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To better elucidate the role of vascular endothelial growth factor (VEGF) 165 in soft tissue sarcoma (STS) growth, metastasis, and chemoresistance, we generated stably transfected human STS cell lines with VEGF 165 to study the effect of VEGF 165 on STS cells in vitro and the effect of culture medium from these cells on human umbilical vascular endothelial cells. Severe combined immunodeficient mice bearing xenografts of transfected cell lines were used to assess the effect of VEGF overexpression and the effect of VEGF receptor (VEGFR) 2 inhibition on STS growth, metastasis, and response to doxorubicin. VEGF 165 -transfected xenografts formed highly vascular tumors with shorter latency, accelerated growth, enhanced chemoresistance, and increased incidence of pulmonary metastases. Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 monoclonal antibody enhanced doxorubicin chemoresponse; this combined biochemotherapy inhibited tumor growth and decreased pulmonary metastases without overt toxicity. Combined therapy reduced microvessel counts while increasing vessel maturation index. VEGF overexpression did not affect on the sarcoma cells per se; however, conditioned medium from VEGF transfectants caused increased endothelial cell proliferation, migration, and chemoresistance. Addition of DC101 induced endothelial cell sensitivity to doxorubicin and suppressed the activity of matrix metalloproteinases secreted by endothelial cells. We therefore conclude that VEGF is a critical determinant of STS growth and metastasis and that STS chemoresistance, in our model, is a process induced by the interplay between STS cells and tumor-associated endothelial cells. STS growth and metastasis can be interrupted by combined low-dose doxorubicin and anti-VEGFR2, a strategy that attacks STS-associated endothelial cells. In the future, such therapeutic approaches may be useful in treating STS before the development of clinically apparent metastases. (Cancer Res 2006; 66(17): 8770-8)

Human soft tissue sarcoma (STS) is a highly lethal malignancy in which control of metastasis determines survival. Little is known about the molecular determinants of STS dissemination. Here, we show that human STS express high levels of matrix metalloproteinase-9 (MMP-9) and that MMP-9 expression levels correlate with sequence analysisdefined p53 mutational status. Reintroduction of wild-type p53 (wtp53) into mutant p53 STS cell lines decreased MMP-9 mRNA and protein levels, decreased zymography-assessed MMP-9 proteolytic activity, and decreased tumor cell invasiveness. Reintroduction of wtp53 into STS xenografts decreased tumor growth and MMP-9 protein expression. Luciferase reporter studies showed that reintroduction of wtp53 into mutant p53 STS cells decreased MMP-9 promoter activity. Deletion constructs of the MMP-9 promoter identified a region containing a p53-responsive element that lacked a p53 consensus binding site but did contain a nuclear factor-KB (NF-KB) site. Mutating this NF-KB binding site eliminated the wtp53-repressive effect. Electrophoretic mobility shift assays confirmed decreased NF-KB binding in STS cells in the presence of wtp53. Our findings suggest a role for MMP-9 in STS progression and expand the role of p53 in molecular control of STS growth and metastasis. Therapeutic interventions in human STS targeting MMP-9 activity directly or via reintroduction of wtp53 merit further investigation. (Mol Cancer Res 2006;4(11):803 -10)

The protein kinase C (PKC) family consists of serine/ threonine protein kinases that play important roles in signal transduction, cell proliferation, and tumor formation. Recent studies found that PKCs are commonly overexpressed in human tumors, including soft tissue sarcoma (STS). Overexpression of PKCs contributes to invasion and migration of tumor cells and induction of angiogenesis. PKC can also phosphorylate the multidrug resistance (MDR) gene-encoded P-glycoprotein and induce MDR phenotype. Our previous studies showed that mutation of p53 enhanced STS metastasis and mediated the MDR phenotype. Restoring wild type (WT) p53 in STS cells containing mutant p53 sensitized the cells to chemotherapy. In the present study, we found that PKC␣ protein expression is inhibited by WT p53 partly due to reduced PKC␣ mRNA expression in STS cells, but p53 does not affect PKC␣ mRNA stability. Deletion and mutation analysis of the PKC␣ promoter fused to the luciferase reporter gene identified a Sp1 binding site (؊244/؊234) in the PKC␣ promoter that is required for p53-mediated inhibition of PKC␣ promoter activity. More importantly, PKC␣ phosphorylates and activates MDR1 P-glycoprotein, whereas inhibition of PKC␣ by p53 leads to decreased MDR1 phosphorylation in STS cells, which sensitizes STS cells to chemotherapeutic agents. These data indicate that WT p53 may resensitize STS to chemotherapeutic agents by reducing MDR1 phosphorylation via transcriptional repression of PKC␣ expression. Thus, molecular-based therapies targeting mutant p53 and PKC␣ may be an effective new strategy to improve chemotherapeutic efficacy in STS.The protein kinase C (PKC) 1 family consists of a large number of serine/threonine kinases that are activated by extracellular signals. In mammalian cells, the PKC family is further divided into three subfamilies, conventional PKCs (␣, ␤I, ␤II, ␥), novel PKCs (␦, ⑀, , ), and atypical PKCs ( , , ) (1). PKC is an additional PKC family member that was discovered recently and is known as protein kinase D (2). A distantly related PKC family includes three isoforms of the PKC-related kinases, known as PKN1, PKN2, and PKN3 (3). Although PKC family members share primary sequence similarities, the subfamily members have different enzymatic activation profiles. Specifically, conventional PKC isoforms are activated by 1,2-diacylglycerol and phosphatidylserine (PS) in a calcium-dependent manner, novel PKC members are activated only by 1,2-diacylglycerol and PS, and atypical PKC members are activated by PS alone as a co-factor (4). In resting cells, PKC predominantly resides in the cytosol in an inactive state and, upon stimulation, translocates as an active kinase to discrete subcellular locations, such as the plasma membrane, membranous vesicles, cytoskeleton, mitochondria, and nucleus (5).PKC functions have been the subject of intense study for many years after identification of PKC as the intracellular receptor for tumor-promoting phorbol esters such as tissuetype plasminogen activator. Studies have shown that PKC a...

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