TACC Domain Research Articles

Abstract 4373: Molecular interactions of human receptor tyrosine kinase oncogenic fusions

Abstract Oncogenic fusions (OFs) play a critical role in cancer development. Despite the exponential increase in identifications, only a small number have been extensively characterized functionally. In this study, we are focused on receptor tyrosine kinase OFs (RTK-OFs) prevalent in aggressive cancers. Our primary objective is to elucidate the underlying molecular mechanisms of 182 in-frame RTK-OFs identified in The Cancer Genome Atlas. An automated high-throughput PCR technique will be used to clone individual gene fusions using the sequences obtained from RNA-Seq data sourced from the TCGA dataset. These clones will be used to establish an RTK-OF-expressing isogenic cell line library. Multiple Approaches Combined (MAC)-tag workflow combining Affinity Purification (AP) and proximity biotin-dependent (BioID) purification within a single construct will be used. Subsequent high-throughput EvoSep One Liquid Chromatography coupled to ultra-fast TimsTOF Pro 2 Mass Spectrometry sample processing enables sensitive and robust analysis. These methods enable the capture of stable protein complexes and transient interactors associated with RTK-OFs. In our proof of concept study, we have identified four in-frame variants of FGFR3::TACC3 oncogenic fusions.FGFR3, part of the fibroblast growth factor (FGF) RTK family, activates cellular proliferation and growth through FGF binding and receptor dimerization. In FGFR3-TACC3 OFs, diverse isoforms are observed, with prevalent rearrangements linking FGFR3 exon 17 to various TACC3 exons. TACC3 is vital for mitotic microtubule organization and stabilizes kinetochore fibers and the mitotic spindle through its conserved C-terminal coiled-coil domain (TACC domain). TACC3 contributes to the fusion with varying sizes of the C-terminal coiled-coil domain which all constitutively activate the FGFR3 tyrosine kinase. In our studies, we show that FGFR3::TACC3 variants have a different localization to their wild-type counterparts. While FGFR3 and TACC3 are found in the cytoplasm all the OF variants strongly localize around the nucleus. Our identified high-confidence interactors represent a comprehensive FGFR3::TACC3 interaction network and reveal central pathways and key interactors related to microtubule assembly and possible deregulation of the cell cycle. The resultant data from these advanced technologies enable the construction of comprehensive interaction networks, shedding light on critical signaling pathways and subnetworks relevant to RTK-OFs. Further in-depth analysis of 182 RTK-OFs may uncover actionable pathways and druggable interactors within these networks. Our multi-faceted research strategy holds significant promise for advancing targeted drug discovery, ultimately contributing to improved cancer treatment strategies. Citation Format: Giovanna Dashi, Markku Varjosalo. Molecular interactions of human receptor tyrosine kinase oncogenic fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4373.

Abstract P4-08-20: Inhibition of TACC3 blocks the growth of highly aggressive breast cancers with centrosome amplification

Abstract Centrosome amplification (CA) is a hallmark of cancer that is strongly associated with highly aggressive disease and worse clinical outcome. Enhanced mitotic progression via clustering of extra centrosomes is a major coping mechanism utilized by cancer cells with CA that would otherwise undergo mitotic cell death due to formation of multipolar spindles. However, the underlying molecular mechanisms have largely been unexplored. Furthermore, mitosis-targeting inhibitors have mostly been unsuccessful in clinical settings with poor efficacy and severe side effects. Therefore, there is a dire need to uncover novel molecular mechanisms of CA-driven tumor growth and identify therapeutic targets playing key roles not only in mitosis, but also in interphase of cancer cells with CA to achieve durable anti-tumor effect with minimal toxicity. Here, we identified Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) as a novel CA-directed dependency, driving highly aggressive cell growth by forming distinct functional interactomes during cell cycle progression. We demonstrated, for the first time, that TACC3 interacts with the Kinesin Family Member C1 (KIFC1) via its TACC domain in mitotic cells with CA to promote centrosome clustering (CC) and facilitate mitotic progression. On the other hand, TACC3 interacts with the members of the nucleosome remodeling and deacetylase (NuRD) complex (HDAC2 and MBD2) in the nucleus of interphase cells with CA, thereby suppressing the transcription of key tumor suppressors to facilitate G1/S progression and cell survival. Inhibiting TACC3 in mitotic cells blocks the formation of TACC3/KIFC1 complex, leading to formation of multipolar spindles and activation of spindle assembly checkpoint (SAC)/CDK1/p-Bcl2 axis that ultimately results in mitotic cell death; whereas TACC3 inhibition in interphase cells blocks TACC3/HDAC2/MBD2 complex, leading to enhanced transcription of cyclin-dependent kinase inhibitors (e.g., p21 and p16) and apoptosis regulators (e.g., APAF1), ultimately causing p53-independent G1 arrest and strong apoptosis. Notably, inducing CA by chemical (cytochalasin D) or genomic (PLK4 overexpression or p53 loss) modulations renders cancer cells highly sensitive to TACC3 inhibition, showing the dependency of cells with CA to TACC3. Targeting TACC3 by small molecule inhibitors or CrispR-CAS9-mediated knock-out significantly reduces colony formation ability, inhibits the growth of organoids of patient-derived xenografts (PDXs) with CA, and strongly inhibits tumor growth in breast cancer cell line xenografts and PDXs with CA. Notably, we demonstrated that high CA tumors express much higher levels of TACC3, and high TACC3 expression, in association with its downstream effectors, KIFC1, HDAC2 and MBD2, leads to drastically worse clinical outcome in cancer patients with CA. Altogether, our results show, for the first time, that TACC3 is a multifunctional driver of the growth of the highly aggressive breast tumors with CA and that targeting TACC3 is a promising approach to tackle this aggressive disease. Citation Format: Ozge Saatci, Ozge Akbulut, Metin Cetin, Vitali Sikirzhytski, Ozgur Sahin. Inhibition of TACC3 blocks the growth of highly aggressive breast cancers with centrosome amplification [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-20.

Nanopore sequencing reveals TACC2 locus complexity and diversity of isoforms transcribed from an intronic promoter

Gene expression is controlled at the transcriptional and post-transcriptional levels. The TACC2 gene was known to be associated with tumors but the control of its expression is unclear. We have reported that activity of the intronic promoter p10 of TACC2 in primary lesion of endometrial cancer is indicative of lymph node metastasis among a low-risk patient group. Here, we analyze the intronic promoter derived isoforms in JHUEM-1 endometrial cancer cells, and primary tissues of endometrial cancers and normal endometrium. Full-length cDNA amplicons are produced by long-range PCR and subjected to nanopore sequencing followed by computational error correction. We identify 16 stable, 4 variable, and 9 rare exons including 3 novel exons validated independently. All variable and rare exons reside N-terminally of the TACC domain and contribute to isoform variety. We found 240 isoforms as high-confidence, supported by more than 20 reads. The large number of isoforms produced from one minor promoter indicates the post-transcriptional complexity coupled with transcription at the TACC2 locus in cancer and normal cells.

Investigating the impact of the phosphorylation status of tyrosine residues within the TACC domain of TACC3 on microtubule behavior during axon growth and guidance.

Axon guidance is a critical process in forming the connections between a neuron and its target. The growth cone steers the growing axon toward the appropriate direction by integrating extracellular guidance cues and initiating intracellular signal transduction pathways downstream of these cues. The growth cone generates these responses by remodeling its cytoskeletal components. Regulation of microtubule dynamics within the growth cone is important for making guidance decisions. TACC3, as a microtubule plus-end binding (EB) protein, modulates microtubule dynamics during axon outgrowth and guidance. We have previously shown that Xenopus laevis embryos depleted of TACC3 displayed spinal cord axon guidance defects, while TACC3-overexpressing spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Tyrosine kinases play an important role in relaying guidance signals to downstream targets during pathfinding events via inducing tyrosine phosphorylation. Here, in order to investigate the mechanism behind TACC3-mediated axon guidance, we examined whether tyrosine residues that are present in TACC3 have any role in regulating TACC3's interaction with microtubules or during axon outgrowth and guidance behaviors. We find that the phosphorylatable tyrosines within the TACC domain are important for the microtubule plus-end tracking behavior of TACC3. Moreover, TACC domain phosphorylation impacts axon outgrowth dynamics such as growth length and growth persistency. Together, our results suggest that tyrosine phosphorylation of TACC3 affects TACC3's microtubule plus-end tracking behavior as well as its ability to mediate axon growth dynamics in cultured embryonic neural tube explants.

Solution NMR assignment of the C-terminal domain of human chTOG

The microtubule regulatory protein colonic and hepatic tumor overexpressed gene (chTOG), also known as cytoskeleleton associated protein 5 (CKAP5) plays an important role in organizing the cytoskeleton and in particular in the assembly of k-fibres in mitosis. Recently, we dissected the hitherto poorly understood C-terminus of this protein by discovering two new domains—a cryptic TOG domain (TOG6) and a smaller, helical domain at the very C-terminus. It was shown that the C-terminal domain is important for the interaction with the TACC domain in TACC3 during the assembly of k-fibres in a ternary complex that also includes clathrin. Here we now present the solution NMR assignment of the chTOG C-terminal domain which confirms our earlier prediction that it is mainly made of α-helices. However, the appearance of the 1H–15N HSQC spectrum is indicative of the presence of a considerable amount of unstructured and possibly flexible portions of protein in the domain.

FGFR3-TACC3 cancer gene fusions cause mitotic defects by removal of endogenous TACC3 from the mitotic spindle.

Fibroblast growth factor receptor 3–transforming acidic coiled-coil containing protein 3 (FGFR3–TACC3; FT3) is a gene fusion resulting from rearrangement of chromosome 4 that has been identified in many cancers including those of the urinary bladder. Altered FGFR3 signalling in FT3-positive cells is thought to contribute to cancer progression. However, potential changes in TACC3 function in these cells have not been explored. TACC3 is a mitotic spindle protein required for accurate chromosome segregation. Errors in segregation lead to aneuploidy, which can contribute to cancer progression. Here we show that FT3-positive bladder cancer cells have lower levels of endogenous TACC3 on the mitotic spindle, and that this is sufficient to cause mitotic defects. FT3 is not localized to the mitotic spindle, and by virtue of its TACC domain, recruits endogenous TACC3 away from the spindle. Knockdown of the fusion gene or low-level overexpression of TACC3 partially rescues the chromosome segregation defects in FT3-positive bladder cancer cells. This function of FT3 is specific to TACC3 as inhibition of FGFR3 signalling does not rescue the TACC3 level on the spindle in these cancer cells. Models of FT3-mediated carcinogenesis should, therefore, include altered mitotic functions of TACC3 as well as altered FGFR3 signalling.

Knockdown of TACC3 inhibits trophoblast cell migration and invasion through the PI3K/Akt signaling pathway.

The insufficient invasion of trophoblasts is known to be correlated with the development of preeclampsia. Transforming acidic coiled‑coil protein 3 (TACC3), a member of the TACC domain family, is important in the regulation of cell differentiation, migration and invasion. However, the role of TACC3 in trophoblast function during placental development remains to be fully elucidated. The present study aimed to determine the expression and function of TACC3 in human placenta and to examine the underlying mechanisms. TACC3 expression was analyzed in preeclamptic placentas using reverse transcription‑quantitative polymerase chain reaction and western blotting. Cell proliferation was determined by the MTT assay, and cell migration and invasion were measured using Transwell assays. The expression levels of TACC3, matrix metalloproteinase (MMP)‑2, MMP‑9, tissue inhibitor of metalloproteinase (TIMP)‑1, TIMP‑2, phosphoinositide 3‑kinase (PI3K), phosphorylated (p)‑PI3K, AKT and p‑AKT were detected by western blotting. The results showed that the expression of TACC3 was downregulated in preeclamptic placentas. The knockdown of TACC3 significantly inhibited HTR8/SVneo cell proliferation, migration and invasion, and inhibited the expression of matrix metalloproteinases. In addition, the knockdown of TACC3 significantly reduced the levels of p‑PI3K and Akt in the HTR8/SVneo cells. Taken together, the results of the present study demonstrated that the knockdown of TACC3 inhibited the migration and invasion of HTR8/SVneo cells through suppression of the PI3K/Akt signaling pathway. Therefore, TACC3 may serve as a novel potential target for treating preeclampsia.

Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast.

Structured summary of protein interactionsAlp14 and Alp7colocalize by fluorescence microscopy (View interaction)Alp4 and Alp7colocalize by fluorescence microscopy (View interaction)Alp7 and Pcp1colocalize by fluorescence microscopy (View interaction)Alp7physically interacts with Pcp1 by anti tag coimmunoprecipitation (View interaction)Pcp1physically interacts with Alp7 by anti bait coip (View interaction)

The Centrosomal Adaptor TACC3 and the Microtubule Polymerase chTOG Interact via Defined C-terminal Subdomains in an Aurora-A Kinase-independent Manner

The cancer-associated, centrosomal adaptor protein TACC3 (transforming acidic coiled-coil 3) and its direct effector, the microtubule polymerase chTOG (colonic and hepatic tumor overexpressed gene), play a crucial function in centrosome-driven mitotic spindle assembly. It is unclear how TACC3 interacts with chTOG. Here, we show that the C-terminal TACC domain of TACC3 and a C-terminal fragment adjacent to the TOG domains of chTOG mediate the interaction between these two proteins. Interestingly, the TACC domain consists of two functionally distinct subdomains, CC1 (amino acids (aa) 414-530) and CC2 (aa 530-630). Whereas CC1 is responsible for the interaction with chTOG, CC2 performs an intradomain interaction with the central repeat region of TACC3, thereby masking the TACC domain before effector binding. Contrary to previous findings, our data clearly demonstrate that Aurora-A kinase does not regulate TACC3-chTOG complex formation, indicating that Aurora-A solely functions as a recruitment factor for the TACC3-chTOG complex to centrosomes and proximal mitotic spindles. We identified with CC1 and CC2, two functionally diverse modules within the TACC domain of TACC3 that modulate and mediate, respectively, TACC3 interaction with chTOG required for spindle assembly and microtubule dynamics during mitotic cell division.

Coordination of adjacent domains mediates TACC3–ch-TOG–clathrin assembly and mitotic spindle binding

A complex of transforming acidic coiled-coil protein 3 (TACC3), colonic and hepatic tumor overexpressed gene (ch-TOG), and clathrin has been implicated in mitotic spindle assembly and in the stabilization of kinetochore fibers by cross-linking microtubules. It is unclear how this complex binds microtubules and how the proteins in the complex interact with one another. TACC3 and clathrin have each been proposed to be the spindle recruitment factor. We have mapped the interactions within the complex and show that TACC3 and clathrin were interdependent for spindle recruitment, having to interact in order for either to be recruited to the spindle. The N-terminal domain of clathrin and the TACC domain of TACC3 in tandem made a microtubule interaction surface, coordinated by TACC3-clathrin binding. A dileucine motif and Aurora A-phosphorylated serine 558 on TACC3 bound to the "ankle" of clathrin. The other interaction within the complex involved a stutter in the TACC3 coiled-coil and a proposed novel sixth TOG domain in ch-TOG, which was required for microtubule localization of ch-TOG but not TACC3-clathrin.

Abstract 5310: Activation of FGFR3 by genomic fusion in urothelial carcinoma of the bladder.

Abstract FGFR3 is frequently activated by mutation in urothelial carcinoma of the bladder. We demonstrate here a further method of activation, by gene fusion following chromosomal re-arrangement. Four fusions with transforming acid coiled-coil 3 (TACC3) resulting from 4p16.3 re-arrangement were identified. A fifth fusion following t(4;7) was with BAI1-associated protein 2-like 1 (BAIAP2L1), also known as IRTKS (Insulin Receptor Kinase Substrate) on chromosome 7. The FGFR3-TACC3 fusions involve four different genomic re-arrangements resulting in three different transcripts. The fusion proteins show constitutive phosphorylation, activation of the MAPK pathway and transform NIH-3T3 cells. All have lost the final exon of FGFR3, which includes the PLCγ1 binding site. Thus PLCγ1 pathway activation and known PLCγ1-induced effects in normal urothelial cells were not induced. The FGFR3-BAIAP2L1 fusion retains most of BAIAP2L1 including the IRSp53/MIM domain (IMD) that mediates actin binding and Rac interaction, while the FGFR3-TACC3 fusions all retain the TACC domain that mediates microtubule binding. As cells containing these fusion genes are highly sensitive to FGFR-selective agents, detection of fusion genes may be useful in selecting patients for targeted therapy. Citation Format: Sarah V. Williams, Carolyn D. Hurst, Margaret A. Knowles. Activation of FGFR3 by genomic fusion in urothelial carcinoma of the bladder. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5310. doi:10.1158/1538-7445.AM2013-5310

Oncogenic FGFR3 gene fusions in bladder cancer

FGF receptor 3 (FGFR3) is activated by mutation or over-expression in many bladder cancers. Here, we identify an additional mechanism of activation via chromosomal re-arrangement to generate constitutively activated fusion genes. FGFR3–transforming acid coiled coil 3 (TACC3) fusions resulting from 4p16.3 re-arrangements and a t(4;7) that generates a FGFR3-BAI1-associated protein 2-like 1 (BAIAP2L1) fusion were identified in 4 of 43 bladder tumour cell lines and 2 of 32 selected tissue samples including the tumour from which one of the cell lines was derived. These are highly activated and transform NIH-3T3 cells. The FGFR3 component is identical in all cases and lacks the final exon that includes the phospholipase C gamma 1 (PLCγ1) binding site. Expression of the fusions in immortalized normal human urothelial cells (NHUC) induced activation of the mitogen-activated protein kinase pathway but not PLCγ1. A protein with loss of the terminal region alone was not as highly activated as the fusion proteins, indicating that the fusion partners are essential. The TACC3 fusions retain the TACC domain that mediates microtubule binding and the BAIAP2L1 fusion retains the IRSp53/MIM domain (IMD) that mediates actin binding and Rac interaction. As urothelial cell lines with FGFR3 fusions are extremely sensitive to FGFR-selective agents, the presence of a fusion gene may aid in selection of patients for FGFR-targeted therapy.

Mos1-Mediated Transgenesis to Probe Consequences of Single Gene Mutations in Variation-Rich Isolates of Caenorhabditis elegans

Caenorhabditis elegans, especially the N2 isolate, is an invaluable biological model system. Numerous additional natural C. elegans isolates have been shown to have unexpected genotypic and phenotypic variations which has encouraged researchers to use next generation sequencing methodology to develop a more complete picture of genotypic variations among the isolates. To understand the phenotypic effects of a genomic variation (GV) on a single gene, in a variation-rich genetic background, one should analyze that particular GV in a well understood genetic background. In C. elegans, the analysis is usually done in N2, which requires extensive crossing to bring in the GV. This can be a very time consuming procedure thus it is important to establish a fast and efficient approach to test the effect of GVs from different isolates in N2. Here we use a Mos1-mediated single-copy insertion (MosSCI) method for phenotypic assessments of GVs from the variation-rich Hawaiian strain CB4856 in N2. Specifically, we investigate effects of variations identified in the CB4856 strain on tac-1 which is an essential gene that is necessary for mitotic spindle elongation and pronuclear migration. We show the usefulness of the MosSCI method by using EU1004 tac-1(or402) as a control. or402 is a temperature sensitive lethal allele within a well-conserved TACC domain (transforming acidic coiled-coil) that results in a leucine to phenylalanine change at amino acid 229. CB4856 contains a variation that affects the second exon of tac-1 causing a cysteine to tryptophan change at amino acid 94 also within the TACC domain. Using the MosSCI method, we analyze tac-1 from CB4856 in the N2 background and demonstrate that the C94W change, albeit significant, does not cause any obvious decrease in viability. This MosSCI method has proven to be a rapid and efficient way to analyze GVs.

BRCA1/BARD1 Orthologs Required for DNA Repair in Caenorhabditis elegans

Inherited germline mutations in the tumor suppressor gene BRCA1 predispose individuals to early onset breast and ovarian cancer [1]. BRCA1 together with its structurally related partner BARD1 is required for homologous recombination and DNA double-strand break repair, but how they perform these functions remains elusive [2, 3]. As part of a comprehensive search for DNA repair genes in C. elegans, we identified a BARD1 ortholog. In protein interaction screens, Ce-BRD-1 was found to interact with components of the sumoylation pathway, the TACC domain protein TAC-1, and most importantly, a homolog of mammalian BRCA1. We show that animals depleted for either Ce-brc-1 or Ce-brd-1 display similar abnormalities, including a high incidence of males, elevated levels of p53-dependent germ cell death before and after irradiation, and impaired progeny survival and chromosome fragmentation after irradiation. Furthermore, depletion of ubc-9 and tac-1 leads to radiation sensitivity and a high incidence of males, respectively, potentially linking these genes to the C. elegans BRCA1 pathway. Our findings support a shared role for Ce-BRC-1 and Ce-BRD-1 in C. elegans DNA repair processes, and this role will permit studies of the BRCA1 pathway in an organism amenable to rapid genetic and biochemical analysis.

The TACC domain identifies a family of centrosomal proteins that can interact with microtubules.

We recently showed that the Drosophila transforming acidic coiled-coil (D-TACC) protein is located in the centrosome, interacts with microtubules, and is required for mitosis in the Drosophila embryo. There are three known human TACC proteins that share a conserved, C-terminal, coiled-coil region with D-TACC. These proteins have all been implicated in cancer, but their normal functions are unknown. We show that all three human TACC proteins are concentrated at centrosomes, but with very different characteristics: TACC1 is weakly concentrated at centrosomes during mitosis; TACC2 is strongly concentrated at centrosomes throughout the cell cycle; and TACC3 is strongly concentrated in a more diffuse region around centrosomes during mitosis. When the C-terminal TACC domain is overexpressed in HeLa cells, it forms large polymers in the cytoplasm that can interact with both microtubules and tubulin. The full-length TACC proteins form similar polymers when overexpressed, but their interaction with microtubules and tubulin is regulated during the cell cycle. At least one of the human TACC proteins appears to increase the number and/or stability of centrosomal microtubules when overexpressed during mitosis. Thus, the TACC domain identifies a family of centrosomal proteins that can interact with microtubules. This may explain the link between the TACC genes and cancer.