Search

PROteolysis TArgeting Chimeras (PROTACs) have been considered the next blockbuster therapies. However, due to their inherent limitations, the efficacy of PROTACs is frequently impaired by limited tissue penetration and particularly insufficient cellular internalization into their action sites. Herein, based on the ultra-pH-sensitive and enzyme-sensitive nanotechnology, a type of polymer PROTAC conjugated and pH/cathepsin B sequential responsive nanoparticles (PSRNs) are deliberately designed, following the construction of the PROTAC for Cyclin-dependent kinase 4 and 6 (CDK4/6). Colorectal cancer (CRC) which hardly responds to many treatments even immune checkpoint blockades was selected as the tumor model in this study. As a result, PSRNs were found to maintain nanostructure (40 nm) in circulation and efficiently accumulated in tumors via enhanced permeation and retention effect. Then, they were dissociated into unimers (<10 nm) in response to an acidic tumor microenvironment, facilitating tumor penetration and cellular internalization. Eventually, the CDK4/6 degrading PROTACs were released intracellularly following the cleavage of cathepsin B. Importantly, PSRNs led to the enhanced degradation of target protein in vitro and in vivo. The degradation of CDK4/6 also augmented the efficacy of immune checkpoint blockades, through the upregulation of programmed cell death-ligand 1 (PD-L1) expression in cancer cells and the suppression of regulatory T cells cell proliferation in tumor microenvironment. By combination with α-PD-1, an enhanced anti-tumor outcome is well achieved in CT26 tumor model. Overall, our study verifies the significance of precise intracellular delivery of PROTACs and introduces a promising therapeutic strategy for the targeted combination treatment of CRC., (© 2024. The Author(s).)

Colorectal cancer (CRC) is one of the most common malignant tumors in the gastrointestinal tract, and has been attracted a great deal attention and extensive investigation due to its high morbidity and mortality rates. The C4orf19 gene encodes a protein with uncharacterized function. Our preliminary exploration of the TCGA database indicated that C4orf19 is markedly downregulated in CRC tissues in comparison to that observed in normal colonic tissues, suggesting its potential association with CRC behaviors. Further studies showed a significant positive correlation between C4orf19 expression levels and CRC patient prognosis. Ectopic expression of C4orf19 inhibited the growth of CRC cells in vitro and tumorigenic ability in vivo. Mechanistic studies showed that C4orf19 binds to Keap1 at near the Lys615, which prevents the ubiquitination of Keap1 by TRIM25, thus protecting the Keap1 protein from degradation. The accumulated Keap1 results in USP17 degradation and in turn leading to the degradation of Elk-1, further attenuates its regulated CDK6 mRNA transcription and protein expression, as well as its mediated proliferation of CRC cells. Collectively, the present studies characterize function of C4orf19 as a tumor suppressor for CRC cell proliferation by targeting Keap1/USP17/Elk-1/CDK6 axis., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Coronary artery disease (CAD) is a long-lasting inflammatory disease characterized by monocyte migration into the vessel wall leading to clinical events like myocardial infarction (MI). However, the role of monocyte subsets, especially their miRNA-driven differentiation in this scenario is still in its infancy. Here, we characterized monocyte subsets in controls and disease phenotypes of CAD and MI patients using flow cytometry and miRNA and mRNA expression profiling using RNA sequencing. We observed major differences in the miRNA profiles between the classical (CD14 ++ CD16 - ) and nonclassical (CD14 + CD16 ++ ) monocyte subsets irrespective of the disease phenotype suggesting the Cyclin-dependent Kinase 6 (CDK6) to be an important player in monocyte maturation. Between control and MI patients, we found a set of miRNAs to be differentially expressed in the nonclassical monocytes and targeting CCND2 (Cyclin D2) that is able to enhance myocardial repair. Interestingly, miRNAs as miR-125b playing a role in vascular calcification were differentially expressed in the classical subset in patients suffering from CAD and not MI in comparison to control samples. In conclusion, our study describes specific peculiarities of monocyte subset miRNA expression in control and diseased samples and provides basis to further functional analysis and to identify new cardiovascular disease treatment targets., (© 2022. The Author(s).)

Despite the development of predictive biomarkers to shape treatment paradigms and outcomes, de novo EGFR TKI resistance advanced non-small cell lung cancer (NSCLC) remains an issue of concern. We explored clinical factors in 332 advanced NSCLC who received EGFR TKI and molecular characteristics through 65 whole exome sequencing of various EGFR TKI responses including; de novo (progression within 3 months), intermediate response (IRs) and long-term response (LTRs) (durability > 2 years). Uncommon EGFR mutation subtypes were significantly variable enriched in de novo resistance. The remaining sensitizing EGFR mutation subtypes (exon 19 del and L858R) accounted for 75% of de novo resistance. Genomic landscape analysis was conducted, focusing in 10 frequent oncogenic signaling pathways with functional contributions; cell cycle, Hippo, Myc, Notch, Nrf2, PI-3-Kinase/Akt, RTK-RAS, TGF-β, p53 and β-catenin/Wnt signaling. Cell cycle pathway was the only significant alteration pathway among groups with the FDR p-value of 6 × 10 -4 . We found only significant q-values of < 0.05 in 7 gene alterations; CDK6, CCNE1, CDK4, CCND3, MET, FGFR4 and HRAS which enrich in de novo resistance [range 36-73%] compared to IRs/LTRs [range 4-22%]. Amplification of CDK4/6 was significant in de novo resistance, contrary to IRs and LTRs (91%, 27.9% and 0%, respectively). The presence of co-occurrence CDK4/6 amplification correlated with poor disease outcome with HR of progression-free survival of 3.63 [95% CI 1.80-7.31, p-value < 0.001]. The presence of CDK4/6 amplification in pretreatment specimen serves as a predictive biomarker for de novo resistance in sensitizing EGFR mutation., (© 2022. The Author(s).)

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.

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.

Yinzhihuang granules (YZHG) is a patented Chinese medicine for the treatment of hepatitis B. This study aimed to investigate the intrinsic mechanisms of YZHG in the treatment of hepatitis B and to provide new evidence and insights for its clinical application. The chemical compounds of YZHG were searched in the CNKI and PUBMED databases, and their putative targets were then predicted through a search of the SuperPred and Swiss Target Prediction databases. In addition, the targets of hepatitis B were obtained from TTD, PharmGKB and DisGeNET. The abovementioned data were visualized using Cytoscape 3.7.1, and network construction identified a total of 13 potential targets of YZHG in the treatment of hepatitis B. Molecular docking verification showed that CDK6, CDK2, TP53 and BRCA1 might be strongly correlated with hepatitis B treatment. Furthermore, GO and KEGG analyses indicated that the treatment of hepatitis B by YZHG might be related to positive regulation of transcription, positive regulation of gene expression, the hepatitis B pathway and the viral carcinogenesis pathway. Network pharmacology intuitively shows the multicomponent, multitarget and multichannel pharmacological effects of YZHG in the treatment of hepatitis B and provides a scientific basis for its mechanism of action.

Cyclin-dependent kinases 4 and 6 (CDK4/6) phosphorylate and inhibit retinoblastoma (RB) family proteins. Hyperphosphorylated RB releases E2F transcription factors, activating a transcriptional program that initiates S phase. Due to the critical role that this pathway has in regulating cell cycle progression, inhibiting CDK4/6 is an attractive therapeutic strategy. Indeed, CDK4/6 inhibitors in combination with antiestrogens produce a significant benefit in patients with ER + /HER2 - breast cancer. Clinical trials are currently investigating if the use of CDK4/6 inhibitors alone or in combination can be extended to other cancer types. Inhibition of CDK4/6 can result in different cell fates such as quiescence, senescence, or apoptosis. Senescence is a stress response that can be induced by stimuli that include oncogenic activation, chemotherapy, irradiation, and targeted therapies such as CDK4/6 inhibitors. Senescent cells undergo a stable cell cycle arrest and produce a bioactive secretome that remodels their microenvironment and engages the immune system. In this review, we analyze the therapeutic relevance of senescence induction by CDK4/6 inhibitors. We also discuss how different therapies, including checkpoint inhibitors and drugs targeting MEK or PI3K, can be used in combination with CDK4/6 inhibitors to reinforce or exploit senescence. Recently, a lot of effort has been put into identifying compounds that selectively kill senescent cells (termed senolytics). Thus, sequential treatment with senolytics might be an additional strategy to potentiate the antitumor effects of CDK4/6 inhibitors.

Combination of CDK4/6 inhibitors and endocrine therapy improves clinical outcome in advanced oestrogen receptor (ER)-positive breast cancer, however relapse is inevitable. Here, we show in model systems that other than loss of RB1 few gene-copy number (CN) alterations are associated with irreversible-resistance to endocrine therapy and subsequent secondary resistance to palbociclib. Resistance to palbociclib occurred as a result of tumour cell re-wiring leading to increased expression of EGFR, MAPK, CDK4, CDK2, CDK7, CCNE1 and CCNE2. Resistance altered the ER genome wide-binding pattern, leading to decreased expression of 'classical' oestrogen-regulated genes and was accompanied by reduced sensitivity to fulvestrant and tamoxifen. Persistent CDK4 blockade decreased phosphorylation of tuberous sclerosis complex 2 (TSC2) enhancing EGFR signalling, leading to the re-wiring of ER. Kinome-knockdown confirmed dependency on ERBB-signalling and G2/M-checkpoint proteins such as WEE1, together with the cell cycle master regulator, CDK7. Noteworthy, sensitivity to CDK7 inhibition was associated with loss of ER and RB1 CN. Overall, we show that resistance to CDK4/6 inhibitors is dependent on kinase re-wiring and the redeployment of signalling cascades previously associated with endocrine resistance and highlights new therapeutic networks that can be exploited upon relapse after CDK4/6 inhibition.

Tumor metastasis is the most common cause of cancer-related deaths, yet it remains poorly understood. The transcription factor zinc-finger E-box binding homeobox 1 (ZEB1) is involved in the epithelial-to-mesenchymal transition (EMT) and plays a pivotal role in tumor metastasis. However, the underlying mechanisms of the posttranslational modification of ZEB1 remain largely unknown. Herein, we demonstrated that specific inhibition of CDK4/6 was able to block tumor metastasis of breast cancer by destabilizing the ZEB1 protein in vitro and in vivo. Mechanistically, we determined that the deubiquitinase USP51 is a bona fide target of CDK4/6. The phosphorylation and activation of USP51 by CDK4/6 is necessary to deubiquitinate and stabilize ZEB1. Moreover, we found a strong positive correlation between the expression of p-RB (an indicator of CDK4/6 activity), p-USP51 and ZEB1 in metastatic human breast cancer samples. Notably, the high expression of p-RB, p-USP51, and ZEB1 was significantly correlated with a poor clinical outcome. Taken together, our results provide evidence that the CDK4/6-USP51-ZEB1 axis plays a key role in breast cancer metastasis and could be a viable therapeutic target for the treatment of advanced human cancers.

CircRNAs have been reported to exert momentous roles in regulating pathophysiological process and guiding clinical diagnosis and treatment in colorectal cancer (CRC). However, there are still a lot of circRNAs that need to be unearthed. In this study, we evaluated the expression profile of circRNAs in 10 CRC tissues and their corresponding normal-appearing tissues (NATs) by microarray, and identified that hsa&#95;circ&#95;101555 (circ101555) was significantly up-regulated in tumor tissues and closely related to the prognosis of CRC patients. A specific close loop structure of circ101555 was described, which was generated by back-splicing of the host gene CSNK1G1 and showed greater stability than the linear RNA. The results in vitro and in vivo showed that silencing circ101555 expression significantly suppressed cell proliferation, induced apoptosis and impaired the DNA repair capacity of CRC cells, while rescue experiments suggested that down-expression of miR-597-5p could significantly attenuate the biological effects of circ101555 knockdown on CRC cells. Subsequent experiments in vitro, including double fluorescence in situ hybridization (D-FISH) analysis, RIP analysis and biotin-coupled probe pull down assay, confirmed that miR-597-5p was effectively enriched by circ101555, and circ101555 might serve as a sponge of miR-597-5p. Moreover, two putative oncogenes (CDK6 and RPA3) were identified as the miR-597-5p potential targets. Taken together, our results proved that circ101555 might function as a competing endogenous RNA of miR-597-5p to up-regulate CDK6 and RPA3 expression in CRC. Circ101555 could be a useful prognostic indicator in patients with CRC, and silence of circ101555 provided a new attractive therapeutic measure for CRC.

Lung cancer (LC) is the leading cause of cancer-related deaths. Downregulation of CDK1, 4 and 6, key regulators of cell cycle progression, correlates with decreased LC cell proliferation. Enforced expression of miRNAs (miRs) is a promising approach to regulate genes. Here, we study the combinatorial treatment of miR-143 and miR-506 to target the CDK1, 4/6 genes, respectively. We analyzed the differential expression of CDK genes by qPCR, and western blot, and evaluated changes in the cell cycle distribution upon combinatorial treatment. We used an antibody microarray analysis to evaluate protein expression, focusing on the cell cycle pathway, and performed RNA-sequencing for pathway analysis. The combinatorial miR treatment significantly downregulated CDK1, 4 and 6 expression, and induced a shift of the cell cycle populations, indicating a G1 and G2 cell cycle block. The two miRs induces strong cytotoxic activity, with potential synergism, and a significant Caspase 3/7 activation. We identified a strong inhibition of tube formation in the presence or absence VEGF in an in vitro angiogenesis model. Together with the pathways analysis of the RNA-sequencing data, our findings establish the combinatorial miR transfection as a viable strategy for lung cancer treatment that merits further investigation.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G 0 /G 1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.

Dysregulation of microRNAs (miRNAs) plays an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). The Eμ-TCL1 transgenic mouse develops a form of leukemia that is similar to the aggressive type of human B-CLL, and this valuable model has been widely used for testing novel therapeutic approaches. Here, we adopted this model to investigate the potential effects of miR-26a, miR-130an and antimiR-155 in CLL therapy. Improved delivery of miRNA molecules into CLL cells was obtained by developing a novel system based on lipid nanoparticles conjugated with an anti-CD38 monoclonal antibody. This methodology has proven to be highly effective in delivering miRNA molecules into leukemic cells. Short- and long-term experiments showed that miR-26a, miR-130a and anti-miR-155 increased apoptosis after in vitro and in vivo treatment. Of this miRNA panel, miR-26a was the most effective in reducing leukemic cell expansion. Following long-term treatment, apoptosis was readily detectable by analyzing cleavage of PARP and caspase-7. These effects could be directly attributed to miR-26a, as confirmed by significant downregulation of its proven targets, namely cyclin-dependent kinase 6 and Mcl1. The results of this study are relevant to two distinct areas. The first is related to the design of a technical strategy and to the selection of CD38 as a molecular target on CLL cells, both consenting efficient and specific intracellular transfer of miRNA. The original scientific finding inferred from the above approach is that miR-26a can elicit in vivo anti-leukemic activities mediated by increased apoptosis.

Therapeutic strategies against KRAS mutant colorectal cancers are developed using cell line models, which do not accurately represent the transcriptome driven by oncogenic KRAS in tumors. We sought to identify a KRAS-associated gene signature from colorectal tumors to develop a precise treatment strategy. Integrative analysis of quantitative KRAS mutation detection and matched gene expression profiling in 55 CRC bulk tumors was carried out to define a gene signature enriched in CRC tumors with high KRAS mutation. The KRAS-associated gene signature identified exhibits functional enrichment in cell cycle and mitosis processes, and includes mitotic transcription factor, FOXM1. Combination treatment of CDK4/6 inhibitor Palbociclib and MEK inhibitor PD0325901 was tested in KRAS-mutant, BRAF-mutant CRC, normal colon epithelial lines and xenografts models to determine their efficacy and toxicity and to monitor the changes in the gene signature. Inhibiting CDK4/6, an upstream regulator of FOXM1, and MEK synergistically depleted FOXM1 and KRAS-associated gene signature, suggesting that CDK4/6 and MEK regulate the KRAS gene signature. The combined inhibition of CDK4/6 and MEK elicited a robust therapeutic response in KRAS-dependent and BRAF-mutant CRC, both in vitro and in vivo and this correlated with downregulation of the KRAS-associated gene signature. Our preclinical study demonstrated the efficacy of Palbociclib and PD0325901 combinatorial treatment selectively in KRAS-dependent and BRAF-mutant CRC but not in normal colon epithelial cells. The KRAS-associated gene signature could facilitate the identification of responsive metastatic CRC to this therapeutic strategy in clinical settings.

Oral verrucous carcinoma (OVC) is a verrucous variant of oral cavity squamous carcinoma (OSCC). The expression of miRNA from OVC and OSCC including their matched normal oral mucosa tissues was profiled through the Affymetrix GeneChip miRNA Arrays. TargetScan and miRanda databases were used to predict the target gene of miRNA-195. The quantitative real-time PCR was applied to validate the expression of miRNA-195. The expression of CDK6 was investigated by the quantitative real-time PCR and immunohistochemistry. In this study, a total of 23 and 35 differentially expressed miRNAs were identified in OVC and OSCC, respectively. Moreover, 44 miRNAs were differentially expressed between OSCC and OVC. In addition, miRNA-195 was significantly decreased in both OVC and OSCC compared to normal oral mucosa. Target gene prediction demonstrated that CDK6 was a potential target gene of miRNA-195. In the quantitative real-time PCR, miR-195 was decreased in OVC and OSCC, which was consistent with the result of miRNA chip analysis. CDK6 was increased in OVC and OSCC, which was opposite to the expression of miRNA-195. In conclusion, miRNA-195 could be the potential diagnosis biomarker and therapy target of OVC.

The phosphatase CDC25A is a key regulator of cell cycle progression by dephosphorylating and activating cyclin-CDK complexes. CDC25A is an unstable protein expressed from G1 until mitosis. CDC25A overexpression, which can be caused by stabilization of the protein, accelerates the G1/S and G2/M transitions, leading to genomic instability and promoting tumorigenesis. Thus, controlling CDC25A protein levels by regulating its stability is a critical mechanism for timing cell cycle progression and to maintain genomic integrity. Herein, we show that CDC25A is phosphorylated on Ser40 throughout the cell cycle and that this phosphorylation is established during the progression from G1 to S phase. We demonstrate that CyclinD-CDK4/CDK6 complexes mediate the phosphorylation of CDC25A on Ser40 during G1 and that these complexes directly phosphorylate this residue in vitro. Importantly, we also find that CyclinD1-CDK4 decreases CDC25A stability in a ßTrCP-dependent manner and that Ser40 and Ser88 phosphorylations contribute to this regulation. Thus our results identify cyclinD-CDK4/6 complexes as novel regulators of CDC25A stability during G1 phase, generating a negative feedback loop allowing control of the G1/S transition.

Despite remarkable progress in cutaneous melanoma genomic profiling, the mutational landscape of primary mucosal melanomas (PMM) remains unclear. Forty-six PMMs underwent targeted exome sequencing of 111 cancer-associated genes. Seventy-six somatic nonsynonymous mutations in 42 genes were observed, and recurrent mutations were noted on eight genes, including TP53 (13%), NRAS (13%), SNX31 (9%), NF1 (9%), KIT (7%) and APC (7%). Mitogen-activated protein kinase (MAPK; 37%), cell cycle (20%) and phosphatidylinositol 3-kinase (PI3K)-mTOR (15%) pathways were frequently mutated. We biologically characterized a novel ZNF767-BRAF fusion found in a vemurafenib-refractory respiratory tract PMM, from which cell line harboring ZNF767-BRAF fusion were established for further molecular analyses. In an independent data set, NFIC-BRAF fusion was identified in an oral PMM case and TMEM178B-BRAF fusion and DGKI-BRAF fusion were identified in two malignant melanomas with a low mutational burden (number of mutation per megabase, 0.8 and 4, respectively). Subsequent analyses revealed that the ZNF767-BRAF fusion protein promotes RAF dimerization and activation of the MAPK pathway. We next tested the in vitro and in vivo efficacy of vemurafenib, trametinib, BKM120 or LEE011 alone and in combination. Trametinib effectively inhibited tumor cell growth in vitro, but the combination of trametinib and BKM120 or LEE011 yielded more than additive anti-tumor effects both in vitro and in vivo in a melanoma cells harboring the BRAF fusion. In conclusion, BRAF fusions define a new molecular subset of PMM that can be targeted therapeutically by the combination of a MEK inhibitor with PI3K or cyclin-dependent kinase 4/6 inhibitors.

Dysregulated activation of the CDK4/6 kinases is a hallmark of most mammary-derived carcinomas. ATP-competitive inhibitors against this complex have been recently advanced in the clinic and have shown significant activity, particularly against tumors driven by the estrogen receptor (ER). However, resistance to these compounds has begun to emerge often months to years after their initiation. We investigated potential mechanisms of resistance using cell line models that are highly sensitive to this class of drugs. After prolonged exposure to the selective and potent CDK4/6 inhibitor LY2835219, clones emerged and several were found to harbor amplification of the CDK6 kinase. Amplification of CDK6 resulted in a marked increase in CDK6 expression and reduced response of the CDK4/6 target, phospho-Rb (pRb), to CDK4/6 inhibitors. Knockdown of CDK6 restored drug sensitivity, while enforced overexpression of CDK6 was sufficient to mediate drug resistance. Not only did CDK6 overexpression mediate resistance to CDK4/6 inhibitors but it also led to reduced expression of the ER and progesterone receptor (PR), and diminished responsiveness to ER antagonism. The reduced ER/PR expression after CDK4/6 inhibitor resistance was additionally observed in tumor biopsy specimens from patients treated with these drugs. Alternative mechanisms of resistance to CDK4/6 inhibitors such as loss of pRb and cyclin E1 overexpression also exhibited decreased hormone responsiveness, suggesting that the clinical paradigm of sequential endocrine-based therapy may be ineffective in some settings of acquired CDK4/6 resistance.

Non-small cell lung cancer (NSCLC) remains one of the leading causes of death worldwide, and thus new molecular targets need to be identified to improve treatment efficacy. Although epidermal growth factor receptor (EGFR)/KRAS mutation-driven lung tumorigenesis is well understood, the mechanism of EGFR/KRAS-independent signal activation remains elusive. Enhanced TFAP2C (transcription factor activating enhancer-binding protein 2C) expression is associated with poor prognosis in some types of cancer patients, but little is known of its relation with the pathogenesis of lung cancer. In the present study, we found that TFAP2C overexpression was associated with cell cycle activation and NSCLC cell tumorigenesis. Interestingly, TFAP2C blocked AKAP12-mediated cyclin D1 inhibition by inducing the overexpression of oncogenic microRNA (miRNA)-183 and simultaneously activated cyclin-dependent kinase 6-mediated cell cycle progression by downregulating tumor-suppressive miRNA-33a. In a mouse xenograft model, TFAP2C promoted lung tumorigenesis and disease aggressiveness via the miR-183 and miR-33a pathways. The study provides a mechanism of mitogenic and oncogenic signaling via two functionally opposed miRNAs and suggests that TFAP2C-induced cell cycle hyperactivation contributes to lung tumorigenesis.

Mutations of the retinoblastoma tumor-suppressor gene (RB1) or components regulating the CDK-RB-E2F pathway have been identified in nearly every human malignancy. Re-establishing cell cycle control through cyclin-dependent kinase (CDK) inhibition has therefore emerged as an attractive option in the development of targeted cancer therapy. The most successful example of this today is the use of the CDK4/6 inhibitor palbociclib combined with aromatase inhibitors for the treatment of estrogen receptor-positive breast cancers. Multiple studies have demonstrated that the CDK-RB-E2F pathway is critical for the control of cell proliferation. More recently, studies have highlighted additional roles of this pathway, especially E2F transcription factors themselves, in tumor progression, angiogenesis and metastasis. Specific E2Fs also have prognostic value in breast cancer, independent of clinical parameters. We discuss here recent advances in understanding of the RB-E2F pathway in breast cancer. We also discuss the application of genome-wide genetic screening efforts to gain insight into synthetic lethal interactions of CDK4/6 inhibitors in breast cancer for the development of more effective combination therapies.

Circular RNAs (circRNAs), a kind of non-coding RNAs, have shown large capabilities in gene regulation. However, the mechanisms underlying circRNAs remain largely unknown so far. Recent studies demonstrated that circRNAs play miRNA sponge effects and regulate gene expression by microRNA response elements. Here, we screened circRNA expression profiles of bladder carcinoma using microarray assay. A total of 469 dysregulated circular transcripts are found in bladder cancer compared with normal tissues, among which 285 were up-regulated and 184 were down-regulated. Six circRNAs were identified to have significant differences by qRT-PCR. We speculated that circRNAs might involve in cancer-related pathways via interactions with miRNA by multiple bioinformatical approaches. Therefore, we further predicted that circTCF25 could sequester miR-103a-3p/miR-107, which potentially lead to the up-regulation of thirteen targets related to cell proliferation, migration and invasion. Subsequently, we demonstrated that over-expression of circTCF25 could down-regulate miR-103a-3p and miR-107, increase CDK6 expression, and promote proliferation and migration in vitro and vivo. This is the first study to exploit circRNA profiling and circRNA/miRNA interactions in bladder cancer. Our work laid the foundation to investigate the functions of circRNAs in cancers. The data also suggest that circTCF25 might be a new promising marker for bladder cancer.

Pluripotent stem cells (PSCs), including embryonic and induced pluripotent stem cells (iPSCs), show atypical cell cycle regulation characterized by a high proliferation rate and a shorter G1 phase compared with somatic cells. The mechanisms by which somatic cells remodel their cell cycle to achieve the high proliferation rate of PSCs during reprogramming are unclear. Here we identify that the Ink4 protein p18, which is expressed at high levels in somatic cells but at low levels in PSCs, is a roadblock to successful reprogramming. Mild inhibition of p18 expression enhances reprogramming efficiency, while ectopic expression of p18 completely blocks reprogramming. Mechanistic studies show that expression of wild-type p18, but not a p18(D68N) mutant which cannot inhibit Cdk4/6, down-regulates expression of Cdk4/6 target genes involved in DNA synthesis (TK, TS, DHFR, PCNA) and cell cycle regulation (CDK1 and CCNA2) and thus inhibits reprogramming. These results indicate that p18 blocks reprogramming by targeting Cdk4/6-mediated cell cycle regulation. Taken together, our results define a novel pathway that inhibits somatic cell reprogramming, and provide a new target to enhance reprogramming efficiency.

It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression.

Type 2 diabetes (T2D) is characterized by insulin resistance and reduced functional β-cell mass. Developmental differences, failure of adaptive expansion and loss of β-cells via β-cell death or de-differentiation have emerged as the possible causes of this reduced β-cell mass. We hypothesized that the proliferative response to mitogens of human β-cells from T2D donors is reduced, and that this might contribute to the development and progression of T2D. Here, we demonstrate that the proliferative response of human β-cells from T2D donors in response to cdk6 and cyclin D3 is indeed dramatically impaired. We show that this is accompanied by increased nuclear abundance of the cell cycle inhibitor, p27(kip1). Increasing nuclear abundance of p27(kip1) by adenoviral delivery decreases the proliferative response of β-cells from non-diabetic donors, mimicking T2D β-cells. However, while both p27(kip1) gene silencing and downregulation by Skp2 overexpression increased similarly the proliferative response of human β-cells, only Skp2 was capable of inducing a significant human β-cell expansion. Skp2 was also able to double the proliferative response of T2D β-cells. These studies define c-Myc as a central Skp2 target for the induction of cell cycle entry, expansion and regeneration of human T2D β-cells.

The G1 cell-cycle kinase CDK6 has long been thought of as a redundant homolog of CDK4. Although the two kinases have very similar roles in cell-cycle progression, it has recently become apparent that they differ in tissue-specific functions and contribute differently to tumor development. CDK6 is directly involved in transcription in tumor cells and in hematopoietic stem cells. These functions point to a role of CDK6 in tissue homeostasis and differentiation that is partially independent of CDK6's kinase activity and is not shared with CDK4. We review the literature on the contribution of CDK6 to transcription in an attempt to link the new findings on CDK6's transcriptional activity to cell-cycle progression. Finally, we note that anticancer therapies based on the inhibition of CDK6 kinase activity fail to take into account its kinase-independent role in tumor development.

Liposarcoma (LPS) is a type of soft tissue sarcoma that mostly occurs in adults, and in humans is characterized by amplifications of MDM2 and CDK4. The molecular pathogenesis of this malignancy is still poorly understood and, therefore, we developed a mouse model with conditional inactivation of PTEN and p53 to investigate these pathways in the progression of the disease. We show that deletion of these two tumor suppressors cooperate in the formation of multiple subtypes of LPS (from well-differentiated LPS to pleomorphic LPS). In addition, progression of the tumors is further characterized by the expression of D cyclins and CDK4/6, which allow for continued cell division. Microarray analysis also revealed novel genes that are differentially expressed between different subtypes of LPS, which could aid in understanding the disease and to unravel potential new therapeutic targets.

The elucidation of the molecular mechanisms underlying the differentiation and proliferation of human adipose tissue-derived stromal cells (hADSCs) represents a critical step in the development of hADSCs-based cellular therapies. To examine the role of the microRNA-103a-3p (miR-103a-3p) in hADSCs functions, miR-103a-3p mimics were transfected into hADSCs in order to overexpress miR-103a-3p. Osteogenic differentiation was induced for 14 days in an osetogenic differentiation medium and assessed by using an Alizarin Red S stain. The regulation of the expression of CDK6 (cyclin-dependent kinase 6), a predicted target of miR-103a-3p, was determined by western blot, real-time PCR and luciferase reporter assays. Overexpression of miR-103a-3p inhibited the proliferation and osteogenic differentiation of hADSCs. In addition, it downregulated protein and mRNA levels of predicted target of miR-103a-3p (CDK6 and DICER1). In contrast, inhibition of miR-103a-3p with 2'O methyl antisense RNA increased the proliferation and osteogenic differentiation of hADSCs. The luciferase reporter activity of the construct containing the miR-103a-3p target site within the CDK6 and DICER1 3'-untranslated regions was lower in miR-103a-3p-transfected hADSCs than in control miRNA-transfected hADSCs. RNA interference-mediated downregulation of CDK6 and DICER1 in hADSCs inhibited their proliferation and osteogenic differentiation. The results of the current study indicate that miR-103a-3p regulates the osteogenic differentiation of hADSCs and proliferation of hADSCs by direct targeting of CDK6 and DICER1 partly. These findings further elucidate the molecular mechanisms governing the differentiation and proliferation of hADSCs.

Cell proliferation and differentiation are closely coupled. However, we previously showed that overexpression of cyclin-dependent kinase (Cdk6) blocks chondrocyte differentiation without affecting cell-cycle progression in vitro. To investigate whether Cdk6 inhibits chondrocyte differentiation in vivo, we generated chondrocyte-specific Cdk6 transgenic mice using Col2a1 promoter. Unexpectedly, differentiation and cell-cycle progression of chondrocytes in the Cdk6 transgenic mice were similar to those in wild-type mice. Then, we generated chondrocyte-specific Ccnd1 transgenic mice and Cdk6/Ccnd1 double transgenic mice to investigate the possibility that Cdk6 inhibits chondrocyte differentiation through E2f activation. Bromodeoxyuridine (BrdU)-positive chondrocytes and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive chondrocytes were increased in number, and chondrocyte maturation was inhibited only in Cdk6/Ccnd1 transgenic mice (K6(H)/D1(H) mice), which showed dwarfism. Retinoblastoma protein (pRb) was highly phosphorylated but p107 was upregulated, and the expression of E2f target genes was dysregulated as shown by upregulation of Cdc6 but downregulation of cyclin E, dihydrofolate reductase (dhfr), Cdc25a and B-Myb in chondrocytes of K6(H)/D1(H) mice. Similarly, overexpression of Cdk6/Ccnd1 in a chondrogenic cell line ATDC5 highly phosphorylated pRb, upregulated p107, induced apoptosis, upregulated Cdc6 and downregulated cyclin E, dhfr and B-Myb and p107 small interfering RNA reversed the expression of downregulated genes. Further, introduction of kinase-negative Cdk6 and cyclin D1 abolished all effects by Cdk6/cyclin D1 in ATDC5 cells, indicating the requirement of the kinase activity on these effects. p53 deletion partially restored the size of the skeleton and almost completely rescued chondrocyte apoptosis, but failed to enhance chondrocyte proliferation in K6(H)/D1(H) mice. These findings indicated that Cdk6/Ccnd1 overexpression inhibited chondrocyte maturation and enhanced G1/S cell-cycle transition by phosphorylating pRb, but the chondrocytes failed to accomplish the cell cycle, and underwent p53-dependent apoptosis probably due to the dysregulation of E2f target genes. Our findings also indicated that p53 deletion in addition to the inactivation of Rb was not sufficient to accelerate chondrocyte proliferation, suggesting the resistance of chondrocytes to sarcomagenesis.

Disruption of contact inhibition and serum afflux that occur after a tissue injury activate cell cycle, which then stops when confluence is reached again. Although the events involved in cell cycle entry have been widely documented, those managing cell cycle exit have remained so far ill defined. We have identified that the final stage of wound closure is preceded in keratinocytes by a strong accumulation of miR-483-3p, which acts as a mandatory signal triggering cell cycle arrest when confluence is reached. Blocking miR-483-3p accumulation strongly delays cell cycle exit, maintains cells into a proliferative state and retards their differentiation program. Using two models of cell cycle synchronization (i.e. mechanical injury and serum addition), we show that an ectopic upregulation of miR-483-3p blocks cell cycle progression in early G1 phase. This arrest results from a direct targeting of the CDC25A phosphatase by miR-483-3p, which can be impeded using an anti-miRNA against miR-483-3p or a protector that blocks the complex formation between miR-483-3p and the 3'-untranslated region (UTR) of CDC25A transcript. We show that the miRNA-induced silencing of CDC25A increases the tyrosine phosphorylation status of CDK4/6 cyclin-dependent kinases which, in turn, abolishes CDK4/6 capacity to associate with D-type cyclins. This prevents CDK4/6 kinases' activation, impairs downstream events such as cyclin E stimulation and sequesters cells in early G1. We propose this new regulatory process of cyclin-CDK association as a general mechanism coupling miRNA-mediated CDC25A invalidation to CDK post-transcriptional modifications and cell cycle control.

E2F1 is a positive regulator of cell cycle progression and also a potent inducer of apoptosis, especially when activated by DNA damage. We identified E2F1-inducible microRNAs (miRNAs) by microarray hybridization and found that the levels of miRNAs 449a and 449b, as well as their host gene CDC20B, are strongly upregulated by E2F1. High miR-449 levels were found in testes, lung, and trachea, but not in testicular and other cancer cells. MiR-449a/b structurally resemble the p53-inducible miRNA 34 family. In agreement with a putative tumor-suppressive role, miR-449a as well as miR-34a reduced proliferation and strongly promoted apoptosis by at least partially p53-independent mechanisms. Both miRNAs reduced the levels of CDK6, implying miR-449 in a negative feedback mechanism for E2F1. Moreover, miR-449a and miR-34a diminished the deacetylase Sirt1 and augmented p53 acetylation. We propose that both miRNAs provide a twofold safety mechanism to avoid excessive E2F1-induced proliferation by cell cycle arrest and by apoptosis. While responding to different transactivators, miRNAs 449 and 34 each repress E2F1, but promote p53 activity, allowing efficient cross-talk between two major DNA damage-responsive gene regulators.

SHP-1, a haematopoietic cell-specific tyrosine phosphatase, is also expressed in human prostate. In this study, we report that SHP-1 depletion in PC-3 cells induced by small interfering RNAs causes G1 phase cell-cycle arrest accompanied by changes in some components of the cell-cycle machinery. SHP-1 knockdown increases p27(Kip1) (p27) protein stability, its nuclear localization and p27 gene transcription. These effects could be mediated by PI3K-AKT pathway as SHP-1 interacts with PI3K regulating its activity and p110 catalytic subunit phosphorylation. The increase in p27 protein stability could also because of reduced cyclin-dependent kinase (CDK2) activity. SHP-1 knockdown decreases the CDK6 levels, inducing retinoblastoma protein hypophosphorylation, downregulation of cyclin E and thereby a decrease in the CDK2 activity. However, the codepletion of SHP-1 and p27 does not produce re-entry into the cycle, implying that p27 is not required to maintain cell-cycle arrest induced by SHP-1 depletion. The maintenance of the PC-3 cell anti-proliferative response after p27 loss could be because of mislocalization of CDK2 induced by SHP-1 knockdown. This study shows that SHP-1 depletion promotes cell-cycle arrest by modulating the activity of cell-cycle regulators and suggests that SHP-1 may be required for the proper functioning of events governing cell-cycle progression.

Human T-cell leukemia virus type I (HTLV-I) can infect a variety of cell types, so the cause of T-cell-specific oncogenesis remains to be elucidated. The trans-activator protein Tax of HTLV-I can promote cell-cycle progression in resting T cells along with induction of cyclin D2 and cyclin-dependent kinase (cdk6) gene expression. Here, we found that Tax cannot induce cell-cycle progression in resting fibroblasts and analysed the molecular basis of the cell-type specificity. Tax activated cyclin D2 and cdk6 promoters in T cells, but not in fibroblasts, depending on its ability to activate the transcription factor nuclear factor (NF)-kappaB. Expression of cyclin D2 and CDK6 activated the transcription factor E2F, which is essential for cell-cycle progression, in both T cells and fibroblasts. Short-hairpin RNA (shRNA)-mediated inhibition of cyclin D2 and CDK6 induction suppressed Tax-induced activation of E2F in T cells. Finally, shRNA-mediated downregulation of NF-kappaB p65 or p100 expression reduced Tax-induced activation of cyclin D2 and/or cdk6 promoters and cell-cycle progression in T cells. These results indicate that Tax-induced cell-cycle progression in T cells is mediated, at least in part, through cell-type-specific activation of the cyclin D2 and cdk6 genes through NF-kappaB and may be important for the cell-type-specific oncogenesis.

In most Dutch melanoma families, a founder deletion in the melanoma susceptibility gene CDKN2A (which encodes p16) is present. This founder deletion (p16-Leiden) accounts for a significant proportion of the increased melanoma risk. However, it does not account for the Atypical Nevus (AN) phenotype that segregates in both p16-Leiden carriers and non-carriers. The AN-affected p16-Leiden family members are therefore a unique valuable resource for unraveling the genetic etiology of the AN phenotype, which is considered both a risk factor and a precursor lesion for melanoma. In this study, we performed a genome-wide scan for linkage in four p16-Leiden melanoma pedigrees, classifying family members with five or more AN as affected. The strongest evidence for an atypical nevus susceptibility gene was mapped to chromosome band 7q21.3 (two-point LOD score=2.751), a region containing candidate gene CDK6.

Loss of JunB has been observed in human leukemia and lymphoma, but it remains unknown, whether this loss is relevant to disease progression. Here, we investigated the consequences of JunB deficiency using Abelson-induced B-lymphoid leukemia as a model system. Mice deficient in JunB expression succumbed to Abelson-induced leukemia with increased incidence and significantly reduced latency. Similarly, bcr/abl p185-transformed JunB-deficient (junB(Delta/Delta)) cells induced leukemia in RAG2(-/-) mice displaying a more malignant phenotype. These observations indicated that cell intrinsic effects within the junB(Delta/Delta) tumor cells accounted for the accelerated leukemia development. Indeed, explantated bcr/abl p185 transformed junB(Delta/Delta) cells proliferated faster than the control cells. The proliferative advantage emerged slowly after the initial transformation process and was associated with increased expression levels of the cell cycle kinase cdk6 and with decreased levels of the cell cycle inhibitor p16(INK4a). These alterations were due to irreversible reprogramming of the cell, because - once established - accelerated disease induced by junB(Delta/Delta) cells was not reverted by re-introducing JunB. Consistent with this observation, we found that the p16 promoter was methylated. Thus, JunB functions as a gatekeeper during tumor evolution. In its absence, transformed leukemic cells acquire an enhanced proliferative capacity, which presages a more malignant disease.