Amy Coxon

t(11;19)(q21;p13) translocation in mucoepidermoid carcinoma creates a novel fusion product that disrupts a Notch signaling pathway

Truncation of Notch1 has been shown to cause a subtype of acute leukemia, and activation of Notch4 has been associated with mammary and salivary gland carcinomas of mice. Here we identify a new mechanism for disrupting Notch signaling in human tumorigenesis, characterized by altered function of a new ortholog of the Drosophila melanogaster Notch co-activator molecule Mastermind. We cloned the t(11;19) translocation that underlies the most common type of human malignant salivary gland tumor. This rearrangement fuses exon 1 from a novel gene of unknown function at 19p13, termed mucoepidermoid carcinoma translocated 1 (MECT1), with exons 2–5 of a novel member of the Mastermind-like gene family (MAML2) at 11q21 (ref. 3). Similar to D. melanogaster Mastermind and MAML1 (refs. 4,5), full-length MAML2 functioned as a CSL (CBF-1, suppressor of hairless and Lag-1)-dependent transcriptional co-activator for ligand-stimulated Notch. In contrast, MECT1–MAML2 activated transcription of the Notch target gene HES1 independently of both Notch ligand and CSL binding sites. MECT1–MAML2 induced foci formation in RK3E epithelial cells, confirming a biological effect for the fusion product. These data suggest a new mechanism to disrupt the function of a Notch co-activator in a common type of malignant salivary gland tumor.

Mect1-Maml2 Fusion Oncogene Linked to the Aberrant Activation of Cyclic AMP/CREB Regulated Genes

Malignant salivary gland tumors can arise from a t(11;19) translocation that fuses 42 residues from Mect1/Torc1, a cyclic AMP (cAMP)/cAMP-responsive element binding protein (CREB)-dependent transcriptional coactivator, with 982 residues from Maml2, a NOTCH receptor coactivator. To determine if the Mect1-Maml2 fusion oncogene mediates tumorigenicity by disrupting cAMP/CREB signaling, we have generated in-frame deletions within the CREB-binding domain of Mect1/Torc1 for testing transformation activity and have also developed a doxycycline-regulated Mect1-Maml2 mammalian expression vector for global gene expression profiling. We observed that small deletions within the CREB-binding domain completely abolished transforming activity in RK3E epithelial cells. Further, we have shown that the ectopic induction of Mect1-Maml2 in HeLa cells strongly activated the expression of a group of known cAMP/CREB-regulated genes. In addition, we detected candidate cAMP-responsive element sites within 100 nucleotides of the transcriptional start sites of other genes activated by Mect1-Maml2 expression. In contrast, we did not observe alterations of known Notch-regulated target genes in these expression array profile experiments. We validated the results by reverse transcription-PCR in transfected HeLa, RK3E, and H2009 lung tumor cells and in mucoepidermoid cancer cells that endogenously express the fusion oncopeptide. Whereas overexpression of components of the cAMP pathway has been associated with a subset of human carcinomas, these data provide a direct genetic link between deregulation of cAMP/CREB pathways and epithelial tumorigenesis and suggest future therapeutic strategies for this group of salivary gland tumors.

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 α-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).

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.

Stch encodes the 'ATPase core' of a microsomal stress 70 protein.

The stress70 protein chaperone family plays a central role in the processing of cytosolic and secretory proteins. We have cloned a human cDNA, designated Stch, that is conserved in rat tissues and which encodes a novel microsome‐associated member of the stress70 protein chaperone family. Stch mRNA is constitutively expressed in all human cell types and is induced by incubation with the calcium ionophore A23187, but not by exposure to heat shock. Inspection of the predicted amino acid sequence reveals that the STCH product contains a unique hydrophobic leader sequence and shares homology within the amino terminal domains of the stress70 gene family, but has a 50 residue insertion within the ATP‐binding domains and truncates the carboxyl terminal peptide‐binding region. Immunofluorescent and subcellular analyses show that STCH migrates predominantly as a 60 kDa species and is enriched in a membrane‐bound microsome fraction. In contrast to purified BiP and dnaK, however, STCH demonstrates ATPase activity that is independent of peptide stimulation. Stch, therefore, encodes a calcium‐inducible, microsome‐associated ATPase activity with properties similar to a proteolytically cleaved N‐terminal HSC70/BiP fragment. This truncated stress70 molecule may allow increased diversity in cellular responses to protein processing requirements.

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.

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.

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.

Enhanced activity of the CREB co-activator Crtc1 in LKB1 null lung cancer

Activation of Crtc1 (also known as Mect1/Torc1) by a t(11;19) chromosomal rearrangement underlies the etiology of malignant salivary gland tumors. As LKB1 is a target for mutational inactivation in lung cancer and was recently shown to regulate hepatic Crtc2/CREB transcriptional activity in mice, we now present evidence suggesting disruption of an LKB1/Crtc pathway in cancer. Although Crtc1 is preferentially expressed in adult brain tissues, we observed elevated levels of steady-state Crtc1 in thoracic tumors. In addition, we show that somatic loss of LKB1 is associated with underphosphorylation of endogenous Crtc1, enhanced Crtc1 nuclear localization and enhanced expression of the Crtc prototypic target gene, NR4A2/Nurr1. Inhibition of NR4A2 was associated with growth suppression of LKB1 null tumors, but showed little effect on LKB1-wildtype cells. These data strengthen the role of dysregulated Crtc as a bona fide cancer gene, present a new element to the complex LKB1 tumorigenic axis, and suggest that Crtc genes may be aberrantly activated in a wider range of common adult malignancies.

Assessment of Cancer Cell Line Representativeness Using Microarrays for Merkel Cell Carcinoma

When using cell lines to study cancer, phenotypic similarity to the original tumor is paramount. Yet, little has been done to characterize how closely Merkel cell carcinoma (MCC) cell lines model native tumors. To determine their similarity to MCC tumor samples, we characterized MCC cell lines via gene expression microarrays. Using whole transcriptome gene expression signatures and a computational bioinformatic approach, we identified significant differences between variant cell lines (UISO, MCC13, and MCC26) and fresh frozen MCC tumors. Conversely, the classic WaGa and Mkl-1 cell lines more closely represented the global transcriptome of MCC tumors. When compared to publicly available cancer lines, WaGa and Mkl-1 cells were similar to other neuroendocrine tumors, but the variant cell lines were not. WaGa and Mkl-1 cells grown as xenografts in mice had histological and immunophenotypical features consistent with MCC, while UISO xenograft tumors were atypical for MCC. Spectral karyotyping and short tandem repeat analysis of the UISO cells matched the original cell line’s description, ruling out contamination. Our results validate the use of transcriptome analysis to assess the cancer cell line representativeness and indicate that UISO, MCC13, and MCC26 cell lines are not representative of MCC tumors, whereas WaGa and Mkl-1 more closely model MCC.