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AurkA controls self-renewal of breast cancer-initiating cells promoting wnt3a stabilization through suppression of miR-128.

Eterno V, Zambelli A, Villani L, Tuscano A, Manera S, Spitaleri A, Pavesi L, Amato A - Sci Rep (2016)

Bottom Line: In our study, AurkA knock-down reduced wnt3a mRNA and suppressed metastatic signature of MDA-MB-231 cells.As a consequence, the amount of BCICs and their migratory capability dramatically decreased.In addition, it suggests a new therapeutic strategy taking advantage of miR-128 to suppress AurkA-Wnt3a signaling.

View Article: PubMed Central - PubMed

Affiliation: Lab of Experimental Oncology &Pharmacogenomics IRCCS Fondazione "Salvatore Maugeri", Pavia, Italy.

ABSTRACT
AurkA overexpression was previously found in breast cancer and associated to its ability in controlling chromosome segregation during mitosis, however whether it may affect breast cancer cells, endorsed with stem properties (BCICs), is still unclear. Surprisingly, a strong correlation between AurkA expression and β-catenin localization in breast cancer tissues suggested a link between AurkA and Wnt signaling. In our study, AurkA knock-down reduced wnt3a mRNA and suppressed metastatic signature of MDA-MB-231 cells. As a consequence, the amount of BCICs and their migratory capability dramatically decreased. Conversely, wnt3a mRNA stabilization and increased CD44(+)/CD24(low/-) subpopulation was found in AurkA-overexpressing MCF7 cells. In vivo, AurkA-overexpressing primary breast cancer cells showed higher tumorigenic properties. Interestingly, we found that AurkA suppressed the expression of miR-128, inhibitor of wnt3a mRNA stabilization. Namely, miR-128 suppression realized after AurkA binding to Snail. Remarkably, a strong correlation between AurkA and miR-128 expression in breast cancer tissues confirmed our findings. This study provides novel insights into an undisclosed role for the kinase AurkA in self-renewal and migration of BCICs affecting response to cancer therapies, metastatic spread and recurrence. In addition, it suggests a new therapeutic strategy taking advantage of miR-128 to suppress AurkA-Wnt3a signaling.

No MeSH data available.


Related in: MedlinePlus

Aurka promote stabilization of wnt3a through repression of miR-128.(A) Q-PCR showed that AurkA knock-down in MDA-MB-231 cells promoted an increase of mir128 expression while reduced levels of mir15 and mir16. Effectively, there was a mechanism of miR128 inhibition by AurkA as revealed by Q-PCR after AurkA overexpression in MCF-7 (MCF-AurkA+) cells of knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (B) miR-128 controls wnt3a. miR-128 inhibitors promoted Wnt3a protein stabilization in MCF-7 (lane 2), conversely specific miR-128 mimics (lane 4) repressed Wnt3a in MDA-MB-231 cells, untreated MCF-7 or MDA-MB-231 cells were considered as control (lane 1 and 3, respectively). (C) miR128 bind to wnt3a 3′UTR. Luciferase activity increased when HEK-293T cells were co-transfected with pMIR-wt-wnt3a and miR128 inhibitors (3rd bar), decreased in presence of pMIR-wt-wnt3a and mimics (mimicking miR128 activity, 2nd bar). In contrast, it was not affected in control cells co-transfected with pMIR-wt-wnt3a and miR128 scramble (1st bar) or co-transfected with pMIR-mut-wnt3a (carrying mutated 3′UTR of wnt3a) and miR128 scramble, mimics or inhibitor (respectively 4th, 5th, 6th bars). At the bottom a schematic view of pMIR-wt-wnt3a and pMIR-mut-wnt3a. Data are representative of biological triplicates. (D) AurkA expression affected Snail transcription. Snail cDNA increased after AurkA overexpression in MCF-7 (MCF-AurkA+) cells and increased after AurkA knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (E) Increased luciferase activity was found when 293T cells were co-transfected with a vector carrying snail and pGL3-wt-Ebox1–2 (2nd gray bar). Conversely, luciferase activity was unaffected when 293T were co-transfected with a snail-vector and pGL3-mut-Ebox1–2 (carrying mutated Ebox1, Ebox2 or both, respectively 3th, 4th and 5th gray bars). Control cells, co-transfected with pGL3-wt-Ebox1–2 or pGL3-mut-Ebox1–2 with pGL3 (missing Snail gene) showed basal luciferase expression.
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f5: Aurka promote stabilization of wnt3a through repression of miR-128.(A) Q-PCR showed that AurkA knock-down in MDA-MB-231 cells promoted an increase of mir128 expression while reduced levels of mir15 and mir16. Effectively, there was a mechanism of miR128 inhibition by AurkA as revealed by Q-PCR after AurkA overexpression in MCF-7 (MCF-AurkA+) cells of knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (B) miR-128 controls wnt3a. miR-128 inhibitors promoted Wnt3a protein stabilization in MCF-7 (lane 2), conversely specific miR-128 mimics (lane 4) repressed Wnt3a in MDA-MB-231 cells, untreated MCF-7 or MDA-MB-231 cells were considered as control (lane 1 and 3, respectively). (C) miR128 bind to wnt3a 3′UTR. Luciferase activity increased when HEK-293T cells were co-transfected with pMIR-wt-wnt3a and miR128 inhibitors (3rd bar), decreased in presence of pMIR-wt-wnt3a and mimics (mimicking miR128 activity, 2nd bar). In contrast, it was not affected in control cells co-transfected with pMIR-wt-wnt3a and miR128 scramble (1st bar) or co-transfected with pMIR-mut-wnt3a (carrying mutated 3′UTR of wnt3a) and miR128 scramble, mimics or inhibitor (respectively 4th, 5th, 6th bars). At the bottom a schematic view of pMIR-wt-wnt3a and pMIR-mut-wnt3a. Data are representative of biological triplicates. (D) AurkA expression affected Snail transcription. Snail cDNA increased after AurkA overexpression in MCF-7 (MCF-AurkA+) cells and increased after AurkA knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (E) Increased luciferase activity was found when 293T cells were co-transfected with a vector carrying snail and pGL3-wt-Ebox1–2 (2nd gray bar). Conversely, luciferase activity was unaffected when 293T were co-transfected with a snail-vector and pGL3-mut-Ebox1–2 (carrying mutated Ebox1, Ebox2 or both, respectively 3th, 4th and 5th gray bars). Control cells, co-transfected with pGL3-wt-Ebox1–2 or pGL3-mut-Ebox1–2 with pGL3 (missing Snail gene) showed basal luciferase expression.

Mentions: By Q-PCR analysis, we were able to assess a correlation between miR-128 and AurkA. Basically, we found that AurkA knock-down in MDA-MB-231 was ineffective for stabilization of miR-15 and reduced miR-16 expression, whereas, induced a significant increase in miR-128 levels (Fig. 5A, left graph).


AurkA controls self-renewal of breast cancer-initiating cells promoting wnt3a stabilization through suppression of miR-128.

Eterno V, Zambelli A, Villani L, Tuscano A, Manera S, Spitaleri A, Pavesi L, Amato A - Sci Rep (2016)

Aurka promote stabilization of wnt3a through repression of miR-128.(A) Q-PCR showed that AurkA knock-down in MDA-MB-231 cells promoted an increase of mir128 expression while reduced levels of mir15 and mir16. Effectively, there was a mechanism of miR128 inhibition by AurkA as revealed by Q-PCR after AurkA overexpression in MCF-7 (MCF-AurkA+) cells of knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (B) miR-128 controls wnt3a. miR-128 inhibitors promoted Wnt3a protein stabilization in MCF-7 (lane 2), conversely specific miR-128 mimics (lane 4) repressed Wnt3a in MDA-MB-231 cells, untreated MCF-7 or MDA-MB-231 cells were considered as control (lane 1 and 3, respectively). (C) miR128 bind to wnt3a 3′UTR. Luciferase activity increased when HEK-293T cells were co-transfected with pMIR-wt-wnt3a and miR128 inhibitors (3rd bar), decreased in presence of pMIR-wt-wnt3a and mimics (mimicking miR128 activity, 2nd bar). In contrast, it was not affected in control cells co-transfected with pMIR-wt-wnt3a and miR128 scramble (1st bar) or co-transfected with pMIR-mut-wnt3a (carrying mutated 3′UTR of wnt3a) and miR128 scramble, mimics or inhibitor (respectively 4th, 5th, 6th bars). At the bottom a schematic view of pMIR-wt-wnt3a and pMIR-mut-wnt3a. Data are representative of biological triplicates. (D) AurkA expression affected Snail transcription. Snail cDNA increased after AurkA overexpression in MCF-7 (MCF-AurkA+) cells and increased after AurkA knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (E) Increased luciferase activity was found when 293T cells were co-transfected with a vector carrying snail and pGL3-wt-Ebox1–2 (2nd gray bar). Conversely, luciferase activity was unaffected when 293T were co-transfected with a snail-vector and pGL3-mut-Ebox1–2 (carrying mutated Ebox1, Ebox2 or both, respectively 3th, 4th and 5th gray bars). Control cells, co-transfected with pGL3-wt-Ebox1–2 or pGL3-mut-Ebox1–2 with pGL3 (missing Snail gene) showed basal luciferase expression.
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f5: Aurka promote stabilization of wnt3a through repression of miR-128.(A) Q-PCR showed that AurkA knock-down in MDA-MB-231 cells promoted an increase of mir128 expression while reduced levels of mir15 and mir16. Effectively, there was a mechanism of miR128 inhibition by AurkA as revealed by Q-PCR after AurkA overexpression in MCF-7 (MCF-AurkA+) cells of knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (B) miR-128 controls wnt3a. miR-128 inhibitors promoted Wnt3a protein stabilization in MCF-7 (lane 2), conversely specific miR-128 mimics (lane 4) repressed Wnt3a in MDA-MB-231 cells, untreated MCF-7 or MDA-MB-231 cells were considered as control (lane 1 and 3, respectively). (C) miR128 bind to wnt3a 3′UTR. Luciferase activity increased when HEK-293T cells were co-transfected with pMIR-wt-wnt3a and miR128 inhibitors (3rd bar), decreased in presence of pMIR-wt-wnt3a and mimics (mimicking miR128 activity, 2nd bar). In contrast, it was not affected in control cells co-transfected with pMIR-wt-wnt3a and miR128 scramble (1st bar) or co-transfected with pMIR-mut-wnt3a (carrying mutated 3′UTR of wnt3a) and miR128 scramble, mimics or inhibitor (respectively 4th, 5th, 6th bars). At the bottom a schematic view of pMIR-wt-wnt3a and pMIR-mut-wnt3a. Data are representative of biological triplicates. (D) AurkA expression affected Snail transcription. Snail cDNA increased after AurkA overexpression in MCF-7 (MCF-AurkA+) cells and increased after AurkA knock-down in MDA-MB-231 and KBr2 cells (MDA-shAurkA and KBr2-shAurka). (E) Increased luciferase activity was found when 293T cells were co-transfected with a vector carrying snail and pGL3-wt-Ebox1–2 (2nd gray bar). Conversely, luciferase activity was unaffected when 293T were co-transfected with a snail-vector and pGL3-mut-Ebox1–2 (carrying mutated Ebox1, Ebox2 or both, respectively 3th, 4th and 5th gray bars). Control cells, co-transfected with pGL3-wt-Ebox1–2 or pGL3-mut-Ebox1–2 with pGL3 (missing Snail gene) showed basal luciferase expression.
Mentions: By Q-PCR analysis, we were able to assess a correlation between miR-128 and AurkA. Basically, we found that AurkA knock-down in MDA-MB-231 was ineffective for stabilization of miR-15 and reduced miR-16 expression, whereas, induced a significant increase in miR-128 levels (Fig. 5A, left graph).

Bottom Line: In our study, AurkA knock-down reduced wnt3a mRNA and suppressed metastatic signature of MDA-MB-231 cells.As a consequence, the amount of BCICs and their migratory capability dramatically decreased.In addition, it suggests a new therapeutic strategy taking advantage of miR-128 to suppress AurkA-Wnt3a signaling.

View Article: PubMed Central - PubMed

Affiliation: Lab of Experimental Oncology &Pharmacogenomics IRCCS Fondazione "Salvatore Maugeri", Pavia, Italy.

ABSTRACT
AurkA overexpression was previously found in breast cancer and associated to its ability in controlling chromosome segregation during mitosis, however whether it may affect breast cancer cells, endorsed with stem properties (BCICs), is still unclear. Surprisingly, a strong correlation between AurkA expression and β-catenin localization in breast cancer tissues suggested a link between AurkA and Wnt signaling. In our study, AurkA knock-down reduced wnt3a mRNA and suppressed metastatic signature of MDA-MB-231 cells. As a consequence, the amount of BCICs and their migratory capability dramatically decreased. Conversely, wnt3a mRNA stabilization and increased CD44(+)/CD24(low/-) subpopulation was found in AurkA-overexpressing MCF7 cells. In vivo, AurkA-overexpressing primary breast cancer cells showed higher tumorigenic properties. Interestingly, we found that AurkA suppressed the expression of miR-128, inhibitor of wnt3a mRNA stabilization. Namely, miR-128 suppression realized after AurkA binding to Snail. Remarkably, a strong correlation between AurkA and miR-128 expression in breast cancer tissues confirmed our findings. This study provides novel insights into an undisclosed role for the kinase AurkA in self-renewal and migration of BCICs affecting response to cancer therapies, metastatic spread and recurrence. In addition, it suggests a new therapeutic strategy taking advantage of miR-128 to suppress AurkA-Wnt3a signaling.

No MeSH data available.


Related in: MedlinePlus