Immunostaining of histological sections of Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> and CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo adenomas (<i>Kras</i>^+/LSLG12Vgeo 6 months) and Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ and CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo adenocarcinomas (<i>Kras</i>^+/LSLG12Vgeo 11 months) with CC10, SP-C, E-Cadherin, Ki67, p53 and LKB1 antibodies.

<p>(*) indicates airways. Bars 500 µm and 80 µm for magnifications.</p

Neonatal activation of BRAF^V600E through the expression of Tyr::<i>Cre</i>^ERT2 upon 4OHTx treatment drives aberrant proliferation of lung cells.

<p>(<b>A</b>) Schematic representation of mouse treatment and expressed proteins. (<b>B</b>) Representative images of Cre-recombinase staining in 4-days-old wild type (WT, n = 3) and Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> (n = 3) mice lungs treated with 4OHTx. Bars 80 µm. (<b>C</b>) Anti-p-ERK1/2 staining of untreated (−4OHTx) and treated (+4OHTx) 4-days-old Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> mice lungs. (<b>D</b>) Schematic representation of the genetic strategy to identify tyrosinase-promoter driven Cre-recombinase lung expressing cells. Representative images of EYFP, SP-C and CC10 expressing cells in Tyr::<i>Cre</i>^ERT2;ROSA-lsl-<i>EYFP</i> mice 3 days after 4OHTx treatment (n = 3 mice) are shown. Bars 80 µm. (<b>E</b>) Ki67 staining of histologically normal lungs in 8-days-old mice showed increased proliferation index in 4OHTx treated Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> mice compared to untreated. Bars 500 µm. Quantification of samples is shown below. 20X fields (n = 8 and n = 11 from 3 different untreated and 4OHTx treated mice respectively) were quantified. <i>p</i>-value was calculated performing Mann-Whitney’s test.</p

Percentage of mice developing adenomas and adenocarcinomas.

<p>Hematoxylin and eosin staining of histological sections showing lung hyperplasia in Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ (<b>A</b>, <b>B</b>) and CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo mice (<b>J</b>, <b>K</b>). Higher magnification is showed in (<b>B</b>, <b>K</b>). Papillary adenomas developed in Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ (<b>C</b>, <b>D</b>) and mixed papillary and solid adenomas developed in CMV-<i>Cre</i>^T/+; <i>K-ras</i>^+/LSLG12Vgeo (<b>L</b>, <b>M</b>). Note <b>C</b> and <b>L</b> tumors in higher magnification (<b>D</b>, <b>M</b>). Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ adenocarcinoma (<b>E</b>) showing papillary (<b>F</b>) and solid (<b>G</b>) regions. Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i>; <i>Lkb1</i>^flox/+ adenocarcinoma showing intra bronchiolar tumor growth (*) (<b>H</b>). Higher magnification showing different cells populations in <b>H.</b> Atypical cells with nuclear hyperchromasia, and contour irregularities (*), cells showed enlarged nuclei displaying prominent nucleoli (**) and cells with hyperchromatic fusiform nuclei (arrows). CMV-<i>Cre</i>^T/+; <i>Kras</i>^+/LSLG12Vgeo adenomas and adenocarcinomas (<b>N</b>). Detail of solid (<b>O</b>) and mucinous (<b>P</b>) tumors. Dashed-lined squares indicate magnified areas. Bars 800 µm (<b>A</b>, <b>C</b>, <b>D</b>, <b>J</b>, <b>L</b> and <b>N</b>), 500 µm (<b>E</b>), 200 µm (<b>K</b>, <b>M</b>, <b>O</b> and <b>P</b>) and 100 µm (<b>B</b>, <b>D</b>, <b>F</b>, <b>G</b> and <b>I</b>).</p

Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).

<p>Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).</p

Neonatal activation of BRAF^V600E in Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+ mice</i> promotes lung adenomas development.

<p><b>(A)</b><b> </b> Kaplan-Meier analysis of lung tumor-free survival in untreated and 4OHTx treated Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> and Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/CA</i> mice. <i>p</i>-value was calculated by Logrank Test. On the right, percentage of mice developing lung adenomas is shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066933#pone-0066933-t001" target="_blank">Table 1</a>). (<b>B</b>, <b>C</b>) Hematoxylin and eosin staining of histological sections of Tyr::<i>Cre</i>^ERT2; <i>Braf^CA/+</i> bronchiolo-alveolar adenoma or (<b>D</b>, <b>E</b>) normal lung from wild-type mice. Note papillary adenomas and normal lung in higher magnification (<b>C</b>, <b>E</b>). Adenomas stain negative for CC10 (<b>F, G</b>) and positive for SP-C (<b>H</b>, <b>I</b>). Bars 500 µm (<b>B</b>, <b>D</b> and <b>F</b>), 300 µm (<b>C</b> and <b>D</b>) and 100 µm for (<b>G</b> and <b>I</b>).</p

Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).

<p>Percentage of mice developing lung adenomas (L. adenomas) or lung adenocarcinomas (L. carcinomas) according to their genotype and treatment (4OHTx).</p

Additional file 1 of Multiple PIK3CA mutation clonality correlates with outcomes in taselisib + fulvestrant-treated ER+/HER2–, PIK3CA-mutated breast cancers

Additional file 1: PDF file, containing the article-associated supplementary tables and figures. Table S1. Clonality subgroup nomenclature and associated definitions for single and multiple PIK3CAPIK3CAmut; Table S2. Clonality estimation and tabulation of multiple PIK3CAPIK3CAmut identified in baseline ctDNA from SANDPIPER participants; Table S3. Clonality estimation and tabulation of single PIK3CAPIK3CAmut identified in baseline ctDNA from SANDPIPER participants; Table S4. Signaling pathway gene lists utilized in pathway-level analyses; Table S5. Fraction of samples with altered signaling pathway genes between SANDPIPER treatment groups; Table S6. Fraction of samples with altered signaling pathway genes between SANDPIPER treatment groups stratified by clonality status; Fig. S1. Gene alteration rates between SANDPIPER baseline ctDNA samples categorized as clonal vs subclonal single PIK3CAPIK3CAmut; Fig. S2. Pathway-level co-alteration analyses between ctDNA samples harboring clonal vs subclonal single PIK3CAPIK3CAmut; Fig. S3. Consort diagram for the clonality analysis of an independent breast tumor tissue dataset; Fig. S4. ORR and PFS of SANDPIPER participants whose baseline ctDNA samples harbored single PIK3CAPIK3CAmut

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

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

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