Greg Plowman

Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis.

Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.

Blocking Neuropilin-2 Function Inhibits Tumor Cell Metastasis

Metastasis, which commonly uses lymphatics, accounts for much of the mortality associated with cancer. The vascular endothelial growth factor (VEGF)-C coreceptor, neuropilin-2 (Nrp2), modulates but is not necessary for developmental lymphangiogenesis, and its significance for metastasis is unknown. An antibody to Nrp2 that blocks VEGFC binding disrupts VEGFC-induced lymphatic endothelial cell migration, but not proliferation, in part independently of VEGF receptor activation. It does not affect established lymphatics in normal adult mice but reduces tumoral lymphangiogenesis and, importantly, functional lymphatics associated with tumors. It also reduces metastasis to sentinel lymph nodes and distant organs, apparently by delaying the departure of tumor cells from the primary tumor. Our results demonstrate that Nrp2, which was originally identified as an axon-guidance receptor, is an attractive target for modulating metastasis.

Chronic DLL4 blockade induces vascular neoplasms

Arising from: J. Ridgway et al. 444, 1083–1087 (2006)10.1038/nature05313Delta-like 4 (DLL4)-mediated Notch signalling has emerged as an attractive target for cancer therapy. However, the potential side effects of blocking this pathway remain uncertain. Here we show that chronic DLL4 blockade causes pathological activation of endothelial cells, disrupts normal organ homeostasis and induces vascular tumours, raising important safety concerns.

Effects of Anti-VEGF Treatment Duration on Tumor Growth, Tumor Regrowth, and Treatment Efficacy

Purpose: Inhibition of the vascular endothelial growth factor (VEGF) axis is the basis of all currently approved antiangiogenic therapies. In preclinical models, anti-VEGF blocking antibodies have shown broad efficacy that is dependent on both tumor context and treatment duration. We aimed to characterize this activity and to evaluate the effects of discontinuation of treatment on the dynamics of tumor regrowth. Experimental Design: We evaluated the effects of anti-VEGF treatment on tumor growth and survival in 30 xenograft models and in genetic mouse models of cancer. Histologic analysis was used to evaluate the effects of treatment on tumor vasculature. We used a variety of treatment regimens to allow analysis of the effects of treatment duration and cessation on growth rate, survival, and vascular density. Results: Preclinical tumor models were characterized for their varied dependence on VEGF, thereby defining models for testing other agents that may complement or augment anti-VEGF therapy. We also found that longer exposure to anti-VEGF monoclonal antibodies delayed tumor growth and extended survival in established tumors from both cell transplants and genetic tumor models and prevented regrowth of a subset of residual tumors following cytoablative therapy. Discontinuation of anti-VEGF in established tumors resulted in regrowth at a rate slower than that in control-treated animals, with no evidence of accelerated tumor growth or rebound. However, more rapid regrowth was observed following discontinuation of certain chemotherapies. Concurrent administration of anti-VEGF seemed to normalize these accelerated growth rates. Conclusions: In diverse preclinical models, continuous VEGF suppression provides maximal benefit as a single agent, combined with chemotherapy, or as maintenance therapy once chemotherapy has been stopped. Clin Cancer Res; 16(15); 3887–900. ©2010 AACR.

Canonical hedgehog signaling augments tumor angiogenesis by induction of VEGF-A in stromal perivascular cells

Hedgehog (Hh) signaling is critical to the patterning and development of a variety of organ systems, and both ligand-dependent and ligand-independent Hh pathway activation are known to promote tumorigenesis. Recent studies have shown that in tumors promoted by Hh ligands, activation occurs within the stromal microenvironment. Testing whether ligand-driven Hh signaling promotes tumor angiogenesis, we found that Hh antagonism reduced the vascular density of Hh-producing LS180 and SW480 xenografts. In addition, ectopic expression of sonic hedgehog in low-Hh–expressing DLD-1 xenografts increased tumor vascular density, augmented angiogenesis, and was associated with canonical Hh signaling within perivascular tumor stromal cells. To better understand the molecular mechanisms underlying Hh-mediated tumor angiogenesis, we established an Hh-sensitive angiogenesis coculture assay and found that fibroblast cell lines derived from a variety of human tissues were Hh responsive and promoted angiogenesis in vitro through a secreted paracrine signal(s). Affymetrix array analyses of cultured fibroblasts identified VEGF-A, hepatocyte growth factor, and PDGF-C as candidate secreted proangiogenic factors induced by Hh stimulation. Expression studies of xenografts and angiogenesis assays using combinations of Hh and VEGF-A inhibitors showed that it is primarily Hh-induced VEGF-A that promotes angiogenesis in vitro and augments tumor-derived VEGF to promote angiogenesis in vivo.

Use of anti-VEGF adjuvant therapy in cancer: challenges and rationale

Several ongoing clinical studies are designed to test the efficacy of antiangiogenic therapies in the adjuvant setting, where the goal is to increase the cure rate in patients who have just undergone surgical resection of all visible disease. Tumors depend on angiogenesis to support their growth and progression and blockade of this process has proven to be a valid strategy for treating multiple types of advanced metastatic cancer. However, results from the first of these clinical adjuvant studies were disappointing, stimulating extensive debate as to the potential of this approach. It will require additional clinical studies before we realize whether the effects of angiogenic blockade are durable, and if they are able to cure a subset of patients with early stage cancer.

Plasma membrane calcium ATPase 4 and the remodeling of calcium homeostasis in human colon cancer cells

A remodeling of calcium homeostasis has been identified as a characterizing feature of some cancers. Possible consequences of this include alterations in many pivotal physiological responses including apoptosis, proliferation and gene transcription. An alteration in calcium homeostasis can occur via changes in the expression of proteins that transport calcium and examples of cancers where this is seen includes the prostate and breast. A specific isoform of the calcium efflux pump, plasma membrane Ca(2+)-ATPase (PMCA) 4, is significantly upregulated during differentiation of the HT-29 colon cancer cell line suggesting that it may also be altered in colon cancer. We now report that differentiated HT-29 colon cancer cells have pronounced plasma membrane PMCA4 localization, consistent with augmented calcium efflux. Assessment of PMCA4 transcription in human colon cancer samples suggests that PMCA4 is significantly (P < 0.000001) downregulated early in the progression of some colon cancers as these cells become less differentiated. Inhibition of PMCA4 using small interfering RNA did not induce cell death or augment sensitivity to the mitochondrial uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or tumor necrosis factor-related apoptosis-inducing ligand. Reversing the colon cancer remodeling of PMCA4 by overexpression reduced cellular proliferation (P < 0.01) and downregulated transcription of the calcium sensitive early response gene FOS. Our studies suggest that the remodeling of the calcium signal in colon cancer is associated with compromised calcium efflux at a level that promotes proliferative pathways while avoiding increased sensitivity to apoptotic stimuli.

Neuropilin-2 mediated β-catenin signaling and survival in human gastro-intestinal cancer cell lines

NRP-2 is a high-affinity kinase-deficient receptor for ligands belonging to the class 3 semaphorin and vascular endothelial growth factor families. NRP-2 has been detected on the surface of several types of human cancer cells, but its expression and function in gastrointestinal (GI) cancer cells remains to be determined. We sought to determine the function of NRP-2 in mediating downstream signals regulating the growth and survival of human gastrointestinal cancer cells. In human gastric cancer specimens, NRP-2 expression was detected in tumor tissues but not in adjacent normal mucosa. In CNDT 2.5 cells, shRNA mediated knockdown NRP-2 expression led to decreased migration and invasion in vitro (p<0.01). Focused gene-array analysis demonstrated that loss of NRP-2 reduced the expression of a critical metastasis mediator gene, S100A4. Steady-state levels and function of β-catenin, a known regulator of S100A4, were also decreased in the shNRP-2 clones. Furthermore, knockdown of NRP-2 sensitized CNDT 2.5 cells in vitro to 5FU toxicity. This effect was associated with activation of caspases 3 and 7, cleavage of PARP, and downregulation of Bcl-2. In vivo growth of CNDT 2.5 cells in the livers of nude mice was significantly decreased in the shNRP-2 group (p<0.05). Intraperitoneal administration of NRP-2 siRNA-DOPC decreased the tumor burden in mice (p = 0.01). Collectively, our results demonstrate that tumor cell–derived NRP-2 mediates critical survival signaling in gastrointestinal cancer cells.

Analysis of multi-arm tumor growth trials in xenograft animals using phase change adaptive piecewise quadratic models

Xenograft trials allow tumor growth in human cell lines to be monitored over time in a mouse model. We consider the problem of inferring the effect of treatment combinations on tumor growth. A piecewise quadratic model with flexible phase change locations is proposed to model the effect of change in therapy over time. Each piece represents a growth phase, with phase changes in response to change in treatment. Piecewise slopes represent phase-specific (log) linear growth rates and curvature parameters represent departure from linear growth. Trial data are analyzed in two stages: (i) subject-specific curve fitting (ii) analysis of slope and curvature estimates across subjects. A least-squares approach with penalty for phase change point location is proposed for curve fitting. In simulation studies, the method is shown to give consistent estimates of slope and curvature parameters under independent and AR (1) measurement error. The piecewise quadratic model is shown to give excellent fit (median R(2)=0.98) to growth data from a six armed xenograft trial on a lung carcinoma cell line.

A comparison of least squares and conditional maximum likelihood estimators under volume endpoint censoring in tumor growth experiments

Measurements in tumor growth experiments are stopped once the tumor volume exceeds a preset threshold: a mechanism we term volume endpoint censoring. We argue that this type of censoring is informative. Further, least squares (LS) parameter estimates are shown to suffer a bias in a general parametric model for tumor growth with an independent and identically distributed measurement error, both theoretically and in simulation experiments. In a linear growth model, the magnitude of bias in the LS growth rate estimate increases with the growth rate and the standard deviation of measurement error. We propose a conditional maximum likelihood estimation procedure, which is shown both theoretically and in simulation experiments to yield approximately unbiased parameter estimates in linear and quadratic growth models. Both LS and maximum likelihood estimators have similar variance characteristics. In simulation studies, these properties appear to extend to the case of moderately dependent measurement error. The methodology is illustrated by application to a tumor growth study for an ovarian cancer cell line.