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Elevated HOXB9 expression promotes differentiation and predicts a favourable outcome in colon adenocarcinoma patients.

Zhan J, Niu M, Wang P, Zhu X, Li S, Song J, He H, Wang Y, Xue L, Fang W, Zhang H - Br. J. Cancer (2014)

Bottom Line: Elevated HOXB9 expression was identified in well-differentiated colon adenocarcinoma patients and was associated with a better overall patients' survival.Overexpression of HOXB9 inhibited colon adenocarcinoma cell growth, migration, invasion in vitro and tumour growth, liver as well as lung metastases in nude mice; whereas silencing HOXB9 promoted these functions.Elevated expression of HOXB9 predicts a longer survival in colon adenocarcinoma patients.

View Article: PubMed Central - PubMed

Affiliation: 1] Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Peking University Health Science Center, Beijing 100191, China [2] State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China [3] Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology and Embryology, Peking University Health Science Center, Beijing 100191, China.

ABSTRACT

Background: Little is known about the tumour suppressive proteins and the underlying mechanisms that suppress colon cancer progression. Homeodomain-containing transcription factor HOXB9 plays an important role in embryogenesis and cancer development. We here aim to uncover the potential role of HOXB9 in the regulation of colon adenocarcinoma progression including epithelial-to-mesenchymal transition.

Methods: HOXB9 expression in colon adenocarcinoma cells and patients was analysed by western blot and immunohistochemistry separately. Correlation between HOXB9 expressions with patients' survival was assessed by Kaplan-Meier analysis. HOXB9-regulated target gene expression was determined by RNA sequencing in HOXB9-overexpressing colon adenocarcinoma cells.

Results: Elevated HOXB9 expression was identified in well-differentiated colon adenocarcinoma patients and was associated with a better overall patients' survival. Overexpression of HOXB9 inhibited colon adenocarcinoma cell growth, migration, invasion in vitro and tumour growth, liver as well as lung metastases in nude mice; whereas silencing HOXB9 promoted these functions. HOXB9 promoted colon adenocarcinoma differentiation via a mechanism that stimulates mesenchymal-to-epithelial transition, involving downregulation of EMT-promoting transcriptional factors including Snail, Twist, FOXC2 and ZEB1 and upregulation of epithelial proteins including E-cadherin, claudins-1, -4, -7, occludin and ZO-1.

Conclusions: HOXB9 is a novel tumour suppressor that inhibits colon adenocarcinoma progression by inducing differentiation. Elevated expression of HOXB9 predicts a longer survival in colon adenocarcinoma patients.

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Related in: MedlinePlus

HOXB9 promotes colon adenocarcinoma cell differentiation by upregulating cellular junction protein expressions. (A) In the gain-of-function experiment, transient expression of HOXB9 upregulated adherens junction protein E-cadherin and tight junction proteins claudin-1, claudin-7, occludin and ZO-1 in SW480 cells (left); E-cadherin, claudin-1 and ZO-1 in SW620 cells (middle) and E-cadherin, claudin-1, claudin-7 and occludin in HCT116 cells (right). (B) In HCT116 cells, stable expression of HOXB9 upregulated E-cadherin, claudin-4, claudin-7 and occludin. (C) In the loss-of-function experiment, stably knocking down of HOXB9 in HCT116 cells led to downregulation of claudin-1 and occludin. (D) HOXB9-regulated genes with 16-fold changes were presented in a heatmap for displaying gene expression profile. (E) Important EMT-related genes SNAI1, SNAI2, FOXC2, ZEB1 ZEB2, Twist, FN1 and VIM were selected to plot in a heatmap presentation, plotted showing that EMT-related transcriptional factors are drastically downregulated, so do mesenchymal markers FN1 and VIM. (F) Confirmation of changes in gene expression for selective key genes in Flag HCT116 or Flag-HOXB9 HCT116 cells. Epithelial marker E-cadherin, mesenchymal marker Vimentin and EMT-stimulating transcriptional factors Twist and ZEB1 were measured by quantitative PCR in the cells expressing HOXB9. Data were expressed as relative expression of mRNAs.
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fig5: HOXB9 promotes colon adenocarcinoma cell differentiation by upregulating cellular junction protein expressions. (A) In the gain-of-function experiment, transient expression of HOXB9 upregulated adherens junction protein E-cadherin and tight junction proteins claudin-1, claudin-7, occludin and ZO-1 in SW480 cells (left); E-cadherin, claudin-1 and ZO-1 in SW620 cells (middle) and E-cadherin, claudin-1, claudin-7 and occludin in HCT116 cells (right). (B) In HCT116 cells, stable expression of HOXB9 upregulated E-cadherin, claudin-4, claudin-7 and occludin. (C) In the loss-of-function experiment, stably knocking down of HOXB9 in HCT116 cells led to downregulation of claudin-1 and occludin. (D) HOXB9-regulated genes with 16-fold changes were presented in a heatmap for displaying gene expression profile. (E) Important EMT-related genes SNAI1, SNAI2, FOXC2, ZEB1 ZEB2, Twist, FN1 and VIM were selected to plot in a heatmap presentation, plotted showing that EMT-related transcriptional factors are drastically downregulated, so do mesenchymal markers FN1 and VIM. (F) Confirmation of changes in gene expression for selective key genes in Flag HCT116 or Flag-HOXB9 HCT116 cells. Epithelial marker E-cadherin, mesenchymal marker Vimentin and EMT-stimulating transcriptional factors Twist and ZEB1 were measured by quantitative PCR in the cells expressing HOXB9. Data were expressed as relative expression of mRNAs.

Mentions: The aforementioned findings have demonstrated that HOXB9 functions as a tumour suppressive transcriptional factor in colon adenocarcinoma cells and its expression correlated with differentiation patterns and overall survival in colon adenocarcinoma patients. However, the mechanism accounting for these inhibitory effects of HOXB9 remained unknown. To this end, we examined changes of a panel of cell differentiation markers in colon adenocarcinomas. In a gain-of-function experiment, we found that transient expression of HOXB9 upregulated cellular junction proteins E-cadherin, claudin-1, claudin-7, ZO-1 and occludin in SW480 cells (Figure 5A left), E-cadherin, claudin-1 and ZO-1 in SW620 cells (Figure 5A middle), and E-cadherin, claudin-1 and occludin in HCT116 cells (Figure 5A right). Similarly, E-cadherin, claudin-4 and -7, and occludin were found upregulated in HCT116 cells stably expressing HOXB9 (Figure 5B). In contrast, in a loss-of-function experiment using HOXB9 siRNAs transient transfection, we found that claudin-1 and occludin were both downregulated in HCT116 cells (Figure 5C). These data together suggested that ectopic expression of HOXB9 promoted colon adenocarcinoma cell differentiation by driving the cells toward an epithelial phenotype, which may involve a mesenchymal-to-epithelial transition (MET) process. To identify the global effect of HOXB9 expression on the colon adenocarcinoma cell phenotypic changes, we performed RNA deep sequencing analyses (Supplementary Table 1). As Figure 5D indicated, genes with 16-fold changes under the HOXB9 regulation were displayed. Furthermore, selective gene expressions which are related to EMT including SNAI1, SNAI2, FOXC2, ZEB1 ZEB2, Twist, FN1 and VIM were shown in the heatmap presentation (Figure 5E). The HOXB9 effect on global expressions of the EMT-related markers was further confirmed in a selective panel of key genes in a quantitative PCR assay (Figure 5F).


Elevated HOXB9 expression promotes differentiation and predicts a favourable outcome in colon adenocarcinoma patients.

Zhan J, Niu M, Wang P, Zhu X, Li S, Song J, He H, Wang Y, Xue L, Fang W, Zhang H - Br. J. Cancer (2014)

HOXB9 promotes colon adenocarcinoma cell differentiation by upregulating cellular junction protein expressions. (A) In the gain-of-function experiment, transient expression of HOXB9 upregulated adherens junction protein E-cadherin and tight junction proteins claudin-1, claudin-7, occludin and ZO-1 in SW480 cells (left); E-cadherin, claudin-1 and ZO-1 in SW620 cells (middle) and E-cadherin, claudin-1, claudin-7 and occludin in HCT116 cells (right). (B) In HCT116 cells, stable expression of HOXB9 upregulated E-cadherin, claudin-4, claudin-7 and occludin. (C) In the loss-of-function experiment, stably knocking down of HOXB9 in HCT116 cells led to downregulation of claudin-1 and occludin. (D) HOXB9-regulated genes with 16-fold changes were presented in a heatmap for displaying gene expression profile. (E) Important EMT-related genes SNAI1, SNAI2, FOXC2, ZEB1 ZEB2, Twist, FN1 and VIM were selected to plot in a heatmap presentation, plotted showing that EMT-related transcriptional factors are drastically downregulated, so do mesenchymal markers FN1 and VIM. (F) Confirmation of changes in gene expression for selective key genes in Flag HCT116 or Flag-HOXB9 HCT116 cells. Epithelial marker E-cadherin, mesenchymal marker Vimentin and EMT-stimulating transcriptional factors Twist and ZEB1 were measured by quantitative PCR in the cells expressing HOXB9. Data were expressed as relative expression of mRNAs.
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fig5: HOXB9 promotes colon adenocarcinoma cell differentiation by upregulating cellular junction protein expressions. (A) In the gain-of-function experiment, transient expression of HOXB9 upregulated adherens junction protein E-cadherin and tight junction proteins claudin-1, claudin-7, occludin and ZO-1 in SW480 cells (left); E-cadherin, claudin-1 and ZO-1 in SW620 cells (middle) and E-cadherin, claudin-1, claudin-7 and occludin in HCT116 cells (right). (B) In HCT116 cells, stable expression of HOXB9 upregulated E-cadherin, claudin-4, claudin-7 and occludin. (C) In the loss-of-function experiment, stably knocking down of HOXB9 in HCT116 cells led to downregulation of claudin-1 and occludin. (D) HOXB9-regulated genes with 16-fold changes were presented in a heatmap for displaying gene expression profile. (E) Important EMT-related genes SNAI1, SNAI2, FOXC2, ZEB1 ZEB2, Twist, FN1 and VIM were selected to plot in a heatmap presentation, plotted showing that EMT-related transcriptional factors are drastically downregulated, so do mesenchymal markers FN1 and VIM. (F) Confirmation of changes in gene expression for selective key genes in Flag HCT116 or Flag-HOXB9 HCT116 cells. Epithelial marker E-cadherin, mesenchymal marker Vimentin and EMT-stimulating transcriptional factors Twist and ZEB1 were measured by quantitative PCR in the cells expressing HOXB9. Data were expressed as relative expression of mRNAs.
Mentions: The aforementioned findings have demonstrated that HOXB9 functions as a tumour suppressive transcriptional factor in colon adenocarcinoma cells and its expression correlated with differentiation patterns and overall survival in colon adenocarcinoma patients. However, the mechanism accounting for these inhibitory effects of HOXB9 remained unknown. To this end, we examined changes of a panel of cell differentiation markers in colon adenocarcinomas. In a gain-of-function experiment, we found that transient expression of HOXB9 upregulated cellular junction proteins E-cadherin, claudin-1, claudin-7, ZO-1 and occludin in SW480 cells (Figure 5A left), E-cadherin, claudin-1 and ZO-1 in SW620 cells (Figure 5A middle), and E-cadherin, claudin-1 and occludin in HCT116 cells (Figure 5A right). Similarly, E-cadherin, claudin-4 and -7, and occludin were found upregulated in HCT116 cells stably expressing HOXB9 (Figure 5B). In contrast, in a loss-of-function experiment using HOXB9 siRNAs transient transfection, we found that claudin-1 and occludin were both downregulated in HCT116 cells (Figure 5C). These data together suggested that ectopic expression of HOXB9 promoted colon adenocarcinoma cell differentiation by driving the cells toward an epithelial phenotype, which may involve a mesenchymal-to-epithelial transition (MET) process. To identify the global effect of HOXB9 expression on the colon adenocarcinoma cell phenotypic changes, we performed RNA deep sequencing analyses (Supplementary Table 1). As Figure 5D indicated, genes with 16-fold changes under the HOXB9 regulation were displayed. Furthermore, selective gene expressions which are related to EMT including SNAI1, SNAI2, FOXC2, ZEB1 ZEB2, Twist, FN1 and VIM were shown in the heatmap presentation (Figure 5E). The HOXB9 effect on global expressions of the EMT-related markers was further confirmed in a selective panel of key genes in a quantitative PCR assay (Figure 5F).

Bottom Line: Elevated HOXB9 expression was identified in well-differentiated colon adenocarcinoma patients and was associated with a better overall patients' survival.Overexpression of HOXB9 inhibited colon adenocarcinoma cell growth, migration, invasion in vitro and tumour growth, liver as well as lung metastases in nude mice; whereas silencing HOXB9 promoted these functions.Elevated expression of HOXB9 predicts a longer survival in colon adenocarcinoma patients.

View Article: PubMed Central - PubMed

Affiliation: 1] Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Peking University Health Science Center, Beijing 100191, China [2] State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing 100191, China [3] Laboratory of Molecular Cell Biology and Tumor Biology, Department of Anatomy, Histology and Embryology, Peking University Health Science Center, Beijing 100191, China.

ABSTRACT

Background: Little is known about the tumour suppressive proteins and the underlying mechanisms that suppress colon cancer progression. Homeodomain-containing transcription factor HOXB9 plays an important role in embryogenesis and cancer development. We here aim to uncover the potential role of HOXB9 in the regulation of colon adenocarcinoma progression including epithelial-to-mesenchymal transition.

Methods: HOXB9 expression in colon adenocarcinoma cells and patients was analysed by western blot and immunohistochemistry separately. Correlation between HOXB9 expressions with patients' survival was assessed by Kaplan-Meier analysis. HOXB9-regulated target gene expression was determined by RNA sequencing in HOXB9-overexpressing colon adenocarcinoma cells.

Results: Elevated HOXB9 expression was identified in well-differentiated colon adenocarcinoma patients and was associated with a better overall patients' survival. Overexpression of HOXB9 inhibited colon adenocarcinoma cell growth, migration, invasion in vitro and tumour growth, liver as well as lung metastases in nude mice; whereas silencing HOXB9 promoted these functions. HOXB9 promoted colon adenocarcinoma differentiation via a mechanism that stimulates mesenchymal-to-epithelial transition, involving downregulation of EMT-promoting transcriptional factors including Snail, Twist, FOXC2 and ZEB1 and upregulation of epithelial proteins including E-cadherin, claudins-1, -4, -7, occludin and ZO-1.

Conclusions: HOXB9 is a novel tumour suppressor that inhibits colon adenocarcinoma progression by inducing differentiation. Elevated expression of HOXB9 predicts a longer survival in colon adenocarcinoma patients.

Show MeSH
Related in: MedlinePlus