Sandra Jensen-Taubman

Matrix Metalloproteinases: Changing Roles in Tumor Progression and Metastasis

Articles on tumor invasion, metastasis, and angiogenesis in normal and disease states have been well represented among the pages of The American Journal of Pathology. In addition to exciting interest in a variety of disease processes, these studies have been central in defining the emerging field in cancer research known as the tumor microenvironment. Early studies in this field established the importance of the extracellular matrix on tumor cell growth and differentiation. With time, the role of the extracellular matrix and matrix metalloproteinases in the regulation of tumor invasion, metastasis, and angiogenesis was recognized, and AJP has published seminal articles in this field. Moreover, recent studies show evidence for a role of matrix metalloproteinases in the regulation of inflammation within tumor lesions, making the targeting of matrix metalloproteinases in cancer therapy even more complex. This review attempts to summarize the contribution of AJP to some of the key changes that have led to the evolution of this field.

TIMP-2 targets tumor-associated myeloid suppressor cells with effects in cancer immune dysfunction and angiogenesis.

Angiogenesis and inflammation are important therapeutic targets in non–small cell lung cancer (NSCLC). It is well known that proteolysis mediated by matrix metalloproteinases (MMPs) promotes angiogenesis and inflammation in the tumor microenvironment. Here, the effects of the MMP inhibitor TIMP-2 on NSCLC inflammation and angiogenesis were evaluated in TIMP-2-deficient (timp2−/−) mice injected subcutaneously (SC) with Lewis lung carcinoma cells and compared with the effects on tumors in wild-type mice. TIMP-2-deficient mice demonstrated increased tumor growth, enhanced expression of angiogenic marker &agr;v&bgr;3 in tumor and endothelial cells, and significantly higher serum vascular endothelial growth factor-A levels. Tumor-bearing timp2−/− mice showed a significant number of inflammatory cells in their tumors, upregulation of inflammation mediators, nuclear factor-kappaB, and Annexin A1, as well as higher levels of serum interleukin (IL)-6. Phenotypic analysis revealed an increase in myeloid-derived suppressor cell (MDSC) cells (CD11b+ and Gr-1+) that coexpressed vascular-endothelial-growth factor receptor 1 (VEGF-R1) and elevated MMP activation present in tumors and spleens from timp2−/− mice. Furthermore, TIMP-2-deficient tumors upregulated expression of the immunosuppressing genes controlling MDSC growth, IL-10, IL-13, IL-11, and chemokine ligand (CCL-5/RANTES), and decreased interferon-&ggr; and increased CD40L. Moreover, forced TIMP-2 expression in human lung adenocarcinoma A-549 resulted in a significant reduction of MDSCs recruited into tumors, as well as suppression of angiogenesis and tumor growth. The increase in MDSCs has been linked to cancer immunosuppression and angiogenesis. Therefore, this study supports TIMP-2 as a negative regulator of MDSCs with important implications for the immunotherapy and/or antiangiogenic treatment of NSCLC.

Endogenous Angiogenesis Inhibitor Blocks Tumor Growth via Direct and Indirect Effects on Tumor Microenvironment

Tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) belongs to a small family of endogenous proteins that inhibits a group of enzymes, the matrix metalloproteinases (MMPs). TIMP-2 inhibits endothelial cell proliferation and migration in vitro and angiogenesis in vivo, through MMP-dependent and -independent mechanisms. However, little is known regarding the contribution of these mechanisms to the antitumor effects of TIMP-2. Using a retroviral delivery system, we stably overexpressed TIMP-2 and its mutant Ala+TIMP-2 (devoid of MMP inhibitory activity) in human adenocarcinoma A549 cells. Using real time PCR, and enzyme-linked immunosorbent assay (ELISA), we confirmed enhanced TIMP-2 expression and its MMP inhibitory activity by reverse zymography. In vitro, growth assays suggested that TIMP-2 and Ala+TIMP-2 did not alter basal cell proliferation rates, however, tumor cell migration and invasion were inhibited. In vivo, both TIMP-2 and Ala+TIMP-2 A549 xenografts exhibited reduced growth rate, CD31 immunostaining indicated decreased intratumoral microvascular density, and TUNEL demonstrated enhanced tumor cell apoptosis. Immunoblotting and immunohistochemical analyses of A549 xenograft tissues with either phospho-FAK (Tyr397) or phospho-AKT (Ser473) showed decreased activation in both TIMP-2 and Ala+TIMP-2 tumor cells. We conclude that TIMP-2-mediated inhibition of tumor growth occurs, at least in part, independently of MMP inhibition, and is a consequence of both direct effects of TIMP-2 on tumor cells and modulation of the tumor microenvironment.

Achaete-scute homolog-1 linked to remodeling and preneoplasia of pulmonary epithelium.

The basic helix–loop–helix protein achaete-scute homolog-1 (ASH1) is involved in lung neuroendocrine (NE) differentiation and tumor promotion in SV40 transgenic mice. Constitutive expression of human ASH-1 (hASH1) in mouse lung results in hyperplasia and remodeling that mimics bronchiolization of alveoli (BOA), a potentially premalignant lesion of human lung carcinomas. We now show that this is due to sustained cellular proliferation in terminal bronchioles and resistance to apoptosis. Throughout the airway epithelium the expression of anti-apoptotic Bcl-2 and c-Myb was increased and Akt/mTOR pathway activated. Moreover, the expression of matrix metalloproteases (MMPs) including MMP7 was specifically enhanced at the bronchiolo-alveolar duct junction and BOA suggesting that MMPs play a key role in this microenvironment during remodeling. We also detected MMP7 in 70% of human BOA lesions. Knockdown of hASH1 gene in human lung cancer cells in vitro suppressed growth by increasing apoptosis. We also show that forced expression of hASH1 in immortalized human bronchial epithelial cells decreases apoptosis. We conclude that the impact of hASH1 is not limited to cells with NE phenotype. Rather, constitutive expression of hASH1 in lung epithelium promotes remodeling through multiple pathways that are commonly activated during lung carcinogenesis. The collective results suggest a novel model of BOA formation via hASH1-induced suppression of the apoptotic pathway. Our study yields a promising new preclinical tool for chemoprevention of peripheral lung carcinomas.

Bronchiolization of the alveoli in lung cancer: Pathology, patterns of differentiation and oncogene expression

We examined the incidence and association of bronchiolization of the alveoli with non‐small cell lung cancer in lung resection specimens from 2 patient groups: those with non‐small cell lung cancer and those diagnosed with a variety of non‐neoplastic lung conditions. We observed marked variation in bronchiolization of the alveoli morphology ranging from normal to severely atypical and developed a classification scheme based on growth pattern, cell number and cytologic criteria. Patterns of differentiation, proliferation and growth factor receptor and oncogene expression were studied using immuno‐histochemical and in situ hybridization techniques. While low‐grade (0–I) bronchiolization of the alveoli lesions demonstrated markers similar to normal bronchiolar epithelium, a significant decrease in the Clara cell 10 kDa protein and tubulin and an increase in surfactant protein‐A expression were observed in high‐grade (II–III) lesions. Focal p53 expression was detected in 2 high‐grade lesions, while c‐ myc mRNA and cJun protein were observed in all grades. No correlation was observed between bronchiolization of the alveoli incidence and histologic tumor type. A comparison of marker expression in lesions and tumors from the same case revealed a negative correlation between cytokeratin‐14 and c‐erbB‐2 immuno‐reactivity. Only one bronchialization of the alveoli lesion was found in the non‐neoplastic patient group. We conclude that up to 12% of non‐small cell lung cancer resection specimens contain bronchiolization of the alveoli lesions which exhibit altered morphology and patterns of differentiation. Int. J. Cancer 75:489–496, 1998. Published 1998 Wiley‐Liss, Inc.

Novel Method for Isolation of Murine Clara Cell Secretory Protein-Expressing Cells with Traces of Stemness

Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP+ cells. Moreover, CCSP+ cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP−expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP+ cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

Combined inhibition of Wee1 and Hsp90 activates intrinsic apoptosis in cancer cells

Heat shock protein 90 (Hsp90) is an essential, evolutionarily conserved molecular chaperone. Cancer cells rely on Hsp90 to chaperone mutated and/or activated oncoproteins, and its involvement in numerous signaling pathways makes it an attractive target for drug development. Surprisingly, however, the impact of Hsp90 inhibitors on cancer cells is frequently cytostatic in nature, and efforts to enhance the antitumor activity of Hsp90 inhibitors in the clinic remain a significant challenge. In agreement with previous data obtained using Wee1 siRNA, we show that dual pharmacologic inhibition of Wee1 tyrosine kinase and Hsp90 causes cancer cells to undergo apoptosis in vitro and in vivo. Gene expression profiling revealed that induction of the intrinsic apoptotic pathway by this drug combination coincided with transcriptional downregulation of Survivin and Wee1, an outcome not seen in cells treated separately with either agent. At the translational level, expression of these two proteins, as well as activated Akt, was completely abrogated. These data support the hypothesis that Wee1 inhibition sensitizes cancer cells to Hsp90 inhibitors; they establish combined Wee1/Hsp90 inhibition as a novel therapeutic strategy; and they provide a mechanistic rationale for enhancing the pro-apoptotic activity of Hsp90 inhibitors.

Molecular mechanisms of tissue inhibitor of metalloproteinase 2 in the tumor microenvironment

There has been a recent paradigm shift in the way we target cancer, drawing a greater focus on the role of the tumor microenvironment (TME) in cancer development, progression and metastasis. Within the TME, there is a crosstalk in signaling and communication between the malignant cells and the surrounding extracellular matrix. Matrix metalloproteinases (MMPs) are zinc-dependent endoproteases that have the ability to degrade the matrix surrounding a tumor and mediate tumor growth, angiogenesis and metastatic disease. Their endogenous inhibitors, the Tissue Inhibitors of Metalloproteinases (TIMPs), primarily function to prevent degradation of the ECM via inhibition of MMPs. However, recent studies demonstrate that TIMP family members also possess MMP-independent functions. One TIMP member in particular, TIMP-2, has many distinct properties and functions, that occur independent of MMP inhibition, including the inhibition of tumor growth and reduction of angiogenesis through decreased endothelial cell proliferation and migration. The MMP-independent molecular mechanisms and signaling pathways elicited by TIMP-2 in the TME are described in this review.

Matrilysin-1 Mediates Bronchiolization of Alveoli, a Potential Premalignant Change in Lung Cancer

Matrilysin-1 (also called matrix metalloproteinase-7) is expressed in injured lung and in cancer but not in normal epithelia. Bronchiolization of the alveoli (BOA), a potential precursor of lung cancer, is a histologically distinct type of metaplasia that is composed of cells resembling airway epithelium in the alveolar compartment. We demonstrate that there is increased expression of matrilysin-1 in human lesions and BOA in the CC10-human achaete-scute homolog-1 transgenic mouse model. Forced expression of the matrilysin-1 gene in immortalized human normal airway epithelial BEAS-2B and HPLD1 cells, which do not normally express matrilysin-1, promoted cellular migration, suggesting a functional link for BOA formation via bronchiolar cell migration. In addition, matrilysin-1 stimulated proliferation and inhibited Fas-induced apoptosis, while a knockdown by RNA interference decreased cell growth, migration, and increased sensitivity to apoptosis. Western blotting demonstrated increased levels of phospho-p38 and phospho-Erk1/2 kinases after matrilysin-1 expression. Gene expression analysis uncovered several genes that were related to cell growth, migration/movement, and death, which could potentially facilitate bronchiolization. In vivo, the formation of BOA lesions was reduced when CC10-human achaete-scute homolog-1 mice were crossed with matrilysin-1 null mice and was correlated with reduced matrilysin-1 expression in BOA. We conclude that matrilysin-1 may play an important role in the bronchiolization of alveoli by promoting proliferation, migration, and attenuation of apoptosis involving multiple genes in the MAP kinase pathway.

Achaete-Scute Homologue-1 Tapers Neuroendocrine Cell Differentiation in Lungs after Exposure to Naphthalene

The basic helix-loop-helix transcription factor achaete-scute homologue-1 (ASH1) plays a critical role in regulating the neuroendocrine (NE) phenotype in normal and neoplastic lung. Transgenic (TG) mice that constitutively express human ASH1 (hASH1) under control of the Clara cell 10-kDa protein (CC10) promoter in non-NE airway lining cells display progressive epithelial hyperplasia and bronchiolar metaplasia or bronchiolization of the alveoli (BOA). However, little is known about the involvement of hASH1 in regeneration of the conducting airway. In this study, we investigated the impact of hASH1 on airway cell injury and repair in the TG mice following an intraperitoneal injection of naphthalene, which specifically ablates bronchiolar Clara cells and induces pulmonary NE cell hyperplasia. We discovered an overall attenuation of NE maturation coupled with increased proliferation in TG mice during post-naphthalene repair. In addition, BOA lesions revealed enhanced epithelial cell proliferation while preserving Clara cell markers CC10 and the principal naphthalene-metabolizing enzyme cytochrome P4502F2. These data suggest that ASH1 may play an important role in maintaining a progenitor phenotype that promotes renewal of both NE and epithelial cells. Moreover, ASH1 may propagate a stem cell microenvironment in BOA where epithelium becomes resistant to naphthalene toxicity.