Linda W. Horton

Augmenting Chemosensitivity of Malignant Melanoma Tumors via Proteasome Inhibition: Implication for Bortezomib (VELCADE, PS-341) as a Therapeutic Agent for Malignant Melanoma

Melanoma poses a great challenge to patients, oncologists, and biologists because of its nearly universal resistance to chemotherapy. Many studies have shown that nuclear factor κB is constitutively activated in melanoma, thereby promoting the proliferation of melanoma cells by inhibiting the apoptotic responses to chemotherapy. Nuclear factor κB activity is regulated by phosphorylation and subsequent degradation of inhibitor of nuclear factor κB by the ubiquitin-proteasome pathway. In this study, we show that the novel proteasome inhibitor, bortezomib, inhibited the growth of melanoma cells in vitro at a concentration range of 0.1–10 nm and in combination with the chemotherapeutic agent temozolomide, the inhibitory effect on melanoma cell growth was even more prominent. Data from a murine model showed reduced tumor growth when bortezomib was administered to human melanoma tumors. Strikingly, animals receiving bortezomib in combination with temozolomide achieved complete remission of palpable tumors after only 30 days of therapy, lasting >200 days. Our data indicate strongly that bortezomib in combination with chemotherapeutic agents should be studied additionally for the treatment of melanoma.

Cytokine Receptor CXCR4 Mediates Estrogen-Independent Tumorigenesis, Metastasis, and Resistance to Endocrine Therapy in Human Breast Cancer

Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1-CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1-CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1-mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.

Potential role for Duffy antigen chemokine-binding protein in angiogenesis and maintenance of homeostasis in response to stress

CXC chemokines, which induce angiogenesis, haveglutamine‐leucine‐arginine amino acid residues (ELR motif) in the aminoterminus and bind CXCR2 and the Duffy antigen chemokine‐bindingprotein. Duffy, a seven transmembrane protein that binds CXC and CCchemokines, has not been shown to couple to trimeric G proteins or totransduce intracellular signals, although it is highly expressed on redblood cells, endothelial cells undergoing neovascularization, andneuronal cells. The binding of chemokines by Duffy could modulatechemokine responses positively or negatively. Positive regulation couldcome through the presentation of chemokine to functional receptors, andnegative regulation could come through Duffy competition withfunctional chemokine receptors for chemokine binding, thus serving as adecoy receptor. To determine whether Duffy has a role in angiogenesisand/or maintenance of homeostasis, we developed transgenic miceexpressing mDuffy under the control of the preproendothelinpromoter/enhancer (PPEP), which directs expression of the transgeneto the endothelium. Two PPEP‐mDuffy‐transgenic founders wereidentified, and expression of the transgene in the endothelium wasverified by Northern blot, RT‐PCR, and immunostaining of tissues. Thephenotype of the mice carrying the transgene appeared normal by allvisual parameters. However, careful comparison of transgenic andnontransgenic mice revealed two phenotypic differences:mDuffy‐transgenic mice exhibited a diminished angiogenic response toMIP‐2 in the corneal micropocket assay, and mDuffy‐transgenic miceexhibited enhanced hepatocellular toxicity and necrosis as comparedwith nontransgenic littermates in response to overdose of acetaminophen(APAP; 400 mg/kg body weight). Morover, APAP treatment was lethal in50% of the mDuffy‐transgenic mice 24 h post challenge, and 100%of the nontransgenic littermates survived this treatment at the 24 h time point. Our data suggest that enhanced expression of mDuffy onendothelial cells can lead to impaired angiogenic response tochemokines and impaired maintenance of homeostasis in response to toxicstresses.

A Phase I Trial of Bortezomib with Temozolomide in Patients with Advanced Melanoma: Toxicities, Antitumor Effects, and Modulation of Therapeutic Targets

Purpose: Preclinical studies show that bortezomib, a proteasome inhibitor, blocks NF-κB activation and, combined with temozolomide, enhances activity against human melanoma xenografts and modulates other critical tumor targets. We initiated a phase I trial of temozolomide plus bortezomib in advanced melanoma. Objectives included defining a maximum tolerated dose for the combination, characterizing biomarker changes reflecting inhibition of both proteasome and NF-κB activity in blood (if possible tumor), and characterizing antitumor activity. Experimental Design: Cohorts were enrolled onto escalating dose levels of temozolomide (50-75 mg/m2) daily, orally, for 6 of 9 weeks and bortezomib (0.75-1.5 mg/m2) by i.v. push on days 1, 4, 8, and 11 every 21 days. Peripheral blood mononuclear cells were assayed at specified time points for proteasome inhibition and NF-κB biomarker activity. Results: Bortezomib (1.3 mg/m2) and temozolomide (75 mg/m2) proved to be the maximum tolerated dose. Dose-limiting toxicities included neurotoxicity, fatigue, diarrhea, and rash. Nineteen melanoma patients were enrolled onto four dose levels. This melanoma population (17 M1c, 10 elevated lactate dehydrogenase, 12 performance status 1-2) showed only one partial response (8 months) and three with stable disease ≥4 months. A significant reduction in proteasome-specific activity was observed 1 hour after infusion at all bortezomib doses. Changes in NF-κB electrophoretic mobility shift assay and circulating chemokines in blood failed to correlate with the schedule/dose of bortezomib, inhibition of proteasome activity, or clinical outcome. Conclusions: We have defined phase II doses for this schedule of temozolomide with bortezomib. Although proteasome activity was inhibited for a limited time in peripheral blood mononuclear cells, we were unable to show consistent effects on NF-κB activation. Clin Cancer Res; 16(1); 348–57

Passage-dependent cancerous transformation of human mesenchymal stem cells under carcinogenic hypoxia

Bone marrow‐derived human mesenchymal stem cells (hMSCs) either promote or inhibit cancer progression, depending on factors that heretofore have been undefined. Here we have utilized extreme hypoxia (0.5% O2) and concurrent treatment with metal carcinogen (nickel) to evaluate the passage‐dependent response of hMSCs toward cancerous transformation. Effects of hypoxia and nickel treatment on hMSC proliferation, apoptosis, gene and protein expression, replicative senescence, reactive oxygen species (ROS), redox mechanisms, and in vivo tumor growth were analyzed. The behavior of late passage hMSCs in a carcinogenic hypoxia environment follows a profile similar to that of transformed cancer cells (i.e., increased expression of oncogenic proteins, decreased expression of tumor suppressor protein, increased proliferation, decreased apoptosis, and aberrant redox mechanisms), but this effect was not observed in earlier passage control cells. These events resulted in accumulated intracellular ROS in vitro and excessive proliferation in vivo. We suggest a mechanism by which carcinogenic hypoxia modulates the activity of three critical transcription factors (c‐MYC, p53, and HIF1), resulting in accumulated ROS and causing hMSCs to undergo cancer‐like behavioral changes. This is the first study to utilize carcinogenic hypoxia as an environmentally relevant experimental model for studying the age‐dependent cancerous transformation of hMSCs.— Crowder, S. W., Horton, L. W., Lee, H. H., McClain, C. M., Hawkins, O. E., Palmer, A. M. Bae, H., Richmond, A., Sung, H.‐J. Passage‐dependent cancerous transformation of human mesenchymal stem cells under carcinogenic hypoxia. FASEB J. 27, 2788‐2798 (2013). www.fasebj.org

Binding and functional properties of a mouse-human chimeric monoclonal antibody of the human IgG1 subclass with specificity for human carcinomas

Recombinant DNA techniques were utilized successfully to join the coding regions for the variable region of a mouse anti-tumor antibody (BA-Br-1) and the human IgG1 constant region for both the light and heavy chains. After insertion into a mouse myeloma host cell line, the chimeric genes were expressed successfully and the resulting antibody (ING-1) was purified. In this study, we describe biochemical, serological, immunohistochemical, and functional properties of the chimeric ING-1 antibody. Analysis of the synthesized antibody revealed that while it was similar in size to the mouse antibody, it had a different pI as determined by isoelectrofocusing. The flow cytometric binding profiles of the new molecule were found to be essentially identical to the parental mouse immunoglobulin. The specificity of the chimeric ING-1 and mouse BA-Br-1 antibodies were compared by extensive immunohistochemical analysis on human normal and tumor tissues. The chimeric antibody retained the same broad carcinoma binding activity, showing strong reactivity with greater than 90% of epithelial tumor tissues, as was previously observed for the mouse BA-Br-1 antibody. The chimeric and mouse antibodies also recognized the same selected normal tissues: primarily glandular epithelia, gastrointestinal mucosa, bile ducts, and thyroid follicles. Analysis of the biological function of the chimeric antibody revealed that it possessed ADCC activity against antigen-bearing tumor targets in vitro which was absent from the mouse form of the antibody. Competent effector cells could be either PBMCs from normal healthy donors, PBMCs from cancer patients receiving LAK/IL-2 therapy, or LAK cells prepared from cancer patients. Enhanced cytotoxicity even in the presence of LAK cell killing was noted with effector cells from the latter two sources. This contrasts sharply with the absence of activity in the same systems when the native murine antibody was used. The in vitro activation of cell-dependent cytolysis observed with the chimeric antibodies when effector cells from both normal and tumor-bearing donors were used strongly suggests that comparable activity would be observed in vivo. These results, along with the broad carcinoma binding activity and minimal normal tissue reactivity, suggest that the ING-1 chimeric antibody may be useful in cancer therapy. The application of the ING-1 chimeric antibody for treatment of tumors thus offers a promising avenue for future research.

Visualization of eosinophil chemotactic factor in abomasal tissue of cattle by immunoperoxidase staining during Ostertagia ostertagi infection

Eosinophil chemotactic factor (ECF) was localized predominantly in the intestinal cells and lateral hypodermal cords of developing fifth stage larvae (L5) of Ostertagia ostertagi within abomasal tissue cross-sections by peroxidase in an antibody sandwich technique using monoclonal antibody to ECF. Cooperia oncophora larvae in tissue cross-sections did not stain using this technique. These experiments demonstrate that ECF is localized in Ostertagia ostertagi organelles and is probably released by the developing L5 into the abomasal tissue surrounding the parasitized gland. The presence of ECF within O. ostertagi larvae in situ and the results of previous experiments demonstrated in vitro and in vivo ECF chemotactic activity help to explain why eosinophils are observed histologically in abomasal tissues from cattle with ostertagiasis.

Human tumor and normal tissue reactivity of the anti-(breast cancer) monoclonal antibody BA-Br-3 and its similarity to the anti-(epithelial membrane antigen) monoclonal antibody E29

SummaryA mouse monoclonal antibody (BA-Br-3) raised against the breast carcinoma cell line CAMA-1 was previously shown to react with a ⩾ 300-kDa globule-like glycoprotein from human milk fat also expressed in the cytoplasm and on the surface of human carcinoma cells of different histological types. In this report the reactivity of this mAb with a large number of normal and malignant human tissues was analyzed using immunoperoxidase techniques. When tested on sections of both fresh-frozen tissues and formalin-fixed, paraffin-embedded tissues, BA-Br-3 reacted with a formalin-resistant antigenic determinant expressed by normal and malignant epithelial cells. Preferential reactivity was observed at the apical portion of ductal epithelial cells in normal breast and in glandular epithelia distributed in several other organs. Reactivity with mucin-like secretions in the lumina of ducts was also found. BA-Br-3 reacted mostly in heterogenous staining patterns with 88% of 49 breast carcinoma specimens tested, regardless of their histological type or whether they were primary or secondary neoplasms. Testing of epithelial malignant tumors other than breast carcinomas with this antibody showed that 127 of 151 (84%) were also reactive. mAb BA-Br-3 and E29 (a commercially available anti-(epithelial membrane antigen) shared very similar staining patterns and distributions of reactivity with breast and other epithelial tumors. However, BA-Br-3 showed a significantly higher percentage of reactivity with melanoma (33% versus 6%,P = 0.003) and a trend toward a higher percentage of reactivity with sarcoma (55% versus 27%,P >0.05). This antibody, therefore, defines a molecule that is a member of the mucin-like epithelial membrane antigen family. Further studies are warranted to determine its usefulness in antibody-directed cancer diagnosis, prognosis, and immunotherapy.

Abstract B12: Synergistic anticancer activity of Aurora A kinase and MDM2 antagonists in melanoma

Senescence-inducing therapies can block proliferation of malignant cells and promote anti-tumor immune activity. However, the risk of tumor relapse remains high due to the long lifespan of senescence cells with potential to escape senescence. Here our preclinical studies demonstrate that combining a senescent-inducing aurora kinase A (AURKA) inhibitor alisertib (MLN8237) with an MDM2 antagonist [(-)-nutlin 3a] effectively induces robust p53 activation in senescent Tp53WT tumors accompanied by: 1) tumor cell proliferation arrest; 2) mitochondrial depolarization and tumor cell apoptosis; and 3) tumor cell clearance via CCL5-, CCL1- and CCL9-mediated recruitment of anti-tumor leukocytes. This combined therapy shows adequate bioavailability and low toxicity to the host in the mouse model. Moreover, the prominent preclinical response of patient-derived melanoma tumors to the co-targeting of MDM2 and AURKA provides rationale for further investigation of alisertib and MDM2 inhibitors. Citation Format: Anna E. Vilgelm, Jeff S. Pawlikowski, Yan Liu, E. Hawkins Oriana, A. Davis Tyler, Kevin P. Weller, Linda W. Horton, Colt M. McClain, Gregory D. Ayers, David Turner, David C. Essaka, Clinton F. Stewart, Jeffrey A. Sosman, Mark C. Kelley, Jeffrey A. Ecsedy, Jeffrey N. Johnston, Ann Richmond. Synergistic anticancer activity of Aurora A kinase and MDM2 antagonists in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B12.

Abstract 4403: Combined antagonism of the IL-4R and IKKα inhibits melanoma tumor growth in immunocompetent mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL With over 8500 deaths each year in the USA, malignant melanoma is the deadliest of all skin cancers. Constitutive activation of the nuclear factor kappa B (NF-κB) is common in melanoma, promoting tumor growth, survival and metastasis. In light of its role in tumorigenesis, targeting of the NF-κB pathway has been considered a possible therapeutic option for the treatment of melanoma. However, NF- αB activation is critical for the development, survival, migration and function of immune cells. We hypothesized that systemic inhibition of the activation of NF-κB might have a deleterious effect on the tumor immune response. Melanoma bearing C57BL/6 mice received 50 mg/kg of BMS-345541, a selective inhibitor of the inhibitor of αB kinase (IKKα), twice daily. Tumors, spleens and bone marrow were harvested at different time points and analyzed by flow cytometry for leukocyte composition and cytokine production. Intracellular cytokine staining revealed an early induction of Interleukin-4 (IL-4) within the myeloid and B cell components of the tumor, as a result of BMS-345541 treatment. The production of IL-4 by these cells suggests an early switch to a tumor promoting T helper 2 (Th2) and M2 driven immune response, which may counteract the anti-tumor effect of IKKα inhibition by generating a pro-tumorigenic microenvironment. These results, in part, explain the modest delay in tumor growth demonstrated. IL-4 receptor (IL-4R) expression on human melanoma cells has been previously documented; however the effects of IL-4 on these cells have generated contradictory reports. IL-4R signaling has been shown to either inhibit growth of tumor cells in vitro or to promote survival of tumor cells. In C57Bl/6 mice bearing a murine melanoma, BMS345541 (75 mg/kg bid) was given in combination with 100 µg of IL-4R blocking antibody on day 4 and day 12 of treatment. This combination significantly inhibited tumor growth compared to BMS345541 treatment alone. These data suggest an important role for IL-4 in promoting melanoma growth in vivo. Dual targeting of IL-4R and NF-κB signaling pathways neutralizes the tumor promoting immune component. Currently, IL-4R antagonists are in clinical trials for the treatment of asthma, which makes the translational prospects of this study extraordinarily viable. We are grateful for support from the NCI (CA116021-07, 5T32CA119925-03) and the Department of Veterans Affairs for these studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4403. doi:1538-7445.AM2012-4403