The autophagy protein Ambra1 regulates gene expression by supporting novel transcriptional complexes.

Ambra1 is considered an autophagy and trafficking protein with roles in neurogenesis and cancer cell invasion. Here, we report that Ambra1 also localizes to the nucleus of cancer cells, where it has a novel nuclear scaffolding function that controls gene expression. Using biochemical fractionation and proteomics, we found that Ambra1 binds to multiple classes of proteins in the nucleus, including nuclear pore proteins, adaptor proteins such as FAK and Akap8, chromatin-modifying proteins, and transcriptional regulators like Brg1 and Atf2. We identified biologically important genes, such as Angpt1, Tgfb2, Tgfb3, Itga8, and Itgb7, whose transcription is regulated by Ambra1-scaffolded complexes, likely by altering histone modifications and Atf2 activity. Therefore, in addition to its recognized roles in autophagy and trafficking, Ambra1 scaffolds protein complexes at chromatin, regulating transcriptional signaling in the nucleus. This novel function for Ambra1, and the specific genes impacted, may help to explain the wider role of Ambra1 in cancer cell biology.

Stem cell enriched lipotransfer reverses the effects of fibrosis in systemic sclerosis.

Oro-facial fibrosis in systemic sclerosis (Scleroderma;SSc) has a major impact on mouth function, facial appearance, and patient quality of life. Lipotransfer is a method of reconstruction that can be used in the treatment of oro-facial fibrosis. The effect of this treatment not only restores oro-facial volume but has also been found to reverse the effects of oro-facial fibrosis. Adipose derived stem cells (ADSCs) within the engrafted adipose tissue have been shown to be anti-fibrotic in SSc and are proposed as the mechanism of the anti-fibrotic effect of lipotransfer. A cohort of 62 SSc patients with oro-facial fibrosis were assessed before and after stem cell enriched lipotransfer treatment. Clinical evaluation included assessment of mouth function using a validated assessment tool (Mouth Handicap in Systemic Sclerosis Scale-MHISS), validated psychological measurements and pre and post-operative volumetric assessment. In addition, to understand the mechanism by which the anti-fibrotic effect of ADSCs occur, SSc derived fibroblasts and ADSCs from this cohort of patients were co-cultured in direct and indirect culture systems and compared to monoculture controls. Cell viability, DNA content, protein secretion of known fibrotic mediators including growth factor- ß1 (TGF ß-1) and connective tissue growth factor (CTGF) using ELISA analysis and fibrosis gene expression using a fibrosis pathway specific qPCR array were evaluated. Mouth function (MHISS) was significantly improved (6.85±5.07) (p<0.0001) after treatment. All psychological measures were significantly improved: DAS 24 (12.1±9.5) (p<0.0001); HADS-anxiety (2.8±3.2) (p<0.0001), HADS-depression (2.0±3.1) (p<0.0001); BFNE (2.9 ± 4.3) (p<0.0001); VAS (3.56±4.1) (p<0.0001). Multiple treatments further improved mouth function (p<0.05), DAS (p<0.0001) and VAS (p = 0.01) scores. SSc fibroblast viability and proliferation was significantly reduced in co-culture compared to monoculture via a paracrine effect over 14 days (p < 0.0001). Protein secretion of transforming growth factor (TGF-ß1) and connective tissue growth factor (CTGF) was significantly reduced in co-culture compared to monoculture (p < 0.0001). Multiple fibrosis associated genes were down regulated in SSc co-culture compared to monoculture after 14 days including Matrix metalloproteinase-8 (MMMP-8), Platelet derived growth factor-ß (PDGF-ß) and Integrin Subunit Beta 6 (ITG-ß6). Autologous stem cell enriched lipotransfer significantly improved the effects of oro-facial fibrosis in SSc in this open cohort study. Lipotransfer may reduce dermal fibrosis through the suppression of fibroblast proliferation and key regulators of fibrogenesis including TG-ß1 and CTGF. Our findings warrant further investigation in a randomised controlled trial.

ß8 Integrin Mediates Pancreatic Cancer Cell Radiochemoresistance.

Pancreatic ductal adenocarcinoma (PDAC) stroma, composed of extracellular matrix (ECM) proteins, promotes therapy resistance and poor survival rate. Integrin-mediated cell/ECM interactions are well known to control cancer cell survival, proliferation, and therapy resistance. Here, we identified ß8 integrin in a high-throughput knockdown screen in three-dimensional (3D), ECM-based cell cultures for novel focal adhesion protein targets as a critical determinant of PDAC cell radiochemoresistance. Intriguingly, ß8 integrin localizes with the golgi apparatus perinuclearly in PDAC cells and resection specimen from PDAC patients. Upon radiogenic genotoxic injury, ß8 integrin shows a microtubule-dependent perinuclear-to-cytoplasmic shift as well as strong changes in its proteomic interactome regarding the cell functions transport, catalysis, and binding. Parts of this interactome link ß8 integrin to autophagy, which is diminished in the absence of ß8 integrin. Collectively, our data reveal ß8 integrin to critically coregulate PDAC cell radiochemoresistance, intracellular vesicle trafficking, and autophagy upon irradiation. IMPLICATIONS: This study identified ß8 integrin as an essential determinant of PDAC cell radiochemosensitivity and as a novel potential cancer target.

ß6 -integrin serves as a novel serum tumor marker for colorectal carcinoma.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide and the need for novel biomarkers and therapeutic strategies to improve diagnosis and surveillance is obvious. This study aims to identify ß6 -integrin (ITGB6) as a novel serum tumor marker for diagnosis, prognosis, and surveillance of CRC. ITGB6 serum levels were validated in retro- and prospective CRC patient cohorts. ITGB6 serum levels were analyzed by ELISA. Using an initial cohort of 60 CRC patients, we found that ITGB6 is present in the serum of CRC, but not in non-CRC control patients. A cut-off of ≥2 ng/mL ITGB6 reveals 100% specificity for the presence of metastatic CRC. In an enlarged study cohort of 269 CRC patients, ITGB6 predicted the onset of metastatic disease and was associated with poor prognosis. Those data were confirmed in an independent, prospective cohort consisting of 40 CRC patients. To investigate whether ITGB6 can also be used for tumor surveillance, serum ITGB6-levels were assessed in 26 CRC patients, pre- and post-surgery, as well as during follow-up visits. After complete tumor resection, ITGB6 serum levels declined completely. During follow-up, a new rise in ITGB6 serum levels indicated tumor recurrence or the onset of new metastasis as confirmed by CT scan. ITGB6 was more accurate for prognosis of advanced CRC and for tumor surveillance as the established marker carcinoembryonic antigen (CEA). Our findings identify ITGB6 as a novel serum marker for diagnosis, prognosis, and surveillance of advanced CRC. This might essentially contribute to an optimized patient care.

Peripheral Th17 cells expressing ß7 intestinal homing receptor in recent and chronic HIV infections.

The objective of this study was to conduct an analysis of peripheral blood Th17 cells with the ability to home to gut mucosa (CD4+ Th17+ ß7+ ) during recent or chronic human immunodeficiency virus (HIV) infections. The relationship between HIV load and systemic inflammation markers was studied. Twenty-five patients with recent (n = 10) or chronic (n = 15) untreated HIV infections; 30 treated HIV-infected patients with undetectable HIV load at the time of inclusion and 30 healthy controls were included. Bacterial translocation markers (16S rDNA), soluble CD14 (sCD14) and interleukin (IL)-6 monocyte activation parameters, CD4/CD8 ratio and T helper type 17 (Th17) subpopulations [CD4+ Th17+ expressing the IL-23 receptor (IL-23R) or ß7] were analysed at baseline and after 6 and 12 months of anti-retroviral therapy (ART). 16S rDNA was detected in all patients. Significantly increased serum levels of sCD14 and IL-6 and a decreased CD4/CD8 ratio were observed in patients. Similar percentages of CD4+ IL-23R+ and CD4+ Th17+ ß7+ cells were observed in healthy controls and patients at baseline. After 12 months of therapy, patients with a recent HIV infection showed significant increases of CD4+ IL-23R+ and CD4+ Th17+ ß7+ cell percentages and a decrease in IL-6 levels, although 16S rDNA continued to be detectable in all patients. No significant differences were observed in Th17 subpopulations in patients with chronic HIV infection after therapy. Early initiation of ART helps to increase the number of Th17 cells with the ability to home to the intestinal mucosa and to partially restore gut mucosal homeostasis. These results provide a rationale for initiating ART during the acute phase of HIV infection.

Novel probiotics isolated from a Japanese traditional fermented food, Funazushi, attenuates DSS-induced colitis by increasing the induction of high integrin αv/ß8-expressing dendritic cells.

BACKGROUND: We isolated two novel probiotics strains (s193 and s292) from Funazushi, which is a traditional Japanese fermented food, and evaluated its effects on DSS-induced colitis to determine the possible underlying mechanisms. METHODS: A single colony from homogenized Funazushi was isolated by its ability to suppress TNF-α in RAW 264.7. Effect of probiotics on colonic inflammation induced by DSS was evaluated. Effect of probiotics on Treg induction by CD11c+ dendritic cells (DCs) of MLNs were analyzed. RESULTS: Two novel probiotics strains classified into the genus Lactobacillus were isolated (s193 and s292), and those strains showed stronger anti-inflammatory effects on DSS-induced colitis than those of L. gasseri isolated from the gut. mRNA expression ß8 integrin in CD11c+DCs of MLNs and the number of Tregs in the large intestine were significantly increased by s193 and s292 administration compared with L. gasseri administration. Bone marrow DCs treated with s193 and s292 highly increased ß8 integrin, and those cells strongly induced differentiation of CD4+ T cells into Tregs. Differentiation of Tregs was remarkably inhibited by anti-ß8 integrin antibody treatment. CONCLUSIONS: Strains s193 and s292 demonstrate strong anti-inflammatory effects on DSS-induced colitis through induction of ß8 integrin expression on DCs. Our results suggested that Japanese traditional fermented foods are valuable sources for probiotics that are effective for IBD therapy and treatment.

Epigenetic regulation of integrin ß6 transcription induced by TGF-ß1 in human oral squamous cell carcinoma cells.

Overexpression of integrin αvß6 is believed to play an important role in the invasion and metastasis of oral squamous cell carcinoma (OSCC). However, little is known about the molecular mechanisms leading to αvß6 upregulation in OSCC. As the integrin ß6 (ITGB6) is the only partner with αv, the expression of αvß6 is dependent on ITGB6, it is, therefore, pivotal to investigate the mechanisms underlying ITGB6 overexpression in OSCC. We previously reported the cloning and characterization of human ITGB6 gene. In the current study, we further investigated the molecular mechanisms of ITGB6 expression and the upregulation by carcinogenesis related cytokine-transforming growth factor-ß1 (TGF-ß1) in OSCC cells. We first demonstrated that TGF-ß1 can induce ITGB6 mRNA and protein express in a time and concentration dependent manner, and the induced-ITGB6 mRNA was not due to increase the mRNA stability, but regulated at transcriptional level. By using a luciferase reporter assay, site-mutation, RNA interference, and chromatin immunoprecipitation assay, we revealed for the first time that JunB, a member of the activator protein-1 (AP-1) family, is involved in the positive regulation to the ITGB6 transcription induced by TGF-ß1 in OSCC cells. Furthermore, our data also demonstrated that histone acetyltransferase (HAT) CBP mediated histone H3 and H4 hyperacetylation, and RNA Polymerase II recruitment to ITGB6 promoter, facilitated the binding of transcription factor JunB to ITGB6 promoter after TGF-ß1 stimulation. Collectively, these findings demonstrate that JunB and CBP-mediated histone hyperacetylation are responsible for TGF-ß1 induced ITGB6 transcription in OSCC cells, suggesting that epigenetic mechanisms are responsible for the active transcription expression of ITGB6 induced by TGF-ß1 in OSCC cells.

IL-4 induces the formation of multinucleated giant cells and expression of ß5 integrin in central giant cell lesion.

BACKGROUND: It is now well established that IL-4 has a central role in the development of monocytes to multinucleated giant cells (MGCs) by inducing the expression of integrins on the surface of monocytes. The aim of this study was to investigate the potential role of IL-4 in induction of ß5 integrin expression in the peripheral blood samples of patients with giant cell granuloma. MATERIAL AND METHODS: Monocytes were isolated from peripheral blood samples of patients with central giant cell granuloma (CGCG) and healthy controls using human Monocyte Isolation Kit II. Isolated monocytes were then cultured in the absence or presence of IL-4 (10 and 20 ng/mL), and following RNA extraction and cDNA synthesis, Real-time PCR was performed to determine the level of ß5 integrin expression. The formation of CGCGs and morphological analyses were done under light microscopy. For confirmation of CGCGs, immunocytochemistry technique was also carried out by anti-RANK (receptor-activator of NF-κB ligand) antibody. RESULTS: In both patient and control groups, ß5 levels were significantly enhanced by increasing the IL-4 dose from 10 to 20 ng/mL. In addition, these differences were significant between patient and control groups without IL-4 treatment. On the other hand, the number of cells which expressed RANK and therefore the number of giant cells were significantly higher in the patient group in comparison to controls, as assessed by immunohistochemistry evaluations. CONCLUSIONS: In this study, we showed an elevation in the expression levels of ß5 integrin when stimulated by IL-4. It is strongly indicated that this integrin acts as an important mediator during macrophage to macrophage fusion and development of giant cells.

Mass cytometry panel optimization through the designed distribution of signal interference.

Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making it a promising technology for innovating biomarker discovery. However, in order for this potential to be fully realized, best practices in panel design need to be further defined in order to achieve consistency and reproducibility in data analysis. Of particular importance are controls that reveal, and panel design principles that mitigate the effects of signal interference or overlap. We observed a disparity between the staining profiles of two noncompeting anti- integrin ß7 mAbs and hypothesized that signal interference was responsible. A mass-minus-one (MMO) control was applied and demonstrated that signal overlap caused the perceived interclonal discrepancy in ß7 expression. Panel redesign in consideration of mass-cytometry specific interference dynamics dramatically improved concordance between both mAbs by redistributing background signals caused by overlap. These studies visualize how signal overlap can complicate mass cytometry data interpretation and demonstrate how the rational distribution of interference can greatly improve panel design and data quality. © 2016 International Society for Advancement of Cytometry.

Water-extracted Perilla frutescens increases endometrial receptivity though leukemia inhibitory factor-dependent expression of integrins.

The leaves and stems of Perilla frutescens var. acuta Kudo (PF) have been used to prevent threatened abortion in traditional medicine in the East Asian countries. Because reduced receptivity of endometrium is a cause of abortion, we analyzed the action of PF on the endometrial receptivity. PF increased the level of leukemia inhibitory factor (LIF), a major cytokine regulating endometrial receptivity, and LIF receptor in human endometrial Ishikawa cells. The PF-induced LIF expression was mediated by c-jun N-terminal kinase (JNK) and p38 pathways. Adhesion between Ishikawa cells and trophoblastic JAr cells stimulated by PF treatment was abolished by knock down of LIF expression or antagonism of LIFR. In addition, the expressions of integrin ß3 and ß5 were increased by PF treatment in Ishikawa cells. The PF-induced expression of integrin ß3 and ß5 was reduced with an LIFR antagonist. Neutralization of both integrins successfully blocked PF-stimulated adhesion of JAr cells and Ishikawa cells. These results suggest that PF enhanced the adhesion between Ishikawa cells and JAr cells by increasing the expression of integrin ß3 and ß5 via an LIF-dependent pathway. Given the importance of endometrial receptivity in successful pregnancy, PF can be a novel and effective candidate for improving pregnancy rate.

Polymerase delta-interacting protein 2 regulates collagen accumulation via activation of the Akt/mTOR pathway in vascular smooth muscle cells.

OBJECTIVES: Polymerase delta interacting protein 2 (Poldip2) has previously been implicated in migration, proliferation and extracellular matrix (ECM) production in vascular smooth muscle cells. To better understand the role of Poldip2 in ECM regulation, we investigated the mechanism responsible for collagen I accumulation in Poldip2(+/-) mouse aortic smooth muscle cells (MASMs). APPROACH AND RESULTS: Protein degradation and protein synthesis pathways were investigated. Depletion of Poldip2 had no effect on proteasome activity, but caused a partial reduction in autophagic flux. However, the rate of collagen I degradation was increased in Poldip2(+/-) vs. Poldip2(+/+) MASMs. Conversely, activation of the PI3K/Akt/mTOR signaling pathway, involved in regulation of protein synthesis, was significantly elevated in Poldip2(+/-) MASMs as was ß1-integrin expression. Suppressing mTOR signaling using Akt inhibitor or rapamycin and reducing ß1-integrin expression using siRNA prevented the increase in collagen I production. While collagen I and fibronectin were increased in Poldip2(+/-) MASMs, overall protein synthesis was not different from that in Poldip2(+/)(+)MASMs, suggesting selectivity of Poldip2 for ECM proteins. CONCLUSIONS: Poldip2(+/-) MASMs exhibit higher ß1-integrin expression and activity of the PI3K/Akt/mTOR signaling pathway, leading to increased ECM protein synthesis. These findings have important implications for vascular diseases in which ECM accumulation plays a role.

Integrin ß6 acts as an unfavorable prognostic indicator and promotes cellular malignant behaviors via ERK-ETS1 pathway in pancreatic ductal adenocarcinoma (PDAC).

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most deadly cancers and is expected to become the second leading cause of cancer death by 2030. Despite extensive efforts to improve surgical treatment, limited progress has been made. Increasing evidence indicates that integrin ß6 plays a crucial role in carcinoma invasion and metastasis. However, the expression and role of ß6 in PDAC remain largely unknown. In the present study, we investigated the expression of ß6 in PDAC and its potential value as a prognostic factor and therapeutic target. ß6 upregulation was identified as an independent unfavorable prognostic indicator. Integrin ß6 markedly promoted the proliferation and invasion of pancreatic carcinoma cells and induced ETS1 phosphorylation in an ERK-dependent manner, leading to the upregulation of matrix metalloprotease-9, which is essential for ß6-mediated invasiveness of pancreatic carcinoma cells. Accordingly, small interfering RNA-mediated silencing of integrin ß6 markedly suppressed xenograft tumor growth in vivo. Taken together, our results suggest that integrin ß6 plays important roles in the progression of pancreatic carcinoma and contributes to reduced survival times, and may serve as a novel therapeutic target for the treatment of PDAC.

The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of integrin-ß8 in prostate cancer cells.

BACKGROUND: The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways. METHODS: We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion. RESULTS: We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin ß8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion. CONCLUSIONS: These results reveal that EphB4 regulates integrin ß8 expression and that integrin ß8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin ß8 may be a new treatment strategy for prostate cancer.

Differential spheroid formation by oral cancer cells.

Squamous cell carcinomas (SCC) make up 96% of all oral cancers. Most laboratory SCC studies grow cells as a monolayer, which does not accurately represent the disease in vivo. We used a more relevant multicellular spheroid (MCS) model to study this disease. The SCC9ß6KDFyn cell line, which expresses full-length ß6 and a kinase dead Fyn formed the largest MCS. Cell adhesive properties are dynamic and N-cadherin was increased in the largest MCS. c-Raf mediates the survival of tumor cells and was consistently expressed both in monolayers and in the MCS by SCC9ß6D1 cells which lack the ß6 cytoplasmic tail and, do not activate Fyn. SCC9ß6KDFyn cells also express high levels of c-Raf when grown as spheroids in which Fyn suppression stimulates MCS formation. Tumor microenvironment and growth patterns modulate cell behavior and suppression of Fyn kinase may promote MCS growth.

Cisplatin at sub-toxic levels mediates integrin switch in lung cancer cells.

BACKGROUND: Resistance to chemotherapeutic agents, as well as enhanced metastasis, have been frequently reported in lung cancer. MATERIALS AND METHODS: Cytotoxicity and proliferative effects of cisplatin on H460 lung cancer cells were evaluated by the MTT assay. Migration capacity was evaluated by the wound healing assay. The number of filopodia per cell were detected by rhodamine-phalloidin staining assay. The changes of protein levels of integrins, and migration-related proteins in response to cisplatin at sub-toxic concentrations were determined by western blotting. RESULTS: Herein we demonstrate for the first time that exposure to low concentrations of cisplatin results in increase of cell motility with the alteration of integrin expression. Cisplatin-treated cells exhibited a significant increase in the number of filopodia per cell in correlation with enhanced migration. Migration regulatory proteins, namely activated forms of focal-adhesion kinase (FAK) and ATP-dependent tyrosine kinase (AKT), were found to significantly be up-regulated in cisplatin-treated cells in comparison to those of the non-treated control. Active Rho A-GTP and Rac-GTP were found to be increased in accordance with activation of FAK/AKT signals. Furthermore, we found that such migration enhancement may be in part due to the integrin switch mediated by cisplatin treatment. Cisplatin induced a dramatic alteration in the integrin expression pattern by up-regulating integrin α4, αv, ß1, and ß5 which were previously reported to increase cell motility, while it had no effect on integrin α5, and ß3. CONCLUSION: As the integrin switch is a hallmark of highly aggressive cancer, these findings may provide insights for better understanding of cancer cell adaptation after exposure to cisplatin.

Effects of negative pressure wound therapy on mesenchymal stem cells proliferation and osteogenic differentiation in a fibrin matrix.

Vacuum-assisted closure (VAC) negative pressure wound therapy (NPWT) has been proven to be an effective therapeutic method for the treatment of recalcitrant wounds. However, its role in bone healing remains to be unclear. Here, we investigated the effects of NPWT on rat periosteum-derived mesenchymal stem cells (P-MSCs) proliferation and osteoblastic differentiation in a 3D fibrin matrix. P-MSCs underwent primary culture for three passages before being used to construct cell clots. The fibrin clots were incubated with NPWT under continuous suction at -125 mmHg in a subatmospheric perfusion bioreactor. Clots exposed to atmospheric pressure served as the static control. Compared to the control group, cell proliferation significantly increased in NPWT group after incubation for 3 days. There was no statistical difference in apoptosis rate between two groups. The ALP activity and mineralization of P-MSCs all increased under continuous suction. The expressions of collagen type 1 and transcription factor Cbfa-1 were higher at the 1-, 3-, and 7-day timepoints and the expressions of osteocalcin and integrin ß5 were higher at the 3-, and 7-day timepoints in the NPWT group. These results indicate that a short time treatment with NPWT, applied with continuous suction at -125 mmHg, can enhance cellular proliferation of P-MSCs and induce the differentiation toward an osteogenic phenotype. The mechanotransduction molecule integrin ß5 was found to be highly expressed after NPWT treatment, which indicates that NPWT may play a positive role in fracture healing through enhance bone formation and decrease bone resorption.

Release of active TGF-ß1 from the latent TGF-ß1/GARP complex on T regulatory cells is mediated by integrin ß8.

Activated T regulatory cells (Tregs) express latent TGF-ß1 on their cell surface bound to GARP. Although integrins have been implicated in mediating the release of active TGF-ß1 from the complex of latent TGF-ß1 and latent TGF-ß1 binding protein, their role in processing latent TGF-ß1 from the latent TGF-ß1/GARP complex is unclear. Mouse CD4(+)Foxp3(+) Treg, but not CD4(+)Foxp3(-) T cells, expressed integrin ß8 (Itgb8) as detected by quantitative RT-PCR. Itgb8 expression was a marker of thymically derived (t)Treg, because it could not be detected on Foxp3(+)Helios(-) Tregs or on Foxp3(+) T cells induced in vitro. Tregs from Itgb8 conditional knockouts exhibited normal suppressor function in vitro and in vivo in a model of colitis but failed to provide TGF-ß1 to drive Th17 or induced Treg differentiation in vitro. In addition, Itgb8 knockout Tregs expressed higher levels of latent TGF-ß1 on their cell surface consistent with defective processing. Thus, integrin αvß8 is a marker of tTregs and functions in a cell intrinsic manner in mediating the processing of latent TGF-ß1 from the latent TGF-ß1/GARP complex on the surface of tTregs.

ß6 integrin induces the expression of metalloproteinase-3 and metalloproteinase-9 in colon cancer cells via ERK-ETS1 pathway.

We previously reported that ß6 integrin played an important role in the progression of colon cancer. In this study, we demonstrated that ß6 integrin induced the expression of MMP-3/MMP-9 and the invasion of colon cancer cells. Moreover, that function was abolished by the inhibition of ERK/MAPK pathways or knockdown of ETS1, an important transcription factor of MMP genes. Here, we showed that ß6 induced phosphorylation of ETS1 via the ERK/MAPK pathways, through which the MMP-3/MMP-9 promoters were stimulated, thereby leading to the up-regulation of MMP-3/MMP-9, and subsequent the invasion of colon cancer cells.

Autocrine galectin-1 promotes collective cell migration of squamous cell carcinoma cells through up-regulation of distinct integrins.

We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and ß5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.

Multiple drug resistant, tumorigenic stem-like cells in oral cancer.

An in vitro cell line model was established to exemplify tumor stem cell concept in oral cancer. We were able to identify CD147 expressing fractions in SCC172 OSCC cell line with differing Hoechst dye efflux activity and DNA content. In vivo tumorigenic assay revealed three fractions enriched with stem-like cells capable of undergoing mesenchymal transition and a non-tumorigenic fraction. The regeneration potential and transition of one fraction to other imitated the phenotypic switch and functional disparities evidenced during oral tumor progression. Knowledge of these additional stem-like subsets will improve understanding of stem cell based oral epithelial tumor progression from normal to malignant lesions.