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PSMD9 expression predicts radiotherapy response in breast cancer.

Langlands FE, Dodwell D, Hanby AM, Horgan K, Millican-Slater RA, Speirs V, Verghese ET, Smith L, Hughes TA - Mol. Cancer (2014)

Bottom Line: However, this conclusion is at odds with the emerging use of proteasome inhibitors as radio-sensitizers.We examined expression of PSMD9 using immunohistochemistry in a cohort of 157 breast cancer patients, including 32 cases (20.4%) that subsequently developed local recurrences.Our conclusion is compatible with use of proteasome inhibitors as radio-sensitizers, and highlights PSMD9 as a potential target for radio-sensitizing drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leeds Institutes of Molecular Medicine, University of Leeds, Leeds, UK. medlsmi@leeds.ac.uk.

ABSTRACT

Background: More than 50% of cancer patients are recommended to receive radiotherapy. Recommendations are based mainly on clinical and pathological factors and not intrinsic tumour radio-sensitivity. Use of radiotherapy according to predictive markers would potentially reduce costly over-treatment, and improve the treatment risk-benefit ratio and cancer outcomes. Tumour expression of the 26S proteasome has been reported to predict radiotherapy response: low expression was associated with higher rates of local recurrence after radiotherapy, suggesting that low proteasome expression and activity was associated with radio-resistance. However, this conclusion is at odds with the emerging use of proteasome inhibitors as radio-sensitizers. Our aim was to further analyse the relevance of 26S proteasome expression, focussing specifically on the PSMD9 subunit, in the largest clinical cohort to date, and to investigate the functional role of PSMD9 in radio-sensitivity in breast cancer cell lines.

Methods: We examined expression of PSMD9 using immunohistochemistry in a cohort of 157 breast cancer patients, including 32 cases (20.4%) that subsequently developed local recurrences. The value of expression as a prognostic or radiotherapy predictive marker was tested using Kaplan-Meier and Cox regression analyses. PSMD9 function was examined in breast cancer cell lines MCF7 and MDA-MB-231 using siRNA knock-downs and colony forming assays after irradiation.

Results: Low tumour PSMD9 expression was significantly associated with a reduced incidence of local recurrence in patients receiving adjuvant radiotherapy (univariate log rank p = 0.02; multivariate regression p = 0.009), but not in those treated without radiotherapy, suggesting that low PSMD9 expression was associated with relative tumour radio-sensitivity. In support of this, reduction of PSMD9 expression using siRNA in breast cancer cell lines in vitro sensitized cells to radiotherapy.

Conclusions: We conclude that PSMD9 expression may predict radiotherapy benefit, with low expression indicative of relative radio-sensitivity, the opposite of previous reports relating to 26S proteasome expression. Our conclusion is compatible with use of proteasome inhibitors as radio-sensitizers, and highlights PSMD9 as a potential target for radio-sensitizing drugs.

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PSMD9 expression is functionally associated with RT response in breast cancer cells. Breast cancer cell lines were transiently transfected with siRNAs targeting PSMD9 or with non-targeting control. A PSMD9 was effectively silenced using siRNA, as demonstrated using Western blotting of MCF7 transfected lysates. B MCF7 cell growth after 48 h was unaffected by PSMD9 knock-down, as demonstrated using MTT survival/proliferation assays. C PSMD9 knock-down enhanced the efficacy of RT in breast cancer cell lines, as demonstrated using colony forming assays. Data points represent means of triplicates (+/- standard deviations).
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Figure 4: PSMD9 expression is functionally associated with RT response in breast cancer cells. Breast cancer cell lines were transiently transfected with siRNAs targeting PSMD9 or with non-targeting control. A PSMD9 was effectively silenced using siRNA, as demonstrated using Western blotting of MCF7 transfected lysates. B MCF7 cell growth after 48 h was unaffected by PSMD9 knock-down, as demonstrated using MTT survival/proliferation assays. C PSMD9 knock-down enhanced the efficacy of RT in breast cancer cell lines, as demonstrated using colony forming assays. Data points represent means of triplicates (+/- standard deviations).

Mentions: In order to determine whether PSMD9 expression was functionally associated with response to RT, we manipulated PSMD expression using siRNA in breast cancer cell lines and assessed sensitivity to RT in vitro. First, MCF7 cells were transiently transfected with siRNAs targeting PSMD9 or with a non-targeting control, and PSMD9 expression was examined using Western blotting (Figure 4A). Expression of PSMD9 was dramatically reduced by the targeted siRNAs. PSMD9 knock-down had no significant influence on cell survival/growth 48 h after transfection (Figure 4B) suggesting PSMD9 expression has little influence on the short-term survival of the bulk population of cells. Next, we performed colony-forming assays with breast cancer cell lines representative of both luminal A (MCF7) and the basal (MDA-MB-231) subtypes after transfection with PSMD9-targeting siRNA, or control, and after different doses of radiation from 0 to 10 Gy (Figure 4C). Cells surviving irradiation and maintaining sustained proliferative potential were quantified by counting individual colonies. Knock-down of PSMD9 significantly sensitized both cell lines to all doses of radiation (p < 0.05; except MCF7 cells at 4 Gy and MDA-MB-231 at 8 Gy, which did not reach statistical significance). The proportion of cells surviving RT was reduced by up to 10-fold after PSMD9 knock-down.


PSMD9 expression predicts radiotherapy response in breast cancer.

Langlands FE, Dodwell D, Hanby AM, Horgan K, Millican-Slater RA, Speirs V, Verghese ET, Smith L, Hughes TA - Mol. Cancer (2014)

PSMD9 expression is functionally associated with RT response in breast cancer cells. Breast cancer cell lines were transiently transfected with siRNAs targeting PSMD9 or with non-targeting control. A PSMD9 was effectively silenced using siRNA, as demonstrated using Western blotting of MCF7 transfected lysates. B MCF7 cell growth after 48 h was unaffected by PSMD9 knock-down, as demonstrated using MTT survival/proliferation assays. C PSMD9 knock-down enhanced the efficacy of RT in breast cancer cell lines, as demonstrated using colony forming assays. Data points represent means of triplicates (+/- standard deviations).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230020&req=5

Figure 4: PSMD9 expression is functionally associated with RT response in breast cancer cells. Breast cancer cell lines were transiently transfected with siRNAs targeting PSMD9 or with non-targeting control. A PSMD9 was effectively silenced using siRNA, as demonstrated using Western blotting of MCF7 transfected lysates. B MCF7 cell growth after 48 h was unaffected by PSMD9 knock-down, as demonstrated using MTT survival/proliferation assays. C PSMD9 knock-down enhanced the efficacy of RT in breast cancer cell lines, as demonstrated using colony forming assays. Data points represent means of triplicates (+/- standard deviations).
Mentions: In order to determine whether PSMD9 expression was functionally associated with response to RT, we manipulated PSMD expression using siRNA in breast cancer cell lines and assessed sensitivity to RT in vitro. First, MCF7 cells were transiently transfected with siRNAs targeting PSMD9 or with a non-targeting control, and PSMD9 expression was examined using Western blotting (Figure 4A). Expression of PSMD9 was dramatically reduced by the targeted siRNAs. PSMD9 knock-down had no significant influence on cell survival/growth 48 h after transfection (Figure 4B) suggesting PSMD9 expression has little influence on the short-term survival of the bulk population of cells. Next, we performed colony-forming assays with breast cancer cell lines representative of both luminal A (MCF7) and the basal (MDA-MB-231) subtypes after transfection with PSMD9-targeting siRNA, or control, and after different doses of radiation from 0 to 10 Gy (Figure 4C). Cells surviving irradiation and maintaining sustained proliferative potential were quantified by counting individual colonies. Knock-down of PSMD9 significantly sensitized both cell lines to all doses of radiation (p < 0.05; except MCF7 cells at 4 Gy and MDA-MB-231 at 8 Gy, which did not reach statistical significance). The proportion of cells surviving RT was reduced by up to 10-fold after PSMD9 knock-down.

Bottom Line: However, this conclusion is at odds with the emerging use of proteasome inhibitors as radio-sensitizers.We examined expression of PSMD9 using immunohistochemistry in a cohort of 157 breast cancer patients, including 32 cases (20.4%) that subsequently developed local recurrences.Our conclusion is compatible with use of proteasome inhibitors as radio-sensitizers, and highlights PSMD9 as a potential target for radio-sensitizing drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Leeds Institutes of Molecular Medicine, University of Leeds, Leeds, UK. medlsmi@leeds.ac.uk.

ABSTRACT

Background: More than 50% of cancer patients are recommended to receive radiotherapy. Recommendations are based mainly on clinical and pathological factors and not intrinsic tumour radio-sensitivity. Use of radiotherapy according to predictive markers would potentially reduce costly over-treatment, and improve the treatment risk-benefit ratio and cancer outcomes. Tumour expression of the 26S proteasome has been reported to predict radiotherapy response: low expression was associated with higher rates of local recurrence after radiotherapy, suggesting that low proteasome expression and activity was associated with radio-resistance. However, this conclusion is at odds with the emerging use of proteasome inhibitors as radio-sensitizers. Our aim was to further analyse the relevance of 26S proteasome expression, focussing specifically on the PSMD9 subunit, in the largest clinical cohort to date, and to investigate the functional role of PSMD9 in radio-sensitivity in breast cancer cell lines.

Methods: We examined expression of PSMD9 using immunohistochemistry in a cohort of 157 breast cancer patients, including 32 cases (20.4%) that subsequently developed local recurrences. The value of expression as a prognostic or radiotherapy predictive marker was tested using Kaplan-Meier and Cox regression analyses. PSMD9 function was examined in breast cancer cell lines MCF7 and MDA-MB-231 using siRNA knock-downs and colony forming assays after irradiation.

Results: Low tumour PSMD9 expression was significantly associated with a reduced incidence of local recurrence in patients receiving adjuvant radiotherapy (univariate log rank p = 0.02; multivariate regression p = 0.009), but not in those treated without radiotherapy, suggesting that low PSMD9 expression was associated with relative tumour radio-sensitivity. In support of this, reduction of PSMD9 expression using siRNA in breast cancer cell lines in vitro sensitized cells to radiotherapy.

Conclusions: We conclude that PSMD9 expression may predict radiotherapy benefit, with low expression indicative of relative radio-sensitivity, the opposite of previous reports relating to 26S proteasome expression. Our conclusion is compatible with use of proteasome inhibitors as radio-sensitizers, and highlights PSMD9 as a potential target for radio-sensitizing drugs.

Show MeSH
Related in: MedlinePlus