CCNE1 Overexpression Research Articles

Abstract 3027: p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas

Abstract High-Grade Serous Ovarian Cancer (HGSOC) is the most common and aggressive gynecological malignancy. It is typically diagnosed in late stages due to lack of diagnostic tests and current therapies available are not efficacious when the tumor is advanced. HGSOC is characterized by ubiquitous loss of functional p53 tumor suppressor protein function, largely due to point mutations that arise very early in carcinogenesis. P53 mutations are detected in pre-neoplastic cells of the fallopian tube mucosa, called p53 signature, and in early precursor lesions called serous tubal intraepithelial carcinoma (STIC). Some mutations promote the misfolding and aggregation of p53, transforming the protein into a cancer-promoting oncogene. Here, we hypothesize that the transition from folded, soluble to aggregated mutant p53 underlies the transformation from a pre-neoplastic lesion (p53 signature, STILs), to STICs and eventually HGSOC. We first set to determine the proportion of cells carrying soluble vs misfolded mutant p53 in fallopian tube tissues collected from n=10 patients undergoing salpingo-oophorectomy. We performed conformation-sensitive staining and quantification of folded and unfolded p53 cases and demonstrated that p53 misfolding, a mandatory precursor to aggregation, is present in STICs and HGSOCs, but notably absent from p53 signatures and surrounding healthy tissue. To better characterize the role of aggregated p53 in the progression of HGSOC, we took advantage of primary human fallopian tube secretory epithelial cells (FTSEC) that mimic early and more advanced stages of carcinogenesis. We utilize the FTSEC cells line FT282, immortalized by introducing hTERT and the aggregation-prone mutant TP53R175H that is found in p53 signature lesions and STILs, as well as the FT282-CCNE1, derived from FT282 cell line with overexpression of CCNE1, representing a more malignant phenotype.We used these models to investigate p53 aggregation, characterize the mutant p53 interactome and test the efficacy of ReACp53, a p53 aggregation-targeting peptide we developed. Our results demonstrated that p53 aggregation is more severe in the FT282-CCNE1 line, with a higher susceptibility to ReACp53 compared to the FT282 cells. Overall, our results demonstrated that aggregation of mutant p53 is a structural defect that can distinguish between pre-malignant and malignant HGSOC lesions and can potentially be targeted, offering a potential window for early intervention in halting ovarian cancer progression. Citation Format: Sara Sartini, Lexi Omholt, Neda A. Moatamed, Alice Soragni. p53 aggregation promotes transformation from non-malignant lesions to high-grade serous ovarian carcinomas [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 3027.

Abstract 5871: Unveiling mechanisms of CDK4/6 inhibitor resistance in ER+ breast cancer models with acquired resistance

Abstract Introduction: Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, including palbociclib, ribociclib, and abemaciclib, have revolutionized the treatment of estrogen receptor-positive (ER+) breast cancer. However, the development of resistance to these inhibitors poses a formidable challenge in the clinical management of ER+ breast cancer. This study aims to explore the common resistance mechanisms in three CDK4/6 inhibitor-resistant models induced by chronic exposure to these agents. Methods: We developed models of resistance to palbociclib, ribociclib, and abemaciclib by repeatedly exposing murine xenografts to these CDK4/6 inhibitors. Resistance was confirmed through cell viability assays and in vivo tumorigenesis studies. The tumors were sequenced using NGS analyses, and genes of interest were validated through Western blotting and qPCR. Results: In all resistant models, we identified a common resistant mechanism via NGS analyses, which was characterized by the overexpression of the FAT1, CCNE1, and AR genes. The upregulation of these genes at mRNA and protein levels was confirmed by qPCR analyses and western blotting, respectively. Our data indicate that upregulation of FAT1, CCNE1, and AR genes may play a critical role in mediating resistance to CDK4/6 inhibitors, namely palbociclib, ribociclib, and abemaciclib. Conclusion: Our investigation uncovered a shared mechanism, characterized by the overexpression of FAT1, CCNE1, and AR genes, that underlay CDK4/6 inhibitors-induced drug resistance in ER+ breast cancer. These findings shed light on the shared pathways that drive CDK4/6 inhibitor resistance and provide insights into potential therapeutic targets for overcoming this resistance in breast cancer patients. Understanding these mechanisms is crucial for the development of more effective treatment strategies for CDK4/6 inhibitor-resistant cancers. Citation Format: Bin Li, Gaoxiang Liu, Ting Ni, Wenting Shi, Qingyang Gu. Unveiling mechanisms of CDK4/6 inhibitor resistance in ER+ breast cancer models with acquired resistance [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 5871.

Abstract B173: The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers

Abstract Cyclin-dependent kinase 2 (CDK2) plays a crucial role in the regulation of the cell cycle and is involved in a variety of biological processes. It is responsible for phosphorylating proteins involved in G1 and S phase cell cycle progression, DNA damage response, intracellular transport, protein degradation, signal transduction, and DNA and RNA metabolism. CDK2 activity is particularly significant in cells exhibiting CCNE1 amplification or overexpression. Additionally, CDK2 activity has been linked to potential resistance against chemotherapy or targeted therapy. Several studies have indicated that abnormal activation of CDK2 may contribute to resistance against targeted therapies, such as CDK4/6 inhibitors and KRAS inhibitors. To investigate this hypothesis, we conducted experiments testing the combined effect of ARTS-021, a highly selective and potent CDK2 inhibitor, with Palbociclib, a CDK4/6 inhibitor, or AMG510, a KRAS-G12C inhibitor, in various xenograft tumor models. The combination of CDK2 inhibition with CDK4/6 inhibition demonstrated superior efficacy compared to either agent alone. This combination induced tumor regression in both Palbociclib-sensitive T47D parental cell line models and Palbociclib-resistant T47D cells overexpressing CCNE1. Similar benefits were also observed when CDK2 inhibition was combined with KRAS G12C inhibition in multiple KRAS-G12C lung cancer cell line xenografts. Importantly, these combination treatments were well tolerated. These findings suggest that CDK2 may play a crucial role in cancer cell resistance to chemotherapy or targeted therapy, and a CDK2 inhibitor may have broader applications beyond CCNE1-amplified cancers, as it can be combined with different treatment modalities. Citation Format: Yaoyu Chen, Jinhong Chen, Yali Guo, Wei Pang, Tingru Zhou, Xipan Liu, Nathan Schomer, Fang Li, Jiaqi Liang, Qing Sheng. The anti-tumor activity of CDK2 inhibition alone or in combination with other anti-cancer agents in human cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B173.

PPM1D activity promotes the replication stress caused by cyclin E1 overexpression.

Oncogene-induced replication stress has been recognized as a major cause of genome instability in cancer cells. Increased expression of cyclin E1 caused by amplification of the CCNE1 gene is a common cause of replication stress in various cancers. Protein phosphatase magnesium-dependent 1 delta (PPM1D) is a negative regulator of p53 and has been implicated in termination of the cell cycle checkpoint. Amplification of the PPM1D gene or frameshift mutations in its final exon promote tumorigenesis. Here, we show that PPM1D activity further increases the replication stress caused by overexpression of cyclin E1. In particular, we demonstrate that cells expressing a truncated mutant of PPM1D progress faster from G1 to S phase and fail to complete licensing of the replication origins. In addition, we show that transcription-replication collisions and replication fork slowing caused by CCNE1 overexpression are exaggerated in cells expressing the truncated PPM1D. Finally, replication speed and accumulation of focal DNA copy number alterations caused by induction of CCNE1 expression was rescued by pharmacological inhibition of PPM1D. We propose that increased activity of PPM1D suppresses the checkpoint function of p53 and thus promotes genome instability in cells expressing the CCNE1 oncogene.

Circular RNA circSEMA5A facilitates colorectal cancer development by regulating microRNA-195-5p to target CCNE1 axis

Colorectal cancer (CRC) is one of the most prevalent malignancies with a high mortality rate worldwide. Circular RNAs (circRNAs) have lately emerged as key molecules involved in cancer development and metastasis. CircSEMA5 is reported to be oncogenic in some cancers, yet its role in the pathogenesis of CRC remains unknown. Herein, we attempted to investigate the functional role and molecular mechanism of circSEMA5A underlying CRC progression. RT-qPCR and RNase R digestion assays were used to evaluate circSEMA5A expression characteristics in CRC cells. Loss-of-function assays were performed to clarify circSEMA5A role in CRC biological processes. Bioinformatics and mechanism experiments were conducted to assess the association of circSEMA5A or CCNE1 with miR-195-5p in CRC cells. Rescue assays were conducted to explore the regulatory function of circSEMA5A-miR-195-5p-CCNE1 in CRC cellular processes. Through bioinformatics and functional screening, we found that circSEMA5A was highly expressed in CRC cells and was mainly localized in the nucleus. CircSEMA5A promoted CRC proliferative, migratory, and invasive capabilities in cultured cells and facilitated the tumorigenic process in xenografts; however, circSEMA5A silencing repressed tumor metastasis in CRC cells. Mechanistically, circSEMA5A was competitively bound with miR-195-5p to upregulate CCNE1 expression. Moreover, the impact of circSEMA5A knockdown on CRC cell proliferative, migratory, and invasive capabilities was countervailed by miR-195-5p inhibitor or CCNE1 overexpression. To summarize, circSEMA5A is a novel circRNA that serves as an oncogene in CRC progression. CircSEMA5A facilitates CRC cell malignancy and tumor growth through sponging miR-195-5p to upregulate CCNE1, thus providing a new direction for CRC diagnosis and targeted therapy.

Abstract 1143: Development of a CDK2-selective small molecule inhibitor INCB123667 for the treatment of CCNE1hi breast cancers

Abstract Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitors have been approved for treatment of metastatic ER+ HER2− breast cancer as monotherapy or in combination with endocrine therapy. However, many patients become resistant to CDK4/6 therapy due to various mechanisms such as loss of tumor suppressor Rb, hyperactivation of CDK4/6, and/or CDK2/Cyclin E1 expression and activity, resulting in a highly unmet clinical need. Triple-negative breast cancers (TNBC), owing to a lack of hormone receptor (HR) expression and extensive heterogeneity, remain notoriously aggressive and unresponsive to current therapies. CCNE1 overexpression or amplification has been shown to be an oncogenic driver associated with poor clinical outcomes in ovarian, gastric, endometrial, and HR+ breast cancers. CCNE1hi cancers are dependent on CDK2 for growth and survival. CDK2 in complex with CCNE1 phosphorylates Rb, resulting in E2F target gene expression and G1 to S-phase cell cycle progression. Using a potent and selective CDK2 inhibitor, INCB123667, we demonstrate the efficacy of CDK2 inhibition in CCNE1hi breast cancer models. Analysis of genome-wide CRISPR screen data identified a subset of CCNE1hi breast cancer models dependent on CDK2 comprised of basal and luminal breast cancer subtypes, including TNBC, ER+ HER2−, and HR− HER2+ models. Two CCNE1hi cell lines, HCC1569 and MDAMB157, were evaluated in cell assays monitoring pRb, S-phase, and growth inhibition, and found to be sensitive to treatment with INCB123667 and insensitive to multiple CDK4/6 inhibitors. Through extensive biochemical studies, INCB123667 was observed to be potently active against CDK2 at subnanomolar range and inactive against other CDKs (CDK1, CDK4, CDK6, CDK7, and CDK9). INCB123667 also selectively induced senescence in CCNE1hi HCC1569 cells. In contrast, CCNE1 nonamplified cell lines, MCF7 and T47D, were inactive in pRb, S-phase, and growth inhibition assays when treated with INCB123667. Significant tumor growth inhibition was observed in CCNE1hi PDX and xenograft models with minimal body weight loss. We also observed dose-dependent inhibition of CDK2 substrates, including pRb S780, both in vitro and in vivo. To further characterize downstream signaling and transcriptional changes with CDK2 inhibition, we identified a discrete gene signature associated with cell cycle regulation, which was notably distinct from CDK4/6 responsive target genes. In conclusion, we have demonstrated CDK2 activity in CCNE1hi breast cancers using INCB123667, a potent and selective small molecule inhibitor of CDK2 currently in clinical development. Treatment resulted in cell growth inhibition and antitumor activity in vitro and in vivo. Our data demonstrate the therapeutic potential of CDK2 inhibition in CCNE1hi HR+ HER2− and TNBC breast cancer models. Acknowledgments: R&D services provided by Pharmaron, Crown Bioscience, and Champions Oncology Citation Format: Saswati Chand, Michael Hansbury, Yvonne Lo, Pat Feldman, Justine Carl, Cynthia Timmers, Joshua Hummel, Susan Wee, Sunkyu Kim. Development of a CDK2-selective small molecule inhibitor INCB123667 for the treatment of CCNE1hi breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1143.

Abstract P4-08-12: Clinical and Genomic Landscape of Breast Cancers Carrying CCNE1 Amplification

Abstract Background: Cyclin E1 (CCNE1) plays a critical role in cell cycle regulation. CCNE1 overexpression and/or gene amplification (amp) have been associated with poor outcome in several tumors, including breast cancer (BC). CCNE1 amp has recently been identified as a potential therapeutic target for novel synthetic lethality-based therapies. Here we sought to define the clinical and genomic features of BCs carrying CCNE1 amp. Methods: Genomic and clinical data from all consecutive BCs, which had been subjected to targeted sequencing using the FDA-authorized MSK-IMPACT assay from April 2014 to December 2021, were retrieved. Allele-specific copy number and fraction genome altered (FGA) were assessed using FACETS. Whole genome doubling (WGD) status was inferred from MSK-IMPACT sequencing data. Samples were categorized as CCNE1 amp or non-amp based on copy number profile assessed by FACETS. Mutual exclusivity and co-occurrence analyses between CCNE1 amp and other genetic alterations were performed using CoMEt. Multiple testing correction using the Benjamini–Hochberg procedure was applied to control for the false discovery rate. Progression-free survival (PFS) was assessed by Kaplan Meier method and Cox proportional-hazards models. Survival analyses were restricted to only patients with available pre-treatment samples. Results: Of 3,753 BCs with full clinical and genomic data, 125 (3.3%) harbored CCNE1 amp. A significant difference in the proportion of CCNE1 amp between treatment-naïve and post-treatment/metastatic samples was observed (2.4% vs 4%, p=0.007). CCNE1 amp was significantly less frequently detected in hormone receptor (HR)+/HER2- BCs than in HR-/HER2+ and HR-/HER2- subtypes (2% vs 7.6% and 7.2%, respectively, p< 0.001), and was particularly rare in invasive lobular BCs (1/452 cases). BCs with CCNE1 amp displayed a higher frequency of WGD (p< 0.001) and higher median FGA (p< 0.001) than non-amp tumors, overall and in different subtypes, suggesting increased genomic instability. No difference in tumor mutational burden (TMB) between CCNE1 amp and non-amp was found. In primary BC (n=1,385), a higher proportion of TP53 alterations was found in cases with CCNE1 amp (odds ratio [OR] 6.0, 95% confidence interval [CI] 2.5-16.6, q< 0.001). Conversely, CCNE1 amp was mutually exclusive with CDH1 alterations (q< 0.001). Comparable results were found in the analysis of post-treatment/metastatic samples (n=2,368). A subset analysis on HR+/HER2- BCs confirmed that TP53 (OR 4.2, 95%CI 2.28-8.11, q< 0.001) and CDH1 (OR 0.09, 95%CI 0.002-0.57, q< 0.1) alterations co-occurred and were mutually exclusive, respectively, with CCNE1 amp. ARID2 alterations were also enriched in HR+/HER2- tumors harboring CCNE1 amp (OR 10.6, 95%CI 2.54-33.93, q< 0.1). CCNE1 amp was significantly associated with reduced median PFS (8.8 vs 15.2 months in CCNE1 amp [n=9] vs CCNE1 non-amp [n=402]; hazard ratio [HR] 2.82, 95% CI 1.38-5.75, p=0.004) on first line treatment with CDK4/6 inhibitor plus endocrine therapy (ET) in HR+/HER2- metastatic BCs, regardless of the ET partner, FGA and TMB. CCNE1 amp was also associated with numerically inferior median PFS (7.3 vs 20.8 months in CCNE1 amp [n=5] vs CCNE1 non-amp [n=106]; HR 3.1, 95% CI 1.24-7.87, p=0.01) on first line trastuzumab/pertuzumab/taxane treatment in HER2+ metastatic BCs, with a trend toward significance after adjusting for FGA and TMB (p=0.09). Conclusions: CCNE1 amp is associated with specific clinicopathological and genomic features in BCs and linked to an increased genomic instability. CCNE1 amp defines a subset of metastatic BCs with marked poor clinical response to available standard-of-care treatments. Further studies testing novel therapeutic approaches, including synthetic lethality-based strategies targeting CCNE1 amp and CDK2-selective inhibition, are warranted. Citation Format: Antonio Marra, Pier Selenica, Yingjie Zhu, Pedram Razavi, Anton Safonov, Emanuela Ferraro, Sarat Chandarlapaty, Jorge Reis-Filho. Clinical and Genomic Landscape of Breast Cancers Carrying CCNE1 Amplification [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-12.

CCNE1 and survival of patients with tubo-ovarian high-grade serous carcinoma: An Ovarian Tumor Tissue Analysis consortium study.

Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC. Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR],1.26; 95% CI, 1.08-1.47, p=.034, and HR,1.18; 95% CI, 1.05-1.32, p=.015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR,1.26; 95% CI, 1.09-1.47, p=.033). CCNE1 mRNA expression was not associated with overall survival (HR,1.00 per 1-SD increase; 95% CI, 0.94-1.06; p=.58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss. This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC.

Preclinical evaluation of CDK4 phosphorylation predicts high sensitivity of pleural mesotheliomas to CDK4/6 inhibition.

Malignant pleural mesothelioma (MPM) is an aggressive cancer with limited therapeutic options. We evaluated the impact of CDK4/6 inhibition by palbociclib in 28 MPM cell lines including 19 patient-derived ones, using various approaches including RNA-sequencing. Palbociclib strongly and durably inhibited the proliferation of 23 cell lines, indicating a unique sensitivity of MPM to CDK4/6 inhibition. When observed, insensitivity to palbociclib was mostly explained by the lack of active T172-phosphorylated CDK4. This was associated with high p16INK4A (CDKN2A) levels that accompany RB1 defects or inactivation, or (unexpectedly) CCNE1 overexpression in the presence of wild-type RB1. Prolonged palbociclib treatment irreversibly inhibited proliferation despite re-induction of cell cycle genes upon drug washout. A senescence-associated secretory phenotype including various potentially immunogenic components was irreversibly induced. Phosphorylated CDK4 was detected in 80% of 47 MPMs indicating their sensitivity to CDK4/6 inhibitors. Its absence in some highly proliferative MPMs was linked to very high p16 (CDKN2A) expression, which was also observed in public datasets in tumours from short-survival patients. Our study supports the evaluation of CDK4/6 inhibitors for MPM treatment, in monotherapy or combination therapy.

CCNE1 Promotes Progression and is Associated with Poor Prognosis in Lung Adenocarcinoma.

Mounting evidence has shown that Cyclin E1 (CCNE1) facilitates various carcinoma progression, but its function in lung adenocarcinoma (LUAD) remains unclear. Our study aims to explore the significance of CCNE1 in clinical progression and study its biological functions in LUAD. CCNE1 expressions in LUAD specimens and cells were detected through quantitative realtime polymerase chain reaction (qRT-RCR) and western blot. An immunohistochemistry technique was used to detect CCNE1 expression to explore its association with clinical parameters. The LUAD cells with stable knockdown of CCNE1 were constructed by small interfering RNA. The effect of CCNE1 on LUAD cells proliferation and apoptosis was evaluated through Cell Counting Kit-8 (CCK-8), colony formation, and Annexin V/propidium iodide (AV-PI) assays, respectively. The cell migration and invasion were evaluated by Wound-healing and Transwell assays, respectively. The xenograft and lung metastasis mouse models were introduced to analyze how CCNE1 knockdown affects tumor growth and tumor metastasis. CCNE1 expression was upregulated in LUAD tissue and cells. CCNE1 knockdown inhibited LUAD cellular malignant behavior in vitro and reduced tumor growth and metastasis in vivo. High expression of CCNE1 was correlated with big tumor size, cancer stage, lymph node metastasis, and poor prognosis. CCNE1 overexpression promotes LUAD growth, metastasis, and forebode poor prognosis: it can serve as a new prognostic marker of LUAD.

LncRNA OXCT1-AS1 promotes the proliferation of non-small cell lung cancer cells by targeting the miR-195/CCNE1 axis.

BackgroundLong non-coding RNAs (lncRNAs) are involved in various biological processes in non-small cell lung cancer (NSCLC). This study aimed to investigate the key lncRNA OXCT1-AS1/miR-195/CCNE1 axis in the development of NSCLC and its potential molecular mechanism.MethodsLncRNA OXCT1-AS1 is considered to be a competitive endogenous RNA (ceRNA) and its potential targeting microRNAs (miRNAs) were predicted through LncBase predicted v.2. The expression of OXCT1-AS1 and miR-195 in NSCLC tissues and cells was detected by reverse transcription polymerase chain reaction (RT-PCR). The Cell Counting Kit-8 (CCK-8) and cell colony-forming test were used to detect the effect of cell proliferation. RT-PCR was used to detect the expression changes of CCND1 and CCNE1. Western Blot was used to detect the changes of the CCNE1 cell cycle protein. Dual luciferase activity was used to determine the potential mechanism of lncRNA OXCT1-AS1.ResultsLncRNA OXCT1-AS1 was highly expressed and miR-195 was lowly expressed in NSCLC tissues and cell lines. LncBase predicted v.2.0 reported a high-scoring binding between OXCT1-AS1 and miR-195. The luciferase reporter assay defined the regulatory relationship between OXTC1-AS1 and miR-195. In NSCLC cells, knockdown of OXCT1-AS1 significantly increased the expression of miR-195, decreased the proliferation and colony formation number of cancer cells, and reduced the expression of CCND1 and CCNE1. Meanwhile, overexpression of miR-195 significantly inhibited the cell proliferation and colony formation number, and reduced the expression of CCND1 and CCNE1. Furthermore, according to the results of the dual-luciferase activity assay, miR-195 targeted the 3' untranslated regions (3' UTRs) of CCNE1, validating that CCNE1 is a direct target of miR-195. Overexpression of CCNE1 restored the role of OXCR1-AS1 in NSCLC cells.ConclusionsLncRNA OXCT1-AS1 can regulate the proliferation of NSCLC cells via miR-195/CCNE1 signaling. Therefore, OXCT1-AS1 may act as a prospective biomarker and therapeutic target for patients with NSCLC.

CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1–4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR–Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB–FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Multi-omics analyses provide novel biological insights to distinguish lobular ductal types of invasive breast cancers.

Invasive lobular carcinoma (ILC) treatment is similar to invasive ductal carcinoma (IDC; now invasive carcinoma-no special type, IBC-NST), based on its intrinsic subtype. However, further investigation is required for an integrative understanding of differentially perturbed molecular patterns and pathways in these histotypes. A dataset of 780 IDC and 201 ILC samples from the TCGA-BRCA project for cross-platform multi-omics was analyzed. We leveraged a consensus approach integrating different bioinformatic algorithms to analyze mutations, CNAs, mRNA, miRNA abundance, methylation, and protein abundance to understand the complex crosstalks that distinguish ILC and IDC samples. A histotype-matched comparison was performed. We performed Cox survival analyses for prognosis based on our identified 53 histotype-specific and four discordant genes. Approximately 90% of ILC cases were of the luminal subtype. Somatic mutations in CDH1 were higher in ILC than in IDC (FDR-adjusted p < 0.01). Fifty-three significant oncogenic or tumor-suppressive DEGs were identified in a single histotype. PPAR signaling and lipolysis regulation in adipocytes were significantly enriched in ILC tumors. CDH1 protein had the highest differential abundance (AUC: 0.85). Moreover, BTG2, GSTA2, GPR37L1, and PGBD5 amplification was associated with poorer OS in ILC compared with no alteration. RIMS2, NACA4P, MYC, ZFPM2, and POU5F1B amplification showed a lower overall survival in patients with IDC. miR-195 showed an IDC-specific downregulation, causing overexpression of CCNE1. Integrative multi-omics supervised analysis identified 296 differentially expressed genes that successfully distinguished IDC and ILC histotypes. Our findings identify novel molecular candidates that potentially drive and modify the disease differentially among these histotypes.

CCNE1 and E2F1 Partially Suppress G1 Phase Arrest Caused by Spliceostatin A Treatment.

The potent splicing inhibitor spliceostatin A (SSA) inhibits cell cycle progression at the G1 and G2/M phases. We previously reported that upregulation of the p27 cyclin-dependent kinase inhibitor encoded by CDKN1B and its C-terminal truncated form, namely p27*, which is translated from CDKN1B pre-mRNA, is one of the causes of G1 phase arrest caused by SSA treatment. However, the detailed molecular mechanism underlying G1 phase arrest caused by SSA treatment remains to be elucidated. In this study, we found that SSA treatment caused the downregulation of cell cycle regulators, including CCNE1, CCNE2, and E2F1, at both the mRNA and protein levels. We also found that transcription elongation of the genes was deficient in SSA-treated cells. The overexpression of CCNE1 and E2F1 in combination with CDKN1B knockout partially suppressed G1 phase arrest caused by SSA treatment. These results suggest that the downregulation of CCNE1 and E2F1 contribute to the G1 phase arrest induced by SSA treatment, although they do not exclude the involvement of other factors in SSA-induced G1 phase arrest.

CCNE1 copy number is a biomarker for response to combination WEE1-ATR inhibition in ovarian and endometrial cancer models

SummaryCCNE1-amplified ovarian cancers (OVCAs) and endometrial cancers (EMCAs) are associated with platinum resistance and poor survival, representing a clinically unmet need. We hypothesized that dysregulated cell-cycle progression promoted by CCNE1 overexpression would lead to increased sensitivity to low-dose WEE1 inhibition and ataxia telangiectasia and Rad3-related (ATR) inhibition (WEE1i-ATRi), thereby optimizing efficacy and tolerability. The addition of ATRi to WEE1i is required to block feedback activation of ATR signaling mediated by WEE1i. Low-dose WEE1i-ATRi synergistically decreases viability and colony formation and increases replication fork collapse and double-strand breaks (DSBs) in a CCNE1 copy number (CN)-dependent manner. Only upon CCNE1 induction does WEE1i perturb DNA synthesis at S-phase entry, and addition of ATRi increases DSBs during DNA synthesis. Inherent resistance to WEE1i is overcome with WEE1i-ATRi, with notable durable tumor regressions and improved survival in patient-derived xenograft (PDX) models in a CCNE1-level-dependent manner. These studies demonstrate that CCNE1 CN is a clinically tractable biomarker predicting responsiveness to low-dose WEE1i-ATRi for aggressive subsets of OVCAs/EMCAs.

Knockdown of SENP1 inhibits HIF-1α SUMOylation and suppresses oncogenic CCNE1 in Wilms tumor

Based on our initial bioinformatics finding of the upregulated expression of sentrin-specific protease 1 (SENP1) and cyclin E1 (CCNE1) in Wilms tumor, this study aimed to illustrate the molecular mechanism of SENP1 in Wilms tumor, which involved the hypoxia-inducible factor 1α (HIF-1α)/stanniocalcin-1 (STC1)/CCNE1 axis. Wilms tumor and adjacent normal tissues were clinically collected. Gain- and loss-of-function assays were performed to evaluate the effects of the regulatory axis on malignant phenotypes of Wilms tumor cells. A mouse model of Wilms tumor xenografts was further established for in vivo substantiation. Overexpression of CCNE1 and SENP1 occurred in Wilms tumor tissues and cells. Silencing SENP1 inhibited viability and enhanced cell-cycle arrest of Wilms tumor cells. SENP1 promoted STC1 expression and upregulated CCNE1 by driving the small ubiquitin-like modifier (SUMO)ylation of HIF-1α, which ultimately promoted the malignant phenotypes of Wilms tumor cells. It was further confirmed that silencing SENP1 downregulated the expression of CCNE1 and restricted tumorigenicity of Wilms tumor cells in vivo. Taken together, SENP1 elevated STC1 expression by driving the SUMOylation of HIF-1α, thereby upregulating the expression of CCNE1 and ultimately promoting the development of Wilms tumor.

Retrospective analysis of the role of cyclin E1 overexpression as a predictive marker for the efficacy of bevacizumab in platinum-sensitive recurrent ovarian cancer.

The relative benefit of bevacizumab in ovarian cancer (OC) patients is greater the more the disease becomes platinum-resistant. Among other mechanisms of action, antiangiogenic agents may induce homologous recombination deficiency. Cyclin E1 (CCNE1) overexpression is a proposed marker of platinum resistance and is mutually exclusive with deficiency in homologous recombination. In this study, we evaluated the predictive value of CCNE1 expression with regard to the efficacy of bevacizumab. We retrospectively evaluated data from patients with platinum-sensitive recurrent OC who were treated with chemotherapy (CT) plus bevacizumab (Bev group) or CT alone (CT group) at a tertiary cancer centre from 2005 to 2017. The two groups were paired according to histology, platinum-free interval (PFI) and number of previous treatment lines. Progression-free survival (PFS) was compared between groups by log rank test and Cox regression. A total of 124 patients were included, with 62 in each group. The groups were well balanced regarding histology, PFI and number of previous treatment lines. Median PFS (mPFS) was 19.5 months for the Bev group versus 16.0 months for CT group (p = 0.150). By multivariate analysis, the HR for PFS was 2.25 (95% CI: 1.10–4.60) for CCNE1 overexpression. The benefit of bevacizumab was larger in the subgroups of patients with PFI 6–12 months (mPFS 18.6 versus 10.4 months, p = 0.002) and CCNE1 overexpression (mPFS 16.3 versus 7.0 months, p = 0.010). In conclusion, CCNE1 overexpression and PFI may suggest which patients will receive the greatest benefit from bevacizumab. These data, if confirmed by other studies, could help better select patients for antiangiogenic therapy.

Overexpression of CCNE1 confers a poorer prognosis in triple-negative breast cancer identified by bioinformatic analysis

BackgroundTriple-negative breast cancer (TNBC) is a major subtype of breast cancer. Due to the lack of effective therapeutic targets, the prognosis is poor. In order to find an effective target, despite many efforts, the molecular mechanisms of TNBC are still not well understood which remain to be a profound clinical challenge.MethodsTo identify the candidate genes in the carcinogenesis and progression of TNBC, microarray datasets GSE36693 and GSE65216 were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified, and functional and pathway enrichment analyses were performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases via DAVID. We constructed the protein-protein interaction network (PPI) and performed the module analysis using STRING and Cytoscape. Then, we reanalyzed the selected DEG genes, and the survival analysis was performed using cBioportal.ResultsA total of 140 DEGs were identified, consisting of 69 upregulated genes and 71 downregulated genes. Three hub genes were upregulated among the selected genes from PPI, and biological process analysis uncovered the fact that these genes were mainly enriched in p53 pathway and the pathways in cancer. Survival analysis showed that only CCNE1 may be involved in the carcinogenesis, invasion, or recurrence of TNBC. The expression levels of CCNE1 were significantly higher in TNBC cells than non-TNBC cells that were detected by qRT-PCR (P < 0.05).ConclusionCCNE1 could confer a poorer prognosis in TNBC identified by bioinformatic analysis and plays key roles in the progression of TNBC which may contribute potential targets for the diagnosis, treatment, and prognosis assessment of TNBC.

Genetically Defined Syngeneic Mouse Models of Ovarian Cancer as Tools for the Discovery of Combination Immunotherapy.

Despite advances in immuno-oncology, the relationship between tumor genotypes and response to immunotherapy remains poorly understood, particularly in high-grade serous tubo-ovarian carcinomas (HGSC). We developed a series of mouse models that carry genotypes of human HGSCs and grow in syngeneic immunocompetent hosts to address this gap. We transformed murine-fallopian tube epithelial cells to phenocopy homologous recombination-deficient tumors through a combined loss of Trp53, Brca1, Pten, and Nf1 and overexpression of Myc and Trp53 R172H, which was contrasted with an identical model carrying wild-type Brca1. For homologous recombination-proficient tumors, we constructed genotypes combining loss of Trp53 and overexpression of Ccne1, Akt2, and Trp53 R172H, and driven by KRAS G12V or Brd4 or Smarca4 overexpression. These lines form tumors recapitulating human disease, including genotype-driven responses to treatment, and enabled us to identify follistatin as a driver of resistance to checkpoint inhibitors. These data provide proof of concept that our models can identify new immunotherapy targets in HGSC. SIGNIFICANCE: We engineered a panel of murine fallopian tube epithelial cells bearing mutations typical of HGSC and capable of forming tumors in syngeneic immunocompetent hosts. These models recapitulate tumor microenvironments and drug responses characteristic of human disease. In a Ccne1-overexpressing model, immune-checkpoint resistance was driven by follistatin.This article is highlighted in the In This Issue feature, p. 211.

Combined CDK2 and CDK4/6 Inhibition Overcomes Palbociclib Resistance in Breast Cancer by Enhancing Senescence.

Simple SummaryCyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used to treat metastatic hormone receptor-positive/human epidermal growth factor receptor 2-negative breast cancer. Despite the effectiveness of CDK4/6 inhibitors, acquired resistance occurs in almost all cases. Strategies to address this issue have not been developed yet. We investigated mechanisms of resistance to CDK4/6 inhibitor in breast cancer and potential therapeutic strategies. We found that cyclin E-CDK2 mediated phosphorylation of C-MYC is responsible for resistance to CDK4/6 inhibitor by suppressing C-MYC induced senescence. On the contrary, the synergistic anti-proliferative effect of the combined inhibition of CDK2 and CDK4/6 overcomes acquired resistance to CDK4/6 inhibitors by enhancing senescence. Our findings could pave the way for the development CDK2-specific kinase inhibitor for the treatment of breast cancers that are resistant to CDK4/6 inhibitor.Breast cancer represents the number one global cancer burden in women and the hormone receptor (HR)-positive subtype comprises approximately 70% of breast cancers. Unfortunately, acquired resistance ultimately occurs in almost all cases, even though cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are a highly effective therapy for HR-positive/human epidermal growth factor receptor 2-negative subtype. Here, we investigated mechanisms of resistance to CDK4/6 inhibitor and potential therapeutic strategies using our palbociclib-resistant preclinical model. We observed that cyclin E was significantly overexpressed in palbociclib-resistant cells, and similar association was also confirmed in pleural effusion samples collected from HR-positive breast cancer patients. After confirmation of cyclin E-CDK2 interaction by co-immunoprecipitation, we demonstrated CDK2 inhibition combined with palbociclib synergistically suppressed proliferation of palbociclib-resistant cells and growth of palbociclib-resistant xenograft in mice. We also proved that enhancing C-MYC-mediated senescence is a novel mechanism behind the synergism created by targeting both CDK2 and CDK4/6. Furthermore, the clinical relevance of cyclin E as a therapeutic target was supported by significant association between CCNE1 overexpression and poor prognosis based on large-scale public gene expression data sets in HR-positive breast cancer patients. Therefore, we propose cyclin E-CDK2 signaling as a promising therapeutic target for overcoming cyclin E-associated resistance to CDK4/6 inhibitor.