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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 (ASCs) isolated from obese women (obASCs) enhance breast cancer cell (BCC) proliferation. a Estrogen receptor positive BCCs were co-cultured (CC) with ASCs isolated from lean women (lnASCs) (n = 6 donors) or obASCs (n = 6 donors) for 7 days and FACS performed based on green fluorescent protein (GFP) expression. BCCs sorted by FACS were counted and data are shown relative to the number of cells cultured alone. b RNA was collected from MCF7 cells, T47D cells, ZR75 cells, control short hairpin RNA (control shRNA) lnASCs, and control shRNA obASCs and analyzed for aromatase expression by quantitative RT-PCR. Data were normalized to the β-actin expression. c Conditioned media (CM) were collected from control shRNA lnASCs or control shRNA obASCs treated with vehicle or letrozole. BCCs were cultured in the CM for 7 days and the number of GFP+ BCCs was counted. d CM were collected from control shRNA lnASCs and control shRNA obASCs treated with vehicle or leptin neutralizing antibody. BCCs were cultured in the CM for 7 days and the number of GFP+ BCCs was counted. Bar represents ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001, relative to no co-culture BCCs; #P <0.05, ##P <0.01 between BCCs co-cultured with lnASCs and obASCs; ψψψP <0.001 relative to lnASCs and obASCs; ΦΦΦP <0.001 between control shRNA obASCs treated with vehicle and leptin neutralizing antibody
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Fig1: Adipose stromal/stem cells (ASCs) isolated from obese women (obASCs) enhance breast cancer cell (BCC) proliferation. a Estrogen receptor positive BCCs were co-cultured (CC) with ASCs isolated from lean women (lnASCs) (n = 6 donors) or obASCs (n = 6 donors) for 7 days and FACS performed based on green fluorescent protein (GFP) expression. BCCs sorted by FACS were counted and data are shown relative to the number of cells cultured alone. b RNA was collected from MCF7 cells, T47D cells, ZR75 cells, control short hairpin RNA (control shRNA) lnASCs, and control shRNA obASCs and analyzed for aromatase expression by quantitative RT-PCR. Data were normalized to the β-actin expression. c Conditioned media (CM) were collected from control shRNA lnASCs or control shRNA obASCs treated with vehicle or letrozole. BCCs were cultured in the CM for 7 days and the number of GFP+ BCCs was counted. d CM were collected from control shRNA lnASCs and control shRNA obASCs treated with vehicle or leptin neutralizing antibody. BCCs were cultured in the CM for 7 days and the number of GFP+ BCCs was counted. Bar represents ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001, relative to no co-culture BCCs; #P <0.05, ##P <0.01 between BCCs co-cultured with lnASCs and obASCs; ψψψP <0.001 relative to lnASCs and obASCs; ΦΦΦP <0.001 between control shRNA obASCs treated with vehicle and leptin neutralizing antibody

Mentions: The impact of ASCs on BCC growth was investigated using a co-culture assay. BCC lines MCF7, ZR75, and T47D were cultured alone or at a 1:1 ratio with either lnASCs or obASCs for 7 days. Prior to co-culture, BCCs were transduced with lentivirus expressing GFP in order to isolate BCCs expressing the fluorochrome following the co-culture period. obASCs increased the proliferation of ER+ BCCs to 2.0 ± 0.2 fold in MCF7 cells, 2.2 ± 0.1 fold in ZR75, and 1.9 ± 0.1 fold in T47D (P <0.05; Fig. 1a). While lnASCs increased the proliferation of ER+ BCCs, the effect was not as robust and was not statistically significant (Fig. 1a). These results suggest that ASCs increase the proliferation of all BCCs; however, obASCs markedly increased the proliferation of ER+ BCCs over lnASCs.Fig. 1


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 (ASCs) isolated from obese women (obASCs) enhance breast cancer cell (BCC) proliferation. a Estrogen receptor positive BCCs were co-cultured (CC) with ASCs isolated from lean women (lnASCs) (n = 6 donors) or obASCs (n = 6 donors) for 7 days and FACS performed based on green fluorescent protein (GFP) expression. BCCs sorted by FACS were counted and data are shown relative to the number of cells cultured alone. b RNA was collected from MCF7 cells, T47D cells, ZR75 cells, control short hairpin RNA (control shRNA) lnASCs, and control shRNA obASCs and analyzed for aromatase expression by quantitative RT-PCR. Data were normalized to the β-actin expression. c Conditioned media (CM) were collected from control shRNA lnASCs or control shRNA obASCs treated with vehicle or letrozole. BCCs were cultured in the CM for 7 days and the number of GFP+ BCCs was counted. d CM were collected from control shRNA lnASCs and control shRNA obASCs treated with vehicle or leptin neutralizing antibody. BCCs were cultured in the CM for 7 days and the number of GFP+ BCCs was counted. Bar represents ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001, relative to no co-culture BCCs; #P <0.05, ##P <0.01 between BCCs co-cultured with lnASCs and obASCs; ψψψP <0.001 relative to lnASCs and obASCs; ΦΦΦP <0.001 between control shRNA obASCs treated with vehicle and leptin neutralizing antibody
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Adipose stromal/stem cells (ASCs) isolated from obese women (obASCs) enhance breast cancer cell (BCC) proliferation. a Estrogen receptor positive BCCs were co-cultured (CC) with ASCs isolated from lean women (lnASCs) (n = 6 donors) or obASCs (n = 6 donors) for 7 days and FACS performed based on green fluorescent protein (GFP) expression. BCCs sorted by FACS were counted and data are shown relative to the number of cells cultured alone. b RNA was collected from MCF7 cells, T47D cells, ZR75 cells, control short hairpin RNA (control shRNA) lnASCs, and control shRNA obASCs and analyzed for aromatase expression by quantitative RT-PCR. Data were normalized to the β-actin expression. c Conditioned media (CM) were collected from control shRNA lnASCs or control shRNA obASCs treated with vehicle or letrozole. BCCs were cultured in the CM for 7 days and the number of GFP+ BCCs was counted. d CM were collected from control shRNA lnASCs and control shRNA obASCs treated with vehicle or leptin neutralizing antibody. BCCs were cultured in the CM for 7 days and the number of GFP+ BCCs was counted. Bar represents ± standard error of the mean. *P <0.05, **P <0.01, ***P <0.001, relative to no co-culture BCCs; #P <0.05, ##P <0.01 between BCCs co-cultured with lnASCs and obASCs; ψψψP <0.001 relative to lnASCs and obASCs; ΦΦΦP <0.001 between control shRNA obASCs treated with vehicle and leptin neutralizing antibody
Mentions: The impact of ASCs on BCC growth was investigated using a co-culture assay. BCC lines MCF7, ZR75, and T47D were cultured alone or at a 1:1 ratio with either lnASCs or obASCs for 7 days. Prior to co-culture, BCCs were transduced with lentivirus expressing GFP in order to isolate BCCs expressing the fluorochrome following the co-culture period. obASCs increased the proliferation of ER+ BCCs to 2.0 ± 0.2 fold in MCF7 cells, 2.2 ± 0.1 fold in ZR75, and 1.9 ± 0.1 fold in T47D (P <0.05; Fig. 1a). While lnASCs increased the proliferation of ER+ BCCs, the effect was not as robust and was not statistically significant (Fig. 1a). These results suggest that ASCs increase the proliferation of all BCCs; however, obASCs markedly increased the proliferation of ER+ BCCs over lnASCs.Fig. 1

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