Timothy Childs

EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer

BackgroundThe epithelial to mesenchymal transition (EMT) is a molecular process through which an epithelial cell undergoes transdifferentiation into a mesenchymal phenotype. The role of EMT in embryogenesis is well-characterized and increasing evidence suggests that elements of the transition may be important in other processes, including metastasis and drug resistance in various different cancers.MethodsAgilent 4 × 44 K whole human genome arrays and selected reaction monitoring mass spectrometry were used to investigate mRNA and protein expression in A2780 cisplatin sensitive and resistant cell lines. Invasion and migration were assessed using Boyden chamber assays. Gene knockdown of snail and slug was done using targeted siRNA. Clinical relevance of the EMT pathway was assessed in a cohort of primary ovarian tumours using data from Affymetrix GeneChip Human Genome U133 plus 2.0 arrays.ResultsMorphological and phenotypic hallmarks of EMT were identified in the chemoresistant cells. Subsequent gene expression profiling revealed upregulation of EMT-related transcription factors including snail, slug, twist2 and zeb2. Proteomic analysis demonstrated up regulation of Snail and Slug as well as the mesenchymal marker Vimentin, and down regulation of E-cadherin, an epithelial marker. By reducing expression of snail and slug, the mesenchymal phenotype was largely reversed and cells were resensitized to cisplatin. Finally, gene expression data from primary tumours mirrored the finding that an EMT-like pathway is activated in resistant tumours relative to sensitive tumours, suggesting that the involvement of this transition may not be limited to in vitro drug effects.ConclusionsThis work strongly suggests that genes associated with EMT may play a significant role in cisplatin resistance in ovarian cancer, therefore potentially leading to the development of predictive biomarkers of drug response or novel therapeutic strategies for overcoming drug resistance.

Regression of cardiac hypertrophy in spontaneously hypertensive rats by enalapril and the expression of contractile proteins.

Several experimental models involving the development of cardiac hypertrophy in adult rats are characterized by the reexpression of the fetal isoform of myosin heavy chain (V3). To determine whether a similar adult-to-fetal shift in the expression of the thin-filament proteins occurs during cardiac hypertrophy, we have examined the expression of the isofonns of myosin, tropomyosin, and troponin T in the left ventricle of young spontaneously hypertensive rats (SHR) with and without treatment using enalapril, an angiotensin converting enzyme inhibitor. Phosphorylation of tropomyosin, which is predominant in the fetal state, was also analyzed. Twelve-week-old SHR were treated with enalapril for 2,5,8, and 9 weeks followed by withdrawal of treatment for 9 weeks. Control SHR, without drug treatment, were weight- and age-matched. After 9 weeks of enalapril treatment, mean arterial blood pressure was reduced (from 166±11 to 89±5 mm Hg), and left ventricular weight/body weight ratio was regressed (from 2.53±0.14 to 1.96±0.05 g/kg) to normotensive levels. During the 9-week treatment period, the percent V3 decreased in SHR substantially from 35±3% to 13±1%. There was a significant correlation between the left ventricular hypertrophy and the percent V3 myosin expression in the SHR during regression (r = 0.697, p < 0.001). However, only the adult isofonns of tropomyosin and troponin T were detected in the SHR with or without enalapril treatment, and the level of tropomyosin phosphorylation remained constant irrespective of the degree of left ventricular hypertrophy. These results suggest that the adult-to-fetal switch in the expression program of myosin isofonns that accompanies the development of left ventricle hypertrophy is not adopted by the thin-filament proteins, tropomyosin and troponin T.

Calponin and tropomyosin interactions

The interaction between chicken gizzard calponin and tropomyosin was examined using viscosity, light scattering, electron microscopy and affinity chromatography. At neutral pH, 10 mM NaCl and in the absence of Mg2+, calponin induced tropomyosin filaments to form paracrystals thus decreasing the viscosity while increasing dramatically the light scattering of the tropomyosin solution. Electron micrographs of the uranyl acetate stained calponin-tropomyosin complex showed the presence of spindle shaped paracrystals with regular striation patterns and repeating units of about 400 A. Under similar conditions, smooth muscle caldesmon also induced tropomyosin to form paracrystals. To localize the calponin-binding site on tropomyosin, binding of fragments of tropomyosin, generated by chemical and mutational means, to a calponin-affinity column was studied. The COOH-terminal tropomyosin fragment Cn1B(142-281) and the NH2-terminal fragment CSM-beta(1/8/12-227) bound to a calponin-affinity column with an affinity similar to that of intact tropomyosin; while the NH2-terminal fragment, Cn1A(11-127), did not bind, indicating that the calponin-binding site(s) resides within residues 142-227 of tropomyosin. To determine the involvement in calponin binding of the area around Cys-190 of tropomyosin, fragments with cleavage sites near or at Cys-190 were used. Thus, while fragments Cy2(190-284) and CSM-beta(1/8/12-200) bound weakly to the calponin-affinity column, fragment Cy1(1-189) did not. These results demonstrate that calponin binds to tropomyosin between residues 142 and 227, and that the integrity of the region around Cys-190 of tropomyosin is important for strong interaction between the two proteins.

Identification of the IGF1/PI3K/NF κB/ERK gene signalling networks associated with chemotherapy resistance and treatment response in high-grade serous epithelial ovarian cancer

BackgroundResistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression profiling to delineate major deregulated pathways and biomarkers associated with the development of intrinsic chemotherapy resistance upon exposure to standard first-line therapy for ovarian cancer.MethodsThe study cohort comprised 28 patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. All 28 patients had advanced stage, high-grade serous ovarian cancer, and were treated with standard platinum-based chemotherapy. Twelve patient tumours demonstrating relative resistance to platinum chemotherapy corresponding to shorter PFS (< eight months) were compared to sixteen tumours from platinum-sensitive patients (PFS > eighteen months). Whole transcriptome profiling was performed using an Affymetrix high-resolution microarray platform to permit global comparisons of gene expression profiles between tumours from the resistant group and the sensitive group.ResultsMicroarray data analysis revealed a set of 204 discriminating genes possessing expression levels which could influence differential chemotherapy response between the two groups. Robust statistical testing was then performed which eliminated a dependence on the normalization algorithm employed, producing a restricted list of differentially regulated genes, and which found IGF1 to be the most strongly differentially expressed gene. Pathway analysis, based on the list of 204 genes, revealed enrichment in genes primarily involved in the IGF1/PI3K/NF κB/ERK gene signalling networks.ConclusionsThis study has identified pathway specific prognostic biomarkers possibly underlying a differential chemotherapy response in patients undergoing standard platinum-based treatment of serous epithelial ovarian cancer. In addition, our results provide a pathway context for further experimental validations, and the findings are a significant step towards future therapeutic interventions.

Blocking of stromal cell-derived factor-1 reduces neoangiogenesis in human endometriosis lesions in a mouse model.

Endometriosis affects 5–10% of women and is characterized by the growth of endometrial tissue outside of the uterus. Establishing new blood supply is a fundamental requirement for endometriosis lesion growth. Endothelial progenitor cells (EPCs), recruited by stromal cell–derived factor‐1 (SDF‐1), contribute to neoangiogenesis in endometriotic lesions. We hypothesized that SDF‐1 is central to the neoangiogenesis and survival of endometriotic lesions, and blocking of SDF‐1 will reduce vascularization of lesions in a mouse model.

STAT1‐associated intratumoural TH1 immunity predicts chemotherapy resistance in high‐grade serous ovarian cancer

High‐grade serous ovarian carcinoma (HGSC) accounts for 70% of all epithelial ovarian cancers but clinical management is challenged by a lack of accurate prognostic and predictive biomarkers of chemotherapy response. This study evaluated the role of Signal Transducer and Activator of Transcription 1 (STAT1) as an independent prognostic and predictive biomarker and its correlation with intratumoural CD8+ T cells in a second independent biomarker validation study. Tumour STAT1 expression and intratumoural CD8+ T cell infiltration were assessed by immunohistochemistry as a multicentre validation study conducted on 734 chemotherapy‐naïve HGSCs. NanoString‐based profiling was performed to correlate expression of STAT1 target genes CXCL9, CXCL10 and CXCL11 with CD8A transcript expression in 143 primary tumours. Multiplexed cytokine analysis of pre‐treatment plasma from resistant and sensitive patients was performed to assess systemic levels of STAT1‐induced cytokines. STAT1 was validated as a prognostic and predictive biomarker in both univariate and multivariate models and its expression correlated significantly with intra‐epithelial CD8+ T cell infiltration in HGSC. STAT1 levels increased the prognostic and predictive value of intratumoural CD8+ T cells, confirming their synergistic role as biomarkers in HGSC. In addition, expression of STAT1 target genes (CXCL9, CXCL10 and CXCL11) correlated significantly with levels of, and CD8A transcripts from intratumoural CD8+ T cells within the resistant and sensitive tumours. Our findings provide compelling evidence that high levels of STAT1, STAT1‐induced chemokines and CD8+ T cells correlate with improved chemotherapy response in HGSC. These results identify STAT1 and its target genes as novel biomarkers of chemosensitivity in HGSC. These findings provide new translational opportunities for patient stratification for immunotherapies based on emerging biomarkers of inflammation in HGSC. An improved understanding of the role of interferon‐inducible genes will be foundational for developing immunomodulatory therapies in ovarian cancer.

Mucinous metaplasia of the endometrium: Current concepts

OBJECTIVES The objective of this review was to discuss the highly variable terminology describing mucinous metaplasia of the endometrium, histologic features and characteristic findings by special stains and immunohistochemistry, differential diagnostic challenges, current treatment and prognosis. METHODS A review of the literature was performed on mucinous metaplasia of the endometrium. RESULTS Mucinous metaplasia is an uncommon type of endometrial epithelial metaplasia, often associated with hyperestrogenic states or hormone replacement therapy. Histologic findings range from simple endocervical-type mucinous epithelium to architecturally complex mucinous proliferations. Mucinous metaplasia often presents a significant diagnostic challenge in endometrial biopsies and should be differentiated from endocervical microglandular hyperplasia, and mucinous or mixed mucinous and endometrioid carcinomas. Simple mucinous metaplasia is believed to have a low risk of associated carcinoma, whereas architecturally complex cases often show invasive malignancy on subsequent biopsy. CONCLUSIONS Complex mucinous proliferations should be managed with dilatation and curettage and repeat biopsy within 6months, and strong consideration should be given to a hysterectomy in persistent mucinous proliferations.

Human Papillomavirus-Related Ovarian Metastasis With Endocervical Adenocarcinoma: Report of 2 Cases and Review of Literature.

Background Most endocervical adenocarcinomas are associated with human papillomavirus (HPV) infection. Studies suggest that synchronous endocervical and ovarian tumors can contain identical HPV subtypes and that, in this setting, the ovarian tumors likely represent metastases from the endocervical adenocarcinoma rather than 2 independent primaries. However, there are still relatively few reports in the literature. Results We describe 2 patients with HPV-related endocervical adenocarcinoma or adenocarcinoma in situ who had metastatic ovarian tumors that simulated primary ovarian neoplasms. After total hysterectomy and bilateral salpingo-oophorectomy, patient 1, in her mid-40s, was diagnosed with endocervical adenocarcinoma in situ and patient 2, in her early 50s, was diagnosed with endocervical adenocarcinoma showing early focal stromal invasion. The ovarian tumors in both cases simulated independent borderline mucinous tumors without evidence of surface involvement or spread beyond the ovary. However, in both cases, the cervical and ovarian tumors showed diffuse, strong P16 staining and contained identical high-risk HPV subtypes (subtypes 16 and 18 in patient 1 and subtype 16 in patient 2). Patient 1 was treated with radiation therapy and remains recurrence free 5 years after diagnosis, and patient 2 has recently completed combined modality treatment with radiation therapy and cisplatin chemotherapy and remains recurrence free 10 months after diagnosis. Conclusions A high index of suspicion and ancillary testing, including P16 immunostaining and molecular genetic testing for HPV, is required to properly diagnose and subclassify HPV-related endocervical adenocarcinoma metastatic to the ovary.

Abstract 441: Association of interferon inducible genes with tumor immune microenvironment and chemotherapy resistance in high-grade serous epithelial ovarian cancer

Chemotherapy resistance is a major hurdle in high-grade serous epithelial ovarian cancer (HGSC) management. We previously reported differential expression of interferon inducible genes in pre-treatment tumours from chemoresistant and sensitive HGSC tumors. STAT1 expression was evaluated as a prognostic and predictive biomarker via immunohistochemistry in Phase I (n = 183) and Phase II (n = 550) biomarker validation studies conducted on HGSC tumours accrued from the Terry Fox Research Institute- Canadian Ovarian Experimental Unified Resource (TFRI-COEUR). Tumor STAT1 expression levels significantly correlated with the density of tumor infiltrating CD8+ T lymphocytes in both Phase I and Phase II cohorts. STAT1 expression significantly associated with progression free survival and response to chemotherapy in both Phase I and Phase II validation studies. Significant positive correlation between STAT1 expression levels and intratumoral CD8+ T cell density was observed. Intratumoral CD8+ T cell infiltration did not associate with progression free survival or response to chemotherapy in both cohorts. Interestingly, the prognostic relevance of CD8+ T cell was enhanced in combination with STAT1 in both cohorts. These findings provide evidence that STAT1 as an independent biomarker and a combination of CD8+ T cell infiltration with STAT1 could be novel immune-based prognostic and predictive biomarkers in HGSC. Findings from the current study will aid in patient stratification for novel immunomodulatory therapies for the management of chemotherapy resistance in HGSC. Citation Format: Katrina K. Au, Liliane Meunier, Cecile Le Page, Charles H. Graham, Andrew WB Craig, Timothy Childs, Julie-Ann Francis, Jeremy Squire, Anne-Marie Mes-Masson, Madhuri Koti. Association of interferon inducible genes with tumor immune microenvironment and chemotherapy resistance in high-grade serous epithelial ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 441.

Abstract 810: Integrative genomic and transcriptomic analysis in idenfitication of biomarkers of chemoresistance in serous epithelial ovarian cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Resistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression and copy number profiling to delineate major deregulated pathways and biomarker networks associated with the development of intrinsic chemotherapy resistance with exposure to standard first-line therapy for ovarian cancer. The study cohort comprised 28 high grade serous ovarian cancer patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. Twelve patient tumors demonstrating relative resistance to platinum based chemotherapy corresponding to shorter PFS (less than 6 months) were compared to 16 tumors from platinum-sensitive patients (PFS more than 18months). Molecular profiling was performed using Affymetrix high-resolution microarray platforms to permit global comparisons of gene expression levels and copy number profiles between tumors from the resistant group with the sensitive group. Microarray data analysis revealed a set of 227 discriminating genes of which expression levels may be influencing differential chemotherapy response between the two groups. Pathway analysis of these genes showed the,PI3K,NFkB and IGF1 networks as some of the significant networks distinguishing the chemotherapy resistant with the sensitive group. Copy number analysis performed using Nexus copy number version 6.1 revealed differences in the chromosomal regions, 4q31.22, 5q13.2, 9p24.3, 2p23.2, 16q21, 6q14.1, 7p22.3, 12p13 and Xq. Integrative copy number and gene expression profiling will delineate the drivers of chemotherapy resistance in patients undergoing standard platinum-based treatment of ovarian cancer. Future studies to validate these markers are necessary to apply this knowledge to biomarker-based clinical trials. Citation Format: Madhuri Koti, Robert J. Gooding, Paulo Nuin, Alexandria Haslehurst, Colleen Crane, Johanne Weberpals, Timothy Childs, Peter Bryson, Moyez Dharsee, Kenneth Evans, Harriet E. Feilotter, Paul C. Park, Jeremy A. Squire. Integrative genomic and transcriptomic analysis in idenfitication of biomarkers of chemoresistance in serous epithelial ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 810. doi:10.1158/1538-7445.AM2013-810