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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 tumor cells exhibit higher DNA repair capability and increased expression/phosphorylation of DNA-dependent protein kinase (DNA-PKCS). aLeft panel: SUM159 chemo-residual cells and parental (untreated) cells were re-challenged with doxorubicin (Dox) (1 μg/ml) for 3 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 at each time point. Cells scored as comet tail-positive are indicated with red arrows in the 72-h (72h) time frame. Right panel: the percentage of cells with comet tails at the indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields. Significance of data points at 24h, 48h and 72 h was determined relative to data reported at 0h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). Cells scored as comet tail-positive are indicated with red arrows in the 72h time frame. b.Left panel: BT549 chemo-residual cells and parental (untreated) cells were challenged with Dox (0.5 μg/ml) for 4 h. DNA damage was assessed at the indicated times using the neutral comet assay as in a. Right panel: the percentage of cells with comet tails at the indicated time points was quantified as in a. Cells scored as comet tail-positive are indicated with red arrows in the 72h time frame. cLeft and middle panel: SUM159 cells (left) and BT549 cells (middle) were treated with Dox as described in Fig. 1a. Nuclear protein from parental and chemo-residual cells was extracted. Equivalent amounts were immunoblotted with phospho (Ser 2056)-DNA-PKCS, DNA-PKCS or Lamin A antibody. Right panel: protein bands from three independent trials (SUM159 cells treated with Dox as described in Fig. 1a) were quantified, and the relative ratio of DNA-PKCS to loading control is shown. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test
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Fig5: Chemo-residual tumor cells exhibit higher DNA repair capability and increased expression/phosphorylation of DNA-dependent protein kinase (DNA-PKCS). aLeft panel: SUM159 chemo-residual cells and parental (untreated) cells were re-challenged with doxorubicin (Dox) (1 μg/ml) for 3 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 at each time point. Cells scored as comet tail-positive are indicated with red arrows in the 72-h (72h) time frame. Right panel: the percentage of cells with comet tails at the indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields. Significance of data points at 24h, 48h and 72 h was determined relative to data reported at 0h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). Cells scored as comet tail-positive are indicated with red arrows in the 72h time frame. b.Left panel: BT549 chemo-residual cells and parental (untreated) cells were challenged with Dox (0.5 μg/ml) for 4 h. DNA damage was assessed at the indicated times using the neutral comet assay as in a. Right panel: the percentage of cells with comet tails at the indicated time points was quantified as in a. Cells scored as comet tail-positive are indicated with red arrows in the 72h time frame. cLeft and middle panel: SUM159 cells (left) and BT549 cells (middle) were treated with Dox as described in Fig. 1a. Nuclear protein from parental and chemo-residual cells was extracted. Equivalent amounts were immunoblotted with phospho (Ser 2056)-DNA-PKCS, DNA-PKCS or Lamin A antibody. Right panel: protein bands from three independent trials (SUM159 cells treated with Dox as described in Fig. 1a) were quantified, and the relative ratio of DNA-PKCS to loading control is shown. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test

Mentions: Elevated DNA repair activity is associated with chemo-resistance in many tumors [31–34]. To compare the DNA double-strand break (DSB) repair capability, we re-challenged untreated parental cells and chemo-residual cells with doxorubicin (a DNA-damaging agent) for 3 h and examined their recovery by neutral comet assay. As shown (Fig. 5a, b) the percent cells with comet tails returned to baseline quicker in chemo-residual cells than in parental cells. Similar results were observed in both SUM159 (Fig. 5a) and BT549 (Fig. 5b) chemo-resistance models. These data indicate that chemo-residual TN tumor cells from our short-term chemotherapy treatment model repaired DNA strand breaks more quickly than parental cells.Fig. 5


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 tumor cells exhibit higher DNA repair capability and increased expression/phosphorylation of DNA-dependent protein kinase (DNA-PKCS). aLeft panel: SUM159 chemo-residual cells and parental (untreated) cells were re-challenged with doxorubicin (Dox) (1 μg/ml) for 3 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 at each time point. Cells scored as comet tail-positive are indicated with red arrows in the 72-h (72h) time frame. Right panel: the percentage of cells with comet tails at the indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields. Significance of data points at 24h, 48h and 72 h was determined relative to data reported at 0h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). Cells scored as comet tail-positive are indicated with red arrows in the 72h time frame. b.Left panel: BT549 chemo-residual cells and parental (untreated) cells were challenged with Dox (0.5 μg/ml) for 4 h. DNA damage was assessed at the indicated times using the neutral comet assay as in a. Right panel: the percentage of cells with comet tails at the indicated time points was quantified as in a. Cells scored as comet tail-positive are indicated with red arrows in the 72h time frame. cLeft and middle panel: SUM159 cells (left) and BT549 cells (middle) were treated with Dox as described in Fig. 1a. Nuclear protein from parental and chemo-residual cells was extracted. Equivalent amounts were immunoblotted with phospho (Ser 2056)-DNA-PKCS, DNA-PKCS or Lamin A antibody. Right panel: protein bands from three independent trials (SUM159 cells treated with Dox as described in Fig. 1a) were quantified, and the relative ratio of DNA-PKCS to loading control is shown. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig5: Chemo-residual tumor cells exhibit higher DNA repair capability and increased expression/phosphorylation of DNA-dependent protein kinase (DNA-PKCS). aLeft panel: SUM159 chemo-residual cells and parental (untreated) cells were re-challenged with doxorubicin (Dox) (1 μg/ml) for 3 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 at each time point. Cells scored as comet tail-positive are indicated with red arrows in the 72-h (72h) time frame. Right panel: the percentage of cells with comet tails at the indicated time points was quantified with a fluorescence microscope. Error bars represent SD, n = 3 fields. Significance of data points at 24h, 48h and 72 h was determined relative to data reported at 0h for the indicated cell population (*p <0.05, **p <0.01, ***p <0.001, two-tailed Student’s t test). Cells scored as comet tail-positive are indicated with red arrows in the 72h time frame. b.Left panel: BT549 chemo-residual cells and parental (untreated) cells were challenged with Dox (0.5 μg/ml) for 4 h. DNA damage was assessed at the indicated times using the neutral comet assay as in a. Right panel: the percentage of cells with comet tails at the indicated time points was quantified as in a. Cells scored as comet tail-positive are indicated with red arrows in the 72h time frame. cLeft and middle panel: SUM159 cells (left) and BT549 cells (middle) were treated with Dox as described in Fig. 1a. Nuclear protein from parental and chemo-residual cells was extracted. Equivalent amounts were immunoblotted with phospho (Ser 2056)-DNA-PKCS, DNA-PKCS or Lamin A antibody. Right panel: protein bands from three independent trials (SUM159 cells treated with Dox as described in Fig. 1a) were quantified, and the relative ratio of DNA-PKCS to loading control is shown. Error bars represent SD, n = 3, **p <0.01, two-tailed Student’s t test
Mentions: Elevated DNA repair activity is associated with chemo-resistance in many tumors [31–34]. To compare the DNA double-strand break (DSB) repair capability, we re-challenged untreated parental cells and chemo-residual cells with doxorubicin (a DNA-damaging agent) for 3 h and examined their recovery by neutral comet assay. As shown (Fig. 5a, b) the percent cells with comet tails returned to baseline quicker in chemo-residual cells than in parental cells. Similar results were observed in both SUM159 (Fig. 5a) and BT549 (Fig. 5b) chemo-resistance models. These data indicate that chemo-residual TN tumor cells from our short-term chemotherapy treatment model repaired DNA strand breaks more quickly than parental cells.Fig. 5

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