Abstract Mutational activation of the PI3K/AKT pathway is among the most common pro-oncogenic events in human cancers. The clinical utility of PI3K and AKT inhibitors has, however, been modest to date. Here, we used CRISPR-mediated gene editing to study the biological consequences of AKT1 E17K mutation by developing an AKT1 E17K–mutant isogenic system in a TP53-null background. AKT1 E17K expression under the control of its endogenous promoter enhanced cell growth and colony formation, but had a paradoxical inhibitory effect on cell migration and invasion. The mechanistic basis by which activated AKT1 inhibited cell migration and invasion was increased E-cadherin expression mediated by suppression of ZEB1 transcription via altered β-catenin subcellular localization. This phenotypic effect was AKT1-specific, as AKT2 activation had the opposite effect, a reduction in E-cadherin expression. Consistent with the opposing effects of AKT1 and AKT2 activation on E-cadherin expression, a pro-migratory effect of AKT1 activation was not observed in breast cancer cells with PTEN loss or expression of an activating PIK3CA mutation, alterations which induce the activation of both AKT isoforms. The results suggest that the use of AKT inhibitors in patients with breast cancer could paradoxically accelerate metastatic progression in some genetic contexts and may explain the frequent coselection for CDH1 mutations in AKT1-mutated breast tumors. Implications: AKT1 E17K mutation in breast cancer impairs migration/invasiveness via sequestration of β-catenin to the cell membrane leading to decreased ZEB1 transcription, resulting in increased E-cadherin expression and a reversal of epithelial–mesenchymal transition.
Abstract Radiation therapy is an effective cancer treatment although damage to healthy tissues is common. Here we characterize the methylomes of healthy human and mouse tissues to establish sequencing-based, cell-type specific reference DNA methylation atlases. Identified cell-type specific DNA blocks were mostly hypomethylated and located within genes intrinsic to cellular identity. Cell-free DNA fragments released from dying cells into the circulation were captured from serum samples by hybridization to CpG-rich DNA panels. The origins of the circulating DNA fragments were inferred from mapping to the established DNA methylation atlases. Thoracic radiation-induced tissue damages in a mouse model were reflected by dose-dependent increases in lung endothelial, cardiomyocyte and hepatocyte methylated DNA in serum. The analysis of serum samples from breast cancer patients undergoing radiation treatment revealed distinct tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type specific effects of radiation and provide a quantitative measure of the biologically effective radiation dose received by healthy tissues. Graphical Abstract
<p>This supplementary data file contains 5 figures pertaining to the tumor distribution of MEK1 hotspot mutations, functional characterization of further MEK1/2 mutants with and without MEK or ERK inhibitor treatment, and the sequence paralogy alignment of MEK1 and MEK2. This file also contains 4 tables detailing hotspot analysis q values for MEK1/2 mutants, tumor incidence of MEK1 in-frame deletions, and a summary of MEK1/2 paralogous residues and their concordant activation status.</p>
// Xiaoling Fu 1 , Weixin Niu 2 , Ji Li 3 , Amber J. Kiliti 4 , Hikmat A. Al-Ahmadie 5 , Gopa Iyer 6,7 , Sizhi Paul Gao 4 and Qi Li 1 1 Department of Medical Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, P.R. China 2 Department of Surgery, Zhongshan Hospital of Fudan University, Shanghai, P.R. China 3 Department of Pancreatic Surgery, Huashan Hospital, Fudan University and Shanghai Medical School, Shanghai, P.R. China 4 Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA 5 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York, USA 6 Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA 7 Weill Cornell Medical College, Cornell University, New York, New York, USA Correspondence to: Qi Li, email: // Sizhi Paul Gao, email: // Keywords : cardiac sarcoma, intimal sarcoma, memorial sloan kettering-integrated mutation profiling of actionable cancer targets, PDGFRA, PDGFRB Received : November 18, 2016 Accepted : July 25, 2017 Published : September 07, 2017 Abstract Cardiac sarcoma is a rare malignant tumor with undefined genetic mutations and no targeted therapy. Here in one rare case of undifferentiated cardiac intimal sarcoma (IS), a next-generation sequencing based assay, MSK-IMPACT (Memorial Sloan Kettering - Integrated Mutation Profiling of Actionable Cancer Targets), identified a somatic, activating mutation in PDGFRB , along with amplification of PDGFRA . This E472D mutation of PDGFRB was discovered for the first time in IS. These findings suggest that concurrent aberrant PDGFRA and PDGFRB signaling may be a diagnostic biomarker and molecular therapeutic target of IS of the heart.
<div>Abstract<p>Despite significant advances in cancer precision medicine, a significant hurdle to its broader adoption remains the multitude of variants of unknown significance identified by clinical tumor sequencing and the lack of biologically validated methods to distinguish between functional and benign variants. Here we used functional data on <i>MAP2K1</i> and <i>MAP2K2</i> mutations generated in real-time within a co-clinical trial framework to benchmark the predictive value of a three-part <i>in silico</i> methodology. Our computational approach to variant classification incorporated hotspot analysis, three-dimensional molecular dynamics simulation, and sequence paralogy. <i>In silico</i> prediction accurately distinguished functional from benign <i>MAP2K1</i> and <i>MAP2K2</i> mutants, yet drug sensitivity varied widely among activating mutant alleles. These results suggest that multifaceted <i>in silico</i> modeling can inform patient accrual to MEK/ERK inhibitor clinical trials, but computational methods need to be paired with laboratory- and clinic-based efforts designed to unravel variabilities in drug response.</p>Significance:<p>Leveraging prospective functional characterization of MEK1/2 mutants, it was found that hotspot analysis, molecular dynamics simulation, and sequence paralogy are complementary tools that can robustly prioritize variants for biologic, therapeutic, and clinical validation.</p><p><i>See related commentary by Whitehead and Sebolt-Leopold, p. 4042</i></p></div>
<div>Abstract<p>Mutational activation of the PI3K/AKT pathway is among the most common pro-oncogenic events in human cancers. The clinical utility of PI3K and AKT inhibitors has, however, been modest to date. Here, we used CRISPR-mediated gene editing to study the biological consequences of AKT1 E17K mutation by developing an AKT1 E17K–mutant isogenic system in a <i>TP53</i>-null background. AKT1 E17K expression under the control of its endogenous promoter enhanced cell growth and colony formation, but had a paradoxical inhibitory effect on cell migration and invasion. The mechanistic basis by which activated AKT1 inhibited cell migration and invasion was increased E-cadherin expression mediated by suppression of <i>ZEB1</i> transcription via altered β-catenin subcellular localization. This phenotypic effect was AKT1-specific, as AKT2 activation had the opposite effect, a reduction in E-cadherin expression. Consistent with the opposing effects of AKT1 and AKT2 activation on E-cadherin expression, a pro-migratory effect of AKT1 activation was not observed in breast cancer cells with PTEN loss or expression of an activating <i>PIK3CA</i> mutation, alterations which induce the activation of both AKT isoforms. The results suggest that the use of AKT inhibitors in patients with breast cancer could paradoxically accelerate metastatic progression in some genetic contexts and may explain the frequent coselection for <i>CDH1</i> mutations in <i>AKT1</i>-mutated breast tumors.</p>Implications:<p>AKT1 E17K mutation in breast cancer impairs migration/invasiveness via sequestration of β-catenin to the cell membrane leading to decreased <i>ZEB1</i> transcription, resulting in increased E-cadherin expression and a reversal of epithelial–mesenchymal transition.</p></div>
<p>S1. Generation of MCF-10A isogenic cells via TP53 knockout and AKT1 E17K knock-in. S2. Frequent co-occurrence of AKT1, TP53 and CDH1 mutations in breast cancers. S3. TP53 CRISPR knockout in MCF-10A cells. S4. Phosphorylation of the AKT1 effector PRAS40 was AKT-dependent in MCF-10A p53ko/E17K cells. S5. Immunofluorescence using a pT308 specific AKT1 antibody of MCF-10A cells following lentiviral infection with V5 tagged (V5 tag) wildtype (WT) or E17K mutant AKT1. S6. Knock-in of the AKT1 E17K mutation did not enhance sensitivity to the pan-AKT kinase inhibitor AZD5363 or the allosteric pan-AKT inhibitor MK2206 in 2Dculture conditions. S7. Survey of previously postulated mechanisms for AKT1-mediated inhibition of cell migration in breast cancer cells. S8. CDH1 promoter methylation status in the MCF-10A isogenic cells. S9. β-catenin interacts with the kinase domain of AKT1. S10. Expression of AKT1 E17K decreased phosphorylation of AKT2 in MCF-10A cells. S11. AKT2 knockdown induces E-cadherin expression in MCF-10A p53ko cells. S12. Model of the mechanism of paradoxical inhibition of cell migration by activated AKT1. Table S1. List of oligonucleotide sequences Table S2. List of antibodies Table S3. List of plasmids</p>
Mutations in the gene ankyrin repeat domain containing 11 (ANKRD11/ANCO1) play a role in neurodegenerative disorders, and its loss of heterozygosity and low expression are seen in some cancers. Here, we show that low ANCO1 mRNA and protein expression levels are prognostic markers for poor clinical outcomes in breast cancer and that loss of nuclear ANCO1 protein expression predicts lower overall survival of patients with triple-negative breast cancer (TNBC). Knockdown of ANCO1 in early-stage TNBC cells led to aneuploidy, cellular senescence, and enhanced invasion in a 3D matrix. The presence of a subpopulation of ANCO1-depleted cells enabled invasion of the overall cell population in vitro and they converted more rapidly to invasive lesions in a xenograft mouse model. In ANCO1-depleted cells, ChIP-seq analysis showed a global increase in H3K27Ac signals that were enriched for AP-1, TEAD, STAT3, and NFκB motifs. ANCO1-regulated H3K27Ac peaks had a significantly higher overlap with known breast cancer enhancers compared to ANCO1-independent ones. H3K27Ac engagement was associated with transcriptional activation of genes in the PI3K-AKT, epithelial-mesenchymal transition (EMT), and senescence pathways. In conclusion, ANCO1 has hallmarks of a tumor suppressor whose loss of expression activates breast-cancer-specific enhancers and oncogenic pathways that can accelerate the early-stage progression of breast cancer.
Abstract Purpose . The cardiovascular biology of proton radiotherapy is not well understood. We aimed to compare the genomic dose-response to proton and gamma radiation of the mouse aorta to assess whether their vascular effects may diverge. Materials and methods. We performed comparative RNA sequencing of the aorta following (4 hrs) total-body proton and gamma irradiation (0.5 - 200 cGy whole body dose, 10 dose levels) of conscious mice. A trend analysis identified genes that showed a dose response. Results. While fewer genes were dose-responsive to proton than gamma radiation (29 vs. 194 genes; q -value ≤ 0.1), the magnitude of the effect was greater. Highly responsive genes were enriched for radiation response pathways (DNA damage, apoptosis, cellular stress and inflammation; p -value ≤ 0.01). Gamma, but not proton radiation induced additionally genes in vasculature specific pathways. Genes responsive to both radiation types showed almost perfectly superimposable dose-response relationships. Conclusions. Despite the activation of canonical radiation response pathways by both radiation types, we detected marked differences in the genomic response of the murine aorta. Models of cardiovascular risk based on photon radiation may not accurately predict the risk associated with proton radiation.
Pancreatic cancer remains largely unresponsive to immune modulatory therapy attributable in part to an immunosuppressive, desmoplastic tumor microenvironment. Here, we analyze mechanisms of cancer cell-autonomous resistance to T cells. We used a 3D co-culture model of cancer cell spheroids from the KPC (LSL-KrasG12D/+/LSL-Trp53R172H/+/p48-Cre) pancreatic ductal adenocarcinoma (PDAC) model, to examine interactions with tumor-educated T cells isolated from draining lymph nodes of PDAC-bearing mice. Subpopulations of cancer cells resistant to these tumor-educated T cells were isolated from the in vitro co-culture and their properties compared with sensitive cancer cells. In co-culture with resistant cancer cell subpopulations, tumor-educated T cells showed reduced effector T cell functionality, reduced infiltration into tumor cell spheroids and decreased induction of apoptosis. A combination of comparative transcriptomic analyses, cytometric and immunohistochemistry techniques allowed us to dissect the role of differential gene expression and signaling pathways between sensitive and resistant cells. A decreased expression of the chemokine CXCL12 (SDF-1) was revealed as a common feature in the resistant cell subpopulations. Adding back CXCL12 reversed the resistant phenotype and was inhibited by the CXCR4 inhibitor AMD3100 (plerixafor). We conclude that reduced CXCL12 signaling contributes to PDAC subpopulation resistance to T cell-mediated attack.
