Calpain-2 expression is associated with response to platinum based chemotherapy, progression-free and overall survival in ovarian cancer

Ovarian cancer is routinely treated with surgery and platinum‐based chemotherapy. Resistance is a major obstacle in the efficacy of this chemotherapy regimen and the ability to identify those patients at risk of developing resistance is of considerable clinical importance. The expression of calpain‐1, calpain‐2 and calpastatin were determined using standard immunohistochemistry on a tissue microarray of 154 primary ovarian carcinomas from patients subsequently treated with platinum‐based adjuvant chemotherapy. High levels of calpain‐2 expression was significantly associated with platinum resistant tumours (P = 0.031). Furthermore, high expression of calpain‐2 was significantly associated with progression‐free (P = 0.049) and overall survival (P = 0.006) in this cohort. The association between calpain‐2 expression and overall survival remained significant in multivariate analysis accounting for tumour grade, stage, optimal debulking and platinum sensitivity (hazard ratio = 2.174; 95% confidence interval = 1.144–4.130; P = 0.018). The results suggest that determining calpain‐2 expression in ovarian carcinomas may allow prognostic stratification of patients treated with surgery and platinum‐based chemotherapy. The findings of this study warrant validation in a larger clinical cohort.     

Variants in Notch signalling pathway genes,PSEN1 and MAML2, predict overall survival in Chinese patients with epithelial ovarian cancer

To identify genetic variants in Notch signalling pathway genes that may predict survival of Han Chinese patients with epithelial ovarian cancer (EOC), we analysed a total of 1273 single nucleotide polymorphisms (SNPs) within 75 Notch genes in 480 patients from a published EOC genomewide association study (GWAS). We found that PSEN1 rs165934 and MAML2 rs76032516 were associated with overall survival (OS) of patients by multivariate Cox proportional hazards regression analysis. Specifically, the PSEN1 rs165934 AA genotype was associated with a poorer survival (adjusted hazards ratio [adjHR] = 1.41, 95% CI = 1.07‐1.84, and P = .014), compared with the CC + CA genotype, while MAML2 rs76032516 AA + AC genotypes were associated with a poorer survival (adjHR = 1.58, 95% CI = 1.16‐2.14, P = .004), compared with the CC genotype. The combined analysis of these two SNPs revealed that the death risk increased as the number of unfavourable genotypes increased in a dose‐dependent manner (P_trend < .001). Additionally, the expression quantitative trait loci analysis revealed that the SNP rs165932 in the rs165934 LD block (r² = .946) was associated with expression levels of PSEN1, which might be responsible for the observed association with SNP rs165934. The associations of PSEN1 rs165934 and MAML2 rs76032516 of the Notch signalling pathway genes with OS in Chinese EOC patients are novel findings, which need to be validated in other large and independent studies.     

Failure of 5-thio-D-glucose to alter cell survival in irradiated or unirradiated EMT6 tumors

The antineoplastic effects of the glucose analog 5-thio-D-glucose (5TG) were tested using EMT6 mouse mammary tumors in vivo and EMT6 cells in cell culture. In vitro, 5TG selectively killed hypoxic EMT6 cells. However, administration of 5TG to mice bearing EMT6 tumors produced no significant toxicity to the cells of unirradiated tumors and did not alter the survival of cells in irradiated tumors. Fasting the mice to lower blood glucose concentrations before administration of 5TG increased the toxicity of the drug to the mice, but did not allow more efficacious treatment of the tumors. The data provided no evidence that 5-thio-D-glucose can be used effectively for the treatment of solid tumors, either as a cytotoxic agent or as a radiosensitizer.     

Intraperitoneal delivery of human natural killer cells for treatment of ovarian cancer in a mouse xenograft model

Background aimsThere is an urgent need for novel therapeutic strategies for relapsed ovarian cancer. Dramatic clinical anti-tumor effects have been observed with interleukin (IL)-2 activated natural killer (NK) cells; however, intravenous delivery of NK cells in patients with ovarian cancer has not been successful in ameliorating disease. We investigated in vivo engraftment of intraperitoneally (IP) delivered NK cells in an ovarian cancer xenograft model to determine if delivery mode can affect tumor cell killing and circumvent lack of NK cell expansion.MethodsAn ovarian cancer xenograft mouse model was established to evaluate efficacy of IP-delivered NK cells. Tumor burden was monitored by bioluminescent imaging of luciferase-expressing ovarian cancer cells. NK cell persistence, tumor burden and NK cell trafficking were evaluated. Transplanted NK cells were evaluated by flow cytometry and cytotoxicity assays.ResultsIP delivery of human NK cells plus cytokines led to high levels of circulating NK and was effective in clearing intraperitoneal ovarian cancer burden in xenografted mice. NK cells remained within the peritoneal cavity 54 days after injection and had markers of maturation. Additionally, surviving NK cells were able to kill ovarian cancer cells at a rate similar to pre-infusion levels, supporting that in vivo functionality of human NK cells can be maintained after IP infusion.ConclusionsIP delivery of NK cells leads to stable engraftment and antitumor response in an ovarian cancer xenograft model. These data support further pre-clinical and clinical evaluation of IP delivery of allogeneic NK cells in ovarian cancer.     

Association of expression of XIAP-associated factor 1 (XAF1) with clinicopathologic factors, overall survival, microvessel density and cisplatin-resistance in ovarian cancer

XIAP-associated factor 1 (XAF1) was identified as a novel X-linked inhibitor of apoptosis (XIAP) binding partner that can reverse the anti-apoptotic effect of XIAP. XAF1 levels are greatly decreased in many cancer tissues and cell lines. The aim of this study was to investigate the expression of XAF1 and XIAP in advanced epithelial ovarian cancer and role of XAF1 in cisplatin resistance of ovarian cancer cells. Tissues from 94 patients with advanced epithelial ovarian cancer (EOC) and 30 ovarian cystadenomas were obtained. We analyzed the association of the immunohistochemical-determined expression of these two factors and clinicopathologic variables, overall survival, and angiogenesis. We established SKOV3 cells stably overexpressing XAF1 and explored the possible functions of XAF1 in ovarian cancer cells in vitro and in vivo. The protein expression of XAF1 was significantly lower and that of XIAP higher in malignant than nonmalignant tissues. Low XAF1 expression was associated with high-grade tumors and poor overall survival for patients. XAF1 expression was associated with microvessel density. Overexpression of XAF1 suppressed cell proliferation and enhanced SKOV3 cells sensitivity to cisplatin, as well as inhibited tumor growth and decreased MVD in vivo. Overexpression of XAF1 induced XIAP inactivation, caspase-3 activation and cytosolic expression of cytochrome c. These results suggested that XAF1 may be involved in ovarian cancer development and up-regulation of XAF1 may confer sensitivity of ovarian cancer cells to cisplatin-mediated apoptosis.     

Conjugated linoleic acids differentially alter polyp number and diameter in the Apc(min/+) mouse model of intestinal cancer

Abstract.   Objective:  Dietary conjugated linoleic acids (CLA) have had many health benefits claimed for them, including antineoplastic actions. Materials and methods:  The effects of the predominant forms of CLA, namely the c9t11 and t10c12 isomers, or a mixture of these on polyp development, were investigated in the Apc ^Min/+ mouse. CLAs have also been linked to altered rates of cell renewal and cell proliferation so this was also studied, as was a further means of increasing tissue mass, namely crypt fission. Results:  The stomach and small intestine were significantly heavier in the t10c12, and in the mixture-treated groups ( P <  0.001). Crypt fission was increased in the middle small intestine by the t10c12 diet while colonic weight was reduced by c9t11 provision and crypts were 20% shorter. The t10c12 and the mixture significantly reduced polyp number in the proximal small intestine but they increased polyp diameter in the middle and distal small intestine, to an extent that the polyp burden was significantly increased at these sites. All CLAs significantly reduced polyp number in the colon, but the mixture significantly increased polyp diameter in the colon. Conclusion : Increased polyp diameter associated with t10c12 diet and especially with the mixture is a cause of concern, as this is the commercially available form. The naturally occurring isomer, c9t11 decreased colonic polyp number and did not increase diameter, suggesting that this natural isomer is the most likely to be protective.     

Expression of death receptor 6 by ovarian tumors in laying hens, a preclinical model of spontaneous ovarian cancer

Tumor-associated neoangiogenesis and suppression of antitumor immunity are hallmarks of tumor development and progression. Death receptor 6 (DR6) has been reported to be associated with suppression of antitumor immunity and tumor progression in several malignancies. However, expression of DR6 by malignant ovarian epithelial tumors at an early stage is unknown. The goals of this study were to determine whether DR6 is expressed by malignant ovarian epithelial tumors at an early stage and to examine whether DR6 expression is associated with ovarian cancer (OVCA) progression in a laying hen model of spontaneous OVCA. Expression of DR6 was examined in normal and malignant ovaries, normal ovarian surface epithelial (OSE) cells, or malignant epithelial cells and in serum of 3-year-old hens. The population of microvessels expressing DR6 was significantly higher in hens with early-stage OVCA than hens with normal ovaries (P < .01) and increased further in late-stage OVCA. The results of this study showed that, in addition to microvessels, tumor cells in the ovary also express DR6 with a significantly higher intensity than normal OSE cells. Similar patterns of DR6 expression were also observed by immunoblot analysis and gene expression studies. Furthermore, DR6 was also detected in the serum of hens. In conclusion, DR6 expression is associated with OVCA development and progression in laying hens. This study may be helpful to examine the feasibility of DR6 as a useful surrogate marker of OVCA, a target for antitumor immunotherapy and molecular imaging and thus provide a foundation for clinical studies.     

Racial disparities in treatment and survival from ovarian cancer

BackgroundBlack women with ovarian cancer in the U.S. have lower survival than whites. We aimed to identify factors associated with racial differences in ovarian cancer treatment and overall survival (OS).MethodsWe examined data from 365 white and 95 black ovarian cancer patients from the Hollings Cancer Center Cancer Registry in Charleston, S.C. between 2000 and 2015. We used unconditional logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs) between race and receipt of surgery and chemotherapy, and Cox proportional hazards regression to estimate hazard ratios (HRs) and 95% CIs between race and OS. Model variables included diagnosis center, stage, histology, insurance status, smoking, age-adjusted Charlson comorbidity index (AACI) and residual disease. Interactions between race and AACI were assessed using −2 log likelihood tests.ResultsBlacks vs. whites were over two-fold less likely to receive a surgery-chemotherapy sequence (multivariable-adjusted OR 2.46, 95% CI 1.43–4.21), particularly if they had a higher AACI (interaction p = 0.008). In multivariable-adjusted Cox models, black women were at higher risk of death (HR 1.81, 95% CI 1.35–2.43) than whites, even when restricted to patients who received a surgery-chemotherapy sequence (HR 1.79, 95% CI 1.10–2.89) and particularly for those with higher AACI (HR 4.70, 95% CI 2.00 − 11.02, interaction p = 0.01).ConclusionsAmong blacks, higher comorbidity associates with less chance of receiving guideline-based treatment and also modifies OS. Differences in receipt of guideline-based care do not completely explain survival differences between blacks and whites with ovarian cancer. These results highlight opportunities for further research.     

The role of alpha 6 integrin in prostate cancer migration and bone pain in a novel xenograft model

Of the estimated 565,650 people in the U.S. who will die of cancer in 2008, almost all will have metastasis. Breast, prostate, kidney, thyroid and lung cancers metastasize to the bone. Tumor cells reside within the bone using integrin type cell adhesion receptors and elicit incapacitating bone pain and fractures. In particular, metastatic human prostate tumors express and cleave the integrin A6, a receptor for extracellular matrix components of the bone, i.e., laminin 332 and laminin 511. More than 50% of all prostate cancer patients develop severe bone pain during their remaining lifetime. One major goal is to prevent or delay cancer induced bone pain. We used a novel xenograft mouse model to directly determine if bone pain could be prevented by blocking the known cleavage of the A6 integrin adhesion receptor. Human tumor cells expressing either the wildtype or mutated A6 integrin were placed within the living bone matrix and 21 days later, integrin expression was confirmed by RT-PCR, radiographs were collected and behavioral measurements of spontaneous and evoked pain performed. All animals independent of integrin status had indistinguishable tumor burden and developed bone loss 21 days after surgery. A comparison of animals containing the wild type or mutated integrin revealed that tumor cells expressing the mutated integrin resulted in a dramatic decrease in bone loss, unicortical or bicortical fractures and a decrease in the ability of tumor cells to reach the epiphyseal plate of the bone. Further, tumor cells within the bone expressing the integrin mutation prevented cancer induced spontaneous flinching, tactile allodynia, and movement evoked pain. Preventing A6 integrin cleavage on the prostate tumor cell surface decreased the migration of tumor cells within the bone and the onset and degree of bone pain and fractures. These results suggest that strategies for blocking the cleavage of the adhesion receptors on the tumor cell surface can significantly prevent cancer induced bone pain and slow disease progression within the bone. Since integrin cleavage is mediated by Urokinase-type Plasminogen Activator (uPA), further work is warranted to test the efficacy of uPA inhibitors for prevention or delay of cancer induced bone pain.     

Vagal nerve activity predicts overall survival in metastatic pancreatic cancer,mediated by inflammation

Recent research findings suggest neuro-modulation of tumors. Finding new modifiable prognostic factors paves the way for additional treatments, which is crucial in advanced cancer, particularly pancreatic cancer. This study examined the relationship between vagal nerve activity, indexed by heart rate variability (HRV), and overall survival (OS) in patients (N = 272) with advanced pancreatic cancer. A “historical prospective” design was employed, where vagal activity and other confounders were retroactively obtained from medical charts at diagnosis, and subsequent OS was examined. HRV was obtained from 10 sec ECGs near diagnosis. Levels of C-reactive protein (CRP) were measured as an inflammatory marker. OS and survival date were obtained from medical charts and the Belgian national registry. Patients with high HRV (>20 msec) survived on average more than double the days (133.5) than those with low HRV (64.0). In a multivariate cox regression, higher initial HRV was significantly correlated with lower risk of death, independent of confounders including age and cancer treatments. This relationship was statistically mediated (accounted for) by CRP levels. Importantly, in patients who lived up to one month from diagnosis only, HRV was unrelated to CRP, while in patients surviving longer, HRV was significantly inversely related to CRP (r = −0.20, p < 0.05). These results are in line with possible vagal nerve protection in a fatal cancer, and propose that the mechanism may involve neuroimmuno-modulation. Future studies must test whether vagal nerve activation may help patients with advanced cancers.     

A novel protease homolog differentially expressed in breast and ovarian cancer.

BACKGROUND: Using differential display (DD), we discovered a new member of the serine protease family of protein-cleaving enzymes, named protease M. The gene is most closely related by sequence to the kallikreins, to prostate-specific antigen (PSA), and to trypsin. The diagnostic use of PSA in prostate cancer suggested that a related molecule might be a predictor for breast or ovarian cancer. This, in turn, led to studies designed to characterize the protein and to screen for its expression in cancer. MATERIALS AND METHODS: The isolation of protease M by DD, the cloning and sequencing of the cDNA, and the comparison of the predicted protein structure with related proteins are described, as are methods to produce recombinant proteins and polyclonal antibody preparations. Protease M expression was examined in mammary, prostate, and ovarian cancer, as well as normal, cells and tissues. Stable transfectants expressing the protease M gene were produced in mammary carcinoma cells. RESULTS: Protease M was localized by fluorescent in situ hybridization analysis to chromosome 19q13.3, in a region to which other kallikreins and PSA also map. The gene is expressed in the primary mammary carcinoma lines tested but not in the corresponding cell lines of metastatic origin. It is strongly expressed in ovarian cancer tissues and cell lines. The enzyme activity could not be established, because of difficulties in producing sufficient recombinant protein, a common problem with proteases. Transfectants were selected that overexpress the mRNA, but the protein levels remained very low. CONCLUSIONS: Protease M expression (mRNA) may be a useful marker in the detection of primary mammary carcinomas, as well as primary ovarian cancers. Other medical applications are also likely, based on sequence relatedness to trypsin and PSA.     

Anti-MUC1 monoclonal antibody (C595) and docetaxel markedly reduce tumor burden and ascites, and prolong survival in an in vivo ovarian cancer model

MUC1 is associated with cellular transformation and tumorigenicity and is considered as an important tumor-associated antigen (TAA) for cancer therapy. We previously reported that anti-MUC1 monoclonal antibody C595 (MAb C595) plus docetaxel (DTX) increased efficacy of DTX alone and caused cultured human epithelial ovarian cancer (EOC) cells to undergo apoptosis. To further study the mechanisms of this combination-mediated apoptosis, we investigated the effectiveness of this combination therapy in vivo in an intraperitoneal (i.p.) EOC mouse model. OVCAR-3 cells were implanted intraperitoneally in female athymic nude mice and allowed to grow tumor and ascites. Mice were then treated with single MAb C595, DTX, combination test (MAb C595 and DTX), combination control (negative MAb IgG(3) and DTX) or vehicle control i.p. for 3 weeks. Treated mice were killed 4 weeks post-treatment. Ascites volume, tumor weight, CA125 levels from ascites and survival of animals were assessed. The expression of MUC1, CD31, Ki-67, TUNEL and apoptotic proteins in tumor xenografts was evaluated by immunohistochemistry. MAb C595 alone inhibited i.p. tumor growth and ascites production in a dose-dependent manner but did not obviously prevent tumor development. However, combination test significantly reduced ascites volume, tumor growth and metastases, CA125 levels in ascites and improved survival of treated mice compared with single agent-treated mice, combination control or vehicle control-treated mice (P<0.05). The data was in a good agreement with that from cultured cells in vitro. The mechanisms behind the observed effects could be through targeting MUC1 antigens, inhibition of tumor angiogenesis, and induction of apoptosis. Our results suggest that this combination approach can effectively reduce tumor burden and ascites, prolong survival of animals through induction of tumor apoptosis and necrosis, and may provide a potential therapy for advanced metastatic EOC.     

Changes in gene expression and cellular architecture in an ovarian cancer progression model

Background

Ovarian cancer is the fifth leading cause of cancer deaths among women. Early stage disease often remains undetected due the lack of symptoms and reliable biomarkers. The identification of early genetic changes could provide insights into novel signaling pathways that may be exploited for early detection and treatment.

Methodology/Principal Findings

Mouse ovarian surface epithelial (MOSE) cells were used to identify stage-dependent changes in gene expression levels and signal transduction pathways by mouse whole genome microarray analyses and gene ontology. These cells have undergone spontaneous transformation in cell culture and transitioned from non-tumorigenic to intermediate and aggressive, malignant phenotypes. Significantly changed genes were overrepresented in a number of pathways, most notably the cytoskeleton functional category. Concurrent with gene expression changes, the cytoskeletal architecture became progressively disorganized, resulting in aberrant expression or subcellular distribution of key cytoskeletal regulatory proteins (focal adhesion kinase, α-actinin, and vinculin). The cytoskeletal disorganization was accompanied by altered patterns of serine and tyrosine phosphorylation as well as changed expression and subcellular localization of integral signaling intermediates APC and PKCβII.

Conclusions/Significance

Our studies have identified genes that are aberrantly expressed during MOSE cell neoplastic progression. We show that early stage dysregulation of actin microfilaments is followed by progressive disorganization of microtubules and intermediate filaments at later stages. These stage-specific, step-wise changes provide further insights into the time and spatial sequence of events that lead to the fully transformed state since these changes are also observed in aggressive human ovarian cancer cell lines independent of their histological type. Moreover, our studies support a link between aberrant cytoskeleton organization and regulation of important downstream signaling events that may be involved in cancer progression. Thus, our MOSE-derived cell model represents a unique model for in depth mechanistic studies of ovarian cancer progression.     

Efficacy of a non-hypercalcemic vitamin-D2 derived anti-cancer agent (MT19c) and inhibition of fatty acid synthesis in an ovarian cancer xenograft model

Background

Numerous vitamin-D analogs exhibited poor response rates, high systemic toxicities and hypercalcemia in human trials to treat cancer. We identified the first non-hypercalcemic anti-cancer vitamin D analog MT19c by altering the A-ring of ergocalciferol. This study describes the therapeutic efficacy and mechanism of action of MT19c in both in vitro and in vivo models.

Methodology/Principal Finding

Antitumor efficacy of MT19c was evaluated in ovarian cancer cell (SKOV-3) xenografts in nude mice and a syngenic rat ovarian cancer model. Serum calcium levels of MT19c or calcitriol treated animals were measured. In-silico molecular docking simulation and a cell based VDR reporter assay revealed MT19c–VDR interaction. Genomewide mRNA analysis of MT19c treated tumors identified drug targets which were verified by immunoblotting and microscopy. Quantification of cellular malonyl CoA was carried out by HPLC-MS. A binding study with PPAR-Y receptor was performed. MT19c reduced ovarian cancer growth in xenograft and syngeneic animal models without causing hypercalcemia or acute toxicity. MT19c is a weak vitamin-D receptor (VDR) antagonist that disrupted the interaction between VDR and coactivator SRC2-3. Genome-wide mRNA analysis and western blot and microscopy of MT19c treated xenograft tumors showed inhibition of fatty acid synthase (FASN) activity. MT19c reduced cellular levels of malonyl CoA in SKOV-3 cells and inhibited EGFR/phosphoinositol-3kinase (PI-3K) activity independently of PPAR-gamma protein.

Significance

Antitumor effects of non-hypercalcemic agent MT19c provide a new approach to the design of vitamin-D based anticancer molecules and a rationale for developing MT19c as a therapeutic agent for malignant ovarian tumors by targeting oncogenic de novo lipogenesis.     

Adipose-derived stem cells in ovarian cancer progression, metastasis,and chemoresistance

Ovarian cancer is the deadliest gynecological malignancy due to its symptomless early stage, metastasis, and high recurrence rate. The tumor microenvironment contributes to the ovarian cancer progression, metastasis, and chemoresistance. Adipose-derived stem cell in the tumor microenvironment of ovarian cancer, as a key player, interacts with ovarian cancer cells to form the cancer-associated fibroblasts and cancer-associated adipocytes, and secretes soluble factors to activate tumor cell signaling, which can promote ovarian cancer metastasis and chemoresistance. We summarize in this review the recent progress in the studies of interactions between adipose-derived stem cell and ovarian cancer, thus, to provide some insight for ovarian cancer therapy through targeting adipose-derived stem cell.     

Conditionally replicating adenovirus expressing TIMP2 increases survival in a mouse model of disseminated ovarian cancer

Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds) are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2) containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs) in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer.