Janine T. Erler

Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling

Tumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening, and increased focal adhesions. Induction of collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibition of integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling, and induced the invasion of a premalignant epithelium. Consistently, reduction of lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy, and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy.

Hypoxia-Induced Lysyl Oxidase Is a Critical Mediator of Bone Marrow Cell Recruitment to Form the Premetastatic Niche

Tumor cell metastasis is facilitated by premetastatic niches formed in destination organs by invading bone marrow-derived cells (BMDCs). Lysyl oxidase (LOX) is critical for premetastatic niche formation. LOX secreted by hypoxic breast tumor cells accumulates at premetastatic sites, crosslinks collagen IV in the basement membrane, and is essential for CD11b+ myeloid cell recruitment. CD11b+ cells adhere to crosslinked collagen IV and produce matrix metalloproteinase-2, which cleaves collagen, enhancing the invasion and recruitment of BMDCs and metastasizing tumor cells. LOX inhibition prevents CD11b+ cell recruitment and metastatic growth. CD11b+ cells and LOX also colocalize in biopsies of human metastases. Our findings demonstrate a critical role for LOX in premetastatic niche formation and support targeting LOX for the treatment and prevention of metastatic disease.

Three-dimensional context regulation of metastasis.

Tumor progression ensues within a three-dimensional microenvironment that consists of cellular and non-cellular components. The extracellular matrix (ECM) and hypoxia are two non-cellular components that potently influence metastasis. ECM remodeling and collagen cross-linking stiffen the tissue stroma to promote transformation, tumor growth, motility and invasion, enhance cancer cell survival, enable metastatic dissemination, and facilitate the establishment of tumor cells at distant sites. Matrix degradation can additionally promote malignant progression and metastasis. Tumor hypoxia is functionally linked to altered stromal-epithelial interactions. Hypoxia additionally induces the expression of pro-migratory, survival and invasion genes, and up-regulates expression of ECM components and modifying enzymes, to enhance tumor progression and metastasis. Synergistic interactions between matrix remodeling and tumor hypoxia influence common mechanisms that maximize tumor progression and cooperate to drive metastasis. Thus, clarifying the molecular pathways by which ECM remodeling and tumor hypoxia intersect to promote tumor progression should identify novel therapeutic targets.

Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK

The extracellular, matrix-modifying enzyme lysyl oxidase (LOX) has recently been linked to colorectal cancer (CRC) progression, in particular to the stages of invasion and metastasis. In this report, we use cell lines expressing a catalytically inactive mutant form of LOX to show that catalytic activity is required for LOX-mediated effects on proliferation and invasion in both in vitro and in vivo models of CRC. Furthermore, we use rheology to measure the relative stiffness of modified collagen matrices and subcutaneous tumors, and show that LOX-induced collagen cross-linking results in stiffening of the matrix both in vitro and in vivo. We observe a strong association between matrix stiffness and activation of the FAK (focal adhesion kinase)/SRC-signaling pathway, with a stiffer environment resulting in increased FAK/SRC phosphorylation and a more proliferative and invasive phenotype. We are the first to show a direct relationship between LOX enzymatic activity and tissue stiffness, and to demonstrate a role for stiffness in driving CRC progression. Our findings provide significant evidence to suggest that therapeutic inhibition of LOX activity may provide a novel effective treatment option for patients with metastatic CRC.

Hypoxia in Models of Lung Cancer: Implications for Targeted Therapeutics

Purpose: To efficiently translate experimental methods from bench to bedside, it is imperative that laboratory models of cancer mimic human disease as closely as possible. In this study, we sought to compare patterns of hypoxia in several standard and emerging mouse models of lung cancer to establish the appropriateness of each for evaluating the role of oxygen in lung cancer progression and therapeutic response. Experimental Design: Subcutaneous and orthotopic human A549 lung carcinomas growing in nude mice as well as spontaneous K-ras or Myc-induced lung tumors grown in situ or subcutaneously were studied using fluorodeoxyglucose and fluoroazomycin arabinoside positron emission tomography, and postmortem by immunohistochemical observation of the hypoxia marker pimonidazole. The response of these models to the hypoxia-activated cytotoxin PR-104 was also quantified by the formation of γH2AX foci in vitro and in vivo. Finally, our findings were compared with oxygen electrode measurements of human lung cancers. Results: Minimal fluoroazomycin arabinoside and pimonidazole accumulation was seen in tumors growing within the lungs, whereas subcutaneous tumors showed substantial trapping of both hypoxia probes. These observations correlated with the response of these tumors to PR-104, and with the reduced incidence of hypoxia in human lung cancers relative to other solid tumor types. Conclusions: These findings suggest that in situ models of lung cancer in mice may be more reflective of the human disease, and encourage judicious selection of preclinical tumor models for the study of hypoxia imaging and antihypoxic cell therapies. Clin Cancer Res; 16(19); 4843–52. ©2010 AACR.

Contrasting effects of sunitinib within in vivo models of metastasis

Sunitinib is a potent and clinically approved tyrosine kinase inhibitor that can suppress tumour growth by inhibiting angiogenesis. However, conflicting data exist regarding the effects of this drug on the growth of metastases in preclinical models. Here we use 4T1 and RENCA tumour cells, which both form lung metastases in Balb/c mice, to re-address the effects of sunitinib on the progression of metastatic disease in mice. We show that treatment of mice with sunitinib prior to intravenous injection of tumour cells can promote the seeding and growth of 4T1 lung metastases, but not RENCA lung metastases, showing that this effect is cell line dependent. However, increased metastasis occurred only upon administration of a very high sunitinib dose, but not when lower, clinically relevant doses were used. Mechanistically, high dose sunitinib led to a pericyte depletion effect in the lung vasculature that correlated with increased seeding of metastasis. By administering sunitinib to mice after intravenous injection of tumour cells, we demonstrate that while sunitinib does not inhibit the growth of 4T1 lung tumour nodules, it does block the growth of RENCA lung tumour nodules. This contrasting response was correlated with increased myeloid cell recruitment and persistent vascularisation in 4T1 tumours, whereas RENCA tumours recruited less myeloid cells and were more profoundly devascularised upon sunitinib treatment. Finally, we show that progression of 4T1 tumours in sunitinib treated mice results in increased hypoxia and increased glucose metabolism in these tumours and that this is associated with a poor outcome. Taken together, these data suggest that the effects of sunitinib on tumour progression are dose-dependent and tumour model-dependent. These findings have relevance for understanding how anti-angiogenic agents may influence disease progression when used in the adjuvant or metastatic setting in cancer patients.

Bioreductively Activated Lysyl Oxidase Inhibitors against Hypoxic Tumours

Most human cancers consist of solid tumours that are significantly less oxygenated than normal tissues. Once their dimensions reach certain values (>1 cm3) they contain hypoxic regions generally surrounding necrotic areas. Hypoxic cells are normally resistant to anticancer chemotherapy and radiotherapy. Hypoxia has also been shown to predispose tumours to increased invasion and metastatic processes,[1] leading to poor prognosis.

3 – The Cellular Microenvironment and Metastases

Abstract One of the most important challenges in clinical oncology is the prevention and treatment of metastatic disease. Tumor metastasis involves tumor cell invasion and migration from the primary tumor, intravasation into the vasculature, dissemination and survival in the circulation, extravasation into distant tissues, survival and metabolic adaptation in the distant tissue, dormancy, reactivation, and overt colonization to form a new macroscopic tumor at a distant site. This process is highly inefficient; it has been estimated that less than 0.01% of tumor cells that enter the circulation develop into metastases. Despite this inefficiency, metastases are responsible for more than 90% of all cancer-related deaths. Understanding the biology and vulnerabilities of metastatic tumor cells is of critical importance if we are to improve overall survival rates in cancer patients. Recent studies have characterized the molecular and phenotypic makeup of metastatic tumor cells. In addition, it is now appreciated that cellular and molecular constituents of the tumor microenvironment also greatly affect metastatic tumor progression. This chapter describes the cellular and molecular traits driving tumor metastasis and discusses how the tumor microenvironment influences this process. Most important, it addresses how this knowledge can be translated into current and future cancer therapies.