Αv Expression Research Articles

Cancer Cell Secreted Legumain Promotes Gastric Cancer Resistance to Anti-PD-1 Immunotherapy by Enhancing Macrophage M2 Polarization.

The interaction between cancer cells and immune cells plays critical roles in gastric cancer (GC) progression and immune evasion. Forced legumain (LGMN) is one of the characteristics correlated with poor prognosis in gastric cancer patients. However, the role of gastric-cancer-secreted LGMN (sLGMN) in modulating the tumor immune microenvironment and the biological effect on the immune evasion of gastric cancer remains unclear. In this study, we found that forced expression of sLGMN in gastric cancer serum correlates with increased M2 macrophage infiltration in GC tissues and predicted resistance to anti-PD-1 immunotherapy. Mechanistically, gastric cancer cells secrete LGMN via binding to cell surface Integrin αvβ3, then activate Integrin αvβ3/PI3K (Phosphatidylinositol-4,5-bisphosphate3-kinase)/AKT (serine/threonine kinase)/mTORC2 (mammalian target of rapamycin complex 2) signaling, promote metabolic reprogramming, and polarize macrophages from the M1 to the M2 phenotype. Either blocking LGMN, Integrin αv, or knocking out Integrin αv expression and abolishing the LGMN/Integrin αvβ3 interaction significantly inhibits metabolic reprogramming and polarizes macrophages from the M1 to the M2 phenotype. This study reveals a critical molecular crosstalk between gastric cancer cells and macrophages through the sLGMN/Integrinαvβ3/PI3K/AKT/mTORC2 axis in promoting gastric cancer immune evasion and resistance to anti-PD-1 immunotherapy, indicating that the sLGMN/Integrinαvβ3/PI3K/AKT/mTORC2 axis may act as a promising therapeutic target.

Dual targeting of αv and α5β1 integrins in triple-negative breast cancer (TNBC).

e15073 Background: Integrin αv has been implicated in a stem-phenotype of breast cancer (1) and is cross-regulated by fibronectin-binding integrin α5β1 in tumor cells (2). We hypothesized that these integrins are expressed in TNBC cells and dual targeting of αv and α5β1 integrins by a bispecific antibody (BsAbαv/α5β1) will be superior to monospecific integrin antibodies (MsAbs) alone or in combination. Methods: A representative panel of basal-subtype TNBC lines was assembled: MDA-MB-231 (P53 mutated, KRAS mutated), MDA-MB-468 (P53 mutated, PTEN deleted, EGFR amplified) and BT-20 (P53 and PIK3CA mutated, EGFR amplified). Integrin expression was determined by flow cytometry and Western blot in cell lines and by IHC in the residual cancer burden (RCB) of TNBC specimens following neoadjuvant chemotherapy (positive: >2+ membranous, >10% cells). The impact of MsAbs targeting αv and α5β1 integrins alone or in combination versus the BsAbαv/α5β1 was assessed in adhesion, migration, and clonogenic survival. The impact of integrin blockade on nuclear localization of basal markers including FAK, YAP, EGFR and beta-catenin, was assessed. Results: Robust integrin αv expression was present in all TNBC lines with variable α5β1 expression. Marked reduction in α5 and in αv expression following BsAbαv/α5β1 was uniquely demonstrated by Western blot and/or flow cytometry compared to MsAbs, likely due to endosomal trafficking and lysosomal degradation of target integrins as reported previously (Joshi, MCR. 2020). Combined α5 and αv neutralization with MsAbs was superior to individual single agents in blocking adhesion and migration across all TNBC lines studied. However, the BsAbα5β1/αv was superior to combinatorial MsAbs in MDA-MB-468 and BT-20 lines in chemotaxis assays. A significant (>50%) reduction in clonogenic cell survival was noted with the BsAbα5β1/αv compared to MsAbs. BsAbαv/α5β1 blocked nuclear localization of FAK, EGFR, activated beta-catenin, and YAP in MDA-MB-231 uniquely compared to MsAbs alone or in combination. RCB tumor cells were strongly positive for membranous αv [7/9, (78%)]. Diffuse expression of integrin α5 (67%) in alpha smooth-muscle actin-expressing fibroblasts with dual staining for integrin αv was found. Marked CD68+ macrophage and CD3+ infiltration was seen in tumor and stroma of all RCB tumors. Conclusions: Combinatorial α5 and αv integrin targeting with BsAbα5/αvβ1 defines a novel therapeutic strategy with a distinct mechanism of action for further investigation in TNBC. Enrichment of integrins αv and α5 in the inflamed RCB of TNBC tumors and stroma respectively point to a potential role of these integrins in epithelial-stromal interactions and therapeutic resistance. 1. Desgrosellier, Dev Cell. 2014. 2. Joshi, MCR. 2020.

Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.

Smooth muscle αv integrins regulate vascular fibrosis via CD109 downregulation of TGF-β signalling.

αv integrins are implicated in fibrosis in a number of organs through their ability to activate TGF-β. However their role in vascular fibrosis and collagen accumulation is only partially understood. Here we have used αv conditional knockout mice and cell lines to determine how αv contributes to vascular smooth muscle cell (VSMC) function in vascular fibrosis and the role of TGF-β in that process. Angiotensin II (Ang II) treatment causes upregulation of αv and β3 expression in the vessel wall, associated with increased collagen deposition. We found that deletion of αv integrin subunit from VSMCs (αv SMKO) protected mice against angiotensin II-induced collagen production and assembly. Transcriptomic analysis of the vessel wall in αv SMKO mice and controls identified a significant reduction in expression of fibrosis and related genes in αv SMKO mice. In contrast, αv SMKO mice showed prolonged expression of CD109, which is known to affect TGF-β signalling. Using cultured mouse and human VSMCs, we showed that overexpression of CD109 phenocopied knockdown of αv integrin, attenuating collagen expression, TGF-β activation, and Smad2/3 signalling in response to angiotensin II or TGF-β stimulation. CD109 and TGF-β receptor were internalized in early endosomes. We identify a role for VSMC αv integrin in vascular fibrosis and show that αv acts in concert with CD109 to regulate TGF-β signalling.

Prognostic and immunological role of sulfatide-related lncRNAs in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver. Long non-coding RNAs (lncRNAs) play important roles in the occurrence and development of HCC through multiple pathways. Our previous study reported the specific molecular mechanism for sulfatide regulation of integrin αV expression and cell adhesion in HCC cells through lncRNA AY927503. Next, it is necessary to identify more sulfatide-related lncRNAs, explore their clinical signifcance, and determine new targeted treatment strategies. Microarrays were used to screen a complete set of lncRNAs with different expression profiles in sulfatide-treated cells. Sulfatide-related lncRNAs expression data and corresponding HCC patient survival information were obtained from the The Cancer Genome Atlas (TCGA) database, and the prognosis prediction model was constructed based on Cox regression analysis. Methylated RNA immunoprecipitation with next generation sequencing (MeRIP-seq) was used to detemine the effect of sulfatide on lncRNAs m6A modification. Tumor Immune Estimation Resource (TIMER) and Gene set nnrichment analysis (GSEA) were utilized to enrich the immune and functional pathways of sulfatide-related lncRNAs. A total of 85 differentially expressed lncRNAs (|Fold Change (FC)|>2, P<0.05) were screened in sulfatide-treated HCC cells. As a result, 24 sulfatide-related lncRNAs were highly expressed in HCC tissues, six of which were associated with poor prognosis in HCC patients. Based on thses data, a sulfatide-related lncRNAs prognosis assessment model for HCC was constructed. According to this risk score analysis, the overall survival (OS) curve showed that the OS of high-risk patients was significantly lower than that of low-risk patients (P<0.05). Notably, the expression difference in sulfatide-related lncRNA NRSN2-AS1 may be related to sulfatide-induced RNA m6A methylation. In addition, the expression level of NRSN2-AS1 was significantly positively correlated with immune cell infiltration in HCC and participated in the peroxisome and Peroxisome proliferator-activated receptor (PPAR) signaling pathways. In conclusion, sulfatide-related lncRNAs might be promising prognostic and therapeutic targets for HCC.

TNF−α Secreted from Macrophages Increases the Expression of Prometastatic Integrin αV in Gastric Cancer

The tumor microenvironment comprising blood vessels, fibroblasts, immune cells, and the extracellular matrix surrounding cancer cells, has recently been targeted for research in cancer therapy. We aimed to investigate the effect of macrophages on the invasive ability of gastric cancer cells, and studied their potential mechanism. In transcriptome analysis, integrin αV was identified as a gene increased in AGS cells cocultured with RAW264.7 cells. AGS cells cocultured with RAW264.7 cells displayed increased adhesion to the extracellular matrix and greater invasiveness compared with AGS cells cultured alone. This increased invasion of AGS cells cocultured with RAW264.7 cells was inhibited by integrin αV knockdown. In addition, the increase in integrin αV expression induced by tumor necrosis factor-α (TNF-α) or by coculture with RAW264.7 cells was inhibited by TNF receptor 1 (TNFR1) knockdown. The increase in integrin αV expression induced by TNF-α was inhibited by both Mitogen-activated protein kinase (MEK) inhibitor and VGLL1 S84 peptide treatment. Finally, transcription of integrin αV was shown to be regulated through the binding of VGLL1 and TEAD4 to the promoter of integrin αV. In conclusion, our study demonstrated that TNFR1-ERK-VGLL1 signaling activated by TNF-α secreted from RAW264.7 cells increased integrin αV expression, thereby increasing the adhesion and invasive ability of gastric cancer cells.

M2-like macrophage-derived exosomes facilitate metastasis in non-small-cell lung cancer by delivering integrin αVβ3.

Metastasis is the most prevalent cause of cancer deaths, and immunological components of the tumor microenvironment, especially tumor-associated macrophages (TAMs), play a vital role in cancer metastasis. However, the underlying mechanisms of TAMs on non-small-cell lung cancer (NSCLC) metastasis remain largely unexplored. Herein, we demonstrated that M2-like TAMs facilitate the migration and invasion of cancer cells in vitro and in vivo through intercellular delivery of M2-like macrophage-derived exosomes (M2-exos). Importantly, we found that M2-exos had considerably higher levels of integrin (ITG) αV and β3. The impact of M2-like macrophage-mediated invasion and migration of NSCLC cells was clearly decreased when ITG αVβ3 was blocked. Mechanistically, exosomal ITG αVβ3 produced from M2-like macrophages successfully triggered the focal adhesion kinase signaling pathway in recipient cells, boosting the migratory and invasive abilities of NSCLC cells. Clinically, we found that metastatic NSCLC patients had greater ITG αV and β3 expression, which was associated with a worse prognosis. This study reveals a novel mechanism by which M2-exos significantly increased NSCLC cell migration and invasion by delivering integrin αVβ3. Exosomal ITG αVβ3 can be used as a potential prognostic marker, and blocking ITG αVβ3 could be a viable treatment option for preventing tumor metastasis.

The human ion channel TRPM2 modulates migration and invasion in neuroblastoma through regulation of integrin expression

Transient receptor potential channel TRPM2 is highly expressed in many cancers and involved in regulation of key physiological processes including mitochondrial function, bioenergetics, and oxidative stress. In Stage 4 non-MYCN amplified neuroblastoma patients, high TRPM2 expression is associated with worse outcome. Here, neuroblastoma cells with high TRPM2 expression demonstrated increased migration and invasion capability. RNA sequencing, RT-qPCR, and Western blotting demonstrated that the mechanism involved significantly greater expression of integrins α1, αv, β1, and β5 in cells with high TRPM2 expression. Transcription factors HIF-1α, E2F1, and FOXM1, which bind promoter/enhancer regions of these integrins, were increased in cells with high TRPM2 expression. Subcellular fractionation confirmed high levels of α1, αv, and β1 membrane localization and co-immunoprecipitation confirmed the presence of α1β1, αvβ1, and αvβ5 complexes. Inhibitors of α1β1, αvβ1, and αvβ5 complexes significantly reduced migration and invasion in cells highly expressing TRPM2, confirming their functional role. Increased pAktSer473 and pERKThr202/Tyr204, which promote migration through mechanisms including integrin activation, were found in cells highly expressing TRPM2. TRPM2 promotes migration and invasion in neuroblastoma cells with high TRPM2 expression through modulation of integrins together with enhancing cell survival, negatively affecting patient outcome and providing rationale for TRPM2 inhibition in anti-neoplastic therapy.

OR09-3 Integrins as Potential Molecular Targets in Thyroid Cancer Imaging and Therapy

Abstract Background Integrins are cell adhesion receptors consisting of 24 transmembrane heterodimers generated from a combination of 18α integrin and 8β integrin subunits. A subset of integrins consists of receptors recognizing Arg-Gly-Asp (RGD) peptide motifs. One of the RGD-recognizing receptors is integrin αvβ3 that has been recently shown to play a role in neovascularization and progression of several cancers. Radiolabeled RGD analogs have emerged as potential imaging and therapeutic options in cancers characterized by a high expression of integrin αvβ3. Therefore, the aim of this study was to establish the expression of integrin αvβ3 in thyroid cancer (TC). Methods We analyzed the mRNA expression of integrin αvβ3 in 496 BRAF-like and RAS-like human TC tissue samples, including 65 paired samples of tumor vs normal tissue based on The Cancer Genome Atlas. We assessed the protein expression of integrin αvβ3 in 70 TC tissue samples and 10 normal thyroid tissues, as well as in 14 TC cell lines. BRAF-like or RAS-like tumor status was determined by BRS score based upon standard expression profiles ranging from -1 to 0 for BRAF-like cancer and 0 to 1 for RAS-like tumors. The association between BRS and αvβ3 expression was tested using the Spearman correlation coefficient (r). T-tests and paired T-tests were used to compare the continuous variables between the two groups as appropriate, and Kruskal-Wallis test was used for multiple group comparisons with an adjusted p-value of ≤ 0.05 as statistically significant. Results αv integrin subunit mRNA expression was significantly higher in TC than normal thyroid (log fold change 0.3, p=0.001), while the expression of the β3 subunit was similar between paired normal and malignant samples (log fold change -0.2, p=0.30). BRAF-like tumors were characterized by a higher mRNA expression of αvβ3 integrins as documented by a moderate negative correlation between BRS and αv (r=-0.5, p&amp;lt;0.001) as well as β3 (r=-0.27, p&amp;lt;0.001). Consistently, the BRAF-like TC cell lines OCUT2 (BRS=-1), TPC1 (BRS=-0.4), K1 (BRS=-0.29), as well as Hurthle cell TC cell line XTC1 (BRS=-0.46), were characterized by the highest αvβ3 mRNA and/or protein expression. Immunostaining revealed αv expression in all malignant samples, with classic papillary TC characterized by the highest expression as compared with follicular TC (p&amp;lt;0.001), poorly-differentiated TC (p=0.006) and normal thyroid (p&amp;lt;0.001). β3 protein expression had lower intensity than αv integrin and was present in 31.8% of papillary TC, 15% of follicular TC and was not detected in poorly-differentiated TC nor normal thyroid. Conclusions TC is characterized by a differential expression of αvβ3 integrin, which is particularly high in the most common type of TC - BRAF-like papillary TC. The αvβ3 integrin could potentially serve as a molecular target for imaging and therapy with radiolabeled RGD analogs in TC. Presentation: Saturday, June 11, 2022 12:00 p.m. - 12:15 p.m.

Integrin Alpha v Beta 6 (αvβ6) and Its Implications in Cancer Treatment.

Integrins are necessary for cell adhesion, migration, and positioning. Essential for inducing signalling events for cell survival, proliferation, and differentiation, they also trigger a variety of signal transduction pathways involved in mediating invasion, metastasis, and squamous-cell carcinoma. Several recent studies have demonstrated that the up- and down-regulation of the expression of αv and other integrins can be a potent marker of malignant diseases and patient prognosis. This review focuses on an arginine-glycine-aspartic acid (RGD)-dependent integrin αVβ6, its biology, and its role in healthy humans. We examine the implications of αVβ6 in cancer progression and the promotion of epithelial-mesenchymal transition (EMT) by contributing to the activation of transforming growth factor beta TGF-β. Although αvβ6 is crucial for proper function in healthy people, it has also been validated as a target for cancer treatment. This review briefly considers aspects of targeting αVβ6 in the clinic via different therapeutic modalities.

The Potential Role of a Surface-Modified Additive-Manufactured Healing Abutment on the Expression of Integrins α2, β1, αv, and β6 in the Peri-Implant Mucosa: A Preliminary Human Study.

The stability of peri-implant soft tissues is essential for long-term success. Integrins play a vital role in biological processes through developing and maintaining cell interactions; however, few studies have evaluated the effects of modifications to abutment surfaces on cell adhesion across integrin expression. Therefore, this pilot study assessed the influence of different surface topographies of titanium healing abutments prepared by additive manufacturing (AM) on the gene expression levels of the integrin subunits α2, β1, αv, and β6 in the human peri-implant mucosa. Thirteen healthy adults were included. Depending on the number of required implants, the subjects were distributed in different groups as a function of healing abutment topography: group 1 (fully rough surface); group 2 (upper machined + lower rough); group 3 (rough upper surface + lower machined); group 4 (fully machined). A total of 40 samples (n = 10/group) of the peri-implant mucosa around the abutments were collected 30 days after implant placement, and subsequently, the gene expression levels were evaluated using real-time PCR. The levels of gene expression of β1-subunit integrin were upregulated for individuals receiving fully rough surface abutments compared with the other surface topographies (p < 0.05). However, the healing abutment topography did not affect the gene expression levels of the α2, αv, and β6 integrin subunits in the human peri-implant mucosa (p > 0.05). This preliminary study suggested that controlled modifications of the surface topography of titanium healing abutments produced by AM may influence the quality of the peri-implant mucosa in the early stages of the soft tissue healing process.

Abstract 1343: CD8+ resident memory T (TRM) cells in tumors

Abstract Tumor-resident memory T (TRM) cells play an important role in cancer diseases. Accumulating evidence indicates that subpopulations of CD3+CD8+ tumor-infiltrating lymphocytes (TIL) are TRM cells, and are emerging as activated tumor-specific T cells. These TRM cells are characterized by expression of CD103 integrin and identify tumor-reactive cytotoxic T lymphocytes (CTL). Human lung tumor CD103+CD8+ TRM cells co-express CD49a and CD69, and are associated with a favorable prognosis. They also express a panel of T-cell inhibitory receptors, including PD-1, and are able to kill autologous tumor cells upon blockade of PD-1 with neutralizing antibodies. This CD103+CD8+ TRM subset also emerges as a predictive biomarker of response to PD-1 blockade and expands during anti-PD-1 treatment in responder, but not in non-responder lung cancer patients. It is now widely admitted that expression of CD103 on activated CD8+ T lymphocytes and persistence of TRM cells in epithelial tissues requires transforming growth factor (TGF)-β. This cytokine is secreted in the tumor microenvironment in its inactive form bound to latency-associated protein (LAP), and is activated by αV integrins on tumor cells and immune cells. Using multiplex immunofluorescence staining on cohorts of anti-PD-(L)1-treated lung cancer patients, we recently showed that decreased expression of tumor αV is associated with improved immunotherapy-related-progression-free survival (PFS) and correlates with an increased density of CD8+CD103+ TIL. Cancer cell αV activates autocrine TGF-β and participates in inducing CD103 on activated CD8+ T cells. Our more recent studies in mouse tumor models and human cancers demonstrate that different CD8+ TRM subsets participate to response different immune checkpoint inhibitors and therapeutic cancer vaccines. Citation Format: Fathia Mami-Chouaib. CD8+ resident memory T (TRM) cells in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1343.

Prognostic value of integrin αV expression and localization pattern in invasive breast carcinomas

Invasion of surrounding stroma is an early event in breast cancer metastatic progression, and involves loss of cell polarity, loss of myoepithelial layer, epithelial-mesenchymal transition (EMT) and remodeling of the extracellular matrix (ECM). Integrins are transmembrane receptors responsible for cell-ECM binding, which triggers signals that regulate many aspects of cell behavior and fate. Changes in the expression, localization and pairing of integrins contribute for abnormal responses found in transformed epithelia. We analyzed 345 human breast cancer samples in tissue microarrays (TMA) from cases diagnosed with invasive breast carcinoma to assess the expression and localization pattern of integrin αV and correlation with clinical parameters. Patients with lower levels of integrin αV staining showed reduced cancer specific survival. A subset of cases presented a peripheral staining of integrin αV surrounding tumor cell clusters, possibly matching the remaining myoepithelial layer. Indeed, the majority of ductal carcinoma in situ (DCIS) components found in the TMA presented integrin αV at their periphery, whereas this pattern was mostly lost in invasive components, even in the same sample. The lack of peripheral integrin αV correlated with decreased cancer specific survival. In addition, we observed that the presence of integrin αV in the stroma was an indicative of poor survival and metastatic disease. Consistently, by interrogating publicly available datasets we found that, although patients with higher mRNA levels of integrin αV had increased risk of developing metastasis, high co-expression of integrin αV and a myoepithelial cell marker (MYH11) mRNA levels correlated with better clinical outcomes. Finally, a 3D cell culture model of non-malignant and malignant cells reproduced the integrin αV pattern seen in patient samples. Taken together, our data indicate that both the expression levels of integrin αV and its tissue localization in primary tumors have prognostic value, and thus, could be used to help predict patients at higher risk of developing metastasis.

Resident memory T cells in antitumor immunity and cancer immunotherapy

Abstract Tumor-resident memory T (TRM) cells play an important role in cancer diseases. Accumulating evidence indicates that subpopulations of CD3+CD8+ tumor-infiltrating lymphocytes (TIL) are TRM cells, and are emerging as activated tumor-specific T cells. These TRM are characterized by expression of CD103 integrin and identify tumor-reactive cytotoxic T lymphocytes. Human lung tumor CD103+CD8+ TRM co-express CD49a and CD69, and are associated with a favorable prognosis. They also express a panel of T-cell inhibitory receptors, including PD-1, and are able to kill autologous tumor cells upon blockade of PD-1 with neutralizing antibodies. This CD103+CD8+ TRM subset also emerges as a predictive biomarker of response to PD-1 blockade and expands during anti-PD-1 treatment in responder, but not in non-responder patients. It is now widely admitted that expression of CD103 on activated CD8+ T lymphocytes and persistence of TRM in epithelial tissues requires TGF-β. This cytokine is secreted in the tumor microenvironment in its inactive form bound to latency-associated protein, and is activated by αV integrins on tumor cells and immune cells. Using multiplex immunofluorescence staining on cohorts of anti-PD-(L)1-treated lung cancer patients, we recently showed that decreased expression of tumor αV is associated with improved immunotherapy-related-progression-free survival and correlates with an increased density of CD8+CD103+ TIL. Cancer cell αV activates autocrine TGF-β and participates in inducing CD103 on activated CD8+ T cells. Our more recent studies in mouse tumor models and human cancers demonstrate that different CD8+ TRM subsets participate to response different immune checkpoint inhibitors and therapeutic cancer vaccines. Supported by ARC, INCa, Ligue Supported by ARC, INCa, Ligue

Fenbendazole Suppresses Growth and Induces Apoptosis of Actively Growing H4IIE Hepatocellular Carcinoma Cells &lt;i&gt;via&lt;/i&gt; p21-Mediated Cell-Cycle Arrest

Bendimidazole anthelmintics (BAs) have gained interest for their anticancer activity. The anticancer activity is mediated via multiple intracellular changes, which are not consistent under different conditions even in the same cells. We investigated the anticancer activity of fenbendazole (FZ, one of BAs) under two different growth conditions. The growth rate of H4IIE cells was dose-dependently decreased by FZ only in actively growing cells but not in fully confluent quiescent cells. Apoptosis-associated changes were also induced by FZ in actively growing cells. Markers of autophagy were not changed by FZ. The number of cells was markedly increased in sub-G1 phase but decreased in S- and G2/M phases by FZ. FZ up-regulated p21 (an inhibitor of cyclin-CDK) but suppressed the expression of cell cycle-promoting proteins (cyclin D1 and cyclin B1). FZ did not affect integrin αV or n-cadherin expression as well as cell migration. Glycolytic changes (glucose consumption and lactate production) and the generation of reactive oxygen species (ROS) were not affected by FZ. Although the activity of mitogen-activated protein kinases (MAPKs) was altered by FZ, the inhibition of MAPKs did not affect the pro-apoptotic activity of FZ. Taken together, FZ selectively suppressed the growth of cells via p21-mediated cell cycle arrest at G1/S and G2/M, and resulted in apoptosis only in actively growing cells but not in quiescent cells. Glucose metabolism, ROS generation, and MAPKs are unlikely targets of FZ at least in H4IIE rat hepatocellular carcinoma cells used in this study.

Effects of dietary supplementation with N-carbamylglutamate on maternal endometrium and fetal development during early pregnancy in Inner Mongolia white cashmere goats.

This study investigated the effects of dietary supplementation with N-carbamylglutamate (NCG) on maternal endometrium and fetal development during early pregnancy of Inner Mongolia white cashmere goats. Forty-eight pregnant Inner Mongolia white cashmere goats (average age 3 years old, average lactation parity 2, and average body weight 43.81 ± 2.66 kg) were randomly allocated to three groups: a basal diet (control group, n = 16), a basal diet plus 0.30-g NCG/d (NCG1 group, n = 16), and a basal diet plus 0.40-g NCG/d (NCG2 group, n = 16). All of the does were housed in individual pens and the NCG treatment was conducted from Days 0 to 90 of pregnancy. At Days 17 and 90 of pregnancy, six representative pregnant does in each group were slaughtered. The current study results demonstrated that maternal NCG administration during early pregnancy effectively increased the arginine family of amino acids and the glucogenic amino acids concentrations and promoted the mRNA expression of osteopontin (OPN), αv and β3 integrins, and endometrial development of Inner Mongolia white cashmere goats. The supplementation improved the fetal brown adipose tissue (BAT) stores and the mRNA expression of UCP-1 and BMP7, thereby helping to the fetal early development.

CCL4 Stimulates Cell Migration in Human Osteosarcoma via the mir-3927-3p/Integrin αvβ3 Axis.

Osteosarcoma is the most common type of primary malignant bone cancer, and it is associated with high rates of pulmonary metastasis. Integrin αvβ3 is critical for osteosarcoma cell migratory and invasive abilities. Chemokine (C-C motif) ligand 4 (CCL4) has diverse effects on different cancer cells through its interaction with its specific receptor, C-C chemokine receptor type 5 (CCR5). Analysis of mRNA expression in human osteosarcoma tissue identified upregulated levels of CCL4, integrin αv and β3 expression. Similarly, an analysis of records from the Gene Expression Omnibus (GEO) dataset showed that CCL4 was upregulated in human osteosarcoma tissue. Importantly, the expression of both CCL4 and integrin αvβ3 correlated positively with osteosarcoma clinical stages and lung metastasis. Analysis of osteosarcoma cell lines identified that CCL4 promotes integrin αvβ3 expression and cell migration by activating the focal adhesion kinase (FAK), protein kinase B (AKT), and hypoxia inducible factor 1 subunit alpha (HIF-1α) signaling pathways, which can downregulate microRNA-3927-3p expression. Pharmacological inhibition of CCR5 by maraviroc (MVC) prevented increases in integrin αvβ3 expression and cell migration. This study is the first to implicate CCL4 as a potential target in the treatment of metastatic osteosarcoma.

Integrin-\u03b1V-mediated activation of TGF-\u03b2 regulates anti-tumour CD8 T cell immunity and response to PD-1 blockade

TGF-β is secreted in the tumour microenvironment in a latent, inactive form bound to latency associated protein and activated by the integrin αV subunit. The activation of latent TGF-β by cancer-cell-expressed αV re-shapes the tumour microenvironment, and this could affect patient responses to PD-1-targeting therapy. Here we show, using multiplex immunofluorescence staining in cohorts of anti-PD-1 and anti-PD-L1-treated lung cancer patients, that decreased expression of cancer cell αV is associated with improved immunotherapy-related, progression-free survival, as well as with an increased density of CD8+CD103+ tumour-infiltrating lymphocytes. Mechanistically, tumour αV regulates CD8 T cell recruitment, induces CD103 expression on activated CD8+ T cells and promotes their differentiation to granzyme B-producing CD103+CD69+ resident memory T cells via autocrine TGF-β signalling. Thus, our work provides the underlying principle of targeting cancer cell αV for more efficient PD-1 checkpoint blockade therapy.

Abstract 2416: Upregulation of amplified in breast cancer 1 contributes to pancreatic ductal adenocarcinoma progression and vulnerability to blockage of hedgehog activation

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and devastating cancers without effective treatments. Amplified in breast cancer 1 (AIB1) is a member of the steroid receptor coactivator family that mediates the transcriptional activities of nuclear receptors. While AIB1 is associated with the initiation and progression of multiple cancers, the mechanism by which AIB1 contributes to PDAC progression remains largely unknown. In this study, we aimed to explore the role of AIB1 in the progression of PDAC and elucidate the underlying mechanisms. Methods: The clinical significance and mRNA level of AIB1 in PDAC were studied by database analysis. To demonstrate whether AIB1 mediates the malignant features of PDAC cells, namely, proliferation, migration, invasion, we performed real-time PCR and Western blot analysis, established xenograft models and used in vivo metastasis assay. With insights into the mechanism of AIB1, we performed RNA sequencing (Seq), ChIP-Seq, luciferase reporter assays and pull-down assays. Furthermore, we analyzed the relationship between AIB1 expression and its target expression in PDAC cells and patients and explored whether PDAC cells with high AIB1 levels are sensitive to inhibitors of its target. Results: We found that AIB1 was significantly upregulated in PDAC and associated with its malignancy. Silencing AIB1 impaired hedgehog (Hh) activation by reducing the expression of smoothened (SMO), leading to cell cycle arrest and the inhibition of PDAC cell proliferation. In addition, AIB1, via upregulation of integrin αv (ITGAV) expression, promoted extracellular matrix (ECM) signaling, which played an important role in PDAC progression. Further studies showed that AIB1 preferably bound to AP-1 related elements and served as a coactivator for enhancing the transcriptional activity of MafB, which promoted the expression of SMO and ITGAV. PDAC cells with high AIB1 levels were sensitive to Hh signaling inhibitors, suggesting that blocking Hh activation is an effective treatment against PDAC with high AIB1 expression. Conclusions: These findings reveal that AIB1 is a crucial oncogenic regulator associated with PDAC progression via Hh and ECM signaling and suggest potential therapeutic targets for PDAC treatment. Citation Format: Licen Li, Jiaolin Bao, Haitao Wang, Haipeng Lei, Peng Cheng, Jianming Zeng, Wenhui Hao, Xu Zhang, Xiaoling Xu, Chundong Yu, Chu-Xia Deng, Qiang Chen. Upregulation of amplified in breast cancer 1 contributes to pancreatic ductal adenocarcinoma progression and vulnerability to blockage of hedgehog activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2416.

The decreased expression of integrin αv is involved in T-2 toxin-induced extracellular matrix degradation in chondrocytes

T-2 toxin is one of the most toxic and common mycotoxins in grains and related products. It is considered a risk factor for Kashin-Beck disease (KBD), an endemic osteoarthritis. Both in vitro and in vivo studies have shown that T-2 toxin can cause extracellular matrix degradation; however, the underlying mechanism is unclear. Integrins have been found to regulate the expression of matrix metalloproteinases (MMPs), the ‘scissors’ of matrix proteins. In this study, we investigated whether integrin αv played a role in T-2 toxin-induced matrix degradation. Results from our study showed that the expression of integrin αv in the cartilage of rats fed T-2 toxin was reduced compared to that in rats fed a normal diet. Integrin αv was downregulated in T-2 toxin-treated C28/I2 chondrocytes, and selenium was found to have a protective effect. The expression of MMP-1, -3, -10, and −13 increased whereas that of type II collagen (Col II) protein decreased in C28/I2 cells treated with an integrin αv inhibitor. In conclusion, T-2 toxin can downregulate integrin αv expression in chondrocytes. Reduced integrin αv signalling could induce the release of MMPs, leading to matrix degradation.