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Oral squamous cell carcinoma (OSCC) is a frequent malignant tumor worldwide. Long non‑coding RNAs (lncRNAs) are known to play key roles in different types of cancer, including OSCC. It was previously reported that lncRNA deleted in lymphocytic leukemia 1 (DLEU1) is notably upregulated in OSCC; however, the role of DLEU1 in OSCC remains unclear. Gene and protein expression levels in OSCC cells were detected by reverse transcription‑quantitative PCR and western blotting, respectively, in the present study. A Transwell assay was performed to measure cell migration and invasion. Flow cytometry was used to detect cell apoptosis, and the dual‑luciferase reporter assay was applied to confirm the interaction between DLEU1, microRNA (miR)‑149‑5p and CDK6 in OSCC cells. DLEU1 expression was negatively associated with the survival rate of patients with OSCC. In addition, silencing of DLEU1 notably inhibited the proliferation of OSCC cells by inducing apoptosis. Meanwhile, DLEU1 directly bound to miR‑149‑5p, and CDK6 was found to be the direct target of miR‑149‑5p. Furthermore, DLEU1 knockdown‑induced inhibition of OSCC cell proliferation was significantly reversed by the miR‑149‑5p antagomir. Knockdown of lncRNA DLEU1 reversed the proliferation of OSCC cells via regulation of the miR‑149‑5p/CDK6 axis. Thus, DLEU1 may serve as a novel target for treating OSCC.

Combining cyclin-dependent kinase (CDK) inhibitors with endocrine therapy improves outcomes for metastatic estrogen receptor positive (ER+) breast cancer patients but its value in earlier stage patients is unclear. We examined evolutionary trajectories of early-stage breast cancer tumors, using single cell RNA sequencing (scRNAseq) of serial biopsies from the FELINE clinical trial (#NCT02712723) of endocrine therapy (letrozole) alone or combined with the CDK inhibitor ribociclib. Despite differences in subclonal diversity evolution across patients and treatments, common resistance phenotypes emerged. Resistant tumors treated with combination therapy showed accelerated loss of estrogen signaling with convergent up-regulation of JNK signaling through growth factor receptors. In contrast, cancer cells maintaining estrogen signaling during mono- or combination therapy showed potentiation of CDK4/6 activation and ERK upregulation through ERBB4 signaling. These results indicate that combination therapy in early-stage ER+ breast cancer leads to emergence of resistance through a shift from estrogen to alternative growth signal-mediated proliferation.

Osteosarcoma is a malignant tumor with high mortality in children and adolescents. The mechanism of osteosarcoma metastasis is currently unclear. Abnormal expression of long non-coding RNA (lncRNA) plays an important role in tumor metastasis. We used bioinformatics to analyze the differences in gene expression between osteosarcoma in situ and osteosarcoma lung metastases. CCK-8 was used to detect the effect of lncRNA LOC100129620 on the proliferation of osteosarcoma cells. The effect of LOC100129620 on the invasion of osteosarcoma cells was assessed by Transwell assay. The regulatory effect of LOC100129620 on miR-335-3p was examined using RNA pull-down and luciferase reporter gene assays. The effect of LOC100129620 on the polarization of macrophages was detected by quantitative real-time fluorescent PCR. The results show that LOC100129620 can promote the proliferation and migration of osteosarcoma cells. LOC100129620 can promote the proliferation of osteosarcoma in vivo . LOC100129620 can bind to miR-335-3p and regulate its function. MiR-335-3p mediates the regulatory effects of LOC100129620 on CDK6. LOC100129620 promotes the formation of blood vessels and the polarization of macrophages. The LOC100129620/miR-335-3p/CDK6 signaling pathway promotes the metastasis of osteosarcoma by regulating the proliferation of osteosarcoma cells, angiogenesis, and macrophage polarization.

Conventional chemotherapy for acute myeloid leukemia regimens generally encompass an intensive induction phase, in order to achieve a morphological remission in terms of bone marrow blasts (<5%). The majority of cases are classified as Primary Induction Response (PIR); unfortunately, 15% of children do not achieve remission and are defined Primary Induction Failure (PIF). This study aims to characterize the gene expression profile of PIF in children with Acute Myeloid Leukemia (AML), in order to detect molecular pathways dysfunctions and identify potential biomarkers. Given that NUP98-rearrangements are enriched in PIF-AML patients, we investigated the association of NUP98-driven genes in primary chemoresistance. Therefore, 85 expression arrays, deposited on GEO database, and 358 RNAseq AML samples, from TARGET program, were analyzed for "Differentially Expressed Genes" (DEGs) between NUP98+ and NUP98-, identifying 110 highly confident NUP98/PIF-associated DEGs. We confirmed, by qRT-PCR, the overexpression of nine DEGs, selected on the bases of the diagnostic accuracy, in a local cohort of PIF patients: SPINK2 , TMA7 , SPCS2 , CDCP1 , CAPZA1 , FGFR1OP2 , MAN1A2 , NT5C3A and SRP54 . In conclusion, the integrated analysis of NUP98 mutational analysis and transcriptome profiles allowed the identification of novel putative biomarkers for the prediction of PIF in AML.

Hyperthermia is one of the most widely employed adjuvant treatments for cancer, especially for hyperthermic intraperitoneal chemotherapy, and has few side effects. Gastric cancer has various hyperthermia sensitivities, but the exact molecular mechanisms remain to be elucidated. In the present study, western blotting was performed to detect differential expression of proteins that have been reported to be upregulated in gastric cancer. Following knockdown of these proteins, apoptosis was measured by Annexin V‑FITC/propidium iodide (PI) double staining and hyperthermia treatment was applied. To evaluate the effect of cyclin‑dependent kinase 6 (CDK6) on hyperthermia‑induced apoptosis, CDK6 was knocked down or inhibited by the addition of a specific inhibitor and subsequent PI staining and cell proliferation, migration and invasion assays were performed. Hyperthermia‑induced protein kinase B (AKT) expression and phosphorylation inhibition were detected. As demonstrated in the present study, the hyperthermia‑induced proteins kinesin family member 11 (KIF11), cyclin‑dependent kinase 6 (CDK6), stromal antigen 2, NIMA‑related kinase 2 and karyopherin subunit α 4 were highly expressed in gastric cancer cells, including SH‑10‑TC and HGC‑27 cells. Knockdown of KIF11 significantly increased apoptosis without hyperthermia treatment and CDK6 significantly increased hyperthermia‑induced apoptosis, prompting the present study to focus on CDK6. It was further confirmed that CDK6 activity was critical for decreasing hyperthermia‑induced apoptosis and for cell proliferation. Hyperthermia‑induced AKT expression and phosphorylation inhibition is potentially the main cause of CDK6 transcriptional upregulation. Taken together, these findings demonstrated that CDK6 is upregulated via hyperthermia‑induced AKT inhibition and subsequently protected gastric cancer cells from hyperthermia‑induced apoptosis, indicating that it is a potential therapeutic target to sensitize gastric cancer cells to hyperthermia‑based therapy.

Altered expressions of proto-oncogenes have been reported during normal lymphocytes mitogenesis and in T and B lymphocytes in patients with autoimmune diseases. We have recently demonstrated a significantly decreased expression of c-kit and c-Myc in NK cells isolated from patients with cancer, which might be related to the functional deficiency of NK cells in the tumor environment. Here, focusing on the regulatory mechanisms of this new clinical phenomenon, we determined expression of c-Myc, Notch1, Notch2, p-53, Cdk6, Rb and phosphorylated Rb in NK cells isolated from the healthy donors and cancer patients. The results of our study revealed a significant down-regulation of expression of Notch receptors and up-regulation of Cdk6 expression in NK cells in cancer, while no significant changes in the expression of p53 and Rb proteins were seen. These data revealed novel signaling pathways altered in NK cells in the tumor environment and support further investigation of the origin of deregulated expression of proto-oncogenes in NK cells patients with different types of cancer.

Background: Alpelisib, a PI3Kα-selective inhibitor and degrader, plus fulvestrant showed efficacy in hormone receptor-positive, HER2-negative, PIK3CA-mutated advanced breast cancer in SOLAR-1; limited data are available in the post-cyclin-dependent kinase 4/6 inhibitor setting. BYLieve aimed to assess alpelisib plus endocrine therapy in this setting in three cohorts defined by immediate previous treatment; here, we report results from cohort A., Methods: This ongoing, phase 2, multicentre, open-label, non-comparative study enrolled patients with hormone receptor-positive, HER2-negative, advanced breast cancer with tumour PIK3CA mutation, following progression on or after previous therapy, including CDK4/6 inhibitors, from 114 study locations (cancer centres, medical centres, university hospitals, and hospitals) in 18 countries worldwide. Participants aged 18 years or older with an Eastern Cooperative Oncology Group performance status of 2 or less, with no more than two previous anticancer treatments and no more than one previous chemotherapy regimen, were enrolled in three cohorts. In cohort A, patients must have had progression on or after a CDK4/6 inhibitor plus an aromatase inhibitor as the immediate previous treatment. Patients received oral alpelisib 300 mg/day (continuously) plus fulvestrant 500 mg intramuscularly on day 1 of each 28-day cycle and on day 15 of cycle 1. The primary endpoint was the proportion of patients alive without disease progression at 6 months per local assessment using Response Evaluation Criteria in Solid Tumors, version 1.1, in patients with a centrally confirmed PIK3CA mutation. This trial is registered with ClinicalTrials.gov, NCT03056755., Findings: Between Aug 14, 2017, and Dec 17, 2019 (data cutoff), 127 patients with at least 6 months' follow-up were enrolled into cohort A. 121 patients had a centrally confirmed PIK3CA mutation. At data cutoff, median follow-up was 11·7 months (IQR 8·5-15·9). 61 (50·4%; 95% CI 41·2-59·6) of 121 patients were alive without disease progression at 6 months. The most frequent grade 3 or worse adverse events were hyperglycaemia (36 [28%] of 127 patients), rash (12 [9%]), and rash maculopapular (12 [9%]). Serious adverse events occurred in 33 (26%) of 127 patients. No treatment-related deaths were reported., Interpretation: BYLieve showed activity of alpelisib plus fulvestrant with manageable toxicity in patients with PIK3CA-mutated, hormone receptor-positive, HER2-negative advanced breast cancer, after progression on a CDK4/6 inhibitor plus an aromatase inhibitor., Funding: Novartis Pharmaceuticals., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Lung cancer (LC) and pancreatic cancer (PC) are the first and fourth leading causes of cancer‑related deaths in the US. Deregulated cell cycle progression is the cornerstone for rapid cell proliferation, tumor development, and progression. Here, we provide evidence that a novel combinatorial miR treatment inhibits cell cycle progression at two phase transitions, through their activity on the CDK4 and CDK1 genes. Following transfection with miR‑143 and miR‑506, we analyzed the differential gene expression of CDK4 and CDK1, using qPCR or western blot analysis, and evaluated cell cycle inhibition, apoptosis and cytotoxicity. The combinatorial miR‑143/506 treatment downregulated CDK4 and CDK1 levels, and induced apoptosis in LC cells, while sparing normal lung fibroblasts. Moreover, the combinatorial miR treatment demonstrated a comparable activity to clinically tested cell cycle inhibitors in inhibiting cell cycle progression, by presenting substantial inhibition at the G 1 /S and G 2 /M cell cycle transitions. More importantly, the miR‑143/506 treatment presented a broader application, effectively downregulating CDK1 and CDK4 levels, and reducing cell growth in PC cells. These findings suggest that the miR‑143/506 combination acts as a promising approach to inhibit cell cycle progression for cancer treatment with minimal toxicity to normal cells.

CDK6 kinase regulates cell-cycle progression in G1, together with CDK4, but has cell-, tissue- and developmentally distinct functions associated with transcription, angiogenesis and metabolism. Although CDK6 makes an attractive cancer biomarker and target, there are no means of assessing its activity in a complex environment. In this study, we describe the design, engineering and characterisation of a fluorescent peptide biosensor derived from 6-phosphofructokinase that reports on CDK6 kinase activity through sensitive changes in fluorescence intensity. This biosensor can report on CDK6 activity in a dose-dependent fashion, thereby enabling quantification of differences in kinase activity in complex and physiologically relevant environments. Further implementation of this biosensor in different lung and melanoma cell lines, as well as in mesothelioma cell lines derived from patients together with a CDK4 biosensor highlighted differences in kinase activity between CDK6 and CDK4 kinase. This work demonstrates the utility of these selective tools for monitoring two closely related kinases comparatively and simultaneously in the same samples, thereby offering attractive perspectives for diagnostic and therapeutic purposes., (© 2020 Wiley-VCH GmbH.)

17β-estradiol (E2) exerts its physiological effects through the estrogen receptor α (i.e., ERα). The E2:ERα signaling allows the regulation of cell proliferation. Indeed, E2 sustains the progression of ERα positive (ERα+) breast cancers (BCs). The presence of ERα at the BC diagnosis drives their therapeutic treatment with the endocrine therapy (ET), which restrains BC progression. Nonetheless, many patients develop metastatic BCs (MBC) for which a treatment is not available. Consequently, the actual challenge is to complement the drugs available to fight ERα+ primary and MBC. Here we exploited a novel anti-estrogen discovery platform to identify new Food and Drug Administration (FDA)-approved drugs inhibiting E2:ERα signaling to cell proliferation in cellular models of primary and MBC cells. We report that the anti-fungal drugs clotrimazole (Clo) and fenticonazole (Fenti) induce ERα degradation and prevent ERα transcriptional signaling and proliferation in cells modeling primary and metastatic BC. The anti-proliferative effects of Clo and Fenti occur also in 3D cancer models (i.e., tumor spheroids) and in a synergic manner with the CDK4/CDK6 inhibitors palbociclib and abemaciclib. Therefore, Clo and Fenti behave as "anti-estrogens"-like drugs. Remarkably, the present "anti-estrogen" discovery platform represents a valuable method to rapidly identify bioactive compounds with anti-estrogenic activity.

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo . CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. SIGNIFICANCE: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects., (©2020 American Association for Cancer Research.)

Acute myeloid leukaemia (AML) with chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukaemia (MLL) is an aggressive subtype with low overall survival. Bortezomib (Bort) is first applied in multiple myeloma. However, whether bort possesses anti-self-renewal and leukemogenesis of leukaemia stem cell (LSC) in AML with MLL rearrangements is still unclear. Here, we found that bort suppressed cell proliferation and decreased colony formation in human and murine leukaemic blasts. Besides, bort reduced the frequency and function of LSC, inhibited the progression, and extended the overall survival in MLL-AF9 (MF9) -transformed leukaemic mice. Furthermore, bort decreased the percentage of human LSC (CD34 + CD38 - ) cells and extended the overall survival in AML blasts-xenografted NOD/SCID-IL2Rγ (NSG) mice. Mechanistically, cyclin dependent kinase 6 (CDK6) was identified as a bort target by RNA sequencing. Bort reduced the expressions of CDK6 by inhibiting NF ĸB recruitment to the promoter of CDK6, leading to the abolishment of NF ĸB DNA-binding activity for CDK6 promoter. Overexpression of CDK6 partially rescued bort-induced anti-leukemogenesis. Most importantly, bort had little side-effect against the normal haematological stem and progenitor cell (HSPC) and did not affect CDK6 expression in normal HSPC. In conclusion, our results suggest that bort selectively targets LSC in MLL rearrangements. Bort might be a prospective drug for AML patients bearing MLL rearrangements., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The aims of this study was to investigate the influences of hsa&#95;circ&#95;0056558/miR-1290/CDK6 axis in ankylosing spondylitis (AS). The differentially expressed has&#95;circ&#95;0056558 and miR-1290 in AS tissue were analysed based on RNA-seq data and microarray data, respectively. qRT-PCR was performed for detection of relative expression levels of hsa&#95;circ&#95;0056558, miR-1290, CDK6, osteogenic differentiation markers (Runx2 and Osterix) and other inflammatory factors (TNF-α, IL-1β, and IL-6). Western blotting analysis was conducted to test the protein levels of CDK6, osteogenic differentiation markers (Runx2 and Osterix), and PI3K/AKT/NF-κB pathway-associated proteins. CCK8 assay and flow cytometry were conducted to determine cell proliferation and cell apoptotic ability, respectively. Targeted relationships were predicted by bioinformatic analysis and verified by dual-luciferase reporter assay. The differentiation of fibroblast cells was analysed by alkaline phosphatase (ALP) activity assay. Our findings revealed that the expression levels of both circ&#95;0056558 and CDK6 in AS tissue were significantly higher than that in normal samples. Besides, hsa&#95;circ&#95;0056558 could suppress cell proliferation and differentiation by facilitating CDK6 expression and suppressing miR-1290 expression in AS. Over-expression of miR-1290 negatively regulated CDK6 expression to enhance cell proliferation. The protein levels of p-AKT, p-NF-κB p65, and p-IκBα were promoted by hsa&#95;circ&#95;0056558 or CDK6 over-expression while suppressed by miR-1290 up-regulation. In conclusion, our study demonstrated that hsa&#95;circ&#95;0056558 and CDK6 suppressed cell proliferation and differentiation while enhanced cell apoptosis by competitive binding to miR-1290 in AS, which might be possibly achieved by PI3K/AKT/NF-κB pathway, providing us novel therapeutic strategy for AS.

Background: Epithelial-to-Mesenchymal Transition (EMT) is considered as a crucial event in disease development and dysregulation of microRNAs (miRNAs) is involved in the regulation of EMT in various human diseases. Emerging evidences congregated over the years have demonstrated that miR-30a-5p was decreased in diseases and its overexpression inhibited the process of diseases via attenuating EMT. Although aberrant expression of miRNAs and occurrence of EMT were previously reported in Nasal Polyps (NPs), the role of miR-30a-5p in EMT of NPs is still remains unclear., Objective: The purpose of our present study was to explore the expression and potential function of miR-30a-5p in EMT of NPs., Methods: The expression of miR-30a-5p and mRNA expression level were detected by quantitative real-time PCR (qRT-PCR) in transforming growth factor β1 (TGF-β1) - induced EMT model and NPs patients. Western Blot (WB) and immunohistochemistry (IHC) were performed to evaluate the protein expression level of EMT markers. The cells mobility was assessed by Wound-Healing assay. Luciferase reporter assay was utilized to verify the relationship between Cyclin-dependent kinase 6 (CDK6) and miR-30a-5p., Results: Firstly, we observed that miR-30a-5p was down-regulated notably, accompanying with the alteration of EMT markers expression in NPs tissues and EMT model induced by TGF-β1 in primary Human Nasal Epithelial Cells (pHNECs) and A549 cells in vitro. Moreover, the functional assays demonstrated that overexpression of miR-30a-5p significantly inhibited EMT and cells mobility. Subsequently, CDK6 was validated as a direct target of miR-30a-5p. Finally, we performed the rescue experiments indicating that overexpression of CDK6 eliminated the suppressive effects of miR-30a-5p in TGF-β1-induced EMT in pHNECs and A549 cells., Conclusion: Taken together, our results suggested that EMT was involved in NPs, and overexpression of miR-30a-5p could attenuate EMT via repressing the expression of the CDK6 in pHNECs and A549 cells.

Intrinsic or acquired resistance to clinically approved CDK4/6 inhibitors has emerged as a major obstacle that hinders their utility beyond ER + breast cancer. In this study, CDK4/6-dependent and -resistant models were employed to identify functional determinants of response to pharmacologic CDK4/6 inhibitors. In all models tested, the activation of RB and inhibition of CDK2 activity emerged as determinants of sensitivity. While depleting CDK4 and 6 was sufficient to limit proliferation in specific resistance settings, RB loss rendered cells completely independent of these kinases. The main downstream target in this context was the activation status of CDK2, which was suppressed with CDK4/6 inhibition in an RB-dependent fashion. Protein levels of p27 were associated with plasticity/rigidity of the cell cycle and correlated with sensitivity to CDK4/6 inhibition. Exogenous overexpression and pharmacologic induction of p27 via inhibition of SKP2 and targeting the MEK/ERK pathway enhanced the cytostatic effect of CDK4/6 inhibitors. Mice bearing ER + xenografts displayed a durable antitumor response to palbociclib; however, over the course of treatment, few cells retained RB phosphorylation, which was associated with limited p27 protein levels as determined by multispectral imaging. Similarly, combination treatment of palbociclib with a MEK inhibitor in pancreatic cancer PDX models upregulated p27 and further enhanced the in vivo tumor response to palbociclib. Collectively, these results suggest that the cell cycle plasticity, which enables tumor models to evade palbociclib-mediated activation of RB, could be targeted using a clinically applicable CDK2 inhibitor. SIGNIFICANCE: This work provides a mechanistic insight toward understanding the functional roles of multiple cell cycle regulators that drive plasticity and sensitivity to CDK4/6 inhibition., (©2020 American Association for Cancer Research.)

Erythropoietin-producing hepatocellular receptor A2 (EphA2) receptor tyrosine kinase plays an important role in tissue organization and homeostasis in normal organs. EphA2 is overexpressed in a variety of types of solid tumours with oncogenic functions. However, the role of EphA2 in cervical cancer (CC) is still needed to be further explored. Here, we examined the role of EphA2 by establishing a stable EphA2 knock-down CC cell lines or a stable EphA2-overexpressed CC cells lines. Overexpression of EphA2 increased cell proliferation and migration of CC while EphA2 knock-down decreased the CC tumorigenicity. In addition, EphA2 knock-down suppressed CC tumour development in the xenograft mouse model. Inhibition of EphA2 by AWL-II-41-27, EphA2-specific tyrosine kinase inhibitor, or knock-down of EphA2 decreased mRNA and protein expression of cyclin-dependent kinase (CDK) 6 in CC cells, which increased cellular susceptibility to epirubicin (EPI), an anti-cancer chemotherapy drug. A clinicopathological study of EphA2 was conducted on a cohort of 158 human CC patients. EphA2 protein expression was positively correlated with CDK6 protein expression, invasion depth, lymph node metastasis and clinicopathological stage (P < .05). This study demonstrates the oncogenic activity of EphA2 in vitro and in vivo, which provides insights into the relevant mechanisms that might lead to novel treatments for CC., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: FGFR1 amplification, but not overexpression, has been related to adverse prognosis in hormone-positive breast cancer (HRPBC). Whether FGFR1 overexpression and amplification are correlated, what is their distribution among luminal A or B HRPBC, and if there is a potential different prognostic role for amplification and overexpression are currently unknown features. The role of FGFR1 inhibitors in HRPBC is also unclear., Methods: FGFR1 amplification (FISH) and overexpression (RNAscope) were investigated in a N = 251 HRPBC patients cohort and the METABRIC cohort; effects on survival and FISH-RNAscope concordance were determined. We generated hormonal deprivation resistant (LTED-R) and FGFR1-overexpressing cell line variants of the ER+ MCF7 and T47-D and the ER+, FGFR1-amplified HCC1428 cell lines. The role of ER, CDK4/6, and/or FGFR1 blockade alone or in combinations in Rb phosphorylation, cell cycle, and survival were studied., Results: FGFR1 overexpression and amplification was non-concordant in > 20% of the patients, but both were associated to a similar relapse risk (~ 2.5-fold; P < 0.05). FGFR1 amplification or overexpression occurred regardless of the luminal subtype, but the incidence was higher in luminal B (16.3%) than A (6.6%) tumors; P < 0.05. The Kappa index for overexpression and amplification was 0.69 (P < 0.001). Twenty-four per cent of the patients showed either amplification and/or overexpression of FGFR1, what was associated to a hazard ratio for relapse of 2.6 (95% CI 1.44-4.62, P < 0.001). In vitro, hormonal deprivation led to FGFR1 overexpression. Primary FGFR1 amplification, engineered mRNA overexpression, or LTED-R-acquired FGFR1 overexpression led to resistance against hormonotherapy alone or in combination with the CDK4/6 inhibitor palbociclib. Blocking FGFR1 with the kinase-inhibitor rogaratinib led to suppression of Rb phosphorylation, abrogation of the cell cycle, and resistance-reversion in all FGFR1 models., Conclusions: FGFR1 amplification and overexpression are associated to similar adverse prognosis in hormone-positive breast cancer. Capturing all the patients with adverse prognosis-linked FGFR1 aberrations requires assessing both features. Hormonal deprivation leads to FGFR1 overexpression, and FGFR1 overexpression and/or amplification are associated with resistance to hormonal monotherapy or in combination with palbociclib. Both resistances are reverted with triple ER, CDK4/6, and FGFR1 blockade.

Breast cancer (BC) is the most common cause of cancer-related death in women worldwide. Therapies targeting molecular pathways altered in BC had significantly enhanced treatment options for BC over the last decades, which ultimately improved the lives of millions of women worldwide. Among various molecular pathways accruing substantial interest for the development of targeted therapies are cyclin-dependent kinases (CDKs)-in particular, the two closely related members CDK4 and CDK6. CDK4/6 inhibitors indirectly trigger the dephosphorylation of retinoblastoma tumor suppressor protein by blocking CDK4/6, thereby blocking the cell cycle transition from the G1 to S phase. Although the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib gained FDA approval for the treatment of hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative BC as they significantly improved progression-free survival (PFS) in randomized clinical trials, regrettably, some patients showed resistance to these therapies. Though multiple molecular pathways could be mechanistically responsible for CDK4/6 inhibitor therapy resistance, one of the most predominant ones seems to be the fibroblast growth factor receptor (FGFR) pathway. FGFRs are involved in many aspects of cancer formation, such as cell proliferation, differentiation, and growth. Importantly, FGFRs are frequently mutated in BC, and their overexpression and/or hyperactivation correlates with CDK4/6 inhibitor resistance and shortened PFS in BC. Intriguingly, the inhibition of aberrant FGFR activity is capable of reversing the resistance to CDK4/6 inhibitors. This review summarizes the molecular background of FGFR signaling and discusses the role of aberrant FGFR signaling during cancer development in general and during the development of CDK4/6 inhibitor resistance in BC in particular, together with other possible mechanisms for resistance to CDK4/6 inhibitors. Subsequently, future directions on novel therapeutic strategies targeting FGFR signaling to overcome such resistance during BC treatment will be further debated.

The role of the androgen receptor (AR) in estrogen receptor (ER)-α-positive breast cancer is controversial, constraining implementation of AR-directed therapies. Using a diverse, clinically relevant panel of cell-line and patient-derived models, we demonstrate that AR activation, not suppression, exerts potent antitumor activity in multiple disease contexts, including resistance to standard-of-care ER and CDK4/6 inhibitors. Notably, AR agonists combined with standard-of-care agents enhanced therapeutic responses. Mechanistically, agonist activation of AR altered the genomic distribution of ER and essential co-activators (p300, SRC-3), resulting in repression of ER-regulated cell cycle genes and upregulation of AR target genes, including known tumor suppressors. A gene signature of AR activity positively predicted disease survival in multiple clinical ER-positive breast cancer cohorts. These findings provide unambiguous evidence that AR has a tumor suppressor role in ER-positive breast cancer and support AR agonism as the optimal AR-directed treatment strategy, revealing a rational therapeutic opportunity.

Objective: Kaempferol has been reported to play an anti-tumor role in various human cancers, while its role in gallbladder cancer (GBC) is unclear., Materials and Methods: We found that kaempferol significantly inhibited the growth, invasion and migration, meanwhile induced apoptosis through cells arrested at G0/G1 phase of GBC cell lines, including GBC-SD and SGC996 cells in vitro., Results: Kaempferol promoted the release of cytochrome C from the mitochondria to cytoplasm, the activation of c-caspase-3 and c-caspase-9 and increased the expression levels of pro-apoptotic factor Bax, meanwhile decreased the expression levels of anti-apoptotic factor Bcl-2. In addition, the expression levels of CDK4, CDK6 and cyclin D1, which are members of the CDK4/CDK6/cyclin D1 signaling pathway, were also decreased by kaempferol. Moreover, kaempferol could efficiently prevent tumor progression of GBC in the xenograft in vivo., Conclusions: Our results demonstrated that kaempferol suppressed GBC progression through activation of the CDK4/CDK6/cyclin D1 signaling pathway, suggesting that it might be a potential anti-tumor agent for clinical treatment of GBC.

The t(8;21) translocation is the most common cytogenetic abnormality in acute myeloid leukemia (AML). Although t(8;21) AML patients have a relatively favorable prognosis, relapse is a frequent occurrence, underscoring the need to develop novel therapeutic approaches. Here, we showed that t(8;21) AML is characterized by frequent mutation and overexpression of CCND2. Analysis of 19 AML cell lines showed that t(8;21) AML cells had lower IC50 values for the selective CDK4/6 inhibitors palbociclib and abemaciclib than non-t(8;21) AML cells. CDK4/6 inhibitors caused cell cycle arrest at G1 phase and impaired cell proliferation in t(8;21) AML cells. CDK4/6 inhibition decreased MAP-ERK and PI3K-AKT-mTOR signaling pathway activity, induced LC3B-I to LC3B-II conversion, and enhanced autophagosome formation, suggesting autophagy induction. Treatment of t(8;21) AML cells with the autophagy inhibitors chloroquine (CQ) or LY294002 in combination with the CDK4/6 inhibitor abemaciclib significantly increased the percentage of apoptotic (Annexin V positive) cells, whereas CQ or LY294002 single treatment had no significant effects. The effectiveness of co-inhibiting CDK4/6 and autophagy was confirmed in primary t(8;21) AML cells. The results suggest that the combination of CDK4/6 and autophagy inhibitors had a synergistic effect on inducing apoptosis, suggesting a novel therapeutic approach for the treatment of t(8;21) AML.

PI3K and CDK4/6 inhibitors (CDK4/6i) are targeted therapies approved to treat advanced breast cancer; CDK4/6is are more widely used. Here, we discuss trials that examine PI3K inhibitors with novel drug combinations, including a CDK4/6i, given data implicating the pathway in CDK 4/6 resistance. See related articles by Lu et al., p. 408, and Tolaney et al., p. 418 ., (©2020 American Association for Cancer Research.)

BACKGROUNDAlthough CDK4/6 inhibitors are an established treatment for hormone receptor-positive, HER2-negative metastatic breast cancers, their benefit in other malignancies remains limited.METHODSWe investigated factors associated with clinical outcomes from CDK4/6 inhibitor-based therapy among patients with G1/S phase cell-cycle alterations (CDK4/6 amplifications, CCND1/2/3 amplifications, or CDKN2A/B alterations).RESULTSOverall, 2457 patients with diverse solid tumors that underwent clinical-grade, next-generation sequencing (182-465 genes) and therapy outcome of (non-breast cancer) patients treated with matched CDK4/6 inhibitors were analyzed. G1/S phase cell-cycle alterations occurred in 20.6% (507 of 2457) of patients; 99% of those patients (n = 501) harbored ≥1 characterized co-alteration (median, 4; range, 0-24). In 40 patients with G1/S phase cell-cycle alterations given CDK4/6 inhibitors as part of their regimen, significantly longer median progression-free survival (PFS) was observed when CDK4/6 inhibitor-based therapies matched a larger proportion of tumor alterations, often because CDK4/6 inhibitors were administered together with other drugs that were matched to genomic co-alterations, hence achieving a high matching score (high vs. low [≥50% vs. <50%] matching score, PFS, 6.2 vs. 2.0 months, P < 0.001 [n = 40] [multivariate]) and higher rate of stable disease ≥6 months or an objective response (57% vs. 21%, P = 0.048).CONCLUSIONIn summary, in cell-cycle-altered cancers, matched CDK4/6 inhibitors, as part of an individualized regimen targeting a majority of genomic alterations, was independently associated with longer PFS.TRIAL REGISTRATIONClinicalTrials.gov NCT02478931.FUNDINGJoan and Irwin Jacobs Fund, National Cancer Institute (P30 CA023100, R01 CA226803), and the FDA (R01 FD006334).

Cyclin-dependent kinase (CDK) 4/6 inhibitors have emerged in the treatment of metastatic hormone receptor (HR)-positive and human epidermal growth factor receptor 2 (HER2)-negative breast cancer. However, most patients will eventually present disease progression, highlighting the inevitable resistance of cancer cells to CDK4/6 inhibition. Several studies have suggested that resistance mechanisms involve aberrations of the molecules that regulate the cell cycle, and the re-wiring of the cell to escape CDK4/6 dependence and turn to alternative pathways. Loss of retinoblastoma function, overexpression of CDK 6, upregulation of cyclin E, overexpression of CDK 7, and dysregulation of several signaling pathways, notably the PI3/AKT/mTOR pathway, have been implicated in the development of resistance to CDK4/6 inhibitors. Overlap with endocrine resistance mechanisms might be possible. Combinational therapeutic strategies should be explored in order to prevent resistance and optimize the management of patients after progression under CDK 4/6 inhibition.

The development of cyclin-dependent kinases (CDK) 4 and 6 inhibitors represented a substantial breakthrough in the treatment of estrogen receptor positive (ER+), human epidermal growth factor receptor 2 (HER2) negative metastatic breast cancer. These drugs showed a significant clinical benefit in pivotal clinical trials. However, resistance eventually occurs, leading to disease progression. Next Generation Sequencing methodologies have been employed to investigate predictive biomarkers of response or resistance to CDK4/6 inhibitors. Whole exome and targeted sequencing of solid and liquid biopsies have revealed several possible genomic alterations associated with resistance. Notably, genomic alterations identified by DNA-sequencing did not fully recapitulate the entire landscape of resistance to CDK4/6 inhibitors. Gene expression analysis, such as RNA-Seq methodologies, have provided insights into transcriptional profiles and may need further application. Herein, we report the main findings derived from the use of NGS analysis in the context of resistance to CDK4/6 inhibitors in ER + breast cancer., (Copyright © 2020. Published by Elsevier B.V.)

Induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRs) have been reported to regulate CM proliferation. In particular, miR‑449a‑5p has been identified to be associated with CM proliferation in previous high throughput functional screening data. However, whether miR‑449a‑5p regulates CM proliferation has not been thoroughly investigated. This study aimed to explore whether miR‑449a‑5p modulates CM proliferation and to identify the molecular mechanism via which miR‑449a‑5p regulates CM proliferation. The current study demonstrated that miR‑449a‑5p expression levels were significantly increased during heart development. Furthermore, the results suggested that miR‑449a‑5p mimic inhibited CM proliferation in vitro as determined via immunofluorescence for ki67 and histone H3 phosphorylated at serine 10 (pH3), as well as the numbers of CMs. However, miR‑449a‑5p knockdown promoted CM proliferation. CDK6 was identified as a direct target gene of miR‑449a‑5p, and CDK6 mRNA and protein expression was suppressed by miR‑449a‑5p. Moreover, CDK6 gain‑of‑function increased CM proliferation. Overexpression of CDK6 also blocked the inhibitory effect of miR‑449a‑5p on CM proliferation, indicating that CDK6 was a functional target of miR‑449a‑5p in CM proliferation. In conclusion, miR‑449a‑5p inhibited CM proliferation by targeting CDK6, which provides a potential molecular target for preventing myocardial injury.

Objective: To investigate the effects of miR-191-5p on cell migration, clone formation and proliferation of gastric cancer (GC) cells. Methods: The level of miR-191-5p expression was detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in 60 paired GC tissues and their adjacent normal tissues. miR-191-5p overexpression was achieved by transfection of construct pcDNA-miR-191-5p into GC cells. The migration, clone formation and proliferation of GC cells were detected by the scratch wound assay, clone formation assay and cell counting kit-8 (CCK-8), respectively. Low expression of miR-191-5p was achieved with miRNA-191-5p inhibitor. The binding sites of cyclin-dependent kinase 6 (CDK6) and miR-191-5p were analyzed using TargetScan software, and the interaction of CDK6 and miR-191-5p was verified using dual-fluorescence reporter gene expression. Western blot (WB) was used to detect the effect of miR-191-5p on the expression of p21 and CDK6 proteins. Results: miR-191-5p decreased in 53 cases (88%) of GC tissues compared to their controls. Furthermore, overexpression of miR-191-5p effectively inhibited the migration, clone formation and proliferation of GC cells ( P <0.05). Dual-fluorescence reporter confirmed that miR-191-5p bound to 3'UTR of CDK6. WB showed that pcDNA-miR-191-5p inhibited the CDK6 expression but promoted the p21. Conclusion: Down-regulation of miR-191-5p has a correlation with the progression of GC. Overexpression of miR-191-5p can decrease the expression of CDK6 and inhibit the growth of GC cells.

The present study aimed to investigate the effect of miR-186 on proliferation, migration, invasion, and epithelialmesenchymal transition (EMT) of hepatocellular carcinoma (HCC). In this work, miR-186 was downregulated in HCC tissues and cells, and low miR-186 level helped predict the occurrence of vascular invasion and poor prognosis in patients with HCC. miR-186 overexpression inhibited cell proliferation and tumor growth in nude mice, repressed migration and invasion abilities, and enhanced apoptosis in HCC cells. miR-186 also retarded progression of EMT. miR-186 directly bound to the 3-untranslated regions of cyclin-dependent kinase 6 (CDK6) to inhibit its expression. Overexpression of CDK6 markedly reversed inhibitory effects of miR-186 on proliferation, apoptosis, migration, and invasion of HCC cells. Conversely, inhibition of CDK6 exerted synergic effect on the biological functions of miR-186. In conclusion, miR-186 represses proliferation, migration, invasion, and EMT, and induces apoptosis through targeting CDK6 in HCC, which may provide a new therapeutic target for HCC.

Non-small-cell lung cancer (NSCLC) is the main pathological type of lung cancer and has a low overall five-year survival rate. miR-187 has been reported to play major roles in various tumor types. In this study, we explored the impact of miR-187 on NSCLC. qRT-PCR results demonstrated that miR-187 expression is lower in NSCLC and cancer cells than normal tissues and normal lung cells. miR-187 expression levels are associated with tumor size, TNM stage and overall survival rate. MTS and colony formation assays showed that high miR-187 expression inhibits NSCLC cell proliferation and colony formation ability, and flow cytometry showed that miR-187 overexpression induces cell cycle arrest at the G0/G1 phase. A luciferase reporter assay showed that FGF9 is a target of miR-187. miR-187 overexpression reduces the expression of FGF9, cyclin D1 CDK4 and CDK6. Therefore, miR-187 may present a new NSCLC treatment target by regulates cyclins-related protein expression.

Acute myeloid leukemia (AML) is an aggressive disease for which only few targeted therapies are available. Using high-throughput RNA interference (RNAi) screening in AML cell lines, we identified LIM kinase 1 (LIMK1) as a potential novel target for AML treatment. High LIMK1 expression was significantly correlated with shorter survival of AML patients and coincided with FLT3 mutations, KMT2A rearrangements, and elevated HOX gene expression. RNAi- and CRISPR-Cas9-mediated suppression as well as pharmacologic inhibition of LIMK1 and its close homolog LIMK2 reduced colony formation and decreased proliferation due to slowed cell-cycle progression of KMT2A-rearranged AML cell lines and patient-derived xenograft (PDX) samples. This was accompanied by morphologic changes indicative of myeloid differentiation. Transcriptome analysis showed upregulation of several tumor suppressor genes as well as downregulation of HOXA9 targets and mitosis-associated genes in response to LIMK1 suppression, providing a potential mechanistic basis for the anti-leukemic phenotype. Finally, we observed a reciprocal regulation between LIM kinases (LIMK) and CDK6, a kinase known to be involved in the differentiation block of KMT2A-rearranged AML, and addition of the CDK6 inhibitor palbociclib further enhanced the anti-proliferative effect of LIMK inhibition. Together, these data suggest that LIMK are promising targets for AML therapy.

The regulation and function of cyclin-dependent kinase 6 (CDK6)- and cyclin-dependent kinase 4 (CDK4)-cyclin complexes are commonly altered with enhanced kinase activity found in hematopoietic malignancies, breast cancer and melanoma making CDK4 and CDK6 attractive targets for therapeutic interference. Although dual CDK4/6 inhibitors have revolutionized treatment of breast cancer patients and reveal promising results in several solid tumors and hematological malignancies, there is a need for novel compounds targeting the versatile kinase-independent functions of CDK6 to improve cancer treatment. The following review summarizes the latest findings on CDK6 in cancer development and discusses novel therapeutic approaches to selectively inhibit CDK6s function as a transcriptional regulator., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Psoriasis is a frequent, inflammatory skin disease characterized by keratinocyte hyperproliferation and a disease-related infiltration of immune cells. Here, we identified a novel proinflammatory signaling pathway driven by cyclin-dependent kinase 4 (CDK4) and CDK6 and the methyltransferase EZH2 as a valid target for psoriasis therapy. Delineation of the pathway revealed that CDK4/6 phosphorylated EZH2 in keratinocytes, thereby triggering a methylation-induced activation of STAT3. Subsequently, active STAT3 resulted in the induction of IκBζ, which is a key proinflammatory transcription factor required for cytokine synthesis in psoriasis. Pharmacological or genetic inhibition of CDK4/6 or EZH2 abrogated psoriasis-related proinflammatory gene expression by suppressing IκBζ induction in keratinocytes. Importantly, topical application of CDK4/6 or EZH2 inhibitors on the skin was sufficient to fully prevent the development of psoriasis in various mouse models by suppressing STAT3-mediated IκBζ expression. Moreover, we found a hyperactivation of the CDK4/6-EZH2 pathway in human and mouse psoriatic skin lesions. Thus, this study not only identifies a novel psoriasis-relevant proinflammatory pathway, but also proposes the repurposing of CDK4/6 or EZH2 inhibitors as a new therapeutic option for patients with psoriasis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly solid tumors in the world. Aerobic glycolysis is among the characteristic features of pancreatic cancer. However, the regulatory process of aerobic glycolysis in pancreatic cancer is too complicated, and the underlying mechanism remains unexplained. Reportedly, CDK4/6 inhibitors repress breast cancer cell proliferation by modulating glucose metabolism. Here, we reveal that the CDK4/6 inhibitor, PD0332991 stabilized FBP1 to hinder aerobic glycolysis in pancreatic cancer. We also show that the CDK4/6-E2 F1 signaling pathway mediated an increase in MAGED1 expression, promoting FBP1 degradation in pancreatic cancer. We, therefore, might have identified a novel mechanism by which the CDK4/6 inhibitor, PD0332991 blocks the Warburg effect of pancreatic cancer by stabilizing FBP1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The present study aimed to investigate the expression of cyclin-dependent kinase 6 (CDK6) and microRNA-126-5p (miR-126-5p) in esophageal cancer tissues and cells, and their effect on esophageal cancer cell proliferation and invasion, and to explore the potential molecular mechanisms. The relative expression levels of CDK6 and miR-126-5p in esophageal cancer tissue, paracancerous tissue, and HEEC and EC109 cells were determined and compared using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A miR-126-5p overexpression vector was constructed and a stable EC109 cell line expressing miR-126-5p was established. The EC109 cell line was transfected with a CDK6 small interfering RNA sequence. The rate of cell proliferation was determined using the WST-8 method, and cell invasion was determined using a Transwell assay. In addition, the relative expression levels of genes were determined using RT-qPCR; the relative expression levels of proteins were determined by western blot analysis; the binding sites of CDK6 and miR-126-5p were analyzed using TargetScan software; and the interaction of CDK6 and miR-126-5p was verified using dual-fluorescence reporter gene expression. Esophageal tissues and EC109 cells expressed higher levels of CDK6 but significantly lower levels of miR-126-5p compared with adjacent tissues and HEEC cells, and their correlation coefficient between esophageal tissues and matched adjacent tissues was -7.526. The overexpression of miR-126-5p and CDK6 knockdown in the EC109 cell line inhibited cell proliferation and invasion compared with the control and NC (negative control) groups. miR-126-5p overexpression reduced the relative expression level of CDK6, and CDK6 knockdown by siRNA increased the expression of miR-126-5p. miR-126-5p regulated CDK6 expression by binding to the 3'-untranslated region of its mRNA. Overexpression miR-126-5p inhibited the proliferation and migration of esophageal cancer cells by targeting CDK6 and negatively regulating its expression. These findings contribute to the understanding of the underlying molecular mechanism of esophageal cancer., (© 2020 American Association for Anatomy.)

Mounting evidence has implicated the vital role of long noncoding RNAs (lncRNAs) in non-small cell lung cancer (NSCLC). This study aims to investigate the mechanism of lncRNA MEG8 on NSCLC progression. The mRNA expressions of MEG8 and miR-107 were examined in tumor and adjacent normal tissues from patients with NSCLC by qRT-PCR. Lung epithelial BEAS-2B cells were transfected with MEG8 overexpression plasmid, and NSCLC A549 and H1299 cells were transfected with MEG8 or miR-107 overexpression/knockdown plasmid to detect the function of MEG8 or miR-107 on cell activity. The function of MEG8 and miR-107 on cell proliferation, cell cycle changes, invasion and migration was separately determined by Cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine staining, flow cytometry, transwell and cell scratch test. Target sites for miR-107 and MEG8, miR-107 and CDK6 were determined and verified by a dual luciferase gene reporter assay. The expression levels of the Rb/E2F3 signal pathway related proteins (p21, p27, E2F3 and Rb) were inspected by Western blot. MEG8 was strongly expressed while miR-107 was lowly expressed in tumor tissues and cells. Overexpression of MEG8 potentiated cell proliferation, migration and invasion in BEAS-2B cells. Silencing MEG8 or overexpression of miR-107 clearly hindered cell progression in A549 and H1299 cells. Mechanistically, MEG8 and CDK6 can competitively bind to miR-107 and together regulate the progression of NSCLC. Additionally, silencing MEG8 or overexpression of miR-107 can inhibit the phosphorylation levels of Rb and E2F3. Evidence in this work indicated that MEG8 regulates miR-107/CDK6 axis to promote NSCLC progression by activating the Rb/E2F3 pathway.

Pancreatic cancer (PC) is a leading cause of cancer mortality and is expected to continue increasing incidence. Abnormally expressed microRNAs have been demonstrated tightly correlated with the development and progression of PC. However, the molecular mechanisms remain largely unknown. In this study through combing both the TCGA database and our two verification PC cohorts, we found the consistent reduction of miR-3613-5p in PC tumors, which significantly correlated with reduced cumulative survival rate among PC patients. PC patients with higher lymph node metastasis rate show reduced miR-3613-5p expression. Through further mechanistic investigation, we demonstrate that miR-3613-5p down-regulated CDK6 in repressing the metastasis capacity of PC cells in vitro and in vivo . Elevated CDK6 were also found in PC samples, which also correlate with poor prognosis. Thus, our study found a novel tumor repressor miR-3613-5p in PC progression.

Cell-cycle entry relies on an orderly progression of signaling events. To start, cells first activate the kinase cyclin D-CDK4/6, which leads to eventual inactivation of the retinoblastoma protein Rb. Hours later, cells inactivate APC/C CDH1 and cross the final commitment point. However, many cells with genetically deleted cyclin Ds, which activate and confer specificity to CDK4/6, can compensate and proliferate. Despite its importance in cancer, how this entry mechanism operates remains poorly characterized, and whether cells use this path under normal conditions remains unknown. Here, using single-cell microscopy, we demonstrate that cells with acutely inhibited CDK4/6 enter the cell cycle with a slowed and fluctuating cyclin E-CDK2 activity increase. Surprisingly, with low CDK4/6 activity, the order of APC/C CDH1 and Rb inactivation is reversed in both cell lines and wild-type mice. Finally, we show that as a consequence of this signaling inversion, Rb inactivation replaces APC/C CDH1 inactivation as the point of no return. Together, we elucidate the molecular steps that enable cell-cycle entry without CDK4/6 activity. Our findings not only have implications in cancer resistance, but also reveal temporal plasticity underlying the G1 regulatory circuit.

Cell cycle dysregulation caused by aberrant cyclin D1 and CDK4 expression is a major determinant for proliferation of cancer cells in mantle cell lymphoma (MCL). Inhibition of CDK4/6 induces G1 arrest of MCL cells in patients, appearing to deepen and prolong the clinical response to partner agents. This article reviews aberrations of cell cycle genes in MCL cells and clinical trials of CDK4/6 inhibitors for MCL. Integrative longitudinal functional genomics is discussed as a strategy to discover genomic drivers for resistance in cancer cells and cancer-immune interactions that potentially contribute to the clinical response to palbociclib combination therapy in MCL., Competing Interests: Conflicts of interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Osteosarcoma (OS) is one of the most commonly diagnosed malignant tumors that mainly affects children and adolescents. The underlying molecular mechanisms that are responsible for the initiation and development of OS are still not clear. Increasing evidence suggested the tumor suppressor role of microRNA-524-5p in a variety of cancers via targeting key pathways involved in tumorigenesis. The aim of this study was to characterize the function of miR-524-5p in OS., Methods: A total 50 paired OS tissues and adjacent normal tissues were collected from OS patients. The expression of miR-524-5p in OS tissues and cells was detected by RT-qPCR. The CCK-8 assay, flow cytometry and transwell assay were applied to determine the proliferation and invasion abilities of OS cells. The targets of miR-524-5p were predicted using the miRDB dataset and confirmed by luciferase reporter assay and western blot analysis., Results: The expression of miR-524-5p was decreased in OS tissues and cell lines. OS patients with lymph node metastasis harbored relative lower level of miR-524-5p. Overexpression of miR-524-5p in OS cells significantly suppressed the proliferation, drove cell cycle arrest and apoptosis. The mechanism investigation revealed that miR-524-5p bound the 3'-untranslated region (UTR) of Cyclin Dependent Kinase 6 (CDK6) and repressed the expression of CDK6 in OS cells. Overexpressed CDK6 was found in OS tissues, which was inversely correlated with that of miR-524-5p. Moreover, forced expression of CDK6 significantly reversed the anti-cancer effects of miR-524-5p on the proliferation, apoptosis and cell cycle arrest of OS cells., Conclusions: Our results identified the tumor-suppressive role of miR-524-5p in OS via targeting CDK6, which may lead to the identification of novel therapeutic target for the treatment of OS., Competing Interests: Declaration of competing interest These authors declare that they have no conflicts of interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Palbociclib, a CDK4/6 inhibitor, has been granted accelerated approval by US FDA for hormone receptor-positive HER2-negative metastatic breast cancer. To determine potential biomarkers of palbociclib sensitivity to assist in patient selection and clinical development, we investigated the effects of palbociclib in a panel of molecularly characterized breast cancer cell lines. We quantified palbociclib sensitivity and c-myc expression in 11 breast cancer cell lines, 124 breast cancer samples, and The Cancer Genome Atlas database. We found non-TNBC subtypes were more sensitive to palbociclib than TNBC. Activation of c-myc led to differential palbociclib sensitivities, and further inhibition of c-myc enhanced palbociclib sensitivity. Moreover, we identified for the first time a c-myc/miR-29b-3p/CDK6 axis in breast cancer that could be responsible for c-myc-induced palbociclib insensitivity, in which c-myc activation resulted in downregulation of miR-29b-3p, further activated CDK6 and inhibited cell-cycle arrest at G 1 phase. Moreover, downregulated (inactived) c-myc-induced oncogenic addiction could increase palbociclib efficacy, using both Xenograft model and patient-derived tumor xenograft (PDTX) model. Our finding extends the concept of combined blockade of the CDK4/6 and c-myc signaling pathways to increase palbociclib sensitivity, making c-myc a promising biomarker for palbociclib sensitivity in breast cancer.

Palbociclib, ribociclib, and abemaciclib belong to the third generation of cyclin-dependent kinases inhibitors (CDKis), an established therapeutic class for advanced and metastatic breast cancer. Interindividual variability in the therapeutic response of CDKis has been reported and some individuals may experience increased and unexpected toxicity. This narrative review aims at identifying the factors potentially concurring at this variability for driving the most appropriate and tailored use of CDKis in the clinic. Specifically, concomitant medications, pharmacogenetic profile, and pathophysiological conditions could influence absorption, distribution, metabolism, and elimination pharmacokinetics. A personalized therapeutic approach taking into consideration all factors potentially contributing to an altered pharmacokinetic/pharmacodynamic profile could better drive safe and effective clinical use.

Background/aims: Studies have found that LncRNA CYTOR is an important regulator of cancer. However, the function of lncRNA CYTOR in pancreatic cancer (PC) is unclear. This study amid to explore the regulation of lncRNA CYTOR in PC., Methods: The expression of CYTOR and miR-205-5p in PC was detected by RT-qPCR. CCK-8 assay, colony formation assay and scratch test were conducted to detect the effects of CYTOR and miR-205-5p on proliferation and migration of PC cells. Target gene prediction and screening and luciferase reporter assays were used to verify downstream target genes of CYTOR and miR-205-5p. The expression of Cyclin-dependent protein kinase 6 (CDK6) was detected by Western blotting. The tumor growth in mice was detected by in vivo experiments in nude mice., Results: The expression of LncRNA CYTOR was significantly elevated in PC. Knockdown of CYTOR significantly inhibited cell proliferation and migration of PC cells. In vivo animal studies showed that CYTOR promoted tumor growth. MiR-205-5p was a direct target of CYTOR, and the expression levels of miR-205-5p were significantly reduced in PC cell lines. Furthermore, co-transfection of shCYTOR with miR-205-5p inhibitor partially abolished the effect of shCYTOR on cell proliferation and migration. In addition, CYTOR was negatively correlated with the expression of miR-205-5p. CDK6 was a direct target of miR-205-5p, and miR-205-5p mimic and sh CYTOR significantly reduced the expression levels of CDK6., Conclusion: CYTOR can promote PC progression by modulating the miR-205-5p/CDK6 axis, which may be a potential therapeutic target for PC., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 IAP and EPC. Published by Elsevier B.V. All rights reserved.)

This study aimed to investigate the function of long non-coding RNA FOXD2 adjacent opposite strand RNA 1 (lncRNA FOXD2-AS1) during the progression of esophagus cancer (EC) and explore its underlying molecular mechanisms. The level of FOXD2-AS1 in EC tissues and paracancerous tissues was detected by using RT-qPCR; ROC curve was used to evaluate the diagnostic value of FOXD2-AS1 for EC. In addition, CCK8 assay and immunofluorescence staining assay were used to detect the proliferation of Eca-109 and TE-1 cells. To investigate the function of FOXD2-AS1 on cell apoptosis and cell cycle, flow cytometry was performed. To detect the invasion ability of EC cells, transwell invasion assay was performed. Starbase3.0 and Targetscan were used to predict the target genes of FOXD2-AS1 and miR-145-5p, and protein expressions were detected with western blot. We found FOXD2-AS1 was significantly upregulated in EC tissues compared with adjacent normal tissues, which was positively correlated with clinicopathological parameters of patients with EC. Downregulation of FOXD2-AS1 inhibited the proliferation and invasion by inducing apoptosis of EC cells. Moreover, FOXD2-AS1 may regulate the expression of CDK6 by targeting miR-145-3p. Meanwhile, silencing of FOXD2-AS1 caused G1 phase arrest of EC cells by reducing the expression of CDK6. In conclusion, silening FOXD2-AS1 significantly inhibited the proliferation and invasion of EC cells by regulating the miR-145-5p/CDK6 axis. Therefore, FOXD2-AS1 might be used as diagnostic biomarker and therapeutic target for EC.

MicroRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level which have been reported to be involved in the pathogenesis of various cancers. In the present study, we found that miR-497 was downregulated in osteosarcoma tissues. Gain and loss of function studies were carried out to investigate the effect of miR-497 on the growth of osteosarcoma cells. The results indicated that miR-497 inhibited the growth of osteosarcoma cells. Furthermore, bioinformatics analysis predicted plexinA4 and CDK6 as targets of miR-497, which was afterward confirmed by luciferase activity assay and rescue experiments. These findings suggested that miR-497, plexinA4 and CDK6 may serve as novel potential makers for osteosarcoma diagnostics and therapy.

MicroRNAs (or miRNAs) are short nucleotide sequences (~17-22 bp long) that play important roles in gene regulation through targeting genes in the 3'untranslated regions (UTRs). Variants located in genomic regions might have different biological consequences in changing gene expression. Exonic variants (e.g., coding variant and 3'UTR variant) are often causative of diseases due to their influence on gene product. Variants harbored in the 3'UTR region where miRNAs perform their targeting function could potentially alter the binding relationships for target pairs, which could relate to disease causation. We gathered miRNA-mRNA targeting pairs from published studies and then employed the database of microRNA Target Site single nucleotide variants (SNVs) (dbMTS) to discover novel SNVs within the selected pairs. We identified a total of 183 SNVs for the 114 pairs of accurate miRNA-mRNA targeting pairs selected. Detailed bioinformatics analysis of the three genes with identified variants that were exclusively located in the 3'UTR section indicated their association with intellectual disability (ID). Our result showed an exceptionally high expression of GPR88 in brain tissues based on GTEx gene expression data, while WNT7A expression data were relatively high in brain tissues when compared to other tissues. Motif analysis for the 3'UTR region of WNT7A showed that five identified variants were well-conserved across three species (human, mouse, and rat); the motif that contains the variant identified in GPR88 is significant at the level of the 3'UTR of the human genome. Studies of pathways, protein-protein interactions, and relations to diseases further suggest potential association with intellectual disability of our discovered SNVs. Our results demonstrated that 3'UTR variants could change target interactions of miRNA-mRNA pairs in the context of their association with ID. We plan to automate the methods through developing a bioinformatics pipeline for identifying novel 3'UTR SNVs harbored by miRNA-targeted genes in the future.

Laboratory research and pharmacoepidemiology provide support for metformin as a potential antitumor agent. However, the lack of a clear understanding of the indications of metformin limits its efficacy. Here, we performed a genome-wide CRISPR knockout negative screen to identify potential targets that might synergize with metformin. Next-generation sequencing of pooled genomic DNAs isolated from surviving cells after 18 days of metformin treatment (T18) compared to those of the untreated cells at day 0 (T0) yielded candidate genes. Knockdown of a group of cyclin-dependent kinases (CDKs), including CDK1, CDK4, and CDK6, confirmed the results of the screen. Combination treatment of the CDKs inhibitor abemaciclib with metformin profoundly inhibited tumor viability in vitro and in vivo. Although cell cycle parameters were not further altered under the combination treatment, investigation of the metabolome revealed significant changes in cell metabolism, especially with regard to fatty acid oxidation, the tricarboxylic acid cycle and aspartate metabolism. Such changes appeared to be mediated through inhibition of the mTOR pathway. Collectively, our study suggests that the combination of CDKs inhibitor with metformin could be recognized as a potential therapy in future clinical applications.

Lysosomes are degradation and signaling organelles that adapt their biogenesis to meet many different cellular demands; however, it is unknown how lysosomes change their numbers for cell division. Here, we report that the cyclin-dependent kinases CDK4/6 regulate lysosome biogenesis during the cell cycle. Chemical or genetic inactivation of CDK4/6 increases lysosomal numbers by activating the lysosome and autophagy transcription factors TFEB and TFE3. CDK4/6 interact with and phosphorylate TFEB/TFE3 in the nucleus, thereby inactivating them by promoting their shuttling to the cytoplasm. During the cell cycle, lysosome numbers increase in S and G2/M phases when cyclin D turnover diminishes CDK4/6 activity. These findings not only uncover the molecular events that direct the nuclear export of TFEB/TFE3, but also suggest a mechanism that controls lysosome biogenesis in the cell cycle. CDK4/6 inhibitors promote autophagy and lysosome-dependent degradation, which has important implications for the therapy of cancer and lysosome-related disorders., (© 2020 Yin et al.)

Background: Germ cell tumours (GCTs) are the most common solid malignancies in young men. Although high cure rates can be achieved, metastases, resistance to cisplatin-based therapy and late toxicities still represent a lethal threat, arguing for the need of new therapeutic options. In this study, we analysed the potential of cyclin-dependent kinase 4/6 (CDK4/6) inhibitors palbociclib and ribociclib (PaRi) as molecular drugs to treat cisplatin-resistant and -sensitive paediatric and adult GCTs., Methods: Ten GCT cell lines, including cisplatin-resistant subclones and non-malignant controls, were treated with PaRi and screened for changes in viability (triphenyl tetrazolium chloride (XTT) assay), apoptosis rates (flow cytometry, caspase assay), the cell cycle (flow cytometry), the transcriptome (RNA-sequencing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and on protein level (western blot). Expression profiling was performed on paediatric and adult GCT tissues (expression microarrays, qRT-PCR, immunohistochemistry, 'The Cancer Genome Atlas' database)., Results: We demonstrate that adult GCTs highly express CDK4, while paediatric GCTs strongly express CDK6 instead. Thus, both GCT types are potentially treatable by PaRi. GCTs presented as highly sensitive towards PaRi, which caused a decrease in viability, cell cycle arrest and apoptosis. Although GCTs mainly arrested in the G1/G0 phase, some embryonal carcinoma cell lines were able to bypass the G1/S checkpoint and progressed to the G2/M phase. We found that upregulation of CDK3 and downregulation of many mitosis regulation factors, like the HAUS genes, might be responsible for bypassing the G1/S checkpoint and termination of mitosis, respectively. We postulate that GCT cells do not tolerate these alterations in the cell cycle and eventually induce apoptosis., Conclusion: Our study highlights PaRi as therapeutic options for cisplatin-resistant and -sensitive paediatric and adult GCTs.

Increasing evidence suggests that circular RNAs are emerging biomarkers or targets for early cancer diagnosis and treatment. However, the studies of circular RNA in osteosarcoma (OS) are limited. In this study we found that circ&#95;ARF3 were highly expressed in osteosarcoma cell lines and tumor tissues. Knocking down circ&#95;ARF3 greatly ceased OS cell growth, impaired cell colony formation and halted cell cycle transition from G1 to S phase. Bioinformatic analysis suggested that miR-1299 is the target of circ&#95;ARF3. Luciferase assay and biotin labeled circ&#95;ARF3 pull down assay confirmed their interactions in OS cells. The regulatory roles of circ&#95;ARF3 on miR-1299 was also investigated. Further bioinformatic analysis showed that CDK6 is the target of miR-1299. Overexpressing miR-1299 in OS cells decreased CDK6 expression and arrested OS cell growth and cell cycle progression. However, the roles of miR-1299 in regulating CDK6 expression, OS cell growth and cell cycle progression were greatly impaired in the presence of circ&#95;ARF3. In general, our study demonstrated that in the OS, highly expressed circ&#95;ARF3 acts as a sponge of miR-1299 to inhibit miR-1299 mediated CDK6 downregulation which further promoted OS pathogenesis. circ&#95;ARF3 could be a potential target for OS treatment in the future., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020. Published by Elsevier Inc.)

Several different mechanisms are implicated in the resistance of lung cancer cells to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), and only few have been functionally investigated. Here, using genetically knocked out EGFR and TKI-resistant lung cancer cells, we show that loss of wild-type EGFR attenuates cell proliferation, migration and 3D-spheroid formation, whereas loss of mutant EGFR or resistance to TKIs reinforces those processes. Consistently, disruption of wild-type EGFR leads to suppression of HER2/HER3, while mutant EGFR ablation or resistance to TKIs increases HER2/HER3 expression, compensating for EGFR loss. Furthermore, HER2/HER3 nuclear translocation mediates overexpression of cyclin D1, leading to tumor cell survival and drug resistance. Cyclin D1/CDK4/6 inhibition resensitizes erlotinib-resistant (ER) cells to erlotinib. Analysis of cyclin D1 expression in patients with non-small cell lung carcinoma (NSCLC) showed that its expression is negatively associated with overall survival and disease-free survival. Our results provide biological and mechanistic insights into targeting EGFR and TKI resistance., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)