RESEARCH ARTICLES
Objective
New prognostic biomarkers, and bio-signatures, are urgently needed to facilitate a precision medicine-based approach to more effectively treat patients with high-grade serous ovarian cancer (HGSC). In this study, we analysed the expression patterns of a series of candidate protein biomarkers.
Methods
The panel of markers which included MyD88, TLR4, MAD2, PR, OR, WT1, p53, p16, CD10 and Ki67 was assessed using immunohistochemistry in a tissue microarray (TMA) cohort of n = 80 patients, composed of stage 3–4 HGSCs. Each marker was analysed for its potential to predict both overall survival (OS) and progression-free survival (PFS).
Results
TLR4 and p53 were found to be individually predictive of poorer PFS (Log Rank, p = 0.017, p = 0.030 respectively). Cox regression analysis also identified high p53 and TLR4 expression as prognostic factors for reduced PFS (p53; HR=1.785, CI=1.036–3.074, p = 0.037 and TLR4; HR=2.175, CI=1.112–4.253, p = 0.023). Multivariate forward conditional Cox regression analysis, examining all markers, identified a combined signature composed of p53 and TLR4 as prognostic for reduced PFS (p = 0.023).
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Conclusion
Combined p53 and TLR4 marker assessment may help to aid treatment stratification for patients diagnosed with advanced-stage HGSC.
P53 AND TLR4 EXPRESSION ARE PROGNOSTIC MARKERS INFORMING PROGRESSION FREE SURVIVAL OF ADVANCED STAGE HIGH GRADE SEROUS OVARIAN CANCER
Bates
2024
ALTERED INFLAMMASOME AND IMMUNE ACTIVATION IN
PAEDIATRIC TRAUMATIC BRAIN INJURY
Introduction
Systemic Inflammation is associated with Traumatic Brain Injury (TBI) and therefore is a potential target for immunomodulation. Dysregulated immune function post-TBI increased susceptibility to infection and post-concussive syndrome. The inflammasome is a protein complex associated with an amplified proinflammatory response and is a potential target for immunomodulation that preserves antimicrobial immunity.
Methods
Samples from children with mild TBI (mTBI; Glasgow coma scale (GCS) 14/15), severe TBI (sTBI; GCS < 8) and control children were collected at baseline and two week follow up and were treated with endotoxin and melatonin. Toll-like receptor (TLR4; marker of endotoxin responses) and CD11b (activation marker) expression on neutrophils and monocytes were evaluated by flow cytometry. Inflammasome-related genes and cytokines were assessed using TaqMan RT-PCR samples ELISA sandwich immunoassay, respectively.
Results
A total of 214 children were enrolled including: TBI (n = 116), with mild TBI (mTBI; Glasgow coma scale (GCS) 14/15) and severe TBI (sTBI; GCS < 8), and (n = 98) control patients collected at baseline and two week follow up. Total monocyte and intermediate monocyte populations were reduced in mTBI at baseline. Neutrophil CD11b and TLR4 expression was decreased in mTBI at 10–14 days. NLRP3 and NLRP1 were downregulated at 10–14 days while IL-1β was increased at baseline at 0–4 days and further elevated by 10–14 days and significantly higher in those with no previous mTBI. Serum cytokines showed lower IL-18 and raised IL-33 in those with mTBI. Prior concussion did not influence serum cytokine levels. In addition, LPS did not stimulate an IL-18 and IL-1β response in the mTBI group at 10–14 days.
Conclusions
Children with mTBI had reduced CD11b and TLR4 expression and NLRP3 inflammasome activation. IL-1β mRNA was raised and continued to rise after injury implicating the innate immune system in the subacute phase of injury. Immune dysregulation post-TBI in children may be a target for immunomodulation following further exploration in vitro of potential mechanisms and therapies.
Ryan
2024
PLASMINOGEN ACTIVATOR INHIBITOR 1 IS ASSOCIATED WITH HIGH-GRADE SEROUS OVARIAN CANCER METASTASIS AND IS REDUCED IN PATIENTS WHO HAVE RECEIVED NEOADJUVANT CHEMOTHERAPY
Introduction
High-grade serous ovarian cancer (HGSOC) is the most prevalent and deadliest subtype of epithelial ovarian cancer (EOC), killing over 140,000 people annually. Morbidity and mortality are compounded by a lack of screening methods, and recurrence is common. Plasminogen-activator-inhibitor 1 (PAI-1, the protein product of SERPIN E1) is involved in hemostasis, extracellular matrix (ECM) remodeling, and tumor cell migration and invasion. Overexpression is associated with poor prognosis in EOC. Platelets significantly increase PAI-1 in cancer cells in vitro, and may contribute to the hematogenous metastasis of circulating tumor cells (CTCs). CTCs are viable tumor cells that intravasate and travel through the circulation–often aided by platelets - with the potential to form secondary metastases. Here, we provide evidence that PAI-1 is central to the platelet-cancer cell interactome, and plays a role in the metastatic cascade.
Methods
SK-OV-3 cells where PAI-1 had been silenced, treated with healthy donor platelets, and treated with platelet-conditioned medium were used as an in vitro model of metastatic EOC. Gene expression analysis was performed using RNA-Seq data from untreated cells and cells treated with PAI-1 siRNA or negative control, each with and without platelets. Four cohorts of banked patient plasma samples (n = 239) were assayed for PAI-1 by ELISA. Treatment-naïve (TN) whole blood (WB) samples were evaluated for CTCs in conjunction with PAI-1 evaluation in matched plasma.
Results and discussion
Significant phenotypic changes occurring when PAI-1 was silenced and when platelets were added to cells were reflected by RNA-seq data, with PAI-1 observed to be central to molecular mechanisms of EOC metastasis. Increased proliferation was observed in cells treated with platelets. Plasma PAI-1 significantly correlated with advanced disease in a TN cohort, and was significantly reduced in a neoadjuvant chemotherapy (NACT) cohort. PAI-1 demonstrated a trend towards significance in overall survival (OS) in the late-stage TN cohort, and correlation between PAI-1 and neutrophils in this cohort was significant. 72.7% (16/22) of TN patients with plasma PAI-1 levels higher than OS cutoff were CTC-positive. These data support a central role for PAI-1 in EOC metastasis, and highlight PAI-1’s potential as a biomarker, prognostic indicator, or gauge of treatment response in HGSOC.
Kelly
2023
DYNAMIC INTERPLAY BETWEEN SORTILIN AND SYNDECAN-1 CONTRIBUTES TO PROSTATE CANCER PROGRESSION
Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions. The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model. Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR. Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.
Lazniewska
2023
PREDICTION OF PROSTATE CANCER BIOCHEMICAL AND CLINICAL RECURRENCE IS IMPROVED BY IHC-ASSISTED GRADING USING APPL1, SORTILIN AND SYNDECAN-1
Prostate cancer (PCa) development and progression relies on the programming of glucose and lipid metabolism, and this involves alterations in androgen receptor expression and signalling. Defning the molecular mechanism that underpins this metabolic programming will have direct signifcance for patients with PCa who have a poor prognosis. Here we show that there is a dynamic balance between sortilin and syndecan-1, that reports on diferent metabolic phenotypes. Using tissue microarrays, we demonstrated by immunohistochemistry that sortilin was highly expressed in low-grade cancer, while syndecan-1 was upregulated in high-grade disease. Mechanistic studies in prostate cell lines revealed that in androgen-sensitive LNCaP cells, sortilin enhanced glucose metabolism by regulating GLUT1 and GLUT4, while binding progranulin and lipoprotein lipase (LPL) to limit lipid metabolism. In contrast, in androgen-insensitive PC3 cells, syndecan-1 was upregulated, interacted with LPL and colocalised with β3 integrin to promote lipid metabolism. In addition, androgen-deprived LNCaP cells had decreased expression of sortilin and reduced glucose-metabolism, but increased syndecan-1 expression, facilitating interactions with LPL and possibly β3 integrin. We report a hitherto unappreciated molecular mechanism for PCa, which may have signifcance for disease progression and how androgen-deprivation therapy might promote castration-resistant PCa.
Logan
2023
ABERRANT PROTEIN EXPRESSION OF APPL1, SORTILIN AND SYNDECAN-1 DURING THE BIOLOGICAL PROGRESSION OF PROSTATE CANCER.
Diagnosis and assessment of patients with prostate cancer is dependent on accurate interpretation and grading of histopathology. However, morphology does not necessarily reflect the complex biological changes occurring in prostate cancer disease progression, and current biomarkers have demonstrated limited clinical utility in patient assessment. This study aimed to develop biomarkers that accurately define prostate cancer biology by distinguishing specific pathological features that enable reliable interpretation of pathology for accurate Gleason grading of patients. Online gene expression databases were interrogated and a pathogenic pathway for prostate cancer was identified. The protein expression of key genes in the pathway, including adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine binding (PTB) domain, and leucine zipper motif 1 (Appl1), Sortilin and Syndecan-1, was examined by immunohistochemistry (IHC) in a pilot study of 29 patients with prostate cancer, using monoclonal antibodies designed against unique epitopes. Appl1, Sortilin, and Syndecan-1 expression was first assessed in a tissue microarray cohort of 112 patient samples, demonstrating that the monoclonal antibodies clearly illustrate gland morphologies. To determine the impact of a novel IHC assisted interpretation (the utility of Appl1, Sortilin, and Syndecan-1 labelling as a panel) of Gleason grading, versus standard haematoxylin and eosin (H&E) Gleason grade assignment, a radical prostatectomy sample cohort comprising 114 patients was assessed. In comparison to H&E, the utility of the biomarker panel reduced subjectivity in interpretation of prostate cancer tissue morphology and improved the reliability of pathology assessment, resulting in Gleason grade redistribution for 41% of patient samples. Importantly, for equivocal IHC-assisted labelling and H&E staining results, the cancer morphology interpretation could be more accurately applied upon re-review of the H&E tissue sections. This study addresses a key issue in the field of prostate cancer pathology by presenting a novel combination of three biomarkers and has the potential to transform clinical pathology practice by standardising the interpretation of the tissue morphology.
Martini
2023
EX VIVO EXPANSION OF CIRCULATING TUMOUR CELLS (CTCS)
Circulating tumour cells (CTCs) are a critical intermediate step in the process of cancer metastasis. The reliability of CTC isolation/purifcation has limited both the potential to report on metastatic progression and the development of CTCs as targets for therapeutic intervention. Here we report a new methodology, which optimises the culture conditions for CTCs using primary cancer cells as a model system. We exploited the known biology that CTCs thrive in hypoxic conditions, with their survival and proliferation being reliant on the activation of hypoxia-inducible factor 1 alpha (HIF-1α). We isolated epithelial-like and quasi-mesenchymal CTC phenotypes from the blood of a cancer patient and successfully cultured these cells for more than 8 weeks. The presence of CTC clusters was required to establish and maintain long-term cultures. This novel methodology for the long-term culture of CTCs will aid in the development of downstream applications, including CTC theranostics.
Mohamed
2023
THE INDUCTION OF A MESENCHYMAL PHENOTYPE BY PLATELET CLOAKING OF CANCER CELLS IS A UNIVERSAL PHENOMENON
Introduction
Tumour metastasis accounts for over 90% of cancer related deaths. The platelet is a key blood component, which facilitates efficient metastasis. This study aimed to understand the molecular mechanisms involved in tumour-platelet cell interactions.
Methods
The interaction between cancer cells and platelets was examined in 15 epithelial cell lines, representing 7 cancer types. Gene expression analysis of EMT-associated and cancer stemness genes was performed by RT-PCR. Whole transcriptome analysis (WTA) was performed using Affymetrix 2.0ST arrays on a platelet co-cultured ovarian model.
Results
Platelet adhesion and activation occurred across all tumour types. WTA identified increases in cellular movement, migration, invasion, adhesion, development, differentiation and inflammation genes and decreases in processes associated with cell death and survival following platelet interaction. Increased invasive capacity was also observed in a subset of cell lines. A cross-comparison with a platelet co-cultured mouse model identified 5 common altered genes; PAI-1, PLEK2, CD73, TNC, and SDPR.​
Conclusion
Platelet cancer cell interactions are a key factor in driving the pro-metastatic phenotype and appear to be mediated by 5 key genes which have established roles in metastasis. Targeting these metastasis mediators could improve cancer patient outcomes.
Spillane
2021
THE ROLE OF THE MAD2-TLR4-MYD88 AXIS IN PACLITAXEL RESISTANCE IN OVARIAN CANCER
Despite the use of front-line anticancer drugs such as paclitaxel for ovarian cancer treatment, mortality rates have remained almost unchanged for the past three decades and the majority of patients will develop recurrent chemoresistant disease which remains largely untreatable. Overcoming chemoresistance or preventing its onset in the first instance remains one of the major challenges for ovarian cancer research. In this study, we demonstrate a key link between senescence and inflammation and how this complex network involving the biomarkers MAD2, TLR4 and MyD88 drives paclitaxel resistance in ovarian cancer. This was investigated using siRNA knockdown of MAD2, TLR4 and MyD88 in two ovarian cancer cell lines, A2780 and SKOV-3 cells and overexpression of MyD88 in A2780 cells. Interestingly, siRNA knockdown of MAD2 led to a significant increase in TLR4 gene expression, this was coupled with the development of a highly paclitaxel-resistant cell phenotype. Additionally, siRNA knockdown of MAD2 or TLR4 in the serous ovarian cell model OVCAR-3 resulted in a significant increase in TLR4 or MAD2 expression respectively. Microarray analysis of SKOV-3 cells following knockdown of TLR4 or MAD2 highlighted a number of significantly altered biological processes including EMT, complement, coagulation, proliferation and survival, ECM remodelling, olfactory receptor signalling, ErbB signalling, DNA packaging, Insulin-like growth factor signalling, ion transport and alteration of components of the cytoskeleton. Cross comparison of the microarray data sets identified 7 overlapping genes including MMP13, ACTBL2, AMTN, PLXDC2, LYZL1, CCBE1 and CKS2. These results demonstrate an important link between these biomarkers, which to our knowledge has never before been shown in ovarian cancer. In the future, we hope that triaging patients into alternative treatment groups based on the expression of these three biomarkers or therapeutic targeting of the mechanisms they are involved in will lead to improvements in patient outcomes and prevent the development of chemoresistance.
Bates
2020
FKBPL-BASED PEPTIDE, ALM201, TARGETS ANGIOGENESIS AND CANCER STEM CELLS IN OVARIAN CANCER
Background
ALM201 is a therapeutic peptide derived from FKBPL that has previously undergone preclinical and clinical development for oncology indications and has completed a Phase 1a clinical trial in ovarian cancer patients and other advanced solid tumours.
Methods
In vitro, cancer stem cell (CSC) assays in a range of HGSOC cell lines and patient samples, and in vivo tumour initiation, growth delay and limiting dilution assays, were utilised. Mechanisms were determined by using immunohistochemistry, ELISA, qRT-PCR, RNAseq and western blotting. Endogenous FKBPL protein levels were evaluated using tissue microarrays (TMA).
Results
ALM201 reduced CSCs in cell lines and primary samples by inducing differentiation. ALM201 treatment of highly vascularised Kuramochi xenografts resulted in tumour growth delay by disruption of angiogenesis and a ten-fold decrease in the CSC population. In contrast, ALM201 failed to elicit a strong antitumour response in non-vascularised OVCAR3 xenografts, due to high levels of IL-6 and vasculogenic mimicry. High endogenous tumour expression of FKBPL was associated with an increased progression-free interval, supporting the protective role of FKBPL in HGSOC.
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Conclusion
FKBPL-based therapy can (i) dually target angiogenesis and CSCs, (ii) target the CD44/STAT3 pathway in tumours and (iii) is effective in highly vascularised HGSOC tumours with low levels of IL-6.
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Annett
2020
EXPOSURE TO TOBACCO SMOKE MEASURED BY URINARY NICOTINE METABOLITES INCREASES RISK OF P16/KI-67 CO-EXPRESSION AND HIGH-GRADE CERVICAL NEOPLASIA IN HPV POSITIVE WOMEN: A TWO YEAR PROSPECTIVE STUDY
Background
Human papillomavirus (HPV) is considered the strongest epidemiologic risk factor for cervical cancer. However, it is not a sufficient cause given the high prevalence of transient infections. We examined the relationship between exposure to tobacco smoke, measured using urinary nicotine metabolite concentrations, and p16/Ki-67 co-expression in cervical smears and subsequent risk of developing CIN2+/CIN3+ lesions in HPV positive women.
Methods
This prospective longitudinal study enrolled women presenting to colposcopy with cytological abnormalities LSIL/ASCUS at the National Maternity Hospital, Dublin. Women gave a urine sample which was used to perform the Nicotine Metabolite Assay (Siemens). HPV positive (HC2) cervical smears were stained by immunocytochemistry for p16/Ki-67 (CINtec PLUS, Roche). Two year follow-up data, including histological diagnosis, was collected for each woman. Crude and adjusted odds ratios were calculated using logistic regression to investigate associations between tobacco smoke, p16/Ki-67 positivity and CIN2+/CIN3 + .
Results
In total, 275 HPV positive women were included. Women with nicotine metabolite concentrations above 500 ng/mL, indicative of smoking, were classified as smokers. Smokers were at an increased risk of testing positive for p16/Ki-67 (OR 1.678; 1.027-2.740) and CIN2+ and CIN3+ (OR 1.816; 1.107-2.977 and OR 2.453; 1.200-5.013) in compared to non-smokers. In p16/Ki-67 positive women, smoking further increased their risk of CIN2+/CIN3+ (OR 2.290; 1.017-5.159 and OR 3.506 (1.534-8.017).
Conclusion
HPV positive women exposed to tobacco smoke are at a higher risk of testing positive for p16/Ki-67 co-expression. Risk of high-grade disease is almost doubled in women who are exposed to tobacco smoke.
White
2020
PREVALENCE OF TUMOR BRCA1 AND BRCA2 DYSFUNCTION IN UNSELECTED PATIENTS WITH OVARIAN CANCER
Objective
The therapeutic benefits of poly(ADP-ribose) polymerase inhibitors highlight the need to evaluate BRCA1/2 defects in tubal/ovarian cancer (OC). We sought to determine the pattern and disease characteristics associated with tumor BRCA1/2 mutations and BRCA1 methylation in women with OC.
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Methods
We obtained 111 OC specimens from 2 university hospitals and assessed BRCA1/2 mutations and BRCA1 methylation in tumor DNA. The frequency and pattern of BRCA1/2 defects were examined. Associations between patient/disease characteristics and BRCA1/2 defects were ascertained (Fisher's exact test). Platinum-free interval (PFI), progression-free survival (PFS), and overall survival (OS) based on the underlying BRCA1/2 defect were determined (Kaplan-Meier analysis [log-rank test]).
Results
We observed a BRCA1/2 dysfunction rate of 40% (28/70) in high-grade serous tubal/ovarian cancer (HGSC), including 14.3% BRCA1 methylation (n=10), 7.1% BRCA1 mutation (n=5), and 18.6% BRCA2 mutation (n=13). Defects in BRCA1/2 genes were associated with stage III/IV HGSC (BRCA1 methylation: P=0.005 [stage III/IV] and P=0.004 [HGSC]; BRCA1/2 mutation: P=0.03 [stage III/IV] and P<0.001 [HGSC]). Patients with BRCA1/2-mutated cancers showed improved OS (hazard ratio [HR], 0.65; 95% confidence interval [CI], 0.43-0.99; P=0.045) and a trend toward improved PFI (HR, 0.48; 95% CI, 0.22-1.06; P=0.07) and PFS (HR, 0.72; 95% CI, 0.51-1.03; P=0.07). No survival differences were observed between BRCA1-methylated and BRCA1/2 wild-type non-BRCA1-methylated cancers.
Conclusion
We observed a high tumor BRCA1/2 dysfunction rate in HGSC with a unique predominance of BRCA2 over BRCA1 mutations. While BRCA1/2 mutations conferred survival benefits in OC, no such association was observed with BRCA1 methylation.
Kalachand
2020
MULTIPLEX PROFILING IDENTIFIES CLINICALLY RELEVANT SIGNALLING PROTEINS IN AN ISOGENIC PROSTATE CANCER MODEL OF RADIORESISTANCE
The exact biological mechanism governing the radioresistant phenotype of prostate tumours at a high risk of recurrence despite the delivery of advanced radiotherapy protocols remains unclear. This study analysed the protein expression profles of a previously generated isogenic 22Rv1 prostate cancer model of radioresistance using DigiWest multiplex protein profling for a selection of 90 signalling proteins. Comparative analysis of the profles identifed ssubstantial change in the expression of 43 proteins. Diferential PARP-1, AR, p53, Notch-3 and YB-1 protein levels were independently validated using Western Blotting. Pharmacological targeting of these proteins was associated with a mild butsignifcant radiosensitisation efect at 4Gy. This study supports the clinical relevance of isogenic in vitro models of radioresistance and clarifes the molecular radiation response of prostate cancer cells.
Inder
2019
CD10 -/ALDH - CELLS ARE THE SOLE CISPLATIN-RESISTANT COMPONENT OF A NOVEL OVARIAN CANCER STEM CELL HIERARCHY
It is long established that tumour-initiating cancer stem cells (CSCs) possess chemoresistant properties. However, little is known of the mechanisms involved, particularly with respect to the organisation of CSCs as stem-progenitor-differentiated cell hierarchies. Here we aimed to elucidate the relationship between CSC hierarchies and chemoresistance in an ovarian cancer model. Using a single cell-based approach to CSC discovery and validation, we report a novel, four-component CSC hierarchy based around the markers cluster of differentiation 10 (CD10) and aldehyde dehydrogenase (ALDH). In a change to our understanding of CSC biology, resistance to chemotherapy drug cisplatin was found to be the sole property of CD10-/ALDH- CSCs, while all four CSC types were sensitive to chemotherapy drug paclitaxel. Cisplatin treatment quickly altered the hierarchy, resulting in a three-component hierarchy dominated by the cisplatin-resistant CD10-/ALDH- CSC. This organisation was found to be hard-wired in a long-term cisplatin-adapted model, where again CD10-/ALDH- CSCs were the sole cisplatin-resistant component, and all CSC types remained paclitaxel-sensitive. Molecular analysis indicated that cisplatin resistance is associated with inherent- and adaptive-specific drug efflux and DNA-damage repair mechanisms. Clinically, low CD10 expression was consistent with a specific set of ovarian cancer patient samples. Collectively, these data advance our understanding of the relationship between CSC hierarchies and chemoresistance, which was shown to be CSC- and drug-type specific, and facilitated by specific and synergistic inherent and adaptive mechanisms. Furthermore, our data indicate that primary stage targeting of CD10-/ALDH- CSCs in specific ovarian cancer patients in future may facilitate targeting of recurrent disease, before it ever develops.
French
2017
MYD88 IS AN ESSENTIAL COMPONENT OF RETINOIC ACID-INDUCED DIFFERENTIATION IN HUMAN PLURIPOTENT EMBRYONAL CARCINOMA CELLS
We have previously reported that myeloid differentiation primary response gene 88 (MyD88) is downregulated during all-trans retinoic acid (RA)-induced differentiation of pluripotent NTera2 human embryonal carcinoma cells (hECCs), whereas its maintained expression is associated with RA differentiation resistance in nullipotent 2102Ep hECCs. MyD88 is the main adapter for toll-like receptor (TLR) signalling, where it determines the secretion of chemokines and cytokines in response to pathogens. In this study, we report that loss of MyD88 is essential for RA-facilitated differentiation of hECCs. Functional analysis using a specific MyD88 peptide inhibitor (PepInh) demonstrated that high MyD88 expression in the self-renewal state inhibits the expression of a specific set of HOX genes. In NTera2 cells, MyD88 is downregulated during RA-induced differentiation, a mechanism that could be broadly replicated by MyD88 PepInh treatment of 2102Ep cells. Notably, MyD88 inhibition transitioned 2102Ep cells into a stable, self-renewing state that appears to be primed for differentiation upon addition of RA. At a molecular level, MyD88 inhibition combined with RA treatment upregulated HOX, RA signalling and TLR signalling genes. These events permit differentiation through a standard downregulation of Oct4-Sox2-Nanog mechanism. In line with its role in regulating secretion of specific proteins, conditioned media experiments demonstrated that differentiated (MyD88 low) NTera2 cell media was sufficient to differentiate NTera2 cells. Protein array analysis indicated that this was owing to secretion of factors known to regulate angiogenesis, neurogenesis and all three branches of TGF-β Superfamily signalling. Collectively, these data offer new insights into RA controlled differentiation of pluripotent cells, with notable parallels to the ground state model of embryonic stem cell self-renewal. These data may provide insights to facilitate improved differentiation protocols for regenerative medicine and differentiation-therapies in cancer treatment.
Sulaiman
2017
TRIAGE OF LSIL/ASC-US WITH P16/KI-67 DUAL STAINING AND HUMAN PAPILLOMAVIRUS TESTING: A 2-YEAR PROSPECTIVE STUDY
Objective
To investigate human papillomavirus (HPV) DNA testing and p16/Ki-67 staining for detecting cervical intraepithelial grade 2 or worse (CIN2+) and CIN3 in women referred to colposcopy with minor abnormal cervical cytology low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of undermined significance (ASC-US). The clinical performance of both tests was evaluated as stand-alone tests and combined, for detection CIN2+ and CIN3 over 2 years.
Methods
ThinPrep® liquid-based cytology (LBC) specimens were collected from 1349 women with repeat LSIL or ASC-US. HPV DNA was performed using Hybrid Capture. Where adequate material remained (n = 471), p16/Ki-67 overexpression was assessed. Clinical performance for detection of histologically diagnosed CIN2+ and CIN3 was calculated.
Results
Approximately 62.2% of the population were positive for HPV DNA, and 30.4% were positive for p16/Ki-67. p16/Ki-67 showed no significant difference in positivity between LSIL and ASC-US referrals (34.3% versus 28.6%; P = 0.189). Women under 30 years had a higher rate of p16/Ki-67 compared to those over 30 years (36.0% versus 26.6%; P = 0.029). Overall HPV DNA testing produced a high sensitivity for detection of CIN3 of 95.8% compared to 79.2% for p16/Ki-67. In contrast, p16/Ki-67 expression offered a higher specificity, 75.2% versus 40.4% for detection of CIN3. Combining p16/Ki-67 with HPV DNA improved the accuracy in distinguishing between CIN3 and <CIN3. The absolute risk of CIN3 increased from 15.6% in women who were HPV DNA positive to 27% in women positive for HPV DNA and p16/Ki-67. Those negative for HPV DNA and p16/Ki-67 had a low risk of 1.2% of CIN3.
Conclusion
The addition of p16/Ki-67 to HPV DNA testing leads to a more accurate stratification of CIN in women presenting with minor cytological abnormalities.
White
2016
THE MYD88+ PHENOTYPE IS AN ADVERSE PROGNOSTIC FACTOR IN EPITHELIAL OVARIAN CANCER
The prognosis of epithelial ovarian cancer is poor in part due to the high frequency of chemoresistance. Recent evidence points to the Toll-like receptor-4 (TLR4), and particularly its adaptor protein MyD88, as one potential mediator of this resistance. This study aims to provide further evidence that MyD88 positive cancer cells are clinically significant, stem-like and reproducibly detectable for the purposes of prognostic stratification. Expression of TLR4 and MyD88 was assessed immunohistochemically in 198 paraffin-embedded ovarian tissues and in an embryonal carcinoma model of cancer stemness. In parallel, expression of TLR4 and MyD88 mRNA and regulatory microRNAs (miR-21 and miR-146a) was assessed, as well as in a series of chemosensitive and resistant cancer cells lines. Functional analysis of the pathway was assessed in chemoresistant SKOV-3 ovarian cancer cells. TLR4 and MyD88 expression can be reproducibly assessed via immunohistochemistry using a semi-quantitative scoring system. TLR4 expression was present in all ovarian epithelium (normal and neoplastic), whereas MyD88 was restricted to neoplastic cells, independent of tumour grade and associated with reduced progression-free and overall survival, in an immunohistological specific subset of serous carcinomas, p<0.05. MiR-21 and miR-146a expression was significantly increased in MyD88 negative cancers (p<0.05), indicating their participation in regulation. Significant alterations in MyD88 mRNA expression were observed between chemosensitive and chemoresistant cells and tissue. Knockdown of TLR4 in SKOV-3 ovarian cells recovered chemosensitivity. Knockdown of MyD88 alone did not. MyD88 expression was down-regulated in differentiated embryonal carcinoma (NTera2) cells, supporting the MyD88+ cancer stem cell hypothesis. Our findings demonstrate that expression of MyD88 is associated with significantly reduced patient survival and altered microRNA levels and suggest an intact/functioning TLR4/MyD88 pathway is required for acquisition of the chemoresistant phenotype. Ex vivo manipulation of ovarian cancer stem cell (CSC) differentiation can decrease MyD88 expression, providing a potentially valuable CSC model for ovarian cancer.