Inhibition of Proliferation by PERK Regulates Mammary Acinar Morphogenesis and Tumor Formation

Endoplasmic reticulum (ER) stress signaling can be mediated by the ER kinase PERK, which phosphorylates its substrate eIF2α. This in turn, results in translational repression and the activation of downstream programs that can limit cell growth through cell cycle arrest and/or apoptosis. These responses can also be initiated by perturbations in cell adhesion. Thus, we hypothesized that adhesion-dependent regulation of PERK signaling might determine cell fate. We tested this hypothesis in a model of mammary acini development, a morphogenetic process regulated in part by adhesion signaling. Here we report a novel role for PERK in limiting MCF10A mammary epithelial cell proliferation during acinar morphogenesis in 3D Matrigel culture as well as in preventing mammary tumor formation in vivo. We show that loss of adhesion to a suitable substratum induces PERK-dependent phosphorylation of eIF2α and selective upregulation of ATF4 and GADD153. Further, inhibition of endogenous PERK signaling during acinar morphogenesis, using two dominant-negative PERK mutants (PERK-ΔC or PERK-K618A), does not affect apoptosis but results instead in hyper-proliferative and enlarged lumen-filled acini, devoid of proper architecture. This phenotype correlated with an adhesion-dependent increase in translation initiation, Ki67 staining and upregulation of Laminin-5, ErbB1 and ErbB2 expression. More importantly, the MCF10A cells expressing PERKΔC, but not a vector control, were tumorigenic in vivo upon orthotopic implantation in denuded mouse mammary fat pads. Our results reveal that the PERK pathway is responsive to adhesion-regulated signals and that it is essential for proper acinar morphogenesis and in preventing mammary tumor formation. The possibility that deficiencies in PERK signaling could lead to hyperproliferation of the mammary epithelium and increase the likelihood of tumor formation, is of significance to the understanding of breast cancer

Analysis of Marker-Defined HNSCC Subpopulations Reveals a Dynamic Regulation of Tumor Initiating Properties

Head and neck squamous carcinoma (HNSCC) tumors carry dismal long-term prognosis and the role of tumor initiating cells (TICs) in this cancer is unclear. We investigated in HNSCC xenografts whether specific tumor subpopulations contributed to tumor growth. We used a CFSE-based label retentions assay, CD49f (α6-integrin) surface levels and aldehyde dehydrogenase (ALDH) activity to profile HNSCC subpopulations. The tumorigenic potential of marker-positive and -negative subpopulations was tested in nude (Balb/c nu/nu) and NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice and chicken embryo chorioallantoic membrane (CAM) assays. Here we identified in HEp3, SQ20b and FaDu HNSCC xenografts a subpopulation of G0/G1-arrested slow-cycling CD49fhigh/ALDH1A1high/H3K4/K27me3low subpopulation (CD49f+) of tumor cells. A strikingly similar CD49fhigh/H3K27me3low subpopulation is also present in primary human HNSCC tumors and metastases. While only sorted CD49fhigh/ALDHhigh, label retaining cells (LRC) proliferated immediately in vivo, with time the CD49flow/ALDHlow, non-LRC (NLRC) tumor cell subpopulations were also able to regain tumorigenic capacity; this was linked to restoration of CD49fhigh/ALDHhigh, label retaining cells. In addition, CD49f is required for HEp3 cell tumorigenicity and to maintain low levels of H3K4/K27me3. CD49f+ cells also displayed reduced expression of the histone-lysine N-methyltransferase EZH2 and ERK1/2phosphorylation. This suggests that although transiently quiescent, their unique chromatin structure is poised for rapid transcriptional activation. CD49f− cells can “reprogram” and also achieve this state eventually. We propose that in HNSCC tumors, epigenetic mechanisms likely driven by CD49f signaling dynamically regulate HNSCC xenograft phenotypic heterogeneity. This allows multiple tumor cell subpopulations to drive tumor growth suggesting that their dynamic nature renders them a “moving target” and their eradication might require more persistent strategies

Measuring the health-related Sustainable Development Goals in 188 countries : a baseline analysis from the Global Burden of Disease Study 2015

Background In September, 2015, the UN General Assembly established the Sustainable Development Goals (SDGs). The SDGs specify 17 universal goals, 169 targets, and 230 indicators leading up to 2030. We provide an analysis of 33 health-related SDG indicators based on the Global Burden of Diseases, Injuries, and Risk Factors Study 2015 (GBD 2015). Methods We applied statistical methods to systematically compiled data to estimate the performance of 33 health-related SDG indicators for 188 countries from 1990 to 2015. We rescaled each indicator on a scale from 0 (worst observed value between 1990 and 2015) to 100 (best observed). Indices representing all 33 health-related SDG indicators (health-related SDG index), health-related SDG indicators included in the Millennium Development Goals (MDG index), and health-related indicators not included in the MDGs (non-MDG index) were computed as the geometric mean of the rescaled indicators by SDG target. We used spline regressions to examine the relations between the Socio-demographic Index (SDI, a summary measure based on average income per person, educational attainment, and total fertility rate) and each of the health-related SDG indicators and indices. Findings In 2015, the median health-related SDG index was 59.3 (95% uncertainty interval 56.8-61.8) and varied widely by country, ranging from 85.5 (84.2-86.5) in Iceland to 20.4 (15.4-24.9) in Central African Republic. SDI was a good predictor of the health-related SDG index (r(2) = 0.88) and the MDG index (r(2) = 0.2), whereas the non-MDG index had a weaker relation with SDI (r(2) = 0.79). Between 2000 and 2015, the health-related SDG index improved by a median of 7.9 (IQR 5.0-10.4), and gains on the MDG index (a median change of 10.0 [6.7-13.1]) exceeded that of the non-MDG index (a median change of 5.5 [2.1-8.9]). Since 2000, pronounced progress occurred for indicators such as met need with modern contraception, under-5 mortality, and neonatal mortality, as well as the indicator for universal health coverage tracer interventions. Moderate improvements were found for indicators such as HIV and tuberculosis incidence, minimal changes for hepatitis B incidence took place, and childhood overweight considerably worsened. Interpretation GBD provides an independent, comparable avenue for monitoring progress towards the health-related SDGs. Our analysis not only highlights the importance of income, education, and fertility as drivers of health improvement but also emphasises that investments in these areas alone will not be sufficient. Although considerable progress on the health-related MDG indicators has been made, these gains will need to be sustained and, in many cases, accelerated to achieve the ambitious SDG targets. The minimal improvement in or worsening of health-related indicators beyond the MDGs highlight the need for additional resources to effectively address the expanded scope of the health-related SDGs.Peer reviewe

The pioneer translation initiation complex is functionally distinct from but structurally overlaps with the steady-state translation initiation complex

The bulk of cellular proteins derive from the translation of eukaryotic translation initiation factor (eIF)4E-bound mRNA. However, recent studies of nonsense-mediated mRNA decay (NMD) indicate that cap-binding protein (CBP)80-bound mRNA, which is a precursor to eIF4E-bound mRNA, can also be translated during a pioneer round of translation. Here, we report that the pioneer round, which can be assessed by measuring NMD, is not inhibited by 4E-BP1, which is known to inhibit steady-state translation by competing with eIF4G for binding to eIF4E. Therefore, at least in this way, the pioneer round of translation is distinct from steady-state translation. eIF4GI, poly(A)-binding protein (PABP)1, eIF3, eIF4AI, and eIF2α coimmunopurify with both CBP80 and eIF4E, which suggests that each factor functions in both modes of translation. Consistent with roles for PABP1 and eIF2α in the pioneer round of translation, PABP-interacting protein 2, which is known to destabilize PABP1 binding to poly(A) and inhibit steady-state translation, as well as inactive eIF2α, which is also known to inhibit steady-state translation, also inhibit NMD. Polysome profiles indicate that CBP80-bound mRNAs are translated less efficiently than their eIF4E-bound counterparts

Suspension Induces Phosphorylation of eIF2α and Translation Repression in Mammary and Kidney Epithelial Cells.

<p>(A) Whole cell lysates from MCF10A (upper left), HEK293T (upper right) and primary HMEC (lower panels) cells grown either in adhered (A) or suspended conditions (S) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000615#s4" target="_blank">methods</a> section for the indicated time points, were immunoblotted for p-eIF2α and total eIF2α levels. Adhered MCF10A or HEK293T cells treated with 2 mM DTT or 5 µg/ml tunicamycin (Tn) respectively, were used as positive controls. (B) Quantification of the rate of DNA synthesis using a BrdU incorporation assay and flow cytometry to measure the percentage of BrdU-positive cells (filled diamonds) at different time points in suspension. The percentage of apoptotic cells was measured using propidium iodide staining and flow cytometry to identify the sub-G0 apoptotic fraction for adhered (dashed line) or suspended (dotted line) MCF10A cells for different time points. Data points show the mean±SD for BrdU–positive cells in each sample as a percentage of the total. (C) Autoradiogram of [³⁵S] Met/Cys incorporation (right panel) into newly synthesized proteins in MCF10A cells adhered or suspended for 24 hrs (two independent samples). Coomassie Blue staining of an identical gel (left panel) shows equal protein loading. (D) Polysome profiles from 24 hr adhered (left) and suspended (right) MCF10A cells showing an increase and decrease in the monosome and polysome peaks, respectively in suspended cells. Absorbance at 254 nm (Y-axis, RNA concentration) was plotted against migration in the sucrose gradient (X-axis, bottom to top). Total RNA was isolated from individual fractions to visualize the 18S and 28S rRNAs by ethidium bromide staining.</p

GADD153 mRNA and Protein Levels Are Strongly Upregulated During Suspension Conditions.

<p>(A) RT-PCR analysis of GADD153 mRNA levels in MCF10A (left panel) and HEK293T (right panel) cells at different time points in either adhered (A) or suspended (S) conditions. Adhered MCF10A cells treated with 2mM DTT for 4h were used as positive control and GAPDH was used as a loading control. (B) MCF10A cells were transiently transfected with a GADD153 promoter-driven-EGFP reporter plasmid and EGFP fluorescence was analyzed 48 h post-transfection by FACS; total events captured: 2×10⁴. The graph shows the number of GFP-positive events in FLH2>10 (mean±SD). (C) Western blot for GADD153 protein in adhered (A) or suspended (S) MCF10A cells. (D and E) Immunofluorescence (D) and FACS (E) analysis of GADD153 (red) expression in MCF10A cells following growth in adhered or suspension conditions for the indicated times. Secondary antibody was used as negative control in E. (F) MCF10A (top) and HEK293T (lower) cells were transiently co-transfected with the GADD153-EGFP reporter plasmid and either a full-length Flag-tagged GADD34 plasmid or an empty vector as control for 24 hrs before being detached and left to reattach or put into suspension for an additional 48 hrs before FACS analysis. GFP fluorescence was analyzed 48 h post transfection by FACS where a total 2×10⁴ events were captured. The graphs show the number of GFP positive events in FLH2>10 or the mean fluorescence intensity (MFI) in the PMT2-FITC channel (mean±SD). (G) RT-PCR for XBP-1 splicing (top panel) in MCF10A cells at different time points either adhered (A) or suspended (S). Adhered MCF10A cells treated with 2mM DTT for 4 hrs was used as positive control and GAPDH, shown in (A) was used as a loading control. Lower panels show RT-PCR for BiP, Hsp47 and Erp72/PDI chaperone mRNA levels in adhered (Adh) or suspended (Sus) MCF10A cells. GAPDH was used as a loading control.</p

Unscheduled Activation of PERK Restricts Acinar Growth and Promotes Apoptosis in 3D Matrigel.

<p>(A) Time-dependent increase in phosphorylation of eIF2α in MCF10A cells expressing an Fv2E-ΔNPERK construct, upon treatment with the dimerizing drug, AP20187 (2 nM). AP20187 has no effect on P-eIF2α levels in β-Gal cells. Total eIF2α was used as loading control. (B) Photomicrographs of Fv2E-ΔNPERK cells in 3D Matrigel treated with 2 nM AP20187 or equal volume of ethanol as control, added every 24 hrs from Day 4 up to Day 6 of morphogenesis; representative phase-contrast images depict the effect of forced PERK activation on acini development; (B-a and B-e) A×10 magnification image of several developing acini; (B-b) Normal acinus, (B-c) 2 cell cluster, (B-f) 4 cell cluster, (B-g) 4 cell cluster containing apoptotic cells. (B-d and h) Confocal images through the equatorial region of Fv2E-ΔNPERK cells in 3D Matrigel immunostained for active caspase-3 (red) or LN-5 (green) with (B-h) and without (B-d) treatment with 2nM AP20187 every 24 hrs, (B-h) cell cluster where the majority of cells have entered apoptosis. (C) Quantitation of phase contrast images of Fv2E-ΔNPERK cells on Day 6, treated every 24 hrs with or without 2nM AP20187. Over 400 acini were visually scored for the presence of apoptotic or growth arrested 2–4 celled acini and calculated as a percentage of the total number of acini; graph shows mean±SD. (D) Photomicrographs of β-Gal vector control cells treated with 2nM AP20187 or with equal volume EtOH as control, every 24 hrs from Day 4 up to Day 6 in Matrigel. Note that AP20187 treatment caused no noticeable changes in acini size or morphology, consistent with the absence of modulation of P-eIF2α levels in the same cells (<i>A</i>). (E) Confocal images showing Fv2E-ΔNPERK cells treated with (+AP) or without (-AP) AP20187 every 12 hrs stained for LN-5 (green) to delineate the acini and active caspase-3 (red). Note that a majority of cells even in large acini can be pushed into apoptosis by strong activation of PERK signaling. Scale bars = 10 µm.</p