Peroxisome Proliferator-Activated Receptor g and Induction of Differentiation in Non-small Cell Lung Cancer
Peroxisome Proliferator-Activated Receptor g and Induction of Differentiation in Non-small Cell Lung Cancer
By Tsg-Hui Chang, and Eva Szabo, MD
Despite recent advances in our understanding of the pathobiology of cancer, as well as the introduction of multiple new chemotherapeutic agents for the clinical treatment of cancer, mortality rates from common epithelial malignancies presenting with local invasion or metastasis essentially remain unchanged. The recognition of cancer as a disease characterized by excessive cell proliferation has led to the utilization of cytotoxic drugs as the primary treatment modalities for disseminated malignancies, although such drugs frequently exhibit substantial toxicities to a wide spectrum of normal tissues as well. However, in addition to being a disorder of the balance between cell proliferation and cell death, cancer is also a disorder of cellular differentiation. Thus, novel approaches to the treatment and prevention of cancer that focus on re-instituting the terminal differentiation program that is abrogated during carcinogenesis are now receiving renewed attention.
Recent Findings
One such recently identified approach involves the induction of differentiation in cancer cells through ligand activation of nuclear hormone receptors such as the peroxisome proliferator-activated receptor g (PPARg). PPARg, a member of a receptor family that also includes PPARa and PPARb/d, is a key regulator of adipocytic differentiation and has an important role in fat metabolism.1 More relevant to solid tumor biology, PPARg has recently been demonstrated to be involved in the induction of growth arrest, differentiation, and apoptosis in multiple human cancer cell lines, including breast, colon, and non-small cell lung cancer (NSCLC) cell types.2-5 In the presence of specific ligands, heterodimers of PPARg and its obligate dimerization partner, retinoid X receptor a (RXRa), bind DNA in a sequence specific manner and regulate transcription of target genes. Forced expression and activation of PPARg in nonadipogenic fibroblasts and myocytes has been shown to lead to the development of an adipogenic phenotype.6,7 Recently, specific ligands that preferentially activate PPARg have been identified, including selected prostaglandins and arachidonic acid metabolites as well as a new class of antidiabetic drugs, the thiazolidinediones.8,9 Availability of these ligands has been instrumental in uncovering the complex biologic functions of PPARg.
Although initially felt to be fat-specific, PPARg expression is not limited to cells of the adipocytic lineage. Detectable levels of PPARg have been reported in multiple tissues such as the heart, liver, large intestine, kidney, breast, colon, lung, ovary, and placenta.2-4,8 Furthermore, PPARg appears to have additional functions in these diverse cell types. For instance, high expression of PPARg has been described in activated macrophages, where ligand activation down regulates inducible nitric oxide synthase and matrix metalloproteinase 9 (MMP-9) production and thereby curbs the inflammatory response.10,11 PPARg activation has also been implicated in atherogenesis by the recent findings that the scavenger receptor CD36 is a PPARg-regulated gene, that oxidized LDL is a naturally occurring ligand, and that receptor activation contributes to monocyte differentiation into foam cells.12,13 On the other hand, ligand activation in vascular smooth muscle cells has been shown to inhibit inducible MMP-9 production and cell migration, thereby suggesting an anti-atherogenic role as well.14 Thus, PPARg appears to have a complex role in a variety of homeostatic mechanisms in diverse cell types.
As a potential target for cancer therapy, treatment of liposarcoma cell lines with the thiazolidinedione class of PPARg ligands results in induction of the mature adipocytic phenotype with terminal withdrawal from the cell cycle.15 The findings that these ligands can also induce growth arrest and molecular changes associated with differentiation in human breast and colon cancer cells suggest that PPARg ligands may induce differentiation of other cell types. Given that in the lung, PPARg expression has been detected in type II pneumocytes that serve as progenitor cells for the pulmonary alveolar epithelium after injury or during carcinogenesis, we examined the expression of PPARg and the effect of ligand activation in NSCLC.3 Our results, as outlined below, indicate that not only is PPARg expressed in NSCLC cell lines and primary tumors, but treatment of NSCLC cells with PPARg ligands also induces growth arrest and changes associated with differentiation.5
Potential Significance of PPARg to NSCLC Differentiation
To determine the potential significance of PPARg to NSCLC differentiation, we first examined the expression of PPARg and the effect of two structurally unrelated PPARg ligands, ciglitizone (a thiazolidinedione) and the prostanoid 15-deoxy- D12,14-prostaglandin J2 (15d-PGJ2), on a panel of 10 well-characterized NSCLC cell lines with varying histology. Protein expression of PPARg and its obligate dimerization partner, RXRa, was found in all cell lines examined, as well as in normal and immortalized human pulmonary epithelial cells. Similarly, immunohistochemical analysis of primary tumors from patients with NSCLC showed positive nuclear staining in approximately 50% of the tumors examined. Treatment of multiple NSCLC cell lines with both PPARg ligands resulted in growth arrest and irreversible loss of capacity for anchorage-independent growth, the latter being a hallmark of the neoplastic phenotype.
Whereas much is known about PPARg and its role in adipocytic differentiation in part because of the identification of well established differentiation markers that can be used to follow the maturation process, the pulmonary epithelium represents a much more complex and challenging system. The lung is composed of multiple epithelial cell lineages with differing proliferative potentials that are characterized by expression of distinct differentiation markers. As a result, no single marker that is pathognomonic of the terminally differentiated state in all lung epithelial cells has been established thus far. Therefore, to determine whether growth arrest induced by PPARg ligands is associated with cellular differentiation, we utilized a panel of "general" differentiation markers that are associated with growth arrest and differentiation in a variety of differentiation model systems (gelsolin, Mad, and p21), as well as "lineage specific" differentiation markers that are associated with specific lung cell lineages (MUC1, CC10, and HTI56). Our data show that PPARg ligand treatment of NSCLC cell lines resulted in modulation of these markers in a manner consistent with differentiation.
Gelsolin, an actin regulatory protein, is a general differentiation marker that is up-regulated in leukemic and epithelial cell lines during in vitro differentiation induced by agents such as phorbol esters and histone deacetylase inhibitors. As compared with histologically normal surrounding lung, lower levels of gelsolin expression has been found in lung cancers.16 Mad is a member of the Myc family of interacting proteins that has been shown to be up-regulated during leukemic and keratinocyte differentiation. p21 (Waf1) is a cyclin-dependent kinase inhibitor that is up-regulated after the in vitro induction of differentiation in leukemic and neuroblastoma cell lines. In our study, treatment of NSCLC cells with PPARg ligands resulted in significantly increased expression of all three of these general differentiation markers: gelsolin, Mad, and p21.
Treatment with PPARg ligands
Treatment with PPARg ligands also resulted in modulation of lineage specific differentiation markers (HTI56, MUC1, and CC10) in a manner consistent with differentiation. In the peripheral lung, the alveolar type II pneumocyte and the bronchiolar Clara cell are the major progenitor cells that repopulate the epithelium after injury or during carcinogenesis. Type II pneumocytes (but not their terminally differentiated counterparts, the type I pneumocytes) express MUC1, which is a transmembrane mucin that is felt to facilitate carcinogenic progression through modulation of cell adhesion and the immune system. Clara cells are characterized by the expression of CC10, a differentiation marker specific to this progenitor cell type that is frequently lost during carcinogenesis.
Treatment of NSCLC cells with PPARg ligands resulted in the down regulation of both MUC1 and CC10 in those cell lines that expressed these markers, suggesting differentiation away from the type II pneumocyte and Clara cell progenitor cell lineages. Simultaneously, the expression of HTI56, a recently described integral membrane protein thus far found exclusively in terminally differentiated type I pneumocytes, was induced by PPARg ligands in NSCLC (but not ovarian cancer) cell lines. Taken in the context of growth arrest, these data indicate a maturation and loss of the progenitor cell phenotype upon treatment with PPARg ligands, with acquisition of a differentiated type I pneumocyte phenotype.
Treatment with PPARg ligands also modulated the neoplastic phenotype in NSCLC in additional ways. De-regulation of cell cycle control protein frequently occurs during carcinogenesis in a variety of cell types. In NSCLC, cyclin D1 is frequently overexpressed while p16, which inhibits the cyclin D1/cdk4 kinase complex, is frequently inactivated by a variety of means. This results in the hyperphosphorylation of the retinoblastoma (Rb) protein, allowing for continuous transit through the cell cycle. Although not pathognomonic of differentiation, inhibition of cyclin D1-associated kinase activity and hypophoshorylation of Rb have been linked to differentiation. Consistent with a more mature, less malignant phenotype, PPARg ligands inhibited the expression of cyclin D1 and led to hypophosphorylation of the retinoblastoma protein in our study.
In a similar fashion, although not specifically associated with differentiation, metalloproteinases, which are a family of zinc-dependent proteases involved in the degradation of the extracellular matrix, have been implicated in tumor invasiveness and metastasis. In lung tumor tissues, the activated form of matrix metalloproteinase-2 (MMP-2) has been shown to be significantly associated with tumor spread. Results from our study indicated that ciglitizone treatment resulted in a significant reduction in the activated form as well as the levels of MMP-2 protein expressed.
Thus, taken together, results from our study showed that treatment of NSCLC cells lines with PPARg ligands leads to growth arrest with inhibition of cyclin D1, irreversible loss of capacity for anchorage-independent growth, decreased activity and expression of a metalloproteinase associated with metastasis, and modulation of multiple protein markers in a manner consistent with maturation and differentiation. These data indicate a potential role for PPARg ligands in the therapy of NSCLC.
Conclusion
Because of the frequent toxic side effects and limited efficacy of conventional chemotherapy, the search for new agents with novel mechanisms of action is of paramount importance in the field of cancer prevention and treatment. Results from our study showed that in an in vitro setting, PPARg ligands promote differentiation and reversal of the transformed phenotype in NSCLC cells, suggesting that PPARg ligands may have clinical utility in the treatment of NSCLC. In an in vivo setting, animal studies have shown that xenograft tumor growth is significantly diminished by PPARg ligand treatment.2,4 Furthermore, evidence of differentiation in biopsied tissues from human beings treated with the PPARg ligand troglitazone for liposarcomas has recently been documented, although whether this will translate to clinical remission remains to be determined.17 The recent observation that combined treatment with conventional chemotherapeutic agents and a differentiation inducer in colon cancer cell lines results in enhanced differentiation and growth inhibition suggests that alternative approaches of combining traditional chemotherapy with differentiation induction also deserve further study.18
Finally, prevention of breast cancer in a rat carcinogenesis model using the novel PPARg ligand GW7845 provides further evidence that PPARg ligands may have even greater utility during earlier phases of carcinogenesis, in addition to control of later stage metastatic disease.19 Given that PPARg is an important target for treating type 2 diabetes and multiple thiazolidinedione ligands are already in clinical use, further investigations of these ligands and the role of PPARg in the treatment and prevention of NSCLC (as well as other epithelial malignancies) are clearly warranted. (Dr. Szabo is Program Director, Lung & Upper Aerodigestive Cancer Research Group, Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Bethesda, MD; and Tsg-Hui Chang is Biologist, Molecular Physiology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD.)
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