Jeanine M.L. Roodhart

Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents

Several hypotheses have been proposed to explain how antiangiogenic drugs enhance the treatment efficacy of cytotoxic chemotherapy, including impairing the ability of chemotherapy-responsive tumors to regrow after therapy. With respect to the latter, we show that certain chemotherapy drugs, e.g., paclitaxel, can rapidly induce proangiogenic bone marrow-derived circulating endothelial progenitor (CEP) mobilization and subsequent tumor homing, whereas others, e.g., gemcitabine, do not. Acute CEP mobilization was mediated, at least in part, by systemic induction of SDF-1alpha and could be prevented by various procedures such as treatment with anti-VEGFR2 blocking antibodies or paclitaxel treatment in CEP-deficient Id mutant mice, both of which resulted in enhanced antitumor effects mediated by paclitaxel, but not by gemcitabine.

The Molecular Basis of Class Side Effects Due to Treatment with Inhibitors of the VEGF/VEGFR Pathway

UNLABELLED Vascular Endothelial Growth Factor (VEGF) is considered to be one of the most important regulators of angiogenesis and a new key target in anti-cancer treatment. Various clinical trials have validated the clinical importance of anti-VEGF or anti-VEGF receptor (VEGFR) therapy. Currently the humanized monoclonal antibody bevacizumab (blocks VEGF-A), and the tyrosine kinase inhibitors sunitinib and sorafenib (inhibit VEGFRs) are approved for patients with various malignancies and several others are expected in the coming years. Unfortunately, anti-VEGF/VEGFR treatment is not void of side effects. An array of unexpected side effects is now seen in clinical practice. Management of these side effects is extremely important in the development of the various anti-VEGF/VEGFR therapies and their optimal use. This review provides an overview of the toxicity profile of this class of agents, the molecular basis behind these side effects and indicates potential options for management. VEGF and its receptors play an important role in normal tissues and are widely expressed. It is likely that interference with this pathway induces an array of side effects due to the lack of normal function of VEGF. A consistent pattern of side effects is now emerging. Hypertension, gastro-intestinal toxicity, hypothyroidism, proteinuria, coagulation disorders and neurotoxicity are side effects observed with both anti-VEGF and anti-VEGFR inhibitors. For these side effects the role of VEGF/VEGFR pathway in normal tissue was reviewed in order to provide a molecular mechanism that linked side effect with physiological activity of VEGF/VEGFR. Insight into the molecular basis may aid specific supportive care measures to ensure optimal use of this class of agents. CONCLUSION Inhibiting the VEGF/VEGFR pathway is an effective approach to treat cancer. It has also provided new insight into the physiological role of this pathway in various organs. Integrating the knowledge in daily oncological practice will be a challenge for the future.

Cytosolic p120-catenin regulates growth of metastatic lobular carcinoma through Rock1-mediated anoikis resistance

Metastatic breast cancer is the major cause of cancer-related death among women in the Western world. Invasive carcinoma cells are able to counteract apoptotic signals in the absence of anchorage, enabling cell survival during invasion and dissemination. Although loss of E-cadherin is a cardinal event in the development and progression of invasive lobular carcinoma (ILC), little is known about the underlying mechanisms that govern these processes. Using a mouse model of human ILC, we show here that cytosolic p120-catenin (p120) regulates tumor growth upon loss of E-cadherin through the induction of anoikis resistance. p120 conferred anchorage independence by indirect activation of Rho/Rock signaling through interaction and inhibition of myosin phosphatase Rho-interacting protein (Mrip), an antagonist of Rho/Rock function. Consistent with these data, primary human ILC samples expressed hallmarks of active Rock signaling, and Rock controlled the anoikis resistance of human ILC cells. Thus, we have linked loss of E-cadherin - an initiating event in ILC development - to Rho/Rock-mediated control of anchorage-independent survival. Because activation of Rho and Rock are strongly linked to cancer progression and are susceptible to pharmacological inhibition, these insights may have clinical implications for the development of tailor-made intervention strategies to better treat invasive and metastatic lobular breast cancer.

Two-protein signature of novel serological markers apolipoprotein-A2 and serum amyloid alpha predicts prognosis in patients with metastatic renal cell cancer and improves the currently used prognostic survival models

BACKGROUND In metastatic renal cell cancer (mRCC), the Memorial Sloan-Kettering Cancer Center (MSKCC) risk model is widely used for clinical trial design and patient management. To improve prognostication, we applied proteomics to identify novel serological proteins associated with overall survival (OS). PATIENTS AND METHODS Sera from 114 mRCC patients were screened by surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF MS). Identified proteins were related to OS. Three proteins were subsequently validated with enzyme-linked immunosorbent assays and immunoturbidimetry. Prognostic models were statistically bootstrapped to correct for overestimation. RESULTS SELDI-TOF MS detected 10 proteins associated with OS. Of these, apolipoprotein A2 (ApoA2), serum amyloid alpha (SAA) and transthyretin were validated for their association with OS (P = 5.5 x 10(-9), P = 1.1 x 10(-7) and P = 0.0004, respectively). Combining ApoA2 and SAA yielded a prognostic two-protein signature [Akaikes Information Criteria (AIC) = 732, P = 5.2 x 10(-7)]. Including previously identified prognostic factors, multivariable Cox regression analysis revealed ApoA2, SAA, lactate dehydrogenase, performance status and number of metastasis sites as independent factors for survival. Using these five factors, categorization of patients into three risk groups generated a novel protein-based model predicting patient prognosis (AIC = 713, P = 4.3 x 10(-11)) more robustly than the MSKCC model (AIC = 729, P = 1.3 x 10(-7)). Applying this protein-based model instead of the MSKCC model would have changed the risk group in 38% of the patients. CONCLUSIONS Proteomics and subsequent validation yielded two novel prognostic markers and survival models which improved prediction of OS in mRCC patients over commonly used risk models. Implementation of these models has the potential to improve current risk stratification, although prospective validation will still be necessary.

Increased Plasma Levels of Chemoresistance-Inducing Fatty Acid 16:4(n-3) After Consumption of Fish and Fish Oil

IMPORTANCE Our research group previously identified specific endogenous platinum-induced fatty acids (PIFAs) that, in picomolar quantities, activate splenic macrophages leading to resistance to chemotherapy in mouse models. Fish oil was shown to contain the PIFA 16:4(n-3) (hexadeca-4,7,10,13-tetraenoic acid) and when administered to mice neutralized chemotherapy activity. OBJECTIVE Because patients with cancer frequently use fish oil supplements, we set out to determine exposure to 16:4(n-3) after intake of fish or fish oil. DESIGN, SETTING, AND PARTICIPANTS (1) In November 2011, 400 patients with cancer undergoing treatment at the University Medical Center Utrecht were surveyed to determine their use of fish oil supplements; 118 patients responded to the questionnaire (30%); (2) pharmacokinetic analysis of the 16:4(n-3) content of 6 fish oils and 4 fishes was carried out; (3) from April through November 2012, a healthy volunteer study was performed to determine 16:4(n-3) plasma levels after intake of 3 different brands of fish oil or 4 different fish species. Thirty healthy volunteers were randomly selected for the fish oil study; 20 were randomly selected for the fish study. These studies were supported by preclinical tumor experiments in mice to determine chemoresistance conducted between September 2011 and December 2012. MAIN OUTCOMES AND MEASURES (1) Rate of use of fish oil supplements among patients undergoing cancer treatment at our institution; (2) levels of 16:4(n-3) present in 3 brands of fish oil and 4 species of fish; and (3) plasma levels of 16:4(n-3) present in healthy volunteers after consuming fish oil or fish. RESULTS Eleven percent of respondents reported using omega-3 supplements. All fish oils tested contained relevant amounts of 16:4(n-3), from 0.2 to 5.7 µM. Mouse experiments showed that addition of 1 µL of fish oil to cisplatin was sufficient to induce chemoresistance, treatment having no impact on the growth rate of tumors compared with vehicle-treated controls (estimated tumor volume difference, 44.1 mm3; P > .99). When the recommended daily amount of 10 mL of fish oil was administered to healthy volunteers, rises in plasma 16:4(n-3) levels were observed, reaching up to 20 times the baseline levels. Herring and mackerel contained high levels of 16:4(n-3) in contrast to salmon and tuna. Consumption of fish with high levels of 16:4(n-3) also resulted in elevated plasma levels of 16:4(n-3). CONCLUSIONS AND RELEVANCE All tested fish oils and herring and mackerel fishes contained relevant levels of fatty acid 16:4(n-3), a fatty acid with chemotherapy-negating effects in preclinical models. After ingestion of these fish oils or fishes, 16:4(n-3) was rapidly taken up in the plasma of human volunteers. Until further data become available, fish oil and fish containing high levels of 16:4(n-3) may best be avoided on the days surrounding chemotherapy.

Analysis of docetaxel therapy in elderly (⩾70 years) castration resistant prostate cancer patients enrolled in the Netherlands Prostate Study

BACKGROUND Prostate cancer truly is an age-associated disease. Due to the increased life expectancy and more sensitive diagnostic techniques in the Western world, prostate cancer is diagnosed more frequently and with rapidly increasing incidence and prevalence rates. However, age above 65 or 70 years has been an exclusion criterion in clinical trials for decades and the knowledge about chemotherapy tolerance in elderly is limited. METHODS We performed a retrospective analysis of data acquired from the recently published Netherlands Prostate Study (NePro) to evaluate the influence of advanced age on docetaxel therapy in elderly men (>70 years) with castration resistant prostate cancer (CRPC) and bone metastases. Statistical analyses were performed stratified for age into four categories: <70 (n=315), 70-74 (n=150), 75-79 (n=85), and ≥80 years old (n=18). RESULTS We analysed 568 patients (median age 68.1 years, range 46-89 years, 44.5% aged ≥70 years). There was no relation between dosage and age (p=0.60). We found no significant differences between the number of dose reductions, time to progression (TTP), overall survival, chemotherapy tolerance and toxicity up to the age of 80 years. However, when compared to younger men, men aged 80 years or above more frequently experienced grade 3/4 toxicity and were five times less likely to complete the first three treatment cycles at the intended dose (Odds ratio (OR) 5.34, p=0.0052) and showed decreased overall survival (15.3 months versus 24.5 months in <80 years group, p=0.020). CONCLUSION In CRPC patients up to the age of 80 years, docetaxel chemotherapy is well tolerated, with toxicity levels and TTP comparable to those of younger patients. For chemotherapeutic treatment of patients above the age of 80 years an individual assessment should be made.

Lysophospholipids secreted by splenic macrophages induce chemotherapy resistance via interference with the DNA damage response

Host responses to systemic anti-cancer treatment play important roles in the development of anti-cancer drug resistance. Here we show that F4/80(+)/CD11b(low) splenocytes mediate the resistance to DNA-damaging chemotherapeutics induced by two platinum-induced fatty acids (PIFAs), 12-S-keto-5,8,10-heptadecatrienoic acid and 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)) in xenograft mouse models. Splenectomy or depletion of splenic macrophages by liposomal clodronate protects against PIFA-induced chemoresistance. In addition, we find that 12-S-HHT, but not 16:4(n-3), functions via leukotriene B4 receptor 2 (BLT2). Genetic loss or chemical inhibition of BLT2 prevents 12-S-HHT-mediated resistance. Mass spectrometry analysis of conditioned medium derived from PIFA-stimulated splenic macrophages identifies several lysophosphatidylcholines as the resistance-inducing molecules. When comparing cisplatin and PIFA-treated tumours with cisplatin alone treated tumours we found overall less γH2AX, a measure for DNA damage. Taken together, we have identified an intricate network of lysophospholipid signalling by splenic macrophages that induces systemic chemoresistance in vivo via an altered DNA damage response.

Phase I evaluation of telatinib, a VEGF receptor tyrosine kinase inhibitor, in combination with bevacizumab in subjects with advanced solid tumors

BACKGROUND Blocking both receptor and ligand of the vascular endothelial growth factor (receptor) VEGF(R) pathway might be feasible and increase antitumor activity. This phase I study investigated telatinib, an oral tyrosine kinase inhibitor targeting VEGFR-2, combined with bevacizumab, in adults with solid tumors. PATIENTS AND METHODS Twenty-six patients were treated in successive cohorts with telatinib (twice-daily continuously, 450-900 mg) or bevacizumab (bi-weekly, starting dose 5 mg/kg). Safety, pharmacokinetics, endothelial (progenitor) cell (E(P)C)/growth factor kinetics and efficacy were assessed. RESULTS Most frequent adverse events were pain, nausea, voice changes and fatigue. Five dose-limiting toxicities (DLTs) occurred: hypertension (cohort I and II), bowel perforation, lipase increase and atrial flutter (cohort III). Cumulative toxicity resulted in a bevacizumab dose reduction to 1 mg/kg (cohort III). Due to three DLTs (n = 14), this cohort represented the best-tolerated dose level. Bevacizumab effectively neutralized plasma VEGF even at 1 mg/kg. Twelve patients had stable disease (clinical benefit 46%). EPC and SDF-1α levels increased during monotherapy telatinib. CONCLUSIONS Telatinib (450 mg b.i.d.) combined with bevacizumab (1 mg/kg bi-weekly) shows antitumor activity, but accumulating constitutional toxicity impedes long-term treatment of patients. Therefore, this combination will not be pursued in a phase II setting.

Notch1 regulates angio-supportive bone marrow–derived cells in mice: relevance to chemoresistance

Host responses to chemotherapy can induce resistance mechanisms that facilitate tumor regrowth. To determine the contribution of bone marrow-derived cells (BMDCs), we exposed tumor-bearing mice to chemotherapeutic agents and evaluated the influx and contribution of a genetically traceable subpopulation of BMDCs (vascular endothelial-cadherin-Cre-enhanced yellow fluorescent protein [VE-Cad-Cre-EYFP]). Treatment of tumor-bearing mice with different chemotherapeutics resulted in a three- to 10-fold increase in the influx of VE-Cad-Cre-EYFP. This enhanced influx was accompanied by a significant increase in angiogenesis. Expression profile analysis revealed a progressive change in the EYFP population with loss of endothelial markers and an increase in mononuclear markers. In the tumor, 2 specific populations of VE-Cad-Cre-EYFP BMDCs were identified: Gr1⁺/CD11b⁺ and Tie2high/platelet endothelial cell adhesion moleculelow cells, both located in perivascular areas. A common signature of the EYFP population that exits the bone marrow is an increase in Notch. Inducible inactivation of Notch in the EYFP⁺ BMDCs impaired homing of these BMDCs to the tumor. Importantly, Notch deletion reduced therapy-enhanced angiogenesis, and was associated with an increased antitumor effect of the chemotherapy. These findings revealed the functional significance of a specific population of supportive BMDCs in response to chemotherapeutics and uncovered a new potential strategy to enhance anticancer therapy.

Liver surgery induces an immediate mobilization of progenitor cells in liver cancer patients: A potential role for G-CSF.

Background: In preclinical models recruitment of bone-marrow derived (endothelial) progenitor cells (BD(E)PCs) contributes to tumor growth and metastasis formation. Here we investigated whether these (E)PCs and mobilizing cytokines are released after partial hepatectomy or radiofrequency ablation (RFA) for liver tumors. In addition, we tested whether G-CSF could play a role in EPC mobilization in mice and in human volunteers. Methods: Before, during and after liver surgery plasma and mononuclear cells were collected from 12 patients undergoing partial hepatectomy or RFA. To explore the role of G-CSF C57Bl/6 mice and 20 human volunteers received G-CSF (0.3 or 3 μg). In all individuals, (E)PC numbers were determined by flow cytometry at predefined timepoints shortly after therapy. Plasma levels of G-CSF, VEGF and SDF-1α were measured by ELISA> Results: Patients undergoing partial hepatectomy or RFA showed a instantaneous release of EPCs following laparotomy and mobilization of the liver. Elevated EPC levels were maintained during the entire procedure, but dropped to near-baseline levels 4 hours after completion of the procedure. Plasma G-CSF levels showed a 5-10-fold increase after the procedure and low-dose G-CSF administration to mice or healthy volunteers was sufficient to induce an immediate release of EPCs. Surgery also caused an increase in the plasma levels of VEGF, but not SDF1. Conclusion: Compliant with previous published data concerning VDA and chemotherapy treatment,liver surgery induces an instantaneous release of EPCs, conceivably in response to elevated G-CSF levels. This suggests the value of exploring therapeutic avenues to prevent this process.