Annexin A2 is regulated by ovarian cancer-peritoneal cell interactions and promotes metastasis

Our recent research identified the protein annexin A2 to be regulated by ovarian cancer-peritoneal cell interactions. This study investigated the role of annexin A2 in ovarian cancer metastasis and its potential utility as a novel therapeutic target, using in vitro and in vivo ovarian cancer models. Annexin A2 expression was examined by qRT-PCR and western blotting in ovarian cancer cell lines and immunohistochemistry in serous ovarian carcinoma tissues. Annexin A2 siRNAs were used to evaluate the effects of annexin A2 suppression on ovarian cancer cell adhesion, motility, and invasion. Furthermore, annexin A2 neutralizing antibodies were used to examine the role of annexin A2 in tumor invasion and metastasis in vivo using a chick chorioallantoic membrane assay and an intraperitoneal xenograft mouse model. Strong annexin A2 immunostaining was observed in 90% (38/42) of the serous ovarian cancer cells and was significantly increased in the cancer-associated stroma compared to non-malignant ovarian tissues. Annexin A2 siRNA significantly inhibited the motility and invasion of serous ovarian cancer cells and adhesion to the peritoneal cells. Annexin A2 neutralizing antibodies significantly inhibited OV-90 cell motility and invasion in vitro and in vivo using the chick chorioallantoic membrane assay. The growth of SKOV-3 cells and their peritoneal dissemination in nude mice was significantly inhibited by annexin A2 neutralizing antibodies. Annexin A2 plays a critical role in ovarian cancer metastasis and is therefore a potential novel therapeutic target against ovarian cancer.Noor A. Lokman, Alison SF. Elder, Miranda P Ween, Carmen E. Pyragius, Peter Hoffmann, Martin K. Oehler, and Carmela Ricciardell

Keratin 5 overexpression is associated with serous ovarian cancer recurrence and chemotherapy resistance

This study investigated the clinical significance of keratin 5 and 6 expression in serous ovarian cancer progression and chemotherapy resistance. KRT5 and KRT6 (KRT6A, KRT6B & KRT6C) gene expression was assessed in publically available serous ovarian cancer data sets, ovarian cancer cell lines and primary serous ovarian cancer cells. Monoclonal antibodies which detect both K5/6 or only K5 were used to assess protein expression in ovarian cancer cell lines and a cohort of high grade serous ovarian carcinomas at surgery (n = 117) and after neoadjuvant chemotherapy (n = 21). Survival analyses showed that high KRT5 mRNA in stage III/IV serous ovarian cancers was significantly associated with reduced progression-free (HR 1.38, P < 0.0001) and overall survival (HR 1.28, P = 0.013) whilst high KRT6 mRNA was only associated with reduced progression-free survival (HR 1.2, P = 0.031). Both high K5/6 (≥ 10%, HR 1.78 95% CI; 1.03−2.65, P = 0.017) and high K5 (≥ 10%, HR 1.90, 95% CI; 1.12−3.19, P = 0.017) were associated with an increased risk of disease recurrence. KRT5 but not KRT6C mRNA expression was increased in chemotherapy resistant primary serous ovarian cancer cells compared to chemotherapy sensitive cells. The proportion of serous ovarian carcinomas with high K5/6 or high K5 immunostaining was significantly increased following neoadjuvant chemotherapy. K5 can be used to predict serous ovarian cancer prognosis and identify cancer cells that are resistant to chemotherapy. Developing strategies to target K5 may therefore improve serous ovarian cancer survival.Carmela Ricciardelli, Noor A Lokman, Carmen E Pyragius, Miranda P Ween, Anne M Macpherson, Andrew Ruszkiewicz, Peter Hoffmann, Martin K Oehle

The relationship between androgens and days per month of period pain, pelvic pain, headache, and TLR4 responsiveness of peripheral blood mononuclear cells in young women with dysmenorrhoea

Purpose: Women bear a disproportionate burden of persistent pain conditions when compared to men. To determine whether the hormonal environment affects the clinical experience of pain, as measured by the days per month of pelvic pain (DPelvicPM), period pain (DPeriodPM), headache (DHeadachePM) or the in vitro EC50 for Interleukin-1β (IL-1β) release following TLR4 stimulation with Lipopolysaccharide from Peripheral Blood Mononuclear Cells (PBMCs). Findings were stratified according to use or non-use of the oral contraceptive pill. Patients and Methods: Fifty-six women aged 16-35 years, with minimal or severe dysmenorrhea, and use or non-use of the OC, were enrolled. Blood was collected on two occasions in a single menstrual cycle: Days 1-2 and Days 7-10. Hormonal analysis for testosterone, dihydrotestosterone, dehydroepiandrosterone, Androstenedione, 3α-Androstanediol, 3β-androstanediol, estradiol, estrone, 17α-hydroxyprogesterone, progesterone, cortisol and sex-hormone binding globulin was undertaken using ultra-sensitive Liquid Chromatography Mass-Spectrometry (LC-MS). PBMCs were exposed to lipopolysaccharide (LPS) and the resulting Interleukin-1β output was determined. Results: Non-users of the OC showed a strongly inverse correlation between a reducing free androgen index (FAI) and increasing DPelvicPM (p=0.0032), DPeriodPM (p=0.013), DHeadachePM (p=0.041). Non-users of the OC showed a significant increase in DPelvicPM (p=0.049) on Days 7-10. Modestly significant associations were found between reduced androgens and potentiated LPS-induced IL-1β (lower EC50). Conclusion: This is the first study to investigate the relationship between the hormonal environment and activation of the immune system in young women with dysmenorrhoea-related pain conditions. Low androgen levels were consistently associated with increased pain. Translational implications for the findings are discussed.Susan F Evans, Yuen Kwok, Ann Solterbeck, Carmen Pyragius, Mary Louise Hull, Mark R Hutchinson, Paul Rola

Chemotherapy-induced hyaluronan production: a novel chemoresistance mechanism in ovarian cancer

Background: Hyaluronan (HA) an important component of the extracellular matrix, has been linked to tumor progression and drug resistance in several malignancies. However, limited data is available for ovarian cancer. This study investigated the role of hyaluronan (HA) and a potential link between the HA-CD44 pathway and membrane ATP binding cassette (ABC) transporter proteins in ovarian cancer chemoresistance. Methods: We investigated the ability of HA to block the cytotoxic effects of the chemotherapy drug carboplatin, and to regulate the expression of ABC transporters in ovarian cancer cells. We also examined HA serum levels in ovarian cancer patients prior to and following chemotherapy and assessed its prognostic relevance. Results: HA increased the survival of carboplatin treated ovarian cancer cells expressing the HA receptor, CD44 (OVCAR-5 and OV-90). Carboplatin significantly increased expression of HAS2, HAS3 and ABCC2 and HA secretion in ovarian cancer cell conditioned media. Serum HA levels were significantly increased in patients following platinum based chemotherapy and at both 1st and 2nd recurrence when compared with HA levels prior to treatment. High serum HA levels (>50 μg/ml) prior to chemotherapy treatment were associated with significantly reduced progression-free (P = 0.014) and overall survival (P = 0.036). HA production in ovarian cancer cells was increased in cancer tissues collected following chemotherapy treatment and at recurrence. Furthermore HA treatment significantly increased the expression of ABC drug transporters (ABCB3, ABCC1, ABCC2, and ABCC3), but only in ovarian cancer cells expressing CD44. The effects of HA and carboplatin on ABC transporter expression in ovarian cancer cells could be abrogated by HA oligomer treatment. Importantly, HA oligomers increased the sensitivity of chemoresistant SKOV3 cells to carboplatin. Conclusions: Our findings indicate that carboplatin chemotherapy induces HA production which can contribute to chemoresistance by regulating ABC transporter expression. The HA-CD44 signaling pathway is therefore a promising target in platinum resistant ovarian cancer.Carmela Ricciardelli, Miranda P Ween, Noor A Lokman, Izza A Tan, Carmen E Pyragius, and Martin K Oehle

Microarray expression analysis of genes and pathways involved in growth plate cartilage injury responses and bony repair

The injured growth plate cartilage is often repaired by a bone bridge which causes bone growth deformities. Whilst previous studies have identified sequential inflammatory, fibrogenic, osteogenic and bone remodelling responses involved in the repair process, the molecular pathways which regulated these cellular events remain unknown. In a rat growth plate injury model, tissue from the injury site was collected across the time-course of bone bridge formation using laser capture microdissection and was subjected to Affymetrix microarray gene expression analysis. Real Time PCR and immunohistochemical analyses were used to confirm changes in levels of expression of some genes identified in microarray. Four major functional groupings of differentially expressed genes with known roles in skeletal development were identified across the time-course of bone bridge formation, including Wnt signalling (SFRP1, SFRP4, β-catenin, Csnk2a1, Tcf7, Lef1, Fzd1, Fzd2, Wisp1 and Cpz), BMP signalling (BMP-2, BMP-6, BMP-7, Chrd, Chrdl2 and Id1), osteoblast differentiation (BMP-2, BMP-6, Chrd, Hgn, Spp1, Axin2, β-catenin, Bglap2) and skeletal development (Chrd, Mmp9, BMP-1, BMP-6, Spp1, Fgfr1 and Traf6). These studies provide insight into the molecular pathways which act cooperatively to regulate bone formation following growth plate cartilage injury and highlight potential therapeutic targets to limit bone bridge formation.Carmen E. Macsai, Kristen R. Georgiou, Bruce K. Foster, Andrew C.W. Zannettino, Cory J. Xia

Structural and molecular analyses of bone bridge formation within the growth plate injury site and cartilage degeneration at the adjacent uninjured area

Injury to the growth plate is common and yet the injured cartilage is often repaired with undesirable bony tissue, leading to bone growth defects in children. Using a rat tibial growth plate injury model, our previous studies have shown sequential inflammatory, fibrogenic, osteogenic and bone maturation responses involved in the bony repair. However, it remains unclear whether there is progressive accumulation of bone within the injury site and any potential degenerative changes at the adjacent non-injured area of the growth plate. This study examined effects of growth plate injury on the structure, composition and some cellular and molecular changes at the injury site and adjacent uninjured area. Micro-CT analysis revealed that while the bone volume within the injury site at day 14 was small, the bone bridge was considerably larger at the injury site by 60 days post-injury. Interestingly, formation of bone bridges in the adjacent uninjured area was detected in 60% of injured animals at day 60. Immunohistochemical analyses revealed reduced chondrocyte proliferation (PCNA labelling) but increased apoptosis (nick translation labelling) in the adjacent uninjured area. RT-PCR analysis on adjacent uninjured growth plate tissue found increased expression of osteocalcin at day 60, differential expression of apoptosis-regulatory genes and alterations in genes associated with chondrocyte proliferation/differentiation, including Sox9 and IGF-I. Therefore, this study has demonstrated progressive changes in the structure/composition of the injury site and adjacent uninjured area and identified cellular and molecular alterations or degeneration in adjacent uninjured growth plate in response to injury.C.E. Macsai, B. Hopwood, R. Chung, B.K. Foster, C.J. Xia

Cellular mechanisms for methotrexate chemotherapy-induced bone growth defects

Crown copyright © 2007 Published by Elsevier Inc.Methotrexate (MTX) is a commonly used anti-metabolite in childhood oncology and is known to cause bone growth arrest and osteoporosis; yet the underlying mechanisms for MTX-induced bone growth defects remain largely unclear. This study characterized damaging effects in young rats of acute chemotherapy with 5 once-daily doses of MTX (0.75 mg/kg) on the cellular activities in the growth plate in producing calcified cartilage and trabecular bone and on activities of osteoblastic cells in the metaphysis. MTX treatment significantly induced chondrocyte apoptosis. MTX also suppressed chondrocyte proliferation and reduced collagen-II mRNA expression and total thickness of the growth plate, with the damage being most obvious on day 9 after the first injection, and with the growth plate histological structure returning normal on day 14. In the adjacent metaphyseal bone, mirroring the decrease in the width of the growth plate, production of primary spongiosa bone was markedly reduced and bone volume of the secondary spongiosa was decreased. Furthermore, MTX treatment significantly induced osteocyte apoptosis in the primary spongiosa and reduced proliferation of osteoblasts and preosteoblasts particularly in the secondary spongiosa. These observations suggest that methotrexate chemotherapy may cause bone growth defects by arresting cellular activities in the growth plate in producing calcified cartilage and primary trabecular bone and by decreasing pools of metaphyseal osteoblastic cells. However, this short-term MTX treatment only caused transit suppressions on growth plate cartilage and trabecular bone, as most cellular and histological parameters had recovered by day 14 or 21.Cory J. Xian, Johanna C. Cool, Michaela A. Scherer, Carmen E. Macsai, Chiaming Fan, Mark Covino and Bruce K. Fosterhttp://www.elsevier.com/wps/find/journaldescription.cws_home/525233/description#descriptio

Reversal of established bone pathology in MPS VII mice following lentiviral-mediated gene therapy

Abstract not availableAinslie L.K. Derrick-Roberts, Kavita Panir, Carmen E. Pyragius, Krystyna H. Zarrinkalam, Gerald J. Atkins, Sharon Byer

Lentiviral-mediated gene therapy results in sustained expression of beta-Glucuronidase for up to 12 Months in the Gus mps/mps and up to 18 Months in the Gus tm(L175F)Sly mouse models of mucopolysaccharidosis type VII

A number of mucopolysaccharidosis type VII (MPS VII) mouse models with different levels of residual enzyme activity have been created replicating the range of clinical phenotypes observed in human MPS VII patients. In this study, a lentivirus encoding murine β-glucuronidase was administered intravenously at birth to both the severe (Gus(mps/mps) strain) and attenuated (Gus(tm(L175F)Sly) strain) mouse models of MPS VII. Circulating enzyme levels were normalized in the Gus(mps/mps) mice and were 3.5-fold higher than normal in the Gus(tm(L175F)Sly) mouse 12 and 18 months after administration. Tissue β-glucuronidase activity increased over untreated levels in all tissues evaluated in both strains at 12 months, and the elevated level was maintained in Gus(tm(L175F)Sly) tissues at 18 months. These elevated enzyme levels reduced glycosaminoglycan storage in the liver, spleen, kidney, and heart in both models. Bone mineral volume decreased toward normal in both models after 12 months of therapy and after 18 months in the Gus(tm(L175F)Sly) mouse. Open-field exploration was improved in 18-month-old treated Gus(tm(L175F)Sly) mice, while spatial learning improved in both 12- and 18-month-old treated Gus(tm(L175F)Sly) mice. Overall, neonatal administration of lentiviral gene therapy resulted in sustained enzyme expression for up to 18 months in murine models of MPS VII. Significant improvements in biochemistry and enzymology as well as functional improvement of bone and behavior deficits in the Gus(tm(L175F)Sly) model were observed. Therapy significantly increased the lifespan of Gus(mps/mps) mice, with 12 months being the longest reported lentiviral treatment for this strain. It is important to assess the long-term outcome on enzyme levels and effect on pathology for lentiviral gene therapy to be a potential therapy for MPS patients.Ainslie L.K. Derrick-Roberts, Carmen E. Pyragius, Xenia M. Kaidonis, Matilda R. Jackson, Donald S. Anson, and Sharon Byer

Skeletal response to lentiviral mediated gene therapy in a mouse model of MPS VII

Mucopolysaccharidosis VII (MPS VII) is an autosomal recessive, lysosomal storage disorder caused by β-glucuronidase (GUSB) deficiency, resulting in the accumulation of glycosaminoglycans (GAGs), in a variety of cell types. Severe, progressive skeletal pathology, termed dysostosis multiplex, is a prominent clinical feature of MPS VII. We have evaluated a gene therapy protocol for its efficacy in preventing the development and progression of bone pathology in MPS VII mice treated with a lentiviral vector at birth or at 7 weeks. Two weeks after injections, high levels of vector expression were observed in liver, spleen and bone marrow and to a lesser extent in kidney, lung and heart. Widespread clearance of GAG storage was observed in somatic tissues of both groups and some clearance of neuronal storage was observed in mice treated from birth. Micro-CT analysis demonstrated a significant decrease in vertebral and femoral bone mineral volume, trabecular number, bone surface density and cortical bone thickness in both treatment groups. Lumbar and femoral bone lengths were significantly decreased in untreated MPS VII mice, while growth plate heights were increased and these parameters did not change upon treatment. Small improvements in performance in the open field and rotarod behaviour tests were noted. Overall, systemic lentiviral-mediated gene therapy results in a measurable improvement in parameters of bone mass and architecture as well as biochemical and enzymatic correction. Conversely, growth plate chondrocytes were not responsive to treatment, as evidenced by the lack of improvement in vertebral and femoral bone length and growth plate height.Carmen E. Macsai, Ainslie L.K. Derrick-Roberts, Xiaodan Ding, Krystyna H. Zarrinkalam, Chantelle McIntyre, Paul H. Anderson, Don S. Anson and Sharon Byer