Expression Of Cell Cycle Proteins Research Articles

Metformin regulates the proliferation and motility of melanoma cells by modulating the LINC00094/miR-1270 axis.

Melanoma is an aggressive tumor with a high mortality rate. Metformin, a commonly prescribed diabetes medication, has shown promise in cancer prevention and treatment. Long noncoding RNAs (lncRNAs) are non-protein-coding RNA molecules that play a key role in tumor development by interacting with cellular chromatins. Despite the benefits of metformin, the anticancer mechanism underlying its effect on the regulation of lncRNAs in melanoma remains unclear. We investigated the lncRNA profiles of human melanoma cells with and without metformin treatment using a next-generation sequencing approach (NGS). Utilizing public databases, we analyzed the expression levels and clinical impacts of LINC00094 and miR-1270 in melanoma. The expression levels of LINC00094 and miR-1270 were verified in human cell lines and clinical samples by real-time PCR and in situ hybridization. The biological roles of LINC00094 and miR-1270 in cell growth, proliferation, cell cycle, apoptosis, and motility were studied using in vitro assays. We identify a novel long noncoding RNA, namely LINC00094, whose expression considerably decreased in melanoma cells after metformin treatment. In situ hybridization analysis revealed substantially higher expression of LINC00094 in cutaneous melanoma tissue compared with adjacent normal epidermis and normal control tissues (P < 0.001). In nondiabetic patients with melanoma, the overall survival of high LINC00094 expression group was shorter than the low LINC00094 expression group with borderline statistical significance (log-rank test, P = 0.057). Coexpression analysis of LINC00094 indicated its involvement in the mitochondrial respiratory pathway, with its knockdown suppressing genes associated with mitochondrial oxidative phosphorylation, glycolysis, antioxidant production, and metabolite levels. Functional analysis revealed that silencing-LINC00094 inhibited the proliferation, colony formation, invasion, and migration of melanoma cells. Cell cycle analysis following LINC00094 knockdown revealed G1 phase arrest with reduced cell cycle protein expression. Combined TargetScan and reporter assays revealed a direct link between miR-1270 and LINC00094. Ectopic miR-1270 expression inhibited melanoma cell growth and motility while inducing apoptosis. Finally, through in silico analysis, we identified two miR-1270 target genes, CD276 and centromere protein M (CENPM), which may be involved in the biological functions of LINC00094. Overall, LINC00094 expression may regulate melanoma cell growth and motility by modulating the expression of miR-1270, and targeting genes of CD276 and CENPM indicating its therapeutic potential in melanoma treatment.

DEK regulates B-cell proliferative capacity and is associated with aggressive disease in low-grade B-cell lymphomas

This study sheds light on the pivotal role of the oncoprotein DEK in B-cell lymphoma. We reveal DEK expression correlates with increased tumor proliferation and inferior overall survival in cases diagnosed with low-grade B-cell lymphoma (LGBCL). We also found significant correlation between DEK expression and copy number alterations in LGBCL tumors, highlighting a novel mechanism of LGBCL pathogenesis that warrants additional exploration. To interrogate the mechanistic role of DEK in B-cell lymphoma, we generated a DEK knockout cell line model, which demonstrated DEK depletion caused reduced proliferation and altered expression of key cell cycle and apoptosis-related proteins, including Bcl-2, Bcl-xL, and p53. Notably, DEK depleted cells showed increased sensitivity to apoptosis-inducing agents, including venetoclax and staurosporine, which underscores the therapeutic potential of targeting DEK in B-cell lymphomas. Overall, our study contributes to a better understanding of DEK’s role as an oncoprotein in B-cell lymphomas, highlighting its potential as both a promising therapeutic target and a novel biomarker for aggressive LGBCL. Further research elucidating the molecular mechanisms underlying DEK-mediated tumorigenesis could pave the way for improved treatment strategies and better clinical outcomes for patients with B-cell lymphoma.

Cell cycle regulated expression of the WHI7 Start repressor gene

ABSTRACT Periodic transcriptional waves along the cell cycle ensure the accurate progression of the different cell cycle phases through the timely regulated expression of cell cycle proteins. The G1/S transition (Start) consists in the activation of a transcriptional program by G1 CDKs through the inactivation of Start transcriptional repressors, Whi5 and Whi7 in yeast or Rb in mammals. Here, we provide a comprehensive characterization of the transcriptional regulation of the Start repressor Whi7 in budding yeast. We found that WHI7 is a cell cycle regulated gene that shows periodic expression peaking in G1. Our results demonstrate that the three cell cycle transcriptional programs related to G1 and their corresponding transcription factors are involved in the transcriptional control of WHI7. Specifically, we identified that the transcriptional regulators Swi5 and Mcm1-Yox1, which are involved in late M and early G1 expression, and the transcription factors MBF and SBF, which are responsible for G1/S expression, are able to associate and regulate the WHI7 gene. In summary, in this work, we provide new mechanisms for the regulation of the Start repressor Whi7, which highlights the precise and complex control of the cell cycle machinery governing the G1/S transition.

Cratoxylum formosum ssp. pruniflorum induces gastric cancer cell apoptosis and pyroptosis through the elevation of ROS and cell cycle arrest.

Cratoxylum formosum ssp. pruniflorum (CF), a traditional medicinal plant in Southern China, is widely recognized as a popular medicinal and tea plant traditionally utilized by diverse linguistic groups in the region for the treatment of gastrointestinal ailments. The objective of this study was to explore the active components and mechanisms of CF against gastric cancer (GC). The chemical ingredients of CF were obtained by using UPLC-MS/MS-based metabolomics. MGC-803 and HGC-27 cells were employed to investigate the direct anti-GC effect. The potential targets and signaling pathway of CF were identified through network pharmacology and proteomics, followed by subsequent experimental validation. Through UPLC-MS/MS metabolomics analysis, a total of 197 chemical ingredients were identified in CF leaves. Network pharmacology and proteomics techniques revealed 25 potential targets for GC, with a protein-protein interaction (PPI) network highlighting 12 cores targets, including CTNNB1, CDK2, et al. Furthermore, seven key CF ingredients - vismione B, feruloylcholine, α-amyrin, vanillic acid, galangin, cinnamic acid, and caffeic acid - were found to mediate anti-GC effects through pathways such as reactive oxygen species (ROS) and cell cycle signaling pathway. In vitro experiments demonstrated that CF significantly inhibited the proliferation and migration of GC cells, increased intracellular reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) levels, arrested the cell cycle at the S-phase, induced apoptosis and pyroptosis, and upregulated expression of apoptosis proteins (Bax, Bax/Bcl-2, cleaved-Caspase-3/Caspase-3), and pyroptosis proteins (GSDMD-N/GSDMD and GSDME-N/GSDME), while downregulating expression of cell cycle proteins (CDK2 and cyclin A1) as well as necroptosis proteins (RIP1 and MLKL). Collectively, these findings reveal CF's therapeutic potential against GC by the augmentation of ROS production, cell cycle arrest, promotion of apoptosis, and pyroptosis, offering valuable evidence for the development and utilization of CF in clinical settings.

Renin and (pro)renin receptors induce vascular smooth muscle cell proliferation and neointimal hyperplasia by activating oxidative stress and inflammation.

Introduction: Renin and prorenin promote the proliferation of vascular smooth muscle cells (VSMCs) through the (pro)renin receptor, or (P)RR, to promote restenosis occurrence. This study aimed to explore whether prorenin promoted the proliferation of VSMCs in a (P)RR-mediated Ang II-independent manner. Methods: Losartan and PD123319 were used to block the interaction between (P)RR and angiotensin in vitro. Cells were treated with renin, platelet-derived growth factor (PDGF), or RNAi-(P)RR, either jointly or individually. Cell proliferation was measured via Cell Counting Kit-8 (CCK-8) and flow cytometry methods; moreover, real-time polymerase chain reaction (RT-PCR) and Western blot (WB) assays were used to detect the expression of cyclin D1, proliferating cell nuclear antigen (PCNA), (P)RR, NOX1, and phosphatidylinositol 3-kinase (PI3K)/AKT signaling proteins. Immunofluorescence staining was conducted to measure the expression of (P)RR, and the levels of renin, PDGF-BB, inflammatory factors, and oxidative stress were determined by using enzyme-linked immunosorbent assay (ELISA). Moreover, a balloon catheter was used to enlarge the carotid artery of the Sprague Dawley rats. PRO20 was applied to identify angiotensin II (Ang II). The hematoxylin and eosin, RT-PCR, and WB results validated the cell assay results. Results: Renin promoted the proliferation of rat VSMCs by enhancing cell viability and cell cycle protein expression when Ang II was blocked, but silencing (P)RR inhibited this effect. Furthermore, renin enhanced NOX1-mediated oxidative stress and inflammation by activating the extracellular signal-regulated kinase 1/2 (ERK1/2)-AKT pathway in vitro. Similarly, the inhibition of (P)RR resulted in the opposite phenomenon. Importantly, the inhibition of (P)RR inhibited neointimal hyperplasia in vivo after common carotid artery injury by restraining NOX1-mediated oxidative stress through the downregulation of the ERK1/2-AKT pathway. The animal study confirmed these findings. Conclusion: Renin and (P)RR induced VSMC proliferation and neointimal hyperplasia by activating oxidative stress, inflammation, and the ERK1/2-AKT pathway in an Ang II-independent manner.

IGFBP1 promotes the proliferation and migration of lung adenocarcinoma cells through the PPARα pathway

BackgroundThe immune status is closely linked to cancer progression, metastasis, and prognosis. Lipid metabolism, crucial for reshaping immune status, plays a key role in regulating the advancement of lung adenocarcinoma (LUAD) and deserves further investigation. MethodsThis study classifies LUAD patients into different immune subtypes based on lipid metabolism-related genes and compares the clinical characteristics among these subtypes. Single-multi COX analysis screens out key genes related to prognosis, and a risk feature and prognostic model are constructed. Cell cloning, scratch, transwell, western blotting and flow cytometry cell cycle analysis to detect the function of key genes. A subcutaneous tumor animal model is used to investigate the in vivo function and molecular mechanisms of key genes. ResultsLUAD patients are classified into three immune subtypes, among which C3 subtype has lower immune status and higher frequency of gene mutations, and show lower immunoreactivity in immunotherapy. COX analysis identified a prognostic model for four lipid metabolism factors (IGFBP1, NR0B2, PPARA, and POMC). IGFBP1, a core gene in this model, is highly expressed in the C3 subtype. Functionally, knocking down IGFBP1 significantly inhibits tumor cell cloning, scratch, and migration abilities, and downregulates the expression of cell cycle and EMT-related proteins. Knocking down IGFBP1 significantly inhibits tumor burden (P < 0.05). Mechanistically, knocking down IGFBP1 inhibits the activation of PPARα to regulate tumor cell growth. ConclusionsThis study found that lipid metabolism genes are closely related to LUAD, and IGFBP1 may be a key gene in regulating tumor growth and development.

Synergistic Efficacy of CDK4/6 Inhibitor Abemaciclib and HDAC Inhibitor Panobinostat in Pancreatic Cancer Cells.

The current 5-year survival rate of pancreatic cancer is about 12%, making it one of the deadliest malignancies. The rapid metastasis, acquired drug resistance, and poor patient prognosis necessitate better therapeutic strategies for pancreatic ductal adenocarcinoma (PDAC). Multiple studies show that combining chemotherapeutics for solid tumors has been successful. Targeting two distinct emerging hallmarks, such as non-mutational epigenetic changes by panobinostat (Pan) and delayed cell cycle progression by abemaciclib (Abe), inhibits pancreatic cancer growth. HDAC and CDK4/6 inhibitors are effective but are prone to drug resistance and failure as single agents. Therefore, we hypothesized that combining Abe and Pan could synergistically and lethally affect PDAC survival and proliferation. Multiple cell-based assays, enzymatic activity experiments, and flow cytometry experiments were performed to determine the effects of Abe, Pan, and their combination on PDAC cells and human dermal fibroblasts. Western blotting was used to determine the expression of cell cycle, epigenetic, and apoptosis markers. The Abe-Pan combination exhibited excellent efficacy and produced synergistic effects, altering the expression of cell cycle proteins and epigenetic markers. Pan, alone and in combination with Abe, caused apoptosis in pancreatic cancer cells. Abe-Pan co-treatment showed relative safety in normal human dermal fibroblasts. Our novel combination treatment of Abe and Pan shows synergistic effects on PDAC cells. The combination induces apoptosis, shows relative safety, and merits further investigation due to its therapeutic potential in the treatment of PDAC.

Synthesis, characterization, and anti-cancer potential of novel p53-mediated Mdm2 and Pirh2 modulators: an integrated In silico and In vitro approach.

Introduction: Leukemia is a global health concern that requires alternative treatments due to the limitations of the FDA-approved drugs. Our focus is on p53, a crucial tumor suppressor that regulates cell division. It appears possible to stabilize p53 without causing damage to DNA by investigating dual-acting inhibitors that target both ligases. The paper aims to identify small molecule modulators of Mdm2 and Pirh2 by using 3D structural models of p53 residues and to further carry out the synthesis and evaluation of hit candidates for anti-cancer potency by in vitro and in silico studies. Methods: We synthesized structural analogues of MMs02943764 and MMs03738126 using a 4,5-(substituted) 1,2,4-triazole-3-thiols with 2-chloro N-phenylacetamide in acetone with derivatives of PAA and PCA were followed. Cytotoxicity assays, including MTT, Trypan Blue Exclusion, and MTS assays, were performed on cancer cell lines. Anti-proliferation activity was evaluated using K562 cells. Cell cycle analysis and protein expression studies of p53, Mdm2, and Pirh2 were conducted using flow cytometry. Results: As for results obtained from our previous studies MMs02943764, and MMs03738126 were selected among the best-fit hit molecules whose structural analogues were further subjected to molecular docking and dynamic simulation. Synthesized compounds exhibited potent anti-proliferative effects, with PAC showing significant cytotoxicity against leukemia cells. PAC induced cell cycle arrest and modulated p53, Mdm2, and Pirh2 protein expressions in K562 cells. Molecular docking revealed strong binding affinity of PAC to p53 protein, further confirmed by molecular dynamics simulation. Discussion: The study presents novel anticancer compounds targeting the p53 ubiquitination pathway, exemplified by PAC. Future perspectives involve further optimization and preclinical studies to validate PAC's potential as an effective anticancer therapy.

BRD4 plays an antiaging role in the senescence of renal tubular epithelial cells.

Age-related kidney failure is often induced by a decrease in the bioavailability of tubular epithelial cells in elderly chronic kidney disease (CKD) patients. BRD4, an epigenetic regulator and a member of the bromodomain and extraterminal (BET) protein family, acts as a super-enhancer (SE) organizing and regulating genes expression during embryogenesis and cancer development. But the physiological function of BRD4 in normal cells has been less studied. This study aimed to research certain biological roles of BRD4 in the process of normal cell aging and discuss the potential mechanisms. In this study, we investigated the biological functions of BRD4 proteins in the aging of renal tubular cells. At first, we used a D-galactose (D-gal) and BRD4 inhibitor (Abbv-075) to replicate kidney senescence in vivo. D-gal and Abbv-075 were then used to measure the aging-related changes, such as changes in cell cycle, β-galactosidase activity, cell migration, and p16 protein expression in vitro. At last, we knocked down and over-expressed BRD4 to investigate the aging-related physiological phenomena in renal tubular cells. In vitro, D-gal treatment induced noticeable aging-related changes such as inducing cell apoptosis and cell cycle arrest, increasing β-galactosidase activity as well as up-regulating p16 protein expression in primary human tubular epithelial cells. In the aging mice model, D-gal significantly induced renal function impairment and attenuated BRD4 protein expression. At the same time, the BRD4 inhibitor (Abbv-075) was able to mimic D-gal-induced cell senescence. In vivo, Abbv-075 also decreased kidney function and up-regulated p21 protein expression. When we knocked down the expression of BRD4, the senescence-associated β-galactosidase (SA-β-gal) activity increased dramatically, cell migration was inhibited, and the proportion of cells in the G0/G1 phase increased. Additionally, the knockdown also promoted the expression of the senescence-related proteins p16. When the renal tubular cells were overexpressed with BRD4, cell aging-related indicators were reversed in the D-gal-induced cell aging model. BRD4 appears to have an active role in the aging of renal tubular cells in vivo and in vitro. The findings also suggest that BRD4 inhibitors have potential nephrotoxic effects for oncology treatment. BRD4 may be a potential therapeutic biomarker and drug target for aging-related kidney diseases, which warrants additional studies.

GuiErBai: a potent inhibitor, exhibiting broadly antitumor effect against cervical cancer in vitro and in vivo.

Introduction: Cervical cancer (CC) ranks as the fourth most prevalent malignant tumor among women worldwide, and is the fourth leading cause of cancer-related mortality. GuiErBai (GEB), a compound preparation developed by our research team, is derived from the ancient Chinese medicine of the Miao nationality and is comprised of podophyllotoxin (PTOX), imperatorin, isoimperatorin, and A. dahurica alkaloids. These individual components have demonstrated notable efficacy in tumor treatment. However, the specific anti-tumor effect of the compound Chinese medicine GEB in the context of CC has yet to be validated. Methods: HeLa and SiHa cell lines were utilized for in vitro experiments and treated with 5mg/mL and 10mg/mL GEB concentrations, respectively. The cell cycle changes after GEB treatment were assessed using flow cytometry. Transmission electron microscopy was employed to observe autophagic bodies and apoptotic bodies, while MDC staining evaluated the occurrence of autophagy. CCK-8 was used to observe the effect of GEB on cell proliferation, and Transwell assays assessed cell migration and invasion. Western blotting detected cell cycle and apoptosis-related protein expression, along with the expression level of autophagy-related protein LC3I/II. Changes in ROS and mitochondrial membrane potential in cervical cancer cells following GEB treatment were determined using ROS detection and mitochondrial membrane potential detection kits. For the in vivo experiment, a nude mouse model of cervical cancer transplantation based on HeLa cells was established. Experimental animals were divided into negative control, positive control, high-dose GEB (10mg/mL), and low-dose GEB (5mg/mL) groups. Results: In HeLa and SiHa cell lines, the G0/G1 phase of tumor cells significantly decreased (p < 0.001), while the G2/M phase increased notably (p < 0.001) following various GEB treatments. Electron microscopy showed GEB promoted apoptotic body and autophagosome formation in both cell lines. Compared to untreated HeLa and SiHa cells, GEB-treated cells exhibited significantly reduced caspase3 protein expression, and substantially increased autophagy-related protein LC3I/II expression. GEB treatment significantly reduced migration and invasion capabilities in both cell lines (p < 0.001), while ROS content and mitochondrial membrane potential were significantly elevated (p < 0.001). GEB effectively inhibited cervical cancer cell proliferation, with the optimal concentration being 10mg/mL. A successful nude mouse model of cervical cancer transplantation was established using HeLa cells. Post-GEB treatment, the tumor volume and weight in nude mice significantly decreased (p < 0.001), with diminished expression of CD34, VEGF, and caspase3 proteins in tumor tissues. Discussion: GEB exhibits a robust antitumor effect against cervical cancer, both in vitro and in vivo, in a concentration-dependent manner, by regulating autophagy and apoptosis of tumor cells.

MicroR-1199-5p targeting SRD5A2 promotes the biological behavior and EMT of hypospadias cells.

Hypospadias, an oft-occurring penis anomaly, ranks among neonatal's foremost birth defects. The SRD5A2 can affect male reproductive system development and is abnormally expressed in its epithelial cells. This study exploration aimed at understanding the role of SRD5A2 in the development of hypospadias from a molecular perspective. SRD5A2 levels in hypospadias primary cells were analyzed by Western blot, while targeted interaction with miR-1199-5p was ascertained by dual-luciferase gene reporter assay. In vitro biological experiments were used to confirm the biological function of SRD5A2 in hypospadias. SRD5A2 expression was significantly upregulated, and miR-1199-5p expression was significantly downregulated in hypospadias primary cells. Intervention of SRD5A2 expression can affect cell proliferation, migration, invasion, EMT, and the expression of cell cycle-related proteins. Additionally, we found that SRD5A2 is regulated by upstream miR-1199-5p and can enhance the effect of SRD5A2 on hypospadias cells. Conclusions Silencing SRD5A2 promotes cell proliferation, invasion, and migration blocks the cell cycle at the G1 phase, and simultaneously promotes EMT, cell cycle, and cell proliferation-related protein expression. The biological function of SRD5A2 in hypospadias cells is regulated by miR-1199-5p. SRD5A2 may be an effective therapeutic target for hypospadias.

Abstract PO1-25-08: Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer

Abstract Background: The human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase overexpressed in approximately 20-25% of breast cancers with varying biological differences according to hormone receptor (HR) positive status. Combination of chemotherapy with dual HER2 blockade and endocrine therapy (adjuvant) or CDK4/6 inhibitor (CDK4/6i) therapy (metastatic) is standard of care for HER2-positive/HR-positive breast cancer. However, de-escalation of chemotherapy remains of significant interest given associated toxicities and, as a result, alternative therapeutic strategies are needed. One such strategy is targeting the signal transducer and activator of transcription 3 (STAT3) which is constitutively activated in the HER-2 positive setting. Moreover, there is preclinical evidence to suggest that phosphorylated STAT3 (pSTAT3) induces overexpression of cyclin D1 (CCND1), which along with CDK4 may mediate resistance to targeted therapy for HER2-positive breast cancer. However, it is unclear if pSTAT3 plays a significant role in tumorigenesis or the development of resistance to therapy in HER2-positive disease. We aim to evaluate the effect of CDK4/6i therapy with palbociclib on tumor growth in an inducible transgenic HER2-positive mouse model and the impact on pSTAT3 and cyclin D1 levels. Methods: Using an inducible transgenic mouse mammary tumor virus (MMTV) model of HER2-positive breast cancer, we evaluated STAT3 phosphorylation in tumors driven by HER2 expression in addition to the impact of CDK4/6 inhibition with palbociclib on tumorigenesis and pSTAT3 activation. To model tumor recurrence, doxycycline was withdrawn from tumor bearing mice and tumor regression was observed. Subsequently, the mice developed recurrent mammary tumors. Given recurrent tumors in this model are driven by a HER2-independent mechanism, we examined expression of cell cycle proteins together with STAT3 phosphorylation to determine association with recurrence. Result: In addition to expressing HER2, the primary tumors expressed estrogen receptor (ERα), Rb, cyclin D1, and CDK4/6 proteins. STAT3 phosphorylation was also observed. Treatment with palbociclib decreased Rb phosphorylation and decreased tumor growth compared to vehicle treated mice (%TGI 79.37% after 21 days). Furthermore, pSTAT3 levels were not altered with palbociclib therapy. When recurrent tumors were analyzed, they lacked HER2 expression but expressed cyclin D1 and CDK4/6 supporting that tumor growth is driven by cyclin D1/CDK4 pathway. Recurrent tumors also demonstrated pSTAT3 despite lack of HER2 expression suggesting that STAT3 phosphorylation in the recurrent tumors is mediated by an alternative mechanism, potentially the CDK4/6 pathway. Conclusion: We demonstrate STAT3 activation in a mouse model of HER2 driven breast cancer and show persistent activation in recurrent tumors lacking HER2 expression rendering them resistant to HER2-targeted therapy. We also demonstrate the anti-tumor effect of palbociclib in primary tumors with HER2-dependent proliferation. Taken together, our findings suggest that pSTAT3 is a viable therapeutic target and provides the rationale for combination therapy with CDK4/6 inhibition using palbociclib and STAT3 inhibition with a novel STAT3 inhibitor in HER2-positive breast cancer. This therapeutic approach may represent a potential chemotherapy de-escalation strategy. Citation Format: Taiwo Adesoye, Linjie Luo, Tuyen Bui, Serena Kim, Hannah Wingate, Debu Tripathy, Kelly Hunt, Khandan Keyomarsi. Evaluating the role of CDK4/6 inhibition on STAT3 activation in a transgenic mouse model of HER2-positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-08.

KNSTRN Is a Prognostic Biomarker That Is Correlated with Immune Infiltration in Breast Cancer and Promotes Cell Cycle and Proliferation

Kinetochore-localized astrin/SPAG5-binding protein (KNSTRN) promotes the progression of bladder cancer and lung adenocarcinoma. However, its expression and biological function in breast cancer remain largely unknown. Therefore, this study aimed to analyze KNSTRN expression, prognoses, correlation with immune infiltration, expression-associated genes, and regulated signaling pathways to characterize its role in regulating the cell cycle using both bioinformatics and in vitro functional experiments. Analyses of The Cancer Genome Atlas, Gene Expression Omnibus, TIMER, and The Human Protein Atlas databases revealed a significant upregulation of KNSTRN transcript and protein levels in breast cancer. Kaplan–Meier survival analyses demonstrated a significant association between high expression of KNSTRN and poor overall survival, relapse-free survival, post-progression survival, and distant metastases-free survival in patients with breast cancer. Furthermore, multivariate Cox regression analyses confirmed that KNSTRN is an independent prognostic factor for breast cancer. Immune infiltration analysis indicated a positive correlation between KNSTRN expression and T regulatory cell infiltration while showing a negative correlation with Tgd and natural killer cell infiltration. Gene set enrichment analysis along with single-cell transcriptome data analysis suggested that KNSTRN promoted cell cycle progression by regulating the expression of key cell cycle proteins. The overexpression and silencing of KNSTRN in vitro, respectively, promoted and inhibited the proliferation of breast cancer cells. The overexpression of KNSTRN enhanced the expression of key cell cycle regulators, including CDK4, CDK6, and cyclin D3, thereby accelerating the G1/S phase transition and leading to aberrant proliferation of breast cancer cells. In conclusion, our study demonstrates that KNSTRN functions as an oncogene in breast cancer by regulating immune response, promoting G1/S transition, and facilitating breast cancer cell proliferation. Moreover, KNSTRN has potential as a molecular biomarker for diagnostic and prognostic prediction in breast cancer.

Inhibition of ACC1 Diminishes the Malignant Phenotype of Hepatobiliary Cancers In Vitro

Hepatobiliary cancers are a collection of malignancies arising from liver and biliary tract cells. These cancers have high anatomical proximity, overlapping symptoms, and share common risk factors. Hepatobiliary cancers are generally rare and often lack effective treatment options due to late diagnoses and limited treatment efficacy. These cancers tend to be highly aggressive, which limits treatment options for patients, especially since diagnosis often occurs at later stages of disease progression. Chronic inflammation is the most significant risk factor for developing these malignancies. Inflammation alters cellular metabolism, which gives them unique metabolism characteristics that enhance their survival and increase malignancy. Acetyl-Coa-carboxylase (ACC) is a metabolic enzyme responsible for the carboxylation of acetyl-CoA (AC) into malonyl-CoA (MC). MC is used in de novo fatty acid synthesis, an upregulated process in human cancers. Acetyl-Coa-carboxylase 1 (ACC1), in particular, is the first rate-limiting step for de novo lipogenesis. Here we delve into the effect of ACC1 inhibition on the malignant phenotypes of hepatobiliary cancers. Our results showed that knockdown of ACC1 slowed proliferation and migration, reduced spheroid formation, and altered cell cycle progression and protein expression in hepatobiliary cancers. In conclusion, our study suggests that ACC1 may contribute to hepatobiliary cancers' malignancy and may be utilized as a therapeutic target for treating such diseases.

TRAIP suppressed apoptosis and cell cycle to promote prostate cancer proliferation via TRAF2-PI3K-AKT pathway activation.

TRAF-interacting protein (TRAIP) is a RING-type E3 ubiquitin ligase, which has been implicated in various cellular processes and participated in various cancers as an oncogene. However, the function and potential mechanism of TRAIP in prostate cancer (PCa) have not been investigated so far. Public TGCA data were used to evaluate the expression profile of TRAIP in prostatic tumors. The relative expression of TRAIP and TRAF2 in PCa tissues and tumor cell lines was detected by qPCR, western blot, and IHC staining. Next, TRAIP knockdown and overexpression plasmids were constructed and transfected into PCa cell lines. Moreover, cell proliferation, invasion, migration, and apoptosis were measured by colony formation, Transwell, wound healing, and flow cytometry assays. Subsequently, cell cycle and signaling pathway-related proteins were tested by western blot. Finally, the effect of TRAIP on PCa was measured based on the nude mouse xenograft model. TRAIP was significantly upregulated in PCa tissues and tumor cell lines. In addition, TRAIP promoted cell proliferation, invasion, and migration of PCa cell lines. Such an oncogenic property was mediated by the cell cycle arrest and the inhibition of apoptosis, as indicated by different functional assays and the expression of cell cycle and apoptosis regulatory proteins in cultured cells. Moreover, TRAIP combined with TRAF2 to activate PI3K/AKT pathway. Finally, TRAIP depletion suppressed the growth of tumors and cell proliferation in vivo. Our study first revealed that TRAIP promoted tumor progression and identified it as a potential therapeutic target for PCa patients in the future.

Profilin1 is required for prevention of mitotic catastrophe in murine and human glomerular diseases.

The progression of proteinuric kidney diseases is associated with podocyte loss, but the mechanisms underlying this process remain unclear. Podocytes reenter the cell cycle to repair double-stranded DNA breaks. However, unsuccessful repair can result in podocytes crossing the G1/S checkpoint and undergoing abortive cytokinesis. In this study, we identified Pfn1 as indispensable in maintaining glomerular integrity - its tissue-specific loss in mouse podocytes resulted in severe proteinuria and kidney failure. Our results suggest that this phenotype is due to podocyte mitotic catastrophe (MC), characterized histologically and ultrastructurally by abundant multinucleated cells, irregular nuclei, and mitotic spindles. Podocyte cell cycle reentry was identified using FUCCI2aR mice, and we observed altered expression of cell-cycle associated proteins, such as p21, p53, cyclin B1, and cyclin D1. Podocyte-specific translating ribosome affinity purification and RNA-Seq revealed the downregulation of ribosomal RNA-processing 8 (Rrp8). Overexpression of Rrp8 in Pfn1-KO podocytes partially rescued the phenotype in vitro. Clinical and ultrastructural tomographic analysis of patients with diverse proteinuric kidney diseases further validated the presence of MC podocytes and reduction in podocyte PFN1 expression within kidney tissues. These results suggest that profilin1 is essential in regulating the podocyte cell cycle and its disruption leads to MC and subsequent podocyte loss.

Histone demethylase KDM4B accelerates the progression of glioblastoma via the epigenetic regulation of MYC stability

BackgroundGlioblastoma (GBM) is the most malignant and invasive human brain tumor. Histone demethylase 4B (KDM4B) is abnormally expressed in GBM, but the molecular mechanisms by which KDM4B affects the malignant tumor progression are not well defined.MethodsGBM cell lines and xenograft tumor samples were subjected to quantitative PCR (qPCR), Western blot, immunohistochemical staining (IHC), as well as ubiquitination, immunoprecipitation (IP), and chromatin immunoprecipitation (ChIP) assays to investigate the role of KDM4B in the progression of GBM.ResultsHere, we report that KDM4B is an epigenetic activator of GBM progression. Abnormal expression of KDM4B is correlated with a poor prognosis in GBM patients. In GBM cell lines, KDM4B silencing significantly inhibited cell survival, proliferation, migration, and invasion, indicating that KDM4B is essential for the anchorage-independent growth and tumorigenic activity of GBM cells. Mechanistically, KDM4B silencing led to downregulation of the oncoprotein MYC and suppressed the expression of cell cycle proteins and epithelial-to-mesenchymal transition (EMT)-related proteins. Furthermore, we found that KDM4B regulates MYC stability through the E3 ligase complex SCFFBXL3+CRY2 and epigenetically activates the transcription of CCNB1 by removing the repressive chromatin mark histone H3 lysine 9 trimethylation (H3K9me3). Finally, we provide evidence that KDM4B epigenetically activates the transcription of miR-181d-5p, which enhances MYC stability.ConclusionsOur study has uncovered a KDM4B-dependent epigenetic mechanism in the control of tumor progression, providing a rationale for utilizing KDM4B as a promising therapeutic target for the treatment of MYC-amplified GBM.

ERK hyperactivation serves as a unified mechanism of escape in intrinsic and acquired CDK4/6 inhibitor resistance in acral lentiginous melanoma.

Patients with metastatic acral lentiginous melanoma (ALM) suffer worse outcomes relative to patients with other forms of cutaneous melanoma (CM), and do not benefit as well to approved melanoma therapies. Identification of cyclin-dependent kinase 4 and 6 (CDK4/6) pathway gene alterations in >60% of ALMs has led to clinical trials of the CDK4/6 inhibitor (CDK4i/6i) palbociclib for ALM; however, median progression free survival with CDK4i/6i treatment was only 2.2 months, suggesting existence of resistance mechanisms. Therapy resistance in ALM remains poorly understood; here we report hyperactivation of MAPK signaling and elevated cyclin D1 expression serve as a mechanism of intrinsic early/adaptive CDK4i/6i resistance. ALM cells that have acquired CDK4i/6i resistance following chronic treatment exposure also exhibit hyperactivation of the MAPK pathway. MEK and/or ERK inhibition increases CDK4i/6i efficacy against therapy naïve and CDK4i/6i-resistant AM cells in xenograft and patient-derived xenograft (PDX) models and promotes a defective DNA repair, cell cycle arrested and apoptotic program. Notably, gene alterations poorly correlate with protein expression of cell cycle proteins in ALM or efficacy of CDK4i/6i, urging additional strategies when stratifying patients for CDK4i/6i trial inclusion. Concurrent targeting of the MAPK pathway and CDK4/6 represents a new approach for patients with metastatic ALM to improve outcomes.

Kinome profiling identifies MARK3 and STK10 as potential therapeutic targets in uveal melanoma

Most uveal melanoma cases harbor activating mutations in either GNAQ or GNA11. Despite activation of the mitogen-activated protein kinase (MAPK) signaling pathway downstream of Gαq/11, there are no effective targeted kinase therapies for metastatic uveal melanoma. The human genome encodes numerous understudied kinases, also called the “dark kinome”. Identifying additional kinases regulated by Gαq/11 may uncover novel therapeutic targets for uveal melanoma. In this study, we treated GNAQ-mutant uveal melanoma cell lines with a Gαq/11 inhibitor, YM-254890, and conducted a kinase signaling proteomic screen using multiplexed-kinase inhibitors followed by mass spectrometry. We observed downregulated expression and/or activity of 22 kinases. A custom siRNA screen targeting these kinases demonstrated that knockdown of microtubule affinity regulating kinase 3 (MARK3) and serine/threonine kinase 10 (STK10) significantly reduced uveal melanoma cell growth and decreased expression of cell cycle proteins. Additionally, knockdown of MARK3 but not STK10 decreased ERK1/2 phosphorylation. Analysis of RNA-sequencing and proteomic data showed that Gαq signaling regulates STK10 expression and MARK3 activity. Our findings suggest an involvement of STK10 and MARK3 in the Gαq/11 oncogenic pathway and prompt further investigation into the specific roles and targeting potential of these kinases in uveal melanoma.

Network Pharmacology Combined with Machine Learning to Reveal the Action Mechanism of Licochalcone Intervention in Liver Cancer

There are reports indicating that licochalcones can inhibit the proliferation, migration, and invasion of cancer cells by promoting the expression of autophagy-related proteins, inhibiting the expression of cell cycle proteins and angiogenic factors, and regulating autophagy and apoptosis. This study aims to reveal the potential mechanisms of licochalcone A (LCA), licochalcone B (LCB), licochalcone C (LCC), licochalcone D (LCD), licochalcone E (LCE), licochalcone F (LCF), and licochalcone G (LCG) inhibition in liver cancer through computer-aided screening strategies. By using machine learning clustering analysis to search for other structurally similar components in licorice, quantitative calculations were conducted to collect the structural commonalities of these components related to liver cancer and to identify key residues involved in the interactions between small molecules and key target proteins. Our research results show that the seven licochalcones molecules interfere with the cancer signaling pathway via the NF-κB signaling pathway, PDL1 expression and PD1 checkpoint pathway in cancer, and others. Glypallichalcone, Echinatin, and 3,4,3′,4′-Tetrahydroxy-2-methoxychalcone in licorice also have similar structures to the seven licochalcones, which may indicate their similar effects. We also identified the key residues (including ASN364, GLY365, TRP366, and TYR485) involved in the interactions between ten flavonoids and the key target protein (nitric oxide synthase 2). In summary, we provide valuable insights into the molecular mechanisms of the anticancer effects of licorice flavonoids, providing new ideas for the design of small molecules for liver cancer drugs.