CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal.

CDK4/6 inhibitors arrest the cell cycle in G1-phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long-lasting effects on tumour growth. Here, we demonstrate that a prolonged G1 arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. Upon release from that arrest, many cells fail to complete DNA replication and exit the cell cycle in a p53-dependent manner. If cells fail to withdraw from the cell cycle following DNA replication problems, they enter mitosis and missegregate chromosomes causing excessive DNA damage, which further limits their proliferative potential. These effects are observed in a range of tumour types, including breast cancer, implying that genotoxic stress is a common outcome of CDK4/6 inhibition. This unanticipated ability of CDK4/6 inhibitors to induce DNA damage now provides a rationale to better predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

Mechanisms of sensitivity and resistance to CDK4/CDK6 inhibitors in hormone receptor-positive breast cancer treatment.

Cell cycle dysregulation is a hallmark of cancer that promotes eccessive cell division. Cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6) are key molecules in the G1-to-S phase cell cycle transition and are crucial for the onset, survival, and progression of breast cancer (BC). Small-molecule CDK4/CDK6 inhibitors (CDK4/6i) block phosphorylation of tumor suppressor Rb and thus restrain susceptible BC cells in G1 phase. Three CDK4/6i are approved for the first-line treatment of patients with advanced/metastatic hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) BC in combination with endocrine therapy (ET). Though this has improved the clinical outcomes for survival of BC patients, there is no established standard next-line treatment to tackle drug resistance. Recent studies suggest that CDK4/6i can modulate other distinct effects in both BC and breast stromal compartments, which may provide new insights into aspects of their clinical activity. This review describes the biochemistry of the CDK4/6-Rb-E2F pathway in HR+ BC, then discusses how CDK4/6i can trigger other effects in BC/breast stromal compartments, and finally outlines the mechanisms of CDK4/6i resistance that have emerged in recent preclinical studies and clinical cohorts, emphasizing the impact of these findings on novel therapeutic opportunities in BC.

Reciprocal regulation of p21 and Chk1 controls the cyclin D1-RB pathway to mediate senescence onset after G2 arrest.

Senescence is an irreversible withdrawal from cell proliferation that can be initiated after DNA damage-induced cell cycle arrest in G2 phase to prevent genomic instability. Senescence onset in G2 requires p53 (also known as TP53) and retinoblastoma protein (RB, also known as RB1) family tumour suppressors, but how they are regulated to convert a temporary cell cycle arrest into a permanent one remains unknown. Here, we show that a previously unrecognised balance between the cyclin-dependent kinase (CDK) inhibitor p21 and the checkpoint kinase Chk1 controls cyclin D-CDK activity during G2 arrest. In non-transformed cells, p21 activates RB in G2 by inhibiting cyclin D1 complexed with CDK2 or CDK4. The resulting G2 exit, which precedes the appearance of senescence markers, is associated with a mitotic bypass, Chk1 downregulation and reduction in the number of DNA damage foci. In p53/RB-proficient cancer cells, a compromised G2 exit correlates with sustained Chk1 activity, delayed p21 induction, untimely cyclin E1 re-expression and genome reduplication. Conversely, Chk1 depletion promotes senescence by inducing p21 binding to cyclin D1- and cyclin E1-CDK complexes and downregulating CDK6, whereas knockdown of the checkpoint kinase Chk2 enables RB phosphorylation and delays G2 exit. In conclusion, p21 and Chk2 oppose Chk1 to maintain RB activity, thus promoting the onset of senescence induced by DNA damage in G2.

The E2F1-HOXB9/PBX2-CDK6 axis drives gastric tumorigenesis and serves as a therapeutic target in gastric cancer.

Homeobox genes include HOX and non-HOX genes. HOX proteins play fundamental roles during ontogenesis by interacting with other non-HOX gene-encoded partners and performing transcriptional functions, whereas aberrant activation of HOX family members drives tumorigenesis. In this study, gastric cancer (GC) expression microarray data indicated that HOXB9 is a prominent upregulated HOX member in GC samples significantly associated with clinical outcomes and advanced TNM stages. However, the functional role of HOXB9 in GC remains contradictory in previous reports, and the regulatory mechanisms are elusive. By in silico and experimental analyses, we found that HOXB9 was upregulated by a vital cell cycle-related transcription factor, E2F1. Depleting HOXB9 causes G1-phase cell cycle arrest by downregulating CDK6 and a subset of cell cycle-related genes. Meanwhile, HOXB9 contributes to cell division and maintains the cytoskeleton in GC cells. We verified that HOXB9 interacts with PBX2 to form a heterodimer, which transcriptionally upregulates CDK6. Knocking down CDK6 can phenocopy the tumor-suppressive effects caused by HOXB9 depletion. Blocking HOXB9 can enhance the anti-tumor effect of CDK6 inhibitors. In conclusion, we elucidate the oncogenic role of HOXB9 in GC and reveal CDK6 as its potent downstream effector. The E2F1-HOXB9/PBX2-CDK6 axis represents a novel mechanism driving gastric carcinogenesis and conveys prognostic and therapeutic implications. © 2023 The Pathological Society of Great Britain and Ireland.

Ring-Contracted Artemisinin Derivatives as Novel CDK 4/6 Inhibitors: Synthesis and Anti-Breast Cancer Evaluation.

The endoperoxide group of artemisinins is universally accepted an essential group for their anti-cancer effects. In this study, a series of D-ring-contracted artemisinin derivatives were constructed by combining ring-contracted artemisinin core with fragments of functional heterocyclic molecules or classical CDK4/6 inhibitors to identify more efficacious breast cancer treatment agents. Twenty-six novel hybridized molecules were synthesized and characterized by HRMS, IR, 1H-NMR and 13C NMR. In antiproliferative activities and kinase inhibitory effects assays, we found that the antiproliferative effects of B01 were close to those of the positive control Palbociclib, with GI50 values of 4.87±0.23 µM and 9.97±1.44 µM towards T47D cells and MDA-MB-436 cells respectively. In addition, the results showed that B01 was the most potent compound against CDK6/cyclin D3 kinase, with an IC50 value of 0.135±0.041 µM, and its activity was approximately 1/3 of the positive control Palbociclib.

Clinicopathological Significance of Cyclin-Dependent Kinase 2 (CDK2) in Ductal Carcinoma In Situ and Early-Stage Invasive Breast Cancers.

Cyclin-dependent kinase 2 (CDK2) is a key cell cycle regulator, with essential roles during G1/S transition. The clinicopathological significance of CDK2 in ductal carcinomas in situ (DCIS) and early-stage invasive breast cancers (BCs) remains largely unknown. Here, we evaluated CDK2's protein expression in 479 BC samples and 216 DCIS specimens. Analysis of CDK2 transcripts was completed in the METABRIC cohort (n = 1980) and TCGA cohort (n = 1090), respectively. A high nuclear CDK2 protein expression was significantly associated with aggressive phenotypes, including a high tumour grade, lymph vascular invasion, a poor Nottingham prognostic index (all p-values < 0.0001), and shorter survival (p = 0.006), especially in luminal BC (p = 0.009). In p53-mutant BC, high nuclear CDK2 remained linked with worse survival (p = 0.01). In DCIS, high nuclear/low cytoplasmic co-expression showed significant association with a high tumour grade (p = 0.043), triple-negative and HER2-enriched molecular subtypes (p = 0.01), Comedo necrosis (p = 0.024), negative ER status (p = 0.004), negative PR status (p < 0.0001), and a high proliferation index (p < 0.0001). Tumours with high CDK2 transcripts were more likely to have higher expressions of genes involved in the cell cycle, homologous recombination, and p53 signaling. We provide compelling evidence that high CDK2 is a feature of aggressive breast cancers. The clinical evaluation of CDK2 inhibitors in early-stage BC patients will have a clinical impact.

The Clinicopathological Significance of the Cyclin D1/E1-Cyclin-Dependent Kinase (CDK2/4/6)-Retinoblastoma (RB1/pRB1) Pathway in Epithelial Ovarian Cancers.

Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.

Unveiling Conformational States of CDK6 Caused by Binding of Vcyclin Protein and Inhibitor by Combining Gaussian Accelerated Molecular Dynamics and Deep Learning.

CDK6 plays a key role in the regulation of the cell cycle and is considered a crucial target for cancer therapy. In this work, conformational transitions of CDK6 were identified by using Gaussian accelerated molecular dynamics (GaMD), deep learning (DL), and free energy landscapes (FELs). DL finds that the binding pocket as well as the T-loop binding to the Vcyclin protein are involved in obvious differences of conformation contacts. This result suggests that the binding pocket of inhibitors (LQQ and AP9) and the binding interface of CDK6 to the Vcyclin protein play a key role in the function of CDK6. The analyses of FELs reveal that the binding pocket and the T-loop of CDK6 have disordered states. The results from principal component analysis (PCA) indicate that the binding of the Vcyclin protein affects the fluctuation behavior of the T-loop in CDK6. Our QM/MM-GBSA calculations suggest that the binding ability of LQQ to CDK6 is stronger than AP9 with or without the binding of the Vcyclin protein. Interaction networks of inhibitors with CDK6 were analyzed and the results reveal that LQQ contributes more hydrogen binding interactions (HBIs) and hot interaction spots with CDK6. In addition, the binding pocket endures flexibility changes from opening to closing states and the Vcyclin protein plays an important role in the stabilizing conformation of the T-loop. We anticipate that this work could provide useful information for further understanding the function of CDK6 and developing new promising inhibitors targeting CDK6.

Comprehensive Landscape of Cyclin Pathway Gene Alterations and Co-occurrence with FGF/FGFR Aberrations Across Urinary Tract Tumors.

BACKGROUND: Cyclin pathway gene alterations are frequent in urothelial tumors and may co-exist with other important aberrations, leading to therapeutic opportunities. We characterized the landscape of cyclin gene alterations in urothelial and non-urothelial urinary tract (UT) malignancies. PATIENTS AND METHODS: Overall, 6842 urothelial and 897 non-urothelial UT cancers were analyzed (hybrid-capture-based comprehensive genomic profile (Foundation Medicine)). Alteration frequency in cyclin-sensitizing and -resistance genes, and co-occurrence with fibroblast growth factor receptor (FGFR) gene abnormalities were evaluated. RESULTS: Cyclin-activating gene alterations were detected in 47.3% of urothelial and 37.9% of non-urothelial UT cancers. Frequency varied by histology and tumor site. CDKN2A and CDKN2B loss were the most frequent alterations in urothelial tumors (present in 38.5% and 30.4% of patients, respectively). Both genes were less frequently altered in adenocarcinomas (15.2% and 8.9%), but commonly altered in squamous cell carcinomas (74.4% and 39%). Tumors of neuroendocrine origin were relatively silent in activating cyclin alterations, but frequently displayed Rb1 alterations (86% and 83.7% of neuroendocrines and small cell carcinomas). Urachal tumors (n = 79) presented a distinct landscape of cyclin alterations relative to other UT cancers, with less frequent alterations overall. FGF/FGFR genes were altered in 34.9% of urothelial (22.1% in FGFR3), and 19.4% of non-urothelial urinary tract tumors (6.8% FGFR3). Cyclin-activating alterations frequently co-occurred with FGF/FGFR alterations but were in general mutually exclusively with cyclin resistance alterations (RB1/CCNE1). CONCLUSIONS: Cyclin pathway activating alterations are common in urinary tract tumors, but frequency varies with histology and tumors sites. Co-occurrence of cyclin and FGFR pathway alterations may inform therapeutic opportunities.

AGA induces sub-G1 cell cycle arrest and apoptosis in human colon cancer cells through p53-independent/p53-dependent pathway.

BACKGROUND: Despite the advancement in chemotherapeutic drugs for colon cancer treatment, it is still a life-threatening disease worldwide due to drug resistance. Therefore, an urgently needed to develop novel drugs for colon cancer therapies. AGA is a combination of traditional Chinese medicine Antler's extract (A), Ganoderma lucidum (G), and Antrodia camphorata (A); it contains a lot of biomolecules like polysaccharides, fatty acids, and triterpenoids that are known to exerting anti-oxidative, anti-inflammatory, anti-microbial and anti-tumor activities in oral cancer. In this study, we investigate AGA anti-proliferative, anti-metastatic and apoptotic activity to explore its anti-cancer activity against colon cancer cells and its underlying mechanism. METHOD: Here, in-vitro studies were performed to determine the antiproliferative activity of AGA through MTT and colony formation assays. Wound healing and transwell migration assay were used to evaluate the metastasis. Flow cytometry and protein expression were used to investigate the involved molecular mechanism by evaluating the cell cycle and apoptosis. The in-vivo anti-cancerous activity of AGA was assessed by xenograft mice model of colon cancer cells. RESULTS: We found that AGA significantly inhibited the proliferative capacity and metastasis of colon cancer cells in-vitro. In addition, AGA induced cell cycle arrest in the sub-G1 phase through upregulating p21 and downregulating CDK2, CDK6 in SW620, and CDK4 in SW480 and HT29, respectively. Annexin-v assay indicated that colon cancer cells had entered early and late apoptosis after treatment with AGA. Furthermore, a mechanistic protein expressions study revealed that AGA in p53-dependent and independent regulated the apoptosis of colon cancer by downregulating the p53 protein expression in SW620 and SW480 cells but upregulating in a dose-dependent manner in HT29 cells and increasing the expression of Bax and caspase-9 to inhibit the colon cancer cells. In vivo study, we found that AGA significantly reduced the xenograft tumor growth in NOD/SCID mice with no adverse effect on the kidney and liver. CONCLUSION: Collectively, AGA has the potential to inhibit colon cancer through inhibiting proliferation, migration, and cell cycle kinase by upregulating p21 protein expression and promoting the apoptotic protein in a p53-dependent and independent manner.

SUZ12 promotes the malignant behavior of gastric cancer cells by CDKs.

Zeste 12 Homolog (SUZ12), as a transcription factor, has been found to be highly expressed in a variety of tumors and promote tumor progression. We focus on revealing its role and mechanism in gastric cancer. Cellular level studies were conducted in mouse gastric cancer MFC cells by performing overexpression of SUZ12, overexpression of CDK6, and treatment with CDK6 inhibitor, respectively. Changes in cell viability, invasion, metastasis and colony formation were detected, and the expression variations of cell cycle regulatory proteins CDK6, P21, and Cyclin D were determined. During the animal experimentation, a mouse xenograft model was established. After transplantation of SUZ12-overexpressing MFC-SUZ12, the tumor growth was compared with that in MFC, and the tissue expressions of CDK-6, SUZ12, and Cyclin D were examined. Overexpression of SUZ12 could enhance the viability of MFC cells while upregulating their migration, invasion and colony formation abilities, which promoted the expression of CDK6, P21, and Cyclin D. CDK6 inhibitor, on the other hand, could suppress the effects of SUZ12 overexpression and weaken the cell viability and malignant behavior. Overexpression of CDK6 also promoted the MFC viability and malignant behavior. We found that SUZ12 exerted its effects by promoting the expression of downstream cyclin CDK6. At the animal level, the mice xenografted with SUZ12-overexpressing MFC cells exhibited larger tumor volumes, as well as elevated cyclin expression. SUZ12 promotes the proliferation and malignant behavior of gastric cancer cells by regulating the expression of downstream CDK6.

Deciphering the Immunomodulatory Role of Cyclin-Dependent Kinase 4/6 Inhibitors in the Tumor Microenvironment.

Cancer is characterized by persistent cell proliferation driven by aberrant cell cycle regulation and stimulation of cyclin-dependent kinases (CDKs). A very intriguing and potential approach for the development of antitumor medicines is the suppression of CDKs that lead to induction of apoptosis and cell cycle arrest. The shift of the cell cycle from the G0/G1 phase to the S phase, which is characterized by active transcription and synthesis, depends on the development of the cyclin D-CDK4/6 complex. A precise balance between anticancer activity and general toxicity is demonstrated by CDK inhibitors, which can specifically block CDK4/6 and control the cell cycle by reducing the G1 to S phase transition. CDK4/6 inhibitors have recently been reported to exhibit significant cell growth inhibition via modulating the tumour microenvironment in cancerous cells. One significant new understanding is that these inhibitors serve important functions in the interaction among tumour cells and the host immune system in addition to being cytostatic. Herein, we discuss the biological significance of CDK4/6 inhibitors in cancer therapeutics, as well as their biological impact on T cells and other important immune cells. Furthermore, we explore the integration of preclinical findings of these pharmaceuticals' ability to enhance antitumor immunity.

miR-3174 Is a New Tumor Suppressor MicroRNA That Inhibits Several Tumor-Promoting Genes in Glioblastoma.

microRNAs (miRNAs) play an important role in the pathology of glioblastoma (GBM), which is the most malignant and most common primary malignant brain tumor. miRNAs can target multiple genes simultaneously and are considered as potential therapeutic agents or targets. This study aimed to determine the role of miR-3174 in the pathobiology of GBM using both in vitro and in vivo approaches. This is the first study deciphering the role of miR-3174 in GBM. We studied the expression of miR-3174 and found it to be downregulated in a panel of GBM cell lines, GSCs and tissues relative to astrocytes and normal brain tissue. This finding led us to hypothesize that miR-3174 has a tumor-suppressive role in GBM. Exogenous expression of miR-3174 inhibited GBM cell growth and invasion, and hampered the neurosphere formation ability of GSCs. miR-3174 downregulated the expression of multiple tumor-promoting genes including CD44, MDM2, RHOA, PLAU and CDK6. Further, overexpression of miR-3174 reduced tumor volume in nude mice with intracranial xenografts. Immuno-histochemical study of brain sections with intracranial tumor xenografts revealed the pro-apoptotic and anti-proliferative activity of miR-3174. In conclusion, we demonstrated that miR-3174 has a tumor-suppressive role in GBM and could be exploited for therapeutic purposes.

Role and mechanism of Circ-PDE7B in the formation of keloid.

The excessive proliferation of keloid fibroblasts is one of the important reasons leading to the formation of keloids. Circular RNA (circRNA) is an important regulator that regulates the biological functions of cells. However, the role and mechanism of circ-PDE7B in keloid formation have not been studied yet. QRT-PCR was used to detect the circ-PDE7B, miR-331-3p and cyclin-dependent kinase 6 (CDK6) expression. The biological functions of keloid fibroblasts were determined by MTT assay, flow cytometry, transwell assay and wound healing assay. Western blot analysis was used to measure the protein levels of extracellular matrix (ECM) markers and CDK6. The interaction between miR-331-3p and circ-PDE7B or CDK6 was confirmed by dual-luciferase reporter assay and RIP assay. Circ-PDE7B was found to be upregulated in keloid tissues and fibroblasts. Downregulation of circ-PDE7B could suppress the proliferation, invasion, migration, ECM accumulation and accelerate the apoptosis of keloid fibroblasts. Circ-PDE7B could serve as a sponge of miR-331-3p, and the regulation of silenced circ-PDE7B on the biological functions of keloid fibroblasts could be abolished by miR-331-3p inhibitor. Additionally, CDK6 was a target of miR-331-3p, and its overexpression could reverse the negative regulation of miR-331-3p on the biological functions of keloid fibroblasts. Circ-PDE7B sponged miR-331-3p to positively regulate CDK6 expression. Taken together, circ-PDE7B promoted the proliferation, invasion, migration and ECM accumulation of keloid fibroblasts by regulating the miR-331-3p/CDK6 axis, suggesting that circ-PDE7B might be a potential target for keloid treatment.

CDK6-PI3K signaling axis is an efficient target for attenuating ABCB1/P-gp mediated multi-drug resistance (MDR) in cancer cells.

BACKGROUND: Multidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1/P-gp) is a major cause of cancer chemotherapy failure, but the regulation mechanisms are largely unknown. METHODS: Based on single gene knockout, we studied the regulation of CDK6-PI3K axis on ABCB1-mediated MDR in human cancer cells. CRISPR/Cas9 technique was performed in KB-C2 cells to knockout cdk6 or cdk4 gene. Western blot, RT-PCR and transcriptome analysis were performed to investigate target gene deletion and expression of critical signaling factors. The effect of cdk4 or cdk6 deficiency on cell apoptosis and the cell cycle was analyzed using flow cytometry. In vivo studies were performed to study the sensitivity of KB-C2 tumors to doxorubicin, tumor growth and metastasis. RESULTS: Deficiency of cdk6 led to remarkable downregulation of ABCB1 expression and reversal of ABCB1-mediated MDR. Transcriptomic analysis revealed that CDK6 knockout regulated a series of signaling factors, among them, PI3K 110α and 110ß, KRAS and MAPK10 were downregulated, and FOS-promoting cell autophagy and CXCL1-regulating multiple factors were upregulated. Notably, PI3K 110α/110ß deficiency in-return downregulated CDK6 and the CDK6-PI3K axis synergizes in regulating ABCB1 expression, which strengthened the regulation of ABCB1 over single regulation by either CDK6 or PI3K 110α/110ß. High frequency of alternative splicing (AS) of premature ABCB1 mRNA induced by CDK6, CDK4 or PI3K 110α/110ß level change was confirmed to alter the ABCB1 level, among them 10 common skipped exon (SE) events were found. In vivo experiments demonstrated that loss of cdk6 remarkably increased the sensitivity of KB-C2 tumors to doxorubicin by increasing drug accumulation of the tumors, resulting in remarkable inhibition of tumor growth and metastasis, as well as KB-C2 survival in the nude mice. CONCLUSIONS: CDK6-PI3K as a new target signaling axis to reverse ABCB1-mediated MDR is reported for the first time in cancers. Pathways leading to inhibition of cancer cell proliferation were revealed to be accompanied by CDK6 deficiency.

In silico development and experimental validation of a novel 7-gene signature based on PI3K pathway-related genes in bladder cancer.

Although bladder cancer (BLCA) is the 10th most common tumor worldwide, particularly practical markers and prognostic models that might guide therapy are needed. We used a non-negative matrix factorization algorithm to classify PI3K pathway-related genes into molecular subtypes. A weighted gene co-expression network analysis (WGCNA) was generated to identify co-expression modules. Univariate Cox regression, least absolute shrinkage sum selection operator-Cox regression, and multivariate Cox regression were utilized to develop a prognostic score model. Kaplan-Meier analysis and receiver operating characteristics were utilized to measure the model's effectiveness. A nomogram was constructed to improve the predictive ability of the model based on clinical parameters and risk. Decision curve analysis (DCA) was used to evaluate the nomogram. To evaluate the immune microenvironment, an estimate algorithm was used. Drug sensitivity was identified using the R package "pRRophetic." UM-UC-3 cell line was used to measure the effect of CDK6 in Western blotting, proliferation assay, and 5-ethynyl-20-deoxyuridine assay. Based on PI3K pathway-related genes, The Cancer Genome Atlas (TCGA)-BLCA and GSE32894 patients were divided into two subtypes. Twenty-five co-expression modules were established using the WGCNA algorithm. A seven-gene signature (CDK6, EGFR, IGF1, ITGB7, PDGFRA, RPS6, and VWF) demonstrated robustness in TCGA and GSE32894 datasets. Expression levels of CDK6 and risk positively correlated with M2 macrophages and IgG. Cisplatin, gemcitabine, methotrexate, mitomycin C, paclitaxel, and vinblastine are sensitive to different groups based on the expression of CDK6 and risk. Functional experiments suggested that CDK6 promotes the proliferation of UM-UC-3 cells. We constructed a seven-gene prognostic signature as an effective marker to predict the outcomes of BLCA patients and guide individual treatment.

Activation of miR-500a-3p/CDK6 axis suppresses aerobic glycolysis and colorectal cancer progression.

BACKGROUND: Colorectal cancer (CRC) is one of the lethal cancers with a high mortality rate worldwide and understanding the mechanisms behind its progression is critical for improving patients' prognosis and developing therapeutics. MiR-500a-3p has been demonstrated to be involved in the progression of several human cancers but its role in CRC remains unclear. The aim of this study is to uncover the expression pattern and mechanisms of action of miR-500a-3p during the CRC progression. METHODS: The expression of miR-500a-3p and Cyclin-dependent kinases 6 (CDK6) in 134 CRC tissues were tested by quantitative PCR (qPCR) and immunohistochemistry staining (IHC), respectively. The effect of miR-500a-3p on cell proliferation was explored in vitro and in vivo. The glycolysis of CRC cells was determined by Mass Spectrometry and Seahorse XF 96 Extracellular Flux Analyzer. A dual-luciferase reporter assay was performed to validate the relationship between miR-500a-3p and CDK6. RESULTS: miR-500a-3p was abnormally downregulated in CRC tissues and cell lines and was negatively associated with a worse prognosis. miR-500a-3p mimics impeded CRC cell proliferation in vitro and in vivo. miR-500a-3p inhibited glucose consumption, lactate and ATP production, and down-regulated the expression of hexokinase2 (HK2). In silico prediction combined with western blot and luciferase assay confirmed that CDK6 is a direct target of miR-500a-3p. Overexpression of CDK6 phenotypically rescued the inhibitory effect of miR-500a-3p on the proliferation and glycolysis of CRC cells. CONCLUSIONS: Our study revealed a potential tumor-suppressive role of miR-500a-3p in CRC, specifically targeting CDK6 and inhibiting cancer cell proliferation and aerobic glycolysis, which may provide new insights into novel prognostic biomarkers and therapeutic targets for CRC.

Cytokeratin 13 promotes radiotherapy sensitivity of nasopharyngeal carcinoma by downregulating the MEK/ERK pathway.

BACKGROUND: Radiation therapy is the first treatment choice for nasopharyngeal carcinoma (NPC), while radiation resistance and recurrence have become the primary factors and are associated with poor prognosis in the clinical treatment of NPC patients. The purpose of the present study was to explore the sensitivity and molecular basis of cytokeratin 13 (CK13) that regulates NPC radiotherapy. METHODS: HNE-3 or C666-1 cell line was used for overexpression and knockdown tests. Under radiotherapy conditions, CCK-8 assay, clone formation assay, and flow cytometry analyzed the effects of CK13 overexpression on cell proliferation, apoptosis, and cell cycle, respectively. In addition, Western blotting detected CK13-mediated downregulation of cell cycle-related genes. The mouse subcutaneous tumor-bearing experiment identified the effects of CK13 overexpression on the treatment of NPC in vivo. Further, Western blotting, CCK-8 assay, and flow cytometry investigated whether the CK13-mediated cell apoptosis involves the MEK/ERK signaling pathway. RESULTS: Overexpression of CK13 significantly inhibited the survival of HNE-3 cells under radiotherapy in vitro and in vivo, and there was a substantial decrease in cyclin-dependent kinase 4 and 6 (CDK4/6) levels promoting the cell percentage number in the G2/M phase and, subsequently, the ratio of the apoptotic cells. In contrast, the knockdown of CK13 showed the opposite partial regulatory effect. Interestingly, CK13 overexpression also showed a reduction in the survival of C666-1 cells and an increased ratio of the apoptotic cells under radiotherapy treatment. Furthermore, higher levels of CK13 downregulated the MEK/ERK signaling pathway, resulting in decreased HNE-3 cell proliferation and increased apoptosis. However, ERK activators were able to rescue the process partially. CONCLUSIONS: Together, these results showed that CK13 promoted the radiosensitivity of NPC cells by downregulating the MEK/ERK signaling pathway. Thus, targeting CK13 provided insights into the treatment of NPC radiotherapy.

Association of CDK6 gene polymorphisms with Behcet's disease in a Han Chinese population.

Cyclin-dependent kinases 4/6 (CDK4/6) and D1-type cyclins (CCND1) can regulate the pro-inflammatory functions of various cytokines during the inflammatory response. This study investigated the association between CDK4/6-CCND1 variants and susceptibility in patients with Behcet's disease (BD). This case-control study enrolled 542 patients with BD and 754 healthy controls. Fourteen tagged single nucleotide polymorphisms (tag SNPs) of the CDK4/6-CCND1 gene were genotyped using the Sequenom MassARRAY system and iPLEX® Pro assay. The results indicated that the frequency of the CDK6 rs2282983 TT genotype was higher in the BD group than the control group (Pc = 0.040, OR = 1.408, 95% CI = 1.124-1.765), and CDK6 rs2282983 CT and rs42034 AG were negatively associated with BD (Pc = 3.647 × 10-4, OR = 0.598, 95% CI = 0.471-0.758; Pc = 0.039, OR = 0.626, 95% CI = 0.459-0.852, respectively). Furthermore, statistical analysis showed that CDK6 rs2282983 TT and CT genotypes were significantly associated with skin lesions in patients with BD (Pc = 0.042, OR = 1.436, 95% CI = 1.130-1.824; Pc = 0.001, OR = 0.594, 95% CI = 0.461-0.764, respectively). This study suggests that the CDK6 loci rs2282983 and rs42034 might confer genetic susceptibility to BD in a Han Chinese population, which could provide new insights into the pathogenesis of BD.

GPR37 promotes cancer growth by binding to CDK6 and represents a new theranostic target in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is associated with poor prognosis. Identifying novel cancer targets and helpful therapeutic strategies remains a serious clinical challenge. This study detected differentially expressed genes in The Cancer Genome Atlas (TCGA) LUAD data collection. We also identified a predictive DNA biomarker, G protein-coupled receptor 37 (GPR37), which was verified as a prognostic biomarker with a critical role in tumor progression. In human LUAD specimens and microarray analyses, we determined that GPR37 was significantly upregulated and associated with a poor prognosis. GPR37 downregulation markedly inhibited the proliferation and migration of LUAD both in vitro and in vivo. Mechanistically, GPR37 could bind to CDK6, thereby facilitating tumor progression in LUAD by inducing cell cycle arrest at the G1 phase. GPR37 also facilitates tumorigenesis in xenograft tumors in vivo. High-throughput screening for GPR37-targeted drugs was performed using the Natural Products Library, which revealed the potential of Hypocrellin B to inhibit GPR37 and cell growth in LUAD. We demonstrated that Hypocrellin B suppressed LUAD cell proliferation and migration both in vitro and in vivo via GPR37 inhibition. Collectively, our findings reveal the role of GPR37 in LUAD progression and migration and the potential of GPR37 as a target for the treatment of LUAD. Thus, the specific inhibition of GPR37 by the natural product Hypocrellin B may possess the potential for the treatment of LUAD.