AKT1E17K activates focal adhesion kinase and promotes melanoma brain metastasis
ABSTRACT: Goal: Comparison of protein and phospho-protein levels in primary tumors based on activation of different AKT paralogs Methods: RPPA performed at the MD Anderson RPPA Core Facility. Detailed procedures available in methods section. Results: Focus of study compared protein and phospho-protein levels of BRAFV600E;Cdkn2a-/-;Pten-/- and BRAFV600E;Cdkn2a-/-;Pten-/-;AKT1E17K cohorts. P-FAK and paxillin were upregulated in tumors that express AKT1E17K compared with controls Conclusion: AKT1E17K has a critical role in upregulating P-FAK and paxillin Overall design: Thirty primary tumor samples included in analyses: Six BRAFV600E;Cdkn2a-/-, Six BRAFV600E;Cdkn2a-/-;Pten-/-, Six BRAFV600E;Cdkn2a-/-;Pten-/-;AKT1E17K, Six BRAFV600E;Cdkn2a-/-;Pten-/-;AKT2E17K, Six BRAFV600E;Cdkn2a-/-;Pten-/-;AKT3E17K
INSTRUMENT(S): University of Utah (Mus musculus) (30 samples) (RPPA 245 antibodies)
Project description:Goal: Comparison of mRNA expression profiles in primary tumors based on activation of different AKT paralogs Methods: RNA sequencing performed at the High-Throughput Genomics and Bioinformatics Analysis Core at the University of Utah. Detailed procedures available in methods and DNA processing pipline sections. Results: Focus of study compared mRNA expression profiles of BRAFV600E;Cdkn2a-/-;Pten-/- and BRAFV600E;Cdkn2a-/-;Pten-/-;AKT1E17K cohorts. PCA and unsupervised heirarchical clustering revealed samples clustered together appropriately. Seventeen of eighteen FA factor genes analyzed were upregulated in the AKT1E17K cohort. Conclusion: AKT1E17K has a critical role in upregulating a large group of FA factors Overall design: Thirty primary tumor samples included in analyses: Six BRAFV600E;Cdkn2a-/-, Six BRAFV600E;Cdkn2a-/-;Pten-/-, Six BRAFV600E;Cdkn2a-/-;Pten-/-;AKT1E17K, Six BRAFV600E;Cdkn2a-/-;Pten-/-;AKT2E17K, Six BRAFV600E;Cdkn2a-/-;Pten-/-;AKT3E17K
Project description:Gene expression profiling was performed to access the changes in gene expression in melanomas from Pdk1-inactivated Brafv600E::Pten-/- mice. The expression profiles of the BrafV600E::Pten-/-::Pdk1-/- were compared to the BrafV600E::Pten-/-::Pdk+/+ genotypes. The analysis has identified several important signaling pathways in Pdk1-dependent melanomagenesis. Melanoma tumors from the BrafV600E::Pten-/-::Pdk1+/+ and BrafV600E::Pten-/-::Pdk1-/- genotypes were harvested and mRNA from each group was pooled to enable four biologically replicates analysis.
Project description:Only a subset of melanoma patients has evidence for spontaneous T cell infiltration into tumor sites, previously associated with clinical responses. However, the molecular mechanisms explaining absence of a T cell response are not yet defined. Analyses of human melanoma metastases have revealed that T cell signature low tumors show alterations in the Wnt/b-catenin signaling pathway. We utilized an inducible mouse model driven by inducible BrafV600E and PTEN-deletion, with or without active b-catenin (CAT-STA) to test if tumor intrinsic active b-catenin can block anti-tumor immunity. While Braf/PTEN melanomas showed presence of a T cell infiltrate, T cells were nearly completely eliminated in tumors expressing active b-catenin. Adoptive transfer experiments revealed defective T cell priming when tumors expressed active b-catenin. Analysis of the antigen-presenting cell compartment revealed a selective decrease in the CD103+ DC subset within the tumor microenvironment, which could be associated with b-catenin depended inhibition of expression of the chemokine CCL4 within tumor cells. Here we used 3 mice of each genotype BrafV600E/PTEN-/- and BrafV600E/PTEN-/-/Cat-STA and compared their gene-expression profiles. we used 3 mice of each genotype BrafV600E/PTEN-/- and BrafV600E/PTEN-/-/Cat-STA and compared their gene-expression profiles.
Project description:Characterized by striking metastatic propensity and chemoresistance, melanoma is among the most lethal cutaneous malignancies. The transcription factor ATF2 was shown to elicit oncogenic activities in melanoma, and its inhibition attenuates melanoma development. Here, a mouse model engineered to express a transcriptionally inactive form of Atf2 (Atf2?8,9) was found to be sufficient to induce nevi formation and, when crossed with BrafV600E animals, to promote melanoma development. The cross of Atf2?8,9 with BrafV600E;Pten-/- mice augmented pigmentation, tumorigenicity, and metastasis. Similar to mouse Atf2?8,9, the human ATF2 splice variant 5 enhanced growth and migration capacity of cultured melanoma and immortalized melanocytes. Induced Melan-A, CXCL9, S100A8, CCR7 expression, seen in Atf2?8,9-driven tumors associate with their enhanced pigmentation, immune infiltration and propensity to metastasize. Notably, elevated ATF2SV5 expression in melanoma specimens coincided with poor prognosis. The gain-of-function activity elicited by the truncated ATF2 form offers unexpected insight into mechanisms underlying melanoma development and progression. Overall design: Compared gene expression profiles of tumor samples from Atf2Δ8,9/Δ8,9;BrafV600E/V600E;Pten–/– and Atf2+/+;BrafV600E/V600E;Pten–/– mice
Project description:From ~1,700 non-small cell lung cancer specimens collected at the MD Anderson Cancer Center over the years 1997 to 2005, we selected 914 tumors with detailed clinical and pathological information. We extracted RNA and DNA from frozen tissues using histology quality control from 700 cases. RNA was examined for quality using Agilent Bioanalyzer and RNA integrity number (RIN) was obtained for all specimens. A final selection of 275 tumor specimens was based on the following criteria: 1) Select mainly adenocarcinomas (n = 183) and squamous carcinomas (n = 80); 2) Tissue material contains at least 70% tumor by section examination; 3) within the tumor section, there should be at least 30% tumor cells (as opposed to stromal cells); 4) RIN should be at least 4.0 (range 0 - 10). Various profiling experiments were then performed including mRNA expression (this study), miRNA profiling, aCGH (Agilent), Reverse-phase protein array (RPPA), mutation analysis of 20 genes (Sequenom) and MS-MALDI-TOFF analysis. 275 lung cancer specimens collected at the MD Anderson Cancer Center were profiled on Illumina WG6-V3 expression arrays. Detailed clinico-pathological information were also collected.
Project description:Here, using mouse squamous cell carcinoma cells, we report a completely new function for the autophagy protein Ambra1 as the first described ‘spatial rheostat’ controlling the Src/FAK pathway. Ambra1 regulates the targeting of active phospho-Src away from focal adhesions into autophagic structures that cancer cells use to survive adhesion stress. Ambra1 binds to both FAK and Src in cancer cells. When FAK is present, Ambra1 is recruited to focal adhesions, promoting FAK-regulated cancer cell direction-sensing and invasion. However, when Ambra1 cannot bind to FAK, abnormally high levels of phospho-Src and phospho-FAK accumulate at focal adhesions, positively regulating adhesion and invasive migration. Spatial control of active Src requires the trafficking proteins Dynactin 1 and IFITM3, which we identified as Ambra1 binding partners by interaction proteomics. We conclude that Ambra1 is a core component of an intracellular trafficking network linked to tight spatial control of active Src and FAK levels, and so crucially regulates their cancer-associated biological outputs.
Project description:In order to assess the impact of sphingosine kinase-1 (SK1) expression on melanoma growth, stable SK1 knockdown cells were generated by shRNA, using Yumm 1.7 cells derived from spontaneous murine melanoma driven by Braf activation as well as Pten and Cdkn2a inactivation. Two cell lines silenced for SK1 (shSK1(1) and shSK1(2)) were obtained.
Project description:11 BRAF inhibitor resistance melanoma cells were treated with PAK inhibitor PF3758309 for 48 hr, the cell lysis were analyzed by RPPA profiling by protein array (RPPA) Overall design: 11 pair of samples were analyzed (control and PF3758309 treatment group) by RPPA, more than 200 of proteins were tested
Project description:3 BRAF/MEK inhibitor resistance melanoma cells were treated with PAK inhibitor PF3758309 for 48 hr, the cell lysis were analyzed by RPPA profiling by protein array (RPPA) Overall design: 3 pair of samples were analyzed (control and PF3758309 treatment group) by RPPA, more than 200 of proteins were tested
Project description:The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor-host interactions, we evaluated three syngeneic murine tumor models, the BRAFV600E-driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAFV600E mutation and PTEN loss (BRAFV600E/PTEN-/-). Administration of anti-PD-1 or anti-PD-L1 antibody therapy had strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. There was no difference in PD-L1 expression between the three models at baseline or upon interferon stimulation. While mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T-cells, as well as CD28 and CD80/86 co-stimulation, with an increase in CD11c+CD11b+MHC-IIhigh dendritic cells and tumor associated macrophages in the tumors after PD-1 blockade. Compared to YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in chemokine-trafficking gene expression levels related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells, and co-stimulation mediated by dendritic cells and macrophages. Overall design: mRNA profiles of YUMM murine melanoma cell lines