Centrosome Amplification Research Articles

STIL overexpression shortens lifespan and reduces tumor formation in mice.

Centrosomes are the major microtubule organizing centers of animal cells. Supernumerary centrosomes are a common feature of human tumors and associated with karyotype abnormalities and aggressive disease, but whether they are cause or consequence of cancer remains controversial. Here, we analyzed the consequences of centrosome amplification by generating transgenic mice in which centrosome numbers can be increased by overexpression of the structural centrosome protein STIL. We show that STIL overexpression induces centrosome amplification and aneuploidy, leading to senescence, apoptosis, and impaired proliferation in mouse embryonic fibroblasts, and microcephaly with increased perinatal lethality and shortened lifespan in mice. Importantly, both overall tumor formation in mice with constitutive, global STIL overexpression and chemical skin carcinogenesis in animals with inducible, skin-specific STIL overexpression were reduced, an effect that was not rescued by concomitant interference with p53 function. These results suggest that supernumerary centrosomes impair proliferation in vitro as well as in vivo, resulting in reduced lifespan and delayed spontaneous as well as carcinogen-induced tumor formation.

PROTAC’ING CENTROSOME AMPLIfiCATION PROTEINS: TARGETED PROTEIN DEGRADATION AS A NOVEL THERAPEUTIC STRATEGY IN GLIOBLASTOMA

Abstract AIMS Glioblastomas (GBMs) are the most common and aggressive brain tumours, notoriously difficult to treat with universal treatment resistance. A key feature of cancer cells is centrosome amplification (CA), leading to chromosomal instability and worse patient outcomes, including metastasis development and treatment resistance. In this work, I identified CA proteins overexpressed in GBM and aimed to investigate how the targeted degradation of proteins associated with centrosome amplification can influence the growth and survival of GBM cells. METHOD Based on well-established literature data concerning CA-related genes, I evaluated the expression level of these targets in different types of samples from the Tumour Cancer Genome Atlas dataset of GBM patient samples. Following the identification of CA targets differentially overexpressed, to test the hypothesis that CA proteins could be involved in the survival of GBM cells and be potential therapeutic targets, I used a novel drug modality, named Proteolysis Targeting Chimeras (PROTACs) to degrade two CA proteins. PROTACs can quickly destroy specific proteins, potentially overcoming resistance to treatment. Target degradation was confirmed via immunoblotting and cell viability and cell cycle analysis were performed to evaluate the impact on GBM cells. RESULTS Both primary and recurrent GBM samples display high levels of CA gene signature when compared to noncancerous samples, and some CA genes are particularly upregulated in recurrent samples. The PROTACs were effective in degrading both CA targets in GBM cells, leading to a significant decrease in cell viability and G2 cell cycle arrest. CONCLUSION This study presents initial evidence that CA protein degradation could be a promising avenue for GBM research and therapy. Further research is needed to fully assess the translational potential of PROTACs in GBM treatment.

KIFC1 depends on TRIM37-mediated ubiquitination of PLK4 to promote centrosome amplification in endometrial cancer

Endometrial cancer (EC), as one of the most common cancers, severely threatens female reproductive health. Our previous study has shown that Kinesin family member C1 (KIFC1) played crucial roles in the progression of EC. In addition, abnormal centrosome amplification, which was reported to be partially regulated by KIFC1, usually occurred in different cancers. However, whether KIFC1 promoted EC through centrosome amplification and the potential mechanism remain to be revealed. The present study demonstrated that overexpressed KIFC1, which exhibited a worse prognosis, had a positive correlation with an increased number of centrosomes in human EC samples. In addition, KIFC1 overexpression in EC cells prompted centrosome amplification, chromosomal instability, and cell cycle progression. Moreover, we demonstrated that KIFC1 inhibited E3 ubiquitin-protein ligase TRIM37 to maintain the stability of PLK4 by reducing its ubiquitination degradation, and finally promoting centrosome amplification and EC progression in vitro. Finally, the contributing role of KIFC1 and the inhibitory effect of TRIM37 on EC development and metastasis was verified in a nude mouse xenograft model. Our study elucidated that KIFC1 depends on TRIM37-mediated reduced ubiquitination degradation of PLK4 to promote centrosome amplification and EC progression, thus providing a potential prognostic marker and promising therapeutic target for EC in the future.

Abstract C141: Inactivating the mitotic kinases TTK and NEK2 as a promising approach for the reduction of metastasis in triple-negative breast cancer in non-Hispanic Blacks and Hispanic/Latinas

Abstract Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer in women worldwide. It is associated with uncontrolled cell proliferation, increased rates of centrosome amplification, genetic instability, and invasive capacity. Advanced stages of TNBC with distant metastasis are virtually incurable, with chemotherapy as the primary treatment option. Non- Hispanic Black (NHB) and Hispanic/Latino (H/L) women have lower 5-year survival rates when compared to non-Hispanic White (NHW) women with breast cancer, in part because their breast tumors are detected at higher stages, and due to elevated rates of TNBC. Women with a higher percentage of African ancestry have been shown to have a higher expression of multiple mitotic kinases. Mitotic kinases, such as TTK, maintain genomic integrity, while NEK2 modulates centrosome separation and microtubule organization. However, their overexpression in breast cancer is associated with cell proliferation, chromosome instability, aneuploidy, and early stages of metastasis (the epithelial-to-mesenchymal transition, or EMT, cell migration and invasion). A tissue microarray confirmed associations between TTK overexpression, proliferation, and EMT markers in breast tumors and the TNBC subtype. Thus, we aim to measure protein and mRNA expression levels of EMT markers upon downregulation of TTK and NEK2. To address this, mesenchymal TNBC cell lines, MDA-MB-231 (NHW) or MDA-MB-157 (NHB), were cultured in DMEM and 10% FBS, followed by TTK siRNA, NEK2 siRNA, or double transfection. TRIzol was then used for protein and RNA extraction. Western blot and qPCR analyses measured EMT markers' protein and mRNA expression levels, respectively. Preliminary results from these experiments showed that silencing of TTK, NEK2, or TTK/NEK2 resulted in a reduction of the mRNA expression levels of Vimentin, N-cadherin, and β-Catenin in MDA-MB- 231 cell lines. Moreover, data showed a decrease in Vimentin in all treatment groups at the protein level. TTK/NEK2 knockdown also reduced N-cadherin and β-Catenin protein expression levels. These preliminary data suggest that silencing of mitotic kinases TTK and NEK2 may be a promising approach for targeting early stages of metastasis in TNBCs. Because of their increased expression in women of African heritage, anti-mitotic kinase inactivation can be a novel approach that can highly benefit women of the West African diaspora with breast cancer, including African American and Caribbean Hispanics, specifically by diminishing cancer cell survival and metastasis. Citation Format: Alexandra N Aquino-Acevedo, Ángel D. Colón-Burgos, Gretchen M. Albarrán-Acosta, Marileana Rodríguez-Ruiz, Joel A. Orengo-Orengo, Melanie E. Cruz- Robles, Harold I. Saavedra. Inactivating the mitotic kinases TTK and NEK2 as a promising approach for the reduction of metastasis in triple-negative breast cancer in non-Hispanic Blacks and Hispanic/Latinas [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C141.

FOXM1 Transcriptionally Co-Upregulates Centrosome Amplification and Clustering Genes and Is a Biomarker for Poor Prognosis in Androgen Receptor-Low Triple-Negative Breast Cancer.

There are currently no approved targeted treatments for quadruple-negative breast cancer [QNBC; ER-/PR-/HER2-/androgen receptor (AR)-], a subtype of triple-negative breast cancer (TNBC). AR-low TNBC is more proliferative and clinically aggressive than AR-high TNBC. Centrosome amplification (CA), a cancer hallmark, is rampant in TNBC, where it induces spindle multipolarity-mediated cell death unless centrosome clustering pathways are co-upregulated to avert these sequelae. We recently showed that genes that confer CA and centrosome clustering are strongly overexpressed in AR-low TNBCs relative to AR-high TNBCs. However, the molecular mechanisms that index centrosome clustering to the levels of CA are undefined. We argue that FOXM1, a cell cycle-regulated oncogene, links the expression of genes that drive CA to the expression of genes that act at kinetochores and along microtubules to facilitate centrosome clustering. We provide compelling evidence that upregulation of the FOXM1-E2F1-ATAD2 oncogene triad in AR-low TNBC is accompanied by CA and the co-upregulation of centrosome clustering proteins such as KIFC1, AURKB, BIRC5, and CDCA8, conferring profound dysregulation of cell cycle controls. Targeting FOXM1 in AR-low TNBC may render cancer cells incapable of clustering their centrosomes and impair their ability to generate excess centrosomes. Hence, our review illuminates FOXM1 as a potential actionable target for AR-low TNBC.

Plk4 regulates centriole duplication in the embryonic development of zebrafish

PLK4 plays a crucial role in centriole duplication, which is essential for maintaining cellular processes such as cell division, cytoskeletal stability, and cilia formation. However, the mechanisms of PLK4 remain incompletely understood, especially in the embryonic development of vertebrate species. In this study, we observed that Plk4 dysfunction led to abnormal embryonic development in zebrafish, characterized by symptoms such as dark and wrinkled skin, microphthalmia, and body axis curvature. In plk4 mutants, defects in centriole duplication led to abnormal cell division, apoptosis, and ciliogenesis defects. Moreover, overexpression of plk4 in zebrafish embryos caused excessive centrosome amplification, disrupting embryonic gastrulation through abnormal cell division and ultimately resulting in embryonic lethality. Furthermore, we identified the "cryptic" polo box (CPB) domain, consisting of two PBs (PB1 and PB2), as the critical centrosome localization domain of Plk4. Surprisingly, overexpression of these two PB domains alone was sufficient to induce embryonic lethality. Additionally, we discovered a truncated form of CPB that localizes to the centrosome without causing defects in embryonic development. Our results demonstrate that Plk4 tightly controls centriole duplication, which is essential for early embryonic development in zebrafish.

Identification of KIFC1 as a putative vulnerability in lung cancers with centrosome amplification

Centrosome amplification (CA), an abnormal increase in the number of centrosomes in the cell, is a recurrent phenomenon in lung and other malignancies. Although CA promotes tumor development and progression by inducing genomic instability (GIN), it also induces mitotic stress that jeopardizes cellular integrity. CA leads to the formation of multipolar mitotic spindles that can cause lethal chromosome segregation errors. To sustain the benefits of CA by mitigating its consequences, malignant cells are dependent on adaptive mechanisms that represent therapeutic vulnerabilities. We aimed to discover genetic dependencies associated with CA in lung cancer. Combining a CRISPR/Cas9 functional genomics screen with tumor genomic analyses, we identified the motor protein KIFC1, also known as HSET, as a putative vulnerability specifically in lung adenocarcinoma (LUAD) with CA. KIFC1 expression was positively correlated with CA in LUAD and associated with worse patient outcomes, smoking history, and indicators of GIN. KIFC1 loss-of-function sensitized LUAD cells with high basal KIFC1 expression to potentiation of CA, which was associated with a diminished ability to cluster extra centrosomes into pseudo-bipolar mitotic spindles. Our work suggests that KIFC1 inhibition represents a novel approach for potentiating GIN to lethal levels in LUAD with CA by forcing cells to divide with multipolar spindles, rationalizing further studies to investigate its therapeutic potential.

The Unkempt RNA binding protein reveals a local translation program in centriole overduplication.

Excess centrosomes cause defects in mitosis, cell-signaling, and cell migration, and therefore their assembly is tightly regulated. Plk4 controls centriole duplication at the heart of centrosome assembly, and elevation of Plk4 promotes centrosome amplification (CA), a founding event of tumorigenesis. Here, we investigate the transcriptional consequences of elevated Plk4 and find Unkempt, a gene encoding an RNA binding protein with roles in translational regulation, to be one of only two upregulated mRNAs. Unk protein localizes to centrosomes and Cep131-positive centriolar satellites and is required for Plk4-induced centriole overduplication in an RNA-binding dependent manner. Translation is enriched at centrosomes and centriolar satellites with Unk and Cep131 promoting this localized translation. A transient centrosomal downregulation of translation occurs early in Plk4-induced CA. CNOT9, an Unk interactor and component of the translational inhibitory CCR4-NOT complex, localizes to centrosomes at this time. In summary, centriolar satellites and Unk promote local translation as part of a translational program that ensures centriole duplication.

Targeting YES1 Disrupts Mitotic Fidelity and Potentiates the Response to Taxanes in Triple-Negative Breast Cancer.

Clinical trials examining broad-spectrum Src family kinase (SFK) inhibitors revealed significant dose-limiting toxicities, preventing advancement for solid tumors. SFKs are functionally heterogeneous, thus targeting individual members is a potential strategy to elicit anti-tumor efficacy while avoiding toxicity. Here, we identified that YES1 is the most highly overexpressed SFK in triple negative breast cancer (TNBC) and is associated with poor patient outcomes. Disrupting YES1, genetically or pharmacologically, induced aberrant mitosis, centrosome amplification, multi-polar spindles, and chromosomal instability (CIN). Mechanistically, YES1 sustained FOXM1 protein levels and elevated expression of FOXM1 target genes that control centrosome function and are essential for effective and accurate mitotic progression. In both in vitro and in vivo TNBC models, YES1 suppression potentiated the efficacy of taxanes, cornerstone drugs for TNBC that require elevated CIN for efficacy. Clinically, elevated expression of YES1 was associated with worse overall survival of TNBC patients treated with taxane and anthracycline combination regimens. Together, this study demonstrates that YES1 is an essential regulator of genome stability in TNBC that can be leveraged to improve taxane efficacy.

CENP-E Inhibition Induces Chromosomal Instability and Synergizes with Diverse Microtubule-Targeting Agents in Breast Cancer.

Drugs that perturb microtubules are commonly used to treat breast cancers of all subtypes in both early stage and metastatic disease, but they are effective in only approximately 50% of patients. High concentrations of microtubule-targeting agents can elicit mitotic arrest in cell culture models; however, recent evidence from primary and metastatic breast cancers has revealed that these agents only accumulate at intratumoral levels capable of inducing abnormal multipolar mitotic spindles, not mitotic arrest. Although the maintenance of multipolar spindles can generate cytotoxic rates of chromosomal instability (CIN), focusing of aberrant multipolar spindles into normal bipolar spindles can dramatically reduce CIN and confer resistance to microtubule poisons. Here, we showed that inhibition of the mitotic kinesin centromeric-associated protein-E (CENP-E) overcomes resistance caused by focusing multipolar spindles. Clinically relevant microtubule-targeting agents used a mechanistically conserved pathway to induce multipolar spindles without requiring centrosome amplification. Focusing could occur at any point in mitosis, with earlier focusing conferring greater resistance to antimicrotubule agents. CENP-E inhibition increased CIN on focused spindles by generating chromosomes that remained misaligned at spindle poles during anaphase, which substantially increased death in the resulting daughter cells. CENP-E inhibition synergized with diverse, clinically relevant microtubule poisons to potentiate cell death in cell lines and suppress tumor growth in orthotopic tumor models. These results suggest that primary resistance to microtubule-targeting drugs can be overcome by simultaneous inhibition of CENP-E. Significance: The increased incidence of polar chromosomes induced by inhibition of the mitotic kinesin CENP-E exacerbates chromosomal instability, reduces daughter cell viability, and improves sensitivity to microtubule-targeting therapies.

Potential involvement of KANK1 haploinsufficiency in centrosome aberrations

KANK1 was found as a tumor suppressor gene based on frequent deletions in renal cell carcinoma and the inhibitory activity of tumor cell proliferation. Previously, we reported that knockdown of KANK1 induced centrosomal amplification, leading to abnormal cell division, through the hyperactivation of RhoA small GTPase. Here, we investigated the loss of KANK1 function by performing CRISPR/Cas9-based genome editing to knockout the gene. After several rounds of genome editing, however, there were no cell lines with complete loss of KANK1, and the less the wild-type KANK1 dosage, the greater the number of cells with abnormal numbers of centrosomes and rates of cell-doubling and apoptosis, suggesting the involvement of KANK1 haploinsufficiency in centrosome aberrations. The rescue of KANK1-knockdown cells with a KANK1-expressing plasmid restored the rates of cells exhibiting centrosomal amplification to the control level. RNA-sequencing analysis of the cells with reduced dosages of functional KANK1 revealed potential involvement of other cell proliferation-related genes, such as EGR1, MDGA2, and BMP3, which have been reported to show haploinsufficiency when they function. When EGR1 protein expression was reduced by siRNA technology, the number of cells exhibiting centrosomal amplification increased, along with the reduction of KANK1 protein expression, suggesting their functional relationship. Thus, KANK1 haploinsufficiency may contribute to centrosome aberrations through the network of haploinsufficiency-related genes.

A first-in-human, phase 1 study evaluating oral TACC3 inhibitor, AO-252, in advanced solid tumors.

TPS3176 Background: TACC3 is the most oncogenic member of the transforming acidic coiled-coil domain-containing protein (TACC) family, which have important roles in microtubule and centrosome-related processes. TACC3 is overexpressed in multiple cancers with centrosome amplification caused by genomic aberrations such as TP53 mutations, leading to chromosome instability and worse prognosis. AO-252 is a computationally designed, small molecule inhibitor of TACC3 protein-protein interactions. Binding assays and kinome screening suggested the specificity of AO-252 to TACC3. AO-252 targets the interactions of TACC3 with Clathrin/KIFC1, BRDA1 and MBD2/HAT complexes regulating mitosis, DNA damage response and epigenetic functions and was confirmed using co-immunoprecipitation (co-IP) assays. Gene expression analysis in multiple cell lines further confirmed the various TACC3 pathways affected by AO-252. In vitro, AO-252 showed low nanomolar potency in >200 cell line panel covering multiple different cancer indications and spared the normal cells. In vivo, AO-252 demonstrated strong tumor growth inhibition or regression in multiple xenograft models including triple-negative breast (TNBC), high-grade serous ovarian (HGSOC) and endometrial cancer with TP53 mutation/loss with a good therapeutic index. Methods: This first-in-human, multicenter, open-label phase I dose escalation and expansion study evaluates the safety, tolerability, maximum tolerated dose, and recommended phase 2 dose (RP2D) of AO-252 in solid tumors (NCT06136884). Patients with unresectable or metastatic TNBC, platinum-resistant HGSOC, primary peritoneal, fallopian-tube and serous endometrial cancers with documented TP53 mutation/loss, who have failed on at least 1 prior line of systemic therapy in advanced/metastatic setting (maximum 4 for TNBC and endometrial carcinoma) are eligible for enrollment. Part 1 follows an accelerated titration dose-escalation for cohort 1 and 2 and a traditional 3+3 study design from cohort 3. A maximum of 54 patients will be treated in the dose escalation, patients receiving AO-252 orally once or twice daily in a 28-day cycle. Part 2 consists of dose-expansion of a maximum of 30 patients into 2 RP2D levels of AO-252. Primary objectives are to evaluate the safety and tolerability as noted by dose-limiting toxicities, adverse events, serious adverse events, laboratory test results, electrocardiograms, vital signs, physical exams, and ECOG performance status. Secondary objectives are to determine the antitumor activity assessed by objective response rate per RECIST1.1, duration of response and disease control rate and to determine the pharmacokinetics of single and repeated doses of AO-252. Potential biomarkers relating to treatment outcome will be evaluated as exploratory endpoints. Enrollment in Cohort 1 is completed and enrollment to Cohort 2 will begin in February 2024. Clinical trial information: NCT06136884 .

Abstract PO4-24-04: Identifying the interactome of TACC3, a major driver in aggressive cancer cells with centrosome amplification

Abstract Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) is a centrosome- and spindle-associated protein that drives the growth of highly aggressive tumors, such as those with centrosome amplification (CA). In recent years, it has become apparent that TACC3 not only functions at the centrosomes and spindles but may also have many other non-canonical functions facilitating the growth of tumors. Given the multifaceted roles of TACC3 in driving tumor aggressiveness, the identification of novel interactors of TACC3 that are responsible for mediating key TACC3-driven processes is crucial. This will not only uncover novel drug targets but will also expand the patient subpopulations that can benefit from TACC3 inhibition. Here, we characterized the cancer cells with CA in terms of their dependency on TACC3 for executing distinct cellular processes depending on the cell cycle phase. We demonstrated that TACC3 is strongly upregulated in cancers with CA at mRNA and protein levels and associated with worse clinical outcome in highly aggressive patient subpopulations, especially in TNBC. We further demonstrated that in mitotic cancer cells with CA, TACC3 is localized at the centrosomes in a complex with KIFC1, thus mediating the clustering of extra centrosomes to enable bipolar spindle formation and faithful mitosis. We showed that inhibiting TACC3 blocks the interaction between TACC3 and KIFC1, thus leading to mitotic cell death in the highly TACC3-dependent cancer cells with CA. While TACC3-KIFC1 complex is needed for mitotic cancer cells with CA, we found that in CA-bearing interphase cells, TACC3 interacts with HDAC2 and MBD2, the two major components of the chromatin remodeling NuRD complex within the nucleus. Inhibiting TACC3 prevented its nuclear localization, thus unloading the NuRD complex from the chromatin, leading to transcription of key tumor suppressors driving G1 arrest and apoptosis. To obtain the proteome-wide interaction network of TACC3 in these highly TACC3-dependent mitotic and interphase cancer cells with CA, we mapped the TACC3 interactome using the state-of-the-art APEX2 method. This uncovered potentially novel partners of TACC3 that could be critical for the survival of mitotic and interphase cancer cells with CA. We performed an extensive bioinformatic analysis to identify the most clinically relevant interactors in the context of cancers with CA. We generated correlation matrices of the mitotic and interphase-specific interactors and coupled them with pathway enrichment analysis to identify the key processes that the interactors are potentially involved in. Altogether, our results map, for the first time, the novel cell cycle-dependent TACC3 interactome in cancer cells with CA. Our results support targeting this multi-functional protein and hold great promise to improve clinical outcome in these highly aggressive cancers given the availability of the first-in-class IND-approved TACC3 inhibitor. Citation Format: Ozge Saatci, Ozge Akbulut, Metin Cetin, Vitali Sikirzhytski, Meral Uner, Deniz Lengerli, Elizabeth C. O’Quinn, Martin J. Romeo, Burcu Caliskan, Erden Banoglu, Sercan Aksoy, Aysegul Uner, Ozgur Sahin. Identifying the interactome of TACC3, a major driver in aggressive cancer cells with centrosome amplification [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-04.

Extra centrosomes delay DNA damage-driven tumorigenesis.

Deregulated centrosome numbers are frequently found in human cancer and can promote malignancies in model organisms. Current research aims to clarify if extra centrosomes are cause or consequence of malignant transformation, and if their biogenesis can be targeted for therapy. Here, we show that oncogene-driven blood cancer is inert to genetic manipulation of centrosome numbers, whereas the formation of DNA damage-induced malignancies is delayed. We provide first evidence that this unexpected phenomenon is connected to extra centrosomes eliciting a pro-death signal engaging the apoptotic machinery. Apoptosis induction requires the PIDDosome multi-protein complex, as it can be abrogated by loss of any of its three components, Caspase-2, Raidd/Cradd, or Pidd1. BCL2 overexpression equally blocks cell death, documenting for the first time induction of mitochondrial apoptosis downstream of extra centrosomes. Our findings demonstrate context-dependent effects of centrosome amplification during transformation and ask to adjust current belief that extra centrosomes are intrinsically pro-tumorigenic.

Centrosomes and associated proteins in pathogenesis and treatment of breast cancer.

Breast cancer is the most prevalent malignancy among women worldwide. Despite significant advances in treatment, it remains one of the leading causes of female mortality. The inability to effectively treat advanced and/or treatment-resistant breast cancer demonstrates the need to develop novel treatment strategies and targeted therapies. Centrosomes and their associated proteins have been shown to play key roles in the pathogenesis of breast cancer and thus represent promising targets for drug and biomarker development. Centrosomes are fundamental cellular structures in the mammalian cell that are responsible for error-free execution of cell division. Centrosome amplification and aberrant expression of its associated proteins such as Polo-like kinases (PLKs), Aurora kinases (AURKs) and Cyclin-dependent kinases (CDKs) have been observed in various cancers, including breast cancer. These aberrations in breast cancer are thought to cause improper chromosomal segregation during mitosis, leading to chromosomal instability and uncontrolled cell division, allowing cancer cells to acquire new genetic changes that result in evasion of cell death and the promotion of tumor formation. Various chemical compounds developed against PLKs and AURKs have shown meaningful antitumorigenic effects in breast cancer cells in vitro and in vivo. The mechanism of action of these inhibitors is likely related to exacerbation of numerical genomic instability, such as aneuploidy or polyploidy. Furthermore, growing evidence demonstrates enhanced antitumorigenic effects when inhibitors specific to centrosome-associated proteins are used in combination with either radiation or chemotherapy drugs in breast cancer. This review focuses on the current knowledge regarding the roles of centrosome and centrosome-associated proteins in breast cancer pathogenesis and their utility as novel targets for breast cancer treatment.

A whale of a tale: whale cells evade the driving mechanism for hexavalent chromium-induced chromosome instability.

Chromosome instability, a hallmark of lung cancer, is a driving mechanism for hexavalent chromium [Cr(VI)] carcinogenesis in humans. Cr(VI) induces structural and numerical chromosome instability in human lung cells by inducing DNA double-strand breaks and inhibiting homologous recombination repair and causing spindle assembly checkpoint (SAC) bypass and centrosome amplification. Great whales are long-lived species with long-term exposures to Cr(VI) and accumulate Cr in their tissue, but exhibit a low incidence of cancer. Data show Cr(VI) induces fewer chromosome aberrations in whale cells after acute Cr(VI) exposure suggesting whale cells can evade Cr(VI)-induced chromosome instability. However, it is unknown if whales can evade Cr(VI)-induced chromosome instability. Thus, we tested the hypothesis that whale cells resist Cr(VI)-induced loss of homologous recombination repair activity and increased SAC bypass and centrosome amplification. We found Cr(VI) induces similar amounts of DNA double-strand breaks after acute (24 h) and prolonged (120 h) exposures in whale lung cells, but does not inhibit homologous recombination repair, SAC bypass, or centrosome amplification, and does not induce chromosome instability. These data indicate whale lung cells resist Cr(VI)-induced chromosome instability, the major driver for Cr(VI) carcinogenesis at a cellular level, consistent with observations that whales are resistant to cancer.

Knockout of Brca1-interacting factor Ola1 in female mice induces tumors with estrogen suppressible centrosome amplification

Obg-like ATPase 1 (OLA1) is a binding protein of Breast cancer gene 1 (BRCA1), germline pathogenic variants of which cause hereditary breast cancer. Cancer-associated variants of BRCA1 and OLA1 are deficient in the regulation of centrosome number. Although OLA1 might function as a tumor suppressor, the relevance of OLA1 deficiency to carcinogenesis is unclear. Here, we generated Ola1 knockout mice. Aged female Ola1+/− mice developed lymphoproliferative diseases, including malignant lymphoma. The lymphoma tissues had low expression of Ola1 and an increase in the number of cells with centrosome amplification. Interestingly, the proportion of cells with centrosome amplification in normal spleen from Ola1+/− mice was higher in male mice than in female mice. In human cells, estrogen stimulation attenuated centrosome amplification induced by OLA1 knockdown. Previous reports indicate that prominent centrosome amplification causes cell death but does not promote tumorigenesis. Thus, in the current study, the mild centrosome amplification observed under estrogen stimulation in Ola1+/− female mice is likely more tumorigenic than the prominent centrosome amplification observed in Ola1+/− male mice. Our findings provide a possible sex-dependent mechanism of the tumor suppressor function of OLA1.

Abstract 527: Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification

Abstract Therapy resistance is one of the most common underlying causes of poor prognosis in cancer patients, ultimately driving fatal outcome. We have previous demonstrated that following chemotherapeutic stress, a subset of cancer cells undergo a mitotic skip and enter an endocycle, causing whole-genome duplication without division. This endocycling cell state is resistant to cytotoxic therapies and thus is an actuator of therapy resistance. Cells in the endocycling state eventually undergo depolyploidization and repopulate the tumor, observed clinically as a recurrence. We also demonstrated that the minus-end directed motor protein KIFC1, a mediator of centrosome clustering, has higher expression in cells following treatment. Centrosome amplification (CA) involves aberrations in centrosome number, where cells have 3 centrosomes. Cells with extra centrosomes create a multi-polar spindle during mitosis, which presents high risk for cell death and arrest, but cancer cells with CA avoid this by clustering extra centrosomes to create a pseudo-bipolar spindle. Based upon this data, we hypothesize that studying centrosome dynamics and abnormalities presents a window into better understanding endocycle biology and this novel mechanism of therapy resistance. Cancer cells were treated with LD50 dose of cisplatin and released from treatment for varying lengths of time. CA in endocycling cells increased from 59.2% to 100% during the treatment recovery period while maintaining identical levels of clustering. Endocycling cancer cells have a nearly 5-fold increase in declustered centrosomes compared to proliferative cells. Centrosomes in endocycling cells are displaced from the perinuclear region and toward the cell periphery. We hypothesize that centrosome number may indicate the number of times a cell has endocycled and provide insight into the timing and mechanism of mitotic bypass. Centrosomes play a major role in microtubule nucleation, affecting cell motility and polarity, and act as signaling platforms, enhancing signaling activation and specificity. Because of this, we hypothesize that CA may be a critical mechanism for endocycling cancer cells to survive and maintain this resistant cell state. Citation Format: Madison Purkerson, Sarah Amend, Kenneth J. Pienta. Cancer cells in the resistant, endocycling cell state exhibit centrosome amplification [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 527.

Abstract 3347: KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe

Abstract Cancer cells often have more than two centrosomes (supernumerary centrosomes) and this is one of the hallmarks of cancer. We previously demonstrated that CDK2 antagonism inhibits clustering of supernumerary centrosomes at mitosis leading to multipolar cell division and apoptotic death in non-small cell lung cancer. This antineoplastic process was designated anaphase catastrophe. Anaphase catastrophe can preferentially affect cancer cells sparing normal cells with two centrosomes. On the other hand, as CDK2 functions in normal cell cycle progression, its inhibition can also affect normal and malignant cells. Identification of a novel drug target that would preferentially cause anaphase catastrophe is needed. The motor protein KIFC1 affects centrosome clustering. Small cell lung cancer (SCLC), known for its poor prognosis with limited therapeutic options, displays disruptions in the cell cycle checkpoint mechanism due to inactivation of p53 and RB. We hypothesized SCLC was susceptible to mitotic abnormalities, including anaphase catastrophe. In this study, we explored the possibility of KIFC1 as a novel therapeutic target that triggers anaphase catastrophe in SCLC. In-silico GEO database analysis revealed elevated levels of KIFC1 and PLK4 in SCLCs compared with other cancers and normal tissues. PLK4 plays a key role in centrosome amplification. Our in-silico analysis results imply that centrosome amplification and their clustering frequently occur in SCLC. We performed in-vitro functional analysis of KIFC1 inhibition in SCLC cell lines (NCI-H146, SHP77, NCI-H209, and NCI-H524 cells), using siRNAs and CRISPR-Cas9 system for genetic inhibition and AZ82, a specific KIFC1 inhibitor, for pharmacologic inhibition of KIFC1. Cell proliferation was measured by the CellTiter-Glo Luminescent Assay and apoptosis was scored by annexin V and PI staining followed by flow cytometry analysis. Cell growth was reduced and apoptosis was statistically significantly increased after genetic inhibition of KIFC1 by siRNAs and by use of the CRISPR-Cas9 system in SCLC cell lines. These antineoplastic effects also occurred after AZ82 treatments, in a dose-dependent manner in these SCLC cell lines. To examine the mitotic status of SCLC cells after KIFC1 inhibition, SCLC cells were stained with α-tubulin antibody and DAPI before analysis with a fluorescence microscope. Besides normal bipolar mitotic cells, multipolar mitotic cells were observed, and their population was increased after KIFC1 knockdown with siRNAs. This indicated that clustering of supernumerary centrosomes was inhibited by targeting KIFC1 in SCLC cells. Taken together, KIFC1 is a potential therapeutic target to induce anaphase catastrophe in cancer cells. Targeting KIFC1 is a novel therapeutic strategy to consider for SCLC that currently has limited treatment options. Citation Format: Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Yuriko Sugiura, Takahiro Iida, Takahiro Ando, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe [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 3347.

MicroRNA‑155‑5p inhibits trophoblast cell proliferation and invasion by disrupting centrosomal function.

Recurrent miscarriage is used to refer to more than three pregnancy failures before 20 weeks of gestation. Defective trophoblast cell growth and invasion are frequently observed in recurrent miscarriage. Several microRNAs (miRs), including miR‑155‑5p, are aberrantly upregulated in recurrent miscarriage; however, the underlying molecular mechanisms remain unclear. The centrosome orchestrates microtubule networks and coordinates cell cycle progression. In addition, it is a base for primary cilia, which are antenna‑like organelles that coordinate signaling during development and growth. Thus, deficiencies in centrosomal functions can lead to several disease, such as breast cancer and microcephaly. In the present study, the signaling cascades were analyzed by western blotting, and the centrosome and primary cilia were observed and analyzed by immunofluorescence staining. The results showed that overexpression of miR‑155‑5p induced centrosome amplification and blocked primary cilia formation in trophoblast cells. Notably, centrosome amplification inhibited trophoblast cell growth by upregulating apoptotic cleaved‑caspase 3 and cleaved‑poly (ADP‑ribose) polymerase in miR‑155‑5p‑overexpressing trophoblast cells. In addition, overexpression of miR‑155‑5p inhibited primary cilia formation, thereby inhibiting epithelial‑mesenchymal transition and trophoblast cell invasion. All phenotypes could be rescued when cells were co‑transfected with the miR‑155‑5p inhibitor, thus supporting the role of miR‑155‑5p in centrosomal functions. It was also found that miR‑155‑5p activated autophagy, whereas disruption of autophagy via the depletion of autophagy‑related 16‑like 1 alleviated miR‑155‑5p‑induced apoptosis and restored trophoblast cell invasion. In conclusion, the present study indicated a novel role of miR‑55‑5p in mediating centrosomal function in recurrent miscarriage.