Tumor Cell Detachment Research Articles

Deregulation of Metalloproteinase Expression in Gray Horse Melanoma Ex Vivo and In Vitro.

The ability of human melanoma cells to switch from an epithelial to a mesenchymal phenotype contributes to the metastatic potential of disease. Metalloproteinases (MPs) are crucially involved in this process by promoting the detachment of tumor cells from the primary lesion and their migration to the vasculature. In gray horse melanoma, epithelial-mesenchymal transition (EMT) is poorly understood, prompting us to address MP expression in lesions versus intact skin by transcriptome analyses and the immunofluorescence staining (IF) of gray horse tumor tissue and primary melanoma cells. RNAseq revealed the deregulation of several MPs in gray horse melanoma and, notably, a 125-fold upregulation of matrix metalloproteinase 1 (MMP1) that was further confirmed by RT-qPCR from additional tumor material. The IF staining of melanoma tissue versus intact skin for MMP1 and tumor marker S100 revealed MMP1 expression in all lesions. The co-expression of S100 was observed at different extents, with some tumors scoring S100-negative. The IF staining of primary tumor cells explanted from the tumors for MMP1 showed that the metalloproteinase is uniformly expressed in the cytoplasm of 100% of tumor cells. Overall, the presented data point to MP expression being deregulated in gray horse melanoma, and suggest that MMP1 has an active role in gray horse melanoma by driving EMT-mediated tumor cell dissemination via the degradation of the extracellular matrix. Whilst S100 is considered a reliable tumor marker in human MM, gray horse melanomas do not seem to regularly express this protein.

Abstract C112: L1CAM targeting antibody-based therapy as a novel approach for L1CAM positive lymphomas

Abstract Background. L1CAM (L1 Cell Adhesion Molecule) is a glycoprotein identified as a key mediator of the metastatic process in many different tumor types. It facilitates the detachment of tumor cells from the primary tumor and their entry into the circulation. By promoting cell-cell interactions, it also directly stimulates the colonization of distant organs by circulating tumor cells. The expression and role of L1CAM in B and T cell lymphomas is poorly explored. The aim of this project was to evaluate L1CAM expression in different subtypes of lymphoma and its role as novel therapeutic target. Methods. RNA was extracted with Monarch Total RNA Miniprep Kit and qPCR performed with QuantiFast SYBR Green RT-PCR. For flow cytometry (FACS), cells were stained either with anti-L1CAM antibody or isotype control at a dilution of 1:1000. For immunohistochemistry (IHC) samples were stained for 20 minutes at a dilution of 1:100. For direct killing assay, cells were exposed six days to compounds, followed by MTT assay. Antibody dependent cellular cytotoxicity (ADCC) was performed with ADCC Reporter Bioassay (Promega). Results. L1CAM mRNA expression was assessed in 59 lymphoma cell lines. Whereas expression in most cell lines appeared low, a subgroup of cells displayed high to very high expression of L1CAM. Surface L1CAM expression was confirmed by FACS. L1CAM mRNA and protein expression levels correlated well across the cell lines and revealed that Sezary syndrome (SS) cell lines displayed the highest expression, followed by mantle cell lymphoma (MCL) and activated B cell diffuse large B cell lymphoma (ABC DLBCL) cell lines. By IHC L1CAM expression was assessed on different tissue microarrays covering multiple lymphoma subtypes; L1CAM expression was observed especially in a subset of cutaneous T-cell lymphoma and MCL. The data confirmed the findings in lymphoma cell lines. Anti-L1CAM monoclonal antibody (mAb) and four L1CAM targeting antibody drug conjugates (ADCs) (ADC1, ADC4, ADC5, ADC6), differing for the linker and payload, were tested in L1CAM positive and negative cell lines. mAB did not show any sign of direct antitumoral activity. Among ADCs, highest activity was observed with ADC6 (SG3199 payload), followed by ADC4 (MMAE payload). ADC1 and ADC5 (also containing MMAE as payload) showed limited activity. ADC6 showed the best correlation between killing activity and L1CAM expression levels, indicating a high level of target specific activity. The in vitro activity of ADC4 and ADC6 was validated in an in vivo xenograft using the MCL cell line Z138. Finally, mAb and a bispecific anti-CD3/L1CAM antibody were tested for their ability to induce ADCC. Both induced strong intracellular signaling in the reporter cell line upon co-incubation with L1CAM expressing lymphoma cells, indicative of the induction of killing activity in effector cells. Conclusions. In summary, we have demonstrated the in vitro and in vivo activity of anti-L1CAM antibody-based therapeutic approach as novel treatment for L1CAM positive lymphomas. Citation Format: Filippo Spriano, Giulio Sartori, Luca Aresu, Luciano Cascione, Elisa Civanelli, Afua A. Mensah, Chiara Tarantelli, Stefano Pileri, Flavio Mehli, Gunther Spohn, Francesco Bertoni. L1CAM targeting antibody-based therapy as a novel approach for L1CAM positive lymphomas [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C112.

Na+ /H+ exchanger NHE1 is active at cell-cell contacts and facilitates cell dissemination during collective migration of melanoma cells.

Tumour cell detachment from the primary tumour is an early and crucial step of the metastatic cascade. At the single cell level, it was already shown that migrating melanoma cells establish both intra- and extracellular pH gradients and that the Na+ /H+ exchanger NHE1 accumulates at the leading edges to strengthen cell-matrix interactions. However, less is known about the role of NHE1 in collective cell migration and the specific pH microenvironment at tumour cell-cell contacts. We used MV3 melanoma cells transfected with a NHE1-expressing vector or a control vector. NHE1 localization at cell-cell contacts was assessed via immunofluorescence imaging. Collective migration was analysed by live-cell imaging. The NHE1 activity and the perimembranous pH were measured both intra- and extracellularly by ratiometric fluorescence microscopy. NHE1 clearly localizes at cell-cell contacts. Its overexpression further increases migratory speed and translocation in multidirectional pathway analyses. NHE1 overexpressing MV3 cells also move further away from their neighbouring cells during wound closure assays. pH measurements revealed that the NHE1 is highly active at cell-cell contacts of melanoma cells. NHE1-mediated pH dynamics at such contact sites are more prominent in NHE1-overexpressing melanoma cells. Our findings highlight the contribution of the NHE1 towards modulation and plasticity of melanoma cell-cell contacts. We propose that its localization and functional activity at cell-cell contacts promotes evasion of single melanoma cells from the primary tumour.

In situ hydrogel enhances non-efferocytic phagocytosis for post-surgical tumor treatment

Post-surgical efferocytosis of tumor associated macrophages (TAMs) originates an immunosuppressive tumor microenvironment and facilitates abscopal metastasis of residual tumor cells. Currently, few strategies could inhibit efferocytosis while recovering the tumor-eliminative phagocytosis of TAMs. Herein, we developed an in situ hydrogel that contains anti-CD47 antibody (aCD47) and apocynin (APO), an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase. This hydrogel amplifies the non-efferocytic phagocytosis of TAMs by (1) blocking the extracellular “Don’t eat me” signal of efferocytosis with aCD47, which enhances the receptor-mediated recognition and engulfment of tumor cells by TAMs in the post-surgical tumor bed, and (2) by utilizing APO to dispose of tumor debris in a non-efferocytic manner, which prevents acidification and maturation of efferosomes and allows for M1-polarization of TAMs, leading to improved antigen presentation ability. With the complementary intervention of extracellular and intracellular, this hydrogel reverses the immunosuppressive effects of efferocytosis, and induces a potent M1-associated Th1 immune response against tumor recurrence. In addition, the in situ detachment and distal colonization of metastatic tumor cells were efficiently restrained due to the intervention of efferocytosis. Collectively, the hydrogel potentiates surgery treatment of tumor by recovering the tumor-elimination ability of post-surgical TAMs.

Functional Profiling of Soft Tissue Sarcoma Using Mechanistic Models.

Soft tissue sarcoma is an umbrella term for a group of rare cancers that are difficult to treat. In addition to surgery, neoadjuvant chemotherapy has shown the potential to downstage tumors and prevent micrometastases. However, finding effective therapeutic targets remains a research challenge. Here, a previously developed computational approach called mechanistic models of signaling pathways has been employed to unravel the impact of observed changes at the gene expression level on the ultimate functional behavior of cells. In the context of such a mechanistic model, RNA-Seq counts sourced from the Recount3 resource, from The Cancer Genome Atlas (TCGA) Sarcoma project, and non-diseased sarcomagenic tissues from the Genotype-Tissue Expression (GTEx) project were utilized to investigate signal transduction activity through signaling pathways. This approach provides a precise view of the relationship between sarcoma patient survival and the signaling landscape in tumors and their environment. Despite the distinct regulatory alterations observed in each sarcoma subtype, this study identified 13 signaling circuits, or elementary sub-pathways triggering specific cell functions, present across all subtypes, belonging to eight signaling pathways, which served as predictors for patient survival. Additionally, nine signaling circuits from five signaling pathways that highlighted the modifications tumor samples underwent in comparison to normal tissues were found. These results describe the protective role of the immune system, suggesting an anti-tumorigenic effect in the tumor microenvironment, in the process of tumor cell detachment and migration, or the dysregulation of ion homeostasis. Also, the analysis of signaling circuit intermediary proteins suggests multiple strategies for therapy.

Fibronectin fragments generated by pancreatic trypsin act as endogenous inhibitors of pancreatic tumor growth

BackgroundThe pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches.Methods and resultsHere we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5β1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models.ConclusionsThis study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer.

457 Plasminogen activating inhibitor-1 could be a key factor for the induction of angiogenesis in cutaneous angiosarcomas

Plasminogen activating inhibitor-1 (PAI-1) is classically known as an angiogenesis promoting factor that is associated with poor clinical outcomes in various cancers, including skin cancer such as cutaneous melanomas. Cutaneous angiosarcoma (CAS) is a vascular tumor histologically characterized by detachment of endothelial cell-derived tumor cells. Since CAS expresses multiple angiogenic growth factors and has increased expressions of angiogenic receptor tyrosine kinase transcripts including VEGFR1/2/3, angiogenesis-promoting factors are potential drug targets in CAS.

Plasminogen activating inhibitor-1 promotes angiogenesis in cutaneous angiosarcomas.

Plasminogen activating inhibitor-1 (PAI-1) is associated with poor clinical outcomes, and elevated levels of PAI-1 in both tissue and serum are correlated with poor response to therapy in various cancers, including skin cancer. Cutaneous angiosarcoma (CAS) is a vascular tumor histologically characterized by detachment of endothelial cell-derived tumor cells. Since CAS expresses multiple angiogenic growth factors and has increased expressions of angiogenic receptor tyrosine kinase transcripts including VEGFR1/2/3, angiogenesis-promoting factors are potential drug targets in CAS. In this study, the expression of PAI-1 was examined in 31 cases of CAS, and the immunomodulatory effects of PAI-1 on a human CAS cell line, ISO-HAS-B, were evaluated. We found that, of the angiogenesis-promoting factors, PAI-1 was expressed in almost all cases of CAS, and PAI-1 increased the mRNA expressions of IL-23p19, VEGF-C, CXCL5 and CCL20 on ISO-HAS-B. Moreover, PAI-1 stimulated ISO-HAS-B culture supernatant promoted favourable tube networks, suggesting that these tumor-derived factors promote the pro-angiogenic effect on tumor development. In addition, IL-23p19 was expressed in 61.3% of cases, whereas VEGF-C was expressed in 41% of cases. The results of the present study suggest that PAI-1 promotes angiogenesis that results in tumor progression in CAS.

Tumor spheroid invasion in epidermal growth factor gradients revealed by a 3D microfluidic device

Epidermal growth factor (EGF), a potent cytokine, is known to promote tumor invasion both in vivo and in vitro. Previously, we observed that single breast tumor cells (MDA-MB-231 cell line) embedded within a 3D collagen matrix displayed enhanced motility but no discernible chemotaxis in the presence of linear EGF gradients using a microfluidic platform. Inspired by a recent theoretical development that clustered mammalian cells respond differently to chemical gradients than single cells, we studied tumor spheroid invasion within a 3D extracellular matrix (ECM) in the presence of EGF gradients. We found that EGF gradients promoted tumor cell detachment from the spheroid core, and the position of the tumor spheroid core showed a mild chemotactic response towards the EGF gradients. For those tumor cells detached from the spheroids, they showed an enhanced motility response in contrast to previous experimental results using single cells embedded within an ECM. No discernible chemotactic response towards the EGF gradients was found for the cells outside the spheroid core. This work demonstrates that a cluster of tumor cells responds differently than single tumor cells towards EGF gradients and highlights the importance of a tumor spheroid platform for tumor invasion studies.

Small Extracellular Vesicles and Metastasis-Blame the Messenger.

Simple SummaryDue to their systemic nature, metastatic lesions are a major problem for curative cancer treatment. According to a common model for metastasis, tumor cells disseminate by local invasion, survival in the blood stream and extravasation into suitable tissue environments. At secondary sites, metastatic cells adapt, proliferate and foster vascularization to satisfy nutrient and oxygen demand. In recent years, tumors were shown to extensively communicate with cells in the local microenvironment and future metastatic sites by secreting small extracellular vesicles (sEVs, exosomes). sEVs deliver bioactive cargos, e.g., proteins, and in particular, several nucleic acid classes to reprogram target cells, which in turn facilitate tumor growth, cell motility, angiogenesis, immune evasion and establishment of pre-metastatic niches. sEV-cargos also act as biomarkers for diagnosis and prognosis. This review discusses how tumor cells utilize sEVs with nucleic acid cargos to progress through metastasis, and how sEVs may be employed for prognosis and treatment.Cancer is a complex disease, driven by genetic defects and environmental cues. Systemic dissemination of cancer cells by metastasis is generally associated with poor prognosis and is responsible for more than 90% of cancer deaths. Metastasis is thought to follow a sequence of events, starting with loss of epithelial features, detachment of tumor cells, basement membrane breakdown, migration, intravasation and survival in the circulation. At suitable distant niches, tumor cells reattach, extravasate and establish themselves by proliferating and attracting vascularization to fuel metastatic growth. These processes are facilitated by extensive cross-communication of tumor cells with cells in the primary tumor microenvironment (TME) as well as at distant pre-metastatic niches. A vital part of this communication network are small extracellular vesicles (sEVs, exosomes) with a size of 30–150 nm. Tumor-derived sEVs educate recipient cells with bioactive cargos, such as proteins, and in particular, major nucleic acid classes, to drive tumor growth, cell motility, angiogenesis, immune evasion and formation of pre-metastatic niches. Circulating sEVs are also utilized as biomarker platforms for diagnosis and prognosis. This review discusses how tumor cells facilitate progression through the metastatic cascade by employing sEV-based communication and evaluates their role as biomarkers and vehicles for drug delivery.

Molecular profiling of epigenetic landscape of cancer cells during extracellular matrix detachment

During cancer, a major challenge faced by oncologists is the treatment of metastasis; a leading cause of cancer-related deaths around the world. Metastasis involves a highly ordered sequence of events starting with the detachment of tumor cells from the extracellular matrix (E.C.M.). In normal cells, detachment from E.C.M. triggers programmed cell death, termed anoikis. However, tumor cells dodge their way to anoikis and spread to distant sites for initiating the metastatic program. In this work, we explored the impact of E.C.M. detachment on the expression of some major oncogenic histone methyltransferases. Results showed both EZH2 expression and its enzymatic activity were significantly increased in E.C.M. detached cancer cells when compared to the attached cells. Inhibition of EZH2 results in a significant reduction in cell proliferation, spheroids size, and induction in apoptosis in E.C.M. detached cells. Furthermore, we observed a reduction in EZH2 expression levels in single cells when compared to clusters of E.C.M. detached cells. Finally, we combined the EZH2 inhibition with AMPK, known to be highly expressed in E.C.M. detached cancer cells and observed antagonistic effects between the two pathways. The observed results clearly showed that E.C.M. detached cancer cells require oncogenic EZH2 and can be targeted by EZH2 inhibitors.

Pre-metastatic Niche Formation byNeutrophils in Different Organs.

Metastasis is a multistep process requiring tumor cell detachment from the primary tumor and migration to secondary target organs through the lymphatic or blood circulatory systems. In certain cancers, specific organs are predisposed to metastases. Metastatic homing to distant organs is orchestrated by the formation of supportive metastatic microenvironment in such organs, called pre-metastatic niche. Formation of pre-metastatic niche depends on the primary tumor-mediated recruitment of bone marrow-derived myeloid cells, including neutrophils. The contribution of neutrophils to the formation of the pre-metastatic niche is recently getting growing attention. Of note, these cells can either stimulate or inhibit metastatic seeding, depending on the activation of these cells. Here, we concentrate on pro-metastatic functions of neutrophils and the mechanisms involved in this process. Pro-tumor neutrophils support the formation of pre-metastatic niche, attract tumor cells, and directly stimulate proliferation of these cells. Moreover, immunosuppressive neutrophils, also called granulocytic MDSC, promote metastatic progression by the inhibition of antitumor T-cells. Altogether, neutrophil pro-tumor properties significantly affect metastatic spread in the host. Here, we provide an up-to-date overview of roles neutrophils play in the regulation of metastatic processes in different organs.

Anchorage independence altered vasculogenic phenotype of melanoma cells through downregulation in aminopeptidase N /syndecan-1/integrin β4 axis.

The detachment of tumor cells from extracellular matrix and survival under anchorage-independence were recognized as the initial step of tumor metastasis. Previously we had demonstrated that anchorage-independence altered gene expressions and showed characteristics of cell invasiveness loss, enhanced chemosensitivity, and enhanced subcutaneous tumor formation. However, whether it affected histological phenotypes in tumor tissues remained unclear.Melanoma metastases were generated in nude mice using adherent or suspended melanoma cells. Examination of melanoma metastases revealed histological features of extensive vascular structures in adherent cell-derived tumors, while not seen in suspended cell-derived tumors. Quantitative proteomic analysis at adherent, suspended, and re-attached melanoma cells suggested that aminopeptidase N was potentially downregulated upon cell suspension or reattachment. Downregulation of aminopeptidase N by gene-specific shRNAs showed reduced cell invasiveness and enhanced subcutaneous tumor formation that was consistent with previous observations. Experiments by suppression or overexpression of aminopeptidase N expression demonstrated that aminopeptidase N regulated syndecan-1 and integrin β4 expression through PKCδ pathway. Histological analysis at melanoma metastases further suggested that CD31+/aminopeptidase N+/syndecan-1+/integrin β4+ phenotypes were associated with vascular structures.In summary, we suggested the expression axis of aminopeptidase N/syndecan-1/integrin β4 in melanoma cells was suppressed by detachment stress, which diminished vascular phenotypes of melanoma metastases.

Searching the Link for Better Therapeutic Combination: The Case of Tumor Cells Migration Pattern and Modality of Immunosuppression Induction at the Metastatic Site.

Cancer is a disease characterized by its high morbidity and mortality, mainly due to its metastatic ability. Metastasis is a multi-step process beginning with detachment of tumor cells from the primary tumor and leading ultimately to the establishment of a new tumoral site. This cascade includes intravascular migration of tumor cells either individually or collectively and the expansion of cancer cells at metastatic sites that is dependent on certain conditions such as an immunosuppressive environment. In this paper, blockers of tumor cell migration and suppressors of immunotolerance at metastatic sites are reviewed as an illustration of early and later phases intervention, respectively. A combination of these two therapeutics will be advocated based on the proposition of correlation between the pattern of tumor cell migration and the mechanism of immunotolerance induction. By extension, the ''delayed complementarity'' will be introduced as an approach to formulate new anticancer drug combinations.

The influence of the gastrointestinal microbiome on colorectal metastasis: a narrative review

The gastrointestinal microbiome has been suggested to contribute to the development of both primary and secondary colorectal cancer. Despite advances in understanding the prognostic and predictive value of clinico-pathological parameters, the underlying mechanisms that result in progression to metastatic disease have yet to be defined. The metastatic cascade involves a number of sequential steps, including detachment of tumour cells from the primary site, intravasation and dissemination within the circulatory and lymphatic systems, with extravasation and proliferation at a secondary site. An analysis of the literature relating to the gastrointestinal microbiome and its role in colorectal metastasis was conducted. This review aims to examine the current evidence supporting a role for the microbiome in colorectal metastasis and to describe the mechanisms by which it may contribute to metastatic progression. The invasive pathways utilized by bacteria and how they may be manipulated by tumour cells for migration and metastasis are presented and the potential of the intestinal microbiome as a therapeutic target in colorectal carcinogenesis and metastasis is detailed here.

Materials-Based Approach for Interrogating Human Prostate Cancer Cell Adhesion and Migratory Potential Using a Fluoroalkylsilica Culture Surface.

OPCT-1 is a heterogeneous prostate cancer cell line derived from primary (rather than metastatic) disease which contains epithelial, mesenchymal, and CD44high/CD24low cancer stem cell (CSC) subpopulations and from which we have previously generated and characterized stable mesenchymal (P4B6B) and epithelial (P5B3) cell subpopulations. In this contribution, we explore the effect of tissue culture surface chemistry (standard tissue culture plastic (TCP) and a fluoroalkylsilica (FS) culture surface with inherently low surface energy) on the phenotype and adherent capacity of mesenchymal and epithelial cell populations. We demonstrate that OPCT-1 cells adherent to FS surfaces comprise both epithelial- and mesenchymal-like populations; a mesenchymal subpopulation derived from OPCT1 (P4B6B) poorly adheres to FS and formed spheroids, whereas an epithelial subpopulation derived from OPCT1 (P5B3) forms an adherent monolayer. In contrast, P4B6B cells do adhere to FS when cocultured with P5B3 cells. Taken together, these findings demonstrate that EMT/cell differentiation status dictates cell adhesive capacity and provide a novel insight into the relationship between epithelial and mesenchymal cell populations in metastasis. Importantly, the differences in adherence capacity between P4B6B and P5B3 are not apparent using standard TCP-based culture, thereby highlighting the value of using alternative culture surfaces for studying cell surface interaction/adhesion phenomena and interrogating mechanisms involved in adhesion and detachment of metastatic tumor cells.

Myeloid Cells in Metastasis.

Metastatic disease is the leading cause of death in patients with solid cancers. The progression to metastasis is a multistep process that involves detachment of tumor cells from their constraining basement membrane at the primary site, migration and intravasation into the circulation, survival in the circulation, extravasation into the secondary organ, and survival and growth at the secondary site. During these steps, tumor and immune cells interact and influence each other both within the tumor microenvironment and systemically. In particular, myeloid cells such as monocytes, macrophages, neutrophils, and myeloid-derived suppressor cells (myeloid regulatory cells) have been shown to play important roles in the metastatic process. These interactions open new avenues for targeting cancer metastasis, especially given the increasing interest in development of cancer immunotherapies. In this review, we describe the currently reported pathways and mechanisms involved in myeloid cell enhancement of the metastatic cascade.

OSM-induced CD44 contributes to breast cancer metastatic potential through cell detachment but not epithelial-mesenchymal transition.

BackgroundHormone receptor status in human breast cancer cells is a strong indicator of the aggressiveness of a tumor. Triple negative breast cancers (TNBC) are aggressive, difficult to treat, and contribute to high incidences of metastasis by possessing characteristics such as increased tumor cell migration and a large presence of the transmembrane protein, cluster of differentiation 44 (CD44) on the cell membrane. Estrogen receptor-positive (ER+) cells are less aggressive and do not migrate until undergoing an epithelial-mesenchymal transition (EMT).MethodsThe relationship between EMT and CD44 during metastatic events is assessed by observing changes in EMT markers, tumor cell detachment, and migration following cytokine treatment on both parental and CD44 knockdown human breast tumor cells.ResultsER+ T47D and MCF-7 human breast cancer cells treated with OSM demonstrate increased CD44 expression and CD44 cleavage. Conversely, ER- MDA-MB-231 human breast cancer cells do not show a change in CD44 expression nor undergo EMT in the presence of OSM. In ER+ cells, knockdown expression of CD44 by shRNA did not prevent EMT but did change metastatic processes such as cellular detachment and migration. OSM-induced migration was decreased in both ER+ and ER- cells with shCD44 cells compared to control cells, while the promotion of tumor cell detachment by OSM was decreased in ER+ MCF7-shCD44 cells, as compared to control cells. Interestingly, OSM-induced detachment in ER- MDA-MB-231-shCD44 cells that normally don’t detach at significant rates.ConclusionOSM promotes both EMT and tumor cell detachment in ER+ breast cancer cells. Yet, CD44 knockdown did not affect OSM-induced EMT in these cells, while independently decreasing OSM-induced cell detachment. These results suggest that regulation of CD44 by OSM is important for at least part of the metastatic cascade in ER+ breast cancer.

Key roles of Rho GTPases, YAP, and Mutant P53 in anti‐neoplastic effects of statins

Emerging epidemiological and preclinical studies have focused on statins and mevalonate pathway to identify potential therapeutic target and clarify the underlying mechanism of the anti-neoplastic effects. Reductions of mevalonate or isoprenoids, caused by statins, would further decrease the isoprenylation of Rho GTPases which is the crucial step for Rho GTPases to anchor on inner cellular membrane. Following anchoring, activated Rho GTPases can mediate a series of cellular activities such as cytoskeleton reprogramming, front-rear polarity, and cell-ECM adhesion. These changes not only facilitate tumor cell detachment and migration but also bring great mechanical changes to directly activate YAP, the major nuclear mechanotransducer, to translocate into nucleus. Recently, statins have been identified as potent inhibitors of YAP. Once entering nucleus, YAP would combine TEADs to promote the transcription of about 100 genes, which are involved in cell proliferation, cell cycle regulation, stemness, invasion, and metastasis. Besides, statins are able to promote the degradation of misfolded mutant p53 (mutp53), which is an oncogene in a variety of human malignancies. Reduction in mevalonate-5-phosphate (MVP), also induced by statins, would impair the stability of DNAJA1-mutp53 complex; then, elevated C terminus of Hsc70-interacting protein (CHIP) mediates the nuclear export and degradation of misfolded mutp53 through ubiquitin-proteasome pathway. It is worth noted that YAP, mutp53, and mevalonate pathway form two positive feedback loops. It is reasonable to believe that Rho GTPases, YAP, and mutp53 are determinants for statins as anti-cancer agents: tumor cells harboring mutp53 and nuclear-located YAP would be more sensitive to statins.

Role of ADAM17 as a regulatory checkpoint of CD16A in NK cells and as a potential target for cancer immunotherapy.

Human NK cell antitumor activities involve Ab-dependent cell-mediated cytotoxicity (ADCC), which is a key mechanism of action for several clinically successful tumor-targeting therapeutic mAbs. Human NK cells exclusively recognize these Abs by the Fcγ receptor CD16A (FcγRIIIA), one of their most potent activating receptors. Unlike other activating receptors on NK cells, CD16A undergoes a rapid down-regulation in expression by a proteolytic process following NK cell activation with various stimuli. In this review, the role of a disintegrin and metalloproteinase-17 (ADAM17) in CD16A cleavage and as a regulatory checkpoint is discussed. Several studies have examined the effects of inhibiting ADAM17 or CD16A cleavage directly during NK cell engagement of Ab-coated tumor cells, which resulted in strengthened Ab tethering, decreased tumor cell detachment, and enhanced CD16A signaling and cytokine production. However, the effects of either manipulation on ADCC have varied between studies, which may be due to dissimilar assays and the contribution of different killing processes by NK cells. Of importance is that NK cells under various circumstances, including in the tumor microenvironment of patients, down-regulate CD16A and this appears to impair their function. Considerable progress has been made in the development of ADAM17 inhibitors, including human mAbs that have advantages of high specificity and increased half-life in vivo. These inhibitors may provide a therapeutic means of increasing ADCC potency and/or antitumor cytokine production by NK cells in an immunosuppressive tumor microenvironment, and if used in combination with tumor-targeting Abs or NK cell-based adoptive immunotherapies may improve their efficacy.