Citron Kinase Research Articles

Zinc finger protein 367 exerts a cancer-promoting role in small cell lung cancer by influencing the CIT/LATS2/YAP signaling cascade

A remarkable cancer-related role of zinc finger protein 367 (ZNF367) has been demonstrated in multiple malignancies. However, whether ZNF367 has a role in small-cell lung cancer (SCLC) remains unexplored. The purpose of this work was to explore the potential role and mechanism of ZNF367 in SCLC. In silico analysis using the Gene Expression Omnibus (GEO) dataset revealed high levels of the ZNF367 transcript in SCLC. Examination of clinical tissues confirmed the significant abundance of ZNF367 in SCLC tissues compared with adjacent non-malignant tissues. The genetic depletion of ZNF367 in SCLC cells led to remarkable alterations in cell proliferation, the cell cycle, colony formation and chemosensitivity. Mechanistically, ZNF367 was shown to regulate the activation of yes-associated protein (YAP) associated with the up-regulation of phosphorylated large tumour suppressor kinase 2 (LATS2). Further investigation revealed that ZNF367 affected the LATS2-YAP cascade by regulating the expression of citron kinase (CIT). Re-expression of constitutively active YAP diminished the tumour-inhibiting function of ZNF367 depletion. Xenograft experiments confirmed the tumour-inhibiting effect of ZNF367 depletion in vivo. In summary, our results demonstrate that the inhibition of ZNF367 displays anticancer effects in SCLC by inhibiting YAP activation, suggesting it as a potential druggable oncogenic target.

Binucleated human hepatocytes arise through late cytokinetic regression during endomitosis M phase.

Binucleated polyploid cells are common in many animal tissues, where they arise by endomitosis, a non-canonical cell cycle in which cells enter M phase but do not undergo cytokinesis. Different steps of cytokinesis have been shown to be inhibited during endomitosis M phase in rodents, but it is currently unknown how human cells undergo endomitosis. In this study, we use fetal-derived human hepatocyte organoids (Hep-Orgs) to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, which is associated with the loss of four cortical anchoring proteins, RacGAP1, Anillin, SEPT9, and citron kinase (CIT-K). Moreover, reduction of WNT activity increases the percentage of binucleated cells in Hep-Orgs, an effect that is dependent on the atypical E2F proteins, E2F7 and E2F8. Together, we have elucidated how hepatocytes undergo endomitosis in human Hep-Orgs, providing new insights into the mechanisms of endomitosis in mammals.

Discovery and Characterization of Selective, First-in-Class Inhibitors of Citron Kinase.

Citron kinase (CITK) is an AGC-family serine/threonine kinase that regulates cytokinesis. Despite knockdown experiments implicating CITK as an anticancer target, no selective CITK inhibitors exist. We transformed a previously reported kinase inhibitor with weak off-target CITK activity into a first-in-class CITK chemical probe, C3TD879. C3TD879 is a Type I kinase inhibitor which potently inhibits CITK catalytic activity (biochemical IC50 = 12 nM), binds directly to full-length human CITK in cells (NanoBRET Kd < 10 nM), and demonstrates favorable DMPK properties for in vivo evaluation. We engineered exquisite selectivity for CITK (>17-fold versus 373 other human kinases), making C3TD879 the first chemical probe suitable for interrogating the complex biology of CITK. Our small-molecule CITK inhibitors could not phenocopy the effects of CITK knockdown in cell proliferation, cell cycle progression, or cytokinesis assays, providing preliminary evidence that the structural roles of CITK may be more important than its kinase activity.

Citron Kinase Is a Druggable Target in Treatment-Resistant Prostate Cancer.

Prolonged treatment with androgen deprivation therapy (ADT) inevitably leads to castration-resistant prostate cancer (CRPC). Development of novel androgen-targeting agents and chemo/radiotherapies has resulted in improved survival. However, metastatic CRPC remains incurable. New therapeutics are greatly needed, and exploration of novel pathways such as the mechanisms underlying prostate cancer cell proliferation could potentially augment the natural course of CRPC. In the latest issue of Cancer Research, Rawat and colleagues delved deeply into the mechanistic role of citron kinase (CIT) in orchestrating prostate cancer proliferation and revealed its catalytic activity as a druggable target for treatment-resistant prostate cancer. The researchers utilized in vitro and in vivo methodologies to elucidate the function of CIT in mediating uncontrolled interphase progression and prostate cancer growth. Furthermore, the authors employed both androgen receptor-dependent and independent models to validate the significance of CIT kinase activity as a crucial factor in driving treatment-resistant prostate cancer growth. At a mechanistic level they determined that the E2F2-Skp2-p27 axis regulates CIT expression. Finally, they defined the landscape of CIT substrates in prostate cancer that encompasses a spectrum of cellular functions that spans key proliferation regulators to alternative splicing events. This comprehensive work provides insights into CIT as a potential biomarker for prostate cancer treatment resistance and disease progression and establishes the CIT kinase domain as a druggable target in CRPC. See related article by Rawat et al., p. 4142.

Prostate Cancer Progression Relies on the Mitotic Kinase Citron Kinase.

The poorly characterized protein kinase citron kinase is a therapeutic target in prostate cancer that drives tumor growth by regulating diverse substrates, which control several hallmarks of aggressive prostate cancer progression. See related commentary by Mishra et al., p. 4008.

Lestaurtinib inhibits Citron kinase activity and medulloblastoma growth through induction of DNA damage, apoptosis and cytokinesis failure.

Medulloblastoma (MB), the most common malignant pediatric brain tumor, is currently treated with surgery followed by radiation and chemotherapy, which is accompanied by severe side effects, raising the need for innovative therapies. Disruption of the microcephaly-related gene Citron kinase (CITK) impairs the expansion of xenograft models as well as spontaneous MB arising in transgenic mice. No specific CITK inhibitors are available. Lestaurtinib, a Staurosporine derivative also known as CEP-701, inhibits CITK with IC50 of 90 nM. We therefore tested the biological effects of this molecule on different MB cell lines, as well as in vivo, injecting the drug in MBs arising in SmoA1 transgenic mice. Similar to CITK knockdown, treatment of MB cells with 100 nM Lestaurtinib reduces phospho-INCENP levels at the midbody and leads to late cytokinesis failure. Moreover, Lestaurtinib impairs cell proliferation through CITK-sensitive mechanisms. These phenotypes are accompanied by accumulation of DNA double strand breaks, cell cycle block and TP53 superfamily activation in vitro and in vivo. Lestaurtinib treatment reduces tumor growth and increases mice survival. Our data indicate that Lestaurtinib produces in MB cells poly-pharmacological effects extending beyond the inhibition of its validated targets, supporting the possibility of repositioning this drug for MB treatment.

Abstract A015: Citron kinase is a novel driver of prostate cancer progression and controls alternative splicing associated with treatment-resistance

Abstract Introduction Prostate cancer (CaP) will cause the deaths of more than 34,000 American men in 2022 because systemic treatments for metastatic CaP eventually fail. There is a need for actionable targets for novel treatments that work via entirely different mechanisms, bypass resistance to current therapies, and control the cellular processes that drive CaP progression to improve patient survival. We examine the contribution of citron kinase (CIT), a key mediator of cytokinesis, to CaP progression and explore the manner by which CIT impacts CaP cell behavior. Experimental procedures Cell proliferation and gene silencing, overexpression and mutation assays were performed in cell line models or PDX-derived organoids (PDXOs) reflecting treatment-resistant progression. Xenografting and tumorigenesis assays using cell lines that allow for CIT induction or repression were done in immunodeficient mice. Clinical relevance and translational potential of CIT was determined using immunohistochemistry and RNA-Seq assays combined with GSEA, MSigDB, rMATS and PCTA computational analyses. Mass spectrometry assays coupled with phospho-enrichment were done to identify CIT substrates; their interactions with CIT and RNA were confirmed via coIP and RNA-IP. Results We found that CIT controls CaP cell cycle progression and cell division and is overexpressed during clinical CaP progression. CIT overexpression was tumorigenic and overcame strategies to inhibit CaP growth. Conversely, CIT silencing inhibited the growth of cell lines and xenografts representing different stages of CaP progression and treatment resistance. Such silencing did not affect proliferation of benign epithelial prostate cells, indicating that CIT action is CaP-specific. CIT’s effects on CaP relied entirely on its kinase activity, which we isolated as druggable. Overexpression of kinase-dead CIT blunted CaP cell proliferation and pharmacological CIT inhibition decreased proliferation of treatment-resistant CaP cells and PDXOs, suggesting that CIT substrates mediate CaP growth. Using state-of-the-art proteomics approaches, we isolated the first CIT kinase substrates and found that over one-third function in RNA binding, metabolism and/or alternative splicing, a hallmark of CaP aggressiveness that is tightly controlled by phosphorylation. Combining RNA-Seq with bioinformatics analyses, we found that CIT induced alternative splicing patterns that were enriched in treatment-resistant castration-resistant CaP and neuroendocrine CaP. Moreover, putative CIT substrates were recruited to CIT-induced splicing sites, and CIT-controlled alternatively spliced transcripts differentially impacted CaP cell proliferation. Conclusion Collectively, these findings isolate CIT, and its kinase action, as a pivotal novel regulator of CaP progression. Moreover, CIT’s CaP driver function was mediated at least in part by clinically relevant alternative splicing events. These results suggest that blocking CIT or its dependent splicing events may overcome CaP-specific treatment resistance and progression. Citation Format: Chitra Rawat, Nidhi Singh, Gaurav Chauhan, Anja Rabljenovic, Salma Ben-Salem, Vishwa Vaghela, Varadha B. Venkadakrishnan, Jonathan MacDonald, Ujjwal Dahiya, Adam D. DePriest, Sanghee Lee, Michelle Muldong, Hyun-Tae Kim, Sangeeta Kumari, Qiang Hu, Eduardo Cortes, Scott M. Dehm, Amina Zoubeidi, Christina A.M. Jamieson, Marlo Nicolas, Jesse McKenney, Belinda Willard, Eric A. Klein, Cristina Magi-Galluzzi, Shaun R. Stauffer, Song Liu, Hannelore V. Heemers. Citron kinase is a novel driver of prostate cancer progression and controls alternative splicing associated with treatment-resistance [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A015.

Evolutionarily conserved midbody remodeling precedes ring canal formation during gametogenesis.

How canonical cytokinesis is altered during germ cell division to produce stable intercellular bridges, called "ring canals," is poorly understood. Here, using time-lapse imaging in Drosophila, we observe that ring canal formation occurs through extensive remodeling of the germ cell midbody, a structure classically associated with its function in recruiting abscission-regulating proteins in complete cytokinesis. Germ cell midbody cores reorganize and join the midbody ring rather than being discarded, and this transition is accompanied by changes in centralspindlin dynamics. The midbody-to-ring canal transformation is conserved in the Drosophila male and female germlines and during mouse and Hydra spermatogenesis. In Drosophila, ring canal formation depends on Citron kinase function to stabilize the midbody, similar to its role during somatic cell cytokinesis. Our results provide important insights into the broader functions of incomplete cytokinesis events across biological systems, such as those observed during development and disease states.

Voltage-Gated T-Type Calcium Channel Modulation by Kinases and Phosphatases: The Old Ones, the New Ones, and the Missing Ones.

Calcium (Ca2+) can regulate a wide variety of cellular fates, such as proliferation, apoptosis, and autophagy. More importantly, changes in the intracellular Ca2+ level can modulate signaling pathways that control a broad range of physiological as well as pathological cellular events, including those important to cellular excitability, cell cycle, gene-transcription, contraction, cancer progression, etc. Not only intracellular Ca2+ level but the distribution of Ca2+ in the intracellular compartments is also a highly regulated process. For this Ca2+ homeostasis, numerous Ca2+ chelating, storage, and transport mechanisms are required. There are also specialized proteins that are responsible for buffering and transport of Ca2+. T-type Ca2+ channels (TTCCs) are one of those specialized proteins which play a key role in the signal transduction of many excitable and non-excitable cell types. TTCCs are low-voltage activated channels that belong to the family of voltage-gated Ca2+ channels. Over decades, multiple kinases and phosphatases have been shown to modulate the activity of TTCCs, thus playing an indirect role in maintaining cellular physiology. In this review, we provide information on the kinase and phosphatase modulation of TTCC isoforms Cav3.1, Cav3.2, and Cav3.3, which are mostly described for roles unrelated to cellular excitability. We also describe possible potential modulations that are yet to be explored. For example, both mitogen-activated protein kinase and citron kinase show affinity for different TTCC isoforms; however, the effect of such interaction on TTCC current/kinetics has not been studied yet.

High Expression of Citron Kinase Contributes to the Development of Esophageal Squamous Cell Carcinoma.

ObjectiveThis study aimed to investigate the role and potential regulatory mechanism of citron kinase (CIT) in esophageal squamous cell carcinoma (ESCC).MethodsCitron kinase (CIT) expression in ESCC tissues was analyzed based on the microarray dataset GSE20347, and CIT expression in ESCC cell lines was analyzed. Eca-109 cells were lentivirally transfected with shRNA-CIT (LV-shCIT) to knock down CIT, followed by investigation of cell proliferation and apoptosis. Nude mouse xenograft experiments were performed to evaluate the tumorigenicity of CIT-knockdown Eca-109 cells. Microarray analysis of Eca-109 cells transfected with LV-shCIT or LV-shNC and subsequent Ingenuity Pathway Analysis (IPA) were performed to identify CIT-related differentially expressed genes (DEGs) and signaling pathways. Furthermore, the expression of key DEGs was validated using the clinical samples of ESCC.ResultsCitron kinase (CIT) was highly expressed in ESCC tissues and cell lines. Knockdown of CIT suppressed Eca-109 cell proliferation and promoted apoptosis in vitro. Moreover, CIT knockdown significantly reduced tumorigenicity of Eca-109 cells in vivo. Microarray and IPA analysis showed that signaling by the Rho family GTPases pathway was significantly activated, and CIT intrinsically interacted with the protein kinase AMP-activated catalytic subunit alpha 1 (PRKAA1), sequestosome 1 (SQSTM1), and interleukin 6 (IL6). Notably, the expression levels of PRKAA1 and SQSTM1 were upregulated in ESCC tissues, while the IL6 expression was downregulated.ConclusionOur findings confirm that CIT functions as an oncogene in ESCC. CIT may contribute to ESCC development by upregulating PRKAA1 and SQSTM1 as well as downregulating IL6. Citron kinase may serve as a promising therapeutic target for ESCC.

Goldberg-Shprintzen syndrome protein KIF1BP is a CITK interactor implicated in cytokinesis.

Goldberg-Shprintzen disease (GOSHS) is a rare microcephaly syndrome accompanied by intellectual disability, dysmorphic facial features, peripheral neuropathy and Hirschsprung disease. It is associated with recessive mutations in the gene encoding kinesin family member 1-binding protein (KIF1BP, also known as KIFBP). The encoded protein regulates axon microtubules dynamics, kinesin attachment and mitochondrial biogenesis, but it is not clear how its loss could lead to microcephaly. We identified KIF1BP in the interactome of citron kinase (CITK, also known as CIT), a protein produced by the primary hereditary microcephaly 17 (MCPH17) gene. KIF1BP and CITK interact under physiological conditions in mitotic cells. Similar to CITK, KIF1BP is enriched at the midbody ring and is required for cytokinesis. The association between KIF1BP and CITK can be influenced by CITK activity, and the two proteins may antagonize each other for their midbody localization. KIF1BP knockdown decreases microtubule stability, increases KIF23 midbody levels and impairs midbody localization of KIF14, as well as of chromosome passenger complex. These data indicate that KIF1BP is a CITK interactor involved in midbody maturation and abscission, and suggest that cytokinesis failure may contribute to the microcephaly phenotype observed in GOSHS.

Abstract B57: A combination of IL7Rα and NRAS mutations sheds light on the oncogenesis of T-cell acute lymphoblastic leukemia in a murine model

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer in children. While most cases of ALL arise from B-cell precursors, 15% originate from T cells in the thymus (T-ALL). With current treatments, survival rate is high, but relapse occurs in 15-20% of children. In addition, most treatments are highly toxic, with severe side effects. Approximately 10% of T-ALL patients have somatic gain-of-function mutations in IL7Rα gene, and 15-22% of ALL patients have mutations in NRAS or KRAS. Furthermore, Ras pathway mutations are responsible for 30%-50% of ALL relapse. Recently, our lab showed that mutations in human IL7Rα alone (c.731_732insTTGTCCCAC) or human NRAS alone (G13D) are not sufficient to induce T-ALL from primary cells. However, a combination of both causes severe T-ALL in mice. Treatment with inhibitors against IL7Rα and NRAS signaling led to prolonged survival in these mice. Our new data indicate that the correct ratio between IL7Rα and NRAS expression is necessary to generate T-ALL in mice. Using RAG-/- mice injected with primary thymocytes transduced with mutated IL7Rα and NRAS in the optimal sequence in a polycistronic vector led to fatal disease 3 weeks after injection. These mice showed a 30-fold increase of leukocytes in the blood, spleen, and thymus, 80-fold increase in the lymph nodes, and a 5-fold increase in spleen weight, all indications of severe T-ALL. There was no indication of disease progression in mice injected with either empty vector or wild-type IL7Rα and mutated NRAS. To identify the pathways involved in T-ALL by these mutations, we used mass spectrometry and RNA sequencing. Our analysis showed significant increases in the well-known antiapoptotic, B-cell lymphoma 2 (BCL-2), mRNA, and protein levels and a major decrease in the proapoptotic BCL-2 antagonist/killer 1 (BAK-1) protein levels. In this manner, combination of both IL7Rα and NRAS mutations resulted in high Bcl-2:Bak-1 ratio, supported also by Western blot analysis. In addition, we observed a significant increase in the protein cytokinesis midbody regulator citron kinase (Cit), potentially leading to increased thymocytes proliferation and driving T-ALL formation. Taken together, our lab developed a strong model to study T-ALL in mice, shedding light on novel pathways of oncogenesis and targets for better therapeutic development. Citation Format: Hila Winer, Gisele Rodrigues, Wenqing Li, Julie Hixon, Scott Durum. A combination of IL7Rα and NRAS mutations sheds light on the oncogenesis of T-cell acute lymphoblastic leukemia in a murine model [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B57.

Low expression of citron kinase is associated with poor patient outcomes in hepatocellular carcinoma.

BackgroundCitron kinase (CIT) is a protein related to cytokinesis and is an important abscission regulator. However, the relationship between CIT and hepatocellular carcinoma (HCC) is unclear. The aim of this study was to investigate the expression CIT in HCC tissues, and explore the connection between this expression and clinicopathological characteristics of HCC.MethodsImmunohistochemistry staining on 235 HCC tissues and 96 non-tumorous liver tissues controls was performed to examine the CIT protein expression. We then analyzed the correlation between protein expression and clinicopathological parameters via χ2 tests, and we performed overall survival analyses via the Kaplan-Meier survival approach. Based on the online Oncomine Expression Array and UALCAN databases, we more broadly compared CIT mRNA expression between normal and HCC tissues. Finally, we compared CIT mRNA expression in these databases to protein expression in our study and explored potential sources for any observe differences.ResultsCompared to normal tissues, CIT expression was significantly lower in HCC tissues. Low CIT expression was found to be related to gender, tumor size, Edmondson Grade, Microvascular invasion, serum AFP levels and poor overall survival. Based on the online databases, CIT mRNA expression was found to be high in HCC tissues and decreased in normal tissues. We hypothesize that this unexpected result is due to a negative feedback loop whereby low protein CIT levels mediate increased CIT mRNA levels.ConclusionsLower CIT protein levels are associated with a poorer prognosis in HCC patients, and lower CIT protein levels may mediate a negative feedback loop leading to increased CIT mRNA levels.

CITK Loss Inhibits Growth of Group 3 and Group 4 Medulloblastoma Cells and Sensitizes Them to DNA-Damaging Agents.

Medulloblastoma (MB) is the most common malignant brain tumor in children, and it is classified into four biological subgroups: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. The current treatment is surgery, followed by irradiation and chemotherapy. Unfortunately, these therapies are only partially effective. Citron kinase protein (CITK) has been proposed as a promising target for SHH MB, whose inactivation leads to DNA damage and apoptosis. D283 and D341 cell lines (Group 3/Group 4 MB) were silenced with established siRNA sequences against CITK, to assess the direct effects of its loss. Next, D283, D341, ONS-76 and DAOY cells were treated with ionizing radiation (IR) or cisplatin in combination with CITK knockdown. CITK depletion impaired proliferation and induced cytokinesis failure and apoptosis of G3/G4 MB cell lines. Furthermore, CITK knockdown produced an accumulation of DNA damage, with reduced RAD51 nuclear levels. Association of IR or cisplatin with CITK depletion strongly impaired the growth potential of all tested MB cells. These results indicate that CITK inactivation could prevent the expansion of G3/G4 MB and increase their sensitivity to DNA-damaging agents, by impairing homologous recombination. We suggest that CITK inhibition could be broadly associated with IR and adjuvant therapy in MB treatment.

High expression of citron kinase predicts poor prognosis of prostate cancer.

Citron kinase (CIT) is a Rho-effector protein kinase that is associated with several types of cancer. However, the role of CIT in prostate cancer (PCa) is unclear. The current study utilized microarray data obtained from The Cancer Genome Atlas, which was analyzed via Biometric Research Program array tools. Additionally, reverse transcription-quantitative (RT-q)PCR was performed to compare the mRNA expression of CIT in PCa tissue and in benign prostatic hyperplasia. The protein expression of CIT was detected in a consecutive cohort via immunochemistry and CIT was screened as a potential oncogene in PCa. The results of RT-qPCR demonstrated that the mRNA expression of CIT was increased in PCa tissues. Furthermore, immunochemistry revealed that CIT protein expression was positively associated with age at diagnosis, Gleason grade, serum PSA, clinical T stage, risk group, lymph node invasion and metastasis. When compared with the low expression group, patients with a high CIT expression exhibited shorter survival rates, cancer specific mortalities (CSM) and biochemical recurrence (BCR). In addition, multivariate analysis revealed that CIT was a potential predictor of CSM and BCR. The results revealed that CIT is overexpressed during the malignant progression of PCa and may be a predictor of a poor patient prognosis.

Abstract 390: Prostate cancer progression depends on the activity of the mitotic kinase citron kinase

Abstract Androgen receptor (AR) is the major driver of prostate cancer (CaP) progression. Despite initially inducing remissions, androgen deprivation therapy (ADT) does not cure CaP and the majority of CaP that recurs under ADT continues to rely on AR. In a subset of patients, administration of novel, more potent ADT drugs leads to emergence of a neuroendocrine (NE) CaP phenotype that is AR-indifferent and even harder to treat. The molecular mechanisms by which AR regulates cell cycle progression and that can be developed into alternative targets to inhibit CaP growth, overcome acquired resistance to ADT, or prevent NE progression, however, remain largely unknown. Here, we identify citron kinase (CIT), which controls cell division, as a novel drugabble target that acts downstream of AR. In multiple CaP models, CIT protein expression was stimulated selectively by low doses of androgens that promote CaP cell proliferation and decreased by AR silencing or short-term administration of AR-antagonist enzalutamide, confirming its AR dependence. Silencing of CIT significantly reduced CaP cell viability and cell proliferation, delayed cell progression, increased the number of multinucleated cells in ADT-naïve and -recurrent CaP cells, and attenuated CaP growth in xenograft models. Overexpression of CIT, transiently or stably, stimulated CaP cell proliferation in androgen-supplemented conditions and under ADT, and this depended entirely on an intact CIT kinase domain. In 2 independent clinical CaP datasets, CIT mRNA levels increased during CaP progression and higher CIT expression correlated with shorter disease-free and overall survival. Using CaP tissue microarrays that contain more than 200 patient specimens, CIT protein expression was significantly higher in primary CaP when compared to adjacent non-neoplastic prostate tissues and correlated with increasing Gleason scores, validating the relevance of CIT overexpression to CaP aggressiveness. Integrated RNA-Seq, MSigDB and GSEA analyses confirmed that the AR- and CIT-dependent transcriptome preferentially controlled CaP cell cycle progression and proliferation. Mechanistically, the use of inhibitors of gene transcription, mRNA translation and proteasome indicated that the androgen-dependence of CIT expression is regulated at the post-transcriptional level. Specifically, CIT protein expression was controlled by the E2F family of cell cycle regulators, with androgen-responsive E2F2 as the major determinant of CaP CIT expression. Our work, thus, isolated a novel role for the mitotic kinase CIT in AR-dependent CaP cell proliferation and clinical progression and identified CIT’s kinase moiety as a novel target for CaP therapy. Funding: NIH/NCI, Case Comprehensive Cancer Center Pilot Research Award Citation Format: Salma Ben-Salem, Salam Bachour, Varadha Balaji Venkadakrishnan, Yixue Su, Eduardo Cortes Gomez, Qiang Hu, Eric Klein, Nicolas Marlo, Cristina Magi-Galluzzi, Liu Song, Hannelore V. Heemers. Prostate cancer progression depends on the activity of the mitotic kinase citron kinase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 390.

Rho-dependent control of the Citron kinase, Sticky, drives midbody ring maturation.

Rho-dependent proteins control assembly of the cytokinetic contractile ring, yet it remains unclear how those proteins guide ring closure and how they promote subsequent formation of a stable midbody ring. Citron kinase is one important component required for midbody ring formation but its mechanisms of action and relationship with Rho are controversial. Here, we conduct a structure–function analysis of the Drosophila Citron kinase, Sticky, in Schneider’s S2 cells. We define two separable and redundant RhoGEF/Pebble-dependent inputs into Sticky recruitment to the nascent midbody ring and show that each input is subsequently required for retention at, and for the integrity of, the mature midbody ring. The first input is via an actomyosin-independent interaction between Sticky and Anillin, a key scaffold also required for midbody ring formation. The second input requires the Rho-binding domain of Sticky, whose boundaries we have defined. Collectively, these results show how midbody ring biogenesis depends on the coordinated actions of Sticky, Anillin, and Rho.

Citron kinase interacts with LATS2 and inhibits its activity by occluding its hydrophobic phosphorylation motif.

The inhibitory effect of large tumor suppressor kinase (LATS1/2) on the activity of the oncoprotein yes-associated protein (YAP) is crucial to maintain tissue homeostasis. Proteomic studies have identified several new regulators of this process. Recently, citron kinase (CIT) was listed as a potential binding candidate of Hippo-related components, suggesting a new connection between CIT and the Hippo pathway. Aside from CIT’s role in cytokinesis, the molecular crosstalk between CIT and the Hippo pathway is largely unknown. Here, we demonstrate a role for CIT as a scaffold protein linking LATS2 and YAP. More importantly, CIT interacts with LATS2 to directly suppress LATS2 phosphorylation at the hydrophobic motif—targeted by MST1, leading to LATS2 inactivation and YAP activation. By studying their genetic interactions, we found that Sticky, the CIT homolog in Drosophila melanogaster, functions with Warts to control Drosophila eye development. Together, our study confirms citron kinase as a novel regulator of the Hippo pathway.

Citron kinase (CIT-K) promotes aggressiveness and tumorigenesis of breast cancer cells in vitro and in vivo: preliminary study of the underlying mechanism

ObjectivesCitron kinase (CIT-K), as a key Rho effector, functions to maintain proper structure of the midbody during cell mitosis. This study assessed CIT-K expression and its role in breast cancer cells.MethodsParaffin-embedded breast cancer and para-tumor tissues from 43 invasive breast cancer patients and 33 normal mammary glands were collected for immunohistochemistry. CIT-K expression knockdown was achieved using lentivirus carrying CIT-K shRNA in a wide range of breast cancer cell lines. Cells were then subjected to Western blot, qRT-PCR, cell proliferation, colony formation, transwell, and flow cytometric assays. The tumorigenicity of CIT-K knocked-down breast cancer cells was assessed using the nude mouse xenograft assay. Microarray analysis was performed to elucidate the underlying gene regulation after CIT-K silencing.ResultsCIT-K protein was overexpressed in breast cancer tissues, which is associated with advanced tumor stage, HER-2 expression and Ki-67 expression, whereas knockdown of CIT-K expression reduced breast cancer cell proliferation and colony formation, but promoted tumor cell apoptosis and cell-cycle arrest. Knockdown of CIT-K expression also inhibited breast cancer cell migration and invasion capacity. Moreover, CIT-K knockdown suppressed the tumorigenicity of breast cancer cells in nude mice. Molecularly, the expression of a variety of signaling genes, such as cyclin D1, EGFR, JAK1, TGF-α, PTK2, RAF1, RALB, SOS1, mTOR, and PTGS2, were altered after CIT-K knockdown.ConclusionsThis study demonstrated that CIT-K is associated with aggressive breast cancer behavior and targeting CIT-K may be a novel strategy for the future control of breast cancer.

Inactivation of Citron Kinase Inhibits Medulloblastoma Progression by Inducing Apoptosis and Cell Senescence.

Medulloblastoma is the most common malignant brain tumor in children. Current treatment for medulloblastoma consists of surgery followed by irradiation of the whole neuraxis and high-dose multiagent chemotherapy, a partially effective strategy associated with highly invalidating side effects. Therefore, identification and validation of novel target molecules capable of contrasting medulloblastoma growth without disturbing brain development is needed. Citron kinase protein (CITK), encoded by primary microcephaly gene MCPH17, is required for normal proliferation and survival of neural progenitors. Constitutive loss of CITK leads to cytokinesis failure, chromosome instability, and apoptosis in the developing brain, but has limited effects on other tissues. On this basis, we hypothesized that CITK could be an effective target for medulloblastoma treatment. In medulloblastoma cell lines DAOY and ONS-76, CITK knockdown increased both cytokinesis failure and DNA damage, impairing proliferation and inducing cell senescence and apoptosis via TP53 or TP73. Similar effects were obtained in the NeuroD-SmoA1 transgenic mouse model, in which CITK deletion increased apoptotic cells and senescence markers such as P21CIP1, P27KIP1, and P16INK4A Most importantly, CITK deletion decreased tumor growth and increased overall survival in these mice, with no apparent side effects. These results suggest that CITK can be a useful molecular target for medulloblastoma treatment.Significance:In vitro and in vivo proof of concept identifies citron kinase protein as a suitable target for medulloblastoma treatment.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4599/F1.large.jpg Cancer Res; 78(16); 4599-612. ©2018 AACR.