Colin R. Lindsay

P-Rex1 is required for efficient melanoblast migration and melanoma metastasis

Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.

A phase I study of continuous oral dosing of OSI-906, a dual inhibitor of insulin-like growth factor-1 and insulin receptors, in patients with advanced solid tumors

Purpose: OSI-906 is a potent inhibitor of insulin-like growth factor-1 receptor (IGF1R) and insulin receptor (IR). The purpose of this study was to determine the MTD, safety, pharmacokinetics, pharmacodynamics, and preliminary activity of OSI-906 in patients with advanced solid tumors. Patients and Methods: This was a nonrandomized, open-label, phase I, dose-escalation study in patients with advanced solid tumors. The study also included a diabetic expansion cohort and a biomarker expansion cohort of patients with colorectal cancer. Patients were treated with OSI-906 by once- or twice-daily continuous dosing schedules. Results: Of 95 patients enrolled in the study, 86 received at least one dose of OSI-906. Dose-limiting toxicities included QTc prolongation, grade 2 abdominal pain and nausea, hyperglycemia, and elevation of aspartate aminotransferase and alanine aminotransferase (all grade 3). The MTDs were established to be 400 mg once daily and 150 mg twice daily. The recommended phase II dose was determined as 150 mg twice daily. OSI-906 was rapidly absorbed with a half-life of 5 hours, and steady-state plasma concentrations were achieved by day 8. Pharmacodynamic effects on IGF1R and IR phosphorylation were levels observed and correlated with plasma concentrations of OSI-906. Thirty-one patients had stable disease as their best response. One patient with melanoma had a radiographic partial response and underwent resection, during which only melanocytic debris but no viable tumor tissue was identified. Conclusions: At the established MTD, OSI-906 was well tolerated and antitumor activity was observed. These results support further evaluation of OSI-906 in solid tumors. Clin Cancer Res; 21(4); 701–11. ©2014 AACR. See related commentary by Yee, p. 667

Melanoma Cells Break Down LPA to Establish Local Gradients That Drive Chemotactic Dispersal

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.

Current status of cediranib: the rapid development of a novel anti-angiogenic therapy

Angiogenesis, the process whereby tumors develop new blood vessels to facilitate growth and metastasis, is a pivotal event in tumorigenesis. It is tightly regulated by the VEGF system. Cediranib (AZD2171, Recentin; AstraZeneca, London, UK) is a novel and potent small-molecule inhibitor of VEGF signaling, with activity against the three VEGF receptors, as well as other targets. This article provides a comprehensive and up-to-date synopsis of all pertinent preclinical and clinical studies detailing this promising new therapy.

A Rac1-Independent Role for P-Rex1 in Melanoblasts

TO THE EDITOR Given the recent discovery of RAC1-activating mutations in melanoma, and our finding that PIP3-dependent Rac-exchanger 1 (PREX1) is overexpressed and drives metastasis in this cancer, an important question is to establish whether the functions of P-Rex1 are mediated specifically by Rac alone (Lindsay et al., 2011; Berger et al., 2012). Here we describe a Rac1-independent in vivo role for P-Rex1 through identification and characterization of a mouse coat color phenotype. P-Rex1 is a guanine-nucleotide exchange factor (GEF) for Rac, whose primary cell function is induction of actin-mediated membrane ruffling and lamellipodia formation at the leading edge of cell migration (Welch et al., 2002; Hill et al., 2005; Barber et al., 2007). To investigate this question we decided to examine the role of Rac1 and P-Rex1 in melanoblast development. Previously, we reported a “white belly” phenotype of mice with Prex1 deletion (Lindsay et al., 2011). Impaired melanoblast migration was mostly responsible for this phenotype, with melanoblasts lacking at the most distal points of migration (belly and paws). Constitutive deletion of Rac1 is embryonically lethal, but a coat color defect of mice with melanocyte-specific RAC1 abrogation (Tyr::Cre Rac1fl/fl) has also been described; these mice have a larger belly spot on their ventral side, suggesting that alternative Rho-GTPases can be activated to enable melanoblast migration to the perimeter of the Tyr::Cre Racfl/fl white belly (Sugihara et al., 1998; Li et al., 2011). A role for Rac1 in proliferation was also observed, as there was a marked reduction of melanoblast numbers in this phenotype. In line with these previous studies, and because mice with melanocyte-specific RAC1 abrogation require euthanization shortly after birth because of neurological problems, we used the same embryonic melanoblast reporter models to assess the downstream effects of P-Rex1 in vivo (Mackenzie et al., 1997; Mort et al., 2010; Li et al., 2011). Melanocyte-specific reporter mouse strains employed were Tyr::Cre Z/EG, which drives green fluorescent protein expression in the melanoblast lineage, and DCT::β-galactosidase (otherwise referred to as DCT-lacZ). First, we hypothesized that, if the effects of P-Rex1 were mediated exclusively via Rac1, double mutant Tyr::Cre Rac1fl/fl; P-Rex1−/− mice would exhibit the same coat color phenotype as Tyr::Cre Racfl/fl mice alone. However, Tyr::Cre Racfl/fl; P-Rex1−/− mice display a dramatic alteration in coat color phenotype from Tyr::Cre Racfl/fl mice (n=7; Figure 1a). The ventral and dorsal coats of these mice are almost entirely white, with hypo-pigmented limbs and tail. Graying pigmented areas were only observed in the head coat. We concluded from this experiment that P-Rex1 and Rac1 together constitute fundamental signaling components of the mouse coat color phenotype, with minimal rescue of melanoblast development conferred by other GEFs or Rho-GTPases. It was also clear that P-Rex1 must be able to exert phenotypic effects other than via Rac1. Figure 1 P-Rex1 and Rac1 are fundamental components of a mouse coat color phenotype. (a) Ventral coats of P-Rex1−/−, Tyr::Cre Rac1fl/fl and P-Rex1−/−;Tyr::Cre Rac1fl/flmice. Final photomicrograph shows dorsal and head coat of Tyr::Cre ... To explore the Rac1-independent effects of P-Rex1 further, we crossed Tyr::Cre Racfl/fl; P-Rex1−/− mice with mice carrying the melanoblast reporter DCT-lacZ transgene (methods detailed in Lindsay et al., 2011). Relative to Tyr::Cre Racfl/flmice or P-Rex1−/− embryos alone, Tyr::Cre Racfl/fl; P-Rex1−/− embryos at E15.5 displayed a substantial reduction in melanoblast numbers across their entire body (Figure 1b and c). To assess whether the cause of this reduction in melanoblast numbers could be accounted for by decreased proliferation ± increased cell death, we next treated our previously described primary immortalized Tyr::CrER2 INK4a−/− Racfl/fl melanocyte cell line with short interfering RNA to P-Rex1 (methods detailed in Li et al., 2012). The use of this model system also allowed us to delete Rac1 function when these cells were treated with 4-hydroxytamoxifen (OHT). We first used western blotting to confirm that efficient P-Rex1 knockdown and OHT-induced Rac deletion were achieved (Figure 1d). There was no increase in cleaved caspase-3 evident in the absence of P-Rex1 and/or Rac, suggesting that the reduced cell numbers observed in Tyr::Cre Racfl/fl; P-Rex1−/− embryos were not accounted for by increased cell death (Figure 1d). Growth curves and anti-BrdU immunofluorescence of the same cell lines confirmed that there was a reduced proliferation in P-Rex1-depleted cells, both in the presence and absence of OHT (Figure 1e; Supplementary Figure S1a and b online). Taken together, these results suggest, in addition to our previously reported effects of Rac1 deletion on mouse coat color, that the loss of a Rac1-independent proliferative effect of P-Rex1 also contributes to the coat color phenotype observed in Tyr::Cre Racfl/fl; P-Rex1−/− mice (Li et al., 2011). In line with our previous characterization of the P-Rex1 knockout phenotype alone, we next decided to delineate whether the coat color phenotype of Tyr::Cre Racfl/fl; P-Rex1−/− mice could also be a consequence of reduced melanoblast migration (Lindsay et al., 2011). To assess this, mice with the Z/EG double reporter transgene were crossed with Tyr::Cre Racfl/fl; P-Rex1−/−mice, driving green fluorescent protein expression in the melanoblast lineage (methods detailed in Lindsay et al., 2011; Figure 2a). Live imaging of melanoblasts was performed using E15.5 embryo skin from each genotype (Figure 2b; Supplementary Movies S1–3 online). Consistent with our previous work, significant reductions in migration speed were observed between wild-type, P-Rex1−/− and Tyr::Cre Racfl/fl melanoblasts (Figure 2c and d). However, there was no significant difference in speed between Tyr::Cre Racfl/fl; P-Rex1−/− and Tyr::Cre Racfl/fl melanoblasts alone, suggesting that there was no change in migratory characteristics to account for the Tyr::Cre Racfl/fl; P-Rex1−/− phenotype (Figure 2c and d). These results were matched by similar differences between the same genotypes when Euclidean distance was measured (Figure 2e), as well as no observable change in cell morphology evident in Tyr::Cre Racfl/fl; P-Rex1−/− compared with Tyr::Cre Racfl/fl melanoblasts alone (Supplementary Figure S1c online; methods detailed in Helmy and Azim, 2012). Finally, no cell death was seen in our melanoblast time-lapse movies of any genotype, again suggesting that P-Rex1 contributes to coat color phenotype by promoting cell proliferation using a Rac1-independent mechanism (Li et al., 2011; Supplementary Movies S1–3 online; methods detailed in Lindsay et al., 2011). Figure 2 P-Rex1 has no additional effect on migration compared with Rac1 alone. All experiments show embryo skin explants at E15.5 (a) Combined Z-stack confocal images of Z/EG melanoblasts from wild-type (control), P-Rex1−/−, Tyr::Cre Rac1fl/fl ... To conclude, we have elucidated a proliferative role of P-Rex1 when Rac1 is deleted. As E15.5 migratory characteristics are not altered in the Tyr::Cre Racfl/fl; P-Rex1−/− double mutant embryos, this suggests that the role of P-Rex1 in migration is almost exclusively mediated via Rac1. Here we focused on E15.5 embryos, a useful time point to observe the late migratory effects observed with previously described Prex and Rac phenotypes (Li et al., 2011; Lindsay et al., 2011). With a greater number of embryos, we would have performed further embryo time-point analyses at E13.5 to ensure there was no earlier melanoblast migratory deficit that could contribute to this phenotype, although even this experiment could not completely exclude such a possibility. One potential Rho-GTPase, RhoG, is a likely candidate for P-Rex1 interaction: it is the most structurally similar Rho-GTPase to Rac and has been shown to cooperate with Rac for induction of cell transformation (Roux et al., 1997). Moreover, there are distinct regulatory and functional similarities between P-Rex1 and Vav proteins, which have been characterized as the predominant GEFs required for RhoG activation (Samson et al., 2010; Lawson et al., 2011). Further studies are underway to investigate the phenotypes of these and other potential Rho-GTPases in melanoblast migration and melanomagenesis. Given that there is now considerable effort to generate Rac1 inhibitors, our data would suggest functions for proteins upstream of Rac1 that may become further therapeutic targets in melanoma. All experiments were conducted and approved in accordance with institutional and UK guidelines, and all animal studies were performed in accordance with local regulatory guidelines.

The insulin-like growth factor system and its receptors: A potential novel anticancer target

The current generation of novel anticancer therapies that are in preclinical and clinical development are based on exploiting our increasing understanding of the molecular and cellular basis of cancer development and progression. Accelerated rates of cell division and proliferation have been postulated to predispose to the development of malignant disease. The insulin-like growth factor (IGF) signaling system has an important physiological role in regulating cellular proliferation and apoptosis. This function has led to considerable interest in its relevance to neoplasia over the last decade. In this review, we give an overview of the IGF system physiology, discuss the epidemiological significance of IGF signaling and neoplasia, and review the preclinical and clinical studies in targeting IGF receptors as cancer therapies.

Blinded by the light: why the treatment of metastatic melanoma has created a new paradigm for the management of cancer.

Until recently, treatment for metastatic melanoma was characterised by a limited availability of treatment options that offer objective survival benefit. Cytotoxic agents fundamentally lack the ability to achieve disease control and cytokine therapy with interleukin-2 has an unacceptably high – for the use across all patient cohorts – rate of toxicities. The validation of braf as an oncogene driving melanoma tumorigenesis, as well as the discovery of the role of CTLA-4 receptor in the evasion of anticancer immune response by melanoma, has revolutionised our treatment options against a disease with dismal prognosis. Quick implementation of translational discoveries brought about BRAF/MEK inhibition in clinic, while at the same time, wider experience with CTLA-4 blockade enabled clinicians to manage previously fatal immune-related toxicities with greater confidence. The suitability for clinical use of other oncogenic drivers such as NRAS and c-kit is currently being tested whilst the PD-1/PD-L1/PD-L2 axis has emerged as a new immunotherapy target with exciting early phase results. The recent exponential progress in treatment of melanoma has set an example of translational medicine and the current review aims to explain why, as well as suggesting new goals for the future.

XELOX in colorectal cancer: a convenient option for the future?

XELOX is a 3-weekly chemotherapy combination of oral capecitabine and intravenous oxaliplatin. The central hypothesis that led to its development was that it would provide a convenient and cost-effective alternative to intravenous fluorouracil-based chemotherapy doublets, without compromising on anti-tumor efficacy. Recently its role in colorectal cancer has become more established in both the metastatic and adjuvant setting. Ongoing investigation of XELOX continues in a number of directions: its combination with novel biological agents, its efficacy and safety in the elderly, and the development of biomarkers that can predict its anti-tumor effect. This article provides a comprehensive and up-to-date synopsis of all pertinent clinical studies detailing this regimen and its promise for the future.

Recombinant human epoetin beta in the treatment of chemotherapy-related anemia

Anemia is a common complication of systemic anti-cancer treatment. In this context epoetin beta, like other erythropoiesis-stimulating agents (ESAs), has demonstrable efficacy in raising Hb concentration and reducing the requirement for red cell transfusion. Consequently ESA therapy has gained increasing prominence in the management of chemotherapy-related anemia. However, recent trial data have suggested a higher rate of thromboembolic events, enhanced tumor progression and reduced survival in some patients with cancer who receive ESA therapy. In response, regulatory authorities have mandated increasingly restrictive label changes. In light of these new developments we consider the current role of epoetin beta in the management of chemotherapy-related anemia.

How do we optimally use cetuximab in first-line treatment for metastatic colorectal cancer?

A number of Phase II/III clinical trials have now been reported assessing the role of cetuximab use in combination with chemotherapy for the first-line treatment of metastatic colorectal cancer. Here we review the current position of cetuximab in this context from two important perspectives. First, we address whether clinicians should prioritize its use with oxaliplatin- or irinotecan-based chemotherapy doublets. Second, we review the trial evidence for the use of cetuximab in patients suffering from colorectal cancer with wild-type and mutant Ras.