Transforming Acidic Coiled-coil 3 Research Articles

Bihemispheric IDH-Wildtype Glioblastoma with Unilateral FGFR3-TACC3 Fusion in Patient with Breast Cancer History: A Case Report and Literature Review

Abstract Introduction/Objective Adult IDH-wild type glioblastomas comprise a heterogeneous spectrum of neoplasms, characterized by poor prognosis and resistance to the chemoradiotherapy. For WHO integrated diagnosis classification and to establish treatment options, molecular analysis is necessary. Herein, we present a case of a glioblastoma, IDH-wildtype with involvement of both cerebral hemispheres and an uncharacteristic in-frame fusion limited to one hemisphere. Methods/Case Report The patient is a 52-year-old woman with past medical history of metastatic breast cancer who presented with headache and worsening facial pain. Brain magnetic resonance imaging brain demonstrated a right frontal lobe lesion with vasogenic edema. The patient was treated with Gamma Knife radiosurgery. Scans revealed enlargement and a new left frontal lobe lesion. Bihemispheric tumor resection and GammaTile placement was undertaken. Results (if a Case Study enter NA) Histologically, the tumor was composed of hypercellular astrocytes, microvascular proliferation, and necrosis with no evidence of metastatic disease. Immunohistochemistically, neoplastic cells showed glial fibrillary acidic protein expression and elevated Ki-67 proliferation index. Molecular examination revealed in the right hemisphere lesion post-radiation (1) FGFR3::TACC3 (exon 17::exon 11) fusion and (2) TERT promoter, while the left hemisphere lesion which did not receive radiation showed clinically relevant variants in (1) TERT promoter, (2) TP53, (3) NF1, and (3) PIK3CA without identification of a fusion. An integrated diagnosis of glioblastoma, IDH- wildtype, CNS WHO grade 4 was established bilaterally. Conclusion The fibroblast growth factor receptor 3 (FGFR3)-transforming acidic coiled-coil 3 (TACC3) fusion is identified as a rare molecular feature in glioblastomas with an estimated prevalence of 4%. Clinical trials suggest that F3T3-positive tumors have a better prognosis than those without the translocation. The identification of the oncogenic FGFR3-TACC3 fusion highlights the possibility of identifying patients potentially responsive to targeted therapy with FGFR kinase inhibitors. The significance of the molecular differences in the right and left hemisphere lesions is uncertain; glioblastomas are heterogeneous tumors with intra-and intertumoral heterogeneity but it may suggest independent origins.

PAK1 Is Involved in the Spindle Assembly during the First Meiotic Division in Porcine Oocytes.

P21-activated kinase 1 (PAK1), as a member of the PAK family, has been implicated in various functions during somatic mitosis; however, less is known about its role during oocyte meiosis. Herein, we highlight the indispensable role of PAK1 in regulating spindle assembly and cell cycle progression during the first meiotic division of porcine oocytes. First, we found that the activated PAK1 expressed dynamically, and its subcellular localization was tightly associated with the spindle dynamics during meiosis in porcine oocytes. Specific inhibition of PAK1 activity by inhibitor targeting PAK1 activation-3 (IPA-3) led to impaired extrusion of the first polar body (PB1); with most of the IPA-3-treated oocytes arrested at germinal vesicle breakdown (GVBD) and subjected to failure of bipolar spindle formation. However, the adverse effects caused by IPA-3 on oocytes could be restored by reducing disulfide bonds between PAK1 and IPA-3 with dithiothreitol (DTT) treatment. Furthermore, the co-immunoprecipitation assay revealed that PAK1 interacted directly with Aurora A and transforming acidic coiled coil 3 (TACC3), providing an additional explanation for the similar localization of Aurora A and activated PAK1. Additionally, inhibiting the activity of PAK1 decreased the expression of p-Aurora A and p-TACC3; however, the reduced activity of Aurora A and TACC3 could be restored by DTT. In conclusion, PAK1 plays a crucial role in the proper assembly of the spindle during the first meiotic division of porcine oocytes, and the regulation of PAK1 is associated with its effects on p-Aurora A and p-TACC3 expression.

Effects of Ran-GTP/importin β inhibition on the meiotic division of porcine oocytes.

The Ran-GTP/importin β pathway has been implicated in a diverse array of mitotic functions in somatic mitosis; however, the possible meiotic roles of Ran-GTP/importin β in mammalian oocyte meiosis are still not fully understood. In the present study, importazole (IPZ), a small molecule inhibitor of the interaction between Ran and importin β was used to explore the potential meiotic roles of Ran-GTP/importin β in porcine oocytes undergoing meiosis. After IPZ treatment, the extrusion rate of the first polar body (PB1) was significantly decreased, and a higher proportion of the oocytes were arrested at the germinal vesicle breakdown (GVBD) stage. Moreover, IPZ treatment led to severe defects in metaphase I (MI) spindle assembly and chromosome alignment during the germinal vesicle (GV)-to-MI stage, as well as failure of metaphase II (MII) spindle reassembly and homologous chromosome segregation during the MI-to-MII stage. Notably, IPZ treatment decreased TPX2 expression and abnormal subcellular localization. Furthermore, the expression levels of aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3) were significantly reduced after IPZ treatment. Collectively, these data indicate that the interaction of Ran-GTP and importin β is essential for proper spindle assembly and successful chromosome segregation during two consecutive meiotic divisions in porcine oocytes, and regulation of this complex might be related to its effect on the TPX2 signaling cascades.

TPX2 deficiency leads to spindle abnormity and meiotic impairment in porcine oocytes

The targeting protein for Xklp2 (TPX2) is a spindle assembly factor, that can stimulate microtubule formation and promote spindle completion during mitosis. However, the role of TPX2 in mammalian oocyte meiotic maturation is still not fully understood. This study was conducted to address the dynamic distribution and potential roles of TPX2 in microtubule nucleation during meiotic maturation in porcine oocytes by microinjecting specific siRNAs. Western blotting results revealed that the expression of TPX2 displayed a lower level from 0 to 22 h of culture, while its expression exhibited a higher level after 28 h of culture. Immunofluorescence staining demonstrated that TPX2 was distributed along the microtubules and enriched in the poles after meiotic spindle formation at the 28 and 44 h of culture. From immunoprecipitation, TPX2 can interact with the microtubule-associated proteins aurora kinase A (AURKA) and transforming acidic coiled-coil 3 (TACC3). Meanwhile, the dynamic changes in the expression and localization of AURKA and TACC3 were highly consistent with TPX2 during meiotic maturation. After knocking down TPX2 by siRNA injection, the proportion of oocytes with aberrant spindles and scattered cytoplasmic actin filaments was significantly increased. In addition, TPX2 depletion markedly downregulated the expression of AURKA and TACC3. Thus, these results suggested that TPX2 is essential for meiotic spindle formation in the porcine oocyte, and that this regulation is related to its effect on AURKA and TACC3 expression.

The prognostic activity of transforming acidic coiled-coil 3 (TACC3) immunohistochemical expression in colon adenocarcinoma patients.

IntroductionColorectal cancer is most common in developed countries. Each year, more than one million people develop colon cancer, and nearly 70,000 people die from the disease. Although medicine has made great strides in the treatment of colorectal cancer, the prognosis of patients is still poor. It is difficult to find the main cause of colon cancer, so it is necessary to introduce new methods that will accurately diagnose the cause of this malignancy.Material and methodsParaffin-embedded colon adenocarcinoma samples (n = 97) were assessed immunohistochemically for TACC3 protein. Connections between TACC3 immunoexpression and clinicopathological factors, including the 5-year overall survival (OS), were evaluated.ResultsImmunohistochemical expression of TACC3 protein in colon adenocarcinoma samples and non-pathological samples of colon tissue was described as weak, moderate, or strong. As demonstrated, the level of the TACC3 immunohistochemical reactivity was not correlated with demographic factors including gender and age, and clinicopathological factors. The average survival time for all the patients was 36.8 months (95% CI: 33.134–40.536). A multivariate analysis demonstrated that the grade of tumour differentiation (HR = 2.740; 95% CI: 1.864–4.027, p < 0.001) and TACC3 immunoexpression in healthy tissues (HR = 1.700; 95% CI: 1.073–2.694) were independent risk factors for worse survival of patients.ConclusionsThe high level of TACC3 immunoexpression in cancerous tissue was not associated with malignancy-related clinicopathological factors and 5-year overall survival of patients.

Inhibiting of TACC3 Promotes Cell Proliferation, Cell Invasion and the EMT Pathway in Breast Cancer.

Accumulating evidences indicate that transforming acidic coiled-coil 3 (TACC3) is a tumor-related gene, was highly expressed in a variety of human cancers, which is involved in cancer development. However, the potential role of TACC3 in breast cancer remains largely unknown. In the present study, we found that TACC3 was highly-expressed in breast cancer tissues, and its level was positively correlated with the clinical features of breast cancer patients. Specifically, TACC3 expression was significantly associated with the estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) status, nodal status, the scarff-bloom-richardson (SBR) grade, nottingham prognostic index (NPI), age, subtypes, and triple-negative and basal-like status, suggesting that TACC3 may be a potential diagnostic indicator of breast cancer. Furthermore, functional studies have shown that inhibition of TACC3 can significantly promote the cell proliferation and viability of breast cancer cells. Moreover, TACC3 knockdown suppressed the expression of E-cadherin, but increased the expression of N-cadherin, Snail, ZEB1, and TWIST, which indicate that TACC3 may impact the migration of breast cancer cells in vitro. Taken together, these findings indicate that TACC3 may serve as a prognostic and therapeutic indicator of breast cancer.

Aurora A site specific TACC3 phosphorylation regulates astral microtubule assembly by stabilizing \u03b3-tubulin ring complex

BackgroundAstral microtubules emanating from the mitotic centrosomes play pivotal roles in defining cell division axis and tissue morphogenesis. Previous studies have demonstrated that human transforming acidic coiled-coil 3 (TACC3), the most conserved TACC family protein, regulates formation of astral microtubules at centrosomes in vertebrate cells by affecting γ-tubulin ring complex (γ-TuRC) assembly. However, the molecular mechanisms underlying such function were not completely understood.ResultsHere, we show that Aurora A site-specific phosphorylation in TACC3 regulates formation of astral microtubules by stabilizing γ-TuRC assembly in human cells. Mutation of the most conserved Aurora A targeting site, Ser 558 to alanine (S558A) in TACC3 results in robust loss of astral microtubules and disrupts localization of the γ-tubulin ring complex (γ-TuRC) proteins at the spindle poles. Under similar condition, phospho-mimicking S558D mutation retains astral microtubules and the γ-TuRC proteins in a manner similar to control cells expressed with wild type TACC3. Time-lapse imaging reveals that S558A mutation leads to defects in positioning of the spindle-poles and thereby causes delay in metaphase to anaphase transition. Biochemical results determine that the Ser 558- phosphorylated TACC3 interacts with the γ-TuRC proteins and further, S558A mutation impairs the interaction. We further reveal that the mutation affects the assembly of γ-TuRC from the small complex components.ConclusionsThe results demonstrate that TACC3 phosphorylation stabilizes γ- tubulin ring complex assembly and thereby regulates formation of centrosomal asters. They also implicate a potential role of TACC3 phosphorylation in the functional integrity of centrosomes/spindle poles.

Long non-coding RNA lnc-PCTST predicts prognosis through inhibiting progression of pancreatic cancer by downregulation of TACC-3.

Pancreatic cancer (PC), which is one of the most lethal of malignancies and a major health burden, is associated with a dismal prognosis despite current therapeutic advances. Numerous long noncoding RNAs (lncRNA) have shown to be essential for PC tumorigenesis and progression. Nevertheless, the exact expression pattern of lnc-PCTST and its clinical significance still remain unclear. This study investigates the expression pattern of lnc-PCTST and its associated mRNA in three paired PC tissues and adjacent non-tumor tissues by Microarray-coarray approach. Briefly, our data demonstrated that lnc-PCTST expression is down-regulated in PC tissues. Also, lnc-PCTST has shown to be negatively correlated with transforming acidic coiled-coil 3 (TACC-3) expression. This expression pattern was further confirmed following qRT-PCR validation of 34 out of 48 paired cancer tissues. Furthermore, lnc-PCTST overexpression in PC cell lines inhibited cell proliferation and invasion in vitro, and tumorigenesis in vivo (using nude mice as animal model), but did not altered cell migration. Moreover, lnc-PCTST overexpression increased E-cadherin and repressed vimentin expression in vitro. Additionally, TACC-3 knockdown simulated the inhibiting effect of lnc-PCTST overexpression on PC cell lines, and the impaired proliferation, invasion effect and E-cadherin, vimentin expression on lnc-PCTST over-expressed cell lines can be rescued by overexpressed TACC-3. Significantly, the expression of lnc-PCTST was closely associated with its genomic neighboring gene TACC-3 and inhibited its promoter activity. In conclusion, lnc-PCTST is a potential tumor suppressor in PC, which inhibits cell proliferation, invasion, tumorigenesis and EMT by modulating TACC-3.

FGFR3-TACC3 is an oncogenic fusion protein in respiratory epithelium.

Structural rearrangements of the genome can drive lung tumorigenesis through the generation of fusion genes with oncogenic properties. Advanced genomic approaches have identified the presence of a genetic fusion between fibroblast growth factor receptor 3 (FGFR3) and transforming acidic coiled-coil 3 (TACC3) in non-small cell lung cancer (NSCLC), providing a novel target for FGFR inhibition. To interrogate the functional consequences of the FGFR3-TACC3 fusion in the transformation of lung epithelial cells, we generated a novel transgenic mouse model that expresses FGFR3-TACC3 concomitant with loss of the p53 tumor suppressor gene. Intra-nasal delivery of an Ad5-CMV-Cre virus promoted seromucinous glandular transformation of olfactory cells lining the nasal cavities of FGFR3-TACC3 (LSL-F3T3) mice, which was further accelerated upon loss of p53 (LSL-F3T3/p53). Surprisingly, lung tumors failed to develop in intra-nasally infected LSL-F3T3 and LSL-F3T3/p53 mice. In line with these observations, we demonstrated that intra-nasal delivery of Ad5-CMV-Cre induces widespread Cre-mediated recombination in the olfactory epithelium. Intra-tracheal delivery of Ad5-CMV-Cre into the lungs of LSL-F3T3 and LSL-F3T3/p53 mice however, resulted in the development of lung adenocarcinomas. Taken together, these findings provide in vivo evidence for an oncogenic function of FGFR3-TACC3 in respiratory epithelium.

TACC3 transcriptionally upregulates E2F1 to promote cell growth and confer sensitivity to cisplatin in bladder cancer

Accumulating evidence has shown that transforming acidic coiled-coil 3 (TACC3) is deregulated in a broad spectrum of cancers. In the present study, we reported that TACC3 was markedly elevated in bladder cancer, especially in muscle-invasive bladder cancers (MIBCs). The upregulation of TACC3 was positively associated with tumor invasiveness, grade, T stage, and progression in patients with bladder cancer. Furthermore, a Kaplan–Meier survival analysis showed that patients with bladder cancer whose tumors had high TACC3 expression experienced a dismal prognosis compared with patients whose tumors had low TACC3 expression. Functional studies have found that TACC3 is a prerequisite for the development of malignant characteristics of bladder cancer cells, including cell proliferation and invasion. Moreover, TACC3 promoted G1/S transition, which was mediated via activation of the transcription of E2F1, eventually enhancing cell proliferation. Notably, the overexpression of TACC3 or E2F1 indicates a high sensitivity to cisplatin. Taken together, these findings define a tumor-supportive role for TACC3, which may also serve as a prognostic and therapeutic indicator in bladder cancers

TACC3 as an independent prognostic marker for solid tumors: a systematic review and meta-analysis.

Recent studies have showed that the transforming acidic coiled coil 3 (TACC3), was aberrantly up-regulated in various solid tumors and was reported to be correlated with unfavorable prognosis in cancer patients. This study aimed to examine the relationship between TACC3 and relevant clinical outcomes. Pubmed, Web of Science, Embase and Cochrane Library were systematically searched to obtain all eligible articles. Pooled hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated to evaluate the influence of TACC3 expression on overall survival (OS) and disease-free survival (DFS) in solid tumors patients. A total of 1943 patients from 11 articles were included. The result indicated that a significantly shorter OS was observed in patients with high expression level of TACC3 (HR=1.90, 95% CI=1.63–2.23). In the subgroup analysis, the association was also observed in patients with cancers of digestive system (HR=1.85, 95% CI=1.53–2.24). Statistical significance was also observed in subgroup meta-analysis stratified by the cancer type, analysis type and sample size. Furthermore, poorer DFS was observed in patients with high expression level of TACC3 (HR=2.67, 95% CI=2.10–3.40). Additionally, the pooled odds ratios (ORs) showed that increased TACC3 expression was also related to positive lymph node metastasis (OR=1.68, 95% CI=1.26–2.25), tumor differentiation (OR=1.90, 95% CI=1.25–2.88) and TNM stage (OR=1.66, 95% CI=1.25-2.20). In conclusion, the increased expression level of TACC3 was associated with unfavorable prognosis, suggesting that it was a valuable prognosis biomarker or a promising therapeutic target of solid tumors. Further studies should be conducted to confirm the clinical utility of TACC3 in human solid tumors.

SNIPER(TACC3) induces cytoplasmic vacuolization and sensitizes cancer cells to Bortezomib

We previously developed a hybrid small molecule SNIPER (Specific and Nongenetic IAP‐dependent Protein ERaser) against transforming acidic coiled‐coil‐3 (TACC3), SNIPER(TACC3), that induces proteasomal degradation of TACC3 protein. In this study, we found that SNIPER(TACC3) induces cytoplasmic vacuolization derived from endoplasmic reticulum (ER) and paraptosis‐like cell death selectively in cancer cells. Mechanistic analysis suggests that accumulation of ubiquitylated protein aggregates that requires X‐linked inhibitor of apoptosis protein (XIAP) induces ER stress, which results in ER‐stress responses involving X‐box binding protein‐1 (XBP‐1) and ER‐derived vacuolization in cancer cells. Importantly, inhibition of proteasome enhanced the SNIPER(TACC3)‐induced vacuolization, and the combination treatment of SNIPER(TACC3) and bortezomib exhibited a synergistic anticancer activity in several cancer cell lines. The induction of paraptosis‐like cell death in cancer cells by SNIPER(TACC3) could be applied to treat cancer cells resistant to undergo apoptosis by overexpression of XIAP.

Abstract 5011: Strong FGFR3 staining is a marker for FGFR3 fusions and poor prognosis in diffuse gliomas

Abstract Inhibitors of fibroblast growth factor receptors (FGFRs) have recently arisen as a promising treatment option for patients with FGFR alterations. Gene fusions involving FGFR3 and transforming acidic coiled coil 3 (TACC3) have been detected in diffuse gliomas and other malignancies, and fusion-positive cases have responded well to FGFR inhibitors. As high FGFR3 expression has been detected in fusion positive tumors, we sought to determine the clinical significance of FGFR3 protein expression level and its potential to indicate FGFR3 fusions. We thus performed FGFR3 IHC on tissue microarrays containing 676 grade II-IV astrocytoma and 116 grade II-III oligodendroglial tumor specimens. Selected cases were further analyzed using targeted sequencing. Moderate-to-strong FGFR3 staining was detected in all tumor grades, was more common in females, and associated with poor survival. Targeted sequencing identified FGFR3-TACC3 fusions and an FGFR3-CAMK2A fusion in 10 of 12 strongly stained cases (staining specificity 86%), whereas no fusions were found in 12 negatively-to-moderately stained cases (staining sensitivity 100%). Fusion-positive cases were predominantly female and negative for IDH, TP53, and EGFR/PDGFRA/MET alterations. Importantly, FGFR3 staining revealed intratumoral heterogeneity, with subclonal negative staining in a subpopulation of fusion-positive cases. Taken together, strong FGFR3 protein expression is indicative of FGFR3 fusions and may serve as a cost-effective predictive marker for FGFR-inhibitor-based treatment regimens. Citation Format: Kirsi J. Granberg, Matti Annala, Birgitta Lehtinen, Juha Kesseli, Joonas Haapasalo, Olli Yli-Harja, Tapio Visakorpi, Hannu Haapasalo, Matti Nykter, Wei Zhang. Strong FGFR3 staining is a marker for FGFR3 fusions and poor prognosis in diffuse gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5011.

TACC3 in personalized medicine.

Cells are constantly exposed to endogenous and exogenous DNA damage events. In order to confront DNA damage and to maintain genomic integrity, cells have evolved a fine-tuned network of cellular pathways, collectively known as the DNA damage response (DDR). The broad components of DDR include surveillance mechanisms, cell cycle checkpoints, DNA repair pathways, and apoptotic programs. Interestingly, the very deficiencies in DDR that lead to cell death, genomic instability, and tumorigenesis, are also the very mechanisms that can be harnessed for cancer's own demise. Such vulnerabilities manifest as deregulated DDR-associated pathways that, with further elucidation, can become prognostic and predictive biomarkers and therapeutic targets in personalized cancer therapy. Transforming Acidic Coiled-Coil 3 (TACC3) is acknowledged for its canonical role in centrosome-microtubule spindle dynamics, and its deregulation has been linked to a variety of human cancers, including breast, colon, liver and lung cancers [1]. Also, high TACC3 has been shown to be strongly associated with poor survival in breast and lung cancer patients [1]. Interestingly, p53-deficient mouse with conditional knockout of Tacc3 showed a remarkable thymus lymphoma regression [2]. Given these associations, it is imperative to clarify the precise mechanistic contribution of TACC3 to tumorigenesis and cancer development. Previously, we demonstrated that TACC3 plays a pivotal role in the process of epithelial-mesenchymal transition (EMT), a key step of tumor progression and metastasis, by promoting phosphoinositide 3-kinase (PI3K)/Akt and extracellular signaling related kinase (ERK) signaling [3]. In our most recent study, we found that high TACC3 induces spontaneous DNA damage and impairs G2/M checkpoint function and repair of DSBs (both homologous recombination [HR] and non-homologous end joining [NHEJ]), thus increasing the frequency of chromosomal aberrations, at least in part, by TACC3-mediated negative regulation of Ataxia telangiectasia mutated (ATM) [1]. Given TACC3's emerging role in DDR regulation, we then sought to investigate TACC3's predictive value for hypersensitivity to radiation and poly(ADPribose)polymerase (PARP) inhibitor treatment. Unsurprisingly, high TACC3 levels confer cellular hypersensitivity to radiation and the PARP inhibitors Olaparib (AZD2281) and NU1025 [1]. In spite of the emerging link between high TACC3 and impaired DDR, unanswered questions need to be uncovered to have a complete knowledge of TACC3 functionality in human cancer. For example, the fact that re-expression of ATM did not completely rescue high TACC3-mediated DNA damage, suggests that there are other mechanisms unaccounted for that might contribute to TACC3-mediated genomic instability. Since Aurora Kinase A (AurA) acts as an upstream of TACC3 [4], and because both TACC3 and Aurora Kinase A disrupt normal cellular response to DNA damage, although TACC3 affects both Chk1 and Chk2 [1] while AurA only affects Chk1 [5], it would be interesting to clarify whether TACC3-mediated disruption of DDR occurs in an AurA-dependent manner. Additionally, a more robust understanding of the upstream regulation of TACC3 is required; this may shed light on the etiology of TACC3 upregulation in various cancers, and provide a starting point for thinking about cancer prevention. Meanwhile, since TACC3 is actually downregulated in a certain subset of cancers [6], it remains unclear if TACC3 is a tumor suppressor, an oncogene, or both. In any case, it is clear that TACC3 deregulation is associated with cancer. While knowledge gaps of TACC3's role in cancer still remain, the clinical-translational significance of TACC3 is becoming increasingly clear. The distribution of TACC3 at the interface of the mitotic spindle-assembling machinery, vital for tumor survival and progression, makes TACC3 a superior therapeutic target for anti-cancer drugs precisely designed to inhibit the mitotic spindle-microtubule of cancer with aberrant TACC3, without interfering with the microtubules activity in non-dividing cell. Furthermore, we discovered that depletion of TACC3 renders cancer cells more sensitive to the anti-microtubule agent paclitaxel [7], a phenomenon consistent with the findings of Schmidt et al. [8]. Taken together, we speculate that patients with high levels of TACC3 may benefit more from radiotherapy, PARP inhibitor therapy, or a combination of both than those with comparatively lower levels of TACC3. On the other hand, patients with low levels of TACC3 may have a better response to paclitaxel. In addition to opening up new avenues for patient stratification, TACC3 also harbors prognostic potential, especially in light of aforementioned evidence of TACC3's role in EMT. Thus, tracking the status of TACC3 levels may also offer an opportunity for monitoring tumor progression and metastasis. As the emerging importance of TACC3 for cancer is increasingly apparent, we are better positioned to strategically address knowledge gaps in cancer etiology, and more effectively reap the harvest of personalized cancer therapy.

156 Behaviour of platinum(IV) complexes with prodrug function in different models of hypoxia

utility. Recently, inhibitors of spindle-regulatory proteins have attracted considerable attention, and transforming acidic coiled-coil-3 (TACC3) is a spindle-regulatory protein overexpressed in many human cancers. We have developed a protein knockdown system to induce degradation of target proteins via the ubiquitin–proteasome system in cells with hybrid molecules named SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser). In this study, we designed and synthesized novel SNIPER(TACC3)s that target TACC3 for degradation, and evaluated their activity in vitro. SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3, and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C mediates the SNIPER(TACC3)-induced degradation of TACC3. Cancer cells express larger amount of TACC3 than do normal fibroblasts, and SNIPER(TACC3) selectively induced cell death in cancer cells. These results suggest protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy to treat cancers overexpressing the TACC3 protein.

Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin–proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/CCDH1 mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

157 Sensitive and specific detection of 1p/19q codeletion in gliomas by next generation sequencing

utility. Recently, inhibitors of spindle-regulatory proteins have attracted considerable attention, and transforming acidic coiled-coil-3 (TACC3) is a spindle-regulatory protein overexpressed in many human cancers. We have developed a protein knockdown system to induce degradation of target proteins via the ubiquitin–proteasome system in cells with hybrid molecules named SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser). In this study, we designed and synthesized novel SNIPER(TACC3)s that target TACC3 for degradation, and evaluated their activity in vitro. SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3, and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C mediates the SNIPER(TACC3)-induced degradation of TACC3. Cancer cells express larger amount of TACC3 than do normal fibroblasts, and SNIPER(TACC3) selectively induced cell death in cancer cells. These results suggest protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy to treat cancers overexpressing the TACC3 protein.

Abstract LB-92: Using immunohistochemistry to evaluate FGFR3 gene fusion recurrence and clinical associations in astrocytomas

Abstract Aberrant activation of fibroblast growth factor receptor 3 (FGFR3) has been reported to be tumorigenic in various cancer types. When FGFR3 is fused with another gene, most commonly with transforming acidic coiled coil 3 (TACC3), it can escape miRNA-mediated regulation and remain constitutively active most likely due to the dimerization via the domains of TACC3 or another fusion partner. Among brain cancers, gene fusions involving FGFR3 have been detected in glioblastoma and certain low grade gliomas. Furthermore, FGFR3-TACC3 fusion positive glioma cells have responded well to targeted therapies. The endogenous FGFR3 protein levels are extremely low in brain partly due to miR-99a-mediated regulation and gene fusions lead to prominent protein expression. In previous studies, FGFR3 fusion recurrence and fusion partners have been analyzed only in limited patient cohorts and there are no reports about their clinical associations. In this study, we have performed immunohistochemistry for FGFR3 on tissue microarrays with over 750 clinical astrocytoma samples (including grade 1-3 astrocytomas and glioblastomas). FGFR3 positive staining has been detected in subpopulations of all included tumor grades. The observed recurrences (1.8%-6.2%, depending on the grade) are consistent with previous sequencing-based studies. To validate immunohistochemistry-based approach for FGFR3 fusion detection, we are using frozen tumor material to confirm the presence of fusion with Western blotting and with PCR followed by Sanger sequencing. Furthermore, we have associated FGFR3 staining scores with clinical records for all the cases. FGFR3 staining is more common in females than males both in astrocytoma (7.6% vs 2.2%, pearson chi-square p-value 0.004, n=752) and glioblastoma cohorts (8.9% vs 2.1%, pearson chi-square p-value 0.008, n=494). In addition, FGFR3-staining negatively associates with aberrant p53 staining indicating p53 mutation (FGFR3-positive cases: 5.8% vs 1.8% in p53 low vs high groups, respectively, pearson chi-square p-value 0.030, n=399). Altogether, this study evaluates the suitability of immunohistochemical methods for FGFR3 fusion detection as well as investigates their recurrence and clinical significance in astrocytomas. Citation Format: Kirsi J. Granberg, Birgitta Lehtinen, Matti J. Annala, Hannu Haapasalo, Matti Nykter, Wei Zhang. Using immunohistochemistry to evaluate FGFR3 gene fusion recurrence and clinical associations in astrocytomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-92. doi:10.1158/1538-7445.AM2014-LB-92

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.

Analysis of Human TACC3 in Cellular Responses to Polycyclic Aromatic Hydrocarbons

Very little is known about the etiology of ovarian cancer. However, studies have shown that occupational exposure to polycyclic aromatic hydrocarbons (PAHs) and tobacco smoking can increase ovarian cancer risk. Previously, we have determined that 100% of ovarian tumors show loss or aberrant subcellular localization of TACC3 (transforming acidic coiled coil 3) relative to the normal ovarian surface epithelium (OSE). In mice, a role for TACC3 in the cellular response to PAH has been defined. However, comparable studies for human TACC3 has not been performed. In this report, we show that specific knockdown of TACC3 in human ovarian cancer cells increases the basal level, and distorts the PAH-induced expression, of genes involved in metabolizing the procarcinogen PAH benzo[a]pyrene to its DNA damaging epoxide. In addition, we demonstrate that PAH stabilizes the TACC3 protein and induces the export of TACC3 and one of its binding partners, Ku70, from the nucleus. This parallels the subcellular distribution of TACC3 in those ovarian cancers that express TACC3. These data suggest that functional downregulation of TACC3 could aid tumor progression by altering cellular responses to chemical carcinogens and the DNA damage that they induce.