Beta1 Integrin Expression Research Articles

Abstract 4059: The bone morphogenic protein 7 (Bmp7) plays a pro-tumorigenic role in pheochromocytoma

Abstract Introduction Rats carrying a germline loss-of-function mutation in p27 (MENX syndrome) develop bilateral pheochromcoytoma (PCC) with complete penetrance. Gene expression profiling of rat PCCs identified genes highly expressed in tumors versus normal adrenal medulla. Several of them were found up-regulated also in human PCCs (both sporadic and familial), including the BMP7 gene encoding a member of the bone morphogenic protein family. BMP7 has been shown to be involved in other human cancers, but its role in PCC tumorigenesis is unknown. Methods We used 3 PCC cell lines: PC12 (rat), having low endogenous levels of Bmp7; MPC (mouse) and its aggressive derivative MTT, both with high levels of Bmp7. We also used primary rat PCC cells with high endogenous levels of Bmp7. A Bmp7-expressing plasmid was transfected in PC12 cells. In contrast, Bmp7 gene knockdown in MPC/MTT cells and in primary rat cultures was performed using lentiviral vector expressing anti-Bmp7 shRNA molecules. In vitro assays assessing proliferation (MTT), migration and invasion (Boyden chamber) were then performed. We analyzed the link between p27 and Bmp7 in embryonic mouse and rat fibroblast cells having different doses of p27 gene and in PC12 cells transfected with anti-p27 siRNA molecules. Integrin beta1 expression following activation of Bmp7 signaling was assessed by western blotting in PCC cell lines. Expression of BMP7 and Integrin beta1 was determined also in snap-frozen human primary PCC tissues by western blotting. Results We observed that up-regulation of Bmp7 enhances proliferation, migration and invasion of the PC12 cells, while down-regulation of Bmp7 impairs these properties in MPC and MTT cells. Knock-down of Bmp7 in primary rat PCC cells reduced their viability compared to cells infected with control vector. Additionally, we found that p27 expression was inversely correlated to that of Bmp7, suggesting a potential functional link between the two molecules. Overexpression of Bmp7 in PC12 cells was associated with an increase in integrin beta1 expression. A good correlation was observed in human primary PCC samples between the expression of BMP7 and that of Integrin beta1. Conclusions In conclusion, we demonstrated for the first time that Bmp7 promotes the migration and invasion of PCC cells. We found that endogenous Bmp7 levels are influenced by the amount of p27. Integrin beta1 seems to mediate the ability of Bmp7 signaling to promote migration and invasion of PCC cells. Bmp7 represents a novel target for therapy of PCC since the knock-down in vitro shows promising impairment of the tumorigenic phenotype. Citation Format: Ines Leinhäuser, Ines Höfig, Natasa Anastasov, Felix Beuschlein, Massimo Mannelli, Michael J. Atkinson, Natalia S. Pellegata. The bone morphogenic protein 7 (Bmp7) plays a pro-tumorigenic role in pheochromocytoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4059. doi:10.1158/1538-7445.AM2014-4059

Interleukin‐8 induces DNA synthesis, migration and down‐regulation of cleaved caspase‐3 in cultured human gingival epithelial cells

Migration of the junctional epithelium occurs in association with the formation of a periodontal pocket. Although the migration of junctional epithelium is known to be related to the proliferation and migration of gingival junctional epithelial cells, the mechanism has not been clarified. In patients with periodontitis, the levels of interleukin-8 (IL-8) in both gingival tissue and gingival crevicular fluid are dramatically increased. IL-8 has broad bioactive functions. In this study, we examined the role of IL-8 in DNA synthesis, migration and protection against apoptosis in cultured human gingival epithelial cells (HGEC). DNA synthesis was estimated by measuring the incorporation of bromodeoxyuridine. The migration of gingival epithelial cells was assessed in a wound-healing assay. The expression of integrin beta-1 was analyzed using immunofluorescence confocal microscopy and western blotting. Cleaved caspase-3 was detected using western blotting and a Caspase-Glo assay kit. IL-8 increased the synthesis of DNA in HGEC, and the maximal effect was seen at 25 or 50 ng/mL of IL-8. In addition, 50 ng/mL of IL-8 induced cell migration, and a neutralizing antibody of integrin beta-1 inhibited the migration. IL-8 also activated expression of integrin beta-1. Furthermore, IL-8 reduced the Aggregatibacter actinomycetemcomitans-induced increase in caspase-3 expression in HGEC. IL-8 may facilitate the migration of gingival junctional epithelium by enhancing DNA synthesis, migration and preventing apoptosis of gingival epithelial cells.

Activation of Integrin β1 Mediates the Increased Malignant Potential of Ovarian Cancer Cells Exerted by Inflammatory Cytokines

Epithelial ovarian cancer (EOC) is a highly lethal gynecological malignancy since it could not be discovered until at late stage. Identifying the molecular phenotype alteration during the development and progression of ovarian cancer is an urgent demand for the targeted intervention therapy. Recently, inflammation and Integrin beta 1(ITGB1), a subunit of heterodimeric transmembrane receptors family, had been pointed out to be involved in promoting ovarian tumorigenesis and cancer progression, respectively. However, the relationship between ITGB1 and the inflammatory mediators in ovarian cancer progression remains obscure. In the present study, ITGB1 was observed to be frequently upregulated in ovarian cancer, overexpression of ITGB1 led to a more invasive and mesenchymal phenotype. Furthermore, our results also provided evidence concerning the role of inflammatory cytokines (IL-6, TGF-β1 and SDF-1) in ITGB1 expression as well as in the malignant potential of ovarian cancer cells. Consistently, sh-RNA mediated knocking down of ITGB1 evidently reduced tumor growth and peritoneal dissemination in in vivo Nod-scid SKOV3 orthotopic xenograft mice. Overall, the present data suggested that ITGB1 upregulation was involved in the regulation of tumorigenesis and disease exacerbation exerted by inflammatory cytokines as IL-6, TGF-β1 and SDF-1, and suggested that targeting ITGB1 and the underlying inflammatory modulator was an attractive strategy for therapeutic intervention during ovarian carcinogenesis.

Expression heterogeneity research of ITGB3 and BCL-2 in lung adenocarcinoma tissue and adenocarcinoma cell line

ObjectiveTo analyze expression heterogeneity of Integrin beta 3 (ITGB3) and B-cell lymphoma 2 (BCL-2) in lung adenocarcinoma tissue and adenocarcinoma cell line and further provide theoretical direction for molecular biological research of lung adenocarcinoma. MethodsTissue microarray was used to observe relation among expression, heterogeneitpy and clinical characteristics of ITGB3 and BCL-2 in lung cancer. ResultsITGB3 and BCL-2 increased significantly in A549 cells in CAFs group withβ-actin as control; the expression level of BCL-2 also increased in ITGB3 transfected cells with GFP plasmid transfected A549 cells as control; immunohistochemistry staining showed that positive rates of ITGB3, ITGB1 and BCL-2 in normal lung tissues were 0, the positive rates in lung adenocarcinoma were 7.04%, 84.51% and 4.23%, respectively; in the results of immunohistochemistry staining, the expression of Girdin protein in lung adenocarcinoma was homogeneous, however protein expression of ITGB3, ITGB1 and BCL-2 showed different patterns in the same location with significant heterogeneity; majority of ITGB3, ITGB1 or BCL-2 positive tissue showed heterogeneity that expression in trailing edge was higher than that of trailing edge in lung adenocarcinoma tissue, the patients with BCL-2 heterogeneity showed higher lymph node metastasis ratio and lower clinical stage (P<0.05); and the expression of ITGB3 and the clinical characteristics of patients were not significant related (P>0.05). ConclusionsExpression of ITGB3 and BCL-2 in lung adenocarcinoma and adenocarcinoma cell line showed heterogeneity that expression in trailing edge was higher than that of trailing edge, which may play an important role in promoting tumor lymph node metastasis and vascular invasion, and provides a new research direction for exploration of lung adenocarcinoma metastasis mechanism.

Reversal of Murine Epidermal Atrophy by Topical Modulation of Calcium Signaling

Cytosolic Ca(2+) signals are performed by Ca(2+) releases from the endoplasmic reticulum and Ca(2+) influx from the extracellular medium. Releases rely on the refilling of the intracellular Ca(2+) stores by the Ca(2+) influx "Store-Operated Calcium Entry" (SOCE) via the channel Orai1. Here we show that Orai1 expression, SOCE amplitude, and epidermal proliferation are decreased in the epidermis of patients with skin fragility when compared with aged nonatrophic skin. Epidermal atrophy was induced in mice by the inhibition of Orai1 with small interfering RNA and the topical application of a SOCE blocker BTP2. The inhibition of Orai1 impaired the heparin-binding epidermal growth factor (HB-EGF)-induced Ca(2+) influxes and fully prevented the mitogen effect of HB-EGF in primary human keratinocytes. Importantly, epidermal proliferation correlated with Orai1 expression in mice. Conversely, the topical application of an Orai1 activator, the benzohydroquinone (BHQ), increased the epidermal thickness and proliferation, whereas the pro-proliferative effect of BHQ was prevented by the inhibition of Orai1. Finally, the topical application of BHQ reversed the epidermal atrophy induced by corticosteroids in mice. The topical modulation of Ca(2+) signals may thus be a promising therapeutic strategy in dermatology.

Aberrant expression and altered cellular localization of desmosomal and hemidesmosomal proteins are associated with aggressive clinicopathological features of oral squamous cell carcinoma.

Disruption of cell adhesion plays a central role in dedifferentiation, invasion, and metastasis of various cancers. The desmosome and hemidesmosome are anchoring junctions that control cell-cell and cell-matrix adhesion, respectively. To clarify their contributions in mediating the biological properties of oral cancer, we immunohistochemically examined the expression of desmoglein 1 (DSG1), DSG2, DSG3, desmocollin 2 (DSC2), integrin beta 4 (ITGB4), laminin gamma chain 2 (LAMC2), and collagen type 17 alpha 1 (COL17A1) in 51 cases of oral squamous cell carcinoma. On normal oral epithelial cells, DSG1, DSG3, DSC2, and COL17A1 were expressed on the plasma membrane, while ITGB4 and mature LAMC2 were present at the basement membrane. In cancer, the expression of DSG1, DSG3, DSC2, and COL17A1 decreased and internalized to the cytoplasm. Cytoplasmic expression of DSG2, ITGB4, and LAMC2 was induced in the cancer cells facing to the stroma. We scored immunohistochemical expression and correlated this to clinicopathological parameters including histologic differentiation, pattern of invasion, and presence of lymph node metastasis. Decrease of DSG3 and DSC2 expression correlated with a more aggressive cancer phenotype: less differentiated and more invasive histologic features and a higher incidence of nodal metastasis. Lower COL17A1 and higher LAMC2 expression were also associated with a more aggressive phenotype. The present study demonstrates that aberrant expression and altered cellular localization of desmosomal and hemidesmosomal proteins are associated with aggressive clinicopathological features of oral cancer. This reinforces the notion that disturbance of the keratin-associated anchoring junctions confers aggressive features to cancer cells.

Anti-tumor efficacy of ultrasonic cavitation is potentiated by concurrent delivery of anti-angiogenic drug in colon cancer

This study investigated the efficacy of concurrent delivery of an anti-angiogenic drug and ultrasonic cavitation therapy in a mouse model of human colon cancer. A biotinylated form of the anti-angiogenic drug Endostar was conjugated to a streptavidin-coated microbubble (MB). Mice bearing subcutaneous tumors (HT29) were divided into 4 groups. Group 1 served as an untreated control. Group 2 served as a cavitation control and received naked microbubbles and sham ultrasonic cavitation (MB+sham cavitation). Group 3 received naked microbubbles and ultrasonic cavitation (MB+cavitation). Group 4 received Endostar loaded microbubbles and ultrasonic cavitation (Endostar-MB+cavitation). Ultrasonic cavitation was performed using a high-power custom built sonicator. Contrast-enhanced ultrasound imaging (CEUS) was used to measure tumor blood flow before and after ultrasonic cavitation. In vivo fluorescence imaging was performed to monitor changes in tumor volume. Immunohistochemistry was performed to assess CD31, VEGFR-2 and alpha-v beta-3 integrin expression within the tumor. Apoptosis of the tumor cells was determined by TUNEL assay, and ultrastructural changes within the tumor were examined by electron microcopy. Ultrasonic cavitation with Endostar-MB demonstrated a significantly greater inhibition of tumor blood flow on day 7 and tumor growth on day 16 compared with naked MB and control groups. The Endostar-MB treated mice showed significantly decreased expression VEGFR-2 and alpha-v beta-3 integrin, and increased apoptosis of tumor cells and degradation of the tumor ultrastructure. Our findings indicated that the anti-vascular and anti-tumor effects of ultrasonic cavitation could be potentiated by simultaneously delivering an anti-angiogenic drug in colon cancer.

Endometrial beta3 integrin profile reflects endometrial receptivity defects in women with unexplained recurrent pregnancy loss.

BackgroundThe pathophysiology of recurrent pregnancy loss (RPL) is still unknown in 50% of the cases. Herein we measure the expression of beta3 integrin subunit, a well-known implantation marker, in women with or without RPL and correlate it with the histological dating of the endometrial tissue.MethodsLH-timed endometrial biopsies were obtained from cases (RPL; n = 21, age 33.9+/-4.7) and healthy controls (n = 29; age 29.8+/-4.1) during the mid-secretory phase (post ovulatory day: 8 to 10). Endometrial samples were timed histologically according to Noyes’ criteria and underwent immunohistochemical staining for beta3 integrin expression. For statistical analysis the semi-quantitative HSCORE was assessed. Type I (beta3 negative in an out-of-phase endometrium) and Type II defect (beta3 negative in an in-phase endometrium) were also analysed. Statistical analysis was done with Student t-test, Mann Whitney U test, ANCOVA and chi square for trend. Significance was set as P < 0.05.ResultsThe mean (SD) age in controls was lower compared to cases [(29.8 (4.1) vs. 33.9 (4.7) – P = 0.001; Student t-test)]. The median (range) expression of beta3 integrin in controls and cases was 1.94 (0 to 3.5) vs. 0.82 (0 to 3.6), respectively (P = 0.001; Mann Whitney U test). Significance was still significant after adjusting for age (P = 0.03;ANCOVA). The normal positive staining > =0.7 of beta3 integrin subunit and in-phase endometrium was seen in 24 out of 29 (82.8%) controls, but in only 6 out of 21 (28.6%) of cases with RPL; Type I and II defects were seen in 10.3 and 6.9% of controls, while present in 52.4 and 19.1% of cases, respectively (P = 0.0005; chi-square).ConclusionsWomen with unexplained RPL had significantly reduced integrin expression compared to controls. Our findings underline the need for further molecular analysis of endometrial tissue in affected women.

Abstract 18954: Novel Role of De Novo Matricellular Protein Cyr61 in Bridging Lysophosphatidic Acid and Integrin Pathways Leading to Smooth Muscle Cell Migration

Lysophosphatidic acid (LPA), a potent bioactive lipid, markedly induces smooth muscle cell (SMC) migration, which is one of the important processes involved in atherosclerotic lesion development. In order to search for key molecules that control LPA-induced SMC migration, we performed gene microarray analysis and attempted to identify LPA-induced extracellular molecules responsible for LPA-triggered cell migration. We observed that LPA selectively induced the expression of the matricellular protein Cyr61, a multifunctional matrix-related protein, which has been implicated in cell angiogenesis, proliferation, adhesion and migration. We hypothesize that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induces temporal and spatial expression of Cyr61 protein in SMCs. LPA-induced de novo Cyr61 proteins promptly accumulate in the Golgi apparatus and then translocate to the extracellular matrix. Cyr61 specific siRNAs diminishes LPA-induced cell migration, indicating the novel regulatory role of the induced matricellular protein Cyr61 in LPA-induced cell migration. Using primary SMCs derived from LPA receptor 1 (LPA1) knockout and LPA2 knockout mice, our data reveal that LPA1 is required for LPA-induced Cyr61 expression in SMCs and cell migration. Furthermore, LPA induces profound activation of focal adhesion kinase (FAK) in SMCs in later time points, coinciding with the accumulation of Cyr61in the extracellular matrix. FAK is an intracellular kinase important in regulating cell migration. Our data demonstrated that knockdown of de novo Cyr61 expression blocked LPA-induced cellular FAK activation and cell migration. Integrin alpha6 and beta1 have been reported to interact with Cyr61 in SMCs. We found that knockdown of the expression of integrin alpha6 or beta1 prevented LPA-induced FAK activation and cell migration. Taken together, these data reveal that a novel LPA/Cyr61 pathway controls cell migration and that de novo Cyr61 in extracellular matrix bridges LPA and integrin signaling, which in turn, activates FAK leading to cell migration. Understanding this novel pathway will provide new insights into mechanisms underlying the development of cell migration related disorders including atherosclerosis and restenosis.

Nerve Growth Factor Promotes Corneal Epithelial Migration by Enhancing Expression of Matrix Metalloprotease-9

Nerve growth factor (NGF) is a neuropeptide essential for the development, survival, growth, and differentiation of corneal cells. Its effects are mediated by both TrkA and p75 receptors. Clinically relevant use of NGF was introduced to treat neurotrophic ulcerations in patients. Herein, we examine the mechanisms by which NGF enhances epithelial wound healing both in vivo and in vitro. An animal model using adult hens was implemented for the in vivo experiments. Laser ablation keratectomy was performed and animals were observed for up to 7 days. Epithelial healing was measured with fluorescein. In addition, proliferation was measured using BrdU incorporation and both TrkA and matrix metalloprotease-9 (MMP-9) expression were measured by immunohistochemistry (IHC) and Western blot (WB). In vitro experiments were carried out with telomerase-immortalized human corneal epithelial cells (HCLE). The rate of proliferation was measured using a colorimetric assay and BrdU incorporation. Real-time migration was evaluated with an inverted microscope. MMP-9 expression was evaluated by immunocytochemistry (ICC), WB, zymography, and RT-PCR. Finally, beta-4 integrin (β4) expression was assessed by ICC and WB. Faster epithelial healing was observed in NGF-treated corneas compared with controls (P < 0.01). These corneas showed increased proliferation, TrkA upregulation, and enhanced MMP-9 presence (P < 0.01). In vitro, faster spreading and migration were observed in response to NGF (P < 0.01). Enhanced proliferation, as well as enhanced TrkA and MMP-9 expression, and decreased β4 levels were observed after adding NGF (P < 0.01). NGF plays a major role during the epithelial healing process by promoting migration, a process that is accelerated by cell spreading. This effect is mediated by both the upregulation of MMP-9 and cleavage of β4 integrin.

Correlation between Beta1 integrin expression and prognosis in clinically localized prostate cancer

Integrins are transmembrane glycoprotein receptors that regulate cell-matrix interactions, thus functioning as sensors from the environment. They also act as cell adhesion molecules that are responsible for the maintenance of the normal epithelial phenotype. Some studies have reported a correlation between carcinogenesis and changes in integrin expression, especially β1 integrin, however its role in prostate cancer (PC) is unclear. The aim of our study was to evaluate the expression of β1 integrin in localized PC and to correlate the pattern of expression with recurrence after surgical treatment. Methods For this case-control study, we retrospectively selected surgical specimens from 111 patients with localized PC who underwent radical prostatectomy. Recurrence was defined as a PSA level exceeding 0.2 ng/mL after surgery, and the median follow-up was 123 months. Integrin expression was evaluated by immunohistochemistry in a tissue microarray containing two samples from each tumor. We employed a semiquantitative analysis and considered a case as positive when the expression was strong and diffusely present. There was a loss of 11 cases during the tissue micro array assembling. β1 expression was positive in 79 of the 100 evaluated cases (79%). The univariate and multivariate analyses showed that the negative expression of β1 integrin was associated with biochemical recurrence (p = 0.047) and time to recurrence after radical prostatectomy (p = 0.023). When β1 was negative, the odds ratio for recurrence was 2.78 times higher than that observed in the positive cases [OR = 2.78, p = 0.047, IC 95% (1.01-7.66)]. The loss of β1 integrin immune expression was correlated with biochemical recurrence in patients treated with radical prostatectomy for localized PC.

SAT0063 Functional analysis of the primary cilium in rheumatoid arthritis synovial fibroblasts

BackgroundThe primary cilium is a microtubule-based, polarized, antenna structure that emanates from the cell surface of most mammalian cell types. It serves as a sensor that mediates reactions to mechanical...

Abstract 2312: Targeting beta1 integrin potentiates antiangiogenic therapy and inhibits growth of bevacizumab-resistant glioblastoma multiforme.

Abstract INTRODUCTION: Antiangiogenic therapies like bevacizumab hold promise for cancer treatment. However, the development of acquired resistance, including an aggressive mesenchymal phenotype, limit utility of these agents. We previously published a report in which microarray and PCR analysis revealed beta1 integrin to be upregulated in bevacizumab-resistant glioblastomas (BRGs). Because the beta1 integrin subunit mediates tumor-microenvironment interactions, we further investigated its role in mesenchymal-type resistance to anti-angiogenic therapy. METHODS: Immunostaining of BRG specimens and genetic or pharmacologic beta1 targeting in BRG cells and BRG-derived xenografts were used to study the role of beta1 integrin in anti-angiogenic therapy resistance in vitro and in vivo. Cells were transduced to express beta1-GFP fusion protein to measure beta1 integrin turnover in focal adhesions using fluorescence recovery after photobleaching (FRAP). RESULTS: Beta1 integrin and downstream beta1-effector focal adhesion kinase immunostaining were upregulated in 75% and 86% of BRGs, resepectively, compared to pre-treatment paired specimens. Flow cytometry revealed 8-fold upregulation of beta1 integrin in BRG-derived primary GBM cells compared to cells from bevacizumab-naïve GBMs (P&amp;lt;0.05). FRAP revealed more rapid beta1 integrin turnover in focal adhesion kinases in BRG-derived cells than in bevacizumab-naïve GBM cells, with 4-fold more time to achieve 37% recovery in bevacizumab-naïve GBM cells than in BRG-derived cells expressing a beta1-GFP fusion protein (95 versus 398 seconds; P&amp;lt;0.05). Hypoxia, which was increased 80% (as evidenced by CA9 immunostaining) (P&amp;lt;0.05) after bevacizumab resistance, increased beta1 integrin expression 30% in cultured bevacizumab-naïve GBM cells (P&amp;lt;0.01). Two hours of incubation with 10 and 100 ng/mL VEGF reduced adhesion of GBM cells to beta1 ligands fibronectin, collagen IV, and laminin by 10-50% in a dose-dependent fashion (P&amp;lt;0.05). BRGs demonstrated aggressive mesenchymal-like phenotype in vitro and growth of subcutaneous BRG xenografts was attenuated by beta1 antibody OS2966 (P&amp;lt;0.05). Addition of OS2966 allowed 20-fold dose reduction of bevacizumab per cycle in subcutaneous GBM-derived xenografts (P&amp;lt;0.05). Intracranial delivery of OS2966 through osmotic pumps in mice carrying intracranial BRG-derived xenografts over 28 days increased tumor cell apoptosis, decreased tumor cell invasiveness, and altered tumor cell morphology (P&amp;lt;0.05). CONCLUSIONS: Beta1 integrin expression and turnover in focal adhesion kinases is upregulated in BRGs due to 2 possible mechanisms - hypoxic upregulation and bevacizumab-induced VEGF depletion eliminating VEGF-mediated inhibition of beta1 activity. Beta1 inhibition is well tolerated in vivo and holds promise for disrupting anti-angiogenic therapy resistance. Citation Format: W. Shawn Carbonell, Michael De Lay, Arman Jahangiri, Catherine C. Park, Manish Aghi. Targeting beta1 integrin potentiates antiangiogenic therapy and inhibits growth of bevacizumab-resistant glioblastoma multiforme. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2312. doi:10.1158/1538-7445.AM2013-2312

Abstract B15: Invasive breast cancer amplifies lymphangiogenic signals in tumor microenvironment through integrin beta4 expressing cancer-associated fibroblasts

Abstract Lymphangiogensis is widely acknowledged as a major route of breast cancer metastasis and a critical alteration of tumor microenvironment (TME) for tumor progression. But, most of it has remained still unknown. Previously, we found that invasive ductal carcinoma (IDC), not ductal carcinoma in situ (DCIS), upregulates integrin alpha6beta4 in the fibrosis adjacent to tumor burden, and that MDA-MB-231 induces integrin beta4 neoexpression in cancer-associated fibroblasts (CAFs) via cell-to-cell contract. Integrin (alpha6)beta4 overexpression is often detected in malignant tumors and negatively associated with patient survivor. In addition, integrin alpha6beta4 was reported to regulate eIF-4E activity leading to vascular endothelial growth factor (VEGF) translation in cancer cells. It is, therefore, plausible that invasive breast cancer cells increase lymphangiogenic signals in TME through driving integrin beta4 expression in CAFs. To verify our hypothesis, we induced integrin beta4 overexpression in CAFs using co-culture with MDA-MB-231 or transfection with pRK5_beta4 plasmid, and then observed tube formation of human dermal lymphatic endothelial cells (HDLEC) by integrin beta4-expressing CAFs in 3D co-culture model. VEGF-D, known as a major factor of lymphangiogenesis, was highly upregulated in integrin beta4-expressing CAFs, which promoted tube formation of HDLEC compared to normal CAFs and MDA-MB-231. Interestingly, the level of VEGF-D expression in MDA-MB-231 was much lower than that of integrin beta4-expressing CAFs before and after co-culture. Taken together, invasive breast cancer may amplify lymphangiogenic signals in TME by inducing integrin beta4-expressing CAFs, and integrin beta4 neoexpression in CAFs by breast cancer may be a pivotal process for lymphangiogensis. Citation Format: Baek Gil Kim, Yoon Pyo Choi, Suki Kang, Joo Hyun Lee, Nam Hoon Cho. Invasive breast cancer amplifies lymphangiogenic signals in tumor microenvironment through integrin beta4 expressing cancer-associated fibroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B15.

Leukocyte Beta2-Integrins; Genes and Disease

Integrins are heterodimeric transmembrane receptors that mediate cell-cell and cell-extracellular matrix interactions. Beta2-integrins are expressed exclusively in leukocytes and mediate many important functions in the immune system. Beta2-integrin genes are important in the pathologies of several diseases and genetic syndromes. These include Leukocyte Adhesion Deficiency (LAD) and Systemic Lupus Erythematosus (SLE), disorders which lie at opposite ends of the spectrum of immunological diseases. In LAD-I and LAD-III syndromes, beta2-integrin expression or function is reduced or absent. In SLE, genetic variants of the ITGAM gene, which encodes for the alpha M/CD11b chain of the beta2-integrin Mac-1,are associated with SLE development. In this mini review we summarise current knowledge regarding the involvement of beta2-integrins in LAD and SLE. Interestingly, dysfunctional beta2-integrins have been linked to both disorders, shedding light on the diverse roles of these receptors in the immune system.

Proteins involved in regulating bone invasion in skull base meningiomas

BackgroundBone invasive skull base meningiomas are a subset of meningiomas that present a unique clinical challenge due to brain and neural structure involvement and limitations in complete surgical resection, resulting in higher recurrence and need for repeat surgery. To date, the pathogenesis of meningioma bone invasion has not been investigated. We investigated immunoexpression of proteins implicated in bone invasion in other tumor types to establish their involvement in meningioma bone invasion.MethodsRetrospective review of our database identified bone invasive meningiomas operated on at our institution over the past 20 years. Using high-throughput tissue microarray (TMA), we established the expression profile of osteopontin (OPN), matrix metalloproteinase-2 (MMP2), and integrin beta-1 (ITGB1). Differential expression in tumor cell and vasculature was evaluated and comparisons were made between meningioma anatomical locations.ResultsMMP2, OPN, and ITGB1 immunoreactivity was cytoplasmic in tumor and/or endothelial cells. Noninvasive transbasal meningiomas exhibited higher vascular endothelial cell MMP2 immunoexpression compared to invasive meningiomas. We found higher expression levels of OPN and ITGB1 in bone invasive transbasal compared to noninvasive meningiomas. Strong vascular ITGB1 expression extending from the endothelium through the media and into the adventitia was found in a subset of meningiomas.ConclusionsWe have demonstrated that key proteins are differentially expressed in bone invasive meningiomas and that the anatomical location of bone invasion is a key determinant of expression pattern of MMP1, OPN, and ITGB1. This data provides initial insights into the pathophysiology of bone invasion in meningiomas and identifies factors that can be pursued as potential therapeutic targets.

Effect of TiO2 scaffolds coated with alginate hydrogel containing a proline‐rich peptide on osteoblast growth and differentiation in vitro

The aim of this study was to investigate the effect of TiO2 scaffold (SC) coated with an alginate hydrogel containing a proline-rich peptide (P2) on osteoblast proliferation and differentiation in vitro. Peptide release was evaluated and a burst release was observed during the first hours of incubation, and then progressively released overtime. No changes were observed in the cytotoxicity after 48 h of seeding MC3T3-E1 cells on the coated and uncoated TiO2 SC. The amount of cells after 7 days was higher on uncoated TiO2 SC than on alginate-coated TiO2 SC, measured by DNA content and scanning electron microscope imaging. In addition, while lower expression of integrin beta1 was detected for alginate-coated TiO2 SC at this time point, similar gene expression was observed for other integrins, fibronectin-1, and several osteoblast differentiation markers. After 21 days, gene expression of integrin beta3, fibronectin-1, osterix, and collagen-I was increased in alginate-coated compared to TiO2 SC. Moreover, increased gene expression of integrin alpha8, bone morphogenetic protein 2, interleukin-6, and collagen-I was found on P2 alginate-coated TiO2 SC compared to alginate-coated TiO2 SC. In conclusion, our results indicate that alginate-coated TiO2 SC can act as a matrix for delivery of proline-rich peptides increasing osteoblast differentiation. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Synthetic Peptide-Acrylate Surface for Self-Renewal of Human Retinal Progenitor Cells

Human retinal progenitor cells (hRPCs), isolated from fetal retina, require extracellular matrix proteins such as fibronectin or laminin for successful attachment and self-renewal in vitro. Here we have shown that a novel synthetic vitronectin-mimicking surface supports self-renewal and multipotency of hRPCs in a chemically defined culture system. The morphology, adhesion, and proliferation of hRPC were equivalent on a novel vitronectin-mimicking surface (Synthemax) compared to a fibronectin-coated surface. When evaluated using real-time polymerase chain reaction, Western blotting, and flow cytometry, both surfaces maintained self-renewal of hRPCs, as shown by similar expression levels of Sox2, Nestin, cMyc, Klf4, and Pax6, with no change in integrin beta1 and integrin alpha5 expression. We suggest that the use of synthetic, xeno-free surfaces such as Synthemax will be useful for basic research studies, as well as development of translational strategies aimed at using stem cell transplantation to treat disease.

TGF-beta-1 up-regulates extra-cellular matrix production in mouse hepatoblasts

Fetal liver is the major embryonic hematopoietic organ and is extrinsically colonized by circulating hematopoietic stem cells (HSCs). Integrin beta-1 expression on HSCs is crucial for colonization, suggesting that interaction of Integrin beta-1 with extra-cellular matrix (ECM) factors promotes HSC adherence to fetal liver. However, little is known about how ECM production is regulated in fetal liver. Here we used flow cytometry to sort fetal liver compartments and detected ECM gene and protein expression predominantly in sorted hepatoblasts. mRNA and protein analysis suggested that TGF-beta-1 expressed by hepatoblasts, sinusoid endothelial cells and hematopoietic cells, binds to the TGF-beta receptor type-2 expressed on hepatoblasts to stimulate ECM production. Intra-cardiac injection of TGF-inhibitors into mouse embryos dramatically decreased fetal liver ECM gene expression. Taken together, our observations suggest that hepatoblasts predominantly produce ECM factors under control of TGF-beta-1 in fetal liver.

Immunophenotypic Identification and Characterization of Tumor Cells and Infiltrating Cell Populations in Meningiomas

Meningiomas are primary tumors of the central nervous system composed of both neoplastic and other infiltrating cells. We determined the cellular composition of 51 meningioma samples by multiparameter flow cytometric (MFC) immunophenotyping and investigated the potential relationship between mRNA and protein expression levels of neoplastic cells. For immunophenotypic, morphologic, and cytogenetic characterization of individual cell populations, a large panel of markers was used together with phagocytic/endocytic functional assays and MFC sorting. Overall, our results revealed coexistence of CD45(-) neoplastic cells and CD45(+) immune infiltrating cells in all meningiomas. Infiltrating cells included tissue macrophages, with an HLA-DR(+)CD14(+)CD45(+)CD68(+)CD16(-/+)CD33(-/+) phenotype and high phagocytic/endocytic activity, and a small proportion of cytotoxic lymphocytes (mostly T CD8(+) and natural killer cells). Tumor cells expressed multiple cell adhesion proteins, tetraspanins, HLA-I/HLA-DR molecules, complement regulatory proteins, cell surface ectoenzymes, and growth factor receptors. Noteworthy, the relationship between mRNA and protein levels was variable, depending on the proteins evaluated and the level of infiltration by immune cells. In summary, our results indicate that MFC immunophenotyping provides a reliable tool for the characterization of the patterns of protein expression of different cell populations coexisting in meningioma samples, with a more accurate measure of gene expression profiles of tumor cells at the functional/protein level than conventional mRNA microarray, independently of the degree of infiltration of the tumor by immune cells.