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By promoting cell differentiation, miR-100 sensitizes basal-like breast cancer stem cells to hormonal therapy.

Petrelli A, Carollo R, Cargnelutti M, Iovino F, Callari M, Cimino D, Todaro M, Mangiapane LR, Giammona A, Cordova A, Montemurro F, Taverna D, Daidone MG, Stassi G, Giordano S - Oncotarget (2015)

Bottom Line: Here we show that miR-100 inhibits maintenance and expansion of BrCSCs in basal-like cancer through Polo-like kinase1 (Plk1) down-regulation.Moreover, miR-100 favors BrCSC differentiation, converting a basal like phenotype into luminal.Our findings indicate a new possible therapeutic strategy, which could make aggressive breast cancers responsive to standard treatments.

View Article: PubMed Central - PubMed

Affiliation: University of Torino School of Medicine, Candiolo Cancer Institute-FPO, IRCCS, Str. Provinciale, Candiolo, Torino, Italy.

ABSTRACT
Basal-like breast cancer is an aggressive tumor subtype with a poor response to conventional therapies. Tumor formation and relapse are sustained by a cell subset of Breast Cancer Stem Cells (BrCSCs). Here we show that miR-100 inhibits maintenance and expansion of BrCSCs in basal-like cancer through Polo-like kinase1 (Plk1) down-regulation. Moreover, miR-100 favors BrCSC differentiation, converting a basal like phenotype into luminal. It induces the expression of a functional estrogen receptor (ER) and renders basal-like BrCSCs responsive to hormonal therapy. The key role played by miR-100 in breast cancer free-survival is confirmed by the analysis of a cohort of patients' tumors, which shows that low expression of miR-100 is a negative prognostic factor and is associated with gene signatures of high grade undifferentiated tumors. Our findings indicate a new possible therapeutic strategy, which could make aggressive breast cancers responsive to standard treatments.

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Ectopic expression of miR-100 reduces stem cell markers, promotes luminal differentiation and renders basal-like BrCSCs responsive to endocrine therapyA, representative confocal microscopy images of immunofluorescence (IF) analysis of ALDH1, Cytokeratins (CK5, CK14, CK8-18) and estrogen receptor (ER) performed in BrCSCs (P5) wt and stably expressing either a control scramble or miR-100. Nuclei were counterstained by Toto-3 (blue). Magnification 40x. B, quantification of the IF staining shown in (A), performed in three independent replicates. ** P<0.01 *** P<0.001. C, representative FACS analysis of Aldefluor assay performed in wt, scramble and miR-100 BrCSCs. Cells were exposed to Aldefluor substrate (BAAA); cells treated with the specific inhibitor of ALDH1 (DEAB) are shown in the insert panels and were used to define the population with low and high (gated region) ALDH1 activity. D, representative analysis of ER-dependent transcriptional activity. The assay was performed in wt, scramble and miR-100 BrCSCs, non transfected (negative control) or transfected (ERE-reporter) with a construct where an Estrogen Responsive Element (ERE) containing promoter drives luciferase expression. Luciferase activity was evaluated in the absence or in the presence of 10nM 17-β-estradiol (E2). MCF7 cells were used as positive control of response to estradiol. E, Analysis of BrCSC viability upon treatment with tamoxifen (tam) and fulvestrant (fulv) at the indicated doses. The experiments were performed in triplicates. ** P<0.01.
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Figure 7: Ectopic expression of miR-100 reduces stem cell markers, promotes luminal differentiation and renders basal-like BrCSCs responsive to endocrine therapyA, representative confocal microscopy images of immunofluorescence (IF) analysis of ALDH1, Cytokeratins (CK5, CK14, CK8-18) and estrogen receptor (ER) performed in BrCSCs (P5) wt and stably expressing either a control scramble or miR-100. Nuclei were counterstained by Toto-3 (blue). Magnification 40x. B, quantification of the IF staining shown in (A), performed in three independent replicates. ** P<0.01 *** P<0.001. C, representative FACS analysis of Aldefluor assay performed in wt, scramble and miR-100 BrCSCs. Cells were exposed to Aldefluor substrate (BAAA); cells treated with the specific inhibitor of ALDH1 (DEAB) are shown in the insert panels and were used to define the population with low and high (gated region) ALDH1 activity. D, representative analysis of ER-dependent transcriptional activity. The assay was performed in wt, scramble and miR-100 BrCSCs, non transfected (negative control) or transfected (ERE-reporter) with a construct where an Estrogen Responsive Element (ERE) containing promoter drives luciferase expression. Luciferase activity was evaluated in the absence or in the presence of 10nM 17-β-estradiol (E2). MCF7 cells were used as positive control of response to estradiol. E, Analysis of BrCSC viability upon treatment with tamoxifen (tam) and fulvestrant (fulv) at the indicated doses. The experiments were performed in triplicates. ** P<0.01.

Mentions: To further validate the role of miR-100 in controlling stemness and differentiation of breast cancer cells, we evaluated either by flow cytometry or immunofluorescence (IF) the expression of putative stem/progenitor and differentiation markers upon ectopic expression of the miRNA. FACS analysis showed that stem cell markers, such as CD44, CD10 and CD49f, were drastically reduced, while the differentiation markers CD24 and EpCAM increased (Fig. 6). We also assessed the expression of additional mammary stem/progenitor markers by immunofluorescence analysis. Early progenitor/stemness markers such as ALDH1, Cytokeratin 5 and myoepithelial Cytokeratin 14 were reduced in miR-100 transduced BrCSCs; conversely, the luminal epithelial markers Cytokeratin 8-18 and ER were de novo expressed (Fig. 7A, B and Supplementary Fig. 7A, B). Expression of ER upon miR-100 transduction was confirmed by FACS analysis as well (Supplementary Fig. 8A). Using the Aldefluor assay, we found that ALDH1 activity was also greatly reduced (Fig. 7C and Supplementary Fig. 7C). Furthermore, the luminal differentiation promoted by miR-100 was observed in CD49fhigh/CD24low sorted BrCSCs as well (Supplementary Fig. 8B), confirming that miR-100 not only interferes with stemness maintenance, but also converts the breast cancer phenotype from basal to luminal-like.


By promoting cell differentiation, miR-100 sensitizes basal-like breast cancer stem cells to hormonal therapy.

Petrelli A, Carollo R, Cargnelutti M, Iovino F, Callari M, Cimino D, Todaro M, Mangiapane LR, Giammona A, Cordova A, Montemurro F, Taverna D, Daidone MG, Stassi G, Giordano S - Oncotarget (2015)

Ectopic expression of miR-100 reduces stem cell markers, promotes luminal differentiation and renders basal-like BrCSCs responsive to endocrine therapyA, representative confocal microscopy images of immunofluorescence (IF) analysis of ALDH1, Cytokeratins (CK5, CK14, CK8-18) and estrogen receptor (ER) performed in BrCSCs (P5) wt and stably expressing either a control scramble or miR-100. Nuclei were counterstained by Toto-3 (blue). Magnification 40x. B, quantification of the IF staining shown in (A), performed in three independent replicates. ** P<0.01 *** P<0.001. C, representative FACS analysis of Aldefluor assay performed in wt, scramble and miR-100 BrCSCs. Cells were exposed to Aldefluor substrate (BAAA); cells treated with the specific inhibitor of ALDH1 (DEAB) are shown in the insert panels and were used to define the population with low and high (gated region) ALDH1 activity. D, representative analysis of ER-dependent transcriptional activity. The assay was performed in wt, scramble and miR-100 BrCSCs, non transfected (negative control) or transfected (ERE-reporter) with a construct where an Estrogen Responsive Element (ERE) containing promoter drives luciferase expression. Luciferase activity was evaluated in the absence or in the presence of 10nM 17-β-estradiol (E2). MCF7 cells were used as positive control of response to estradiol. E, Analysis of BrCSC viability upon treatment with tamoxifen (tam) and fulvestrant (fulv) at the indicated doses. The experiments were performed in triplicates. ** P<0.01.
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Figure 7: Ectopic expression of miR-100 reduces stem cell markers, promotes luminal differentiation and renders basal-like BrCSCs responsive to endocrine therapyA, representative confocal microscopy images of immunofluorescence (IF) analysis of ALDH1, Cytokeratins (CK5, CK14, CK8-18) and estrogen receptor (ER) performed in BrCSCs (P5) wt and stably expressing either a control scramble or miR-100. Nuclei were counterstained by Toto-3 (blue). Magnification 40x. B, quantification of the IF staining shown in (A), performed in three independent replicates. ** P<0.01 *** P<0.001. C, representative FACS analysis of Aldefluor assay performed in wt, scramble and miR-100 BrCSCs. Cells were exposed to Aldefluor substrate (BAAA); cells treated with the specific inhibitor of ALDH1 (DEAB) are shown in the insert panels and were used to define the population with low and high (gated region) ALDH1 activity. D, representative analysis of ER-dependent transcriptional activity. The assay was performed in wt, scramble and miR-100 BrCSCs, non transfected (negative control) or transfected (ERE-reporter) with a construct where an Estrogen Responsive Element (ERE) containing promoter drives luciferase expression. Luciferase activity was evaluated in the absence or in the presence of 10nM 17-β-estradiol (E2). MCF7 cells were used as positive control of response to estradiol. E, Analysis of BrCSC viability upon treatment with tamoxifen (tam) and fulvestrant (fulv) at the indicated doses. The experiments were performed in triplicates. ** P<0.01.
Mentions: To further validate the role of miR-100 in controlling stemness and differentiation of breast cancer cells, we evaluated either by flow cytometry or immunofluorescence (IF) the expression of putative stem/progenitor and differentiation markers upon ectopic expression of the miRNA. FACS analysis showed that stem cell markers, such as CD44, CD10 and CD49f, were drastically reduced, while the differentiation markers CD24 and EpCAM increased (Fig. 6). We also assessed the expression of additional mammary stem/progenitor markers by immunofluorescence analysis. Early progenitor/stemness markers such as ALDH1, Cytokeratin 5 and myoepithelial Cytokeratin 14 were reduced in miR-100 transduced BrCSCs; conversely, the luminal epithelial markers Cytokeratin 8-18 and ER were de novo expressed (Fig. 7A, B and Supplementary Fig. 7A, B). Expression of ER upon miR-100 transduction was confirmed by FACS analysis as well (Supplementary Fig. 8A). Using the Aldefluor assay, we found that ALDH1 activity was also greatly reduced (Fig. 7C and Supplementary Fig. 7C). Furthermore, the luminal differentiation promoted by miR-100 was observed in CD49fhigh/CD24low sorted BrCSCs as well (Supplementary Fig. 8B), confirming that miR-100 not only interferes with stemness maintenance, but also converts the breast cancer phenotype from basal to luminal-like.

Bottom Line: Here we show that miR-100 inhibits maintenance and expansion of BrCSCs in basal-like cancer through Polo-like kinase1 (Plk1) down-regulation.Moreover, miR-100 favors BrCSC differentiation, converting a basal like phenotype into luminal.Our findings indicate a new possible therapeutic strategy, which could make aggressive breast cancers responsive to standard treatments.

View Article: PubMed Central - PubMed

Affiliation: University of Torino School of Medicine, Candiolo Cancer Institute-FPO, IRCCS, Str. Provinciale, Candiolo, Torino, Italy.

ABSTRACT
Basal-like breast cancer is an aggressive tumor subtype with a poor response to conventional therapies. Tumor formation and relapse are sustained by a cell subset of Breast Cancer Stem Cells (BrCSCs). Here we show that miR-100 inhibits maintenance and expansion of BrCSCs in basal-like cancer through Polo-like kinase1 (Plk1) down-regulation. Moreover, miR-100 favors BrCSC differentiation, converting a basal like phenotype into luminal. It induces the expression of a functional estrogen receptor (ER) and renders basal-like BrCSCs responsive to hormonal therapy. The key role played by miR-100 in breast cancer free-survival is confirmed by the analysis of a cohort of patients' tumors, which shows that low expression of miR-100 is a negative prognostic factor and is associated with gene signatures of high grade undifferentiated tumors. Our findings indicate a new possible therapeutic strategy, which could make aggressive breast cancers responsive to standard treatments.

Show MeSH
Related in: MedlinePlus