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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

Selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) decreases the survival of chemo-residual tumor cells and subsequent colony formation. a SUM159 cells were treated with doxorubicin (1 μg/ml) plus dimethyl sulfoxide (DMSO) or a selective DNA-PK inhibitor (NU7441), at a non-cytotoxic concentration [36] (1 μM or 5 μM) for 2 days, as described in Fig. 1a. Fresh medium was added after treatment removal. Upper panel: pictures of chemo-residual cells were taken on day 7. Magnification ×40. Lower panel: Chemo-residual cell number on day 7 was determined by trypan blue exclusion. Error bars represent SD, n = 3, *p <0.05, **p <0.01, two-tailed Student’s t test. b Colonies (containing >50 cells) were quantified on days 14, 16, and 18, respectively. Error bars represent SD, n = 3. Significance was determined relative to DMSO-treated cells at each time point using the two-tailed Student’s t test (*p <0.05, ***p <0.001). Similar results were obtained in at least three independent trials
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Fig6: Selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) decreases the survival of chemo-residual tumor cells and subsequent colony formation. a SUM159 cells were treated with doxorubicin (1 μg/ml) plus dimethyl sulfoxide (DMSO) or a selective DNA-PK inhibitor (NU7441), at a non-cytotoxic concentration [36] (1 μM or 5 μM) for 2 days, as described in Fig. 1a. Fresh medium was added after treatment removal. Upper panel: pictures of chemo-residual cells were taken on day 7. Magnification ×40. Lower panel: Chemo-residual cell number on day 7 was determined by trypan blue exclusion. Error bars represent SD, n = 3, *p <0.05, **p <0.01, two-tailed Student’s t test. b Colonies (containing >50 cells) were quantified on days 14, 16, and 18, respectively. Error bars represent SD, n = 3. Significance was determined relative to DMSO-treated cells at each time point using the two-tailed Student’s t test (*p <0.05, ***p <0.001). Similar results were obtained in at least three independent trials

Mentions: NU7441 is a specific inhibitor of DNA-PK with 100-fold selectivity for DNA-PK, compared to other PI3K kinase family members [36, 37]. To determine whether DNA-PK inhibition reduces TN chemo-residual tumor cell survival and regrowth, we simultaneously treated SUM159 TN breast tumor cells with doxorubicin and NU7441 at either of two non-cytotoxic concentrations [36]. NU7441 significantly decreased the number of chemo-residual cells (Fig. 6a) and subsequent colony formation (Fig. 6b) in a concentration-dependent manner. Previous preclinical studies indicate that the DNA-PK inhibitor NU7441 synergizes with chemotherapy to reduce tumor growth in a colon cancer model [36]. Our results suggest the importance of testing the efficacy of combination therapy (NU7441 + chemotherapy) for TNBC in future preclinical studies.Fig. 6


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)

Selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) decreases the survival of chemo-residual tumor cells and subsequent colony formation. a SUM159 cells were treated with doxorubicin (1 μg/ml) plus dimethyl sulfoxide (DMSO) or a selective DNA-PK inhibitor (NU7441), at a non-cytotoxic concentration [36] (1 μM or 5 μM) for 2 days, as described in Fig. 1a. Fresh medium was added after treatment removal. Upper panel: pictures of chemo-residual cells were taken on day 7. Magnification ×40. Lower panel: Chemo-residual cell number on day 7 was determined by trypan blue exclusion. Error bars represent SD, n = 3, *p <0.05, **p <0.01, two-tailed Student’s t test. b Colonies (containing >50 cells) were quantified on days 14, 16, and 18, respectively. Error bars represent SD, n = 3. Significance was determined relative to DMSO-treated cells at each time point using the two-tailed Student’s t test (*p <0.05, ***p <0.001). Similar results were obtained in at least three independent trials
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491247&req=5

Fig6: Selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) decreases the survival of chemo-residual tumor cells and subsequent colony formation. a SUM159 cells were treated with doxorubicin (1 μg/ml) plus dimethyl sulfoxide (DMSO) or a selective DNA-PK inhibitor (NU7441), at a non-cytotoxic concentration [36] (1 μM or 5 μM) for 2 days, as described in Fig. 1a. Fresh medium was added after treatment removal. Upper panel: pictures of chemo-residual cells were taken on day 7. Magnification ×40. Lower panel: Chemo-residual cell number on day 7 was determined by trypan blue exclusion. Error bars represent SD, n = 3, *p <0.05, **p <0.01, two-tailed Student’s t test. b Colonies (containing >50 cells) were quantified on days 14, 16, and 18, respectively. Error bars represent SD, n = 3. Significance was determined relative to DMSO-treated cells at each time point using the two-tailed Student’s t test (*p <0.05, ***p <0.001). Similar results were obtained in at least three independent trials
Mentions: NU7441 is a specific inhibitor of DNA-PK with 100-fold selectivity for DNA-PK, compared to other PI3K kinase family members [36, 37]. To determine whether DNA-PK inhibition reduces TN chemo-residual tumor cell survival and regrowth, we simultaneously treated SUM159 TN breast tumor cells with doxorubicin and NU7441 at either of two non-cytotoxic concentrations [36]. NU7441 significantly decreased the number of chemo-residual cells (Fig. 6a) and subsequent colony formation (Fig. 6b) in a concentration-dependent manner. Previous preclinical studies indicate that the DNA-PK inhibitor NU7441 synergizes with chemotherapy to reduce tumor growth in a colon cancer model [36]. Our results suggest the importance of testing the efficacy of combination therapy (NU7441 + chemotherapy) for TNBC in future preclinical studies.Fig. 6

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