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Breast cancers with high DSS1 expression that potentially maintains BRCA2 stability have poor prognosis in the relapse-free survival.

Rezano A, Kuwahara K, Yamamoto-Ibusuki M, Kitabatake M, Moolthiya P, Phimsen S, Suda T, Tone S, Yamamoto Y, Iwase H, Sakaguchi N - BMC Cancer (2013)

Bottom Line: Patient survival was compared using Kaplan-Meier method.DSS1 high-expression reduces the susceptibility of MCF7 cells to DNA-damaging drugs, as observed in cell cycle and apoptosis analyses.DSS1 is necessary to rescue cells from DNA damage, but high DSS1 expression increases drug resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1, Honjo, Chuo-ku, Kumamoto 860-8556, Japan. nobusaka@kumamoto-u.ac.jp.

ABSTRACT

Background: Genetic BRCA2 insufficiency is associated with breast cancer development; however, in sporadic breast cancer cases, high BRCA2 expression is paradoxically correlated with poor prognosis. Because DSS1, a mammalian component of the transcription/RNA export complex, is known to stabilize BRCA2, we investigated how the expression of DSS1 is associated with clinical parameters in breast cancers.

Methods: DSS1 mRNA and p53 protein were examined by RT-PCR and immunohistochemical staining of breast cancer specimens to classify DSS1(high) and DSS1(low) or p53(high) and p53(low) groups. Patient survival was compared using Kaplan-Meier method. DSS1(high) or DSS1(low) breast cancer cells were prepared by retroviral cDNA transfection or DSS1 siRNA on proliferation, cell cycle progression, and survival by flow cytometric analyses with or without anti-cancer drugs.

Results: In comparison to patients with low levels of DSS1, high-DSS1 patients showed a poorer prognosis, with respect to relapse-free survival period. The effect of DSS1 was examined in breast cancer cells in vitro. DSS1 high-expression reduces the susceptibility of MCF7 cells to DNA-damaging drugs, as observed in cell cycle and apoptosis analyses. DSS1 knockdown, however, increased the susceptibility to the DNA-damaging drugs camptothecin and etoposide and caused early apoptosis in p53 wild type MCF7 and p53-insufficient MDA-MB-231 cells. DSS1 knockdown suppresses the proliferation of drug-resistant MDA-MB-231 breast cancer cells, particularly effectively in combination with DNA-damaging agents.

Conclusion: Breast cancers with high DSS1 expression have worse prognosis and shorter relapse-free survival times. DSS1 is necessary to rescue cells from DNA damage, but high DSS1 expression increases drug resistance. We suggest that DSS1 expression could be a useful marker for drug resistance in breast cancers, and DSS1 knockdown can induce tumor apoptosis when used in combination with DNA-damaging drugs.

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Effect of DSS1 over-expression on sensitivity to DNA damage. Effect of DSS1 over-expression on drug-induced DNA injury was measured using the alkaline comet assay after treatment of cells with CPT (50 μM) for 3 hr. DMSO was used as a solvent control. The DNA content pattern observed under fluorescent microscopy is shown (left), and the DNA damage rate was measured as the tail moment in the fluorescence image using CometScore software. DSS-over-expressing MCF7 (A) or MDA-MB-231 (B) cells were compared with GFP-expressing controls. Data obtained by counting more than 100 cells are shown as a graph, which represents more than three independent experiments (right). Statistical significance was calculated using Student’s t-test, with **P < 0.01 as significant. Scale bar indicates 100 μm.
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Figure 3: Effect of DSS1 over-expression on sensitivity to DNA damage. Effect of DSS1 over-expression on drug-induced DNA injury was measured using the alkaline comet assay after treatment of cells with CPT (50 μM) for 3 hr. DMSO was used as a solvent control. The DNA content pattern observed under fluorescent microscopy is shown (left), and the DNA damage rate was measured as the tail moment in the fluorescence image using CometScore software. DSS-over-expressing MCF7 (A) or MDA-MB-231 (B) cells were compared with GFP-expressing controls. Data obtained by counting more than 100 cells are shown as a graph, which represents more than three independent experiments (right). Statistical significance was calculated using Student’s t-test, with **P < 0.01 as significant. Scale bar indicates 100 μm.

Mentions: For molecular analysis, we used two breast cancer cell lines, MCF7 (ERα+PgR+ with wild type p53) and MDA-MB-231 (ERα-PgR- with p53 mutation). The effect of DSS1 over-expression on cell proliferation and cell cycle progression was examined in breast cancer cells that had been engineered to over-express DSS1 (MCF7DSS1 or MDA-MB-231DSS1) by retroviral transfection (Additional file2: Figure S2). Stable transfectants that expressed high levels of DSS1 mRNA did not show any changes in cell cycle or cell proliferation compared with the control (GFP only) transfectants (Figure 2A). DSS1-over-expressing cells showed no abnormalities in proliferation. Trastuzumab is a first choice for treatment of HER2-positive breast cancer in our cohort, but camptothecin (CPT) is often chosen as a regular regimen for HER2-negative breast cancer cases (Table 1). The susceptibility of DSS1-over-expressing breast cancer cells to the DNA-damage inducing agents CPT and etoposide (ETP) was examined. DSS1 over-expression renders breast cancer cells more resistant to treatment with CPT, an inhibitor of type 1 topoisomerase (which cleaves one strand of double-stranded DNA), compared with the GFP-control MCF7 transfectants (Figure 2B), while no change was detected in DSS1 over-expressing MDA-MB-231 transfectants (Figure 2C). The effect of DSS1 over-expression was masked in both MCF7 and MDA-MB-231 cells upon exposure to ETP, an inhibitor of topoisomerase II (which cleaves two strands of double-stranded DNA and induces cell cycle arrest at G2/M) (Additional file3: Figure S3A and B). Therefore, DNA damage was examined using the alkaline comet assay (Figure 3A and B). DSS1 over-expression reduced CPT-induced DNA damage in MCF7 and MDA-MB-231 cells, suggesting that DSS1 over-expression increases drug resistance in breast cancers.


Breast cancers with high DSS1 expression that potentially maintains BRCA2 stability have poor prognosis in the relapse-free survival.

Rezano A, Kuwahara K, Yamamoto-Ibusuki M, Kitabatake M, Moolthiya P, Phimsen S, Suda T, Tone S, Yamamoto Y, Iwase H, Sakaguchi N - BMC Cancer (2013)

Effect of DSS1 over-expression on sensitivity to DNA damage. Effect of DSS1 over-expression on drug-induced DNA injury was measured using the alkaline comet assay after treatment of cells with CPT (50 μM) for 3 hr. DMSO was used as a solvent control. The DNA content pattern observed under fluorescent microscopy is shown (left), and the DNA damage rate was measured as the tail moment in the fluorescence image using CometScore software. DSS-over-expressing MCF7 (A) or MDA-MB-231 (B) cells were compared with GFP-expressing controls. Data obtained by counting more than 100 cells are shown as a graph, which represents more than three independent experiments (right). Statistical significance was calculated using Student’s t-test, with **P < 0.01 as significant. Scale bar indicates 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219476&req=5

Figure 3: Effect of DSS1 over-expression on sensitivity to DNA damage. Effect of DSS1 over-expression on drug-induced DNA injury was measured using the alkaline comet assay after treatment of cells with CPT (50 μM) for 3 hr. DMSO was used as a solvent control. The DNA content pattern observed under fluorescent microscopy is shown (left), and the DNA damage rate was measured as the tail moment in the fluorescence image using CometScore software. DSS-over-expressing MCF7 (A) or MDA-MB-231 (B) cells were compared with GFP-expressing controls. Data obtained by counting more than 100 cells are shown as a graph, which represents more than three independent experiments (right). Statistical significance was calculated using Student’s t-test, with **P < 0.01 as significant. Scale bar indicates 100 μm.
Mentions: For molecular analysis, we used two breast cancer cell lines, MCF7 (ERα+PgR+ with wild type p53) and MDA-MB-231 (ERα-PgR- with p53 mutation). The effect of DSS1 over-expression on cell proliferation and cell cycle progression was examined in breast cancer cells that had been engineered to over-express DSS1 (MCF7DSS1 or MDA-MB-231DSS1) by retroviral transfection (Additional file2: Figure S2). Stable transfectants that expressed high levels of DSS1 mRNA did not show any changes in cell cycle or cell proliferation compared with the control (GFP only) transfectants (Figure 2A). DSS1-over-expressing cells showed no abnormalities in proliferation. Trastuzumab is a first choice for treatment of HER2-positive breast cancer in our cohort, but camptothecin (CPT) is often chosen as a regular regimen for HER2-negative breast cancer cases (Table 1). The susceptibility of DSS1-over-expressing breast cancer cells to the DNA-damage inducing agents CPT and etoposide (ETP) was examined. DSS1 over-expression renders breast cancer cells more resistant to treatment with CPT, an inhibitor of type 1 topoisomerase (which cleaves one strand of double-stranded DNA), compared with the GFP-control MCF7 transfectants (Figure 2B), while no change was detected in DSS1 over-expressing MDA-MB-231 transfectants (Figure 2C). The effect of DSS1 over-expression was masked in both MCF7 and MDA-MB-231 cells upon exposure to ETP, an inhibitor of topoisomerase II (which cleaves two strands of double-stranded DNA and induces cell cycle arrest at G2/M) (Additional file3: Figure S3A and B). Therefore, DNA damage was examined using the alkaline comet assay (Figure 3A and B). DSS1 over-expression reduced CPT-induced DNA damage in MCF7 and MDA-MB-231 cells, suggesting that DSS1 over-expression increases drug resistance in breast cancers.

Bottom Line: Patient survival was compared using Kaplan-Meier method.DSS1 high-expression reduces the susceptibility of MCF7 cells to DNA-damaging drugs, as observed in cell cycle and apoptosis analyses.DSS1 is necessary to rescue cells from DNA damage, but high DSS1 expression increases drug resistance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1, Honjo, Chuo-ku, Kumamoto 860-8556, Japan. nobusaka@kumamoto-u.ac.jp.

ABSTRACT

Background: Genetic BRCA2 insufficiency is associated with breast cancer development; however, in sporadic breast cancer cases, high BRCA2 expression is paradoxically correlated with poor prognosis. Because DSS1, a mammalian component of the transcription/RNA export complex, is known to stabilize BRCA2, we investigated how the expression of DSS1 is associated with clinical parameters in breast cancers.

Methods: DSS1 mRNA and p53 protein were examined by RT-PCR and immunohistochemical staining of breast cancer specimens to classify DSS1(high) and DSS1(low) or p53(high) and p53(low) groups. Patient survival was compared using Kaplan-Meier method. DSS1(high) or DSS1(low) breast cancer cells were prepared by retroviral cDNA transfection or DSS1 siRNA on proliferation, cell cycle progression, and survival by flow cytometric analyses with or without anti-cancer drugs.

Results: In comparison to patients with low levels of DSS1, high-DSS1 patients showed a poorer prognosis, with respect to relapse-free survival period. The effect of DSS1 was examined in breast cancer cells in vitro. DSS1 high-expression reduces the susceptibility of MCF7 cells to DNA-damaging drugs, as observed in cell cycle and apoptosis analyses. DSS1 knockdown, however, increased the susceptibility to the DNA-damaging drugs camptothecin and etoposide and caused early apoptosis in p53 wild type MCF7 and p53-insufficient MDA-MB-231 cells. DSS1 knockdown suppresses the proliferation of drug-resistant MDA-MB-231 breast cancer cells, particularly effectively in combination with DNA-damaging agents.

Conclusion: Breast cancers with high DSS1 expression have worse prognosis and shorter relapse-free survival times. DSS1 is necessary to rescue cells from DNA damage, but high DSS1 expression increases drug resistance. We suggest that DSS1 expression could be a useful marker for drug resistance in breast cancers, and DSS1 knockdown can induce tumor apoptosis when used in combination with DNA-damaging drugs.

Show MeSH
Related in: MedlinePlus