Investigating CIC. A gene associated with the development of Brain Cancer.

The New Year has seen me undertake a small research project as part of my master’s degree. The topic in question, or rather the gene in question, is a transcription factor called CIC. This gene has been associated with the development of a particular brain cancer known as Oligodendroglioma. Patients with this cancer do not survive and those that are diagnosed die within 2-15years[1]. A noteworthy 77% of Oligodendrogliomas have been shown to harbour a CIC mutation[2,3,4]. Some mutations can result in the perturbation of a gene’s functionality. If the disruption of a gene has the potential to contribute to cancer, the gene will fall into one of two categories. Oncogenes are determined when mutation leads to a gain of function in the encoded protein, where the newly acquired function aids cancer development [5]. A good example would be the Myc gene where mutations can cause irreversible activation of the protein. Activated Myc results in increased cellular proliferation; a key step in tumourigenesis [6]. The other type is referred to as a tumour suppressor (TS). Knudson’s two hit hypothesis was established, through the study of retinoblastoma genetics, to help define a TS. If a mutation causes a loss of function and the cellular consequences promote cancer development. The gene is regarded as a TS.  In order to completely lose protein functionality, two genetic events (hits) are required to ensure both alleles are affected [7].

The first recurrent genetic aberration found in ODG was the codeletion of the short arm of chromosome 1 and long arm of chromosome 19 (1p19q codeletion). This is seen in nearly two thirds of Oligodendroglomas and is regarded as a defining feature for this brain cancer [1]. Interestingly the CIC gene is located on the same arm of chromosome 19 that is lost during the codeletion. As both 1p19q codeletions and CIC mutations are so common to Oligodendroglioma, it has been speculated that CIC might be a TS [8]. It is easy to see how CIC could meet the two hit hypothesis in such tumours. The loss of an entire CIC allele via the 1p19q codeletion would act as the first hit. If the mutation in CIC’s remaining allele is deleterious then this would suffice a second hit (Figure1). But does a loss of CIC function actually contribute to cancer development? This is a difficult question to answer because of a lack of understanding in CIC’s conventional cellular functions. Without a clear understanding of how something works normally, you cannot fully understand the ramifications of when it goes wrong.

2 hit hypothesis

Figure 1: Knudson’s two-hit hypothesis applied to the CIC gene in 1p19 codeleted ODGs. The purple and green chromosomes correspond to chromosomes 1 and 19 respectively. Chromosome arms p and q are indicated with white lettering. The yellow rectangles represent wild type CIC alleles. The first allele is lost (1st Hit) due to an imbalanced reciprocal translocation between chromosomes 1 and 19. The second allele is disrupted (2nd Hit) by a somatic mutation capable of inhibiting protein function. This mutation is indicated by a red cross. With no functional CIC alleles remaining the gene is effectively lost and tumourigenesis can begin (Figure by Luke Webster).

The majority of what we do know about CIC has been established through the extrapolation of previous work on the Drosophila orthologue; Capicua. The primary function of Capicua is to act as a transcriptional repressor, turning off target genes. This repressor activity is fundamental to the anteroposterior patterning and wing vein differentiation of Drosophila development [9]. For Capicua to function as a transcriptional repressor the presence of co-repressor Groucho is required. CIC has also been shown to possess a transcriptional repressor activity in humans with the ability to target a group of genes called PEA3 [10, 11]. Furthermore, interaction with other proteins can heighten and encourage CIC’s repressor activity, much like Drosophila and Groucho. There are two domains that are vital for Capicua’s functionality. The first is a common DNA binding domain called the HMG box. This allows Capicua to interact with its target genes. The HMG box is highly conserved in mammalian CIC and actually facilitated CIC’s discovery during a neuronal development study [12]. The other is a C-terminal domain associated with the transcriptional repressor activity of Capicua. Within this domain are two conserved motifs. The first (C1) is thought to be the interface between Capicua and Groucho [13]. The second motif (C2) allows the inhibition of Capicua’s repressor activity in response to signalling cascades that phosphorylate C2. Human CIC shares this trait with Capicua as its repressor activity is also blocked in response to cell signalling dependent phosphorylation [11].

So of what interest is all of this to us? After assessing all the known recorded CIC mutations found in Oligodendrogliomas there are two regions within which nearly all missense mutations reside [2, 3,4,8,14]. These correspond to the two known functional domains; the HMG box and the C-terminal protein interaction domain (CPID). Furthermore protein structure simulation software predicted that these mutations were “probably damaging” to protein function [8]. This is all very encouraging as the identified functional domains do appear key to CIC’s cellular roles, and these domains are being disrupted in Oligodendrogliomas. If CIC is a TS then the loss of function that promotes the development of cancer is likely to be sourced from these recurrently mutated domains.

Previous work from my lab has shown that mammalian CIC can also interact with Groucho and suggests that such an interaction induces a repressor activity in CIC. The thesis goes on to provide some evidence that the CIC/Groucho interaction is in the N-terminal region[15]. This is rather contrasting to the Capicua literature. I wanted to assess whether CIC’s CPID could facilitate a Groucho interaction as the homology with Capicua suggests. I also wanted to see whether the CPID mutations seen in Oligodendroglioma could prevent a CIC Groucho interaction. My running hypothesis is that CIC is a TS and mutations in the CPID prevent a CIC Groucho interaction. Unable to bind its co-repressor, CIC loses the ability to repress target genes. The resulting cellular consequences encourage the formation of Oligodendroglioma.

To test some of my ideas I designed a nuclear translocation assay. CIC contains a nuclear localisation site (NLS) and is therefore believed to favour a nuclear residence within the cell. This is coherent with the idea that CIC is a transcription factor. Most forms of Groucho also contain an NLS however Groucho5 does not. This implies Groucho5 has a cytoplasmic subcellular localisation. I took a GFP fused Groucho5 (Grg5-GFP) and transfected it into COS7 cells. Fluorescent microscopy allowed the visualisation Grg5-GFP within the COS7 cells. Groucho5 did indeed reside in the cytoplasm. Next I co-transfected the Grg5-GFP along with CIC. This time the green fluorescence was predominantly nuclear, implying that CIC interacted with Groucho and facilitated its translocation to the nucleus. My next steps are to use a mutagenesis kit to create CIC mutants that mimic those seen in oligodendroglioma. When these CPID mutations are synthesised I will co-transfect the mutant CIC genes into COS7 cells along with Grg5-GFP. If a mutation disrupts the CIC Groucho interaction then the cells will display a cytoplasmic localisation of Grg5-GFP. If a mutation does not disrupt the CIC/Grg5 interaction then the green fluorescence will be seen predominantly in the nucleus. With this experiment there is unfortunately no way to tell the difference between a CIC mutant that blocks the Grg5-GFP interaction, and a failed co-transfection where the CIC mutant has not been successfully incorporated into the COS7 cells.

A solution to this problem would be to immunostain for the CIC mutants so that the CIC proteins fluoresce a different colour to the GFP. Unfortunately after many attempts and different antibodies I could not get specific binding for CIC. To show whether CIC genes have been successfully incorporated I will extract the RNA from co-tranfected cells. If CIC is successfully incorporated there will be CIC RNA within the cell. Converting this to DNA allows me to use qPCR which can tell me how much, and if any, CIC protein is being synthesised in the co-transfected cells.

Due to the time constraints on lab availability I will not be able to accomplish much more than the experiments described here. I hope my results can make a contribution, however small, to our understanding of CIC. Only once we have established the normal workings of CIC, can we move forward to assess CIC’s status as a TS, and its role in the formation of Oligodendroglioma.

Glossary

1p19q codeletion: This deletion of chromosome arms 1p and 19q occurs via an an imbalanced reciprocal translocation event. Both chromosomal breakpoints occur near the centromeres and homologies between these regions probably facilitate the translocation.

Domain: conserved region of a protein that can evolve function.

GFP:green florescent protein is from a deep sea jellyfish and is commonly used in Biology to tag proteins.

Missense mutation: Where a single base change causes a change in the translated amino acid.

Orthologue: Genes in different species that eveolved from a common ancestral gene. They usually retain the same or similar functions across species.

Phosphorylation: The addition of a phosphate group to a molecule. This can often cause an activation or inactivation of a protein.

Retinoblastoma: A malignant intraocular cancer that often arises in children.

Signalling cascade: This often starts in response to extracellular signalling that is picked up by cell surface receptor. The receptor becomes activated which often causes the phosphorylation of an intracellular protein. This protein phohorylates another, which in turn phospohrylates another and so on until the message reaches effectors. These effectors are usually transcription factors

Transcription factor: These proteins bind DNA and influence the level of transcription of its targets. Transcription factors may work alone, in protein complexes and may activate, repress or either their target gene.

Sources

  1. ALENTORN, A., SANSON, M. & IDBAIH, A. 2012. Oligodendrogliomas: new insights from the genetics and perspectives.Current Opinion in Oncology, 24, 687-693.
  2. BETTEGOWDA, C., AGRAWAL, N., JIAO, Y., SAUSEN, M., WOOD, L. D., HRUBAN, R. H., RODRIGUEZ, F. J., CAHILL, D. P., MCLENDON, R., RIGGINS, G., VELCULESCU, V. E., OBA-SHINJO, S. M., MARIE, S. K. N., VOGELSTEIN, B., BIGNER, D., YAN, H., PAPADOPOULOS, N. & KINZLER, K. W. 2011. Mutations in CIC and FUBP1 Contribute to Human Oligodendroglioma.Science, 333, 1453-1455.
  3. YIP, S., BUTTERFIELD, Y. S., MOROZOVA, O., CHITTARANJAN, S., BLOUGH, M. D., AN, J., BIROL, I., CHESNELONG, C., CHIU, R., CHUAH, E., CORBETT, R., DOCKING, R., FIRME, M., HIRST, M., JACKMAN, S., KARSAN, A., LI, H., LOUIS, D. N., MASLOVA, A., MOORE, R., MORADIAN, A., MUNGALL, K. L., PERIZZOLO, M., QIAN, J., ROLDAN, G., SMITH, E. E., TAMURA-WELLS, J., THIESSEN, N., VARHOL, R., WEISS, S., WU, W., YOUNG, S., ZHAO, Y., MUNGALL, A. J., JONES, S. J. M., MORIN, G. B., CHAN, J. A., CAIRNCROSS, J. G. & MARRA, M. A. 2012. Concurrent CIC mutations, IDH mutations, and 1p/19q loss distinguish oligodendrogliomas from other cancers.Journal of Pathology, 226, 7-16.
  4. SAHM, F., KOELSCHE, C., MEYER, J., PUSCH, S., LINDENBERG, K., MUELLER, W., HEROLD-MENDE, C., VON DEIMLING, A. & HARTMANN, C. 2012. CIC and FUBP1 mutations in oligodendrogliomas, oligoastrocytomas and astrocytomas.Acta Neuropathologica, 123, 853-860.
  5. SCOTTING, P. 2010. Cancer A Beginner’s Guide. Oneworld Publications.
  6. HSIAO, H. H., NATH, A., LIN, C. Y., FOLTA-STOGNIEW, E. J., RHOADES, E. & BRADDOCK, D. T. 2010. Quantitative Characterization of the Interactions among c-myc Transcriptional Regulators FUSE, FBP, and FIR.Biochemistry, 49, 4620-4634.
  7. BERGER, A. H., KNUDSON, A. G. & PANDOLFI, P. P. 2011. A continuum model for tumour suppression.Nature, 476, 163-169.
  8. EISENREICH, S., ABOU-EL-ARDAT, K., SZAFRANSKI, K., VALENZUELA, J. A. C., RUMP, A., NIGRO, J. M., BJERKVIG, R., GERLACH, E.-M., HACKMANN, K., SCHROECK, E., KREX, D., KADERALI, L., SCHACKERT, G., PLATZER, M. & KLINK, B. 2013. Novel CIC Point Mutations and an Exon-Spanning, Homozygous Deletion Identified in Oligodendroglial Tumors by a Comprehensive Genomic Approach Including Transcriptome Sequencing.Plos One,
  9. JIMENEZ, G., SHVARTSMAN, S. Y. & PAROUSH, Z. 2012. The Capicua repressor – a general sensor of RTK signaling in development and disease.Journal of Cell Science, 125, 1383-1391.
  10. KAWAMURA-SAITO, M., YAMAZAKI, Y., KANEKO, K., KAWAGUCHI, N., KANDA, H., MUKAI, H., GOTOH, T., MOTOI, T., FUKAYAMA, M., ABURATANI, H., TAKIZAWA, T. & NAKAMURA, T. 2006. Fusion between CIC and DUX4 up-regulates PEA3 family genes in Ewing-like sarcomas with t(4 ; 19)(q35 ; q13) translocation.Human Molecular Genetics,15, 2125-2137
  11. DISSANAYAKE, K., TOTH, R., BLAKEY, J., OLSSON, O., CAMPBELL, D. G., PRESCOTT, A. R. & MACKINTOSH, C. 2011. ERK/p90(RSK)/14-3-3 signalling has an impact on expression of PEA3 Ets transcription factors via the transcriptional repressor capicua.Biochemical Journal, 433, 515-525.
  12. LEE, C. J., CHAN, W. I., CHEUNG, M., CHENG, Y. C., APPLEBY, V. J., ORME, A. T. & SCOTTING, P. J. 2002. CIC, a member of a novel subfamily of the HMG-box superfamily, is transiently expressed in developing granule neurons.Molecular Brain Research, 106, 151-156.
  13. AJURIA, L., NIEVA, C., WINKLER, C., KUO, D., SAMPER, N., ANDREU, M. J., HELMAN, A., GONZALEZ-CRESPO, S., PAROUSH, Z., COUREY, A. J. & JIMENEZ, G. 2011. Capicua DNA-binding sites are general response elements for RTK signaling in Drosophila.Development, 138, 915-924.
  14. JIAO, Y. C., KILLELA, P. J., REITMAN, Z. J., RASHEED, B. A., HEAPHY, C. M., DE WILDE, R. F., RODRIGUEZ, F. J., ROSEMBERG, S., OBA-SHINJO, S. M., MARIE, S. K. N., BETTEGOWDA, C., AGRAWAL, N., LIPP, E., PIROZZI, C. J., LOPEZ, G. Y., HE, Y. P., FRIEDMAN, H. S., FRIEDMAN, A. H., RIGGINS, G. J., HOLDHOFF, M., BURGER, P., MCLENDON, R. E., BIGNER, D. D., VOGELSTEIN, B., MEEKER, A. K., KINZLER, K. W., PAPADOPOULOS, N., DIAZ, L. A. & YAN, H. 2012. Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas.Oncotarget, 3, 709-722.
  15. WAI, I.-C. 2004. Characterisation of CIC – A Novel Sox-like Factor. PhD Thesis. University of Nottingham: United Kingdom.

Featured image from: https://www.google.co.uk/search?q=oligodendroglioma&rlz=1C1CHFX_en-GBGB450GB450&espv=2&biw=1366&bih=629&source=lnms&tbm=isch&sa=X&ei=BrMaVeTuNOne7AathIHQAQ&sqi=2&ved=0CAYQ_AUoAQ#imgdii=_&imgrc=KN69e1p2x1gJiM%253A%3BexphyI-G7VZ5AM%3Bhttp%253A%252F%252Fwww.merckmanuals.com%252Fmedia%252Fprofessional%252Fphotos%252Fmedia%252Fphotos%252Foligodendroglioma_low_grade.jpg%3Bhttp%253A%252F%252Fwww.merckmanuals.com%252Fprofessional%252Fsf%252Fmultimedia%252Fv1047076%252Fn%252Fx.html%3B512%3B512

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