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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

INT6-repressed HMFs enhance transformation phenotypes of breast cancer cells. (A) HMFs were transfected with either control siRNA or anti-INT6 siRNA. A fraction of these cells was analyzed by Western blot to confirm the reduction in Int6 and α-SMA levels (data not shown). The rest were mixed with MCF7 cells before seeding in triplicate (n = 3). The colonies in each well (a representative area from each is shown below the graph) were counted after 15 days. We note that HMFs seeded alone do not form colonies in soft agar (column 4 and column 5). To confirm that the emerged colonies are of cancer cells, we tagged MCF7 cells with mCherry and found that all colonies were enriched with mCherry-positive cells (right). The HMFs were already tagged by GFP (marked by white arrow heads) [31], but we did not detect large colonies full of GFP-positive cells. (B) MCF10AT cells were examined similarly as in panel A, except that we did not include the HMF-alone control. (C) SUM102 cells were examined similarly as in panel B. (D) MCF7 cells were loaded in an invasion chamber and submerged in conditioned medium from control or INT6-repressed HMFs. Invaded cancer cells from five different areas (n = 5) on each insert membrane were counted. (E) Normal and INT6-repressed HMFs were mixed with MCF7 cells and seeded in triplicate (n = 3) into soft agar with or without AMD3100 (500 ng/mL). Colonies were counted after 15 days. (F)INT6-repressed HMFs were co-cultured with MCF7 cells for 4 days before AMD3100 was added (500 ng/mL). After 24 hours, the α-SMA levels in HMFs were measured by Western blot. α-SMA levels, normalized by the loading control GAPDH from the control cells, were set as 1 (n = 4 separate experiments).
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Fig3: INT6-repressed HMFs enhance transformation phenotypes of breast cancer cells. (A) HMFs were transfected with either control siRNA or anti-INT6 siRNA. A fraction of these cells was analyzed by Western blot to confirm the reduction in Int6 and α-SMA levels (data not shown). The rest were mixed with MCF7 cells before seeding in triplicate (n = 3). The colonies in each well (a representative area from each is shown below the graph) were counted after 15 days. We note that HMFs seeded alone do not form colonies in soft agar (column 4 and column 5). To confirm that the emerged colonies are of cancer cells, we tagged MCF7 cells with mCherry and found that all colonies were enriched with mCherry-positive cells (right). The HMFs were already tagged by GFP (marked by white arrow heads) [31], but we did not detect large colonies full of GFP-positive cells. (B) MCF10AT cells were examined similarly as in panel A, except that we did not include the HMF-alone control. (C) SUM102 cells were examined similarly as in panel B. (D) MCF7 cells were loaded in an invasion chamber and submerged in conditioned medium from control or INT6-repressed HMFs. Invaded cancer cells from five different areas (n = 5) on each insert membrane were counted. (E) Normal and INT6-repressed HMFs were mixed with MCF7 cells and seeded in triplicate (n = 3) into soft agar with or without AMD3100 (500 ng/mL). Colonies were counted after 15 days. (F)INT6-repressed HMFs were co-cultured with MCF7 cells for 4 days before AMD3100 was added (500 ng/mL). After 24 hours, the α-SMA levels in HMFs were measured by Western blot. α-SMA levels, normalized by the loading control GAPDH from the control cells, were set as 1 (n = 4 separate experiments).

Mentions: To determine whether INT6-repressed HMFs can functionally affect transforming phenotypes of breast cancer cells, we first analyzed colony formation in soft agar with or without co-cultured HMFs. As shown in Figure 3A, while parental HMFs can weakly enhance colony formation by MCF7 cells in soft agar, when INT6 was repressed in HMFs, colony formation increased 5 fold. We have obtained similar results with a variant of MCF7 cells [21] (data not shown). To further investigate this concept, we examined two preinvasive cell lines, MCF10AT [28] (Figure 3B) and SUM102 cells [29,30] (Figure 3C), and found that INT6-repressed HMFs can also readily enhance colony formation in soft agar. Next, we analyzed cell invasiveness using a Matrigel-coated invasion chamber and found that INT6-repressed HMFs can more efficiently attract MCF7 cells across the Matrigel, suggesting that the invasiveness of cancer cells can be enhanced by INT6-repressed HMFs (Figure 3D). To determine whether SDF-1 is a key signaling molecule secreted by INT6-repressed HMFs to influence transforming activities in cancer cells, we added the SDF-1 receptor antagonist AMD3100 and found that HMF-induced colony formation in soft agar was greatly reduced (Figure 3E), with a concurrent reduction of the CAF marker α-SMA (Figure 3F). Collectively, these results demonstrate that INT6-repressed HMFs can function like CAFs to enhance transforming phenotypes of several breast cancer cell lines.Figure 3


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)

INT6-repressed HMFs enhance transformation phenotypes of breast cancer cells. (A) HMFs were transfected with either control siRNA or anti-INT6 siRNA. A fraction of these cells was analyzed by Western blot to confirm the reduction in Int6 and α-SMA levels (data not shown). The rest were mixed with MCF7 cells before seeding in triplicate (n = 3). The colonies in each well (a representative area from each is shown below the graph) were counted after 15 days. We note that HMFs seeded alone do not form colonies in soft agar (column 4 and column 5). To confirm that the emerged colonies are of cancer cells, we tagged MCF7 cells with mCherry and found that all colonies were enriched with mCherry-positive cells (right). The HMFs were already tagged by GFP (marked by white arrow heads) [31], but we did not detect large colonies full of GFP-positive cells. (B) MCF10AT cells were examined similarly as in panel A, except that we did not include the HMF-alone control. (C) SUM102 cells were examined similarly as in panel B. (D) MCF7 cells were loaded in an invasion chamber and submerged in conditioned medium from control or INT6-repressed HMFs. Invaded cancer cells from five different areas (n = 5) on each insert membrane were counted. (E) Normal and INT6-repressed HMFs were mixed with MCF7 cells and seeded in triplicate (n = 3) into soft agar with or without AMD3100 (500 ng/mL). Colonies were counted after 15 days. (F)INT6-repressed HMFs were co-cultured with MCF7 cells for 4 days before AMD3100 was added (500 ng/mL). After 24 hours, the α-SMA levels in HMFs were measured by Western blot. α-SMA levels, normalized by the loading control GAPDH from the control cells, were set as 1 (n = 4 separate experiments).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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Fig3: INT6-repressed HMFs enhance transformation phenotypes of breast cancer cells. (A) HMFs were transfected with either control siRNA or anti-INT6 siRNA. A fraction of these cells was analyzed by Western blot to confirm the reduction in Int6 and α-SMA levels (data not shown). The rest were mixed with MCF7 cells before seeding in triplicate (n = 3). The colonies in each well (a representative area from each is shown below the graph) were counted after 15 days. We note that HMFs seeded alone do not form colonies in soft agar (column 4 and column 5). To confirm that the emerged colonies are of cancer cells, we tagged MCF7 cells with mCherry and found that all colonies were enriched with mCherry-positive cells (right). The HMFs were already tagged by GFP (marked by white arrow heads) [31], but we did not detect large colonies full of GFP-positive cells. (B) MCF10AT cells were examined similarly as in panel A, except that we did not include the HMF-alone control. (C) SUM102 cells were examined similarly as in panel B. (D) MCF7 cells were loaded in an invasion chamber and submerged in conditioned medium from control or INT6-repressed HMFs. Invaded cancer cells from five different areas (n = 5) on each insert membrane were counted. (E) Normal and INT6-repressed HMFs were mixed with MCF7 cells and seeded in triplicate (n = 3) into soft agar with or without AMD3100 (500 ng/mL). Colonies were counted after 15 days. (F)INT6-repressed HMFs were co-cultured with MCF7 cells for 4 days before AMD3100 was added (500 ng/mL). After 24 hours, the α-SMA levels in HMFs were measured by Western blot. α-SMA levels, normalized by the loading control GAPDH from the control cells, were set as 1 (n = 4 separate experiments).
Mentions: To determine whether INT6-repressed HMFs can functionally affect transforming phenotypes of breast cancer cells, we first analyzed colony formation in soft agar with or without co-cultured HMFs. As shown in Figure 3A, while parental HMFs can weakly enhance colony formation by MCF7 cells in soft agar, when INT6 was repressed in HMFs, colony formation increased 5 fold. We have obtained similar results with a variant of MCF7 cells [21] (data not shown). To further investigate this concept, we examined two preinvasive cell lines, MCF10AT [28] (Figure 3B) and SUM102 cells [29,30] (Figure 3C), and found that INT6-repressed HMFs can also readily enhance colony formation in soft agar. Next, we analyzed cell invasiveness using a Matrigel-coated invasion chamber and found that INT6-repressed HMFs can more efficiently attract MCF7 cells across the Matrigel, suggesting that the invasiveness of cancer cells can be enhanced by INT6-repressed HMFs (Figure 3D). To determine whether SDF-1 is a key signaling molecule secreted by INT6-repressed HMFs to influence transforming activities in cancer cells, we added the SDF-1 receptor antagonist AMD3100 and found that HMF-induced colony formation in soft agar was greatly reduced (Figure 3E), with a concurrent reduction of the CAF marker α-SMA (Figure 3F). Collectively, these results demonstrate that INT6-repressed HMFs can function like CAFs to enhance transforming phenotypes of several breast cancer cell lines.Figure 3

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