E. Aubrey Thompson

Akt interacts directly with Smad3 to regulate the sensitivity to TGF-β-induced apoptosis

Transforming growth factor β (TGF-β) induces both apoptosis and cell-cycle arrest in some cell lines, but only growth arrest in others. It is not clear how this differential response to TGF-β is specified. Smad proteins are critical mediators of TGF-β signalling. After stimulation by TGF-β, Smad2 and Smad3 become phosphorylated by the activated TGF-β receptor kinases, oligomerize with Smad4, translocate to the nucleus and regulate the expression of TGF-β target genes. Here we report that the sensitivity to TGF-β-induced apoptosis is regulated by crosstalk between the Akt/PKB serine/threonine kinase and Smad3 through a mechanism that is independent of Akt kinase activity. Akt interacts directly with unphosphorylated Smad3 to sequester it outside the nucleus, preventing its phosphorylation and nuclear translocation. This results in inhibition of Smad3-mediated transcription and apoptosis. Furthermore, the ratio of Smad3 to Akt correlates with the sensitivity of cells to TGF-β-induced apoptosis. Alteration of this ratio changes the apoptotic, but not the growth-inhibitory, responses of cells to TGF-β. These findings identify an important determinant of sensitivity to TGF-β-induced apoptosis that involves crosstalk between the TGF-β and phosphatidylinositol-3-OH kinase (PI(3)K) pathways.

Protein kinase Cι is required for Ras transformation and colon carcinogenesis in vivo

Protein kinase C ι (PKCι) has been implicated in Ras signaling, however, a role for PKCι in oncogenic Ras-mediated transformation has not been established. Here, we show that PKCι is a critical downstream effector of oncogenic Ras in the colonic epithelium. Transgenic mice expressing constitutively active PKCι in the colon are highly susceptible to carcinogen-induced colon carcinogenesis, whereas mice expressing kinase-deficient PKCι (kdPKCι) are resistant to both carcinogen- and oncogenic Ras-mediated carcinogenesis. Expression of kdPKCι in Ras-transformed rat intestinal epithelial cells blocks oncogenic Ras-mediated activation of Rac1, cellular invasion, and anchorage-independent growth. Constitutively active Rac1 (RacV12) restores invasiveness and anchorage-independent growth in Ras-transformed rat intestinal epithelial cells expressing kdPKCι. Our data demonstrate that PKCι is required for oncogenic Ras- and carcinogen-mediated colon carcinogenesis in vivo and define a procarcinogenic signaling axis consisting of Ras, PKCι, and Rac1.

Protein Kinase C (PKC) βII Induces Cell Invasion through a Ras/Mek-, PKCι/Rac 1-dependent Signaling Pathway

Protein kinase C βII (PKCβII) promotes colon carcinogenesis. Expression of PKCβII in the colon of transgenic mice induces hyperproliferation and increased susceptibility to colon cancer. To determine molecular mechanisms by which PKCβII promotes colon cancer, we established rat intestinal epithelial (RIE) cells stably expressing PKCβII. Here we show that RIE/PKCβII cells acquire an invasive phenotype that is blocked by the PKCβ inhibitor LY379196. Invasion is not observed in RIE cells expressing a kinase-deficient PKCβII, indicating that PKCβII activity is required for the invasive phenotype. PKCβII induces activation of K-Ras and the Ras effector, Rac1, in RIE/PKCβII cells. PKCβII-mediated invasion is blocked by the Mek inhibitor, U0126, and by expression of either dominant negative Rac1 or kinase-deficient atypical PKCι. Expression of constitutively active Rac1 induces Mek activation and invasion in RIE cells, indicating that Rac1 is the critical downstream effector of PKCβII-mediated invasion. Taken together, our results define a novel PKCβII → Ras → PKCι /Rac1 → Mek signaling pathway that induces invasion in intestinal epithelial cells. This pathway provides a plausible mechanism by which PKCβII promotes colon carcinogenesis.

The PRKCI and SOX2 Oncogenes Are Coamplified and Cooperate to Activate Hedgehog Signaling in Lung Squamous Cell Carcinoma

We report that two oncogenes coamplified on chromosome 3q26, PRKCI and SOX2, cooperate to drive a stem-like phenotype in lung squamous cell carcinoma (LSCC). Protein kinase Cι (PKCι) phosphorylates SOX2, a master transcriptional regulator of stemness, and recruits it to the promoter of Hedgehog (Hh) acyltransferase (HHAT) that catalyzes the rate-limiting step in Hh ligand production. PKCι-mediated SOX2 phosphorylation is required for HHAT promoter occupancy, HHAT expression, and maintenance of a stem-like phenotype. Primary LSCC tumors coordinately overexpress PKCι, SOX2, and HHAT and require PKCι-SOX2-HHAT signaling to maintain a stem-like phenotype. Thus, PKCι and SOX2 are genetically, biochemically, and functionally linked in LSCC, and together they drive tumorigenesis by establishing a cell-autonomous Hh signaling axis.

MicroRNA signatures: clinical biomarkers for the diagnosis and treatment of breast cancer

Recognition that breast cancer is a heterogeneous disease in which each patients tumor has specific characteristics has led to a search for biomarkers and combinations of markers (signatures) to improve the diagnosis, prognostic classification and prediction of therapeutic benefit versus toxicity for individual tumors and patients. Many microRNAs (miRNAs) are aberrantly expressed in cancer and seem to influence tumor behavior and progression. miRNAs have great potential to evolve into effective biomarkers in the clinic because of their extreme stability and ease of detection. However, there are several major technical challenges as well as numerous discrepancies among currently reported miRNA signatures. In this review, we discuss the use of miRNA signatures for breast cancer treatment and discuss the challenges in the field.

Integrated Analysis of Gene Expression, CpG Island Methylation, and Gene Copy Number in Breast Cancer Cells by Deep Sequencing

We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+) and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A), and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER− cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER− cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5′ end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER− breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations.

Genome-wide analysis reveals recurrent structural abnormalities of TP63 and other p53-related genes in peripheral T-cell lymphomas

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.

Genomic Analysis Reveals That Immune Function Genes Are Strongly Linked to Clinical Outcome in the North Central Cancer Treatment Group N9831 Adjuvant Trastuzumab Trial

PURPOSE To develop a genomic signature that predicts benefit from trastuzumab in human epidermal growth factor receptor 2-positive breast cancer. PATIENTS AND METHODS DASL technology was used to quantify mRNA in samples from 1,282 patients enrolled onto the Combination Chemotherapy With or Without Trastuzumab in Treating Women With Breast Cancer (North Central Cancer Treatment Group N9831 [NCCTG-N9831]) adjuvant trastuzumab trial. Cox proportional hazard ratios (HRs), adjusted for significant clinicopathologic risk factors, were used to determine the association of each gene with relapse-free survival (RFS) for 433 patients who received chemotherapy alone (arm A) and 849 patients who received chemotherapy plus trastuzumab (arms B and C). Network and pathway analyses were used to identify key biologic processes linked to RFS. The signature was built by using a voting scheme. RESULTS Network and functional ontology analyses suggested that increased RFS was linked to a subset of immune function genes. A voting scheme model was used to define immune gene enrichment based on the expression of any nine or more of 14 immune function genes at or above the 0.40 quantile for the population. This model was used to identify immune gene-enriched tumors in arm A and arms B and C. Immune gene enrichment was linked to increased RFS in arms B and C (HR, 0.35; 95% CI, 0.22 to 0.55; P < .001), whereas arm B and C patients who did not exhibit immune gene enrichment did not benefit from trastuzumab (HR, 0.89; 95% CI, 0.62 to 1.28; P = .53). Enriched immune function gene expression as defined by our predictive signature was not associated with increased RFS in arm A (HR, 0.90; 95% CI, 0.60 to 1.37; P = .64). CONCLUSION Increased expression of a subset of immune function genes may provide a means of predicting benefit from adjuvant trastuzumab.

THE CYCLIN E PROMOTER IS ACTIVATED BY HUMAN CYTOMEGALOVIRUS 86-KDA IMMEDIATE EARLY PROTEIN

Human cytomegalovirus (HCMV) activates cyclin E/Cdk2, which regulates cell cycle progression in G1and S phase of the cell cycle. HCMV activation of cyclin E/Cdk2 can be demonstrated in cells that are refractory to normal mitotic stimuli. This observation suggests that the virus has some means to overcome the stringent control on expression of cell cycle progression factors that is characteristic of cells in the G0 state. One of the mechanisms involved in activation of cyclin E/Cdk2 is the induction of cyclin E expression. We report here that HCMV induces cyclin E expression through a transcriptional mechanism. The cyclin E gene is activated by the HCMV 86-kDa immediate early gene product (IE86), which directly binds to nucleotide sequences within the cyclin E promoter. An IE86 DNA-binding mutant neither binds nor activates the cyclin E promoter. IE86-binding sites within the cyclin E promoter are required for IE86-mediated activation, and deletion of the IE86-binding site inhibits IE86 activation of the cyclin E promoter. We also demonstrate that mutation of the known E2F-binding sites in the cyclin E promoter does not block activation by HCMV or IE86. These data provide a molecular mechanism for HCMV induction of cyclin E and represent the first report of IE86 directly binding to a cellular promoter.

Toll-like receptor 4 mediates cross-talk between peroxisome proliferator-activated receptor γ and nuclear factor-κB in macrophages

The peroxisome proliferator‐activated receptor γ (PPARγ) is expressed in macrophages and plays an important role in suppressing the inflammatory response. Lipopolysaccharides (LPS), which activate Toll‐like receptor 4 (TLR4), reduced PPARγ expression and function in peritoneal macrophages and macrophage cell lines. Moreover, pretreatment with the synthetic PPARγ ligand, rosiglitazone did not prevent LPS‐mediated downregulation of PPARγ. Inhibition of PPARγ expression was not blocked by cycloheximide, indicating that de novo protein synthesis is not required for LPS‐mediated suppression of PPARγ. Destabilization of PPARγ messenger RNA (mRNA) was not observed in LPS‐stimulated macrophages, suggesting that LPS regulates the synthesis of PPARγ mRNA. LPS had no effect on PPARγ expression in macrophages from TLR4 knockout mice, whereas LPS inhibited PPARγ expression in cells that had been reconstituted to express functional TLR4. Targeting the TLR4 pathway with inhibitors of MEK1/2, p38, JNK and AP‐1 had no effect on PPARγ downregulation by LPS. However, inhibitors that target NEMO, IκB and NF‐κB abolished LPS‐mediated downregulation of PPARγ in LPS‐stimulated macrophages. Our data indicate that activation of TLR4 inhibits PPARγ mRNA synthesis by an NF‐κB‐dependent mechanism. Low‐density genomic profiling of macrophage‐specific PPARγ knockout cells indicated that PPARγ suppresses inflammation under basal conditions, and that loss of PPARγ expression is sufficient to induce a proinflammatory state. Our data reveal a regulatory feedback loop in which PPARγ represses NF‐κB‐mediated inflammatory signalling in unstimulated macrophages; however, upon activation of TLR4, NF‐κB drives down PPARγ expression and thereby obviates any potential anti‐inflammatory effects of PPARγ in LPS‐stimulated macrophages.