fluorescence-molecular-tomography-for-vivo-imaging-glioblastoma

Fluorescence Molecular Tomography for <em>In Vivo</em> Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Ludwig Institute for Cancer Research

Chemotherapy and molecularly targeted approaches represent two very different modes of cancer treatment and each is associated with unique benefits and limitations. Both types of therapy share the overarching limitation of the emergence of drug resistance, which prevents these drugs from eliciting lasting clinical benefit. This review will provide an overview of the various mechanisms of resistance to each of these classes of drugs and examples of drug combinations that have been tested clinically. This analysis supports the contention that understanding modes of resistance to both chemotherapy and molecularly targeted therapies may be very useful in selecting those drugs of each class that will have complementing mechanisms of sensitivity and thereby represent reasonable combination therapies.

A tale of two approaches: complementary mechanisms of cytotoxic and targeted therapy resistance may inform next-generation cancer treatments.

mTOR pathway hyperactivation occurs in approximately 90% of glioblastomas, but the allosteric mTOR inhibitor rapamycin has failed in the clinic. Here, we examine the efficacy of the newly discovered ATP-competitive mTOR kinase inhibitors CC214-1 and CC214-2 in glioblastoma, identifying molecular determinants of response and mechanisms of resistance, and develop a pharmacologic strategy to overcome it.

The mTOR kinase inhibitors, CC214-1 and CC214-2, preferentially block the growth of EGFRvIII-activated glioblastomas.

Receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR) regulate cellular homeostatic processes. EGFR activates downstream signaling cascades that promote tumor cell survival, proliferation and migration. Dysregulation of EGFR signaling as a consequence of overexpression, amplification and mutation of the EGFR gene occurs frequently in several types of cancers and many become dependent on EGFR signaling to maintain their malignant phenotypes. Consequently, concerted efforts have been mounted to develop therapeutic agents and strategies to effectively inhibit EGFR. However, limited therapeutic benefits to cancer patients have been derived from EGFR-targeted therapies. A well-documented obstacle to improved patient survival is the presence of EGFR-inhibitor resistant tumor cell variants within heterogeneous tumor cell masses. Here, we summarize the mechanisms by which tumors resist EGFR-targeted therapies and highlight the emerging role of microRNAs (miRs) as downstream effector molecules utilized by EGFR to promote tumor initiation, progression and that play a role in resistance to EGFR inhibitors. We also examine evidence supporting the utility of miRs as predictors of response to targeted therapies and novel therapeutic agents to circumvent EGFR-inhibitor resistance mechanisms. 

Therapeutic resistance in cancer: microRNA regulation of EGFR signaling networks.

The EGF receptor (EGFR) is amplified and mutated in glioblastoma, in which its common mutation (ΔEGFR, also called EGFRvIII) has a variety of activities that promote growth and inhibit death, thereby conferring a strong tumor-enhancing effect. This range of activities suggested to us that ΔEGFR might exert its influence through pleiotropic effectors, and we hypothesized that microRNAs might serve such a function. Here, we report that ΔEGFR specifically suppresses one such microRNA, namely miR-9, through the Ras/PI3K/AKT axis that it is known to activate. Correspondingly, expression of miR-9 antagonizes the tumor growth advantage conferred by ΔEGFR. Silencing of FOXP1, a miR-9 target, inhibits ΔEGFR-dependent tumor growth and, conversely, de-repression of FOXP1, as a consequence of miR-9 inhibition, increases tumorigenicity. FOXP1 was sufficient to increase tumor growth in the absence of oncogenic ΔEGFR signaling. The significance of these findings is underscored by our finding that high FOXP1 expression predicts poor survival in a cohort of 131 patients with glioblastoma. Collectively, these data suggest a novel regulatory mechanism by which ΔEGFR suppression of miR-9 upregulates FOXP1 to increase tumorigenicity.

Suppression of microRNA-9 by mutant EGFR signaling upregulates FOXP1 to enhance glioblastoma tumorigenicity.

Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCβ mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCβ is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.

Cancer-associated protein kinase C mutations reveal kinase's role as tumor suppressor.

Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive tumor growth are not well understood. Here, through integrated epigenome and transcriptome analyses of cell lines, genotyped clinical samples, and TCGA data, we show that EGFR mutations remodel the activated enhancer landscape of GBM, promoting tumorigenesis through a SOX9 and FOXG1-dependent transcriptional regulatory network in vitro and in vivo. The most common EGFR mutation, EGFRvIII, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in a SOX9, FOXG1-dependent manner. These results identify the role of transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy. 

EGFR Mutation Promotes Glioblastoma through Epigenome and Transcription Factor Network Remodeling.

With the evolution of technology, there is now a deeper understanding of glioblastoma as an inter- and intraheterogeneous disease comprising a multitude of genetically and epigenetically different cancer cells. Greater characterization of glioblastoma at the molecular level has improved its initial pathophysiological staging and classification. With this knowledge comes the hope that more efficacious therapies to combat this highly lethal disease are on the horizon. One possibility for intervention is represented by the targeting of epidermal growth factor receptor (EGFR), which is amplified and mutated in a large subset of patients. In this review, we provide a brief overview of EGFR and its mutated form, EGFR variant III, describing the downstream cellular pathways activated by each receptor, available animal models, therapeutic strategies to inhibit the receptor, and possible intervention routes to efficiently target this receptor and prevent the emergence of resistant mechanisms which to date have hampered a successful therapeutic outcome.

Epidermal growth factor receptor targeting and challenges in glioblastoma.

Small-molecule inhibitors targeting growth factor receptors have failed to show efficacy for brain cancers, potentially due to their inability to achieve sufficient drug levels in the CNS. Targeting non-oncogene tumor co-dependencies provides an alternative approach, particularly if drugs with high brain penetration can be identified. Here we demonstrate that the highly lethal brain cancer glioblastoma (GBM) is remarkably dependent on cholesterol for survival, rendering these tumors sensitive to Liver X receptor (LXR) agonist-dependent cell death. We show that LXR-623, a clinically viable, highly brain-penetrant LXRα-partial/LXRβ-full agonist selectively kills GBM cells in an LXRβ- and cholesterol-dependent fashion, causing tumor regression and prolonged survival in mouse models. Thus, a metabolic co-dependency provides a pharmacological means to kill growth factor-activated cancers in the CNS.

An LXR-Cholesterol Axis Creates a Metabolic Co-Dependency for Brain Cancers.

Glioblastoma (GBM) is the most lethal type of human brain cancer, where deletions and mutations in the tumour suppressor gene PTEN (phosphatase and tensin homolog) are frequent events and are associated with therapeutic resistance. Herein, we report a novel chromatin-associated function of PTEN in complex with the histone chaperone DAXX and the histone variant H3.3. We show that PTEN interacts with DAXX and, in turn PTEN directly regulates oncogene expression by modulating DAXX-H3.3 association on the chromatin, independently of PTEN enzymatic activity. Furthermore, DAXX inhibition specifically suppresses tumour growth and improves the survival of orthotopically engrafted mice implanted with human PTEN-deficient glioma samples, associated with global H3.3 genomic distribution changes leading to upregulation of tumour suppressor genes and downregulation of oncogenes. Moreover, DAXX expression anti-correlates with PTEN expression in GBM patient samples. Since loss of chromosome 10 and PTEN are common events in cancer, this synthetic growth defect mediated by DAXX suppression represents a therapeutic opportunity to inhibit tumorigenesis specifically in the context of PTEN deletion.

PTEN regulates glioblastoma oncogenesis through chromatin-associated complexes of DAXX and histone H3.3.

In glioblastoma (GBM), heterogeneous expression of amplified and mutated epidermal growth factor receptor (EGFR) presents a substantial challenge for the effective use of EGFR-directed therapeutics. Here we demonstrate that heterogeneous expression of the wild-type receptor and its constitutively active mutant form, EGFRvIII, limits sensitivity to these therapies through an interclonal communication mechanism mediated by interleukin-6 (IL-6) cytokine secreted from EGFRvIII-positive tumor cells. IL-6 activates a NF-κB signaling axis in a paracrine and autocrine manner, leading to bromodomain protein 4 (BRD4)-dependent expression of the prosurvival protein survivin (BIRC5) and attenuation of sensitivity to EGFR tyrosine kinase inhibitors (TKIs). NF-κB and survivin are coordinately up-regulated in GBM patient tumors, and functional inhibition of either protein or BRD4 in in vitro and in vivo models restores sensitivity to EGFR TKIs. These results provide a rationale for improving anti-EGFR therapeutic efficacy through pharmacological uncoupling of a convergence point of NF-κB-mediated survival that is leveraged by an interclonal circuitry mechanism established by intratumoral mutational heterogeneity.

Ciro Zanca

Ludwig Institute for Cancer Research

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts