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Background
Ovarian cancer (OvCA) tissues show abundant expression of the ectonucleotidases CD39 and CD73 which generate immunomodulatory adenosine, thereby inhibiting cytotoxic lymphocytes. Little, however, is known about the effect of adenosine on myeloid cells. Considering that tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) constitute up to 20 % of OvCA tissue, we investigated the effect of adenosine on myeloid cells and explored a possible contribution of myeloid cells to adenosine generation in vitro and ex vivo.
Methods
Monocytes were used as human blood-derived myeloid cells. After co-incubation with SK-OV-3 or OAW-42 OvCA cells, monocyte migration was determined in transwell assays. For conversion into M2-polarized “TAM-like” macrophages, monocytes were co-incubated with OAW-42 cells. Ex vivo TAMs were obtained from OvCA ascites. Macrophage phenotypes were investigated by intracellular staining for IL-10 and IL-12. CD39 and CD73 expression were assessed by FACS analysis both on in vitro-induced TAM-like macrophages and on ascites-derived ex situ-TAMs. Myeloid cells in solid tumor tissue were analyzed by immunohistochemistry. Generation of biologically active adenosine by TAM-like macrophages was measured in luciferase-based reporter assays. Functional effects of adenosine were investigated in proliferation-experiments with CD4+ T cells and specific inhibitors.
Results
When CD39 or CD73 activity on OvCA cells were blocked, the migration of monocytes towards OvCA cells was significantly decreased. In vivo, myeloid cells in solid ovarian cancer tissue were found to express CD39 whereas CD73 was mainly detected on stromal fibroblasts. Ex situ-TAMs and in vitro differentiated TAM-like cells, however, upregulated the expression of CD39 and CD73 compared to monocytes or M1 macrophages. Expression of ectonucleotidases also translated into increased levels of biologically active adenosine. Accordingly, co-incubation with these TAMs suppressed CD4+ T cell proliferation which could be rescued via blockade of CD39 or CD73.
Conclusion
Adenosine generated by OvCA cells likely contributes to the recruitment of TAMs which further amplify adenosine-dependent immunosuppression via additional ectonucleotidase activity. In solid ovarian cancer tissue, TAMs express CD39 while CD73 is found on stromal fibroblasts. Accordingly, small molecule inhibitors of CD39 or CD73 could improve immune responses in ovarian cancer.
Background: Genes that, when mutated, cause Fanconi anemia or greatly increase breast cancer risk encode for proteins that converge on a homology-directed DNA damage repair process. Mutations in the SLX4 gene, which encodes for a scaffold protein involved in the repair of interstrand cross-links, have recently been identified in unclassified Fanconi anemia patients. A mutation analysis of SLX4 in German or Byelorussian familial cases of breast cancer without detected mutations in BRCA1 or BRCA2 has been completed, with globally negative results.
Methods: The genomic region of SLX4, comprising all exons and exon-intron boundaries, was sequenced in 94 Spanish familial breast cancer cases that match a criterion indicating the potential presence of a highly-penetrant germline mutation, following exclusion of BRCA1 or BRCA2 mutations.
Results: This mutational analysis revealed extensive genetic variation of SLX4, with 21 novel single nucleotide variants; however, none could be linked to a clear alteration of the protein function. Nonetheless, genotyping 10 variants (nine novel, all missense amino acid changes) in a set of controls (138 women and 146 men) did not detect seven of them. Conclusions: Overall, while the results of this study do not identify clearly pathogenic mutations of SLX4 contributing to breast cancer risk, further genetic analysis, combined with functional assays of the identified rare variants, may be warranted to conclusively assess the potential link with the disease.
The ectonucleotidases CD39 and CD73 degrade ATP to adenosine which inhibits immune responses via the \(A_{2A}\) adenosine receptor (ADORA2A) on T and NK cells. The current study investigates the potential therapeutic use of the specific anti CD39- and anti CD73-antibodies A1 (CD39) and 7G2 (CD73) as these two ectonucleotidases are overexpressed in ovarian cancer (OvCA). As expected, NK cell cytotoxicity against the human ovarian cancer cell lines OAW-42 or SK-OV-3 was significantly increased in the presence of A1 or 7G2 antibody. While this might partly be due to antibody-dependent cell-mediated cytotoxicity, a luciferase-dependent assay for quantifying biologically active adenosine further showed that A1 and 7G2 can inhibit CD39 and CD73-dependent adenosine-generation. In turn, the reduction in adenosine levels achieved by addition of A1 and 7G2 to OAW-42 or SK-OV-3 cells was found to de-inhibit the proliferation of \(CD4^+\) T cells in coculture with OvCA cells. Likewise, blocking of CD39 and CD73 on OvCA cells via A1 and 7G2 led to an increased cytotoxicity of alloreactive primed T cells. Thus, antibodies like A1 and 7G2 could improve targeted therapy in ovarian cancer not only by specifically labeling overexpressed antigens but also by blocking adenosine-dependent immune evasion in this immunogenic malignancy.
Background
Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial.
Methods
To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants.
Results
BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02–36.57, P < 0.0001) and in the subgroup of index patients with late-onset OC (OR = 29.91, 95% CI = 14.99–59.66, P < 0.0001). No significant association of BRIP1 LoF mutations with familial BC was observed (OR = 1.81 95% CI = 1.00–3.30, P = 0.0623). In the subgroup of familial BC index patients without a family history of OC there was also no apparent association (OR = 1.42, 95% CI = 0.70–2.90, P = 0.3030). In 1027 familial BC index patients with a family history of OC, the BRIP1 mutation prevalence was significantly higher than that observed in controls (OR = 3.59, 95% CI = 1.43–9.01; P = 0.0168). Based on the negative association between BRIP1 LoF mutations and familial BC in the absence of an OC family history, we conclude that the elevated mutation prevalence in the latter cohort was driven by the occurrence of OC in these families. Compared with controls, predicted damaging rare missense variants were significantly more prevalent in OC (P = 0.0014) but not in BC (P = 0.0693) patients.
Conclusions
To avoid ambiguous results, studies aimed at assessing the impact of candidate predisposition gene mutations on BC risk might differentiate between BC index patients with an OC family history and those without. In familial cases, we suggest that BRIP1 is a high-risk gene for late-onset OC but not a BC predisposition gene, though minor effects cannot be excluded.
Introduction: Several common alleles have been shown to be associated with breast and/or ovarian cancer risk for BRCA1 and BRCA2 mutation carriers. Recent genome-wide association studies of breast cancer have identified eight additional breast cancer susceptibility loci: rs1011970 (9p21, CDKN2A/B), rs10995190 (ZNF365), rs704010 (ZMIZ1), rs2380205 (10p15), rs614367 (11q13), rs1292011 (12q24), rs10771399 (12p11 near PTHLH) and rs865686 (9q31.2).
Methods: To evaluate whether these single nucleotide polymorphisms (SNPs) are associated with breast cancer risk for BRCA1 and BRCA2 carriers, we genotyped these SNPs in 12,599 BRCA1 and 7,132 BRCA2 mutation carriers and analysed the associations with breast cancer risk within a retrospective likelihood framework.
Results: Only SNP rs10771399 near PTHLH was associated with breast cancer risk for BRCA1 mutation carriers (per-allele hazard ratio (HR) = 0.87, 95% CI: 0.81 to 0.94, P-trend = 3 x 10\(^{-4}\)). The association was restricted to mutations proven or predicted to lead to absence of protein expression (HR = 0.82, 95% CI: 0.74 to 0.90, P-trend = 3.1 x 10\(^{-5}\), P-difference = 0.03). Four SNPs were associated with the risk of breast cancer for BRCA2 mutation carriers: rs10995190, P-trend = 0.015; rs1011970, P-trend = 0.048; rs865686, 2df P = 0.007; rs1292011 2df P = 0.03. rs10771399 (PTHLH) was predominantly associated with estrogen receptor (ER)-negative breast cancer for BRCA1 mutation carriers (HR = 0.81, 95% CI: 0.74 to 0.90, P-trend = 4 x 10\(^{-5}\)) and there was marginal evidence of association with ER- negative breast cancer for BRCA2 mutation carriers (HR = 0.78, 95% CI: 0.62 to 1.00, P-trend = 0.049).
Conclusions: The present findings, in combination with previously identified modifiers of risk, will ultimately lead to more accurate risk prediction and an improved understanding of the disease etiology in BRCA1 and BRCA2 mutation carriers.
Single Nucleotide Polymorphisms (SNPs) in genes involved in the DNA Base Excision Repair (BER) pathway could be associated with cancer risk in carriers of mutations in the high-penetrance susceptibility genes BRCA1 and BRCA2, given the relation of synthetic lethality that exists between one of the components of the BER pathway, PARP1 (poly ADP ribose polymerase), and both BRCA1 and BRCA2. In the present study, we have performed a comprehensive analysis of 18 genes involved in BER using a tagging SNP approach in a large series of BRCA1 and BRCA2 mutation carriers. 144 SNPs were analyzed in a two stage study involving 23,463 carriers from the CIMBA consortium (the Consortium of Investigators of Modifiers of BRCA1 and BRCA2). Eleven SNPs showed evidence of association with breast and/or ovarian cancer at p<0.05 in the combined analysis. Four of the five genes for which strongest evidence of association was observed were DNA glycosylases. The strongest evidence was for rs1466785 in the NEIL2 (endonuclease VIII-like 2) gene (HR: 1.09, 95% CI (1.03-1.16), p = 2.7x10(-3)) for association with breast cancer risk in BRCA2 mutation carriers, and rs2304277 in the OGG1 (8-guanine DNA glycosylase) gene, with ovarian cancer risk in BRCA1 mutation carriers (HR: 1.12 95% CI: 1.03-1.21, p = 4.8x10(-3)). DNA glycosylases involved in the first steps of the BER pathway may be associated with cancer risk in BRCA1/2 mutation carriers and should be more comprehensively studied.
Background
Ovarian cancer is mostly associated with pathologically regulated permeability of peritoneal vessels, leading to ascites. Here, we investigated the molecular regulation of endothelial permeability by the vascular endothelial growth factor (VEGF) and both tight and adherens junction proteins (VE-cadherin and claudin 5) with regards to the tumor biology of different ovarian cancer types.
Methods
Serum and ascites samples before and after surgery, as well as peritoneal biopsies of 68 ovarian cancer patients and 20 healthy controls were collected. In serum and ascites VEGF protein was measured by ELISA. In peritoneal biopsies co-localization of VE-cadherin and claudin 5 was investigated using immunohistochemical dual staining. In addition, the gene expression of VE-cadherin and claudin 5 was quantified by Real-time PCR. Differences in VEGF levels, VE-cadherin and claudin 5 gene expression were analyzed in relation to various tumor characteristics (tumor stage, grading, histological subtypes, resection status after surgery) and then compared to controls. Furthermore, human primary ovarian cancer cells were co-cultured with human umbilical vein endothelial cells (HUVEC) and changes in VE-cadherin and claudin 5 were investigated after VEGF inhibition.
Results
VEGF was significantly increased in tumor patients in comparison to controls and accumulates in ascites. The highest VEGF levels were found in patients diagnosed with advanced tumor stages, with tumors of poor differentiation, or in the group of solid / cystic-solid tumors. Patients with residual tumor after operation showed significantly higher levels of VEGF both before and after surgery as compared to tumor-free resected patients. Results of an immunohistochemical double-staining experiment indicated co-localization of VE-cadherin and claudin 5 in the peritoneal vasculature. Compared to controls, expression of VE-cadherin and claudin 5 was significantly suppressed in peritoneal vessels of tumor patients, but there were no significant differences regarding VE-cadherin and claudin 5 expression in relation to different tumor characteristics. A significant positive correlation was found between VE-cadherin and claudin 5 expression. VEGF inhibition in vitro was associated with significant increase in VE-cadherin and claudin 5.
Conclusions
Our results indicate that increased peritoneal permeability in ovarian cancer is due to down-regulation of adhesion proteins via tumor derived VEGF. Advanced ovarian cancer with aggressive tumor biology may be associated with early dysregulation of vascular permeability leading to ascites. These patients may benefit from therapeutic VEGF inhibition.
Ovarian cancer is the second most common gynecological malignancy in women. More than 70% of the cases are diagnosed at the advanced stage, presenting as primary peritoneal metastasis, which results in a poor 5-year survival rate of around 40%. Mechanisms of peritoneal metastasis, including adhesion, migration, and invasion, are still not completely understood and therapeutic options are extremely limited. Therefore, there is a strong requirement for a 3D model mimicking the in vivo situation. In this study, we describe the establishment of a 3D tissue model of the human peritoneum based on decellularized porcine small intestinal submucosa (SIS) scaffold. The SIS scaffold was populated with human dermal fibroblasts, with LP-9 cells on the apical side representing the peritoneal mesothelium, while HUVEC cells on the basal side of the scaffold served to mimic the endothelial cell layer. Functional analyses of the transepithelial electrical resistance (TEER) and the FITC-dextran assay indicated the high barrier integrity of our model. The histological, immunohistochemical, and ultrastructural analyses showed the main characteristics of the site of adhesion. Initial experiments using the SKOV-3 cell line as representative for ovarian carcinoma demonstrated the usefulness of our models for studying tumor cell adhesion, as well as the effect of tumor cells on endothelial cell-to-cell contacts. Taken together, our data show that the novel peritoneal 3D tissue model is a promising tool for studying the peritoneal dissemination of ovarian cancer.
Population-based genome wide association studies have identified a locus at 9p22.2 associated with ovarian cancer risk, which also modifies ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. We conducted fine-scale mapping at 9p22.2 to identify potential causal variants in BRCA1 and BRCA2 mutation carriers. Genotype data were available for 15,252 (2,462 ovarian cancer cases) BRCA1 and 8,211 (631 ovarian cancer cases) BRCA2 mutation carriers. Following genotype imputation, ovarian cancer associations were assessed for 4,873 and 5,020 SNPs in BRCA1 and BRCA 2 mutation carriers respectively, within a retrospective cohort analytical framework. In BRCA1 mutation carriers one set of eight correlated candidate causal variants for ovarian cancer risk modification was identified (top SNP rs10124837, HR: 0.73, 95%CI: 0.68 to 0.79, p-value 2× 10−16). These variants were located up to 20 kb upstream of BNC2. In BRCA2 mutation carriers one region, up to 45 kb upstream of BNC2, and containing 100 correlated SNPs was identified as candidate causal (top SNP rs62543585, HR: 0.69, 95%CI: 0.59 to 0.80, p-value 1.0 × 10−6). The candidate causal in BRCA1 mutation carriers did not include the strongest associated variant at this locus in the general population. In sum, we identified a set of candidate causal variants in a region that encompasses the BNC2 transcription start site. The ovarian cancer association at 9p22.2 may be mediated by different variants in BRCA1 mutation carriers and in the general population. Thus, potentially different mechanisms may underlie ovarian cancer risk for mutation carriers and the general population.
Nectin‐2 is an adhesion molecule that has been reported to play a role in tumor growth, metastasis and tumor angiogenesis. Herein, we investigated Nectin‐2 in ovarian cancer patients and in cell culture. Tumor as well as peritoneal biopsies of 60 ovarian cancer patients and 22 controls were dual stained for Nectin‐2 and CD31 using immunohistochemistry. Gene expression of Nectin‐2 was quantified by real‐time PCR and differences analyzed in relation to various tumor characteristics. In the serum of patients, vascular endothelial growth factor (VEGF) was quantified by ELISA. Effect of VEGF on Nectin‐2 expression as well as permeability was investigated in HUVEC. In tumor biopsies, Nectin‐2 protein was mainly localized in tumor cells, whereas in peritoneal biopsies, clear colocalization was found in the vasculature. T3 patients had a significantly higher percentage of positive lymph nodes and this correlated with survival. Nectin‐2 was significantly upregulated in tumor biopsies in patients with lymph node metastasis and with residual tumor >1 cm after surgery. Nectin‐2 expression was significantly suppressed in the peritoneal endothelium of patients associated with significantly increased VEGF serum levels. In cell culture, VEGF stimulation led to a significant downregulation of Nectin‐2 which was reversed by VEGF‐inhibition. In addition, Nectin‐2 knockdown in endothelial cells was associated with significantly increased endothelial permeability. Nectin‐2 expression in ovarian cancer may support tumor cell adhesion, leading to growth and lymph node metastasis. In addition, VEGF‐induced Nectin‐2 suppression in peritoneal endothelium may support an increase in vascular permeability leading to ascites production.