A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer.

New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and DI) domains. In proof-of-concept studies, we demonstrated that a monoclonal antibody (mAb) that simultaneously inhibits both domains represents a promising therapeutic approach. Here, we screened a hybridoma library using a multistep selection strategy, including flow cytometry for Ab binding to native conformation protein and in vitro cell-based functional assays to isolate a novel panel of highly specific human ADAM8 dual MP and DI inhibitory mAbs, called ADPs. The screening of four top candidates for in vivo anti-cancer activity in an orthotopic MDA-MB-231 TNBC model of ADAM8-driven primary growth identified two lead mAbs, ADP2 and ADP13. Flow cytometry, hydrogen/deuterium exchange-mass spectrometry (HDX-MS) and alanine (ALA) scanning mutagenesis revealed that dual MP and DI inhibition was mediated via binding to the DI. Further testing in mice showed ADP2 and ADP13 reduce aggressive TNBC characteristics, including locoregional regrowth and metastasis, and improve survival, demonstrating strong therapeutic potential. The continued development of these mAbs into an ADAM8-targeted therapy could revolutionize TNBC treatment.

ADAM8 is expressed widely in breast cancer and predicts poor outcome in hormone receptor positive, HER-2 negative patients.

BACKGROUND: Breast malignancies are the predominant cancer-related cause of death in women. New methods of diagnosis, prognosis and treatment are necessary. Previously, we identified the breast cancer cell surface protein ADAM8 as a marker of poor survival, and a driver of Triple-Negative Breast Cancer (TNBC) growth and spread. Immunohistochemistry (IHC) with a research-only anti-ADAM8 antibody revealed 34.0% of TNBCs (17/50) expressed ADAM8. To identify those patients who could benefit from future ADAM8-based interventions, new clinical tests are needed. Here, we report on the preclinical development of a highly specific IHC assay for detection of ADAM8-positive breast tumors. METHODS: Formalin-fixed paraffin-embedded sections of ADAM8-positive breast cell lines and patient-derived xenograft tumors were used in IHC to identify a lead antibody, appropriate staining conditions and controls. Patient breast cancer samples (n = 490) were used to validate the assay. Cox proportional hazards models assessed association between survival and ADAM8 expression. RESULTS: ADAM8 staining conditions were optimized, a lead anti-human ADAM8 monoclonal IHC antibody (ADP2) identified, and a breast staining/scoring control cell line microarray (CCM) generated expressing a range of ADAM8 levels. Assay specificity, reproducibility, and appropriateness of the CCM for scoring tumor samples were demonstrated. Consistent with earlier findings, 36.1% (22/61) of patient TNBCs expressed ADAM8. Overall, 33.9% (166/490) of the breast cancer population was ADAM8-positive, including Hormone Receptor (HR) and Human Epidermal Growth Factor Receptor-2 (HER2) positive cancers, which were tested for the first time. For the most prevalent HR-positive/HER2-negative subtype, high ADAM8 expression identified patients at risk of poor survival. CONCLUSIONS: Our studies show ADAM8 is widely expressed in breast cancer and provide support for both a diagnostic and prognostic value of the ADP2 IHC assay. As ADAM8 has been implicated in multiple solid malignancies, continued development of this assay may have broad impact on cancer management.

Reciprocal control of Forkhead box O 3a and c-Myc via the phosphatidylinositol 3-kinase pathway coordinately regulates p27Kip1 levels.

B cell receptor (BCR) engagement of murine WEHI 231 immature B lymphoma cells leads sequentially to a drop in NF-kappa B and c-Myc, and induction of the p27(Kip1) cyclin-dependent kinase inhibitor, which promotes growth arrest and apoptosis. BCR engagement was recently shown to induce a drop in phosphatidylinositol 3-kinase (PI3K)/Akt signaling, preceding the increase in p27. As induction of p27 is due to an increase in gene transcription, we investigated the role of the Forkhead box O (FOXO) transcription factor family, which has been shown to potently induce p27 promoter activity. We demonstrate that pharmacologic inhibitors of PI3K or BCR engagement lead to decreased inactive cytoplasmic levels and increased active functional nuclear FOXO3a. In contrast, inhibition of PI3K/Akt signaling decreased the levels of NF-kappa B and c-Myc, which has been shown to repress p27 promoter activity. To test the effects of ectopic c-Myc on endogenous p27 levels, WEHI 231 cells stably expressing c-Myc or empty vector DNA were prepared. Ectopic c-Myc blocked the induction of p27 expression upon either inhibition of PI3K or BCR engagement. Thus, p27(Kip1) is coordinately regulated via two arms of a signaling pathway that are inversely controlled upon inhibition of PI3K: induction of the activator FOXO3a and down-regulation of the repressor c-Myc.

Lysyl oxidase inhibits ras-mediated transformation by preventing activation of NF-kappa B.

Lysyl oxidase (LO), which catalyzes the oxidation of lysine residues, was previously shown to have anti-oncogenic activity on ras-transformed cells. Since oncogenic Ras mediates transformation, in part, through the activation of the transcription factor nuclear factor-kappa B (NF-kappa B), we tested here the effects of LO on NF-kappa B activity. Expression of LO in ras-transformed NIH 3T3 cells led to decreased NF-kappa B binding and activity, as well as the expression of the NF-kappa B target gene c-myc. Importantly, ectopic expression of LO led to a dramatic decrease in colony formation by ras-transformed NIH 3T3 cells, a finding comparable to the expression of the I kappa B alpha dominant-negative mutant, which could be rescued by p65/p50 NF-kappa B subunit expression. LO was unable to directly inhibit the activity of ectopically expressed p65 and c-Rel NF-kappa B subunits, suggesting that LO affected an upstream signaling pathway(s) induced by Ras. Consistent with this hypothesis, LO expression decreased both the rate of I kappa B alpha turnover and the activities of IKK alpha and IKK beta. Moreover, the ectopic expression of a constitutively active version of either kinase reversed the negative effects of LO. Ras can induce NF-kappa B via both the phosphatidylinositol 3-kinase (PI3K)/Akt and Raf/MEK pathways. LO potently downregulated the PI3K and Akt kinases, while partially inhibiting MEK kinase activity. Expression of a constitutively activated, myristylated Akt or PDK1 was able to counteract the effect of LO on NF-kappa B, whereas constitutively activated Raf was only partially effective. Importantly, LO blocked membrane localization of Akt and PDK1 in Ras-transformed cells. Overall, these results strongly argue that the anti-oncogenic effects of LO on ras-mediated transformation are due to its ability to inhibit signaling pathways that lead to activation of NF-kappa B.

Green tea polyphenol epigallocatechin-3 gallate inhibits Her-2/neu signaling, proliferation, and transformed phenotype of breast cancer cells.

Overexpression of the epidermal growth factor receptor family member Her-2/neu in breast cancer is associated with poor prognosis. With evidence accumulating for a chemopreventive role of green tea polyphenols, the effects of epigallocatechin-3 gallate (EGCG) on Her-2/neu-overexpressing breast cancer cells were examined. EGCG inhibited mouse mammary tumor virus (MMTV)-Her-2/neu NF639 cell growth in culture and soft agar. EGCG reduced signaling via the phosphatidylinositol 3- kinase, Akt kinase to NF-kappaB pathway because of inhibition of basal Her-2/neu receptor tyrosine phosphorylation. EGCG similarly inhibited basal receptor phosphorylation in SMF and Ba/F3 2 + 4 cells, which suggests the potential beneficial use of EGCG in adjuvant therapy of tumors with Her-2/neu overexpression.