Elucidating Novel Targets for Ovarian Cancer Antibody-Drug Conjugate Development: Integrating In Silico Prediction and Surface Plasmon Resonance to Identify Targets with Enhanced Antibody Internalization Capacity.

Antibody-drug conjugates (ADCs) constitute a rapidly expanding category of biopharmaceuticals that are reshaping the landscape of targeted chemotherapy. The meticulous process of selecting therapeutic targets, aided by specific monoclonal antibodies' high specificity for binding to designated antigenic epitopes, is pivotal in ADC research and development. Despite ADCs' intrinsic ability to differentiate between healthy and cancerous cells, developmental challenges persist. In this study, we present a rationalized pipeline encompassing the initial phases of the ADC development, including target identification and validation. Leveraging an in-house, computationally constructed ADC target database, termed ADC Target Vault, we identified a set of novel ovarian cancer targets. We effectively demonstrate the efficacy of Surface Plasmon Resonance (SPR) technology and in vitro models as predictive tools, expediting the selection and validation of targets as ADC candidates for ovarian cancer therapy. Our analysis reveals three novel robust antibody/target pairs with strong binding and favourable antibody internalization rates in both wild-type and cisplatin-resistant ovarian cancer cell lines. This approach enhances ADC development and offers a comprehensive method for assessing target/antibody combinations and pre-payload conjugation biological activity. Additionally, the strategy establishes a robust platform for high-throughput screening of potential ovarian cancer ADC targets, an approach that is equally applicable to other cancer types.

The Potent G-Quadruplex-Binding Compound QN-302 Downregulates S100P Gene Expression in Cells and in an In Vivo Model of Pancreatic Cancer.

The naphthalene diimide compound QN-302, designed to bind to G-quadruplex DNA sequences within the promoter regions of cancer-related genes, has high anti-proliferative activity in pancreatic cancer cell lines and anti-tumor activity in several experimental models for the disease. We show here that QN-302 also causes downregulation of the expression of the S100P gene and the S100P protein in cells and in vivo. This protein is well established as being involved in key proliferation and motility pathways in several human cancers and has been identified as a potential biomarker in pancreatic cancer. The S100P gene contains 60 putative quadruplex-forming sequences, one of which is in the promoter region, 48 nucleotides upstream from the transcription start site. We report biophysical and molecular modeling studies showing that this sequence forms a highly stable G-quadruplex in vitro, which is further stabilized by QN-302. We also report transcriptome analyses showing that S100P expression is highly upregulated in tissues from human pancreatic cancer tumors, compared to normal pancreas material. The extent of upregulation is dependent on the degree of differentiation of tumor cells, with the most poorly differentiated, from more advanced disease, having the highest level of S100P expression. The experimental drug QN-302 is currently in pre-IND development (as of Q1 2023), and its ability to downregulate S100P protein expression supports a role for this protein as a marker of therapeutic response in pancreatic cancer. These results are also consistent with the hypothesis that the S100P promoter G-quadruplex is a potential therapeutic target in pancreatic cancer at the transcriptional level for QN-302.

Discovery of Targets for Immune-Metabolic Antitumor Drugs Identifies Estrogen-Related Receptor Alpha.

Drugs that kill tumors through multiple mechanisms have the potential for broad clinical benefits. Here, we first developed an in silico multiomics approach (BipotentR) to find cancer cell-specific regulators that simultaneously modulate tumor immunity and another oncogenic pathway and then used it to identify 38 candidate immune-metabolic regulators. We show the tumor activities of these regulators stratify patients with melanoma by their response to anti-PD-1 using machine learning and deep neural approaches, which improve the predictive power of current biomarkers. The topmost identified regulator, ESRRA, is activated in immunotherapy-resistant tumors. Its inhibition killed tumors by suppressing energy metabolism and activating two immune mechanisms: (i) cytokine induction, causing proinflammatory macrophage polarization, and (ii) antigen-presentation stimulation, recruiting CD8+ T cells into tumors. We also demonstrate a wide utility of BipotentR by applying it to angiogenesis and growth suppressor evasion pathways. BipotentR (http://bipotentr.dfci.harvard.edu/) provides a resource for evaluating patient response and discovering drug targets that act simultaneously through multiple mechanisms. SIGNIFICANCE: BipotentR presents resources for evaluating patient response and identifying targets for drugs that can kill tumors through multiple mechanisms concurrently. Inhibition of the topmost candidate target killed tumors by suppressing energy metabolism and effects on two immune mechanisms. This article is highlighted in the In This Issue feature, p. 517.

Dinaciclib as an effective pan-cyclin dependent kinase inhibitor in platinum resistant ovarian cancer.

Background: Ovarian cancer (OC) is amongst the most lethal of common cancers in women. Lacking in specific symptoms in the early stages, OC is predominantly diagnosed late when the disease has undergone metastatic spread and chemotherapy is relied on to prolong life. Platinum-based therapies are preferred and although many tumors respond initially, the emergence of platinum-resistance occurs in the majority of cases after which prognosis is very poor. Upregulation of DNA damage pathways is a common feature of platinum resistance in OC with cyclin dependent kinases (CDKs) serving as key regulators of this process and suggesting that CDK inhibitors (CDKis) could be effective tools in the treatment of platinum resistant and refractory OC. Aim: The aim of this study was to evaluate the efficacy of CDKis in platinum resistant OC models and serve as a predictor of potential clinical utility. Methods: The efficacy of CDKi, dinaciclib, was determined in wildtype and platinum resistant cell line pairs representing different OC subtypes. In addition, dinaciclib was evaluated in primary cells isolated from platinum-sensitive and platinum-refractory tumors to increase the clinical relevance of the study. Results and conclusions: Dinaciclib proved highly efficacious in OC cell lines and primary cells, which were over a thousand-fold more sensitive to the CDKi than to cisplatin. Furthermore, cisplatin resistance in these cells did not influence sensitivity to dinaciclib and the two drugs combined additively in both platinum-sensitive and platinum-resistant OC cells suggesting a potential role for pan-CDKis (CDKis targeting multiple CDKs), such as dinaciclib, in the treatment of advanced and platinum-resistant OC.

Attenuating Adaptive VEGF-A and IL8 Signaling Restores Durable Tumor Control in AR Antagonist-Treated Prostate Cancers.

Inhibiting androgen signaling using androgen signaling inhibitors (ASI) remains the primary treatment for castrate-resistant prostate cancer. Acquired resistance to androgen receptor (AR)-targeted therapy represents a major impediment to durable clinical response. Understanding resistance mechanisms, including the role of AR expressed in other cell types within the tumor microenvironment, will extend the clinical benefit of AR-targeted therapy. Here, we show the ASI enzalutamide induces vascular catastrophe and promotes hypoxia and microenvironment adaptation. We characterize treatment-induced hypoxia, and subsequent induction of angiogenesis, as novel mechanisms of relapse to enzalutamide, highlighting the importance of two hypoxia-regulated cytokines in underpinning relapse. We confirmed AR expression in CD34+ vascular endothelium of biopsy tissue and human vascular endothelial cells (HVEC). Enzalutamide attenuated angiogenic tubule formation and induced cytotoxicity in HVECs in vitro, and rapidly induced sustained hypoxia in LNCaP xenografts. Subsequent reoxygenation, following prolonged enzalutamide treatment, was associated with increased tumor vessel density and accelerated tumor growth. Hypoxia increased AR expression and transcriptional activity in prostate cells in vitro. Coinhibition of IL8 and VEGF-A restored tumor response in the presence of enzalutamide, confirming the functional importance of their elevated expression in enzalutamide-resistant models. Moreover, coinhibition of IL8 and VEGF-A resulted in a durable, effective resolution of enzalutamide-sensitive prostate tumors. We conclude that concurrent inhibition of two hypoxia-induced factors, IL8 and VEGF-A, prolongs tumor sensitivity to enzalutamide in preclinical models and may delay the onset of enzalutamide resistance. IMPLICATIONS: Targeting hypoxia-induced signaling may extend the therapeutic benefit of enzalutamide, providing an improved treatment strategy for patients with resistant disease.

Targeting the p300/CBP Axis in Lethal Prostate Cancer.

Resistance to androgen receptor (AR) blockade in castration-resistant prostate cancer (CRPC) is associated with sustained AR signaling, including through alternative splicing of AR (AR-SV). Inhibitors of transcriptional coactivators that regulate AR activity, including the paralog histone acetyltransferase proteins p300 and CBP, are attractive therapeutic targets for lethal prostate cancer. Herein, we validate targeting p300/CBP as a therapeutic strategy for lethal prostate cancer and describe CCS1477, a novel small-molecule inhibitor of the p300/CBP conserved bromodomain. We show that CCS1477 inhibits cell proliferation in prostate cancer cell lines and decreases AR- and C-MYC-regulated gene expression. In AR-SV-driven models, CCS1477 has antitumor activity, regulating AR and C-MYC signaling. Early clinical studies suggest that CCS1477 modulates KLK3 blood levels and regulates CRPC biopsy biomarker expression. Overall, CCS1477 shows promise for the treatment of patients with advanced prostate cancer. SIGNIFICANCE: Treating CRPC remains challenging due to persistent AR signaling. Inhibiting transcriptional AR coactivators is an attractive therapeutic strategy. CCS1477, an inhibitor of p300/CBP, inhibits growth and AR activity in CRPC models, and can affect metastatic CRPC target expression in serial clinical biopsies.See related commentary by Rasool et al., p. 1011.This article is highlighted in the In This Issue feature, p. 995.

Asymmetrically Substituted Quadruplex-Binding Naphthalene Diimide Showing Potent Activity in Pancreatic Cancer Models.

Targeting of genomic quadruplexes is an approach to treating complex human cancers. We describe a series of tetra-substituted naphthalene diimide (ND) derivatives with a phenyl substituent directly attached to the ND core. The lead compound (SOP1812) has 10 times superior cellular and in vivo activity compared with previous ND compounds and nanomolar binding to human quadruplexes. The pharmacological properties of SOP1812 indicate good bioavailability, which is consistent with the in vivo activity in xenograft and genetic models for pancreatic cancer. Transcriptome analysis shows that it down-regulates several cancer gene pathways, including Wnt/ß-catenin signaling.

Improved early detection of ovarian cancer using longitudinal multimarker models.

BACKGROUND: Ovarian cancer has a poor survival rate due to late diagnosis and improved methods are needed for its early detection. Our primary objective was to identify and incorporate additional biomarkers into longitudinal models to improve on the performance of CA125 as a first-line screening test for ovarian cancer. METHODS: This case-control study nested within UKCTOCS used 490 serial serum samples from 49 women later diagnosed with ovarian cancer and 31 control women who were cancer-free. Proteomics-based biomarker discovery was carried out using pooled samples and selected candidates, including those from the literature, assayed in all serial samples. Multimarker longitudinal models were derived and tested against CA125 for early detection of ovarian cancer. RESULTS: The best performing models, incorporating CA125, HE4, CHI3L1, PEBP4 and/or AGR2, provided 85.7% sensitivity at 95.4% specificity up to 1 year before diagnosis, significantly improving on CA125 alone. For Type II cases (mostly high-grade serous), models achieved 95.5% sensitivity at 95.4% specificity. Predictive values were elevated earlier than CA125, showing the potential of models to improve lead time. CONCLUSIONS: We have identified candidate biomarkers and tested longitudinal multimarker models that significantly improve on CA125 for early detection of ovarian cancer. These models now warrant independent validation.

Discovery of non-invasive biomarkers for the diagnosis of endometriosis.

BACKGROUND: Endometriosis is a common gynaecological disorder affecting 5-10% of women of reproductive age who often experience chronic pelvic pain and infertility. Definitive diagnosis is through laparoscopy, exposing patients to potentially serious complications, and is often delayed. Non-invasive biomarkers are urgently required to accelerate diagnosis and for triaging potential patients for surgery. METHODS: This retrospective case control biomarker discovery and validation study used quantitative 2D-difference gel electrophoresis and tandem mass tagging-liquid chromatography-tandem mass spectrometry for protein expression profiling of eutopic and ectopic endometrial tissue samples collected from 28 cases of endometriosis and 18 control patients undergoing surgery for investigation of chronic pelvic pain without endometriosis or prophylactic surgery. Samples were further sub-grouped by menstrual cycle phase. Selected differentially expressed candidate markers (LUM, CPM, TNC, TPM2 and PAEP) were verified by ELISA in a set of 87 serum samples collected from the same and additional women. Previously reported biomarkers (CA125, sICAM1, FST, VEGF, MCP1, MIF and IL1R2) were also validated and diagnostic performance of markers and combinations established. RESULTS: Cycle phase and endometriosis-associated proteomic changes were identified in eutopic tissue from over 1400 identified gene products, yielding potential biomarker candidates. Bioinformatics analysis revealed enrichment of adhesion/extracellular matrix proteins and progesterone signalling. The best single marker for discriminating endometriosis from controls remained CA125 (AUC = 0.63), with the best cross-validated multimarker models improving the AUC to 0.71-0.81, depending upon menstrual cycle phase and control group. CONCLUSIONS: We have identified menstrual cycle- and endometriosis-associated protein changes linked to various cellular processes that are potential biomarkers and that provide insight into the biology of endometriosis. Our data indicate that the markers tested, whilst not useful alone, have improved diagnostic accuracy when used in combination and demonstrate menstrual cycle specificity. Tissue heterogeneity and blood contamination is likely to have hindered biomarker discovery, whilst a small sample size precludes accurate determination of performance by cycle phase. Independent validation of these biomarker panels in a larger cohort is however warranted, and if successful, they may have clinical utility in triaging patients for surgery.

The unidirectional hypoxia-activated prodrug OCT1002 inhibits growth and vascular development in castrate-resistant prostate tumors.

BACKGROUND: OCT1002 is a unidirectional hypoxia-activated prodrug (uHAP) OCT1002 that can target hypoxic tumor cells. Hypoxia is a common feature in prostate tumors and is known to drive disease progression and metastasis. It is, therefore, a rational therapeutic strategy to directly target hypoxic tumor cells in an attempt to improve treatment for this disease. Here we tested OCT1002 alone and in combination with standard-of-care agents in hypoxic models of castrate-resistant prostate cancer (CRPC). METHODS: The effect of OCT1002 on tumor growth and vasculature was measured using murine PC3 xenograft and dorsal skin fold (DSF) window chamber models. The effects of abiraterone, docetaxel, and cabazitaxel, both singly and in combination with OCT1002, were also compared. RESULTS: The hypoxia-targeting ability of OCT1002 effectively controls PC3 tumor growth. The effect was evident for at least 42 days after exposure to a single dose (30 mg/kg) and was comparable to, or better than, drugs currently used in the clinic. In DSF experiments OCT1002 caused vascular collapse in the PC3 tumors and inhibited the revascularization seen in controls. In this model OCT1002 also enhanced the anti-tumor effects of abiraterone, cabazitaxel, and docetaxel; an effect which was accompanied by a more prolonged reduction in tumor vasculature density. CONCLUSIONS: These studies provide the first evidence that OCT1002 can be an effective agent in treating hypoxic, castrate-resistant prostate tumors, either singly or in combination with established chemotherapeutics for prostate cancer.

Effects of ErbB2 Overexpression on the Proteome and ErbB Ligand-specific Phosphosignaling in Mammary Luminal Epithelial Cells.

Most breast cancers arise from luminal epithelial cells, and 25-30% of these tumors overexpress the ErbB2/HER2 receptor that correlates with disease progression and poor prognosis. The mechanisms of ErbB2 signaling and the effects of its overexpression are not fully understood. Herein, stable isotope labeling by amino acids in cell culture (SILAC), expression profiling, and phosphopeptide enrichment of a relevant, non-transformed, and immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGF receptor (EGF treatment) and ErbB3 (HRG1ß treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signaling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signaling, and we identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion, and enhanced motility. Moreover, we define a novel mechanism by which ErbB signaling suppresses basal interferon signaling that would promote the survival and proliferation of mammary luminal epithelial cells. Numerous novel sites of growth factor-regulated phosphorylation were identified that were enhanced by ErbB2 overexpression, and we putatively link these to altered cell behavior and also highlight the importance of performing parallel protein expression profiling alongside phosphoproteomic analysis.

Targeting Hypoxic Prostate Tumors Using the Novel Hypoxia-Activated Prodrug OCT1002 Inhibits Expression of Genes Associated with Malignant Progression.

Purpose: To understand the role of hypoxia in prostate tumor progression and to evaluate the ability of the novel unidirectional hypoxia-activated prodrug OCT1002 to enhance the antitumor effect of bicalutamide.Experimental Design: The effect of OCT1002 on prostate cancer cells (LNCaP, 22Rv1, and PC3) was measured in normoxia and hypoxia in vitroIn vivo, tumor growth and lung metastases were measured in mice treated with bicalutamide, OCT1002, or a combination. Dorsal skin fold chambers were used to image tumor vasculature in vivo Longitudinal gene expression changes in tumors were analyzed using PCR.Results: Reduction of OCT1002 to its active form (OCT1001) decreased prostate cancer cell viability. In LNCaP-luc spheroids, OCT1002 caused increased apoptosis and decreased clonogenicity. In vivo, treatment with OCT1002 alone, or with bicalutamide, showed significantly greater tumor growth control and reduced lung metastases compared with controls. Reestablishment of the tumor microvasculature following bicalutamide-induced vascular collapse is inhibited by OCT1002. Significantly, the upregulation of RUNX2 and its targets caused by bicalutamide alone was blocked by OCT1002.Conclusions: OCT1002 selectively targets hypoxic tumor cells and enhances the antitumor efficacy of bicalutamide. Furthermore, bicalutamide caused changes in gene expression, which indicated progression to a more malignant genotype; OCT1002 blocked these effects, emphasizing that more attention should be attached to understanding genetic changes that may occur during treatment. Early targeting of hypoxic cells with OCT1002 can provide a means of inhibiting prostate tumor growth and malignant progression. This is of importance for the design and refinement of existing androgen-deprivation regimens in the clinic. Clin Cancer Res; 23(7); 1797-808. ©2016 AACR.

Disulfide-activated protein kinase G Iα regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response.

The Frank-Starling mechanism allows the amount of blood entering the heart from the veins to be precisely matched with the amount pumped out to the arterial circulation. As the heart fills with blood during diastole, the myocardium is stretched and oxidants are produced. Here we show that protein kinase G Iα (PKGIα) is oxidant-activated during stretch and this form of the kinase selectively phosphorylates cardiac phospholamban Ser16-a site important for diastolic relaxation. We find that hearts of Cys42Ser PKGIα knock-in (KI) mice, which are resistant to PKGIα oxidation, have diastolic dysfunction and a diminished ability to couple ventricular filling with cardiac output on a beat-to-beat basis. Intracellular calcium dynamics of ventricular myocytes isolated from KI hearts are altered in a manner consistent with impaired relaxation and contractile function. We conclude that oxidation of PKGIα during myocardial stretch is crucial for diastolic relaxation and fine-tunes the Frank-Starling response.

Nitric Oxide Up-Regulates RUNX2 in LNCaP Prostate Tumours: Implications for Tumour Growth In Vitro and In Vivo.

Aberrant expression of the transcription factor RUNX2 in prostate cancer has a number of important consequences including increased resistance to apoptosis, invasion and metastasis to bone. We previously demonstrated that hypoxia up-regulated RUNX2 in tumour cells, which in turn up-regulated the anti-apoptotic factor Bcl-2. Here, we investigate the impact of nitric oxide (NO) on RUNX2 and Bcl-2 expression in prostate cancer and further, how RUNX2 over-expression can impact tumour growth, angiogenesis and oxygenation in vivo. The effect of NO levels on RUNX2 and thus Bcl-2 expression was examined in prostate cancer cells in vitro using methods including gene and protein expression analyses, nitrite quantitation, protein-DNA interaction assays (ChIP) and viability assays (XTT). The effect of RUNX2 over-expression on tumour physiology (growth, oxygenation and angiogenesis) was also assessed in vivo using LNCaP xenografts. A low (but not high) concentration of NO (10 µM) induced expression of RUNX2 and Bcl-2, conferring resistance to docetaxel. These effects were induced via the ERK and PI3K pathways and were dependent on intact AP-1 binding sites in the RUNX2 promoter. RUNX2 over-expression in LNCaP tumours in vivo decreased the time to tumour presentation and increased tumour growth. Moreover, these tumours exhibited improved tumour angiogenesis and oxygenation. Low levels of NO increase expression of RUNX2 and Bcl-2 in LNCaP prostate tumour cells, and in vivo up-regulation of RUNX2 created tumours with a more malignant phenotype. Collectively, our data reveals the importance of NO-regulation of key factors in prostate cancer disease progression.

Identification of novel candidate biomarkers of epithelial ovarian cancer by profiling the secretomes of three-dimensional genetic models of ovarian carcinogenesis.

Epithelial ovarian cancer (EOC) is still considered the most lethal gynecological malignancy and improved early detection of ovarian cancer is crucial to improving patient prognoses. To address this need, we tested whether candidate EOC biomarkers can be identified using three-dimensional (3D) in vitro models. We quantified changes in the abundance of secreted proteins in a 3D genetic model of early-stage EOC, generated by expressing CMYC and KRAS(G) (12) (V) in TERT-immortalized normal ovarian epithelial cells. Cellular proteins were labeled in live cells using stable isotopic amino acid analogues, and secreted proteins identified and quantified using liquid chromatography-tandem mass spectrometry. Thirty-seven and 55 proteins were differentially expressed by CMYC and CMYC+KRAS(G) (12) (V) expressing cells respectively (p < 0.05; >2-fold). We evaluated expression of the top candidate biomarkers in ∼210 primary EOCs: CHI3L1 and FKBP4 are both expressed by >96% of primary EOCs, and FASN and API5 are expressed by 86 and 75% of cases. High expression of CHI3L1 and FKBP4 was associated with worse patient survival (p = 0.042 and p = 0.002, respectively). Expression of LGALS3BP was positively associated with recurrence (p = 0.0001) and suboptimal debulking (p = 0.018) suggesting that these proteins may be novel prognostic biomarkers. Furthermore, within early stage tumours (I/II), high expression of API5, CHI3L1 and FASN was associated with high tumour grade (p = 3 × 10(-4) , p = 0.016, p = 0.010, respectively). We show in vitro cell biology models of early-stage cancer development can be used to identify novel candidate biomarkers for disease, and report the identification of proteins that represent novel potential candidate diagnostic and prognostic biomarkers for this highly lethal disease.

Discovery of serum biomarkers of ovarian cancer using complementary proteomic profiling strategies.

PURPOSE: Ovarian cancer is a devastating disease and biomarkers for its early diagnosis are urgently required. Serum may be a valuable source of biomarkers that may be revealed by proteomic profiling. Herein, complementary serum protein profiling strategies were employed for discovery of biomarkers that could discriminate cases of malignant and benign ovarian cancer. EXPERIMENTAL DESIGN: Identically collected and processed serum samples from 22 cases of invasive epithelial ovarian cancer, 45 benign ovarian neoplasms, and 64 healthy volunteers were subjected to immunodepletion and protein equalization coupled to 2D-DIGE/MS and multidimensional fractionation coupled to SELDI-TOF profiling with MS/MS for protein identification. Selected candidates were verified by ELISA in samples from malignant (n = 70) and benign (n = 89) cases and combined marker panels tested against serum CA125. RESULTS: Both profiling platforms were complementary in identifying biomarker candidates, four of which (A1AT, SLPI, APOA4, VDBP) significantly discriminated malignant from benign cases. However, no combination of markers was as good as CA125 for diagnostic accuracy. SLPI was further tested as an early marker using prediagnosis serum samples. While it rose in cases toward diagnosis, it did not discriminate prediagnosis cases from controls. CONCLUSIONS AND CLINICAL RELEVANCE: The candidate biomarkers warrant further validation in independent sample sets.

Virgin olive oil phenolics extract inhibit invasion of HT115 human colon cancer cells in vitro and in vivo.

The decreased cancer risk associated with consumption of olive oil may be due to the presence of phenolics which can modulate pathways including apoptosis and invasion that are relevant to carcinogenesis. We have previously shown that a virgin olive oil phenolics extract (OVP) inhibited invasion of HT115 colon cancer cells in vitro. In the current study we assessed the in vitro effects of OVP (25 µg mL(-1)) on HT115 cell migration, spreading and integrin expression. Furthermore, the anti-metastatic activity of OVP - at a dose equivalent to 25 mg per kg per day for 2, 8 or 10 weeks - was assessed in a Severe Combined ImmunoDeficiency (SCID) Balb-c mouse model. After 24 h OVP did not inhibit cell migration but significantly reduced cell spreading on fibronectin (65% of control; p < 0.05) and expression of a range of α and ß integrins was modulated. In vivo, OVP by gavage significantly (p < 0.05) decreased not only tumour volume but also the number of metastases in SCID Balb-c mice. Collectively, the data suggest that - possibly through modulation of integrin expression - OVP decreases invasion in vitro and also inhibits metastasis in vivo.

Protection of corneal epithelial stem cells prevents ultraviolet A damage during corneal collagen cross-linking treatment for keratoconus.

BACKGROUND/AIMS: Cross-linking of the cornea is usually carried out at a young age as a treatment to manage ectasia. The corneal limbal region contains delicate long-lived stem cells, which could potentially be deleteriously affected by Ultraviolet A (UV-A) radiation. Damage to these stem cells may not demonstrate as a clinical problem for many years subsequent to cross-linking treatment. UV-A radiation is known to have potential mutagenic effects upon mammalian DNA and can result in cancer. METHODS: Cultured corneal epithelial cells and ex vivo corneal tissue were treated with the standard clinical cross-linking protocol for UV-A irradiation. 8-hydroxydeoxyguansoine (8-OHdG) and cyclin-dependent kinase inhibitor genes (CDKN1A and CDKN2A) were assayed as markers of DNA damage using immunohistochemistry, ELISA and quantitative real time PCR. RESULTS: Staining of treated limbal tissue demonstrated the presence of 8-OHdG within p63 positive basal limbal cells. Levels of 8-OHdG and CDKN1A mRNA were found to be significantly increased in cultured corneal epithelial cells and limbal epithelial cells but no increase was demonstrated with the use of a polymethyl methylacrylate protective cover. CONCLUSIONS: This study provides evidence that oxidative nuclear DNA damage can occur through cross-linking in layers of corneal epithelial cells at the limbus and that this can be easily prevented by covering the limbus.

The anti-migratory effects of FKBPL and its peptide derivative, AD-01: regulation of CD44 and the cytoskeletal pathway.

FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity in vitro and in vivo and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development in vivo suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks.

Androgen deprivation results in time-dependent hypoxia in LNCaP prostate tumours: informed scheduling of the bioreductive drug AQ4N improves treatment response.

Androgen withdrawal induces hypoxia in androgen-sensitive tissue; this is important as in the tumour microenvironment, hypoxia is known to drive malignant progression. Our study examined the time-dependent effect of androgen deprivation therapy (ADT) on tumour oxygenation and investigated the role of ADT-induced hypoxia on malignant progression in prostate tumours. LNCaP xenografted tumours were treated with anti-androgens and tumour oxygenation measured. Dorsal skin fold (DSF) chambers were used to image tumour vasculature in vivo. Quantitative PCR (QPCR) identified differential gene expression following treatment with bicalutamide. Bicalutamide-treated and vehicle-only-treated tumours were re-established in vitro, and invasion and sensitivity to docetaxel were measured. Tumour growth delay was calculated following treatment with bicalutamide combined with the bioreductive drug AQ4N. Tumour oxygenation measurements showed a precipitate decrease following initiation of ADT. A clinically relevant dose of bicalutamide (2 mg/kg/day) decreased tumour oxygenation by 45% within 24 hr, reaching a nadir of 0.09% oxygen (0.67 ± 0.06 mmHg) by Day 7; this persisted until Day 14 when it increased up to Day 28. Using DSF chambers, LNCaP tumours treated with bicalutamide showed loss of small vessels at Days 7 and 14 with revascularisation occurring by Day 21. QPCR showed changes in gene expression consistent with the vascular changes and malignant progression. Cells from bicalutamide-treated tumours were more malignant than vehicle-treated controls. Combining bicalutamide with AQ4N (50 mg/kg, single dose) caused greater tumour growth delay than bicalutamide alone. Our study shows that bicalutamide-induced hypoxia selects for cells that show malignant progression; targeting hypoxic cells may provide greater clinical benefit.