Helen Sheldon

hsa-miR-210 Is Induced by Hypoxia and Is an Independent Prognostic Factor in Breast Cancer

Purpose: MicroRNA (miRNA) expression alterations have been described in cancer. Many cancers are characterized by areas of hypoxia, enhanced hypoxia-inducible factor (HIF) levels, and increased expression of hypoxically regulated genes, all of which correlate with patient outcome. We examined hypoxia-induced miRNA expression changes to identify markers of survival in breast cancer. Experimental Design: We used microarrays to analyze miRNA expression changes induced by hypoxia in MCF7 breast cancer cell lines and validated results by quantitative-PCR (Q-PCR). Small interfering RNA against HIF-1α and HIF-2α, and RCC4 cells transfected with the von Hippel-Lindau (VHL) protein were used to investigate HIF dependency of miRNA expression. miRNA Q-PCR assays were done on 219 early breast cancer samples with long-term follow-up. Correlation of expression with clinical variables was done using Pearson and Spearmans rank tests, univariate, and Cox multivariate analysis. Results: hsa-miR-210 induction was the most significant change under hypoxia by microarray analysis (3.4-fold, P < 0.001). hsa-miR-210 expression changes were validated by Q-PCR and detected in other cancer cell lines. Using small interfering RNAs and RCC4 cells transfected with VHL, we showed that the regulation by hypoxia of hsa-miR-210 was mediated by the HIF-1α/VHL transcriptional system but not HIF-2α. hsa-miR-210 expression levels in breast cancer samples correlated directly with a hypoxia score based on the expression of 99 genes. hsa-miR-210 expression levels showed an inverse correlation with disease-free and overall survival, significant in both univariate and multivariate analyses. Conclusions: We show that hsa-miR-210 overexpression is induced by hypoxia in a HIF-1α– and VHL-dependent fashion and its expression levels in breast cancer samples are an independent prognostic factor.

New mechanism for Notch signaling to endothelium at a distance by Delta-like 4 incorporation into exosomes

Notch signaling is an evolutionary conserved pathway that is mediated by cell-cell contact. It is involved in a variety of developmental processes and has an essential role in vascular development and angiogenesis. Delta-like 4 (Dll4) is a Notch ligand that is up-regulated during angiogenesis. It is expressed in endothelial cells and regulates the differentiation between tip cells and stalk cells of neovasculature. Here, we present evidence that Dll4 is incorporated into endothelial exosomes. It can also be incorporated into the exosomes of tumor cells that overexpress Dll4. These exosomes can transfer the Dll4 protein to other endothelial cells and incorporate it into their cell membrane, which results in an inhibition of Notch signaling and a loss of Notch receptor. Transfer of Dll4 was also shown in vivo from tumor cells to host endothelium. Addition of Dll4 exosomes confers a tip cell phenotype on the endothelial cell, which results in a high Dll4/Notch-receptor ratio, low Notch signaling, and filopodia formation. This was further evidenced by increased branching in a tube-formation assay and in vivo. This reversal in phenotype appears to enhance vessel formation and is a new form of signaling for Notch ligands that expands their signaling potential beyond cell-cell contact.

microRNA associated progression pathways and potential therapeutic targets identified by integrated mRNA and microRNA expression profiling in breast cancer

microRNA expression profiling plays an emerging role in cancer classification and identification of therapeutic strategies. In this study, we have evaluated the benefits of a joint microRNA-mRNA analysis in breast cancer. Matched mRNA and microRNA global expression profiling was conducted in a well-annotated cohort of 207 cases with complete 10-year follow-up. Penalized Cox regression including microRNA expression, mRNA expression, and clinical covariates was used to identify microRNAs associated with distant relapse-free survival (DRFS) that provide independent prognostic information, and are not simply surrogates of previously identified prognostic covariates. Penalized regression was chosen to prevent overfitting. Furthermore, microRNA-mRNA relationships were explored by global expression analysis, and exploited to validate results in several published cohorts (n = 592 with DRFS, n = 1,050 with recurrence-free survival). Four microRNAs were independently associated with DRFS in estrogen receptor (ER)-positive (3 novel and 1 known; miR-128a) and 6 in ER-negative (5 novel and 1 known; miR-210) cases. Of the latter, miR-342, -27b, and -150 were prognostic also in triple receptor-negative tumors. Coordinated expression of predicted target genes and prognostic microRNAs strengthened these results, most significantly for miR-210, -128a, and -27b, whose targets were prognostic in meta-analysis of several cohorts. In addition, miR-210 and -128a showed coordinated expression with their cognate pri-microRNAs, which were themselves prognostic in independent cohorts. Our integrated microRNA-mRNA global profiling approach has identified microRNAs independently associated with prognosis in breast cancer. Furthermore, it has validated known and predicted microRNA-target interactions, and elucidated their association with key pathways that could represent novel therapeutic targets.

The small-nucleolar RNAs commonly used for microRNA normalisation correlate with tumour pathology and prognosis.

Background:To investigate small-nucleolar RNAs (snoRNAs) as reference genes when measuring miRNA expression in tumour samples, given emerging evidence for their role in cancer.Methods:Four snoRNAs, commonly used for normalisation, RNU44, RNU48, RNU43 and RNU6B, and miRNA known to be associated with pathological factors, were measured by real-time polymerase chain reaction in two patient series: 219 breast cancer and 46 head and neck squamous cell carcinoma (HNSCC). SnoRNA and miRNA were then correlated with clinicopathological features and prognosis.Results:Small-nucleolar RNA expression was as variable as miRNA expression (miR-21, miR-210, miR-10b). Normalising miRNA PCR expression data to these recommended snoRNAs introduced bias in associations between miRNA and pathology or outcome. Low snoRNA expression correlated with markers of aggressive pathology. Low levels of RNU44 were associated with a poor prognosis. RNU44 is an intronic gene in a cluster of highly conserved snoRNAs in the growth arrest specific 5 (GAS5) transcript, which is normally upregulated to arrest cell growth under stress. Low-tumour GAS5 expression was associated with a poor prognosis. RNU48 and RNU43 were also identified as intronic snoRNAs within genes that are dysregulated in cancer.Conclusion:Small-nucleolar RNAs are important in cancer prognosis, and their use as reference genes can introduce bias when determining miRNA expression.

DLL4-Notch Signaling Mediates Tumor Resistance to Anti-VEGF Therapy In Vivo

Resistance to VEGF inhibitors is emerging as a major clinical problem. Notch signaling has been implicated in tumor angiogenesis. Therefore, to investigate mechanisms of resistance to angiogenesis inhibitors, we transduced human glioblastoma cells with retroviruses encoding Notch delta-like ligand 4 (DLL4), grew them as tumor xenografts and then treated the murine hosts with the VEGF-A inhibitor bevacizumab. We found that DLL4-mediated tumor resistance to bevacizumab in vivo. The large vessels induced by DLL4-Notch signaling increased tumor blood supply and were insensitive to bevacizumab. However, blockade of Notch signaling by dibenzazepine, a γ-secretase inhibitor, disrupted the large vessels and abolished the tumor resistance. Multiple molecular mechanisms of resistance were shown, including decreased levels of hypoxia-induced VEGF and increased levels of the VEGF receptor VEGFR1 in the tumor stroma, decreased levels of VEGFR2 in large blood vessels, and reduced levels of VEGFR3 overall. DLL4-expressing tumors were also resistant to a VEGFR targeting multikinase inhibitor. We also observed activation of other pathways of tumor resistance driven by DLL4-Notch signaling, including the FGF2-FGFR and EphB4-EprinB2 pathways, the inhibition of which reversed tumor resistance partially. Taken together, our findings show the importance of classifying mechanisms involved in angiogenesis in tumors, and how combination therapy to block DLL4-Notch signaling may enhance the efficacy of VEGF inhibitors, particularly in DLL4-upregulated tumors, and thus provide a rational base for the development of novel strategies to overcome antiangiogenic resistance in the clinic.

MicroRNA-10b and breast cancer metastasis

Arising from: L. Ma, J. Teruya-Feldstein & R. A. Weinberg 449, 682–688 (2007)10.1038/nature06174; Ma et al. replyMicroRNAs regulate messenger RNA expression but are frequently dysregulated in tumours. Ma et al. report that overexpression of microRNA-10b (miR-10b) initiates invasion and metastasis in models of breast cancer and that its expression in primary breast carcinomas correlates with clinical progression. We tested this in patients with primary breast cancer, of whom 92 had nodal metastases at diagnosis and 127 were node-negative. We found no significant association between miR-10b levels and metastasis or prognosis. Although we concede that miR-10b may have a biological effect in a few cells at the growing edge of a tumour, we believe that it is unlikely to correlate in whole tumour samples with clinical progression.

Recurrent PTPRB and PLCG1 mutations in angiosarcoma

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation-promoting factors

This study aimed to further elucidate the function of Roundabout proteins in endothelium. We show that both Robo1 and Robo4 are present in human umbilical vein endothelial cells (HUVECs) and have knocked expression down using small interfering RNA (siRNA) technology. Roundabout knockout endothelial cells were then studied in a variety of in vitro assays. We also performed a yeast 2‐hybrid analysis using the intracellular domain of Robo4 as bait to identify interacting proteins and downstream signaling. Both Robo1 and Robo4 siRNA knockdown and transfection of Robo4‐green fluorescent protein inhibited endothelial cell movement and disrupted tube formation on Matrigel. Consistent with a role in regulating cell movement, yeast 2‐hybrid and glutathione‐S‐transferase pulldown analyses show Robo4 binding to a Wiskott‐Aldrich syndrome protein (WASP), neural Wiskott‐Aldrich syndrome protein, and WASP‐interacting protein actin‐nucleating complex. We have further shown that Robo1 forms a heterodimeric complex with Robo4, and that transfection of Robo4GFP into HUVECs induces filopodia formation. We finally show using Robo1 knockdown cells that Robo1 is essential for Robo4‐mediated filopodia induction. Our results favor a model whereby Slit2 binding to a Robo1/Robo4 heterodimer activates actin nucleation‐promoting factors to promote endothelial cell migration.—Sheldon, H., Andre, M., Legg, J. A., Heal, P., Herbert, J. M., Sainson, R, Sharma, A. S., Kitajewski, J. K., Heath, V. L., Bicknell, R. Active involvement of Robo1 and Robo4 in filopodia formation and endothelial cell motility mediated via WASP and other actin nucleation‐promoting factors. FASEB J. 23, 513–522 (2009)

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

Phase I/II Trial of Bevacizumab and Radiotherapy for Locally Advanced Inoperable Colorectal Cancer: Vasculature-Independent Radiosensitizing Effect of Bevacizumab

Purpose: Antivascular endothelial growth factor therapy enhances the activity of radiotherapy in experimental models, and bevacizumab has therapeutic activity in patients with metastatic colorectal cancer. Experimental Design: Twenty-two patients with locally advanced inoperable colorectal carcinomas (LA/I-CRC) were treated with conformal hypofractionated (3.4 Gy/fraction 15) split-course accelerated radiotherapy (biological equivalent dose, 67.2 Gy) supported with amifostine, capecitabine (600 mg/m2 daily, 5 days/week), and bevacizumab (5 mg/kg every 2 weeks, five cycles). Biopsies from nine patients, performed before and 1 week after bevacizumab administration, were analyzed for changes in mRNA expression with Illumina gene arrays. Results: No serious grade 3 chemotherapy-related side effects were recorded. There was low acute toxicity, with moist perineal desquamation noted in 2 of 22 patients, diarrhea grade 2 to 3 in 5 of 22 patients, and severe proctalgia in 2 of 22 patients. One patient died from Fourniers gangrene before treatment completion. Within a median follow-up of 18 months, two patients with preradiotheraphy direct involvement of adjacent organs expressed recto-vaginal/perineal fistula. Out of 19 evaluable cases, 13 (68.5) showed complete response and 4 showed (21.1) partial response. Fourteen patients are alive with no evidence of loco-regional relapse. In the gene array analysis, 30 known genes associated with transcription factors, DNA repair, and proliferation were downregulated by bevacizumab. DUSP1 gene was the most consistently downregulated transcript. Conclusions: The combination of radiotherapy with bevacizumab is feasible and results in a high rate of durable complete responses in patients with LA/I-CRC. Radiosensitization may occur through a direct effect on tumor cells followed by a wide scale suppression of transcription factors and genes involved in DNA repair and proliferation. (Clin Cancer Res 2009;15(22):706976)