The biology of brain metastasis.

BACKGROUND: It is estimated that at least 200 000 cases of brain metastases occur each year in the US, which is 10 times the number of patients diagnosed with primary brain tumors. Brain metastasis is associated with poor prognosis, neurological deterioration, diminished quality of life, and extremely short survival. Favorable interactions between tumor cells and cerebral microvascular endothelial cells encourage tumor growth in the central nervous system, while tumor cell interactions with astrocytes protect brain metastases from the cytotoxic effects of chemotherapy. CONTENT: We review the pathogenesis of brain metastasis and emphasize the contributions of microvascular endothelial cells and astrocytes to disease progression and therapeutic resistance. Animal models used to study brain metastasis are also discussed. SUMMARY: Brain metastasis has many unmet clinical needs. There are few clinically relevant tumor models and no targeted therapies specific for brain metastases, and the mean survival for untreated patients is 5 weeks. Improved clinical outcomes are dependent on an enhanced understanding of the metastasis-initiating population of cells and the identification of microenvironmental factors that encourage disease progression in the central nervous system.

Crosstalk between the DNA damage response pathway and microRNAs.

MicroRNAs (miRNAs) are a family of small, non-coding RNAs that control gene expression at the post-transcriptional level by destabilizing and inhibiting translation of their target messenger RNAs. MiRNAs are involved in the regulation of a number of fundamental biological processes, and their dysregulation is thought to contribute to several disease processes. Emerging evidence suggests that miRNAs also play a critical role in protecting the heritable genome by contributing to the regulation of the DNA damage response. Consequently, much recent investigative effort has been directed towards an improved understanding of how miRNAs are regulated in response to DNA damage. In this review, we discuss the most recent findings regarding the regulation of miRNA expression and the functional roles of miRNAs in the DNA damage response.

Selection of brain metastasis-initiating breast cancer cells determined by growth on hard agar.

An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44(+) and CD133(+) and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice.

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 Paget's 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.

Tumor cell-organ microenvironment interactions in the pathogenesis of cancer metastasis.

The process of cancer metastasis is sequential and selective and contains stochastic elements. The growth of metastases represents the endpoint of many lethal events that few tumor cells can survive. Primary tumors consist of multiple subpopulations of cells with heterogeneous metastatic properties, and the outcome of metastasis depends on the interplay of tumor cells with various host factors. The findings that different metastases can originate from different progenitor cells account for the biological diversity that exists among various metastases. Even within a solitary metastasis of proven clonal origin, however, heterogeneity of biological characteristics can develop rapidly. The pathogenesis of metastasis depends on multiple interactions of metastatic cells with favorable host homeostatic mechanisms. Interruption of one or more of these interactions can lead to the inhibition or eradication of cancer metastasis. For many years, all of our efforts to treat cancer have concentrated on the inhibition or destruction of tumor cells. Strategies both to treat tumor cells (such as chemotherapy and immunotherapy) and to modulate the host microenvironment (including the tumor vasculature) should offer additional approaches for cancer treatment. The recent advances in our understanding of the biological basis of cancer metastasis present unprecedented possibilities for translating basic research to the clinical reality of cancer treatment.

Estrous cycle modulates ovarian carcinoma growth.

PURPOSE: The effects of reproductive hormones on ovarian cancer growth are not well understood. Here, we examined the effects of estrous cycle variation and specific reproductive hormones on ovarian cancer growth. EXPERIMENTAL DESIGN: We investigated the role of reproductive hormones in ovarian cancer growth using both in vivo and in vitro models of tumor growth. RESULTS: In vivo experiments using the HeyA8 and SKOV3ip1 ovarian cancer models showed that tumor cell inoculation during proestrus significantly increased tumor burden (251-273%) compared with injection during the estrus phase. Treatment of ovariectomized mice with 17beta-estradiol resulted in a 404% to 483% increase in tumor growth compared with controls. Progestins had no significant effect, but did block estrogen-stimulated tumor growth. Tumors collected from mice sacrificed during proestrus showed increased levels of vascular endothelial growth factor (VEGF) and microvessel density compared with mice injected during estrus. HeyA8, SKOV3ip1, and mouse endothelial (MOEC) cells expressed estrogen receptor alpha and beta and progesterone receptor at the protein and mRNA levels, whereas 2774 ovarian cancer cells were estrogen receptor-negative. In vitro assays showed that 17beta-estradiol significantly increased ovarian cancer cell adhesion to collagen in estrogen receptor-positive, but not in estrogen receptor-negative cells. Additionally, 17beta-estradiol increased the migratory potential of MOEC cells, which was abrogated by the mitogen-activated protein kinase (MAPK) inhibitor, PD 09859. Treatment with 17beta-estradiol activated MAPK in MOEC cells, but not in HeyA8 or SKOV3ip1 cells. CONCLUSION: Our data suggest that estrogen may promote in vivo ovarian cancer growth, both directly and indirectly, by making the tumor microenvironment more conducive for cancer growth.

Modification of the primary tumor microenvironment by transforming growth factor alpha-epidermal growth factor receptor signaling promotes metastasis in an orthotopic colon cancer model.

The transforming growth factor alpha (TGFalpha)/epidermal growth factor receptor (EGFR) signaling pathway appears to play a critical role in colon cancer progression, but the cellular and molecular mechanisms that contribute to metastasis remain unknown. KM12C colon cancer cell clones expressing high (C9) or negligible (C10) levels of TGFalpha were implanted into the cecal walls of nude mice. C9 tumors formed autocrine and paracrine EGFR networks, whereas C10 tumors were unable to signal through EGFR. The tumor microenvironment of C9, but not C10, contained cells enriched in vascular endothelial growth factor (VEGF) A, interleukin-8, and matrix metalloproteinases-2 and -9 and had a high vascular surface area. C9 tumors recruited a macrophage population that co-expressed F4/80 and lymphatic vessel endothelial hyaluronic acid receptor and produced VEGFC. The mean lymphatic density of C9 tumors was threefold higher than that of C10 tumors. C9, but not C10, tumor cells metastasized to regional lymph nodes in all mice and to the liver in 5 of 10 mice. Forced expression of TGFalpha in C10 tumor cells led to the generation of autocrine and paracrine EGFR signaling, macrophage recruitment, enhanced blood and lymphatic vascular surface areas, and increased lymphatic metastasis. Collectively, these data show that activation of TGFalpha-EGFR signaling in colon cancer cells creates a microenvironment that is conducive for metastasis, providing a rationale for efforts to inhibit EGFR signaling in TGFalpha-positive colon cancers.

Biobehavioral influences on matrix metalloproteinase expression in ovarian carcinoma.

PURPOSE: Stromal cells in the tumor microenvironment, such as macrophages, play an active role in tumor growth and angiogenesis. However, little is known about relationships of biobehavioral factors with angiogenic cytokines and matrix metalloproteinases (MMP) produced by stromal cells. This study examined distress, MMPs, and angiogenic cytokines in ovarian cancer patients and in vitro. EXPERIMENTAL DESIGN: Patients suspected of ovarian cancer completed preoperative questionnaires. At surgery, 56 were confirmed to have epithelial ovarian cancer. Tumor samples were analyzed for macrophage (CD68(+)) and tumor cell levels of MMP-2, MMP-9, and vascular endothelial growth factor. In vitro stimulation of isolated macrophage cells by the stress hormones norepinephrine and cortisol was done to assess effects on MMP-9. RESULTS: Depressed patients showed significant elevations of MMP-9 in CD68(+) cells, adjusting for stage (P<0.0001). Patients with higher levels of current stress (P=0.01), life stress over the last 6 months (P=0.004), and general negative affect (P=0.007) also showed significantly greater MMP-9 in CD68(+) cells. In contrast, higher social support was associated with lower levels of MMP-9 (P=0.023) and vascular endothelial growth factor (P=0.036) in tumor cells. In vitro analyses showed that macrophage MMP-9 production could be directly enhanced (up to a 2-fold increase) by the stress hormones norepinephrine and cortisol. CONCLUSIONS: Ovarian cancer patients with elevated depressive symptoms, chronic stress, and low social support showed elevations in MMP-9 in tumor-associated macrophages. Direct in vitro enhancement of stromal MMP-9 production by stress hormones was also shown. These findings may have implications for patient outcomes in ovarian cancer.

A selective small molecule inhibitor of c-Met, PHA-665752, reverses lung premalignancy induced by mutant K-ras.

The c-Met receptor tyrosine kinase has been implicated in cellular transformation induced by mutant Ras, a commonly activated proto-oncogene in non-small cell lung cancer (NSCLC). However, the role of c-Met has not been defined in K-ras-mutant NSCLC, a disease for which no effective targeted therapeutic options currently exist. To acquire a greater understanding of its role, we used genetic and pharmacologic approaches to inhibit c-Met in mice and cultured cells. In Kras(LA1) mice, which develop premalignant lung lesions that progress to multifocal lung adenocarcinomas owing to somatic mutations in K-ras, c-Met was expressed in multiple cell types within premalignant lung lesions, and high concentrations of HGF were detected in bronchoalveolar lavage samples. Short-term treatment with PHA-665752, a c-Met inhibitor, decreased the numbers of premalignant lung lesions and induced apoptosis in tumor cells and vascular endothelial cells within lesions. In cell culture, PHA-665752 induced apoptosis of a lung adenocarcinoma cell line derived from Kras(LA1) mice (LKR-13) and a murine lung endothelial cell line (MEC). c-Met depletion by siRNA transfection induced apoptosis of MECs but not LKR-13 cells. Collectively, these findings suggest that apoptosis was an on-target effect of PHA-665752 in MECs but not in LKR-13 cells. We conclude that PHA-665752 inhibited lung tumorigenesis in Kras(LA1) mice and may provide a novel therapeutic approach to the prevention of K-ras-mutant NSCLC.

Targeting the EGFR, VEGFR, and PDGFR on colon cancer cells and stromal cells is required for therapy.

Immunohistochemical analysis of human colon cancers growing in the cecal walls of nude mice revealed that epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) were expressed by different tumor cells and tumor-associated endothelial cells, whereas platelet-derived growth factor receptor (PDGFR)beta was expressed by tumor-associated endothelial cells and pericytes. We hypothesized that treatment of nude mice with AEE788 (an inhibitor of EGFR and VEGFR phosphorylation) and STI571 (an inhibitor of PDGFRbeta phosphorylation) combined with irinotecan would overcome the intratumoral heterogeneity of these growth factors and efficiently inhibit colon cancer growth and metastasis. We implanted HT29 and KM12SM cells into the cecal walls of nude mice. Two weeks later, the mice were treated with oral vehicle solution; oral AEE788, oral STI571, or intraperitoneal injection of irinotecan as single agents; or the various combinations of these agents. We then assessed the mice for tumor growth and metastasis. Immunohistochemical analyses revealed that oral AEE788 suppressed proliferation and increased apoptosis of tumor cells and tumor-associated endothelial cells. Oral STI571 increased apoptosis of tumor-associated endothelial cells and pericytes. The combination of AEE788, STI571, and irinotecan produced the greatest inhibition of primary tumor growth and metastasis. Collectively, these data demonstrate that only targeting multiple tyrosine kinase receptors on colon cancer cells and tumor-associated stromal cells can overcome the effects of biologic heterogeneity for resistance to treatment and has the potential to improve therapeutic outcome for patients with this disease.

Impact of vessel maturation on antiangiogenic therapy in ovarian cancer.

OBJECTIVE: The purpose of this study was to examine the functional and therapeutic significance of pericytes in ovarian cancer vasculature. STUDY DESIGN: Tumor vessel morphologic condition and efficacy of endothelial and pericyte targeting were examined with the use of in vivo ovarian cancer models. The expression of platelet-derived growth factor (PDGF) ligands and receptors was examined in endothelial, pericyte-like, and ovarian cancer cells. RESULTS: Relative to normal vessels, tumor vasculature was characterized by loosely attached pericytes in reduced density. PDGF-BB was expressed predominantly by the endothelial and cancer cells, whereas PDGFRbeta was present in pericyte-like cells. PDGF-BB significantly increased the migration of and VEGF production by pericyte-like cells; PDGFRbeta blockade abrogated these effects. Dual VEGF (VEGF-Trap) and PDGF-B (PDGF-Trap) targeted therapy was more effective in inhibiting in vivo tumor growth than either agent alone. CONCLUSION: Aberrations in the tumor microenvironment contribute to endothelial cell survival. Strategies that target both endothelial cells and pericytes should be considered for clinical trials.

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.

Targeted therapy of orthotopic human lung cancer by combined vascular endothelial growth factor and epidermal growth factor receptor signaling blockade.

The outcome for patients with lung cancer has not changed significantly for more than two decades. Several studies show that the overexpression of vascular endothelial growth factor (VEGF)/vascular permeability factor and epidermal growth factor (EGF) and their receptors correlates with the clinical outcome for lung cancer patients. However, clinical trials of agents that target either of these pathways alone have been disappointing. We hypothesize that targeting both the tumor and its vasculature by simultaneously blocking the VEGFR and EGFR pathways will improve the treatment of locoregional lung cancer. Human lung cancer specimens were first examined for the activation of VEGF receptor 2 (VEGFR2) and EGF receptor (EGFR) for tumor and tumor-associated endothelial cells, and both were found to be activated. The effects of ZD6474 (ZACTIMA), a small-molecule inhibitor of VEGFR2 and EGFR tyrosine kinases, were then studied in vitro using human lung cancer and microvascular endothelial cells. In vitro, ZD6474 inhibited EGFR, VEGFR2, mitogen-activated protein kinase and Akt phosphorylation, EGF- and VEGF-induced proliferation, and endothelial cell tube formation and also induced apoptosis. ZD6474 was further studied in vivo using an orthotopic mouse model of non-small cell lung cancer using NCI-H441 human lung adenocarcinoma cells. The inhibition of both VEGFR2 and EGFR signaling pathways by ZD6474 resulted in profound antiangiogenic, antivascular, and antitumor effects. These results provide a basis for the development of clinical strategies for the combination of selective protein tyrosine kinase inhibitors that block both EGFR and VEGFR signaling as part of the management of locally advanced lung cancer.

Expression of epidermal growth factor (EGF)/transforming growth factor-alpha by human lung cancer cells determines their response to EGF receptor tyrosine kinase inhibition in the lungs of mice.

Epidermal growth factor receptor (EGFR) has been extensively targeted in the treatment of non-small cell lung cancer, producing responses in a small number of patients. To study the role of ligand expression in mediating response to EGFR antagonism, we injected NCI-H441 [EGFR and EGF/transforming growth factor-alpha (TGF-alpha) positive] or PC14-PE6 (EGFR positive and EGF/TGF-alpha negative) human lung adenocarcinoma cells into the lungs of nude mice. We randomized the mice to receive treatment with the EGFR tyrosine kinase inhibitors gefitinib or AEE788 or vehicle. Treatment of mice bearing NCI-H441 but not PC14-PE6 lung tumors resulted in a significant reduction in primary tumor growth, pleural effusion, and lymph node metastasis. Immunohistochemical analyses revealed that NCI-H441 and PC14-PE6 cells expressed EGFR but that the expression of EGF/TGF-alpha was high in NCI-H441 cells and very low in PC14-PE6 cells. Consequently, EGFR was activated in both tumor and tumor-associated endothelial cells in the NCI-H441 tumors but not in the PC14-PE6 tumors. Antagonism of EGFR signaling by treatment of mice with AEE788 decreased proliferation and increased apoptosis of both tumor cells and tumor-associated endothelial cells in NCI-H441 tumors but not in PC14-PE6 tumors. However, after transfection of PC14-PE6 cells with TGF-alpha, lung tumors derived from the transfected cells expressed and activated EGFR in both tumor and tumor-associated endothelial cells and tumors responded to treatment with AEE788. Collectively, these results strongly suggest that the response of human lung cancers growing orthotopically in mice to the inhibition of EGFR signaling is determined by ligand (EGF/TGF-alpha) expression by tumor cells. Our findings provide an additional explanation for the susceptibility of lung cancers to treatment with EGFR tyrosine kinase inhibitors.

Inflammatory cytokines induce MAdCAM-1 in murine hepatic endothelial cells and mediate alpha-4 beta-7 integrin dependent lymphocyte endothelial adhesion in vitro.

BACKGROUND: MAdCAM-1 plays a central role in T-lymphocyte homing to the gut, but its role in chronic liver inflammation remains unknown. Therefore, this study measured MAdCAM-1 expression, regulation, and function in cultured murine hepatic endothelial cells. METHODS: Cultures of hepatic endothelial cells (HEC) were prepared from mice expressing a temperature-sensitive SV40 large T antigen (H-2Kb-tsA58) under the control of an IFN-gamma promoter. Time and dose dependent expression of MAdCAM-1 in response to TNF-alpha, IL-1 beta and IFN-gamma was studied by immunoblotting. Lymphocyte adhesion was studied using alpha 4 beta 7 integrin expressing lymphocytes (TK-1) +/- anti-MAdCAM-1 mAb. RESULTS: TNF-alpha induced MAdCAM-1 dose-and time-dependently with maximum expression at 20 ng/ml and at 48 hours. IL-1 beta also induced MAdCAM-1 to a lesser extent compared to TNF-alpha; IFN-gamma did not induce MAdCAM-1. TNF-alpha significantly increased lymphocyte-endothelial adhesion (P < 0.01), which was reversed by anti-MAdCAM-1 antibody. MAdCAM-1 expression was also reduced by N-acetylcysteine and by two NO donors (SperNO, DETANO) suggesting that hepatic endothelial MAdCAM-1 is oxidant and NO regulated. CONCLUSION: MAdCAM-1 is a major determinant of leukocyte recruitment in chronic inflammation and is expressed by HEC in response to IL-1 beta and TNF-alpha. This system may provide a useful model for studying inflammatory mechanisms in liver disease and help determine if controlled MAdCAM-1 expression might influence inflammation in liver disease.

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.

Modulation of bone microenvironment with zoledronate enhances the therapeutic effects of STI571 and paclitaxel against experimental bone metastasis of human prostate cancer.

Prostate cancer cells metastasize to the bone where their interaction with osteoclasts and osteoblasts can lead to alterations in the structure of the bone. We determined whether the systemic administration of the bisphosphonate, zoledronate, could prevent bone lysis and halt the proliferation of human prostate cancer cells injected into the tibia of nude mice. Zoledronate did not affect the in vitro proliferation of human prostate cancer PC-3MM2 cells. The in vivo administration of zoledronate produced significant bone preservation but did not inhibit the progressive growth of PC-3MM2 cells. The systemic administration of STI571 (imatinib mesylate, Gleevec), an inhibitor of phosphorylation of the platelet-derived growth factor receptor, in combination with paclitaxel, produced apoptosis of tumor cells and bone- and tumor-associated endothelial cells. The systemic administration of zoledronate with STI571 and paclitaxel produced a significant preservation of bone structure, a decrease in tumor incidence and weight, and a decrease in incidence of lymph node metastasis. This therapeutic activity was correlated with inhibition of osteoclast function, inhibition of tumor cell proliferation, and induction of apoptosis in tumor-associated endothelial cells and tumor cells. Cancer is a heterogeneous disease that requires multimodality therapy. The present data recommend the combination of a bisphosphonate agent with protein tyrosine kinase inhibitor and an anticycling drug for the treatment of prostate cancer bone metastasis.

The HGF/c-MET Pathway Is a Driver and Biomarker of VEGFR-inhibitor Resistance and Vascular Remodeling in Non-Small Cell Lung Cancer.

Purpose: Resistance to VEGFR inhibitors is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). We investigated the cellular mechanisms mediating resistance of NSCLCs to VEGFR tyrosine kinase inhibitors.Experimental Design: We generated murine models of human NSCLC and performed targeted inhibition studies with the VEGFR TKIs cediranib and vandetanib. We used species-specific hybridization of microarrays to compare cancer (human) and stromal (mouse) cell transcriptomes of TKI-sensitive and -resistant tumors. We measured tumor microvascular density and vessel tortuosity to characterize the effects of therapy on the tumor vascular bed. Circulating cytokine and angiogenic factor levels in patients enrolled in VEGFR TKI trials were correlated with clinical outcomes.Results: Murine xenograft models of human lung adenocarcinoma were initially sensitive to VEGFR TKIs, but developed resistance to treatment. Species-specific microarray analysis identified increased expression of stromal-derived hepatocyte growth factor (HGF) as a candidate mediator of TKI resistance and its receptor, c-MET, was activated in cancer cells and tumor-associated stroma. A transient increase in hypoxia-regulated molecules in the initial response phase was followed by adaptive changes resulting in a more tortuous vasculature. Forced HGF expression in cancer cells reduced tumor sensitivity to VEGFR TKIs and produced tumors with tortuous blood vessels. Dual VEGFR/c-MET signaling inhibition delayed the onset of the resistant phenotype and prevented the vascular morphology alterations. In patients with cancer receiving VEGFR TKIs, high pretreatment HGF plasma levels correlated with poorer survival.Conclusions: HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC. Clin Cancer Res; 23(18); 5489-501. ©2017 AACR.

Activation of the platelet-derived growth factor-receptor enhances survival of murine bone endothelial cells.

The activation of the microvascular endothelial cell platelet-derived growth factor (PDGF) receptor (PDGF-R) by PDGF has been implicated in neoplastic angiogenesis. Here, we established cultures of murine bone microvascular endothelial cells and examined their response to stimulation with PDGF BB ligand and to blockade of PDGF-R signaling with the tyrosine kinase inhibitor STI571 (Gleevec). The addition of STI571 to cultures of bone endothelial cells blocked PDGF BB-induced phosphorylation in a dose-dependent manner and completely abrogated the activation of downstream targets Akt and ERK1/2. Coadministration of STI571 and Taxol also induced the activation of procaspase-3 and significant apoptosis. These data suggest that phosphorylation of PDGF-R stimulates survival pathways in bone endothelial cells and that by selectively inhibiting PDGF-R signaling with STI571, the cells are rendered sensitive to Taxol treatment. The therapeutic combination of STI571 and Taxol may be a powerful tool for targeting tumor-associated endothelial cells in the skeletal compartment.

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-R alpha and -beta 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.