Lynn M. Matrisian

Matrix metalloproteinases: they're not just for matrix anymore!

The matrix metalloproteinases (MMPs) have been viewed as bulldozers, destroying the extracellular matrix to permit normal remodeling and contribute to pathological tissue destruction and tumor cell invasion. More recently, the identification of specific matrix and non-matrix substrates for MMPs and the elucidation of the biological consequence of cleavage indicates that perhaps MMPs should be viewed more as pruning shears, playing sophisticated roles in modulating normal cellular behavior, cell-cell communication and tumor progression.

Matrix metalloproteinases: multifunctional contributors to tumor progression

Matrix metalloproteinases (MMPs) are a family of extracellular matrix degrading proteinases. Owing to their matrix-degrading abilities and high expression in advanced tumors, MMPs were originally implicated in invasion and metastasis during cancer progression. However, recent work extends a role for MMPs during multiple stages of tumor progression to include other functions such as growth, angiogenesis and migration. Based on studies in animal models implicating MMP activity in cancer, synthetic MMP inhibitors are currently being tested in a clinical setting.

Role of β-arrestin 1 in the metastatic progression of colorectal cancer

G protein-coupled receptor ligand-dependent transactivation of growth factor receptors has been implicated in human cancer cell proliferation, migration, and cell survival. For example, prostaglandin E2 (PGE2)-induced transactivation of the EGF receptor (EGFR) in colorectal carcinoma cells is mediated by means of a c-Src-dependent mechanism and regulates cell proliferation and migration. Recent evidence indicates that β-arrestin 1 may act as an important mediator in G protein-coupled receptor-induced activation of c-Src. Whether β-arrestin 1 serves a functional role in these events is, however, unknown. We investigated the effects of PGE2 on colorectal cancer cells expressing WT and mutant β-arrestin 1. Here we report that PGE2 induces the association of a prostaglandin E receptor 4/β-arrestin 1/c-Src signaling complex resulting in the transactivation of the EGFR and downstream Akt (PKB) signaling. The interaction of β-arrestin 1 and c-Src is critical for the regulation of colorectal carcinoma cell migration in vitro as well as metastatic spread of disease to the liver in vivo. These results show that the prostaglandin E/β-arrestin 1/c-Src signaling complex is a crucial step in PGE2-mediated transactivation of the EGFR and may play a pivotal role in tumor metastasis. Furthermore, our data implicate a functional role for β-arrestin 1 as a mediator of cellular migration and metastasis.

Increased diacylglycerols characterize hepatic lipid changes in progression of human nonalcoholic fatty liver disease; comparison to a murine model.

Background and Aims The spectrum of nonalcoholic fatty liver disease (NAFLD) includes steatosis, nonalcoholic steatohepatitis (NASH), and progression to cirrhosis. While differences in liver lipids between disease states have been reported, precise composition of phospholipids and diacylglycerols (DAG) at a lipid species level has not been previously described. The goal of this study was to characterize changes in lipid species through progression of human NAFLD using advanced lipidomic technology and compare this with a murine model of early and advanced NAFLD. Methods Utilizing mass spectrometry lipidomics, over 250 phospholipid and diacylglycerol species (DAGs) were identified in normal and diseased human and murine liver extracts. Results Significant differences between phospholipid composition of normal and diseased livers were demonstrated, notably among DAG species, consistent with previous reports that DAG transferases are involved in the progression of NAFLD and liver fibrosis. In addition, a novel phospholipid species (ether linked phosphatidylinositol) was identified in human cirrhotic liver extracts. Conclusions Using parallel lipidomics analysis of murine and human liver tissues it was determined that mice maintained on a high-fat diet provide a reproducible model of NAFLD in regards to specificity of lipid species in the liver. These studies demonstrated that novel lipid species may serve as markers of advanced liver disease and importantly, marked increases in DAG species are a hallmark of NAFLD. Elevated DAGs may contribute to altered triglyceride, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) levels characteristic of the disease and specific DAG species might be important lipid signaling molecules in the progression of NAFLD.

Increased plasma MMP9 in integrin α1-null mice enhances lung metastasis of colon carcinoma cells

Inhibitors of matrix metalloproteinases (MMPs) were developed as anticancer agents based on the observation that MMPs facilitate local tumor spread and metastasis by promoting matrix degradation and cell migration. Unfortunately, these inhibitors were unsuccessful in the clinical treatment of several cancers, including lung cancer. A possible reason contributing to their failure is that MMP activity is critical for the generation of inhibitors of tumor angiogenesis, including angiostatin. Thus, MMPs might play opposing roles in tumor vascularization and invasion. To determine which effect of elevated MMP levels dominates in the progression of metastatic cancer, experimental lung metastasis assays were performed in integrin α1‐null mice, a genetic model for increased plasma levels of MMP9 and MMP9‐generated angiostatin (Pozzi et al., Proc. Natl. Acad. Sci. USA 2000;97:2202–7). We show that while the number of lung colonies in integrin α1‐null mice was significantly increased compared to their wild‐type counterparts, tumor volume was markedly reduced. In vivo treatment with the MMP inhibitor doxycycline resulted in a significant decrease in the number of lung colonies in both genotypes, but the tumors that formed were bigger and more vascularized. Increased tumor vascularization paralleled decreased plasma levels of MMP9 and consequent decreased angiostatin synthesis. These results demonstrate that while inhibition of MMPs prevents and/or reduces tumor invasion and lung metastasis, it has the paradoxical effect of increasing the size and vascularization of metastatic tumors due to decreased generation of inhibitors of endothelial cell proliferation. The continued growth of these large well‐vascularized tumors may explain the poor efficacy of MMP inhibitors in lung cancer clinical trials.

An Orthotopic Model of Lung Cancer to Analyze Primary and Metastatic NSCLC Growth in Integrin α1-Null Mice

The role of matrix metalloproteinase (MMP)9 in lung cancer progression is controversial. MMP9 promotes local tumor progression and distant metastasis in mouse models by enhancing extracellular matrix degradation, releasing VEGF from extracellular matrix and promoting vascular pericyte recruitment. Furthermore, increased plasma MMP9 expression levels in human subjects with metastatic non-small cell lung cancer (NSCLC) inversely correlates with survival. In contrast, MMP9 can benefit the host by generating inhibitors of endothelial cell proliferation such as angiostatin and NC1 domains of collagen IV. To better understand the role of host MMP9 on the primary growth and metastatic potential of NSCLC, we performed an orthotopic model of NSLC in integrin α1-null mice (a genetic model for increased MMP9). In these mice we observed decreased number, size and vascularization of primary NSCLC tumors when compared to wild type controls. In addition, decreased number and size of NSCLC-derived metastases were evident in the α1-null mice. Furthermore, pharmacological inhibition of MMPs in the α1-null mice at the time of tumor cell injection resulted in an increase in the number of both primary and metastatic lung cancer as compared to untreated mice, suggesting that primary growth and metastases of NSCLC are worsened by the early inhibition of MMPs. In conclusion, although MMP9 may potentially promote tumor growth and metastasis, production of MMP-dependent anti-angiogenic factors seems to override these effects and protects the host from NSCL growth and progression.

Matrix Metalloproteinases in Cancer

The production of proteinase activity has long been thought to be an essential property of tumor cells that allow them to invade and metastasize to distant sites. The “three step theory of invasion” proposed by Liotta and colleagues (1) suggests that potentially invasive cells must first attach to basement membrane proteins via cell-surface receptors, i.e., the integrins. Localized, extracellular proteolytic activity then clears a path for the cell. Finally, the cell has to move into the cleared region, a locomotive process which probably depends on specific chemotactic factors. This invasion process first occurs as a tumor cell breaches the basement membrane at the primary tumor site—an event which signifies a malignant lesion. In order to result in a growth at a secondary site, the process has to be repeated as tumor cells penetrate blood vessels through a process referred to as intravasation. They can be carried to a new location, where a third invasive event must occur to extravasate into the parenchyma of the distant organ. Thus, proteolysis of basement membrane (BM) and extracellular matrix (ECM) components has been viewed as an essential step in tumor invasion and metastasis. Since metastasis is the principal cause of cancerassociated mortality, the tumor proteases responsible for BM and ECM degradation have been viewed as accessible targets for therapeutic intervention.

Role of transforming growth factor‐β1 and epidermal growth factor in the wound‐healing process: an in vivo biomechanical evaluation

The purpose of this study was to assess and evaluate the role of transforming growth factor‐β1, epidermal growth factor, and their respective carriers (collagen and liposomes) in the early phases of the wound‐healing process in linear incision wounds; we used an in vivo biomechanical testing system. One hundred twenty specific pathogen—free male Sprague‐Dawley rats were divided into five experimental groups (n = 24), including one group receiving no treatment at all. Each animal received one abdominal midline incision. At 3 and 5 days after wounding, 12 animals per group were randomly selected for in vivo biomechanical evaluation. Specimens were also randomly obtained from nondisrupted tissues for histologic analysis. Statistical analysis comparing groups revealed that transforming growth factor‐β1 significantly increased wound strength at day 5, and liposomes decreased wound strength at day 3. There were no other significant differences among groups for each of the time intervals studied. Our results suggest that in vivo biomechanical evaluation of tissue response to injury and treatments will add new dimension to future studies of skin and wound healing.

Abstract 1947: Self-reporting dendritic nanoparticles (nanodendrons) for drug delivery targeted to the tumor microenvironment and with reduced neurotoxicity

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnProteinases, including matrix metalloproteinases (MMPs), contribute to cancer progression and other pathologies. Selective MMP expression can be used to distinguish benign from malignant tumors and identify aggressive tumors associated with poor outcome. MMP9, a basement membrane-degrading type-IV collagenase/gelatinase, is associated with tumor invasion and metastasis. In this project, we describe a new class of dendritic nanoparticles, nanodendrons (NDs), with MMP molecular recognition and targeting capabilities. These NDs can be studied as individual dendrons tuned for specific functions such as enhanced imaging or targeted drug treatments. Additionally, the NDs can be coupled to facilitate multifunctional purposes such as in NDs that can self-report drug delivery to tumors. The prototypical system presented here describes NDs that are activated by MMP9: 1) ND-PB, a near infrared imaging beacon; 2) ND-PXL, a therapeutic that delivers paclitaxel (PXL) and 3) NDPB-NDPXL, a bi-functional agent. In vivo studies in two orthotopic models of breast cancer demonstrate efficacy of these NDs to image and treat breast cancer. The proteinase-activated prodrug, NDPXL, delivers PXL to breast cancer through release of the drug in the tumor microenvironment and increases therapeutic efficacy while reducing systemic toxicity (including peripheral neuropathy). The delivery of PXL using the proteolytically activated ND-PXL is effective in inhibiting tumor growth in two orthotopic models of breast cancer (PyVT-R221A and MDA-MB231). Daily treatment of MDA-MB231 tumors with 12.5 mg/kg PXL as either ND-PXL or Abraxane® (Abx), showed similar reduction in tumor growth as compared with vehicle-treated animals. Further investigation of the NDPXL in a fully immunocompetent mouse model (PyVT-R221A) with treatments given on alternate days at a dose of 12.5 mg/kg (ND-PXL or Abx) yielded similar results: an average reduction in tumor growth of 58% and 53% in ND-PXL and Abx cohorts, respectively. Peripheral nerve toxicity, a debilitating, long term side effect of Abx therapy, was assessed in both tumor and non-tumor mice through monitoring behavior indicative of peripheral nerve damage before, during and after administration of each drug. Peripheral neurotoxicity is markedly reduced in the ND-PXL-treated versus Abraxane®-treated mice as evident in a number of behavioral assessments. The development of this novel class of NDs expands upon the current capabilities of modern proteinase-based optical beacons and prodrugs and is a step forward in treatment of both primary and metastatic cancer. [Supported in part by Susan G. Komen for the Cure®]nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1947. doi:1538-7445.AM2012-1947

Proteinase optical imaging tools for cancer detection and response to therapy

A variety of physiological processes such as wound healing and tissue remodeling are mediated by a plethora of proteinases – enzymes that can hydrolyze peptide bonds – of which as many as 622 have been identified in the human genome. These proteinases are classified into five of the seven clans of peptidases with known catalytic type: S (serine), C (cysteine), A (aspartyl), M (metallo), and T (threonine) [MEROPS, http://merops.sanger.ac.uk; (Rawlings et al. 2008) (Fig. 1)]. In many physiological processes, the proteinases mediate and/or regulate both intercellular signaling, such as in the release and/or processing of chemokines, and intracellular pathways, such as in the apoptotic pathways leading to programmed cell death. Dysregulation of the temporal and/or spatial co-ordination of these intracellular and/or intercellular pathways disrupts the normal physiology and rhythm of life that can be manifest in unregulated growth such as occurs in tumors and their metastatic progeny.