Different Biological Action of Oleic Acid in ALDH^high and ALDH^low Subpopulations Separated from Ductal Carcinoma <i>In Situ</i> of Breast Cancer

<div><p>The mechanisms underlying breast cancer progression of ductal carcinoma in situ <b>(</b>DCIS) associated with fatty acids are largely unknown. In the present study, we compared the action of oleic acid (OA) on two human DCIS cell lines, MCF10DCIS.COM (ER/PR/HER2-negative) and SUM225 (HER2 overexpressed). OA led to a significant increase in proliferation, migration, lipid accumulation and the expression of lipogenic proteins, such as SREBP-1, FAS and ACC-1, in MCF10DCIS.COM cells but not SUM225 cells. The ALDH^high subpopulation analyzed by the ALDEFLUOR assay was approximately 39.2±5.3% of MCF10DCIS.COM cells but was small (3.11±0.9%) in SUM225 cells. We further investigated the different biological action of OA in the distinct ALDH^low and ALDH^high subpopulations of MCF10DCIS.COM cells. OA led to an increase in the expression of ALDH1A1, ALDH1A2 and ALDH1A3 in MCF10DCIS.COM cells. SREBP-1 and ACC-1 were highly expressed in ALDH^high cells relative to ALDH^low cells, whereas FAS was higher in ALDH^low cells. In the presence of OA, ALDH^high cells were more likely to proliferate and migrate and displayed significantly high levels of SREBP-1 and FAS and strong phosphorylation of FAK and AKT relative to ALDH^low cells. This study suggests that OA could be a critical risk factor to promote the proliferation and migration of ALDH^high cells in DCIS, leading to breast cancer progression.</p></div

Oleic acid (OA) further promotes the proliferation and migration abilities and upregulates lipogenic proteins in ALDH^high cells.

<p>(A) Quantitative real-time RT-PCR of ALDH1A1, ALDH1A2 and ALDH1A3 in MCF10DCIS.COM cells. All subtypes of ALDH1 were significantly increased by OA. Notably, OA led to a remarkable increase in ALDH1A2 in MCF10DCIS.COM cells. (B) MTT assay of cell proliferation in ALDH^high and ALDH^low cells incubated with OA. OA-induced proliferation of ALDH^high cells was greater than that of ALDH^low cells. (C) Trans-well assay of cell migration in ALDH^high and ALDH^low cells. The OA-induced migration ability was higher in ALDH^high cells than ALDH^low cells. (D) Representative Western blot of SREBP-1, FAS and ACC-1 in ALDH^high cells and ALDH^low cells. (E, F and G) Analysis of expression levels of SREBP-1, FAS and ACC-1. Significantly higher expression of SERBP-1 and ACC-1 was observed in ALDH^high cells, whereas FAS was significantly higher in ALDH^low cells. OA led to the significant upregulation of SREBP-1 and FAS in ALDH^high cells and the significant upregulation of SREBP-1 and downregulation of FAS in ALDH^low cells. All experiments were performed at least in triplicate, and the values are reported as the means ± standard error. *<i>p</i><0.05, **<i>p</i><0.01.</p

Oleic acid (OA) promotes the proliferation and migration ability of MCF10DCIS.COM cells but not SUM225 cells, whereas palmitic acid (PA) leads to cell death in both cells.

<p>(A and B) MTT assay of cell proliferation in MCF10DCIS.COM and SUM225 cells incubated with increasing OA or PA. OA induced significantly increased viability in MCF10DCIS.COM cells but led to cell death in SUM225 cells. PA induced the death of both MCF10DCIS.COM and SUM225 cells. (C) Trans-well assay of cell migration in MCF10DCIS.COM and SUM 225 cells. OA significantly promoted the migration of MCF10DCIS.COM cell but not SUM225 cells. (D) Wound healing assay of lateral migration of MCF10DCIS.COM cells incubated with OA. OA significantly enhanced the lateral migration of MCF10DCIS.COM cells. All the experiments were performed at least in triplicate and the values are reported as the means ± standard error. *<i>p</i><0.05, **<i>p</i><0.01.</p

Oleic acid (OA) promotes the viability and migration through the FAK, PI3K/AKT, and MEK/ERK signaling pathway in MCF10DCIS.COM cells.

<p>(A and B) Representative Western blot and quantitative analysis of phosphorylated FAK, AKT and ERK1/2 in MCF10DCIS.COM cells incubated with OA. OA induced a significant increase in the phosphorylation of FAK, AKT and ERK1/2. (C) MTT assay of cell proliferation in MCF10DCIS.COM cells incubated with OA in the presence of FAK (PF573228), PI3K/AKT (LY294002) and MEK/ERK (PD98059) inhibitors. All kinase inhibitors induced cell death, and OA-promoted proliferation was reduced in the presence of all kinase inhibitors. (D) Trans-well assay of cell migration in MCF10DCIS.COM cells incubated with OA in the presence of FAK, PI3K/AKT and MEK/ERK inhibitors. OA-induced migration was suppressed by the presence of all kinase inhibitors. All the experiments were performed at least in triplicate, and the values are reported as the means ± standard error. *<i>p</i><0.05, **<i>p</i><0.01.</p

Oleic acid (OA) induces lipid accumulation and the upregulation of lipogenic proteins in MCF10DCIS.COM cells but not SUM225 cells.

<p>(A) Representative Oil Red O staining in MCF10DCIS.COM and SUM 225 cells incubated with OA. A large number of lipid droplets were observed in MCF10DCIS.COM cells but not SUM225 cells. (B) Quantitative analysis of intracellular lipid contents from Oil Red O staining. OA led to lipid accumulation in MCF10DCIS.COM cells. (C) Representative Western blot of SREBP-1, FAS and ACC-1 in MCF10DCIS.COM and SUM 225 cells incubated with OA. (D) Quantitative analysis of lipogenic protein levels in MCF10DCIS.COM and SUM225 cells. A significantly higher level of SERBP-1 was observed in MCF10DCIS.COM cells relative to SUM225 cells. The FAS level was significantly higher in SUM225 cells than MCF10DCIS.COM cells. The level of ACC-1 was similar between MCF10DCIS.COM cells and SUM225 cells (upper). OA resulted in the significant upregulation of SREBP-1, FAS and ACC-1 in MCF10DCIS.COM cells (middle) but led to the downregulation of SREBP-1 and the upregulation of ACC-1 significantly in SUM225 cells (lower). All experiments were performed at least in triplicate, and the values are reported as the means ± standard error. *<i>p</i><0.05, **<i>p</i><0.01.</p

Distinct subpopulations of ALDH1^high and ALDH1^low cells were separated from MCF10DCIS.COM cells.

<p>(A) Representative flow cytometry for ALDEFLUOR assay showing the percentage of ALDH^high cells in MCD10DCIS.COM and SUM225 cells. Graph showed that the ALDH^high cell population obtained from experiments performed at least in triplicate. Cells exhibiting high ALDH activity were higher in MCF10DCIS.COM cells relative to SUM225 cells. (B) Quantitative real-time RT-PCR of ALDH1A1, ALDH1A2 and ALDH1A3 in ALDH1^high and ALDH1^low subpopulation cells separated from MCF10DCIS.COM cells. Significantly higher expression levels of ALDH1A2 and ALDH1A3 mRNAs were detected in ALDH^high cells relative to ALDH^low cells. The experiments were performed at least in triplicate, and the values are reported as the means ± standard error. *<i>p</i><0.05, **<i>p</i><0.01. (C) RT-PCR analysis of CD24 and CD44 mRNAs in ALDH^high and ALDH^low cells. CD44 mRNA was higher in ALDH1^high cells than ALDH1^low cells. (D) Flow cytometric analysis of CD44 and CD24. Of MCF10DCIS.COM cells, 70% exhibited the CD44+/CD24- phenotype. CD44+/CD24- cell populations were higher in separated ALDH1^high cells than ALDH1^low cells. (E) Immunofluorescence staining of CD44, CD24 and ALDH1. ALDH1^high cells expressed a high level of ALDH1 and CD44, whereas ALDH1^low cells displayed a high level of CD24 and a low level of ALDH1 and CD44.</p

Downregulation of Choline Kinase-Alpha Enhances Autophagy in Tamoxifen-Resistant Breast Cancer Cells

<div><p>Choline kinase-α (Chk-α) and autophagy have gained much attention, as they relate to the drug-resistance of breast cancer. Here, we explored the potential connection between Chk-α and autophagy in the mechanisms driving to tamoxifen (TAM) resistance, in estrogen receptor positive (ER+) breast cancer cells (BCCs). Human BCC lines (MCF-7 and TAM-resistant MCF-7 (MCF-7/TAM) cells) were used. Chk-α expression and activity was suppressed by the transduction of shRNA (shChk-α) with lentivirus and treatment with CK37, a Chk-α inhibitor. MCF-7/TAM cells had higher Chk-α expression and phosphocholine levels than MCF-7 cells. A specific downregulation of Chk-α by the transduction of shChk-α exhibited a significant decrease in phosphocholine levels in MCF-7 and MCF-7/TAM cells. The autophagy-related protein, cleaved microtubule-associated protein light chain 3 (LC3) and autophagosome-like structures were significantly increased in shChk-α-transduced or CK37-treated MCF-7 and MCF-7/TAM cells. The downregulation of Chk-α attenuated the phosphorylation of AKT, ERK1/2, and mTOR in both MCF-7 and MCF-7/TAM cells. In MCF-7 cells, the downregulation of Chk-α resulted in an induction of autophagy, a decreased proliferation ability and an activation of caspase-3. In MCF-7/TAM cells, despite a significant decrease in proliferation ability and an increase in the percentage of cells in the G0/G1 phase of the cell cycle, the downregulation of Chk-α did not induced caspase-dependent cell death and further enhanced autophagy and G0/G1 phase arrest. An autophagy inhibitor, methyladenine (3-MA) induced death and attenuated the level of elevated LC3 in MCF-7/TAM cells. Elucidating the interplay between choline metabolism and autophagy will provide unique opportunities to identify new therapeutic targets and develop novel treatment strategies that preferentially target TAM-resistance.</p></div

Establishment of choline kinase-α (Chk-α)-downregulated breast cancer cells and analysis of choline-containing metabolites.

<p>(A and B) Images of MCF-7-shChk-α and MCF-7/TAM-shChk-α cells transduced with lentivirus containing Chk-α shRNA and GFP. (C and D) RT-PCR analysis of Chk-α and β. A selective and significant decrease in Chk-α mRNA levels was detected in MCF-7-shChk-α and MCF-7/TAM-shChk-α cells. (E and F) Western blot analysis of Chk-α. Chk-α was significantly downregulated in MCF-7-shChk-α and MCF-7/TAM-shChk-α. (G and H) Analysis of 1H-NMR spectra of choline-containing metabolites, choline (Cho), phosphocholine (PC) and glycerophosphocholine (GPC). PC level remarkably decreased in MCF-7-shChk-α and MCF-7/TAM-shChk-α as compared to MCF-7 and MCF-7/TAM cells. Data are presented as the mean ± standard deviation of 5 independent experiments. * <i>p</i><0.05 and ** <i>p</i><0.001. shChk-α transduced vs control.</p

Choline kinase-α (Chk-α) expression and choline-containing metabolites increased in tamoxifen-resistant breast cancer cells, MCF-7/TAM.

<p>(A) RT-PCR analysis of Chk-α and β in MCF-7 and MCF-7/TAM cells. The level of Chk-α mRNA was higher in MCF-7/TAM cells and there was no different in the level of Chk-β mRNA between MCF-7 cells and MCF-7/TAM cells. (B) Western blot analysis of Chk-α in MCF-7 and MCF-7/TAM cells. Chk-α expression was significantly increased in MCF-7/TAM cells compared to MCF-7 cells. (C) A representative 1H-NMR spectra of choline-containing metabolites (choline (Cho), phosphocholine (PC) and glycerophosphocholine (GPC)) of MCF-7 and MCF-7/TAM cells. Higher PC level was observed in MCF-7/TAM cells relative to MCF-7 cells. Data are presented as the mean ± standard deviation of 3 independent experiments. ** <i>p</i><0.001.</p

Downregulation of choline kinase-α (Chk-α) expression and activity enhanced the expression of the autophagosome marker, LC3 and P62 and the formation of autophagosomes in MCF-7/TAM cells.

<p>(A and B) Images of autophagic structure in MCF-7-shChk-α and MCF-7/TAM-shChk-α cells. Chk-α downregulation induced autophagic structures (arrow) in MCF-7 cells as well as MCF-7/TAM cells. (C and D) Western blot analysis of LC3. MCF-7-shChk-α and MCF-7/TAM-shChk-α cells exhibited a significantly higher LC3 as compared to MCF-7 and MCF-7/TAM cells. (E and F) Immunofluorescence images for LC3. Chk-α downregulation led to an increase in the number of autophagosomes stained with LC3 antibody. (G and H) Western blot analysis of p62. MCF-7/TAM-shChk-α cells exhibited a significantly higher p62 as compared to MCF-7/TAM cells. (I and J) Western blot analysis of LC3. Inhibition of Chk-α activity by treatment with CK37 significantly increased LC3 expression. (K and L) Immunofluorescence images for LC3. CK37 treatment resulted in an increase in the number of autophagosomes stained with LC3 antibody. (M and N) Western blot analysis of p62. CK37 treatment did not change the level of p62 as compared to untreated cells. Data are presented as the mean ± standard deviation of 5 independent experiments. *<i>p</i><0.05. shChk-α transduced vs control and CK37-treated vs control.</p