Wang Y et al. (2020), The critical role of dysregulated Hh-FOXM1-TPX2...

XB-ART-57218

Cell Commun Signal 2020 Jul 28;181:116. doi: 10.1186/s12964-020-00628-4.

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The critical role of dysregulated Hh-FOXM1-TPX2 signaling in human hepatocellular carcinoma cell proliferation.

Wang Y , Wang H , Yan Z , Li G , Hu G , Zhang H , Huang D , Wang Y , Zhang X , Yan Y , Lu Q , Cheng M , Luo S .

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BACKGROUND: Aberrant activation of the Hedgehog (Hh) signaling pathway is frequently observed in hepatocellular carcinoma (HCC), nevertheless, the precise molecular mechanism remains unclear. Forkhead box M1 (FOXM1), a target of the Hh pathway, is a key oncofetal transcription factor and a master cell cycle regulator. Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is an oncogene critical for mitosis. However, how these molecular events affect HCC progression remains unclear. METHODS: Realtime PCR, immunohistochemistry, western blotting, and analyses of datasets TCGA and Gene Expression Omnibus (GEO) were conducted to assess the expression of TPX2 and FOXM1 at the mRNA and protein levels in HCC samples or HCC cells. Expression and knockdown of TPX2 and FOXM1 were performed to assess their role in regulating HCC cell proliferation in vitro and in vivo. Dual luciferase report assay and chromosome immunoprecipitation (ChIP) were investigated to seek the FOXM1 binding sites in the promoter of TPX2. RESULTS: Specific antagonists (cyclopamine and GANT61) of the Hh pathway down-regulated TPX2, whereas activation of Hh signaling stimulated TPX2 expression. Furthermore, TPX2 over-expression accelerated HCC cell proliferation when upstream events of Hh signaling were inhibited, and TPX2 knockdown significantly alleviated Sonic Hh ligand (Shh)-induced HCC cell proliferation. Reporter assays and ChIP showed that FOXM1 bound to the TPX2 promoter, confirming that TPX2 is a direct downstream target of FOXM1. Xenograft model further verified the cell function and expression regulation of TPX2 and FOXM1 in vivo. Furthermore, FOXM1 regulated TPX2 activity to drive HCC proliferation. Immunohistochemical (IHC) analysis indicated that FOXM1 and TPX2 were highly-expressed in HCC samples and cohort study revealed that FOXM1 and TPX2 may act as negative predictors for the prognosis of patients with HCC. CONCLUSIONS: TPX2 acts as a novel downstream target and effector of the Hh pathway, and Hh signaling contributes to HCC proliferation via regulating the FOXM1-TPX2 cascade, suggesting that this signaling axis may be a novel therapeutic target for HCC.

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Species referenced: Xenopus
Genes referenced: foxm1 gli2 shh tpx2

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	Fig. 1. TPX2 expression is regulated by the Hh signaling pathway. a. Venn diagrams of differentially expressed genes (DEGs) in Huh7 and HepG2 cells after treating with GANT61 versus genes enriched in “Cell Cycle” gene set. b. Representative candidate genes derived from Venn diagrams in Fig. 1a were represented in a heat map. Red signal denotes higher expression and blue signal denotes lower expression. Gene names marked in red are previously reported genes regulated by FOXM1. c. Hep3B cells were treated with GANT61 (10 ~ 20 μM) for 48 h and harvested for real-time PCR analysis with the indicated primers. d. Hep3B cells were treated with GANT61 (left panel) or cyclopamine (right panel) (10 ~ 20 μM) for 48 h and harvested for WB analysis with the indicated antibodies. e. Hep3B cells were treated with cyclopamine (10 ~ 20 μM) for 48 h and harvested for real-time PCR analysis with the indicated primers. f-g. Hep3B cells transfected with shRNA-control or shRNA-GLI2 was harvested for real-time PCR analysis with the indicated primers (f) and for WB analysis with the indicated antibodies (g). h-i. Huh7cells transfected with Lv-control or Lv-GLI2A were subjected to real-time PCR analysis with the indicated primers (h) as well as WB analysis with the indicated antibodies (i). j. Huh7 cells incubated with or without N-Shh were subjected to real-time PCR analysis with the indicated primers. k. Huh7 cells incubated with N-Shh for 0, 12, and 24 h were harvested for WB analysis with the indicated antibodies. Data was shown as mean ± SD (n = 3). , p < 0.05; *, p < 0.01
	Fig. 2. Over-expression of TPX2 promotes HCC proliferation despite inhibition of Hh/GLI signaling. a-b. Validation of HepG2 cells stably over-expressing TPX2 using WB analysis with the indicated antibodies (a) and real-time PCR with the indicated primers (b). c. Comparison of the proliferative ability of Lv-control + DMSO, Lv-control + GANT61 (2 μM), Lv-TPX2 + DMSO, and Lv-TPX2 + GANT61 (2 μM) in HepG2 cells treated with EdU. Scale bar, 100 μm. d. The ratio of EdU-positive cells was quantified using the ImageJ software (n = 3). e. Cell growth curves of Lv-control + DMSO, Lv-control + GANT61 (2 μM), Lv-TPX2 + DMSO, and Lv-TPX2 + GANT61 (2 μM) in HepG2 cells. f-i. Comparison of the proliferative ability of Lv-control + DMSO, Lv-control + GANT61 (2 μM), Lv-TPX2 + DMSO, and Lv-TPX2 + GANT61 (2 μM) in HepG2 cells using soft-agar colony formation assays (f) and plate colony formation assay (h). Soft-agar colonies (g) and plate colonies (i) were counted using the ImageJ software. Scale bar, 100 μm. Data was shown as mean ± SD (n = 3). , p < 0.05; *, p < 0.01, N.S. denotes not significant
	Fig. 3. TPX2 abrogation inhibits Hh signaling-induced HCC cell proliferation. a-b. HEK293 cells were transfected with shRNA-Control or three shRNA-TPX2 constructs for 48 h and were harvested for WB analysis with the indicated antibodies (a) and real-time PCR analysis with the indicated primers (b). c. The most efficient shRNA sequence for knocking down TPX2 was selected to construct lentivirus systems. Validation of Huh7 sh-TPX2 stable cell lines using WB analysis (left panel) and quantitative real-time PCR (right panel). d. TPX2 abrogation inhibited Hh signaling-induced HCC cell proliferation as determined using EdU staining. Scale bar, 100 μm. e. The ratio of EdU-positive cells was quantified using the ImageJ software (n = 3). f. Cell growth curves of sh-Control / Shh (−), sh-Control / Shh (+), sh-TPX2 / Shh (−), and sh-TPX2 / Shh (+) in Huh7 cells. g-j. Comparison of the proliferative ability of sh-Control / Shh(−), sh-Control / Shh (+), sh-TPX2 / Shh(−), and sh-TPX2 / Shh (+) in Huh7 cells using soft-agar colony formation assays (g) and plate colony formation assay (i). Soft-agar colonies (h) and plate colonies (j) were counted using the ImageJ software. Scale bar, 100 μm. Data was shown as mean ± SD (n = 3). , p < 0.05; *, p < 0.01, N.S. denotes not significant
	Fig. 4. FOXM1 directly activates TPX2 transcription. a. Consensus sequence of FOXM1-binding site, sourced from MotifMap [33]. b. Predicted FOXM1-binding sites within the genomic sequence adjacent to the transcription start site of TPX2. The underlined sequences with red lines are core sequence of FOXM1-binding sites. c. Schematic diagram showing candidate FOXM1-binding site within the TPX2 promoter, the full-length luciferase reporter construct, and its deletion variants with different FOXM1-binding sites. d. Dual luciferase assay of TPX2 promoter-luciferase reporters in FOXM1-expressing or control HepG2 cells. e-g. Dual luciferase assay of sets of TPX2 promoter truncation-luciferase reporters in FOXM1-expressing or control HepG2 cells. h. ChIP assay in Huh7 cells to detect FOXM1 binding to the TPX2 promoter. Data was represented as mean ± SD of three independent experiments. **, p < 0.01
	Fig. 5. TPX2 expression is regulated by Hh-FOXM1 signaling. a. Protein expression levels of TPX2 and FOXM1 were assessed after stable knock down of FOXM1 in Huh7 and Hep3B cells using WB analysis. b. WB analysis to determine protein expression levels of TPX2 and FOXM1 in Huh7 and HepG2 cells stably over-expressing FOXM1. c-e. FOXM1 depletion suppresses tumor growth in vivo. Huh7 cell lines (2 × 107 cells) that expressing sh-FOXM1 or sh-Con were subcutaneously injected into nude mice on each side of the inguinal region. Mice were administered 2 μg/mL doxycycline and 5% sucrose in sterile drinking water. Xenografts were harvested after 3 weeks. Tumor sizes on either side were monitored every other day (c), and tumor size (d) and weight (e) are shown. Data are presented as mean ± SD (n = 5). , p < 0.05; , p < 0.01. f-g. FOXM1 depletion down-regulates TPX2 expression in vivo. Protein level of TPX2 in tumors were checked by western blot (f) and IHC (g). h. Huh7 cells transfected with shRNA-control or shRNA-FOXM1 were incubated with or without N-Shh conditional medium for 48 h and were harvested for WB analysis with the indicated antibodies. i. Huh7 cells stably infected with Lv-control or Lv-GLI2A were infected twice with shRNA-control or shRNA-FOXM1 and were harvested for WB with the indicated antibodies. j. Hep3B cells transfected with Lv-control or Lv-FOXM1 were treated with DMSO or 20 μM GANT61 for 48 h and were harvested for WB analysis with the indicated antibodies. Data was shown as mean ± SD (n = 3). , p < 0.05; **, p < 0.01
	Fig. 6. FOXM1 promotes HCC cell proliferation through TPX2. a. WB analysis of Huh7 cells infected with FOXM1 overexpression and TPX2 knockdown lentiviral vectors. b. Comparison of the proliferative ability of Lv-control + sh-Con, Lv-control + sh-TPX2, Lv-FOXM1 + sh-Con, and Lv-FOXM1 + sh-TPX2 in Huh7 cells using the EdU assay. Scale bar, 100 μm. c. The ratio of EdU-positive cells was quantified using the ImageJ software (n = 3). d. Growth curves of Lv-control + sh-Con, Lv-control + sh-TPX2, Lv-FOXM1 + sh-Con, and Lv-FOXM1 + sh-TPX2 of Huh7 cells. e-h. Comparison of the proliferative ability (f) and plate colony formation assay (h) of Lv-control + sh-Con, Lv-control + sh-TPX2, Lv-FOXM1 + sh-Con, and Lv-FOXM1 + sh-TPX2 in Huh7 cells. Scale bar, 100 μm. Soft agar formation (g) and plate colony formation (I) were quantified using the ImageJ software. i. Huh7 cells stably expressing FOXM1 + shTPX2 were subjected to cell cycle analysis (left panel) and fraction of cells in each phase quantified (right panel). j. Quantification (%) of multinucleated Huh7 cells stably expressing FOXM1 + shTPX2. Data was represented as mean ± SD of three independent experiments. , p < 0.05; *, p < 0.01

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	Fig. 1. TPX2 expression is regulated by the Hh signaling pathway. a. Venn diagrams of differentially expressed genes (DEGs) in Huh7 and HepG2 cells after treating with GANT61 versus genes enriched in “Cell Cycle” gene set. b. Representative candidate genes derived from Venn diagrams in Fig. 1a were represented in a heat map. Red signal denotes higher expression and blue signal denotes lower expression. Gene names marked in red are previously reported genes regulated by FOXM1. c. Hep3B cells were treated with GANT61 (10 ~ 20 μM) for 48 h and harvested for real-time PCR analysis with the indicated primers. d. Hep3B cells were treated with GANT61 (left panel) or cyclopamine (right panel) (10 ~ 20 μM) for 48 h and harvested for WB analysis with the indicated antibodies. e. Hep3B cells were treated with cyclopamine (10 ~ 20 μM) for 48 h and harvested for real-time PCR analysis with the indicated primers. f-g. Hep3B cells transfected with shRNA-control or shRNA-GLI2 was harvested for real-time PCR analysis with the indicated primers (f) and for WB analysis with the indicated antibodies (g). h-i. Huh7cells transfected with Lv-control or Lv-GLI2A were subjected to real-time PCR analysis with the indicated primers (h) as well as WB analysis with the indicated antibodies (i). j. Huh7 cells incubated with or without N-Shh were subjected to real-time PCR analysis with the indicated primers. k. Huh7 cells incubated with N-Shh for 0, 12, and 24 h were harvested for WB analysis with the indicated antibodies. Data was shown as mean ± SD (n = 3). , p < 0.05; *, p < 0.01
	Fig. 2. Over-expression of TPX2 promotes HCC proliferation despite inhibition of Hh/GLI signaling. a-b. Validation of HepG2 cells stably over-expressing TPX2 using WB analysis with the indicated antibodies (a) and real-time PCR with the indicated primers (b). c. Comparison of the proliferative ability of Lv-control + DMSO, Lv-control + GANT61 (2 μM), Lv-TPX2 + DMSO, and Lv-TPX2 + GANT61 (2 μM) in HepG2 cells treated with EdU. Scale bar, 100 μm. d. The ratio of EdU-positive cells was quantified using the ImageJ software (n = 3). e. Cell growth curves of Lv-control + DMSO, Lv-control + GANT61 (2 μM), Lv-TPX2 + DMSO, and Lv-TPX2 + GANT61 (2 μM) in HepG2 cells. f-i. Comparison of the proliferative ability of Lv-control + DMSO, Lv-control + GANT61 (2 μM), Lv-TPX2 + DMSO, and Lv-TPX2 + GANT61 (2 μM) in HepG2 cells using soft-agar colony formation assays (f) and plate colony formation assay (h). Soft-agar colonies (g) and plate colonies (i) were counted using the ImageJ software. Scale bar, 100 μm. Data was shown as mean ± SD (n = 3). , p < 0.05; *, p < 0.01, N.S. denotes not significant
	Fig. 3. TPX2 abrogation inhibits Hh signaling-induced HCC cell proliferation. a-b. HEK293 cells were transfected with shRNA-Control or three shRNA-TPX2 constructs for 48 h and were harvested for WB analysis with the indicated antibodies (a) and real-time PCR analysis with the indicated primers (b). c. The most efficient shRNA sequence for knocking down TPX2 was selected to construct lentivirus systems. Validation of Huh7 sh-TPX2 stable cell lines using WB analysis (left panel) and quantitative real-time PCR (right panel). d. TPX2 abrogation inhibited Hh signaling-induced HCC cell proliferation as determined using EdU staining. Scale bar, 100 μm. e. The ratio of EdU-positive cells was quantified using the ImageJ software (n = 3). f. Cell growth curves of sh-Control / Shh (−), sh-Control / Shh (+), sh-TPX2 / Shh (−), and sh-TPX2 / Shh (+) in Huh7 cells. g-j. Comparison of the proliferative ability of sh-Control / Shh(−), sh-Control / Shh (+), sh-TPX2 / Shh(−), and sh-TPX2 / Shh (+) in Huh7 cells using soft-agar colony formation assays (g) and plate colony formation assay (i). Soft-agar colonies (h) and plate colonies (j) were counted using the ImageJ software. Scale bar, 100 μm. Data was shown as mean ± SD (n = 3). , p < 0.05; *, p < 0.01, N.S. denotes not significant
	Fig. 4. FOXM1 directly activates TPX2 transcription. a. Consensus sequence of FOXM1-binding site, sourced from MotifMap [33]. b. Predicted FOXM1-binding sites within the genomic sequence adjacent to the transcription start site of TPX2. The underlined sequences with red lines are core sequence of FOXM1-binding sites. c. Schematic diagram showing candidate FOXM1-binding site within the TPX2 promoter, the full-length luciferase reporter construct, and its deletion variants with different FOXM1-binding sites. d. Dual luciferase assay of TPX2 promoter-luciferase reporters in FOXM1-expressing or control HepG2 cells. e-g. Dual luciferase assay of sets of TPX2 promoter truncation-luciferase reporters in FOXM1-expressing or control HepG2 cells. h. ChIP assay in Huh7 cells to detect FOXM1 binding to the TPX2 promoter. Data was represented as mean ± SD of three independent experiments. **, p < 0.01
	Fig. 5. TPX2 expression is regulated by Hh-FOXM1 signaling. a. Protein expression levels of TPX2 and FOXM1 were assessed after stable knock down of FOXM1 in Huh7 and Hep3B cells using WB analysis. b. WB analysis to determine protein expression levels of TPX2 and FOXM1 in Huh7 and HepG2 cells stably over-expressing FOXM1. c-e. FOXM1 depletion suppresses tumor growth in vivo. Huh7 cell lines (2 × 107 cells) that expressing sh-FOXM1 or sh-Con were subcutaneously injected into nude mice on each side of the inguinal region. Mice were administered 2 μg/mL doxycycline and 5% sucrose in sterile drinking water. Xenografts were harvested after 3 weeks. Tumor sizes on either side were monitored every other day (c), and tumor size (d) and weight (e) are shown. Data are presented as mean ± SD (n = 5). , p < 0.05; , p < 0.01. f-g. FOXM1 depletion down-regulates TPX2 expression in vivo. Protein level of TPX2 in tumors were checked by western blot (f) and IHC (g). h. Huh7 cells transfected with shRNA-control or shRNA-FOXM1 were incubated with or without N-Shh conditional medium for 48 h and were harvested for WB analysis with the indicated antibodies. i. Huh7 cells stably infected with Lv-control or Lv-GLI2A were infected twice with shRNA-control or shRNA-FOXM1 and were harvested for WB with the indicated antibodies. j. Hep3B cells transfected with Lv-control or Lv-FOXM1 were treated with DMSO or 20 μM GANT61 for 48 h and were harvested for WB analysis with the indicated antibodies. Data was shown as mean ± SD (n = 3). , p < 0.05; **, p < 0.01
	Fig. 6. FOXM1 promotes HCC cell proliferation through TPX2. a. WB analysis of Huh7 cells infected with FOXM1 overexpression and TPX2 knockdown lentiviral vectors. b. Comparison of the proliferative ability of Lv-control + sh-Con, Lv-control + sh-TPX2, Lv-FOXM1 + sh-Con, and Lv-FOXM1 + sh-TPX2 in Huh7 cells using the EdU assay. Scale bar, 100 μm. c. The ratio of EdU-positive cells was quantified using the ImageJ software (n = 3). d. Growth curves of Lv-control + sh-Con, Lv-control + sh-TPX2, Lv-FOXM1 + sh-Con, and Lv-FOXM1 + sh-TPX2 of Huh7 cells. e-h. Comparison of the proliferative ability (f) and plate colony formation assay (h) of Lv-control + sh-Con, Lv-control + sh-TPX2, Lv-FOXM1 + sh-Con, and Lv-FOXM1 + sh-TPX2 in Huh7 cells. Scale bar, 100 μm. Soft agar formation (g) and plate colony formation (I) were quantified using the ImageJ software. i. Huh7 cells stably expressing FOXM1 + shTPX2 were subjected to cell cycle analysis (left panel) and fraction of cells in each phase quantified (right panel). j. Quantification (%) of multinucleated Huh7 cells stably expressing FOXM1 + shTPX2. Data was represented as mean ± SD of three independent experiments. , p < 0.05; *, p < 0.01