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.

A polymorphism in the lysyl oxidase propeptide domain accelerates carcinogen-induced cancer.

The propeptide (LOX-PP) domain of the lysyl oxidase proenzyme was shown to inhibit the transformed phenotype of breast, lung and pancreatic cells in culture and the formation of Her2/neu-driven breast cancer in a xenograft model. A single nucleotide polymorphism (SNP, rs1800449) positioned in a highly conserved region of LOX-PP results in an Arg158Gln substitution (humans). This arginine (Arg)→glutamine (Gln) substitution profoundly impaired the ability of LOX-PP to inhibit the invasive phenotype and xenograft tumor formation. To study the effect of the SNP in vivo, here we established a knock in (KI) mouse line (LOX-PPGln mice) expressing an Arg152Gln substitution corresponding to the human Arg158Gln polymorphism. Breast cancer was induced in wild-type (WT) and LOX-PPGln female mice beginning at 6 weeks of age by treatment with 7,12-dimethylbenz(a)anthracene (DMBA) in combination with progesterone. Time course analysis of tumor development demonstrated earlier tumor onset and shorter overall survival in LOX-PPGln versus WT mice. To further compare the tumor burden in WT and LOX-PPGln mice, inguinal mammary glands from both groups of mice were examined for microscopic lesion formation. LOX-PPGln glands contained more lesions (9.6 versus 6.9 lesions/#4 bilateral). In addition, more DMBA-treated LOX-PPGln mice had increased leukocyte infiltrations in their livers and were moribund compared with DMBA-treated WT mice. Thus, these data indicate that the Arg→Gln substitution in LOX-PP could be an important marker associated with a more aggressive cancer phenotype and that this KI model is ideal for further mechanistic studies regarding the tumor suppressor function of LOX-PP.

UXT Is a LOX-PP Interacting Protein That Modulates Estrogen Receptor Alpha Activity in Breast Cancer Cells.

The lysyl oxidase proenzyme propeptide region (LOX-PP) is a tumor suppressor protein whose mechanism of action is not completely understood. Here, the Ubiquitously expressed Transcript (UXT) was identified in a yeast two-hybrid assay with LOX-PP as bait and confirmed as a novel LOX-PP associating protein. UXT, a prefoldin-like protein, is ubiquitous in human and mouse. Since UXT modulates androgen receptor transcriptional activity in prostate cancer, we studied its role in breast cancer. Breast tumors and derived cell lines overexpressed UXT. UXT was able to associate with the estrogen receptor alpha (ER) and decrease its transcriptional activity and target gene expression. Conversely, UXT knockdown increased ER element-dependent transcriptional activity. Ectopic LOX-PP relocalized UXT to the cytoplasm and decreased its stability. UXT ubiquitination and depletion in the presence of LOX-PP was rescued by a proteasomal inhibitor. In summary, proteasome-mediated turnover of UXT upon interaction with LOX-PP releases repression of ER transcriptional activity. J. Cell. Biochem. 118: 2347-2356, 2017. © 2017 Wiley Periodicals, Inc.

Endogenous light scattering as an optical signature of circulating tumor cell clusters.

Circulating tumor cell clusters (CTCCs) are significantly more likely to form metastases than single tumor cells. We demonstrate the potential of backscatter-based flow cytometry (BSFC) to detect unique light scattering signatures of CTCCs in the blood of mice orthotopically implanted with breast cancer cells and treated with an anti-ADAM8 or a control antibody. Based on scattering detected at 405, 488, and 633 nm from blood samples flowing through microfluidic devices, we identified 14 CTCCs with large scattering peak widths and intensities, whose presence correlated strongly with metastasis. These initial studies demonstrate the potential to detect CTCCs via label-free BSFC.

miR-720 is a downstream target of an ADAM8-induced ERK signaling cascade that promotes the migratory and invasive phenotype of triple-negative breast cancer cells.

BACKGROUND: ADAM8 (a disintegrin and metalloproteinase 8) protein promotes the invasive and metastatic phenotype of triple-negative breast cancer (TNBC) cells. High ADAM8 expression in breast cancer patients is an independent predictor of poor prognosis. Here, we investigated whether ADAM8 regulates specific miRNAs, their roles in aggressive phenotype, and potential use as biomarkers of disease. METHODS: Microarray analysis was performed on RNA from MDA-MB-231 cells after transient ADAM8 knockdown using TaqMan miRNA cards. Changes in miRNA levels were confirmed using two ADAM8 siRNAs in TNBC cell lines. Kinase inhibitors, ß1-integrin antagonist antibody, and different forms of ADAM8 were employed to elucidate the signaling pathway required for miR-720 expression. miR-720 levels were modulated using a specific antagomiR or a mimic, and effects on aggressive phenotype of TNBC cells were determined using Boyden chamber and 3D-Matrigel outgrowth assays. Plasma was isolated from mice before and after implantation of MDA-MB-231 cells and analyzed for miR-720 levels. Serum samples of TNBC patients were evaluated for their ADAM8 and miR-720 levels. RESULTS: We identified 68 miRNAs differentially regulated upon ADAM8 knockdown, including decreased levels of secreted miR-720. Ectopic overexpression of wild-type ADAM8 or forms that lack metalloproteinase activity similarly induced miR-720 levels. The disintegrin and cysteine-rich domains of ADAM8 were shown to induce miR-720 via activation of a ß1-integrin to ERK signaling cascade. Knockdown of miR-720 led to a significant decrease in migratory and invasive abilities of TNBC cells. Conversely, miR-720 overexpression rescued these properties. A profound increase in plasma levels of miR-720 was detected 7 days after TNBC cell inoculation into mouse mammary fat pads when tumors were barely palpable. Concordantly, miR-720 levels were found to be significantly higher in serum samples of TNBC patients with high ADAM8 expression. CONCLUSIONS: We have shown for the first time that miR-720 is induced by ADAM8 signaling via ERK and plays an essential role in promoting the aggressive phenotype of TNBCs. miR-720 is elevated in serum of patients with ADAM8-high TNBC and, in a group with other miRNAs downstream of ADAM8, holds promise as a biomarker for early detection of or treatment response of ADAM8-positive TNBCs.

N-glycosylation regulates ADAM8 processing and activation.

The transmembrane ADAM8 (A Disintegrin And Metalloproteinase 8) protein is abundantly expressed in human breast tumors and derived metastases compared with normal breast tissue, and plays critical roles in aggressive Triple-Negative breast cancers (TNBCs). During ADAM8 maturation, the inactive proform dimerizes or multimerizes and autocatalytically removes the prodomain leading to the formation of the active, processed form. ADAM8 is a glycoprotein; however, little was known about the structure or functional role of these sugar moieties. Here, we report that in estrogen receptor (ER)α-negative, but not -positive, breast cancer cells ADAM8 contains N-glycosylation, which is required for its correct processing and activation. Consistently ADAM8 dimers were detected on the surface of ERα-negative breast cancer cells but not on ERα-positive ones. Site-directed mutagenesis confirmed four N-glycosylazhytion sites (Asn-67, Asn-91, Asn-436, and Asn-612) in human ADAM8. The Asn-67 and Asn-91 prodomain sites contained high mannose, whereas complex type N-glycosylation was observed on Asn-436 and Asn-612 in the active and remnant forms. The Asn-91 and Asn-612 sites were essential for its correct processing and cell surface localization, in particular its exit from the Golgi and endoplasmic reticulum, respectively. The N436Q mutation led to decreased ADAM8 stability due to enhanced lysosomal degradation. In contrast, mutation of the Asn-67 site had only modest effects on enzyme stability and processing. Thus, N-glycosylation is essential for processing, localization, stability, and activity of ADAM8.

ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis.

The transmembrane metalloprotease-disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple-negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF-A and transendothelial cell migration via ß1-integrin activation. In vivo, treatment with an anti-ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non-essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.

Inhibition of CIN85-mediated invasion by a novel SH3 domain binding motif in the lysyl oxidase propeptide.

The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the "Cbl-interacting protein of 85-kDa" (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.

Epigallocatechin-3-gallate inhibits stem-like inflammatory breast cancer cells.

Inflammatory Breast Cancer (IBC) is a highly aggressive form of cancer characterized by high rates of proliferation, lymphangiogenesis and metastasis, and an overall poor survival. As regular green tea consumption has been associated with improved prognosis of breast cancer patients, including decreased risk of recurrence, here the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) were tested on two IBC lines: SUM-149 and SUM-190. EGCG decreased expression of genes that promote proliferation, migration, invasion, and survival. Consistently, growth, invasive properties, and survival of IBC cells were reduced by EGCG treatment. EGCG also reduced lymphangiogenesis-promoting genes, in particular VEGF-D. Conditioned media from EGCG-treated IBC cells displayed decreased VEGF-D secretion and reduced ability to promote lymphangiogenesis in vitro as measured by hTERT-HDLEC lymphatic endothelial cell migration and tube formation. Tumorsphere formation by SUM-149 cells was robustly inhibited by EGCG, suggesting effects on self-renewal ability. Stem-like SUM-149 cells with high aldehyde dehydrogenase (ALDH) activity, previously implicated in poor patient prognosis, were isolated. EGCG treatment reduced growth and induced apoptosis of the stem-like SUM-149 cells in culture. In an orthotopic mouse model, EGCG decreased growth of pre-existing tumors derived from ALDH-positive stem-like SUM-149 cells and their expression of VEGF-D, which correlated with a significant decrease in peritumoral lymphatic vessel density. Thus, EGCG inhibits the overall aggressive IBC phenotype. Reduction of the stem-like cell compartment by EGCG may explain the decreased risk of breast cancer recurrence among green tea drinkers. Recent clinical trials demonstrate the efficacy of green tea polyphenol extracts in treatment of prostate cancer and lymphocytic leukemia with low toxicity. Given the poor prognosis of IBC patients, our findings suggest further exploration of EGCG or green tea in combinatorial treatments against active IBC disease or in maintenance regimens to avoid recurrence is warranted.

Epithelial-to-mesenchymal transition induced by TGF-ß1 is mediated by Blimp-1-dependent repression of BMP-5.

Induction of epithelial-to-mesenchymal transition (EMT) by TGF-ß1 requires Ras signaling. We recently identified the transcriptional repressor Blimp-1 (PRDM1) as a downstream effector of the NF-κB, RelB/Bcl-2/Ras-driven pathway that promotes breast cancer cell migration. As the RelB/Blimp-1 pathway similarly required Ras signaling activation, we tested whether Blimp-1 plays a role in TGF-ß1-mediated EMT. Here, TGF-ß1 treatment of untransformed NMuMG mammary epithelial and MDA-MB-231 breast cancer cells was shown to induce Blimp-1 expression, which promoted an EMT signature and cell migration. TGFB1 and BLIMP1 RNA levels were correlated in patient breast tumors. BLIMP1 gene transcription was activated by TGF-ß1 via a c-Raf (RAF1) to AP-1 pathway. Blimp-1 induced expression of the EMT master regulator Snail (SNAI1) via repressing BMP-5, which inhibited Snail expression upon TGF-ß1 treatment. Interestingly, a similar cascade was observed during postnatal mouse mammary gland development. RelB expression was detected early in pregnancy followed progressively by Blimp-1 and then Snail; whereas, BMP-5 levels were high in nulliparous and regressing glands. Finally, lower BMP5 RNA levels were detected in patient breast tumors versus normal tissues, and correlated with cancer recurrence. Thus, the Ras effector Blimp-1 plays an essential role in TGF-ß1-induced EMT via repression of BMP-5 in breast cancer.

Blimp1 activation by AP-1 in human lung cancer cells promotes a migratory phenotype and is inhibited by the lysyl oxidase propeptide.

B lymphocyte-induced maturation protein 1 (Blimp1) is a master regulator of B cell differentiation, and controls migration of primordial germ cells. Recently we observed aberrant Blimp1 expression in breast cancer cells resulting from an NF-κB RelB to Ras signaling pathway. In order to address the question of whether the unexpected expression of Blimp1 is seen in other epithelial-derived tumors, we selected lung cancers as they are frequently driven by Ras signaling. Blimp1 was detected in all five lung cancer cell lines examined and shown to promote lung cancer cell migration and invasion. Interrogation of microarray datasets demonstrated elevated BLIMP1 RNA expression in lung adenocarcinoma, pancreatic ductal carcinomas, head and neck tumors as well as in glioblastomas. Involvement of Ras and its downstream kinase c-Raf was confirmed using mutant and siRNA strategies. We next addressed the issue of mechanism of Blimp1 activation in lung cancer. Using knockdown and ectopic expression, the role of the Activator Protein (AP)-1 family of transcription factors was demonstrated. Further, chromatin immunoprecipitation assays confirmed binding to identified AP-1 elements in the BLIMP1 promoter of ectopically expressed c-Jun and of endogenous AP-1 subunits following serum stimulation. The propeptide domain of lysyl oxidase (LOX-PP) was identified as a tumor suppressor, with ability to reduce Ras signaling in lung cancer cells. LOX-PP reduced expression of Blimp1 by binding to c-Raf and inhibiting activation of AP-1, thereby attenuating the migratory phenotype of lung cancer cells. Thus, Blimp1 is a mediator of Ras/Raf/AP-1 signaling that promotes cell migration, and is repressed by LOX-PP in lung cancer.

Recombinant lysyl oxidase propeptide protein inhibits growth and promotes apoptosis of pre-existing murine breast cancer xenografts.

Lysyl oxidase propeptide (LOX-PP) ectopic overexpression inhibits the growth of cancer xenografts. Here the ability and mode of action of purified recombinant LOX-PP (rLOX-PP) protein to inhibit the growth of pre-existing xenografts was determined. Experimental approaches employed were direct intratumoral injection (i.t.) of rLOX-PP protein into murine breast cancer NF639 xenografts, and application of a slow release formulation of rLOX-PP implanted adjacent to tumors in NCR nu/nu mice (n = 10). Tumors were monitored for growth, and after sacrifice were subjected to immunohistochemical and Western blot analyses for several markers of proliferation, apoptosis, and for rLOX-PP itself. Direct i.t. injection of rLOX-PP significantly reduced tumor volume on days 20, 22 and 25 and tumor weight at harvest on day 25 by 30% compared to control. Implantation of beads preloaded with 35 micrograms rLOX-PP (n = 10) in vivo reduced tumor volume and weight at sacrifice when compared to empty beads (p<0.05). A 30% reduction of tumor volume on days 22 and 25 (p<0.05) and final tumor weight on day 25 (p<0.05) were observed with a reduced tumor growth rate of 60% after implantation. rLOX-PP significantly reduced the expression of proliferation markers and Erk1/2 MAP kinase activation, while prominent increases in apoptosis markers were observed. rLOX-PP was detected by immunohistochemistry in harvested rLOX-PP tumors, but not in controls. Data provide pre-clinical findings that support proof of principle for the therapeutic anti-cancer potential of rLOX-PP protein formulations.

The lysyl oxidase propeptide interacts with the receptor-type protein tyrosine phosphatase kappa and inhibits ß-catenin transcriptional activity in lung cancer cells.

The propeptide region of the lysyl oxidase proenzyme (LOX-PP) has been shown to inhibit Ras signaling in NIH 3T3 and lung cancer cells with activated RAS, but its mechanism of action is poorly understood. Here, a yeast two-hybrid assay of LOX-PP-interacting proteins identified a clone encoding the intracellular phosphatase domains of receptor-type protein tyrosine phosphatase kappa (RPTP-κ), and the interaction of the two proteins in mammalian cells was confirmed. RPTP-κ is proteolytically processed to isoforms that have opposing effects on ß-catenin activity. The RPTP-κ transmembrane P subunit interacts with and sequesters ß-catenin at the cell membrane, where it can associate with E-cadherin and promote intercellular interactions. At high cell density, further processing of the P subunit yields a phosphatase intracellular portion (PIC) subunit, which chaperones ß-catenin to the nucleus, where it can function to activate transcription. Lung cancer cells were found to contain higher PIC levels than untransformed lung epithelial cells. In H1299 lung cancer cells, ectopic LOX-PP expression reduced the nuclear levels of PIC by increasing its turnover in the lysosome, thereby decreasing the nuclear levels and transcriptional activity of ß-catenin while increasing ß-catenin membrane localization. Thus, LOX-PP is shown to negatively regulate pro-oncogenic ß-catenin signaling in lung cancer cells.

The Ras signaling inhibitor LOX-PP interacts with Hsp70 and c-Raf to reduce Erk activation and transformed phenotype of breast cancer cells.

The lysyl oxidase gene (LOX) inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162-amino-acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibits Erk signaling, motility, and tumor formation in a breast cancer xenograft model; however, its mechanism of action is largely unknown. Here, a copurification-mass spectrometry approach was taken using ectopically expressed LOX-PP in HEK293T cells and the heat shock/chaperone protein Hsp70 identified. Hsp70 interaction with LOX-PP was confirmed using coimmunoprecipitation of intracellularly and bacterially expressed and endogenous proteins. The interaction was mapped to the Hsp70 peptide-binding domain and to LOX-PP amino acids 26 to 100. LOX-PP association reduced Hsp70 chaperone activities of protein refolding and survival after heat shock. LOX-PP interacted with the Hsp70 chaperoned protein c-Raf. With the use of ectopic expression of LOX-PP wild-type and deletion proteins, small interfering RNA (siRNA) knockdown, and Lox(-/-) mouse embryo fibroblasts, LOX-PP interaction with c-Raf was shown to decrease downstream activation of MEK and NF-κB, migration, and anchorage-independent growth and reduce its mitochondrial localization. Thus, the interaction of LOX-PP with Hsp70 and c-Raf inhibits a critical intermediate in Ras-induced MEK signaling and plays an important role in the function of this tumor suppressor.

Lysyl oxidase propeptide sensitizes pancreatic and breast cancer cells to doxorubicin-induced apoptosis.

RAS mutations or its activation by upstream receptor tyrosine kinases are frequently associated with poor response of carcinomas to chemotherapy. The 18 kDa propeptide domain of lysyl oxidase (LOX-PP) released from the secreted precursor protein (Pro-LOX) has been shown to inhibit RAS signaling and the transformed phenotype of breast, pancreatic, lung, and prostate cancer cells in culture, and formation of tumors by Her-2/neu-driven breast cancer cells in a mouse xenograft model. Here, we tested the effects of LOX-PP on MIA PaCa-2 pancreatic cancer cells, driven by mutant RAS. In MIA PaCa-2 cells in culture, LOX-PP attenuated the ERK and AKT activities and decreased the levels of the NF-κB p65 and RelB subunits and cyclin D1, which are activated by RAS signaling. In mouse xenograft growth, LOX-PP reduced growth of tumors by these pancreatic cancer cells, and the nuclear levels of the p65 NF-κB subunit and cyclin D1 proteins. While biological agents attenuate tumor growth when used alone, often they have additive or synergistic effects when used in combination with chemotherapeutic agents. Thus, we next tested the hypotheses that LOX-PP sensitizes pancreatic and breast cancer cells to the chemotherapeutic agent doxorubicin. Purified LOX-PP enhanced the cytotoxic effects of doxorubicin in pancreatic and breast cancer cells, as judged by ATP production, Cell Death ELISA assays, caspase 3 activation, PARP cleavage, and Annexin V staining. Thus, LOX-PP potentiates the cytotoxicity of doxorubicin on breast and pancreatic cancer cells, warranting additional studies with a broader spectrum of current cancer treatment modalities.

Characterization of recombinant lysyl oxidase propeptide.

Lysyl oxidase enzyme activity is critical for the biosynthesis of mature and functional collagens and elastin. In addition, lysyl oxidase has tumor suppressor activity that has been shown to depend on the propeptide region (LOX-PP) derived from pro-lysyl oxidase (Pro-LOX) and not on lysyl oxidase enzyme activity. Pro-LOX is secreted as a 50 kDa proenzyme and then undergoes biosynthetic proteolytic processing to active approximately 30 kDa LOX enzyme and LOX-PP. The present study reports the efficient recombinant expression and purification of rat LOX-PP. Moreover, using enzymatic deglycosylation and DTT derivatization combined with mass spectrometry technologies, it is shown for the first time that rLOX-PP and naturally occurring LOX-PP contain both N- and O-linked carbohydrates. Structure predictions furthermore suggest that LOX-PP is a mostly disordered protein, which was experimentally confirmed in circular dichroism studies. Due to its high isoelectric point and its disordered structure, we propose that LOX-PP can associate with extracellular and intracellular binding partners to affect its known biological activities as a tumor suppressor and inhibitor of cell proliferation.

TAp63 is a transcriptional target of NF-kappaB.

The p53 homologue p63 encodes multiple protein isoforms either with (TA) or without (DeltaN) the N-terminal transactivation domain. Accumulating evidence indicates that TAp63 plays an important role in various biological processes, including cell proliferation, differentiation, and apoptosis. However, how TAp63 is regulated remains largely unclear. In this study, we demonstrate that NF-kappaB induces TAp63 gene expression. The responsible elements for NF-kappaB-mediated TAp63 induction are located within the region from -784 to -296 bp in the TAp63 promoter, which contains two NF-kappaB binding sites. Ectopic expression of RelA stimulates TAp63 promoter-driven reporter activity and increases endogenous TAp63 mRNA levels. Inhibition of NF-kappaB by IkappaBalpha super-repressor or with a chemical inhibitor leads to down regulation of TAp63 mRNA expression and activity. In addition, mutations in the critical NF-kappaB-binding sites significantly abolish the effects of NF-kappaB on TAp63. Activation of NF-kappaB by TNFalpha enhances p50/RelA binding to the NF-kappaB binding sites. Furthermore, we show that an Sp1 site adjacent to the NF-kappaB sites plays a role in NF-kappaB-mediated upregulation of TAp63. Taken together, these data reveal that TAp63 is a transcriptional target of NF-kappaB, which may play a role in cell proliferation, differentiation and survival upon NF-kappaB activation by various stimuli.

A loss-of-function polymorphism in the propeptide domain of the LOX gene and breast cancer.

The lysyl oxidase (LOX) gene reverted Ras transformation of NIH 3T3 fibroblasts and tumor formation by gastric cancer cells, which frequently carry mutant RAS genes. The secreted lysyl oxidase proenzyme is processed to a propeptide (LOX-PP) and a functional enzyme (LOX). Unexpectedly, the tumor suppressor activity mapped to the LOX-PP domain, which inhibited tumor formation and the invasive phenotype of NF639 breast cancer cells driven by human epidermal growth factor receptor-2/neu, which signals via Ras. A single-nucleotide polymorphism, G473A (rs1800449), resulting in an Arg158Gln substitution in a highly conserved region within LOX-PP, occurs with an average 473A allele carrier frequency of 24.6% in the HapMap database, but was present in many breast cancer cell lines examined. Here, we show that the Arg-to-Gln substitution profoundly impairs the ability of LOX-PP to inhibit the invasive phenotype and tumor formation of NF639 cells in a xenograft model. LOX-PP Gln displayed attenuated ability to oppose the effects of LOX, which promoted a more invasive phenotype. In a case-control study of African American women, a potential association of the Gln-encoding A allele was seen with increased risk of estrogen receptor (ER)-alpha-negative invasive breast cancer in African American women. Consistently, LOX gene expression was higher in ER-negative versus ER-positive primary breast cancers, and LOX-PP Gln was unable to inhibit invasion by ER-negative cell lines. Thus, these findings identify for the first time genetic polymorphism as a mechanism of impaired tumor suppressor function of LOX-PP and suggest that it may play an etiologic role in ER-negative breast cancer.

RelB NF-kappaB represses estrogen receptor alpha expression via induction of the zinc finger protein Blimp1.

Aberrant constitutive expression of NF-kappaB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERalpha)-negative breast cancers versus ERalpha-positive ones, due in part to repression of RelB synthesis by ERalpha signaling. Notably, RelB promoted a more invasive phenotype in ERalpha-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERalpha synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERalpha (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERalpha-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-kappaB subunit mediates repression, specifically of ERalpha, thereby promoting a more migratory phenotype.