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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

Chemo-residual triple-negative (TN) breast cancer cells from a short-term chemotherapy treatment model exhibit increased expression of a nuclear isoform of basic fibroblast growth factor (bFGF). a Increased bFGF RNA in chemo-residual SUM159 cells after doxorubicin treatment as described in Fig. 1a. Total RNA was extracted from parental and chemo-residual cells. bFGF mRNA expression was quantified by qRT-PCR, and is shown as fold increase relative to β-actin. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test. bLeft panel: increased expression of nuclear, but not cytosolic bFGF in chemo-residual SUM159 cells after doxorubicin or docetaxel treatment (as described in Fig. 1a). Nuclear or cytosolic protein was extracted from parental and chemo-residual cells. Equivalent amounts were immunoblotted with bFGF, Lamin A, or GAPDH antibody, followed by IrDye-conjugated secondary antibodies. Protein bands were detected by infrared imaging. Right panel: protein bands from three independent trials (doxorubicin treatment, as described in Fig. 1a) were quantified using Image J software (NIH), and the relative ratio of nuclear bFGF to loading control is shown for parental and chemo-residual SUM159 cells. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. c SUM159 and BT549 cells were treated with doxorubicin as described in Fig. 1a. Parental and chemo-residual cells were fixed and stained with Hoechst (blue) and bFGF antibody (red) to demonstrate the increased nuclear localization of bFGF in chemo-residual TN breast cancer cells. Magnification ×400.
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Fig2: Chemo-residual triple-negative (TN) breast cancer cells from a short-term chemotherapy treatment model exhibit increased expression of a nuclear isoform of basic fibroblast growth factor (bFGF). a Increased bFGF RNA in chemo-residual SUM159 cells after doxorubicin treatment as described in Fig. 1a. Total RNA was extracted from parental and chemo-residual cells. bFGF mRNA expression was quantified by qRT-PCR, and is shown as fold increase relative to β-actin. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test. bLeft panel: increased expression of nuclear, but not cytosolic bFGF in chemo-residual SUM159 cells after doxorubicin or docetaxel treatment (as described in Fig. 1a). Nuclear or cytosolic protein was extracted from parental and chemo-residual cells. Equivalent amounts were immunoblotted with bFGF, Lamin A, or GAPDH antibody, followed by IrDye-conjugated secondary antibodies. Protein bands were detected by infrared imaging. Right panel: protein bands from three independent trials (doxorubicin treatment, as described in Fig. 1a) were quantified using Image J software (NIH), and the relative ratio of nuclear bFGF to loading control is shown for parental and chemo-residual SUM159 cells. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. c SUM159 and BT549 cells were treated with doxorubicin as described in Fig. 1a. Parental and chemo-residual cells were fixed and stained with Hoechst (blue) and bFGF antibody (red) to demonstrate the increased nuclear localization of bFGF in chemo-residual TN breast cancer cells. Magnification ×400.

Mentions: bFGF signaling has been implicated in tumor resistance to targeted therapies [10–15]. Accordingly, we investigated bFGF expression in chemo-residual TN tumor cells from our short-term chemotherapy treatment model. As shown in Fig. 2a, we observed significantly increased bFGF mRNA expression in chemo-residual tumor cells compared to parental cells. To further elucidate the connection between different isoforms of bFGF and chemo-resistance, we measured nuclear and cytosolic bFGF levels in chemo-residual TN tumor cells. On western blots, significantly increased levels of nuclear bFGF isoforms (22, 24 kDa), but not the cytosolic bFGF isoform (18 kDa), were detected in chemo-residual cells compared to parental cells (Fig. 2b). This trend was observed regardless of the chemotherapy class studied (doxorubicin or docetaxel, Fig. 2b). By immunofluorescence, we confirmed increased nuclear bFGF in chemo-residual cells relative to parental cells for both SUM159 and BT549 TN breast cancer cells (Fig. 2c) and for two other TN breast cancer cell lines (HS578T and MDA-MB-231, data not shown). These results suggest an association between nuclear bFGF expression and TN breast cancer chemo-resistance.Fig. 2


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)

Chemo-residual triple-negative (TN) breast cancer cells from a short-term chemotherapy treatment model exhibit increased expression of a nuclear isoform of basic fibroblast growth factor (bFGF). a Increased bFGF RNA in chemo-residual SUM159 cells after doxorubicin treatment as described in Fig. 1a. Total RNA was extracted from parental and chemo-residual cells. bFGF mRNA expression was quantified by qRT-PCR, and is shown as fold increase relative to β-actin. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test. bLeft panel: increased expression of nuclear, but not cytosolic bFGF in chemo-residual SUM159 cells after doxorubicin or docetaxel treatment (as described in Fig. 1a). Nuclear or cytosolic protein was extracted from parental and chemo-residual cells. Equivalent amounts were immunoblotted with bFGF, Lamin A, or GAPDH antibody, followed by IrDye-conjugated secondary antibodies. Protein bands were detected by infrared imaging. Right panel: protein bands from three independent trials (doxorubicin treatment, as described in Fig. 1a) were quantified using Image J software (NIH), and the relative ratio of nuclear bFGF to loading control is shown for parental and chemo-residual SUM159 cells. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. c SUM159 and BT549 cells were treated with doxorubicin as described in Fig. 1a. Parental and chemo-residual cells were fixed and stained with Hoechst (blue) and bFGF antibody (red) to demonstrate the increased nuclear localization of bFGF in chemo-residual TN breast cancer cells. Magnification ×400.
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Fig2: Chemo-residual triple-negative (TN) breast cancer cells from a short-term chemotherapy treatment model exhibit increased expression of a nuclear isoform of basic fibroblast growth factor (bFGF). a Increased bFGF RNA in chemo-residual SUM159 cells after doxorubicin treatment as described in Fig. 1a. Total RNA was extracted from parental and chemo-residual cells. bFGF mRNA expression was quantified by qRT-PCR, and is shown as fold increase relative to β-actin. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test. bLeft panel: increased expression of nuclear, but not cytosolic bFGF in chemo-residual SUM159 cells after doxorubicin or docetaxel treatment (as described in Fig. 1a). Nuclear or cytosolic protein was extracted from parental and chemo-residual cells. Equivalent amounts were immunoblotted with bFGF, Lamin A, or GAPDH antibody, followed by IrDye-conjugated secondary antibodies. Protein bands were detected by infrared imaging. Right panel: protein bands from three independent trials (doxorubicin treatment, as described in Fig. 1a) were quantified using Image J software (NIH), and the relative ratio of nuclear bFGF to loading control is shown for parental and chemo-residual SUM159 cells. Error bars represent SD, n = 3, ***p <0.001, two-tailed Student’s t test. c SUM159 and BT549 cells were treated with doxorubicin as described in Fig. 1a. Parental and chemo-residual cells were fixed and stained with Hoechst (blue) and bFGF antibody (red) to demonstrate the increased nuclear localization of bFGF in chemo-residual TN breast cancer cells. Magnification ×400.
Mentions: bFGF signaling has been implicated in tumor resistance to targeted therapies [10–15]. Accordingly, we investigated bFGF expression in chemo-residual TN tumor cells from our short-term chemotherapy treatment model. As shown in Fig. 2a, we observed significantly increased bFGF mRNA expression in chemo-residual tumor cells compared to parental cells. To further elucidate the connection between different isoforms of bFGF and chemo-resistance, we measured nuclear and cytosolic bFGF levels in chemo-residual TN tumor cells. On western blots, significantly increased levels of nuclear bFGF isoforms (22, 24 kDa), but not the cytosolic bFGF isoform (18 kDa), were detected in chemo-residual cells compared to parental cells (Fig. 2b). This trend was observed regardless of the chemotherapy class studied (doxorubicin or docetaxel, Fig. 2b). By immunofluorescence, we confirmed increased nuclear bFGF in chemo-residual cells relative to parental cells for both SUM159 and BT549 TN breast cancer cells (Fig. 2c) and for two other TN breast cancer cell lines (HS578T and MDA-MB-231, data not shown). These results suggest an association between nuclear bFGF expression and TN breast cancer chemo-resistance.Fig. 2

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