Limits...
Cancer-associated fibroblasts induce high mobility group box 1 and contribute to resistance to doxorubicin in breast cancer cells.

Amornsupak K, Insawang T, Thuwajit P, O-Charoenrat P, Eccles SA, Thuwajit C - BMC Cancer (2014)

Bottom Line: Cancer-associated fibroblasts and high mobility group box 1 (HMGB1) protein have been suggested to mediate cancer progression and chemotherapy resistance.Extracellular HMGB1 was strongly expressed in the CM after Dox-induced MDA-MB-231 cell death and was higher in cells pre-treated with BCF-CM than NTF-CM.Recombinant HMGB1 was shown to increase Dox resistance and this was associated with evidence of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. cthuwajit@yahoo.com.

ABSTRACT

Background: Cancer-associated fibroblasts and high mobility group box 1 (HMGB1) protein have been suggested to mediate cancer progression and chemotherapy resistance. The role of such fibroblasts in HMGB1 production in breast cancer is unclear. This study aimed to investigate the effects of cancer-associated fibroblasts on HMGB1 expression in breast cancer cells and its role in chemotherapeutic response.

Methods: Breast cancer-associated fibroblasts (BCFs) and non-tumor-associated fibroblasts (NTFs) were isolated from human breast cancers or adjacent normal tissues and established as primary cultures in vitro. After confirmation of the activated status of these fibroblasts, conditioned-media (CM) were collected and applied to MDA-MB-231 human triple negative breast cancer cells. The levels of intracellular and extracellular HMGB1 were measured by real-time PCR and/or Western blot. The response of BCF-CM-pre-treated cancer cells to doxorubicin (Dox) was compared with those pre-treated with NTF-CM or control cultures. The effect of an HMGB1 neutralizing antibody on Dox resistance induced by extracellular HMGB1 from non-viable Dox-treated cancer cells or recombinant HMGB1 was also investigated.

Results: Immunocytochemical analysis revealed that BCFs and NTFs were alpha-smooth muscle actin (ASMA) positive and cytokeratin 19 (CK19) negative cells: a phenotype consistent with that of activated fibroblasts. We confirmed that the CM from BCFs (but not NTFs), could significantly induce breast cancer cell migration. Intracellular HMGB1 expression was induced in BCF-CM-treated breast cancer cells and also in Dox-treated cells. Extracellular HMGB1 was strongly expressed in the CM after Dox-induced MDA-MB-231 cell death and was higher in cells pre-treated with BCF-CM than NTF-CM. Pre-treatment of breast cancer cells with BCF-CM induced a degree of resistance to Dox in accordance with the increased level of secreted HMGB1. Recombinant HMGB1 was shown to increase Dox resistance and this was associated with evidence of autophagy. Anti-HMGB1 neutralizing antibody significantly reduced the effect of extracellular HMGB1 released from dying cancer cells or of recombinant HMGB1 on Dox resistance.

Conclusions: These findings highlight the potential of stromal fibroblasts to contribute to chemoresistance in breast cancer cells in part through fibroblast-induced HMGB1 production.

Show MeSH

Related in: MedlinePlus

HMGB1 expression in MDA-MB-231 cells treated with fibroblast CM. Real time PCR for HMGB1 expression in MDA-MB-231 cells treated with NTF-CMs and BCF-CMs for 48 h using paired fibroblasts isolated from the same patient.The levels of HMGB1 transcript (A) and protein levels (B) are shown after normalization against the internal control β-actin. Controls (Ctl) are cells cultured in fresh medium with no CM treatment. Bars represent the mean ± SD of triplicate experiments. $ = p-value of less than 0.05. * = p-value of less than 0.05 compared to the average HMGB1 of the two NTFs-CM treatment conditions whereas # = p-value of less than 0.05 compared to HMGB1 of the matched NTF-CM treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4301465&req=5

Fig4: HMGB1 expression in MDA-MB-231 cells treated with fibroblast CM. Real time PCR for HMGB1 expression in MDA-MB-231 cells treated with NTF-CMs and BCF-CMs for 48 h using paired fibroblasts isolated from the same patient.The levels of HMGB1 transcript (A) and protein levels (B) are shown after normalization against the internal control β-actin. Controls (Ctl) are cells cultured in fresh medium with no CM treatment. Bars represent the mean ± SD of triplicate experiments. $ = p-value of less than 0.05. * = p-value of less than 0.05 compared to the average HMGB1 of the two NTFs-CM treatment conditions whereas # = p-value of less than 0.05 compared to HMGB1 of the matched NTF-CM treatment.

Mentions: BCF-CM significantly induced intracellular HMGB1 protein expression in MDA-MB-231 breast cancer cells as detected by Western blot analysis at all time points tested (Figure 3). The effect was time-dependent and since the greatest differential induction (BCF-CM vs NTF-CM) was observed at 48 h, this time period was selected for further studies. As a further quality control, the CMs of BCF and NTF isolated from the same patient were used to treat MDA-MB-231 cells and HMGB1 gene expression was analyzed by real time PCR. The results showed that BCF-CM induced HMGB1 mRNA to a significantly greater degree than NTF-CM (Figure 4A). Western blot analysis confirmed that the protein levels of HMGB1 induced by BCF-CM were statistically significantly higher than those induced by patient-matched NTF-CM (Figure 4B). In addition, HMGB1 protein levels in MDA-MB-231 cells treated with BCF-CMs from different patients were consistently significantly higher than those treated with NTF-CMs.Figure 3


Cancer-associated fibroblasts induce high mobility group box 1 and contribute to resistance to doxorubicin in breast cancer cells.

Amornsupak K, Insawang T, Thuwajit P, O-Charoenrat P, Eccles SA, Thuwajit C - BMC Cancer (2014)

HMGB1 expression in MDA-MB-231 cells treated with fibroblast CM. Real time PCR for HMGB1 expression in MDA-MB-231 cells treated with NTF-CMs and BCF-CMs for 48 h using paired fibroblasts isolated from the same patient.The levels of HMGB1 transcript (A) and protein levels (B) are shown after normalization against the internal control β-actin. Controls (Ctl) are cells cultured in fresh medium with no CM treatment. Bars represent the mean ± SD of triplicate experiments. $ = p-value of less than 0.05. * = p-value of less than 0.05 compared to the average HMGB1 of the two NTFs-CM treatment conditions whereas # = p-value of less than 0.05 compared to HMGB1 of the matched NTF-CM treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4301465&req=5

Fig4: HMGB1 expression in MDA-MB-231 cells treated with fibroblast CM. Real time PCR for HMGB1 expression in MDA-MB-231 cells treated with NTF-CMs and BCF-CMs for 48 h using paired fibroblasts isolated from the same patient.The levels of HMGB1 transcript (A) and protein levels (B) are shown after normalization against the internal control β-actin. Controls (Ctl) are cells cultured in fresh medium with no CM treatment. Bars represent the mean ± SD of triplicate experiments. $ = p-value of less than 0.05. * = p-value of less than 0.05 compared to the average HMGB1 of the two NTFs-CM treatment conditions whereas # = p-value of less than 0.05 compared to HMGB1 of the matched NTF-CM treatment.
Mentions: BCF-CM significantly induced intracellular HMGB1 protein expression in MDA-MB-231 breast cancer cells as detected by Western blot analysis at all time points tested (Figure 3). The effect was time-dependent and since the greatest differential induction (BCF-CM vs NTF-CM) was observed at 48 h, this time period was selected for further studies. As a further quality control, the CMs of BCF and NTF isolated from the same patient were used to treat MDA-MB-231 cells and HMGB1 gene expression was analyzed by real time PCR. The results showed that BCF-CM induced HMGB1 mRNA to a significantly greater degree than NTF-CM (Figure 4A). Western blot analysis confirmed that the protein levels of HMGB1 induced by BCF-CM were statistically significantly higher than those induced by patient-matched NTF-CM (Figure 4B). In addition, HMGB1 protein levels in MDA-MB-231 cells treated with BCF-CMs from different patients were consistently significantly higher than those treated with NTF-CMs.Figure 3

Bottom Line: Cancer-associated fibroblasts and high mobility group box 1 (HMGB1) protein have been suggested to mediate cancer progression and chemotherapy resistance.Extracellular HMGB1 was strongly expressed in the CM after Dox-induced MDA-MB-231 cell death and was higher in cells pre-treated with BCF-CM than NTF-CM.Recombinant HMGB1 was shown to increase Dox resistance and this was associated with evidence of autophagy.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. cthuwajit@yahoo.com.

ABSTRACT

Background: Cancer-associated fibroblasts and high mobility group box 1 (HMGB1) protein have been suggested to mediate cancer progression and chemotherapy resistance. The role of such fibroblasts in HMGB1 production in breast cancer is unclear. This study aimed to investigate the effects of cancer-associated fibroblasts on HMGB1 expression in breast cancer cells and its role in chemotherapeutic response.

Methods: Breast cancer-associated fibroblasts (BCFs) and non-tumor-associated fibroblasts (NTFs) were isolated from human breast cancers or adjacent normal tissues and established as primary cultures in vitro. After confirmation of the activated status of these fibroblasts, conditioned-media (CM) were collected and applied to MDA-MB-231 human triple negative breast cancer cells. The levels of intracellular and extracellular HMGB1 were measured by real-time PCR and/or Western blot. The response of BCF-CM-pre-treated cancer cells to doxorubicin (Dox) was compared with those pre-treated with NTF-CM or control cultures. The effect of an HMGB1 neutralizing antibody on Dox resistance induced by extracellular HMGB1 from non-viable Dox-treated cancer cells or recombinant HMGB1 was also investigated.

Results: Immunocytochemical analysis revealed that BCFs and NTFs were alpha-smooth muscle actin (ASMA) positive and cytokeratin 19 (CK19) negative cells: a phenotype consistent with that of activated fibroblasts. We confirmed that the CM from BCFs (but not NTFs), could significantly induce breast cancer cell migration. Intracellular HMGB1 expression was induced in BCF-CM-treated breast cancer cells and also in Dox-treated cells. Extracellular HMGB1 was strongly expressed in the CM after Dox-induced MDA-MB-231 cell death and was higher in cells pre-treated with BCF-CM than NTF-CM. Pre-treatment of breast cancer cells with BCF-CM induced a degree of resistance to Dox in accordance with the increased level of secreted HMGB1. Recombinant HMGB1 was shown to increase Dox resistance and this was associated with evidence of autophagy. Anti-HMGB1 neutralizing antibody significantly reduced the effect of extracellular HMGB1 released from dying cancer cells or of recombinant HMGB1 on Dox resistance.

Conclusions: These findings highlight the potential of stromal fibroblasts to contribute to chemoresistance in breast cancer cells in part through fibroblast-induced HMGB1 production.

Show MeSH
Related in: MedlinePlus