Delayed development of ossification centers in the tibia of prenatal and early postnatal MPS VII mice.

Short stature is a characteristic feature of most of the mucopolysaccharidoses, a group of inherited lysosomal storage disorders caused by a single enzyme deficiency. MPS patients present with progressive skeletal defects from an early age, including short stature due to impaired cartilage-to-bone conversion (endochondral ossification). The aim of this study was to determine which murine MPS model best reproduces the bone length reduction phenotype of human MPS and use this model to determine the earliest developmental stage when disrupted endochondral ossification first appears. Gusmps/mps mice representing severe MPS VII displayed the greatest reduction in bone elongation and were chosen for histopathological analysis. Tibial development was assessed from E12.5 to 6 months of age. Chondrocytes in the region of the future primary ossification center became hypertrophic at a similar age to normal in the MPS VII mouse fetus, but a delay in bone deposition was observed with an approximate 1 day delay in the formation of the primary ossification centre. Likewise, chondrocytes in the region of the future secondary ossification center also became hypertrophic at the same age as normal in the MPS VII early postnatal mouse. Bone deposition in the secondary ossification centre was delayed by two days in the MPS VII proximal tibia (observed at postnatal day 14 (P14) compared to P12 in normal). The thickness of the tibial growth plate was larger in MPS VII mice from P9 onwards. Abnormal endochondral ossification starts in utero in MPS VII and worsens with age. It is characterized by a normal timeframe for chondrocyte hypertrophy but a delay in the subsequent deposition of bone in both the primary and secondary ossification centres, accompanied by an increase in growth plate thickness. This suggests that the signals for vascular invasion and bone deposition, some of which are derived from hypertrophic chondrocytes, are altered in MPS VII.

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

Severe, progressive skeletal dysplasia is a major symptom of multiple mucopolysaccharidoses (MPS) types. While a gene therapy approach initiated at birth has been shown to prevent the development of bone pathology in different animal models of MPS, the capacity to correct developed bone disease is unknown. In this study, ex vivo micro-computed tomography was used to demonstrate that bone mass and architecture of murine MPS VII L5 vertebrae were within the normal range at 1month of age but by 2months of age were significantly different to normal. The difference between normal and MPS VII BV/TV increased with age reaching a maximal difference at approximately 4months of age. In mature MPS VII bone BV/TV is increased (51.5% versus 21.5% in normal mice) due to an increase in trabecular number (6.2permm versus 3.8permm in normal mice). The total number of osteoclasts in the metaphysis of MPS VII mice was decreased, as was the percentage of osteoclasts attached to bone. MPS VII osteoblasts produced significantly more osteoprotegerin (OPG) than normal osteoblasts and supported the production of fewer osteoclasts from spleen precursor cells than normal osteoblasts in a co-culture system. In contrast, the formation of osteoclasts from MPS VII spleen monocytes was similar to normal in vitro, when exogenous RANKL and m-CSF was added to the culture medium. Administration of murine ß-glucuronidase to MPS VII mice at 4months of age, when bone disease was fully manifested, using lentiviral gene delivery resulted in a doubling of osteoclast numbers and a significant increase in attachment capacity (68% versus 29.4% in untreated MPS VII animals). Bone mineral volume rapidly decreased by 39% after gene therapy and fell within the normal range by 6months of age. Collectively, these results indicate that lentiviral-mediated gene therapy is effective in reversing established skeletal pathology in murine MPS VII.

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.

Aberrant lipid metabolism: an emerging diagnostic and therapeutic target in ovarian cancer.

Ovarian cancer remains the most lethal gynaecological cancer. A better understanding of the molecular pathogenesis of ovarian cancer is of critical importance to develop early detection tests and identify new therapeutic targets that would increase survival. Cancer cells depend on de novo lipid synthesis for the generation of fatty acids to meet the energy requirements for increased tumour growth. There is increasing evidence that lipid metabolism is deregulated in cancers, including ovarian cancer. The increased expression and activity of lipogenic enzymes is largely responsible for increased lipid synthesis, which is regulated by metabolic and oncogenic signalling pathways. This article reviews the latest knowledge on lipid metabolism and the alterations in the expression of lipogenic enzymes and downstream signalling pathways in ovarian cancer. Current developments for exploiting lipids as biomarkers for the detection of early stage ovarian cancer and therapeutic targets are discussed. Current research targeting lipogenic enzymes and lipids to increase the cytotoxicity of chemotherapy drugs is also highlighted.

Novel ex vivo ovarian cancer tissue explant assay for prediction of chemosensitivity and response to novel therapeutics.

The majority of ovarian cancer patients present with advanced disease and despite aggressive treatment, prognosis remains poor. Response to first-line carboplatin-containing chemotherapy is usually good, however, recurrence rates and subsequent chemoresistance are very high and ultimately responsible for the fatal outcome of the disease. To improve treatment outcomes pre-clinical models that can predict individual patient response to 1st line chemotherapy and novel therapeutics are urgently required. In this study, we employed an ex vivo ovarian cancer tissue explant assay to assess response to carboplatin and an inhibitor of the extracellular matrix molecule, hyaluronan (4-methylubelliferone, 4-MU), shown to inhibit cancer metastasis. Cryopreserved ovarian cancer tissues were cultured on gelatine sponges for 48-120 h with increasing concentrations of carboplatin (0-400 µM) or 4-MU (1 mM) alone or the combination of both drugs. Effects on apoptosis and proliferation were assessed by immunohistochemistry using antibodies to cleaved caspase 3 or Ki67, respectively. The ex vivo tissue explant assay maintained viable tumor cells in an intact tumor microenvironment similar to the in vivo situation over the 120 h culture period. Carboplatin treatment promoted apoptosis in chemosensitive (P = 0.0047) but not chemoresistant cancer tissues. The combination of 4-MU (1 mM) and carboplatin (100 µM) significantly increased apoptosis (P = 0.0111) and reduced proliferation (P = 0.0064) in chemoresistant tissues. Overall, our results show that the ex vivo explant assay is a robust and cost effective model to assess chemosensitivity and the effect of novel therapeutics in ovarian cancer.

Substrate Deprivation Therapy to Reduce Glycosaminoglycan Synthesis Improves Aspects of Neurological and Skeletal Pathology in MPS I Mice.

Mucopolysaccharidosis type I (MPS I) is the most common form of the MPS group of genetic diseases. MPS I results from a deficiency in the lysosomal enzyme α-l-iduronidase, leading to accumulation of undegraded heparan and dermatan sulphate glycosaminoglycan (GAG) chains in patient cells. MPS children suffer from multiple organ failure and die in their teens to early twenties. In particular, MPS I children also suffer from profound mental retardation and skeletal disease that restricts growth and movement. Neither brain nor skeletal disease is adequately treated by current therapy approaches. To overcome these barriers to effective therapy we have developed and tested a treatment called substrate deprivation therapy (SDT). MPS I knockout mice were treated with weekly intravenous injections of 1 mg/kg rhodamine B for six months to assess the efficacy of SDT. Mice were assessed using biochemistry, micro-CT and a battery of behaviour tests to determine the outcome of treatment. A reduction in female bodyweight gain was observed with the treatment as well as a decrease in lung GAG. Behavioural studies showed slight improvements in inverted grid and significant improvements in learning ability for female MPS I mice treated with rhodamine B. Skeletal disease also improved with a reduction in bone mineral volume observed. Overall, rhodamine B is safe to administer to MPS I knockout mice where it had an effect on improving aspects of neurological and skeletal disease symptoms and may therefore provide a potential therapy or adjunct therapy for MPS I patients.

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.

Annexin A2 and S100A10 are independent predictors of serous ovarian cancer outcome.

Annexin A2, a calcium phospholipid binding protein, has been shown to play an important role in ovarian cancer metastasis. This study examined whether annexin A2 and S100A10 can be used as prognostic markers in serous ovarian cancer. ANXA2 and S100A10 gene expressions were assessed in publicly available ovarian cancer data sets and annexin A2 and S100A10 protein expressions were assessed by immunohistochemistry in a uniform cohort of stage III serous ovarian cancers (n = 109). Kaplan-Meier and Cox regression analyses were performed to assess the relationship between annexin A2 or S100A10 messenger RNA (mRNA) and protein expressions with clinical outcome. High ANXA2 mRNA levels in stage III serous ovarian cancers were associated with reduced progression-free survival (PFS; P = 0.023) and overall survival (OS; P = 0.0038), whereas high S100A10 mRNA levels predicted reduced OS (P = 0.0019). Using The Cancer Genome Atlas data sets, ANXA2 but not S100A10 expression was associated with higher clinical stage (P = 0.005), whereas both ANXA2 and S100A10 expressions were associated with the mesenchymal molecular subtype (P < 0.0001). Kaplan-Meier and Cox regression analyses showed that high stromal annexin A2 immunostaining was significantly associated with reduced PFS (P = 0.013) and OS (P = 0.044). Moreover, high cytoplasmic S100A10 staining was significantly associated with reduced OS (P = 0.027). Multivariate Cox regression analysis showed stromal annexin A2 (P = 0.009) and cytoplasmic S100A10 (P = 0.016) levels to be independent predictors of OS. Patients with high stromal annexin A2 and high cytoplasmic S100A10 expressions had a 3.4-fold increased risk of progression (P = 0.02) and 7.9-fold risk of ovarian cancer death (P = 0.04). Our findings indicate that together annexin A2 and S100A10 expressions are powerful predictors of serous ovarian cancer outcome.

Transketolase is upregulated in metastatic peritoneal implants and promotes ovarian cancer cell proliferation.

Ovarian cancer, the most lethal gynaecological cancer, is characterised by the shedding of epithelial cells from the ovarian surface, followed by metastasis and implantation onto the peritoneal surfaces of abdominal organs. Our proteomic studies investigating the interactions between peritoneal (LP-9) and ovarian cancer (OVCAR-5) cells found transketolase (TKT) to be regulated in the co-culture system. This study characterized TKT expression in advanced stage (III/IV) serous ovarian cancers (n = 125 primary and n = 54 peritoneal metastases), normal ovaries (n = 6) and benign serous cystadenomas (n = 10) by immunohistochemistry. In addition, we also evaluated the function of TKT in ovarian cancer cells in vitro. Nuclear TKT was present in all primary serous ovarian cancer tissues examined (median 82.0 %, range 16.5-100 %) and was significantly increased in peritoneal metastases compared with matching primary cancers (P = 0.01, Wilcoxon Rank test). Kaplan-Meier survival and Cox regression analyses showed that high nuclear TKT positivity in peritoneal metastases (>94 %) was significantly associated with reduced overall survival (P = 0.006) and a 2.8 fold increased risk of ovarian cancer death (95 % CI 1.29-5.90, P = 0.009). Knockdown of TKT by siRNAs significantly reduced SKOV-3 cell proliferation but had no effect on their motility or invasion. Oxythiamine, an inhibitor of TKT activity, significantly inhibited the proliferation of four ovarian cancer cell lines (OV-90, SKOV-3, OVCAR-3 and OVCAR-5) and primary serous ovarian cancer cells isolated from patient ascites. In conclusion, these findings indicate that TKT plays an important role in the proliferation of metastatic ovarian cancer cells and could be used as novel therapeutic target for advanced disease.

Lentiviral-mediated gene therapy results in sustained expression of ß-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 ß-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 ß-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.

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.