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Metabolic reprogramming of metastatic breast cancer and melanoma by let-7a microRNA.

Serguienko A, Grad I, Wennerstrøm AB, Meza-Zepeda LA, Thiede B, Stratford EW, Myklebost O, Munthe E - Oncotarget (2015)

Bottom Line: Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes.To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin.Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

ABSTRACT
Let-7 microRNAs (miRNAs) are highly conserved well-established promoters of terminal differentiation that are expressed in healthy adult tissues and frequently repressed in cancer cells. The tumor suppressive role of let-7 in a variety of cancers in vitro and in vivo has been widely documented and prompted these miRNAs to be candidate genes for miRNA replacement therapy. In this study we described a new role of let-7a in reprogramming cancer metabolism, recently identified as a new hallmark of cancer. We show that let-7a down-regulates key anabolic enzymes and increases both oxidative phosphorylation and glycolysis in triple-negative breast cancer and metastatic melanoma cell lines. Strikingly, the accelerated glycolysis coexists with drastically reduced cancer features. Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes. To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin. In both cancer types let-7a increased cell sensitivity to doxorubicin. Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway. We thus have demonstrated that let-7a plays a prominent role in regulating energy metabolism in cancer cells, further expanding its therapeutic potential.

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Related in: MedlinePlus

Let-7a represses cell proliferation and clonogenic capacity of MDA-MB-231 cellsA, protein levels of CCND1, HMGA2, and LIN28A on day 3 post transfection and densitometric quantification of proteins normalized to α-tubulin, n=3, SD. B, phase-contrast images of cells on day 3 post transfection, magnification 10X. C, cell confluence-based growth curve. D, count of cells using Trypan Blue on day 3 post transfection, n=3, SD. E, colonies generated after 10 days of incubation in the methylcellulose-based medium. F, quantification of colonies by counting of MTT stained colonies, n=4, SD.
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Figure 1: Let-7a represses cell proliferation and clonogenic capacity of MDA-MB-231 cellsA, protein levels of CCND1, HMGA2, and LIN28A on day 3 post transfection and densitometric quantification of proteins normalized to α-tubulin, n=3, SD. B, phase-contrast images of cells on day 3 post transfection, magnification 10X. C, cell confluence-based growth curve. D, count of cells using Trypan Blue on day 3 post transfection, n=3, SD. E, colonies generated after 10 days of incubation in the methylcellulose-based medium. F, quantification of colonies by counting of MTT stained colonies, n=4, SD.

Mentions: To assess the validity of our model system, we first investigated the protein level of three known let-7 targets, CCND1, HMGA2 and LIN28A upon let-7a transfection. Cells transfected with let-7a mimics (hereinafter let-7a) or with negative control oligos (hereinafter negative control) were lysed on day 3 post transfection and extracts were subjected to Western blot analysis. The levels of all three proteins were reduced to approximately 50% in let-7a transfected cells compared to the negative control (Fig. 1A). Interestingly, we observed that, in contrast to the spindle-shaped, mesenchymal-like morphology of untreated and negative control cells, the cells transfected with let-7a became more columnar and epithelial-like already 48 hours post transfection (Fig. 1B). Next, we investigated the effect of let-7a overexpression on cell growth. Cell growth was assessed by time-lapse live-cell imaging based on cellular confluence. Cells transfected with let-7a showed a time-dependent decrease in confluence compared to the negative control cells (Fig. 1C). The cell number and viability were assessed using Trypan Blue. As expected, let-7a significantly reduced the final number of cells in culture (Fig. 1D) with no effect on viability (Fig. S1A). Further analysis of caspase-3/7 activity to check for apoptosis revealed no difference between let-7a transfected and negative control cells (Fig. S1B). We performed colony formation assay to determine whether ectopic expression of let-7a impairs the clonogenic capability of MDA-MB-231 cells. The number of colonies generated from single cells was drastically reduced in let-7a treated samples (Fig. 1E and F), while the size of the colonies did not differ (Fig. S1C).


Metabolic reprogramming of metastatic breast cancer and melanoma by let-7a microRNA.

Serguienko A, Grad I, Wennerstrøm AB, Meza-Zepeda LA, Thiede B, Stratford EW, Myklebost O, Munthe E - Oncotarget (2015)

Let-7a represses cell proliferation and clonogenic capacity of MDA-MB-231 cellsA, protein levels of CCND1, HMGA2, and LIN28A on day 3 post transfection and densitometric quantification of proteins normalized to α-tubulin, n=3, SD. B, phase-contrast images of cells on day 3 post transfection, magnification 10X. C, cell confluence-based growth curve. D, count of cells using Trypan Blue on day 3 post transfection, n=3, SD. E, colonies generated after 10 days of incubation in the methylcellulose-based medium. F, quantification of colonies by counting of MTT stained colonies, n=4, SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385863&req=5

Figure 1: Let-7a represses cell proliferation and clonogenic capacity of MDA-MB-231 cellsA, protein levels of CCND1, HMGA2, and LIN28A on day 3 post transfection and densitometric quantification of proteins normalized to α-tubulin, n=3, SD. B, phase-contrast images of cells on day 3 post transfection, magnification 10X. C, cell confluence-based growth curve. D, count of cells using Trypan Blue on day 3 post transfection, n=3, SD. E, colonies generated after 10 days of incubation in the methylcellulose-based medium. F, quantification of colonies by counting of MTT stained colonies, n=4, SD.
Mentions: To assess the validity of our model system, we first investigated the protein level of three known let-7 targets, CCND1, HMGA2 and LIN28A upon let-7a transfection. Cells transfected with let-7a mimics (hereinafter let-7a) or with negative control oligos (hereinafter negative control) were lysed on day 3 post transfection and extracts were subjected to Western blot analysis. The levels of all three proteins were reduced to approximately 50% in let-7a transfected cells compared to the negative control (Fig. 1A). Interestingly, we observed that, in contrast to the spindle-shaped, mesenchymal-like morphology of untreated and negative control cells, the cells transfected with let-7a became more columnar and epithelial-like already 48 hours post transfection (Fig. 1B). Next, we investigated the effect of let-7a overexpression on cell growth. Cell growth was assessed by time-lapse live-cell imaging based on cellular confluence. Cells transfected with let-7a showed a time-dependent decrease in confluence compared to the negative control cells (Fig. 1C). The cell number and viability were assessed using Trypan Blue. As expected, let-7a significantly reduced the final number of cells in culture (Fig. 1D) with no effect on viability (Fig. S1A). Further analysis of caspase-3/7 activity to check for apoptosis revealed no difference between let-7a transfected and negative control cells (Fig. S1B). We performed colony formation assay to determine whether ectopic expression of let-7a impairs the clonogenic capability of MDA-MB-231 cells. The number of colonies generated from single cells was drastically reduced in let-7a treated samples (Fig. 1E and F), while the size of the colonies did not differ (Fig. S1C).

Bottom Line: Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes.To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin.Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, Institute of Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway.

ABSTRACT
Let-7 microRNAs (miRNAs) are highly conserved well-established promoters of terminal differentiation that are expressed in healthy adult tissues and frequently repressed in cancer cells. The tumor suppressive role of let-7 in a variety of cancers in vitro and in vivo has been widely documented and prompted these miRNAs to be candidate genes for miRNA replacement therapy. In this study we described a new role of let-7a in reprogramming cancer metabolism, recently identified as a new hallmark of cancer. We show that let-7a down-regulates key anabolic enzymes and increases both oxidative phosphorylation and glycolysis in triple-negative breast cancer and metastatic melanoma cell lines. Strikingly, the accelerated glycolysis coexists with drastically reduced cancer features. Moreover, let-7a causes mitochondrial ROS production concomitant with the up-regulation of oxidative stress responsive genes. To exploit these increased ROS levels for therapeutic purposes, we combined let-7a transfection with the chemotherapeutic drug doxorubicin. In both cancer types let-7a increased cell sensitivity to doxorubicin. Pre-treatment with N-acetyl cysteine (NAC) totally abolished this effect, indicating that the increased doxorubicin sensitivity of let-7a cells depends on the redox pathway. We thus have demonstrated that let-7a plays a prominent role in regulating energy metabolism in cancer cells, further expanding its therapeutic potential.

Show MeSH
Related in: MedlinePlus