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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: 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.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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MiR-100 impairs CSC properties by down-regulating Plk1A, Western blot analysis of Plk1 expression in wt, scramble or miR-100 transduced BrCSCs; a total protein lysate of HeLa cells was used as a positive control. B, colony forming efficiency of wt and miR-100 expressing BrCSCs transduced with either an empty vector (mock) or Plk1, assessed by soft agar assay; histogram shows the quantitative analysis. Data are average + SD of 3 independent experiments. C, representative fluorescence microscopy images of BrCSCs transduced as in (B) and labelled with PKH-26 (upper left). Flow cytometry analysis of PKH-26 in cells transduced as in (B) after 14 days of culture (bottom left) and the corresponding quantification (right). D, Analysis of BrCSC mortality in bulk and CD49fhigh/CD24low sorted BrCSCs upon treatment with the Plk1 inhibitor BI2536 (10nM) for 72 hours. The experiments were performed in triplicates. UT: untreated.
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Figure 5: MiR-100 impairs CSC properties by down-regulating Plk1A, Western blot analysis of Plk1 expression in wt, scramble or miR-100 transduced BrCSCs; a total protein lysate of HeLa cells was used as a positive control. B, colony forming efficiency of wt and miR-100 expressing BrCSCs transduced with either an empty vector (mock) or Plk1, assessed by soft agar assay; histogram shows the quantitative analysis. Data are average + SD of 3 independent experiments. C, representative fluorescence microscopy images of BrCSCs transduced as in (B) and labelled with PKH-26 (upper left). Flow cytometry analysis of PKH-26 in cells transduced as in (B) after 14 days of culture (bottom left) and the corresponding quantification (right). D, Analysis of BrCSC mortality in bulk and CD49fhigh/CD24low sorted BrCSCs upon treatment with the Plk1 inhibitor BI2536 (10nM) for 72 hours. The experiments were performed in triplicates. UT: untreated.

Mentions: In an attempt to untangle the molecular mechanisms underlying miR-100 induced phenotype, the expression of Plk1, a known miR-100 target gene recently shown to be involved in the regulation of stem cell proliferation and differentiation [33, 34], was analyzed. MiR-100 transduced BrCSCs displayed a significant reduction of Plk1 protein (Fig. 5A). Rescue experiments were performed by re-introducing Plk1 in miR-100 expressing BrCSCs and evaluating their self-renewing ability. Upon Plk1 expression, colony forming efficiency was partially recovered (Fig. 5B), while self-renewal was restored at a level comparable to wild type BrCSCs (Fig. 5C). Consistently, the Plk1 inhibitor BI2536 impaired viability both in the bulk population of BrCSCs and in the CD49fhigh/CD24low sorted cells (Fig. 5D). These results indicate that Plk1 plays a key role in mediating miR-100 induced phenotype.


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)

MiR-100 impairs CSC properties by down-regulating Plk1A, Western blot analysis of Plk1 expression in wt, scramble or miR-100 transduced BrCSCs; a total protein lysate of HeLa cells was used as a positive control. B, colony forming efficiency of wt and miR-100 expressing BrCSCs transduced with either an empty vector (mock) or Plk1, assessed by soft agar assay; histogram shows the quantitative analysis. Data are average + SD of 3 independent experiments. C, representative fluorescence microscopy images of BrCSCs transduced as in (B) and labelled with PKH-26 (upper left). Flow cytometry analysis of PKH-26 in cells transduced as in (B) after 14 days of culture (bottom left) and the corresponding quantification (right). D, Analysis of BrCSC mortality in bulk and CD49fhigh/CD24low sorted BrCSCs upon treatment with the Plk1 inhibitor BI2536 (10nM) for 72 hours. The experiments were performed in triplicates. UT: untreated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385854&req=5

Figure 5: MiR-100 impairs CSC properties by down-regulating Plk1A, Western blot analysis of Plk1 expression in wt, scramble or miR-100 transduced BrCSCs; a total protein lysate of HeLa cells was used as a positive control. B, colony forming efficiency of wt and miR-100 expressing BrCSCs transduced with either an empty vector (mock) or Plk1, assessed by soft agar assay; histogram shows the quantitative analysis. Data are average + SD of 3 independent experiments. C, representative fluorescence microscopy images of BrCSCs transduced as in (B) and labelled with PKH-26 (upper left). Flow cytometry analysis of PKH-26 in cells transduced as in (B) after 14 days of culture (bottom left) and the corresponding quantification (right). D, Analysis of BrCSC mortality in bulk and CD49fhigh/CD24low sorted BrCSCs upon treatment with the Plk1 inhibitor BI2536 (10nM) for 72 hours. The experiments were performed in triplicates. UT: untreated.
Mentions: In an attempt to untangle the molecular mechanisms underlying miR-100 induced phenotype, the expression of Plk1, a known miR-100 target gene recently shown to be involved in the regulation of stem cell proliferation and differentiation [33, 34], was analyzed. MiR-100 transduced BrCSCs displayed a significant reduction of Plk1 protein (Fig. 5A). Rescue experiments were performed by re-introducing Plk1 in miR-100 expressing BrCSCs and evaluating their self-renewing ability. Upon Plk1 expression, colony forming efficiency was partially recovered (Fig. 5B), while self-renewal was restored at a level comparable to wild type BrCSCs (Fig. 5C). Consistently, the Plk1 inhibitor BI2536 impaired viability both in the bulk population of BrCSCs and in the CD49fhigh/CD24low sorted cells (Fig. 5D). These results indicate that Plk1 plays a key role in mediating miR-100 induced phenotype.

Bottom Line: 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.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