Sustained expression of Mect1-Maml2 is essential for tumor cell growth in salivary gland cancers carrying the t(11;19) translocation.

Mucoepidermoid (MEC) salivary gland tumors arise from a t(11;19) rearrangement which generates a fusion oncogene, Mect1-Maml2, that functions to activate CREB-responsive target genes. To determine if sustained expression of Mect1-Maml2 is required for tumor cell growth, we first showed that ectopic expression of Mect1-Maml2 in rat epithelial RK3E cells is tumorigenic in vivo in nude mice and that excised xenografts continue to express the fusion oncogene. We then generated a hairpin RNAi vector that selectively suppressed the fusion peptide and showed that ectopic expression in either parotid or pulmonary MEC tumor cell lines containing the t(11;19) rearrangement resulted in at least 90% colony growth inhibition. In contrast, single nucleotide changes within this RNAi sequence abolished the ability to suppress Mect1-Maml2 protein and abolished all growth inhibition of these MEC tumor lines. In addition, the RNAi-specific vector had no effect on colony growth of non-MEC tumors including a lung tumor or two other salivary gland cell lines that do not express Mect1-Maml2. We also generated a mutant Mect1-Maml2 expression plasmid that carried silent nucleotide changes within the RNAi target sequence and observed that co-transfection of this mutant, but not wild-type Mect1-Maml2, could partially rescue RNAi growth inhibition in the MEC tumor line. The recent detection of acquired fusion oncogenes in epithelial solid tumors has suggested new possibilities for the diagnosis and therapy of these cancers. Our data show that the 'gain-of-function' activity from aberrant Mect1-Maml2 expression is a candidate therapeutic target for this group of malignant salivary gland tumors.

Protein expression of the RB-related gene family and SV40 large T antigen in mesothelioma and lung cancer.

Mutational inactivation of the RB-related gene RBL2/p130 has been reported as a common and important prognostic factor in human lung cancer. To examine the role of the RB-related gene family in lung cancer we analysed the protein expression of the RB gene in cell lines obtained from 83 patients with small cell lung cancer (SCLC) and 114 patients with non-SCLC that included 21 novel lung tumor samples. While we detected five new SCLC with mutant RB expression (RB inactivation in 75/83; 90.4%), we did not detect any RB mutations in the new non-SCLC cell lines (RB inactivation in 13/114 non-SCLC and mesothelioma; 11.4%). In addition, we detected expression of a full-length RBL1/p107 and RBL2/p130 species in every sample tested (RBL1 or RBL2 inactivation in 0/69) and confirmed that both RB-related gene products retain functional binding activity to the E1A viral oncoprotein. Since expression of SV40 Large T antigen (Tag) has been reported in a subset of human lung tumors where it may inactivate RBL1 and RBL2, we also examined mesothelioma and non-mesothelioma lung tumors for Tag expression. Although we detected a faint 85 kDa protein species using specific anti-Tag antibodies, this signal migrated slightly faster than Tag extracted from Cos7 cells and did not exhibit binding activity to the RB or RBL1 proteins. Finally, we subjected 11 lung cancer cell lines to nucleotide sequencing and did not detect mutations within the C-terminal RBL2 exons 19-22 as recently reported. While the RB/p16 tumor suppressor pathway is targeted for mutations in 100% of lung cancers, mutational inactivation of the related RBL1 and RBL2 genes is a rare event.

Temperature-sensitive RB mutations linked to incomplete penetrance of familial retinoblastoma in 12 families.

The tumor-suppressor activity of the retinoblastoma protein (RB) is encoded within a protein-binding ("pocket") domain that is targeted for mutations in all cases of familial retinoblastoma and in many common adult cancers. Although familial retinoblastoma is a paradigm for a highly penetrant, recessive model of tumorigenesis, the molecular basis for the phenotype of incomplete penetrance of familial retinoblastoma is undefined. We studied the RB pocket-binding properties of three independent, mutant RB alleles that are present in the germline of 12 kindreds with the phenotype of incomplete penetrance of familial retinoblastoma. Each arises from alterations of single codons within the RB pocket domain (designated "delta 480," "661W," or "712R"). Under the same conditions, we studied the properties of wild-type (WT) RB, an RB point mutant isolated from a lung carcinoma sample (706F) and an adjacent, in vitro-generated point mutant (707W). The delta 480, 661W, and 712R mutants lack pocket protein-binding activity in vitro but retain the WT ability to undergo cyclin-mediated phosphorylation in vivo. Each of the low-penetrant RB mutants exhibits marked enhancement of pocket protein binding when the cells are grown at reduced temperature. In contrast, in this temperature range, no change in binding activity is seen with WT RB, the 706F mutant, or the 707W mutant. We have demonstrated that many families with incomplete penetrance of familial retinoblastoma carry unstable, mutant RB alleles with temperature-sensitive pocket protein-binding activity. The variable frequency for tumor development in these families may result from reversible fluctuations in a threshold level of RB pocket-binding activity.

RET cooperates with RB/p53 inactivation in a somatic multi-step model for murine thyroid cancer.

Mice bred to carry germline Rb and p53 null alleles are associated with a tumor spectrum that overlaps with the inherited multiple endocrine neoplasia-1 (MEN1) and MEN2 syndromes in humans, including medullary thyroid cancer (MTC). To study the genetic basis for these tumors, we microdissected MTC specimens or obtained fresh MTC tissue from nine independent Rb(+/-) p53(+/-) mice, amplified the region of the Ret gene known to be mutated in human MTC, and detected acquired missense Ret mutations in four different mice. These mutations were localized to a group of tandem cysteines which are analogous to activating germline mutations observed in human MEN2A and familial MTC (FMTC). To determine whether the remaining wild type Rb allele was inactivated in these murine MTC samples, we subjected tumor tissue to immunohistochemical staining with an Rb antibody, and demonstrated the absence of RB staining in murine MTC, while normal tissue retained RB nuclear staining. These findings demonstrate the ability of the gene knockout model to recapitulate somatic multi-step tumorigenesis and suggest that the development of a murine neuroendocrine tumor requires mutational dysregulation within both receptor tyrosine kinase and nuclear tumor suppressor gene pathways.

Incomplete penetrance of familial retinoblastoma linked to germ-line mutations that result in partial loss of RB function.

To study the molecular basis for the clinical phenotype of incomplete penetrance of familial retinoblastoma, we have examined the functional properties of three RB mutations identified in the germ line of five different families with low penetrance. RB mutants isolated from common adult cancers and from classic familial retinoblastoma (designated as classic RB mutations) are unstable and generally do not localize to the nucleus, do not undergo cyclin-dependent kinase (cdk)-mediated hyperphosphorylation, show absent protein "pocket" binding activity, and do not suppress colony growth of RB(-) cells. In contrast, two low-penetrant alleles (661W and "deletion of codon 480") retained the ability to localize to the nucleus, showed normal cdk-mediated hyperphosphorylation in vivo, exhibited a binding pattern to simian virus 40 large T antigen using a quantitative yeast two-hybrid assay that was intermediate between classic mutants (null) and wild-type RB, and had absent E2F1 binding in vitro. A third, low-penetrant allele, "deletion of RB exon 4," showed minimal hyperphosphorylation in vivo but demonstrated detectable E2F1 binding in vitro. In addition, each low-penetrant RB mutant retained the ability to suppress colony growth of RB(-) tumor cells. These findings suggest two categories of mutant, low-penetrant RB alleles. Class 1 alleles correspond to promoter mutations, which are believed to result in reduced or deregulated levels of wild-type RB protein, whereas class 2 alleles result in mutant proteins that retain partial activity. Characterization of the different subtypes of class 2 low-penetrant genes may help to define more precisely functional domains within the RB product required for tumor suppression.

Apoptosis is associated with cleavage of a 5 kDa fragment from RB which mimics dephosphorylation and modulates E2F binding.

Dephosphorylation of the RB protein has been reported to be associated with apoptosis. In contrast, we show that treatment of HL60 cells with etoposide or cytosine arabinoside or treatment of breast epithelial cells with alpha-FAS is associated with the cleavage of a 5 kDa fragment from the C-terminus of RB, resulting in a truncated product that we have designated as p100cl. This cleavage event coincides with the activation of cysteine proteases at the onset of apoptosis, is blocked by the addition of iodoacetamide to cells prior to the onset of apoptosis, and results in the expression of faster migrating protein species which can mimic dephosphorylated RB. The free 5 kDa fragment is detected only during apoptosis, predicts a cleavage site that we have mapped to a unique CPP32-like recognition sequence which is present at the C-terminus of all reported RB homologues, and results in a truncated RB protein with enhanced E2F binding affinity. While the causality for this cleavage event in the apoptotic process is still under investigation, our findings suggest distinct post-translational pathways for the RB product between cells examined during growth arrest (p105 hypophosphorylated RB) or apoptosis (p100cl).

CDKN2 gene silencing in lung cancer by DNA hypermethylation and kinetics of p16INK4 protein induction by 5-aza 2'deoxycytidine.

Absent expression of the cyclin dependent kinase-inhibitor, p16INK4, is observed in a wide range of primary human cancers. Although homozygous deletions and point mutations have been reported in a subset of these tumors, the molecular basis for absent p16INK4 in other samples is unknown. We have examined 33 tumor cell lines and have shown that hypermethylation of a G:C-rich region within exon 1 of the CDKN2 gene was present in 100% of samples with wildtype RB expression and no detectable CDKN2 mutations. Treatment for at least 4 hours with the demethylating agent 5-aza 2'deoxycytidine, but not 5-azacytidine or 6-azacytidine, induces the prolonged expression of p16INK4 protein in each of these samples following a discrete 24-48 hour lag period. Consistent with the hypothesis that hypermethylation of the CDKN2 gene is a tumor-specific mechanism for gene inactivation, we observed hypomethylation at the exon 1 site exclusively in tumor lines that expressed p16INK4 or that had sustained inactivating point mutations within the CDKN2 open reading frame. These findings demonstrate a link between DNA methylation and the p16INK4:RB tumor suppressor pathway.

Differential specificity for binding of retinoblastoma binding protein 2 to RB, p107, and TATA-binding protein.

The growth suppressor activities of the RB and p107 products are believed to be mediated by the reversible binding of a heterogeneous family of cellular proteins to a conserved T/E1A pocket domain that is present within both proteins. To study the functional role of these interactions, we examined the properties of cellular retinoblastoma binding protein 2 (RBP2) binding to RB, p107, and the related TATA-binding protein (TBP) product. We observed that although RBP2 bound exclusively to the T/E1A pocket of p107, it could interact with RB through independent T/E1A and non-T/E1A domains and with TBP only through the non-T/E1A domain. Consistent with this observation, we found that a mutation within the Leu-X-Cys-X-Glu motif of RBP2 resulted in loss of ability to precipitate p107, while RB- and TBP-binding activities were retained. We located the non-T/E1A binding site of RBP2 on a 15-kDa fragment that is independent from the Leu-X-Cys-X-Glu motif and encodes binding activity for RB and TBP but does not interact with p107. Despite the presence of a non-T/E1A binding site, however, recombinant RBP2 retained the ability to preferentially precipitate active hypophosphorylated RB from whole-cell lysates. In addition, we found that cotransfection of RBP2 can reverse in vivo RB-mediated suppression of E2F activity. These findings confirm the differential binding specificities of the related RB, p107, and TBP proteins and support the presence of multifunctional domains on the nuclear RBP2 product which may allow complex interactions with the cellular transcription machinery.

Partial inactivation of the RB product in a family with incomplete penetrance of familial retinoblastoma and benign retinal tumors.

While familial retinoblastoma has served as the paradigm for the two-hit theory of tumorigenesis and for the concept of the tumor suppressor gene, the etiology of incomplete penetrance of familial retinoblastoma is poorly understood. To address the molecular basis for this phenotype we have studied the functional properties of a mutant Rb gene identified in a kindred with incomplete penetrance of familial retinoblastoma and evidence for regressed retinal lesions (retinomas). In contrast to all previously isolated RB mutant proteins, we demonstrated that the mutant product from this kindred retained the wildtype properties of nuclear localization, the ability to undergo hyperphosphorylation in vivo, and the capacity to suppress growth of RB(-) cells. Protein binding ('pocket') activity, however, was defective defining a new class of RB mutant with partial inactivation. The presence of this unique RB mutant in the germline of obligate carriers with incomplete penetrance and regressed retinal lesions suggests a molecular basis for this phenotype and supports the hypothesis that a minimum 'RB threshold' level of protein binding activity is required to suppress tumorigenesis.