Limits...
Int6 reduction activates stromal fibroblasts to enhance transforming activity in breast epithelial cells.

Suo J, Medina D, Herrera S, Zheng ZY, Jin L, Chamness GC, Contreras A, Gutierrez C, Hilsenbeck S, Umar A, Foekens JA, Hanash S, Schiff R, Zhang XH, Chang EC - Cell Biosci (2015)

Bottom Line: We analyzed INT6-repressed HMFs and found an increase in the levels of a key carcinoma-associated fibroblast (CAF) marker, smooth muscle actin.Intriguingly, when mesenchymal stem cells (MSCs) were induced to form CAFs, Int6 levels were reduced.These data suggest that besides enhancing transforming activity in epithelial cells, INT6 repression can also induce fibroblasts, and possibly MSCs as well, via mesenchymal-mesenchymal transitions to promote the formation of CAFs, leading to a proinvasive microenvironment for tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, 77030 TX USA ; Department of Molecular and Cellular Biology and Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, 77030 TX USA.

ABSTRACT

Background: The INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; however, INT6's role in the development of human breast cancer remains largely unknown. By gene silencing, we have previously shown that repressing INT6 promotes transforming activity in untransformed human mammary epithelial cells. In the present study, guided by microarray data of human tumors, we have discovered a role of Int6 in stromal fibroblasts.

Results: We searched microarray databases of human tumors to assess Int6's role in breast cancer. While INT6 expression levels, as expected, were lower in breast tumors than in adjacent normal breast tissue samples, INT6 expression levels were also substantially lower in tumor stroma. By immunohistochemistry, we determined that the low levels of INT6 mRNA observed in the microarray databases most likely occurs in stromal fibroblasts, because far fewer fibroblasts in the tumor tissue showed detectable levels of the Int6 protein. To directly investigate the effects of Int6 repression on fibroblasts, we silenced INT6 expression in immortalized human mammary fibroblasts (HMFs). When these INT6-repressed HMFs were co-cultured with breast cancer cells, the abilities of the latter to form colonies in soft agar and to invade were enhanced. We analyzed INT6-repressed HMFs and found an increase in the levels of a key carcinoma-associated fibroblast (CAF) marker, smooth muscle actin. Furthermore, like CAFs, these INT6-repressed HMFs secreted more stromal cell-derived factor 1 (SDF-1), and the addition of an SDF-1 antagonist attenuated the INT6-repressed HMFs' ability to enhance soft agar colony formation when co-cultured with cancer cells. These INT6-repressed HMFs also expressed high levels of mesenchymal markers such as vimentin and N-cadherin. Intriguingly, when mesenchymal stem cells (MSCs) were induced to form CAFs, Int6 levels were reduced.

Conclusion: These data suggest that besides enhancing transforming activity in epithelial cells, INT6 repression can also induce fibroblasts, and possibly MSCs as well, via mesenchymal-mesenchymal transitions to promote the formation of CAFs, leading to a proinvasive microenvironment for tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

Reduction of Int6 in the fibroblasts in human breast tumors. (A) Left: gene expression data from the breast cancer TCGA project were directly exported from Oncomine, in which mRNA levels of INT6 in normal breast tissue and invasive ductal carcinomas were compared. INT6 mRNA levels were reported to be 50% lower in the latter. Right: stroma gene expression data in the Finak study available in Oncomine were analyzed to show that INT6 mRNA levels were approximately 42 times higher in the tissue surrounding the normal adjacent ducts than in the stroma in the tumor. (B) Control or INT6-repressed MCF7 cells were analyzed by Western blot (left) or IHC (right) by an anti-Int6 antibody. We note that agreeing with our previous finding using GFP-tagging [26], Int6 is mainly cytoplasmic. (C) This is a typical IHC experiment examining fibroblasts in the adjacent normal and tumor region from the same human tumor sample. The top pictures were captured using a 10× objective, and one area in each was then examined by a 40× objective to reveal more details. Closed and open arrowheads mark Int6-positive vs. Int6-negative fibroblasts. (D) The histoscore differences between the normal and tumor regions were compared by the Wilcoxon signed rank test. While there was no difference in Int6 intensity between the normal and tumor regions (p = 0.66), a much lower percentage of Int6-positive fibroblasts was found in the tumor. As a result, all but two samples (marked red) show lower values for tumor fibroblasts. The mean normal and tumor histoscores are marked orange. On the right is a boxplot of the differences between normal and tumor histoscores (individual values shown as circles, mean difference shown in orange). Mean difference ± SEM = 27.0 ± 7.2 (p < 10−3, Wilcoxon signed rank test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4359526&req=5

Fig1: Reduction of Int6 in the fibroblasts in human breast tumors. (A) Left: gene expression data from the breast cancer TCGA project were directly exported from Oncomine, in which mRNA levels of INT6 in normal breast tissue and invasive ductal carcinomas were compared. INT6 mRNA levels were reported to be 50% lower in the latter. Right: stroma gene expression data in the Finak study available in Oncomine were analyzed to show that INT6 mRNA levels were approximately 42 times higher in the tissue surrounding the normal adjacent ducts than in the stroma in the tumor. (B) Control or INT6-repressed MCF7 cells were analyzed by Western blot (left) or IHC (right) by an anti-Int6 antibody. We note that agreeing with our previous finding using GFP-tagging [26], Int6 is mainly cytoplasmic. (C) This is a typical IHC experiment examining fibroblasts in the adjacent normal and tumor region from the same human tumor sample. The top pictures were captured using a 10× objective, and one area in each was then examined by a 40× objective to reveal more details. Closed and open arrowheads mark Int6-positive vs. Int6-negative fibroblasts. (D) The histoscore differences between the normal and tumor regions were compared by the Wilcoxon signed rank test. While there was no difference in Int6 intensity between the normal and tumor regions (p = 0.66), a much lower percentage of Int6-positive fibroblasts was found in the tumor. As a result, all but two samples (marked red) show lower values for tumor fibroblasts. The mean normal and tumor histoscores are marked orange. On the right is a boxplot of the differences between normal and tumor histoscores (individual values shown as circles, mean difference shown in orange). Mean difference ± SEM = 27.0 ± 7.2 (p < 10−3, Wilcoxon signed rank test).

Mentions: To determine whether INT6 may act as a tumor suppressor for breast cancer, we searched Oncomine for gene expression changes by focusing on studies in which normal and tumor tissues were compared. INT6 levels were found to be significantly higher in normal tissues compared with invasive breast tumors and premalignant ductal carcinoma in situ (DCIS), according to data from the TCGA project (Figure 1A, left) [24] and Curtis et al. (data not shown) [25], respectively.Figure 1


Int6 reduction activates stromal fibroblasts to enhance transforming activity in breast epithelial cells.

Suo J, Medina D, Herrera S, Zheng ZY, Jin L, Chamness GC, Contreras A, Gutierrez C, Hilsenbeck S, Umar A, Foekens JA, Hanash S, Schiff R, Zhang XH, Chang EC - Cell Biosci (2015)

Reduction of Int6 in the fibroblasts in human breast tumors. (A) Left: gene expression data from the breast cancer TCGA project were directly exported from Oncomine, in which mRNA levels of INT6 in normal breast tissue and invasive ductal carcinomas were compared. INT6 mRNA levels were reported to be 50% lower in the latter. Right: stroma gene expression data in the Finak study available in Oncomine were analyzed to show that INT6 mRNA levels were approximately 42 times higher in the tissue surrounding the normal adjacent ducts than in the stroma in the tumor. (B) Control or INT6-repressed MCF7 cells were analyzed by Western blot (left) or IHC (right) by an anti-Int6 antibody. We note that agreeing with our previous finding using GFP-tagging [26], Int6 is mainly cytoplasmic. (C) This is a typical IHC experiment examining fibroblasts in the adjacent normal and tumor region from the same human tumor sample. The top pictures were captured using a 10× objective, and one area in each was then examined by a 40× objective to reveal more details. Closed and open arrowheads mark Int6-positive vs. Int6-negative fibroblasts. (D) The histoscore differences between the normal and tumor regions were compared by the Wilcoxon signed rank test. While there was no difference in Int6 intensity between the normal and tumor regions (p = 0.66), a much lower percentage of Int6-positive fibroblasts was found in the tumor. As a result, all but two samples (marked red) show lower values for tumor fibroblasts. The mean normal and tumor histoscores are marked orange. On the right is a boxplot of the differences between normal and tumor histoscores (individual values shown as circles, mean difference shown in orange). Mean difference ± SEM = 27.0 ± 7.2 (p < 10−3, Wilcoxon signed rank test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4359526&req=5

Fig1: Reduction of Int6 in the fibroblasts in human breast tumors. (A) Left: gene expression data from the breast cancer TCGA project were directly exported from Oncomine, in which mRNA levels of INT6 in normal breast tissue and invasive ductal carcinomas were compared. INT6 mRNA levels were reported to be 50% lower in the latter. Right: stroma gene expression data in the Finak study available in Oncomine were analyzed to show that INT6 mRNA levels were approximately 42 times higher in the tissue surrounding the normal adjacent ducts than in the stroma in the tumor. (B) Control or INT6-repressed MCF7 cells were analyzed by Western blot (left) or IHC (right) by an anti-Int6 antibody. We note that agreeing with our previous finding using GFP-tagging [26], Int6 is mainly cytoplasmic. (C) This is a typical IHC experiment examining fibroblasts in the adjacent normal and tumor region from the same human tumor sample. The top pictures were captured using a 10× objective, and one area in each was then examined by a 40× objective to reveal more details. Closed and open arrowheads mark Int6-positive vs. Int6-negative fibroblasts. (D) The histoscore differences between the normal and tumor regions were compared by the Wilcoxon signed rank test. While there was no difference in Int6 intensity between the normal and tumor regions (p = 0.66), a much lower percentage of Int6-positive fibroblasts was found in the tumor. As a result, all but two samples (marked red) show lower values for tumor fibroblasts. The mean normal and tumor histoscores are marked orange. On the right is a boxplot of the differences between normal and tumor histoscores (individual values shown as circles, mean difference shown in orange). Mean difference ± SEM = 27.0 ± 7.2 (p < 10−3, Wilcoxon signed rank test).
Mentions: To determine whether INT6 may act as a tumor suppressor for breast cancer, we searched Oncomine for gene expression changes by focusing on studies in which normal and tumor tissues were compared. INT6 levels were found to be significantly higher in normal tissues compared with invasive breast tumors and premalignant ductal carcinoma in situ (DCIS), according to data from the TCGA project (Figure 1A, left) [24] and Curtis et al. (data not shown) [25], respectively.Figure 1

Bottom Line: We analyzed INT6-repressed HMFs and found an increase in the levels of a key carcinoma-associated fibroblast (CAF) marker, smooth muscle actin.Intriguingly, when mesenchymal stem cells (MSCs) were induced to form CAFs, Int6 levels were reduced.These data suggest that besides enhancing transforming activity in epithelial cells, INT6 repression can also induce fibroblasts, and possibly MSCs as well, via mesenchymal-mesenchymal transitions to promote the formation of CAFs, leading to a proinvasive microenvironment for tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Cancer Prevention, The University of Texas MD Anderson Cancer Center, Houston, 77030 TX USA ; Department of Molecular and Cellular Biology and Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, 77030 TX USA.

ABSTRACT

Background: The INT6 gene was first discovered as a site of integration in mouse mammary tumors by the mouse mammary tumor virus; however, INT6's role in the development of human breast cancer remains largely unknown. By gene silencing, we have previously shown that repressing INT6 promotes transforming activity in untransformed human mammary epithelial cells. In the present study, guided by microarray data of human tumors, we have discovered a role of Int6 in stromal fibroblasts.

Results: We searched microarray databases of human tumors to assess Int6's role in breast cancer. While INT6 expression levels, as expected, were lower in breast tumors than in adjacent normal breast tissue samples, INT6 expression levels were also substantially lower in tumor stroma. By immunohistochemistry, we determined that the low levels of INT6 mRNA observed in the microarray databases most likely occurs in stromal fibroblasts, because far fewer fibroblasts in the tumor tissue showed detectable levels of the Int6 protein. To directly investigate the effects of Int6 repression on fibroblasts, we silenced INT6 expression in immortalized human mammary fibroblasts (HMFs). When these INT6-repressed HMFs were co-cultured with breast cancer cells, the abilities of the latter to form colonies in soft agar and to invade were enhanced. We analyzed INT6-repressed HMFs and found an increase in the levels of a key carcinoma-associated fibroblast (CAF) marker, smooth muscle actin. Furthermore, like CAFs, these INT6-repressed HMFs secreted more stromal cell-derived factor 1 (SDF-1), and the addition of an SDF-1 antagonist attenuated the INT6-repressed HMFs' ability to enhance soft agar colony formation when co-cultured with cancer cells. These INT6-repressed HMFs also expressed high levels of mesenchymal markers such as vimentin and N-cadherin. Intriguingly, when mesenchymal stem cells (MSCs) were induced to form CAFs, Int6 levels were reduced.

Conclusion: These data suggest that besides enhancing transforming activity in epithelial cells, INT6 repression can also induce fibroblasts, and possibly MSCs as well, via mesenchymal-mesenchymal transitions to promote the formation of CAFs, leading to a proinvasive microenvironment for tumorigenesis.

No MeSH data available.


Related in: MedlinePlus