Publications by authors named "Yutaka Amemiya"

32 Publications

Delayed MRI Enhancement of Colorectal Cancer Liver Metastases Is Associated With Metastatic Mutational Profile.

Cancer Genomics Proteomics 2021 Sep-Oct;18(5):627-635

Department of Medical Imaging, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, Canada.

Background/aim: Individual tumor genomics plays a key role in determining patient prognosis, response to chemotherapy and in guiding therapy. In prior studies, it was shown that the degree of late enhancement of colorectal liver metastases (CRCLM) target tumor enhancement (TTE) as seen on magnetic resonance imaging (MRI) was associated with overall survival. In order to better understand the relationship between MRI enhancement and survival, the aim of this study was to characterize genomic profiles of tumors clustered by MRI TTE, and investigate the association between TTE and genetic mutations.

Materials And Methods: Matched tumor and normal tissue samples from patients with weak TTE and strong TTE were analyzed by Next-generation sequencing (NGS) technology using a custom colorectal cancer panel.

Results: We discovered a total of 42 non-synonymous somatic mutations from 10 patients with weak TTE and 26 with 10 patients with strong TTE. Adenomatosis Polyposis Coli (APC) was the most commonly altered gene, 18 of those APC mutations were found in the weak TTE and 9 in the strong TTE group.

Conclusion: An association exists between TTE and mutational status of CRCLM, which may offer some explanation as to why TTE is associated with overall survival in patients with CRCLM.
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http://dx.doi.org/10.21873/cgp.20285DOI Listing
May 2021

MiR-139 Induces an Interferon-β Response in Prostate Cancer Cells by Binding to RIG-1.

Cancer Genomics Proteomics 2021 May-Jun;18(3):197-206

Platform Biological Sciences, Sunnybrook Research Institute, University of Toronto, Toronto, ON, Canada;

Background: We previously identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. Expression of one of the down-regulated miRNAs, miR-139-5p, was significantly associated with a lower incidence of biochemical recurrence and metastasis. Transcriptome profiling of miR-139-expressing prostate cancer cells revealed up-regulation of genes involved in interferon (IFN) stimulation. The association between miR-139 and IFN-β was further explored in this study.

Materials And Methods: We examined miR-139 transfected PC3, Du145 and LNCaP cells and the associated IFN response by transcriptome sequencing, immunoblotting and pulldown assays.

Results: Treatment of prostate cancer cells by miR-139 resulted in the up-regulation of IFN-related genes. Specifically, miR-139 induced expression of the IFN-β protein. The ability of miR-139 to induce IFN-β was due to its binding to RIG-1 and the induction of IFN-related genes was found to be dependent on RIG-1 expression.

Conclusion: miR-139 acts as an immune agonist of RIG-1 to enhance IFN-β response in prostate cancer cells.
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http://dx.doi.org/10.21873/cgp.20252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126337PMC
March 2021

MR-guided focused ultrasound liquid biopsy enriches circulating biomarkers in patients with brain tumors.

Neuro Oncol 2021 Mar 10. Epub 2021 Mar 10.

Sunnybrook Research Institute, Toronto, Ontario, Canada.

Background: Liquid biopsy is promising for early detection, monitoring of response and recurrence of cancer. The blood-brain barrier (BBB) limits the shedding of biomarker, such as cell-free DNA (cfDNA), into the blood, and their detection by conventional assays. Transcranial MR-guided focused ultrasound (MRgFUS) can safely and transiently open the BBB, providing an opportunity for less-invasive access to brain pathology. We hypothesized MRgFUS can enrich the signal of circulating brain-derived biomarkers to aid in liquid biopsy.

Methods: Nine patients were treated in a prospective single-arm, open-label trial to investigate serial MRgFUS and adjuvant temozolomide combination in patients with glioblastoma (NCT03616860). Blood samples were collected as an exploratory measure within the hours before and after sonication, with control samples from non-brain tumor patients undergoing BBB opening alone (NCT03739905).

Results: Brain regions averaging 7.8±6.0 cm 3 (range 0.8-23.1 cm 3) were successful treated within 111±39 minutes without any serious adverse events. We found MRgFUS acutely enhanced plasma cfDNA (2.6±1.2 fold, p<0.01, Wilcoxon signed-rank test), neuron-derived extracellular vesicles (3.2±1.9 fold, p<0.01), and brain specific protein S100b (1.4±0.2 fold, p<0.01). Further comparison of the cfDNA methylation profiles suggests a signature that is disease and post-BBB opening specific, in keeping with our hypothesis. We also found cfDNA mutant copies of isocitrate dehydrogenase 1 (IDH1) increased, although this was in only one patient known to harbour the tumor mutation.

Conclusions: This first-in-human proof-of concept study shows MRgFUS enriches the signal of circulating brain-derived biomarkers, demonstrating the potential of the technology to support liquid biopsy for the brain.
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http://dx.doi.org/10.1093/neuonc/noab057DOI Listing
March 2021

Mir-139 Regulates Autophagy in Prostate Cancer Cells Through Beclin-1 and mTOR Signaling Proteins.

Anticancer Res 2020 Dec 7;40(12):6649-6663. Epub 2020 Dec 7.

Platform Biological Sciences, Sunnybrook Research Institute, University of Toronto, Toronto, ON, Canada;

Background/aim: We previously identified a panel of five miRNAs (including miR-139) associated with biochemical recurrence and metastasis in prostate cancer patients.

Materials And Methods: We examined miR-139 transfected PC3, DU145 and LNCaP cells by morphology as well as by cell-based assays, confocal microscopy and immunoblotting.

Results: We found that treatment of prostate cancer cells with miR-139 resulted in phenotypic changes characteristic of autophagic cells. MiR-139 increased the autophagy-related conversion of the microtubule-associated protein light chain 3 (LC3-I to LC3-II) that was specifically inhibited by the miR-139 antagomir. The upregulation of LC3 II was further confirmed by confocal microscopy. miR-139 regulated activation of both mTOR and Beclin1 the two important autophagy-related molecules. We found that upon miR-139 treatment, the cargo adaptor protein p62 which is degraded during autophagy, accumulates.

Conclusion: These results suggest that miR-139 is inducing autophagic flux blockade leading to apoptosis in prostate cancer cells through the mTOR and Beclin-1 proteins.
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http://dx.doi.org/10.21873/anticanres.14689DOI Listing
December 2020

CTNNB1 Mutations and Aberrant β-Catenin Expression in Ovarian Endometrioid Carcinoma: Correlation With Patient Outcome.

Am J Surg Pathol 2021 01;45(1):68-76

Department of Laboratory Medicine and Pathobiology, University of Toronto.

CTNNB1 mutations and aberrant β-catenin expression have adverse prognosis in endometrial endometrioid carcinoma, and recent evidence suggests a prognostic role of β-catenin in ovarian endometrioid carcinoma. Thus, we aimed to determine the prognostic value of the CTNNB1 mutational status, and its correlation with β-catenin expression, in a well-annotated cohort of 51 ovarian endometrioid carcinomas. We performed immunohistochemistry for β-catenin and developed an 11-gene next-generation sequencing panel that included whole exome sequencing of CTNNB1 and TP53. Results were correlated with clinicopathologic variables including disease-free and disease-specific survival. Tumor recurrence was documented in 14 patients (27%), and cancer-related death in 8 patients (16%). CTNNB1 mutations were found in 22 cases (43%), and nuclear β-catenin in 26 cases (51%). CTNNB1 mutation highly correlated with nuclear β-catenin (P<0.05). Mutated CTNNB1 status was statistically associated with better disease-free survival (P=0.04, log-rank test) and approached significance for better disease-specific survival (P=0.07). It also correlated with earlier International Federation of Gynecology and Obstetrics stage (P<0.05). Nuclear β-catenin, TP53 mutations, age, ProMisE group, surface involvement, tumor grade and stage also correlated with disease-free survival. There was no association between membranous β-catenin expression and disease-free or disease-specific survival. CTNNB1 mutations and nuclear β-catenin expression are associated with better progression-free survival in patients with OEC. This relationship may be in part due to a trend of CTNNB1-mutated tumors to present at early stage. β-catenin immunohistochemistry may serve as a prognostic biomarker and a surrogate for CTNN1B mutations in the evaluation of patients with ovarian endometrioid neoplasia, particularly those in reproductive-age or found incidentally without upfront staging surgery.
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http://dx.doi.org/10.1097/PAS.0000000000001553DOI Listing
January 2021

Differentiated exophytic vulvar intraepithelial lesion: Clinicopathologic and molecular analysis documenting its relationship with verrucous carcinoma of the vulva.

Mod Pathol 2020 10 19;33(10):2011-2018. Epub 2020 May 19.

Sunnybrook Health Sciences Centre and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.

Verruciform proliferations of the vulva unrelated to HPV infection are rare. The term differentiated exophytic vulvar intraepithelial lesion (DEVIL) was recently proposed for these lesions, which harbor recurrent PIK3CA mutations. It is still unclear whether DEVIL is related to verrucous carcinoma, a neoplasm characterized by persistence and local recurrence but nil risk of distant spread. Specimens identified using the words "verruciform" and "verrucous" were reviewed. Diagnosis of DEVIL required verruciform acanthosis, hyper and/or parakeratosis, hypogranulosis, cytoplasmic pallor, and bland nuclei. Verrucous carcinoma required, in addition, discontinuous, bulbous, puzzle-like nests in the stroma. A targeted next-generation sequencing using a custom 11-gene panel was performed. Eighteen specimens corresponding to ten patients with DEVIL and/or verrucous carcinoma were included. Median age at presentation was 66 years for DEVIL and 70 years for verrucous carcinoma. A similar spectrum of prevalent mutations was found in both lesions involving HRAS, PIK3CA, and BRAF. DEVIL preceded verrucous carcinoma and/or was diagnosed concurrently or in subsequent follow-up in five patients. In four of these, the same mutation was identified in DEVIL and synchronous or metachronous carcinoma. All cases showed wild-type 53 staining and lacked pathogenic TP53 mutations. DEVIL is a rare form of squamous proliferation characterized by prevalent PIK3CA and HRAS mutations. Its temporal relationship with verrucous carcinoma and their shared mutational profile in some patients suggest that DEVIL is a precursor of verrucous carcinoma. Moreover, given their morphologic and molecular overlap and the nil risk of verrucous carcinoma for distant spread, it is conceivable that DEVIL and verrucous carcinoma represent a spectrum of the same entity.
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http://dx.doi.org/10.1038/s41379-020-0573-5DOI Listing
October 2020

Pilot Study on the Utility of Circulating HER2/Neu Levels in the Serum of Breast Cancer Patients.

Anticancer Res 2019 Oct;39(10):5345-5352

Department of Laboratory Medicine and Molecular Diagnostics, Division of Anatomic Pathology, Sunnybrook Health Sciences Centre, Toronto, ON, Canada

Background/aim: Accurate and timely assessment of the human epidermal growth factor receptor 2 (HER2/neu) overexpression is pivotal for the identification of breast cancer (BC) patients that could benefit from HER2-targeted therapy. Currently approved tissue-based HER2 assays (tHER2) are limited to testing HER2 status on tumor samples obtained at a few points in time during the course of the disease. Herein, we assessed serum HER2 (sHER2) status longitudinally in 81 serial samples prospectively collected from 43 consenting patients pre- and post-therapy to revisit the idea of serum testing in the follow-up of BC patients.

Patients And Methods: The cohort included 11 patients with early BC (EBC), 17 with locally advanced BC (LABC), and 15 with metastatic BC (MBC). sHER2 concentrations were measured using a quantitative ELISA-based technique, using 15 ng/ml as the cut-off for positivity.

Results: At baseline, sHER2 was negative in all EBC patients while positive in 1 LABC and 5 MBC patients. Sixteen BC patients (10 LABC, 1 EBC, and 5 MBC) were tHER2 positive. sHER2 and tHER2 results were discordant in 14 patients. Among the 16 tHER2 positive patients, 9 LABC, 1 EBC and 2 MBC patients were sHER2 negative. Conversely, 2 MBC patients were sHER2 positive, despite being tHER2 negative. A rise or drop of sHER2 by >20% correlated with disease progression or pathological response to therapy, respectively.

Conclusion: The study demonstrated the technical validity and feasibility of the sHER2 assay. Findings suggest that post initial tissue diagnosis (tHER2), sHER2 assay may supplement subsequent tissue tests to monitor disease status and response to therapy. Further studies to assess the role of HER2 targeted therapies in sHER-positive/tHER2-negative cases upon disease progression are warranted.
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http://dx.doi.org/10.21873/anticanres.13727DOI Listing
October 2019

MicroRNA-139 is a predictor of prostate cancer recurrence and inhibits growth and migration of prostate cancer cells through cell cycle arrest and targeting IGF1R and AXL.

Prostate 2019 09 3;79(12):1422-1438. Epub 2019 Jul 3.

Platform Biological Sciences, Sunnybrook Health Sciences Centre, Sunnybrook Research Institute, University of Toronto, Toronto, Ontario, Canada.

Background: We previously identified a panel of five microRNAs (miRNAs) associated with biochemical recurrence and metastasis following prostatectomy from prostate cancer patients using next-generation sequencing-based whole miRNome sequencing and quantitative polymerase chain reaction-based validation analysis. In this study, we examined the mechanism of action of miR-139-5p, one of the downregulated miRNAs identified in the panel.

Methods: Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-139 (dichotomized around the median) using the Kaplan-Meier method and Cox proportional hazard models. We validated these results using The Cancer Genome Atlas (TCGA) data. We created cell lines that overexpressed miR-139 to confirm its targets as well as examine pathways through which miR-139 may function using cell-based assays.

Results: Low miR-139 expression was significantly associated with a variety of prognostic factors in prostate cancer, including Gleason score, pathologic stage, margin positivity, and lymph node status. MiR-139 expression was associated with prognosis: the cumulative incidence of biochemical recurrence and metastasis were significantly lower among patients with high miR-139 expression (P = .0004 and .038, respectively). Validation in the TCGA data set showed a significant association between dichotomized miR-139 expression and biochemical recurrence (odds ratio, 0.52; 95% confidence interval, 0.33-0.82). Overexpression of miR-139 in prostate cancer cells led to a significant reduction in cell proliferation and migration compared with control cells, with cells arrested in G2 of cell cycle. IGF1R and AXL were identified as potential targets of miR-139 based on multiple miRNA-binding sites in 3'-untranslated regions of both the genes and their association with prostate cancer growth pathways. Luciferase assays verified AXL and IGF1R as direct targets of miR-139. Furthermore, immunoblotting of prostate cancer cells demonstrated IGF1R and AXL protein expression were inhibited by miR-139 treatment, which was reversed by the addition of miR-139 antagomir. Examination of the molecular mechanism of growth inhibition by miR-139 revealed the downregulation of activated AKT and cyclin D1, with upregulation of the CDK inhibitor p21.

Conclusions: miR-139 is associated with improved prognosis in patients with localized prostate cancer, which may be mediated through downregulation of IGF1R and/or AXL and associated signaling pathway components.
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http://dx.doi.org/10.1002/pros.23871DOI Listing
September 2019

Identification of a Novel MicroRNA Panel Associated with Metastasis Following Radical Prostatectomy for Prostate Cancer.

Anticancer Res 2018 Sep;38(9):5027-5034

Sunnybrook Research Institute Genomics Facility, Toronto, Canada

Background/aim: This is a case control study designed to identify one or more novel microRNA sequences associated with metastasis following radical prostatectomy for clinically localized prostate cancer.

Materials And Methods: Samples were obtained from patients with clinical evidence of metastatic disease following surgery (cases) and patients who showed no evidence of metastasis or biochemical recurrence at least 5 years following surgery (controls) as identified from a single-center, institutional database. Cases and controls were matched for tumor grade and duration of follow-up.

Results: Whole miRNome analysis identified 2,792 expressed miRNAs in 19 patient pairs. The 497 miRNA sequences with reads per million over 10, were used for analysis, bootstrapping with backward selection identified a panel of 5-miRNA (miR-17-3p, miR-27a-3p, miR-200a-3p, miR-375, and miR-376b-3p) with a risk score strongly associated with metastasis (AUC=89.5%, 95%CI=79.5-99.5%). Methodologically, most studies use the magnitude of differential expression with or without clinical judgement for selection of predictors for inclusion in panels. In order to strengthen the predictive model, a selection strategy was employed, bootstrapping with automated backwards selection, which relied on the strength of association for inclusion.

Conclusion: A genome-wide analysis of microRNA expression identified a panel of 5 miRNAs strongly associated with prostate cancer metastasis following radical prostatectomy.
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http://dx.doi.org/10.21873/anticanres.12821DOI Listing
September 2018

MicroRNA-652 induces NED in LNCaP and EMT in PC3 prostate cancer cells.

Oncotarget 2018 Apr 10;9(27):19159-19176. Epub 2018 Apr 10.

Platform Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada.

MicroRNAs (miRNAs) are small noncoding RNA molecules that post-transcriptionally regulate gene expression. Dysregulation of miRNAs is frequently associated with disease and, in particular, is involved in prostate cancer progression. Next generation miRNA sequencing identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. High expression of one of these five miRNAs, miR-652, correlated significantly with an increased rate of prostate cancer biochemical recurrence. Overexpression of miR-652 in prostate cancer cells, PC3 and LNCaP, resulted in increased growth, migration and invasion. Prostate cancer cell xenografts overexpressing miR-652 showed increased tumorigenicity and metastases. We found that miR-652 directly targets the B" regulatory subunit, PPP2R3A, of the tumor suppressor PP2A, inducing epithelial-mesenchymal transition (EMT) in PC3 cells and neuroendocrine-like differentiation (NED) in LNCaP cells. The mesenchymal marker N-cadherin increased and epithelial marker E-cadherin decreased in PC3 cells overexpressing miR-652. In LNCaP cells and xenografted tumors, overexpression of miR-652 increased markers of NED, including chromogranin A, neuron specific enolase, and synaptophysin. MiR-652 may contribute to prostate tumor progression by promoting NED through decreased PP2A function. MiR-652 expression could serve as a biomarker for aggressive prostate cancer, as well as provide an opportunity for novel therapy in prostate cancer.
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http://dx.doi.org/10.18632/oncotarget.24937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922385PMC
April 2018

Direct Reprogramming of Spiral Ganglion Non-neuronal Cells into Neurons: Toward Ameliorating Sensorineural Hearing Loss by Gene Therapy.

Front Cell Dev Biol 2018 14;6:16. Epub 2018 Feb 14.

Biological Sciences, Sunnybrook Research Institute, Toronto, ON, Canada.

Primary auditory neurons (PANs) play a critical role in hearing by transmitting sound information from the inner ear to the brain. Their progressive degeneration is associated with excessive noise, disease and aging. The loss of PANs leads to permanent hearing impairment since they are incapable of regenerating. Spiral ganglion non-neuronal cells (SGNNCs), comprised mainly of glia, are resident within the modiolus and continue to survive after PAN loss. These attributes make SGNNCs an excellent target for replacing damaged PANs through cellular reprogramming. We used the neurogenic pioneer transcription factor Ascl1 and the auditory neuron differentiation factor NeuroD1 to reprogram SGNNCs into induced neurons (iNs). The overexpression of both and generated iNs at high efficiency. Transcriptome analyses revealed that iNs displayed a transcriptome profile resembling that of endogenous PANs, including expression of several key markers of neuronal identity: Tubb3, Map2, Prph, Snap25, and Prox1. Pathway analyses indicated that essential pathways in neuronal growth and maturation were activated in cells upon neuronal induction. Furthermore, iNs extended projections toward cochlear hair cells and cochlear nucleus neurons when cultured with each respective tissue. Taken together, our study demonstrates that PAN-like neurons can be generated from endogenous SGNNCs. This work suggests that gene therapy can be a viable strategy to treat sensorineural hearing loss caused by degeneration of PANs.
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http://dx.doi.org/10.3389/fcell.2018.00016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817057PMC
February 2018

High-grade Müllerian Adenosarcoma: Genomic and Clinicopathologic Characterization of a Distinct Neoplasm With Prevalent TP53 Pathway Alterations and Aggressive Behavior.

Am J Surg Pathol 2017 Nov;41(11):1513-1522

Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, University of Toronto, Toronto, ON, Canada.

Müllerian adenosarcoma harbors low malignant potential, except in cases with myometrial invasion or sarcomatous overgrowth. The presence of a high-grade stromal component has been proposed as an important pathologic predictor of outcome. We hypothesized that high-grade adenosarcoma has distinct clinical and molecular features, distinct from low-grade adenosarcoma. We analyzed the clinicopathologic features and follow-up of 9 high-grade adenosarcomas and a control group of 9 low-grade adenosarcomas. Comprehensive genomic analysis of the high-grade group was performed targeting exons of 409 oncogenes and tumor suppressor genes. In 1 case, the high-grade and low-grade components were separately sequenced. High-grade and low-grade adenosarcomas were comparable in patient age, myometrial invasion, and stage at presentation. Sarcomatous overgrowth was observed in 2/9 (22%) low-grade and 8/9 (89%) high-grade adenosarcomas. Six of 9 (67%) patients with high-grade adenosarcoma developed rapid recurrence; 1 died of her disease. Conversely, no low-grade tumors recurred or metastasized. Sequencing of high-grade adenosarcomas revealed frequent TP53 pathway alterations, identified in 7/9 (78%) cases. p53 expression by immunohistochemistry highly correlated with mutation status. Copy number variations occurred at a mean of 28.8 per tumor; most frequently involved genes included CDK4, MDM2, GNAS, SGK1, and DICER1. High-grade adenosarcoma is an aggressive neoplasm with propensity for short-interval recurrence and metastasis. The proportion of copy number alterations is similar to that reported for adenosarcoma with sarcomatous overgrowth. However, the high frequency of TP53 abnormalities is a novel finding, indicating that high-grade adenosarcoma is a distinct subset with driver TP53 pathway alterations. p53 immunohistochemistry can be used to confirm the presence of a high-grade component. Given its aggressive potential, the presence of any high-grade component in an adenosarcoma should be reported, even in the absence of sarcomatous overgrowth.
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http://dx.doi.org/10.1097/PAS.0000000000000907DOI Listing
November 2017

Genomic abnormalities in invasive endocervical adenocarcinoma correlate with pattern of invasion: biologic and clinical implications.

Mod Pathol 2017 11 21;30(11):1633-1641. Epub 2017 Jul 21.

Department of Pathology, Sunnybrook Health Sciences Centre, Toronto, ON, Canada.

The pattern-based classification system for HPV-related endocervical adenocarcinoma, which classifies tumors based on the destructiveness of stromal invasion, is predictive of the risk of nodal metastases and adverse outcome. Previous studies have demonstrated clinically important molecular alterations in endocervical adenocarcinoma, including KRAS and PIK3CA mutations; however, correlation between the molecular landscape and pathological variables including pattern of invasion has not been thoroughly explored. In this study, 20 endocervical adenocarcinomas were classified using the pattern-based classification system and were subjected to targeted sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 (ThermoFisher Scientific, Waltham, MA, USA) that surveys hotspot regions of 50 oncogenes and tumor suppressor genes. Single-nucleotide polymorphisms were correlated with clinical and pathologic variables including pattern of invasion. Five (25%), six (30%), and nine (45%) cases were classified as patterns A, B, and C respectively. Lymph node metastases, advanced stage at presentation and mortality from disease were exclusively seen in destructively invasive tumors (patterns B or C). Prevalent mutations in the cohort involved PIK3CA (30%), KRAS (30%), MET (15%), and RB1 (10%). Most (94%) relevant genomic alterations were present in destructively invasive tumors with PIK3CA, KRAS, and RB1 mutations seen exclusively in pattern B or C subgroups. KRAS mutations correlated with advanced stage at presentation (FIGO stage II or higher). Our findings indicate that the pattern of stromal invasion correlates with genomic abnormalities detected by next-generation sequencing, suggesting that tumors without destructive growth (pattern A) are biologically distinct from those with destructive invasion (patterns B and C), and that pattern B endocervical adenocarcinoma is more closely related to its pattern C counterpart. The pattern-based classification may be used as a triage tool when considering molecular testing for prognostic or therapeutic purposes.
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http://dx.doi.org/10.1038/modpathol.2017.80DOI Listing
November 2017

MiR-301a regulates E-cadherin expression and is predictive of prostate cancer recurrence.

Prostate 2016 07 15;76(10):869-84. Epub 2016 Mar 15.

Department of Anatomic Pathology, Platform Biological Sciences, Sunnybrook Health Sciences Centre, Sunnybrook Research Institute, University of Toronto, Toronto, Ontario, Canada.

Background: MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression post-transcriptionally. Dysregulation of miRNA has been implicated in the development and progression of prostate cancer. Through next generation miRNA sequencing, we recently identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. Of the five miRNAs, miR-301a had the strongest association with prostate cancer recurrence. Overexpression of miR-301a in prostate cancer cells, PC3, and LNCaP resulted in increased growth both in vitro and in xenografted tumors. We therefore sought to examine its role in prostate carcinogenesis in greater detail.

Methods: We examined the effect of miR-301a expression on biochemical recurrence and metastasis among 585 men treated with radical prostatectomy for prostate cancer. We examined the mechanism of growth deregulation by miR-301a in prostate cancer cells using analysis of the miRome of prostate cancer cell lines, quantitative PCR, and Western blotting.

Results: High levels of miR-301a (above the median) were associated with an increased risk of biochemical recurrence (adjusted hazard ratio [aHR] 1.42, 95% confidence interval (CI) 1.06-1.90, P = 0.002) but not of metastasis (aHR 0.84, 95%CI 0.41-1.70, P = 0.6) after adjustment for known prognostic factors. RNA transcriptome sequencing analysis of miR-301a overexpressing prostate cancer cell lines identified the tumor suppressor p63 as a potential direct miR-301a target. Transcriptome sequencing, qPCR and Western blotting showed that miR-301a induced epithelial-mesenchymal transition (EMT) in prostate cancer cells through a pathway initiated by p63 inhibition. Luciferase assay verified p63 as a direct target of miR-301a. Loss of p63 resulted in miR-205 downregulation, releasing Zeb1 and Zeb2 from inhibition, culminating in Zeb1/Zeb2 suppression of E-cadherin. This pathway of growth alteration mediated by miR-301a upregulation was shown to be valid in prostate cancer cell lines and patient-derived tumors.

Conclusions: These data indicate that miR-301a functions as an oncogene in prostate cancer by directly targeting the p63 tumor suppressor leading to loss of E-cadherin and EMT. Hence, miR-301a may serve as a novel biomarker in prostate cancer as well as a therapeutic target for prostate cancer management. Prostate 76:869-884, 2016. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/pros.23177DOI Listing
July 2016

Identification and Validation of a Five MicroRNA Signature Predictive of Prostate Cancer Recurrence and Metastasis: A Cohort Study.

J Cancer 2015 15;6(11):1160-71. Epub 2015 Sep 15.

2. Department of Anatomic Pathology, Platform Biological Sciences, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, University of Toronto, 2075 Bayview Ave., Toronto, ON, M4N3M5, Canada.

Background: MicroRNA (miRNA) have been shown to be important in regulating gene expression in prostate cancer. We used next generation miRNA sequencing to conduct a whole miRNome analysis to identify miRNAs associated with prostate cancer metastasis.

Methods: We conducted discovery and validation analyses of miRNAs among a total of 546 men who underwent surgery for prostate cancer using the development of metastasis as an endpoint. Genome wide analysis was conducted among the discovery group (n=31) to identify new miRNAs associated with prostate cancer metastasis. Selected miRNAs were then analyzed using qPCR on prostatectomy specimens from an independent cohort (n=515) to determine whether their expression could predict the development of metastasis after surgery. To examine the biology underlying these associations, we created prostate cancer cell lines which overexpressed miR-301a for in vitro and in vivo functional assays.

Results: We identified 33 miRNAs associated with prostate cancer metastasis and selected a panel comprising miRs-301a, 652, 454, 223 and 139 which strongly predicted metastasis (AUC=95.3%, 95%C.I.:84%-99%). Among the validation cohort, the 15-year metastasis-free survival was 77.5% (95% C.I.:63.9%-86.4%) for patients with a high miRNA panel score and 98.8% (95% C.I.:94.9%-99.7%, p<0.0001 for difference) for those with a low score. After adjusting for grade, stage, and PSA, the hazard ratio for metastasis was 4.3 (95% C.I.: 1.7-11.1, p=0.002) for patients with a high miRNA panel score, compared to those with a low score. Prostate cancer cell lines overexpressing miR-301a had in significantly higher tumor growth and metastasis in a xenograft mouse model.

Conclusions: A panel of miRNAs is associated with prostate cancer metastasis. These could be used as potential new prognostic factors in the surgical management of prostate cancer.
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http://dx.doi.org/10.7150/jca.13397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615353PMC
October 2015

A Comparative Analysis of Breast and Ovarian Cancer-related Gene Mutations in Canadian and Saudi Arabian Patients with Breast Cancer.

Anticancer Res 2015 May;35(5):2601-10

Genomics Core Facility, Toronto, ON, Canada Sunnybrook Research Institute, Toronto, ON, Canada Department of Anatomic Pathology, Sunnybrook Health Sciences Centre, Toronto, ON, Canada

Previous reports have indicated that patients with breast cancer who are from the Eastern Province of Saudi Arabia have a different gene expression profile from that known for their age-matched North American population. In the present study, breast tumor samples from Canadian and Saudi Arabian patients were screened for known and unknown mutations within BRCA1 and BRCA2 as well as 21 additional genes, including, ATM, BARD1, CDH1, P53, EPCAM, MSH6, and RAD50, which have been implicated in breast and ovarian cancer predisposition. A total of 129 non-synonymous mutations were identified by Ion Torrent amplicon sequencing. Forty-one mutations in 18 genes were unique to the Canadian population and 59 mutations in 20 genes were unique to the Saudi Arabian population. A total of 55/129 unique mutations in 22 genes were not previously reported in the database. Twenty-nine mutations in 16 genes were common to both populations; one of these mutations was not previously reported in the database. The most frequently mutated gene in both populations was the BRCA2 gene, followed by BRCA1 and TP53. Unique to this work is the identification of mutations frequently found in the Saudi Arabian population that are rare in the Canadian population. This work will allow direction of genetic analysis resources toward the clinical needs of each particular population.
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May 2015

Multiplex Ligation-dependent Probe Amplification Can Clarify HER2 Status in Gastric Cancers with "Polysomy 17".

J Cancer 2015 26;6(5):403-8. Epub 2015 Feb 26.

1. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada; ; 4. Department of Pathology, Sunnybrook Health Sciences Centre, Toronto, Canada.

Therapy with trastuzumab confers a survival benefit in HER2 positive advanced gastric and gastroesophageal adenocarcinoma. HER2 status is evaluated by immunohistochemistry (IHC) and in situ hybridization (ISH). An ISH ratio of HER2 to centromere 17 (CEP17) ≥2.0 is considered amplified. This assumes that CEP17 reflects chromosomal copy number. Cases where CEP17 exceeds 3 are classified as polysomic, but it's unknown if they represent true polysomy or centromeric amplification. This has implications on the validity of current ISH criteria. Multiplex ligation-dependent probe amplification (MLPA) allows simultaneous quantification of multiple loci and can distinguish between true polysomy and centromeric amplification. We selected 13 gastric cancers with CEP17 counts ≥3.0 (polyCEP17), and 8 non-polyCEP17 gastric cancer controls. Silver ISH for HER2 and CEP17 were performed and scored by manufacturer guidelines. We also performed an MLPA HER2 assay that evaluates 22 genes on chromosome 17. MLPA identified HER2 amplification in 7 polyCEP17 cases compared to 2 identified by ISH. Overall, 9 of 13 polyCEP17 cases had amplification of the peri-centromeric gene WSB1, compared to 1 of 8 non-polyCEP17 controls (p=0.02). This could account for ISH CEP17 counts ≥3.0. MLPA did not show any cases of complete chromosome 17 duplication and peri-centromeric amplification can explain most cases of ISH polyCEP17. Current ISH criteria may under-diagnose HER2 amplification in polyCEP17 cases due to flawed assumptions about polysomy. MLPA can detect HER2 amplification missed by IHC and ISH, and thus may be an effective ancillary technique in evaluating HER2 status.
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http://dx.doi.org/10.7150/jca.11424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392047PMC
April 2015

Molecular cloning and functional characterisation of chicken Atonal homologue 1: a comparison with human Atoh1.

Biol Cell 2015 Feb 13;107(2):41-60. Epub 2015 Jan 13.

Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON, M4N 3M5, Canada.

Background Information: The vertebrate basic helix-loop-helix transcription factor Atoh1 is essential for maturation and survival of mechanosensory hair cells of the inner ear, neurogenesis, differentiation of the intestine, homeostasis of the colon and is implicated in cancer progression. Given that mutations in Atoh1 are detected in malignant tumours, study of functionally different Atoh1 alleles and homologues might yield useful avenues for investigation. The predicted sequence of chicken Atoh1 (cAtoh1) has large regions of dissimilarity to that of mammalian Atoh1 homologues. We hypothesise that cAtoh1 might have intrinsic functional differences to mammalian Atoh1.

Results: In this study, we cloned and sequenced the full open reading frame of cAtoh1. In overexpression experiments, we show that this sequence is sufficient to generate a cAtoh1 protein capable of inducing hair cell markers when expressed in nonsensory regions of the developing inner ear, and that morpholino-mediated knock-down using a section of the sequence 5' to the start codon inhibits differentiation of hair cells in the chicken basilar papilla. Furthermore, we compare the behaviour of cAtoh1 and human Atoh1 (hAtoh1) in embryonic mouse cochlear explants, showing that cAtoh1 is a potent inducer of hair cell differentiation and that it can overcome Sox2-mediated repression of hair cell differentiation more effectively than hAtoh1.

Conclusions: cAtoh1 is both necessary and sufficient for avian mechanosensory hair cell differentiation. The non-conserved regions of the cAtoh1 coding region have functional consequences on its behaviour.
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http://dx.doi.org/10.1111/boc.201400078DOI Listing
February 2015

Loss of Igfbp7 causes precocious involution in lactating mouse mammary gland.

PLoS One 2014 4;9(2):e87858. Epub 2014 Feb 4.

Biological Sciences Platform, Sunnybrook Research Institute and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

Background: Insulin like growth factors (IGFs) and their binding proteins (IGFBPs) are secreted peptides that play major roles in regulating the normal development and maturation of mammary gland. While Igfbp7 has been shown to decrease breast tumor growth, its role in regulating the normal mammary gland development has not been studied. To this end, we generated Igfbp7-null mice and examined the development and maturation of mammary glands in the virgin, pregnant and lactating animals.

Results: We report here that loss of Igfbp7 significantly retards mammary gland development in the virgin animals. More significantly, the pregnant Igfpb7-null glands contained fewer alveolar structures and that during lactation these glands exhibit the morphological changes that are associated with involution. The transcriptome profile of the Igfbp7-null glands on the lactation day 3 revealed a distinct involution-related gene signature compared to the lactating WT glands. Interestingly, we found that the lactating Igfbp7-null glands exhibit increased expression of Stat3 and enhanced activation of (phosphorylated) Stat3, combined with decreased expression of Stat5 suggesting that the absence of Igfbp7 accelerates the onset of involution. We also found that in absence of Igfpb7, the lactating glands contain increased Igfbp5 protein along with decreased expression of IGF-1 Receptor and Akt activation. Finally, we show that during the normal course of involution, Igfbp7 expression is significantly decreased in the mammary gland.

Conclusion: Our data suggest that loss of Igfbp7 induces precocious involution possibly through diminished cell survival signals. Our findings identify Igfbp7 as major regulator of involution in the mammary gland.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087858PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3913705PMC
December 2014

Whole-transcriptome analysis reveals established and novel associations with TMPRSS2:ERG fusion in prostate cancer.

Anticancer Res 2012 Sep;32(9):3629-41

Department of Laboratory Medicine and Pathobiology, University of Toronto, ON, Canada.

Background/aim: Shortcomings of current methods of prostate cancer detection call for improved biomarkers. The transmembrane protease, serine 2:ets-related gene (TMPRSS2:ERG) gene fusion leads to the overexpression of ERG, an E-twenty six (ETS) family transcription factor, and is the most prevalent genetic lesion in prostate cancer, but its clinical utility remains unclear.

Materials And Methods: Two radical prostatectomy samples were analysed by next-generation whole-transcriptome sequencing. The chosen samples differed in fusion gene status, as previously determined by reverse transcription polymerase chain reaction (RT-PCR).

Results: Next-generation sequencing identified the involvement of novel and previously reported prostate cancer-related transcripts, the WNT signalling pathway, evasion of p53-mediated anti-proliferation and several ETS-regulated pathways in the prostate cancer cases examined. Overexpression of Rho GDP-dissociation inhibitor (RhoGDIB), a gene associated with fusion-positive prostate cancer, was found to elicit spindle-shaped morphology, faster cell migration and increased cell proliferation, phenotypic changes suggestive of cancer progression.

Conclusion: The present findings confirm the value of comprehensive sequencing for biomarker development and provide potential avenues of future study.
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September 2012

Effects of partner proteins on BCA2 RING ligase activity.

BMC Cancer 2012 Feb 8;12:63. Epub 2012 Feb 8.

Sunnybrook Research Institute, Toronto, ON, Canada.

Background: BCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2.

Methods: Here, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays.

Results: Ten unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways.

Conclusions: The interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression.
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http://dx.doi.org/10.1186/1471-2407-12-63DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3298473PMC
February 2012

IGFBP7 reduces breast tumor growth by induction of senescence and apoptosis pathways.

Breast Cancer Res Treat 2012 Jun 14;133(2):563-73. Epub 2011 Oct 14.

Division of Molecular and Cellular Biology, Sunnybrook Research Institute, Toronto, ON, Canada.

Insulin-like growth factor binding protein 7 (IGFBP7) has been shown to be a tumor suppressor in a variety of cancers. We previously have shown that IGFBP7 expression is inversely correlated with disease progression and poor outcome in breast cancer. Overexpression of IGFBP7 in MDA-MB-468, a triple-negative breast cancer (TNBC) cell line, resulted in inhibition of growth and migration. Xenografted tumors bearing ectopic IGFBP7 expression were significantly growth-impaired compared to IGFBP7-negative controls, which suggested that IGFBP7 treatment could inhibit breast cancer cell growth. To confirm this notion, 14 human patient primary breast tumors were analyzed by qRTPCR for IGFBP7 expression. The TNBC tumors expressed the lowest levels of IGFBP7 expression, which also correlated with higher tumorigenicity in mice. Furthermore, when breast cancer cell lines were treated with IGFBP7, only the TNBC cell lines were growth inhibited. Treatment of NOD/SCID mice harboring xenografts of TNBC cells with IGFBP7 systemically every 3-4 days inhibited tumorigenesis, with associated anti-angiogenic effects, together with increased apoptosis. Upon examining the mechanism of IGFBP7-mediated growth inhibition in TNBC cells, we found that cells not only were arrested in G1 phase of the cell cycle but also underwent senescence as a result of treatment with IGFBP7. Interestingly, IGFBP7 treatment was also associated with strong activation of the stress-associated p38 MAPK pathway, together with upregulation of p53 and the cyclin-dependent protein kinase (CDK) inhibitor, p21(cip1). Prolonged treatment of cells with IGFBP7 resulted in increased cell death, marked by an increase in apoptotic cells and associated cleaved PARP. This is the first study showing that exogenous IGFBP7 inhibits TNBC cell growth both in vitro and in vivo. Taken together, these results suggest IGFBP7 treatment might have therapeutic potential for TNBC.
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http://dx.doi.org/10.1007/s10549-011-1816-4DOI Listing
June 2012

Role of the BCA2 ubiquitin E3 ligase in hormone responsive breast cancer.

Open Cancer J 2010 ;3(1):116-123

Barbara Ann Karmanos Cancer Institute and Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA.

The BCA2 protein contains a RING H2 finger and a Zn finger near the N-terminus and has E3 ligase activity. RING finger proteins play critical roles in mediating the transfer of ubiquitin and ubiquitin like modifiers to heterologous substrates as well as to the RING finger proteins themselves. Protein modification by ubiquitin and small ubiquitin-related modifier (SUMO) plays a pivotal role in protein homeostasis and is critical to regulating basic cellular processes such as proliferation, differentiation, apoptosis, intracellular signaling, and gene-transcriptional regulation. The addition of ubiquitin or SUMO can modulate the ability of proteins to interact with their partners, alter their patterns of sub-cellular localization and control their stability. It is clear that SUMO influences many different biological processes however recent data suggest that it is specifically important in the regulation of transcription. BCA2 is an E3 ligase that interacts with the SUMO conjugating enzyme Ubc9. It could therefore function as an E3 in the sumoylation of various transcription factors. We have found that the BCA2 is co-expressed with the estrogen receptor in 74% of ER-positive invasive ductal carcinomas from a 635 member breast cancer cohort (p = 0.004). At the cellular level, BCA2 co-localizes with ER and it appears that at the transcriptional level BCA2 mRNA expression is regulated by estrogen. Bioinformatic analysis of the BCA2 promoter region revealed ER and PR binding sites as well as that of other more general transcription factors. The data presented here provides an overview of the potential involvement of the BCA2 in hormone responsive breast cancer and opens up avenues that should be exploited to better understand the regulation of ER expression, growth of breast cancer cells, and the importance of BCA2.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004234PMC
http://dx.doi.org/10.2174/1874079001003010116DOI Listing
January 2010

A human bone NOD/SCID mouse model to distinguish metastatic potential in primary breast cancers.

Cancer Biol Ther 2009 Jun 6;8(11):1010-7. Epub 2009 Jun 6.

Division of Molecular and Cellular Biology, Sunnybrook Research Institute, University of Toronto, ON, CA.

In this study, we created a clinically relevant model using NOD/SCID mice engrafted with human bone fragments in both right and left flanks, and show that the human bone implants are viable and functional for more than 6 mo. To investigate the growth and metastatic behavior of breast cancer, human primary breast tumor specimens were transplanted into human bone grafts under only the right flanks of the human-bone NOD/SCID mice. Some of the engrafted tumors proliferated extensively with massive neo-vascularization and also metastasized to the initially tumor-free left flank bone grafts. We show for the first time that this mouse model can be used to distinguish between primary breast tumors that do or do not proliferate and metastasize to contralateral human bone implants that were initially tumor-free.
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http://dx.doi.org/10.4161/cbt.8.11.8343DOI Listing
June 2009

Autoubiquitination of BCA2 RING E3 ligase regulates its own stability and affects cell migration.

Mol Cancer Res 2008 Sep;6(9):1385-96

Division of Molecular and Cellular Biology, Sunnybrook Research Institute, Toronto, Ontario, Canada M4N 3M5.

Accumulating evidence suggests that ubiquitination plays a role in cancer by changing the function of key cellular proteins. Previously, we isolated BCA2 gene from a library enriched for breast tumor mRNAs. The BCA2 protein is a RING-type E3 ubiquitin ligase and is overexpressed in human breast tumors. In order to deduce the biochemical and biological function of BCA2, we searched for BCA2-binding partners using human breast and fetal brain cDNA libraries and BacterioMatch two-hybrid system. We identified 62 interacting partners, the majority of which were found to encode ubiquitin precursor proteins including ubiquitin C and ubiquitin A-52. Using several deletion and point mutants, we found that the BCA2 zinc finger (BZF) domain at the NH(2) terminus specifically binds ubiquitin and ubiquitinated proteins. The autoubiquitination activity of BCA2, RING-H2 mutant, BZF mutant, and various lysine mutants of BCA2 were investigated. Our results indicate that the BCA2 protein is strongly ubiquitinated and no ubiquitination is detected with the BCA2 RING-H2 mutant, indicating that the RING domain is essential for autoubiquitination. Mutation of the K26 and K32 lysines in the BZF domain also abrogated autoubiquitination activity. Interestingly, mutation of the K232 and K260 lysines in and near the RING domain resulted in an increase in autoubiquitination activity. Additionally, in cellular migration assays, BCA2 mutants showed altered cell motility compared with wild-type BCA2. On the basis of these findings, we propose that BCA2 might be an important factor regulating breast cancer cell migration/metastasis. We put forward a novel model for BCA2 E3 ligase-mediated cell regulation.
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http://dx.doi.org/10.1158/1541-7786.MCR-08-0094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2814348PMC
September 2008

Novel RING E3 ubiquitin ligases in breast cancer.

Neoplasia 2006 Aug;8(8):689-95

Sunnybrook Research Institute and Department of Anatomic Pathology, Sunnybrook Health Sciences Center, Toronto, Ontario, Canada.

Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases, including cancer, because of their central role in the control of diverse signaling pathways. RING E3 ligases promote the ubiquitination of proteins that are essential to a variety of cellular events. Identification of which ubiquitin ligases specifically affect distinct cellular processes is essential to the development of targeted therapeutics for these diseases. Here we discuss two novel RING E3 ligases, BCA2 and RNF11, that are closely linked to human breast cancer. BCA2 E3 ligase is coregulated with estrogen receptor and plays a role in the regulation of epidermal growth factor receptor (EGF-R) trafficking. RNF11 is a small RING E3 ligase that affects transforming growth factorbeta and EGF-R signaling and is overexpressed in invasive breast cancers. These two proteins demonstrate the complexity of RING E3 ligase interactions in breast cancer and are potential targets for therapeutic interventions.
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http://dx.doi.org/10.1593/neo.06469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1601945PMC
August 2006

Cloning of stanniocalcin (STC) cDNAs of divergent teleost species: Monomeric STC supports monophyly of the ancient teleosts, the osteoglossomorphs.

Gen Comp Endocrinol 2006 Oct 8;149(1):100-7. Epub 2006 May 8.

Department of Zoology (Scarborough), University of Toronto, Toronto, Ont., Canada M1C 1A4.

Molecular cloning of teleost stanniocalcin (STC) cDNAs was undertaken in two species of order Osteoglossiformes of subdivision Osteoglossomorpha and one species of each of orders Cypriniformes and Perciformes within the subdivision Euteleostei. The elephantnose (Gnathonemus petersii) and the butterflyfish (Pantadon buchholzi) are basal teleosts in different osteoglossiforme suborders yet their 218 amino acid (aa) mature hormones, from prehormones of 249 and 251aa, respectively, have only 10 cysteine residues. A substitution for cysteine at the intermonomeric disulfide linkage site, implies that their STCs exist as monomeric peptides, as is the case with STC from another osteoglossormorph, arawana [Amemiya, Y., Marra, L.E., Reyhani, N., Youson, J.H., 2002. Stanniocalcin from an ancient teleost: a monomeric form of the hormone and a possible extracorpuscular distribution. Mol. Cell. Endocrinol. 188, 141-150]. The STC cDNA of the generalized teleost and cyprinid, the white sucker (Catostomus commersoni), encodes a prehormone of 249aa with a signal peptide of 31aa and a mature protein of 218aa that possesses 11 cysteine residues. The latter feature is consistent with a previous analysis that white sucker mature STC is a glycosylated, homodimeric peptide [Amemiya, Y., Marra, L.E., Reyhani, N., Youson, J.H., 2002. Stanniocalcin from an ancient teleost: a monomeric form of the hormone and a possible extracorpuscular distribution. Mol. Cell. Endocrinol. 188, 141-150]. An open reading frame of the STC cDNA of the derived teleost and perciforme, the smallmouth bass (Micropterus dolomieui), encodes a prehormone of 255aa with a signal peptide of 33aa and a mature protein of 222aa. The position of the 11 cysteines in smallmouth bass STC suggests that it exists as a homodimeric peptide. A phylogenetic analysis, using the new STC-1 amino acid sequences and those in the gene data base provided strong support for monophyly of the Osteoglossomorpha and indicated, with positioning of white sucker and smallmouth bass, that this molecule has some utility as a taxonomic marker. This analysis also suggested that two STC-1 gene sequences exist in multiple fish genomes, and that they may be a product of the fish-specific genome duplication. The mutation in the osteoglossomorph STC likely occurred after the appearance of the first teleosts and before movement of the tectonic plates.
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http://dx.doi.org/10.1016/j.ygcen.2006.03.015DOI Listing
October 2006

A novel RING-type ubiquitin ligase breast cancer-associated gene 2 correlates with outcome in invasive breast cancer.

Cancer Res 2005 Nov;65(22):10401-12

Laboratories of Molecular Pathology, Department of Anatomic Pathology, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ontario, Canada.

The RING finger family of proteins possess ubiquitin ligase activity and play pivotal roles in protein degradation and receptor-mediated endocytosis. In this study, we examined whether the breast cancer-associated gene 2 (BCA2), a novel RING domain protein, has E3 ubiquitin ligase activity and investigated its expression status in breast tumors. The full-length BCA2 gene was cloned from the human breast cancer cell line MDA-MB-468. It encodes an open reading frame of 304 amino acids and contains a RING-H2 domain. BCA2 maps to chromosome 1q21.1, a region known to harbor cytogenetic aberrations in breast cancers. We found that the BCA2 protein has an intrinsic autoubiquitination activity, the hallmark of E3 ligases, whereas mutant RING protein is not autoubiquitinated. This indicates that the BCA2 ubiquitin ligase activity is dependent on the RING-H2 domain. Using tissue microarrays and immunohistochemistry, we found strong to intermediate BCA2 staining in 56% of 945 invasive breast cancers cases, which was significantly correlated with positive estrogen receptor status [odds ratio (OR), 1.51; P = 0.004], negative lymph node status (OR, 0.73; P = 0.02), and an increase in disease-free survival for regional recurrence (OR, 0.45; P = 0.03). Overexpression of BCA2 increased proliferation and small interfering RNA inhibited growth of T47D human breast cancer cells and NIH3T3 mouse cells. The autoubiquitination activity of BCA2 indicates that it is a novel RING-type E3 ligase. Its association with clinical measures and its effects on cell growth indicate that BCA2 may be important for the ubiquitin modification of proteins crucial to breast carcinogenesis and growth.
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http://dx.doi.org/10.1158/0008-5472.CAN-05-2103DOI Listing
November 2005

Nucleotide sequence and expression of three subtypes of proopiomelanocortin mRNA in barfin flounder.

Gen Comp Endocrinol 2005 May;141(3):291-303

School of Fisheries Sciences, Kitasato University, Sanriku, Ofunato, Iwate 022-0101, Japan.

Melanophore-stimulating hormone (MSH) has been shown to be associated with food intake in addition to body color change in teleosts. MSH is encoded by a proopiomelanocortin (POMC) gene together with endorphin (END). To assess the significance of MSH to biological activities, we determined the structure and evaluated the expression of POMC mRNA in barfin flounder (bf), Verasper moseri, a member of a group of teleosts, Pleuronectiformes. Three subtypes of POMC cDNAs (A, B, and C) were amplified from bf pituitary glands. These bfPOMCs contained segments for N-POMC, alpha-MSH, beta-MSH, and beta-END as do other teleost POMCs, while POMC-C showed remarkable variations in the segments corresponding to N-POMC and beta-END. A phylogenetic tree of ray-finned fish POMCs constructed by the neighbor joining method revealed that the three POMC subtypes may have appeared as a result of duplication events occurring at least twice during the course of bf evolution. The first duplication may have generated the lineage leading to an ancestor of bfPOMC-A and -B and that leading to bfPOMC-C, and then the lineage of bfPOMC-A may have diverged from that of bfPOMC-B. All peptides flanked by processing signals excluding N-POMC-C (1-14) were identified in a single pituitary extract by mass spectrometry, and the cDNAs of three POMCs were amplified from a single pituitary by reverse transcription polymerase chain reaction. These results demonstrated that the three POMC genes are expressed in a single individual. While the bfPOMC-A gene was exclusively expressed in the pituitary, the bfPOMC-B and -C genes were expressed in non-pituitary tissues such as brain, gill, heart, spleen, liver, stomach, intestine, testis, muscle, blood, and skin in addition to the pituitary. The expression levels of the POMC-A, -B, and -C genes in pituitary neurointermediate lobe were greater in the fish reared with a black background than the fish reared with a white background, indicating that MSH derived from all of the three bfPOMC genes was associated with body color change. No difference was observed in the expression levels of bfPOMC-C in the brain in response to feeding status.
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http://dx.doi.org/10.1016/j.ygcen.2005.01.010DOI Listing
May 2005

Primary structure of stanniocalcin in two basal Actinopterygii.

Gen Comp Endocrinol 2004 Jan;135(2):250-7

Department of Zoology, University of Toronto at Scarborough, Toronto, ON, Canada M1C 1A4.

The primary structure of stanniocalcin (STC), the principal product of the corpuscles of Stannius (CS) in ray-finned fishes, was deduced from STC cDNA clones for two species of holostean, the gar, Lepisosteus osseus and the bowfin, Amia calva. Overlapping partial cDNA clones were amplified by polymerase chain reaction (PCR) from single-strand cDNA of the CS. Excluding the poly(A) tail, the cDNAs of 1863 base pairs [bp] (gar) and 914 bp (bowfin) contained the 5' untranslated region followed by the coding region and the 3' untranslated region. Both the gar and bowfin STC cDNA encode a prehormone of 252 amino acids (aa) with a signal peptide of 32 aa and a mature protein of 220 aa. The deduced aa sequence of gar STC shows 87% identity with bowfin STC, 60-72% identity with most vertebrate STCs and 26% identity with mouse STC2. Phylogenetic analysis of the sequences support a view that the gar and bowfin form a monophyletic holostean clade. RT-PCR revealed in the gar and bowfin that, just as in mammals and rainbow trout, the expression of STC mRNA is widely spread in many tissues and organs. Since the gar and bowfin are representatives of the most ancient fishes known to possess CS, the corpuscular-derived STC molecule in fish has had a conserved evolution.
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http://dx.doi.org/10.1016/j.ygcen.2003.09.019DOI Listing
January 2004
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