Loss of the Tumor Suppressor PML in Human Cancers of Multiple Histologic Origins

Carmela Gurrieri 0 Paola Capodieci 0 Rosa Bernardi 0 Pier Paolo Scaglioni 0 Khedoudja Nafa 0 Laura J. Rush 0 David A. Verbel 0 Carlos Cordon-Cardo 0 Pier Paolo Pandolfi ) 0 0 Affiliations of authors: Molecular Biology Program and Department of Pa- thology (CG, RB, PPP), Department of Pathology (PC, CCC), Molecular Biol- ogy Program and Departments of Pathology and Medicine (PPS), Department of Medicine (KN), Department of Epidemiology and Biostatistics (DAV), Memo- rial Sloan-Kettering Cancer Center, Sloan-Kettering Division, Graduate School of Medical Sciences, Cornell University , New York , NY; Department of Vet- erinary Biosciences, The Ohio State University , Columbus , OH (LJR). gram and Department of Pathology, Sloan-Kettering Institute, Memorial Sloan- Kettering Cancer Center , Box 110, 1275 York Ave., New York, NY 10021 ( Background: The PML gene is fused to the RAR gene in the vast majority of acute promyelocytic leukemias (APL) and has been implicated in the control of key tumor-suppressive pathways. However, its role in the pathogenesis of human cancers other than APL is still unclear. We therefore assessed the status and expression of the PML gene in solid tumors of multiple histologic origins. Methods: We created tumor tissue microarrays (TTMs) with samples from patients with colon adenocarcinoma (n 109), lung carcinoma (n 19), prostate adenocarcinoma (n 36), breast carcinoma (n 38), central nervous system (CNS) tumors (n 51), germ cell tumors (n 60), thyroid carcinoma (n 32), adrenal cortical carcinoma (n 12), and non-Hodgkin's lymphoma (n 251) and from normal tissue corresponding to each histotype and analyzed PML protein and mRNA expression by immunohistochemistry and in situ hybridization, respectively. Tumor cell lines (n 64) of various histologic origins were analyzed for PML protein and mRNA expression by immunofluorescence and northern blotting, respectively. DNA from microdissected tumor samples and cell lines was analyzed for PML mutations and loss of heterozygosity (LOH). For some tumor types, the association between PML expression and tumor stage and grade was analyzed. Statistical tests were two-sided. Results: All normal tissues expressed PML protein. PML protein expression was reduced or abolished in prostate adenocarcinomas (63% [95% confidence interval {CI} 48% to 78%] and 28% [95% CI 13% to 43%], respectively), colon adenocarcinomas (31% [95% CI 22% to 40%] and 17% [95% CI 10% to 24%]), breast carcinomas (21% [95% CI 8% to 34%] and 31% [95% CI 16% to 46%]), lung carcinomas (36% [95% CI 15% to 57%] and 21% [95% 3% to 39%]), lymphomas (14% [95% CI 10% to 18%] and 69% [95% CI 63% to 75%]), CNS tumors (24% [95% CI 13% to 35%] and 49% [95% CI 36% to 62%]), and germ cell tumors (36% [95% CI 24% to 48%] and 48% [95% CI 36% to 60%]) but not in thyroid or adrenal carcinomas. Loss of PML protein expression was associated with tumor progression in prostate cancer (the progression from prostatic intraepithelial neoplasia to invasive carcinoma was associated with complete PML loss; P<.001), breast cancer (complete PML loss was associated with lymph node metastasis; P .01), and CNS tumors (complete PML loss was associated with high-grade tumors ; P .003). PML mRNA was expressed in all tumor and cell line samples. The PML gene was rarely mutated and was not subject to LOH. Conclusions: PML protein expression is frequently lost in human cancers of various histologic origins, and its loss associates with tumor grade and progression in some tumor histotypes. [J Natl Cancer Inst 2004;96:269 -79] - PML (formerly known as Myl) has become the object of intense research due to its involvement in the pathogenesis of acute promyelocytic leukemia (APL) (1). In the vast majority of APL patients, the PML gene (on chromosome 15) is fused to the retinoic acid receptor alpha (RAR) gene (on chromosome 17) as a consequence of reciprocal and balanced chromosomal translocation resulting in the production of a PMLRAR fusion protein. PML encodes a founding member of a growing family of proteins that all contain a distinctive C3HC4 zinc-binding domain termed RING finger. Some members of this family (e.g., BRCA1) have also been implicated in tumor suppression and control of genomic stability (2). PML is typically found in multiprotein speckled subnuclear structures termed PML nuclear bodies (2,3). To date, more than 50 proteins have been reported to co-localize with PML in the nuclear body, either transiently or constitutively, including p53, pRb, Daxx, and CBP (2,3). In APL blast cells, PMLRAR causes the de-localization of PML into microspeckled nuclear structures and the consequent disruption of the PML nuclear bodies. In Pml/ primary cells, nuclear body components acquire an aberrant nuclear localization pattern that can be restored to normal when PML is added back (4,5). Thus, PML is essential for the formation and stability of the nuclear body. This conclusion implies, in turn, that PML may regulate the nuclear body associated functions of multiple nuclear body components and that these functions may be impaired in APL blasts or in cells lacking PML function. In vivo analyses have underscored the importance of the functional disruption of PML and the nuclear body in tumorigenesis. For instance, when expressed in the promyelocytic/ myeloid compartment of transgenic mice, PMLRAR caused leukemia with APL-like features (6,7). Moreover, the progressive reduction of the dose of PML obtained by crossing PML RAR transgenic mice with Pml/ mice resulted in a dramatic increase in the incidence of leukemia and in an acceleration of leukemia onset (7,8). Finally, Pml/ mice are highly susceptible to developing tumors in several in vivo models of physically or chemically induced carcinogenesis (9,10). Until recently, little was known about the biologic and biochemical roles of PML in tumorigenesis and tumor progression. It is now becoming apparent that PML and the PML nuclear body are essential for critical tumor-suppressive pathways (9,1115). PML acts as a p53 transcriptional coactivator and is required for p53-dependent induction of apoptosis and cellular senescence upon exposure to ionizing radiations and oncogenic transformation (1215). Moreover, Pml/ mice and cells are protected from multiple caspase-dependent apoptotic stimuli, such as Fas, tumor necrosis factor, ceramide, and interferon (9). PML is also required for transcriptional repression mediated by the tumor suppressors Mad and Rb (16,17). Thus, at the cellular level, PML controls, at least in part as a result of its localization in the nuclear body, functionssuch as induction of apoptosis, growth suppression, and cellular senescence in response to oncogenic transformation and DNA damagethat are essential for tumor suppression (11). However, it remains to be determined whether PML loss is a critical event in human tumorigenesis. We conducted a comprehensive analysis of the status of the PML gene, including its mRNA (...truncated)