Abstract:The PI3K pathway is activated in approximately 70% of breast cancers. PIK3CA gene mutations or amplifications that affect the PI3K p110a subunit account for activation of this pathway in 20% to 40% of cases, particularly in estrogen receptor alpha (ERa)-positive breast cancers. AKT family of kinases, AKT1-3, are the downstream targets of PI3K and these kinases activate ERa. Although several inhibitors of PI3K have been developed, none has proven effective in the clinic, partly due to an incomplete understandin… Show more
“…Currently, several PI3Kα inhibitors are under development and in clinical trials 12 . Alone or in combination with other drugs, PI3Kα inhibitors prevent tumor growth particularly in estrogen receptor alpha (ERα)-positive breast cancers, but depending on the type of the PI3Kα mutation different therapeutic regimes must be followed 13 . Figure 1 PI3Kα crystal structure and organization in functional domains.…”
Phosphoinositide 3-kinase alpha (PI3Kα) is involved in fundamental cellular processes including cell proliferation and differentiation and is frequently mutated in human malignancies. One of the most common mutations is E545K, which results in an amino acid substitution of opposite charge. It has been recently proposed that in this oncogenic charge-reversal mutation, the interactions between the protein catalytic and regulatory subunits are abrogated, resulting in loss of regulation and constitutive PI3Kα activity, which can lead to oncogenesis. To assess the mechanism of the PI3Kα E545K activating mutation, extensive Molecular Dynamics simulations were performed to examine conformational changes differing between the wild type (WT) and mutant proteins as they occur in microsecond simulations. In the E545K mutant PI3Kα, we observe a spontaneous detachment of the nSH2 PI3Kα domain (regulatory subunit, p85α) from the helical domain (catalytic subunit, p110α) causing significant loss of communication between the regulatory and catalytic subunits. We examine the allosteric network of the two proteins and show that a cluster of residues around the mutation is important for delivering communication signals between the catalytic and regulatory subunits. Our results demonstrate the dynamical and structural effects induced by the p110α E545K mutation in atomic level detail and indicate a possible mechanism for the loss of regulation that E545K confers on PI3Kα.
“…Currently, several PI3Kα inhibitors are under development and in clinical trials 12 . Alone or in combination with other drugs, PI3Kα inhibitors prevent tumor growth particularly in estrogen receptor alpha (ERα)-positive breast cancers, but depending on the type of the PI3Kα mutation different therapeutic regimes must be followed 13 . Figure 1 PI3Kα crystal structure and organization in functional domains.…”
Phosphoinositide 3-kinase alpha (PI3Kα) is involved in fundamental cellular processes including cell proliferation and differentiation and is frequently mutated in human malignancies. One of the most common mutations is E545K, which results in an amino acid substitution of opposite charge. It has been recently proposed that in this oncogenic charge-reversal mutation, the interactions between the protein catalytic and regulatory subunits are abrogated, resulting in loss of regulation and constitutive PI3Kα activity, which can lead to oncogenesis. To assess the mechanism of the PI3Kα E545K activating mutation, extensive Molecular Dynamics simulations were performed to examine conformational changes differing between the wild type (WT) and mutant proteins as they occur in microsecond simulations. In the E545K mutant PI3Kα, we observe a spontaneous detachment of the nSH2 PI3Kα domain (regulatory subunit, p85α) from the helical domain (catalytic subunit, p110α) causing significant loss of communication between the regulatory and catalytic subunits. We examine the allosteric network of the two proteins and show that a cluster of residues around the mutation is important for delivering communication signals between the catalytic and regulatory subunits. Our results demonstrate the dynamical and structural effects induced by the p110α E545K mutation in atomic level detail and indicate a possible mechanism for the loss of regulation that E545K confers on PI3Kα.
“…6b ). We recently reported that AKT1 but not AKT2 is active in MCF7 cells [ 17 ]. Immunoblotting using isoform-specific phospho-antibodies showed upregulation of pAKT1 but not pAKT2 in sh-UNC5A cells compared with sh-Control cells (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…Treatments consisted vehicle, heregulin-β1 (HRG-β1, R&D systems), E2, 4-hydroxy-tamoxifen (OHT), or ICI-182,780 (Sigma-Aldrich). The immunoblotting has been previously described [ 17 ] and details of antibodies are provided in Additional file 1 . Although the majority of immunoblots were reprobed with antibodies against ACTB (β-actin) as a loading control, only representative data per batch of cell lysates are shown.…”
BackgroundThe majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown.MethodsWe used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses.ResultsKnockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ∆Np63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2− tumors, was associated with poor outcome.ConclusionThese studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers.Electronic supplementary materialThe online version of this article (10.1186/s13058-018-0963-5) contains supplementary material, which is available to authorized users.
“…While these studies used other cancer models to study the role of BMAL1 , the findings are likely relevant to breast cancer. p53 mutations in breast cancer are relatively frequent (~20%) ( 85 , 87 ), and the PI3K/Akt pathway is commonly affected (~70%) ( 88 ). However, the same study found that p21 (a p53 target protein) and c-myc exhibited different expression levels in various BMAL1 -knockdown colon cancer cells, indicating that the relationships among BMAL1, p21, and c-myc are probably cell-type specific ( 71 ).…”
Section: Molecular Studies Of Circadian Clocks and Breast Cancermentioning
Circadian clocks are fundamental, time-tracking systems that allow organisms to adapt to the appropriate time of day and drive many physiological and cellular processes. Altered circadian rhythms can result from night-shift work, chronic jet lag, exposure to bright lights at night, or other conditioning, and have been shown to lead to increased likelihood of cancer, metabolic and cardiovascular diseases, and immune dysregulation. In cases of cancer, worse patient prognoses and drug resistance during treatment have also been observed. Breast, colon, prostate, lung, and ovarian cancers and hepatocellular carcinoma have all been linked in one way or another with altered circadian rhythms. Critical elements at the molecular level of the circadian system have been associated with cancer, but there have been fairly few studies in this regard. In this mini-review, we specifically focus on the role of altered circadian rhythms in breast cancer, providing an overview of studies performed at the epidemiological level through assessments made in animal and cellular models of the disease. We also address the disparities present among studies that take into account the rhythmicity of core clock and other proteins, and those which do not, and offer insights to the use of small molecules for studying the connections between circadian rhythms and cancer. This article will provide the reader with a concise, but thorough account of the research landscape as it pertains to altered circadian rhythms and breast cancer.
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