Modeling glioblastoma.

Establishing a zebrafish model of a deadly type of brain tumor highlights the role of the immune system in the early stages of the disease.

Potential roles for efferocytosis in glioblastoma immune evasion.

Glioblastoma is an aggressive and incurable brain cancer. This cancer establishes both local and systemic immunosuppression that creates a major obstacle to effective immunotherapies. Many studies point to tumor-resident myeloid cells (primarily microglia and macrophages) as key mediators of this immunosuppression. Myeloid cells exhibit a high level of plasticity with respect to their phenotype and are capable of both stimulating and repressing immune responses. How glioblastomas recruit myeloid cells and exploit them to avoid the immune system is an active area of research. Macrophages can acquire an immunosuppressive phenotype as a consequence of exposure to cytokines such as TGFB1 or IL4; in addition, macrophages can acquire an immunosuppressive phenotype as a consequence of the engulfment of apoptotic cells, a process referred to as efferocytosis. There is substantial evidence that glioblastoma cells are able to secrete cytokines and other factors that induce an immunosuppressive phenotype in macrophages and microglia. However, less is known about the contribution of efferocytosis to immunosuppression in glioblastoma. Here I review the literature in this area and discuss the potential of efferocytosis inhibition to improve glioblastoma response to immunotherapy.

Pevonedistat, a first-in-class NEDD8-activating enzyme inhibitor, sensitizes cancer cells to VSVΔ51 oncolytic virotherapy.

The clinical efficacy of VSVΔ51 oncolytic virotherapy has been limited by tumor resistance to viral infection, so strategies to transiently repress antiviral defenses are warranted. Pevonedistat is a first-in-class NEDD8-activating enzyme (NAE) inhibitor currently being tested in clinical trials for its antitumor potential. In this study, we demonstrate that pevonedistat sensitizes human and murine cancer cells to increase oncolytic VSVΔ51 infection, increase tumor cell death, and improve therapeutic outcomes in resistant syngeneic murine cancer models. Increased VSVΔ51 infectivity was also observed in clinical human tumor samples. We further identify the mechanism of this effect to operate via blockade of the type 1 interferon (IFN-1) response through neddylation-dependent interferon-stimulated growth factor 3 (ISGF3) repression and neddylation-independent inhibition of NF-κB nuclear translocation. Together, our results identify a role for neddylation in regulating the innate immune response and demonstrate that pevonedistat can improve the therapeutic outcomes of strategies using oncolytic virotherapy.

Engineered cells as glioblastoma therapeutics.

In spite of significant recent advances in our understanding of the genetics and cell biology of glioblastoma, to date, this has not led to improved treatments for this cancer. In addition to small molecule, antibody, and engineered virus approaches, engineered cells are also being explored as glioblastoma therapeutics. This includes CAR-T cells, CAR-NK cells, as well as engineered neural stem cells and mesenchymal stem cells. Here we review the state of this field, starting with clinical trial studies. These have established the feasibility and safety of engineered cell therapies for glioblastoma and show some evidence for activity. Next, we review the preclinical literature and compare the strengths and weaknesses of various starting cell types for engineered cell therapies. Finally, we discuss future directions for this nascent but promising modality for glioblastoma therapy.

Avelumab in newly diagnosed glioblastoma.

BACKGROUND: Glioblastoma (GBM) is known to use both local and systemic immunosuppressive strategies. One such strategy is the expression of the immune checkpoint protein programmed cell death ligand-1 (PD-L1) by both tumor cells and tumor-associated immune cells. Recent phase III trials using IgG4 antibodies targeting PD-1, the ligand for PD-L1, failed to show any benefit. Avelumab is an IgG1 monoclonal antibody targeting PD-L1. In contrast to the previously tested immune checkpoint inhibitors, it can directly bind tumor cells and immune cells expressing PD-L1 and can induce antibody-dependent cellular cytotoxicity. METHODS: We conducted a single center, open label, phase II study where avelumab 10 mg/kg IV Q2W was added concurrently to the first monthly temozolomide cycle in patients with newly diagnosed GBM. Immunohistochemical analyses were performed on surgery samples. The primary objective was safety. Secondary objectives were efficacy outcomes according to the immunotherapy Response Assessment in Neuro Oncology criteria, progression free survival (PFS), and overall survival (OS). Exploratory objectives aimed at determining prognostic biomarkers. RESULTS: Thirty patients were started on therapy and two were lost to follow-up. Median follow-up time (reverse Kaplan-Meier) was 41.7 months (IQR: 28.3-43.4). Three (10.0%) patients had a related or possibly related treatment emergent adverse event that lead to transient or permanent discontinuation of avelumab. Eight (26.7%) patients had one or more immune-related adverse events, and 8 (26.7%) patients had an infusion-related reaction. The overall response rate was 23.3%, median PFS was 9.7 months, and the median OS was 15.3 months. No pretreatment biomarkers showed any predictive value. CONCLUSIONS: The addition of avelumab to standard therapy in patients with GBM was not associated with any new safety signal. There was no apparent improvement in OS. TRIAL REGISTRATION: NCT03047473 Registered February 9, 2017.

Identification of Rac guanine nucleotide exchange factors promoting Lgl1 phosphorylation in glioblastoma.

The protein Lgl1 is a key regulator of cell polarity. We previously showed that Lgl1 is inactivated by hyperphosphorylation in glioblastoma as a consequence of PTEN tumour suppressor loss and aberrant activation of the PI 3-kinase pathway; this contributes to glioblastoma pathogenesis both by promoting invasion and repressing glioblastoma cell differentiation. Lgl1 is phosphorylated by atypical protein kinase C that has been activated by binding to a complex of the scaffolding protein Par6 and active, GTP-bound Rac. The specific Rac guanine nucleotide exchange factors that generate active Rac to promote Lgl1 hyperphosphorylation in glioblastoma are unknown. We used CRISPR/Cas9 to knockout PREX1, a PI 3-kinase pathway-responsive Rac guanine nucleotide exchange factor, in patient-derived glioblastoma cells. Knockout cells had reduced Lgl1 phosphorylation, which was reversed by re-expressing PREX1. They also had reduced motility and an altered phenotype suggestive of partial neuronal differentiation; consistent with this, RNA-seq analyses identified sets of PREX1-regulated genes associated with cell motility and neuronal differentiation. PREX1 knockout in glioblastoma cells from a second patient did not affect Lgl1 phosphorylation. This was due to overexpression of a short isoform of the Rac guanine nucleotide exchange factor TIAM1; knockdown of TIAM1 in these PREX1 knockout cells reduced Lgl1 phosphorylation. These data show that PREX1 links aberrant PI 3-kinase signaling to Lgl1 phosphorylation in glioblastoma, but that TIAM1 is also to fill this role in a subset of patients. This redundancy between PREX1 and TIAM1 is only partial, as motility was impaired in PREX1 knockout cells from both patients.

Aberrant Rac pathway signalling in glioblastoma.

Glioblastoma is an aggressive and incurable form of brain cancer. Both mutation analysis in human glioblastoma and mouse modelling studies have shown that aberrant activation of the PI 3-kinase pathway is a central driver of glioblastoma malignancy. The small GTPase Rac is activated downstream of this pathway, mediating a subset of the effects of aberrant PI 3-kinase pathway activation. Here I discuss the current state of our knowledge on Rac activation mechanisms in glioblastoma. Current knowledge on roles for specific PI 3-kinase pathway responsive Rac guanine nucleotide exchange factors in glioblastoma is reviewed. Rac is best known for its role in promoting cell motility and invasion, but there is also evidence for roles in multiple other cellular processes with cancer relevance, including proliferation, differentiation, apoptosis, DNA damage responses, metabolism, angiogenesis and immunosuppression. I review what is known about the role of Rac in these processes in glioblastoma. Finally, I assess possible strategies to inhibit this pathway in glioblastoma through either direct inhibition of Rac or inhibition of upstream activators or downstream mediators of Rac signalling.

Engineering PTEN-L for Cell-Mediated Delivery.

The tumor suppressor PTEN is frequently inactivated in glioblastoma. PTEN-L is a long form of PTEN produced by translation from an alternate upstream start codon. Unlike PTEN, PTEN-L has a signal sequence and a tract of six arginine residues that allow PTEN-L to be secreted from cells and be taken up by neighboring cells. This suggests that PTEN-L could be used as a therapeutic to restore PTEN activity. However, effective delivery of therapeutic proteins to treat CNS cancers such as glioblastoma is challenging. One method under evaluation is cell-mediated therapy, where cells with tumor-homing abilities such as neural stem cells are genetically modified to express a therapeutic protein. Here, we have developed a version of PTEN-L that is engineered for enhanced cell-mediated delivery. This was accomplished by replacement of the native leader sequence of PTEN-L with a leader sequence from human light-chain immunoglobulin G (IgG). This version of PTEN-L showed increased secretion and an increased ability to transfer to neighboring cells. Neural stem cells derived from human fibroblasts could be modified to express this version of PTEN-L and were able to deliver catalytically active light-chain leader PTEN-L (lclPTEN-L) to neighboring glioblastoma cells.

Induction of senescence in primary glioblastoma cells by serum and TGFß.

Glioblastoma is the most common type of adult brain tumour and has a median survival after diagnosis of a little more than a year. Glioblastomas have a high frequency of mutations in the TERT promoter and CDKN2A locus that are expected to render them resistant to both replicative and oncogene-induced senescence. However, exposure of PriGO8A primary glioblastoma cells to media with 10% serum induced a senescence-like phenotype characterized by increased senescence-associated ß galactosidase activity, PML bodies and p21 and morphological changes typical of senescence. Microarray expression analysis showed that 24 h serum exposure increased the expression of genes associated with the TGFß pathway. Treatment of PriGO8A cells with TGFß was sufficient to induce senescence in these cells. The response of PriGO8A cells to serum was dependent on basal expression of the TGFß activator protein thrombospondin. Primary glioblastoma cells from three additional patients showed a variable ability to undergo senescence in response to serum. However all were able to undergo senescence in response to TGFß, although for cells from one patient this required concomitant inhibition of Ras pathway signalling. Primary glioblastoma cells therefore retain a functional senescence program that is inducible by acute activation of the TGFß signalling pathway.

PREX1 integrates G protein-coupled receptor and phosphoinositide 3-kinase signaling to promote glioblastoma invasion.

A defining feature of the brain cancer glioblastoma is its highly invasive nature. When glioblastoma cells are isolated from patients using serum free conditions, they accurately recapitulate this invasive behaviour in animal models. The Rac subclass of Rho GTPases has been shown to promote invasive behaviour in glioblastoma cells isolated in this manner. However the guanine nucleotide exchange factors responsible for activating Rac in this context have not been characterized previously. PREX1 is a Rac guanine nucleotide exchange factor that is synergistically activated by binding of G protein αγ subunits and the phosphoinositide 3-kinase pathway second messenger phosphatidylinositol 3,4,5 trisphosphate. This makes it of particular interest in glioblastoma, as the phosphoinositide 3-kinase pathway is aberrantly activated by mutation in almost all cases. We show that PREX1 is expressed in glioblastoma cells isolated under serum-free conditions and in patient biopsies. PREX1 promotes the motility and invasion of glioblastoma cells, promoting Rac-mediated activation of p21-associated kinases and atypical PKC, which have established roles in cell motility. Glioblastoma cell motility was inhibited by either inhibition of phosphoinositide 3-kinase or inhibition of G protein ßγ subunits. Motility was also inhibited by the generic dopamine receptor inhibitor haloperidol or a combination of the selective dopamine receptor D2 and D4 inhibitors L-741,626 and L-745,870. This establishes a role for dopamine receptor signaling via G protein ßγ subunits in glioblastoma invasion and shows that phosphoinositide 3-kinase mutations in glioblastoma require a context of basal G protein-coupled receptor activity in order to promote this invasion.

Control of glioblastoma tumorigenesis by feed-forward cytokine signaling.

EGFRvIII-STAT3 signaling is important in glioblastoma pathogenesis. Here, we identified the cytokine receptor OSMR as a direct target gene of the transcription factor STAT3 in mouse astrocytes and human brain tumor stem cells (BTSCs). We found that OSMR functioned as an essential co-receptor for EGFRvIII. OSMR formed a physical complex with EGFRvIII, and depletion of OSMR impaired EGFRvIII-STAT3 signaling. Conversely, pharmacological inhibition of EGFRvIII phosphorylation inhibited the EGFRvIII-OSMR interaction and activation of STAT3. EGFRvIII-OSMR signaling in tumors operated constitutively, whereas EGFR-OSMR signaling in nontumor cells was synergistically activated by the ligands EGF and OSM. Finally, knockdown of OSMR strongly suppressed cell proliferation and tumor growth of mouse glioblastoma cells and human BTSC xenografts in mice, and prolonged the lifespan of these mice. Our findings identify OSMR as a critical regulator of glioblastoma tumor growth that orchestrates a feed-forward signaling mechanism with EGFRvIII and STAT3 to drive tumorigenesis.

PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma.

Cellular senescence is a tumor suppressor mechanism where cells enter a permanent growth arrest following cellular stress. Oncogene-induced senescence (OIS) is induced in non-malignant cells following the expression of an oncogene or inactivation of a tumor suppressor. Previously, we have shown that protein kinase C iota (PKCι) depletion induces cellular senescence in glioblastoma cells in the absence of a detectable DNA damage response. Here we demonstrate that senescent glioblastoma cells exhibit an aberrant centrosome morphology. This was observed in basal levels of senescence, in p21-induced senescence, and in PKCι depletion-induced senescence. In addition, senescent glioblastoma cells are polyploid, Ki-67 negative and arrest at the G1/S checkpoint, as determined by expression of cell cycle regulatory proteins. These markers are all consistent with cells that have undergone mitotic slippage. Failure of the spindle assembly checkpoint to function properly can lead to mitotic slippage, resulting in the premature exit of mitotic cells into the G1 phase of the cell cycle. Although in G1, these cells have the replicated DNA and centrosomal phenotype of a cell that has entered mitosis and failed to divide. Overall, we demonstrate that PKCι depletion initiates mitotic slippage-induced senescence in glioblastoma cells. To our knowledge, this is the first evidence of markers of mitotic slippage directly in senescent cells by co-staining for senescence-associated ß-galactosidase and immunofluorescence markers in the same cell population. We suggest that markers of mitotic slippage be assessed in future studies of senescence to determine the extent of mitotic slippage in the induction of cellular senescence.

Atypical PKCι as target for glioblastoma therapy.

Glioblastoma (grade IV astrocytoma) is an aggressive and incurable form of brain tumor. It invariably shows extensive invasion at the time of diagnosis, often involving both hemispheres. Recent studies have given us a very detailed picture of glioblastoma genetics. These paint a picture of a disease with extensive heterogeneity, both between patients and within individual patients. This within patient heterogeneity presents a major challenge in the design of targeted therapies. One approach is to identify targets that are common downstream elements in signaling pathways that are aberrantly activated in glioblastoma. Here we review the evidence that the atypical protein kinase C family member PKCι may fulfill this role. Our current understanding of PKCι activation mechanisms is discussed and related to common genetic changes in glioblastoma. The data showing an essential role for PKCι in multiple aspects of glioblastoma pathology are also reviewed. Finally, data on the role of PKCι in normal brain function are reviewed for insights into potential side effects of PKCι inhibition in the central nervous system.

Inhibition of glioblastoma malignancy by Lgl1.

lethal giant larvae (lgl) was first identified as a tumor suppressor in Drosophila, where its loss repressed the differentiation and promoted the invasion of neuroblasts, the Drosophila equivalent of the neural stem cell. Recently we have shown that a human homolog of Lgl, Lgl1 (LLGL1), is constitutively phosphorylated and inactivated in glioblastoma cells; this occurs as a downstream consequence of PTEN loss, one of the most frequent genetic events in glioblastoma. Here we have investigated the consequences of this loss of functional Lgl1 in glioblastoma in vivo. We used a doxycycline-inducible system to express a non-phosphorylatable, constitutively active version of Lgl1 (Lgl3SA) in either a glioblastoma cell line or primary glioblastoma cells isolated under neural stem cell culture conditions from patients. In both types of cells, expression of Lgl3SA, but not wild type Lgl1, inhibited cell motility in vitro. Induction of Lgl3SA in intracerebral xenografts markedly reduced the in vivo invasion of primary glioblastoma cells. Lgl3SA expression also induced the differentiation of glioblastoma cells in vitro and in vivo along the neuronal lineage. Thus the central features of Lgl function as a tumor suppressor in Drosophila are conserved in human glioblastoma.

PIK3CA, BRAF, and PTEN status and benefit from cetuximab in the treatment of advanced colorectal cancer--results from NCIC CTG/AGITG CO.17.

PURPOSE: Cetuximab improves survival in patients with K-ras wild-type advanced colorectal cancer. We examined the predictive and prognostic significance of additional biomarkers in this setting, in particular BRAF, PIK3CA, and PTEN. EXPERIMENTAL DESIGN: Available colorectal tumor samples were analyzed from the CO.17 study. BRAF mutations were identified in tumor-derived DNA by direct sequencing and PIK3CA mutations were identified using a high-resolution melting screen with confirmation by sequencing. PTEN expression by immunohistochemistry (IHC) was performed on tissue microarrays. For each biomarker, prognostic and predictive effects were examined using a Cox model with tests for treatment-biomarker interaction. RESULTS: A total of 572 patients with pretreated colorectal cancer were randomly assigned to receive cetuximab or best supportive care (BSC). Of 401 patients assessed for BRAF status, 13 (3.2%) had mutations. Of 407 patients assessed for PIK3CA status, 61 (15%) had mutations. Of 205 patients assessed for PTEN, 148 (72%) were negative for IHC expression. None of BRAF, PIK3CA, or PTEN was prognostic for overall or progression-free survival in the BSC arm. None was predictive of benefit from cetuximab, either in the whole study population or the K-ras wild-type subset. In the K-ras wild-type subgroup, the overall survival adjusted HR according to BRAF mutation status was 1.39 (interaction P = 0.69), PIK3CA mutation status HR = 0.79 (interaction P = 0.63), and PTEN expression HR = 0.75 (interaction P = 0.61). CONCLUSIONS: In chemotherapy-refractory colorectal cancer, neither PIK3CA mutation status nor PTEN expression were prognostic, nor were they predictive of benefit from cetuximab. Evaluation of predictive significance of BRAF mutations requires a larger sample size.

Loss of PDCD4 contributes to enhanced chemoresistance in Glioblastoma multiforme through de-repression of Bcl-xL translation.

Glioblastoma multiforme (GBM) is the most common and aggressive form of tumor of the central nervous system. Despite significant efforts to improve treatments, patient survival rarely exceeds 18 months largely due to the highly chemoresistant nature of these tumors. Importantly, misregulation of the apoptotic machinery plays a key role in the development of drug resistance. We previously demonstrated that Bcl-xL, an important anti-apoptotic protein, is regulated at the level of translation by the tumor suppressor programmed cell death 4 (PDCD4). We report here a strong correlation between low expression of PDCD4 and high expression of Bcl-xL in adult de novo GBM, GBM tumor initiating cells, and established GBM cell lines. Importantly, high Bcl-xL expression correlated significantly with poor progression and patient survival. We demonstrate that re-expression of PDCD4 in GBM cells down-regulated Bcl-xL expression and decreased cell viability. Finally, we show that direct inhibition of Bcl-xL by small molecule antagonist ABT-737 sensitizes GBM cells to doxorubicin. Our results identify Bcl-xL as a novel marker of GBM chemoresistance and advocate for the combined use of Bcl-xL antagonists and existing chemotherapeutics as a treatment option for this aggressive tumor.

PTEN loss represses glioblastoma tumor initiating cell differentiation via inactivation of Lgl1.

Glioblastoma multiforme is an aggressive and incurable type of brain tumor. A subset of undifferentiated glioblastoma cells, known as glioblastoma tumor initiating cells (GTICs), has an essential role in the malignancy of this disease and also appears to mediate resistance to radiation therapy and chemotherapy. GTICs retain the ability to differentiate into cells with reduced malignant potential, but the signaling pathways controlling differentiation are not fully understood at this time. PTEN loss is a very common in glioblastoma multiforme and leads to aberrant activation of the phosphoinositide 3-kinase pathway. Increased signalling through this pathway leads to activation of multiple protein kinases, including atypical protein kinase C. In Drosophila, active atypical protein kinase C has been shown to promote the self-renewal of neuroblasts, inhibiting their differentiation along a neuronal lineage. This effect is mediated by atypical protein kinase c-mediated phosphorylation and inactivation of Lgl, a protein that was first characterized as a tumour suppressor in Drosophila. The effects of the atypical protein kinase C/Lgl pathway on the differentiation status of GTICs, and its potential link to PTEN loss, have not been assessed previously. Here we show that PTEN loss leads to the phosphorylation and inactivation of Lgl by atypical protein kinase C in glioblastoma cells. Re-expression of PTEN in GTICs promoted their differentiation along a neuronal lineage. This effect was also seen when atypical protein kinase C was knocked down using RNA interference, and when a non-phosphorylatable, constitutively active form of Lgl was expressed in GTICs. Thus PTEN loss, acting via atypical protein kinase C activation and Lgl inactivation, helps to maintain GTICs in an undifferentiated state.

Gefitinib versus placebo in completely resected non-small-cell lung cancer: results of the NCIC CTG BR19 study.

PURPOSE: Survival of patients with completely resected non-small-cell lung cancer (NSCLC) is unsatisfactory, and in 2002, the benefit of adjuvant chemotherapy was not established. This phase III study assessed the impact of postoperative adjuvant gefitinib on overall survival (OS). PATIENTS AND METHODS: Patients with completely resected (stage IB, II, or IIIA) NSCLC stratified by stage, histology, sex, postoperative radiotherapy, and chemotherapy were randomly assigned (1:1) to receive gefitinib 250 mg per day or placebo for 2 years. Study end points were OS, disease-free survival (DFS), and toxicity. RESULTS: As a result of early closure, 503 of 1,242 planned patients were randomly assigned (251 to gefitinib and 252 to placebo). Baseline factors were balanced between the arms. With a median of 4.7 years of follow-up (range, 0.1 to 6.3 years), there was no difference in OS (hazard ratio [HR], 1.24; 95% CI, 0.94 to 1.64; P = .14) or DFS (HR, 1.22; 95% CI, 0.93 to 1.61; P = .15) between the arms. Exploratory analyses demonstrated no DFS (HR, 1.28; 95% CI, 0.92 to 1.76; P = .14) or OS benefit (HR, 1.24; 95% CI, 0.90 to 1.71; P = .18) from gefitinib for 344 patients with epidermal growth factor receptor (EGFR) wild-type tumors. Similarly, there was no DFS (HR, 1.84; 95% CI, 0.44 to 7.73; P = .395) or OS benefit (HR, 3.16; 95% CI, 0.61 to 16.45; P = .15) from gefitinib for the 15 patients with EGFR mutation-positive tumors. Adverse events were those expected with an EGFR inhibitor. Serious adverse events occurred in ≤ 5% of patients, except infection, fatigue, and pain. One patient in each arm had fatal pneumonitis. CONCLUSION: Although the trial closed prematurely and definitive statements regarding the efficacy of adjuvant gefitinib cannot be made, these results indicate that it is unlikely to be of benefit.

Detection of PIK3CA Mutations in Breast Cancer Bone Metastases.

Background. An important goal of personalized cancer therapy is to tailor specific therapies to the mutational profile of individual patients. However, whole genome sequencing studies have shown that the mutational profiles of cancers evolve over time and often differ between primary and metastatic sites. Activating point mutations in the PIK3CA gene are common in primary breast cancer tumors, but their presence in breast cancer bone metastases has not been assessed previously. Results. Fourteen patients with breast cancer bone metastases were biopsied by three methods: CT-guided bone biopsies; bone marrow trephine biopsies; and bone marrow aspiration. Samples that were positive for cancer cells were obtained from six patients. Three of these patients had detectable PIK3CA mutations in bone marrow cancer cells. Primary tumor samples were available for four of the six patients assessed for PIK3CA status in their bone metastases. For each of these, the PIK3CA mutation status was the same in the primary and metastatic sites. Conclusions. PIK3CA mutations occur frequently in breast cancer bone metastases. The PIK3CA mutation status in bone metastases samples appears to reflect the PIK3CA mutation status in the primary tumour. Breast cancer patients with bone metastases may be candidates for treatment with selective PIK3CA inhibitors.

Stage-dependent differential expression of microRNAs in colorectal cancer: potential role as markers of metastatic disease.

MicroRNAs (miRs) are short non-coding RNAs that bind complementary sequences in mRNA resulting in translation repression and/or mRNA degradation. We investigated expression of the reported metastasis-associated miRs-335, 206, 135a, 146a, 146b, 10b, 21, let7a and let7b in normal mucosa, non-metastatic and metastatic colorectal cancer (CRC). Expression of target miRs in micro-dissected paraffin embedded tissues was evaluated in 15 primary tumours with adjacent normal tissue from patients that were disease-free at 4 years (cohort A) and 19 paired primary tumours with corresponding liver metastases (cohort B) by quantitative real-time PCR. Increased expression of miR-21, mir-135a and miR-335 was associated with clinical progression of CRC, while miR-206 demonstrated an opposite trend. The levels of mir-21 did not associate with the expression of PTEN, an important tumour suppressor in CRC and one of many putative targets of miR-21, but interestingly was associated with stage of disease in the PTEN expressing tumours. Surprisingly, let7a, a KRAS-targeting miR, showed elevated expression in metastatic disease compared to normal mucosa or non-metastatic disease, and only in KRAS mutation positive tumors. Finally, a prognostic signature of miR 21,135a, 335, 206 and let-7a for detecting the presence of metastases had a specificity of 87% and sensitivity of 76% for the presence of metastases. In summary, we have shown stage-associated differential expression of five out of nine tested metastasis-associated miRs. We have further found that an analysis of these five miRs expression levels in primary tumors significantly correlates with the presence of metastatic disease, making this a potential clinically useful prognostic tool.