Therapeutic targeting of integrin αvβ6 in breast cancer

BACKGROUND: Integrin ?v?6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of ?v?6 has yet to be elucidated in breast cancer.METHODS: Protein expression of integrin subunit beta6 (?6) was measured in breast cancers by immunohistochemistry (n &gt; 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of ?6 in breast cell lines, the role of ?v?6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ? 3), respectively. A student's t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni's Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided.RESULTS: High expression of either the mRNA or protein for the integrin subunit ?6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of ?6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P &lt; .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts.CONCLUSIONS: Targeting ?v?6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients.<br/

The integrin αvβ6 drives pancreatic cancer through diverse mechanisms and represents an effective target for therapy

Pancreatic ductal adenocarcinoma (PDAC) has a five‐year survival rate of &lt;4% and desperately needs novel effective therapeutics. Integrin αvβ6 has been linked with poor prognosis in cancer but its potential as a target in PDAC remains unclear. We report that transcriptional expression analysis revealed high levels of β6 mRNA correlated strongly with significantly poorer survival (n=491 cases, p= 3.17x10‐8). In two separate cohorts we showed that over 80% of PDAC expressed αvβ6 protein and that paired metastases retained αvβ6 expression. In vitro, integrin αvβ6 promoted PDAC cell growth, survival, migration and invasion. Treatment of both αvβ6‐positive human PDAC xenografts and transgenic mice bearing αvβ6‐positive PDAC with the αvβ6 blocking antibody 264RAD, combined with gemcitabine, significantly reduced tumour growth (p&lt;0.0001) and increased survival (Log‐rank test, p&lt;0.05). Antibody therapy was associated with suppression of both tumour cell activity (suppression of pErk growth signals, increased apoptosis seen as activated Caspase 3) and suppression of the pro‐tumourigenic microenvironment (suppression of TGFβ signalling, fewer αSMA‐positive myofibroblasts, decreased blood vessel density). These data show that αvβ6 promotes PDAC growth through both tumour cell and tumour microenvironment mechanisms and represents a valuable target for PDAC therapy

Targeting of Aberrant αvβ6 Integrin Expression in Solid Tumors Using Chimeric Antigen Receptor-Engineered T Cells.

Expression of the αvβ6 integrin is upregulated in several solid tumors. In contrast, physiologic expression of this epithelial-specific integrin is restricted to development and epithelial re-modeling. Here, we describe, for the first time, the development of a chimeric antigen receptor (CAR) that couples the recognition of this integrin to the delivery of potent therapeutic activity in a diverse repertoire of solid tumor models. Highly selective targeting αvβ6 was achieved using a foot and mouth disease virus-derived A20 peptide, coupled to a fused CD28+CD3 endodomain. To achieve selective expansion of CAR T cells ex vivo, an IL-4-responsive fusion gene (4αβ) was co-expressed, which delivers a selective mitogenic signal to engineered T cells only. In vivo efficacy was demonstrated in mice with established ovarian, breast, and pancreatic tumor xenografts, all of which express αvβ6 at intermediate to high levels. SCID beige mice were used for these studies because they are susceptible to cytokine release syndrome, unlike more immune-compromised strains. Nonetheless, although the CAR also engages mouse αvβ6, mild and reversible toxicity was only observed when supra-therapeutic doses of CAR T cells were administered parenterally. These data support the clinical evaluation of αvβ6 re-targeted CAR T cell immunotherapy in solid tumors that express this integrin

The integrin αvβ6: a novel target for CAR T-cell immunotherapy?

Immunotherapy of cancer using chimeric antigen receptor (CAR) T-cells is a rapidly expanding field. CARs are fusion molecules that couple the binding of a tumour-associated cell surface target to the delivery of a tailored T-cell activating signal. Re-infusion of such genetically engineered T-cells to patients with haematological disease has demonstrated unprecedented response rates in Phase I clinical trials. However, such successes have not yet been observed using CAR T-cells against solid malignancies and this is, in part, due to a lack of safe tumour-specific targets. The αvβ6 integrin is strongly up-regulated in multiple solid tumours including those derived from colon, lung, breast, cervix, ovaries/fallopian tube, pancreas and head and neck. It is associated with poorer prognosis in several cancers and exerts pro-tumorigenic activities including promotion of tumour growth, migration and invasion. By contrast, physiologic expression of αvβ6 is largely restricted to wound healing. These attributes render this epithelial-specific integrin a highly attractive candidate for targeting using immunotherapeutic strategies such as CAR T-cell adoptive immunotherapy. This mini-review will discuss the role and expression of αvβ6 in cancer, as well as its potential as a therapeutic target.</jats:p

Two-dimensional heteronuclear saturation transfer difference NMR reveals detailed integrin alphavbeta6 protein-peptide interactions

We report the first example of peptide-protein heteronuclear two-dimensional (2D) saturation transfer difference nuclear magnetic resonance (STD NMR). This method, resulting in dramatically reduced overlap, was applied to the interaction of the integrin alphavbeta6 with a known peptide ligand and highlights novel contact points between the substrate and target protein

In vivo retargeting of adenovirus type 5 to ?v?6 integrin results in reduced hepatotoxicity and improved tumor uptake following systemic delivery

A key impediment to successful cancer therapy with adenoviral vectors is the inefficient transduction of malignant tissue in vivo. Compounding this problem is the lack of cancer-specific targets, coupled with a shortage of corresponding high-efficiency ligands, permitting selective retargeting. The epithelial cell-specific integrin ?v?6 represents an attractive target for directed therapy since it is generally not expressed on normal epithelium but is upregulated in numerous carcinomas, where it plays a role in tumor progression. We previously have characterized a high-affinity, ?v?6-selective peptide (A20FMDV2) derived from VP1 of foot-and-mouth disease virus. We generated recombinant adenovirus type 5 (Ad5) fiber knob, incorporating A20FMDV2 in the HI loop, for which we validated the selectivity of binding and functional inhibition of ?v?6. The corresponding ?v?6-retargeted virus Ad5-EGFPA20 exhibited up to 50-fold increases in coxsackievirus- and-adenovirus-receptor-independent transduction and up to 480-fold-increased cytotoxicity on a panel of ?v?6-positive human carcinoma lines compared with Ad5-EGFPWT. Using an ?v?6-positive (DX3-?6) xenograft model, we observed a ~2-fold enhancement in tumor uptake over Ad5-EGFPWT following systemic delivery. Furthermore, ~5-fold-fewer Ad5-EGFPA20 genomes were detected in the liver (P = 0.0002), correlating with reduced serum transaminase levels and E1A expression. Warfarin pretreatment, to deplete coagulation factors, did not improve tumor uptake significantly with either virus but did significantly reduce liver sequestration and hepatic toxicity. The ability of Ad5-EGFPA20 to improve delivery to ?v?6, combined with its reduced hepatic tropism and toxicity, highlights its potential as a prototype virus for future clinical investigation

Biological and clinical implications of nicastrin expression in invasive breast cancer

Nicastrin is an essential component of the gamma secretase (GS) enzyme complex, required for its synthesis and recognition of substrates for proteolytic cleavage. The purpose of this study was to investigate whether nicastrin has prognostic value or potential as a therapeutic target in breast cancer (BC). The suitability of nicastrin as a target in BC was assessed using BC tissue microarrays (TMAs) (n = 1050), and its biological role in vitro was evaluated in BC cell lines following gene silencing. Nicastrin blocking antibodies were developed and evaluated for their suitability as potential clinical therapeutics. TMA and cell line analysis confirmed that nicastrin expression was upregulated in BC compared to normal breast cells. In TMA patient samples, high nicastrin expression was observed in 47.5% of cases and correlated with ERa expression, patient age, and tumor grade. In pre-defined subset analysis, high nicastrin expression predicted for worse BC specific survival in the ERa -ve cohort. In vitro gene silencing of nicastrin resulted in disruption of the GS complex and a decrease in notch1 cleavage. This was sufficient to increase E-cadherin expression and its co-localization with p120 catenin at cell-cell junctions in MCF7 cells. Nicastrin silencing in invasive MDA-MB-231 cells resulted in loss of vimentin expression and a marked reduction in both cell motility and invasion; which was concomitant with the de novo formation of cell-cell junctions characterized by the colocalization of p120 catenin and F-actin. These data indicate that nicastrin can function to maintain epithelial to mesenchymal transition during BC progression. Anti-nicastrin polyclonal and monoclonal antibodies were able to decrease notch1 and vimentin expression and reduced the invasive capacity of BC cells in vitro. This supports our hypothesis that a nicastrin blocking antibody could be used to limit metastatic dissemination in invasive BC