Elimination of dormant, autophagic ovarian cancer cells and xenografts through enhanced sensitivity to anaplastic lymphoma kinase inhibition.

BACKGROUND: Poor outcomes for patients with ovarian cancer relate to dormant, drug-resistant cancer cells that survive after primary surgery and chemotherapy. Ovarian cancer (OvCa) cells persist in poorly vascularized scars on the peritoneal surface and depend on autophagy to survive nutrient deprivation. The authors have sought drugs that target autophagic cancer cells selectively to eliminate residual disease. METHODS: By using unbiased small-interfering RNA (siRNA) screens, the authors observed that knockdown of anaplastic lymphoma kinase (ALK) reduced the survival of autophagic OvCa cells. Small-molecule ALK inhibitors were evaluated for their selective toxicity against autophagic OvCa cell lines and xenografts. Autophagy was induced by reexpression of GTP-binding protein Di-Ras3 (DIRAS3) or serum starvation and was evaluated with Western blot analysis, fluorescence imaging, and transmission electron microscopy. Signaling pathways required for crizotinib-induced apoptosis of autophagic cells were explored with flow cytometric analysis, Western blot analysis, short-hairpin RNA knockdown of autophagic proteins, and small-molecule inhibitors of STAT3 and BCL-2. RESULTS: Induction of autophagy by reexpression of DIRAS3 or serum starvation in multiple OvCa cell lines significantly reduced the 50% inhibitory concentration of crizotinib and other ALK inhibitors. In 2 human OvCa xenograft models, the DIRAS3-expressing tumors treated with crizotinib had significantly decreased tumor burden and long-term survival in 67% to 79% of mice. Crizotinib treatment of autophagic cancer cells further enhanced autophagy and induced autophagy-mediated apoptosis by decreasing phosphorylated STAT3 and BCL-2 signaling. CONCLUSIONS: Crizotinib may eliminate dormant, autophagic, drug-resistant OvCa cells that remain after conventional cytoreductive surgery and combination chemotherapy. A clinical trial of ALK inhibitors as maintenance therapy after second-look operations should be seriously considered.

Mechanism of thioamide drug action against tuberculosis and leprosy.

Thioamide drugs, ethionamide (ETH) and prothionamide (PTH), are clinically effective in the treatment of Mycobacterium tuberculosis, M. leprae, and M. avium complex infections. Although generally considered second-line drugs for tuberculosis, their use has increased considerably as the number of multidrug resistant and extensively drug resistant tuberculosis cases continues to rise. Despite the widespread use of thioamide drugs to treat tuberculosis and leprosy, their precise mechanisms of action remain unknown. Using a cell-based activation method, we now have definitive evidence that both thioamides form covalent adducts with nicotinamide adenine dinucleotide (NAD) and that these adducts are tight-binding inhibitors of M. tuberculosis and M. leprae InhA. The crystal structures of the inhibited M. leprae and M. tuberculosis InhA complexes provide the molecular details of target-drug interactions. The purified ETH-NAD and PTH-NAD adducts both showed nanomolar Kis against M. tuberculosis and M. leprae InhA. Knowledge of the precise structures and mechanisms of action of these drugs provides insights into designing new drugs that can overcome drug resistance.

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.

Circulating monocytes expressing CD31: implications for acute and chronic angiogenesis.

To identify the roles of various circulating cells (eg, endothelial and/or stem and progenitor cells) in angiogenesis, we parabiosed a wild-type syngeneic mouse with a transgenic syngeneic green fluorescent protein mouse. Following the establishment of a common circulation between these parabionts, we investigated acute (7 to 10 days), subacute (2 to 3 weeks), and chronic (4 to 6 weeks) phases of angiogenesis in wild-type mice using wound healing, implanted gel foam fragments, and subcutaneous tumor assays, respectively. We found that under in vitro conditions, circulating murine monocytes expressed F4/80, CD31, and vascular endothelial growth factor receptor 2, but neither CD133 nor von Willebrand factor, whereas murine endothelial cells expressed CD31, vascular endothelial growth factor receptor 2, and von Willebrand factor, but neither CD133 nor F4/80. Immunofluorescence analysis revealed that green fluorescent protein-positive cells in the walls of new vessels in wounds, gel foam blocks, and tumors expressed both F4/80 and CD31, that is, macrophages. Pericytes, cells that express both CD31 and desmin, were found both in the walls of tumor-associated vessels and within tumors. Collectively, these data demonstrate that monocytes (ie, cells that express both CD31 and F4/80) may be recruited to the site of tissue injury and directly contribute to angiogenesis, reaffirming the close relationships between various cell types within the reticuloendothelial system and suggesting possible targets for anticancer treatments.

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.

Neoadjuvant platelet derived growth factor receptor inhibitor therapy combined with docetaxel and androgen ablation for high risk localized prostate cancer.

PURPOSE: Platelet derived growth factor receptor inhibitor therapy improves the efficacy of taxane chemotherapy in preclinical models of prostate cancer. Men with high risk localized prostate cancer were treated with platelet derived growth factor receptor inhibitor therapy, docetaxel and hormone ablation in the preoperative setting, and clinicopathological outcomes were evaluated. MATERIALS AND METHODS: A total of 36 men with cT2 or greater disease, Gleason grade 8-10, serum prostate specific antigen more than 20 ng/ml or cT2b and prostate specific antigen more than 10 ng/ml and Gleason 7 disease, without radiological evidence of metastases, were scheduled to receive intramuscular leuprolide, 600 mg daily oral imatinib and 30 mg/m(2) weekly docetaxel x 4 every 42 days for 3 cycles before radical prostatectomy (beta [0.02, 1.98] prior on the possibility of pathological complete remission). Unresectable disease, postoperative prostate specific antigen 0.2 ng/ml or greater, or administration of postoperative radiation or hormones were defined as treatment failure. RESULTS: A total of 39 men were registered over 15 months. Median patient age was 57 years (range 44 to 71). Risk factors included T3 disease (22 of 39), Gleason 8-10 disease (31 of 39) and prostate specific antigen more than 20 ng/ml (12 of 39). Three men were ineligible or declined therapy, 29 of 36 (81%) received 3 cycles of therapy and 7 of 36 (19%) discontinued therapy related to toxicity. Grades 3-4 toxicity included rash (4), diarrhea (4), fatigue (6) and neutropenia (1). The surgical approach was feasible, without excessive or unusual complications such as wound dehiscence. No pathological complete remissions were defined. At a median followup of 39 months 53% were free from progression. CONCLUSIONS: Evidence for a favorable impact of platelet derived growth factor receptor inhibitor therapy on the efficacy of neoadjuvant docetaxel and hormonal ablation in high risk localized prostate cancer was not obtained.

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.

Antitumor and antivascular effects of AVE8062 in ovarian carcinoma.

The purpose of this study was to examine the therapeutic efficacy and underlying mechanisms of action of a vascular-disrupting agent, AVE8062, and to determine its effects on tumor metabolic activity. The in vitro and in vivo effects of AVE8062 alone and in combination with docetaxel were tested in chemotherapy-sensitive and chemotherapy-resistant ovarian cancer models. Tumors were analyzed for necrosis, microvessel density, endothelial cell apoptosis, and proliferation following treatment. The effect of AVE8062 on tumor regression and metabolic activity was examined by magnetic resonance (MR) or by [18F]fluorodeoxyglucose ([18F]FDG) uptake by positron emission tomography (PET) with MR imaging, respectively. AVE8062 monotherapy was effective in inhibiting tumor growth in all models (range 43-51% versus control; P < 0.05). Combination therapy was even more effective in inhibiting tumor growth (range 76-90% compared with controls, P < 0.01). AVE8062 in combination with chemotherapy significantly prolonged survival in HeyA8-injected mice (P < 0.001) compared with other groups. AVE8062-based therapy resulted in rapid development of central tumor necrosis, decreased microvessel density, decreased proliferation, and induction of apoptosis of tumor-associated endothelial cells. MR imaging showed regression of established HeyA8 ovarian tumors and [18F]FDG PET with MR showed rapid decrease in metabolic activity after AVE8062 therapy. Combination of AVE8062 plus docetaxel results in potent inhibition of ovarian cancer growth. These results suggest that AVE8062 may be useful as a clinical therapeutic approach for ovarian cancer patients and that functional [18F]FDG PET imaging may predict clinical response before an anatomic reduction in tumor size.

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.

Amino Acid Deprivation-Induced Autophagy Requires Upregulation of DIRAS3 through Reduction of E2F1 and E2F4 Transcriptional Repression.

Failure to cure ovarian cancer relates to the persistence of dormant, drug-resistant cancer cells following surgery and chemotherapy. "Second look" surgery can detect small, poorly vascularized nodules of persistent ovarian cancer in ~50% of patients, where >80% are undergoing autophagy and express DIRAS3. Autophagy is one mechanism by which dormant cancer cells survive in nutrient poor environments. DIRAS3 is a tumor suppressor gene downregulated in >60% of primary ovarian cancers by genetic, epigenetic, transcriptional and post-transcriptional mechanisms, that upon re-expression can induce autophagy and dormancy in a xenograft model of ovarian cancer. We examined the expression of DIRAS3 and autophagy in ovarian cancer cells following nutrient deprivation and the mechanism by which they are upregulated. We have found that DIRAS3 mediates autophagy induced by amino acid starvation, where nutrient sensing by mTOR plays a central role. Withdrawal of amino acids downregulates mTOR, decreases binding of E2F1/4 to the DIRAS3 promoter, upregulates DIRAS3 and induces autophagy. By contrast, acute amino acid deprivation did not affect epigenetic regulation of DIRAS3 or expression of miRNAs that regulate DIRAS3. Under nutrient poor conditions DIRAS3 can be transcriptionally upregulated, inducing autophagy that could sustain dormant ovarian cancer cells.

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.

Identification of a type III thioesterase reveals the function of an operon crucial for Mtb virulence.

Rv0098 is part of an operon, Rv0096-Rv0101, from Mycobacterium tuberculosis (Mtb) that is essential for Mtb's survival in mouse macrophages. This operon also contains an acyl carrier protein and one of the only two nonribosomal peptide synthases in Mtb. Rv0098 is annotated in the genome as a hypothetical protein and was proposed to be an acyl-coenzyme A (CoA) dehydratase. The structure of Rv0098, together with subsequent biochemical analysis, indicated that Rv0098 is a long-chain fatty acyl-CoA thioesterase (FcoT). However, FcoT lacks a general base or a nucleophile that is always found in the catalytic site of type II and type I thioesterases, respectively. The active site of Mtb FcoT reveals the structural basis for its substrate specificity for long-chain acyl-CoA and allows us to propose a catalytic mechanism for the enzyme. The characterization of Mtb FcoT provides a putative function of this operon that is crucial for Mtb pathogenicity.

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.

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.