George N. Naumov

LKB1 modulates lung cancer differentiation and metastasis.

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz–Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.

PF00299804, an Irreversible Pan-ERBB Inhibitor, Is Effective in Lung Cancer Models with EGFR and ERBB2 Mutations that Are Resistant to Gefitinib

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors gefitinib and erlotinib are effective treatments for a subset of non-small cell lung cancers. In particular, cancers with specific EGFR-activating mutations seem to be the most sensitive to these agents. However, despite their initial response, such cancers almost invariably develop resistance. In 50% of such cancers, a secondary EGFR mutation, T790M, has been identified that renders gefitinib and erlotinib ineffective inhibitors of EGFR kinase activity. Thus, there is a clinical need to develop novel EGFR inhibitors that can effectively inactivate T790M-containing EGFR proteins. In this study, we evaluate the effectiveness of a novel compound, PF00299804, an irreversible pan-ERBB inhibitor. The results from these studies show that PF00299804 is a potent inhibitor of EGFR-activating mutations as well as the EGFR T790M resistance mutation both in vitro and in vivo. Additionally, PF00299804 is a highly effective inhibitor of both the wild-type ERBB2 and the gefitinib-resistant oncogenic ERBB2 mutation identified in lung cancers. These preclinical evaluations support further clinical development of PF00299804 for cancers with mutations and/or amplifications of ERBB family members.

Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR -amplified lung cancer

EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alpha E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.

Role of angiogenesis in human tumor dormancy: animal models of the angiogenic switch.

Tumor progression depends on sequential events, including a switch to the angiogenic phenotype (i.e. initial recruitment of blood vessels). Failure of a microscopic tumor to complete one or more early steps in this process may lead to delayed clinical manifestation of the cancer. Microscopic human cancers can remain in an asymptomatic, non-detectable, and occult state for the life of a person. Clinical and experimental evidence suggest that human tumors can persist for long periods of time as microscopic lesions that are in a state of dormancy (i.e. not expanding in tumor mass). Because it is well established that tumor growth beyond the size of 1-2 mm is angiogenesis-dependent, we hypothesized that presentation of large tumors is attributed to a switch to the angiogenic phenotype in otherwise microscopic, dormant tumors. Although clinically important, the biology of human tumor dormancy is poorly understood. The development of animal models which recapitulate the clinically observed timing and proportion of dormant tumors which switch to the angiogenic phenotype are reviewed here. The contributing molecular mechanisms involved in the angiogenic switch and different strategies for isolation of both angiogenic and nonangiogenic tumor cell populations from otherwise heterogeneous human tumor cell lines or surgical specimens are also summarized. Several imaging techniques have been utilized for the qualitative and quantitative detection of microscopic tumors in mice and their strengths and limitations are discussed. The animal models employed here permitted further studies of the angiogenic switch. These models also allowed development of an angiogenesis-based panel of blood and urine biomarkers that can be quantified and used to detect microscopic tumors before or during the angiogenic switch. If the information obtained from these animal models is translatable to the clinic, it may be possible in the future to liberate the management of cancer from a dependency on anatomical site years before it becomes symptomatic and detectable.

Combined Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor (EGFR) Blockade Inhibits Tumor Growth in Xenograft Models of EGFR Inhibitor Resistance

Purpose: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non–small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification. Experimental Design: We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation. Results: Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects. Conclusion: These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients.

Differential effects of vascular endothelial growth factor receptor-2 inhibitor ZD6474 on circulating endothelial progenitors and mature circulating endothelial cells: Implications for use as a surrogate marker of antiangiogenic activity

Purpose: Circulating endothelial cells (CEC) comprise at least two distinct populations: bone marrow–derived circulating endothelial progenitors (CEP) and mature CECs derived from existing vasculature. We hypothesized that antiangiogenic agents may have differential effects on CEPs and mature CECs and that these changes may serve as a marker of biological activity. Experimental Design: The effect of angiogenesis inhibitors on CECs was evaluated by flow cytometry after vascular endothelial growth factor (VEGF)–induced mobilization and in mice bearing Lewis lung carcinoma (LLC). Tumor angiogenesis was evaluated in parallel by immunohistochemistry. Results: In nontumor-bearing mice, VEGF administration increased both mature CECs and CEPs. This increase was inhibited by the VEGF receptor 2 inhibitor ZD6474 as well as the VEGF inhibitor–soluble Flt-1. ZD6474 had no significant effect on CECs in the absence of exogenous VEGF stimulation. In contrast, LLC-bearing mice had an increase in mature CECs but not CEPs after 3 days of treatment with ZD6474. The increase in mature CECs was dose-dependent, accompanied by a decrease in tumor microvessel density, and preceded reduction in tumor volume. Treatment of LLC-bearing mice with the vascular targeting agent ZD6126 also increased mature CECs. Conclusions: VEGF inhibitors can have differential effects on mature CECs and CEPs, and agents inhibiting tumor angiogenesis may cause a concomitant increase in mature CECs. This increase occurs in tumor-bearing but not in nontumor-bearing mice, suggesting that tumor endothelium is a potential source of mature CECs. Therefore, assessing both mature CECs and CEPs may provide insights into the mechanism of antiangiogenic agents and serve as an early surrogate marker of biological activity.

HIF2α cooperates with RAS to promote lung tumorigenesis in mice

Members of the hypoxia-inducible factor (HIF) family of transcription factors regulate the cellular response to hypoxia. In non–small cell lung cancer (NSCLC), high HIF2α levels correlate with decreased overall survival, and inhibition of either the protein encoded by the canonical HIF target gene VEGF or VEGFR2 improves clinical outcomes. However, whether HIF2α is causal in imparting this poor prognosis is unknown. Here, we generated mice that conditionally express both a nondegradable variant of HIF2α and a mutant form of Kras (KrasG12D) that induces lung tumors. Mice expressing both Hif2a and KrasG12D in the lungs developed larger tumors and had an increased tumor burden and decreased survival compared with mice expressing only KrasG12D. Additionally, tumors expressing both KrasG12D and Hif2a were more invasive, demonstrated features of epithelial-mesenchymal transition (EMT), and exhibited increased angiogenesis associated with mobilization of circulating endothelial progenitor cells. These results implicate HIF2α causally in the pathogenesis of lung cancer in mice, demonstrate in vivo that HIF2α can promote expression of markers of EMT, and define HIF2α as a promoter of tumor growth and progression in a solid tumor other than renal cell carcinoma. They further suggest a possible causal relationship between HIF2α and prognosis in patients with NSCLC.

Tumor dormancy due to failure of angiogenesis: role of the microenvironment

Tumor progression is dependent on a number of sequential steps, including initial recruitment of blood vessels (i.e., angiogenic switch). Failure of a microscopic tumor to complete one or more of these early steps may lead to delayed clinical manifestation of the cancer. In this review we summarize some of the clinical and experimental evidence suggesting that microscopic human cancers can remain in an asymptomatic, non-detectable, and occult state for the life of a person or animal. We present three clinical cases where tumors present shortly after an accidental trauma in otherwise healthy individuals. We also review current experimental human tumor dormancy models with special emphasis on the angiogenic switch which closely recapitulates clinically observed delay in tumor recurrence.

Tumor-vascular interactions and tumor dormancy.

Tumor progression is dependent on a number of sequential steps, including initial tumor‐vascular interactions and recruitment of blood vessels (i.e., the angiogenic switch), as well as tumor cells interacting with the surrounding microenvironment and its different components. Failure of a microscopic tumor to complete one or more of these early stages may lead to delayed clinical manifestation of the cancer and a state of stable non‐progressing disease (i.e., tumor dormancy). In this review, some of the clinical and experimental evidence is summarized, suggesting that microscopic human cancers, either primary, recurrent or metastatic, can remain in an asymptomatic, non‐detectable, and occult state for a long period of time. We also review current experimental human tumor dormancy models which closely recapitulate clinically observed delay in tumor progress.

Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesis

The disease state of cancer appears late in tumor development. Before being diagnosed, a tumor can remain for prolonged periods of time in a dormant state. Dormant human cancer is commonly defined as a microscopic tumor that does not expand in size and remains asymptomatic. Dormant tumors represent an early stage in tumor development and may therefore be a potential target for nontoxic, antiangiogenic therapy that could prevent tumor recurrence. Here, we characterize an experimental model that recapitulates the clinical dormancy of human tumors in mice. We demonstrate that these microscopic dormant cancers switch to the angiogenic phenotype at a predictable time. We further show that while angiogenic liposarcomas expand rapidly after inoculation of tumor cells in mice, nonangiogenic dormant liposarcomas remain microscopic up to one‐third of the normal severe combined immune deficiency (SCID) mouse life span, although they contain proliferating tumor cells. Nonangiogenic dormant tumors follow a similar growth pattern in subcutaneous (s.c.) and orthotopic environments. Throughout the dormancy period, development of intratumoral vessels is impaired. In nonangogenic dormant tumors, small clusters of endothelial cells without lumens are observed early after tumor cell inoculation, but the nonangiogenic tumor cannot sustain these vessels, and they disappear within weeks. There is a concomitant decrease in microvessel density, and the nonangiogenic dormant tumor remains harmless to the host. In contrast, microvessel density in tumors increases rapidly after the angiogenic switch and correlates with rapid expansion of tumor mass. Both tumor types cultured in vitro contain fully transformed cells, but only cells from the nonangiogenic human liposarcoma secrete relatively high levels of the angiogenesis inhibitors thrombospondin‐1 and TIMP‐1. This model suggests that as improved blood or urine molecular biomarkers are developed, the microscopic, nonangiogenic, dormant phase of human cancer may be vulnerable to antiangiogenic therapy years before symptoms, or before anatomical location of a tumor can be detected, by conventional methods.—Almog, N., Henke, V., Flores, L., Hlatky, L., Kung, A. L., Wright, R. D., Berger, R., Hutchinson, L., Naumov, G., Bender, E., Akslen, L., Achilles, E.‐G., Folkman, J. Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesis. FASEB J. 20, E1–E10 (2006)