Additional file 2: Figure S1. of Clusterization in head and neck squamous carcinomas based on lncRNA expression: molecular and clinical correlates

Kaplan-Meier plot of TCGA HNSCC patients stratified by lncRNA clusters using recurrence as endpoint. P val was calculated with the log-rank test. n: number of patients with available follow-up information. (PDF 472 kb

Vav proteins are involved in the engagement and signaling efficiency of keratinocyte autocrine/paracrine loops.

<p>(A) Quantification by flow cytometry of the apoptosis induced in pure (No) and genotypically mixed (Yes) cultures of wild-type and <i>Vav2</i>^−/−;<i>Vav3</i>^−/− keratinocytes upon 12 h in complete media with DMBA or under serum-free media conditions. (B) Quantification by flow cytometry of the apoptosis observed in wild-type and <i>Vav2</i>^−/−;<i>Vav3</i>^−/− keratinocytes kept for 12 h with serum, without serum, or without serum plus the indicated extracellular factors (<i>n</i> = 3). (C) Determination by flow cytometry of the percentage of wild-type and <i>Vav2</i>^−/−;<i>Vav3</i>^−/− keratinocytes that have entered S phase after 4.5 h under the indicated culture conditions (<i>n</i> = 3). (D–F) Phosphorylation and expression status of Erk and Stat3 in serum-starved wild-type and <i>Vav2</i>^−/−;<i>Vav3</i>^−/− keratinocytes stimulated with either EGF (D) or IL6 (E–G) for the indicated periods of time. In panels (F) and (G), keratinocytes were transduced with empty and Vav-encoding lentiviruses prior to the starvation and stimulation steps as indicated in the respective panel (<i>n</i> = 3). (H) Tyrosine phosphorylation levels of endogenous Vav2 (top panel) in wild-type keratinocytes treated with the indicated agents for 5 min (<i>n</i> = 2). Due to problems with the detection of the endogenous proteins after blot stripping, the loading control shown in the second panel from top was made using a parallel immunoprecipitation with the same anti-Vav2 antibody.</p

Progressive upr-egulation of EGF family ligands and Vav family-dependent genes in skin tumors developed in <i>Vav2</i>^−/−;<i>Vav3</i>^−/− mice.

<p>(A–D) qRT-PCR experiments showing the relative abundance of indicated mRNAs in DMBA/TPA-induced papillomas (A, C) and DMBA/DMBA-induced carcinomas (B, D) in wild-type and <i>Vav2</i>^−/−;<i>Vav3</i>^−/− FVB mice. Values for each mRNA are given in relation to the levels seen in samples from control mice (which were given an arbitrary value of 1) (<i>n</i> = 4 mice per genotype). (E) Summary of the results obtained in this work. (Left panel) The Vav2/Vav3-dependent biological programs in the skin that favor tumor initiation and promotion. (Right panel) The above programs upon the deletion of <i>Vav2</i> and <i>Vav3</i> genes. Numbered gray circles indicate the three main signaling functions of Vav proteins. The intracellular and extracellular signaling programs are indicated on the left boxes. Intracellular and autocrine/paracrine signaling is indicated by black and gray arrows, respectively. The oncogenic route is shown in red. Stimulation steps are shown as green stars. Defective receptor signaling is induced as a stop sign. Normal and defective processes are shown in bold and normal font types, respectively.</p

Vav proteins play pleiotropic roles during the skin tumor promotion phase.

<p>(A) Scheme of the experiments made in this section. (B–D) Example of immunofluorescence experiments (B; scale bar, 100 µm) and quantification of the hyperplasia (C, green color cells) and number of BrdU⁺ keratinocytes (D) in the epidermis of TPA-treated mice of indicated genotypes (<i>n</i> = 6). In (B), sections were stained with antibodies to keratin 14 (ID number: 16664), DAPI, and rhodamine-phalloidin to reveal K14 (green color), cell nuclei (blue color), and F-actin (red color), respectively. TPA-1 refers to tissue samples harvested either 24 (C) or 17 (D) h after the TPA stimulation. ET, epidermal thickness. (E) Quantification of the number of BrdU⁺ keratinocytes at the indicated times after a single topic application of TPA in animals of indicated genotypes (<i>n</i> = 3). (F) Phosphorylation and expression status of indicated proteins in the epidermis of wild-type and <i>Vav2</i>^−/−;<i>Vav3</i>^−/− mice at the indicated times after a single topic application of TPA. Tubulin α (ID number: 22142) was used as loading control. WB, Western blot. (G–I) Example (G; scale bar, 100 µm) and quantification of the neutrophil infiltration (H, green color) and edema (I) induced by TPA in mice of indicated genotypes (<i>n</i> = 3). In (G), the sections were stained with antibodies to myeloperoxydase (MPO, ID number: 17523), DAPI, and rhodamine-phalloidin to decorate neutrophils (green color), nuclei (blue color), and F-actin (red color), respectively. TST, total skin thickness.</p

Vav proteins participate in the initiation phase of skin tumors.

<p>(A) Scheme of these experiments. (B, C) Example (B; scale bar, 100 µm) and quantification (C) of apoptotic cells found in the epidermis of mice of the indicated genotypes 24 h upon the application of DMBA (<i>n</i> = 4). In (B), sections were stained with antibodies to the cleaved fragment of caspase 3 (ID number: 12367) and 14, 4′,6-diamidino-2-phenylindole (DAPI) to reveal apoptotic cells (red color) and cell nuclei (blue color), respectively. (D, E) Example (D; scale bar, 100 µm) and quantification (E) of phospho-histone H2AX⁺ keratinocytes (brown color) present in the epidermis of short-term DMBA-treated mice of indicated genotypes (<i>n</i> = 4). p-, phosphorylated. (F) Number of apoptotic (annexin V⁺) wild-type and <i>Vav2</i>^−/−;<i>Vav3</i>^−/− keratinocytes induced after 8 (left panel) and 12 (right panel) h in the indicated culture conditions (<i>n</i> = 4). Bleo, bleomycin; DTT, dithiothreitol; NS, no statistically significant. (G, H) Example of a flow cytometry experiment (G) and subsequent quantification (H) of the level of apoptosis induced by either DMBA (G, H) or serum starvation (H) in wild-type and <i>Vav2</i>^−/−;<i>Vav3</i>^−/− keratinocytes ectopically expressing GFP either alone (G, H) or in combination with HA-Vav2 (H) or Myc-Vav3 (G, H) (<i>n</i> = 3). In (G), only the gated GFP⁺ cells are shown.</p

The defective proliferation of the epidermis of <i>Vav2</i>^−/−;<i>Vav3</i>^−/− mice is not due to a defective hematopoietic system.

<p>(A) Scheme of the bone marrow reconstitution experiments used to generate data in panels (B) and (C). BM, bone marrow. (B, C) Example (B; scale bar, 100 µm) and quantification (C) of the epithelial hyperplasia induced upon four daily applications of TPA in the skin of wild-type and <i>Vav2</i>^−/−;<i>Vav3</i>^−/− C57BL/10 mice previously reconstituted with a wild-type hematopoietic system (C57BL/6-Ly5.1 genetic background) (<i>n</i> = 3 animals/genotype). In (B), sections were stained with antibodies to K14, DAPI, and rhodamine-phalloidin to reveal K14 (green color), cell nuclei (blue color), and F-actin (red color), respectively. (D) Scheme of the bone marrow reconstitution experiments used to generate data in panels (E) and (F). (E, F) Example (E; scale bar, 100 µm) and quantification (F) of the epidermal thickness induced by four daily applications of TPA in the skin of wild-type C57BL/6-Ly5.1 mice whose hematopoietic systems had been reconstituted with either wild-type or <i>Vav2</i>^−/−;<i>Vav3</i>^−/− bone marrow cells (C57BL/10 genetic background) (<i>n</i> = 3 animals/genotype). Panels shown in (E) were stained as in panel (B).</p

Expression of Vav2 and Vav3 is important for CST development.

<p>(A, B) Incidence (A) and number (B) of tumors in DMBA/TPA-treated FVB mice of indicated genotypes. The differences in tumor incidence up to week 10 (A) and in tumor number (B) were statistically significant (<i>p</i>≤0.001, <i>n</i> = 15 animals per genotype in each experiment). (C) Example of animals subjected to the above treatments for 16 wk. (D, E) Incidence (D) and number (E) of tumors in DMBA/TPA-treated C57BL/10 mice of indicated genotypes. The differences in tumor incidence up to week 15 (D) and in tumor number (E) were statistically significant (<i>p</i>≤0.001, <i>n</i> = 15 animals per genotype in each experiment). (F) Example of C57BL/10 mice of the indicated genotypes that were subjected to the above treatment for 16 wk. (G, H) Incidence (G) and number (H) of tumors in DMBA/DMBA-treated FVB mice of indicated genotypes. The differences in tumor incidence up to week 13 (G) and in tumor number (H) were statistically significant (<i>p</i>≤0.001, <i>n</i> = 15 animals per genotype in each experiment). (I) Example of animals subjected to the above treatments for 16 wk.</p

Histopathology of skin transplants.

<p>Histopathology of skin transplants.</p

Induction of p21 and cyclin A in E7-grafts.

<p>Sections of paraffin-embedded grafts were processed for immunohistochemistry staining with antibodies to cell cycle inhibitor p21 and cell cycle regulator cyclin A. With respect to control vector grafts (<b>A</b> and <b>D</b>), both p21 and cyclin A proteins are induced in suprabasal cells of HPV16 E7 transplants (<b>C</b> and <b>F</b>) and in focal HPV5 E7 areas (<b>B</b> and <b>E</b>). In the inserts, dotted lines indicate the location of the basal membrane.</p

E7-grafts showed no significant apoptosis.

<p>Sections of paraffin-embedded grafts were processed for immunofluorescence staining with antibodies to the apoptosis markers caspase-3 active and p53. Caspase-3 active is not expressed in control vector samples (<b>A</b>), but some scattered positive cells are observed in HPV5 E7 (<b>B</b>) and HPV16 E7 grafts (<b>C</b> and <b>D</b>). No p53 staining was observed in control and HPV5 E7 samples (not shown), but eventual, p53-positive cells were observed in the HPV16 E7-transplants (<b>D</b>). Dotted line indicates the location of the basal membrane. Inserts in panels <b>B</b>–<b>D</b> show magnified areas of staining. DAPI was used to visualize cell nuclei.</p