沃度保兒謨ニ因セル濕疹

Evaluation of CNA-IC50 correlations by known cancer driver mutations. Distributions of PCCs for cancer cell lines with mutated or wild-type proto-oncogenes or tumor suppressors as depicted in each figure. The rank position of negatively correlated drugs (p < 0.10) is shown. (EPS 975 kb

A Mouse Model Uncovers LKB1 as an UVB-Induced DNA Damage Sensor Mediating CDKN1A (p21^WAF1/CIP1) Degradation

<div><p>Exposure to ultraviolet (UV) radiation from sunlight accounts for 90% of the symptoms of premature skin aging and skin cancer. The tumor suppressor serine-threonine kinase <i>LKB1</i> is mutated in Peutz-Jeghers syndrome and in a spectrum of epithelial cancers whose etiology suggests a cooperation with environmental insults. Here we analyzed the role of LKB1 in a UV-dependent mouse skin cancer model and show that <i>LKB1</i> haploinsufficiency is enough to impede UVB-induced DNA damage repair, contributing to tumor development driven by aberrant growth factor signaling. We demonstrate that LKB1 and its downstream kinase NUAK1 bind to CDKN1A. In response to UVB irradiation, LKB1 together with NUAK1 phosphorylates CDKN1A regulating the DNA damage response. Upon UVB treatment, <i>LKB1</i> or <i>NUAK1</i> deficiency results in CDKN1A accumulation, impaired DNA repair and resistance to apoptosis. Importantly, analysis of human tumor samples suggests that <i>LKB1</i> mutational status could be a prognostic risk factor for UV-induced skin cancer. Altogether, our results identify LKB1 as a DNA damage sensor protein regulating skin UV-induced DNA damage response.</p></div

<i>Hgf^Tg; Lkb1^+/−</i> mice are highly prone to neonatal UVB-induced SCCs.

<p>(<b>A</b>) Kaplan–Meier analysis of neonatal UVB irradiated wild type (WT), <i>Hgf^Tg, Lkb1^+/−</i> and <i>Hgf</i>^Tg; <i>Lkb1</i>^+/− mice documenting the development of SCC. <i>Hgf^Tg, Lkb1^+/−</i> mice showed significant differences in UVB-induced tumor development, P<0.0001). (<b>B</b>) (i to iii), gross image and progression of SCC in an <i>Hgf</i>^Tg; <i>Lkb1</i>^+/− mouse after UVB irradiation. (<b>C</b>) Histology of cutaneous SCC. Hematoxilin-Eosin staining of mouse tumor samples and immunostaining of SCC for involucrin keratin-14, β-catenin, p-C-MET, LKB1 and cyclin D1. Bars 200 µm, Inset bar 50 µm. (<b>D</b>) Penetrance of skin-SCC in neonatal UVB-irradiated vs. non-irradiated mice. <i>P</i>-value was calculated using a fisher's exact test between UVB-irradiated vs. non-irradiated mice. (<b>E</b>) Hematoxilin-Eosin staining of mouse and human samples showing histological similarities. Bars upper panels 150 µm, bars lower panels 50 µm.</p

LKB1 expression in human skin-SCC.

<p>(<b>A</b>) Representative images of differentiated, moderately differentiated and poorly differentiated human skin SCC, showing high expression and low expression amounts of LKB1. A positive control of LKB1 specific staining (Muscle) is shown. (<b>B</b>) Distribution of human tumor samples (n = 51) according to their stage of differentiation and the Hscore for LKB1. (<b>C</b>) Distribution of the same samples in respect to the exposure of the samples to UV according to their anatomical distribution. (<b>D</b>) Distribution of low LKB1 expression samples within the different tumor stages and according to their UV status. (<b>E</b>) Model for the role of LKB1 in UVB-induced DNA damage response. In response to low doses of UV radiation, LKB1 becomes phosphorylated by ATR and induces CDKN1A degradation through its phosphorylation liberating PCNA and its recruitment to chromatin for DNA repair. In the absence of LKB1, CDKN1A is not phosphorylated and accumulates, contributing to UV-induced mutagenesis and resistance to apoptosis. According to the animal model this UVB-induced DNA damage cooperates with aberrant growth factor signaling for tumor development.</p

LKB1 and NUAK1 phosphorylate CDKN1A.

<p>(<b>A</b>) In vitro kinase assay using recombinant heterotrimer His-LKB1/GST-STRADα/GST-Mo25α and recombinant human <i>Hs</i>-GST-CDKN1A. Autoradiography shows in vitro phosphorylation of CDKN1A by <i>LKB1</i>. Western-blot shows the loading for GST-tagged proteins. Assays were performed in triplicates. Mass spectrometry analysis of <i>in vitro</i> phosphorylated CDKN1A by LKB1. (<b>B</b>) LKB1 phosphorylates CDKN1A in vivo. 293T cells were transfected with CDKN1A and/or equimolar amounts of Flag-<i>Lkb1</i>^WT, Flag-<i>STRADα</i> and <i>MO25α</i>. After in vivo labeling with [³²P], CDKN1A and Flag-LKB1 were immunoprecipitated and the amount of CDKN1A phosphorylated determined by autoradiography. Western blots show the immunoprecipitated CDKN1A and LKB1 in one representative experiment out of three. (<b>C</b>) In vitro kinase assay using recombinant heterotrimer His-LKB1/GST-STRADα/GST-Mo25α, NUAK1 and recombinant human (<i>Hs</i>) and mouse (<i>Mm</i>) GST-CDKN1A. Below, mass spectrometry analysis of <i>in vitro</i> phosphorylated CDKN1A by LKB1 or NUAK1. One out of four experiments is shown. (<b>D</b>) HaCat cells were transfected with scrambled (Scr. shRNA), <i>LKB1</i> (shRNA) or <i>NUAK1</i> (siRNA). Western-blot shows the amounts of CDKN1A, LKB1 and NUAK1. GAPDH is used as loading control. (<b>E</b>) HaCat cells were transiently transfected with either HA-<i>NUAK1</i>^WT, HA-<i>NUAK1</i>^T211A and treated with UVB for the indicated time points. Amounts of CDKN1A and NUAK1 proteins are showed. GAPDH is the loading control. (<b>F</b>) HaCat cells stably infected with <i>shLKB1</i> were transfected with HA-<i>NUAK1</i> and treated with UVB for the indicated times. Variation of the amount of CDKN1A was assessed by western-blot. GAPDH is shown as loading control.</p

UVB-induced phosphorylation of LKB1^T366 mediates CDKN1A degradation and DNA repair.

<p>(<b>A</b>) Mouse skin from non irradiated and UVB irradiated mouse were stained with anti p-LKB1^T366 antibody. Dashed squares indicate amplified areas. Bar represent 100 µm. (<b>B</b>) HeLa cells were transfected with <i>CDKN1A</i>, <i>MO25α</i>, Flag-<i>STRADα</i> and either Flag-<i>Lkb1</i>^WT, Flag-<i>Lkb1</i>^KD or Flag-<i>Lkb1</i>^T366A mutant and treated with UVB (30 J/m²) and lysed 30 min after UVB irradiation. Western-blot shows the expression of LKB1, STRADα and CDKN1A. Graph shows the quantifications of the bands normalized against GAPDH. One representative experiment out of three is shown. (<b>C</b>) On the left HaCat cells were transfected either with Flag-<i>Lkb1</i>^WT, Flag-<i>Lkb1</i>^KD or Flag-<i>Lkb1</i>^T366A together with Flag-<i>STRADα</i> and <i>Mo25α</i>. Western-blot shows the amount of LKB1 and endogenous CDKN1A 30 min after UVB irradiation (30 J/m²). Graph show quantifications under the different conditions. (n = 3 experiments). Error bars represent mean ± SD. <i>P</i>-value was calculated performing a student's <i>t</i>-test. On the right NHEK were transfected with either Flag-<i>Lkb1</i>^WT or Flag-<i>Lkb1</i>^T366A mutant and treated with UVB (30 J/m²) and lysed at the indicated time points. Western-blot shows the amount of LKB1 and endogenous CDKN1A. Fold induction of CDKN1A expression normalized against GAPDH is showed. One representative experiment out of three is shown. (<b>D</b>) Global UVB-induced DNA damage repair assay. LKB1 knockdown HaCat cells were transfected and treated as in (<b>C</b>). Graphs show normalized quantification (n = 3 experiments) of DNA damage repair 48 h after UVB irradiation. <i>P</i>-value was calculated performing a student's <i>t</i>-test. (<b>E</b>) HaCat cells and <i>LKB1</i> HaCat knockdown cells were transiently transfected with <i>CDKN1A</i> siRNA and treated with UVB (30 J/m²). Western-blot shows the amounts of CDKN1A. Graph shows percentage of UVB-induced DNA damage repair.</p

Loss of LKB1 and accumulation of CDKN1A in response to UVB contributes to keratinocyte transformation and resistance to UVB-induced apoptosis.

<p>(<b>A</b>) HaCat cells infected either with scrambled or <i>shLKB1</i> were irradiated with UVB (30 J/m²). Then, at 48 h, EGFP-Annexin V and PI (propidium iodide) positive cells were analyzed by flow cytometry. Histograms show the result from FACS analysis. (<b>B</b>) Time course of UVB irradiated (30 J/m²) HaCat cells infected either with scrambled or two different <i>shLKB1</i> (#1 and #2). Western-blot shows the amounts of BIM, PUMA, CDKN1A and LKB1. GAPDH is shown as loading control. Graph shows quantification of bands normalized by GAPDH. (<b>C</b>) Immunohistochemistry showing Bim and cleaved caspase-3 staining in wild type (WT), <i>Lkb1</i>^+/− (L), <i>Hgf</i>^Tg (H) and <i>Hgf</i>^Tg; <i>Lkb1</i>^+/− (HL) mice. Bar represent 100 µm. Graphs show quantification of positive cells in mouse skin (at least 25 fields (20×)/genotype). Bars represent mean values. <i>P</i>-values were calculated using a student's t-test.</p

<i>Lkb1</i> haploinsufficiency induces CDKN1A accumulation after UVB-mediated DNA damage.

<p>(<b>A</b>) Representation of the average amounts of p-CHK2 and CDKN1A in the skin of mice from different genotypes at 48 h post irradiation. (WT, <i>Lkb1</i>^+/− (L), <i>Hgf</i>^Tg (H) and <i>Hgf</i>^Tg; <i>Lkb1</i>^+/− (HL)). <i>P</i>-values were calculated performing a student's <i>t</i>-test. (<b>B</b>) Global genomic UVB-induced DNA repair analysis performed in skin DNA from WT <i>Lkb1^+/−</i> and <i>Lkb1^+/−; Hgf^Tg</i> mice. Graphs show the average repair at different time point. At least five mice per genotype and time point were analyzed. Error bars represent mean ± SD. <i>P</i>-values were calculated performing a student's <i>t</i>-test. (<b>C</b>) CDKN1A degradation is induced after UVB DNA damage in Normal Human Epidermal Keratinocytes (NHEK). NHEK were pretreated for 2 h with MG132 (200 nM) and treated with UVB (30 J/m²). Western-blot shows the level of p-ATR, CDKN1A, LKB1. β-Actin is shown as a loading control. One representative experiment of three is shown. (<b>D</b>) Depletion of LKB1 in normal human epidermal keratinocytes (NHEK) and immortalized normal keratinocytes (HaCat cells) induced the accumulation of CDKN1A after UVB irradiation. Cells stably infected with lentiviral constructs expressing either scramble shRNA (<i>Scr</i>.) or two different shRNAs sequences against human <i>LKB1</i> (<i>shLKB1#1, shLKB1#2</i>). Western blot show the amount of LKB1, CDKN1A and GAPDH after UVB treatment (30 J/m²). (<i>n</i> = 3 experiments). On the right panel, LKB1 depletion does not induce the transcriptional up regulation of <i>CDKN1A</i>. <i>CDKN1A</i> and <i>LKB1</i> mRNA abundance were determined after UVB irradiation (30 J/m²) by qRT-PCR. Error bars represent mean ± SD. Measurements were normalized against <i>18S</i> mRNA and <i>GAPDH</i> (<i>n</i> = 3 experiments). (<b>E</b>) UVB irradiation induces CDKN1A accumulation in <i>Lkb1^+/−</i> and <i>Hgf</i>^Tg; <i>Lkb1</i>^+/− mice. Isolated keratinocytes from different mouse skin genotypes were UVB irradiated (30 J/m²) (WT, <i>Lkb1</i>^+/− (L), <i>Hgf</i>^Tg (H) and <i>Hgf</i>^Tg; <i>Lkb1</i>^+/− (HL)). Western blot shows the amount of LKB1, CDKN1A and GAPDH at the indicated time points post-irradiation. Graph show the normalized quantification of bands. (<b>F</b>) Global genomic UVB-induced DNA repair analysis. HaCat cells infected with scrambled shRNA (Scr.), <i>shLKB1#1</i> or <i>shLKB1#2</i> were irradiated with (30 J/m²). Graphs show the quantification of the modification's repair normalized by the amount of DNA from at least three independent experiments. Error bars represent mean ± SD. <i>P</i>-value was calculated doing a student's <i>t</i>-test.</p

Additional file 2: Table S2. of Cancer network activity associated with therapeutic response and synergism

Normalized CNA values for all cancer cell lines. (XLSX 55 kb

Additional file 5: Figure S3. of Cancer network activity associated with therapeutic response and synergism

The CNA-IC50 correlation differences are not observed when a random, null network model that preserves degree distribution and connectedness is analyzed. (PDF 148 kb