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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

Transfection of basic fibroblast growth factor (bFGF) knockdown cells with 23-kDa bFGF (nuclear bFGF) vector restores chemo-residual cell survival and subsequent colony formation. a bFGF shRNA-transfected SUM159 cells were transfected with vectors expressing low molecular weight (LMW) (cytosolic) bFGF, high molecular weight (HMW) (nuclear) bFGF, or pCI as a vector control (ctrl). The expression of addback constructs in stable transfectants was confirmed and compared to control shRNA-transfected cells by immunoblotting equivalent amounts of nuclear (upper two lanes) or cytosolic (lower two lanes) extract with bFGF antibody. Protein loading was assessed using Lamin A or GAPDH antibody. b SUM159 cells expressing control shRNA, bFGF shRNA, or bFGF shRNA plus indicated addback constructs were treated with doxorubicin as described in Fig. 1a. The number of chemo-residual cells was determined on day 7 by trypan blue exclusion. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. c Colonies (containing >50 cells) were quantified on the indicated days. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. d Colonies were fixed and stained with crystal violet on day 20. Similar results were obtained in at least three independent trials
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Fig4: Transfection of basic fibroblast growth factor (bFGF) knockdown cells with 23-kDa bFGF (nuclear bFGF) vector restores chemo-residual cell survival and subsequent colony formation. a bFGF shRNA-transfected SUM159 cells were transfected with vectors expressing low molecular weight (LMW) (cytosolic) bFGF, high molecular weight (HMW) (nuclear) bFGF, or pCI as a vector control (ctrl). The expression of addback constructs in stable transfectants was confirmed and compared to control shRNA-transfected cells by immunoblotting equivalent amounts of nuclear (upper two lanes) or cytosolic (lower two lanes) extract with bFGF antibody. Protein loading was assessed using Lamin A or GAPDH antibody. b SUM159 cells expressing control shRNA, bFGF shRNA, or bFGF shRNA plus indicated addback constructs were treated with doxorubicin as described in Fig. 1a. The number of chemo-residual cells was determined on day 7 by trypan blue exclusion. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. c Colonies (containing >50 cells) were quantified on the indicated days. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. d Colonies were fixed and stained with crystal violet on day 20. Similar results were obtained in at least three independent trials

Mentions: To determine which bFGF isoform facilitates chemo-residual tumor cell survival and colony formation in our model, we transfected bFGF shRNA-expressing cells with a vector expressing 18-kDa rat bFGF, 23-kDa rat bFGF, or an empty control vector (Fig. 4a). The 18-kDa and 23-kDa rat bFGF constructs exhibit 97 % and 82 % homology with human 18-kDa and 24-kDa nuclear bFGF, respectively [30]. The addback of the 23-kDa rat nuclear bFGF, but not the 18-kDa rat cytosolic bFGF, to bFGF shRNA transfectants increased the number of chemo-residual tumor cells to that observed in control cells (Fig. 4b). Likewise addback of the 23-kDa bFGF isoform restored the ability of bFGF shRNA-transfected chemo-residual cells to establish colonies (Fig. 4c, d) following short-term doxorubicin treatment. Of note, similar transfection of SUM159 cells expressing the control shRNA did not influence cell viability (data not shown). Collectively, our results demonstrate that high molecular weight (nuclear) bFGF, but not low molecular weight (cytosolic) bFGF, is sufficient to maintain the viability of chemo-residual tumor cells and promote subsequent colony growth after chemotherapy withdrawal.Fig. 4


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)

Transfection of basic fibroblast growth factor (bFGF) knockdown cells with 23-kDa bFGF (nuclear bFGF) vector restores chemo-residual cell survival and subsequent colony formation. a bFGF shRNA-transfected SUM159 cells were transfected with vectors expressing low molecular weight (LMW) (cytosolic) bFGF, high molecular weight (HMW) (nuclear) bFGF, or pCI as a vector control (ctrl). The expression of addback constructs in stable transfectants was confirmed and compared to control shRNA-transfected cells by immunoblotting equivalent amounts of nuclear (upper two lanes) or cytosolic (lower two lanes) extract with bFGF antibody. Protein loading was assessed using Lamin A or GAPDH antibody. b SUM159 cells expressing control shRNA, bFGF shRNA, or bFGF shRNA plus indicated addback constructs were treated with doxorubicin as described in Fig. 1a. The number of chemo-residual cells was determined on day 7 by trypan blue exclusion. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. c Colonies (containing >50 cells) were quantified on the indicated days. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. d Colonies were fixed and stained with crystal violet on day 20. Similar results were obtained in at least three independent trials
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Related In: Results  -  Collection

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Fig4: Transfection of basic fibroblast growth factor (bFGF) knockdown cells with 23-kDa bFGF (nuclear bFGF) vector restores chemo-residual cell survival and subsequent colony formation. a bFGF shRNA-transfected SUM159 cells were transfected with vectors expressing low molecular weight (LMW) (cytosolic) bFGF, high molecular weight (HMW) (nuclear) bFGF, or pCI as a vector control (ctrl). The expression of addback constructs in stable transfectants was confirmed and compared to control shRNA-transfected cells by immunoblotting equivalent amounts of nuclear (upper two lanes) or cytosolic (lower two lanes) extract with bFGF antibody. Protein loading was assessed using Lamin A or GAPDH antibody. b SUM159 cells expressing control shRNA, bFGF shRNA, or bFGF shRNA plus indicated addback constructs were treated with doxorubicin as described in Fig. 1a. The number of chemo-residual cells was determined on day 7 by trypan blue exclusion. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. c Colonies (containing >50 cells) were quantified on the indicated days. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. d Colonies were fixed and stained with crystal violet on day 20. Similar results were obtained in at least three independent trials
Mentions: To determine which bFGF isoform facilitates chemo-residual tumor cell survival and colony formation in our model, we transfected bFGF shRNA-expressing cells with a vector expressing 18-kDa rat bFGF, 23-kDa rat bFGF, or an empty control vector (Fig. 4a). The 18-kDa and 23-kDa rat bFGF constructs exhibit 97 % and 82 % homology with human 18-kDa and 24-kDa nuclear bFGF, respectively [30]. The addback of the 23-kDa rat nuclear bFGF, but not the 18-kDa rat cytosolic bFGF, to bFGF shRNA transfectants increased the number of chemo-residual tumor cells to that observed in control cells (Fig. 4b). Likewise addback of the 23-kDa bFGF isoform restored the ability of bFGF shRNA-transfected chemo-residual cells to establish colonies (Fig. 4c, d) following short-term doxorubicin treatment. Of note, similar transfection of SUM159 cells expressing the control shRNA did not influence cell viability (data not shown). Collectively, our results demonstrate that high molecular weight (nuclear) bFGF, but not low molecular weight (cytosolic) bFGF, is sufficient to maintain the viability of chemo-residual tumor cells and promote subsequent colony growth after chemotherapy withdrawal.Fig. 4

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