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Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

Li S, Payne S, Wang F, Claus P, Su Z, Groth J, Geradts J, de Ridder G, Alvarez R, Marcom PK, Pizzo SV, Bachelder RE - Breast Cancer Res. (2015)

Bottom Line: The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies.Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance.Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, N.C., 27710, USA. shenduo.li@duke.edu.

ABSTRACT

Introduction: Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)].

Methods: Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy.

Results: TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells.

Conclusion: These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

No MeSH data available.


Related in: MedlinePlus

Nuclear basic fibroblast growth factor (bFGF) drives DNA repair and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression. aLeft panel: BT549 cells transfected with bFGF shRNA or control (ctrl) shRNA were challenged with doxorubicin (Dox) (0.25 μg/ml) for 2 h. Fresh medium was added after chemotherapy removal. DNA damage at sequential time points after chemotherapy treatment was analyzed by neutral comet assay. Representative images are shown for each time point. Cells scored as comet tail-positive are indicated with red arrows in the 48-h time frame. Right panel: percentage of cells with comet tails at indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields (each field containing >50 cells). Significance of data points at 24 and 48 h was determined relative to data reported at 0 h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). b SUM159 cells expressing control shRNA, bFGF shRNA, or bFGF shRNA plus indicated addback constructs (as in Fig. 4a) were challenged with doxorubicin (0.25 μg/ml) for 2 h. Fresh medium was added after chemotherapy removal. DNA damage at sequential time points after chemotherapy treatment was analyzed by neutral comet assay. Percentage of cells with comet tails at the indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields (each field containing >50 cells). Significance of data points at 24 and 48 h was determined relative to data reported at 0 h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). c SUM159 and BT549 cells transfected with bFGF shRNA or ctrl shRNA were treated with doxorubicin as in Fig. 1a. Nuclear protein from chemo-residual cells was extracted. Equivalent amounts were immunoblotted with DNA-PKCS and lamin A antibodies. Protein bands were quantified, and the relative ratio of DNA-PKCS to loading control is shown for each lane. dLeft panel: bFGF shRNA-transfected SUM159 cells expressing indicated addback constructs were treated with doxorubicin as in Fig. 1a. Nuclear protein from chemo-residual cells was extracted. Equivalent amounts were immunoblotted with DNA-PKCS and Lamin A antibodies. Right panel: protein bands from three independent trials were quantified and the relative ratio of DNA-PKCS to loading control is shown for each line. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test
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Fig7: Nuclear basic fibroblast growth factor (bFGF) drives DNA repair and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression. aLeft panel: BT549 cells transfected with bFGF shRNA or control (ctrl) shRNA were challenged with doxorubicin (Dox) (0.25 μg/ml) for 2 h. Fresh medium was added after chemotherapy removal. DNA damage at sequential time points after chemotherapy treatment was analyzed by neutral comet assay. Representative images are shown for each time point. Cells scored as comet tail-positive are indicated with red arrows in the 48-h time frame. Right panel: percentage of cells with comet tails at indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields (each field containing >50 cells). Significance of data points at 24 and 48 h was determined relative to data reported at 0 h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). b SUM159 cells expressing control shRNA, bFGF shRNA, or bFGF shRNA plus indicated addback constructs (as in Fig. 4a) were challenged with doxorubicin (0.25 μg/ml) for 2 h. Fresh medium was added after chemotherapy removal. DNA damage at sequential time points after chemotherapy treatment was analyzed by neutral comet assay. Percentage of cells with comet tails at the indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields (each field containing >50 cells). Significance of data points at 24 and 48 h was determined relative to data reported at 0 h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). c SUM159 and BT549 cells transfected with bFGF shRNA or ctrl shRNA were treated with doxorubicin as in Fig. 1a. Nuclear protein from chemo-residual cells was extracted. Equivalent amounts were immunoblotted with DNA-PKCS and lamin A antibodies. Protein bands were quantified, and the relative ratio of DNA-PKCS to loading control is shown for each lane. dLeft panel: bFGF shRNA-transfected SUM159 cells expressing indicated addback constructs were treated with doxorubicin as in Fig. 1a. Nuclear protein from chemo-residual cells was extracted. Equivalent amounts were immunoblotted with DNA-PKCS and Lamin A antibodies. Right panel: protein bands from three independent trials were quantified and the relative ratio of DNA-PKCS to loading control is shown for each line. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test

Mentions: We next investigated the effects of bFGF knockdown on DNA repair in chemotherapy-challenged TN breast tumor cells. Twenty-four hours after doxorubicin challenge, SUM159 cells expressing a bFGF shRNA had a similar level of DNA damage to that of cells expressing a control shRNA, with approximately 70 % of cells having a comet tail (Fig. 7a). However, control shRNA-expressing cells exhibited more rapid DNA repair than bFGF shRNA transfectants, with only approximately 30 % of control shRNA-expressing cells having comet tails at 48 h post challenge (compared to approximately 60 % of bFGF shRNA-expressing cells having comet tails at this time) (Fig. 7a). To determine which bFGF isoform drives repair in these cells, we next performed comet assays on knockdown cells reconstituted with low molecular weight (LMW) or high molecular weight (HMW) bFGF. As shown in Fig. 7b, bFGF knockdown cells exhibited significantly slower DNA repair than control cells. Moreover, expression of HMW bFGF, but not LMW bFGF in knockdown cells restored DNA repair to the level observed in control shRNA cells (Fig. 7b).Fig. 7


Nuclear basic fibroblast growth factor regulates triple-negative breast cancer chemo-resistance.

Li S, Payne S, Wang F, Claus P, Su Z, Groth J, Geradts J, de Ridder G, Alvarez R, Marcom PK, Pizzo SV, Bachelder RE - Breast Cancer Res. (2015)

Nuclear basic fibroblast growth factor (bFGF) drives DNA repair and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression. aLeft panel: BT549 cells transfected with bFGF shRNA or control (ctrl) shRNA were challenged with doxorubicin (Dox) (0.25 μg/ml) for 2 h. Fresh medium was added after chemotherapy removal. DNA damage at sequential time points after chemotherapy treatment was analyzed by neutral comet assay. Representative images are shown for each time point. Cells scored as comet tail-positive are indicated with red arrows in the 48-h time frame. Right panel: percentage of cells with comet tails at indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields (each field containing >50 cells). Significance of data points at 24 and 48 h was determined relative to data reported at 0 h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). b SUM159 cells expressing control shRNA, bFGF shRNA, or bFGF shRNA plus indicated addback constructs (as in Fig. 4a) were challenged with doxorubicin (0.25 μg/ml) for 2 h. Fresh medium was added after chemotherapy removal. DNA damage at sequential time points after chemotherapy treatment was analyzed by neutral comet assay. Percentage of cells with comet tails at the indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields (each field containing >50 cells). Significance of data points at 24 and 48 h was determined relative to data reported at 0 h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). c SUM159 and BT549 cells transfected with bFGF shRNA or ctrl shRNA were treated with doxorubicin as in Fig. 1a. Nuclear protein from chemo-residual cells was extracted. Equivalent amounts were immunoblotted with DNA-PKCS and lamin A antibodies. Protein bands were quantified, and the relative ratio of DNA-PKCS to loading control is shown for each lane. dLeft panel: bFGF shRNA-transfected SUM159 cells expressing indicated addback constructs were treated with doxorubicin as in Fig. 1a. Nuclear protein from chemo-residual cells was extracted. Equivalent amounts were immunoblotted with DNA-PKCS and Lamin A antibodies. Right panel: protein bands from three independent trials were quantified and the relative ratio of DNA-PKCS to loading control is shown for each line. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test
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Related In: Results  -  Collection

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Fig7: Nuclear basic fibroblast growth factor (bFGF) drives DNA repair and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression. aLeft panel: BT549 cells transfected with bFGF shRNA or control (ctrl) shRNA were challenged with doxorubicin (Dox) (0.25 μg/ml) for 2 h. Fresh medium was added after chemotherapy removal. DNA damage at sequential time points after chemotherapy treatment was analyzed by neutral comet assay. Representative images are shown for each time point. Cells scored as comet tail-positive are indicated with red arrows in the 48-h time frame. Right panel: percentage of cells with comet tails at indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields (each field containing >50 cells). Significance of data points at 24 and 48 h was determined relative to data reported at 0 h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). b SUM159 cells expressing control shRNA, bFGF shRNA, or bFGF shRNA plus indicated addback constructs (as in Fig. 4a) were challenged with doxorubicin (0.25 μg/ml) for 2 h. Fresh medium was added after chemotherapy removal. DNA damage at sequential time points after chemotherapy treatment was analyzed by neutral comet assay. Percentage of cells with comet tails at the indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields (each field containing >50 cells). Significance of data points at 24 and 48 h was determined relative to data reported at 0 h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). c SUM159 and BT549 cells transfected with bFGF shRNA or ctrl shRNA were treated with doxorubicin as in Fig. 1a. Nuclear protein from chemo-residual cells was extracted. Equivalent amounts were immunoblotted with DNA-PKCS and lamin A antibodies. Protein bands were quantified, and the relative ratio of DNA-PKCS to loading control is shown for each lane. dLeft panel: bFGF shRNA-transfected SUM159 cells expressing indicated addback constructs were treated with doxorubicin as in Fig. 1a. Nuclear protein from chemo-residual cells was extracted. Equivalent amounts were immunoblotted with DNA-PKCS and Lamin A antibodies. Right panel: protein bands from three independent trials were quantified and the relative ratio of DNA-PKCS to loading control is shown for each line. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test
Mentions: We next investigated the effects of bFGF knockdown on DNA repair in chemotherapy-challenged TN breast tumor cells. Twenty-four hours after doxorubicin challenge, SUM159 cells expressing a bFGF shRNA had a similar level of DNA damage to that of cells expressing a control shRNA, with approximately 70 % of cells having a comet tail (Fig. 7a). However, control shRNA-expressing cells exhibited more rapid DNA repair than bFGF shRNA transfectants, with only approximately 30 % of control shRNA-expressing cells having comet tails at 48 h post challenge (compared to approximately 60 % of bFGF shRNA-expressing cells having comet tails at this time) (Fig. 7a). To determine which bFGF isoform drives repair in these cells, we next performed comet assays on knockdown cells reconstituted with low molecular weight (LMW) or high molecular weight (HMW) bFGF. As shown in Fig. 7b, bFGF knockdown cells exhibited significantly slower DNA repair than control cells. Moreover, expression of HMW bFGF, but not LMW bFGF in knockdown cells restored DNA repair to the level observed in control shRNA cells (Fig. 7b).Fig. 7

Bottom Line: The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies.Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance.Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, N.C., 27710, USA. shenduo.li@duke.edu.

ABSTRACT

Introduction: Chemotherapy remains the only available treatment for triple-negative (TN) breast cancer, and most patients exhibit an incomplete pathologic response. Half of patients exhibiting an incomplete pathologic response die within five years of treatment due to chemo-resistant, recurrent tumor growth. Defining molecules responsible for TN breast cancer chemo-resistance is crucial for developing effective combination therapies blocking tumor recurrence. Historically, chemo-resistance studies have relied on long-term chemotherapy selection models that drive genetic mutations conferring cell survival. Other models suggest that tumors are heterogeneous, being composed of both chemo-sensitive and chemo-resistant tumor cell populations. We previously described a short-term chemotherapy treatment model that enriches for chemo-residual TN tumor cells. In the current work, we use this enrichment strategy to identify a novel determinant of TN breast cancer chemotherapy resistance [a nuclear isoform of basic fibroblast growth factor (bFGF)].

Methods: Studies are conducted using our in vitro model of chemotherapy resistance. Short-term chemotherapy treatment enriches for a chemo-residual TN subpopulation that over time resumes proliferation. By western blotting and real-time polymerase chain reaction, we show that this chemotherapy-enriched tumor cell subpopulation expresses nuclear bFGF. The importance of bFGF for survival of these chemo-residual cells is interrogated using short hairpin knockdown strategies. DNA repair capability is assessed by comet assay. Immunohistochemistry (IHC) is used to determine nuclear bFGF expression in TN breast cancer cases pre- and post- neoadjuvant chemotherapy.

Results: TN tumor cells surviving short-term chemotherapy treatment express increased nuclear bFGF. bFGF knockdown reduces the number of chemo-residual TN tumor cells. Adding back a nuclear bFGF construct to bFGF knockdown cells restores their chemo-resistance. Nuclear bFGF-mediated chemo-resistance is associated with increased DNA-dependent protein kinase (DNA-PK) expression and accelerated DNA repair. In fifty-six percent of matched TN breast cancer cases, percent nuclear bFGF-positive tumor cells either increases or remains the same post- neoadjuvant chemotherapy treatment (compared to pre-treatment). These data indicate that in a subset of TN breast cancers, chemotherapy enriches for nuclear bFGF-expressing tumor cells.

Conclusion: These studies identify nuclear bFGF as a protein in a subset of TN breast cancers that likely contributes to drug resistance following standard chemotherapy treatment.

No MeSH data available.


Related in: MedlinePlus