Beta1 Integrin Activation Research Articles

Enzymatic Digestion of Cell-surface Heparan Sulfate Alters the Radiation Response in Triple-negative Breast Cancer Cells

Radiation resistance represents a major challenge in the treatment of breast cancer. As heparan sulfate (HS) chains are known to contribute to tumorigenesis, we aimed to investigate the interplay between HS degradation and radiation response in triple-negative breast cancer (TNBC) cells. HS chains were degraded in vitro as TNBC cells MDA-MB-231 and HCC1806 were treated with heparinase I and III. Subsequently, radioresistance was determined via colony formation assay after doses of 2, 4 and 6 Gy. Cell cycle profile, stem cell characteristics, expression of HS, activation of beta integrins, and apoptosis were determined by flow cytometry. Additionally, cell motility was analyzed via wound-healing assays, and expression and activation of FAK, CDK-6, Src, and Erk1/2 were quantified by western blot pre- and post-irradiation. Finally, the expression of cytokines was analyzed using a cytokine array. Radiation promoted cell cycle changes, while heparinase treatment induced apoptosis in both cell lines. Colony formation assays showed significantly increased radio-resistance for both cell lines after degradation of HS. Cell migration was similarly upregulated after degradation of HS compared to controls. This effect was even more prominent after irradiation. Interestingly, FAK, a marker of radioresistance, was significantly activated in the heparinase-treated group. Additionally, we found Src to be dysregulated in MDA-MB-231 cells. Finally, we observed differential secretion of GRO, CXCL1, IGFBP1, IL8, Angiogenin, and Osteoprotegerin after HS degradation and radiotherapy. Our results suggest an influence of HS chains on the development of radioresistance in TNBC.

Involvement of integrin-activating peptides derived from tenascin-C in colon cancer progression

Tenascin-C (TNC) is an adhesion modulatory protein present in the extracellular matrix that is highly expressed in several malignancies, including colon cancer. Although TNC is considered a negative prognostic factor for cancer patients, the substantial role of the TNC molecule in colorectal carcinogenesis and its malignant progression is poorly understood. We previously found that TNC has a cryptic functional site and that a TNC peptide containing this site, termed TNIIIA2, can potently and persistently activate beta1-integrins. In contrast, the peptide FNIII14, which contains a cryptic bioactive site within the fibronectin molecule, can inactivate beta1-integrins. This review presents the role of TNC in the development of colitis-associated colorectal cancer and in the malignant progression of colon cancer, particularly the major involvement of its cryptic functional site TNIIIA2. We propose new possible prophylactic and therapeutic strategies based on inhibition of the TNIIIA2-induced beta1-integrin activation by peptide FNIII14.

Mitofusin-2 regulates leukocyte adhesion through the maturation of beta2 integrin activation in differentiation

Abstract Neutrophils are critical in inflammation and innate immunity. Neutrophil adhesion to vascular endothelium is a crucial step in neutrophil recruitment. Mitofusin-2 is required for neutrophil adhesion, but molecular details are unclear. Here, we demonstrated that β2-integrin-mediated slow-rolling and arrest, but not PSGL-1-mediated cell rolling, are defective in mitofusin-2-deficient neutrophil-like HL60 cells. This adhesion defect is associated with reduced expression of fMLP receptors and β2 integrins as well as the inhibited β2 integrin activation, as assessed by conformational-specific antibodies. Mitofusin-2 deficiency limited the maturation of β2 integrin activation during the neutrophil-directed differentiation of HL60 cells, which is indicated by identified markers CD35 and CD87. Reversed to results in overall HL60 cells, mitofusin-2 knockdown in β2-integrin-activation-matured cells (CD87high population) enhanced integrin activation. Our study illustrates the function of mitofusin-2 in leukocyte adhesion and may provide new insights into the development and treatment of mitofusin-2-deficiency-related disease.

Abstract A08: PI3Kbeta regulates beta-1 integrin signaling in invadopodia through formation of PI(3,4)P2

Abstract The PI3Kbeta isoform of PI 3-kinase, which is activated downstream from both RTKs and GPCRs, is an important regulator of invadopodia formation. We have previously reported that in MDA-MB-231 breast cancer cells, replacement of endogenous PI3Kbeta with kinase dead (KD) or GPCR-uncoupled (KK-DD) mutants leads to defects in invadopodia-mediated gelatin degradation. We have now studied the mechanism by which loss of PI3Kbeta signaling affects invadopodia. While the number of invadopodia precursors is unaffected by mutation of PI3Kbeta, formation of mature invadopodia is reduced in cells expressing KK-DD and KD PI3Kbeta mutants. Previous studies have demonstrated a role for beta-1 integrins in invadopodia maturation, and PI3Kbeta has been implicated in integrin signaling in platelets. We therefore tested whether mutation of PI3Kbeta would affect integrin signaling in breast cancer cells. In both haptotaxis and cell spreading assays on collagen I, cells expressing KD or KK-DD PI3Kbeta showed a significant impairment. Haptotaxis and cell spreading was also inhibited by TGX221 (PI3Kbeta inhibitor) and pertussis toxin (PTX), but not by specific inhibitors of other Class I PI3Ks. To distinguish whether PI3Kbeta was acting on integrins by inside-out versus outside-in mechanisms, we treated cells with the activating beta-1 integrin antibody TS2/16, which bypasses inside-out regulation of integrin binding. TS2/16 increased cell spreading on gelatin, but this was blocked by TGX221, suggesting that PI3Kbeta acts downstream of beta-1 integrins. Importantly, haptotaxis and cell spreading were not affected by an Akt inhibitor, but were blocked by an inhibitor of SHIP2, which hydrolyzes PIP3 to produce PI[3,4]P2. These data suggest that integrin signaling requires the coupling of Gbeta/gamma-mediated activation of PI3Kbeta to the production of PI[3,4]P2. Finally, to determine whether PI3Kbeta was involved in beta-1 integrin signaling in invadopodia, we plated starved cells on high-density fibrillar collagen (HDFC), which drives invadopodia formation through beta-1 integrin activation. Collagen degradation on HDFC was blocked by inhibition of PI3Kbeta or SHIP2, but not PI3Kalpha. Moreover, recruitment of the PI(3,4)P2 binding protein lamellipodin to invadopodia was reduced by inhibition of PI3Kbeta. In summary, beta-1 integrin signaling in the context of invadopodia maturation requires signaling through PI3Kbeta. We propose that heterotrimeric G-protein activation of PI3Kbeta is necessary for formation of PI(3,4)P2 in invadopodia, which is required for invadopodial maturation. Citation Format: Zahra Erami, Anne R. Bresnick, Jonathan M. Backer. PI3Kbeta regulates beta-1 integrin signaling in invadopodia through formation of PI(3,4)P2 [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A08.

Abstract GMM-041: OVARIAN CANCER CELLS INDUCE A FEEDBACK RESPONSE TO PLATINUM-BASED CHEMOTHERAPY BY ACTIVATING AN ONCOGENIC RSK-EPHA2-GPRC5A SIGNALING SWITCH

Abstract Despite increased understanding of chemotherapy resistance, the effects of the chemotherapy itself on the oncogenic cancer cell properties and adaptive resistance mechanisms of the therapy-escaping metastatic cancer cells remain largely undefined. In this study, we show that the platinum-based chemotherapeutic drugs cisplatin and carboplatin induce a switch from the canonical EphA2 receptor tyrosine kinase activity to the stemness and invasion-associated, ligand independent signaling via EphA2 S897 phosphorylation. In patient-derived high grade serous ovarian cancer (HGSOC) cells and established ovarian cancer cell lines, the chemotherapy treatment led to a significant increase in the ratio between S897 and Y588 phosphorylation of EphA2 in conjunction with GPRC5A protein upregulation and beta1-integrin activation. Notably, high protein levels of GPRC5A were significantly associated to poor survival in patients with metastatic HGSOC, revealing a previously unknown association of this EphA2 interacting receptor with the clinical HGSOC outcome. Moreover, pharmacological inhibition of RSK serine-threonine kinases blocked both the EphA2 S897 phosphorylation and the GPRC5A induction coincident with markedly increased cisplatin and carboplatin sensitivity of the HGSOC cells ex vivo. These results identify a novel mechanism of chemotherapy-induced adaptive resistance via the RSK-EphA2-GPRC5A oncogenic signaling axis in HGSOC. Citation Format: Lidia Moyano-Galceran, Siri Pauliina Turunen, Elina Pietilä, Twana Alkasalias, Sara Corvigno, Elisabet Hjerpe, Ulrika Joneborg, Daria Bulanova, Mina Eriksson, Hanna Dahlstrand, Joseph Carlson, Kaisa Lehti. OVARIAN CANCER CELLS INDUCE A FEEDBACK RESPONSE TO PLATINUM-BASED CHEMOTHERAPY BY ACTIVATING AN ONCOGENIC RSK-EPHA2-GPRC5A SIGNALING SWITCH [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-041.

Abstract 60: Heparan sulfate proteoglycans can mediate cancer cell oriented migration through integrin independent Rac 1 activation

Abstract Cancer cell membranes and tumor associated extracellular matrix are characterized by a predominant presence of highly sulfated Heparan Sulfate Proteoglycans (HSPGs), which have already been identified as potential tumor markers in different cancers. We previously reported on stable tetra-branched peptides named NT4, which selectively bind different human cancer tissues and cells, thanks to their high affinity binding to sulfated glycosaminoglycans (GAGs). Using drug-conjugated NT4 peptides as selective tumor targeting agents, we obtained a significant reduction in tumor growth, compared with animals treated with the unconjugated drug under identical conditions. Drug-conjugated NT4 can also by-pass drug resistance mediated by membrane transporters (1-8). Besides being potential tumor markers, HSPG are also interesting potential drug targets since they appear to take part in many crucial events of cancer progression, such as epithelial mesenchymal transition, migration and invasion. We identified a putative sulfated oligosaccharide motif allowing high-affinity binding of NT4 to sulfated GAGs. NT4 was then used as a specific tool to analyze the role of sulfated GAGs in signaling events regulating oriented cancer cell migration. NT4 binding exerts different effects on adhesion and migration in different cancer cells. In PANC-1 human pancreatic adenocarcinoma cells, NT4 binding to sulfated GAGs abolishes cell adhesion and dramatically affects directional migration, inducing disorganization of actin filaments and stress fibers and increasing the number filopodia. This occurs without any effect on activation of beta1 integrins or focal adhesion kinase, but with a clear reduction in Rac1 activity. Otherwise, in TE-671 human rhabdomyosarcoma cells, binding of NT4 to sulfated GAGs has little effect on cell adhesion and cell migration. Untreated TE671 have a slow and scarcely oriented migration and a general morphology which recalls that assumed by PANC-1 upon treatment with NT4, with many filopodia and scarcely organized stress fibers. Interestingly, activated Rac1 is not detectable in untreated migrating TE671 cells. PANC-1 and TE671 have a very different expression of syndecans, particularly syndecan 2 and syndecan 4. Our results suggest that HSPGs can work as primary players in directional cell migration and actin filaments assembly, by regulating Rac1 activity through an integrin-independent signaling pathway, which seems to require the expression of syndecan 4. 1. Falciani C et al Mol. Cancer Ther. 2007; 6:2441−2448. 2. Falciani C et al ChemMedChem. 2010; 5:567−574. 3. Falciani C et al Curr. Cancer Drug Targets. 2010; 10:695−704. 4. Falciani C et a. ChemMedChem. 2011; 6:678−685. 5. Falciani C et al J Med Chem. 2013; 56:5009-18. 6. Brunetti J et al Sci Rep. 2016; 6:27174. 7. Brunetti J et al Sci Rep. 2015; 5:17736. 8. Depau L et al Oncotarget. 2017; 8:76141-76152. Note: This abstract was not presented at the meeting. Citation Format: Luisa Bracci, Lorenzo Depau, Jlenia Brunetti, Giulia Riolo, Elisabetta Mandarini, Marta Zanchi, Fabrizia Zevolini, Alessandro Pini, Chiara Falciani. Heparan sulfate proteoglycans can mediate cancer cell oriented migration through integrin independent Rac 1 activation [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 60.

Detection of beta1 integrin activation in chondrocytes

Purpose: Integrins are heterodimeric transmembrane receptors linking the actin cytoskeleton to the ECM. Their activity is regulated via an allosteric switch between low- and high-affinity states, induced by effector binding to the cytoplasmic tail of the α- and β-subunits or by ligand binding to the ectodomain. β1 integrin activity in chondrocytes has been shown to increase during osteoarthritis leading to increased catabolism, however β1 integrin signalling is required during chondrogenic differentiation. Integrin signalling is involved in mechanisms of cell survival, growth, differentiation and matrix remodelling, however the exact mechanisms by which β1 integrin signalling may interact with other homeostatic signalling pathways in chondrocytes remains unknown. Here, we aim to examine the expression pattern of β1 integrins within their activated state in chondrocytes. Methods: Costal chondrocytes obtained from 8 to 12-week-old male wild type (WT) mice were cultured under standard conditions. β1 integrin within an active conformation was detected by immunofluorescence staining using a Purified Rat Anti-Mouse CD29 Clone 9EG7 antibody. Activation levels were compared in chondrocytes plated for 24 hours on either fibronectin, collagen type II or uncoated glass coverslips. Chondrocytes in non-coated wells were stimulated in a time course with 1 mM Mn2+ to act as a positive control. To further investigate β1 integrin activation as part of attachment behaviour, chondrocytes were added to fibronectin coated and uncoated coverslips for 1 hour, 6 hours and 24 hours before β1 integrin activation was assessed by immunocytochemistry. Integrin-specific downstream signalling was analysed by immunoblotting of p-FAK/FAK and PKC in lysates of treated chondrocytes. Results: β1 integrins within an active conformation could be detected in wild type murine chondrocytes in vitro. The 9EG7 epitope was detected firstly at low levels in chondrocytes grown in standard conditions, however activation levels were found to increase significantly when chondrocytes were cultured on either fibronectin or type II collagen coatings. Mn2+ added to chondrocytes cultured in uncoated wells as a positive control led to increased levels of activated β1 integrin. Activated integrin β1 in chondrocytes grown on fibronectin and type II collagen exhibited a specific activation pattern, which was not found in chondrocytes grown on uncoated surfaces. Immunoblotting demonstrated that activation of β1 integrins via manganese or through interaction with RGD sequence containing ECM substrates led to increased phosphorylation of the focal adhesion kinase and protein kinase C. Conclusions: β1 integrins within an active conformation were able to be detected in in vitro cultured chondrocytes using immunocytochemistry for the 9EG7 epitope of β1 integrin. The activated state was increased both by Mn2+ and when chondrocytes were grown on either fibronectin or type II collagen substrates. This detection of β1 integrin activation state allows for the further investigation of integrin signalling within the context of interactions with homeostatic growth factor and chemokine signalling pathways.

In Vivo Blockade of Beta-1 and Beta-2 Integrin Activity Inhibits Splenomegaly in JAK2-V617F Positive Myeloproliferative Disease

Introduction:Recently, we showed that in chronic myeloproliferative neoplasia (CMN), β1 and β2 integrins are overactivated on leukocytes in patients and in a JAK2-V617F knock-in mouse model (Gupta et al., Leukemia, 2017; Edelmann-Stephan et al., JCI, In-Press Preview) resulting in increased adhesion of leukocytes to vascular cell adhesion molecule 1 (VCAM-1) and to intercellular cell adhesion molecule 1 (ICAM-1). VCAM-1 and ICAM-1 are abundantly expressed on endothelial cells and in spleen tissue. In JAK2-V617F induced CMN, current therapeutic strategies for symptomatic splenomegaly as hydroxyurea, JAK inhibitors or allogeneic stem cell transplantation (SCT) are effective, however, often limited in duration of efficacy or in eligibility for SCT. Therefore, there is a need to deeper understand the pathophysiology of splenomegaly in CMN in order to develop novel therapeutic approaches. We sought to investigate the effects of in vivo blockade of β1 and β2 integrin overactivation on spleen size, cellular composition of spleen and peripheral blood counts.Material and Methods:11 weeks old Vav1-Cre x JAK2+/V617F mice were treated with a combination of 200µg neutralizing anti-α4β1-integrin (VLA4) antibody (BioXCell, USA) plus 200µg neutralizing anti-β2 integrin antibody (BD Pharmingen, USA) (N=6) and with isotype controls (each 200µg) (N=3), respectively. Animals were injected i.p. on day 1 and day 8. Small animal MRI was used to serially assess the development of spleen volume on days 1, 8, and 15. Abdominal MRI-images employing a slice thickness of 1-2 mm were obtained. The volume of spleens were computed according to radiology standards. Peripheral blood was collected via retro-orbital bleeding and blood counts were measured on days 1, 8, and 15. Thereafter, mice were sacrificed by cervical dislocation and spleens were removed for further analysis by FACS.Results and Discussion:The applied antibody injections were well tolerated and all animals survived the planned observation period. At the start of the experiments (day 1), splenomegaly was confirmed by MRI. As expected, the spleen volumes were similar in the cohorts of isotype and β1/β2 integrin antibody treated JAK2+/V617F mice: 550±76 mm3 (mean±SD) and 518±96 mm3, respectively (Fig. 1). At day 8, upon a single injection of β1/β2 integrin antibodies, a significant decrease in mean spleen volume from baseline 518±96 mm3 to 414±32 mm3 (p=0.0302) was observed (Fig. 1). The correspondent mean±SD for the isotype antibody treated animals was 594±63 mm3. Thus, on day 8, a 30.3% reduction in spleen volume was demonstrated upon β1/β2 integrin antibody treatment in comparison to isotype treatment (p=0.0006). On day 15, the mean spleen volumes of isotype-treated and β1/β2 integrin antibody-treated mice were 665±187 mm3 and 460±69 mm3 (p=0.0410). These results demonstrate that in JAK2-V617F induced disease, blocking of overactivated β1/β2 integrins reduces splenomegaly within a short period of time (8 days). Longer application schedules may build up even stronger and stable decrease in spleen size.In addition to MRI imaging, we also analyzed the composition of peripheral blood cells. At the start of the experiments on day 1, blood parameters showed the typical abnormalities of myeloproliferative disease in both cohorts. Mean counts in isotype treated animals were: WBC = 14,300/µL, hematocrit = 80.3 %, PLT = 1283/nL. Mean counts in β1/β2 integrin antibody treated animals were: WBC = 13,850/µL, hematocrit = 76.0 %, PLT = 1348/nL. This confirms that the JAK2-V617F mouse model used indeed resembles polycythemia vera. In general, the changes observed upon β1/β2 integrin antibody treatment in blood were subtle: the WBC slightly decreased from 13,850/µL to 12,520/µL on day 15, whereas RBC, HCT and PLT remained nearly unchanged. This indicates tolerability of β1/β2 integrin antibody treatment at the level of the hematopoietic system. In isotype treated mice, peripheral blood counts also stayed stable. Data obtained by FACS analysis of peripheral blood and of spleen tissue (composition in granulocytes, B-cells and T-cells) will be presented.Overall, our results indicate a novel role of JAK2-V617F induced overactivation of β1/β2 integrins in development of splenomegaly. Thus, modulation of integrin activity may have therapeutic potential to reduce the spleen volume in CMN patients insufficiently responding to standard treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract B096: CDCP1, a potential noninvasive biomarker in circulating tumor cells for treatment of prostate cancer with FAK inhibitors

Abstract Background: Tumor metastasis requires dynamic changes in cell adhesion mediated through beta 1-integrin and focal adhesion kinase (FAK). In particular, FAK is responsible for counteracting anoikis and promoting survival of CTCs. The Cub-Domain Containing Protein–1 (CDCP1) is a transmembrane glycoprotein expressed in epithelial cells that regulates cell adhesion. CDCP1 loss leads to activation of FAK in nonadherent cancer cells (na-PCC) (Spassov, 2011). As we demonstrated, another consequence of loss of CDCP1 is the inhibition of inside-out activation of beta 1-integrin in na-PCC, suggesting that FAK activation can occur in the absence of active beta 1-integrin. The main goal of this project is to determine how FAK becomes activated in na-PCC upon CDCP1 silencing and to establish that CDCP1 loss sensitizes to treatment with FAK inhibitors. Methods: CTCs were collected from nine patients with castrate-resistant prostate cancer (CRPC) (Schehr, 2016). CTC mRNA was eluted and used for CDCP1 qPCR. CDCP1 was stably silenced in DU145 with short hairpins (Spassov, 2013). In the anoikis assay, 5 x 105 DU145 shControl and shCDCP1 cells were suspended on HEMA coated plates for up to 6 hrs. Western blot membranes were probed for pMAPK1/2, MAPK1/2, FAKpY397, FAKpY925, and FAK. For MTT assays, 5,000 DU145 shControl and shCDCP1 were incubated for 24 hrs on HEMA-coated 96-well plates in regular culture medium +/- Saracatinib, FAKi-PF573228 (kinase domain inhibitor) and FAKi-LGA (FAT domain inhibitor) and/or MEKi-PD98059. A kinase dead mutant of PIP5K-D316A from the Addgene PIP5K plasmid was generated by site directed mutagenesis. Results: We find reduced CDCP1 expression in CTCs from patients with CRPC. Six patients did not express detectable CDCP1 mRNA in CTCs, 1 patient demonstrated CDCP1 expression at the limit of detection, and in 2 patients CDCP1 mRNA levels were above the limit of detection. Silencing CDCP1 in DU145 and PC3 cells results in 3.4-fold higher proliferation rates and 4.4-fold greater anchorage-independent growth rates. CDCP1-negative tumors grew in 100% of mice, versus 30% growth of CDCP1 positive control tumors. In na-PCC, CDCP1 silencing triggered FAK activation, as demonstrated by de novo pY397 and pY925. In addition, 73% of CDCP1-negative cells expressed FAK-stimulated pMAPK. FAK activation required a factor in human plasma and involved a trypsin-sensitive receptor in na-PCC. CDCP1 loss also conferred increased sensitivity to treatment with the PF573228 FAK-kinase inhibitor. At the 4uM concentration 98.1% of CDCP1-positive cells survived compared to 32.9% of CDCP1-silenced cells. An inhibitor treatment matrix showed a synergistic treatment effect of dual targeting of SRC, FAK, and MEK with up to 3-fold increased cytotoxicity of FAKi+SRCi or FAKi+MEKi. The largest treatment effect was observed when all 3 inhibitors were combined. To better understand the role of the FAK-MEK pathway in anoikis, a novel C-terminal FAT domain inhibitor was investigated and compared to PF573228. To determine the mechanism of FAK activation in na-PCC, we expressed a dominant negative PIP5K-D316A mutant. Expression of this mutant is expected to reduce CDCP1-induced FAK activation and also diminish SRC activity. Conclusion: We discovered a synergistic treatment effect between FAK and SRC and/or MEK inhibition in CDCP1-negative nonadherent cells. Expression of pFAK and pMAPK downstream of CDCP1 loss overcomes anoikis and depends on a factor in human plasma. By silencing beta 1-integrin and activating FAK, CDCP1-negative cancer cells may gain easy access to the circulation and survive during the CTC phase of cancer dissemination. CDCP1 loss may serve as a biomarker to identify patients who benefit from treatment with FAK inhibitors. Funding: This work was supported by Department of Defense Synergistic Idea Development Award W81XWH-08-1-0268. Citation Format: Sara Pollan, Jamie Sperger, Joshua Lang, Fangjin Huang, Jie Zheng, Colm Morrissey, Anne E. Cress, Danislav Spassov, Mark Moasser, William Carter, Beatrice Knudsen. CDCP1, a potential noninvasive biomarker in circulating tumor cells for treatment of prostate cancer with FAK inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B096.

Abstract 3165: PI 3-kinase-beta regulates invadopodia maturation and beta-1 integrin signaling

Abstract The invasion of tumors cells during metastasis is mediated by invadopodia, actin rich protrusive organelles that secrete matrix metalloproteases and mediate matrix degradation. The PI3K-beta isoform of PI 3-kinase, which is regulated by both receptor tyrosine kinases and GPCRs, is an important regulator of invadopodia formation. We have previously reported that in MDA-MB-231 breast cancer cells, replacement of endogenous PI3K-beta with kinase dead (p110-betaK799R; KD) or GPCR-uncoupled (p110-beta526KK-DD; KK-DD) mutants leads to defects in gelatin degradation. We have now studied the mechanism by which loss of PI3K-beta signaling affects invadopodia formation and matrix degradation. While the number of invadopodia precursors (cortactin-Tks5 positive, degradation negative) is unaffected by mutation of PI3K-beta, formation of mature invadopodia (cortactin-Tks5 positive, degradation positive) is reduced in cells expressing KK-DD and KD PI3K-beta. Previous studies have demonstrated a role for beta-1 integrins in invadopodia maturation, and PI3K-beta has been implicated in integrin signaling in platelets. We therefore tested whether mutation of PI3K-beta would affect integrin signaling in breast cancer cells. In a haptotaxis assay, MDA-MB-231 cells expressing KD or KK-DD PI3K-beta showed a significant decrease in migration. Consistent with these data, cell spreading on collagen I was significantly impaired in cells expressing KD or KK-DD PI3K-beta, and was inhibited by TGX221 (PI3K-beta inhibitor) and LY294002 (pan-PI3K inhibitor), but not by specific inhibitors of other Class I PI3Ks (alpha, delta and gamma). Spreading was also impaired in cells treated with pertussis toxin, which blocks GPCR activation of PI3K-beta. To distinguish whether PI3K-beta was acting on integrins by inside-out versus outside-in mechanisms, we treated cells with the activating beta-1 integrin antibody TS2/16, which bypasses inside-out regulation of integrin binding. TS2/16 increased cell spreading on collagen I, but TS2/16-stimulated spreading was blocked by treatment of cells with TGX221, suggesting that PI3K-beta acts downstream of beta-1 integrins. Finally, to determine whether PI3K-beta was involved in beta-1 integrin signaling in invadopodia, we plated cells on high density fibrillar collagen (HDFC), which enhances invadopodia formation through beta-1 integrin activation. Invadopodia formation on HDFC was blocked by treatment of cells with TGX221. In summary, beta-1 integrin signaling in the context of invadopodia requires GPCR signaling to PI3K-beta. We propose that GPCR activation of PI3K-beta is a critical downstream component of beta-1 integrin signaling in invadopodia. Citation Format: Zahra Erami, Anne R. Bresnick, Jonathan M. Backer. PI 3-kinase-beta regulates invadopodia maturation and beta-1 integrin signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3165.

Eosinophil Activation Status in Separate Compartments and Association with Asthma.

Asthma is frequently characterized by eosinophil-rich airway inflammation. Airway eosinophilia is associated with asthma exacerbations and likely plays a part in airway remodeling. Eosinophil recruitment from the bloodstream depends on circulating eosinophils becoming activated, which leads to eosinophil arrest on activated endothelium, extravasation, and continued movement through the bronchial tissue by interaction with the extracellular matrix (ECM). Circulating eosinophils can exist at different activation levels, which include non-activated or pre-activated (sensitized or “primed”). Further, the bloodstream may lack pre-activated cells, due to such eosinophils having arrested on endothelium or extravasated into tissue. Increased expression, and in some instances, decreased expression of cell-surface proteins, including CD44, CD45, CD45R0, CD48, CD137, neuropeptide S receptor, cytokine receptors, Fc receptors, and integrins (receptors mediating cell adhesion and migration by interacting with ligands on other cells or in the ECM), and activated states of integrins or Fc receptors on blood eosinophils have been reported to correlate with aspects of asthma. A subset of these proteins has been reported to respond to intervention, e.g., with anti-interleukin (IL)-5. How these surface proteins and the activation state of the eosinophil respond to other interventions, e.g., with anti-IL-4 receptor alpha or anti-IL-13, is unknown. Eosinophil surface proteins suggested to be biomarkers of activation, particularly integrins, and reports on correlations between eosinophil activation and aspects of asthma are described in this review. Intermediate activation of beta1 and beta2 integrins on circulating eosinophils correlates with decreased pulmonary function, airway inflammation, or airway lumen eosinophils in non-severe asthma. The correlation does not appear in severe asthma, likely due to a higher degree of extravasation of pre-activated eosinophils in more severe disease. Bronchoalveolar lavage (BAL) eosinophils have highly activated integrins and other changes in surface proteins compared to blood eosinophils. The activation state of eosinophils in lung tissue, although likely very important in asthma, is largely unknown. However, some recent articles, mainly on mice but partly on human cells, indicate that tissue eosinophils may have a surface phenotype(s) different from that of sputum or BAL eosinophils.

NG2 Proteoglycan Enhances Brain Tumor Progression by Promoting Beta-1 Integrin Activation in both Cis and Trans Orientations.

By physically interacting with beta-1 integrins, the NG2 proteoglycan enhances activation of the integrin heterodimers. In glioma cells, co-localization of NG2 and α3β1 integrin in individual cells (cis interaction) can be demonstrated by immunolabeling, and the NG2-integrin interaction can be confirmed by co-immunoprecipitation. NG2-dependent integrin activation is detected via use of conformationally sensitive monoclonal antibodies that reveal the activated state of the beta-1 subunit in NG2-positive versus NG2-negative cells. NG2-dependent activation of beta-1 integrins triggers downstream activation of FAK and PI3K/Akt signaling, resulting in increased glioma cell proliferation, motility, and survival. Similar NG2-dependent cis activation of beta-1 integrins occurs in microvascular pericytes, leading to enhanced proliferation and motility of these vascular cells. Surprisingly, pericyte NG2 is also able to promote beta-1 integrin activation in closely apposed endothelial cells (trans interaction). Enhanced beta-1 signaling in endothelial cells promotes endothelial maturation by inducing the formation of endothelial junctions, resulting in increased barrier function of the endothelium and increased basal lamina assembly. NG2-dependent beta-1 integrin signaling is therefore important for tumor progression by virtue of its affects not only on the tumor cells themselves, but also on the maturation and function of tumor blood vessels.

Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models.

ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells.

Abstract # 1813 G-alpha-s-protein coupled receptor signaling suppresses integrin-mediated cell adhesion of antigen-specific human CD8+ T cells

An efficient immune response requires a selective and strong interaction of T cells with endothelium or target cells. These interactions are strengthened by the rapid induction of beta2-integrin-mediated adhesion upon chemokine receptor or T-cell receptor (TCR) stimulation, respectively. We and others have shown that an increase in G-alpha-s-protein coupled receptor (GalphasPCR) signaling, such as via beta2-adrenergic receptors counteracts chemokine-induced beta2-integrin activation and promotes de-adhesion and mobilization of effector CD8+ T cells from the marginal pool into the circulation. We asked whether GalphasPCR agonists also inhibit integrin activation induced by TCR stimulation of human antigen-specific T cells. Using soluble multimeric ICAM1-Fc-F(ab)2 fluorescent complexes, we developed a flow cytometry assay to identify integrin activation on virus-specific CD8+ T cells following short-term stimulation and staining with cytomegalovirus, Epstein–Barr virus, or influenza virus peptide-major histocompatibility complex class I HLA-multimers. From the GalphasPCR agonists used, epinephrine, norepinephrine, prostaglandins E2/D2 and adenosine strongly down-modulated the beta2-integrin activation on all virus-specific T cells and subsets of different stages of activation, while dopamine, serotonin and histamine had no effects. This active down-modulation of integrin activation through GalphasPCR signaling might be an essential mechanism by which the immune response is suppressed in a variety of pathologies characterized by increased levels of catecholamines (e.g., sleep deprivation), prostaglandins (e.g., tumor growth), or adenosine (e.g., hypoxia).

G-alpha-s-coupled receptor signaling controls circadian rhythm in integrin-mediated cell adhesion of virus-specific human CD8+ T cells

Abstract Beta2-integrin activation in response to inside-out signaling by chemokines plays a major role in T-cell adhesion to the endothelium and the following extravasation into the inflamed tissue. We and others have previously shown that daytime increase in G-alpha-s-coupled receptor (GαsPCR) signaling, such as via beta2-adrenergic receptors counteracts integrin activation and promotes mobilization of effector CD8+ T cells from the marginal pool into the recirculation. In T cells, integrin activation is also rapidly induced by stimulation of antigen-specific T-cell receptors (TCR), resulting in the formation of stable immunological synapses. Therefore, we asked whether daytime increase in GαsPCR signaling inhibits integrin activation induced by TCR stimulation of human virus-specific T cells. Using ligand-complex-based adhesion assay to measure beta2-integrin activation, we show that affinity of integrins, which were activated by soluble cytomegalovirus virus peptide-major histocompatibility complex class I HLA-multimers is minimal during daytime. Additional in vitro experiments with GαsPCR agonists showed that epinephrine, norepinephrine, histamine, adenosine, and prostaglandins E2/D2 down-modulate the beta2-integrin activation; however, only epinephrine did so in physiological daytime concentrations, suggesting its mayor role in circadian regulation of T-cell adhesion. This active down-modulation of integrin activation through GαsPCR might be an essential mechanism of circadian immune defense regulation.

Abstract 444: Semaphorin3A Reduces Atherosclerotic Plaque Formation in ApoE Knock Out Mice Through Regulation of M2 Type Macrophage Migration

Objective: Recent data point to an important immunomodulatory role for neural guidance molecules, including semaphorin 3a (Sema-3A), in inflammatory diseases. Sema-3A is a secreted member of the Sema family and is produced by several immune cells including macrophages. We found that Sema-3A receptors are expressed in macrophages and in atherosclerotic plaques. Approach and results: To investigate the role of Sema-3A in atherosclerosis, ApoE-/- mice were administered with a Sema-3A overexpressing or a control plasmid and were fed a high fat diet for 9 weeks. Sema-3A receptors were expressed in macrophages and atherosclerotic plaques. Our results show that Sema-3A overexpressing mice had significantly smaller atherosclerotic plaques than control mice in the aortic sinus (0.3±0.02 vs. 0.4±0.03 mm 2 ), the brachiocephalic artery (0.04±0.01 vs. 0.1±0.01 mm 2 ) and the aorta (9.5±1.4 vs 15.3±2.9%), assessed by oil red O staining. No differences were observed in plaque stability, measured by collagen and smooth muscle cell alpha-actin staining. However, there was significantly less (2-fold) macrophage content in the plaques of Sema-3A compared to control mice, associated with decreased circulating monocytes determined by flow cytometry as cd11b positive and Gr-1 negative cells (4.97±0.74 vs. 7.2±0.62%). To better define the involved mechanisms, we investigated macrophage function In vitro and found that recombinant Sema-3A increased by 4 fold migration of M2 but not M1 macrophages. In addition, active beta-1 integrin expression was significantly enhanced (2-fold) by Sema-3A in human M2 macrophages. Importantly, Sema-3A induced a significant increase (by 50%) of focal adhesion kinase phosphorylation. Conclusions: Our data show that Sema-3A prevents atherosclerotic plaque formation in ApoE -/- mice. This may be due in part to enhanced motility and function of M2 macrophages through regulation of beta-1 integrin.

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin.

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55–89] μm2 for uninfected and 41 [34–51] μm2 for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 μm2 s−1 ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 μm2 s−1 ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.

451 Distinct Roles of AKT Isoforms in Regulating Beta1-integrin Activity, Migration and Invasion in Prostate Cancer

451 Distinct Roles of AKT Isoforms in Regulating Beta1-integrin Activity, Migration and Invasion in Prostate Cancer

Atomic force microscope studies demonstrate enhanced beta1‐integrin adhesion as a factor assocatiated with age‐related increases in vascular smooth muscle stiffness

Beta1-integrin, an important vascular smooth muscle cell-extracelluar matrix (VSMC-ECM) adhesion molecule, shows enhanced VSMC expression associated with age-related increases in aortic stiffness. We investigated whether active beta 1-integrin adhesion to the ECM protein collagen type 1 (COL1) and fibronectin (FN) was enhanced in VSMC from old and young male monkey thoracic aortas using the atomic force microscopic (AFM). As the abundance of beta1-integrin is significantly higher in old compared to young monkeys, we hypothesized that enhanced beta 1-integrin adhesion could provide a mechanism for accentuating aortic stiffness by strengthening VSMC-ECM connections. As evidence of increased beta 1-integrin expression in old vs young VSMC, AFM probes functionalized with FN or COL1 exhibited significantly increased adhesion to the beta1-integrin in old vs young VSMC. In old vs young, COL1 binding probability was 70±1% vs 63±1% and adhesion force was 72±1.3 pN vs 50±2 pN whereas for FN binding probability was 43±1% vs 33±1% and adhesion force 61±1.4 pN vs 39 ± 1 pN. Our results support the involvement of beta1-integrin as a potential factor in the increased stiffness of VSMC observed during aging that could contribute to enhanced stiffness of the aortic wall. (NIH R01HL102472 and P01HL095486).

An in vitro correlation of mechanical forces and metastatic capacity

Mechanical forces have a major influence on cell migration and are predicted to significantly impact cancer metastasis, yet this idea is currently poorly defined. In this study we have asked if changes in traction stress and migratory properties correlate with the metastatic progression of tumor cells. For this purpose, four murine breast cancer cell lines derived from the same primary tumor, but possessing increasing metastatic capacity, were tested for adhesion strength, traction stress, focal adhesion organization and for differential migration rates in two-dimensional and three-dimensional environments. Using traction force microscopy (TFM), we were surprised to find an inverse relationship between traction stress and metastatic capacity, such that force production decreased as the metastatic capacity increased. Consistent with this observation, adhesion strength exhibited an identical profile to the traction data. A count of adhesions indicated a general reduction in the number as metastatic capacity increased but no difference in the maturation as determined by the ratio of nascent to mature adhesions. These changes correlated well with a reduction in active beta-1 integrin with increasing metastatic ability. Finally, in two dimensions, wound healing, migration and persistence were relatively low in the entire panel, maintaining a downward trend with increasing metastatic capacity. Why metastatic cells would migrate so poorly prompted us to ask if the loss of adhesive parameters in the most metastatic cells indicated a switch to a less adhesive mode of migration that would only be detected in a three-dimensional environment. Indeed, in three-dimensional migration assays, the most metastatic cells now showed the greatest linear speed. We conclude that traction stress, adhesion strength and rate of migration do indeed change as tumor cells progress in metastatic capacity and do so in a dimension-sensitive manner.