrl + scramble.

Mentions: Twist1 has been shown to mediate cancer migration through triggering Rac1 activation [27]. Twist also enhanced hepatocellular carcinoma EMT and invasion through activating matrix metalloproteinase (MMP)-2 and MMP-9 expression [28]. HMGA2 plays important roles in promoting cancer metastasis by downregulating miR-200b expression and increasing the level of lysyl oxidase (LOX) [29–31]. Therefore, we asked whether cordycepin can inhibit melanoma migration by targeting miR-33b and downregulating HMGA2 and Twist1 expression. We employed wound healing assays to assess the melanoma migratory capacities. 200 μg/ml cordycepin treatment inhibited the 24 hr wound closure capacities of melanoma cells (Figure 4A). sh-miR-33b transfection abrogated the suppressive effect of cordycepin on cell motility. Melanoma cells briskly migrated into the wound area with 24 hr after wound scratch. Quantitative analysis of the gap closure suggests that cordycepin significantly attenuated gap closure capacities of Lu1205 and A375 cells in a dose-dependent manner (p < 0.05) (Figure 4B). Ectopic expression of miR-33b significantly increased the cell-free gap area for Lu1205 and A375 cell lines (Figure 4C). In the context of cordycepin treatment, knockdown of miR-33b promoted the wound healing capacities (Figure 4C). The wound area in cordycepin/sh-miR-33b group reached 70% and 50% sealing for Lu1205 and A375 after 24 hr of wound scratch. In sharp contrast, miR-200b, miR-200c, miR-205 and miR-211 knockdown did not significantly alter the motility of melanoma, suggesting that miR-33b would be the sole miRNA responsible for cordycepin-mediated suppression of melanoma migration (Figure 4C). Silencing LXRβ/RXRα significantly ameliorated cordycepin-mediated inhibition of wound healing capacities (p < 0.05) (Figure 4D). Focal adhesion kinase (FAK), Src, myosin light chain (MLC) and RhoA activations are important for cancer motility. Cordycepin treatment suppressed the phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA without affecting the total FAK, Src, MLC and RhoA expressions (Figure 4E). Knockdown of miR-33b in cordycepin-treated Lu1205 and A375 melanoma cells increased the levels of phosphorylated FAK, Src, and MLC and RhoA-GTP. This implies that cordycepin downregulates cell migratory machinery and cytoskeletal remodeling by targeting miR-33b. To determine whether the phenotypes related to cordycepin treatment can be reversed by restoration of HMGA2 and Twist1, we transfected cells with either mock (vector control), HMGA2 or Twist1 overexpression plasmids. HMGA2 or Twist1 overexpression reverted cordycepin-mediated reduction of melanoma motility (Figure 4F). They reduced the 24-hr cell-free area by 30%–55% in cordycepin-treated A375 and Lu1205 monolayer.


Cordycepin (3'-deoxyadenosine) suppressed HMGA2, Twist1 and ZEB1-dependent melanoma invasion and metastasis by targeting miR-33b.

Zhang P, Huang C, Fu C, Tian Y, Hu Y, Wang B, Strasner A, Song Y, Song E - Oncotarget (2015)

Cordycepin suppresses melanoma migration through miR-33b(A–C) sh-miR-33b reverted cordycepin-mediated suppression of Lu1205 melanoma migration capacities as measured by wound healing assay. (A) Representative images photographed at 0 and 24 hr post-wounding were shown at magnification of 20X. (B) Increasing cordycepin concentration reduced Lu1205 and A375 cell motility. (C) sh-miR-33b but not sh-miR-200b, sh-miR-200c, sh-miR-205 and sh-miR-211 reverted cordycepin-mediated suppression of Lu1205 and A375 motility. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with control. †p < 0.05 compared with sh-NT. (D) siLXRβ/RXRα cordycepin-mediated suppression of Lu1205 and A375 motility. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with control. (E) sh-miR-33b reverted cordycepin-mediated suppression of phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA in Lu1205 and A375 cells. Cells were untransfected or transfected with sh-NT or sh-miR-33b for 24 hr before being treated or untreated with 200 μg/ml cordycepin for 24 hr. Cells were lysed and subjected to Western blotting analysis of phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA. Total FAK, Src, MLC and RhoA were used as loading controls. (F) HMGA2 and Twist1 overexpression reverted cordycepin-mediated suppression of Lu1205 migration as measured by wound healing assay. Lu1205 monolayer were transfected with either mock, HMGA2 or Twist1 construct before being treated with 200 μg/ml cordycepin for 24 hr. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with cordycepin + mock. (G) Cordycepin inhibited Lu1205 melanoma stress fiber and focal adhesion formation via miR-33b. Lu1205 cells were transfected with a variety of miRNA mimic, sh-miRNA, and/or overexpressing constructs before being treated or untreated with cordycepin. Lu1205 cells were stained with rhodamine-phalloidin and paxillin antibody (Geen = paxillin, red = F-actin). Bar = 10 μm. (H–I) Quantification of the average number and size (μm2) of paxillin-containing focal adhesions in Lu1205 cells using ImageJ software.12 cells were analyzed per condition in each experiment. Data are expressed as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 compared with ctrl + scramble.
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Figure 4: Cordycepin suppresses melanoma migration through miR-33b(A–C) sh-miR-33b reverted cordycepin-mediated suppression of Lu1205 melanoma migration capacities as measured by wound healing assay. (A) Representative images photographed at 0 and 24 hr post-wounding were shown at magnification of 20X. (B) Increasing cordycepin concentration reduced Lu1205 and A375 cell motility. (C) sh-miR-33b but not sh-miR-200b, sh-miR-200c, sh-miR-205 and sh-miR-211 reverted cordycepin-mediated suppression of Lu1205 and A375 motility. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with control. †p < 0.05 compared with sh-NT. (D) siLXRβ/RXRα cordycepin-mediated suppression of Lu1205 and A375 motility. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with control. (E) sh-miR-33b reverted cordycepin-mediated suppression of phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA in Lu1205 and A375 cells. Cells were untransfected or transfected with sh-NT or sh-miR-33b for 24 hr before being treated or untreated with 200 μg/ml cordycepin for 24 hr. Cells were lysed and subjected to Western blotting analysis of phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA. Total FAK, Src, MLC and RhoA were used as loading controls. (F) HMGA2 and Twist1 overexpression reverted cordycepin-mediated suppression of Lu1205 migration as measured by wound healing assay. Lu1205 monolayer were transfected with either mock, HMGA2 or Twist1 construct before being treated with 200 μg/ml cordycepin for 24 hr. The cell free area were quantified at 24 hr and normalized against that at 0 hr. Data are expressed as mean ± SEM from three independent experiments. *p < 0.05 compared with cordycepin + mock. (G) Cordycepin inhibited Lu1205 melanoma stress fiber and focal adhesion formation via miR-33b. Lu1205 cells were transfected with a variety of miRNA mimic, sh-miRNA, and/or overexpressing constructs before being treated or untreated with cordycepin. Lu1205 cells were stained with rhodamine-phalloidin and paxillin antibody (Geen = paxillin, red = F-actin). Bar = 10 μm. (H–I) Quantification of the average number and size (μm2) of paxillin-containing focal adhesions in Lu1205 cells using ImageJ software.12 cells were analyzed per condition in each experiment. Data are expressed as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 compared with ctrl + scramble.
Mentions: Twist1 has been shown to mediate cancer migration through triggering Rac1 activation [27]. Twist also enhanced hepatocellular carcinoma EMT and invasion through activating matrix metalloproteinase (MMP)-2 and MMP-9 expression [28]. HMGA2 plays important roles in promoting cancer metastasis by downregulating miR-200b expression and increasing the level of lysyl oxidase (LOX) [29–31]. Therefore, we asked whether cordycepin can inhibit melanoma migration by targeting miR-33b and downregulating HMGA2 and Twist1 expression. We employed wound healing assays to assess the melanoma migratory capacities. 200 μg/ml cordycepin treatment inhibited the 24 hr wound closure capacities of melanoma cells (Figure 4A). sh-miR-33b transfection abrogated the suppressive effect of cordycepin on cell motility. Melanoma cells briskly migrated into the wound area with 24 hr after wound scratch. Quantitative analysis of the gap closure suggests that cordycepin significantly attenuated gap closure capacities of Lu1205 and A375 cells in a dose-dependent manner (p < 0.05) (Figure 4B). Ectopic expression of miR-33b significantly increased the cell-free gap area for Lu1205 and A375 cell lines (Figure 4C). In the context of cordycepin treatment, knockdown of miR-33b promoted the wound healing capacities (Figure 4C). The wound area in cordycepin/sh-miR-33b group reached 70% and 50% sealing for Lu1205 and A375 after 24 hr of wound scratch. In sharp contrast, miR-200b, miR-200c, miR-205 and miR-211 knockdown did not significantly alter the motility of melanoma, suggesting that miR-33b would be the sole miRNA responsible for cordycepin-mediated suppression of melanoma migration (Figure 4C). Silencing LXRβ/RXRα significantly ameliorated cordycepin-mediated inhibition of wound healing capacities (p < 0.05) (Figure 4D). Focal adhesion kinase (FAK), Src, myosin light chain (MLC) and RhoA activations are important for cancer motility. Cordycepin treatment suppressed the phosphorylation of FAK, Src and MLC and GTP-coupling of RhoA without affecting the total FAK, Src, MLC and RhoA expressions (Figure 4E). Knockdown of miR-33b in cordycepin-treated Lu1205 and A375 melanoma cells increased the levels of phosphorylated FAK, Src, and MLC and RhoA-GTP. This implies that cordycepin downregulates cell migratory machinery and cytoskeletal remodeling by targeting miR-33b. To determine whether the phenotypes related to cordycepin treatment can be reversed by restoration of HMGA2 and Twist1, we transfected cells with either mock (vector control), HMGA2 or Twist1 overexpression plasmids. HMGA2 or Twist1 overexpression reverted cordycepin-mediated reduction of melanoma motility (Figure 4F). They reduced the 24-hr cell-free area by 30%–55% in cordycepin-treated A375 and Lu1205 monolayer.

Bottom Line: Cordycepin, the active component of Cordyceps spp., has been identified to have anti-metastatic effect on tumor progression and thus possesses pharmacological and therapeutic potentials.Cordycepin-mediated miR-33b expression was dependent on LXR-RXR heterodimer activation. miR-33b directly binds to HMGA2, Twist1 and ZEB1 3'-UTR to suppress their expression.In spontaneous metastasis models, cordycepin suppressed tumor metastasis without altering primary tumor growth.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.

ABSTRACT
Malignant melanoma, the most deadly form of skin cancer, has a high propensity for metastatic spread and is notoriously chemotherapy-resistant. Cordycepin, the active component of Cordyceps spp., has been identified to have anti-metastatic effect on tumor progression and thus possesses pharmacological and therapeutic potentials. However, the mechanisms of anti-metastatic effects of cordycepin at cellular levels remain elusive. We analyzed the effect of cordycepin on human melanoma miRNA expression profiles by miRNAarray and found that miR-33b was upregulated in highly-metastatic melanoma cell lines following cordycepin exposure. Cordycepin-mediated miR-33b expression was dependent on LXR-RXR heterodimer activation. miR-33b directly binds to HMGA2, Twist1 and ZEB1 3'-UTR to suppress their expression. The negative correlations between miR-33b levels and HMGA2, Twist1 or ZEB1 expression were detected in 72 patient melanoma tissue samples. By targeting HMGA2 and Twist1, miR-33b attenuated melanoma migration and invasiveness upon cordycepin exposure. miR-33b knockdown or ZEB1 overexpression reverted cordycepin-mediated mesenchymal-epithelial transition (MET), triggering the expression of N-cadherin. In spontaneous metastasis models, cordycepin suppressed tumor metastasis without altering primary tumor growth. We showed for the first time that targeting miRNA by cordycepin indicates a new mechanism of cordycepin-induced suppression of tumor metastasis and miR-33b/HMGA2/Twist1/ZEB1 axis plays critical roles in regulating melanoma dissemination.

No MeSH data available.


Related in: MedlinePlus