Charles H. Graham

Hypoxia-driven selection of the metastatic phenotype.

Intratumoral hypoxia is an independent indicator of poor patient outcome and increasing evidence supports a role for hypoxia in the development of metastatic disease. Studies suggest that the acquisition of the metastatic phenotype is not simply the result of dysregulated signal transduction pathways, but instead is achieved through a stepwise selection process driven by hypoxia. Hypoxia facilitates disruption of tissue integrity through repression of E-cadherin expression, with concomitant gain of N-cadherin expression which allows cells to escape anoikis. Through upregulation of urokinase-type plasminogen activator receptor (uPAR) expression, hypoxia enhances proteolytic activity at the invasive front and alters the interactions between integrins and components of the extracellular matrix, thereby enabling cellular invasion through the basement membrane and the underlying stroma. Cell motility is increased through hypoxia-induced hepatocyte growth factor (HGF)-MET receptor signaling, resulting in cell migration towards the blood or lymphatic microcirculation. Hypoxia-induced vascular endothelial growth factor (VEGF) activity also plays a critical role in the dynamic tumor–stromal interactions required for the subsequent stages of metastasis. VEGF promotes angiogenesis and lymphangiogenesis in the primary tumor, providing the necessary routes for dissemination. VEGF-induced changes in vascular integrity and permeability promote both intravasation and extravasation, while VEGF-induced angiogenesis in the secondary tissue is essential for cell proliferation and establishment of metastatic lesions. Through regulation of these critical molecular targets, hypoxia promotes each step of the metastatic cascade and selects tumor cell populations that are able to escape the unfavorable microenvironment of the primary tumor.

Hypoxia-mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression.

Tumor hypoxia and high levels of expression of the urokinase‐type plasminogen activator (uPA) receptor (uPAR) represent a poor clinical outcome for patients with various cancers. Here, we examined the effect of hypoxia on in vitro invasion of extracellular matrix and uPAR expression by human carcinoma cells. Compared with culture under 20% O2, culture for up to 24 hr under 1% or 4% O2 resulted in increased cell surface uPAR. However, the highest uPAR levels were observed in cells cultured under 1% O2. Culture of MDA‐MB‐231 breast carcinoma cells under hypoxia also resulted in increased uPAR mRNA levels. Furthermore, incubation with cobalt chloride or with an iron chelator also resulted in elevated uPAR expression, while presence of 30% carbon monoxide in the hypoxic atmosphere reduced the hypoxia‐mediated uPAR mRNA upregulation. Increased uPAR expression was paralleled by higher cell‐associated uPA levels and lower levels of secreted uPA as determined by gel zymography performed on cell extracts and culture‐conditioned media. In addition, the in vitro invasiveness of MDA‐MB‐231 breast carcinoma cells was significantly higher when the invasion assay was performed under hypoxic conditions. This effect of hypoxia on invasion was abrogated by including in the assay a monoclonal, function‐blocking anti‐uPAR antibody or by the presence of 30% carbon monoxide in the hypoxic atmosphere. Our findings indicate that hypoxia stimulates carcinoma cell invasiveness by upregulating uPAR expression on the cell surface through a mechanism that requires a putative heme protein. Through a similar mechanism, hypoxia may stimulate tumor invasion and metastasis in vivo. Int. J. Cancer 80:617–623, 1999.

Characteristic's of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture

We developed a method of propagating pure first trimester human trophoblast cells growing out of primary explants of mechanically derived chorionic villus fragments (Yagel et al, 1989; Graham et al, 1992). We have now extensively characterized these cells during their initial outgrowth and in long-term culture, employing a variety of markers and techniques as outlined below. By double label immunofluorescence using epithelial (cytokeratin) and mesenchymal (vimentin) cell markers, we identified the chorionic villus migrant cell populations as pure trophoblast (39 per cent of outgrowths) or a mixture of trophoblast and fibroblast (61 per cent). Further phenotyping of the pure trophoblast outgrowths by double label immunostaining using anti-cytokeratin antibody and a panel of other primary antisera revealed that these cells exhibit a variety of markers characteristic of extravillous invasive trophoblast cells in situ: insulin-like growth factor (IGF)-II, NDOG-5, proliferating cell nuclear antigen (PCNA), human leucocyte antigen framework antigen (W6/32) and a distinct set of integrins including alpha 1, alpha 3, alpha 5, alpha v and beta 1 subunits and alpha v beta 3/beta 5 vitonectin receptor. They were negative for alpha 6 and beta 4 integrin subunits. Immunogold electron microscopy of explants grown on type IV collagen gel revealed the production of conventional and oncofetal types of fibronectin by mononucleate trophoblast cells and human placental lactogen by multinucleate cells. Immunolabelling, flow cytometry and immunoprecipitation revealed that this phenotypic profile was retained with complete fidelity in the long-term culture; thus, trophoblasts migrating out of first trimester chorionic villus explants and their propagated progeny belong to the invasive extravillous trophoblast of the placenta.

A mechanism of hypoxia-mediated escape from adaptive immunity in cancer cells

Immune escape is a fundamental trait of cancer in which mechanistic knowledge is incomplete. Here, we describe a novel mechanism by which hypoxia contributes to tumoral immune escape from cytotoxic T lymphocytes (CTL). Exposure of human or murine cancer cells to hypoxia for 24 hours led to upregulation of the immune inhibitory molecule programmed cell death ligand-1 (PD-L1; also known as B7-H1), in a manner dependent on the transcription factor hypoxia-inducible factor-1α (HIF-1α). In vivo studies also demonstrated cellular colocalization of HIF-1α and PD-L1 in tumors. Hypoxia-induced expression of PD-L1 in cancer cells increased their resistance to CTL-mediated lysis. Using glyceryl trinitrate (GTN), an agonist of nitric oxide (NO) signaling known to block HIF-1α accumulation in hypoxic cells, we prevented hypoxia-induced PD-L1 expression and diminished resistance to CTL-mediated lysis. Moreover, transdermal administration of GTN attenuated tumor growth in mice. We found that higher expression of PD-L1 induced in tumor cells by exposure to hypoxia led to increased apoptosis of cocultured CTLs and Jurkat leukemia T cells. This increase in apoptosis was prevented by blocking the interaction of PD-L1 with PD-1, the PD-L1 receptor on T cells, or by addition of GTN. Our findings point to a role for hypoxia/HIF-1 in driving immune escape from CTL, and they suggest a novel cancer immunotherapy to block PD-L1 expression in hypoxic-tumor cells by administering NO mimetics.

Hypoxia-induced resistance to anticancer drugs is associated with decreased senescence and requires hypoxia-inducible factor-1 activity

Hypoxia in solid tumors is associated with the development of chemoresistance. Although many studies have focused on the effect of hypoxia on drug-induced apoptosis, the effect of nonapoptotic pathways on hypoxia-induced drug resistance has not been previously investigated. Here, we determined the effects of hypoxia on multiple forms of drug-induced death in human MDA-MB-231 breast carcinoma cells. Clonogenic assays showed that preexposure to hypoxia leads to resistance to various classes of chemotherapeutic agents, including anthracyclines (daunorubicin and doxorubicin), epipodophyllotoxins (etoposide), and anthracenediones (mitoxantrone). Results revealed a high degree of heterogeneity in nuclear and cytoplasmic alterations in response to acute drug exposure; however, the majority of exposed cells displayed morphologic and biochemical changes consistent with drug-induced senescence. Hypoxia decreased only the proportion of cells in the senescent population, whereas the small proportion of cells exhibiting features of apoptosis or mitotic catastrophe were unaffected. Similar results were obtained with human HCT116 colon carcinoma cells, indicating that the protective effect of hypoxia on drug-induced senescence is not unique to MDA-MB-231 cells. Treatment of MDA-MB-231 cells with small interfering RNA targeting the α-subunit of hypoxia-inducible factor-1 (HIF-1), a key regulator of cellular adaptations to hypoxia, prevented hypoxia-induced resistance. HIF-1α small interfering RNA also selectively abolished the hypoxia-induced changes in the senescent population, indicating that the increased survival was due to protection against drug-induced senescence. These results support a requirement for HIF-1 in the adaptations leading to drug resistance and reveal that decreased drug-induced senescence is also an important contributor to the development of hypoxia-induced resistance. [Mol Cancer Ther 2008;7(7):1961–73]

Inflammation in rat pregnancy inhibits spiral artery remodeling leading to fetal growth restriction and features of preeclampsia

Abnormal maternal inflammation leads to TNF-mediated fetal growth restriction and some features of preeclampsia that can be ameliorated with the nitric oxide mimetic nitroglycerin.

Effect of transforming growth factor-β on the plasminogen activator system in cultured first trimester human cytotrophoblasts

Plasminogen activators (PAs) play an important role in facilitating trophoblast invasion of the uterus and in the maintenance of blood fluidity within the placental intervillous spaces. We previously found that transforming growth factor-beta (TGF-beta), produced mainly by the decidua, inhibits first trimester trophoblast invasiveness at least partly via induction of tissue inhibitor of metalloproteinases-1 production and secretion by the trophoblasts. The present study examined whether TGF-beta 1 affects PA and plasminogen activator inhibitor-1 (PAI-1) production by cultured human cytotrophoblasts. Immortalized first trimester human cytotrophoblasts (HTR-8/SVneo) were cultured in the absence or presence of TGF-beta 1 (0-10 ng/ml) for 24 h, after which the levels of urokinase-type PA (uPA), PAI-1 and uPA activity in the serum-free conditioned media were determined by enzyme-linked immunosorbent assay (ELISA), protein zymography, and a chromogenic uPA activity assay. Cellular PAI-1 mRNA levels were also determined in cultures following a 24-h incubation with a single dose (5 ng/ml) of TGF-beta 1. Presence of TGF-beta 1 at 1 ng/ml resulted in a greater than 12-fold reduction in the levels of total uPA as determined by ELISA. Furthermore, released uPA activity levels in similar cultures incubated with 5 ng/ml of TGF-beta 1 were reduced to 35 per cent of control values. In contrast, cultures incubated with as little as 0.1 ng/ml TGF-beta exhibited a 63 per cent increase in the levels of secreted PAI-1 protein. Similarly, both the 2.2- and 3.0-kb PAI-1 cellular mRNA species were elevated in trophoblast cells incubated with 5 ng/ml TGF-beta 1 when compared with control cells. Thus, it appears that the reduced uPA activity observed in the cultures incubated with TGF-beta 1 is due to reduced secretion of uPA and increased PAI-1 production and secretion. These results suggest that TGF-beta may also exert its anti-invasive effect by down-regulating trophoblast-derived PA activity. Through its effects on the PA system, TGF-beta may also play an indirect role in the regulation of uteroplacental blood flow.

Activated Macrophages Inhibit Human Cytotrophoblast Invasiveness In Vitro

Abstract Pre-eclampsia is associated with inadequate cytotrophoblast invasion and remodeling of the uterine spiral arterioles, as well as by an aberrant maternal immune response. This study determined the effect of activated macrophages and one of its products, tumor necrosis factor (TNF)-alpha, on cytotrophoblast invasiveness. Coculture with human lipopolysaccharide-activated macrophages decreased the ability of immortalized HTR-8/ SVneo human trophoblast cells to invade through reconstituted extracellular matrix (P < 0.05). This effect of activated macrophages on trophoblast invasiveness was paralleled by abrogation of a 55-kDa caseinolytic activity corresponding to prourokinase plasminogen activator (pro-uPA) and an increased secretion of plasminogen activator inhibitor 1 (PAI1), as determined by gel zymography and ELISA, respectively. Coculture with nonactivated macrophages did not significantly affect trophoblast invasiveness or pro-uPA and PAI1 secretion. Activated macrophages secreted detectable levels of TNF, and administration of exogenous TNF significantly decreased trophoblast invasiveness (P < 0.05), increased the secretion of PAI1 (P < 0.01), and completely inhibited the pro-uPA-associated caseinolytic activity by binding to the TNF receptor 1. Moreover, addition of up to 10 ng/ml of TNF did not increase the rate of apoptosis in HTR-8/SVneo cells. Finally, the increased secretion of PAI1 by trophoblast cells cocultured with activated macrophages was significantly inhibited when a neutralizing anti-TNF antibody was added to the cocultures. These results suggest that the aberrant presence of activated macrophages around uterine vessels may contribute to inadequate trophoblast invasion and remodeling of the uterine spiral arterioles. Thus, the presence of activated macrophages may be important in the etiology of pre-eclampsia.

Chemosensitization of Cancer In vitro and In vivo by Nitric Oxide Signaling

Purpose: Hypoxia contributes to drug resistance in solid cancers, and studies have revealed that low concentrations of nitric oxide (NO) mimetics attenuate hypoxia-induced drug resistance in tumor cells in vitro. Classic NO signaling involves activation of soluble guanylyl cyclase, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinase. Here, we determined whether chemosensitization by NO mimetics requires cGMP-dependent signaling and whether low concentrations of NO mimetics can chemosensitize tumors in vivo. Experimental Design: Survival of human prostate and breast cancer cells was assessed by clonogenic assays following exposure to chemotherapeutic agents. The effect of NO mimetics on tumor chemosensitivity in vivo was determined using a mouse xenograft model of human prostate cancer. Drug efflux in vitro was assessed by measuring intracellular doxorubicin-associated fluorescence. Results: Low concentrations of the NO mimetics glyceryl trinitrate (GTN) and isosorbide dinitrate attenuated hypoxia-induced resistance to doxorubicin and paclitaxel. Similar to hypoxia-induced drug resistance, inhibition of various components of the NO signaling pathway increased resistance to doxorubicin, whereas activation of the pathway with 8-bromo-cGMP attenuated hypoxia-induced resistance. Drug efflux was unaffected by hypoxia and inhibitors of drug efflux did not significantly attenuate hypoxia-induced chemoresistance. Compared with mice treated with doxorubicin alone, tumor growth was decreased in mice treated with doxorubicin and a transdermal GTN patch. The presence of GTN and GTN metabolites in plasma samples was confirmed by gas chromatography. Conclusion: Tumor hypoxia induces resistance to anticancer drugs by interfering with endogenous NO signaling and reactivation of NO signaling represents a novel approach to enhance chemotherapy.

Spontaneous Pregnancy Loss Mediated by Abnormal Maternal Inflammation in Rats Is Linked to Deficient Uteroplacental Perfusion

Abnormal maternal inflammation during pregnancy is associated with spontaneous pregnancy loss and intrauterine fetal growth restriction. However, the mechanisms responsible for these pregnancy outcomes are not well understood. In this study, we used a rat model to demonstrate that pregnancy loss resulting from aberrant maternal inflammation is closely linked to deficient placental perfusion. Administration of LPS to pregnant Wistar rats on gestational day 14.5, to induce maternal inflammation, caused fetal loss in a dose-dependent manner 3–4 h later, and surviving fetuses were significantly growth restricted. Pregnancy loss was associated with coagulopathy, structural abnormalities in the uteroplacental vasculature, decreased placental blood flow, and placental and fetal hypoxia within 3 h of LPS administration. This impairment in uteroplacental hemodynamics in LPS-treated rats was linked to increased uterine artery resistance and reduced spiral arteriole flow velocity. Pregnancy loss induced by LPS was prevented by maternal administration of the immunoregulatory cytokine IL-10 or by blocking TNF-α activity after treatment with etanercept (Enbrel). These results indicate that alterations in placental perfusion are responsible for fetal morbidities associated with aberrant maternal inflammation and support a rationale for investigating a potential use of immunomodulatory agents in the prevention of spontaneous pregnancy loss.