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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 increases invasion of breast cancer cells (BCCs). BCC cells were co-cultured (CC) with adipose stromal/stem cells isolated from lean women (lnASCs) or obASCs stably transfected with a control short hairpin RNA (control shRNA) or leptin shRNA (leptin shRNA) for 7 days. Green fluorescent protein-positive BCC cells were isolated with FACS and plated in the top chamber of a, an uncoated 8-μm pore membrane, and assessed after 4 hours for migratory potential or b, a Matrigel-coated 8-μm pore membrane and assessed after 24 hours for invasive capacity. Medium containing FBS was plated in the lower chamber and served as a chemoattractant. Data are shown relative to cells without prior exposure to lnASCs or obASCs. Bar represents ± standard error of the mean: *P <0.05, ***P <0.001 relative to BCCs cultured alone; ##P <0.01 for comparison between BCCs after co-culture with control shRNA lnASCs and control shRNA obASCs; ΦP <0.05, ΦΦP <0.01 between BCCs after co-culture with control shRNA obASCs and letpin shRNA obASCs
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Fig3: Adipose stromal/stem cells isolated from obese women (obASC)-derived leptin increases invasion of breast cancer cells (BCCs). BCC cells were co-cultured (CC) with adipose stromal/stem cells isolated from lean women (lnASCs) or obASCs stably transfected with a control short hairpin RNA (control shRNA) or leptin shRNA (leptin shRNA) for 7 days. Green fluorescent protein-positive BCC cells were isolated with FACS and plated in the top chamber of a, an uncoated 8-μm pore membrane, and assessed after 4 hours for migratory potential or b, a Matrigel-coated 8-μm pore membrane and assessed after 24 hours for invasive capacity. Medium containing FBS was plated in the lower chamber and served as a chemoattractant. Data are shown relative to cells without prior exposure to lnASCs or obASCs. Bar represents ± standard error of the mean: *P <0.05, ***P <0.001 relative to BCCs cultured alone; ##P <0.01 for comparison between BCCs after co-culture with control shRNA lnASCs and control shRNA obASCs; ΦP <0.05, ΦΦP <0.01 between BCCs after co-culture with control shRNA obASCs and letpin shRNA obASCs

Mentions: BCCs were directly co-cultured for 7 days with lnASCs or obASCs transfected with control shRNA or leptin shRNA and collected by detection of GFP expression using FACS. Sorted cells were re-suspended in serum-free medium and plated on top of a transwell and assessed for migration after 4 hours or plated on top of a Matrigel-coated transwell and assessed for invasion after 24 hours. The BCCs collected after co-culture demonstrated enhanced migration towards chemoattractants by at least 2.7-fold relative to non-co-cultured BCCs, following direct co-culture of BCCs with lnASCs or obASCs (Fig. 3a). Silencing of leptin slightly diminished the capacity of both lnASCs and obASCs, to affect BCC migration; however, these results were not statistically significant (Fig. 3a). The invasive capacity of BCCs was enhanced after direct exposure to lnASCs and obASCs. The impact of obASCs on BCC invasion was more robust than the effect of lnASC on BCC invasion. lnASCs enhanced the invasion of MCF7, ZR75, and T47D cells by 4.1 ± 1.1, 3.9 ± 0.8, and 3.8 ± 1.0 times, respectively, while obASCs increased the invasive potential of MCF7, ZR75, and T47D cells by 10.2±2.3, 9.2±1.1, and 11.0±1.2 times, respectively (P <0.05; Fig. 3b). Inhibiting leptin expression in obASCs significantly decreased the levels of BCC invasion, while reducing leptin expression in lnASCs did not have the same impact. Leptin inhibition in obASCs reduced MCF7, ZR75, and T47D cell invasion by 27.5 %, 29.3 %, and 28.1 %, respectively (P <0.05; Fig. 3b). These results suggest that while obASCs have a greater capacity to enhance the migration and invasion of BCCs compared to lnASCs, obASC-derived leptin only plays an integral role in BCC invasion.Fig. 3


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 increases invasion of breast cancer cells (BCCs). BCC cells were co-cultured (CC) with adipose stromal/stem cells isolated from lean women (lnASCs) or obASCs stably transfected with a control short hairpin RNA (control shRNA) or leptin shRNA (leptin shRNA) for 7 days. Green fluorescent protein-positive BCC cells were isolated with FACS and plated in the top chamber of a, an uncoated 8-μm pore membrane, and assessed after 4 hours for migratory potential or b, a Matrigel-coated 8-μm pore membrane and assessed after 24 hours for invasive capacity. Medium containing FBS was plated in the lower chamber and served as a chemoattractant. Data are shown relative to cells without prior exposure to lnASCs or obASCs. Bar represents ± standard error of the mean: *P <0.05, ***P <0.001 relative to BCCs cultured alone; ##P <0.01 for comparison between BCCs after co-culture with control shRNA lnASCs and control shRNA obASCs; ΦP <0.05, ΦΦP <0.01 between BCCs after co-culture with control shRNA obASCs and letpin shRNA obASCs
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Adipose stromal/stem cells isolated from obese women (obASC)-derived leptin increases invasion of breast cancer cells (BCCs). BCC cells were co-cultured (CC) with adipose stromal/stem cells isolated from lean women (lnASCs) or obASCs stably transfected with a control short hairpin RNA (control shRNA) or leptin shRNA (leptin shRNA) for 7 days. Green fluorescent protein-positive BCC cells were isolated with FACS and plated in the top chamber of a, an uncoated 8-μm pore membrane, and assessed after 4 hours for migratory potential or b, a Matrigel-coated 8-μm pore membrane and assessed after 24 hours for invasive capacity. Medium containing FBS was plated in the lower chamber and served as a chemoattractant. Data are shown relative to cells without prior exposure to lnASCs or obASCs. Bar represents ± standard error of the mean: *P <0.05, ***P <0.001 relative to BCCs cultured alone; ##P <0.01 for comparison between BCCs after co-culture with control shRNA lnASCs and control shRNA obASCs; ΦP <0.05, ΦΦP <0.01 between BCCs after co-culture with control shRNA obASCs and letpin shRNA obASCs
Mentions: BCCs were directly co-cultured for 7 days with lnASCs or obASCs transfected with control shRNA or leptin shRNA and collected by detection of GFP expression using FACS. Sorted cells were re-suspended in serum-free medium and plated on top of a transwell and assessed for migration after 4 hours or plated on top of a Matrigel-coated transwell and assessed for invasion after 24 hours. The BCCs collected after co-culture demonstrated enhanced migration towards chemoattractants by at least 2.7-fold relative to non-co-cultured BCCs, following direct co-culture of BCCs with lnASCs or obASCs (Fig. 3a). Silencing of leptin slightly diminished the capacity of both lnASCs and obASCs, to affect BCC migration; however, these results were not statistically significant (Fig. 3a). The invasive capacity of BCCs was enhanced after direct exposure to lnASCs and obASCs. The impact of obASCs on BCC invasion was more robust than the effect of lnASC on BCC invasion. lnASCs enhanced the invasion of MCF7, ZR75, and T47D cells by 4.1 ± 1.1, 3.9 ± 0.8, and 3.8 ± 1.0 times, respectively, while obASCs increased the invasive potential of MCF7, ZR75, and T47D cells by 10.2±2.3, 9.2±1.1, and 11.0±1.2 times, respectively (P <0.05; Fig. 3b). Inhibiting leptin expression in obASCs significantly decreased the levels of BCC invasion, while reducing leptin expression in lnASCs did not have the same impact. Leptin inhibition in obASCs reduced MCF7, ZR75, and T47D cell invasion by 27.5 %, 29.3 %, and 28.1 %, respectively (P <0.05; Fig. 3b). These results suggest that while obASCs have a greater capacity to enhance the migration and invasion of BCCs compared to lnASCs, obASC-derived leptin only plays an integral role in BCC invasion.Fig. 3

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