USP10 promotes migration and cisplatin resistance in esophageal squamous cell carcinoma cells.

Cisplatin-based chemotherapy is the main treatment option for advanced or metastatic esophageal squamous cell carcinoma (ESCC). However, most ESCC patients develop drug resistance within 2 years after receiving cisplatin chemotherapy. Ubiquitin-specific protease 10 (USP10) is abnormally expressed in a variety of cancers, but the mechanistic roles of USP10 in ESCC are still obscure. Here, the effects of USP10 on the migration and cisplatin resistance of ESCC in vivo and in vitro and the underlying mechanisms have been investigated by bioinformatics analysis, RT-PCR, western blotting, immunoprecipitation, immunohistochemistry, cell migration and MTS cell proliferation assays, deubiquitination assay, and mouse tail vein injection model. USP10 was significantly up-regulated in ESCC tissues compared with adjacent normal tissues in both public databases and clinical samples and was closely associated with overall survival. Subsequent results revealed that USP10 contributed to the migration and cisplatin resistance of ESCC cells, while knocking down USP10 in cisplatin-resistant cells exhibited opposite effects in vitro and in vivo. Further Co-IP experiments showed that integrin ß1 and YAP might be targets for USP10 deubiquitination. Moreover, deficiency of USP10 significantly inhibited the migrative and chemo-resistant abilities of ESCC cells, which could be majorly reversed by integrin ß1 or YAP reconstitution. Altogether, USP10 was required for migration and cisplatin resistance in ESCC through deubiquinating and stabilizing integrin ß1/YAP, highlighting that inhibition of USP10 may be a potential therapeutic strategy for ESCC.

Combined use of CDAI and blood indices for assessing endoscopic activity in ileocolic Crohn's disease.

BACKGROUND: Mucosal healing has become the primary treatment target for patients with Crohn's disease (CD). We aimed to develop a noninvasive and convenient tool to evaluate the endoscopic activity in patients with ileocolic CD. METHODS: A retrospective multicenter study including 300 CD patients (training, 210 patients; test, 90 patients) was conducted at two tertiary referral centers. Independent risk factors associated with endoscopic activity were explored, which were then combined into a comprehensive index. The predictive performance was evaluated with the area under receiver operating characteristic curve (ROC). Cohen's Kappa was adopted to examine the consistency between each indicator and endoscopic activity. RESULTS: A total of 210 CD patients were recruited in the training cohort. We found that Crohn's Disease Activity Index (CDAI), C-reactive protein (CRP) and platelet-to-lymphocyte percentage ratio (PLpR) were independently associated with endoscopic activity. Additionally, the comprehensive index generated from the above three indices achieved good discrimination and performed better than CDAI in AUC (0.849 vs. 0.769, P < 0.05). This was further well demonstrated by the external test cohort, which showed good discrimination (AUC: 0.84, 95% CI: 0.744-0.936). Intra-individual comparison revealed the comprehensive index to be superior in the prediction of endoscopic activity. In the subgroup analysis, the AUC of comprehensive index was significantly higher than CDAI especially in inflammatory phenotype (0.824 vs. 0.751, P < 0.05). CONCLUSION: Combining CDAI, CRP and PLpR significantly improved the accuracy for predicting endoscopic activity in ileocolic CD, which can help better monitor an endoscopic flare.

Prognostic, Diagnostic, and Clinicopathological Significance of Circular RNAs in Pancreatic Cancer: A Systematic Review and Meta-Analysis.

Pancreatic cancer (PC) is a highly aggressive malignant tumor with a high mortality rate. It is urgent to find optimal molecular targets for the early diagnosis and treatment of PC. Here, we aimed to systematically analyze the prognostic, diagnostic, and clinicopathological significance of circular RNAs (circRNAs) in PC. Relevant studies were screened through PubMed, Web of Science, and other databases. The prognostic value of PC-associated circRNAs was assessed using the composite hazard ratio (HR), the diagnostic performance was assessed using the area under the summary receiver operator characteristic (SROC) curve (AUC), and the correlation with clinicopathological characteristics using the composite odds ratio (OR) was explored. In our study, 48 studies were included: 34 for prognosis, 11 for diagnosis, and 30 for correlation with clinicopathological characteristics. For prognosis, upregulated circRNAs were associated with poorer overall survival (OS) (HR = 2.02) and disease-free survival/progression-free survival (HR = 1.84) while downregulated circRNAs were associated with longer OS (HR = 0.55). Notably, the combination of circRNAs, including hsa_circ_0064288, hsa_circ_0000234, hsa_circ_0004680, hsa_circ_0071036, hsa_circ_0000677, and hsa_circ_0001460, was associated with worse OS (HR = 2.35). For diagnosis, the AUC was 0.83, and the pooled sensitivity and specificity were 0.79 and 0.73, respectively. For clinicopathologic characteristics, upregulated circRNAs were associated with poorer tumor differentiation, more nerve and vascular invasion, higher T stage, lymphatic metastasis, distant metastasis, advanced TNM stage, and higher preoperative CA19-9 level. In contrast, downregulated circRNAs were negatively associated with PC differentiation and lymphatic metastasis. Overall, our results showed that circRNAs are closely related to the prognosis and clinicopathological characteristics of PC patients and could be utilized for early diagnosis; thus, they are promising biomarkers for clinical application in PC.

Colorectal Cancer in Ulcerative Colitis: Mechanisms, Surveillance and Chemoprevention.

Patients with ulcerative colitis (UC) are at a two- to three-fold increased risk of developing colorectal cancer (CRC) than the general population based on population-based data. UC-CRC has generated a series of clinical problems, which are reflected in its worse prognosis and higher mortality than sporadic CRC. Chronic inflammation is a significant contributor to the development of UC-CRC, so comprehending the relationship between the proinflammatory factors and epithelial cells together with downstream signaling pathways is the core to elucidate the mechanisms involved in developing of CRC. Clinical studies have shown the importance of early prevention, detection and management of CRC in patients with UC, and colonoscopic surveillance at regular intervals with multiple biopsies is considered the most effective way. The use of endoscopy with targeted biopsies of visible lesions has been supported in most populations. In contrast, random biopsies in patients with high-risk characteristics have been suggested during surveillance. Some of the agents used to treat UC are chemopreventive, the effects of which will be examined in cancers in UC in a population-based setting. In this review, we outline the current state of potential risk factors and chemopreventive recommendations in UC-CRC, with a specific focus on the proinflammatory mechanisms in promoting CRC and evidence for personalized surveillance.

Adverse Impacts of Temporomandibular Disorders Symptoms and Tooth Loss on Psychological States and Oral Health-Related Quality of Life During the COVID-19 Pandemic Lockdown.

Background: Emotion and quality of life may have been impacted by the coronavirus disease 2019 (COVID-19) crisis, especially in the lockdown. The impact of temporomandibular disorders (TMD) symptoms and tooth loss on mental status and Oral Health-Related Quality of Life (OHRQoL) are not fully understood in a stressful situation. Objectives: We aimed to investigate whether TMD and tooth loss were the impaired risks of psychological states and OHRQoL in COVID-19 lockdown, and attempt to explore other potential risk factors. Methods: This cross-sectional study surveyed residents via an online self-reported questionnaire, when Yangzhou was in lockdown. Demographic data, clinical information, the level of anxiety, depression and OHRQoL were collected and analyzed. Results: Painful TMD symptoms and tooth loss are the risks of more severe anxiety and depression. TMD symptoms and tooth loss worsened OHRQoL. Lower education degree (OR: 6.31, P = 0.019), TMD-related pain symptoms (OR: 10.62, P = 0.005), tooth loss (OR: 3.12, P = 0.035), sleep disorders (OR: 2.92, P = 0.049) and relatively close contacts (OR: 3.95, P = 0.020) were verified as risk factors for increased level of anxiety. With respect to depression, low socio-economic status (OR: 6.22, P = 0.021), TMD-related pain (OR: 7.35, P = 0.012), tooth loss (OR: 4.48, P = 0.009), sleep disorders (OR: 5.13, P = 0.007) and relatively close contacts (OR: 12.94, P = 0.001) were identified as independent factors for developing depression. Additionally, drinking (B: -2.584, P = 0.013) and never going to the dental clinic (B: -3.675, P = 0.024) were relevant to better OHRQoL, while TMD without pain (B: 2.797, P = 0.008), TMD-related pain (B: 12.079, P < 0.001), tooth loss (B: 2.546, P = 0.006), sleep disorders (B: 2.598, P = 0.003) were independent factors for impaired OHRQoL. Conclusion: Painful TMD symptoms, tooth loss and sleep disorders were the impaired risks of psychological states. TMD symptoms and tooth loss damaged OHRQoL when the city was in lockdown. Therefore, individualized psychological counseling is supposed to maintain control of mental health and OHRQoL under the stressful event.

Integrin ß1 in Pancreatic Cancer: Expressions, Functions, and Clinical Implications.

Pancreatic cancer (PC) is characterized by rapid progression and a high mortality rate. The current treatment is still based on surgical treatment, supplemented by radiotherapy and chemotherapy, and new methods of combining immune and molecular biological treatments are being explored. Despite this, the survival rate of PC patients is still very disappointing. Therefore, clarifying the molecular mechanism of PC pathogenesis and developing precisely targeted drugs are key to improving PC prognosis. As the most common ß subunit of the integrin family, integrin ß1 has been proved to be closely related to the vascular invasion, distant metastasis, and survival of PC patients, and treatment targeting integrin ß1 in PC has gained initial success in animal models. In this review, we summarize the various signaling pathways by which integrins are involved in PC, focusing on the roles of integrin ß1 in the malignant behaviors of PC. Additionally, recent studies regarding the feasibility of integrin ß1 as a diagnostic and prognostic biomarker in PC are also discussed. Finally, we present the progress of several integrin ß1-based clinical trials to highlight the potential of integrin ß1 as a target for personalized therapy in PC.

TM4SF1 promotes esophageal squamous cell carcinoma metastasis by interacting with integrin α6.

Transmembrane-4 L-six family member-1 (TM4SF1) is a member of the L6 family and functions as a signal transducer to regulate tumor cell behaviors. However, the function and mechanism of TM4SF1 in esophageal squamous cell carcinoma (ESCC) metastasis remains unclear. Here, we find that TM4SF1 expression is increased and positively correlated with clinical TNM stage, N classification, differentiation, tumor size, and poor prognosis in ESCC patients. Interestingly, we demonstrate that TM4SF1 promotes ESCC cell adhesion, spreading, migration, and invasion, but not cell proliferation, in a laminin-dependent manner by interacting with integrin α6. Mechanistically, the TM4SF1/integrin α6/FAK axis signal pathway mediates cell migration under laminin-coating condition. Inhibiting FAK or knocking down TM4SF1 can attenuate TM4SF1-mediated cell migration and lung metastasis. Clinically, the TM4SF1/integrin α6/FAK axis positively correlates with ESCC. Altogether, these findings reveal a new mechanism of TM4SF1 in promoting ESCC metastasis via binding to integrin α6 and suggest that the cross-talk between TM4SF1 and integrin α6 may serve as a therapeutic target for ESCC.

Long Non-Coding RNAs in Pancreatic Cancer: Biologic Functions, Mechanisms, and Clinical Significance.

Despite tremendous efforts devoted to research in pancreatic cancer (PC), the mechanism underlying the tumorigenesis and progression of PC is still not completely clear. Additionally, ideal biomarkers and satisfactory therapeutic strategies for clinical application in PC are still lacking. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) might participate in the pathogenesis of diverse cancers, including PC. The abnormal expression of lncRNAs in PC is considered a vital factor during tumorigenesis that affects tumor cell proliferation, migration, invasion, apoptosis, angiogenesis, and drug resistance. With this review of relevant articles published in recent years, we aimed to summarize the biogenesis mechanism, classifications, and modes of action of lncRNAs and to review the functions and mechanisms of lncRNAs in PC. Additionally, the clinical significance of lncRNAs in PC was discussed. Finally, we pointed out the questions remaining from recent studies and anticipated that further investigations would address these gaps in knowledge in this field.

Impact of the SARS-CoV-2 Delta Variant on the Psychological States and Health-Related Quality of Life in Patients With Crohn's Disease.

Background: Since the outbreak of the coronavirus disease 2019 (COVID-19) pandemic first reported in Wuhan, China, several research on the psychological impact of the pandemic on patients with Crohn's disease (CD) have been conducted. However, with the progression of the global pandemic and the emergence of the SARS-CoV-2 B.1.617.2 (Delta) variant, follow-up studies need to be performed to monitor the alterations of psychological status and health-related quality of life (HRQoL) among CD patients. Aims: We aimed to evaluate the impact of the SARS-CoV-2 Delta variant on the mental health and life quality among the CD population and tried to explore potent risk factors. Methods: This observational study included 153 CD patients who responded to our pre-designed self-reported questionnaire. Demographic, clinical, and psychological information were collected and analyzed. Results: Quite a number of CD patients were confronted with different levels of anxiety and depression, with incidence of 28.10 and 31.37% for anxiety and depression, respectively. Compared with non-pandemic circumstances, the life quality of CD patients due to the present situation was more often compromised. Isolation [odds ratio (OR): 4.71, P = 0.007] was verified as a risk factor for anxiety while use of telemedicine could help relieve anxiety (OR: 0.22, P < 0.001). Worsening of symptoms (OR: 4.92, P = 0.006), isolation (OR: 5.75, P = 0.005), and drug withdrawn (OR: 2.66, P = 0.026) were identified to be independent factors for developing depression. Likewise, use of telemedicine (OR: 0.13, P < 0.001) was negatively related to depression. Considering life quality, vaccination (OR: 3.07, P = 0.021) together with no medication (OR: 7.73, P = 0.010) was relevant to better life quality while worsening of symptoms (OR: 0.09, P = 0.034) were an independent risk factor for impaired life quality. Conclusion: Many CD patients suffered from symptoms of anxiety and depression and impaired life quality during the COVID-19 pandemic. Those in isolation or with worsening of symptoms and drug withdrawn were more prone to experience psychological stress. Individualized management such as drug delivery and telemedicine should be promoted to maintain control of mental health and life quality during the pandemic.

Roles of Integrins in Gastrointestinal Cancer Metastasis.

Integrins are a large family of heterodimeric transmembrane receptors which mediate cell adhesion and transmit signals to the cell interior. The mechanistic roles of integrins have long been an enigma in cancer, given its complexity in regulating different cellular behaviors. Recently, however, increasing research is providing new insights into its function and the underlying mechanisms, which collectively include the influences of altered integrin expression on the aberrant signaling pathways and cancer progression. Many studies have also demonstrated the potentiality of integrins as therapeutic targets in cancer treatment. In this review, we have summarized these recent reports and put a particular emphasis on the dysregulated expression of integrins and how they regulate related signaling pathways to facilitate the metastatic progression of gastrointestinal cancer, including gastric cancer (GC) and colorectal cancer (CRC), which will address the crucial roles of integrins in gastrointestinal cancer.

Recent Progress in the Neoadjuvant Treatment Strategy for Locally Advanced Esophageal Cancer.

Neoadjuvant therapies, primarily chemotherapy and chemoradiotherapy, are able to improve the overall survival (OS) in patients with locally advanced resectable esophageal cancer (EC) based on the results of several randomized clinical trials. The advantage of neoadjuvant therapy is chiefly attributed to the decreased risk of local-regional recurrence and distant metastasis. Thus, it has been recommended as standard treatment for patients with resectable EC. However, several fundamental problems remain. First, the combination of neoadjuvant chemotherapy (nCT), neoadjuvant chemoradiotherapy (nCRT), and surgery for EC patients with different histological types remain controversial. Furthermore, to reduce the toxicity of preoperative chemotherapy and the risk of complications caused by preoperative radiation therapy, the treatment protocols of nCT and nCRT still need to be investigated and optimized by prospective trials. Moreover, for patients with complete clinical response following neoadjuvant therapy, it is worth ascertaining whether a "watch and wait" surveillance plus surgery-as-needed policy is more favorable, as well as, in addition to preoperative chemoradiotherapy, whether immunotherapy, especially when combined with the traditional neoadjuvant therapy regimens, brings new prospects for EC treatment. In this review, we summarize the recent insights into the research progress and existing problems of neoadjuvant therapy for locally advanced resectable EC.

Identification of the minimal N-glycosylation on integrin α5ß1 required for its inhibitory effect on EGFR signaling and cell proliferation.

The N-glycosylation of integrin α5ß1 is involved in multiple cell biological functions. Our group previously reported that the N-glycosylation of the Calf-1,2 domain on α5 subunit (S3-5,10-14) was important for its inhibitory effect on EGFR signaling through regulating α5-EGFR complex formation. In this follow-up study, we provide evidence that the N-glycosylation on integrin ß1 subunit suppress cell growth by promoting its association with EGFR under fibronectin (FN)-coated conditions. Expression of wild-type (WT) ß1, but not the N-glycosylation mutant S4-6 ß1, which contains fewer N-glycans, inhibited EGFR signaling and cell proliferation after cell adhesion to FN. Furthermore, consistent restoration of the N-glycans on sites 1-3 of ß1 reinstated the inhibitory effects. Mechanistically, the N-glycosylation mutant of ß1 (S4-6+1-3) inhibited the EGFR response upon EGF stimulation via facilitating the α5ß1-EGFR complex formation. Moreover, we identified the N-glycosylation of sites 10-14 on α5 and 1-3 on ß1 were most important for EGFR signaling. Taken together, these data indicate that α5S3-5+10-14ß1S4-6+1-3 mutant represents the minimal N-glycosylation required for its regulation on EGFR signaling and cell proliferation, providing a plausible mechanism for the crosstalk between with α5ß1 and EGFR.

The label-free detection and distinction of CYP2C9-expressing and non-expressing cells by surface-enhanced Raman scattering substrates based on bimetallic AuNPs-AgNWs.

Cytochrome P450 2C9 (CYP2C9) is capable of catalyzing the biotransformation of endogenous compounds in cells, indicating that this enzyme could change the intracellular environment and is related to the pathogenesis of diseases. Currently, it is still a challenge to study the differences in cellular components between CYP2C9-expressing and non-expressing cells. In this study, employing a Au nanoparticles-Ag nanowires (AuNPs-AgNWs) decorated silicon wafer as a novel non-destructive and label-free tool, we applied surface-enhanced Raman scattering (SERS) spectroscopy to detect and distinguish the cellular composition of CYP2C9-expressing cells (293T-Mig-2C9) and non-expressing cells (293T-Mig-R1). AgNWs with high surface roughness were formed by modification of AuNPs onto their surface by electrostatic interactions, which enabled them to exhibit greatly enhanced SERS ability. Then, they were employed to fabricate SERS substrates via an electrostatically assisted 3-aminopropyltriethoxysilane (APTES)-functionalized surface-assembly method. The SERS substrates exhibited high sensitivity with a detection limit of 1 × 10-9 M for 4-mercaptobenzoic acid (4-MBA). Meanwhile, the SERS substrates exhibited good uniformity and reproducibility. The cytotoxicity assay demonstrated that the SERS substrates displayed good biocompatibility with 293T cells. Before SERS measurements, CYP2C9 constantly expressed cells (293T-Mig-2C9 cells) and control cells (293T-Mig-R1 cells) were constructed. The expression of CYP2C9 and the catalytic activity in the cells were checked. Using the AuNPs-AgNWs substrates as a high-performance in vitro sensing platform allowed us to obtain fingerprint spectra of 293T-Mig-R1 and 293T-Mig-2C9 cells. The difference spectra between the two cell lines were studied to interpret the spectral differences and gain insight into the biochemical variations. Finally, principal component analysis (PCA) score plots of the SERS spectra were also used to better view the differences between the two cell lines. SERS detection based on the AuNPs-AgNWs substrates provides a sensitive, non-destructive and label-free method for differentiation between 293T-Mig-R1 and 293T-Mig-2C9 cells.

Integrin α5 promotes migration and cisplatin resistance in esophageal squamous cell carcinoma cells.

Cisplatin, as one of the front-line chemotherapeutic drugs, is employed for the treatment of esophageal squamous cell carcinoma (ESCC). However, the occurrence of cisplatin resistance and metastasis remain as challenges in clinical therapy. To investigate the mechanism involved in cisplatin resistance, in this study, we established cisplatin resistant cell lines (Res) from Eca109 and TE-1 parental cells (Par), and we observed that fibronectin (FN)-mediated cell migration and spreading abilities are significantly increased in Res cells when compared to Par cells. Furthermore, we found that the integrin α5 expression is remarkably upregulated in Res cells, and inhibition of α5 results in more apoptosis and endows the Res cells resensitize to cisplatin in vitro and in vivo. In a mechanistic manner, we identified the expression of BARD1 is significantly increased in Res cells, and silencing of BARD1 reverse the effects of α5 on cisplatin resistance. Moreover, we found that the α5/FAK/PI3K/AKT signal axis is activated in Res cells, which mediates the increased expression of BARD1, as well as the cisplatin resistance and cell survival. Thus, our results demonstrate that α5 is required for cisplatin resistance through the promotion of FAK/PI3K/AKT/BARD1 signaling to prevent cells from apoptosis and enhance the DNA damage repair ability. Taken together, our study provides plausible mechanisms of α5-mediated cisplatin resistance in ESCC cells, highlighting that inhibition of α5 may be a potential target for improving efficacy in cisplatin-based chemotherapy.

Inhibition of fucosylation by 2-fluorofucose suppresses human liver cancer HepG2 cell proliferation and migration as well as tumor formation.

Core fucosylation is one of the most important glycosylation events in the progression of liver cancer. For this study, we used an easily handled L-fucose analog, 2-fluoro-L-fucose (2FF), which interferes with the normal synthesis of GDP-fucose, and verified its potential roles in regulating core fucosylation and cell behavior in the HepG2 liver cancer cell line. Results obtained from lectin blot and flow cytometry analysis clearly showed that 2FF treatment dramatically inhibited core fucosylation, which was also confirmed via mass spectrometry analysis. Cell proliferation and integrin-mediated cell migration were significantly suppressed in cells treated with 2FF. We further analyzed cell colony formation in soft agar and tumor xenograft efficacy, and found that both were greatly suppressed in the 2FF-treated cells, compared with the control cells. Moreover, the treatment with 2FF decreased the core fucosylation levels of membrane glycoproteins such as EGF receptor and integrin ß1, which in turn suppressed downstream signals that included phospho-EGFR, -AKT, -ERK, and -FAK. These results clearly described the roles of 2FF and the importance of core fucosylation in liver cancer progression, suggesting 2FF shows promise for use in the treatment of hepatoma.

A Key Regulator of Cell Adhesion: Identification and Characterization of Important N-Glycosylation Sites on Integrin α5 for Cell Migration.

The N-glycosylation of integrin α5ß1 is thought to control many fundamental aspects of cell behavior, including cell adhesion and migration. However, the mechanism of how N-glycans function remains largely obscure. Here, we used a loss-of-function approach. Wild-type (WT) integrin α5 and N-glycosylation mutant S3-5 (sites 3 to 5) integrin α5, which contains fewer N-glycans, were stably reconstituted in α5 knockout cancer cells. We found that the migration ability of S3-5 cells was decreased in comparison with that of the WT. Interestingly, the levels of phosphorylated focal adhesion kinase and actin stress fiber formation were greatly enhanced in the S3-5 mutant. In a mechanistic manner, the internalization of active but not total integrin α5ß1 was inhibited in S3-5 cells, which is a process that is related to the enhanced expression of active integrin α5ß1 on the cell surface. Importantly, restoration of N-glycosylation on the ß-propeller domain of α5 reinstated the cell migration ability, active α5ß1 expression, and internalization. Moreover, these N-glycans are critical for α5-syndecan-4 complex formation. These findings indicate that N-glycosylation on the ß-propeller domain functions as a molecular switch to control the dynamics of α5ß1 on the cell surface that in turn is required for optimum adhesion for cell migration.

N-Glycosylation of integrin α5 acts as a switch for EGFR-mediated complex formation of integrin α5ß1 to α6ß4.

N-Glycosylation of integrin α5ß1 is involved in multiple cell behaviors. We previously reported that the N-glycosylations of the calf domain on integrin α5 (S3-5,10-14) are essential for its inhibitory effect on EGFR signaling in regulating cell proliferation. However, the importance of the individual N-glycosylation and the underlying mechanisms of inhibition remain unclear. Here, we characterize the S3-5,10-14 mutants in detail and found that the N-glycosylation of site-11 (Asn712) is key for cell growth. The restoration of site-11, unlike the other individual sites, significantly suppressed cell growth and EGFR signaling in a manner that was similar to that of wild-type (WT). Mechanistically, this N-glycosylation inhibited the response abilities upon EGF stimulation and EGFR dimerization. Interestingly, we found this N-glycosylation controlled the EGFR complex formation with integrin α5ß1 or α6ß4; i.e., the loss of site-11 switched EGFR-α5ß1 to EGFR-α6ß4, which is well known to promote cellular signaling for cell growth. Moreover, the site-11 N-glycan exhibited a more branching structure compared with other sites, which may be required for EGFR-α5ß1 formation. Taken together, these data clearly demonstrate that the site-11 N-glycosylation on α5 is most important for its inhibitory effect on EGFR signaling, which may provide a novel regulatory mechanism for crosstalks between integrins and EGFR.

Importance of membrane-proximal N-glycosylation on integrin ß1 in its activation and complex formation.

N-Glycosylation of integrin α5ß1 plays important roles in cell biologic functions; however, the mechanisms that underlie those roles remain poorly understood. Here, we present evidence that the membrane-proximal N-glycosylation on integrin ß1 could positively regulate cell migration by promoting ß1 activation. The S4-6 ß1 mutant contains only 3 N-glycosylation sites, which are essential for α5 and ß1 heterodimer formation, and despite only a small difference in expression levels of α5ß1 between wild-type and S4-6 mutant, cell spreading and migration of the S4-6 mutant was significantly decreased compared with that of control. Consistent with these phenotypes, ß1-mediated cellular signaling and its activation were clearly suppressed in the S4-6 mutant. Of note, these developments could be rescued by restoration of N-glycosylation sites in the membrane-proximal domain. Further study on the regulatory mechanisms suggested that membrane-proximal N-glycosylation is critical for intermolecular interactions between integrin ß1 and other cell membrane proteins, such as syndecan-4 and epidermal growth factor receptor. Moreover, α2,6-sialylation is required for ß1 activation. These data suggest a novel regulatory mechanism wherein N-glycosylation near the cell membrane on ß1 may serve as a platform that facilitates its complex formation on the cell membrane, thereby affecting integrin-mediated functions.-Hou, S., Hang, Q., Isaji, T., Lu, J., Fukuda, T., Gu, J. Importance of membrane-proximal N-glycosylation on integrin ß1 in its activation and complex formation.

Distinct effects of ß1 integrin on cell proliferation and cellular signaling in MDA-MB-231 breast cancer cells.

An aberrant expression of integrin ß1 has been implicated in breast cancer progression. Here, we compared the cell behaviors of wild-type (WT), ß1 gene deleted (KO), and ß1 gene restored (Res) MDA-MB-231 cells. Surprisingly, the expression of ß1 exhibited opposite effects on cell proliferation. These effects were dependent on cell densities, and they showed an up-regulation of cell proliferation when cells were cultured under sparse conditions, and a down-regulation of cell growth under dense conditions. By comparison with WT cells, the phosphorylation levels of ERK in KO cells were consistently suppressed under sparse culture conditions, but consistently up-regulated under dense culture conditions. The phosphorylation levels of EGFR were increased in the KO cells. By contrast, the phosphorylation levels of AKT were decreased in the KO cells. The abilities for both colony and tumor formation were significantly suppressed in the KO cells, suggesting that ß1 plays an important role in cell survival signaling for tumorigenesis. These aberrant phenotypes in the KO cells were rescued in the Res cells. Taken together, these results clearly showed the distinct roles of ß1 in cancer cells: the inhibition of cell growth and the promotion of cell survival, which may shed light on cancer therapies.

Integrin α5 Suppresses the Phosphorylation of Epidermal Growth Factor Receptor and Its Cellular Signaling of Cell Proliferation via N-Glycosylation.

Integrin α5ß1-mediated cell adhesion regulates a multitude of cellular responses, including cell proliferation, survival, and cross-talk between different cellular signaling pathways. Integrin α5ß1 is known to convey permissive signals enabling anchorage-dependent receptor tyrosine kinase signaling. However, the effects of integrin α5ß1 on cell proliferation are controversial, and the molecular mechanisms involved in the regulation between integrin α5ß1 and receptor tyrosine kinase remain largely unclear. Here we show that integrin α5 functions as a negative regulator of epidermal growth factor receptor (EGFR) signaling through its N-glycosylation. Expression of WT integrin α5 suppresses the EGFR phosphorylation and internalization upon EGF stimulation. However, expression of the N-glycosylation mutant integrin α5, S3-5, which contains fewer N-glycans, reversed the suppression of the EGFR-mediated signaling and cell proliferation. In a mechanistic manner, WT but not S3-5 integrin α5 forms a complex with EGFR and glycolipids in the low density lipid rafts, and the complex formation is disrupted upon EGF stimulation, suggesting that the N-glycosylation of integrin α5 suppresses the EGFR activation through promotion of the integrin α5-glycolipids-EGFR complex formation. Furthermore, consistent restoration of those N-glycans on the Calf-1,2 domain of integrin α5 reinstated the inhibitory effects as well as the complex formation with EGFR. Taken together, these data are the first to demonstrate that EGFR activation can be regulated by the N-glycosylation of integrin α5, which is a novel molecular paradigm for the cross-talk between integrins and growth factor receptors.