Loss of STAT6 leads to anchorage-independent growth and trastuzumab resistance in HER2+ breast cancer cells.

Approximately 20% of breast cancers are HER2-positive. Trastuzumab has improved patient outcomes significantly for these cancers. However, acquired resistance remains a major hurdle in the clinical management of these patients. Therefore, identifying molecular changes that cause trastuzumab resistance is worthwhile. STAT6 is a transcription factor that regulates a variety of genes involved in cell cycle regulation, growth inhibition, and apoptosis. STAT6 expression is lost in approximately 3% of breast cancers, but little work has been done in the context of trastuzumab resistance in breast cancer. In isogenic cell line pairs, we observed that trastuzumab-resistant cells expressed significantly lower levels of STAT6 compared to trastuzumab-sensitive cells. Therefore, in order to study the consequences of STAT6 loss in HER2+ breast cancer, we knocked out both alleles of the STAT6 gene using somatic cell gene targeting. Interestingly, loss of STAT6 resulted in anchorage-independent growth and changes in several genes involved in epithelial to mesenchymal transition. This study suggests that STAT6 may play a role in the pathophysiology of HER2+ human breast cancer

Evaluation of [Co(gly)3]- as a 35Cl- NMR Shift Reagent for Cellular Studies

We studied the efficacy of the tris-glycinatocobaltate(II) complex ([Co(gly)3]-) as a shift reagent (SR) for chloride by 35Cl NMR spectroscopy and compared to that of Co2+(aq). Due to the relatively low thermodynamic stability of [Co(gly)3]-, a 1:3 Co(II)/gly stoichiometric solution at physiological pH is approximately a 2:1 mixture of [Co(gly)2(H2O)2] and [Co(gly)(H2O)4]+. This SR was found to be stable up to higher pH values than Co2+(aq), better preventing Co(OH)2 formation at alkaline pH. No significant differences in the 35Cl- NMR chemical shift induced by Co(II)/gly or Co2+(aq) were observed in the presence of physiological concentrations of either Ca2+ or Mg2+, or of either Na+ or K+. Although Co2+(aq) was almost twice as effective as Co(II)/gly in shifting the 35Cl- NMR resonance at the same high ρ ([SR]/[Cl-]) value and low ionic strength, Co2+(aq) showed a significant decrease (p < 0.05) in the 35Cl- chemical shift at higher ionic strength. Line widths at half-height were significantly (p < 0.05) less for Co(II)/gly than for Co2+(aq) at ρ values in the range 0.066−0.40. Intracellular chloride was clearly detectable by 35Cl NMR spectroscopy in human skin fibroblast cells suspended in medium containing 40 mM Co(II)/gly SR. We determined that, although Co2+(aq) provides a larger shift than Co(II)/gly at the same ρ value, there are significant advantages for using Co(II)/gly, such as pH stability, ionic strength independent chemical shifts, narrow 35Cl- NMR resonances, and reduced cellular toxicity, as a SR in biological systems

Somatic loss of PIK3R1 may sensitize breast cancer to inhibitors of the MAPK pathway

Purpose: The PI3K pathway, which includes the PI3K catalytic subunits p110α (PIK3CA) and the PI3K regulatory subunit p85α (PIK3R1), is the most frequently altered pathway in cancer. We encountered a breast cancer patient whose tumor contained a somatic alteration in PIK3R1. Some commercial sequencing platforms suggest that somatic mutations in PIK3R1 may sensitize cancers to drugs that inhibit the mammalian target of rapamycin (mTOR). However, a review of the preclinical and clinical literature did not find evidence substantiating that hypothesis. The purpose of this study was to knock out PIK3R1 in order to determine the optimal therapeutic approach for breast cancers lacking p85α. Methods: We created an isogenic cellular system by knocking out both alleles of the PIK3R1 gene in the non-tumorigenic human breast cell line MCF-10A. Knockout cells were compared with wild-type cells by measuring growth, cellular signaling, and response to drugs. Results: We observed hyperphosphorylation of MEK in these knockouts, which sensitized PIK3R1-null cells to a MEK inhibitor, trametinib. However, they were not sensitized to the mTOR inhibitor, everolimus. Conclusions: Our findings suggest that breast cancers with loss of p85α may not respond to mTOR inhibition, but may be sensitive to MEK inhibition

Effects of Li+ transport and Li+ immobilization on Li+/Mg2+ competition in cells: implications for bipolar disorder

Li+/Mg2+ competition has been implicated in the therapeutic action of Li+ treatment in bipolar illness. We hypothesized that this competition depended on cell-specific properties. To test this hypothesis, we determined the degree of Li+ transport, immobilization, and Li+/Mg2+ competition in lymphoblastomas, neuroblastomas, and erythrocytes. During a 50 mmol/L Li+-loading incubation, Li+ accumulation at 30 min (mmoles Li+/L cells) was the greatest in lymphoblastomas (11.1±0.3), followed by neuroblastomas (9.3±0.5), and then erythrocytes (4.0±0.5). Li+ binding affinities to the plasma membrane in all three cell types were of the same order of magnitude; however, Li+ immobilization in intact cells was greatest in neuroblastomas and least in erythrocytes. When cells were loaded for 30 min in a 50 mmol/L Li+-containing medium, the percentage increase in free intracellular [Mg2+] in neuroblastoma and lymphoblastoma cells (~55 and ~52%, respectively) was similar, but erythrocytes did not exhibit any substantial increase (~6%). With the intracellular [Li+] at 15 mmol/L, the free intracellular [Mg2+] increased by the greatest amount in neuroblastomas (~158%), followed by lymphoblastomas (~75%), and then erythrocytes (~50%). We conclude that Li+ immobilization and transport are related to free intracellular [Mg2+] and to the extent of Li+/Mg2+ competition in a cell-specific manner.http://www.sciencedirect.com/science/article/B6T4P-49JHRTX-1/1/a707c6f5021c0af8d926e0ee947843b

Identification of Li+ binding sites and the effect of Li+ treatment on phospholipid composition in human neuroblastoma cells: a 7Li and 31P NMR study

Li+ binding in subcellular fractions of human neuroblastoma SH-SY5Y cells was investigated using 7Li NMR spin-lattice (T1) and spin-spin (T2) relaxation measurements, as the T1/T2 ratio is a sensitive parameter of Li+ binding. The majority of Li+ binding occurred in the plasma membrane, microsomes, and nuclear membrane fractions as demonstrated by the Li+ binding constants and the values of the T1/T2 ratios, which were drastically larger than those observed in the cytosol, nuclei, and mitochondria. We also investigated by 31P NMR spectroscopy the effects of chronic Li+ treatment for 4-6 weeks on the phospholipid composition of the plasma membrane and the cell homogenate and found that the levels of phosphatidylinositol and phosphatidylserine were significantly increased and decreased, respectively, in both fractions. From these observations, we propose that Li+ binding occurs predominantly to membrane domains, and that chronic Li+ treatment alters the phospholipid composition at these membrane sites. These findings support those from clinical studies that have indicated that Li+ treatment of bipolar patients results in irregularities in Li+ binding and phospholipid metabolism. Implications of our observations on putative mechanisms of Li+ action, including the cell membrane abnormality, the inositol depletion and the G-protein hypotheses, are discussed.http://www.sciencedirect.com/science/article/B6T1Y-4GTVVV4-1/1/78da35833eca5a0d76829b754d64687

PIK3CA mutations and EGFR overexpression predict for lithium sensitivity in human breast epithelial cells

A high frequency of somatic mutations has been found in breast cancers within the gene encoding the catalytic p110α subunit of PI3K, PIK3CA. Using isogenic human breast epithelial cells, we have previously demonstrated that oncogenic PIK3CA “hotspot” mutations predict for response to the toxic effects of lithium. However, other somatic genetic alterations occur within this pathway in breast cancers, and it is possible that these changes may also predict for lithium sensitivity. We overexpressed the epidermal growth factor receptor (EGFR) into the non-tumorigenic human breast epithelial cell line MCF-10A, and compared these cells to isogenic cell lines previously created via somatic cell gene targeting to model Pten loss, PIK3CA mutations, and the invariant AKT1 mutation, E17K. EGFR overexpressing clones were capable of cellular proliferation in the absence of EGF and were sensitive to lithium similar to the results previously seen with cells harboring PIK3CA mutations. In contrast, AKT1 E17K cells and PTEN−/− cells displayed resistance or partial sensitivity to lithium, respectively. Western blot analysis demonstrated that lithium sensitivity correlated with significant decreases in both PI3K and MAPK signaling that were observed only in EGFR overexpressing and mutant PIK3CA cell lines. These studies demonstrate that EGFR overexpression and PIK3CA mutations are predictors of response to lithium, whereas Pten loss and AKT1 E17K mutations do not predict for lithium sensitivity. Our findings may have important implications for the use of these genetic lesions in breast cancer patients as predictive markers of response to emerging PI3K pathway inhibitors