Effects of the synthetic vasopressin analog desmopressin in a mouse model of colon cancer

Experimental and clinical data indicated that perioperative administration of the hemostatic peptide desmopressin (DDAVP) can inhibit progression of residual metastatic cells. The compound seems to act by inducing an agonist effect on specific V2 vasopressin membrane receptors present in both tumor cells and endothelial cells. Here we explored the antitumor effects of DDAVP in cultured colon carcinoma cells and in a syngeneic Balb/c mouse model. Both human Colo-205 and mouse CT-26 colon carcinoma cell lines expressed the V2 receptor, as revealed by immunofluorescence. DDAVP (at doses ranging from 100 ng/ml to 1 μg/ml) exerted a modest but significant antiproliferative effect on cultured CT-26 and Colo-205 cells. In vivo, DDAVP (2 intravenous doses of 2 μg/kg) reduced accumulation of ascites and formation of intestinal tumor nodules in mice intraperitoneally inoculated with CT-26 cells. Perioperative administration of DDAVP significantly inhibited tumor progression in animals surgically implanted in the spleen with CT-26 cells, and caused some reduction in liver metastasis. Although DDAVP and 5-fluorouracil demonstrated additive cytostatic effects in vitro, no antitumor effects were observed in this study in mice receiving a single cycle of chemotherapy (25 mg/kg) in combination with the peptide. Our data suggest that DDAVP may be potentially used to minimize spread or survival of residual malignant cells during surgical procedures for colon and other gastrointestinal tumors.     

Desmopressin inhibits lung and lymph node metastasis in a mouse mammary carcinoma model of surgical manipulation

BACKGROUND AND OBJECTIVES: Desmopressin (DDAVP) is a synthetic derivative of vasopressin with hemostatic and fibrinolytic properties that has been used during surgery in patients with bleeding disorders. Our aim was to investigate the effect of DDAVP on lung and lymph node metastatic cell colonization using a preclinical mouse mammary carcinoma model of subcutaneous tumor manipulation and surgical excision. METHODS: Female BALB/c mice bearing the highly aggressive F3II mammary carcinoma were subjected to repeated manipulations of primary tumors (0.5 kg/cm(2) during 2 min), followed (or not) by surgical excision. DDAVP was administered intravenously 30 min before and 24 h after each manipulation or surgery, at a dose of 2 microg/kg. At the end of the experiment, mice were sacrificed and necropsied. RESULTS: Tumor manipulation induced dissemination to the axillary nodes and increased up to 6-fold the number of metastatic lung nodules. Perioperative treatment with DDAVP dramatically reduced regional metastasis. The incidence of lymph node involvement in manipulated animals was 12% with DDAVP and 87% without treatment (P < 0.02). Histopathological analysis of axillary nodes from DDAVP-treated animals showed sinusal histiocytosis and no evidence of cancer cells. Metastatic lung nodules were also reduced about 65% in animals treated with DDAVP (P = 0.026). CONCLUSIONS: Our results suggest a potential clinical application of DDAVP in the management of breast cancer, as well as other aggressive solid tumors. DDAVP may be useful to reduce the risk of metastatic cell colonization both during and after surgical manipulation.     

Aryl Hydrocarbon Receptor in Breast Cancer—A Newly Defined Prognostic Marker

Aryl hydrocarbon receptor (AhR) has been reported to exert various anticancer effects upon breast carcinoma cells in vitro but its details have remained largely unknown. Therefore, we first examined the AhR status in 90 invasive ductal carcinoma patients using immunohistochemistry. We then performed in vitro studies including wound healing assay, invasion assay, and matrix metalloproteinase (MMP) protein array in order to further elucidate the roles of AhR signaling in breast carcinoma. The status of AhR immunoreactivity was inversely correlated with histological grade (P?=?0.0135) and Ki-67 labeling index (LI; P?=?0.0087) of the patients. In addition, results of both uni- and multivariate analyses revealed that AhR in carcinoma cells turned out an independent prognostic factor with a protective relative risk (P?=?0.0179). An administration of 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand of AhR, significantly decreased Ki-67 LI in an AhR-dependent fashion in MCF-7, T47D, ZR75-1, and MDA-MB-231. Wound healing and invasion assays performed in T47D and ZR75-1 further demonstrated that 10 nM TCDD inhibited estrogen-induced migration and invasion of cells. MMP proteins associated with AhR in breast carcinoma cells were also firstly identified. These results demonstrated that AhR in breast carcinoma cells is considered a newly defined histological prognostic parameter of the breast cancer patients and effects of AhR activation on proliferation and MMPs expression may be related to the relatively good clinical outcome of AhR-positive breast cancer patients.     

Desmopressin and other synthetic vasopressin analogues in cancer treatment

Desmopressin (DDAVP) is a well tolerated and convenient haemostatic agent that can be used in a number of clinical conditions with bleeding diathesis. It has several effects on the haemostatic system, causing endogenous release of coagulation factor VIII, von Willebrand factor and tissue-type plasminogen activator, among others. In this review we present a growing body of evidence showing that DDAVP treatment may impair spread of cancer cells and contribute to encapsulation of tumour tissue. Our data in preclinical animal models suggest a potential application of DDAVP in the perioperative management of aggressive solid tumours. Novel vasopressin analogues with improved antitumor effects are currently in development.     

Angiostatin generation by human tumor cell lines: involvement of plasminogen activators

Angiostatin is a tumor-derived angiogenesis inhibitor consisting of an internal fragment of plasminogen. Little is known about the production of angiostatin by human tumors. In this study, we examined the in vitro angiostatin-generating capacities of a panel of human tumor cell lines (total n = 75) and the proteolytic molecule(s) involved. Angiostatin formation was determined by assessing the level of plasminogen digestion in conditioned medium by Western-blot analysis. We found that the capacity to produce angiostatin is a common feature of many cell lines, depending on the tumor type. All 6 bladder-carcinoma and 6 out of 7 prostate-carcinoma cell lines showed intermediate to potent angiostatin-generating activity. In contrast, only 2 out of 7 colon-carcinoma and 2 out of 9 renal-cell carcinoma cell lines were able to generate angiostatin at intermediate levels. Out of 25 melanoma cell lines, only one line failed to generate angiostatin. In the other cell-line groups (cervix, breast and ovary), angiostatin formation varied. Remarkably, angiostatin bands were not of equal size in all plasminogen digests. Since reported data have indicated that plasminogen activators (uPA and tPA) were able to excise the angiostatin fragment from the plasminogen parent molecule via plasmin generation, we determined levels of uPA and tPA and PAI-1 antigen in the conditioned media, and correlated the results with angiostatin-generating capacity. Whereas prostate- and bladder-carcinoma lines capable of generating high levels of angiostatin showed high uPA levels, angiostatin generation in melanoma cell lines was correlated with tPA levels. Generally, angiostatin non-producers did not express uPA or tPA. In 6 out of 75 cell lines, however, we found angiostatin generation combined with low or absent levels of plasminogen activator, suggesting the involvement of alternative proteolytic pathways in the generation of angiostatin.     

Antimetastatic effect of desmopressin in a mouse mammary tumor model

We have investigated the effects of desmopressin (DDAVP), a synthetic analog of the natural hormone vasopressin, on experimental lung colonization of mammary tumor cells using a syngeneic BALB/c mouse model. Coinjection of DDAVP (1–2g/kg body weight) at the time of i.v. inoculation of F3II carcinoma cells or LM3 adenocarcinoma cells significantly inhibited the formation of experimental lung metastases. In both cases, the number of pulmonary nodules was reduced about 70%. Inhibition of metastasis was also obtained with i.v. administration of DDAVP 24h after tumor cell inoculation. Interestingly, the inhibition of lung metastasis was not due to direct cytotoxic effects of DDAVP on mammary tumor cells. The in vitro formation of multicellular aggregates in the presence of citrated plasma from control and DDAVPtreated mice was also examined. Control plasma rapidly induced a significant tumor cell aggregation. In contrast, in the presence of plasma from DDAVPtreated mice, tumor cells remained as a single cell suspension. DDAVP may help to dissolve the protective fibrin shield of circulating tumor cells. Our data suggest, for the first time, that adjuvant DDAVP therapy may impair successful implantation of circulating mammary tumor cells.     

The accumulation of angiostatin-like fragments in human prostate carcinoma.

PURPOSE: Angiostatin, a potent inhibitor of angiogenesis and, hence, the growth of tumor cell metastasis, is generated by a proteolytic enzyme from plasminogen. However, its localization and specific enzymes have yet to be ascertained in human tissue. EXPERIMENTAL DESIGN: To elucidate the generation and the localization of angiostatin in prostate carcinoma, we examined angiostatin generation in a panel of human prostate cancer cell lines and performed immunohistochemistry with the antibodies to angiostatin and prostate-specific antigen (PSA), a potent proteolytic enzyme of angiostatin in 55 cases of prostate carcinoma. RESULTS: We demonstrated that the lysates of human prostate carcinoma cell lines could generate angiostatin-like fragments from purified human plasminogen but could not generate angiostatin in the absence of exogenous plasminogen. The fragmented proteins were reacted with the monoclonal antibody specific for plasminogen lysine-binding site 1 (LBS-1). Immunohistochemically, the intracytoplasmic immunostaining of LBS-1 was positive in 87.3% (48 of 55) of prostate carcinoma cases, and the immunostaining of miniplasminogen was negative in all cases. There was a significant relationship between the positive immunostaining of LBS-1 and Gleason score (P = 0.0007). The intracytoplasmic immunostaining of PSA was positive in 37.0% (20 of 54) of prostate carcinoma cases, but there was no significant relationship between the expression of PSA and Gleason score, or between the positive immunostaining of LBS-1 and PSA. CONCLUSIONS: These findings suggest that angiostatin is generated by prostate carcinoma cells and is accumulated within the cytoplasm. In addition, the generation of angiostatin-like fragments was correlated with tumor grade; however, PSA may not be the only enzyme for angiostatin generation in human prostate carcinoma.     

Role of 20-Hydroxyeicosatetraenoic Acid (20-HETE) in Androgen-Mediated Cell Viability in Prostate Cancer Cells

20-Hydroxyeicosatetraenoic acid (20-HETE) is generated intracellularly through the ω-hydroxylation of arachidonic acid by the cytochrome P450 (in humans, CYP4A11 and CYP4F2). 20-HETE induces mitogenic responses in different cancer cells. The aim of this study was to analyze how 20-HETE impacts cell survival, proliferation, and apoptosis in prostate cancer cells. Incubation of the human androgen-sensitive cells (LNCaP) with 1–10 μM HET0016 (a selective inhibitor of 20-HETE synthesis) reduced cell viability by 49*–64%* (*p < 0.05 vs. control). This was explained by a reduction in cell proliferation (vehicle, 46 ± 3%; 1 μM, 23 ± 3%*; 10 μM, 28 ± 3%*) and by an increase in apoptosis (vehicle, 2.1 ± 0%; 1 μM, 16 ± 4%*; 10 μM, 31 ± 3%*). Furthermore, the increase in LNCaP cell viability induced by dihydrotestosterone (DHT, 0.1 nM) was abrogated by 30*–42%* by 1–10 μM HET0016. Incubation with 20-HETE (5–1000 nM) increased LNCaP cell viability up to 50%*, together with a 70%* reduction in apoptosis. PC-3 (androgen-insensitive) cell viability was not affected by either HET0016 or 20-HETE. In LNCaP cells, HET0016 (10 μM) diminished the expression of androgen receptors (AR): messenger RNA (mRNA) (40%*) and protein (50%*). DHT (10 nM) augmented CYP4F2 protein expression (1.9-fold*) and 20-HETE levels (50%*). Oppositely, enzalutamide (AR antagonist) reduced CYP4F2 mRNA and protein expressions by 30 and 25%, respectively. Thus, intracellular availability of 20-HETE is necessary to sustain LNCaP cell viability. 20-HETE may act as a signaling molecule in the pathways involved in LNCaP cell viability upon stimulation of the AR. This effect may be partially attributed to its role on securing normal AR expression levels that in turn contribute to maintain intracellular levels of 20-HETE.     

Interaction of vasopressin and oxytocin with human breast carcinoma cells

The arginine vasopressin and oxytocin content of normal and cancerous human breast tissue were measured using radioimmunoassay. Both peptides were present in amounts greater than that found in the circulation, but no difference between normal and malignant tissues was found. Binding of [3H]oxytocin and [3H]vasopressin were characterized in human breast carcinoma cells (MCF7 cells). Binding of both hormones to MCF7 cells was specific and saturable, the vasopressin receptor found to be of the V1 subtype. Scatchard analyses of the data were linear, indicating a single high affinity, low capacity binding site for each hormone (vasopressin: KD = 47.4 +/- 1.6 nmol/liter, Bmax = 27,300 +/- 6,500 sites/cell; oxytocin: KD = 51.3 +/- 0.4 nmol/liter, Bmax = 87,000 +/- 4,000 sites/cell). The effects of vasopressin and oxytocin on the growth of MCF7 cells were assessed using protein accumulation and cell numbers. Vasopressin at 10-1000 pmol/liter was mitogenic for MCF7 cells, but higher doses (10 nmol/liter) were growth inhibitory. Oxytocin was also mitogenic for MCF7 cells but to a lesser extent than vasopressin. In conclusion, we suggest that vasopressin and possibly oxytocin may be important modulators of the growth of some human breast carcinomas.     

Provasopressin expression by breast cancer cells: implications for growth and novel treatment strategies

Summary The arginine vasopressin (AVP) gene is expressed in certain cancers such as breast cancer, where it is believed to act as an autocrine growth factor. However, little is known about the regulation of the AVP protein precursor (proAVP) or AVP-mediated signaling in breast cancer and this study was undertaken to address some of the basic issues. The cultured cell lines examined (Mcf7, Skbr3, BT474, ZR75, Mcf10a) and human breast cancer tissue extract were found to express proAVP mRNA. Western analysis revealed multiple forms of proAVP protein were present in cell lysates, corresponding to those detected in human hypothalamus extracts. Monoclonal antibodies directed against different regions of proAVP bound to intact live Mcf7 and Skbr3 cells. Dexamethasone increased the amount of proAVP-associated glycopeptide (VAG) secreted by Skbr3 cells and a combination of dexamethasone, IBMX and 8br-cAMP increased cellular levels of VAG. Exogenous AVP (1, 10, and 100 nM) elevated phospho-ERK1/2 levels, and increased cell proliferation was observed in the presence of 10 nM AVP. Concurrent treatment with the V1a receptor antagonist SR49059 reduced the effects of AVP on proliferation in Mcf7 cells, and abolished it in Skbr3 cells. Results here show that proAVP components are found at the surface of Skbr3 and Mcf7 cells and are also secreted from these cells. In addition, they show that AVP promotes cancer cell growth, apparently through a V1-type receptor-mediated pathway and subsequent ERK1/2 activation. Thus, strategies for targeting proAVP should be examined for their effectiveness in diagnosing and treating breast cancer.     

Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275.

BACKGROUND: Previous studies have shown that polyhydroxylated flavonoids such as quercetin and genistein can inhibit tumor cell growth in vitro, and preliminary in vivo studies of the flavone L86-8275 have shown growth inhibition of LX529 and A549 lung carcinomas. L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)- piperidinyl]-4H-1-benzopyran-4-one] is a flavone of novel structure. PURPOSE: The purpose of this study was to determine in vitro whether L86-8275 is a more potent inhibitor of growth in breast carcinoma and lung carcinoma cells than quercetin or genistein. METHODS: We studied the effects of L86-8275 on cell growth in seven breast carcinoma cell lines and five lung carcinoma cell lines. MDA468 breast carcinoma was then selected for further study. Cell proliferation was measured by a colorimetric dye reduction assay; synthesis of DNA, RNA, and protein by incorporation of the radioactive metabolic precursors thymidine, uridine, or leucine, respectively; adenosine triphosphate (ATP) content by a luciferase-mediated bioluminescence reaction; and cell cycle progression by the use of cell-synchronizing drugs (aphidicolin and nocodazole) and flow cytometry. RESULTS: L86-8275 was not cytotoxic to stationary-phase cells but reversibly inhibited the growth of cells in exponential growth phase. At concentrations of 25-160 nM, L86-8275 inhibited growth of human breast and lung carcinoma cell lines by 50%. MDA468 breast carcinoma cells were 60-fold and 400-fold more sensitive to L86-8275 than to quercetin and genistein, respectively. By 24 hours after addition of L86-8275, DNA synthesis in MDA468 cells was inhibited by greater than 95%, protein synthesis by 80%, and RNA synthesis by 40%-60%, under conditions that preserved cellular ATP levels at approximately 80%-90% of control values. When MDA468 cells released from aphidicolin-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed the S phase but arrested in G2. When cells released from nocodazole-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed mitosis but arrested in G1. CONCLUSIONS: L86-8275 is a potent, yet reversible, growth-inhibitory flavone that can selectively block cell cycle progression in vitro at more than one point in the cell cycle. IMPLICATIONS: These findings suggest that L86-8275 is a candidate for further preclinical development, as well as a model for the synthesis of other flavonoids that might potently delay cell cycle progression to achieve inhibition of tumor growth. Future studies need to address optimal schedules for antiproliferative activity in vivo and inhibition of clonogenic activity.     

Modulation of growth and urokinase secretion by vasopressin and closely related nonapeptides in metastatic mouse mammary tumor cells

We examined the effects of the neuropeptide hormones vasopressin and oxytocin, and their respective synthetic derivatives desmopressin and isotocin, on F3II mouse mammary carcinoma cells. Vasopressin and desmopressin at concentrations ranging from 0.1 to 1 mu M were mitogenic for F3II cells and induced protein accumulation. On the contrary, oxytocin and isotocin were moderately growth inhibitory at similar doses. In confluent monolayers vasopressin stimulated the secretion of urokinase, a profibrinolytic enzyme involved in hematogenous metastasis. However, the net effect of the peptide on tumor-derived proteolytic activity was dependent on cell density. Stimulation of cell growth and urokinase production by vasopressin was strongly linked with calcium mobilization. These data suggest that vasopressin and its synthetic analog desmopressin may be important modulators of the behavior of metastatic mammary tumor cells.     

Inhibition of tumor growth correlates with the expression level of a human angiostatin transgene in transfected B16F10 melanoma cells

Although the therapeutic value of angiostatin, a proteolytic fragment of plasminogen, has been recognized for the treatment of cancer, the production of bioactive angiostatin remains a difficult task. Here we report that expression of a cDNA encoding a secreted, four-kringle human angiostatin inhibited tumor growth of B16F10 melanoma cells in mice but did not suppress tumor cell growth in culture. After transfection and selection, stable expression of the angiostatin cDNA was demonstrated in several B16F10 clones by quantitative mRNA analysis using the Taqman method. Cells that expressed angiostatin at either a low, medium, or high level were injected into C57BL/6 mice. s.c. Growth of B16F10 tumors was diminished by the angiostatin transgene, and the inhibition was directly proportional to the expression level of angiostatin in the transfected cells. However, suppression of s.c. tumor growth was transient, and eventually, tumors emerged with a strongly decreased expression of the transgene. Angiostatin expression also reduced lung metastasis from i.v.-injected B16F10 cells. Our data indicate that a cDNA encoding bioactive human angiostatin is potentially useful for gene therapy of human cancers, but the delivery of the transgene may require repeated dosing to achieve sustained dormancy of primary tumors and cancer metastases.     

Murine Mammary Carcinoma Induces Chronic Systemic Inflammation and Immunosuppression in BALB/c Mice

PurposeThe F3II cell line is a highly invasive variant of mammary carcinoma. Although it is frequently used as a model to evaluate the efficacy of immunotherapy, its impact on the immune system remains poorly understood. The main objectives of this study were to evaluate the effects of F3II tumors on the development of chronic inflammation and to characterize tumor-associated immunosuppression.MethodsFollowing the experimental implantation of F3II tumors in BALB/c mice, alterations in the liver and spleen anatomy and the numbers of circulating leukocytes, myeloid-derived suppressor cells (MDSCs), and regulatory T cells were measured using hematological techniques, histopathological analysis, and flow cytometry. The capacity of the F3II tumor-bearing mice to reject MB16F10 allogeneic tumor transplantation was also evaluated. In addition, the restoration of immune parameters in tumor-bearing mice was evaluated after standard breast cancer chemotherapy and surgical tumor excision.ResultsF3II tumor implantation increased the levels of chronic inflammatory markers, such as the neutrophil-to-lymphocyte and platelet-to-lymphocyte ratios, and caused myeloid alterations, including extramedullary granulopoiesis and megakaryopoiesis, along with the recruitment of MDSCs to the spleen. Chemotherapy or surgical F3II tumor removal completely rescued the tumor-associated extramedullary granulopoiesis and megakaryopoiesis. Notably, the presence of F3II tumors reduced the capacity of BALB/c mice to reject MB16F10 allogeneic tumor transplantation.ConclusionThese results support the occurrence of F3II tumor-mediated immune cell dysfunction, which mimics the immune alterations characterized by chronic systemic inflammation and immunosuppression observed in breast cancer in clinical settings. Thus, the F3II tumor model is relevant for evaluating novel breast cancer immunotherapies and combinations in preclinical studies. This model could also be useful for identifying appropriate therapeutic targets and developing proof-of-concept experiments in the future.     

ICI 182,780 (Faslodex): development of a novel, "pure" antiestrogen

BACKGROUND: The nonsteroidal antiestrogen tamoxifen is well established as an effective treatment for patients with breast carcinoma, both for the treatment of metastatic disease and as an adjuvant to surgery for patients with primary breast carcinoma. In addition to exerting antagonistic effects on the estrogen receptor, tamoxifen and its derivatives act as partial agonists on certain tissues. These agonistic effects, for example, endometrial stimulation and stimulation of tumor growth after previous response to tamoxifen, may limit their clinical efficacy. ICI 182,780 (Faslodex) from AstraZeneca (Cheshire, United Kingdom) is a novel, steroidal estrogen antagonist that was designed to be devoid of estrogen agonist activity in preclinical models. METHODS: ICI 182,780 was tested in a large number of in vitro and in vivo preclinical models, and its value was assessed clinically when administered before surgery for breast carcinoma and hysterectomy for benign conditions and after failure of tamoxifen in patients with advanced breast carcinoma. RESULTS: All data indicated that ICI 182,780 is devoid of agonist activity in preclinical models and in clinical trials. It inhibits growth of the breast and endometrium. In animal models, it does not cross the blood-brain barrier and appears to be neutral with respect to lipids and bone. ICI 182,780 down-regulates the estrogen receptor and is active in tamoxifen-resistant breast carcinoma. In a small, Phase II study, durable responses were seen: Phase III clinical trials are in progress comparing ICI 182,780 with anastrozole and tamoxifen in the treatment of patients with advanced breast carcinoma. CONCLUSIONS: ICI 182,780 specifically down-regulates the estrogen receptor and, thus, represents the first of a new class of therapeutic agents. In this report, the authors present the current evidence that distinguishes ICI 182,780 from tamoxifen and related nonsteroidal compounds and establishes ICI 182,780 as the first in a new class of therapeutic agents.     

Antitumor and antiangiogenic activity of soy isoflavone genistein in mouse models of melanoma and breast cancer

Tumor invasion, angiogenesis and metastasis involve secretion of proteolytic enzymes and cell migration into blood vessels. Tumor cells are capable of degrading the extracellular matrix via a proteolytic cascade that includes urokinase-type plasminogen activator (uPA) and matrix metalloproteases (MMPs). We have investigated the antitumor and antiangiogenic properties of soy isoflavone genistein in B16 melanoma and F3II mammary carcinoma mouse models. At non-cytotoxic concentrations (0.1-50 microM) genistein induced dose-dependent spindle-cell morphology and significantly reduced motility in both cell lines. Genistein inhibited uPA secreted by F3II cell monolayers, while inducing an increase in the proteolytic activity of B16 cells. On the contrary, the compound did not modify the MMP-9 and -2 produced by tumor cells. In vivo, i.p. administration of genistein at a dose of 10 mg/kg/day reduced tumor-induced angiogenesis in syngeneic mice implanted with B16 or F3II cells. Similar antiangiogenic effects were obtained with a soybean-based diet. This data suggest that tumor cell migration and proteolysis may be associated with the antitumor and antiangiogenic activity of soy isoflavone genistein.     

Expression of uPA and its receptor by both neoplastic and stromal cells during xenograft invasion

Recent studies have shown that molecules involved in generation and regulation of extracellular proteolytic activity are often expressed by non-malignant stromal cells during human cancer invasion. We have studied the expression of the urokinase-type plasminogen activator and the urokinase-type plasminogen activator cell-surface receptor in xenografts of human MDA-MB-231 mammary carcinoma cells growing invasively in nude mice. Northern analysis showed the presence of both human and mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA in tumor extracts. By in situ hybridization, mRNA for human urokinase-type plasminogen activator and its receptor was detected in virtually all the cancer cells, while mouse urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor mRNA was expressed by tumor-infiltrating fibroblast-like and macrophage-like cells. In invasive areas the cells expressing the 2 murine mRNAs were either the same or located immediately adjacent to each other. This model system has several advantages for studies of the mechanism by which cancer cells induce or recruit stromal cells to produce molecules involved in proteolysis. © 1994 Wiley-Liss, Inc.     

A phase I trial of the IGF-1R antibody Cixutumumab in combination with temsirolimus in patients with metastatic breast cancer

The mammalian target of rapamycin (mTOR) plays a critical role in promoting tumor cell growth and is frequently activated in breast cancer. In preclinical studies, the antitumor activity of mTOR inhibitors is attenuated by feedback up-regulation of AKT mediated in part by Insulin-like growth factor type 1 receptor (IGF-1R). We designed a phase I trial to determine the maximum-tolerated dose (MTD) and pharmacodynamic effects of the IGF-1R antibody Cixutumumab in combination with temsirolimus in patients with metastatic breast cancer refractory to standard therapies. A 3 + 3 Phase I design was chosen. Temsirolimus and Cixutumumab were administered intravenously on days 1, 8, 15, and 22 of a 4-week cycle. Of the 26 patients enrolled, four did not complete cycle 1 because of disease progression (n = 3) or comorbid condition (n = 1) and were replaced. The MTD was determined from the remaining 22 patients, aged 34–72 (median 48) years. Most patients (86 %) had estrogen receptor positive cancer. The median number of prior chemotherapy regimens for metastatic disease was 3. The MTD was determined to be Cixutumumab 4 mg/kg and temsirolimus 15 mg weekly. Dose-limiting toxicities (DLTs) included mucositis, neutropenia, and thrombocytopenia. Other adverse events included grade 1/2 fatigue, anemia, and hyperglycemia. No objective responses were observed, but four patients experienced stable disease that lasted for at least 4 months. Compared with baseline, there was a significant increase in the serum levels of IGF-1 (p < 0.001) and IGFBP-3 (p = 0.019) on day 2. Compared with day 2, there were significant increases in the serum levels of IGF-1 (p < 0.001), IGF-2 (p = 0.001), and IGFBP-3 (p = 0.019) on day 8. A phase II study in women with metastatic breast cancer is ongoing.     

Systemic Sclerosis-Endothelial Cell Antiangiogenic Pentraxin 3 and Matrix Metalloprotease 12 Control Human Breast Cancer Tumor Vascularization and Development in Mice

We have previously shown that endothelial cell matrix metalloprotease 12 (MMP12) and pentraxin 3 (PTX3) overproduction is the main alteration accounting for reduced proneness to angiogenesis in systemic sclerosis (SSc). On this basis, we stably transfected MMP12 and PTX3 in two breast cancer cell lines expressing very low amounts of the target molecules when compared with normal breast epithelial cells, relying on the hypothesis that antiangiogenic molecules released by cancer cells could confer an SSc-like antiangiogenic pattern on target endothelial cells. In Matrigel Boyden chamber invasion and capillary morphogenesis studies, transfected clones reduced endothelial cell invasion and capillary tube formation, which were abolished by tumor cell populations expressing both molecules. The Matrigel sponge assay, performed in vivo in C57/BL6 mice by injecting aliquots of lyophilized culture medium of transfected clones, indicated a similar reduction in angiogenesis. Functional studies have shown that endothelial cells treated with a culture medium of MMP12-expressing clones underwent cleavage of urokinase-type plasminogen activator receptor domain 1 which is indispensable to angiogenesis. We did not observe angiostatin production from plasminogen under the same experimental conditions. PTX3-overexpressing clones showed a powerful anti-fibroblast growth factor 2 (FGF2) activity in FGF2-dependent capillary morphogenesis. We have injected control and transfected clones into nude nu/nu (CD-1) BR mice to study the differential tumor growth pattern. We observed a reduction of tumor growth in transfected clones, which was basically complete when clones expressing both molecules were simultaneously injected. The extent of tumor necrosis suggested an antiangiogenesis-dependent inhibition of tumor development.