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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

Basic fibroblast growth factor (bFGF) knockdown in triple-negative (TN) breast tumor cells reduces the number of chemo-residual cells and blocks subsequent colony formation. a SUM159 and BT549 cells were transfected stably with a bFGF shRNA or control (ctrl) shRNA. The knockdown of nuclear bFGF was confirmed by immunoblotting equivalent amounts of nuclear extract with bFGF antibody. Protein loading was accessed using Lamin A antibody. Protein bands were quantified using Image J software (NIH), and the relative ratio of bFGF to loading control is shown for each lane. b and c SUM159 cells (b) and BT549 cells (c) transfected stably with a bFGF shRNA or control shRNA were treated with doxorubicin as described in Fig. 1a. Upper panel: pictures of remaining chemo-residual cells were taken on day 7. Magnification ×20. Lower panel: numbers of chemotherapy (chemo)-enriched chemo-residual cells on day 7 were determined by trypan blue exclusion; n = 3, error bars represent SD, ***p <0.001, two-tailed Student’s t test. d and e SUM159 cells (d) and BT549 cells (e) transfected stably with a bFGF shRNA or control (ctrl) shRNA were treated with doxorubicin as described in Fig. 1a. Upper panel: colonies (containing >50 cells) were quantified on the indicated days. Error bars represent SD, n = 3, **p <0.01, ***p <0.001, two-tailed Student’s t test. Lower panel: colonies were fixed and stained with crystal violet on day 22 (SUM159 cell) and day 24 (BT549 cell). Similar results were obtained in at least three independent trials
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Fig3: Basic fibroblast growth factor (bFGF) knockdown in triple-negative (TN) breast tumor cells reduces the number of chemo-residual cells and blocks subsequent colony formation. a SUM159 and BT549 cells were transfected stably with a bFGF shRNA or control (ctrl) shRNA. The knockdown of nuclear bFGF was confirmed by immunoblotting equivalent amounts of nuclear extract with bFGF antibody. Protein loading was accessed using Lamin A antibody. Protein bands were quantified using Image J software (NIH), and the relative ratio of bFGF to loading control is shown for each lane. b and c SUM159 cells (b) and BT549 cells (c) transfected stably with a bFGF shRNA or control shRNA were treated with doxorubicin as described in Fig. 1a. Upper panel: pictures of remaining chemo-residual cells were taken on day 7. Magnification ×20. Lower panel: numbers of chemotherapy (chemo)-enriched chemo-residual cells on day 7 were determined by trypan blue exclusion; n = 3, error bars represent SD, ***p <0.001, two-tailed Student’s t test. d and e SUM159 cells (d) and BT549 cells (e) transfected stably with a bFGF shRNA or control (ctrl) shRNA were treated with doxorubicin as described in Fig. 1a. Upper panel: colonies (containing >50 cells) were quantified on the indicated days. Error bars represent SD, n = 3, **p <0.01, ***p <0.001, two-tailed Student’s t test. Lower panel: colonies were fixed and stained with crystal violet on day 22 (SUM159 cell) and day 24 (BT549 cell). Similar results were obtained in at least three independent trials

Mentions: Studies in Fig. 2 indicate that nuclear bFGF is upregulated in chemo-residual TN breast cancer cells. To determine whether bFGF is required for TN breast cancer chemo-resistance, we knocked down bFGF expression in SUM159 and BT549 cells by stable bFGF shRNA transfection (Fig. 3a). Cells transfected with bFGF or control shRNA were treated for 2 days with doxorubicin as in Fig. 1a. The number of doxorubicin-enriched chemo-residual cells on day 7 was significantly decreased in bFGF shRNA transfectants compared to control shRNA transfectants (Fig. 3b, c). Moreover, bFGF shRNA-transfected chemo-residual cells formed dramatically fewer colonies after chemotherapy removal than control shRNA transfectants (Fig. 3d, e). Similar results were observed in two TN breast cancer cell lines (SUM159, BT549; Fig. 3d, e). These results indicate that bFGF is necessary for the survival of chemo-residual tumor cells after doxorubicin challenge and for their subsequent proliferation upon chemotherapy withdrawal.Fig. 3


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)

Basic fibroblast growth factor (bFGF) knockdown in triple-negative (TN) breast tumor cells reduces the number of chemo-residual cells and blocks subsequent colony formation. a SUM159 and BT549 cells were transfected stably with a bFGF shRNA or control (ctrl) shRNA. The knockdown of nuclear bFGF was confirmed by immunoblotting equivalent amounts of nuclear extract with bFGF antibody. Protein loading was accessed using Lamin A antibody. Protein bands were quantified using Image J software (NIH), and the relative ratio of bFGF to loading control is shown for each lane. b and c SUM159 cells (b) and BT549 cells (c) transfected stably with a bFGF shRNA or control shRNA were treated with doxorubicin as described in Fig. 1a. Upper panel: pictures of remaining chemo-residual cells were taken on day 7. Magnification ×20. Lower panel: numbers of chemotherapy (chemo)-enriched chemo-residual cells on day 7 were determined by trypan blue exclusion; n = 3, error bars represent SD, ***p <0.001, two-tailed Student’s t test. d and e SUM159 cells (d) and BT549 cells (e) transfected stably with a bFGF shRNA or control (ctrl) shRNA were treated with doxorubicin as described in Fig. 1a. Upper panel: colonies (containing >50 cells) were quantified on the indicated days. Error bars represent SD, n = 3, **p <0.01, ***p <0.001, two-tailed Student’s t test. Lower panel: colonies were fixed and stained with crystal violet on day 22 (SUM159 cell) and day 24 (BT549 cell). Similar results were obtained in at least three independent trials
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: Basic fibroblast growth factor (bFGF) knockdown in triple-negative (TN) breast tumor cells reduces the number of chemo-residual cells and blocks subsequent colony formation. a SUM159 and BT549 cells were transfected stably with a bFGF shRNA or control (ctrl) shRNA. The knockdown of nuclear bFGF was confirmed by immunoblotting equivalent amounts of nuclear extract with bFGF antibody. Protein loading was accessed using Lamin A antibody. Protein bands were quantified using Image J software (NIH), and the relative ratio of bFGF to loading control is shown for each lane. b and c SUM159 cells (b) and BT549 cells (c) transfected stably with a bFGF shRNA or control shRNA were treated with doxorubicin as described in Fig. 1a. Upper panel: pictures of remaining chemo-residual cells were taken on day 7. Magnification ×20. Lower panel: numbers of chemotherapy (chemo)-enriched chemo-residual cells on day 7 were determined by trypan blue exclusion; n = 3, error bars represent SD, ***p <0.001, two-tailed Student’s t test. d and e SUM159 cells (d) and BT549 cells (e) transfected stably with a bFGF shRNA or control (ctrl) shRNA were treated with doxorubicin as described in Fig. 1a. Upper panel: colonies (containing >50 cells) were quantified on the indicated days. Error bars represent SD, n = 3, **p <0.01, ***p <0.001, two-tailed Student’s t test. Lower panel: colonies were fixed and stained with crystal violet on day 22 (SUM159 cell) and day 24 (BT549 cell). Similar results were obtained in at least three independent trials
Mentions: Studies in Fig. 2 indicate that nuclear bFGF is upregulated in chemo-residual TN breast cancer cells. To determine whether bFGF is required for TN breast cancer chemo-resistance, we knocked down bFGF expression in SUM159 and BT549 cells by stable bFGF shRNA transfection (Fig. 3a). Cells transfected with bFGF or control shRNA were treated for 2 days with doxorubicin as in Fig. 1a. The number of doxorubicin-enriched chemo-residual cells on day 7 was significantly decreased in bFGF shRNA transfectants compared to control shRNA transfectants (Fig. 3b, c). Moreover, bFGF shRNA-transfected chemo-residual cells formed dramatically fewer colonies after chemotherapy removal than control shRNA transfectants (Fig. 3d, e). Similar results were observed in two TN breast cancer cell lines (SUM159, BT549; Fig. 3d, e). These results indicate that bFGF is necessary for the survival of chemo-residual tumor cells after doxorubicin challenge and for their subsequent proliferation upon chemotherapy withdrawal.Fig. 3

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