Adhesion Of Breast Cancer Cells Research Articles

Short-Term Exposure to Foodborne Xenoestrogens Affects Breast Cancer Cell Morphology and Motility Relevant for Metastatic Behavior In Vitro.

Breast cancer is highly susceptible to metastasis formation. During the time of disease progression, tumor pathophysiology can be impacted by endogenous factors, like hormonal status, as well as by environmental exposures, such as those related to diet and lifestyle. New lines of evidence point toward a potential role for foodborne endocrine disruptive chemicals in this respect; however, mechanistic understanding remains limited. At the molecular level, crucial steps toward metastasis formation include cell structural changes, alteration of adhesion, and reorganization of cytoskeletal proteins involved in motility. Hence, this study investigates the potential of dietary xenoestrogens to impact selected aspects of breast cancer cell mechanotransduction. Taking the onset of the metastatic cascade as a model, experiments focused on cell-matrix adhesion, single-cell migration, and adaptation of cell morphology. Dietary mycoestrogens alternariol (AOH, 1 μM) and α-zearalenol (α-ZEL, 10 nM), soy isoflavone genistein (GEN, 1 μM), and food packaging plasticizer bisphenol A (BPA, 10 nM) were applied as single compounds or in mixtures. Pursuing the hypothesis that endocrine active molecules could affect cell functions beyond the estrogen receptor-dependent cascade, experiments were performed comparing the MCF-7 cell line to the triple negative breast cancer cells MDA MB-231. Indeed, the four compounds functionally affected the motility and the adhesion of both cell types. These responses were coherent with rearrangements of the actin cytoskeleton and with the modulation of the expression of integrin β1 and cathepsin D. Mechanistically, molecular dynamics simulations confirmed a potential interaction with fragments of the α1 and β1 integrin subunits. In sum, dietary xenoestrogens proved effective in modifying the motility and adhesion of breast cancer cells, as predictive end points for metastatic behavior in vitro. These effects were measurable after short incubation times (1 or 8 h) and contribute to shed novel light on the activity of compounds with hormonal mimicry potential in breast cancer progression.

The anti-tumor effects of cosmosiin through regulating AhR/CYP1A1-PPARγ in breast cancer.

Breast cancer is one of the threatening malignant tumors with the highest mortality and incidence rate over the world. There are a lot of breast cancer patients dying every year due to the lack of effective and safe therapeutic drugs. Therefore, it is highly necessary to develop more effective drugs to overcome breast cancer. As a glycoside derivative of apigenin, cosmosiin is characterized by low toxicity, high water solubility, and wide distribution in nature. Additionally, cosmosiin has been shown to perform anti-tumor effects in cervical cancer, hepatocellular carcinoma and melanoma. However, its pharmacological effects on breast cancer and its mechanisms are still unknown. In our study, the anti-breast cancer effect and mechanism of cosmosiin were investigated by using breast cancer models invivo and invitro. The results showed that cosmosiin inhibited the proliferation, migration, and adhesion of breast cancer cells invitro and suppressed the growth of tumor invivo through binding with AhR and inhibiting it, thus regulating the downstream CYP1A1/AMPK/mTOR and PPARγ/Wnt/β-catenin signaling pathways. Collectively, our findings have made contribution to the development of novel drugs against breast cancer by targeting AhR and provided a new direction for the research in the field of anti-breast cancer therapy.

The novel ECM protein SNED1 mediates cell adhesion via α5β1 integrin.

The extracellular matrix (ECM) is a complex meshwork comprising over 100 proteins. It serves as an adhesive substrate for cells and, hence, plays critical roles in health and disease. We have recently identified a novel ECM protein, SNED1, and have found that it is required for neural crest cell migration and craniofacial morphogenesis during development and in breast cancer, where it is necessary for the metastatic dissemination of tumor cells. Interestingly, both processes involve the dynamic remodeling of cell-ECM adhesions via cell surface receptors. Sequence analysis revealed that SNED1 contains two amino acid motifs, RGD and LDV, known to bind integrins, the largest class of ECM receptors. We thus sought to investigate the role of SNED1 in cell adhesion. Here, we report that SNED1 mediates breast cancer and neural crest cell adhesion via its RGD motif. We further demonstrate that cell adhesion to SNED1 is mediated by α5β1integrin. These findings are a first step toward identifying the signaling pathways activated downstream of the SNED1-integrin interactions guiding craniofacial morphogenesis and breast cancer metastasis.

The F-actin bundler SWAP-70 promotes tumor metastasis.

Dynamic rearrangements of the F-actin cytoskeleton are a hallmark of tumor metastasis. Thus, proteins that govern F-actin rearrangements are of major interest for understanding metastasis and potential therapies. We hypothesized that the unique F-actin binding and bundling protein SWAP-70 contributes importantly to metastasis. Orthotopic, ectopic, and short-term tail vein injection mouse breast and lung cancer models revealed a strong positive dependence of lung and bone metastasis on SWAP-70. Breast cancer cell growth, migration, adhesion, and invasion assays revealed SWAP-70's key role in these metastasis-related cell features and the requirement for SWAP-70 to bind F-actin. Biophysical experiments showed that tumor cell stiffness and deformability are negatively modulated by SWAP-70. Together, we present a hitherto undescribed, unique F-actin modulator as an important contributor to tumor metastasis.

IQGAP1 and NWASP promote human cancer cell dissemination and metastasis by regulating β1-integrin via FAK and MRTF/SRF

Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic outgrowth. Breast and prostate cancers are common malignancies in women and men, respectively. Here, we observe that β1-integrin is required for human prostate and breast cancer cell adhesion to ECs under shear-stress conditions invitro and to lung blood vessel ECs invivo. We identify IQGAP1 and neural Wiskott-Aldrich syndrome protein (NWASP) as regulators of β1-integrin transcription and protein expression in prostate and breast cancer cells. IQGAP1 and NWASP depletion in cancer cells decreases adhesion to ECs invitro and retention in the lung vasculature and metastatic lung nodule formation invivo. Mechanistically, NWASP and IQGAP1 act downstream of Cdc42 to increase β1-integrin expression both via extracellular signal-regulated kinase (ERK)/focal adhesion kinase signaling at the protein level and by myocardin-related transcription factor/serum response factor (SRF) transcriptionally. Our results identify IQGAP1 and NWASP as potential therapeutic targets to reduce early metastatic dissemination.

Heparan Sulfate Modulation Affects Breast Cancer Cell Adhesion and Transmigration across In Vitro Blood-Brain Barrier.

The disruption of endothelial heparan sulfate (HS) is an early event in tumor cell metastasis across vascular barriers, and the reinforcement of endothelial HS reduces tumor cell adhesion to endothelium. Our recent study showed that while vascular endothelial growth factor (VEGF) greatly reduces HS at an in vitro blood-brain barrier (BBB) formed by human cerebral microvascular endothelial cells (hCMECs), it significantly enhances HS on a breast cancer cell, MDA-MB-231 (MB231). Here, we tested that this differential effect of VEGF on the HS favors MB231 adhesion and transmigration. We also tested if agents that enhance endothelial HS may affect the HS of MB231 and reduce its adhesion and transmigration. To test these hypotheses, we generated an in vitro BBB by culturing hCMECs on either a glass-bottom dish or a Transwell filter. We first quantified the HS of the BBB and MB231 after treatment with VEGF and endothelial HS-enhancing agents and then quantified the adhesion and transmigration of MB231 across the BBB after pretreatment with these agents. Our results demonstrated that the reduced/enhanced BBB HS and enhanced/reduced MB231 HS increase/decrease MB231 adhesion to and transmigration across the BBB. Our findings suggest a therapeutic intervention by targeting the HS-mediated breast cancer brain metastasis.

Cell Line-Dependent Adhesion and Inhibition of Proliferation on Carbon-Based Nanofilms.

Disorganisation of the extracellular matrix (ECM) is strongly connected to tumor progression. Even small-scale changes can significantly influence the adhesion and proliferation of cancer cells. Therefore, the use of biocompatible nanomaterials capable of supporting and partially replenishing degraded ECM might be essential to recover the niche after tumor resection. The objective of this study was to evaluate the influence of graphene, graphene oxide, fullerene, and diamond nanofilms on breast cancer and glioblastoma grade IV cell lines. Nanomaterials were characterized using SEM and TEM techniques; zeta potential analysis was also performed. Nanofilms of graphene, fullerene, and diamond nanoparticles were also characterized using AFM. The toxicity was tested on breast cancer MDA.MB.231 and glioblastoma grade IV U-87 MG cell lines, using LDH assay and by counting stained dead cells in bioprinted 3D models. The following parameters were analyzed: proliferation, adhesion to the nanofilm, and adhesion to particular ECM components covered with diamond nanoparticles. Our studies demonstrated that nanofilms of graphene and diamond nanoparticles are characterized by cell-specific toxicity. Those nanomaterials were non-toxic to MDA.MB.231 cells. After applying bioprinted 3D models, diamond nanoparticles were not toxic for both cell lines. Nanofilms made of diamond nanoparticles and graphene inhibit the proliferation of MDA.MB.231 cells after 48 and 72 hours. Increased adhesion on nanofilm made of diamond nanoparticles was only observed for MDA.MB.231 cells after 30 and 60 minutes from seeding the cells. However, analysis of adhesion to certain ECM components coated with diamond nanoparticles revealed enhanced adhesion to tenascin and vitronectin for both tested cell lines. Our studies show that nanofilm made of diamond nanoparticles is a non-toxic and pro-adhesive nanomaterial that might stabilize and partially replenish the niche after breast tumor resection as it enhances the adhesion of breast cancer cells and inhibits their proliferation.

(Invited) Development of Cell-Based Biosensor Platform for Monitoring the Effect of Functional Nanocarriers As Drug Delivery System on Breast Cancer Cells

With the rapid emerging research of nanotechnology and engineering in biomedical applications, scientists start to develop state-of-the-art diverse biosensing platforms. Here, in-vitro cell-based biosensor (CBB) was fabricated using nano/micro technologies to electrophysiologically observe the effects of drug treatment on human breast cancer cells. Indium tin oxide coated vertically aligned silicon nanowires (ITO-coated VA-SiNWs) were applied as a cell monitoring CBB platform to investigate the growth and adhesion of MCF-7 human breast cancer cells via impedimetric method. Monitoring was performed by measuring the non-Faradaic electrical impedance of the cell–APTMS-modified ITO-coated VA-SiNWs system using a precision LCR meter. Our APTMS-modified ITO-coated VA-SiNWs exhibited changes in impedance parameters during cell growth. It was observed that during MCF-7 cell growth, the impedance magnitude increased. By contrast, the cytolysis of the cells led to significant changes in impedance parameters. Impedimetric cell monitoring platform has the ability to observe MCF-7 cell growth and adhesion and investigate anomalous and non-electrogenic cell behavior in toxin and drug screenings. With the aid of CBB platform, the real-time electrical signals can be applied to monitor the therapeutic efficacy of the nanocarriers in drug delivery system and screening large libraries of potential drugs for cancer treatment. Moreover, our pilot study indicates that our CBB could be a feasible platform to reduce/replace the use of animals based on the “Three R principles” in animal-based research in the future. Figure 1

BCKDK regulates breast cancer cell adhesion and tumor metastasis by inhibiting TRIM21 ubiquitinate talin1

Breast cancer is the most common malignant cancer in women worldwide. Cancer metastasis is the major cause of cancer-related deaths. BCKDK is associated with various diseases, including proliferation, migration, and invasion in multiple types of human cancers. However, the relevance of BCKDK to the development and progression of breast cancers and its function is unclear. This study found that BCKDK was overexpressed in breast cancer, associated with poor prognosis, and implicated in tumor metastasis. The downregulation of BCKDK expression inhibited the migration of human breast cancer cells in vitro and diminished lung metastasis in vivo. BCKDK perturbed the cadherin-catenin complex at the adherens junctions (AJs) and assembled focal adhesions (FAs) onto the extracellular matrix, thereby promoting the directed migration of breast cancer cells. We observed that BCKDK acted as a conserved regulator of the ubiquitination of cytoskeletal protein talin1 and the activation of the FAK/MAPK pathway. Further studies revealed that BCKDK inhibited the binding of talin1 to E3 ubiquitin ligase-TRIM21, leading to the decreased ubiquitination/degradation of talin1. In conclusion, identifying BCKDK as a biomarker for breast cancer metastasis facilitated further research on diagnostic biomarkers. Elucidating the mechanism by which BCKDK exerted its biological effect could provide a new theoretical basis for developing new markers for breast cancer metastasis and contribute to developing new therapies for the clinical treatment of breast cancer patients.

Role of glycocalyx in breast cancer cell adhesion and transmigration across an in vitro blood-brain barrier (BBB)

On the surface of every mammalian cell, there is a matrix-like glycocalyx (GCX) consisting of proteoglycans and glycosaminoglycans (GAGs). Disruption of endothelial cell (EC) GCX by vascular endothelial growth factor (VEGF, VEGF-A165), a tumor secretion, was found to be an early event in tumor cell (TC) metastasis across vascular barriers. But how the TC secretion VEGF affects its own GCX to affect its adhesion and transmigration across EC barriers is unknown. Our recent study by employing Super-resolution Optical Microscopy (STORM) showed that while 1 nM VEGF greatly reduces the length and coverage of heparan sulfate (HS) of GCX at an in vitro BBB formed by human cerebral microvascular endothelial cells (hCMEC), it significantly enhances the coverage of HS on a malignant breast cancer cell, MDA-MB-231 (MB231). In the current study, we want to test the hypothesis that the differential effects of VEGF on the GCX of MB231 and that of the BBB favor MB231 adhesion and transmigration across the BBB for the metastasis. To test this hypothesis, we first generated an in vitro BBB by culturing hCMECs on a Transwell filter with 8 um pores. For adhesion, we added MB231 cells to the BBB under 1) no treatment; 2) after VEGF treatment for MB231 for 2h; 2) after VEGF treatment for the BBB for 2h; and 4) after VEGF treatment for both for 2h. After 1h adhesion, the number of adherent MB231 cells increased by 1.7-fold,1.5-fold and 2.4-fold, respectively for 2), 3) and 4) VEGF treatments, compared to no treatment. For transmigration, we add MB231 cells to the BBB under the same conditions as for adhesion. After 6 h, the number of transmigrated MB231 cells increased by 1.3-fold, 1.4-fold, and 1.6-fold, respectively, for 2), 3) and 4) VEGF treatments, compared to no treatment. Our results conform to our hypothesis that the reduced BBB GCX and enhanced MB231 GCX by VEGF favor MB231 adhesion to and transmigration across the BBB. Our findings also suggest a therapeutic intervention by targeting the glycocalyx-mediated breast cancer brain metastasis. Partially supported by NIH RO1NS101362 and 1UG3UH3TR002151. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Silencing RAB27a inhibits proliferation, invasion and adhesion of triple-negative breast cancer cells

To investigate the effect of inhibition of RAB27 protein family, which plays a pivotal role in exosome secretion, on biological behaviors of triple-negative breast cancer cells. Quantitative real-time PCR and Western blotting were used to examine the expressions of RAB27 family and exosome secretion in 3 triple-negative breast cancer cell lines (MDA-MB-231, MDA-MB-468, and Hs578T) and a normal breast epithelial cell line (MCF10A). The effect of small interfering RNA (siRNA)-mediated silencing of RAB27a and RAB27b on exosome secretion in the 3 breast cancer cell lines was detected using Western blotting, and the changes in cell proliferation, invasion and adhesion were evaluated. Compared with normal breast epithelial cells, the 3 triple-negative breast cancer cell lines exhibited more active exosome secretion (P < 0.001) and showed significantly higher expressions of RAB27a and RAB27b at both the mRNA and protein levels (P < 0.01). Silencing of RAB27a in the breast cancer cells significantly down-regulated exosome secretion (P < 0.001), while silencing of RAB27b did not significantly affect exosome secretion. The 3 breast cancer cell lines with RAB27a silencing-induced down-regulation of exosome secretion showed obvious inhibition of proliferation, invasion and adhesion (P < 0.01) as compared with the cell lines with RAB27b silencing. RAB27a plays central role in the exosome secretion in triple-negative breast cancer cells, and inhibiting RAB27a can inhibit the proliferation, invasion and adhesion of the cells.

L-carvone decreases breast cancer cells adhesion, migration, and invasion by suppressing FAK activation

Breast cancer is one of the most common types of cancer in the world and current therapeutic strategies present severe drawbacks. l-carvone (CRV), a monoterpene found in Mentha spicata (spearmint), has been reported to have potent anti-inflammatory activity. Here, we examined the role of CRV in breast cancer cell adhesion, migration and invasion in vitro and how this component could suppress the growth of Ehrlich carcinoma-bearing mice. In vivo, treatment with CRV significantly decreased tumor growth, increased tumor necrosis area, and reduced the expression of VEGF and HIF-1α in Ehrlich carcinoma-bearing mice. Furthermore, the anticancer efficacy of CRV was similar to currently used chemotherapy (Methotrexate), and the combination of CRV with MTX potentiated the chemotherapy effects. Further mechanistic investigation in vitro revealed that CRV modulates the interaction of breast cancer cells with the extracellular matrix (ECM) by disrupting focal adhesion, which was shown by scanning electron microscopy (SEM) and immunofluorescence. Moreover, CRV caused a decrease in β1-integrin expression and inhibited focal adhesion kinase (FAK) activation. FAK is one of the most important downstream activators of several metastatic processes, including MMP-2 mediated invasion and HIF-1α/VEGF angiogenesis stimulus, both of which were found to be reduced in MDA-MB-231 cells exposed to CRV. Our results provide new insight about targeting β1-integrin/FAK signaling pathway with CRV, which could be a new potential agent in the treatment of breast cancer.

Truncation of GalNAc-type O-glycans suppresses CD44-mediated osteoclastogenesis and bone metastasis in breast cancer.

The glycoprotein CD44 is a key regulator of malignant behaviors in breast cancer cells. To date, hyaluronic acid (HA)-CD44 signaling pathway has been widely documented in the context of metastatic bone diseases. Core 1 β1,3-galactosyltransferase (C1GALT1) is a critical enzyme responsible for the elongation of O-glycosylation. Aberrant O-glycans are recognized as a hallmark in cancers. However, the effects of C1GALT1 on CD44 signaling and bone metastasis remain unclear. In this study, immunohistochemical analysis indicated that C1GALT1 expression positively correlates with CD44 in breast cancer. Silencing C1GALT1 accumulates the Tn antigen on CD44, which decreases CD44 levels and osteoclastogenic signaling. Mutations in the O-glycosites on the stem region of CD44 impair its surface localization as well as suppress cell-HA adhesion and osteoclastogenic effects of breast cancer cells. Furthermore, in vivo experiments demonstrated the inhibitory effect of silencing C1GALT1 on breast cancer bone metastasis and bone loss. In conclusion, our study highlights the importance of O-glycans in promoting CD44-mediated tumorigenic signals and indicates a novel function of C1GALT1 in driving breast cancer bone metastasis. Implications: Truncation of GalNAc-type O-glycans by silencing C1GALT1 suppresses CD44-mediated osteoclastogenesis and bone metastasis in breast cancer; targeting the O-glycans on CD44 may serve as a potential therapeutic target for blocking cancer bone metastasis.

Breast tumors interfere with endothelial TRAIL at the premetastatic niche to promote cancer cell seeding.

Endothelial cells (ECs) grant access of disseminated cancer cells to distant organs. However, the molecular players regulating the activation of quiescent ECs at the premetastatic niche (PMN) remain elusive. Here, we find that ECs at the PMN coexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate death receptor 5 (DR5). Unexpectedly, endothelial TRAIL interacts intracellularly with DR5 to prevent its signaling and preserve a quiescent vascular phenotype. In absence of endothelial TRAIL, DR5 activation induces EC death and nuclear factor κB/p38-dependent EC stickiness, compromising vascular integrity and promoting myeloid cell infiltration, breast cancer cell adhesion, and metastasis. Consistently, both down-regulation of endothelial TRAIL at the PMN by proangiogenic tumor-secreted factors and the presence of the endogenous TRAIL inhibitors decoy receptor 1 (DcR1) and DcR2 favor metastasis. This study discloses an intracrine mechanism whereby TRAIL blocks DR5 signaling in quiescent endothelia, acting as gatekeeper of the vascular barrier that is corrupted by the tumor during cancer cell dissemination.

Natural product Eriocalyxin B suppressed triple negative breast cancer metastasis both in vitro and in vivo

Breast cancer is the most commonly diagnosed cancer among women, and its metastasis to distant organs accounts for the majority of death. Eriocalyxin B (Eri B), an ent-kaurane diterpenoid isolating from Isodon eriocalyx var. laxiflora, has previously been reported to have anti-tumor and anti-angiogenic effects in breast cancer. Here, we investigated the effect of Eri B on cell migration and adhesion in triple negative breast cancer (TNBC) cells, as well as aldehyde dehydrogenases 1 family member A1 (ALDH1A1) expression, colony- and sphere-formation in cancer stem cell (CSC) enriched MDA-MB-231 cells. The in vivo anti-metastatic activities of Eri B were determined in 3 different breast tumor-bearing mouse models. Our results indicated that Eri B inhibited TNBC cell migration and adhesion to extracellular matrix proteins, and also reduced ALDH1A1 expression and colony formation in CSC-enriched MDA-MB-231 cells. The metastasis-related pathways, such as epidermal growth factor receptor/ mitogen-activated protein kinase kinases 1/2/ extracellular regulated protein kinase signaling altered by Eri B was firstly shown in MDA-MB-231 cells. The potent anti-metastatic efficacies of Eri B were demonstrated in breast xenograft-bearing mice and syngeneic breast tumor-bearing mice. Gut microbiome analysis results revealed the change in the diversity and composition of microbiome after Eri B treatment, and the potential pathways that are involved in the anti-cancer efficacy of Eri B. In conclusion, Eri B was shown to inhibit breast cancer metastasis in both in vitro and in vivo models. Our findings further support the development of Eri B as an anti­metastatic agent for breast cancer.

Graphene-based 3D-Printed nanocomposite bioelectronics for monitoring breast cancer cell adhesion

This work examines the suitability of graphene-based 3D-printed nanocomposite bioelectronics as innovative systems to in situ monitor and evaluate both breast cancer cell adhesion and the chemosensitivity of anti-cancer drugs. With this aim, 3D-printed nanocomposite graphene electrodes (3D-nGEs) —made of a commercially available graphene/polylactic acid filament— have been covalently biofunctionalized with an extracellular matrix protein (i.e., fibronectin) by exploiting the carbon reactivity of 3D-nGEs. The specificity and selectivity of the developed electrochemical system to monitor breast cancer cell adhesion has been tested via electrochemical impedance spectroscopy (EIS). Importantly, the resulting 3D-printed bioelectronic system displayed excellent accuracy for the rapid screening of anti-cancer drugs, which exactly corresponded with the results achieved by the standard optical method, while having the advantage of employing a label-free approach. In light of the current state-of-the-art in the field, this proof-of-concept connects electronics to biological systems within 3D printing technology, providing the bases for the sustainable and cost-effective manufacturing of graphene-based 3D-printed nanocomposite bioelectronics to simulate in vivo microenvironments using in situ and real time electronic output signals.

Homotypic and heterotypic adhesion of cancer cells revealed by force-induced remnant magnetization spectroscopy.

Intercellular interaction has tremendous impacts on physiological processes, while unsuccessful cell-cell interaction causes diseases, such as tumorigenesis and metastasis. In-depth investigation of cell-cell adhesions is of great significance to understand the pathological state of cells, and for the rational design of drugs and therapies. Herein, we developed a force-induced remnant magnetization spectroscopy (FIRMS) method to measure cell-cell adhesion in a high throughput way. Our results showed that FIRMS is capable of quantifying and identifying cell-cell adhesion with high detection efficiency. Specifically, we quantified homotypic and heterotypic adhesion forces during tumor metastasis using breast cancer cell lines. We observed that homotypic and heterotypic adhesion forces of cancer cells were associated with degrees of malignancy. In addition, we revealed that CD43-ICAM-1 was a ligand-receptor pair mediating heterotypic adhesion of breast cancer cells to endothelial cells. These findings contribute to advance in-depth understanding of the process of cancer metastasis and provide insight into targeting intercellular adhesion molecules as a potential strategy to inhibit cancer metastasis.

SPAD-1, a serine proteinase associated disintegrin from Russell's viper venom disrupts adhesion of MCF7 human breast cancer cells

Serine Proteinase Associated Disintegrin-1 (SPAD-1) is a low molecular mass (26 kDa) positively charged protein purified from Russell's viper venom (RVV) possessing cytotoxic activity on MCF7, human breast cancer cells. Primary sequence analysis of the protein confirms that it is a novel Snake Venom Serine Proteinase (SVSP) and a member of the trypsin family. SPAD-1 contains a conserved triad of Histidine (H), Aspartic acid(D) and Serine(S) residues at its active site for proteinase activity and also an adjacent histidine-glycine-aspartic acid (HGD) disintegrin-like motif. The serine proteinase and disintegrin parts are functionally active and independent. SPAD-1 showed proteolytic digestion of fibrinogen and fibronectin, but laminin digestion was below the detectable limit. Proteolytically inactivated SPAD-1 inhibited collagen and ADP-induced platelet aggregation. This study proposes considering Serine Proteinase Associated Disintegrin (SPAD) as a new group of snake venom proteins. Members of this group contain a serine proteinase catalytic triad and a disintegrin-like motif. SPAD-1 caused visible morphological changes in MCF7 cells, including a reduction of the cell-to-cell attachments, rounding of cell shape and death, in vitro. SPAD-1 also showed a dose-dependent significant decrease in the invasive potency of breast cancer cells. Confocal microscopic analysis revealed the breakage of nuclei of the SPAD-1-treated cells. SPAD-1 also increased cell detachment from the poly L-lysine-coated, laminin-coated and fibronectin-coated culture plate matrices, confirming the disintegrin activity. This study concludes that SPAD-1 may be a good candidate for anti-tumour drug design in the future.

JAG1 promotes migration, invasion, and adhesion of triple-negative breast cancer cells by promoting angiogenesis

To investigate the effect of JAG1 on the malignant phenotype of triple-negative breast cancer (TNBC) and its role in angiogenesis in breast cancer microenvironment. The expressions of Notch molecules were detected in human TNBC 231 and 231B cells using RT-qPCR. Five female nude mice were inoculated with 231 cells and another 5 with 231B cells into the mammary fat pads, and 4-6 weeks later, the tumors were collected for immunohistochemical and immunofluorescence tests. 231 cells and 231B cells were treated with recombinant JAG (rJAG) protein and DAPT, respectively, and changes in their malignant phenotypes were assessed using CCK-8 assay, Hoechst 33258 staining, wound healing assay, Transwell chamber assay and endothelial cell adhesion assay. Western blotting was used to detect the changes in the expressions of proteins related with the malignant phenotypes of 231 and 231B cells. The effects of conditioned medium (CM) derived from untreated 231 and 231 B cells, rJAG1-treated 231 cells and DAPT-treated 231B cells on proliferation and tube formation ability of cultured human umbilical vein endothelial cells (HUVECs) were evaluated using CCK-8 assay and tube-forming assay. The expression of JAG1 was higher in 231B cells than in 231 cells (P < 0.05). Tumor 231B showed higher expression of VEGFA and CD31. Compared with 231-Blank group, the migration, invasion and adhesion of 231 cells in 231-rJAG1 were significantly enhanced (P < 0.05). Protein levels of Twist1 and Snail increased (P < 0.01), anti-apoptotic protein Bcl-2 increased (P < 0.05), while DAPT inhibited the related phenomena and indicators of 231B. The 231-rJAG1-CM increased the cell number and tubule number of HUVEC (P < 0.05). JAG1 may affect the malignant phenotype of TNBC and promote angiogenesis in the tumor microenvironment.

P-235 Elevated protein tyrosine phosphatase kappa expression is associated with disease progression and poor prognosis of pancreatic cancer

Protein tyrosine phosphatase kappa (PTPRK) is a phosphatase which is recognised as a tumour suppressor. PTPRK can promote the internalization of ZNRF3 by dephosphorylating CD133. It is a negative regulator of the invasion and adhesion of breast cancer cells, what is more, it is also enhance the activation of STAT3 in lung cancer. Pancreatic exocrine cancer is an extremely aggressive tumour with a mortality being nearly equal to its incidence. The aim of present study is to assess the involvement of PTPRK in the disease progression of pancreatic cancer. PTPRK transcripts were determined in pancreatic cancers (n=201) and adjacent non-tumour pancreatic tissues (n=201) using real time PCR. The pancreatic tissue samples were collected immediately after surgery at Peking University Cancer Hospital. All procedures used are approved by the Peking University Cancer Hospital Research Ethics Committee. The implication of PTPRK in disease progression and prognosis was analysed which was also further supported by analyses of RNAseq data of pancreatic cancer (The Cancer Genome Atlas, TCGA_PAAD) and a gene array data (GSE15471 and GSE71729). Further bioinformatic analysis has been done to determine which pathway is regulated by PTPRK. Both the public dataset GSE15471 and the Beijing clinical cohort shows increased PTPRK expression in the tumours compared with adjacent normal tissue. PTPRK expression in tumours of the earliest stage (T1) is lower than more invasive tumours (T2, T3 and T4). However, PTPRK tends to be lower in primary tumours that presented lymph node metastases. There is no significant change of PTPRK expression in the primary tumours that developed distant metastases. Moreover, PTPRK is down regulated in distant metastatic tumours from pancreatic cancer (GSE71729). Compared with lesions in the tail of pancreas, the PTPRK has also been found has a higher expression level in pancreatic head and body. Pancreatic cancer patients with a higher PTPRK level had poor overall and relapse free survival compared with patients with tumours of low PTPRK expression. In the TCGA PAAD cohort, PTPRK is positively correlated with some RTKs family gene, including EGFR, KDR, FGF and PDGF. However, neither protein level of EGFR nor phosphorylation of EGFR (Y1068 and Y1173) presented obvious difference according to PTPRK transcripts in the corresponding tumours. The PTPRK is involved in pancreatic cancer progression, it is associated with a poor prognosis. Exact role played by PTPRK in pancreatic cancer and its predictive and therapeutic potential are yet to be fully investigated using both in vitro and in vivo models.