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Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers.

Strong AL, Ohlstein JF, Biagas BA, Rhodes LV, Pei DT, Tucker HA, Llamas C, Bowles AC, Dutreil MF, Zhang S, Gimble JM, Burow ME, Bunnell BA - Breast Cancer Res. (2015)

Bottom Line: BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction.Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver.These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, SL-99, New Orleans, LA, 70112, USA. alin1@tulane.edu.

ABSTRACT

Introduction: The steady increase in the incidence of obesity among adults has been paralleled with higher levels of obesity-associated breast cancer. While recent studies have suggested that adipose stromal/stem cells (ASCs) isolated from obese women enhance tumorigenicity, the mechanism(s) by which this occurs remains undefined. Evidence suggests that increased adiposity results in increased leptin secretion from adipose tissue, which has been shown to increased cancer cell proliferation. Previously, our group demonstrated that ASCs isolated from obese women (obASCs) also express higher levels of leptin relative to ASCs isolated from lean women (lnASCs) and that this obASC-derived leptin may account for enhanced breast cancer cell growth. The current study investigates the impact of inhibiting leptin expression in lnASCs and obASCs on breast cancer cell (BCC) growth and progression.

Methods: Estrogen receptor positive (ER+) BCCs were co-cultured with leptin shRNA lnASCs or leptin shRNA obASCs and changes in the proliferation, migration, invasion, and gene expression of BCCs were investigated. To assess the direct impact of leptin inhibition in obASCs on BCC proliferation, MCF7 cells were injected alone or mixed with control shRNA obASCs or leptin shRNA obASCs into SCID/beige mice.

Results: ER+ BCCs were responsive to obASCs during direct co-culture, whereas lnASCs were unable to increase ER(+) BCC growth. shRNA silencing of leptin in obASCs negated the enhanced proliferative effects of obASC on BCCs following direct co-culture. BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction. Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver. These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs.

Conclusion: This study provides mechanistic insight as to how obesity enhances the proliferation and metastasis of breast cancer cells; specifically, obASC-derived leptin contributes to the aggressiveness of breast cancer in obese women.

No MeSH data available.


Related in: MedlinePlus

Adipose stromal/stem cells isolated from obese women (obASC)-derived leptin induces SERPINE1 and matrix metalloproteinase-2 (MMP2) expression in MCF7 xenografts. MCF7 cells were prepared alone or co-injected with control short hairpin RNA (control shRNA) obASCs or leptin shRNA obASCs (1:1 ratio) into the mammary fat pad of SCID/beige mice (n = 5 mice/group). After 36 days, tumors were harvested for analysis. a Total cellular RNA was isolated from tumors and analyzed for the expression of SERPINE1, MMP-2, and IL-6. b Western blot analysis of tumor lysate. A total of 20 μg of protein was separated by SDS-PAGE under reducing conditions, blotted, and probed with indicated antibodies. Blots were stripped and probed with actin antibody for normalization. c Densitometry analysis of SERPINE1 and MMP2 bands, normalized to actin. d Representative images of SERPINE1 staining and MMP-2 staining of tumor sections visualized at × 10 and × 40 magnification (inset). Scale bar represents 50 μm. Bar represents ± SD. *P <0.05, **P <0.01, ***P <0.0001 relative to MCF7 xenografts; ΦP <0.05, ΦΦP <0.01, ΦΦΦP <0.0001 between MCF7 plus control shRNA obASC xenografts and MCF7 plus leptin shRNA obASC xenografts
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Fig6: Adipose stromal/stem cells isolated from obese women (obASC)-derived leptin induces SERPINE1 and matrix metalloproteinase-2 (MMP2) expression in MCF7 xenografts. MCF7 cells were prepared alone or co-injected with control short hairpin RNA (control shRNA) obASCs or leptin shRNA obASCs (1:1 ratio) into the mammary fat pad of SCID/beige mice (n = 5 mice/group). After 36 days, tumors were harvested for analysis. a Total cellular RNA was isolated from tumors and analyzed for the expression of SERPINE1, MMP-2, and IL-6. b Western blot analysis of tumor lysate. A total of 20 μg of protein was separated by SDS-PAGE under reducing conditions, blotted, and probed with indicated antibodies. Blots were stripped and probed with actin antibody for normalization. c Densitometry analysis of SERPINE1 and MMP2 bands, normalized to actin. d Representative images of SERPINE1 staining and MMP-2 staining of tumor sections visualized at × 10 and × 40 magnification (inset). Scale bar represents 50 μm. Bar represents ± SD. *P <0.05, **P <0.01, ***P <0.0001 relative to MCF7 xenografts; ΦP <0.05, ΦΦP <0.01, ΦΦΦP <0.0001 between MCF7 plus control shRNA obASC xenografts and MCF7 plus leptin shRNA obASC xenografts

Mentions: To assess the mechanism by which obASC-derived leptin affects BCC signaling in vivo, RNA was isolated from tumors and assessed with the custom breast cancer PCR array (Additional file 6). Assessment of the tumors showed increased expression of SERPINE1, MMP-2, and IL-6 in tumors formed with control shRNA obASCs mixed with leptin (P <0.05; Fig. 6a; Additional file 6). Inhibition of leptin in obASCs negated the gene induction of SERPINE1 and MMP-2 caused by obASCs in tumors. Western blot analysis was conducted on tumor samples to validate the expression of SERPINE1 and MMP-2 proteins. Tumors formed with MCF7 cells mixed with control shRNA obASCs demonstrated a 3.0-fold (±0.5) induction of SERPINE1 while tumors formed with MCF7 cells mixed with leptin shRNA obASCs demonstrated a 1.6-fold (±0.1) increase in SERPINE1, relative to MCF7 cells unexposed to ASCs (Fig. 6c). Likewise, tumors formed with MCF7 cells mixed with control shRNA obASCs demonstrated a 4.5-fold (±0.2) increase in MMP-2. Meanwhile, tumors formed with MCF7 cells mixed with leptin shRNA obASCs showed no difference from MCF7 cells without prior exposure to ASCs (Fig. 6c). Confirmation by immunohistochemical analysis demonstrated increased expression of SERPINE1 and MMP-2 in tumors formed with MCF7 cells and control shRNA ASCs compared to tumors formed with MCF7 cells and leptin shRNA ASCs (Fig. 6d).Fig. 6


Leptin produced by obese adipose stromal/stem cells enhances proliferation and metastasis of estrogen receptor positive breast cancers.

Strong AL, Ohlstein JF, Biagas BA, Rhodes LV, Pei DT, Tucker HA, Llamas C, Bowles AC, Dutreil MF, Zhang S, Gimble JM, Burow ME, Bunnell BA - Breast Cancer Res. (2015)

Adipose stromal/stem cells isolated from obese women (obASC)-derived leptin induces SERPINE1 and matrix metalloproteinase-2 (MMP2) expression in MCF7 xenografts. MCF7 cells were prepared alone or co-injected with control short hairpin RNA (control shRNA) obASCs or leptin shRNA obASCs (1:1 ratio) into the mammary fat pad of SCID/beige mice (n = 5 mice/group). After 36 days, tumors were harvested for analysis. a Total cellular RNA was isolated from tumors and analyzed for the expression of SERPINE1, MMP-2, and IL-6. b Western blot analysis of tumor lysate. A total of 20 μg of protein was separated by SDS-PAGE under reducing conditions, blotted, and probed with indicated antibodies. Blots were stripped and probed with actin antibody for normalization. c Densitometry analysis of SERPINE1 and MMP2 bands, normalized to actin. d Representative images of SERPINE1 staining and MMP-2 staining of tumor sections visualized at × 10 and × 40 magnification (inset). Scale bar represents 50 μm. Bar represents ± SD. *P <0.05, **P <0.01, ***P <0.0001 relative to MCF7 xenografts; ΦP <0.05, ΦΦP <0.01, ΦΦΦP <0.0001 between MCF7 plus control shRNA obASC xenografts and MCF7 plus leptin shRNA obASC xenografts
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4541745&req=5

Fig6: Adipose stromal/stem cells isolated from obese women (obASC)-derived leptin induces SERPINE1 and matrix metalloproteinase-2 (MMP2) expression in MCF7 xenografts. MCF7 cells were prepared alone or co-injected with control short hairpin RNA (control shRNA) obASCs or leptin shRNA obASCs (1:1 ratio) into the mammary fat pad of SCID/beige mice (n = 5 mice/group). After 36 days, tumors were harvested for analysis. a Total cellular RNA was isolated from tumors and analyzed for the expression of SERPINE1, MMP-2, and IL-6. b Western blot analysis of tumor lysate. A total of 20 μg of protein was separated by SDS-PAGE under reducing conditions, blotted, and probed with indicated antibodies. Blots were stripped and probed with actin antibody for normalization. c Densitometry analysis of SERPINE1 and MMP2 bands, normalized to actin. d Representative images of SERPINE1 staining and MMP-2 staining of tumor sections visualized at × 10 and × 40 magnification (inset). Scale bar represents 50 μm. Bar represents ± SD. *P <0.05, **P <0.01, ***P <0.0001 relative to MCF7 xenografts; ΦP <0.05, ΦΦP <0.01, ΦΦΦP <0.0001 between MCF7 plus control shRNA obASC xenografts and MCF7 plus leptin shRNA obASC xenografts
Mentions: To assess the mechanism by which obASC-derived leptin affects BCC signaling in vivo, RNA was isolated from tumors and assessed with the custom breast cancer PCR array (Additional file 6). Assessment of the tumors showed increased expression of SERPINE1, MMP-2, and IL-6 in tumors formed with control shRNA obASCs mixed with leptin (P <0.05; Fig. 6a; Additional file 6). Inhibition of leptin in obASCs negated the gene induction of SERPINE1 and MMP-2 caused by obASCs in tumors. Western blot analysis was conducted on tumor samples to validate the expression of SERPINE1 and MMP-2 proteins. Tumors formed with MCF7 cells mixed with control shRNA obASCs demonstrated a 3.0-fold (±0.5) induction of SERPINE1 while tumors formed with MCF7 cells mixed with leptin shRNA obASCs demonstrated a 1.6-fold (±0.1) increase in SERPINE1, relative to MCF7 cells unexposed to ASCs (Fig. 6c). Likewise, tumors formed with MCF7 cells mixed with control shRNA obASCs demonstrated a 4.5-fold (±0.2) increase in MMP-2. Meanwhile, tumors formed with MCF7 cells mixed with leptin shRNA obASCs showed no difference from MCF7 cells without prior exposure to ASCs (Fig. 6c). Confirmation by immunohistochemical analysis demonstrated increased expression of SERPINE1 and MMP-2 in tumors formed with MCF7 cells and control shRNA ASCs compared to tumors formed with MCF7 cells and leptin shRNA ASCs (Fig. 6d).Fig. 6

Bottom Line: BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction.Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver.These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell Research and Regenerative Medicine, Tulane University School of Medicine, 1430 Tulane Avenue, SL-99, New Orleans, LA, 70112, USA. alin1@tulane.edu.

ABSTRACT

Introduction: The steady increase in the incidence of obesity among adults has been paralleled with higher levels of obesity-associated breast cancer. While recent studies have suggested that adipose stromal/stem cells (ASCs) isolated from obese women enhance tumorigenicity, the mechanism(s) by which this occurs remains undefined. Evidence suggests that increased adiposity results in increased leptin secretion from adipose tissue, which has been shown to increased cancer cell proliferation. Previously, our group demonstrated that ASCs isolated from obese women (obASCs) also express higher levels of leptin relative to ASCs isolated from lean women (lnASCs) and that this obASC-derived leptin may account for enhanced breast cancer cell growth. The current study investigates the impact of inhibiting leptin expression in lnASCs and obASCs on breast cancer cell (BCC) growth and progression.

Methods: Estrogen receptor positive (ER+) BCCs were co-cultured with leptin shRNA lnASCs or leptin shRNA obASCs and changes in the proliferation, migration, invasion, and gene expression of BCCs were investigated. To assess the direct impact of leptin inhibition in obASCs on BCC proliferation, MCF7 cells were injected alone or mixed with control shRNA obASCs or leptin shRNA obASCs into SCID/beige mice.

Results: ER+ BCCs were responsive to obASCs during direct co-culture, whereas lnASCs were unable to increase ER(+) BCC growth. shRNA silencing of leptin in obASCs negated the enhanced proliferative effects of obASC on BCCs following direct co-culture. BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction. Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver. These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs.

Conclusion: This study provides mechanistic insight as to how obesity enhances the proliferation and metastasis of breast cancer cells; specifically, obASC-derived leptin contributes to the aggressiveness of breast cancer in obese women.

No MeSH data available.


Related in: MedlinePlus