Publications by authors named "Fares Al-Ejeh"

51 Publications

G9a Inhibition Enhances Checkpoint Inhibitor Blockade Response in Melanoma.

Clin Cancer Res 2021 05 15;27(9):2624-2635. Epub 2021 Feb 15.

QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.

Purpose: G9a histone methyltransferase exerts oncogenic effects in several tumor types and its inhibition promotes anticancer effects. However, the impact on checkpoint inhibitor blockade response and the utility of G9a or its target genes as a biomarker is poorly studied. We aimed to examine whether G9a inhibition can augment the efficacy of checkpoint inhibitor blockade and whether , a G9a target gene, can predict treatment response.

Experimental Design: Clinical potential of LC3B as a biomarker of checkpoint inhibitor blockade was assessed using patient samples including tumor biopsies and circulating tumor cells from liquid biopsies. Efficacy of G9a inhibition to enhance checkpoint inhibitor blockade was examined using a mouse model.

Results: Patients with melanoma who responded to checkpoint inhibitor blockade were associated with not only a higher level of tumor LC3B but also a higher proportion of cells expressing LC3B. A higher expression of or LC3B protein was associated with longer survival and lower incidence of acquired resistance to checkpoint inhibitor blockade, suggesting LC3B as a potential predictive biomarker. We demonstrate that G9a histone methyltransferase inhibition is able to not only robustly induce LC3B level to augment the efficacy of checkpoint inhibitor blockade, but also induces melanoma cell death.

Conclusions: Checkpoint inhibitor blockade response is limited to a subset of the patient population. These results have implications for the development of LC3B as a predictive biomarker of checkpoint inhibitor blockade to guide patient selection, as well as G9a inhibition as a strategy to extend the proportion of patients responding to immunotherapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3463DOI Listing
May 2021

Differential gene expression of tumor-infiltrating CD4 T cells in advanced versus early stage colorectal cancer and identification of a gene signature of poor prognosis.

Oncoimmunology 2020 09 30;9(1):1825178. Epub 2020 Sep 30.

Cancer Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), Doha, Qatar.

Tumor-infiltrating lymphocytes (TILs) play indispensable roles in the progression and response to treatment of solid tumors. However, the prognostic significance of CD4 TILs is not fully disclosed in cancers generally and in CRC in particular, mainly due to the existence of different functional subsets of CD4 T cells. We performed transcriptomic profiling of CD4 TILs isolated from CRC patients in order to identify differentially expressed genes and their functional pathways in early versus advanced disease stages. We found that in advanced stages, genes related to immune and inflammatory responses, in particular Th1-mediated immune response and cytotoxicity-mediated genes, were downregulated; while epigenetic-mediated silencing genes were upregulated. Interestingly, we identified genes, which were steadily upregulated or downregulated in CD4 TILs with CRC progression from stage I to IV. Additionally, of the top 200 deregulated genes, 43 upregulated and 64 downregulated genes showed similar deregulation trends in the cancer genome atlas CRC dataset. From these 97 deregulated genes, we identified a "poor prognosis CD4 gene signature (ppCD4sig)". Patients with high ppCD4sig score showed shorter disease-specific survival (DSS) and progression-free interval (PFI). The ppCD4sig was an independent prognostic indicator for DSS (HR = 1.73, 95% CI 1.32-2.27, = 0.0001) and PFI (HR = 1.75, 95% CI 1.3-2.35, = 0.0016). Additionally, patients at advanced stages and at a younger age (<55 years) were more likely to have a high ppCD4sig score. Altogether, our data provide novel insights and a unique prognostic gene signature of CD4 TILs in the CRC microenvironment.
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http://dx.doi.org/10.1080/2162402X.2020.1825178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553563PMC
September 2020

Clinicopathologic significance of nuclear HER4 and phospho-YAP(S) in human breast cancers and matching brain metastases.

Ther Adv Med Oncol 2020 31;12:1758835920946259. Epub 2020 Jul 31.

The University of Queensland Faculty of Medicine, UQ Centre for Clinical Research, Herston, Qld, Australia.

Background: Human epidermal growth factor receptor-4 (HER4) and yes-associated protein-1 (YAP) are candidate therapeutic targets in oncology. YAP's transcriptional coactivation function is modulated by the HER4 intracellular domain (HER4-ICD) , but the clinical relevance of this has not been established. This study investigated the potential for targeting the HER4-YAP pathway in brain metastatic breast cancer.

Methods: We performed immuno-phenotypic profiling of pathway markers in a consecutive breast cancer series with 25 years of clinical follow up ( = 371), and patient-matched breast and metastatic brain tumours ( = 91; 30 pairs).

Results: Membrane localisation of phospho-HER4 [pHER4(Y)] was infrequent in primary breast cancer, but very frequent in brain metastases (5.9% 75% positive), where it was usually co-expressed with pHER3(Y) ( < 0.05). The presence of YAP in tumour cell nuclei was associated directly with nuclear pERK5(T/Y) ( = 0.003). However, relationships with disease-specific survival depended on oestrogen receptor (ER) status. Nuclear pYAP(S) was associated with smaller, good prognostic ER+ breast tumours (log-rank hazard-ratio 0.53;  = 9.6E), but larger, poor prognostic triple-negative cancers (log-rank hazard-ratio 2.78;  = 1.7E), particularly when co-expressed with nuclear HER4-ICD ( = 0.02). This phenotype was associated with stemness and mitotic instability markers (vimentin, SOX9, ID1, SPAG5, TTK, geminin;  < 0.05). YAP expression in brain metastases was higher than matched primary tumours; specifically, nuclear pYAP(S) in ER-negative cases ( < 0.05). Nuclear YAP was detected in ~70% of ER-negative, HER4-activated brain metastases.

Discussion: Our findings suggest that the canonical-mechanism where Hippo pathway-mediated phosphorylation of YAP ostensibly excludes it from the nucleus is dysfunctional in breast cancer. The data are consistent with pYAP(S) having independent transcriptional functions, which may include transducing neuregulin signals in brain metastases. Consistent with mechanistic studies implicating it as an ER co-factor, nuclear pYAP(S) associations with breast cancer clinical outcomes were dependent on ER status.

Conclusion: Preclinical studies investigating HER4 and nuclear YAP combination therapy strategies are warranted.
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http://dx.doi.org/10.1177/1758835920946259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7517995PMC
July 2020

Differential gene expression of tumor-infiltrating CD8 T cells in advanced versus early-stage colorectal cancer and identification of a gene signature of poor prognosis.

J Immunother Cancer 2020 09;8(2)

Cancer Research Center, Qatar Biomedical Research Institute (QBRI), Hamad Bin Khalifa University (HBKU), Qatar Foundation (QF), Doha, Qatar

Background: Cytotoxic CD8 T cell-mediated response is the most important arm of adaptive immunity, which dictates the capacity of the host immune response in eradicating tumor cells. Due to tumor intrinsic and/or extrinsic factors, the density and function of CD8 tumor-infiltrating lymphocytes (TILs) could be compromised, leading to poor prognosis and survival.

Methods: Using RNA-Seq, transcriptomes of sorted CD3CD8 TILs from treatment-naïve colorectal cancer (CRC) patients at advanced stages (III and IV) were compared with those from patients with early stages (I and II). A signature referred to as 'poor prognosis CD8 gene signature (ppCD8sig)' was identified and analyzed in The Cancer Genome Atlas CRC dataset. Scores for the ppCD8sig were calculated and classified as high, intermediate and low, and its prognostic significance was assessed using multivariate analysis and Cox proportional hazard model. Densities of CD3 and CD8 T cell infiltration in tumors from patients with high and low ppCD8sig scores were assessed by flow cytometry and immunostaining.

Results: Genes related to epigenetic regulation and response to hypoxia were upregulated in CD8 TILs from patients with advanced stages, while genes related to T cell activation, cell proliferation and cell cycle were downregulated. Patients with high ppCD8sig score had poorer disease-specific survival (DSS) and shorter progression-free interval (PFI). The ppCD8sig was an independent prognostic indicator for DSS (HR 1.83, 95% CI 1.40 to 2.38, p<0.0001) and PFI (HR 1.42, 95% CI 1.04 to 1.93, p=0.026). Additionally, patients with high ppCD8sig score were more likely to have advanced stages (χ p<0.0001) and residual disease after primary therapy (χ p=0.046). Patients with high ppCD8sig score had reduced levels of CD3 and CD8 TILs and low Immunoscores (IS), compared to patients with low ppCD8sig score.

Conclusions: Our data provided insights into the altered regulation of biological mechanisms and signaling pathways in CD8 TILs during CRC progression, and revealed a gene signature as an independent prognostic indicator. Patients with high ppCD8sig score had lower levels of TILs and low IS. These data further confirm the prognostic value of the identified ppCD8sig and potentially highlight its clinical relevance.
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http://dx.doi.org/10.1136/jitc-2020-001294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511623PMC
September 2020

G9a-mediated repression of CDH10 in hypoxia enhances breast tumour cell motility and associates with poor survival outcome.

Theranostics 2020 15;10(10):4515-4529. Epub 2020 Mar 15.

QIMR Berghofer Medical Research Institute, Herston Rd, Herston, QLD 4006, Australia.

: Epigenetic mechanisms are fundamental processes that can modulate gene expression, allowing cellular adaptation to environmental conditions. Hypoxia is an important factor known to initiate the metastatic cascade in cancer, activating cell motility and invasion by silencing cell adhesion genes. G9a is a histone methyltransferase previously shown to accumulate in hypoxic conditions. While its oncogenic activity has been previously reported, not much is known about the role G9a plays in the hypoxia-mediated metastatic cascade. : The role of G9a in cell motility in hypoxic condition was determined by inhibiting G9a either by short-hairpin mediated knock down or pharmacologically using a small molecule inhibitor. Through gene expression profiling, we identified CDH10 to be an important G9a target that regulates breast cancer cell motility. Lung metastasis assay in mice was used to determine the physiological significance of G9a. : We demonstrate that, while hypoxia enhances breast cancer migratory capacity, blocking G9a severely reduces cellular motility under both normoxic and hypoxic conditions and prevents the hypoxia-mediated induction of cellular movement. Moreover, inhibition of G9a histone methyltransferase activity in mice using a specific small molecule inhibitor significantly reduced growth and colonisation of breast cancer cells in the lung. We identify the type-II cadherin as being a novel hypoxia-dependent gene, directly repressed by G9a through histone methylation. CDH10 overexpression significantly reduces cellular movements in breast cancer cell lines and prevents the hypoxia-mediated increase in cell motility. In addition, we show that expression is prognostic in breast cancer and that it is inversely correlated to (G9a) transcript levels in many tumor-types, including breast cancer. : We propose that G9a promotes cellular motility during hypoxic stress through the silencing of the cell adhesion molecule CDH10 and we describe as a novel prognostic biomarker for breast cancer.
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http://dx.doi.org/10.7150/thno.41453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150496PMC
April 2021

Overexpression of miRNA-25-3p inhibits Notch1 signaling and TGF-β-induced collagen expression in hepatic stellate cells.

Sci Rep 2019 06 12;9(1):8541. Epub 2019 Jun 12.

Hepatic Fibrosis Group, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

During chronic liver injury hepatic stellate cells (HSCs), the principal source of extracellular matrix in the fibrotic liver, transdifferentiate into pro-fibrotic myofibroblast-like cells - a process potentially regulated by microRNAs (miRNAs). Recently, we found serum miRNA-25-3p (miR-25) levels were upregulated in children with Cystic Fibrosis (CF) without liver disease, compared to children with CF-associated liver disease and healthy individuals. Here we examine the role of miR-25 in HSC biology. MiR-25 was detected in the human HSC cell line LX-2 and in primary murine HSCs, and increased with culture-induced activation. Transient overexpression of miR-25 inhibited TGF-β and its type 1 receptor (TGFBR1) mRNA expression, TGF-β-induced Smad2 phosphorylation and subsequent collagen1α1 induction in LX-2 cells. Pull-down experiments with biotinylated miR-25 revealed Notch signaling (co-)activators ADAM-17 and FKBP14 as miR-25 targets in HSCs. NanoString analysis confirmed miR-25 regulation of Notch- and Wnt-signaling pathways. Expression of Notch signaling pathway components and endogenous Notch1 signaling was downregulated in miR-25 overexpressing LX-2 cells, as were components of Wnt signaling such as Wnt5a. We propose that miR-25 acts as a negative feedback anti-fibrotic control during HSC activation by reducing the reactivity of HSCs to TGF-β-induced collagen expression and modulating the cross-talk between Notch, Wnt and TGF-β signaling.
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http://dx.doi.org/10.1038/s41598-019-44865-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561916PMC
June 2019

Proteomic Analysis of the Breast Cancer Brain Metastasis Microenvironment.

Int J Mol Sci 2019 May 22;20(10). Epub 2019 May 22.

Faculty of Medicine, the University of Queensland, Centre for Clinical Research, Herston 4029, QLD, Australia.

Patients with brain-metastatic breast cancer face a bleak prognosis marked by morbidity and premature death. A deeper understanding of molecular interactions in the metastatic brain tumour microenvironment may inform the development of new therapeutic strategies. In this study, triple-negative MDA-MB-231 breast cancer cells or PBS (modelling traumatic brain injury) were stereotactically injected into the cerebral cortex of NOD/SCID mice to model metastatic colonization. Brain cells were isolated from five tumour-associated samples and five controls (pooled uninvolved and injured tissue) by immunoaffinity chromatography, and proteomic profiles were compared using the Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) discovery platform. Ontology and cell type biomarker enrichment analysis of the 125 differentially abundant proteins ( < 0.05) showed the changes largely represent cellular components involved in metabolic reprogramming and cell migration (min = 4.59 × 10), with high-throughput PubMed text mining indicating they have been most frequently studied in the contexts of mitochondrial dysfunction, oxidative stress and autophagy. Analysis of mouse brain cell type-specific biomarkers suggested the changes were paralleled by increased proportions of microglia, mural cells and interneurons. Finally, we orthogonally validated three of the proteins in an independent xenograft cohort, and investigated their expression in craniotomy specimens from triple-negative metastatic breast cancer patients, using a combination of standard and fluorescent multiplex immunohistochemistry. This included 3-Hydroxyisobutyryl-CoA Hydrolase (HIBCH), which is integral for gluconeogenic valine catabolism in the brain, and was strongly induced in both graft-associated brain tissue (13.5-fold by SWATH-MS; = 7.2 × 10), and areas of tumour-associated, reactive gliosis in human clinical samples. HIBCH was also induced in the tumour compartment, with expression frequently localized to margins and haemorrhagic areas. These observations raise the possibility that catabolism of valine is an effective adaptation in metastatic cells able to access it, and that intermediates or products could be transferred from tumour-associated glia. Overall, our findings indicate that metabolic reprogramming dominates the proteomic landscape of graft-associated brain tissue in the intracranial MDA-MB-231 xenograft model. Brain-derived metabolic provisions could represent an exploitable dependency in breast cancer brain metastases.
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http://dx.doi.org/10.3390/ijms20102524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6567270PMC
May 2019

Genome-wide association and transcriptome studies identify target genes and risk loci for breast cancer.

Nat Commun 2019 04 15;10(1):1741. Epub 2019 Apr 15.

Molecular Oncology Laboratory, CIBERONC, Hospital Clinico San Carlos, IdISSC (Instituto de Investigación Sanitaria del Hospital Clínico San Carlos), 28040, Madrid, Spain.

Genome-wide association studies (GWAS) have identified more than 170 breast cancer susceptibility loci. Here we hypothesize that some risk-associated variants might act in non-breast tissues, specifically adipose tissue and immune cells from blood and spleen. Using expression quantitative trait loci (eQTL) reported in these tissues, we identify 26 previously unreported, likely target genes of overall breast cancer risk variants, and 17 for estrogen receptor (ER)-negative breast cancer, several with a known immune function. We determine the directional effect of gene expression on disease risk measured based on single and multiple eQTL. In addition, using a gene-based test of association that considers eQTL from multiple tissues, we identify seven (and four) regions with variants associated with overall (and ER-negative) breast cancer risk, which were not reported in previous GWAS. Further investigation of the function of the implicated genes in breast and immune cells may provide insights into the etiology of breast cancer.
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http://dx.doi.org/10.1038/s41467-018-08053-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6465407PMC
April 2019

Secreted cellular prion protein binds doxorubicin and correlates with anthracycline resistance in breast cancer.

JCI Insight 2019 02 26;5. Epub 2019 Feb 26.

Tumor Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.

Anthracyclines are amongst the most effective chemotherapeutics ever developed, but they produce grueling side-effects, serious adverse events and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular Prion protein (PrPC), blocking their cytotoxic activity. This effect was dose-dependent using either cell line-conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pre-treatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly amongst doxorubicin-treated patients with residual disease at surgery (p=2.8E-08). These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by immunohistochemistry, particularly soluble isoforms released into the extracellular environment by shedding (p=0.015). These findings have important potential clinical implications for frontline regimen decision-making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemo-sensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.
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http://dx.doi.org/10.1172/jci.insight.124092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6483007PMC
February 2019

EphA3 Pay-Loaded Antibody Therapeutics for the Treatment of Glioblastoma.

Cancers (Basel) 2018 Dec 17;10(12). Epub 2018 Dec 17.

Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Brisbane 4006, Australia.

The EphA3 receptor has recently emerged as a functional tumour-specific therapeutic target in glioblastoma (GBM). EphA3 is significantly elevated in recurrent disease, is most highly expressed on glioma stem cells (GSCs), and has a functional role in maintaining self-renewal and tumourigenesis. An unlabelled EphA3-targeting therapeutic antibody is currently under clinical assessment in recurrent GBM patients. In this study, we assessed the efficacy of EphA3 antibody drug conjugate (ADC) and radioimmunotherapy (RIT) approaches using orthotopic animal xenograft models. Brain uptake studies, using positron emission tomography/computed tomography (PET/CT) imaging, show EphA3 antibodies are effectively delivered across the blood-tumour barrier and accumulate at the tumour site with no observed normal brain reactivity. A robust anti-tumour response, with no toxicity, was observed using EphA3, ADC, and RIT approaches, leading to a significant increase in overall survival. Our current research provides evidence that GBM patients may benefit from pay-loaded EphA3 antibody therapies.
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http://dx.doi.org/10.3390/cancers10120519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6316644PMC
December 2018

Breast cancer metastasis to gynaecological organs: a clinico-pathological and molecular profiling study.

J Pathol Clin Res 2019 01 22;5(1):25-39. Epub 2018 Oct 22.

Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Brisbane, Australia.

Breast cancer metastasis to gynaecological organs is an understudied pattern of tumour spread. We explored clinico-pathological and molecular features of these metastases to better understand whether this pattern of dissemination is organotropic or a consequence of wider metastatic dissemination. Primary and metastatic tumours from 54 breast cancer patients with gynaecological metastases were analysed using immunohistochemistry, DNA copy-number profiling, and targeted sequencing of 386 cancer-related genes. The median age of primary tumour diagnosis amongst patients with gynaecological metastases was significantly younger compared to a general breast cancer population (46.5 versus 60 years; p < 0.0001). Median age at metastatic diagnosis was 54.4, time to progression was 4.8 years (range 0-20 years), and survival following a diagnosis of metastasis was 1.95 years (range 0-18 years). Patients had an average of five involved sites (most frequently ovary, fallopian tube, omentum/peritoneum), with fewer instances of spread to the lungs, liver, or brain. Invasive lobular histology and luminal A-like phenotype were over-represented in this group (42.8 and 87.5%, respectively) and most patients had involved axillary lymph nodes (p < 0.001). Primary tumours frequently co-expressed oestrogen receptor cofactors (GATA3, FOXA1) and harboured amplifications at 8p12, 8q24, and 11q13. In terms of phenotype conversion, oestrogen receptor status was generally maintained in metastases, FOXA1 increased, and expression of progesterone receptor, androgen receptor, and GATA3 decreased. ESR1 and novel AR mutations were identified. Metastasis to gynaecological organs is a complication frequently affecting young women with invasive lobular carcinoma and luminal A-like breast cancer, and hence may be driven by sustained hormonal signalling. Molecular analyses reveal a spectrum of factors that could contribute to de novo or acquired resistance to therapy and disease progression.
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http://dx.doi.org/10.1002/cjp2.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317061PMC
January 2019

A transcriptome-wide association study of 229,000 women identifies new candidate susceptibility genes for breast cancer.

Nat Genet 2018 07 18;50(7):968-978. Epub 2018 Jun 18.

Department of Oncology, Helsinki University Hospital, University of Helsinki, Helsinki, Finland.

The breast cancer risk variants identified in genome-wide association studies explain only a small fraction of the familial relative risk, and the genes responsible for these associations remain largely unknown. To identify novel risk loci and likely causal genes, we performed a transcriptome-wide association study evaluating associations of genetically predicted gene expression with breast cancer risk in 122,977 cases and 105,974 controls of European ancestry. We used data from the Genotype-Tissue Expression Project to establish genetic models to predict gene expression in breast tissue and evaluated model performance using data from The Cancer Genome Atlas. Of the 8,597 genes evaluated, significant associations were identified for 48 at a Bonferroni-corrected threshold of P < 5.82 × 10, including 14 genes at loci not yet reported for breast cancer. We silenced 13 genes and showed an effect for 11 on cell proliferation and/or colony-forming efficiency. Our study provides new insights into breast cancer genetics and biology.
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http://dx.doi.org/10.1038/s41588-018-0132-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314198PMC
July 2018

Characterization of a novel breast cancer cell line derived from a metastatic bone lesion of a breast cancer patient.

Breast Cancer Res Treat 2018 Jul 21;170(1):179-188. Epub 2018 Feb 21.

QIMR Berghofer Medical Research Institute, Royal Brisbane Hospital, Locked Bag 2000, Brisbane, QLD, 4029, Australia.

Purpose: We aimed to generate and characterize a novel cell line from a breast cancer bone metastasis to better study the progression of the disease.

Methods: The cell line, P7731, was derived from a metastatic bone lesion of a breast cancer patient and assessed for marker expression. P7731 was analyzed for DNA copy number variation, somatic mutations, and gene expression and was compared with the primary tumor.

Results: P7731 cells are negative for estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 (triple-negative); strongly express vimentin (100% of cells positive) and also express cytokeratins 8/18 and 19 but at lower frequencies. Flow cytometry indicates P7731 cells are predominantly CD44/CD49f/EpCAM, consistent with a primitive, mesenchymal-like phenotype. The cell line is tumorigenic in immunocompromised mice. Exome sequencing identified a total of 45 and 76 somatic mutations in the primary tumor and cell line, respectively, of which 32 were identified in both samples and included mutations in known driver genes PIK3CA, TP53, and ARID1A. P7731 retains the DNA copy number alterations present in the matching primary tumor. Homozygous deletions detected in the cell line and in the primary tumor were found in regions containing three known (CDKN2A, CDKN2B, and CDKN1B) and 23 putative tumor suppressor genes. Cell line-specific gene amplification coupled with mRNA expression analysis revealed genes and pathways with potential pro-metastatic functions.

Conclusion: This novel human breast cancer-bone metastasis cell line will be a useful model to study aspects of breast cancer biology, particularly metastasis-related changes from breast to bone.
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http://dx.doi.org/10.1007/s10549-018-4719-9DOI Listing
July 2018

Identification of ten variants associated with risk of estrogen-receptor-negative breast cancer.

Nat Genet 2017 Dec 23;49(12):1767-1778. Epub 2017 Oct 23.

Department of Epidemiology, University of California, Irvine, Irvine, California, USA.

Most common breast cancer susceptibility variants have been identified through genome-wide association studies (GWAS) of predominantly estrogen receptor (ER)-positive disease. We conducted a GWAS using 21,468 ER-negative cases and 100,594 controls combined with 18,908 BRCA1 mutation carriers (9,414 with breast cancer), all of European origin. We identified independent associations at P < 5 × 10 with ten variants at nine new loci. At P < 0.05, we replicated associations with 10 of 11 variants previously reported in ER-negative disease or BRCA1 mutation carrier GWAS and observed consistent associations with ER-negative disease for 105 susceptibility variants identified by other studies. These 125 variants explain approximately 16% of the familial risk of this breast cancer subtype. There was high genetic correlation (0.72) between risk of ER-negative breast cancer and breast cancer risk for BRCA1 mutation carriers. These findings may lead to improved risk prediction and inform further fine-mapping and functional work to better understand the biological basis of ER-negative breast cancer.
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http://dx.doi.org/10.1038/ng.3785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808456PMC
December 2017

Multidimensional phenotyping of breast cancer cell lines to guide preclinical research.

Breast Cancer Res Treat 2018 01 9;167(1):289-301. Epub 2017 Sep 9.

Faculty of Medicine, The University of Queensland, Herston, QLD, Australia.

Purpose: Cell lines are extremely useful tools in breast cancer research. Their key benefits include a high degree of control over experimental variables and reproducibility. However, the advantages must be balanced against the limitations of modelling such a complex disease in vitro. Informed selection of cell line(s) for a given experiment now requires essential knowledge about molecular and phenotypic context in the culture dish.

Methods: We performed multidimensional profiling of 36 widely used breast cancer cell lines that were cultured under standardised conditions. Flow cytometry and digital immunohistochemistry were used to compare the expression of 14 classical breast cancer biomarkers related to intrinsic molecular profiles and differentiation states: EpCAM, CD24, CD49f, CD44, ER, AR, HER2, EGFR, E-cadherin, p53, vimentin, and cytokeratins 5, 8/18 and 19.

Results: This cell-by-cell analysis revealed striking heterogeneity within cultures of individual lines that would be otherwise obscured by analysing cell homogenates, particularly amongst the triple-negative lines. High levels of p53 protein, but not RNA, were associated with somatic mutations (p = 0.008). We also identified new subgroups using the nanoString PanCancer Pathways panel (730 transcripts representing 13 canonical cancer pathways). Unsupervised clustering identified five groups: luminal/HER2, immortalised ('normal'), claudin-low and two basal clusters, distinguished mostly by baseline expression of TGF-beta and PI3-kinase pathway genes.

Conclusion: These features are compared with other published genotype and phenotype information in a user-friendly reference table to help guide selection of the most appropriate models for in vitro and in vivo studies, and as a framework for classifying new patient-derived cancer cell lines and xenografts.
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http://dx.doi.org/10.1007/s10549-017-4496-xDOI Listing
January 2018

MITF and BRN2 contribute to metastatic growth after dissemination of melanoma.

Sci Rep 2017 09 7;7(1):10909. Epub 2017 Sep 7.

Cancer Drug Mechanisms Group, Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Herston, Queensland, 4006, Australia.

Melanoma tumors are highly heterogeneous, comprising of different cell types that vary in their potential for growth and invasion. Heterogeneous expression of the Microphthalmia-associated Transcription Factor (MITF) and the POU domain transcription factor BRN2 (POU3F2) has been found in malignant melanoma. Changing expression of these transcription factors as the disease progresses has been linked to the metastatic mechanism of phenotype switching. We therefore investigated the effects of MITF and BRN2 expression in melanoma growth and metastasis. Depletion of MITF resulted in a cell population that had a slowed cell cycle progression, was less invasive in vitro and had hindered tumor and metastasis forming ability in mouse xenograft studies. BRN2 depletion left a cell population with intact proliferation and invasion in vitro; however metastatic growth was significantly reduced in the mouse xenograft model. These results suggest that the proliferative population within melanoma tumors express MITF, and both MITF and BRN2 are important for metastatic growth in vivo. This finding highlights the importance of BRN2 and MITF expression in development of melanoma metastasis.
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http://dx.doi.org/10.1038/s41598-017-11366-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589904PMC
September 2017

Long Noncoding RNAs CUPID1 and CUPID2 Mediate Breast Cancer Risk at 11q13 by Modulating the Response to DNA Damage.

Am J Hum Genet 2017 Aug;101(2):255-266

Cancer Division, QIMR Berghofer Medical Research Institute, Brisbane, QLD 4006, Australia. Electronic address:

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.
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http://dx.doi.org/10.1016/j.ajhg.2017.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544418PMC
August 2017

G9a drives hypoxia-mediated gene repression for breast cancer cell survival and tumorigenesis.

Proc Natl Acad Sci U S A 2017 07 19;114(27):7077-7082. Epub 2017 Jun 19.

QIMR Berghofer Medical Research Institute, Herston, QLD 4006, Australia;

G9a is an epigenetic regulator that methylates H3K9, generally causing repression of gene expression, and participates in diverse cellular functions. G9a is genetically deregulated in a variety of tumor types and can silence tumor suppressor genes and, therefore, is important for carcinogenesis. Although hypoxia is recognized to be an adverse factor in tumor growth and metastasis, the role of G9a in regulating gene expression in hypoxia has not been described extensively. Here, we show that G9a protein stability is increased in hypoxia via reduced proline hydroxylation and, hence, inefficient degradation by the proteasome. This inefficiency leads to an increase in H3K9me2 at its target promoters. Blocking the methyltransferase activity of G9a inhibited cellular proliferation and migration in vitro and tumor growth in vivo. Furthermore, an increased level of G9a is a crucial factor in mediating the hypoxic response by down-regulating the expression of specific genes, including , , , , , and This down-regulation can be rescued by a small molecule inhibitor of G9a. Based on the hypothesis that the changes in gene expression would influence patient outcomes, we have developed a prognostic G9a-suppressed gene signature that can stratify breast cancer patients. Together, our findings provide an insight into the role G9a plays as an epigenetic mediator of hypoxic response, which can be used as a diagnostic marker, and proposes G9a as a therapeutic target for solid cancers.
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http://dx.doi.org/10.1073/pnas.1618706114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502591PMC
July 2017

Quinazolinone derivatives as inhibitors of homologous recombinase RAD51.

Bioorg Med Chem Lett 2017 07 15;27(14):3096-3100. Epub 2017 May 15.

Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia; Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. Electronic address:

RAD51 is a vital component of the homologous recombination DNA repair pathway and is overexpressed in drug-resistant cancers, including aggressive triple negative breast cancer (TNBC). A proposed strategy for improving therapeutic outcomes for patients is through small molecule inhibition of RAD51, thereby sensitizing tumor cells to DNA damaging irradiation and/or chemotherapy. Here we report structure-activity relationships for a library of quinazolinone derivatives. A novel RAD51 inhibitor (17) displays up to 15-fold enhanced inhibition of cell growth in a panel of TNBC cell lines compared to compound B02, and approximately 2-fold increased inhibition of irradiation-induced RAD51 foci formation. Additionally, compound 17 significantly inhibits TNBC cell sensitivity to DNA damage, implying a potentially targeted therapy for cancer treatment.
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http://dx.doi.org/10.1016/j.bmcl.2017.05.039DOI Listing
July 2017

Ssb1 and Ssb2 cooperate to regulate mouse hematopoietic stem and progenitor cells by resolving replicative stress.

Blood 2017 05 7;129(18):2479-2492. Epub 2017 Mar 7.

QIMR Berghofer Medical Research Institute, Herston, QLD, Australia.

Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both and were constitutively or conditionally deleted. Constitutive double knockout (DKO) caused early embryonic lethality, whereas conditional double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of or Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of -loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.
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http://dx.doi.org/10.1182/blood-2016-06-725093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418634PMC
May 2017

'Omics Approaches in Breast Cancer Research and Clinical Practice.

Adv Anat Pathol 2016 Nov;23(6):356-367

*UQ Centre for Clinical Research, The Royal Brisbane & Women's Hospital §School of Medicine, The University of Queensland †QIMR Berghofer Medical Research Institute ‡Pathology Queensland, Royal Brisbane Women's Hospital, Herston, Qld, Australia.

Our understanding of the natural history of breast cancer has evolved alongside technologies to study its genomic, transcriptomic, proteomic, and metabolomics landscapes. These technologies have helped decipher multiple molecular pathways dysregulated in breast cancer. First-generation 'omics analyses considered each of these dimensions individually, but it is becoming increasingly clear that more holistic, integrative approaches are required to fully understand complex biological systems. The 'omics represent an exciting era of discovery in breast cancer research, although important issues need to be addressed to realize the clinical utility of these data through precision cancer care. How can the data be applied to predict response to molecular-targeted therapies? When should treatment decisions be based on tumor genetics rather than histology? And with the sudden explosion of "big data" from large 'omics consortia and new precision clinical trials, how do we now negotiate evidence-based pathways to clinical translation through this apparent sea of opportunity? The aim of this review is to provide a broad overview of 'omics technologies used in breast cancer research today, the current state-of-play in terms of applying this new knowledge in the clinic, and the practical and ethical issues that will be central to the public discussion on the future of precision cancer care.
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http://dx.doi.org/10.1097/PAP.0000000000000128DOI Listing
November 2016

RAD51 inhibition in triple negative breast cancer cells is challenged by compensatory survival signaling and requires rational combination therapy.

Oncotarget 2016 09;7(37):60087-60100

Tumor Microenvironment Laboratory, QIMR Berghofer, Herston Rd, Herston QLD 4006, Australia.

The molecular rationale to induce synthetic lethality, by targeting defective homologous recombination repair in triple negative breast cancer (TNBC), has proven to have several shortcomings. Not meeting the expected minimal outcomes in clinical trials has highlighted common clinical resistance mechanisms including; increased expression of the target gene PARP1, increased expression or reversion mutation of BRCA1, or up-regulation of the compensatory homologous recombination protein RAD51. Indeed, RAD51 has been demonstrated to be an alternative synthetic lethal target in BRCA1-mutated cancers. To overcome selective pressure on DNA repair pathways, we examined new potential targets within TNBC that demonstrate synthetic lethality in association with RAD51 depletion. We confirmed complementary targets of PARP1/2 and DNA-PK as well as a new synthetic lethality combination with p38. p38 is considered a relevant target in breast cancer, as it has been implicated in resistance to chemotherapy, including tamoxifen. We show that the combination of targeting RAD51 and p38 inhibits cell proliferation both in vitro and in vivo, which was further enhanced by targeting of PARP1. Analysis of the molecular mechanisms revealed that depletion of RAD51 increased ERK1/2 and p38 signaling. Our results highlight a potential compensatory mechanism via p38 that limits DNA targeted therapy.
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http://dx.doi.org/10.18632/oncotarget.11065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312370PMC
September 2016

Long-range regulators of the lncRNA HOTAIR enhance its prognostic potential in breast cancer.

Hum Mol Genet 2016 08 4;25(15):3269-3283. Epub 2016 Jul 4.

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia

Predicting response to endocrine therapy and survival in oestrogen receptor positive breast cancer is a significant clinical challenge and novel prognostic biomarkers are needed. Long-range regulators of gene expression are emerging as promising biomarkers and therapeutic targets for human diseases, so we have explored the potential of distal enhancer elements of non-coding RNAs in the prognostication of breast cancer survival. HOTAIR is a long non-coding RNA that is overexpressed, promotes metastasis and is predictive of decreased survival. Here, we describe a long-range transcriptional enhancer of the HOTAIR gene that binds several hormone receptors and associated transcription factors, interacts with the HOTAIR promoter and augments transcription. This enhancer is dependent on Forkhead-Box transcription factors and functionally interacts with a novel alternate HOTAIR promoter. HOTAIR expression is negatively regulated by oestrogen, positively regulated by FOXA1 and FOXM1, and is inversely correlated with oestrogen receptor and directly correlated with FOXM1 in breast tumours. The combination of HOTAIR and FOXM1 enables greater discrimination of endocrine therapy responders and non-responders in patients with oestrogen receptor positive breast cancer. Consistent with this, HOTAIR expression is increased in cell-line models of endocrine resistance. Analysis of breast cancer gene expression data indicates that HOTAIR is co-expressed with FOXA1 and FOXM1 in HER2-enriched tumours, and these factors enhance the prognostic power of HOTAIR in aggressive HER2+ breast tumours. Our study elucidates the transcriptional regulation of HOTAIR, identifies HOTAIR and its regulators as novel biomarkers of patient response to endocrine therapy and corroborates the importance of transcriptional enhancers in cancer.
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http://dx.doi.org/10.1093/hmg/ddw177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5179926PMC
August 2016

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.

J Pathol 2015 Nov 19;237(3):363-78. Epub 2015 Aug 19.

QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia.

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2)  = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.
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http://dx.doi.org/10.1002/path.4583DOI Listing
November 2015

EphA2 Is a Therapy Target in EphA2-Positive Leukemias but Is Not Essential for Normal Hematopoiesis or Leukemia.

PLoS One 2015 17;10(6):e0130692. Epub 2015 Jun 17.

QIMR Berghofer Medical Research Institute, Brisbane, Australia; The University of Queensland, Brisbane, Australia.

Members of the Eph family of receptor tyrosine kinases and their membrane bound ephrin ligands have been shown to play critical roles in many developmental processes and more recently have been implicated in both normal and pathological processes in post-embryonic tissues. In particular, expression studies of Eph receptors and limited functional studies have demonstrated a role for the Eph/ephrin system in hematopoiesis and leukemogenesis. In particular, EphA2 was reported on hematopoietic stem cells and stromal cells. There are also reports of EphA2 expression in many different types of malignancies including leukemia, however there is a lack of knowledge in understanding the role of EphA2 in hematopoiesis and leukemogenesis. We explored the role of EphA2 in hematopoiesis by analyzing wild type and EphA2 knockout mice. Mature, differentiated cells, progenitors and hematopoietic stem cells derived from knockout and control mice were analyzed and no significant abnormality was detected. These studies showed that EphA2 does not have an obligatory role in normal hematopoiesis. Comparative studies using EphA2-negative MLL-AF9 leukemias derived from EphA2-knockout animals showed that there was no detectable functional role for EphA2 in the initiation or progression of the leukemic process. However, expression of EphA2 in leukemias initiated by MLL-AF9 suggested that this protein might be a possible therapy target in this type of leukemia. We showed that treatment with EphA2 monoclonal antibody IF7 alone had no effect on tumorigenicity and latency of the MLL-AF9 leukemias, while targeting of EphA2 using EphA2 monoclonal antibody with a radioactive payload significantly impaired the leukemic process. Altogether, these results identify EphA2 as a potential radio-therapeutic target in leukemias with MLL translocation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130692PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4470658PMC
May 2016

Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib.

PLoS One 2015 13;10(5):e0125232. Epub 2015 May 13.

QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

Background: Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown.

Materials And Methods: Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed.

Results: Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer.

Conclusions: Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125232PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430383PMC
February 2016

MEK5-ERK5 pathway associates with poor survival of breast cancer patients after systemic treatments.

Oncoscience 2015 20;2(2):99-101. Epub 2015 Feb 20.

QIMR Berghofer Medical Research Institute, Herston QLD, Australia.

The MEK5-ERK5 pathway is a mammalian mitogen-activated protein (MAP) kinase cascade that is not well studied compared to other MAP kinase cascades. Two independent studies by Al-Ejeh et al. and Ortiz-Ruiz et al. published in Oncotarget last year concluded that ERK5 is an attractive target in triple negative breast cancer. In this perspective, we briefly describe the findings of these studies and propose the use of pharmacological inhibition of ERK5 in combination with chemotherapy against triple negative breast cancer because MEK5-ERK5 overexpression associates with poor survival of patients treated with chemotherapy.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381702PMC
http://dx.doi.org/10.18632/oncoscience.135DOI Listing
April 2015

Eph family co-expression patterns define unique clusters predictive of cancer phenotype.

Growth Factors 2014 Dec 20;32(6):254-64. Epub 2014 Nov 20.

Brain Cancer Research Unit & Leukaemia Foundation Research Unit, QIMR Berghofer Medical Research Institute , Brisbane, Queensland , Australia.

The Eph genes are the largest sub-family of receptor tyrosine kinases; however, it is most likely the least understood and the arena for many conflicting reports. In this tribute to Prof. Martin Lackmann and Prof. Tony Pawson, we utilized The Cancer Genome Atlas resources to shed new light on the understanding of this family. We found that mutation and expression analysis define two clusters of co-expressed Eph family genes that relate to aggressive phenotypes across multiple cancer types. Analysis of signal transduction pathways using reverse-phase protein arrays revealed a network of interactions, which associates cluster-specific Eph genes with epithelial-mesenchymal transition, metabolism, DNA-damage repair and apoptosis. Our findings support the role of the Eph family in modulating cancer progression and reveal distinct patterns of Eph expression, which correlate with disease outcome. These observations provide further rationale for seeking cancer therapies, which target the Eph/ephrin system.
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http://dx.doi.org/10.3109/08977194.2014.984807DOI Listing
December 2014

Gemcitabine and CHK1 inhibition potentiate EGFR-directed radioimmunotherapy against pancreatic ductal adenocarcinoma.

Clin Cancer Res 2014 Jun 16;20(12):3187-97. Epub 2014 May 16.

Authors' Affiliations: Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Herston; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland; The Kinghorn Cancer Centre, Cancer Division, Garvan Institute of Medical Research; St Vincent's Clinical School, Faculty of Medicine, University of NSW; Department of Anatomical Pathology, SYDPATH, St Vincent's Hospital, Darlinghurst, New South Wales; Australian Pancreatic Cancer Genome Initiative, for the full list of contributors see http://www.pancreaticcancer.net.au/apgi/collaborators; Cancer Clinical Trials Unit, Royal Adelaide Hospital Cancer Centre, and Centre for Cancer Biology, SA Pathology; School of Medicine, University of Adelaide, Adelaide, Australia; and Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, Scotland, United KingdomAuthors' Affiliations: Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Herston; Queensland Centre for Medical Genomics, Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland; The Kinghorn Cancer Centre, Cancer Division, Garvan Institute of Medical Research; St Vincent's Clinical School, Faculty of Medicine, University of NSW; Department of Anatomical Pathology, SYDPATH, St Vincent's Hospital, Darlinghurst, New South Wales; Australian Pancreatic Cancer Genome Initiative, for the full list of contributors see http://www.pancreaticcancer.net.au/apgi/collaborators; Cancer Clinical Trials Unit, Royal Adelaide Hospital Cancer Centre, and Centre for Cancer Biology, SA Pathology; School of Medicine, University of Adelaide, Adelaide, Australia; and Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom.

Purpose: To develop effective combination therapy against pancreatic ductal adenocarcinoma (PDAC) with a combination of chemotherapy, CHK1 inhibition, and EGFR-targeted radioimmunotherapy.

Experimental Design: Maximum tolerated doses were determined for the combination of gemcitabine, the CHK1 inhibitor PF-477736, and Lutetium-177 ((177)Lu)-labeled anti-EGFR antibody. This triple combination therapy was investigated using PDAC models from well-established cell lines, recently established patient-derived cell lines, and fresh patient-derived xenografts. Tumors were investigated for the accumulation of (177)Lu-anti-EGFR antibody, survival of tumor-initiating cells, induction of DNA damage, cell death, and tumor tissue degeneration.

Results: The combination of gemcitabine and CHK1 inhibitor PF-477736 with (177)Lu-anti-EGFR antibody was tolerated in mice. This triplet was effective in established tumors and prevented the recurrence of PDAC in four cell line-derived and one patient-derived xenograft model. This exquisite response was associated with the loss of tumor-initiating cells as measured by flow cytometric analysis and secondary implantation of tumors from treated mice into treatment-naïve mice. Extensive DNA damage, apoptosis, and tumor degeneration were detected in the patient-derived xenograft. Mechanistically, we observed CDC25A stabilization as a result of CHK1 inhibition with consequent inhibition of gemcitabine-induced S-phase arrest as well as a decrease in canonical (ERK1/2 phosphorylation) and noncanonical EGFR signaling (RAD51 degradation) as a result of EGFR inhibition.

Conclusions: Our study developed an effective combination therapy against PDAC that has potential in the treatment of PDAC.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-0048DOI Listing
June 2014

SCF-FBXO31 E3 ligase targets DNA replication factor Cdt1 for proteolysis in the G2 phase of cell cycle to prevent re-replication.

J Biol Chem 2014 Jun 14;289(26):18514-25. Epub 2014 May 14.

Signal Transduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4006, Australia

FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor.
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http://dx.doi.org/10.1074/jbc.M114.559930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4140253PMC
June 2014
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