Donna L. Livant

EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells

The Polycomb Group Protein EZH2 is a transcriptional repressor involved in controlling cellular memory and has been linked to aggressive prostate cancer. Here we investigate the functional role of EZH2 in cancer cell invasion and breast cancer progression. EZH2 transcript and protein were consistently elevated in invasive breast carcinoma compared with normal breast epithelia. Tissue microarray analysis, which included 917 samples from 280 patients, demonstrated that EZH2 protein levels were strongly associated with breast cancer aggressiveness. Overexpression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion. EZH2-mediated cell invasion required an intact SET domain and histone deacetylase activity. This study provides compelling evidence for a functional link between dysregulated cellular memory, transcriptional repression, and neoplastic transformation.

WISP3 is a novel tumor suppressor gene of inflammatory breast cancer

Inflammatory breast cancer (IBC) is an aggressive form of breast cancer with a 5-year disease-free survival of less than 45%. Little is known about the genetic alterations that result in IBC. In our previous work, we found that WISP3 was specifically lost in human IBC tumors when compared to stage-matched, non-IBC tumors. We hypothesize that WISP3 has tumor suppressor function in the breast and that it may be a key genetic alteration that contributes to the unique IBC phenotype. The full-length WISP3 cDNA was sequenced and cloned into an expression vector. The resulting construct was introduced in to the SUM149 cell line that was derived from a patient with IBC and lacks WISP3 expression. In soft agar, stable WISP3 transfectants formed significantly fewer colonies than the controls. Stable WISP3 transfectants lost their ability to invade and had reduced angiogenic potential. WISP3 transfection was effective in suppressing in vivo tumor growth in nude mice. Mice bearing WISP3 expressing tumors had a significantly longer survival than those with vector-control transfectant tumors. Our data demonstrate that WISP3 acts as a tumor suppressor gene in the breast. Loss of WISP3 expression contributes to the phenotype of IBC by regulating tumor cell growth, invasion and angiogenesis.

The PHSRN sequence induces extracellular matrix invasion and accelerates wound healing in obese diabetic mice

The PHSRN sequence of the plasma fibronectin (pFn) cell-binding domain induces human keratinocytes and fibroblasts to invade the naturally serum-free extracellular matrices of sea urchin embryos. The potency of acetylated, amidated PHSRN (Ac-PHSRN-NH(2)) is significantly increased, making it more active on a molar basis than the 120-kDa cell-binding domain of pFn. Arginine is important to this activity because PHSAN and PHSEN are inactive, as is a randomized sequence peptide, Ac-HSPNR-NH(2). One treatment with Ac-PHSRN-NH(2) stimulates reepithelialization and contraction of dermal wounds in healing-impaired, obese diabetic C57BL6/KsJ db/db mice. Wound closure is equally rapid in treated db/db and db/+ mice and may be more rapid than in untreated nondiabetic db/+ littermates. In contrast, treatment with either Ac-HSPNR-NH(2) or normal saline (NS) has no effect. Analysis of sectioned db/db wounds shows that, in contrast to treatment with Ac-HSPNR-NH(2) or NS, a single Ac-PHSRN-NH(2) treatment stimulates keratinocyte and fibroblast migration into wounds, enhances fibroplasia and vascularization in the provisional matrix, and stimulates the formation of prominent fibers that may be associated with wound contraction.

Integrin Fibronectin Receptors in Matrix Metalloproteinase-1-Dependent Invasion by Breast Cancer and Mammary Epithelial Cells

Integrins contribute to progression in many cancers, including breast cancer. For example, the interaction of α5β1 with plasma fibronectin causes the constitutive invasiveness of human prostate cancer cells. Inhibition of this process reduces tumorigenesis and prevents metastasis and recurrence. In this study, naturally serum-free basement membranes were used as invasion substrates. Immunoassays were used to compare the roles of α5β1 and α4β1 fibronectin receptors in regulating matrix metalloproteinase (MMP)-1–dependent invasion by human breast cancer and mammary epithelial cells. We found that a peptide consisting of fibronectin PHSRN sequence, Ac-PHSRN-NH2, induces α5β1-mediated invasion of basement membranes in vitro by human breast cancer and mammary epithelial cells. PHSRN-induced invasion requires interstitial collagenase MMP-1 activity and is suppressed by an equimolar concentration of a peptide consisting of the LDV sequence of the fibronectin connecting segment, Ac-LHGPEILDVPST-NH2, in mammary epithelial cells, but not in breast cancer cells. This sequence interacts with α4β1, an integrin that is often down-regulated in breast cancer cells. Immunoblotting shows that the PHSRN peptide stimulates MMP-1 production by serum-free human breast cancer and mammary epithelial cells and that the LDV peptide represses PHSRN-stimulated MMP-1 production only in mammary epithelial cells. Furthermore, PHSRN stimulates MMP-1 activity in breast cancer cells and mammary epithelial cells with a time course that closely parallels invasion induction. Thus, down-regulation of surface α4β1 during oncogenic transformation may be crucial for establishment of the α5β1-induced, MMP-1–dependent invasive phenotype of breast cancer cells.

Radiation Increases Invasion of Gene-Modified Mesenchymal Stem Cells into Tumors

PURPOSE Mesenchymal stem cells (MSCs) are multipotent cells in the bone marrow that have been found to migrate to tumors, suggesting a potential use for cancer gene therapy. MSCs migrate to sites of tissue damage, including normal tissues damaged by radiation. In this study, we investigated the effect of tumor radiotherapy on the localization of lentivirus-transduced MSCs to tumors. METHODS AND MATERIALS MSCs were labeled with a lipophilic dye to investigate their migration to colon cancer xenografts. Subsequently, the MSCs were transduced with a lentiviral vector to model gene therapy and mark the infused MSCs. LoVo tumor xenografts were treated with increasing radiation doses to assess the effect on MSC localization, which was measured by quantitative polymerase chain reaction. MSC invasion efficiency was determined in an invasion assay. RESULTS MSCs migrated to tumor xenografts of various origins, with few cells found in normal tissues. A lentiviral vector efficiently transduced MSCs in the presence, but not the absence, of hexadimethrine bromide (Polybrene). When LoVo tumors were treated with increasing radiation doses, more MSCs were found to migrate to them than to untreated tumors. Irradiation increased MSC localization in HT-29 and MDA-MB-231, but not UMSCC1, xenografts. Monocyte chemotactic protein-1 expression in tumors did not correlate with the basal levels of MSC infiltration; however, monocyte chemotactic protein-1 was modestly elevated in irradiated tumors. Media from irradiated LoVo cells stimulated MSC invasion into basement membranes. CONCLUSION These findings suggest that radiation-induced injury can be used to target MSCs to tumors, which might increase the effectiveness of MSC cancer gene therapy. The production of tumor-derived factors in response to radiation stimulates MSC invasion.

Monocyte-Fibronectin Interactions, Via α5β1 Integrin, Induce Expression of CXC Chemokine-Dependent Angiogenic Activity

Monocyte-derived macrophages are important sources of angiogenic factors in cancer and other disease states. Upon extravasation from vasculature, monocytes encounter the extracellular matrix. We hypothesized that interaction with extracellular matrix proteins leads monocytes to adopt an angiogenic phenotype. We performed endothelial cell chemotaxis assays on conditioned medium (CM) from monocytes that had been cultured in vitro on various matrix substrates (collagen I, laminin, Matrigel, fibronectin), in the presence of autologous serum, or on tissue culture plastic alone. Monocytes cultured on Matrigel and on fibronectin were the most potent inducers of angiogenic activity compared with tissue culture plastic or autologous serum-differentiated monocytes. This increased angiogenic activity was associated with increased expression of angiogenic CXC chemokines (IL-8, epithelial neutrophil-activating peptide-78, growth-related oncogene α, and growth-related oncogene γ) but not of vascular endothelial growth factor. Additionally, CM from monocytes cultured on fibronectin-depleted Matrigel (MGFN−) induced significantly less angiogenic activity than CM from monocytes cultured on control-depleted Matrigel. ELISA analysis of CM from monocytes cultured on MGFN− revealed a significant decrease in GRO-α and GRO-γ compared with CM from monocytes cultured on MG. Incubation of monocytes before adherence on fibronectin with PHSCN (a competitive peptide inhibitor of the PHSRN sequence of fibronectin binding via α5β1 integrin) results in diminished expression of angiogenic activity and CXC chemokines compared with control peptide. These data suggest that fibronectin, via α5β1 integrin, promotes CXC chemokine-dependent angiogenic activity from monocytes.

p38MAPK induces cell surface α4 integrin downregulation to facilitate erbB-2-mediated invasion

We have previously shown that human breast cancer cells that overexpress erbB-2 are growth factor-independent. In order to test the contribution of erbB-2 to this and other transformed phenotypes without the genetic instability of cancer cells, erbB-2 was overexpressed in human mammary epithelial (HME) cells. ErbB-2-overexpressing HME cells exhibit several transformed phenotypes including cell surface alpha(4) integrin downregulation and invasiveness. We formulated a model for invasiveness that depends on a cells ability to downregulate alpha(4) integrin. As small G-proteins play a role in cytoskeleton remodeling and as this is a likely route for alpha(4) integrin trafficking, we investigated the role of small G-proteins and their downstream signals in mediating alpha(4) integrin downregulation and invasiveness using Rac 1. Dominant-negative Rac 1 blocked erbB-2-mediated invasion and reversed erbB-2-mediated alpha(4) integrin downregulation. In addition, constitutively active Rac 1 induced alpha(4) integrin downregulation and invasiveness. In erbB-2-overexpressing and in constitutively active Rac 1-expressing cells, a p38MAP kinase (p38MAPK) inhibitor blocked invasiveness and reversed alpha(4) integrin downregulation. These data suggest a model in which erbB-2 signaling activates Rac 1, which, in turn, activates p38MAPK, leading to the downregulation of alpha(4) integrin. These data strengthen the model where loss of alpha(4) integrin at the cell surface, leading to reduced alpha(4) integrin binding to plasma fibronectin, plays a role in erbB-2-mediated invasiveness.

Phenylbutyrate inhibits the invasive properties of prostate and breast cancer cell lines in the sea urchin embryo basement membrane invasion assay.

Histone deacetylase inhibitors, such as phenylbutyrate, are currently undergoing clinical trials as potential anticancer agents. Phenylbutyrate can induce cell differentiation and apoptosis in a number of cancer cell types and can act in synergy with ionizing radiation and chemotherapy to induce apoptosis. We used the sea urchin embryo basement membrane invasion assay to show that phenylbutyrate potently inhibited the invasive properties of both prostate and breast cancer cells at clinically achievable doses. This inhibition was dose‐dependent and persisted for at least 24 hr after the drug was removed. These results suggest that in addition to activating apoptosis in cancer cells, phenylbutyrate may be used in prevention of metastatic disease.

Abstract 3890: The PhScN peptide as a highly potent inhibitor of basement membrane invasion in vitro and lung colonization in nude mice by metastatic human prostate cancer cells

Surgery and radiation can cure localized prostate cancer, but not metastatic disease. Our research has shown that activated Δ5α1 integrin fibronectin receptors mediate metastatic invasion by human prostate cancer cell lines, DU 145 and PC-3, as well as angiogenic invasion by human microvascular endothelial cells (hmvec). Thus, activated Δ5α1 receptors are key to metastatic progression. To inhibit metastatic invasion, we devised the Ac-PHSCN-NH2 peptide, PHSCN, licensed as ATN-161. Systemic ATN-161 monotherapy was well tolerated in Phase I trial, and halted metastatic progression for 4-14 months in 35% of patients; however, it was not potent enough for easy administration. Thus, there are some major difficulties with ATN-161 as a drug. Also, PHSCN can be degraded by endoproteinases, enzymes that cleave between its linked L-amino acids. Since tumors secrete abundant endoproteinases, we devised a potent, exo- and endoproteinase-resistant, Δ5α1αtargeted invasion inhibitor, the Ac-PhScN-NH2 peptide (PhScN). Because it contains mirror image D-isomers of His (h) and Cys (c) separating L-isomers of Pro, Ser, and Asn, is resistant to endoproteolytic degradation. Our results suggest that PhScN is 27,000 to 340,000-fold more potent than PHSCN at blocking Δ5α1-mediated, serum-induced, or serum-free PHSRN peptide-induced invasion in vitro by metastatic human DU 145 and PC-3 prostate cancer cells. Our results also indicate that a single pretreatment of suspended DU 145 and PC-3 cells with varying concentrations of the PhScN peptide prior to intravenous injection reduces lung colony formation in athymic, nude mice by 100,000 to 300,000-fold, relative to lung metastasis inhibition by the parental PHSCN peptide. Because Δ5α1 integrin also supports survival, we determined the effects of elevated PhScN concentrations on clonogenic DU 145 and PC-3 survival in vitro. We found that PhScN has no appreciable effect on clonogenic survival, suggesting that its potent anti-metastatic activity is due to invasion inhibition in lung microvasculature. Consistent with this hypothesis, our preliminary results suggest that PhScN pretreatment is also significantly more potent method for preventing extravasation into lung tissue than pretreatment with the parental PHSCN peptide. Since our published results show that the PHSCN polylysine dendrimer (PHSCNGGK-MAP) is 100-fold more potent than the PHSCN peptide at inhibiting DU 145 and PC-3 lung colonization, the results presented here suggest that D-amino acid substitution in the PHSCN sequence, thereby forming the PhScN peptide or dendrimer (PhScNGGK-MAP) may be an excellent strategy for the synthesis of highly potent Δ5α1-targeted inhibitors of metastatic invasion and lung colonization. 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 3890. doi:1538-7445.AM2012-3890