Search

Additional file 1.

Additional file 5: Supplementary Table 1. Complete list of antibodies used in this study.

Additional file 3: Supplementary Figure 3. Che-1 phosphorylation is required for its pro-proliferative ability. A: WB analysis with the indicated antibodies showing the transfection efficiency of the experiment described in Fig. 3A. B, C and D: Cell proliferation analysis (left) and relative WB analysis (right) of KMS27, HeLa and 293 T cells transiently transfected with Myc-Che-1, Myc-Che-1 3S or control vector (pCS2MT). Bar plot shows the average number of cells observed in these experiments (n = 5). E: Cell number-normalized total RNA quantification of the indicated cell line transiently transfected with Myc-Che-1 wt, 3S mutant or control vector. Error bars represent the SD of triplicate experiments (n = 3). F: Nuclear extracts from HCT116 cells transiently transfected with Myc-Che-1 wt or 3S and subjected to IP with H3 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. G: Cell proliferation analysis of KMS27 cells transiently transfected with the indicated peptides. Bar plot shows the average number of cells observed in these experiments (n = 3). Statistical significance is indicated by asterisks as follow: *P

Additional file 4: Supplementary Figure 4. CK2 phosphorylates Che-1. A: Nuclear extracts from HCT116 cells were subjected to IP with CK2 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. B and C: Representative WB analyses of total cell extracts showing the transfection efficiency of the experiments described in Fig. 5B and C. D: The panel shows the MS/MS spectrum of the phosphopeptide YLVDGTKPNAGSEEISSEDDELVEEK identifying the phosphorylation of Che-1 residues S320 and S321. E: Representative WB analyses of total cell extracts showing the transfection efficiency of the experiment shown in Fig. 5D. F: Nuclear extracts from HCT116 cells treated or not with 80 μM TBB for 4 h and then subjected to IP with Che-1 antibody. Immunoprecipitated complexes were then analysed by WB with the indicated antibodies. Input corresponds to 10% of the nuclear extracts used for IP. G: WB of the IP experiments used for densitometryc analysis shown in Fig. 5E.

Additional file 2: Supplementary Figure 2. Che-1 is highly phosphorylated. A: Two different replicates of 2D- experiments of total cell extracts from HeLa cells treated or not with λ-PP related to Fig. 2B. B: Two different replicates of 2D-Gel electrophoresis (top) and representative WB (bottom) of HeLa cells transiently transfected with Myc-Che-1 or Myc-Che-1 3S, related to Fig. 2D.

Additional file 5: Figure S1. High level workflow for ICI biomarker validation and combination. The N biomarkers were individually tested for each dataset, this test serving as a primary validation of the proposed performance. Mean accuracy was computed for each classifier in all available datasets. Combinatorial analysis was carried out with majority voting and Generalized Linear Models. The E[X] formula stands as an example of the model to create when estimating the a0â ŚaN linear factors.

Tables S1. Primers used for resequencing of B16 neoantigens. Table S2. B16 selected neoantigens. Table S3. Summary results of RNAseq of in vitro and in vivo MC38 cells. Table S4. M285 selected peptides. Binding affinity prediction of M285 neoantigens according to NetMHC-4 and RNAseq values. Table S5. U11 selected peptides. Binding affinity prediction of U11 neoantigens according to NetMHC-4 and RNAseq values. (DOCX 116 kb)

Additional file 1. Methods.

Figure S1. Representative gating strategy for the identification of neoantigen specific immune responses. Figure S2. Comparison of CEA specific immune responses induced by the M1 vaccine vector vs. the full length CEA protein delivered by DNA-EP. Figure S3. Comparison in peripheral blood by IFN-Îł ICS analysis for the Reps1 neoantigen and cognate WT peptide. Figure S4. M2 specific memory T cells. Figure S5. M3 specific T cells. Figure S6. Immuno modulators prevent MC38 tumor growth. (PPTX 1226 kb)

Additional file 2. Representative examples of immunohistochemical expression of TAZ and YAP in gastric cancer. Two cases are presented with combined nuclear expression of both TAZ and YAP (Aâ D).

Additional file 1. Baseline characteristics of gastric cancer (GC) patients included in this study (N = 83).

Additional file 1. First-line chemotherapy regimens and schedules (NÂ =Â 86).

Additional file 3. Multivariate Cox regression model for progression-free survival (PFS) evaluating three different molecular signatures (N = 83).

The Mdr2(-/-) mouse develops early chronic cholestatic hepatitis and hepatocellularcarcinoma (HCC) when adult. We tested the effects of a restricted-calorie diet on HCC development in Mdr2(-/-) mice. BACKGROUND/AIM: The Mdr2(-/-) mouse develops early chronic cholestatic hepatitis and hepatocellularcarcinoma (HCC) when adult. We tested the effects of a restricted-calorie diet on HCC development in Mdr2(-/-) mice.MATERIALS AND METHODS: Mdr2(-/-) mice (n=40, divided into two groups of 20 mice each) were randomized to receive ad libitum diet or restricted-calorie diet. Two mice from each group were sacrificed at 3 and 6 months, and liver tissue samples were removed for analysis. The remaining mice were fed their respective diets until the age of 30 months, at which time they were euthanized and livers were collected for analysis.RESULTS: The restricted-calorie diet had partial chemopreventive effect on the development of HCC in Mdr2(-/-) mice. Moreover, mice with ad libitum diet had a median survival of 361 days, while the restricted-calorie group had a median survival of 500 days (p=0.0001).CONCLUSION: A restricted diet might reduce the chance of developing HCC in patients at risk and could increase the protective action of anti-inflammatory agents.

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G

Different mRNAs associated to eEF1BÎł. (DOC 42 kb)

A. Chromatin immunoprecipitation was performed in hSH-SY5Y cells using anti-eEF1Bγ rabbit polyclonal antibodies or no-Ab as a control. Immunoprecipitates from each sample were analyzed by PCR performed with primers specific for the human Che-1 promoter and for the human TP53 promoter. The thymidine kinase human promoter was amplified as a negative control. A sample representing linear amplification of the total input chromatin (input) was included in the PCR as a control. B. Co-localization of endogenous Che-1, performed with the anti-Che-1 rat polyclonal antibody (green), and the mitochondrial marker Tom20 (red), in hSH-SY5Y cells. Extensive co-localization (yellow) between Che-1 and Tom20 is visualized by the merged-color image. The boxed area represents a high magnification image of co-localization. Nuclei were labeled with DAPI (blue). Scale bars: 10 μm. C. Western blot analysis of hSH-SY5Y whole-cell lysate and mitochondrial enriched fraction. The quality of mitochondrial-enriched fractions was monitored using anti-HSP60 monoclonal antibodies and anti-Tom20 rabbit polyclonal antibodies. D. Quantitative real time PCR (qPCR) analysis of the eEF1Bγ and Che-1 mRNAs in hSH-SY5Y cells (siRNA-Control and siRNA-eEF1Bγ). The gene expression ratio between eEF1Bγ and GAPDH and between Che-1 and GAPDH are shown as the mean ± SD from three independent experiments performed in triplicate. E. Representative Western blot of hSH-SY5Y whole-cell lysates treated or un-treated with siRNA as shown. The antibodies that were used are indicated. (PDF 199 kb)

Oligos used in the present study. (DOC 56 kb)

A. RIP assay output was analyzed by semi-quantitative RT-PCR with specific primers to validate some of mRNAs co-immunoprecipitated with eEF1Bγ and they are listed in Table S2. B. The myc-eEF1Bγ and MS2-GFP fusion proteins were expressed in HeLa cells with the report mRNA carrying both the MS2 binding site and the indicated 3′ UTR. C. RIP assay-eEF1Bγ mRNAs (Additional file 2: Table S2) analyzed by quantitative real time PCR (qPCR) in HeLa whole-cell lysates treated with siRNA as shown. The gene expression ratio between mRNAs and GAPDH are shown as the mean ± SD from three independent experiments performed in triplicate. D. Representative western blot of HeLa whole-cell lysates treated or un-treated with siRNA as shown. The antibodies that were used are indicated. Densitometric analysis represents the mean ± S.D. of 3 independent experiments (right panel). (PDF 208 kb)

Background Metastases are the major cause of cancer-related morbidity and mortality. By the time cancer cells detach from their primary site to eventually spread to distant sites, they need to acquire the ability to survive in non-adherent conditions and to proliferate within a new microenvironment in spite of stressing conditions that may severely constrain the metastatic process. In this study, we gained insight into the molecular mechanisms allowing cancer cells to survive and proliferate in an anchorage-independent manner, regardless of both tumor-intrinsic variables and nutrient culture conditions. Methods 3D spheroids derived from lung adenocarcinoma (LUAD) and breast cancer cells were cultured in either nutrient-rich or -restricted culture conditions. A multi-omics approach, including transcriptomics, proteomics, and metabolomics, was used to explore the molecular changes underlying the transition from 2 to 3D cultures. Small interfering RNA-mediated loss of function assays were used to validate the role of the identified differentially expressed genes and proteins in H460 and HCC827 LUAD as well as in MCF7 and T47D breast cancer cell lines. Results We found that the transition from 2 to 3D cultures of H460 and MCF7 cells is associated with significant changes in the expression of genes and proteins involved in metabolic reprogramming. In particular, we observed that 3D tumor spheroid growth implies the overexpression of ALDOC and ENO2 glycolytic enzymes concomitant with the enhanced consumption of glucose and fructose and the enhanced production of lactate. Transfection with siRNA against both ALDOC and ENO2 determined a significant reduction in lactate production, viability and size of 3D tumor spheroids produced by H460, HCC827, MCF7, and T47D cell lines. Conclusions Our results show that anchorage-independent survival and growth of cancer cells are supported by changes in genes and proteins that drive glucose metabolism towards an enhanced lactate production. Notably, this finding is valid for all lung and breast cancer cell lines we have analyzed in different nutrient environmental conditions. broader Validation of this mechanism in other cancer cells of different origin will be necessary to broaden the role of ALDOC and ENO2 to other tumor types. Future in vivo studies will be necessary to assess the role of ALDOC and ENO2 in cancer metastasis.

Multiple myeloma (MM) is a hematologic malignancy produced by a clonal expansion of plasma cells and characterized by abnormal production and secretion of monoclonal antibodies. This pathology exhibits an enormous heterogeneity resulting not only from genetic alterations but also from several epigenetic dysregulations. Here we provide evidence that Che-1/AATF (Che-1), an interactor of RNA polymerase II, promotes MM proliferation by affecting chromatin structure and sustaining global gene expression. We found that Che-1 depletion leads to a reduction of “active chromatin” by inducing a global decrease of histone acetylation. In this context, Che-1 directly interacts with histones and displaces histone deacetylase class I members from them. Strikingly, transgenic mice expressing human Che-1 in plasma cells develop MM with clinical features resembling those observed in the human disease. Finally, Che-1 downregulation decreases BRD4 chromatin accumulation to further sensitize MM cells to bromodomain and external domain inhibitors. These findings identify Che-1 as a promising target for MM therapy, alone or in combination with bromodomain and external domain inhibitors.

Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che-1, a RNA polymerase II-binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che-1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress-induced autophagy. Strikingly, Che-1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.

International audience; Poly(ADP-ribose) polymerase 1 (PARP-1) catalyzes a post-translational modification that plays a crucial role in coordinating the signalling cascade in response to stress stimuli. During the DNA damage response, phosphorylation by ataxia telangiectasia mutated (ATM) kinase and checkpoint kinase Chk2 induces the stabilization of Che-1 protein, which is critical for the maintenance of G2/M arrest. In this study we showed that poly(ADP-ribosyl)ation, beyond phosphorylation, is involved in the regulation of Che-1 stabilization following DNA damage. We demonstrated that Che-1 accumulation upon doxorubicin treatment is reduced after the inhibition of PARP activity in HCT116 cells and in PARP-1 knock-out or silenced cells. In accordance, impairment in Che-1 accumulation by PARP inhibition reduced Che-1 occupancy at p21 promoter and affected the expression of the corresponding gene. Epistasis experiments showed that the effect of poly(ADP-ribosyl)ation on Che-1 stabilization is independent from ATM kinase activity. Indeed we demonstrated that Che-1 protein co-immunoprecipitates with ADP-ribose polymers and that PARP-1 directly interacts with Che-1, promoting its modification in vitro and in vivo.