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Physical Intimacy of Breast Cancer Cells with Mesenchymal Stem Cells Elicits Trastuzumab Resistance through Src Activation.

Daverey A, Drain AP, Kidambi S - Sci Rep (2015)

Bottom Line: The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors.To our knowledge, this is the first report that showed PTEN loss without the use of chemical inhibitors, matrix stiffness, or silencing RNAs.Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, NE, 68588.

ABSTRACT
The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors. Here, we demonstrate that the physical contact of breast cancer cells with mesenchymal stem cells (MSCs) is a potential modulator of trastuzumab response by activation of nonreceptor tyrosine kinase c-Src and down regulation of phosphatase and tensin homolog (PTEN). Using an in vitro patterned breast cancer/MSC co-culture model, we find that the presence of MSCs results in Src activation that is missing in cancer cells monoculture, transwell co-culture, and cells treated with MSCs conditioned media. Interestingly, the co-culture model also results in PTEN loss and activation of PI3K/AKT pathway that has been demonstrated as fundamental proliferative and survival pathways in clinical settings. To our knowledge, this is the first report that showed PTEN loss without the use of chemical inhibitors, matrix stiffness, or silencing RNAs. In addition, breast cancer cells in co-culture with MSCs conferred trastuzumab resistance in vitro as observed in the lack of inhibition of proliferative and migrative properties of the cancer cells. Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients.

No MeSH data available.


Related in: MedlinePlus

MSCs activates pathways regulating tumor growth and trastuzumab resistance.(a) Left, Immunostaining of HER-2 (Texas red) to assess the expression of HER-2 in patterned co-culture. Right, quantification of fluorescence intensity of HER-2 expression using imageJ. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture; ***p < 0.001 compared with monoculture. (b) Top, immunoblots of HER-2 expression in BT-474 and 21MT-1 cells sorted after co-culture with MSCs, and after exposed to MSCs-CM. Bottom, respective densitometry of HER-2 expression normalized with monoculture after GAPDH correction. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture. (c) Top, immunoblots of PTEN expression in BT-474 and 21MT-1 cells sorted after co-culture with MSCs, transwell co-culture and after exposed to MSCs-CM. Bottom, respective densitometry of PTEN expression normalized with monoculture after GAPDH correction. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture. (d) Top, western blot analysis of PI3K, pAKT and AKT. Bottom, respective densitometry of bands normalized with monoculture after loading control (GAPDH) correction. Expression of pAKT was further normalized with AKT (pAKT/AKT). Data are mean ± SD; n = 3 independent experiments; *p < 0.05 compared with monoculture; **p < 0.01 compared with monoculture.
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f4: MSCs activates pathways regulating tumor growth and trastuzumab resistance.(a) Left, Immunostaining of HER-2 (Texas red) to assess the expression of HER-2 in patterned co-culture. Right, quantification of fluorescence intensity of HER-2 expression using imageJ. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture; ***p < 0.001 compared with monoculture. (b) Top, immunoblots of HER-2 expression in BT-474 and 21MT-1 cells sorted after co-culture with MSCs, and after exposed to MSCs-CM. Bottom, respective densitometry of HER-2 expression normalized with monoculture after GAPDH correction. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture. (c) Top, immunoblots of PTEN expression in BT-474 and 21MT-1 cells sorted after co-culture with MSCs, transwell co-culture and after exposed to MSCs-CM. Bottom, respective densitometry of PTEN expression normalized with monoculture after GAPDH correction. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture. (d) Top, western blot analysis of PI3K, pAKT and AKT. Bottom, respective densitometry of bands normalized with monoculture after loading control (GAPDH) correction. Expression of pAKT was further normalized with AKT (pAKT/AKT). Data are mean ± SD; n = 3 independent experiments; *p < 0.05 compared with monoculture; **p < 0.01 compared with monoculture.

Mentions: Src activation has been demonstrated to play a direct role in the progression of aggressive HER-2 positive breast cancer67. To explore the effect of MSCs on tumor growth, we examined whether MSCs in contact with tumor cells has any effect on HER-2 expression, an important biomarker and indicator of tumor growth and progression12. Immunostaining of HER-2 demonstrated increased fluorescence in patterned co-culture as compared to the monoculture and cancer cells exposed to MSC conditioned media (Fig. 4A and Supplementary Fig. 4). Quantification of fluorescence intensity indicated higher HER-2 expression in breast cancer cells in co-culture compared to both monoculture and breast cancer cells exposed to MSCs-CM. To further confirm that MSCs mediated Src activation regulate HER-2, we examined the protein expression in breast cancer cells after co-culture using western blotting (Fig. 4B). A notably increased HER-2 expression in both BT-474 and 21MT-1 cells were observed in co-culture with MSCs compared to monoculture and MSCs-CM treated cells. Also, upregulation of HER-2 expression was more prominent in 21MT-1 cells, which are derived from metastatic cancer patients, suggesting the potential role of MSCs in more aggressive tumors.


Physical Intimacy of Breast Cancer Cells with Mesenchymal Stem Cells Elicits Trastuzumab Resistance through Src Activation.

Daverey A, Drain AP, Kidambi S - Sci Rep (2015)

MSCs activates pathways regulating tumor growth and trastuzumab resistance.(a) Left, Immunostaining of HER-2 (Texas red) to assess the expression of HER-2 in patterned co-culture. Right, quantification of fluorescence intensity of HER-2 expression using imageJ. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture; ***p < 0.001 compared with monoculture. (b) Top, immunoblots of HER-2 expression in BT-474 and 21MT-1 cells sorted after co-culture with MSCs, and after exposed to MSCs-CM. Bottom, respective densitometry of HER-2 expression normalized with monoculture after GAPDH correction. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture. (c) Top, immunoblots of PTEN expression in BT-474 and 21MT-1 cells sorted after co-culture with MSCs, transwell co-culture and after exposed to MSCs-CM. Bottom, respective densitometry of PTEN expression normalized with monoculture after GAPDH correction. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture. (d) Top, western blot analysis of PI3K, pAKT and AKT. Bottom, respective densitometry of bands normalized with monoculture after loading control (GAPDH) correction. Expression of pAKT was further normalized with AKT (pAKT/AKT). Data are mean ± SD; n = 3 independent experiments; *p < 0.05 compared with monoculture; **p < 0.01 compared with monoculture.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4561910&req=5

f4: MSCs activates pathways regulating tumor growth and trastuzumab resistance.(a) Left, Immunostaining of HER-2 (Texas red) to assess the expression of HER-2 in patterned co-culture. Right, quantification of fluorescence intensity of HER-2 expression using imageJ. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture; ***p < 0.001 compared with monoculture. (b) Top, immunoblots of HER-2 expression in BT-474 and 21MT-1 cells sorted after co-culture with MSCs, and after exposed to MSCs-CM. Bottom, respective densitometry of HER-2 expression normalized with monoculture after GAPDH correction. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture. (c) Top, immunoblots of PTEN expression in BT-474 and 21MT-1 cells sorted after co-culture with MSCs, transwell co-culture and after exposed to MSCs-CM. Bottom, respective densitometry of PTEN expression normalized with monoculture after GAPDH correction. Mean ± SD; n = 3 independent experiments; **p < 0.01 compared with monoculture. (d) Top, western blot analysis of PI3K, pAKT and AKT. Bottom, respective densitometry of bands normalized with monoculture after loading control (GAPDH) correction. Expression of pAKT was further normalized with AKT (pAKT/AKT). Data are mean ± SD; n = 3 independent experiments; *p < 0.05 compared with monoculture; **p < 0.01 compared with monoculture.
Mentions: Src activation has been demonstrated to play a direct role in the progression of aggressive HER-2 positive breast cancer67. To explore the effect of MSCs on tumor growth, we examined whether MSCs in contact with tumor cells has any effect on HER-2 expression, an important biomarker and indicator of tumor growth and progression12. Immunostaining of HER-2 demonstrated increased fluorescence in patterned co-culture as compared to the monoculture and cancer cells exposed to MSC conditioned media (Fig. 4A and Supplementary Fig. 4). Quantification of fluorescence intensity indicated higher HER-2 expression in breast cancer cells in co-culture compared to both monoculture and breast cancer cells exposed to MSCs-CM. To further confirm that MSCs mediated Src activation regulate HER-2, we examined the protein expression in breast cancer cells after co-culture using western blotting (Fig. 4B). A notably increased HER-2 expression in both BT-474 and 21MT-1 cells were observed in co-culture with MSCs compared to monoculture and MSCs-CM treated cells. Also, upregulation of HER-2 expression was more prominent in 21MT-1 cells, which are derived from metastatic cancer patients, suggesting the potential role of MSCs in more aggressive tumors.

Bottom Line: The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors.To our knowledge, this is the first report that showed PTEN loss without the use of chemical inhibitors, matrix stiffness, or silencing RNAs.Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, NE, 68588.

ABSTRACT
The development of resistance to trastuzumab is a major obstacle for lasting effective treatment of patients with ErbB2-overexpressing tumors. Here, we demonstrate that the physical contact of breast cancer cells with mesenchymal stem cells (MSCs) is a potential modulator of trastuzumab response by activation of nonreceptor tyrosine kinase c-Src and down regulation of phosphatase and tensin homolog (PTEN). Using an in vitro patterned breast cancer/MSC co-culture model, we find that the presence of MSCs results in Src activation that is missing in cancer cells monoculture, transwell co-culture, and cells treated with MSCs conditioned media. Interestingly, the co-culture model also results in PTEN loss and activation of PI3K/AKT pathway that has been demonstrated as fundamental proliferative and survival pathways in clinical settings. To our knowledge, this is the first report that showed PTEN loss without the use of chemical inhibitors, matrix stiffness, or silencing RNAs. In addition, breast cancer cells in co-culture with MSCs conferred trastuzumab resistance in vitro as observed in the lack of inhibition of proliferative and migrative properties of the cancer cells. Our findings show that MSCs are potent mediators of resistance to trastuzumab and might reveal targets to enhance trastuzumab efficacy in patients.

No MeSH data available.


Related in: MedlinePlus