Research Highlights

Dicer and microRNAs Regulate Glioma Immunoresistance

Evaluation of: Ueda R, Kohanbash G, Sasaki K et al.: Dicer-regulated microRNAs 222 and 339 promote resistance of cancer cells to cytotoxic T-lymphocytes by down-regulation of ICAM-1. Proc. Natl Acad. Sci. USA 106(26), 10746–10751 (2009).

MicroRNAs (miRNAs) are a class of small noncoding RNA molecules (19–25 nucleotides in length) that function to alter gene expression at a post-transcriptional level [1,2]. Before they can affect gene expression, miRNAs must undergo modification. The RNAse III endonuclease, Dicer, plays a key role in the generation of mature, functional, miRNAs from long primary miRNA transcripts [3].

Abnormal expression of miRNAs and Dicer occurs in a variety of malignancies, including chronic lymphocytic leukemia [4], hepatocellular carcinoma [5], lung cancer [6] and glioblastoma multiforme [7]. Reports indicate that depending on the target mRNA and/or cancer type, miRNAs and Dicer appear to be able to function as tumor suppressors or oncogenes [8]. Dicer‘s role in tumor biology, coupled with its central role in regulatory T-cell development, led Ueda et al. to hypothesize that Dicer expression in cancer cells may regulate immunosurveillance via miRNAs [9,10].

In a series of elegant experiments, Ueda and colleagues use antigen-specific cytolytic T lymphocyte (CTL) assays to demonstrate that Dicer-mediated elaboration of miRNAs 222 and 339 promotes the immunoresistance of cancer cells through regulation of intercellular adhesion molecule (ICAM)-1 expression [10]. In both human colorectal carcinoma cells and glioblastoma cell lines, they found that inhibition of Dicer expression increased tumor cell susceptibility to lysis by CTLs. Enhanced cytolysis of the Dicer- disrupted cells was due to an upregulation of cell surface ICAM-1 and, consequently, a higher frequency of tumor–CTL conjugates compared with Dicer-nondisrupted cells. A requirement for ICAM-1 in this process was demonstrated by inhibiting Dicer- disrupted cell–CTL conjugates with an ICAM-1 monoclonal antibody.

The mechanism of Dicer regulation of ICAM-1 expression was further evaluated. Among the miRNAs specifically downregulated by disruption of Dicer expression in both the colorectal and glioblastoma cell lines were miRNAs 222 and 339. Both miRNA 222 and 339 possess ‘seed‘ sequences that match nucleotide sequences within the 3´ UTR of human ICAM-1. To directly demonstrate that miRNA 222 and 339 can regulate ICAM-1 expression, Ueda and colleagues utilized luciferase reporter constructs containing the wildtype or mutant 3´ UTR target sequences for each miRNA [10]. In each case, the presence of the wildtype sequence only resulted in decreased ICAM-1 expression when cotransfected with the corresponding miRNA. Decreased expression occurred without any change in relative abundance of ICAM-1 mRNA, indicating that regulation was occurring at the level of translation. Finally, immunostaining of primary human malignant gliomas revealed the same inverse correlation of Dicer and miRNA 222 and 339 expression and ICAM-1 as observed in vitro.

These results suggest the possibility that the role of Dicer-regulated miRNAs 222 and 339 in promoting immunoresistance may be exploited to develop novel miRNA-targeted therapies to enhance cytolysis of tumor cells in a variety of malignancies, including glioblastoma multiforme.

Inhibiting Glioblastoma Multiforme PI3K/mTOR Signaling without Impacting T-Cell Function

Evaluation of: Crane C, Panner A, Pieper RO et al.: Honokiol-mediated inhibition of PI3K/mTOR pathway: a potential strategy to overcome immunoresistance in glioma, breast, and prostate carcinoma without impacting T-cell function. J. Immunother. 32(6), 585-592 (2009).

Cancer cells can adapt and evade immunity by methods such as MHC downregulation and overexpression of immunosuppressive factors [1]. One of these factors, B7-H1, is a surface glycoprotein that promotes apoptosis of T cells, and is overexpressed in malignancies of the lung, colon, ovary and brain [2,3]. Recent reports have highlighted a link between oncogenesis and immunoresistance in which activation of the PI3K/mammalian target of rapamycin (mTOR) pathway not only promotes tumor cell survival, but also enhances tumor cell immunoresistance through increased expression of the T-cell ‘coinhibitory‘ factor B7-H1 [4,5]. While currently available PI3K/mTOR inhibitors such as rapamycin can effectively block tumor cell survival signaling and eliminate B7-H1-mediated immunoresistance, they are also well known to inhibit T-cell mediated immune responses and, therefore, would be contraindicated as components of immunotherapy [6]. Novel inhibitors with differential effects on T-cell responses and the PI3K/mTOR pathway in a tumor cell would be of potential benefit in the treatment of tumors such as glioblastoma multiforme.

Crane and colleagues investigated the relatively new drug Honokiol (HNK), a product isolated from the Magnolia grandiflora seed cones. HNK was recently demonstrated to inhibit PI3K/mTOR signaling in breast cancer cells [7]. In their study, Crane et al. evaluated the effect of HNK on T-cell function and PI3K/mTOR signaling. The investigators first determined the relative effectiveness of HNK-mediated PI3K/mTOR inhibition compared to established inhibitors such as rapamycin and wortmannin. Using glioma, breast and prostate cancer cell lines they demonstrated that all inhibitors, including HNK, blocked the activation of the downstream effector of PI3K/mTOR signaling, S6 kinase. As predicted, inhibition of tumor cell PI3K/mTOR signaling reduced B7-H1 expression and blocked T-cell apoptosis in a mixed tumor cell-activated T-cell coculture assay.

In addition to blocking PI3K/mTOR signaling, rapamycin and its analogs are potent inhibitors of activated T-cell proliferation. Crane and coworkers demonstrated that while HNK was a potent inhibitor of PI3K/mTOR signaling, it was distinguished from other inhibitors by its lack of effect on activated T-cell proliferation. Furthermore, unlike the other inhibitors, HNK treatment of the activated T cells did not impair their ability to produce the necessary proinflammatory cytokines, IL-17 and IFN-γ. Interestingly, when investigators examined one of the downstream effectors of PI3K/mTOR, S6 kinase, in HNK-treated T cells, they could not demonstrate any significant difference in activation compared with untreated controls, suggesting that S6 may be activated through a different mechanism in T cells, which allows their function to remain intact.

One key experiment that is still necessary to more fully assess the potential of HNK to overcome B7-H1-mediated immunoresistance without inhibiting T-cell function, is to demonstrate that T-cell-mediated anti-tumor activity can be enhanced by HNK treatment in animal models of disease. Nonetheless, these data raise the intriguing possibility of using HNK in the treatment of cancers where other mTOR inhibitors have failed.

Targeting EGFRvIII Usingadoptive Immunotherapy

Evaluation of: Bullain SS, Sahin A, Szentirmai O et al.: Genetically engineered T  cells to target EGFRvIII expressing glioblastoma. J. Neurooncol. 94, 373-382 (2009).

The principles behind cancer immunotherapy are simple. The specific antigen must be tumor-specific, widely expressed in tumor tissues and absent in normal tissues, and have appropriate immunogenicity to be a suitable and effective target for immunotherapy. The EGF-receptor variant III (EGFRvIII) embodies all the aforementioned qualities. EGFRvIII mutation is an in-frame deletion that results in constitutive tyrosine kinase activity [1]. EGFRvIII is present in many different malignancies such as breast, lung and prostate cancer, but was first discovered in, and is most commonly found in, primary glioblastoma [2–4]. More importantly, EGFRvIII has not been found in normal tissues [4]. Targeting EGFRvIII also has the added benefit of directly affecting a key factor in the oncogenic phenotype. Several investigators have chosen to focus on EGFRvIII in glioblastoma because it is the most common mutation in a disease that has had little progress in therapeutics in the last 25 years.

While several groups are currently involved in the development of an EGFRvIII-specific cancer vaccine, Bullain and coworkers chose to look at adoptive immunotherapy with genetically modified T cells. Among the barriers to effective EGFRvIII-specific anti-tumor immunity is the MHC-restricted nature of normal T-cell-receptor mediated responses. MHC molecules are known to be downregulated in glioblastoma multiforme [5], thus limiting the potential for T-cell activation. Bullain hypothesized that by engineering T cells expressing an MHC-independent anti-EGFRvIII-specific chimeric immune receptor (CIR), they could effectively target the cytolysis of EGFRvIII-expressing tumor cells [6]. The CIRs are comprised of three domains: a tumor-antigen-binding domain, a transmembrane linker domain and a cytoplasmic signaling domain [7]. In this study, the experimental and control CIRs included a full-length anti-EGFRvIII binding domain, a second with a mutated anti-EGFRvIII binding domain and a final signaling-deficient variant. T cells expressing all three CIR constructs were successfully expanded. Anti-tumor responses were measured by IFN-γ release and cytolysis when CIR-expressing T cells were cocultured with EGFRvIII⁺ glioma cells in vitro. Tumor-specific responses were evident only with T cells expressing the full-length anti-EGFRvIII CIR. Furthermore, the administration of full-length anti-EGFRvIII-T cells slowed tumor growth and enhanced survival in mice bearing glioma xenografts.

While these studies are encouraging, the effect on survival was most pronounced when the T cells and glioma cells were implanted simultaneously. Under these conditions, there was 100% survival at 29 days. By contrast, when T-cell administration occurred just 3 days after glioma cell injection, mice began to succumb to disease at day 13. Simultaneous injections may target tumor cells that have not yet engrafted and integrated into their microenvironment. This may not be directly applicable to humans where gliomas have characteristically infiltrated extensively into surrounding brain tissue prior to their detection. Nonetheless, these data have important implications for the future of targeted immunotherapies in which anti-EGFRvIII T cells may function as a bridging therapy for maximal anti-tumor effects in the setting of EGFRvIII vaccine therapy.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.