Stanislawa Weremowicz

Integrative Genomic Approaches Identify IKBKE as a Breast Cancer Oncogene

The karyotypic chaos exhibited by human epithelial cancers complicates efforts to identify mutations critical for malignant transformation. Here we integrate complementary genomic approaches to identify human oncogenes. We show that activation of the ERK and phosphatidylinositol 3-kinase (PI3K) signaling pathways cooperate to transform human cells. Using a library of activated kinases, we identify several kinases that replace PI3K signaling and render cells tumorigenic. Whole genome structural analyses reveal that one of these kinases, IKBKE (IKKepsilon), is amplified and overexpressed in breast cancer cell lines and patient-derived tumors. Suppression of IKKepsilon expression in breast cancer cell lines that harbor IKBKE amplifications induces cell death. IKKepsilon activates the nuclear factor-kappaB (NF-kappaB) pathway in both cell lines and breast cancers. These observations suggest a mechanism for NF-kappaB activation in breast cancer, implicate the NF-kappaB pathway as a downstream mediator of PI3K, and provide a framework for integrated genomic approaches in oncogene discovery.

A novel ALK secondary mutation and EGFR signaling cause resistance to ALK kinase inhibitors

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKI), including crizotinib, are effective treatments in preclinical models and in cancer patients with ALK-translocated cancers. However, their efficacy will ultimately be limited by the development of acquired drug resistance. Here we report two mechanisms of ALK TKI resistance identified from a crizotinib-treated non-small cell lung cancer (NSCLC) patient and in a cell line generated from the resistant tumor (DFCI076) as well as from studying a resistant version of the ALK TKI (TAE684)-sensitive H3122 cell line. The crizotinib-resistant DFCI076 cell line harbored a unique L1152R ALK secondary mutation and was also resistant to the structurally unrelated ALK TKI TAE684. Although the DFCI076 cell line was still partially dependent on ALK for survival, it also contained concurrent coactivation of epidermal growth factor receptor (EGFR) signaling. In contrast, the TAE684-resistant (TR3) H3122 cell line did not contain an ALK secondary mutation but instead harbored coactivation of EGFR signaling. Dual inhibition of both ALK and EGFR was the most effective therapeutic strategy for the DFCI076 and H3122 TR3 cell lines. We further identified a subset (3/50; 6%) of treatment naive NSCLC patients with ALK rearrangements that also had concurrent EGFR activating mutations. Our studies identify resistance mechanisms to ALK TKIs mediated by both ALK and by a bypass signaling pathway mediated by EGFR. These mechanisms can occur independently, or in the same cancer, suggesting that the combination of both ALK and EGFR inhibitors may represent an effective therapy for these subsets of NSCLC patients.

Disruption of the architectural factor HMGI-C: DNA-binding AT hook motifs fused in lipomas to distinct transcriptional regulatory domains

Lipomas are one of the most common mesenchymal neoplasms in humans. They are characterized by consistent cytogenetic aberrations involving chromosome 12 in bands q14-15. Interestingly, this region is also the site of rearrangement for other mesenchymally derived tumors. This study demonstrates that HMGI-C, an architectural factor that functions in transcriptional regulation, has been disrupted by rearrangement at the 12q14-15 chromosomal breakpoint in lipomas. Chimeric transcripts were isolated from two lipomas in which HMGI-C DNA-binding domains (AT hook motifs) are fused to either a LIM or an acidic transactivation domain. These results, identifying a gene rearranged in a benign neoplastic process that does not proceed to a malignancy, suggest a role for HMGI-C in adipogenesis and mesenchyme differentiation.

Regulation of In Situ to Invasive Breast Carcinoma Transition

The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma is a poorly understood key event in breast tumor progression. Here, we analyzed the role of myoepithelial cells and fibroblasts in the progression of in situ carcinomas using a model of human DCIS and primary breast tumors. Progression to invasion was promoted by fibroblasts and inhibited by normal myoepithelial cells. Molecular profiles of isolated luminal epithelial and myoepithelial cells identified an intricate interaction network involving TGFbeta, Hedgehog, cell adhesion, and p63 required for myoepithelial cell differentiation, the elimination of which resulted in loss of myoepithelial cells and progression to invasion.

Mutations in the protein kinase A R1α regulatory subunit cause familial cardiac myxomas and Carney complex

Cardiac myxomas are benign mesenchymal tumors that can present as components of the human autosomal dominant disorder Carney complex. Syndromic cardiac myxomas are associated with spotty pigmentation of the skin and endocrinopathy. Our linkage analysis mapped a Carney complex gene defect to chromosome 17q24. We now demonstrate that the PRKAR1alpha gene encoding the R1alpha regulatory subunit of cAMP-dependent protein kinase A (PKA) maps to this chromosome 17q24 locus. Furthermore, we show that PRKAR1alpha frameshift mutations in three unrelated families result in haploinsufficiency of R1alpha and cause Carney complex. We did not detect any truncated R1alpha protein encoded by mutant PRKAR1alpha. Although cardiac tumorigenesis may require a second somatic mutation, DNA and protein analyses of an atrial myxoma resected from a Carney complex patient with a PRKAR1alpha deletion revealed that the myxoma cells retain both the wild-type and the mutant PRKAR1alpha alleles and that wild-type R1alpha protein is stably expressed. However, in this atrial myxoma, we did observe a reversal of the ratio of R1alpha to R2beta regulatory subunit protein, which may contribute to tumorigenesis. Further investigation will elucidate the cell-specific effects of PRKAR1alpha haploinsufficiency on PKA activity and the role of PKA in cardiac growth and differentiation.

Functional and molecular characterization of the human neutral solute channel aquaporin-9

In metabolically active cells, the coordinated transport of water and solutes is important for maintaining osmotic homeostasis. We recently identified a broad selective-neutral solute channel, AQP9, from rat liver that allows the passage of a wide variety of water and neutral solutes (H. Tsukaguchi, C. Shayakul, U. V. Berger, B. Mackenzie, S. Devidas, W. B. Guggino, A. N. van Hoek, and M. A. Hediger. J. Biol. Chem. 273: 24737-24743, 1998). A human homolog (hAQP9) with 76% amino acid sequence identity to rat AQP9 (rAQP9) was described, but its permeability was found to be restricted to water and urea (K. Ishibashi, M. Kuwahara, Y. Gu, Y. Tanaka, F. Marumo, and S. Sasaki. Biochem. Biophys. Res. Commun. 244: 268-274, 1998). Here we report a reevaluation of the functional characteristics of hAQP9, its tissue distribution, the structure of its gene, and its chromosomal localization. When expressed in Xenopus oocytes, hAQP9 allowed passage of a wide variety of noncharged solutes, including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercurial-sensitive manner. These functional characteristics are similar to those of rAQP9. Based on Northern blot analysis, both rat and human AQP9 are abundantly expressed in liver, whereas, in contrast to rAQP9, hAQP9 is also expressed in peripheral leukocytes and in tissues that accumulate leukocytes, such as lung, spleen, and bone marrow. The human AQP9 gene is composed of 6 exons and 5 introns distributed over approximately approximately 25 kb. The gene organization is strikingly similar to that reported for human AQP3 and AQP7, suggesting their evolution from a common ancestral gene. The promoter region contains putative tonicity and glucocorticoid-responsive elements, suggesting that AQP9 may be regulated by osmolality and catabolism. Fluorescence in situ hybridization assigned its locus to chromosome 15 q22.1-22.2. Our data show that hAQP9 serves as a promiscuous solute channel expressed in both liver and peripheral leukocytes, where it is ideally suited to transport of metabolites and/or nutrients into and out of these cellsIn metabolically active cells, the coordinated transport of water and solutes is important for maintaining osmotic homeostasis. We recently identified a broad selective-neutral solute channel, AQP9, from rat liver that allows the passage of a wide variety of water and neutral solutes (H. Tsukaguchi, C. Shayakul, U. V. Berger, B. Mackenzie, S. Devidas, W. B. Guggino, A. N. van Hoek, and M. A. Hediger. J. Biol. Chem. 273: 24737-24743, 1998). A human homolog (hAQP9) with 76% amino acid sequence identity to rat AQP9 (rAQP9) was described, but its permeability was found to be restricted to water and urea (K. Ishibashi, M. Kuwahara, Y. Gu, Y. Tanaka, F. Marumo, and S. Sasaki. Biochem. Biophys. Res. Commun. 244: 268-274, 1998). Here we report a reevaluation of the functional characteristics of hAQP9, its tissue distribution, the structure of its gene, and its chromosomal localization. When expressed in Xenopus oocytes, hAQP9 allowed passage of a wide variety of noncharged solutes, including carbamides, polyols, purines, and pyrimidines in a phloretin- and mercurial-sensitive manner. These functional characteristics are similar to those of rAQP9. Based on Northern blot analysis, both rat and human AQP9 are abundantly expressed in liver, whereas, in contrast to rAQP9, hAQP9 is also expressed in peripheral leukocytes and in tissues that accumulate leukocytes, such as lung, spleen, and bone marrow. The human AQP9 gene is composed of 6 exons and 5 introns distributed over approximately ∼25 kb. The gene organization is strikingly similar to that reported for human AQP3 and AQP7, suggesting their evolution from a common ancestral gene. The promoter region contains putative tonicity and glucocorticoid-responsive elements, suggesting that AQP9 may be regulated by osmolality and catabolism. Fluorescence in situ hybridization assigned its locus to chromosome 15 q22.1-22.2. Our data show that hAQP9 serves as a promiscuous solute channel expressed in both liver and peripheral leukocytes, where it is ideally suited to transport of metabolites and/or nutrients into and out of these cells.

USP6 (Tre2) Fusion Oncogenes in Aneurysmal Bone Cyst

Aneurysmal bone cyst (ABC) is a locally aggressive osseous lesion that typically occurs during the first two decades of life. ABC was regarded historically as a nonneoplastic process, but recent cytogenetic data have shown clonal rearrangements of chromosomal bands 16q22 and 17p13, indicating a neoplastic basis in at least some ABCs. Herein we show that a recurring ABC chromosomal translocation t(16;17)(q22;p13) creates a fusion gene in which the osteoblast cadherin 11 gene (CDH11) promoter region on 16q22 is juxtaposed to the entire ubiquitin-specific protease USP6 (Tre2) coding sequence on 17p13. CDH11-USP6 fusion transcripts were demonstrated only in ABC with t(16;17) but other ABCs had CDH11 or USP6 rearrangements resulting from alternate cytogenetic mechanisms. CDH11 is expressed strongly in bone, and our findings implicate a novel oncogenic mechanism in which deregulated USP6 transcription results from juxtaposition to the highly active CDH11 promoter.

Discrimination of complete hydatidiform mole from its mimics by immunohistochemistry of the paternally imprinted gene product p57KIP2.

The p57 KIP2 protein is a cell cycle inhibitor and tumor suppressor encoded by a strongly paternally imprinted gene. We explored the utility of p57 KIP2 as a diagnostic marker in hydatidiform mole, a disease likely the result of abnormal dosage and consequent misexpression of imprinted genes. Using a monoclonal antibody on paraffin-embedded, formalin-fixed tissue sections, the authors evaluated p57 KIP2 expression in normal placenta and in 149 gestations including 59 complete hydatidiform moles, 39 PHMs, and 51 spontaneous losses with hydropic changes. p57 KIP2 was strongly expressed in cytotrophoblast and villous mesenchyme in normal placenta, all cases of partial hydatidiform moles (39 of 39) and all spontaneous losses with hydropic changes (51 of 51). In contrast, p57 KIP2 expression in cytotrophoblast and villous mesenchyme was absent or markedly decreased in 58 of 59 complete hydatidiform moles. In all gestations p57 KIP2 was strongly expressed in decidua and in intervillous trophoblast islands, which served as internal positive controls for p57 KIP2 immunostaining. p57 KIP2 immunohistochemistry can reliably identify most cases of complete hydatidiform mole irrespective of gestational age and is thus a useful diagnostic adjunct, complementary to ploidy analysis, in the diagnosis of hydatidiform mole.

Combined cDNA array comparative genomic hybridization and serial analysis of gene expression analysis of breast tumor progression

To identify genetic changes involved in the progression of breast carcinoma, we did cDNA array comparative genomic hybridization (CGH) on a panel of breast tumors, including 10 ductal carcinoma in situ (DCIS), 18 invasive breast carcinomas, and two lymph node metastases. We identified 49 minimal commonly amplified regions (MCRs) that included known (1q, 8q24, 11q13, 17q21-q23, and 20q13) and several uncharacterized (12p13 and 16p13) regional copy number gains. With the exception of the 17q21 (ERBB2) amplicon, the overall frequency of copy number alterations was higher in invasive tumors than that in DCIS, with several of them present only in invasive cancer. Amplification of candidate loci was confirmed by quantitative PCR in breast carcinomas and cell lines. To identify putative targets of amplicons, we developed a method combining array CGH and serial analysis of gene expression (SAGE) data to correlate copy number and expression levels for each gene within MCRs. Using this approach, we were able to distinguish a few candidate targets from a set of coamplified genes. Analysis of the 12p13-p12 amplicon identified four putative targets: TEL/ETV6, H2AFJ, EPS8, and KRAS2. The amplification of all four candidates was confirmed by quantitative PCR and fluorescence in situ hybridization, but only H2AFJ and EPS8 were overexpressed in breast tumors with 12p13 amplification compared with a panel of normal mammary epithelial cells. These results show the power of combined array CGH and SAGE analysis for the identification of candidate amplicon targets and identify H2AFJ and EPS8 as novel putative oncogenes in breast cancer.

Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner.

The human globin locus control region-binding protein, NF-E2, was purified by DNA affinity chromatography. Its tissue-specific component, p45 NF-E2, was cloned by use of a low-stringency library screen with murine p45 NF-E2 cDNA (N. C. Andrews, H. Erdjument-Bromage, M. B. Davidson, P. Tempst, and S. H. Orkin, Nature [London] 362:722-728, 1993). The human p45 NF-E2 gene was localized to chromosome 12q13 by fluorescent in situ hybridization. Human p45 NF-E2 and murine p45 NF-E2 are highly homologous basic region-leucine zipper (bZIP) proteins with identical DNA-binding domains. Immunoprecipitation experiments demonstrated that p45 NF-E2 is associated in vivo with an 18-kDa protein (p18). Because bZIP proteins bind DNA as dimers, we infer that native NF-E2 must be a heterodimer of 45- and 18-kDa subunits. Although AP-1 and CREB copurified with NF-E2, no evidence was found for heterodimer formation between p45 NF-E2 and proteins other than p18. Thus, p18 appears to be the sole specific partner of p45 NF-E2 in erythroid cells. Cloning of human p45 NF-E2 should permit studies of the role of NF-E2 in globin gene regulation and erythroid differentiation.