CDK6 mRNA Research Articles

Cooperation of long noncoding RNA LOC100909675 and transcriptional regulator CTCF modulates Cdk1 transcript to control astrocyte proliferation

Astrocyte activation and proliferation contribute to glial scar formation during spinal cord injury (SCI), which limits nerve regeneration. LncRNAs are involved in astrocyte proliferation and act as novel epigenetic regulators. Here, we found that lncRNA-LOC100909675 (LOC9675) expression promptly increased after SCI and that reducing its expression decreased the proliferation and migration of the cultured spinal astrocytes. Depletion of LOC9675 reduced astrocyte proliferation and facilitated axonal regrowth after SCI. FISH detection and RT‒qPCR assays using nuclear/cytoplasmic RNAs revealed LOC9675 mainly localized in astrocytic nuclei. We next used RNA-seq to analyze gene expression profile alterations in LOC9675-depleted astrocytes and identified the Cdk1 gene as a hub candidate. Our RNA pull-down and RNA immunoprecipitation assays showed that LOC9675 directly interacted with the transcriptional regulator CTCF. Luciferase reporter and ChIP assays, together with down/upregulated expression investigation, revealed that CTCF is a novel regulator of the Cdk1 gene. Interestingly, we found that with the simultaneous overexpression of CTCF and LOC9675 in astrocytes, the Cdk1 transcript was restored to the normal level. We then designed the deletion construct of LOC9675 by removing its interacting region with CTCF, and found this effect disappeared. A transcription inhibition assay using actinomycin D revealed that LOC9675 could stabilize Cdk1 mRNA, while LOC9675 depletion or binding with CTCF reduced Cdk1 mRNA stability. These data suggest that the cooperation between CTCF and LOC9675 regulates Cdk1 transcription at a steady level, thereby strictly controlling astrocyte proliferation. This study provides a novel perspective on the regulation of the Cdk1 gene transcript by lncRNA LOC9675.

Huannao Yicong decoction ameliorates cognitive deficits in APP/PS1/tau triple transgenic mice by interfering with neurotoxic interaction of Aβ-tau

Ethnopharmacological relevanceHuannao Yicong decoction (HYD) has been used in the study of AD for many years, which consists of Polygonum multiflorum Thunb., Panax ginseng C.A.Mey., Acorus gramineus Aiton, Coptis chinensis Franch., and Conioselinum acuminatum (Franch.) Lavrova. Previous studies have found that HYD could reduce β-Amyloid (Aβ) deposition and tau hyperphosphorylation which are the two critical pathological factors of AD. However, the mechanism of the neurotoxic interaction between Aβ and tau in AD remains unclear. Thus, the underlying mechanisms for HYD improving cognitive function of AD by interfering with the neurotoxic interaction between Aβ and tau remain to be explored. Aim of the studyThe main objective of this study is to clarify the specific mechanisms of HYD on interfering with the neurotoxic interaction between Aβ and tau of AD both in vivo and in vitro. Materials and methodsAPP/PS1/tau triple transgenic mice were randomly divided into 4 groups, namely model group, memantine group, HYD low-dose group (HYD-L), and HYD high-dose group (HYD-H) with 28 mice in each group, while 28 C57BL/6J mice as the control group. Gavage was applied to all the mice daily for 24 weeks. SH-SY5Y model cells overexpressing Aβ and tau proteins as the intervention object in vitro experiments. Morris water maze was used to observe the learning and memory ability of APP/PS1/tau mice. Aβ deposition was detected by immunohistochemistry, and the levels of Aβ1-40 and Aβ1-42 were detected by enzyme-linked immunosorbent assay (ELISA). Neurofibrillary tangles (NFTs) were observed by silver staining and the levels of phosphorylated tau proteins were detected by Western blot. The GSK-3β and CDK-5 mRNA expression were detected by real-time polymerase chain reaction (RT-PCR). Besides, the levels of PSD95, GluR1, NR2A, and NR2B were detected by Western blot. Meanwhile, cell experiments were performed to further verify the effect of HYD on tau phosphorylation related kinases (GSK-3β, CDK-5, and PP2A), which further to clarify the mechanism of HYD intervention on the neurotoxic interaction between Aβ and tau. ResultsHYD improved the learning and memory ability of APP/PS1/tau mice. HYD decreased the levels of Aβ1-40 and Aβ1-42 and inhibited tau hyperphosphorylation, which reduced Aβ deposition and NFTs forming. In addition, HYD inhibited the activity of kinases GSK-3β and CDK-5, and enhancing the activity of kinase PP2A. Moreover, HYD inhibited the overexpression of NR2A and NR2B, and increased the expression of GluR1 and postsynaptic density protein-95 (PSD95). ConclusionsHYD can improve the cognitive deficits by interfering with the neurotoxic interaction between Aβ and tau. In addition, HYD can inhibit the overactivation of NMDARs and increase the levels of GluR1 and PSD95, which may play a role in alleviating neuronal excitotoxicity and improving synaptic function.

RNA expression levels from peripheral immune cells, a minimally invasive liquid biopsy source to predict response to therapy, survival and immune-related adverse events in patients with triple negative breast cancer enrolled in the GeparNuevo trial.

1011 Background: The impact of immune checkpoint therapy on the tumor microenvironment including tumor-infiltrating immune cells is well described. However, little is known about the circulating immune repertoire and its association with treatment outcome. Hence, we set out to investigate the RNA phenotype of peripheral immune cells before and during treatment of patients enrolled in the GeparNuevo trial (Loibl S et al. Annals Oncol 2022). Methods: The GeparNuevo trial investigated the outcome after neoadjuvant nabP-EC-chemotherapy in combination with the anti-PD-L1 immune checkpoint inhibitor durvalumab versus placebo in patients with non-metastatic triple negative breast cancer. Blood was collected before therapy (baseline), after window, before epirubicin/cyclophosphamide and at end of treatment. RNA from circulating leucocytes of 117 patients was extracted and analyzed using a custom NanoString nCounter CodeSet. Expression of 290 immune-related genes was quantified. The association of RNA expression and signatures with outcome parameters like pathologic complete response (pCR), distant disease-free survival (DDFS), invasive disease-free survival (iDFS), overall survival (OS), and immune-related adverse events (irAEs) were investigated. Results: Immune cell type scores representing macrophages and neutrophils significantly increased during treatment, while B cell, T and Th1 cell scores decreased (p < 0.0001, respectively) regardless of treatment arm. Multivariate logistic respectively multivariate Cox regression analysis revealed a significant association of each baseline DPP4 and MYC gene expression with pCR, DDFS, iDFS, and OS in patients. CDK2, F5 and HLA-DR mRNA expressions were associated with the presence of irAEs. The signature score for TNFR2 non-canonical NF-kB pathway, which is known for its protective capacity, was inversely associated with irAEs in the durvalumab arm (OR = 0.454, 95% CI 0.231-0.892, p = 0.0220). Multiple immune-related signatures at baseline were associated with tumor mutational burden. Conclusions: Our study indicates the importance of the peripheral immune phenotype for treatment response and survival. This association between RNA expression levels of circulating immune cells and outcome seems to be not only relevant for patients receiving immune checkpoint therapy, but also for those under standard chemotherapy alone. Clinical trial information: NCT02685059 .

Astragaloside IV promotes keratinocyte proliferation and migration through upregulating lncRNA H19 recruited ILF3 to enhance the stability of CDK4 mRNA.

Skin is the first line of the body to resist pathogen invasion. A potentially fatal infection may result from problems with wound healing. Small molecule drugs like astragaloside IV (AS-IV) show pro-healing activities, but the mechanisms are not fully understood. Using real-time quantitative PCR and a western blot assay, the amount of gene expression was evaluated. The proliferation and migration of keratinocytes were determined by MTS and wound healing assay, respectively. The binding of lncRNA H19 to RBP protein ILF3 and the binding of ILF3 protein to CDK4 mRNA were confirmed by RNA immunoprecipitation. Treatment with AS-IV enhanced the expression of lncRNA H19, ILF3, and CDK4 and improved the proliferation and migration of keratinocytes HaCaT. Additionally, apoptosis of keratinocytes was attenuated by AS-IV. Further studies showed that both lncRNA H19 and ILF3 were important for AS-IV-mediated keratinocyte growth and migration. In addition, lncRNA H19 recruited ILF3 to increase CDK4 mRNA level and enhanced cell proliferation. We discovered a lncRNA H19/ILF3/CDK4 axis that is activated by AS-IV to promote keratinocyte migration and proliferation. These results elucidate the mechanism of action of AS-IV and justify its application in further application in wound healing treatment.

C4orf19 inhibits colorectal cancer cell proliferation by competitively binding to Keap1 with TRIM25 via the USP17/Elk-1/CDK6 axis.

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.

The c-Myc targeting hnRNPAB promotes lung adenocarcinoma cell proliferation via stabilization of CDK4 mRNA.

The c-Myc oncoprotein plays a pivotal role in tumorigenesis. The deregulated expression of c-Myc has been linked to a variety of human cancers including lung adenocarcinoma. The oncogenic function of c-Myc has been largely attributed to its intrinsic nature as a transcription factor. Here we reported the RNA binding protein hnRNPAB as a direct transcriptional target of c-Myc by performing quantitative real-time polymerase chain reaction (qRT-PCR), western blot, chromatin immunoprecipitation (ChIP), and luciferase reporter analyses. Flow cytometry, colony formation, and RNA immunoprecipitation (RIP) assays were used to investigate the role of hnRNPAB in lung adenocarcinoma cell proliferation, as well as the underlying mechanism. HnRNPAB was functionally shown to promote lung adenocarcinoma cell proliferation by accelerating G1/S cell cycle progression. Mechanistically, hnRNPAB interacted with and stabilized CDK4 mRNA, thereby increasing CDK4 expression. Moreover, hnRNPAB was able to promote G1/S cell cycle progression and cell proliferation via the regulation of CDK4. HnRNPAB was also revealed as a mediator of the promoting effect of c-Myc on cell proliferation. Together, these findings demonstrate that hnRNPAB is an important regulator of lung adenocarcinoma cell proliferation. They also add new insights into the mechanisms of how c-Myc promotes tumorigenesis.

Abstract P6-12-06: Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study

Abstract Background: Despite the wide-ranging clinical success of human epidermal growth factor receptor 2 (HER2)-directed therapies, many HER2-positive breast cancer patients eventually progress because of the development of primary or acquired resistance. PIK3CA is found often mutated in breast cancer (>30%, TCGA dataset) and responsible for HER2-directed treatment failure. Purpose: Inhibition of pan-tyrosine kinases of HER-receptors along with the blockade of estrogen receptor (ER) prevents the activation of downstream effectors and crosstalk between HER2 and ER, leading to tumor cell death. Similarly, the combined inhibition of HER2-receptor signaling with cell cycle arrest leads to efficient tumor cell apoptosis. Here, we evaluate the mechanistic efficacy and comparability of neratinib (N) (an irreversible pan-ERBB tyrosine kinase inhibitor) in combination with fulvestrant (F) against ribociclib (R) plus trastuzumab (T) in HER2-amplified breast cancer cells. Methods: HER2+ breast cancer cell lines with Rb- wild-type- BT474 (ER+), SKBR3 (ER-), and MDA-MB453 (ER-, PIK3CA mutated [H1047R]) were used for the study. Cells were treated with N+F (F added in ER+ cell line only) or R or T as a single-agent or combination and assessed for real-time proliferation, 3D ON-TOP assay, changes in mitochondrial potential, and apoptosis. Cells treated with R and/or T were additionally examined for cell cycle arrest and changes in CDK4 mRNA transcription. Baseline CCND1 mRNA transcripts were quantified by RT-qPCR. Immunohistochemistry was used to assess baseline BCL2 expression in HER2+ tumor microarrays (TMA). Western blot was used to evaluate the effects on key downstream signaling proteins in response to the above treatment or combination. Results: High CCND1 expression was found in HER2+ cell lines that show promise for CDK4/6 inhibition. High BCL2 expression was found in HER2+ TMA that confirmed the natural resistance to programmed cell death. BT474 and MDA-MB453 were highly sensitive to N (+F), and high growth inhibition was evident at lower doses (< 20 nM). SKBR3 was comparatively less sensitive to N monotherapy and required dosing >160 nM to induce marked cell death. BT474 and MDA-MB453 had pronounced cytostatic responses to R or T+R, while a moderate response was observed in SKBR3. Substantial reduction in CDK4 transcription was noted following R or R+T treatment in MDA-MB453/BT474 but not in SKBR3. Apoptosis assays confirmed enhanced cell death with the R+T combination in BT474 and MDA-MB453 but not in SKBR3. Inhibition of key downstream oncogenic signaling (p-AKT/p-S6RP/p-ERK) was more evident with N compared to R, T, or R+T combination. Attenuated p-Rb expression (Ser 780 & Ser 807/811) was detected in all cell lines from R administration, indicating interruption in cell cycle progression. Conclusion: Mechanistically, N+F was superior to T+R in terms of inhibition of HER2 and its downstream signaling in the HER2+/ER+ BC model. N+F induced significantly more apoptosis and inhibited cell proliferation compared to T+R. Additionally, N monotherapy was highly effective in HER2-amplified/ER-negative breast cancer cells with PIK3CA mutation. Citation Format: Nischal Koirala, Jennifer Aske, Xiaoqian Lin, Nandini Dey, Pradip De. Neratinib vs. Trastuzumab plus Ribociclib in ERBB2-positive breast cancer: Preclinical mechanistic efficacy study [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-06.

Prognostic significance of CDK6 amplification in esophageal squamous cell carcinoma

Dysregulation of CDK6 plays crucial roles in the carcinogenesis of many kinds of human malignancies. However, the role of CDK6 in esophageal squamous cell carcinoma (ESCC) is not well known. We investigated the frequency and prognostic value of CDK6 amplification to improve the risk stratification in patients with ESCC. Pan-cancer analysis of CDK6 was conducted on The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and Gene Expression Omnibus (GEO) databases. CDK6 amplification was detected in 502 ESCC samples by Fluorescence in situ hybridization (FISH) through tissue microarrays (TMA). Pan-cancer analysis revealed that CDK6 mRNA level was much higher in multiple kinds of cancers and higher CDK6 mRNA level indicated a better prognosis in ESCC. In this study, CDK6 amplification was detected in 27.5% (138/502) of patients with ESCC. CDK6 amplification was significantly correlated with tumor size (p = 0.044). Patients with CDK6 amplification tended to have a longer disease-free survival (DFS) (p = 0.228) and overall survival (OS) (p = 0.200) compared with patients without CDK6 amplification but of no significance. When further divided into I–II and III–IV stage, CDK6 amplification was significantly associated with longer DFS and OS in III-IV stage group (DFS, p = 0.036; OS, p = 0.022) rather than in I-II stage group (DFS, p = 0.776; OS, p = 0.611). On univariate and multivariate analysis of Cox hazard model, differentiation, vessel invasion, nerve invasion, invasive depth, lymph node metastasis and clinical stage were significantly associated with DFS and OS. Moreover, invasion depth was an independent factor for ESCC prognosis. Taken together, for ESCC patients in III-IV stage, CDK6 amplification indicated a better prognosis.

Secoisolariciresinol diglucoside lignan concentrate of flaxseeds exhibits chemoprotective role in non-melanoma skin cancer through inhibition of CDK4 and upregulation of p53

Cyclin-dependent kinases (CDKs) serve as target for various cancers including skin cancer. Secoisolariciresinoldiglucoside (SDG) and lignans exert anticancer effect on colon cancer through inhibition of CDKs. However, reports of SDG lignan concentrate (SLC) of Linum usitatissimum (L.) on skin cancer are not available. Hence, in this study, we evaluated the effect of SLC of L. usitatissimum on skin cancer, and determined the mechanism of action. Cell viability studies were done using the A-375 cell line. Skin cancer was induced by dimethyl Benz(a) anthracene and croton oil in female balb/c mice. SLC (5%) was administered from the 7th to 16th week after which we evaluated serum and tissue parameters. The IC50 value of SLC was found to be 93.7 μg/mL on the A-375 cell line. Skin cancer control animals exhibited increased tumor volume and burden and an increase in non-specific serum markers and tissue markers. Treatment with SLC decreased tumor volume and burden, and serum and tissue markers. Histopathological studies also depicted protection with SLC treatment. Docking studies revealed that SDG exhibits a good binding score with CDK4. Skin cancer control mice showed significantly increased CDK4 mRNA and decreased p53 mRNA levels which were prevented by SLC treatment. SLC exhibited a chemopreventive effect in skin cancer depicted by a reduction in serum biomarker, oxidative stress, collagen levels, tumor volume, tumor burden, and histopathological studies. These effects are mediated through the inhibition of CDK4 and upregulation of p53.

Correction for: circACTA2 mediates Ang II-induced VSMC senescence by modulation of the interaction of ILF3 with CDK4 mRNA.

Correction for: circACTA2 mediates Ang II-induced VSMC senescence by modulation of the interaction of ILF3 with CDK4 mRNA.

The pivotal regulatory factor circBRWD1 inhibits arsenic exposure-induced lung cancer occurrence by binding mRNA and regulating its stability

Multiple studies have indicated that circular RNAs (circRNAs) play a regulatory role in different stages of tumors by interacting with various molecules. With continuous in-depth research on the biological functions of circRNAs, increasing evidence has shown that circRNAs play important roles in carcinogenesis caused by environmental pollutants. However, the function and mechanism of circRNAs in arsenic exposure-induced lung cancer occurrence have not been reported. In this study, RNA sequencing and qPCR assays revealed that the expression of circBRWD1 was decreased in BEAS-2B-As cells and multiple lung cancer cell lines. Silencing circBRWD1 promoted cell viability and proliferation, inhibited cell apoptosis, and accelerated the G0/G1 phase transition in BEAS-2B-As cells; however, these functions were abrogated by circBRWD1 overexpression. Mechanistically, under arsenic exposure, expression of decreased circBRWD1 led to enhanced stability of the mRNA to which it directly binds (c-JUN, c-MYC, and CDK6 mRNA), increasing its expression. This mechanism promotes the malignant transformation of lung cells and ultimately leads to lung cancer. Our findings thus reveal the molecular mechanism of arsenic carcinogenesis.

Whole transcriptome and proteome analyses identify potential targets and mechanisms underlying tumor treating fields against glioblastoma

Glioblastoma (GBM) is one of the most malignant types of brain cancer. Tumor treating fields (TTFields) is the up-to-date treatment for GBM. However, its molecular mechanism requires additional investigation. Herein, a novel TTFields system was developed (CL-301A) and its efficiency in suppressing GBM cell proliferation and inducing cell apoptosis was demonstrated. Through the whole proteomic and transcriptomic analyses, a multitude of differentially expressed proteins (1243), mRNAs (4191), miRtNAs (47), lncRNAs (4286), and circRNAs (13,903) were identified. Bioinformatic analysis indicated that TTFields mainly affected nuclear proteins and interrupt cell mitosis-related events. Moreover, the inhibition of autophagy could significantly enhance the anti-GBM activity of TTFields. And CDK2-AS1 might be a target of TTFields to mediate cell cycle arrest via regulating CDK2 mRNA stability. This study provided valuable resources for understanding the mechanism of TTFields, which might further assist the investigation of TTFields in GBM treatment.

Abstract 5649: Bromodomain inhibition reduces CDK6 expression and reverses CDK4/6 inhibitor resistance in ER+ breast cancer

Abstract Introduction: CDK4/6 inhibitor (CDK4/6i) resistance is a pressing clinical problem for patients with ER+ breast cancer who are treated with CDK4/6 inhibitors combined with hormonal therapy. Our previous work identified CDK6 overexpression as a critical determinant of acquired resistance to CDK4/6i. Bromodomain and extra-terminal domain (BET) family proteins (BRD2, BRD3, BRD4, and BRDT) play important roles in regulating the expression of oncogenes in multiple cancer types. BET inhibitors (BETi), such as JQ1, have been extensively studied for the treatment of cancers. It has been shown that BRD4 is recruited to the CDK6 gene promoter and that BET inhibition reduces CDK6 mRNA and protein expression. In this study, we investigated the potential of BET inhibition to reverse CDK4/6i resistance of ER+ breast cancer. Methods: By long-term exposure to increasing concentrations of a CDK4/6i, we developed MCF7 and T47D ER+ breast cancer cell lines with acquired resistance to several FDA-approved CDK4/6 inhibitors demonstrating marked CDK6 overexpression. Cell viability and survival assays were performed to assess the in vitro efficacy of BETi. Flow cytometry was performed to evaluate cell cycle alterations as well as cell death. Gene expression was analyzed by Western blotting and qPCR to examine cell cycle-related molecular changes. Markers of apoptosis were also evaluated by Western blotting. CDK4/6i-resistant ER+ tumors were established in immunodeficient mouse models to explore the in vivo efficacy of BETi. Results: Our data showed that JQ1 significantly reduced cell viability and survival of CDK4/6i-resistant ER+ breast cancer cells in the presence of palbociclib, a commonly used CDK4/6i for the treatment of metastatic ER+ HER2- breast cancer. JQ1 also induced marked G1 cell cycle arrest and apoptosis in CDK4/6 inhibitor-resistant MCF7 and T47D cell lines in a time and dose-dependent manner. Analyses of key molecular mediators of cell cycle progression identified CDK6 and cyclin D1 as the major targets of JQ1 in CDK4/6i-resistant cells. Furthermore, we demonstrated that JQ1 suppressed CDK6 expression at the mRNA level. Importantly, we determined that growth suppression and killing of CDK4/6i-resistant cells by BETi were largely the consequence of CDK6 downregulation by BET inhibition. We have also established CDK4/6i-resistant xenografts successfully from MCF7 cells to test the in vivo efficacy of BETi in reversing CDK4/6 inhibitor resistance. Conclusions: Our results suggest that BET inhibition has potent efficacy in reversing acquired CDK4/6i resistance of ER+ breast cancer, mainly through its inhibitory regulation of CDK6. BET inhibitors are therefore promising candidate therapeutic agents for the treatment of CDK4/6i-resistant ER+ breast cancer. Citation Format: Renyan Liu, Constantia Pantelidou, Heta Jadhav, Geoffrey Shapiro. Bromodomain inhibition reduces CDK6 expression and reverses CDK4/6 inhibitor resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5649.

Abstract 1553: miR-1 targets Cdk6 and controls cell cycle progression and apoptosis in colitis-associated dysplasia

Abstract Detection of colorectal dysplasia during surveillance colonoscopy is currently the best method of determining risk of colitis-associated colorectal cancer (CAC). An understanding of the early molecular changes associated with the development of these lesions will inform the identification of new biomarkers for earlier detection. miRNAs (miRs), highly conserved noncoding RNAs, show great promise as stable, tissue-specific biomarkers of neoplasia. We previously identified 12 miRs that are differentially-expressed in colitis-associated dysplasias (flat and polypoid) vs. inflamed colonic mucosa from mice treated with AOM/DSS. miR-1, a putative tumor suppressor, was downregulated in colitis-associated dysplasias. Analysis of the mRNA expression profile of AOM/DSS-induced dysplasias and prediction of the interactions between the miRs and their targets led to the selection of the Cdk6 as the target of miR-1 to be further investigated, based on its: 1) upregulation in AOM/DSS-induced dysplasia; and 2) association with cell cycle progression and inflammatory signaling. The goal of the present study was to validate the predicted interaction between miR-1 and Cdk6 and assess the biological function of miR-1 in vitro. The ability of miR-1 to interact with the 3’UTR of Cdk6 mRNA was assessed using a dual luciferase assay. Co-transfection of HCT116 or RKO colon carcinoma cells with Cdk6-WT and miR-1 mimics led to a significant reduction in relative luciferase activity in both cell lines (30%, p=0.0117 and 47%, p=0.0269; respectively). Transfection with the Cdk6-Mut did not alter relative luciferase activity, confirming the Cdk6 binding site was specific for miR-1. The biological function of miR-1 was assessed in HCT116 and RKO cells reverse-transfected with miR-1 and cel-miR-67 (negative control) for 48 hrs. Apoptosis (AnnexinV+ cells) and cell cycle progression (% of cells in G0/G1, G2/M and S phase) were evaluated by flow cytometry, and proliferation by cell count (Trypan Blue). HCT116 and RKO transfected with the miR-1 mimic exhibited a higher proportion of apoptotic cells than the negative control (30% and 20% increase, p=0.0022 and 0.0013, respectively). Cell cycle analyses revealed the miR-1 mimic induced cell cycle arrest (G0/G1) in both cell lines (p<0.05). In HCT116 cells, this was accompanied by a reduction in the percentage of cells in G2/M (p=0.002) and S (p<0.001) phase. No effect of miR-1 on total cell number was observed. These results demonstrate that Cdk6 is a direct target of miR-1, and suggest that downregulation of this miR in dysplastic lesions contributes to CAC by inducing cell cycle progression and inhibiting apoptosis. These data provide novel insight into the early molecular changes that accompany the development of colitis-associated dysplasia and may serve as biomarkers for early detection of neoplasia. Supported by the Timothy P. and Aurora M. Hughes Fund for Colon Cancer Research. Citation Format: Mariana F. Fragoso, Geysson J. Fernandez, Lisa Vanderveer, Harry S. Cooper, Michael Slifker, Margie L. Clapper. miR-1 targets Cdk6 and controls cell cycle progression and apoptosis in colitis-associated dysplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1553.

Phase II trial of palbociclib in advanced sarcoma overexpressing CDK4 gene excluding dedifferentiated liposarcoma (DD LPS): A study from the Spanish Group for Research on Sarcoma (GEIS).

11511 Background: CDK4/6 inhibitors showed a favorable progression-free survival (PFS) in DD LPS, a sarcoma bearing 12q 13-15 amplicon that implies CDK4 amplification. The median PFS was 4 and 7 months (m) for palbociclib and abemaciclib, respectively. Preclinical experiments in 10 sarcoma cell lines and 6 PDX models, including only one DD LPS, showed higher efficacy of anti-CDK4 in cases with high expression of CDK4 and low expression of p16. This rationale supported the design of a phase II trial exploring palbociclib in a wide range of sarcomas, excluding DD LPS. Methods: Progressing pretreated advanced soft tissue sarcoma, excluding DD LPS, or osteosarcoma adult patients (pts), whose tumors overexpressed CDK4 and underexpressed CDKN2A mRNA in a baseline mandatory biopsy, were enrolled. CDK4 and CDKN2A expression were assessed by qRT-PCR, using an external control as reference (Universal human reference RNA; Agilent Technologies). The primary endpoint was 6-m PFS rate. Minimax Simon’s two-stage with type 1 and 2 errors of 10%, and null and alternative hypothesis of H0 15%, H1 40%, 6-month PFS rates were specified. The study will warrant further investigation if 6 or more pts had a PFS > 6 m from 21 evaluable pts. Palbociclib was administered orally at 125 mg/ day 21 out of 28 days. Pre-screening intended to increase the probability of positive profile in the baseline biopsy. Results: A total of 214 pts with 236 CDK4/ CDKN2A determinations were assessed for enrolment; 141 for prescreening, in archive tumor sample, and 95 for screening, in a baseline biopsy. There were 38/141 (27%) and 28/95 (29%) pts with favorable mRNA profile from pre and screening, respectively. Twenty-two pts were enrolled with a median of previous systemic lines of 3 (1-5). There were 9 different sarcoma subtypes, including 2 osteosarcomas. With a median FU of 10 m (0.4-23.3), the median PFS was 4.2 m (95% CI 0.9-7.4), while the 6- and 12-m PFS rates were 30% (95% CI 9-51) and 18% (95% CI 12-48) respectively. From 19 evaluable pts (1 early death by COVID, 1 withdrew consent and for 1 it was too early to be assessed) 11 had stable disease (58%) and 8 progressed (42%) as the best response. Patients with CDK4 expression above the median value had significantly longer mPFS in the univariate analysis: 5.9 m (95% CI 1.4-10.4) vs 1.9 m (95% CI 0.6-3.2), p = 0.046; and longer OS: 15.5 m (95% CI 6.8-24.3) vs 10.6 m (95% CI 0-23.2), p = 0.047, respectively. The probability to find a positive profile in the screening was 29%, but this proportion increased up to 41% if in pre-screening had been positive. Conclusions: Palbociclib showed to be effective in a wide variety of sarcoma subtypes, other than DD LPS, selected by CDK4/CDKN2A biomarkers. Clinical trial information: NCT03242382.

CDK6 Immunophenotype Implicates Potential Therapeutic Application of CDK4/6 Inhibitors in Urothelial Carcinoma.

BackgroundInfiltrating bladder urothelial carcinoma is the most common bladder malignancy with limited therapeutic options and poor prognosis. Identifying new therapeutic targets or strategies has important clinical significance. The data from public sources indicate poor prognosis in urothelial carcinoma cases with high CDK6 mRNA levels. Furthermore, studies have shown that CDK6 expression is elevated in urothelial carcinoma tissue compared to the surrounding urothelium, thus presenting a case for performing CDK4/6 inhibitor targeted research in urothelial carcinoma. However, a phase II trial showed that CDK4/6 inhibitors are not effective for advanced urothelial carcinoma, suggesting that case screening is important for targeted therapy.ObjectiveImmunohistochemistry (IHC) is simple and easy to perform and can be used to screen urothelial carcinoma cases with high CDK6 expression in clinical practice. The aim of this study was to determine the CDK6 expression threshold for positive cases.MethodsWe evaluated the correlation between the H-score of CDK6 protein expression and survival or CDK6 mRNA level using RNA sequencing. The effects of different CDK4/6 inhibitors were tested on bladder carcinoma cell lines with different CDK6 expression levels.ResultsThe H-score, which predicts poor prognosis and reflects a high CDK6 mRNA level, was determined as the selection criterion for positive cases. Furthermore, we found that urothelial carcinoma cell lines with higher CDK6 expression levels displayed greater sensitivity to CDK4/6 inhibitors than cells with lower expression levels.ConclusionsIHC staining for CDK6 protein in urothelial carcinoma is proposed as a promising screening platform for CDK4/6 inhibitor targeted therapy.

Dendritic distribution of CDK5 mRNA and p35 mRNA, and a glutamate-responsive increase of CDK5/p25 complex contribute to tau hyperphosphorylation

BackgroundIn Alzheimer's disease (AD), abnormally phosphorylated tau in the somatodendrite compartment of brain neurons causes synaptic loss, resulting in neuron death. Although the mechanism by which hyperphosphorylated tau appears in dendrites remains unclear, we have previously reported that local translation of tau mRNA and GSK3β mRNA in response to glutamatergic stimulation triggers an increase of tau protein and initiation of a cycle for amplification of reactivated preexisting GSK3β, respectively. In this study, we investigated the mechanism responsible for neural excitation-dependent activation of another major tau kinase, CDK5, within dendrites. MethodsPrimary hippocampal neurons were treated with glutamate and examined by in situ hybridization, immunocytochemistry and Western blotting. ResultsThe mRNAs for both CDK5 and its neural-specific activator, p35, were found to be constitutively distributed in dendrites. Glutamate treatment induced immediate local dendritic translation of these proteins as well as conversion of p35 to p25, which forms the hyper-activated CDK5/p25 complex. This neural excitation-dependent tau phosphorylation by CDK5 was suppressed in the presence of a calpain inhibitor or a NMDA receptor antagonist. ConclusionOur results indicate that in addition to an increase of dendritic tau and reactivation of preexisting GSK3β, increase and hyper-activation of CDK5 are evoked by translation of dendrite-distributed mRNAs upon NMDA receptor-mediated neural excitation. General significanceHyperphosphorylated tau with AD epitopes is locally produced in dendrites via translational activation of dendrite-distributed mRNAs in response to glutamatergic stimulation. Therefore, tau hyperphosphorylation may play a crucial role in synaptic transduction.

Correlation between CDK1 protein and CDK1 mRNA during oocyte maturation in mouse.

The aim of this study was to investigate the correlation between CDK1 protein and CDK1 mRNA during oocyte maturation in vivo in mouse. GV, GVBD, MI and MII oocytes were obtained from mice, respectively. Western blot validated that the CDK1 protein expression increased continuously and significantly with oocyte maturation in vivo (P<0.05). Real-time qRT-PCR showed that CDK1 mRNA expression was down-regulated significantly during transformation from GV to MI stages (P<0.05), and up-regulated significantly during transformation from MI to MII stages (P<0.05). The level of CDK1 mRNA peaked at MII stages. Spearman correlation analysis indicated that CDK1 protein expression was poor correlation with CDK1 mRNA expression during oocyte maturation in vivo (R=0.200). This finding suggested that the increase of CDK1 protein during oocyte maturation in vivo was not entirely caused by the change of transcription level. The results provide new food for thought for further research on the molecular mechanism of oocyte maturation in vivo.

A heat shock protein 90 inhibitor reduces oncoprotein expression and induces cell death in heterogeneous glioblastoma cells with EGFR, PDGFRA, CDK4, and NF1 aberrations

AimsGlioblastoma (GBM) is a highly malignant brain tumor. After treatment with the first-line drug temozolomide, only 50% of patients are responsive. Recent literature shows that the difficulty in treating GBM is mainly due to the heterogeneity of its four major cellular states, which are characterized by differences in EGFR, PDGFRA, CDK4, and NF1. Therefore, development of a multitarget drug is a potential strategy for treating heterogeneous GBM. Main methodsIn this study, the antitumor ability of a potent heat shock protein 90 inhibitor, NVP-AUY922 (AUY922), was evaluated in GBM cell lines (U-87 MG and T98G cells) and patient-derived GBM cell lines [P#5 and P#5 temozolomide-resistant (TMZ-R) cells]. Key findingsWe found that AUY922 significantly reduced cell viability and colony formation in four GBM cell lines. AUY922 also significantly induced apoptosis by increasing PARP1 cleavage and the number of annexin V-positive cells. The autophagy indicators as MAP1LC3B cleavage and MAP1LC3B puncta were increased after AUY922 treatment. AUY922-induced cell death could be partially reversed by pharmacological inhibition of either apoptotic inhibitor or autophagy inhibitor. Moreover, AUY922 reduced the mRNA and protein expressions of EGFR, PDGFRA, CDK4, and NF1, which contribute to the four cellular state subtypes in GBM cells. In addition, the downstream signaling proteins of these four proteins, AKT/p-AKT, MAPK/p-MAPK, and BRAF, were downregulated after AUY922 treatment. SignificanceTaken together, AUY922 led to GBM cell death via apoptosis and autophagy, and reduced the mRNA and protein expression of EGFR, PDGFRA, CDK4, and NF1in heterogeneous GBM cells.

CDK1, CCNB1 and NDC80 are associated with prognosis and progression of hepatitis B virus-associated hepatocellular carcinoma: a bioinformatic analysis

To identify the key genes involved in the transformation of hepatitis B virus (HBV) into hepatocellular carcinoma (HCC) and explore the underlying molecular mechanisms. We analyzed the mRNA microarray data of 119 HBV-related HCC tissues and 252 HBV-related non-tumor tissues in GSE55092, GSE84044 and GSE121248 from the GEO database, and the "sva" R package was used to remove the batch effects. Integration analysis was performed to identify the differentially expressed genes (DEGs) in HBV-related liver cancer and liver tissues with HBV infection. The significant DEGs were functionally annotated using GO and KEGG analyses, and the most important modules and hub genes were explored with STRING analysis. Kaplan-Meier and Oncomine databases were used to verify the HCC gene expression data in the TCGA database to explore the correlations of the hub genes with the occurrence, progression and prognosis of HCC. We also examined the expressions of the hub genes in 17 pairs of surgical specimens of HCC and adjacent tissues using RT-qPCR. We identified a total of 121 DEGs and 3 genetic markers in HCC (P < 0.01). These DEGs included cyclin1 (CDK1), cyclin B1 (CCNB1), and nuclear division cycle 80 (NDC80), which participated in cell cycle, pyrimidine metabolism and DNA replication and were highly correlated (P < 0.05). Analysis of the UALCAN database confirmed high expressions of these 3 genes in HCC tissues, which were correlated with a low survival rate of the patients, as shown by Kaplan-Meier analysis of the prognostic data from the UALCAN database. CDK1, CCNB1 and NDC80 were all correlated with the clinical grading of HCC (P < 0.05). The results of RT-qPCR on the surgical specimens verified significantly higher expressions of CDK1, CCNB1 and NDC80 mRNA in HCC tissues than in the adjacent tissues. CDK1, CCNB1 and NDC80 genes can be used as prognostic markers of HBV-related HCC and may serve as potential targets in preclinical studies and clinical treatment of HCC.