Robert R. Langley

The seed and soil hypothesis revisited--the role of tumor-stroma interactions in metastasis to different organs.

The fact that certain tumors exhibit a predilection for metastasis to specific organs has been recognized for well over a century now. An extensive body of clinical data and experimental research has confirmed Stephen Pagets original “seed and soil” hypothesis that proposed the organ‐preference patterns of tumor metastasis are the product of favorable interactions between metastatic tumor cells (the “seed”) and their organ microenvironment (the “soil”). Indeed, many of the first‐line therapeutic regimens, currently in use for the treatment of human cancer are designed to target cancer cells (such as chemotherapy) and also to modulate the tumor microenvironment (such as antiangiogenic therapy). While some types of tumors are capable of forming metastases in virtually every organ in the body, the most frequent target organs of metastasis are bone, brain, liver and the lung. In this review, we discuss how tumor‐stromal interactions influence metastasis in each of these organs.

Regulation of Tumor Angiogenesis by EZH2

Although VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we show that increased Zeste homolog 2 (EZH2) expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohibin1 (vash1). Ezh2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with ezh2 silencing in tumor cells. Collectively, these data support the potential for targeting ezh2 as an important therapeutic approach.

Interleukin-6, secreted by human ovarian carcinoma cells, is a potent proangiogenic cytokine.

Angiogenesis, a key rate-limiting step in the growth and dissemination of malignant tumors, is regulated by the balance between positive and negative effectors. Recent studies indicate that the pleiotropic cytokine interleukin-6 (IL-6) may contribute to the vascularization of some tumors by disrupting the equilibrium between positive and negative angiogenic regulatory molecules. We determined whether IL-6 participates in the angiogenesis observed during the progression of ovarian carcinoma. We measured IL-6 production by human ovarian cancer cell lines in vitro and in vivo. Not all cell lines secreted IL-6 in vitro; however, when the cell lines were implanted into the peritoneal cavity of female nude mice, every line secreted IL-6. Most human ovarian carcinoma cell lines tested secreted significant levels of the soluble IL-6 receptor (sIL-6R). Endothelial cell lines established from the ovary and mesentery of female H-2K(b)-tsA58 mice were tested for response to IL-6. Both endothelial cell lines expressed the IL-6R and their stimulation with the exogenous ligand significantly enhanced cell migration and activated the downstream signaling molecule signal transducers and activators of transcription 3. Dual immunohistochemical staining for IL-6R and CD31 revealed IL-6R expression on human endothelial cells within normal ovary and carcinoma specimens. Gelfoam sponges containing 0.4% agarose and IL-6 or basic fibroblast growth factor and implanted into the subcutis of BALB/c mice were vascularized to the same extent. Collectively, the data indicate that ovarian tumor cells secreted IL-6, a highly angiogenic cytokine that supports progression of disease.

Effect of Interleukin-8 Gene Silencing With Liposome-Encapsulated Small Interfering RNA on Ovarian Cancer Cell Growth

BACKGROUND Interleukin-8 (IL-8) is a proangiogenic cytokine that is overexpressed in many human cancers. We investigated the clinical and biologic significance of IL-8 in ovarian carcinoma using human samples and orthotopic mouse models. METHODS Tumor expression of IL-8 was assessed by immunohistochemistry among ovarian cancer patients (n = 102) with available clinical and survival data. We examined the effect of IL-8 gene silencing with small interfering RNAs incorporated into neutral liposomes (siRNA-DOPCs), alone and in combination with docetaxel, on in vivo tumor growth, angiogenesis (microvessel density), and tumor cell proliferation in mice (n = 10 per treatment group) bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) and taxane-resistant (SKOV3ip2.TR) ovarian tumors. All statistical tests were two-sided. RESULTS Of the 102 cancer specimens, 43 (42%) had high IL-8 expression and 59 (58%) had low or no IL-8 expression; high IL-8 expression was associated with advanced tumor stage (P = .019), high tumor grade (P = .031), and worse survival (median survival for patients with high vs low IL-8 expression: 1.62 vs 3.79 years; P < .001). Compared with empty liposomes, IL-8 siRNA-DOPC reduced the mean tumor weight by 32% (95% confidence interval [CI] = 14% to 50%; P = .03) and 52% (95% CI = 27% to 78%; P = .03) in the HeyA8 and SKOV3ip1 mouse models, respectively. In all three mouse models, treatment with IL-8 siRNA-DOPC plus the taxane docetaxel reduced tumor growth the most compared with empty liposomes (77% to 98% reduction in tumor growth; P < .01 for all). In the HeyA8 and SKOV3ip1 models, tumors from mice treated with IL-8 siRNA-DOPC alone had lower microvessel density than tumors from mice treated with empty liposomes (HeyA8: 34% lower, 95% CI = 32% to 36% lower [P = .002]; SKOV3ip1: 39% lower, 95% CI = 34% to 44% lower [P = .007]). Compared with empty liposomes, IL-8 siRNA-DOPC plus docetaxel reduced tumor cell proliferation by 35% (95% CI = 25% to 44%; P < .001) and 38% (95% CI = 28% to 48%; P < .001) in the HeyA8 and SKOV3ip1 models, respectively. CONCLUSIONS Increased IL-8 expression is associated with poor clinical outcome in human ovarian carcinoma, and IL-8 gene silencing decreases tumor growth through antiangiogenic mechanisms.

Vimentin Is a Novel Anti-Cancer Therapeutic Target; Insights from In Vitro and In Vivo Mice Xenograft Studies

Background Vimentin is a ubiquitous mesenchymal intermediate filament supporting mechano-structural integrity of quiescent cells while participating in adhesion, migration, survival, and cell signaling processes via dynamic assembly/disassembly in activated cells. Soft tissue sarcomas and some epithelial cancers exhibiting “epithelial to mesenchymal transition” phenotypes express vimentin. Withaferin-A, a naturally derived bioactive compound, may molecularly target vimentin, so we sought to evaluate its effects on tumor growth in vitro and in vivo thereby elucidating the role of vimentin in drug-induced responses. Methods and Findings Withaferin-A elicited marked apoptosis and vimentin cleavage in vimentin-expressing tumor cells but significantly less in normal mesenchymal cells. This proapoptotic response was abrogated after vimentin knockdown or by blockade of caspase-induced vimentin degradation via caspase inhibitors or overexpression of mutated caspase-resistant vimentin. Pronounced anti-angiogenic effects of Withaferin-A were demonstrated, with only minimal effects seen in non-proliferating endothelial cells. Moreover, Withaferin-A significantly blocked soft tissue sarcoma growth, local recurrence, and metastasis in a panel of soft tissue sarcoma xenograft experiments. Apoptosis, decreased angiogenesis, and vimentin degradation were all seen in Withaferin-A treated specimens. Conclusions In light of these findings, evaluation of Withaferin-A, its analogs, or other anti-vimentin therapeutic approaches in soft tissue sarcoma and “epithelial to mesenchymal transition” clinical contexts is warranted.

Cardiomyocyte PDGFR-β signaling is an essential component of the mouse cardiac response to load-induced stress

PDGFR is an important target for novel anticancer therapeutics because it is overexpressed in a wide variety of malignancies. Recently, however, several anticancer drugs that inhibit PDGFR signaling have been associated with clinical heart failure. Understanding this effect of PDGFR inhibitors has been difficult because the role of PDGFR signaling in the heart remains largely unexplored. As described herein, we have found that PDGFR-beta expression and activation increase dramatically in the hearts of mice exposed to load-induced cardiac stress. In mice in which Pdgfrb was knocked out in the heart in development or in adulthood, exposure to load-induced stress resulted in cardiac dysfunction and heart failure. Mechanistically, we showed that cardiomyocyte PDGFR-beta signaling plays a vital role in stress-induced cardiac angiogenesis. Specifically, we demonstrated that cardiomyocyte PDGFR-beta was an essential upstream regulator of the stress-induced paracrine angiogenic capacity (the angiogenic potential) of cardiomyocytes. These results demonstrate that cardiomyocyte PDGFR-beta is a regulator of the compensatory cardiac response to pressure overload-induced stress. Furthermore, our findings may provide insights into the mechanism of cardiotoxicity due to anticancer PDGFR inhibitors.

High Expression of Ligands for Chemokine Receptor CXCR2 in Alveolar Epithelial Neoplasia Induced by Oncogenic Kras

CXCL8, a ligand for the chemokine receptor CXCR2, was recently reported to be a transcriptional target of Ras signaling, but its role in Ras-induced tumorigenesis has not been fully defined. Here, we investigated the role of KC and MIP-2, the murine homologues of CXCL8, in Kras(LA1) mice, which develop lung adenocarcinoma owing to somatic activation of the KRAS oncogene. We first investigated biological evidence of CXCR2 ligands in Kras(LA1) mice. Malignant progression of normal alveolar epithelial cells to adenocarcinoma in Kras(LA1) mice was associated with enhanced intralesional vascularity and neutrophilic inflammation, which are hallmarks of chemoattraction by CXCR2 ligands. In in vitro migration assays, supernatants of bronchoalveolar lavage samples from Kras(LA1) mice chemoattracted murine endothelial cells, alveolar inflammatory cells, and the LKR-13 lung adenocarcinoma cell line derived from Kras(LA1) mice, an effect that was abrogated by pretreatment of the cells with a CXCR2-neutralizing antibody. CXCR2 and its ligands were highly expressed in LKR-13 cells and premalignant alveolar lesions in Kras(LA1) mice. Treatment of Kras(LA1) mice with a CXCR2-neutralizing antibody inhibited the progression of premalignant alveolar lesions and induced apoptosis of vascular endothelial cells within alveolar lesions. Whereas the proliferation of LKR-13 cells in vitro was resistant to treatment with the antibody, LKR-13 cells established as syngeneic tumors were sensitive, supporting a role for the tumor microenvironment in the activity of CXCR2. Thus, high expression of CXCR2 ligands may contribute to the expansion of early alveolar neoplastic lesions induced by oncogenic KRAS.

The role of the organ microenvironment in the biology and therapy of cancer metastasis

By the time of diagnosis, primary neoplasms are biologically heterogeneous and contain subpopulations of cells with different metastatic potentials. The pathogenesis of a metastasis consists of many sequential steps that must be completed to produce clinically relevant lesions. During any of these steps, tumor cells interact with host factors in the microenvironment that the tumor cells can usurp. Treatment of metastasis can be directed against tumor cells and/or microenvironmental factors that support tumor growth, such as tumor‐associated blood vessels. J. Cell. Biochem. 101: 927–936, 2007.

Upregulated stromal EGFR and vascular remodeling in mouse xenograft models of angiogenesis inhibitor–resistant human lung adenocarcinoma

Angiogenesis is critical for tumor growth and metastasis, and several inhibitors of angiogenesis are currently in clinical use for the treatment of cancer. However, not all patients benefit from antiangiogenic therapy, and those tumors that initially respond to treatment ultimately become resistant. The mechanisms underlying this, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. Here, using species-specific profiling of mouse xenograft models of human lung adenocarcinoma, we have shown that gene expression changes associated with acquired resistance to the VEGF inhibitor bevacizumab occurred predominantly in stromal and not tumor cells. In particular, components of the EGFR and FGFR pathways were upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Importantly, dual targeting of the VEGF and EGFR pathways reduced pericyte coverage and increased progression-free survival. These findings demonstrated that alterations in tumor stromal pathways, including the EGFR and FGFR pathways, are associated with, and may contribute to, resistance to VEGF inhibitors and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens.

Simultaneous blockade of platelet-derived growth factor-receptor and epidermal growth factor-receptor signaling and systemic administration of paclitaxel as therapy for human prostate cancer metastasis in bone of nude mice.

Once prostate cancer metastasizes to bone, conventional chemotherapy is largely ineffective. We hypothesized that inhibition of phosphorylation of the epidermal growth factor receptor (EGF-R) and platelet-derived growth factor receptor (PDGF-R) expressed on tumor cells and tumor-associated endothelial cells, which is associated with tumor progression, in combination with paclitaxel would inhibit experimental prostate cancer bone metastasis and preserve bone structure. We tested this hypothesis in nude mice, using human PC-3MM2 prostate cancer cells. PC-3MM2 cells growing adjacent to bone tissue and endothelial cells within these lesions expressed phosphorylated EGF-R and PDGF-Rα and -β on their surfaces. The percentage of positive endothelial cells and the intensity of receptor expression directly correlated with proximity to bone tissue. Oral administration of PKI166 inhibited the phosphorylation of EGF-R but not PDGF-R, whereas oral administration of STI571 inhibited the phosphorylation of PDGF-R but not EGF-R. Combination therapy using oral PKI166 and STI571 with i.p. injections of paclitaxel induced a high level of apoptosis in tumor vascular endothelial cells and tumor cells in parallel with inhibition of tumor growth in the bone, preservation of bone structure, and reduction of lymph node metastasis. Collectively, these data demonstrate that blockade of phosphorylation of EGF-R and PDGF-R coupled with administration of paclitaxel significantly suppresses experimental human prostate cancer bone metastasis.