PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.

BACKGROUND: Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. METHODS: We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. RESULTS: Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. CONCLUSIONS: Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.

Non-invasive molecular imaging for preclinical cancer therapeutic development.

Molecular and non-invasive imaging are rapidly emerging fields in preclinical cancer drug discovery. This is driven by the need to develop more efficacious and safer treatments, the advent of molecular-targeted therapeutics, and the requirements to reduce and refine current preclinical in vivo models. Such bioimaging strategies include MRI, PET, single positron emission computed tomography, ultrasound, and optical approaches such as bioluminescence and fluorescence imaging. These molecular imaging modalities have several advantages over traditional screening methods, not least the ability to quantitatively monitor pharmacodynamic changes at the cellular and molecular level in living animals non-invasively in real time. This review aims to provide an overview of non-invasive molecular imaging techniques, highlighting the strengths, limitations and versatility of these approaches in preclinical cancer drug discovery and development.

Polysialyltransferase: a new target in metastatic cancer.

Polysialic acid (polySia) is a carbohydrate polymer critical for neuronal cell migration and axon pathfinding in embryonic development. Besides brain regions requiring persistent neuronal plasticity, polySia is essentially absent from the adult body. However, polySia is aberrantly re-expressed on many tumours, where it decorates the surface of NCAM (neuronal cell adhesion molecule) and modulates cell adhesion, migration and invasion. PolySia-NCAM expression is strongly associated with poor clinical prognosis and correlates with aggressive and invasive disease in many cancers, including lung cancer, neuroblastoma and gliomas. The synthesis of polySia is mediated by two polysialyltransferases (polySTs), ST8SiaIV (PST) and particularly ST8SiaII (STX) in cancer cells. The demonstration that polyST knock-down negates events associated with tumour cell dissemination indicates that PST and STX are validated targets. Selective inhibition of polySTs therefore presents a therapeutic opportunity to inhibit tumour invasion and metastasis.

Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo.

Hypoxia is an important factor in tumor growth. It is associated with resistance to conventional anticancer treatments. Gene therapy targeting hypoxic tumor cells therefore has the potential to enhance the efficacy of treatment of solid tumors. Transfection of a panel of tumor cell lines with plasmid constructs containing hypoxia-responsive promoter elements from the genes, vascular endothelial growth factor (VEGF) and erythropoietin, linked to the minimal cytomegalovirus (mCMV) or minimal interleukin-2 (mIL-2) promoters showed optimum hypoxia-inducible luciferase reporter gene expression with five repeats of VEGF hypoxic-response element linked to the mCMV promoter. Adenoviral vectors using this hypoxia-inducible promoter to drive therapeutic transgenes produced hypoxia-specific cell kill of HT1080 and HCT116 cells in the presence of prodrug with both herpes simplex virus thymidine kinase/ganciclovir and nitroreductase (NTR)/CB1954 prodrug-activating systems. Significant cytotoxic effects were also observed in patient-derived human ovarian cancer cells. The NTR/CB1954 system provided more readily controllable transgene expression and so was used for in vivo experiments of human HCT116 xenografts in nude mice. Subjects treated intratumorally with Ad-VEGFmCMV-NTR and intraperitoneal injection of CB1954 demonstrated a statistically significant reduction in tumor growth. Immunohistochemistry of treated xenografts showed a good correlation between transgene expression and hypoxic areas. Further investigation of these hypoxia-inducible adenoviral vectors, alone or in combination with existing modalities of cancer therapy, may aid in the future development of successful Gene-Directed Enzyme Prodrug Therapy systems, which are much needed for targeting solid tumors.

Small molecule FGF receptor inhibitors block FGFR-dependent urothelial carcinoma growth in vitro and in vivo.

BACKGROUND: Activating mutations of FGFR3 are frequently identified in superficial urothelial carcinoma (UC) and increased expression of FGFR1 and FGFR3 are common in both superficial and invasive UC. METHODS: The effects of inhibition of receptor activity by three small molecule inhibitors (PD173074, TKI-258 and SU5402) were investigated in a panel of bladder tumour cell lines with known FGFR expression levels and FGFR3 mutation status. RESULTS: All inhibitors prevented activation of FGFR3, and inhibited downstream MAPK pathway signalling. Response was related to FGFR3 and/or FGFR1 expression levels. Cell lines with the highest levels of FGFR expression showed the greatest response and little or no effect was measured in normal human urothelial cells or in UC cell lines with activating RAS gene mutations. In sensitive cell lines, the drugs induced cell cycle arrest and/or apoptosis. IC(50) values for PD173074 and TKI-258 were in the nanomolar concentration range compared with micromolar concentrations for SU5402. PD173074 showed the greatest effects in vitro and in vivo significantly delayed the growth of subcutaneous bladder tumour xenografts. CONCLUSION: These results indicate that inhibition of FGFR1 and wild-type or mutant FGFR3 may represent a useful therapeutic approach in patients with both non-muscle invasive and muscle invasive UC.

Retargeted adenoviral cancer gene therapy for tumour cells overexpressing epidermal growth factor receptor or urokinase-type plasminogen activator receptor.

We have assessed the ability of bispecific fusion proteins to improve adenovirus-mediated transfer of therapeutic and marker transgenes. We constructed an expression vector that can be easily modified to synthesize a variety of fusion proteins for retargeting adenoviral gene therapy vectors to cell surface markers, which are differentially expressed between normal and cancer cells. Adenoviral transduction can be improved in a number of tumour cell lines which overexpress EGFR (epidermal growth factor receptor) or uPAR (urokinase-type plasminogen activator receptor), but which have only low levels of endogenous hCAR (human coxsackie B and adenovirus receptor) expression. Up to 40-fold improvement in beta-galactosidase transgene expression was seen using an EGFR retargeting protein, and up to 16-fold using a second fusion protein targeting uPAR. In vitro, our uPAR retargeting fusion protein improved the sensitivity to adenoviral herpes simplex virus thymidine kinase/ganciclovir by an order of magnitude, whereas in vivo, our EGFR retargeting protein is able to significantly delay tumour growth in rodent animal models in a dose-dependent manner. The 'cassette' design of our fusion protein constructs offers a flexible method for the straightforward synthesis of multiple adenoviral retargeting proteins, directed against a variety of tumour-associated antigens, for use in clinical trials.

Reducing the cost of screening novel agents using the hollow fibre assay.

Although the in vivo hollow fibre assay (HFA) as utilised by the National Cancer Institute is a highly effective screening tool, it has not been adopted en masse in the cancer pharmacology field. However, in laboratories which have adopted it, the effectiveness of HFA has also been confirmed. If immunocompetent mice could be used with the HFA, thereby reducing the cost of the assay, accessibility would increase and reductions in the cost of selecting appropriate agents for early clinical trials would result. It was demonstrated here that there was no difference in terms of cell growth and response to chemotherapy for cancer cells in hollow fibres in immunocompetent compared with immunodeficient mice. The HFA can thus be performed in these less expensive and more easily available mice with the implication of considerable savings to the preclinical cancer pharmacology community.

Development of a modified hollow fibre assay for studying agents targeting the tumour neovasculature.

BACKGROUND: Previous studies have shown extensive vascularisation surrounding subcutaneously implanted fibres when the duration of the US National Cancer Institute (NCI) hollow fibre assay was prolonged. MATERIALS AND METHODS: The feasibility of adapting the NCI assay for evaluating agents targeting the tumour vasculature was investigated in vitro and in vivo. Finally, in the optimised assay, changes in neovasculature formation around the fibres following treatment with the anti-vascular agent paclitaxel were quantified by immunohistochemistry. RESULTS: Correlations between cell number seeded, time in culture and vascular endothelial growth factor (VEGF) secretion were seen. In vivo studies showed that transplanting single rather than 3 fibres at a site reduced inflammation, reducing the length of the fibre transplanted, as did without any significant loss in cell growth over 21 days. A statistically significant reduction in neovascularisation surrounding the fibres was seen accompanying paclitaxel treatment. CONCLUSION: Modifications made here to the NCI hollow fibre assay demonstrate its potential for analysing anti-tumour vasculature agents.

Quantitative angiogenesis assays in vivo--a review.

The development of agents that target tumour vasculature is ultimately dependent on the availability of appropriate preclinical screening assays. Several quantitative angiogenesis assays exist, each with its own unique characteristics and disadvantages. In this review we discuss some of the commonly used assays, their methodological pitfalls and current use. The corneal micropocket and the CAM assay are well established. However, the matrix-implant assays have the potential advantage of replicating the hypoxic tumour microenvironment, thus making them suitable for the study of tumour angiogenesis. The ideal quantitative angiogenesis assay does not exist and the use of two complimentary quantitative assays, such as a matrix implant assay and a microcirculatory preparation like the CAM or corneal micropocket assay, provides the best compromise. Newer models like the hollow-fibre assay are being developed and older ones refined. Assay systems should reflect distinct disease processes. Thus it is appropriate to develop assays that study exclusively pro- or anti-angiogenic compounds or anti-vascular agents. Criticisms of currently available screening systems are that the predictive value of current screening systems remains to be established as anti-angiogenic agents are still in clinical development. Anti-angiogenic agents are likely to be most effective as chronic therapy for remission maintenance in the metastatic setting or as adjuvant therapy in patients at high risk of relapse, an important clinical aspect not addressed in animal models of tumour angiogenesis. Histological analysis still provides the most detailed information on in vivo angiogenesis. However, angiogenesis is a dynamic process and assays that permit continuous monitoring of the angiogenic response and provide information on the physiological characteristics of new vessels will be distinctly advantageous over older systems. The development of non-invasive techniques for quantitation of angiogenesis will greatly facilitate this process.

Combretastatin A-1 phosphate potentiates the antitumour activity of cisplatin in a murine adenocarcinoma model.

BACKGROUND: The tubulin depolymerizing drug Combretastatin A-1 phosphate (CA1P), a water-soluble derivative of combretastatin A-1, has been recently shown to have a better efficacy in experimental models than the clinically active, close structural analogue, combretastatin A-4 phosphate (CA4P). Previous studies with CA4P in combination with standard anti-cancer agents have demonstrated improved efficacy relative to the standard agents. MATERIALS AND METHODS: In this study the synergistic effects of administering CA1P in combination with cisplatin (CPL) in a well-differentiated transplantable murine colon model (MAC 29) was evaluated. RESULTS: CA1P at 100 mgkg-1 significantly potentiated the anti-tumour effects of CPL. The effect with CPL was similar to that seen for CA1P at its maximum tolerated dose (MTD) alone. CONCLUSION: These data demonstrate that the combination of CA1P and CPL has significant preclinical antitumour activity against a transplantable murine adenocarcinoma model that is related to the antivascular effects of CA1P.

Vinflunine potentiates the activity of cisplatin but not 5-fluorouracil in a transplantable murine adenocarcinoma model.

BACKGROUND: Vinflunine is a novel Vinca alkaloid currently undergoing Phase II clinical trials, which have previously demonstrated anti-vascular effects in a transplantable murine colon adenocarcinoma model. Previous studies with compounds showing similar effects in combination with standard anti-cancer agents have demonstrated an improved efficacy relative to the standard agents. MATERIALS AND METHODS: In this study the synergistic effects of administering vinflunine in combination with either Cisplatin (CPL) or 5-fluorouracil (5-FU) were investigated in a well-differentiated transplantable murine colon adenocarcinoma model (MAC 29). RESULTS: Vinflunine significantly potentiated the anti-tumour effects of CPL, but had little effect in combination with 5-FU. Using Hoescht 33342 dye labelling of the functional vasculature, clear evidence of vascular shutdown was seen for treatment groups including vinflunine. CONCLUSION: These data demonstrate that the combination of vinflunine and CPL has significant preclinical anti-tumour activity against a transplantable murine adenocarcinoma model that is related to the anti-vascular effects of vinflunine.

Combretastatin A-1 phosphate a novel tubulin-binding agent with in vivo anti vascular effects in experimental tumours.

In view of the clinical potential of a number of natural products, Combretastatin A-1 phosphate was developed as a water-soluble derivative of combretastatin A-1. This study examined the anti-tumour activity of this compound against an experimental colon tumour (MAC29) in mice. A comparison was made with the clinically active combretastatin A-4 phosphate. The new compound was well-tolerated up to a dose of 250 mg/kg and was more effective at producing tumour growth delays than the A-4 analogue. Significant growth delays were seen at a dose of 50 mg/kg whereas the A-4 phosphate produced no measurable growth delay until a dose of 150 mg/kg was administered. Histological examination of treated tumours indicated that combretastatin A-1 phosphate caused very severe haemorrhagic necrosis in the tumour tissue and analysis of the sections indicated that almost 94 percent of the tumour was dead within 24 hours of treatment. The mechanism of action of combretastatin A-1 phosphate appears to be similar to the A-4 phosphate in that tumour vascular shutdown occurs within 4 hours of treatment. In summary combretastatin A-1 phosphate, the water-soluble analogue of combretastatin A-1, is more potent against a well-vascularised murine colon tumour than its predecessor, combretastatin A-4 phosphate. These data suggest this compound may have potential for clinical development.

In vitro and in vivo activity of LS 4477 and LS 4559, novel analogues of the tubulin binder estramustine.

LS 4477 and LS 4559, two of a series of N-acyl-aminoalkyl phenyl ethers, are rationally designed compounds based on the tubulin binder estramustine. This study investigated their mechanism of action and compared their effectiveness in relation to estramustine in vitro against a panel of human and murine cell lines and in vivo against two murine colon tumour models (MAC). At biologically relevant concentrations, LS 4477 and LS 4559 caused a 59.9 and 56% reduction in tubulin assembly, respectively, compared with a 28.4% reduction in tubulin assembly by estramustine. The analogues were approximately 100 times more potent in chemosensitivity tests in vitro than the parent compound. Both analogues were orally active against the MAC 15A murine tumour model, to a greater extent than estramustine, producing significant growth delays (P<0.01). Significant activity was also shown against the slower growing MAC 26 tumour for LS 4577 (the soluble pro-drug of LS 4559). The results presented in this study suggest these compounds warrant further development with a view to assessing their clinical activity.

P-glycoprotein and metallothionein expression and resistance to chemotherapy in osteosarcoma.

The expression of the drug resistance (DR) mediators P-glycoprotein (P-gp) and the metallothioneins (MT) was assessed immunohistochemically in biopsy material from patients with high-grade malignant osteosarcoma (OS). No significant difference was found in survival rate between expressors of both P-gp and MT and non-expressors. Thus, it was concluded that lack of expression of these two drug resistance-related proteins does not appear to confer any advantage in terms of patient survival in osteosarcoma.