Rui M. Reis

Frequency of TERT promoter mutations in human cancers

Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Here we report the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%), bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. Our results show that TERT promoter mutations are relatively frequent in specific types of human cancers, where they lead to enhanced expression of telomerase.

MicroRNA history: discovery, recent applications, and next frontiers.

Since 1993, when the first small non-coding RNA was identified, our knowledge about microRNAs has grown exponentially. In this review, we focus on the main progress in this field and discuss the most important findings under a historical perspective. In addition, we examine microRNAs as markers of disease diagnosis and prognosis, and as new therapeutic targets.

EGFR amplification and lack of activating mutations in metaplastic breast carcinomas

Metaplastic breast carcinomas are reported to harbour epidermal growth factor receptor (EGFR) overexpression in up to 80% of the cases, but EGFR gene amplification is the underlying genetic mechanism in around one‐third of these. In this study, EGFR gene amplification as defined by chromogenic in situ hybridization and protein overexpression was examined in a cohort of 47 metaplastic breast carcinomas. Furthermore, the presence of activating EGFR mutations in exons 18, 19, 20, and 21 was investigated. Thirty‐two cases showed EGFR overexpression and of these, 11 (34%) harboured EGFR gene amplification. In addition, EGFR amplification showed a statistically significant association with EGFR overexpression (p < 0.0094) and was restricted to carcinomas with homologous metaplasia. Ten cases, five with and five without EGFR amplification, were subjected to microarray‐based CGH, which demonstrated that EGFR copy number gain may occur by amplification of a discrete genomic region or by gains of the short arm of chromosome 7 with a breakpoint near the EGFR gene locus, the minimal region of amplification mapping to EGFR, LANCL2, and SEC61G. No activating EGFR mutations were identified, suggesting that this is unlikely to be a common alternative underlying genetic mechanism for EGFR expression in metaplastic breast carcinomas. Given that metaplastic breast carcinomas are resistant to conventional chemotherapy or hormone therapy regimens and that tumours with EGFR amplification are reported to be sensitive to EGFR tyrosine kinase inhibitors, these findings indicate that further studies are warranted to explore EGFR tyrosine kinase inhibitors as potential therapeutic agents for metaplastic breast carcinomas harbouring amplification of 7p11.2. Copyright

Overexpression of platelet-derived growth factor receptor α in breast cancer is associated with tumour progression

IntroductionReceptor tyrosine kinases have been extensively studied owing to their frequently abnormal activation in the development and progression of human cancers. Platelet-derived growth factor receptors (PDGFRs) are receptors with intrinsic tyrosine kinase activity that regulate several functions in normal cells and are widely expressed in a variety of malignancies. After the demonstration that gastrointestinal stromal tumours without c-Kit mutations harbour PDGFR-α-activating mutations and that PDGFR-α is also a therapeutic target for imatinib mesylate, the interest for this receptor has increased considerably. Because breast cancer is one of the most frequent neoplasias in women worldwide, and only one study has reported PDGFR-α expression in breast carcinomas, the aim of this work was to investigate the potential significance of PDGFR-α expression in invasive mammary carcinomas.MethodsWe used immunohistochemistry to detect PDGFR-α overexpression on a series of 181 formalin-fixed paraffin-embedded invasive ductal breast carcinomas and in two breast cancer cell lines: MCF-7 and HS578T. We associated its expression with known prognostic factors and we also performed polymerase chain reaction–single-stranded conformational polymorphism and direct sequencing to screen for PDGFR-α mutations.ResultsPDGFR-α expression was observed in 39.2% of the breast carcinomas and showed an association with lymph node metastasis (P = 0.0079), HER-2 expression (P = 0.0265) and Bcl2 expression (P = 0.0121). A correlation was also found with the expression of platelet-derived growth factor A (PDGF-A; P = 0.0194). The two cell lines tested did not express PDGFR-α. Screening for mutations revealed alterations in the PDGFR-α gene at the following locations: 2500A→G, 2529T→A and 2472C→T in exon 18 and 1701G→A in exon 12. We also found an intronic insertion IVS17-50insA at exon 18 in all sequenced cases. None of these genetic alterations was correlated with PDGFR-α expression. The cell lines did not reveal any alterations in the PDGFR-α gene sequence.ConclusionPDGFR-α is expressed in invasive breast carcinomas and is associated with biological aggressiveness. The genetic alterations described were not correlated with protein expression, but other mechanisms such as gene amplification or constitutive activation of a signalling pathway inducing this receptor could still sustain PDGFR-α as a potential therapeutic target.

Histone H3.3 Mutations Drive Pediatric Glioblastoma through Upregulation of MYCN

UNLABELLED Children and young adults with glioblastoma (GBM) have a median survival rate of only 12 to 15 months, and these GBMs are clinically and biologically distinct from histologically similar cancers in older adults. They are defined by highly specific mutations in the gene encoding the histone H3.3 variant H3F3A , occurring either at or close to key residues marked by methylation for regulation of transcription—K27 and G34. Here, we show that the cerebral hemisphere-specific G34 mutation drives a distinct expression signature through differential genomic binding of the K36 trimethylation mark (H3K36me3). The transcriptional program induced recapitulates that of the developing forebrain, and involves numerous markers of stem-cell maintenance, cell-fate decisions, and self-renewal.Critically, H3F3A G34 mutations cause profound upregulation of MYCN , a potent oncogene that is causative of GBMs when expressed in the correct developmental context. This driving aberration is selectively targetable in this patient population through inhibiting kinases responsible for stabilization of the protein. SIGNIFICANCE We provide the mechanistic explanation for how the fi rst histone gene mutation inhuman disease biology acts to deliver MYCN, a potent tumorigenic initiator, into a stem-cell compartment of the developing forebrain, selectively giving rise to incurable cerebral hemispheric GBM. Using synthetic lethal approaches to these mutant tumor cells provides a rational way to develop novel and highly selective treatment strategies

A Distinct Spectrum of Copy Number Aberrations in Pediatric High-Grade Gliomas

Purpose: As genome-scale technologies begin to unravel the complexity of the equivalent tumors in adults, we can attempt detailed characterization of high-grade gliomas in children, that have until recently been lacking. Toward this end, we sought to validate and extend investigations of the differences between pediatric and adult tumors. Experimental Design: We carried out copy number profiling by array comparative genomic hybridization using a 32K bacterial artificial chromosome platform on 63 formalin-fixed paraffin-embedded cases of high-grade glioma arising in children and young people (<23 years). Results: The genomic profiles of these tumors could be subclassified into four categories: those with stable genomes, which were associated with a better prognosis; those with aneuploid and those with highly rearranged genomes; and those with an amplifier genotype, which had a significantly worse clinical outcome. Independent of this was a clear segregation of cases with 1q gain (more common in children) from those with concurrent 7 gain/10q loss (a defining feature of adults). Detailed mapping of all the amplification and deletion events revealed numerous low-frequency amplifications, including IGF1R, PDGFRB, PIK3CA, CDK6, CCND1, and CCNE1, and novel homozygous deletions encompassing unknown genes, including those at 5q35, 10q25, and 22q13. Despite this, aberrations targeting the “core signaling pathways” in adult glioblastomas are significantly underrepresented in the pediatric setting. Conclusions: These data highlight that although there are overlaps in the genomic events driving gliomagenesis of all ages, the pediatric disease harbors a distinct spectrum of copy number aberrations compared with adults. Clin Cancer Res; 16(13); 3368–77. ©2010 AACR.

MGMT-Independent Temozolomide Resistance in Pediatric Glioblastoma Cells Associated with a PI3-Kinase–Mediated HOX/Stem Cell Gene Signature

Sensitivity to temozolomide is restricted to a subset of glioblastoma patients, with the major determinant of resistance being a lack of promoter methylation of the gene encoding the repair protein DNA methyltransferase MGMT, although other mechanisms are thought to be active. There are, however, limited preclinical data in model systems derived from pediatric glioma patients. We screened a series of cell lines for temozolomide efficacy in vitro, and investigated the differential mechanisms of resistance involved. In the majority of cell lines, a lack of MGMT promoter methylation and subsequent protein overexpression were linked to temozolomide resistance. An exception was the pediatric glioblastoma line KNS42. Expression profiling data revealed a coordinated upregulation of HOX gene expression in resistant lines, especially KNS42, which was reversed by phosphoinositide 3-kinase pathway inhibition. High levels of HOXA9/HOXA10 gene expression were associated with a shorter survival in pediatric high-grade glioma patient samples. Combination treatment in vitro of pathway inhibition and temozolomide resulted in a highly synergistic interaction in KNS42 cells. The resistance gene signature further included contiguous genes within the 12q13-q14 amplicon, including the Akt enhancer PIKE, significantly overexpressed in the KNS42 line. These cells were also highly enriched for CD133 and other stem cell markers. We have thus shown an in vitro link between phosphoinositide 3-kinase-mediated HOXA9/HOXA10 expression, and a drug-resistant, progenitor cell phenotype in MGMT-independent pediatric glioblastoma.

IDENTIFICATION IN HUMAN BRAIN TUMORS OF DNA SEQUENCES SPECIFIC FOR SV40 LARGE T ANTIGEN

Simian virus 40 (SV40) sequences have recently been identified in a variety of human neoplasms, including mesothelioma, osteosarcoma, and brain tumors, but significant discrepancies exist regarding the frequency at which this occurs. The SV40 genome is 70% homologous to JC and BK, two related polyomaviruses that are highly prevalent in humans and which may cause in immune‐compromised patients progressive multifocal leukoencephalopathy (PML) and cystitis, respectively. We have established a specific and sensitive method to identify SV40 sequence in DNA extracted from histological sections, using PCR followed by Southern hybridization to probes specific to the large T region. We found SV40 largeT antigen sequences in all brain tumor types investigated. High frequencies were found in low‐grade astrocytomas, anaplastic astro‐cytomas and secondary glioblastomas derived thereof (13/22, 59%) while somewhat lower frequencies were found in gemistocytic astrocytomas (9/28, 32%) and oligodendrogliomas (3/12, 25%). Primary glioblastomas, giant cell glioblastomas, and gliosarcomas, which clinically develop de novo, contained SV40 sequences in 11–25% of cases. Presence of viral DNA was also observed in pediatric brain tumors, including ependymomas (9/16, 56%), choroid plexus papillomas (6/16, 38%), and medulloblastomas (5/17, 29%). In 8 tumor biopsies with SV40 sequences, the adjacent normal brain tissue was also analyzed but was devoid of viral DNA in all but one case. BK and JC virus sequences were rarely detected, the overall frequencies being 3% and 2%, respectively. It remains to be shown whether the presence of SV40 contributes significantly to malignant transformation or whether certain human neoplasms provide a microenvironment that favors viral replication in humans with latent SV40 infection.

Monocarboxylate transporters (MCTs) in gliomas: Expression and exploitation as therapeutic targets

BACKGROUND Gliomas exhibit high glycolytic rates, and monocarboxylate transporters (MCTs) play a major role in the maintenance of the glycolytic metabolism through the proton-linked transmembrane transport of lactate. However, their role in gliomas is poorly studied. Thus, we aimed to characterize the expression of MCT1, MCT4, and their chaperone CD147 and to assess the therapeutic impact of MCT inhibition in gliomas. METHODS MCTs and CD147 expressions were characterized by immunohistochemistry in nonneoplastic brain and glioma samples. The effect of CHC (MCT inhibitor) and MCT1 silencing was assessed in in vitro and in vivo glioblastoma models. RESULTS MCT1, MCT4, and CD147 were overexpressed in the plasma membrane of glioblastomas, compared with diffuse astrocytomas and nonneoplastic brain. CHC decreased glycolytic metabolism, migration, and invasion and induced cell death in U251 cells (more glycolytic) but only affected proliferation in SW1088 (more oxidative). The effectiveness of CHC in glioma cells appears to be dependent on MCT membrane expression. MCT1 downregulation showed similar effects on different glioma cells, supporting CHC as an MCT1 inhibitor. There was a synergistic effect when combining CHC with temozolomide treatment in U251 cells. In the CAM in vivo model, CHC decreased the size of tumors and the number of blood vessels formed. CONCLUSIONS This is the most comprehensive study reporting the expression of MCTs and CD147 in gliomas. The MCT1 inhibitor CHC exhibited anti-tumoral and anti-angiogenic activity in gliomas and, of importance, enhanced the effect of temozolomide. Thus, our results suggest that development of therapeutic approaches targeting MCT1 may be a promising strategy in glioblastoma treatment.

Strand-Specific miR-28-5p and miR-28-3p Have Distinct Effects in Colorectal Cancer Cells

BACKGROUND & AIMS MicroRNAs (miRNAs) can promote or inhibit tumor growth and are therefore being developed as targets for cancer therapies. They are diverse not only in the messenger RNAs (mRNA) they target, but in their production; the same hairpin RNA structure can generate mature products from each strand, termed 5p and 3p, that can bind different mRNAs. We analyzed the expression, functions, and mechanisms of miR-28-5p and miR-28-3p in colorectal cancer (CRC) cells. METHODS We measured levels of miR-28-5p and miR-28-3p expression in 108 CRC and 49 normal colorectal samples (47 paired) by reverse transcription, quantitative real-time polymerase chain reaction. The roles of miR-28 in CRC development were studied using cultured HCT116, RKO, and SW480 cells and tumor xenograft analyses in immunodeficient mice; their mRNA targets were also investigated. RESULTS miR-28-5p and miR-28-3p were down-regulated in CRC samples compared with normal colon samples. Overexpression of miRNAs in CRC cells had different effects and the miRNAs interacted with different mRNAs: miR-28-5p altered expression of CCND1 and HOXB3, whereas miR-28-3p bound NM23-H1. Overexpression of miR-28-5p reduced CRC cell proliferation, migration, and invasion in vitro, whereas miR-28-3p increased CRC cell migration and invasion in vitro. CRC cells overexpressing miR-28 developed tumors more slowly in mice compared with control cells, but miR-28 promoted tumor metastasis in mice. CONCLUSION miR-28-5p and miR-28-3p are transcribed from the same RNA hairpin and are down-regulated in CRC cells. Overexpression of each has different effects on CRC cell proliferation and migration. Such information has a direct application for the design of miRNA gene therapy trials.