Roeliene C. Kruizinga

A review on CXCR4/CXCL12 axis in oncology : No place to hide

Classical chemotherapeutic anti-cancer treatments induce cell death through DNA damage by taking advantage of the proliferative behaviour of cancer cells. The more recent approach of targeted therapy (usually protein-targeted) has led to many treatments that are currently available or are under development, all of which are designed to strike at the critical driving forces of cancer cells. The interaction of the cancer cells with their microenvironment is one of these fundamental features of neoplasms that could be targeted in such cancer treatments. Haematological and solid tumour cells interact with their microenvironment through membrane chemokine receptors and their corresponding ligands, which are expressed in the tumour microenvironment. Important representatives of this system are the chemokine ligand CXCL12 and its receptor chemokine receptor 4 (CXCR4). This interaction can be disrupted by CXCR4 antagonists, and this concept is being used clinically to harvest haematopoietic stem/progenitor cells from bone marrow. CXCR4 and CXCL12 also have roles in tumour growth and metastasis, and more recently their roles in cancer cell-tumour microenvironment interaction and angiogenesis have been studied. Our review focuses on these roles and summarises strategies for treating cancer by disrupting this interaction with special emphasis on the CXCR4/CXCL12 axis. Finally, we discuss ongoing clinical trials with several classes of CXCR4 inhibitors, and their potential additive value for patients with a (therapy resistant) malignancy by sensitising cancer cells to conventional therapy.

Role of Chemokines and Their Receptors in Cancer

Metastases are the cause of 90% of human cancer deaths. The current treatment of cancer with chemo,- and/or radiotherapy is based on cell death by DNA damage neglecting the fact that cancer cell invasion into surrounding tissues and metastasizing are fundamental features of neoplasms and the major reason for treatment failure. Metastasis is the result of several sequential steps and represents a highly organized, non-random, and organ-selective process. A number of in vitro and in vivo models show that tumor cells use chemokine-mediated mechanisms during this metastasizing process, comparable to those observed in the regulation of leukocyte trafficking. Furthermore, chemokines modulate tumor behavior such as the regulation of tumor-associated angiogenesis, activation of host tumor-specific immunological responses, and direct stimulation of tumor cell proliferation in an autocrine fashion. These findings may lead to new drugs that target chemokines or their receptors and will likely be of great additional value for treatment of cancer patients.

The chemokine network, a newly discovered target in high grade gliomas

Chemokines are small cytokines, characterised by their ability to induce directional migration of cells by binding to chemokine receptors. They are known to play a role in tumour development, angiogenesis and metastasis. Interestingly, the chemokine network also contributes to the progression of gliomas, mainly by intensifying their characteristic invasive character. The main hurdle in treatment of these tumours is their infiltration of surrounding tissues, hampering complete surgical tumour removal. Standard postsurgical treatment with radio- and chemotherapy is of limited effect. Therefore drugs that target the chemokine system in high grade gliomas might fill the gap existing in the current approach. This review presents the current knowledge of the role of chemokine network in the development of the central nervous system, in brain physiology and the involvement in brain tumour progression. Finally, current studies exploring new compounds targeting the chemokine network in cancer patient are discussed.

Pregnancy-related hemangioblastoma progression and complications in von Hippel-Lindau disease

Objective: We studied the reciprocal effect of pregnancy and von Hippel-Lindau (VHL) disease by analyzing the influence of pregnancy on VHL disease-related lesions and VHL disease on pregnancy outcome. Methods: Medical charts and imaging reports from the VHL disease expertise centers in the Netherlands were used to retrospectively assess lesion progression score before and after pregnancy and to obtain data on pregnancy outcome and VHL disease-related lesions. The Friedman test was used for analysis (p ≤ 0.05). Twenty-nine patients were studied (48 pregnancies, 49 newborns). Results: The progression score of cerebellar hemangioblastomas significantly changed between the single MRI scan before and the 2 scans after pregnancy (p = 0.049) (n = 12). Fetal mortality rate was 2% (n = 1) caused by maternal pheochromocytoma. Maternal VHL disease-related complications occurred in 17% (n = 8) of all pregnancies. In 4 patients, a life-threatening situation emerged: hydrocephalus due to cerebellar hemangioblastoma (n = 2) and pheochromocytoma (n = 2). Conclusions: Pregnancy in patients with VHL disease induces cerebellar hemangioblastoma progression and causes a high VHL disease-related pregnancy complication rate. We recommend intensified surveillance of patients with VHL disease, especially of cerebellar hemangioblastomas during preconception care and pregnancy.

Calculating optimal surveillance for detection of von Hippel–Lindau-related manifestations

von Hippel-Lindau (VHL) mutation carriers develop benign and malignant tumors, requiring regular surveillance. The aim of this study was to calculate the optimal organ-specific age to initiate surveillance and optimal intervals to detect initial and subsequent VHL-related manifestations. In this study, we compare these results with the current VHL surveillance guidelines. We collected data from 82 VHL mutation carriers in the Dutch VHL surveillance program. The cumulative proportion of carriers diagnosed with a first VHL-related manifestation was estimated by the Kaplan-Meier method. The Poisson distribution model was used to calculate average time to detection of the first VHL-related manifestation and subsequent manifestations. We used this to calculate the optimal organ-specific age to initiate surveillance and the surveillance interval that results in a detection probability of 5%. The calculated organ-specific ages to initiate surveillance were 0 years (birth) for adrenal glands, 7 years for the retina, 14 years for the cerebellum, 15 years for the spinal cord, 16 years for pancreas, and 18 years for the kidneys. The calculated surveillance intervals were 4 years for the adrenal glands, biennially for the retina and pancreas, and annually for the cerebellum, spinal cord, and kidneys. Compared with current VHL guidelines, the calculated starting age of surveillance was 6 years later for the retina and 5 years earlier for adrenal glands. The surveillance intervals were two times longer for the retina and four times longer for the adrenal glands. To attain a 5% detection probability rate per organ, our mathematical model indicates that several modifications of current VHL surveillance guidelines should be considered.

Vascular Endothelial Growth Factor A isoform mRNA expression in pediatric acute myeloid leukemia

In AML high VEGFA protein expression correlates with poor overall and relapse‐free survival (OS/RFS). To date, the relevance of the various VEGFA isoforms is unclear. We determined VEGF121, VEGF145, VEGF148, VEGF165, VEGF183, and VEGF189 mRNA expression in pediatric AML samples and investigated the relation between VEGFA isoform expression and clinicopatholologic characteristics and outcome. A significant co‐expression of VEGF121, VEGF165, VEGF183, and VEGF189 isoforms was apparent (mean rho = 0.716, P < 0.0001). This co‐expression justifies measuring a single VEGFA isoform (e.g., 121, 165, 183, and 189) as representative expression of all VEGFA isoforms in future studies designed to determine the prognostic importance of VEGFA isoforms. Pediatr Blood Cancer 2011;56:294–297.

CXCR4 expression in VHL-related and sporadic hemangioblastomas.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background & aim. Central nervous system hemangioblastomas are sporadically as well in patients with von Hippel-Lindau (VHL) disease. In VHL disease a germline mutation in the VHL- gene leads to a defect VHL protein. This results in enhanced transcription of vascular endothelial growth factor (VEGF)-A, chemokine receptor 4 (CXCR4) and its ligand (CXCL12). The aim of this study was to analyze whether VEGF-A, CXCR4, and CXCL12 protein expression differ between VHL-related and sporadic hemangioblastomas. Methods. Freshly frozen tissue of patients operated in the University Medical Center Groningen the Netherlands, for hemangioblastomas between 1995-2010 was retrieved. Thirtythree samples of 27 patients were available, including 11 patients diagnosed with VHL-disease. Immunohistochemical analysis of 33 hemangioblastoma specimens (4 VHL patients and 1 sporadic patient were included more than once) was performed for hematoxylin & eosin, VEGF-A, CXCR4 and CXCL12 expression and compared with normal surrounding brain tissue. VEGF-A was evaluated describing morphology in relation to positive cells. A semi quantitative evaluation was performed for CXCL12; negative, positive (>1% positive cells) or strongly positive (>50 % positive cells or high intensity). CXCR4 was scored quantitatively by counting percentage of positive cells per specimen. Results. VEGF-A was equally expressed in all sporadic and VHL-related hemangioblastomas, and present in hemangioblastoma cells and endothelial cells. CXCL12 was strongly expressed in 78% of hemangioblastoma tissues and did not differ between sporadic and VHL-related hemangioblastomas. In all hemangioblastomas CXCR4 was overexpressed as compared to the normal surrounding brain tissue (found in 15 specimens). Interestingly, in sporadic hemangioblastomas the mean percentage of CXCR4 positive cells per specimen (15%) was higher as compared to VHL-related hemangioblastomas (7%, p=0.001). Conclusions. VEGF-A and CXCL12 protein expression was overexpressed in the majority of VHL-related and sporadic hemangioblastomas. This makes targeting VEGF-A a potential strategy for hemangioblastoma treatment. In addition, targeting CXCR4 could be of additional value as it was overexpressed in all hemangioblastomas, although differences in expression between sporadic and VHL related hemangioblastomas do exist. Supported by a grant from the Jan Kornelis de Cock Stichting and a UEF-JSM Talent Grant to RC Kruizinga. Citation Format: Roeliene C. Kruizinga, Wilfred F.A. den Dunnen, Elisabeth G.E. de Vries, Thera P. Links, Annemiek M.E. Walenkamp. CXCR4 expression in VHL-related and sporadic hemangioblastomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 93. doi:10.1158/1538-7445.AM2013-93

Abstract 1672: Mitochondrial and bacterial peptides act on the formyl peptide receptor (FPR) to promote migration and proliferation in high grade glioblastoma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Recently several aberrant molecular pathways and underlying genetic defects are elucidated in astrocytomas, among which the role of the formylpeptide receptor (FPR) malignant behavior. In a highly malignant human astrocytoma cell line FPR promotes cell motility, growth, and angiogenesis by interacting with host-derived agonists originating from necrotic cell material. Until now the only known ligands of FPR are N-formyl peptides, which are cleavage products of bacterial proteins like N-formyl-methionyl-leucyl-phenylalanine (fMLF). In humans the only conserved N-formyl peptides are the cleavage products of mitochondrial proteins. This study aims to identify these peptides as responsible ligands for FPR expressing high grade glioma cells. Methods: We used FITC-fMLF and flow cytometry to confirm binding to FPR-expressing high grade human gliobastoma cell line U87. In addition, U937 cells (human promonocytic cell line) transfected with the FPR (U937FPR) and empty expression vectors were generated. The hallmark of chemokine receptor activation is a rapid and transient increase in the free intracellular Ca2+ level upon ligand binding. Formylated mitochondrial peptides fMLKLIV/fMLALV and FPRs natural peptide ligand fMLF were used to induce Ca2+ mobilization, chemotaxis as determined by the Neuro Probe® system and proliferation in U87 cells, analyzed by MTT assay. Results: FITC-fMLF bound to FPR expressing U87 and U937FPR cells. Increasing concentrations of fMLF (10−8 M-10−5 M) caused a dose dependent calcium mobilization in FPR expressing U87 cells. Increasing concentrations of fMLF/fMLKLIV/fMLALV (10−8 M- 10−5M) were tested in U937FPR cells, again showing dose dependent calcium mobilization. In the chemotaxis assay, respectively 22, 20 and 24% of U937FPR cells migrated towards fMLF/fMLKLIV/fMLALV as compared to 4% towards 0.05% FCS. A 2.5 fold increase in migration of U87 cells towards 10−7 M fMLF was found as compared to 0.05% FCS. Respectively 20, 21 and 15% increase in U87 cell proliferation was found when cells were stimulated with 10−6 M fMLF/fMLKLIV/fMLALV. Conclusions: These results indicate that mitochondrial peptides present in necrotic tumor material serve as ligands for FPR, expressed on high grade human gliobastoma cell line U87. Activation of FPR via N-formyl peptides causes increased cell growth and motility and is a potential target for anti-cancer therapy. Supported by grant RUG 4622 of the Dutch Cancer Society Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1672. doi:10.1158/1538-7445.AM2011-1672

Abstract 380: Sunitinib treatment induces up-regulation of CXCR4

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Chemokine receptors expressed on solid tumor cells and their corresponding ligands are an example of a network which is used by these cells to interact with their microenvironment to sustain survival, proliferation, invasive growth, angiogenesis, and to promote organ-specific localization of distant metastases. Indeed, CXCR4/CXCL12 signaling can induce angiogenesis and progression of tumors, for example by increasing the expression of vascular endothelial growth factor (VEGF). Inversely, it is hypothesized that anti-angiogenic treatment increases the expression of CXCR4/CXCL12 which could explain the in preclinical models observed invasive growth characteristics of tumors. Methods: A human A2780 ovarian xenograft mouse model was used. When the tumor was established, mice were treated with vehicle or sunitinib (60 mg/kg i.p.) daily for 2 weeks. CXCR4 expression was assessed by immunohistochemistry at baseline and after 1 and 2 weeks of treatment and compared with markers like HIF1α, VEGF, Ki67, VEGFR2, mean vessel density (MVD) and blood vessel diameter. In addition human plasma VEGF levels were analyzed. Results: Moderate cell membrane CXCR4 expression was seen in untreated tumors. One week of sunitinib treatment resulted in decrease angiogenice markers and tumor proliferation (Ki67) in both the tumor center and rim. Two weeks of sunitinib treatment resulted in up regulation of CXCR4 with a 2.2 fold increase (P = 0.046) particularly in the tumor rim. This coincided with normalization of blood vessel diameters, intense VEGF staining and increased tumor proliferation (Ki67) to baseline levels in the tumor rim. In contrast, an increase of HIF1α (P = 0.017) and decrease of angiogenic markers like VEGFR2 (P = 0.019) and MVD (P = 0.016) was still observed in the tumor center. Conclusion: This study shows that anti-angiogenic treatment with sunitinib up regulates CXCR4 expression, mainly in the rim of the tumor. CXCR4 is a potential target to block anti-angiogenic induced invasive growth/metastasis. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 380.