Sonja Markwart

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.

Inhibition of mTOR Radiosensitizes Soft Tissue Sarcoma and Tumor Vasculature

Purpose: The PI3K/Akt/mTOR prosurvival pathway is frequently up-regulated in soft tissue sarcoma. Mammalian target of rapamycin (mTOR) inhibitors, such as rapamycin, have recently shown clinical benefit in soft tissue sarcoma, and mTOR inhibition has also been associated with radiosensitization of carcinoma and endothelial cells. This study tested the hypothesis that rapamycin radiosensitizes soft tissue sarcoma and endothelial cells in vitro and in vivo through the inhibition of mTOR. Experimental Design: Colony formation assays were done to determine the radiosensitizing properties of rapamycin on three human soft tissue sarcoma cell lines (SK-LMS-1, SW-872, and HT-1080) and human dermal microvascular endothelial cells (HDMEC). The functional effects of rapamycin and radiation on the endothelial compartment were evaluated with microvascular sprouting assays. The in vivo radiosensitizing activity of rapamycin was assessed with s.c. SK-LMS-1 nude mice xenografts treated with concurrent daily rapamycin, radiation, or both for three weeks. Results:In vitro radiosensitization was shown in all three soft tissue sarcoma cell lines with minimally cytotoxic doses of rapamycin. SK-LMS-1 xenografts displayed significant tumor growth delay with rapamycin and radiation compared with either treatment alone. Radiation resulted in transient increased mTOR function, whereas rapamycin abolished this signaling in irradiated and unirradiated samples. In HDMEC, rapamycin and radiation reduced microvessel sprouting, but did not alter colony formation. Conclusions: Minimally cytotoxic concentrations of rapamycin inhibited the mTOR cascade in culture and in vivo while radiosensitizing soft tissue sarcoma, and produced synergistic effects with radiation on HDMEC microvessel formation. By targeting both tumor and endothelial compartments, rapamycin produced potent radiosensitization of soft tissue sarcoma xenografts. Clinical trials combining rapamycin and radiotherapy in soft tissue sarcoma are warranted.

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.

Loss of Raf Kinase Inhibitory Protein Induces Radioresistance in Prostate Cancer

PURPOSE External beam radiotherapy (RT) is often used in an attempt to cure localized prostate cancer (PCa), but it is only palliative against disseminated disease. Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in approximately 50% of localized PCa tissues and is absent in metastases. Chemotherapeutic agents have been shown to induce tumor apoptosis through induction of RKIP expression. Our goal was to test whether RT similarly induces apoptosis through induction of RKIP expression. METHODS AND MATERIALS The C4-2B PCa cell line was engineered to overexpress or underexpress RKIP. The engineered cells were tested for apoptosis in cell culture and tumor regression in mice after RT. RESULTS RT induced both RKIP expression and apoptosis of PCa cells. Overexpression of RKIP sensitized PCa cells to radiation-induced apoptosis. In contrast, short-hairpin targeting of RKIP, so that RT could not induce RKIP expression, protected cells from radiation-induced apoptosis. In a murine model, knockdown of RKIP in PCa cells diminished radiation-induced apoptosis. Molecular concept mapping of genes altered on manipulation of RKIP expression revealed an inverse correlation with the concept of genes altered by RT. CONCLUSION The data presented in this report indicate that the loss of RKIP, as seen in primary PCa tumors and metastases, confers protection against radiation-induced apoptosis. Therefore, it is conceivable that the loss of RKIP confers a growth advantage on PCa cells at distant sites, because the loss of RKIP would decrease apoptosis, favoring proliferation.

Evaluation of d-Methionine as a Novel Oral Radiation Protector for Prevention of Mucositis

Purpose: Oral mucositis is a common acute morbidity associated with radiation and/or chemotherapy treatment for cancer. d-Methionine (d-Met), the dextro-isomer of the common amino acid l-methionine, has been documented to protect normal tissues from a diverse array of oxidative insults. Experimental Design: We evaluated if d-Met could selectively prevent radiation-induced oral mucositis using in vitro cell culture models as well as an in vivo model of radiation injury to the oral mucosa in C3H mice. Results: Unlike free-radical scavengers, which protected both normal and transformed tumor cells in vitro from radiation-induced cell death, treatment with d-Met in culture protected nontransformed primary human cells from radiation-induced cell death (protective factor between 1.2 and 1.6; P < 0.05) whereas it did not confer a similar protection on transformed tumor cells. d-Met treatment also provided significant protection to normal human fibroblasts, but not to tumor cell lines, from radiation-induced loss of clonogenicity (protection factor, 1.6 ± 0.15). d-Met treatment did not alter DNA damage (as measured by histone phosphorylation) following irradiation but seemed to selectively mitigate the loss of mitochondrial membrane potential in nontransformed cells, whereas it did not provide a similar protection to tumor cells. Tumor control of implanted xenografts treated with radiation or concurrent cisplatin and radiation was not altered by d-Met treatment. Pharmacokinetics following administration of a liquid suspension of d-Met in rats showed 68% bioavailability relative to i.v. administration. Finally, in a murine model of mucositis, a dose-dependent increase in protection was observed with the protective factor increasing from 1.6 to 2.6 over a range of oral d-Met doses between 200 and 500 mg/kg (P < 0.0003). Conclusions:d-Met protected normal tissues, but not tumor cells, in culture from radiation-induced cell death; it also protected normal cells from radiation-induced mucosal injury in a murine model but did not alter tumor response to therapy. Further studies on the use of d-Met to protect from oral mucositis are warranted.

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.