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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 induces CAF-like properties in normal human mammary fibroblasts. (A) HMFs were transfected with control or anti-INT6 siRNA. On the left, protein samples were analyzed over time by Western blots using antibodies against α-SMA and Int6. GAPDH was the loading control. On the right, a quantification of Western blots from three separate experiments was graphed. (B) HMFs were treated with control or anti-INT6 siRNA for 5 days and then immunostained with antibodies against α-SMA (red) and tubulins (green), and counter-stained by DAPI (blue) to mark the nuclei. Cells containing α-SMA cables were counted (n = 100 cells). White arrow indicates a typical α-SMA cable in a cell. Quantification of cells containing α-SMA cables is shown on the right. (C) On the left, semiquantitative RT-PCR was performed to measure CXCL12 (encoding SDF-1) mRNA levels in parental and Int6-repressed HMFs. CXCL12 mRNA levels were normalized to those of ACTB, and the normalized CXCL12 mRNA levels in the control were set to 1. (D) The growth media of the cells from panel C were analyzed by ELISA to measure SDF-1 levels 7 days after seeding. SDF-1 levels in the control were set to 1 (n = 3 separate experiments).
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Fig2: Reduction of Int6 induces CAF-like properties in normal human mammary fibroblasts. (A) HMFs were transfected with control or anti-INT6 siRNA. On the left, protein samples were analyzed over time by Western blots using antibodies against α-SMA and Int6. GAPDH was the loading control. On the right, a quantification of Western blots from three separate experiments was graphed. (B) HMFs were treated with control or anti-INT6 siRNA for 5 days and then immunostained with antibodies against α-SMA (red) and tubulins (green), and counter-stained by DAPI (blue) to mark the nuclei. Cells containing α-SMA cables were counted (n = 100 cells). White arrow indicates a typical α-SMA cable in a cell. Quantification of cells containing α-SMA cables is shown on the right. (C) On the left, semiquantitative RT-PCR was performed to measure CXCL12 (encoding SDF-1) mRNA levels in parental and Int6-repressed HMFs. CXCL12 mRNA levels were normalized to those of ACTB, and the normalized CXCL12 mRNA levels in the control were set to 1. (D) The growth media of the cells from panel C were analyzed by ELISA to measure SDF-1 levels 7 days after seeding. SDF-1 levels in the control were set to 1 (n = 3 separate experiments).

Mentions: To investigate whether Int6 reduction in fibroblasts can induce CAF-like activities, we repressed INT6 expression using siRNA in h-TERT-immortalized normal human mammary fibroblasts (HMFs) and then measured a key CAF marker, α-SMA. As shown in Figure 2A, repressing INT6 steadily increased α-SMA levels over a 5-day period after gene silencing. To determine whether the increase in α-SMA levels resulted in more efficient formation of α-SMA cables in the cells, we performed immunostaining. As shown in Figure 2B, greater than 2.5 times more INT6-repressed HMFs contained α-SMA cables.Figure 2


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 induces CAF-like properties in normal human mammary fibroblasts. (A) HMFs were transfected with control or anti-INT6 siRNA. On the left, protein samples were analyzed over time by Western blots using antibodies against α-SMA and Int6. GAPDH was the loading control. On the right, a quantification of Western blots from three separate experiments was graphed. (B) HMFs were treated with control or anti-INT6 siRNA for 5 days and then immunostained with antibodies against α-SMA (red) and tubulins (green), and counter-stained by DAPI (blue) to mark the nuclei. Cells containing α-SMA cables were counted (n = 100 cells). White arrow indicates a typical α-SMA cable in a cell. Quantification of cells containing α-SMA cables is shown on the right. (C) On the left, semiquantitative RT-PCR was performed to measure CXCL12 (encoding SDF-1) mRNA levels in parental and Int6-repressed HMFs. CXCL12 mRNA levels were normalized to those of ACTB, and the normalized CXCL12 mRNA levels in the control were set to 1. (D) The growth media of the cells from panel C were analyzed by ELISA to measure SDF-1 levels 7 days after seeding. SDF-1 levels in the control were set to 1 (n = 3 separate experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Reduction of Int6 induces CAF-like properties in normal human mammary fibroblasts. (A) HMFs were transfected with control or anti-INT6 siRNA. On the left, protein samples were analyzed over time by Western blots using antibodies against α-SMA and Int6. GAPDH was the loading control. On the right, a quantification of Western blots from three separate experiments was graphed. (B) HMFs were treated with control or anti-INT6 siRNA for 5 days and then immunostained with antibodies against α-SMA (red) and tubulins (green), and counter-stained by DAPI (blue) to mark the nuclei. Cells containing α-SMA cables were counted (n = 100 cells). White arrow indicates a typical α-SMA cable in a cell. Quantification of cells containing α-SMA cables is shown on the right. (C) On the left, semiquantitative RT-PCR was performed to measure CXCL12 (encoding SDF-1) mRNA levels in parental and Int6-repressed HMFs. CXCL12 mRNA levels were normalized to those of ACTB, and the normalized CXCL12 mRNA levels in the control were set to 1. (D) The growth media of the cells from panel C were analyzed by ELISA to measure SDF-1 levels 7 days after seeding. SDF-1 levels in the control were set to 1 (n = 3 separate experiments).
Mentions: To investigate whether Int6 reduction in fibroblasts can induce CAF-like activities, we repressed INT6 expression using siRNA in h-TERT-immortalized normal human mammary fibroblasts (HMFs) and then measured a key CAF marker, α-SMA. As shown in Figure 2A, repressing INT6 steadily increased α-SMA levels over a 5-day period after gene silencing. To determine whether the increase in α-SMA levels resulted in more efficient formation of α-SMA cables in the cells, we performed immunostaining. As shown in Figure 2B, greater than 2.5 times more INT6-repressed HMFs contained α-SMA cables.Figure 2

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