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The transcription factor ETV7 is an oncoprotein that is up-regulated in all breast cancer (BC) types. We have recently demonstrated that ETV7 promoted breast cancer progression by increasing cancer cell proliferation and stemness and was also involved in the development of chemo- and radio-resistance. However, the roles of ETV7 in breast cancer inflammation have yet to be studied. Gene ontology analysis previously performed on BC cells stably over-expressing ETV7 demonstrated that ETV7 was involved in the suppression of innate immune and inflammatory responses. To better decipher the involvement of ETV7 in these signaling pathways, in this study, we identified TNFRSF1A, encoding for the main receptor of TNF-α, TNFR1, as one of the genes down-regulated by ETV7. We demonstrated that ETV7 directly binds to the intron I of this gene, and we showed that the ETV7-mediated down-regulation of TNFRSF1A reduced the activation of NF-κB signaling. Furthermore, in this study, we unveiled a potential crosstalk between ETV7 and STAT3, another master regulator of inflammation. While it is known that STAT3 directly up-regulates the expression of TNFRSF1A, here we demonstrated that ETV7 reduces the ability of STAT3 to bind to the TNFRSF1A gene via a competitive mechanism, recruiting repressive chromatin remodelers, which results in the repression of its transcription. The inverse correlation between ETV7 and TNFRSF1A was confirmed also in different cohorts of BC patients. These results suggest that ETV7 can reduce the inflammatory responses in breast cancer through the down-regulation of TNFRSF1A.

Background The immunological composition of the tumor microenvironment has a decisive influence on the biological course of cancer and is therefore of profound clinical relevance. In this study, we analyzed the cooperative effects of integrin β4 (ITGB4) on tumor cells and E-/P-selectin on endothelial cells within the tumor stroma for regulating tumor growth by shaping the local and systemic immune environment. Methods We used several preclinical mouse models for different solid human cancer types (xenograft and syngeneic) to explore the role of ITGB4 (shRNA-mediated knockdown in tumor cells) and E-/P-selectins (knockout in mice) for tumor growth; effects on apoptosis, proliferation and intratumoral signaling pathways were determined by histological and biochemical methods and 3D in vitro experiments; changes in the intratumoral and systemic immune cell composition were determined by flow cytometry and immunohistochemistry; chemokine levels and their attracting potential were measured by ELISA and 3D invasion assays. Results We observed a very robust synergism between ITGB4 and E-/P-selectin for the regulation of tumor growth, accompanied by an increased recruitment of CD11b+ Gr-1Hi cells with low granularity (i.e., myeloid-derived suppressor cells, MDSCs) specifically into ITGB4-depleted tumors. ITGB4-depleted tumors undergo apoptosis and actively attract MDSCs, well-known to promote tumor growth in several cancers, via increased secretion of different chemokines. MDSC trafficking into tumors crucially depends on E-/P-selectin expression. Analyses of clinical samples confirmed an inverse relationship between ITGB4 expression in tumors and number of tumor-infiltrating leukocytes. Conclusions These findings suggest a distinct vulnerability of ITGB4Lo tumors for MDSC-directed immunotherapies. Graphical Abstract

Background The disruption of epithelial features represents a critical step during breast cancer spread. In this context, the dysregulation of desmosomal proteins has been associated with malignant progression and metastasis formation. Curiously, both tumour suppressive and pro-metastatic roles have been attributed to desmosomal structures in different cancer entities. In the present study, we describe the pro-metastatic role of the desmosomal protein desmocollin 2 (DSC2) in breast cancer. Methods We analysed the prognostic role of DSC2 at mRNA and protein level using microarray data, western blot analysis and immunohistochemistry. Functional consequences of DSC2 overexpression and DSC2 knock down were investigated in the triple negative breast cancer (TNBC) cell line MDA-MB-231 and its brain-seeking subline MDA-MB-231-BR, respectively in vitro and in vivo. Results We found a significantly higher DSC2 expression in the more aggressive molecular subtypes HER2-positive and TNBC than in luminal breast cancers, as well as a significant correlation between increased DSC2 expression and a shorter disease-free—also in multivariate analysis—and overall survival. Additionally, a significant association between DSC2 expression in the primary tumour and an increased frequency of cerebral and lung metastasis could be observed. In vitro, ectopic DSC2 expression or DSC2 down-regulation in MDA-MB-231 and MDA-MB-231-BR led to a significant tumour cell aggregation increase and decrease, respectively. Furthermore, tumour cells displaying higher DSC2 levels showed increased chemoresistance in 3D structures, but not 2D monolayer structures, suggesting the importance of cell aggregation as a means for reduced drug diffusion. In an in vivo brain dissemination xenograft mouse model, reduced expression of DSC2 in the brain-seeking TNBC cells led to a decreased amount of circulating tumour cells/clusters and, in turn, to fewer and smaller brain metastatic lesions. Conclusion We conclude that high DSC2 expression in primary TNBC is associated with a poorer prognosis, firstly by increasing tumour cell aggregation, secondly by reducing the diffusion and effectiveness of chemotherapeutic agents, and, lastly, by promoting the circulation and survival of tumour cell clusters, each of which facilitates distant organ colonisation.

Background: Hyaluronan (HA), a component of the extracellular matrix, is frequently increased under pathological conditions including cancer. Not only stroma cells but also cancer cells themselves synthesize HA, and the interaction of HA with its cognate receptors promotes malignant progression and metastasis. Methods: In the present study, HA deposition in tissue sections was analyzed by hyaluronan-binding protein (HABP) ligand histochemistry in 17 borderline tumors and 102 primary and 20 recurrent ovarian cancer samples. The intensity and, particularly, localization of the HA deposition were recorded: for the localization, the pericellular deposition around the ovarian cancer cells was distinguished from the deposition within the stromal compartment. These histochemical data were correlated with clinical and pathological parameters. Additionally, within a reduced subgroup of ovarian cancer samples (n = 70), the RNA levels of several HA-associated genes were correlated with the HA localization and intensity. Results: Both stroma-localized and pericellular tumor-cell-associated HA deposition were observed. Cancer-cell pericellular HA deposition, irrespective of its staining intensity, was significantly associated with malignancy, and in the primary ovarian cancer cohort, it represents an independent unfavorable prognostic marker for overall survival. Furthermore, a significant association between high CD44, HAS2 and HAS3 mRNA levels and a cancer-cell pericellular HA-deposition pattern was noted. In contrast, stromal hyaluronan deposition had no impact on ovarian cancer prognosis. Conclusions: In conclusion, the site of HA deposition is of prognostic value, but the amount deposited is not. The significant association of only peritumoral cancer-cell HA deposition with high CD44 mRNA expression levels suggests a pivotal role of the CD44–HA signaling axis for malignant progression in ovarian cancer.

Background Despite recent progress in liquid biopsy technologies, early blood-based detection of breast cancer is still a challenge. Methods We analyzed secretion of the protein cellular communication network factor 1 (CCN1, formerly cysteine-rich angiogenic inducer 61) in breast cancer cell lines by an enzyme-linked immunosorbent assay (ELISA). Soluble CCN1 in the plasma (2.5 µL) of 544 patients with breast cancer and 427 healthy controls was analyzed by ELISA. The breast cancer samples were acquired at the time of primary diagnosis prior to neoadjuvant therapy or surgery. A classifier was established on a training cohort of patients with breast cancer and age-adapted healthy controls and further validated on an independent cohort comprising breast cancer patients and healthy controls. Samples from patients with benign breast diseases were investigated as additional controls. Samples from patients with acute heart diseases (n = 127) were investigated as noncancer controls. The diagnostic accuracy was determined by receiver operating characteristic using the parameters area under the curve, sensitivity, and specificity. Results CCN1 was frequently secreted by breast cancer cell lines into the extracellular space. Subsequent analysis of clinical blood samples from patients with breast cancer and age-adjusted healthy controls revealed an overall specificity of 99.0% and sensitivity of 80.0% for cancer detection. Remarkably, 81.5% of small T1 cancers were already CCN1-positive, while CCN1 concentrations in patients with benign breast lesions were below the threshold for breast cancer detection. Conclusions Circulating CCN1 is a potentially novel blood biomarker for the detection of breast cancer at the earliest invasive stage.

Cervical cancer is the fourth most common cancer in women, which is associated in >95% with a high-risk human papillomavirus (HPV) infection. Methylation of specific genes has been closely associated with the progress of cervical high-grade dysplastic lesions to invasive carcinomas. Therefore, DNA methylation has been proposed as a triage for women infected with high-risk HPV. Methylation analyses of cervical cancer tissue have shown that cell adhesion molecule 1 (CADM1) and myelin and lymphocyte protein (MAL) methylation are present in over 90% of all cervical high-grade neoplasias and invasive cervical cancers. Here, we established a liquid biopsy-based assay to detect MAL and CADM1 methylation in cell free (cf)DNA of cervical cancer. Methylation of the target gene was validated on bisulfite converted smear-DNA from cervical dysplasia patients and afterward applied to cfDNA using quantitative real-time PCR. In 52 smears, a combined analysis of CADM1 and/or MAL (CADM1/MAL) showed methylation in 86.5% of the cases. In cfDNA samples of 24 cervical cancer patients, CADM1/MAL methylation was detected in 83.3% of the cases. CADM1/MAL methylation was detected already in 81.8% of stage I-II patients showing the high sensitivity of this liquid biopsy assay. In combination with a specificity of 95.5% towards healthy donors (HD) and an area under the curve (AUC) of 0.872 in the receiver operating characteristic (ROC) analysis, CADM1/MAL cfDNA methylation detection might represent a novel and promising liquid biopsy marker in cervical cancer.

Background: Over 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Molecular screening approaches for HPV detection are gaining promising data over cytologic analysis. Similarly, liquid biopsy-based approaches are becoming a powerful tool for cancer detection and monitoring. Materials and Methods: An HPV panel detecting 24 HPV types was developed suitable for liquid biopsy approaches. Here, HPV measurement was performed with an iPLEX multiplex PCR-based workflow combined with mass spectrometry-based analysis (MassARRAY System, Agena Bioscience). The assay was validated on HPV positive plasmid controls and cell lines. Furthermore, 52 cervical smear samples from HSIL positive women and 40 cfDNA samples (peripheral blood) from cervical cancer patients were screened using this approach. Results: According to already published ring trial results, the HPV types HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 58 and 59 were considered proficient with the required detection level of 50 and 5 international units (IU)/5 µl for HPV 16 and 18, and 500 and 50 HPV genome equivalents (GE)/5µl for the other HPV types (Arroyo Mühr et al., 2022). Further analyses showed a sensitivity of 0.03% for HPV16 and 0.33% HPV18 and 0.33% for the other tested HPV types. HPV DNA was detected in 94% of (49/52) smear samples analyzed. 86.5% were found positive for at least one of the high-risk HPV (hrHPV) types in the study cohort. Two of the three HPV negative samples were truly negative according to clinical data. In 15 samples, multiple HPV types were found, with positivity in up to five different HPV types. For smear samples a sensitivity of 98.14% and a positive predictive value of 0.98 (49/50) were thus obtained. The overall agreement between the MassARRAY and Seegene (from clinical records) HPV tests was 97.2% (κ=0.84). 27 of 40 (67.5%) cfDNA cervical cancer samples were tested positive for any HPV type. The distribution of HPV types showed most infections being due to hrHPV (25/27 plasma HPV positive). HPV positive samples were found in all FIGO stages, with 56.5% positivity in FIGO stage I and 87.5% in FIGO stage III and IV. Conclusions: The HPV assay showed reliable results for detecting a large number of HPV types in a multiplex mass spectrometry-based assay. A high sensitivity was achieved for both, cervical smear samples of HSIL patients as well as cfDNA from blood samples of cervical cancer patients. Citation Format: Johanna Herbst, Vanessa Vohl, Maroje Krajina, Katharina Prieske, Anna Jaeger, Markus Leffers, Leticia Oliveira-Ferrer, Yvonne Goy, Klaus Pantel, Alexander Sartori, Caren Vollmert, Linn Wölber, Katharina Effenberger, Harriet Wikman. Development of a liquid biopsy cfDNA assay for detection of multiple HPV types in cervical neoplasias [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6688.

Phenotypic characterization of γδ T cells in the MALs (malignant ascites lymphocytes), TILs (tumor infiltrating lymphocytes), and PBLs (peripheral blood lymphocytes) of ovarian cancer (OvCA) patients is lacking. Therefore, we quantified γδ T cell prevalence in MAL, TIL, and PBL specimens from n = 18 OvCA patients and PBL from age-matched healthy donors (HD, n = 14). Multicolor flow cytometry was performed to evaluate the expression of inhibitory receptors (TIGIT, PD-1 and TIM-3), stimulatory receptors (Ox40), and purinergic ectoenzymes (CD39 and CD73) on γδ T cell subsets. We identified an abundant infiltration of Vδ1 T cells in the MALs and TILs. These cells varied in their differentiation: The majority of Vδ1 TILs displayed an effector memory (EM) phenotype, whereas Vδ1 MALs had a more mature phenotype of terminally differentiated effector memory cells (TEMRA) with high CD45RA expression. TIGIT and TIM-3 were abundantly expressed in both MALs and PBLs, whereas Vδ1 TILs exhibited the highest levels of PD-1, CD39, and Ox40. We also observed specific clusters on mature differentiation stages for the analyzed molecules. Regarding co-expression, Vδ1 TILs showed the highest levels of cells co-expressing TIGIT with PD-1 or CD39 compared to MALs and PBLs. In conclusion, the Vδ1 T cell population showed a high prevalence in the MALs and primary tumors of OvCA patients. Due to their (co-)expression of targetable immune receptors, in particular TIGIT with PD-1 and CD39 in TILs, Vδ1 T cell-based approaches combined with the inhibition of these targets might represent a promising strategy for OvCA.

Background The survival rate is poor in breast cancer patients with brain metastases. Thus, new concepts for therapeutic approaches are required. During metastasis, the cytoskeleton of cancer cells is highly dynamic and therefore cytoskeleton-associated proteins are interesting targets for tumour therapy. Methods Screening for genes showing a significant correlation with brain metastasis formation was performed based on microarray data from breast cancer patients with long-term follow up information. Validation of the most interesting target was performed by MTT-, Scratch- and Transwell-assay. In addition, intracellular trafficking was analyzed by live-cell imaging for secretory vesicles, early endosomes and multiple vesicular bodies (MVB) generating extracellular vesicles (EVs). EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Western blotting, mass spectrometry, and ingenuity pathway analysis (IPA). Effect of EVs on the blood-brain-barrier (BBB) was examined by incubating endothelial cells of the BBB (hCMEC/D3) with EVs, and permeability as well as adhesion of breast cancer cells were analyzed. Clinical data of a breast cancer cohort was evaluated by χ2-tests, Kaplan-Meier-Analysis, and log-rank tests while for experimental data Student’s T-test was performed. Results Among those genes exhibiting a significant association with cerebral metastasis development, the only gene coding for a cytoskeleton-associated protein was Tubulin Tyrosine Ligase Like 4 (TTLL4). Overexpression of TTLL4 (TTLL4plus) in MDA-MB231 and MDA-MB468 breast cancer cells (TTLL4plus cells) significantly increased polyglutamylation of β-tubulin. Moreover, trafficking of secretory vesicles and MVBs was increased in TTLL4plus cells. EVs derived from TTLL4plus cells promote adhesion of MDA-MB231 and MDA-MB468 cells to hCMEC/D3 cells and increase permeability of hCMEC/D3 cell layer. Conclusions These data suggest that TTLL4-mediated microtubule polyglutamylation alters exosome homeostasis by regulating trafficking of MVBs. The TTLL4plus-derived EVs may provide a pre-metastatic niche for breast cancer cells by manipulating endothelial cells of the BBB.

Despite increasing incidence, vulvar squamous cell carcinoma (VSCC) is still a rare disease. Until now, two etiological pathways have been described: a high-risk human papillomavirus (HPV)-dependent route and an HPV-independent pathway often associated with lichen sclerosus. To date, therapeutic strategies in VSCC are not influenced by molecular pathological information and therapeutic options for advanced or recurrent disease are limited.Whole exome sequencing of DNA, isolated from 34 VSCC samples and matched normal tissue for each individual was performed on an Illumina HiSeq4000. Short variant discovery was carried out using BWA mem and FreeBayes. Variants were annotated using ANNOVAR.FIGO stages were: IB (n = 7), II (n = 11), III (n = 8), and IVA (n = 3), (n = 5 unknown). TP53 missense mutations were most commonly detected with 56% (19/34). 12/34 (35.3%) samples were HPV positive (all HPV16), HPV positivity and TP53 mutations were mutually exclusive (p .0001). Additionally, we observed mutations in known cancer relevant genes, like NBPF1 (n = 7), MACF1 (n = 5), SYNE2 (n = 5), DOCK2 (n = 4), KMT2D (n = 4), MAP2 (n = 4), NACA (n = 4), PIK3CA (n = 4), SYNE1 (n = 4), FBWX7 (n = 3), MSH6 (n = 3), NSD1 (n = 3), POLE (n = 3), TSC2, (n = 3) and CDKN2A (n = 2), but at considerably lower frequencies. For the total cohort 1848 cancer related mutations were detected (median of 54.4 per sample).The key mutation in HPV negative vulvar carcinoma affects TP53. While a multitude of cancer related mutations was detected in various samples, only few mutations recur and/or affect concurrent signaling pathways.

Background Brain metastasis (BM) in non-small-cell lung cancer (NSCLC) has a very poor prognosis. Recent studies have demonstrated the importance of cell adhesion molecules in tumor metastasis. The aim of our study was to investigate the role of activated leukocyte cell adhesion molecule (ALCAM) in BM formation in NSCLC. Methods Immunohistochemical analysis was performed on 143 NSCLC primary tumors and BM. A correlation between clinicopathological parameters and survival was developed. Biological properties of ALCAM were assessed in vitro by gene ablation using CRISPR/Cas9 technology in the NCI-H460 NSCLC cell line and in vivo by intracranial and intracardial cell injection of NCI-H460 cells in NMRI-Foxn1nu/nu mice. Results ALCAM expression was significantly upregulated in NSCLC brain metastasis (P = 0.023) with a de novo expression of ALCAM in 31.2% of BM. Moderate/strong ALCAM expression in both primary NSCLC and brain metastasis was associated with shortened survival. Functional analysis of an ALCAM knock-out (KO) cell line showed a significantly decreased cell adhesion capacity to human brain endothelial cells by 38% (P = 0.045). In vivo studies showed significantly lower tumor cell dissemination in mice injected with ALCAM-KO cells in both mouse models, and both the number and size of BM were significantly diminished in ALCAM depleted tumors. Conclusions Our findings suggest that elevated levels of ALCAM expression promote BM formation in NSCLC through increased tumor cell dissemination and interaction with the brain endothelial cells. Therefore, ALCAM could be targeted to reduce the occurrence of BM. Key Points 1. ALCAM expression associates with poor prognosis and brain metastasis in NSCLC. 2. ALCAM mediates interaction of NSCLC tumor cells with brain vascular endothelium. 3. ALCAM might represent a novel preventive target to reduce the occurrence of BM in NSCLC.

Brain metastases (BM) represent a growing problem for breast cancer (BC) patients. Recent studies have demonstrated a strong impact of the BC molecular subtype on the incidence of BM development. This study explores the interaction between BC cells of different molecular subtypes and the blood–brain barrier (BBB). We compared the ability of BC cells of different molecular subtypes to overcome several steps (adhesion to the brain endothelium, disruption of the BBB, and invasion through the endothelial layer) during cerebral metastases formation, in vitro as well as in vivo. Further, the impact of these cells on the BBB was deciphered at the molecular level by transcriptome analysis of the triple-negative (TNBC) cells themselves as well as of hBMECs after cocultivation with BC cell secretomes. Compared to luminal BC cells, TNBC cells have a greater ability to influence the BBB in vitro and consequently develop BM in vivo. The brain-seeking subline and parental TNBC cells behaved similarly in terms of adhesion, whereas the first showed a stronger impact on the brain endothelium integrity and increased invasive ability. The comparative transcriptome revealed potential brain-metastatic-specific key regulators involved in the aforementioned processes, e.g., the angiogenesis-related factors TNXIP and CXCL1. In addition, the transcriptomes of the two TNBC cell lines strongly differed in certain angiogenesis-associated factors and in several genes related to cell migration and invasion. Based on the present study, we hypothesize that the tumor cell’s ability to disrupt the BBB via angiogenesis activation, together with increased cellular motility, is required for BC cells to overcome the BBB and develop brain metastases.

Simple Summary The number of women, especially younger women, diagnosed with vulvar cancer, has been rising mainly due to the infection with human papilloma virus (HPV) over the last years. In contrast to other tumor entities, limited information on the underlying genetic changes is available, and thus treatment advances, especially the development of personalized treatments, are hampered. We aimed to explore the RNA expression profiles in a group of 24 vulvar cancer samples in order to detect potential prognostic markers and therapeutic targets in order to establish to a more profound understanding of vulvar cancer carcinogenesis. Abstract To date, therapeutic strategies in vulvar squamous cell carcinoma (VSCC) are lacking molecular pathological information and targeted therapy hasn’t been approved in the treatment of VSCC, yet. Two etiological pathways are widely accepted: HPV induced vs. HPV independent, associated with chronic skin disease, often harboring TP53 mutations (mut). The aim of this analysis was to analyze the RNA expression patterns for subtype stratification on VSCC samples that can be integrated into the previously performed whole exome sequencing data for the detection of prognostic markers and potential therapeutic targets. We performed multiplex gene expression analysis (NanoString) with 770 genes in 24 prior next generation sequenced samples. An integrative data analysis was performed. Here, 98 genes were differentially expressed in TP53mut vs. HPV+ VSCC, in the TP53mut cohort, where 56 genes were upregulated and 42 were downregulated in comparison to the HPV+ tumors. Aberrant expression was primarily observed in cell cycle regulation, especially in HPV+ disease. Within the TP53mut group, a distinct cluster was identified that was correlated to a significantly worse overall survival (p = 0.017). The RNA expression profiles showed distinct patterns with regard to the known VSCC subtypes and could potentially enable further subclassification in the TP53mut groups

Methylation of the BRCA1 promoter is an epigenetic gene expression regulator and is frequently observed in ovarian cancer; however, conversion of methylation status is thought to drive disease recurrence. Therefore, longitudinal monitoring of methylation status by liquid biopsy in cell‐free DNA may be a predictive marker. In total, 135 plasma samples were collected from 69 ovarian cancer patients before and during systemic treatment. Our liquid biopsy assay could detect down to a single molecule of methylated DNA in a high background of normal DNA (0.03%) with perfect specificity in control samples. We found that 60% of the cancer patients exhibited BRCA1 promoter hypermethylation at one point, although 24% lost hypermethylation during treatment. Multivariate survival analyses indicate that relapses are independent events and that hypermethylation and methylation conversion are independently correlated to longer relapse‐free survival. We present a highly sensitive and specific methylation‐specific quantitative PCR‐based liquid biopsy assay. BRCA1 promoter hypermethylation is frequently found in ovarian cancer and is often reversed upon recurrence, indicating the selection of therapy‐resistant clones and unfavorable clinical outcome., In patients with ovarian cancer (OC) undergoing platinum‐based chemotherapy and PARP‐inhibitor treatment, BRCA1 deficiency is used as major predictive marker for estimating response to therapy. Here, we present a highly sensitive and specific liquid biopsy assay that assesses BRCA1 gene promoter hypermethylation on circulating tumor DNA (ctDNA) isolated from blood plasma. Our results suggest that hypermethylation and methylation conversion of the BRCA1 promoter in ctDNA are independently correlated to longer progression‐free survival of patients with OC.

Breast cancer (BC) is the second-most common cause of brain metastases (BM) and BCBM patients have a reduced quality of life and a poor prognosis. Hyaluronan (HA), and in particular the hyaluronidase Hyal-1, has been already linked to the development of BCBM, and therefore presents an interesting opportunity to develop new effective therapeutic options. HA metabolism was further discovered by the CRISPR/Cas9-mediated knockout of HYAL1 and the shRNA-mediated down-regulation of HA-receptor CD44 in the brain-seeking triple-negative breast cancer (TNBC) cell line MDA-MB-231-BR. Therefore, the impact of Hyal-1 on adhesion, disruption, and invasion through the brain endothelium, both in vitro and in vivo, was studied. Our analysis points out a key role of Hyal-1 and low-molecular-weight HA (LMW-HA) in the formation of a pericellular HA-coat in BC cells, which in turn promotes tumor cell adhesion, disruption, and migration through the brain endothelium in vitro as well as the extent of BM in vivo. CD44 knockdown in MDA-MB-231-BR significantly reduced the pericellular HA-coat on these cells, and, consequently, tumor cell adhesion and invasion through the brain endothelium. Thus, the interaction between Hyal-1-generated LMW-HA fragments and the HA-receptor CD44 might represent a potential target for future therapeutic options in BC patients with a high risk of cerebral metastases formation.

Objective At present, maintenance therapy with the antiangiogenic agent bevacizumab or with PARP-inhibitors represent two options for BRCA-wildtype ovarian cancer patients, after platinum-based first line chemotherapy. The identification of molecular markers to predict patient response to different maintenance therapies remains a major challenge. In the present study we analyzed the predictive potential of vascular endothelial growth factor C (VEGF-C) to identify ovarian cancer patients that might benefit from an antiangiogenic therapy. Methods 101 patients with primary epithelial ovarian cancer were analyzed for serum levels of VEGF-A,–C and CA-125 by ELISA. Serum levels were compared between patients with low pT-stage (pT1a-pT2c n = 11), healthy individuals (n = 27) and patients with higher pT-stage (> = pT3 n = 90). Adjusted ROC curves and an adjusted logistic regression model were carried out to evaluate the potential impact of VEGF-A and -C, as well as CA-125 serum level concentration on bevacizumab-therapy response, under consideration of covariates such as FIGO, pM, pN and residual tumor after surgery. Results A patient which has in comparison twice the VEGF-C concentration in serum, has a significant increased chance of response to bevacizumab by a factor of 2.79. Further, only VEGF-C serum levels were significantly higher in the group of patients with lower pT-stage compared to healthy individuals, whereas VEGF-A or CA-125 serum levels could not discriminate between healthy individuals and patients with ovarian cancer at low pT-stages. Conclusion VEGF-C serum level might serve as as a biomarker to evaluate treatment response under bevacizumab.

Simple Summary Breast cancer, the most common malignant tumor among women worldwide, remains an incurable disease once it has spread to the brain. Past research has shown that a primary breast cancer’s biology is an important determining factor predisposing its ability to form brain metastases. This review summarizes our current understanding of which genes, mutations, and molecules cause this increased ability to spread to and survive in the brain, specifically focusing on the different stages of this process. This knowledge may help us develop more effective, tumor-specific therapies and, as such, increase the chance of recovery for patients with breast cancer brain metastases. Abstract Breast cancer (BC) is the most frequent cause of cancer-associated death for women worldwide, with deaths commonly resulting from metastatic spread to distant organs. Approximately 30% of metastatic BC patients develop brain metastases (BM), a currently incurable diagnosis. The influence of BC molecular subtype and gene expression on breast cancer brain metastasis (BCBM) development and patient prognosis is undeniable and is, therefore, an important focus point in the attempt to combat the disease. The HER2-positive and triple-negative molecular subtypes are associated with an increased risk of developing BCBM. Several genetic and molecular mechanisms linked to HER2-positive and triple-negative BC breast cancers appear to influence BCBM formation on several levels, including increased development of circulating tumor cells (CTCs), enhanced epithelial-mesenchymal transition (EMT), and migration of primary BC cells to the brain and/or through superior local invasiveness aided by cancer stem-like cells (CSCs). These specific BC characteristics, together with the ensuing developments at a clinical level, are presented in this review article, drawing a connection between research findings and related therapeutic strategies aimed at preventing BCBM formation and/or progression. Furthermore, we briefly address the critical limitations in our current understanding of this complex topic, highlighting potential focal points for future research.

BackgroundMaturation of complex N-glycans involves the action of Golgi mannosidases and plays a major role in cancer progression. We recently showed a favourable prognostic role of α-mannosidase MAN1A1 in breast cancer mainly caused by alteration of certain adhesion molecules.MethodsWe analysed the protein expression of MAN1A1 in ovarian cancer (n = 204) using western blot and studied the impact of MAN1A1 itself and of MAN1A1-related glycosylation on the prognostic relevance of two adhesion molecules. Functional consequences of mannosidase inhibition using kifunensine and MAN1A1 knock out were investigated in ovarian cancer cells in vitro.ResultsPatients with high MAN1A1 expression in tumours showed significantly shorter RFS than those with low-MAN1A1 levels. Moreover, high MAN1A1 expression correlated significantly with advanced stage, lymph node involvement and distant metastasis. Further, the glycosylated adhesion molecule ALCAM reveals a significant adverse prognostic effect only in the presence of high MAN1A1 expression. In spheroid-formation assays, mannosidase inhibition and especially MAN1A1 knock out led to strong reduction of tumour cell aggregation.ConclusionsOur study demonstrates the unfavourable prognostic role of MAN1A1 in ovarian cancer, probably caused by an altered ability of spheroid formation, and the strong influence of this glycosylation enzyme on the prognostic impact of ALCAM.

The RAS family comprises three proto-oncogenes (H-RAS, K-RAS, and N-RAS) and is among the most widely studied of oncogenes. The present study aimed at investigating the clinical relevance of mRNA levels of the three isoforms in a large group of breast cancer patients with a long-term follow-up. 198 previously untreated patients were enrolled in the study. mRNA levels of K-RAS, H-RAS, and N-RAS were measured using microarray (Affymetrix HG-U133A). Elevated H-RAS levels were found significantly more frequently in patients with larger (p = 0.021) and ER-positive tumors (p = 0.048), while elevated K-RAS levels were associated with nodal positivity (p = 0.001) and HER2-positivity (p = 0.010). Patients with high N-RAS mRNA levels were more likely to be diagnosed with triple-negativity (p

Background: Ovarian cancer (OvCa) cells disseminate primarily intraperitoneally. Here, detached tumor cell aggregates (spheroids) from the primary tumor are generally regarded as “metastatic units”, which exhibit a survival benefit, probably due to the protective microenvironment and their unique molecular characteristics. Hence, current therapeutic concepts such as classical chemotherapy are not sufficient preventing growth and spread of OvCa spheroids. Methods: In the current study we analyzed the cellular composition of ascites from ovarian cancer patients using flow cytometry and the tumorigenic potential of the different subpopulations in an intraperitoneal mouse model. Comparative transcriptome analyses (RNAseq) from ascites-derived tumor cells spheroids (n=10) vs. tumor samples from different metastatic sites (n=30) were further performed in order to identify key molecular players responsible for the special cellular characteristics of OvCa spheroids. Results: In vitro culture of ascites-derived cells gave rise to two different subpopulations: an adherent cell population (ADs) including mainly CD90+ cells with highly proliferative rates in vitro but no tumorigenic potential in vivo, and a non-adherent cell population (NADs) containing principally EpCAM+/CD24+ cells with low proliferative potential in vitro. NADs included cell aggregates and single cells, the first showing a high content (> 80%) of tumor cells (EpCAM+/CD24+). Enriched tumor cell spheroids from the ascites using cell strainers showed higher tumorigenic potential in vivo in comparison to the original ascites-derived cell population. Interestingly, the different metastatic spread patterns observed in the mice resembled the tumor dissemination pattern found in the corresponding patients. RNAseq analyses from tumor-spheroids revealed up-regulation of genes involved in chemoresistance (TGM1, HSPAs, MT1s), cell-adhesion and cell barrier (PKP3, CLDNs, PPL) and the oxidative phosphorylation (OXPHOS) process compared to the solid tumor tissue samples. Additionally, down-regulation of extracellular matrix components and angiogenesis-related genes could be observed. Targeting OXPHOS by metformin treatment led to reduced viability of ascites-derived spheroids from OvCa patients, showing to some extent a synergistic effect with cisplatin treatment. Conclusions: the actual study contributes to a better understanding of the biology of ovarian cancer spheroids and to the identification of new treatment opportunities in advanced ovarian cancer.

The incidence of brain metastases (BM) in breast cancer patients is increasing [...]

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis, and cancer invasion and is often hijacked by viral infections 1. Unlike clathrin-mediated endocytosis, which exploits cargo-specific adaptors for selective protein internalization, the clathrin and dynamin-independent endocytic pathway (CLIC-GEEC, CG-pathway) has until now been considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids 2,3. Although the core molecular players of CG endocytosis have been recently defined, no cargo-specific adaptors are known and evidence of selective protein uptake into the pathway is lacking 3. Here, we identify the first cargo-specific adaptor for CG-endocytosis and demonstrate its clinical relevance in breast cancer progression. By combining unbiased molecular characterization and super-resolution imaging, we identified the actin-binding protein swiprosin-1 (EFHD2) as a cargo-specific adaptor regulating integrin internalization via the CG-pathway. Swiprosin-1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery, IRSp53 and actin. Swiprosin-1 is critical for integrin endocytosis, but not for other CG-cargo and supports integrin-dependent cancer cell migration and invasion, with clinically relevant implications for breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG-pathway and opens the possibility to discover more adaptors regulating it.

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.

Specific microRNAs (miRNAs) are packaged in exosomes that regulate processes in tumor development and progression. The current study focuses on the influence of exosomal miRNAs in the pathogenesis of epithelial ovarian cancer (EOC). MiRNA profiles were determined in exosomes from plasma of 106 EOC patients, eight ovarian cystadenoma patients, and 29 healthy women by TaqMan real-time PCR-based miRNA array cards containing 48 different miRNAs. In cell culture experiments, the impact of miR-200b and miR-320 was determined on proliferation and apoptosis of ovarian cancer cell lines. We report that miR-21 (P = 0.0001), miR-100 (P = 0.034), miR-200b (P = 0.008), and miR-320 (P = 0.034) are significantly enriched, whereas miR-16 (P = 0.009), miR-93 (P = 0.014), miR-126 (P = 0.012), and miR-223 (P = 0.029) are underrepresented in exosomes from plasma of EOC patients as compared to those of healthy women. The levels of exosomal miR-23a (P = 0.009, 0.008) and miR-92a (P = 009, 0.034) were lower in ovarian cystadenoma patients than in EOC patients and healthy women, respectively. The exosomal levels of miR-200b correlated with the tumor marker CA125 (P = 0.002) and patient overall survival (P = 0.019). MiR-200b influenced cell proliferation (P = 0.0001) and apoptosis (P < 0.008). Our findings reveal specific exosomal miRNA patterns in EOC and ovarian cystadenoma patients, which are indicative of a role of these miRNAs in the pathogenesis of EOC.

Loading of microRNAs (miRNAs) into exosomes that are involved in cellular communication is a selective process. The current study investigates whether the enrichment of miRNAs in exosomes reflects the pathogenesis of breast cancer (BC) and ductal carcinoma in situ (DCIS). The levels of miRNAs were quantified in exosomes from plasma of 32 BC patients, 8 DCIS patients and 8 healthy women by TaqMan real-time PCR-based miRNA array cards containing 47 different miRNAs. Then, exosomal miR-16, miR-30b and miR-93 that displayed deregulation in the arrays were selected and analyzed in 111 BC patients, 42 DCIS patients and 39 healthy women by TaqMan real-time PCR. Identification of exosomes was performed by Western blot. The levels of exosomal miR-16 were higher in plasma of BC (p = 0.034) and DCIS (p = 0.047) patients than healthy women, and were associated with estrogen (p = 0.004) and progesterone (p = 0.008) receptor status. Particularly, in estrogen-positive patients miR-16 was significantly enriched in exosomes (p = 0.0001). Lower levels of exosomal miR-30b were associated with recurrence (p = 0.034). Exosomal miR-93 was upregulated in DCIS patients (p = 0.001). Our findings suggest that different signatures of miR-16, miR-30b and miR-93 in exosomes from BC and DCIS patients are associated with a particular biology of breast tumors.

Elderly patients are underrepresented in clinical trials in gynecological cancer, even though they are disproportionally often affected. This study aimed to evaluate the disposition and apprehension of elderly patients toward study participation. 112 elderly gynecological cancer patients (median age 70) were surveyed in a multicenter cross-sectional study. Besides fitness, state of disease, education and domestic situation, questions aimed at the general willingness to participate in a clinical trial. Personal reasons for refusal and anticipated advantages/disadvantages that might evolve from participation were inquired. Willingness to participate in a clinical study was generally high (72%, 74/102). Reasons for potential study participation were: ‘better monitoring of the disease’ (67.1%), ‘better medical care’ (46.1%), ‘to help medical research’ (44.7%), ‘better medication’ (35.5%) and ‘because of my doctor’s recommendation’ (22.4%). Reasons for potential refusal were: ‘too time consuming’ (24.4%), ‘fear of side effects’ (21.8%), ‘misuse as experimental animal’ (18%), ‘long distance to clinic’ (14.1%) and ‘too little or unclear information’ (10.3%). 37.2% (29/78) of the patients stated that they had ‘no objection’ at all against study participation. The question if patients anticipated having a longer life due to study participation was answered with ‘yes’ or ‘rather yes’ in 42% (38/90); 28.9% answered ‘no’ or ‘rather no’ (29% undecided). No statistical significant relation between willingness to participate in a study and general fitness (p = 0.133), education (p = 0.122), age (p = 0.474) or domestic situation (p = 0.123) could be observed in a multivariate logistic regression model. Elderly patients are generally willing to participate in clinical studies, in our cohort regardless of their fitness. Benefits of participation seem to be unclear among a majority of potential study participants. Therefore, it might be decisive to provide more general information regarding benefits and safety for elderly patients in a clinical trial.

Aberrant sialylation of glycoproteins has been detected in many tumors, and upregulation of the beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6GAL1) has been implicated with tumor aggressiveness and chemoresistance in experimental models. In our present study, we aimed to study the prognostic or predictive role of ST6GAL1 in ovarian carcinoma, using two independent ovarian cancer cohorts. ST6GAL1 mRNA levels were retrieved from a publicly available database (n = 517), and ST6GAL1 protein levels were analyzed by western blot analysis in a cohort of 204 ovarian tumor samples. The results were correlated with clinical and histological tumor parameters and follow-up information. High ST6GAL1 mRNA levels significantly correlated with lymphovascular invasion and shorter survival, whereas high ST6GAL1 protein expression was associated with advanced stage, distant metastasis and shorter recurrence-free intervals. In both cohorts the prognostic role was most pronounced in tumors without macroscopically visible residual tumor after surgery. In these cases, ST6GAL1 expression levels might help to identify cases with a higher risk of chemoresistance and metastatic relapse that might require an adapted therapeutic regime.

Background: Alterations in protein glycosylation have been related to malignant transformation and tumour progression. We recently showed that low mRNA levels of Golgi alpha-mannosidase MAN1A1 correlate with poor prognosis in breast cancer patients. Methods: We analysed the role of MAN1A1 on a protein level using western blot analysis (n=105) and studied the impact of MAN1A1-related glycosylation on the prognostic relevance of adhesion molecules involved in breast cancer using microarray data (n=194). Functional consequences of mannosidase inhibition using the inhibitor kifunensine or MAN1A1 silencing were investigated in breast cancer cells in vitro. Results: Patients with low/moderate MAN1A1 expression in tumours showed significantly shorter disease-free intervals than those with high MAN1A1 levels (P=0.005). Moreover, low MAN1A1 expression correlated significantly with nodal status, grading and brain metastasis. At an mRNA level, membrane proteins ALCAM and CD24 were only significantly prognostic in tumours with high MAN1A1 expression. In vitro, reduced MAN1A1 expression or mannosidase inhibition led to a significantly increased adhesion of breast cancer cells to endothelial cells. Conclusions: Our study demonstrates the prognostic role of MAN1A1 in breast cancer by affecting the adhesive properties of tumour cells and the strong influence of this glycosylation enzyme on the prognostic impact of some adhesion proteins.

Background: There are 2 known pathways for tumorigenesis of vulvar squamous cell carcinoma—a human papillomavirus–dependent pathway characterized by p16 overexpression and a human papillomavirus–independent pathway linked to lichen sclerosus, characterized by TP53 mutation. A correlation of human papillomavirus dependency with a favorable prognosis has been proposed.Objective: The objective of the study was to further understand the role of human papillomavirus and p53 status in vulvar squamous cell carcinoma and characterize its clinical relevance.Study Design: The Arbeitsgemeinschaft Gynaecological Oncology Chemo and Radiotherapy in Epithelial Vulvar Cancer-1 study is a retrospective cohort study of 1618 patients with primary vulvar squamous cell carcinoma Fédération Internationale de Gynécologie et d'Obstétrique stage ≥1B treated at 29 gynecologic cancer centers in Germany between 1998 and 2008. For this translational substudy, formalin-fixed paraffin-embedded tissue was collected. A tissue microarray was constructed (n=652 samples); p16 and p53 expression was determined by immunohistochemistry. Human papillomavirus status and subtype were analyzed by polymerase chain reaction.Results: p16 immunohistochemistry was positive in 166 of 550 tumors (30.2%); p53 staining in 187 of 597 tumors (31.3%). Only tumors with available information regarding p16 and p53 immunohistochemistry and without p53 silent expression pattern were further analyzed (n=411); 3 groups were defined: p53+ (n=163), p16+/p53− (n=132), and p16−/p53− (n=116). Human papillomavirus DNA was detected in 85.6% of p16+/p53− tumors; human papillomavirus-16 was the most common subtype (86.3%). Patients with p16+ tumors were younger (64 vs 72 years for p53+, respectively, 69 years for p16−/p53− tumors; PConclusion: p16 overexpression is associated with an improved prognosis whereas p53 positivity is linked to an adverse outcome. Our data support the hypothesis of a clinically relevant third subgroup of vulvar squamous cell carcinoma with a p53−/p16− phenotype showing an intermediate prognosis that needs to be further characterized.

Purpose Ovarian cancer (OvCa) progression mainly takes place by intraperitoneal spread. Adhesion of tumor cells to the mesothelial cells which form the inner surface of the peritoneum is a crucial step in this process. Cancer cells use in principle different molecules of the leukocyte adhesion cascade to facilitate adhesion. This cascade is initiated by selectin-ligand interactions followed by integrin - extracellular matrix protein interactions. Here we address the question whether all tumor cells predominantly employ selectin-dependent leukocyte-like adhesion cascade (SDAC) or whether they use integrin mediated adhesion for OvCa progression as well. Methods A comparative transcriptomic analysis of the human OvCa cell lines OVCAR8 and SKOV3 was performed. Intraperitoneal xenograft model of OVCAR8 cells was used to determine whether there is a correlation between SDAC gene expression and the metastatic potential of the control cells and the cells overexpressing c-Fos. Transcriptomic analysis of OVCAR8 and SKOV3 samples was performed using microarrays. Results One-third of the protein-coding genes involved in SDAC exhibited lower expression levels in OVCAR8 than in SKOV3 cells. In contrast to SKOV3 cells, c-Fos overexpression in OVCAR8 cells did not significantly influence the expression of SDAC genes. Intraperitoneal xenograft model of OVCAR8 cells unexpectedly demonstrated that the aggressiveness of OVCAR8 tumors was not depended on the c-Fos expression level and was comparable to that of SKOV3 control tumors. Gene expression analysis of tumors suggests that SKOV3-derived tumor progression was mainly depended on SDAC. Progression of OVCAR8 tumors relied on other cell adhesion molecules that do not interact with selectins. Conclusions High expression of c-Fos in ovarian cancer cells is not always associated with reduced metastatic potential. Low expression level of SDAC genes may not ensure low OvCa metastatic potential hence alternative adhesion mechanisms involving laminin-integrin interactions exist as well.

// Katharina Prieske 1, * , Stefan Prieske 1, * , Simon A. Joosse 2 , Fabian Trillsch 3 , Donata Grimm 1 , Eike Burandt 4 , Sven Mahner 3 , Barbara Schmalfeldt 1 , Karin Milde-Langosch 1 , Leticia Oliveira-Ferrer 1 and Linn Woelber 1 1 Department of Gynecology and Gynecologic Oncology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany 2 Department of Tumor Biology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany 3 Department of Gynecology and Obstetrics, University of Munich, 81377 Munich, Germany 4 Department of Pathology, University Medical Centre Hamburg-Eppendorf, 20246 Hamburg, Germany * These authors have contributed equally to this work Correspondence to: Katharina Prieske, email: k.prieske@uke.de Keywords: BRCA methylation, ovarian cancer, platinum, recurrence, high-grade Received: May 30, 2017 Accepted: July 25, 2017 Published: September 15, 2017 ABSTRACT Background: Approximately 20-25% of ovarian cancers are attributable to germline or somatic BRCA1/2 mutations, resulting in defects in the homologous recombination pathway. Inactivation of these genes can also be mediated by epigenetic changes, e.g., hypermethylation of CpG islands in the promoter regions. In such homologous recombination deficient tumors, platinum based chemotherapy is in general effective, however, loss of hypermethylation might lead to refractory disease. The aim of this study was to evaluate the stability of BRCA1 promoter hypermethylation in recurrent disease after platinum based chemotherapy. Methods: Tumor tissue from 76 patients with primary and 48 patients with platinum-sensitive recurrent high-grade ovarian cancer was collected. In a subgroup of 12 patients, ‘paired’ tumor tissue from primary and recurrent surgery was available. BRCA1 promoter methylation status was assessed using methylation specific polymerase chain reaction and was verified by Sanger Sequencing. Results: 73.7% (56/76) of primary and 20.8% (10/48) of recurrent tumors displayed BRCA1 promoter hypermethylation. BRCA1 promoter methylation status was not associated with progression-free- or overall survival. In the paired subgroup 83.3% (10/12) of the primary vs. 16.7% (2/12) of the recurrent tumors showed hypermethylation. In eight patients loss of BRCA1 hypermethylation was observed, whereas two patients had stable methylation status. Conclusions: Loss of BRCA1 promoter methylation may be a mechanism to restore BRCA1 function in recurrent disease. However, currently the clinical significance is still unclear and should be evaluated in prospective clinical trials.

Although altered glycosylation has been detected in human cancer cells decades ago, only investigations in the last years have enormously increased our knowledge about the details of protein glycosylation and its role in tumour progression. Many proteins, which are heavily glycosylated, i.e. adhesion proteins or proteases, play an important role in cancer metastasis that represents the crucial and frequently life-threatening step in progression of most tumour types. Compared to normal tissue, tumour cells often show altered glycosylation patters with appearance of new tumour-specific antigens. In this review, we give an overview about the role of glycosylation in tumour metastasis, describing recent results about O-glycans, N-glycans and glycosaminoglycans. We show that glycan structures, glycosylated proteins and glycosylation enzymes have influence on different steps of the metastatic process, including epithelial-mesenchymal transition (EMT), migration, invasion/intravasation and extravasation of tumour cells. Regarding the important role of cancer metastasis for patients survival, further knowledge about the consequences of altered glycosylation patterns in tumour cells is needed which might eventually lead to the development of novel therapeutic approaches.

// Sascha Kuerti 1, * , Leticia Oliveira-Ferrer 1, * , Karin Milde-Langosch 1 , Barbara Schmalfeldt 1 , Karen Legler 1 , Linn Woelber 1 , Katharina Prieske 1 , Sven Mahner 2 and Fabian Trillsch 2 1 Department of Gynaecology and Gynaecologic Oncology, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany 2 Department of Obstetrics and Gynaecology, University of Munich, Munich 81377, Germany * These authors have contributed equally to this work Correspondence to: Fabian Trillsch, email: Fabian.Trillsch@med.uni-muenchen.de Keywords: ovarian cancer, (VEGF)-A, -C, -D, mesenchymal phenotype, tumour dissemination, lymph node metastases Received: February 20, 2017 Accepted: March 31, 2017 Published: May 18, 2017 ABSTRACT Background: Peritoneal dissemination and retroperitoneal lymph node involvement are main routes for progression of epithelial ovarian cancer (EOC). Vascular endothelial growth factor (VEGF) mediated angiogenesis has been identified as an important mechanism promoting tumour progression. Methods: Tumour tissue of 100 patients with EOC was analysed for protein expression of vascular endothelial growth factor (VEGF)-A, -C, -D by Western Blot analysis. Expression patterns in patients with ‘extensive intraperitoneal’ metastases (pT3c pN1 and pT3b-pT3c pN0, n=80) were compared to patients with ‘predominantly retroperitoneal’ metastases (pT1a-pT3b, pN1, n=20). Overall and progression-free survival was analysed by Kaplan-Meier method. Results: While no significant differences in expression levels among the different modes of metastases were noted for VEGF-A and -D, VEGF-C expression was significantly higher in the group of predominantly retroperitoneal metastases compared to the group with extensive intraperitoneal metastases. Patients with high VEGF-C expression had a significantly worse overall survival compared to patients with low expression levels. Conclusions: Retroperitoneal tumour progression in EOC patients is associated with high VEGF-C expression. VEGF-C may serve as a molecular marker to identify patients with assumed high risk for lymphatic metastases, who might benefit from specific treatment strategies.

Background Ligands of the C-type lectin CLEC10A such as Tn and sialyl-Tn representing early intermediates of O-glycosylation are hallmarks of many human malignancies. A variety of regulatory mechanisms underlying their expression are being discussed. Methods CLEC10A ligands were detected in various tissues and cells using the recombinant glycan-binding domain of CLEC10A. In normal breast and endometrium, presence of ligands was correlated to the female cycle. Estrogen- and stress dependent induction of CLEC10A ligands was analyzed in MCF7 and T47D cells exposed to 4-hydroxy-tamoxifen (Tam), zeocin and hydrogen peroxide. The expression and localization of CLEC10A ligands was analyzed by Western blot and immunofluorescence. In breast cancer patients CLEC10A ligand expression and survival was correlated by Kaplan-Meyer analysis. Result We observed binding of CLEC10A in normal endometrial and breast tissues during the late phase of the female hormonal cycle suggesting a suppressive effect of female sex hormones on CLEC10A ligand expression. Accordingly, CLEC10A ligands were induced in MCF7- and T47D breast cancer cells after Tam treatment and accumulated on the cell surface and in the endosomal/lysosomal compartment. Phagocytosis experiments indicate that macrophages preferentially internalize CLEC10A ligands coated beads and Tam treated MCF7 cells. CLEC10A ligands were also expressed after the addition of zeocin and hydrogen-peroxide. Each substance induced the production of ROS indicating reactive oxygen species as a unifying mechanism of CLEC10A ligand induction. Mechanistically, increased expression of GalNAc-transferase 6 (GalNT6) and translocation of GalNT2 and GalNT6 from cis- towards trans-Golgi compartment was observed, while protein levels of COSMC and T-synthase remained unaffected. In breast cancer patients, positivity for CLEC10A staining in tumor tissues was associated with improved outcome and survival. Conclusion CLEC10A ligands are inducible by hormone depletion, 4-hydroxy-tamoxifen and agents inducing DNA damage and oxidative stress. Our results indicate that CLEC10A acts as a receptor for damaged and dead cells and may play an important role in the uptake of cell debris by macrophages and dendritic cells. Graphical Abstract Electronic supplementary material The online version of this article (10.1186/s12964-019-0420-9) contains supplementary material, which is available to authorized users.

Immune checkpoints are intensively investigated as targets in cancer therapy. T-cell immunoreceptor with immunoglobulin (Ig) and ITIM domains (TIGIT) and its ligand poliovirus receptor (PVR) are recently emerging as novel promising targets in immunotherapy. Here, we show that high expression of PVR represents an independent prognostic marker being associated with poor outcome for breast cancer patients. Furthermore, PVR mRNA, as well as protein expression, is associated with more aggressive breast cancer subtypes such as HER2 positive and triple-negative breast cancer. In vitro, blocking TIGIT or PVR resulted in enhanced immune cell-mediated lysis of breast cancer cell lines SKBR-3, MDA-MB-231, MDA-MB-468, and BT549 and additionally increased the cytotoxic effects of a bispecific T cell engager BiTE® antibody construct targeting EGFR. Taken together, our data identify the immune checkpoint factor PVR as a novel prognostic marker in breast cancer and indicate that blocking the TIGIT-PVR axis might represent a novel therapeutic option for the treatment of breast cancer patients.

Background: Peritoneal dissemination and retroperitoneal lymph node involvement are main routes for tumour spread of epithelial ovarian cancer (EOC), possibly determined by the intercellular connecting protein E-Cadherin (E-Cad) and its fragments. Methods: Tumour tissue of 105 advanced EOC patients was evaluated for protein expression of E-Cad, β-Catenin and Calpain by western blotting and immunohistochemistry. Expression patterns were compared between tumours with solely intraperitoneal (pT3c, pN0; n=41) and tumours with retroperitoneal metastases (pT1a-3c, pN1; n=64). Lysates of the EOC cell line SKOV3 and tumour tissue from the intraperitoneal group were tested for E-Cad expression following Calpain treatment. Results: E-Cad full-length (E-Cad-FL, 120 kDa) and two major fragments at 85 kDa (E-Cad-85) and 23 kDa (E-Cad-23) were detected by western blotting. E-Cad-85 expression was significantly higher in tumours with solely intraperitoneal metastases and correlated strongly with E-Cad-23 and the protease Calpain. Calpain-mediated cleavage was identified as a potential mechanism to generate E-Cad-85 from E-Cad-FL by treating lysates from SKOV3 cells and tumour tissue with this enzyme. Increased cytoplasmic localisation of β-Catenin in tumours with high E-Cad-85 expression corroborates that E-Cad-85 loses the binding site for β-Catenin after fragmentation, enabling tumour cluster formation and peritoneal dissemination. Conclusions: Calpain-mediated E-Cad fragmentation appears to promote intraperitoneal EOC progression. Understanding these mechanisms might eventually lead to new tailored subtype-specific diagnostic and therapeutic interventions.

During the last few years, diverse studies have shown that tumors can actively interact with the lymphatic system and promote metastases development. In order to examine the molecular mechanisms involved in this interaction, we co-cultured tumor and lymphatic endothelial cells (LEC) and subsequently analyzed the molecular alterations of LECs. Therefore, LECs were co-cultivated with either a highly or weakly metastatic breast cancer cell line using contact (mixture) and non-contact (transwell) co-cultures. mRNA profiles from LECs were subsequently analyzed for genes specifically induced by highly metastatic tumor cells (&ldquo, metastatic specific&rdquo, ). Among the up-regulated &ldquo, genes, we found candidates involved in cell cycle, cell adhesion and motility (BST2, E-selectin, and HMMR), cytokines (CCL7, CXCL6, CXCL1, and CSF2) and factors of the complement system (C1R, C3, and CFB). Among the down-regulated genes, we detected the hyaluronan receptor STAB2, angiogenic factor apelin receptor (APLNR), and the glycosylation enzyme MAN1A1. In an additional prostate cancer co-culture model, we could confirm a &ldquo, upregulation of E-selectin and CCL7 in LECs after interaction with the prostate cancer cell lines LNCAP (highly metastatic) and DU145 (weakly metastatic). These data allowed us to identify a set of genes regulated in LECs during in vitro communication with cancer cells, which might subsequently facilitate lymphatic metastasis.

Long noncoding RNAs (lncRNAs) are frequently precursor RNAs of microRNAs (miRNAs) or act as competing endogenous RNAs (ceRNAs) to interact with miRNAs. To better understand the shared impact of lncRNAs and miRNAs in the regulatory post-transcriptional network, we focused here on the relationships between (a) lncRNA H19 and miR-675, (b) NEAT1 and miR-204, and (c) HOTAIR and miR-331 in plasma of early breast cancer (BC) patients. We quantified each RNA in plasma samples of 63 BC patients and 10 healthy women by quantitative real-time PCR. In cell culture experiments, the influence of these noncoding RNAs (ncRNAs) on proliferation and apoptosis of BC cell line MCF-7 was examined. Plasma levels of H19 (P = 0.030), NEAT1 (P = 0.030), and miR-331 (P = 0.012) were deregulated in BC patients compared with healthy women. In both cohorts, the concentrations of H19 correlated with those of miR-675 (P = 0.0001). Higher H19 (P = 0.001) along with lower miR-675 (P = 0.007) levels and higher miR-204 (P = 0.017) along with lower NEAT1 (P = 0.030) levels were detected in plasma of HER2-positive patients compared with the other BC subgroups. Whereas the expression of HOTAIR was below the detection level, miR-331 levels correlated with nodal status (P = 0.002) and recurrence (P = 0.012). In cell culture experiments, a competitive impact on cell proliferation and apoptosis by these ncRNAs was also documented. Our findings describe a relationship of the plasma levels of H19/miR-675 and NEAT1/miR-204 in the different BC subtypes; in addition, they reveal an interplay between these lncRNAs and miRNAs in the regulatory network in MCF-7 cells, which should also be considered in the search for new diagnostic and therapeutic markers.

Loading of microRNAs (miRNAs) into exosomes that are involved in cellular communication is a selective process. The current study investigates whether the enrichment of miRNAs in exosomes reflects the pathogenesis of breast cancer (BC) and ductal carcinoma in situ (DCIS). The levels of miRNAs were quantified in exosomes from plasma of 32 BC patients, 8 DCIS patients and 8 healthy women by TaqMan real-time PCR-based miRNA array cards containing 47 different miRNAs. Then, exosomal miR-16, miR-30b and miR-93 that displayed deregulation in the arrays were selected and analyzed in 111 BC patients, 42 DCIS patients and 39 healthy women by TaqMan real-time PCR. Identification of exosomes was performed by Western blot. The levels of exosomal miR-16 were higher in plasma of BC (p = 0.034) and DCIS (p = 0.047) patients than healthy women, and were associated with estrogen (p = 0.004) and progesterone (p = 0.008) receptor status. Particularly, in estrogen-positive patients miR-16 was significantly enriched in exosomes (p = 0.0001). Lower levels of exosomal miR-30b were associated with recurrence (p = 0.034). Exosomal miR-93 was upregulated in DCIS patients (p = 0.001). Our findings suggest that different signatures of miR-16, miR-30b and miR-93 in exosomes from BC and DCIS patients are associated with a particular biology of breast tumors.

Ovarian cancer has a high mortality rate and up to now no reliable molecular prognostic biomarkers have been established. During malignant progression, the cytoskeleton is strongly altered. Hence we analyzed if expression of certain cytoskeleton-associated proteins is correlated with clinical outcome of ovarian cancer patients. First, in silico analysis was performed using the cancer genome atlas (TCGA), the human expression atlas and Pubmed. Selected candidates were validated on 270 ovarian cancer patients by qRT-PCR and/or by western blotting. In silico analysis revealed that mRNAs of 214 cytoskeleton-associated proteins are detectable in ovarian cancer tissue. Among these, we selected 17 proteins that participate in cancer disease progression and cytoskeleton modulation: KIF14, KIF20A, KIF18A, ASPM, CEP55, DLGAP5, MAP9, EB1, KATNA1, DIAPH1, ANLN, SCIN, CCDC88A, FSCN1, GSN, VASP and CDC42. The first ten candidates interact with microtubules (MTs) and the others bind to actin filaments. Validation on clinical samples of ovarian cancer patients revealed that the expression levels of DIAPH1, EB1, KATNA1, KIF14 and KIF18A significantly correlated with clinical and histological parameters of ovarian cancer. High DIAPH1, EB1, KATNA1 and KIF14 protein levels were associated with increased overall survival (OAS) of ovarian cancer patients, while high DIAPH1 and EB1 protein levels were also associated with low differentiation of respective tumors (G2/3). Moreover, DIAPH1 was the only protein, whose expression significantly correlated with increased recurrence-free interval (RFI). Mainly the expression levels of the MT-associated proteins analyzed in this study, correlated with prolonged survival of ovarian cancer patients. From > 200 genes initially considered, 17 cytoskeletal proteins are involved in cancer progression according to the literature. Among these, four proteins significantly correlated with improved survival of ovarian cancer patients.

The underlying mechanisms of ovarian cancer (OvCa) dissemination are still poorly understood, and novel molecular markers for this cancer type are urgently needed. In search of adhesion molecules with prognostic relevance in OvCa, we compared tumors with good outcome (alive > 3 years) and those with poor outcome (dead n=517) as well as CEACAM1 protein expression by Western blot analysis in a cohort of 242 tumor samples. Further, CEACAM1 localization in tumour tissue was evaluated by immunohistochemistry and CEACAM1 splice variants by RT-PCR in representative tumours. In Kaplan–Meier analysis, high CEACAM1 mRNA levels significantly correlated with longer survival (p=0.008). By Western blot analysis in the second cohort, similar associations of high CEACAM1 protein levels with longer recurrence-free survival (RFS, p=0.035) and overall survival (OAS, p=0.004) were observed. In multivariate Cox regression analysis including clinical prognostic parameters, CEACAM1 mRNA or protein expression turned out as independent prognostic markers. Stratified survival analysis showed that high CEACAM1 protein expression was prognostic in node-negative tumors (p=0.045 and p=0.0002 for DFS and OAS) but lost prognostic significance in node-positive carcinomas. Similarly, high CEACAM1 mRNA expression did not show prognostic relevance in tumors with lymphatic invasion (L1) but was associated with longer survival in cases without lymphovascular involvement. Further analysis showed a predominance of 4S and 4L isoforms and mostly membraneous CEACAM1 localization in ovarian tumours. Our results suggest that CEACAM1 might be an independent favorable prognostic marker in OvCa, especially in the subgroup of patients with solely intraperitoneal metastasis.

Background: In a recent study based on cDNA microarray data of breast cancer patients, we could show that the mRNA expression of some glycosylation enzymes involved in synthesis and trimming of N-glycansis significantly associated with metastasis and recurrence in breast cancer patients suggesting an important role of N-glycosylation in tumour progression (Milde-Langosch et al., Breast Cancer Res Treat 2014). The aim of the present study was to further investigate this issue on a protein and glycoprotein level (1.) by western blot analysis of selected glycosylation enzymes and (2.) by lectin histochemistry using lectins which bind to O- and/or N-glycans. Methods: In order to confirm the role of two selected glycosylation enzymes, ribophorin (RPN1) and mannosidase (MAN1A1) on protein level, we analyzed their expression in a cohort of 196 breast cancer samples by western blot analysis. In addition, we studied tumour cell binding of various lectins, which recognize diverse O- or N-glycans (UEA1, HPA, PNA, GNA, PHA-L), using a tissue microarray with up to 293 evaluable breast cancer samples. These results were correlated with recurrence-free survival (RFS) and overall survival (OS) and data on vascular and lymphatic invasion. Results: By western blot analysis, the negative prognostic significance of ribophorin (RPN1), a key enzyme of N-glycosylation, was corroborated on a protein level. For MAN1A1 which is involved in trimming of N-glycan structures, two isoforms with different molecular weight could be detected on western blots, showing contradictory associations with OS. Protein levels of RPN1 and total MAN1A1 showed significant correlations to the respective mRNA expression data. By lectin histochemistry, we found significant correlations of lectin binding to lymph node metastasis, vascular invasion and lymphangiosis in breast cancer samples. Binding of PHA-L and GNA (binding to N-glycans) and PNA (binding to O- and N-glycans) was significantly associated with vascular invasion, whereas PNA and PHA-L binding correlated with lymph node involvement. In addition, PNA and HPA reactivity was associated with microscopically detected lymphangiosis. PNA binding also correlated with shorter RFS and OS. Conclusion: While the role of O-glycans in breast cancer metastasis has already been described, our data point to an additional strong impact of N-glycosylation on metastasis and progression of mammary carcinomas. Citation Format: Karin Milde-Langosch, Tosca Karius, Dina Schütze, Harriet Wikman, Isabell Witzel, Leticia Oliveira-Ferrer, Volkmar Müller, Udo Schumacher. N-glycosylation changes in tumour cells are associated with vascular invasion and metastasis in breast cancer patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-21.

Fra-1, a component of the transcription factor AP-1 family, has been found to be overexpressed in carcinomas with high metastatic potential such as in triple-negative breast cancer. The effect of Fra-1 on the morphology, motility and invasive potential of breast cancer cells has been previously described. Since tumor cell adhesion plays an essential role in the metastatic process, especially for extravasation from blood vessels, we investigated the influence of Fra-1 on breast cancer cell interactions with the endothelium. Using the human breast cancer cell lines MCF7 (weakly invasive, estrogen receptor (ER)-positive) and MDA MB 231 (strongly invasive, ER-negative) with stable Fra-1 overexpression, we performed dynamic cell-flow adhesion assays on surfaces coated with E-selectin or with human pulmonary microvascular endothelial cells (HPMEC). Our analyses revealed increased adhesion of Fra-1 overexpressing MCF7 cells to E-selectin but also to activated endothelial cells, whereas the per se highly invasive and Fra-1-positive MDA MB 231 cell line showed enhanced cell rolling and tethering on E-selectin and endothelium-coated surfaces, but no increased firm adhesion after Fra-1 overexpression. These different behaviors correspond to an up-regulation of various adhesion-related proteins such as CD44, Integrin α5 and CEACAM6 in Fra-1 overexpressing MCF7 cells measured by microarray analysis and flow cytometry in comparison to weaker differences in the expression of adhesion molecules found in the Fra-1 overexpressing MDA MB 231 cell line. In line with these results and based on cDNA microarray data of breast cancer patients (n=175) high Fra-1 expression significantly correlates with shorter overall survival and higher rate of lung metastasis in ER-positive breast cancer patients (n=130), but has no impact on the prognosis of patients with ER-negative tumors. Thus, in addition to its pro-invasive and pro-migratory effect, Fra-1 might influence the metastatic potential of breast cancer cells by changing the expression of adhesion molecules resulting in increased adherence to endothelial cells under flow conditions. Citation Format: Leticia Oliveira-Ferrer, Melanie Kürschner, Vera Labitzky, Daniel Wicklein, Volkmar Müller, Udo Schumacher, Karin Milde-Langosch, Christine Schröder. Transcription factor Fra-1 modulates the adhesive properties of breast cancer cells contributing to a metastatic phenotype [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-17.

Background Approximately 20-25% of ovarian cancers are attributable to germline or somatic BRCA1/2 mutations, resulting in defects in the homologous recombination pathway. Inactivation of these genes can also be mediated by epigenetic changes, e.g., hypermethylation of CpG islands in the promoter regions. In such homologous recombination deficient tumors, platinum based chemotherapy is in general effective, however, loss of hypermethylation might lead to refractory disease. The aim of this study was to evaluate the stability of BRCA1 promoter hypermethylation in recurrent disease after platinum based chemotherapy. Methods Tumor tissue from 76 patients with primary and 48 patients with platinum-sensitive recurrent high-grade ovarian cancer was collected. In a subgroup of 12 patients, ‘paired’ tumor tissue from primary and recurrent surgery was available. BRCA1 promoter methylation status was assessed using methylation specific polymerase chain reaction and was verified by Sanger Sequencing. Results 73.7% (56/76) of primary and 20.8% (10/48) of recurrent tumors displayed BRCA1 promoter hypermethylation. BRCA1 promoter methylation status was not associated with progression-free- or overall survival. In the paired subgroup 83.3% (10/12) of the primary vs. 16.7% (2/12) of the recurrent tumors showed hypermethylation. In eight patients loss of BRCA1 hypermethylation was observed, whereas two patients had stable methylation status. Conclusions Loss of BRCA1 promoter methylation may be a mechanism to restore BRCA1 function in recurrent disease. However, currently the clinical significance is still unclear and should be evaluated in prospective clinical trials.

High expression of the actin bundling protein Fascin increases the malignancy of tumor cells. Here we show that fascin expression is up-regulated in more malignant sub-cell lines of MDA-MB-231 cells as compared to parental cells. Since also parental MDA-MB-231 cells exhibit high fascin levels, increased fascin expression was termed as "hyperexpression". To examine the effect of fascin hyperexpression, fascin was hyperexpressed in parental MDA-MB-231 cells and metastasis was analyzed in NOD scid gamma (NSG) mice. In addition, the effect of fascin mutants with inactive or constitutively active actin bundling activity was examined. Unexpectedly, we found that hyperexpression of both, wildtype (wt) and mutant fascin strongly increased metastasis in vivo, showing that the effect of fascin hyperexpression did not depend on its actin bundling activity. Cellular assays revealed that hyperexpression of wt and mutant fascin increased adhesion of MDA-MB-231 cells while transmigration and proliferation were not affected. Since it has been shown that fascin controls adhesion by directly interacting with microtubules (MTs), we analyzed if fascin hyperexpression affects MT dynamics. We found that at high concentrations fascin significantly increased MT dynamics in cells and in cell-free approaches. In summary our data show that strong expression of fascin in breast cancer cells increases metastasis independent of its actin bundling activity. Thus, it seems that the mechanism of fascin-stimulated metastasis depends on its concentration.

Glycosylation of cellular proteins has important impact on their stability and functional properties, and glycan structures strongly influence cell adhesion. Many enzymes are involved in glycoconjugate synthesis and degradation, but there is only limited information about their role in breast cancer progression. Therefore, we retrieved RNA expression data of 202 glycosylation genes generated by microarray analysis (Affymetrix HG-U133A) in a cohort of 194 mammary carcinomas with long-term follow-up information. After univariate and multivariate Cox regression analysis, genes with independent prognostic value were identified. These were further analysed by Kaplan-Meier analysis and log-rank tests, and their prognostic value was validated in a second cohort of 200 tumour samples from patients without systemic therapy. In our first cohort, we identified 24 genes with independent prognostic value, coding for sixteen anabolic and eight catabolic enzymes. Functionally, these genes are involved in all important glycosylation pathways, namely O-glycosylation, N-glycosylation, O-fucosylation, synthesis of glycosaminoglycans and glycolipids. Eighteen genes also showed prognostic significance in chemotherapy-treated patients. In the second cohort, six of the 24 relevant genes were of prognostic significance (FUT1, FUCA1, POFUT1, MAN1A1, RPN1 and DPM1), whereas a trend was observed for three additional probesets (GCNT4, ST3GAL6 and UGCG). In a stratified analysis of molecular subtypes combining both cohorts, great differences appeared suggesting a predominant role of N-glycosylation in luminal cancers and O-glycosylation in triple-negative ones. Correlations of gene expression with metastases of various localizations point to a role of glycan structures in organ-specific metastatic spread. Our results indicate that various glycosylation reactions influence progression and metastasis of breast cancer and might thus represent potential therapeutic targets.