Mechanism of Transformation by the WWTR1/CAMTA1 Fusion Protein in Epithelioid Hemangioendothelioma |
An ESUN Experimental Plan
Introduction
Epithelioid hemangioendothelioma (EHE) is a rare vascular (endothelial cell) sarcoma that can arise in soft tissue, bone, and parenchymal organs such as liver and lung. Endothelial cells are the cells that line blood vessels. Benign endothelial cell neoplasms (hemangiomas) are very common while malignant endothelial cell tumors are very rare. EHE is characterized by a reciprocal t(1;3)(p36;q25) translocation (see box 1).1 EHE may arise as a solitary lesion but also has a tendency to present with multifocal/metastatic involvement, especially when it arises in the liver and lung. 61% of liver EHE develop metastasis (spread to other tissues/organs). Disease-specific mortality has been estimated around 13-18% for EHE arising in soft tissue, while the disease-specific mortality rate for EHE in the lung and liver is 40% and 65% respectively.2 Although localized EHE can be resected, there is no medical treatment for multifocal/metastatic EHE, underscoring the need to develop targeted therapy. EHE is a difficult diagnosis histologically and can be confused with carcinoma and other epithelioid vascular neoplasms. Since as a group, the prognosis of epithelioid vascular tumors vary greatly, from benign lesions such as epithelioid hemangioma without any metastatic potential to very aggressive cancers such as epithelioid angiosarcoma, it is important to classify these lesions precisely. Because EHE is difficult to classify correctly, there is a need for a specific and sensitive biomarker to objectively confirm the diagnosis of EHE.
What is a chromosomal translocation? Chromosomes, are composed of two arms, a short or “p” arm and a long or “q” arm. A translocation occurs when two chromosomes swap arms due to an error in DNA replication or repair (Figure 1). When two chromosomes swap arms there is always the potential for fusion of two genes at the site of chromosome fusion since the chromosome breaks can occur directly in the middle of genes. Disease-defining translocations, such as the t(1;3) translocation which fuses the short arm of chromosome 1 to the long arm of chromosome 3 in EHE, are common in sarcomas. Generally, the fusion genes created at these chromosomal fusion sites are the initiating tumorigenic events that cause these cancers. That is why it is so important to study them. They are the “big bang” that causes the cancers, so understanding what they do to cause cancer is central to coming up with cures.
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Previous Studies by Our Group
Identification of the WWTR1/CAMTA1 gene fusion in EHE
We used an integrative genomic approach which combined cytogenetics (i.e. knowledge of the t(1;3) translocation in EHE) with next generation, deep transcriptomic sequencing (see Box 2) to determine the genes involved in the t(1;3)(p36;q25) translocation. The transcriptome of a single case of EHE known to have the translocation was evaluated. A recently described algorithm, Fusion-Seq,3 was used to analyze the deep sequencing data, which identified a fusion transcript containing WWTR1 (3q25) (WW domain-containing transcription regulator 1) and CAMTA1 (1p36) (calmodulin binding transcription activator 1), two genes present at the exact chromosomal bands involved in the t(1;3) translocation.4 We integrated the karyotype with the sequence data because sequence data generates a lot of “noise”. In our case, there were hundreds of translocations and mutations identified by sequencing and only one of the translocations was the one that we were after. By knowing the exact location of the translocation t(1;3)(p36;q25), we were able to discard all of the other findings and focus on the WWTR1/CAMTA1 gene fusion. This finding has been confirmed by a different research group headed by Dr. Cristina Antonescu of Memorial Sloan Kettering who also identified the WWTR1/CAMTA1 gene fusion in epithelioid hemangioendothelioma by an entirely different research method.5
What is next generation, deep transcriptomic sequencing? Next generation sequencing refers to recently developed technologies that can determine the sequence of a tremendous amount of DNA or RNA. It took several years to originally figure out the DNA sequence of a single individual. With next generation sequencing, this can be done in days. Transcriptomic sequencing means that RNA is sequenced instead of DNA. This is an advantage because RNA encodes only the expressed, functional part of genomic DNA, which comprises only 1-2% of the total genome. Sequencing of RNA provides information only about DNA that has been transcribed into RNA; thus representing a focused sequencing approach. Deep sequencing implies that each piece of RNA is sequenced many times (approximately 50 times) so that nothing is missed.
Validation of WWTR1/CAMTA1 breakpoint and gene fusion at RNA and DNA levels
We validated the presence of the WWTR1/CAMTA1 fusion transcript in several additional EHE cases. The fusion transcripts resulted in fusion of either exon 2 or 3 of WWTR1 to a breakpoint within exon 9 of CAMTA1, giving rise to type 1 or 2 fusion transcripts, respectively. WWTR1 was fused “in-frame” to CAMTA1 in all five tumors, suggesting that a functional protein was being produced. Using RNA in-situ hybridization, we confirmed expression of the WWTR1/CAMTA1 transcript within EHE tumor cells, but not in epithelioid hemangioma. RNA in-situ hybridization also demonstrated that epithelioid hemangioma did not contain expression of CAMTA1, which is an important point below for our proposal to use immunohistochemistry (IHC) for CAMTA1 as a diagnostic aid for EHE.
Development of FISH diagnostic assay and determination of the incidence of the WWTR1/CAMTA 1 fusion gene in EHE and other vascular tumors
Break-apart DNA FISH assays were developed for use in formalin-fixed paraffin embedded (FFPE) tissue using fluorescent-labeled bacterial artificial chromosome (BAC) probes to determine the incidence and specificity of the WWTR1/CAMTA1 gene fusion in 165 vascular tumors including 42 additional EHE cases (see Box 3). We found that WWTR1 and CAMTA1 gene rearrangements were present in 87% and 89% of EHE respectively, indicating that the WWTR1/CAMTA1 gene fusion is sensitive for the diagnosis of EHE. WWTR1 and CAMTA1 gene rearrangements were absent from all other vascular neoplasms (26 different vascular tumor entities/118 total cases), demonstrating the WWTR1/CAMTA1 fusion gene to be a highly specific, disease-defining genetic alteration in EHE.
What is a “break apart” FISH assay? Break apart FISH assays are commonly used in molecular diagnostics to show that a particular gene region is rearranged, indicating that the region is involved in a chromosomal translocation. DNA is a duplex composed of two complementary strands and each strand contains infinite combinations of only four bases, known as adenine (A), cytosine (C), guanine (G) and thymine (T). There are rules about how the bases can pair in opposite strands. A binds to T and C binds to G. So if the sequence of one strand is ATTT, then the opposite strand has to be AAAT due to complementary base pairing. You can imagine that a gene that is thousands of bases long has a very specific sequence. It is this sequence specificity that allows us to make DNA “probes” that bind to only one region of the DNA. In the case of a break apart FISH assay, the probes are several hundred thousand to around a million bases in length in general, generating tremendous specificity and signal. To visualize the probes, they are labeled with fluorescently labeled nucleotides which cause them to glow when visualized under a fluorescent microscope. Probes on both sides of the gene of interest are generated. The probe before the gene (the 5’ probe) is labeled with DNA nucleotides that will fluoresce one color and the probe after the gene (the 3’ probe) is labeled with DNA nucleotides that will fluoresce another color (Figure 2). Typically the probes are labeled DNA nucleotides that will fluoresce red/orange or green. When the gene is not rearranged or broken apart, a single yellow signal (red/orange and green combined) is observed. When the gene is rearranged, a red and a green signal are observed. Each nucleus within the tumor is scored as to whether there is a together or a broken apart signal to determine whether the tumor contains the translocation.
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Structure of the putative WWTR1/CAMTA1 fusion protein
WWTR1 and CAMTA1 are both poorly-studied transcription factors (See box 4). WWTR1 is composed of 7 exons encoding a protein containing a 14-3-3 binding site (exon 2), a WW domain (exons 2-3), a coiled coil domain (exons 4-5), and a PDZ binding motif (exon 7).6 CAMTA1 has 23 exons encoding a protein containing a CG-1 domain (exons 3-7), a TIG domain (exons 9-11), a series of ankyrin repeats (exons 13-15), and calmodulin binding/IQ domains (exons 19-20).7 The protein resulting from the fusion 1 transcript is expected to contain the 14-3-3 binding site and a portion of the WW domain (protein binding domain) from WWTR1 fused to the TIG domain (putative DNA binding domain), ankyrin repeats, and calmodulin binding domains/IQ domains of CAMTA1. The protein resulting from the fusion 2 transcript is essentially the same, but would contain the entire WW domain from WWTR1 (Figure 3).
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Transcription factors are proteins that are critical to cell function because they regulate transcription, the process by which DNA is transcribed into RNA. RNA encodes proteins so transcription factors indirectly control the spectrum of proteins produced by a cell. The spectrum of proteins produced by each cell controls that cells behavior, theoretically dictating whether a cell is “normal” or a cancer cell.
CAMTA1 is not normally expressed in endothelial cells
Using a bioinformatics approach to mine the expression array literature, we found evidence that WWTR1 and CAMTA1 are differentially expressed in endothelial cells. While WWTR1 is expressed strongly, CAMTA1 is not expressed in endothelial cells. Indeed, CAMTA1 appears to be expressed exclusively in the central nervous system.8-10 To validate the bioinformatics data, we performed quantitative real-time PCR on primary endothelial cells, which demonstrated that WWTR1 is expressed at 103 to 106 times higher levels than CAMTA1, depending on which endothelial cells are evaluated (data not shown). Since we performed absolute quantitation, we know that CAMTA1 is expressed at 100 to 100,000 copies per microgram of total RNA, which is towards the lower level of detection of the assay and indicates very poor expression in endothelial cells.
Hypothesis
We hypothesize that the WWTR1-CAMTA1 fusion gene is oncogenic due to a ‘promoter switch mechanism’ whereby the most of the CAMTA1 protein is expressed at much higher levels than normal in the precursor cell that gives rise to EHE, since CAMTA1 expression is placed under the control of an active WWTR1 promoter (Figure 4). Inherent in this hypothesis is that deregulation of CAMTA1 drives oncogenesis. Because CAMTA1 is thought to act as a transcriptional regulator, it is likely that CAMTA1 drives an oncogenic transcriptional program that transforms EHE precursor cells.
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Purpose
The aims of the proposed work are:
- To implement an IHC-based diagnostic assay for the diagnosis of EHE, based on aberrant expression of CAMTA1, and
- To determine the molecular mechanism that causes the WWTR1-CAMTA1 fusion protein to transform EHE precursor cells.
Research Plan and Experimental Design
Development of an Immunohistochemistry-Based Bioassay for the Diagnosis of EHE
Based on our preliminary studies, we believe that CAMTA1 protein expression will be a sensitive and specific bioassay for the diagnosis of EHE. It is useful to develop an IHC-based bioassay for the diagnosis of EHE since many pathology laboratories do not have direct access to molecular diagnostics, while they do have access to IHC. An IHC-based assay for CAMTA1 expression would allow pathologists to confirm the diagnosis of EHE in their own laboratories. To accomplish our first aim, we will screen commercially available CAMTA1 antibodies against EHE in FFPE. If these antibodies fail to detect CAMTA1, we will develop our own monoclonal antibodies for this purpose. The success of this aim relies on the ability to develop antibodies that readily recognize CAMTA1 in FFPE. If commercial antibodies or our own CAMTA1 antibodies can successfully detect CAMTA1 in FFPE, we will screen a comprehensive set of vascular tumors for expression of CAMTA1. Because we identified CAMTA1 gene rearrangements solely in EHE, and because endothelial cells and endothelial cell tumors do not express CAMTA1 or express it at very low levels (as demonstrated above by quantitative RT-PCR on primary endothelial cells and RNA in-situ hybridization), we predict that detection of CAMTA1 protein expression by IHC will be a sensitive and specific biomarker for the diagnosis of EHE. In the event that we are unable to identify or develop diagnostic antibodies, we can still rely on our FISH-based and RT-PCR-based molecular assays that we have already developed and validated.
What is immunohistochemistry? Immunohistochemistry describes a technique where antibodies are used to detect the presence/absence of specific proteins in tissue sections. By using antibodies that are generated to CAMTA1, we can detect whether or not CAMTA1 is present or not in tumor cells. CD31 is an endothelial specific, but not EHE specific protein that is present in EHE. The brown staining indicates that CD31 is present within the tumor cells as is shown in Figure 5.
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Determination of How WWTR1/CAMTA1 Causes EHE
In order to determine the molecular mechanism by which the WWTR1/CAMTA1 fusion protein transforms EHE precursor cells, we will need to develop cell-based models of EHE. EHE cell lines are not currently available; they simply do not exist. Hence, we will need to manufacture them in our laboratory. We will do this by taking a variety of endothelial and other “normal” tissue cell lines and use virally-mediated gene transfer to express the WWTR1-CAMTA1 gene fusion in these cells. After successful expression of WWTR1-CAMTA1, we will need to determine if the behavior of the cells has been altered, indicating that they have at least partially been transformed. To monitor cell behavior, we will analyze our EHE cell models for alterations in the Hallmarks of Cancer (Figure 6), as originally proposed by Hanahan and Weinberg in 201011 and more recently updated in 2011.12 The Hallmarks of Cancer, a landmark cancer publication, organized various activities of cancer cells into thematic groups to understand cancer behavior. The groups are: evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis and resisting cell death (Figure 6). These activities are all hallmark characteristics of cancer cells. To investigate whether our EHE cell line models have acquired any of these hallmarks, we will perform the following assays: We will examine them for alterations in senescence (enabling replicative immortality), proliferation (sustaining proliferative signaling), resistance to apoptosis (resisting cell death), growth factor independence (sustaining proliferative signaling), anchorage independent growth (evading growth suppressors, sustaining proliferative signaling, activating invasion and metastasis) and cell migration (activating invasion and metastasis). Once we have determined whether any of these properties have been altered, this will give us a sense of what WWTR1/CAMTA1 is doing to transform EHE precursor cells. For instance, if the cells exhibit increased proliferation and inhibition of senescence and proliferation, then we will conclude that the WWTR1/CAMTA1 fusion protein transforms EHE precursors by activating proliferation and inhibiting senescence and cell death.
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Because WWTR1/CAMTA1 is most likely a transcriptional activator, it is unlikely that this protein functions directly to transform EHE precursor cells. It is much more likely that this protein transforms these cells by activating the expression of (“turning on”) a group of genes/proteins that directly transform EHE precursor cells. To determine the list of genes that are regulated by WWTR1/CAMTA1, we will use gene expression microarray chips, which can simultaneously measure the levels of each gene in the human genome and report on variations in gene levels. Once we have determined the list of genes that are controlled by WWTR1/CAMTA1, we can narrow down the list of candidate cancer causing/transforming genes by integrating that list with lists of genes that fall into behavior categories identified in our hallmarks of cancer experiments (Fig. 6). This integrative analysis where we use one set of experiments to filter the results of another set of experiments is a powerful method to limit the number of genes/proteins identified for further study, and allows us to focus only on the most important candidate genes/proteins.
At the end of these experiments we hope to have the following deliverables: 1. An IHC-based bioassay for the diagnosis of EHE. 2. A proposed mechanism for how EHE transforms EHE precursor cells including a list of genes that are involved in this process.
Impact and Clinical Relevance
The development of an easy, sensitive, and specific bioassay for the diagnosis of EHE will facilitate accurate diagnosis of EHE. Correct diagnosis is the first step towards obtaining the correct therapy and disease management. Identification of the mechanism of transformation, including the relevant genes/proteins that play important roles in EHE tumorigenesis is a first step towards identifying pharmacological/medical therapies. This is especially important for a cancer like EHE where multifocal disease and metastasis are common, limiting surgical options.
Editor's Note: This study is funded by a $50,000 grant from the Liddy Shriver Sarcoma Initiative.
References
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