Metastasis is the leading cause of cancer mortality. The metastatic cascade represents a multi-step process which includes local tumor cell invasion, entry into the vasculature followed by the exit ...of carcinoma cells from the circulation and colonization at the distal sites. At the earliest stage of successful cancer cell dissemination, the primary cancer adapts the secondary site of tumor colonization involving the tumor–stroma crosstalk. The migration and plasticity of cancer cells as well as the surrounding environment such as stromal and endothelial cells are mandatory. Consequently, the mechanisms of cell movement are of utmost relevance for targeted intervention of which three different types have been reported. Tumor cells can migrate either collectively, in a mesenchymal or in an amoeboid type of movement and intravasate the blood or lymph vasculature. Intravasation by the interaction of tumor cells with the vascular endothelium is mechanistically poorly understood. Changes in the epithelial plasticity enable carcinoma cells to switch between these types of motility. The types of migration may change depending on the intervention thereby increasing the velocity and aggressiveness of invading cancer cells. Interference with collective or mesenchymal cell invasion by targeting integrin expression or metalloproteinase activity, respectively, resulted in an amoeboid cell phenotype as the ultimate exit strategy of cancer cells. There are little mechanistic details reported in vivo showing that the amoeboid behavior can be either reversed or efficiently inhibited. Future concepts of metastasis intervention must simultaneously address the collective, mesenchymal and amoeboid mechanisms of cell invasion in order to advance in anti-metastatic strategies as these different types of movement can coexist and cooperate. Beyond the targeting of cell movements, the adhesion of cancer cells to the stroma in heterotypic circulating tumor cell emboli is of paramount relevance for anti-metastatic therapy.
Three-dimensional (3D) cancer models are used as preclinical systems to mimic physiologic drug responses. We provide evidence for strong changes of proliferation and metabolic capacity in three ...dimensions by systematically analyzing spheroids of colon cancer cell lines. Spheroids showed relative lower activities in the AKT, mammalian target of rapamycin (mTOR) and S6K (also known as RPS6KB1) signaling pathway compared to cells cultured in two dimensions. We identified spatial alterations in signaling, as the level of phosphorylated RPS6 decreased from the spheroid surface towards the center, which closely coordinated with the tumor areas around vessels in vivo These 3D models displayed augmented anti-tumor responses to AKT-mTOR-S6K or mitogen-activated protein kinase (MAPK) pathway inhibition compared to those in 2D models. Inhibition of AKT-mTOR-S6K resulted in elevated ERK phosphorylation in 2D culture, whereas under these conditions, ERK signaling was reduced in spheroids. Inhibition of MEK1 (also known as MAP2K1) led to decreased AKT-mTOR-S6K signaling in 3D but not in 2D culture. These data indicate a distinct rewiring of signaling in 3D culture and during treatment. Detached tumor-cell clusters in vessels, in addition to circulating single tumor cells, play a putative role in metastasis in human cancers. Hence, the understanding of signaling in spheroids and the responses in the 3D models upon drug treatment might be beneficial for anti-cancer therapies.
In the last decade, microRNAs (miRs or miRNAs) became of great interest in cancer research due to their multifunctional and active regulation in a variety of vital cellular processes. In this review, ...we discuss the miR-200 family, which is composed of five members (miR-141, miR-200a/200b/200c and miR-429). Although being among the best investigated miRNAs in the field, there are still many open issues. Here, we describe the potential role of miR-200 as prognostic and/or predictive biomarker, its influence on motility and cell migration as well as its role in epithelial to mesenchymal transition (EMT) and metastasis formation in different tumour types. Recent studies also demonstrated the influence of miR-200 on drug resistance and described a correlation between miR-200 expression levels and overall survival of patients. Despite intense research in this field, the full role of the miR-200 family in cancer progression and metastasis is not completely understood and seems to differ between different tumour types and different cellular backgrounds. To elucidate these differences further, a finer characterisation of the role of the individual miRNA-200 family members is currently under investigation.
A new flexible germacranolide (
, lobatolide H) was isolated from the aerial parts of
. The structure elucidation was performed by classical NMR experiments and DFT NMR calculations. Altogether, 80 ...theoretical level combinations with existing
C NMR scaling factors were tested, and the best performing ones were applied on
.
H and
C NMR scaling factors were also developed for two combinations utilizing known exomethylene containing derivatives, and the results were complemented by homonuclear coupling constant (
) and TDDFT-ECD calculations to elucidate the stereochemistry of
. Lobatolide H possessed remarkable antiproliferative activity against human cervical tumor cell lines with different HPV status (SiHa and C33A), induced cell cycle disturbance and exhibited a substantial antimigratory effect in SiHa cells.
In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form ...initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.
The health effects of plant phenolics in vegetables and other food and the increasing evidence of the preventive potential of flavonoids in "Western Diseases" such as cancer, neurodegenerative ...diseases and others, have gained enormous interest. This prompted us to investigate the effects of 20 different flavonoids of the groups of flavones, flavonols and flavanones in 3D in vitro systems to determine their ability to inhibit the formation of circular chemorepellent induced defects (CCIDs) in monolayers of lymph- or blood-endothelial cells (LECs, BECs; respectively) by 12(S)-HETE, which is secreted by SW620 colon cancer spheroids. Several compounds reduced the spheroid-induced defects of the endothelial barriers. In the SW620/LEC model, apigenin and luteolin were most active and acacetin, nepetin, wogonin, pinocembrin, chrysin and hispidulin showed weak effects. In the SW620/BEC model acacetin, apigenin, luteolin, wogonin, hispidulin and chrysin exhibited weak activity.
Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Unlike many other cancers, PM is mostly characterized by inactivation of tumor suppressor genes. Its highly malignant nature ...in absence of tumor driving oncogene mutations indicates an extrinsic supply of stimulating signals by cells of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are an abundant cell type of the TME and have been shown to drive the progression of several malignancies. The aim of the current study was to isolate and characterize patient-derived mesothelioma-associated fibroblasts (Meso-CAFs), and evaluate their impact on PM cells.
Meso-CAFs were isolated from surgical specimens of PM patients and analyzed by array comparative genomic hybridization, next generation sequencing, transcriptomics and proteomics. Human PM cell lines were retrovirally transduced with GFP. The impact of Meso-CAFs on tumor cell growth, migration, as well as the response to small molecule inhibitors, cisplatin and pemetrexed treatment was investigated in 2D and 3D co-culture models by videomicroscopy and automated image analysis.
Meso-CAFs show a normal diploid genotype without gene copy number aberrations typical for PM cells. They express CAF markers and lack PM marker expression. Their proteome and secretome profiles clearly differ from normal lung fibroblasts with particularly strong differences in actively secreted proteins. The presence of Meso-CAFs in co-culture resulted in significantly increased proliferation and migration of PM cells. A similar effect on PM cell growth and migration was induced by Meso-CAF-conditioned medium. Inhibition of c-Met with crizotinib, PI3K with LY-2940002 or WNT signaling with WNT-C59 significantly impaired the Meso-CAF-mediated growth stimulation of PM cells in co-culture at concentrations not affecting the PM cells alone. Meso-CAFs did not provide protection of PM cells against cisplatin but showed significant protection against the EGFR inhibitor erlotinib.
Our study provides the first characterization of human patient-derived Meso-CAFs and demonstrates a strong impact of Meso-CAFs on PM cell growth and migration, two key characteristics of PM aggressiveness, indicating a major role of Meso-CAFs in driving PM progression. Moreover, we identify signaling pathways required for Meso-CAF-mediated growth stimulation. These data could be relevant for novel therapeutic strategies against PM.
Flavonoids, present in fruits, vegetables and traditional medicinal plants, show anticancer effects in experimental systems and are reportedly non-toxic. This is a favorable property for long term ...strategies for the attenuation of lymph node metastasis, which may effectively improve the prognostic states in breast cancer. Hence, we studied two flavonoids, apigenin and luteolin exhibiting strong bio-activity in various test systems in cancer research and are readily available on the market. This study has further advanced the mechanistic understanding of breast cancer intravasation through the lymphatic barrier. Apigenin and luteolin were tested in a three-dimensional (3-D) assay consisting of MDA-MB231 breast cancer spheroids and immortalized lymph endothelial cell (LEC) monolayers. The 3-D model faithfully resembles the intravasation of breast cancer emboli through the lymphatic vasculature. Western blot analysis, intracellular Ca
determination, EROD assay and siRNA transfection revealed insights into mechanisms of intravasation as well as the anti-intravasative outcome of flavonoid action. Both flavonoids suppressed pro-intravasative trigger factors in MDA-MB231 breast cancer cells, specifically MMP1 expression and CYP1A1 activity. A pro-intravasative contribution of FAK expression in LECs was established as FAK supported the retraction of the LEC monolayer upon contact with cancer cells thereby enabling them to cross the endothelial barrier. As mechanistic basis, MMP1 caused the phosphorylation (activation) of FAK at Tyr397 in LECs. Apigenin and luteolin prevented MMP1-induced FAK activation, but not constitutive FAK phosphorylation. Luteolin, unlike apigenin, inhibited MMP1-induced Ca
release. Free intracellular Ca
is a central signal amplifier triggering LEC retraction through activation of the mobility protein MLC2, thereby enhancing intravasation. FAK activity and Ca
levels did not correlate. This implicates that the pro-intravasative contribution of FAK and of Ca
release in LECs was independent of each other and explains the better anti-intravasative effects of luteolin
. In specific formulations, flavonoid concentrations causing significant anti-intravasative effects, can certainly be achieved
. As the therapeutic strategy has to be based on permanent flavonoid treatment both the beneficial and adverse effects have to be investigated in future studies.
Cancer in general, and specifically gynaecological neoplasms, represents a major public health issue worldwide. Based on the effect of sex hormones on breast tumorigenesis and prognosis, as well as ...on the development of breast cancer metastases, modification of the steroid skeleton is a hotspot of research for novel anticancer agents. Numerous recent studies support that minor modifications of the androstane skeleton yield potent antiproliferative and antimetastatic drug candidates. The aim of the present study was to assess the antitumor and antimetastatic properties, as well as the mechanism of action of a D-ring-modified exo-heterocyclic androstadiene derivative named 17APAD. The test compound was found to be highly selective towards human breast cancer-derived cell lines (MCF-7, T47D, MDA-MB-361, MDA-MB-231) compared to non-cancerous fibroblast cells (NIH/3T3), and exerted superior effect compared to the clinically applied reference drug cisplatin. Changes in MCF-7- and MDA-MB-231 cell morphology and membrane integrity induced by the test substance were assessed by fluorescent double staining. Cell cycle disturbances were analyzed by flow cytometry, and concentration-dependent alterations were detected on breast cancer cell lines. Mitochondrial apoptosis induced by the test compound was demonstrated by JC-1 staining. Inhibitory effects on metastasis formation, including the inhibition of migration, invasion and intravasation were investigated in 2D and 3D models. Significant anti-migratory and anti-invasive effects on MCF-7 and MDA-MB-231 cells were detected after 24 h exposure in 2D wound healing and Boyden-chamber assays. The anti-intravasative properties of 17APAD were evident after 4 h of incubation in a co-culture 3D circular chemorepellent-induced defects (CCID) assay, and the level of inhibition at concentrations ≥2 µM was comparable to that exerted by the focal adhesion kinase inhibitor defactinib. Single cell mass cytometry revealed that chemosensitive subpopulations of MDA-MB-231 cells engaged to apoptosis were less positive for EGFR, CD274, and CD326, while the percentage of cells positive for GLUT1, MCT4, Pan-Keratin, CD66(a,c,e), Galectin-3 and TMEM45A increased in response to 17APAD treatment. Finally, the novel androstane analogue 17APAD had an outstanding inhibitory effect on tumour growth in the 4T1 orthotopic murine breast cancer model in vivo after 2 weeks of intraperitoneal administration. These findings support that substitution of the androsta-5,16-diene framework with a N-containing heterocyclic moiety at C17 position yields a molecular entity rational to be considered for design and synthesis of novel, effective antitumor agents, and 17APAD is worth further investigation as a promising anticancer drug candidate.
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•Androstane analogue (17APAD) exerted antiproliferative effects on breast cancer.•17APAD induced mitochondrial apoptosis and cell cycle disturbances.•17APAD inhibited several steps of metastasis development in vitro.•17APAD changed expression levels of tumor markers.•17APAD decelerated tumour growth rate in 4T1 mouse breast cancer model in vivo.
Since cancer cells, when grown as spheroids, display drug sensitivity and radiation resistance patterns such as seen in vivo we recently established a three‑dimensional (3D) in vitro model ...recapitulating colorectal cancer (CRC)-triggered lymphatic endothelial cell (LEC)‑barrier breaching to study mechanisms of intra‑/extravasation. CRC metastasizes not only through lymphatics but also through blood vessels and here we extend the 3D model to the interaction of blood endothelial cells (BECs) with naïve and 5‑fluorouracil (5‑FU)‑resistant CRC CCL227 cells. The 3D model enabled quantifying effects of tumour‑derived microRNA200 (miR200) miR200a, miR200b, miR200c, miR141 and miR429 regarding the induction of so-called 'circular chemorepellent‑induced defects' (CCIDs) within the BEC‑barrier, which resemble gates for tumour transmigration. For this, miR200 precursors were individually transfected and furthermore, the modulation of ZEB family expression was analysed by western blotting. miR200c, miR141 and miR429, which are contained in exosomes from naïve CCL227 cells, downregulated the expression of ZEB2, SNAI and TWIST in BECs. The exosomes of 5‑FU‑resistant CCL227‑RH cells, which are devoid of miR200, accelerated CCID formation in BEC monolayers as compared to exosomes from naïve CCL227 cells. This confirmed the reported role of ZEB2 and SNAI in CRC metastasis and highlighted the active contribution of the stroma in the metastatic process. CCL227 spheroids affected the integrity of BEC and LEC barriers alike, which was in agreement with the observation that CRC metastasizes via blood stream (into the liver) as well as via lymphatics (into lymph nodes and lungs). This further validated the CRC/LEC and CRC/BEC in vitro model to study mechanisms of CRC spreading through vascular systems. Treatment of CCL227‑RH cells with the HDAC inhibitors mocetinostat and sulforaphane reduced CCID formation to the level triggered by naïve CCL227 spheroids, however, without significantly influencing miR200 expression in CCL227-RH cells.
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