Regulate Tumor Cell Migration Research Articles

K-Ras(V12) differentially affects the three Akt isoforms in lung and pancreatic carcinoma cells and upregulates E-cadherin and NCAM via Akt3

K-Ras is the most frequently mutated Ras variant in pancreatic, colon and non-small cell lung adenocarcinoma. Activating mutations in K-Ras result in increased amounts of active Ras-GTP and subsequently a hyperactivation of effector proteins and downstream signaling pathways. Here, we demonstrate that oncogenic K-Ras(V12) regulates tumor cell migration by activating the phosphatidylinositol 3-kinases (PI3-K)/Akt pathway and induces the expression of E-cadherin and neural cell adhesion molecule (NCAM) by upregulation of Akt3. In vitro interaction and co-precipitation assays identified PI3-Kα as a bona fide effector of active K-Ras4B but not of H-Ras or N-Ras, resulting in enhanced Akt phosphorylation. Moreover, K-Ras(V12)-induced PI3-K/Akt activation enhanced migration in all analyzed cell lines. Interestingly, Western blot analyses with Akt isoform-specific antibodies as well as qPCR studies revealed, that the amount and the activity of Akt3 was markedly increased whereas the amount of Akt1 and Akt2 was downregulated in EGFP-K-Ras(V12)-expressing cell clones. To investigate the functional role of each Akt isoform and a possible crosstalk of the isoforms in more detail, each isoform was stably depleted in PANC-1 pancreatic and H23 lung carcinoma cells. Akt3, the least expressed Akt isoform in most cell lines, is especially upregulated and active in Akt2-depleted cells. Since expression of EGFP-K-Ras(V12) reduced E-cadherin-mediated cell-cell adhesion by induction of polysialylated NCAM, Akt3 was analyzed as regulator of E-cadherin and NCAM. Western blot analyses revealed pronounced reduction of E-cadherin and NCAM in the Akt3-kd cells, whereas Akt1 and Akt2 depletion upregulated E-cadherin, especially in H23 lung carcinoma cells. In summary, we identified oncogenic K-Ras4B as a key regulator of PI3-Kα-Akt signaling and Akt3 as a crucial regulator of K-Ras4B-induced modulation of E-cadherin and NCAM expression and localization.

Phosphoproteomic Changes Induced by Cell-Derived Matrix and Their Effect on Tumor Cell Migration and Cytoskeleton Remodeling.

Increased fibrotic extracellular matrix (ECM) deposition promotes tumor invasion, which is the first step of the metastatic cascade. Yet, the underlying mechanisms are poorly understood as conventional studies of tumor cell migration are often performed in 2D cultures lacking the compositional and structural complexity of native ECM. Moreover, these studies frequently focus on select candidate pathways potentially overlooking other relevant changes in cell signaling. Here, we combine a cell-derived matrix (CDM) model with phosphotyrosine phosphoproteomic analysis to investigate tumor cell migration on fibrotic ECM relative to standard tissue culture plastic (TCP). Our results suggest that tumor cells cultured on CDMs migrate faster and in a more directional manner than their counterparts on TCP. These changes in migration correlate with decreased cell spreading and increased cell elongation. While the formation of phosphorylated focal adhesion kinase (pFAK)+ adhesion complexes did not vary between TCP and CDMs, time-dependent phosphoproteomic analysis identified that the SRC family kinase LYN may be differentially regulated. Pharmacological inhibition of LYN decreased tumor cell migration and cytoskeletal rearrangement on CDMs and also on TCP, suggesting that LYN regulates tumor cell migration on CDMs in combination with other mechanisms. These data highlight how the combination of physicochemically complex in vitro systems with phosphoproteomics can help identify signaling mechanisms by which the fibrotic ECM regulates tumor cell migration.

Modeling the crosstalk between malignant B cells and their microenvironment in B-cell lymphomas: challenges and opportunities.

B-cell lymphomas are a group of heterogeneous neoplasms resulting from the clonal expansion of mature B cells arrested at various stages of differentiation. Specifically, two lymphoma subtypes arise from germinal centers (GCs), namely follicular lymphoma (FL) and GC B-cell diffuse large B-cell lymphoma (GCB-DLBCL). In addition to recent advances in describing the genetic landscape of FL and GCB-DLBCL, tumor microenvironment (TME) has progressively emerged as a central determinant of early lymphomagenesis, subclonal evolution, and late progression/transformation. The lymphoma-supportive niche integrates a dynamic and coordinated network of immune and stromal cells defining microarchitecture and mechanical constraints and regulating tumor cell migration, survival, proliferation, and immune escape. Several questions are still unsolved regarding the interplay between lymphoma B cells and their TME, including the mechanisms supporting these bidirectional interactions, the impact of the kinetic and spatial heterogeneity of the tumor niche on B-cell heterogeneity, and how individual genetic alterations can trigger both B-cell intrinsic and B-cell extrinsic signals driving the reprogramming of non-malignant cells. Finally, it is not clear whether these interactions might promote resistance to treatment or, conversely, offer valuable therapeutic opportunities. A major challenge in addressing these questions is the lack of relevant models integrating tumor cells with specific genetic hits, non-malignant cells with adequate functional properties and organization, extracellular matrix, and biomechanical forces. We propose here an overview of the 3D in vitro models, xenograft approaches, and genetically-engineered mouse models recently developed to study GC B-cell lymphomas with a specific focus on the pros and cons of each strategy in understanding B-cell lymphomagenesis and evaluating new therapeutic strategies.

Hsa-Circ-0002360 Promotes the Development of Non-Small Cell Lung Cancer Cells by Regulating Specific E3 Ubiquitin Protein Ligase 2 Pathway Through miRNA-511-5p

Lung cancer is one of the most common causes of cancer-related deaths for cancer patients, the specific role of cyclic RNA in non-small cell lung cancer still needs further exploration. The effect of Hsa-Circ-0002360 and the relative pathway participated in the development of non-small cell lung cancer cells, lentivirus expression vector shcirc0002360, miRNA-511-5p mimics and recombinant protein SMURF2 were used in this study. The function of cell proliferation, migration and apoptosis were compared by flow cytometry, wound healing, RT-qPCR and western blot. We found circRNA 0002360 knockout significantly inhibited A549 cell functions. After further overexpression of miR-511-5p, the migration function, proliferation function, and cell viability of cells are significantly decreased while apoptosis rate increased, and quite the opposite after adding SMURF2. Smurf2 promotes the development of non-small cell lung cancer cells by regulating tumor cell migration, dissemination, and apoptosis, and this process is also regulated by Hsa-cic-000236 and miR-511-5p.

Autocrine motility factor receptor promotes the malignancy of glioblastoma by regulating cell migration and invasion

ABSTRACTObjectiveOne of the important causes of death in cancer patients is malignant metastasis, invasion, and metastasis of tumor cells. Metastasis is also the most basic physiological characteristics and pathogenesis of various tumors. Previously published studies have suggested that autocrine motor factor receptor (AMFR) is the key regulator of tumor cell migration and invasion. Meanwhile, AMFR is highly expressed in esophageal tumors, gastrointestinal tumors, and bladder cancer, and it is also involved in its pathogenesis. However, the role of AMFR in glioblastoma has not been reported.MethodsIn order to study the role of AMFR in the cell migration and invasion of glioblastoma, AMFR was silenced using siRNA and overexpressed using cDNA. Immunoblotting analysis and real-time quantitative polymerase chain reaction (PCR) were employed to assess the expression of AMFR. We conducted wound healing assay, cell migration assay, and tumorsphere formation assay to detect the invasion and metastatic ability of glioblastoma.ResultsThis study found that the level of AMFR expression was significantly correlated with the malignant degree of glioma tissue in clinic samples. AMFR silencing decreased cell migration and invasion of LN229. Overexpression of AMFR significantly increased cell migration and invasion of U251.ConclusionThis study suggests that AMFR could be used as a therapeutic strategy for the clinical treatment of glioblastoma.

Fiber Microarchitecture in Interpenetrating Collagen-Alginate Hydrogel with Tunable Mechanical Plasticity Regulates Tumor Cell Migration.

The fiber structures of tumor microenvironment (TME) are well-known in regulating tumor cell behaviors, and the plastic remolding of TME has recently been suggested to enhance tumor metastasis as well. However, the interrelationship between the fiber microarchitecture and matrix plasticity is inextricable by existing in vitro models. The individual roles of fiber microarchitecture and matrix plasticity in tuning tumor cell behaviors remain elusive. This study develops an interpenetrating collagen-alginate hydrogel platform with independently tunable matrix plasticity and fiber microarchitecture through an interpenetrating strategy of alginate networks and collagen I networks. With this hydrogel platform, it is demonstrated that tumor cells in high plasticity hydrogels are more extensive and aggressive than in low plasticity hydrogels and fiber structures only have influence in high plasticity hydrogels. The study further elucidates the underlying mechanisms through analyzing the distribution of forces within the matrix and tracking the focal adhesions (FAs) and finds that highly plastic hydrogels can activate the FAs formation, whereas the maturation and stability of FAs are dominated by fiber dispersion. This study not only establishes new ideas on how cells interact with TME cues but also would help to further finely tailor engineered hydrogel platforms for studying tumor behaviors in vitro.

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.

CCL21-CCR7 signaling promotes microglia/macrophage recruitment and chemotherapy resistance in glioblastoma

Glioblastoma (GBM) is the most common and fatal primary tumor of the central nervous system (CNS) and current treatments have limited success. Chemokine signaling regulates both malignant cells and stromal cells of the tumor microenvironment (TME), constituting a potential therapeutic target against brain cancers. Here, we investigated the C–C chemokine receptor type 7 (CCR7) and the chemokine (C–C-motif) ligand 21 (CCL21) for their expression and function in human GBM and then assessed their therapeutic potential in preclinical mouse GBM models. In GBM patients, CCR7 expression positively associated with a poor survival. CCL21–CCR7 signaling was shown to regulate tumor cell migration and proliferation while also controlling tumor associated microglia/macrophage recruitment and VEGF-A production, thereby controlling vascular dysmorphia. Inhibition of CCL21–CCR7 signaling led to an increased sensitivity to temozolomide-induced tumor cell death. Collectively, our data indicate that drug targeting of CCL21–CCR7 signaling in tumor and TME cells is a therapeutic option against GBM.

The 3q Oncogene SEC62 Predicts Response to Neoadjuvant Chemotherapy and Regulates Tumor Cell Migration in Triple Negative Breast Cancer.

In the absence of targeted treatment options, neoadjuvant chemotherapy (NACT) is applied widely for triple-negative breast cancer (TNBC). Response to NACT is an important parameter predictive of oncological outcomes (progression-free and overall survival). An approach to the evaluation of predictive markers enabling therapy individualization is the identification of tumor driver genetic mutations. This study was conducted to investigate the role of SEC62, harbored at 3q26 and identified as a driver of breast cancer pathogenesis, in TNBC. We analyzed SEC62 expression in The Cancer Genome Atlas database, and immunohistologically investigated SEC62 expression in pre- and post-NACT tissue samples from 64 patients with TNBC treated at the Department of Gynecology and Obstetrics/Saarland University Hospital/Homburg between January 2010 and December 2018 and compared the effect of SEC62 on tumor cell migration and proliferation in functional assays. SEC62 expression dynamics correlated positively with the response to NACT (p ≤ 0.01) and oncological outcomes (p ≤ 0.01). SEC62 expression stimulated tumor cell migration (p ≤ 0.01). The study findings indicate that SEC62 is overexpressed in TNBC and serves as a predictive marker for the response to NACT, a prognostic marker for oncological outcomes, and a migration-stimulating oncogene in TNBC.

Vasoactive intestinal peptide-VIPR2 signaling regulates tumor cell migration.

Phosphoinositide metabolism is critically involved in human cancer cell migration and metastatic growth. The formation of lamellipodia at the leading edge of migrating cells is regulated by metabolism of the inositol phospholipid PI(4,5)P2 into PI(3,4,5)P3. The synthesized PI(3,4,5)P3 promotes the translocation of WASP family verprolin homologous protein 2 (WAVE2) to the plasma membrane and regulates guanine nucleotide exchange factor Rac-mediated actin filament remodeling. Here, we investigated if VIPR2, a receptor for vasoactive intestinal peptide (VIP), has a potential role in regulating cell migration via this pathway. We found that silencing of VIPR2 in MDA-MB-231 and MCF-7 human breast cancer cells inhibited VIP-induced cell migration. In contrast, stable expression of exogenous VIPR2 promoted VIP-induced tumor cell migration, an effect that was inhibited by the addition of a PI3-kinase (PI3K)γ inhibitor or a VIPR2-selective antagonist. VIPR2 stably-expressing cells exhibited increased PI3K activity. Membrane localization of PI(3,4,5)P3 was significantly attenuated by VIPR2-silencing. VIPR2-silencing in MDA-MB-231 cells suppressed lamellipodium extension; in VIPR2-overexpressing cells, VIPR2 accumulated in the cell membrane on lamellipodia and co-localized with WAVE2. Conversely, VIPR2-silencing reduced WAVE2 level on the cell membrane and inhibited the interaction between WAVE2, actin-related protein 3, and actin. These findings suggest that VIP–VIPR2 signaling controls cancer migration by regulating WAVE2-mediated actin nucleation and elongation for lamellipodium formation through the synthesis of PI(3,4,5)P3.

Prognosis of gastric adenocarcinoma associated with girdin, Akt, and cortactin.

BACKGROUND: The actin-binding protein girdin regulates tumor cell migration and invasion by maintaining actin structure. PI3K/Akt signaling is an important actin-remodeling pathway. The protein cortactin acts directly on microfilaments and promotes tumor invasion and metastasis by rearranging the cytoskeleton. However, there are few reports on the co-expression of girdin, Akt, and cortactin in gastric adenocarcinoma (GAC).OBJECTIVES: Evaluate girdin, Akt, and cortactin expression in GAC tissues and assess their relationship to the prognosis of GAC patients.DESIGN: Survival analysisSETTING: Medical college in ChinaPATIENTS AND METHODS: We compared survival in 110 paraffin-preserved GAC with corresponding normal gastric mucosa tissues in relationship to girdin, Akt, and cortactin expression levels.MAIN OUTCOME MEASURE: Expression levels of the proteins.SAMPLE SIZE: 110RESULTS: The expression of girdin, Akt, and cortactin were all upregulated in GAC tissues compared with corresponding normal tissues (66.4% vs 36.3%, 57.3% vs 28.2% and 69.1% vs 22.7%, respectively; P<.05) and expression was mutually positive (all P<.05). Overall survival in the girdin, Akt, and cortactin high expression groups was reduced. Multivariate analysis showed that girdin, Akt, cortactin, lymph node metastasis (LNM) and TNM stages were independent factors affecting GAC patients prognosis (P<.05).CONCLUSIONS: Girdin and cortactin may promote GAC invasion and metastasis via the PI3-K/Akt signaling pathway. Girdin, Akt, and cortactin co-expression might serve as a novel molecular target for GAC therapy and improve the prognosis of patients with this disease.LIMITATIONS: A small sample size and lack of related research on molecular mechanisms.CONFLICT OF INTEREST: None.

The Solute Carrier MFSD1 Decreases the Activation Status of β1 Integrin and Thus Tumor Metastasis.

Solute carriers are increasingly recognized as participating in a plethora of pathologies, including cancer. We describe here the involvement of the orphan solute carrier Major Facilitator Superfamily Domain-containing protein 1 (MFSD1) in the regulation of tumor cell migration. Loss of MFSD1 enabled higher levels of metastasis in experimental and spontaneous metastasis mouse models. We identified an increased migratory potential in MFSD1−/− tumor cells which was mediated by increased focal adhesion turnover, reduced stability of mature inactive β1 integrin, and the resulting increased integrin activation index. We show that MFSD1 promoted recycling to the cell surface of endocytosed inactive β1 integrin and thereby protected β1 integrin from proteolytic degradation; this led to dampening of the integrin activation index. Furthermore, downregulation of MFSD1 expression was observed during the early steps of tumorigenesis, and higher MFSD1 expression levels correlate with a better cancer patient prognosis. In sum, we describe a requirement for endolysosomal MFSD1 in efficient β1 integrin recycling to suppress tumor cell dissemination.

Identification of TRPM2 as a Marker Associated With Prognosis and Immune Infiltration in Kidney Renal Clear Cell Carcinoma.

TRPM2 (transient receptor potential melastatin-2), a Ca2+ permeable, non-selective cation channel, is highly expressed in cancers and regulates tumor cell migration, invasion, and proliferation. However, no study has yet demonstrated the association of TRPM2 with the prognosis of cancer patients or tumor immune infiltration, and the possibility and the clinical basis of TRPM2 as a prognostic marker in cancers are yet unknown. In the current study, we first explored the correlation between the mRNA level of TRPM2 and the prognosis of patients with different cancers across public databases. Subsequently, the Tumor Immune Estimation Resource (TIMER) platform and the TISIDB website were used to assess the correlation between TRPM2 and tumor immune cell infiltration level. We found that 1) the level of TRPM2 was significantly elevated in most tumor tissues relative to normal tissues; 2) TRPM2 upregulation was significantly associated with adverse clinical characteristics and poor survival of kidney renal clear cell carcinoma (KIRC) patients; 3) the level of TRPM2 was positively related to immune cell infiltration. Moreover, TRPM2 was closely correlated to the gene markers of diverse immune cells; 4) a high TRPM2 expression predicted worse prognosis in KIRC based on different enriched immune cell cohorts; and 5) TRPM2 was mainly implemented in the T-cell activation process indicated by Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. In conclusion, TRPM2 can serve as a marker to predict the prognosis and immune infiltration in KIRC through the regulation of T-cell activation. The current data may provide additional information for further studies surrounding the function of TRPM2 in KIRC.

A new insight into the role of aryl hydrocarbon receptor (AhR) in the migration of glioblastoma by AhR-IL24 axis regulation

Cancer occurrence and development are closely related to the environment. Aryl hydrocarbon receptor (AhR) is an important receptor mediating the toxic effects of many environmental compounds, and is also involved in regulating tumor cell migration. Glioblastoma is the most malignant glioma and exhibits high motility, but the effects of AhR on the migration of glioblastoma are still unclear. We aimed to understand the role of AhR in the migration of this type of tumor cell and to explore the underlying molecular mechanism. In cultured human neuroblastoma cells (U87), we found that AhR overexpression or knockdown increased or suppressed the migration ability of U87 cells, respectively. Furthermore, inhibition of basal activation of the AhR pathway suppressed migration ability, suggesting a positive correlation between endogenous activity of the AhR pathway and cell migration. When the AhR pathway was activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or 6-formyl [3,2-b] carbazole (FICZ), the migration of U87 cells was inhibited by inducing the expression of a tumor suppressor, IL24, which is a downstream responsive gene of AhR activation. Moreover, a similar AhR-IL24-dependent mechanism for migration inhibition of TCDD was documented in a breast cancer cell line and a lung cancer cell line. This study demonstrated that AhR plays important roles in regulating the migration of glioblastoma, and the induction of the AhR-IL24 axis mediates the inhibition of migration in response to TCDD or FICZ treatment.

The Role of Tumor Microenvironment in Mycosis Fungoides and Sézary Syndrome.

Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common subtypes of cutaneous T-cell lymphomas (CTCLs). Most cases of MF display an indolent course during its early stage. However, in some patients, it can progress to the tumor stage with potential systematic involvement and a poor prognosis. SS is defined as an erythrodermic CTCL with leukemic involvements. The pathogenesis of MF and SS is still not fully understood, but recent data have found that the development of MF and SS is related to genetic alterations and possibly to environmental influences. In CTCL, many components interacting with tumor cells, such as tumor-associated macrophages, fibroblasts, dendritic cells, mast cells, and myeloid-derived suppressor cells, as well as with chemokines, cytokines and other key players, establish the tumor microenvironment (TME). In turn, the TME regulates tumor cell migration and proliferation directly and indirectly and may play a critical role in the progression of MF and SS. The TME of MF and SS appear to show features of a Th2 phenotype, thus dampening tumor-related immune responses. Recently, several studies have been published on the immunological characteristics of MF and SS, but a full understanding of the CTCL-related TME remains to be determined. This review focuses on the role of the TME in MF and SS, aiming to further demonstrate the pathogenesis of the disease and to provide new ideas for potential treatments targeted at the microenvironment components of the tumor.

Abstract 2729: Novel functions of the UPS in the control of tumor cells invasiveness

Abstract Despite formidable advance in the prevention, early detection and treatment of a great number of cancers, the development of metastasis foci in patients suffering from this disease still represents a significant reduction in their survival and life quality. Although metastatic cells have partially been characterized, there is yet not an effective treatment for this pathological condition. Metastasis is a distinctive trait of triple negative breast cancers (TNBC), which lack targeted therapies and therefore represent an unmet clinical need.The Ubiquitin-Proteasome System (UPS) plays a fundamental role in the maintenance of protein homeostasis both in normal and stressed conditions. This enzymatic cascade represents one of the most important metabolic protein degradation pathways and plays a fundamental role in the control of almost every single cellular process. Since alterations in the ubiquitylation cascade have been shown to be associated with malignant transformation, invasive potential of cells and metastasis, we sought to investigate the role of the UPS in the regulation of tumor-cell migration and invasion. To this end, we performed a genetic screen using an shRNA library against UPS genes, and Boyden chambers to analyze the migrating potential of breast cancer cells infected with this library. After the selection process, we characterized the non-migrating cell population and obtained a list of 30 candidate positive migration regulator genes, half of which had already been associated with the regulation of migration, invasion, tumorigenic processes or metastasis. Among the candidates, we focused on a specific DUB and demonstrated that its silencing reduces the migratory and invasive potential of different TNBC cell lines. Since silenced cells proliferation was impaired using in vitro three-dimensional setups, we furthered our investigation with in vivo studies. We demonstrated that NOD/SCID mice inoculated with silenced cells present Kaplan Meier curves for tumor free survival with a clear separation within the control group, as well as a delay in the onset of the tumor formation compared to the tumors generated by control cells. In addition, our results showed a significant impairment in the generation of metastatic foci, indicating that tumor cells niche colonization might be impaired. Overexpression experiments in poorly migrating BC cells further validated our findings, plus indicating that the catalytic activity is important for the observed phenotypes. Finally, we performed a retrospective clinical study which demonstrated that this DUB's protein expression is a prognostic predictor of distant relapse free survival in patients with BC. Altogether, these findings demonstrate that shRNA screens using Boyden chambers are useful for finding novel genes that regulate migration and invasion, which might represent novel therapeutic targets for the development or improvement of cancer treatments. Citation Format: Fabiana A. Rossi, Juliana H. Enriqué Steinberg, Ezequiel H. Calvo Roitberg, Joaquín M. Espinosa, Vincenzo De Laurenzi, Gianluca Sala, Rossano Lattanzio, Mario Rossi. Novel functions of the UPS in the control of tumor cells invasiveness [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2729.

Paxillin family of focal adhesion adaptor proteins and regulation of cancer cell invasion.

The paxillin family of proteins, including paxillin, Hic-5, and leupaxin, are focal adhesion adaptor/scaffolding proteins which localize to cell-matrix adhesions and are important in cell adhesion and migration of both normal and cancer cells. Historically, the role of these proteins in regulating the actin cytoskeleton through focal adhesion-mediated signaling has been well documented. However, studies in recent years have revealed additional functions in modulating the microtubule and intermediate filament cytoskeletons to affect diverse processes including cell polarization, vesicle trafficking and mechanosignaling. Expression of paxillin family proteins in stromal cells is also important in regulating tumor cell migration and invasion through non-cell autonomous effects on the extracellular matrix. Both paxillin and Hic-5 can also influence gene expression through a variety of mechanisms, while their own expression is frequently dysregulated in various cancers. Accordingly, these proteins may serve as valuable targets for novel diagnostic and treatment approaches in cancer.

Abstract LB-036: Tetraspanin 5 expressing macrophages promote mammary tumor growth

Abstract Rationale: Tetraspanins (TSPANs) have emerged as key players in regulating tumor cell migration, invasion, and metastasis. Whilst we know TSPANs are expressed by macrophages very little is known regarding their role in Tumor Associated Macrophages (TAMs). We have shown that all 33 TSPAN members are differentially expressed by macrophages; however, following exposure to tumor-derived factors we see a significant increase in TSPAN5. We hypothesize that TSPAN5 plays a role in supporting the pro-tumor activities of TAMs and targeting tetraspanins in cancer may be a novel therapeutic approach. Objectives: To determine TSPAN5 expression by macrophages in vitro and in situ in human breast cancer tissue and investigate the role of TSPAN5 in regulating the pro-tumor phenotype of TAMs. Methodology: Expression of TSPAN5 was confirmed at the gene and protein level by qPCR, flow cytometry, western blotting, immunocytochemistry and by immunohistochemistry in formalin fixed breast cancer tissue. Accell siRNA knockdown was performed to determine the role of TSPAN5 in macrophage phenotype and function. NextSeq500 was performed to identify how TSPAN5 influences gene expression in BMDMs prepared from C57Bl/6 wild-type (WT) and TSPAN5 knockout (KO) mice. The role of TSPAN5 in a mammary carcinoma model in C57BL/6 WT and TSPAN5KO mice was also determined. Findings: TSPAN5 was up-regulated by tumor-conditioned macrophages both in vitro and in vivo. This expression was also evident in TAMs located in patient derived breast cancer tissue (n=30). Interestingly, 64% of TAMs expressed TSPAN5 and these cells were the main source of TSPAN5 in human breast tumors (77%). Knockdown of TSPAN5 promoted a pro-inflammatory macrophage phenotype and NextSeq500 (n=3) revealed significant changes in genes involved in cell adhesion (Fn1, Cdh11, Lamb1, Cyr61, Postn and Wisp2) and immune regulation (PTGS2, GPX8 and Crip2) in BMDMs from TSPAN5KO mice compared to WT mice. Genetic and antibody ablation of TSPAN5 in C57BL/6 significantly reduced primary EO771 tumors and enhanced the survival of tumor bearing mice. Interestingly, clodronate depletion of macrophages in TSPAN5KO mice promoted mammary tumor growth. Conclusion: Our study demonstrates for the first-time expression of TSPAN5 by TAMs in breast cancers and the pro-tumor properties of TSPAN5 in primary and metastatic mammary tumor growth. Citation Format: Mohammed Ridha Moamin, Peter Monk, Angela Cox, Penelope Ottewell, Julien Saint-Pol, Eric Rubinstein, Munitta Muthana. Tetraspanin 5 expressing macrophages promote mammary tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-036.

STRIP2 silencing inhibits vascular smooth muscle cell proliferation and migration via P38-AKT-MMP-2 signaling pathway.

STRIP2 (FAM40B) was reported to regulate tumor cell migration. Our study aims to discuss the effect of STRIP2 in mouse aortic smooth muscle cell (MOVAS) proliferation and migration processes, which contributes greatly to atherosclerosis formation. In MOVAS cells, STRIP2 depletion suppressed cell proliferation and migration, which were related to a remarkable decrease in matrix metalloproteinases-2 (MMP-2)/MMP-9 expression. Additionally, P38mitogen-activated protein kinases and Protein kinase B (AKT) are inactivated while extracellular signal-regulated kinase (ERK1/2) and jun N-terminal kinase (JNK) are activated upon STRIP2 silencing. SB203580 (P38 inhibitor) further reduced AKT phosphorylation (p-AKT) while dehydrocorydaline chloride (Dc; P38 activator) reversed this effect. Furthermore, Dc significantly recovered MMP-2 expression in STRIP2-knockdown cells. As expected, overexpressing STRIP2 exhibited a contrary effect. Dc and AKT activator SC79 reversed the inhibition of cell proliferation and migration induced by STRIP2 silencing. Interestingly, STRIP2 depletion increased vascular endothelial growth factor level significantly. Taken together, STRIP2 contributed to cell proliferation and migration through P38-AKT-MMP-2 signaling in MOVAS cells, indicating the importance of STRIP2 in atherosclerosis.

Heat Shock Protein 27 Phosphorylation Regulates Tumor Cell Migration under Shear Stress

Heat shock protein 27 (HSP27) is a multifunctional protein that undergoes significant changes in its expression and phosphorylation in response to shear stress stimuli, suggesting that it may be involved in mechanotransduction. However, the mechanism of HSP27 affecting tumor cell migration under shear stress is still not clear. In this study, HSP27-enhanced cyan fluorescent protein (ECFP) and HSP27-Ypet plasmids are constructed to visualize the self-polymerization of HSP27 in living cells based on fluorescence resonance energy transfer technology. The results show that shear stress induces polar distribution of HSP27 to regulate the dynamic structure at the cell leading edge. Shear stress also promotes HSP27 depolymerization to small molecules and then regulates polar actin accumulation and focal adhesion kinase (FAK) polar activation, which further promotes tumor cell migration. This study suggests that HSP27 plays an important role in the regulation of shear stress-induced HeLa cell migration, and it also provides a theoretical basis for HSP27 as a potential drug target for metastasis.