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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

INT6repression may induce MMT. (A) HMFs were transfected with control or anti-INT6 siRNA for 3 days, and their lysates were analyzed by Western blots to detect the indicated proteins. Shown here is a representative experiment in which levels of mesenchymal markers increased when INT6 was silenced. Protein levels normalized to GAPDH in the control cells were set to 1. (B) Human MSCs were incubated in conditioned medium from MDA-MD-231 cells for 10 days before they were analyzed by Western blot to detect α-SMA and Int6 as in panel A. (C) Protein levels of α-SMA and Int6 in control or INT6-repressed MSCs were measured by Western blot over time (left). The data from day 5 are shown as an example (right).
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Fig4: INT6repression may induce MMT. (A) HMFs were transfected with control or anti-INT6 siRNA for 3 days, and their lysates were analyzed by Western blots to detect the indicated proteins. Shown here is a representative experiment in which levels of mesenchymal markers increased when INT6 was silenced. Protein levels normalized to GAPDH in the control cells were set to 1. (B) Human MSCs were incubated in conditioned medium from MDA-MD-231 cells for 10 days before they were analyzed by Western blot to detect α-SMA and Int6 as in panel A. (C) Protein levels of α-SMA and Int6 in control or INT6-repressed MSCs were measured by Western blot over time (left). The data from day 5 are shown as an example (right).

Mentions: The mechanism(s) by which CAFs are generated are poorly understood. However, Int6 reduction has been shown to induce EMT [15], suggesting that Int6 reduction may induce more mesenchymal traits in the cell. We thus investigated whether INT6 repression promotes CAF-like properties by inducing mesenchymal traits in HMFs. As shown in Figure 4A, when INT6 expression was silenced, protein levels of two mesenchymal markers, vimentin and N-cadherin, showed an average increase of 1.5 and 2.5 times respectively, in two experiments.Figure 4


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)

INT6repression may induce MMT. (A) HMFs were transfected with control or anti-INT6 siRNA for 3 days, and their lysates were analyzed by Western blots to detect the indicated proteins. Shown here is a representative experiment in which levels of mesenchymal markers increased when INT6 was silenced. Protein levels normalized to GAPDH in the control cells were set to 1. (B) Human MSCs were incubated in conditioned medium from MDA-MD-231 cells for 10 days before they were analyzed by Western blot to detect α-SMA and Int6 as in panel A. (C) Protein levels of α-SMA and Int6 in control or INT6-repressed MSCs were measured by Western blot over time (left). The data from day 5 are shown as an example (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: INT6repression may induce MMT. (A) HMFs were transfected with control or anti-INT6 siRNA for 3 days, and their lysates were analyzed by Western blots to detect the indicated proteins. Shown here is a representative experiment in which levels of mesenchymal markers increased when INT6 was silenced. Protein levels normalized to GAPDH in the control cells were set to 1. (B) Human MSCs were incubated in conditioned medium from MDA-MD-231 cells for 10 days before they were analyzed by Western blot to detect α-SMA and Int6 as in panel A. (C) Protein levels of α-SMA and Int6 in control or INT6-repressed MSCs were measured by Western blot over time (left). The data from day 5 are shown as an example (right).
Mentions: The mechanism(s) by which CAFs are generated are poorly understood. However, Int6 reduction has been shown to induce EMT [15], suggesting that Int6 reduction may induce more mesenchymal traits in the cell. We thus investigated whether INT6 repression promotes CAF-like properties by inducing mesenchymal traits in HMFs. As shown in Figure 4A, when INT6 expression was silenced, protein levels of two mesenchymal markers, vimentin and N-cadherin, showed an average increase of 1.5 and 2.5 times respectively, in two experiments.Figure 4

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