CA : A Cancer Journal for Clinicians

Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, and OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements and are sufficient to fully reprogram differentiated GBM cells to "induced" TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies candidate therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies. PAPERCLIP:

Reconstructing and Reprogramming the Tumor-Propagating Potential of Glioblastoma Stem-like Cells

Jak Stat signaling and cancer: Opportunities, benefits and side effects of targeted inhibition.

MicroRNAs (miRNAs) are endogenous, small non-coding RNAs known to regulate expression of protein-coding genes. A large proportion of miRNAs are highly conserved, localized as clusters in the genome, transcribed together from physically adjacent miRNAs and show similar expression profiles. Since a single miRNA can target multiple genes and miRNA clusters contain multiple miRNAs, it is important to understand their regulation, effects and various biological functions. Like protein-coding genes, miRNA clusters are also regulated by genetic and epigenetic events. These clusters can potentially regulate every aspect of cellular function including growth, proliferation, differentiation, development, metabolism, infection, immunity, cell death, organellar biogenesis, messenger signalling, DNA repair and self-renewal, among others. Dysregulation of miRNA clusters leading to altered biological functions is key to the pathogenesis of many diseases including carcinogenesis. Here, we review recent advances in miRNA cluster research and discuss their regulation and biological functions in pathological conditions.

Clustered miRNAs and their role in biological functions and diseases

Glioblastoma multiforme (GBM) is the most common and lethal cancer of the adult brain, remaining incurable with a median survival time of only 15 months. In an effort to identify new targets for GBM diagnostics and therapeutics, recent studies have focused on molecular phenotyping of GBM subtypes. This has resulted in mounting interest in microRNAs (miRNAs) due to their regulatory capacities in both normal development and in pathological conditions such as cancer. miRNAs have a wide range of targets, allowing them to modulate many pathways critical to cancer progression, including proliferation, cell death, metastasis, angiogenesis, and drug resistance. This review explores our current understanding of miRNAs that are differentially modulated and pathologically involved in GBM as well as the current state of miRNA-based therapeutics. As the role of miRNAs in GBM becomes more well understood and novel delivery methods are developed and optimized, miRNA-based therapies could provide a critical step forward in cancer treatment.

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MicroRNAs in glioblastoma multiforme pathogenesis and therapeutics.

The emergence of low molecular weight kinase inhibitors as “targeted” drugs has led to remarkable advances in the treatment of cancer patients. The clinical benefits of these tumor therapies, however, vary widely in patient populations and with duration of treatment. Intrinsic and acquired resistance against such drugs limits their efficacy. In addition to the well studied mechanisms of resistance based upon drug transport and metabolism, genetic alterations in drug target structures and the activation of compensatory cell signaling have received recent attention. Adaptive responses can be triggered which counteract the initial dependence of tumor cells upon a particular signaling molecule and allow only a transient inhibition of tumor cell growth. These compensating signaling mechanisms are often based upon the relief of repression of regulatory feedback loops. They might involve cell autonomous, intracellular events or they can be mediated via the secretion of growth factor receptor ligands into the tumor microenvironment and signal induction in an auto- or paracrine fashion. The transcription factors Stat3 and Stat5 mediate the biological functions of cytokines, interleukins and growth factors and can be considered as endpoints of multiple signaling pathways. In normal cells this activation is transient and the Stat molecules return to their non-phosphorylated state within a short time period. In tumor cells the balance between activating and de-activating signals is disturbed resulting in the persistent activation of Stat3 or Stat5. The constant activation of Stat3 induces the expression of target genes, which cause the proliferation and survival of cancer cells, as well as their migration and invasive behavior. Activating components of the Jak-Stat pathway have been recognized as potentially valuable drug targets and important principles of compensatory signaling circuit induction during targeted drug treatment have been discovered in the context of kinase inhibition studies in HNSCC cells [1]. The treatment of HNSCC with a specific inhibitor of c-Src, initially resulted in reduced Stat3 and Stat5 activation and subsequently an arrest of cell proliferation and increased apoptosis. However, the inhibition of c-Src only caused a persistent inhibition of Stat5, whereas the inhibition of Stat3 was only transient. The activation of Stat3 was restored within a short time period in the presence of the c-Src inhibitor. This process is mediated through the suppression of P-Stat5 activity and the decrease in the expression of the Stat5 dependent target gene SOCS2, a negative regulator of Jak2. Jak2 activity is enhanced upon SOCS2 downregulation and causes the reactivation of Stat3. A similar observation has been made upon inhibition of Bmx, bone marrow kinase x-linked, activated in the murine glioma cell lines Tu-2449 and Tu-9648. Its inhibition resulted in a transient decrease of P-Stat3 and the induction of a compensatory Stat3 activation mechanism, possibly through the relief of negative feedback inhibition and Jak2 activation. These observations indicate that the inhibition of a single tyrosine kinase might not be sufficient to induce lasting therapeutic effects in cancer patients. Compensatory kinases and pathways might become activated and maintain the growth and survival of tumor cells. The definition of these escape pathways and their preemptive inhibition will suggest effective new combination therapies for cancer.

Resistance of Cancer Cells to Targeted Therapies Through the Activation of Compensating Signaling Loops

Cellular transformation is initiated by the activation of oncogenes and a closely associated developmental reprogramming of the epigenetic landscape. Transcription factors, regulators of chromatin states and microRNAs influence cell fates in development and stabilize the phenotypes of normal, differentiated cells and of cancer cells. The miR‐302/367 cluster, predominantly expressed in human embryonic stem cells (hESs), can promote the cellular reprogramming of human and mouse cells and contribute to the generation of iPSC. We have used the epigenetic reprogramming potential of the miR‐302/367 cluster to “de‐program” tumor cells, that is, hift their gene expression pattern towards an alternative program associated with more benign cellular phenotypes. Induction of the miR‐302/367 cluster in extensively mutated U87MG glioblastoma cells drastically suppressed the expression of transformation related proteins, for example, the reprogramming factors OCT3/4, SOX2, KLF4 and c‐MYC, and the transcription factors POU3F2, SALL2 and OLIG2, required for the maintenance of glioblastoma stem‐like tumor propagating cells. It also diminished PI3K/AKT and STAT3 signaling, impeded colony formation in soft agar and cell migration and suppressed pro‐inflammatory cytokine secretion. At the same time, the miR‐302/367 cluster restored the expression of neuronal markers of differentiation. Most notably, miR‐302/367 cluster expressing cells lose their ability to form tumors and to establish liver metastasis in nude mice. The induction of the miR‐302/367 cluster in U87MG glioblastoma cells suppresses the expression of multiple transformation related genes, abolishes the tumor and metastasis formation potential of these cells and can potentially become a new approach for cancer therapy.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/ijc.29606

Expression of the miR‐302/367 cluster in glioblastoma cells suppresses tumorigenic gene expression patterns and abolishes transformation related phenotypes

Distinct gene expression patterns, accompanied by particular epigenetic states, provide the basis for different stages of cellular differentiation. The programming of cells usually proceeds from stem cells to progenitor cells to differentiated progeny. The process, however, is not irreversible, and pluripotency can be reestablished in terminally differentiated cells through the expression of the reprogramming factors (RFs) octamer-binding transcription factor 3/4 (Oct4), sex-determining region Y box 2 (Sox2), Kruppel-like factor 4 (Klf4), and c-Myc. Tumor cell formation is characterized by partial differentiation, epigenetic alterations, and drastic changes in the transcriptional program. As the RF can cause pluripotency through cellular dedifferentiation and epigenetic alterations, it is possible that the activation of their genes might contribute to cellular transformation. The shared capacity for self-renewal between pluripotent stem cells and cancer stem cells is in line with this assumption. The deregulation of RF has been observed in poorly differentiated, high-grade breast cancer and is associated with unfavorable prognosis. Signal transducer and activator of transcription 3 (Stat3) mediates a wide variety of cellular functions including survival, proliferation, and differentiation and is constitutively activated in tumor cells of diverse origins. Stat3 is also accelerates somatic cell reprogramming. We investigated the connection between Stat3 activation and the expression of RF in the breast cancer cell lines MCF-7, SK-BR-3, MDA-MB-231, and MDA-MB-468 and the normal mammary epithelial cell line MCF-10A by real-time quantitative PCR and immunoblot analyses. We detected strong expression of Sox2 and Klf4 messenger RNA (mRNA) in MCF-7 cells and the expression of Oct4 mRNA in all the four cell lines. Immunoblot analyses revealed the strong protein expression of homeobox protein Nanog (Nanog), Oct4, and Sox2 in MCF-7 cells. This cell line only contains a low level of phosphorylated Stat3. We also examined the effect of the Stat3 inhibitor Stattic on the expression of RF and observed that it suppressed mRNA and protein expression of RF in MCF-7 cells. The expression levels of reprogramming proteins can strongly differ from their mRNA expression levels and do not necessarily correspond with the extent of Stat3 activation in the cell lines compared.

Chul Min Yang

Papers

Resistance of Cancer Cells to Targeted Therapies Through the Activation of Compensating Signaling Loops

Expression of the miR‐302/367 cluster in glioblastoma cells suppresses tumorigenic gene expression patterns and abolishes transformation related phenotypes

Expression of reprogramming factors in breast cancer cell lines and the regulation by activated Stat3.