Genes & Cancer

Increasing evidence suggests that processes termed epithelial-mesenchymal transitions (EMTs) play a key role in therapeutic resistance, tumor recurrence, and metastatic progression. NF-κB signaling has been previously identified as an important pathway in the regulation of EMT in a mouse model of tumor progression. However, it remains unclear whether there is a broad requirement for this pathway to govern EMT and what the relative contribution of IKK family members acting as upstream NF-κB activators is toward promoting EMT and metastasis. To address this question, we have used a novel, small-molecule inhibitor of IκB kinase 2 (IKK2/IKKβ), termed BI 5700. We investigated the role of IKK2 in a number of mouse models of EMT, including TGFβ-induced EMT in the mammary epithelial cell line EpRas, CT26 colon carcinoma cells, and 4T1 mammary carcinoma cells. The latter model was also used to evaluate in vivo activities of BI 5700.We found that BI 5700 inhibits IKK2 with an IC₅₀ of 9 nM and was highly selective as compared to other IKK family members (IKK1, IKKε, and TBK1) and other kinases. BI 5700 effectively blocks NF-κB activity in EpRas cells and prevents TGFβ-induced EMT. In addition, BI 5700 reverts EMT in mesenchymal CT26 cells and prevents EMT in the 4T1 model. Oral application of BI 5700 significantly interferes with metastasis after mammary fat-pad injection of 4T1 cells, yielding fewer, smaller, and more differentiated metastases as compared to vehicle-treated control animals. We conclude that IKK2 is a key regulator of both the induction and maintenance of EMT in a panel of mouse tumor progression models and that the IKK2 inhibitor BI 5700 constitutes a promising candidate for the treatment of metastatic cancers.

Within healthy human somatic cells, retrotransposition by long interspersed nuclear element-1 (also known as LINE-1 or L1) is thought to be held in check by a variety of mechanisms, including DNA methylation and RNAi. The expression of L1-ORF1 protein, which is rarely found in normal tissue, was assayed using antibodies with a variety of clinical cancer specimens and cancer cell lines. L1-ORF1p expression was detected in nearly all breast tumors that the authors examined, and the protein was also present in a high percentage of ileal carcinoids, bladder, and pancreatic neuroendocrine tumors, as well as in a smaller percentage of prostate and colorectal tumors. Tumors generally demonstrated cytoplasmic L1-ORF1p; however, in several breast cancers, L1-ORF1p was nuclear. Patients with breast tumors displaying nuclear L1-ORF1p had a greater incidence of both local recurrence and distal metastases and also showed poorer overall survival when compared with patients with tumors displaying cytoplasmic L1-ORF1p. These data suggest that expression of L1-ORF1p is widespread in many cancers and that redistribution from cytoplasm to nucleus could be a poor prognostic indicator during breast cancer. High expression and nuclear localization of L1-ORF1p may result in a higher rate of L1 retrotransposition, which could increase genomic instability.

Factors associated with tumor sensitivity to epidermal growth factor receptor (EGFR) inhibitors in the context of wild-type EGFR remain elusive. This study investigates the mechanistic basis of responsiveness to EGFR inhibitors in the RIP1-Tag2 (RT2) mouse model of pancreatic neuroendocrine tumorigenesis (PNET). Upon treatment of RT2 mice with EGFR inhibitors, PNET tumors harboring wild-type, nonamplified alleles of Egfr grow at a markedly reduced rate and display a significant increase in tumor cell apoptosis, as well as reduced neovascularization. The authors identify Tgf-α and Hb-egf as key limiting mediators of separable pathological functions of Egfr in neuroendocrine tumor progression: Tgf-α mutant tumors present with an elevated apoptotic index, whereas Hb-egf mutant lesions exhibit decreased angiogenic switching and neovascularization. This study not only associates Tgf-α and Hb-egf expression with wild-type Egfr oncogenicity but also ascribes the proangiogenic activity of Egfr in this tumor model to a novel mesenchymal Hb-egf/Egfr signaling axis, whereby endothelial and pericyte-derived Hb-egf activates Egfr specifically in tumor-associated perivascular cells, leading to increased pericyte coverage of the tumor endothelium and enhanced angiogenesis.

Synthetic lethal interaction between oncogenic Ha-ras and loss of PKC has been demonstrated. Recently, the authors reported that the concurrent knockdown of PKC α and β, via upregulating PKC δ, sensitizes cells with aberrant Ras signaling to apoptosis. As a continuation of the study, using shRNA, the authors demonstrate that loss of PKC δ causes a lethal reaction in NIH3T3/Hras or prostate cancer DU145 cells that overexpress JNK. In this apoptotic process, PKC α and β are upregulated and then associated with RACK1 (an adaptor for activated PKC) and JNK. Immunoblotting analysis shows that JNK is phosphorylated, accompanied with caspase 8 cleavage. The inhibition of JNK abrogates this apoptotic process triggered by PKC δ knockdown. Interestingly, without blocking PKC δ, the concurrent overexpression of wt- or CAT-PKC α and β is insufficient to induce apoptosis in the cells. Together with the authors’ previous findings, the data suggest that PKC α/β and δ function oppositely to maintain a balance that supports cells expressing v-ras to survive and prevents them from being eliminated through oncogenic stress-induced apoptosis.

Clear cell renal cell carcinoma (ccRCC) is the predominant RCC subtype, but even within this classification, the natural history is heterogeneous and difficult to predict. A sophisticated understanding of the molecular features most discriminatory for the underlying tumor heterogeneity should be predicated on identifiable and biologically meaningful patterns of gene expression. Gene expression microarray data were analyzed using software that implements iterative unsupervised consensus clustering algorithms to identify the optimal molecular subclasses, without clinical or other classifying information. ConsensusCluster analysis identified two distinct subtypes of ccRCC within the training set, designated clear cell type A (ccA) and B (ccB). Based on the core tumors, or most well-defined arrays, in each subtype, logical analysis of data (LAD) defined a small, highly predictive gene set that could then be used to classify additional tumors individually. The subclasses were corroborated in a validation data set of 177 tumors and analyzed for clinical outcome. Based on individual tumor assignment, tumors designated ccA have markedly improved disease-specific survival compared to ccB (median survival of 8.6 vs 2.0 years, P = 0.002). Analyzed by both univariate and multivariate analysis, the classification schema was independently associated with survival. Using patterns of gene expression based on a defined gene set, ccRCC was classified into two robust subclasses based on inherent molecular features that ultimately correspond to marked differences in clinical outcome. This classification schema thus provides a molecular stratification applicable to individual tumors that has implications to influence treatment decisions, define biological mechanisms involved in ccRCC tumor progression, and direct future drug discovery.

The FLT3 tyrosine kinase receptor is involved in both hematopoiesis and hematological malignancies. The Wnt/β-catenin pathway has been shown to participate in the self-renewal of hematopoietic stem cells and to cooperate with the mutant FLT3 receptors in leukemic transformation. However, the detailed biological impact of such a constitutively activated Wnt pathway remains to be further explored. Here, the authors report that activating mutations of FLT3 constitutively activate β-catenin by inhibition of GSK-3β in a PI3 kinase pathway–dependent manner. Ectopic expression of a dominant negative form of GSK-3β in FLT3-ITD–expressing cells activated β-catenin and blocked the downregulation of the TCF/β-catenin transcriptional activity induced by inhibition of FLT3 kinase. Furthermore, inhibition of cell proliferation and colony formation induced by such suppression of FLT3 kinase activity could be partially reversed by knockdown of GSK-3β and restored by knockdown of either TCF4 or β-catenin. Moreover, exogenous activation of the Wnt pathway also attenuated such inhibitory effect. These findings indicate that the potencies of the inhibitors of FLT3 kinase activity could be modulated by the activity of the Wnt/β-catenin pathway in the cells harboring FLT3-ITD mutations, and FLT3-ITDs signal through GSK-3β to activate β-catenin that this is likely to directly contribute to the leukemic phenotype.

ETS proteins are a family of transcription factors that play important roles in the development of cancer. The Ewing’s sarcoma EWS/ETS fusion oncoproteins control a number of cancer-relevant phenotypes in that disease. We recently demonstrated that EWS/FLI, the most common EWS/ETS fusion in Ewing’s sarcoma, regulates a portion of its target genes, including the critical target NR0B1, via GGAA-containing microsatellites in their promoters. Given the unusual nature of microsatellites as EWS/FLI response elements, we sought to elucidate the mechanism of EWS/FLI activity at these sites. We found that the ability to bind GGAA microsatellites is shared by multiple ETS family members from distinct phylogenetic subfamilies. Importantly, however, only EWS/ETS-containing fusions are capable of mediating transcriptional activation via these elements, highlighting a neomorphic function of the Ewing’s sarcoma fusion proteins. Additional analysis revealed that the GGAA microsatellite binds EWS/FLI with an affinity that is 2 to 3 orders of magnitude lower than previously identified high-affinity consensus/redundant binding sites. The stoichiometry of this interaction is 2 protein molecules for each DNA molecule, suggesting that EWS/FLI binds these elements as a homodimer. The isolated FLI ETS domain bound microsatellite sequences in a nearly identical fashion to full-length EWS/FLI, thus indicating that residues required for homodimeric binding are localized to the ETS domain. These data suggest a new paradigm for an ETS family member binding to DNA at cancer-relevant genetic loci and highlight emergent properties of EWS/FLI that are required for the development of Ewing’s sarcoma.