Anterior gradient protein 2 (AGR2), a member of the protein disulfide isomerase family, plays a critical role in endoplasmic reticulum proteostasis and has been implicated in breast cancer progression. However, the downstream regulatory programs and signaling pathways governed by AGR2 remain incompletely defined. Here, we employed CRISPR-Cas9-mediated knockout of AGR2 in breast cancer cells to systematically investigate the functional and transcriptional consequences of AGR2 loss. AGR2 depletion resulted in significant suppression of cell migration, invasion, and chemoresistance. Unbiased transcriptomic profiling by RNA sequencing revealed extensive differential gene expression, implicating AGR2 in receptor-mediated signaling, oxidative stress responses, and cell adhesion pathways. Protein-protein interaction network analysis identified several highly connected hub genes within the AGR2-regulated transcriptome, including estrogen receptor alpha (ESR1/ERα), cadherin 1 (CDH1), androgen receptor (AR), lymphocyte cell-specific protein-tyrosine kinase (LCK), S100 calcium binding protein P (S100P), parkin RBR E3 ubiquitin ligase (PRKN), and decay-accelerating factor (CD55). Notably, ERα emerged as a prominent node within this network, consistent with prior reports linking ERα and AGR2 biology. Integration with publicly available epigenomic datasets further supports a potential regulatory connection between ERα-associated chromatin landscapes and AGR2 expression. Together, these findings define AGR2-dependent transcriptional networks in breast cancer and identify ESR1-associated signaling as a key pathway perturbed upon AGR2 loss, providing a foundation for future mechanistic studies targeting this regulatory interaction.

Super enhancers (SEs) are involved in regulating cell identity and lineage‑specific gene expression, and drive cancer‑associated gene expression. The transcription factor Kruppel‑like factor 6 (KLF6) promotes the growth and progression of clear cell renal cell carcinoma (ccRCC), with its high expression driven by one of the strongest SEs in ccRCC. However, the mechanisms that establish and maintain KLF6 SE activity, particularly the roles of epigenetic regulators bromodomain-containing 4 (BRD4) and p300, remain poorly understood. This study investigated the roles of BRD4 and p300 in modulating KLF6 SE activity. The effects of JQ1‑mediated BRD4 and A‑485‑mediated p300 inhibition were assessed using cell viability and colony formation assays. Reverse transcription‑quantitative (q)PCR and ChIP‑qPCR were employed to evaluate the impact of BRD4 and p300 inhibition, as well as CRISPR‑mediated deacetylation of individual constituent enhancers, on KLF6 expression and SE activity. Chemical inhibition of BRD4 and p300 significantly reduced ccRCC cell viability and colony formation, and decreased KLF6 expression and levels of acetylation at lysine 27 of histone H3 (H3K27ac) at KLF6 enhancer regions SE_1, SE_2 and SE_3, suggesting decreased chromatin accessibility. On the other hand, deacetylation of SE_1 using dead Cas9 fused to histone deacetylase 3, led to KLF6 downregulation, which was associated with decreased H3K27ac signals at this region. The present results demonstrated that BRD4 and p300 are key for maintaining KLF6 SE activity and driving high KLF6 expression in ccRCC. However, deacetylation of  individual enhancer regions using CRISPR was insufficient to fully suppress KLF6 transcription, emphasizing the robustness of the KLF6 SE and its modular role in sustaining high KLF6 expression. Overall, the present study deepens the understanding of growth‑promoting KLF6 transcriptional networks in ccRCC and offers insights to support the development of diagnostic or therapeutic strategies.

Esophageal cancer is a formidable malignancy, presenting a significant health challenge due to its widespread prevalence and associated high mortality rates. Epithelial cell adhesion molecule (EpCAM), a pro-oncogenic glycoprotein, has been identified as an upregulated protein in esophageal adenocarcinoma (ESCA) through multi-OMICS platforms. However, its functional role in ESCA remains relatively understudied. Here, we investigated the contribution of EpCAM to ESCA pathogenesis using an EpCAM-null ESCA cell line, FLO-1, as a gain-of-function model. Introduction of a recombinant EpCAM-GFP fusion construct into FLO-1 cells resulted in enhanced cell migration, adhesion, clonogenic survival, and invasive capacity, supporting a pro-tumorigenic role for EpCAM. To define EpCAM-associated regulatory networks, RNA sequencing was performed on EpCAM-overexpressing cells, revealing 797 differentially expressed genes. Functional enrichment analyses indicated significant involvement of pathways related to cell adhesion, cell motility, transmembrane activity, and neuronal-associated processes, with enrichment in plasma membrane, focal adhesion, and neuron projection terminus compartments. Protein-protein interaction network analysis identified key hub genes, including SOX2, COL1A1, LOX, COL3A1, LUM, PXDN, BDNF, NCAM1, TLR2, and CCL5, linking EpCAM signaling to PI3K-Akt, ECM-receptor interaction, and focal adhesion pathways. Importantly, quantitative polymerase chain reaction (qPCR) validation of selected hub genes confirmed significant upregulation of the extracellular matrix components COL1A1 and PXDN in EpCAM-overexpressing FLO-1 cells, supporting the transcriptomic predictions and implicating ECM remodeling as a downstream consequence of EpCAM signaling. Collectively, these findings demonstrate that EpCAM promotes aggressive cellular phenotypes in ESCA and drives transcriptional programs associated with adhesion, invasion, and extracellular matrix regulation, highlighting potential therapeutic vulnerabilities in EpCAM-driven ESCA.

Cell division control protein 42 homolog (Cdc42) is a small Rho GTPase that cycles between active GTP-bound and inactive GDP-bound states to regulate cytoskeletal dynamics and immune signalling. Pathogenic variants of Cdc42 have been linked to rare immunological disorders, including a recently described C81Y mutation associated with primary immunodeficiency (PID) and Hodgkin lymphoma. Although C81Y shares phenotypic similarities with the non-oncogenic C81F variant, it is uniquely associated with thrombocytopenia, chronic inflammation, and malignancy, suggesting distinct molecular consequences that remain poorly characterised. In this study, we first assessed the impact of the C81Y mutation expressed in HEK293T cells using biochemical approaches. Cdc42C81Y exhibited reduced interaction with canonical downstream effectors, particularly WASp and N-WASp, indicating attenuation of classical Cdc42 signalling outputs. To gain mechanistic insight into these biochemical findings, we next applied molecular modelling approaches. Computational analyses revealed that the C81Y mutation alters nucleotide-dependent conformational dynamics, stabilizes the GDP-bound state, and modifies surface electrostatic properties, consistent with impaired effector engagement. Functional assays in HEK293T cells demonstrated that Cdc42C81Y enhanced cellular migration without significantly affecting proliferation and was associated with increased secretion of IL-6 and IL-10. Together, these data suggest that the Cdc42C81Y mutation disrupts canonical Cdc42 signalling while promoting context-dependent cellular and inflammatory responses. This study provides mechanistic insight into how Cdc42C81Y may contribute to PID-associated malignancy and underscores its role at the intersection of cytoskeletal regulation and immune dysregulation.

Although purine metabolism is one of the most impacted pathways in colorectal cancer (CRC), little is known about the role of equilibrative nucleoside transporter 2 (ENT2) in CRC development and its association with the altered purine metabolism pathway. This study aimed to determine the role of ENT2 in altered purine metabolism in the early and late stages of CRC using CRISPR/Cas9 gene editing tools and a variety of functional experiments. The expression of ENT2 was significantly higher (P < 0.001) in all CRC cell lines as compared to the normal colon cells. The two CRC cell lines with the highest ENT2 expression, the early stage HT29 cells and the late stage DLD1 cells, were knocked out (KO) using the CRISPR/Cas9 tool. The hypoxanthine (HPX) level and the xanthine oxidase (XO) activity were significantly higher in both HT29/KO and DLD1/KO single cell-derived clones (P < 0.01). The increase in HPX level and XO activity were associated with an elevation in the reactive oxygen species (ROS) level. These data suggest that the ENT2 KO elevated the ROS levels induced apoptosis and impaired the cell proliferation of the early stage of CRC cell line, i.e., HT29/KO clonal cells. In this context, targeting ENT2 gene might be a potential strategy in CRC treatment by increasing the production of ROS and hence, inducing the apoptosis pathway.

MicroRNAs (miRNAs) contribute to the progression of vascular complications in diabetes. Metformin whether this effect was mediated through the modulation of miRNAs. To gain insight into the effects of metformin on miRNA profile in endothelial cells treated with high glucose, we utilised next gene sequencing (NGS) and further conducted the bioinformatic analyses to explore the associated are crucial in mediating the involvement of metformin in diabetes-induced oxidative stress and to diabetes-related pathways, such as type II diabetes mellitus and phosphatidylinositol signaling These findings could help develop more targeted therapeutic interventions for diabetes.

Kemunculan teknologi penyuntingan genom, terutamanya CRISPR/Cas9, telah mengubah dan merevolusikan landskap bidang genetik dan biologi molekul dengan begitu drastik. Sistem CRISPR/Cas9 diadaptasi daripada sistem adaptasi imuniti bakteria, menggunakan enzim Cas9 yang dipandu oleh sgRNA untuk penyingkiran gen atau penyuntingan genom secara jitu. Walau bagaimanapun, cabaran seperti kesan luar sasaran (off-target effects) telah mendorong pembangunan beberapa varian Cas9 seperti dCas9. dCas9 diubah suai untuk tidak memiliki sebarang aktiviti endonuklease dan kini dCas9 telah digunakan untuk pelbagai aplikasi yang melangkaui fungsi tradisional CRISPR/Cas9 sebagai kaedah pengeditan genom. Selain itu, teknologi terkini prime editing telah menggabungkan enzim Cas9 yang diubahsuai bersama reverse transcriptase untuk meningkatkan lagi tahap keberkesanan penyuntingan genom secara jitu. Walaupun wujud kerisauan terhadap pertimbangan etika dan kebimbangan terkait keselamatan, namun teknologi ini menjanjikan dampak yang besar di dalam menangani penyakit genetik serta aplikasi dalam bidang precision medicine. Memahami dan mengoptimumkan potensi CRISPR/Cas9 dan teknologi prime editing menandakan bermula era baharu dalam bidang penyelidikan berkaitan biologi dan perubatan, dan seterusnya menyediakan satu platform untuk penyuntingan genom yang tepat dan pengawalseliaan proses transkripsi gen.

Managing women with diminished ovarian reserve for in vitro fertilization (IVF) is challenging, often resulting in low oocyte yield and cycle failures. We hypothesize that coupling in vitro fertilization (IVF) with rescue in vitro maturation (r-IVM) can improve overall maturation rates without compromising the overall IVF outcome. Our study compared DOR and normal ovarian reserve (NOR) cohorts by evaluating 15 immature oocyte progressions following r-IVM. We analyzed the gene expression of cumulus cells related to GREM1, PTGS2, and HAS2 to correlate with OQ, EQ, and overall IVF outcome. Significant differences were noted in AMH levels, AFCs, and oocyte numbers (p < 0.05). Following r-IVM, the DOR cohort achieved a 50% maturation rate with improved overall quality; however, the difference was not statistically significant (p > 0.05). Fertilization rates were comparable, but EQ was better in DOR. All genes in DOR were upregulated post-r-IVM, whereas NOR showed downregulation of PTGS2 and GREM1 (p < 0.05). Otherwise, DOR exhibited higher pregnancy rates and live birth rates, although the difference was not statistically significant (p > 0.05). Overall, our findings suggest that r-IVM could provide improved fertility outcomes for DOR women in standard IVF cycles.

Offspring of mothers with hypertensive disorders of pregnancy (HDP) are at increased risk of developing endothelial dysfunction and cardiovascular disease (CVD) in adulthood. MicroRNAs (miRNAs), as key regulators of endothelial cells, may contribute to the early onset of endothelial dysfunction. However, there are limited studies characterizing the miRNA profile of endothelial cells in offspring of HDP. Therefore, this study aims to determine the miRNA expression profile of human umbilical vein endothelial cells (HUVECs) isolated from the offspring of HDP. HUVECs were obtained from both normal and hypertensive umbilical cords. RNA sequencing analysis revealed that eight miRNAs were significantly upregulated in HUVECs from HDP (p < 0.05). The target genes of these miRNAs were then predicted using four databases: miRDB, TargetScan, DIANA-microT-CDS, and miRWalk. Gene ontology, pathway enrichment, and protein-protein interaction network analyses revealed that the target genes of these miRNAs are involved in cellular functions and pathways related to angiogenesis and cellular senescence, which may contribute to endothelial dysfunction and CVD. The most significantly upregulated miRNA, hsa-miR-196a-5p expression was then validated through stem-loop RT-qPCR where its expression was significantly upregulated in hypertensive HUVEC by 6-fold as compared to normal HUVEC (p < 0.01). These findings offer insights into the role of miRNAs in the development of CVD in offspring exposed to HDP, highlighting their potential as predictive markers and therapeutic targets in the future.

Obesity is a major global health problem and increases the risk of various chronic diseases, such as cancer and type 2 diabetes. Understanding the genetic factors contributing to obesity is essential for developing effective intervention strategies. Thus, the study aimed to identify obesity-associated genetic loci in the Malaysian youth population. We conducted a genome-wide association analysis of 203 Malaysian youths (18-30 years of age) to identify susceptibility loci associated with body mass index-based obesity using a genotyped dataset imputed from a list of Asian-specific, obesity-associated loci, and the 1000 Genomes data. Statistical analysis, including linear regression and case-control association, was conducted to determine the association between single nucleotide polymorphisms (SNPs) and obesity. While no single SNP achieved genomewide significance (p = 5x10(-8)), 34 SNPs exhibited suggestive significance (p <= 5x10(-5)). Of note, three chromosomal regions stood out (3q29, 7p11.2 and 17p13.1), harboring multiple suggestive significant associated SNPs. Although we were not able to identify the precise mechanisms and/or metabolic pathways underlying obesity in this particular target population, the study advances our knowledge of the genetic aspects of obesity in a Malaysian multi-ethnic population, which provides a baseline dataset for future studies.

Our study aims to consolidate appropriate cumulus cells (CCs) collection methods by comparing sample handling obtained from diminished ovarian reserved (DOR) and normal ovarian reserved (NOR) women, aiming for a good yield of ribonucleic acid (RNA) for better gene expression analysis. We compared the sample collection by mechanical versus enzymatic method, standard rotor-stator homogeniser (RSH) versus disposable easy grind homogeniser for sample preparation technique and subsequently the cDNA synthesis and qPCR for final evaluation. We also conducted a systematic review to consolidate our findings with current evidence of RNA extraction technique. The optimal strategy was via mechanical denudation without an enzymatic process, followed by RNA stabiliser prior to RNA extraction. Subsequently, utilising a manual pressure control with filter, is paramount for a pure and homogenised sample and comparable for both NOR and DOR women CCs. These sample preparation and extraction techniques yielded an optimum RNA concentration and successful gene expression analysis. Our review also added value as a current strategy for optimising RNA extraction in CCs for experimental studies. Our findings contribute to formulating a better strategy for optimising RNA extraction in human CCs for transcriptomic studies.

Background/Objectives: Diminished ovarian reserve (DOR) poses significant challenges in the reproductive field, resulting in fewer mature and more low-quality eggs. Methods: We studied r-IVM in addition to standard in vitro fertilization (IVF) and compared the embryological outcomes between both DOR and NOR women. Results: We recruited 90 women (45 NOR; 45 DOR) with a younger age seen in NOR (35.2 vs. 36.5 years old) women. Otherwise, DOR women had lower levels of AMH and AFC, thus fewer retrieved follicles and collected oocytes. Most of the group presented with primary subfertility, with 55.6% in the NOR group diagnosed with polycystic ovary syndrome (PCOS), while 37.8% in the DOR group presented with aging and cancer survivorship issues. Most women in the NOR group used hCG as a trigger (82.2%), while 17.8% of the DOR group opted for a decapeptide. A total of 719 oocytes were retrieved, with 72.3% of eggs being mature in the NOR group compared to 64.9% in the DOR group. Following r-IVM, 47.69% of NOR eggs were matured compared to 60% in DOR eggs. The fertilization rates (FRs) following r-IVM were higher in the DOR group (66.7% vs. 37.8%). Overall, higher numbers and quality of D3 embryos were seen in the DOR group. Our analysis revealed that the trigger type, hCG, was the only significant factor linked to successful oocyte maturation rates. Conclusions: Our study suggests that r-IVM may enhance outcomes for women with DOR, including better egg maturity, FR, and embryo quality than NOR women.

An overall in vitro fertilization (IVF) outcome is dynamic. Good oocyte quality is important for achieving a better IVF pregnancy. Various methods have been used for OQ assessment (OQA). However, due to inconclusive evidence, one method’s superiority has not been proposed over others. Thus, OQA should be consolidated based on the current evidence for this matter. Our study aims to summarize the current OQA used and compare their recommended methods. Our results revealed that the optimal OQA for cost-effectiveness and clinical implementation is still via morphological assessment. The current method most opted for is total oocyte scoring. Although other methods—follicular fluid evaluation, mitochondria spindle assessment, or live zona imaging—can be applied with better precision, due to cost, additional tools, and expertise, and they should cater to research purposes and selected centers. Our results added value as a current strategy for optimizing OQA in IVF practice, aiming at cost-effectiveness and reproducibility for better clinical implementation.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing is evolving into an essential tool in the field of biological and medical research. Notably, the development of catalytically deactivated Cas9 (dCas9) enzyme has substantially broadened its traditional boundaries in gene editing or perturbation. The conjugation of dCas9 with various molecular effectors allows precise control over transcriptional processes, epigenetic modifications, visualization of chromosomal dynamics, and several other applications. This expanded repertoire of CRISPR-Cas9 applications has emerged as an invaluable molecular tool kit that empowers researchers to comprehensively interrogate and gain insights into health and diseases. This review delves into the advancements in Cas9 protein engineering, specifically on the generation of various dCas9 tools that have significantly enhanced the CRISPR-based technology capability and versatility. We subsequently discuss the multifaceted applications of dCas9, especially in interrogating the regulation and function of genes that involve in supporting cancer pathogenesis. In addition, we also delineate the designing and utilization of dCas9-based tools as well as highlighting its current constraints and transformative potentials in cancer research.

Aim: Rs16851030, a single-nucleotide variant located in the 3 '-untranslated region of the ADORA1 gene, has been proposed as a potential marker of caffeine sensitivity in apnea of prematurity. Besides, it is associated with aspirin-induced asthma and the development of acute chest syndrome. However, its functional significance is still unconfirmed. This study aimed to elucidate the functional impact of rs16851030 by using CRISPR/Cas9 approach to induce the DNA variant and attendant physiological changes.Methods: Rs16851030 was introduced into HEK293 cells via homology-directed repair (HDR). Edited cells were fluorescence-enriched, sorted, isolated, and expanded into single-cell-derived clones. The edit was confirmed by Sanger sequencing. RNA sequencing was used to analyze affected pathways.Results: Rs16851030-mutant cells showed increased susceptibility to hypoxia, a condition related to apnea of prematurity. After 24 h of hypoxia, the viability of mutant clones 1 and 2 was low compared with wild-type cells (75.45% and 74.47% vs. 96.34%). RNA sequencing revealed transcriptomic changes linked to this increased vulnerability.Conclusion: Rs16851030 impairs cellular resistance to hypoxia, suggesting its role in conditions like apnea of prematurity. Further research should investigate the molecular mechanisms and transcriptomic alterations caused by rs16851030 under hypoxic conditions.

The leading indicator for successful outcomes in in-vitro fertilization (IVF) is the quality of gametes in oocytes and sperm. Thus, advanced research aims to highlight the parameter in assessing these qualities – DNA fragmentation in sperm and oocyte development capacity (ODC) via evaluation of microenvironments involving its maturation process. Regarding oocytes, most evidence reveals the role of cumulus cells as non-invasive methods in assessing their development competency, mainly via gene expression evaluation. Our review aims to consolidate the evidence of GDF-9 derivatives, the HAS2, GREM1, and PTGS2 gene expression in cumulus cells used as ODC markers in relevant publications and tailored to current IVF outcomes. In addition to that, we also added the bioinformatic analysis in our review to strengthen the evidence aiming for a better understanding of the pathways and cluster of the genes of interest - HAS2, GREM1, and PTGS2 in cumulus cell level. Otherwise, the current non-invasive method can be used in exploring various causes of infertility that may affect these gene expressions at the cumulus cell level. Nevertheless, this method can also be used in assessing the ODC in various cohorts of women or as an improvement of markers following targeted tools or procedures by evaluating the advancement of these gene expressions following the targeted intervention.

Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an apoptosis inducer that exhibits an ideal therapeutic safety profile with less adverse effects than conventional chemotherapy. However, the occurrence of TRAIL resistance has been reported in various cancers including colorectal cancer (CRC). Substantial efforts have been channelled towards managing TRAIL resistance including identifying molecular targets. Interleukins (ILs) have been recently shown to play critical roles in modulating TRAIL sensitivity in cancer cells. This study investigated the roles of two ILs, IL-8 and IL⍺, in TRAIL resistance in CRC. TRAIL-resistant HT-29 and TRAIL-sensitive HCT 116 cells, were treated with human recombinant IL-8 and IL-1⍺. The results indicated that treatment with IL-8 (2.5 ng/mL) significantly protected TRAIL-sensitive HCT 116 cells from TRAIL-induced cell death (p < 0.05). However, IL-1⍺ did not play a role in modulating CRC cells’ responses to TRAIL. Data from RT-qPCR and Western blotting revealed the molecular regulations of IL-8 on TRAIL decoy receptor genes (OPG) and autophagy-related genes (BECN1 and LC3B) expression. The activation of the phosphoinositide 3-kinase (PI3K) pathway was shown to counteract TRAIL-induced cell death. By inhibiting its activation with wortmannin, the protective role of IL-8 against TRAIL treatment was reversed, suggesting the involvement of the PI3K pathway. Collectively, findings from this study identified the role of IL-8 and PI3K in modulating CRC cells’ sensitivity to TRAIL. Further validation of these two potential molecular targets is warranted to overcome TRAIL resistance in CRC.

RNA extraction from human cumulus cells (CCs) is considered challenging due to low cell’s pop-ulation and small cell. The successful gene expression analysis from cumulus cells ultimately de-pends on an excellent concentration of Ribonucleic acid (RNA) yield by an appropriate technique. Our study aims to consolidate appropriate cumulus cell collection method, preparation and ex-traction techniques to ensure a good yield of RNA for better gene expression analysis. From the results obtained, the optimal strategy found involved mechanical denudation without an enzy-matic process, followed by the use of RNA stabilizer prior to RNA extraction. Subsequently, uti-lizing a smaller homogenizer, preferably manual pressure control with filter (Bio-Masher III®) is paramount for a pure and homogenize sample. Subsequently, during RNA extraction, the use of RNeasy micro kit and RNA carriers is recommended. These sample collection, preparation, and extraction techniques yielded an excellent-quality RNA concentration and successful gene expres-sion analysis. We also conducted a systematic review to consolidate our findings with current evidence of RNA extraction technique. Our results added a value as a current strategy for opti-mizing RNA extraction in CCs for experimental studies. Thus, our findings can contribute to for-mulating a better strategy for optimizing the RNA extraction in CCs for transcriptomic studies.

Accounting for approximately 75% of all renal cancer cases, clear cell renal cell carcinoma (ccRCC) is characterized by the bi-allelic inactivation of the VHL tumour suppressor gene, leading to the abnormal stabilization of hypoxia-inducible factor alpha (HIFα).1Hsieh J.J. Purdue M.P. Signoretti S. et al.Renal cell carcinoma.Nat Rev Dis Primers. 2017; 317009https://doi.org/10.1038/nrdp.2017.9Crossref PubMed Scopus (1588) Google Scholar Of the two major HIFα subunits, HIF2α is responsible for driving ccRCC growth while HIF1α may exhibit suppressive effects on ccRCC progression.2Shen C. Kaelin W.G. The VHL/HIF axis in clear cell renal carcinoma.Semin Cancer Biol. 2013; 23: 18-25https://doi.org/10.1016/j.semcancer.2012.06.001Crossref PubMed Scopus (288) Google Scholar Moreover, ccRCC has widespread genetic heterogeneities with VHL inactivation being the truncal event observed in ∼90% of sporadic ccRCC cases.3Turajlic S. Xu H. Litchfield K. et al.Deterministic evolutionary trajectories influence primary tumor growth: TRACERx renal.Cell. 2018; 173: 595-610.e11https://doi.org/10.1016/j.cell.2018.03.043Summary Full Text Full Text PDF PubMed Scopus (390) Google Scholar Another distinctive feature of ccRCC is the accumulation of lipid droplet (LD), predominantly in the form of cholesteryl ester (CE) and triglycerides.4Gebhard R.L. Clayman R.V. Prigge W.F. et al.Abnormal cholesterol metabolism in renal clear cell carcinoma.J Lipid Res. 1987; 28: 1177-1184Summary Full Text PDF PubMed Google Scholar,5Saito K. Arai E. Maekawa K. et al.Lipidomic signatures and associated transcriptomic profiles of clear cell renal cell carcinoma.Sci Rep. 2016; 628932https://doi.org/10.1038/srep28932Crossref Scopus (88) Google Scholar Notably, aberrant stabilization of HIFα has been implicated in driving the LD formation in ccRCC by regulating the expression of PLIN2 and CPT1A.6Qiu B. Ackerman D. Sanchez D.J. et al.HIF2α-dependent lipid storage promotes endoplasmic reticulum homeostasis in clear-cell renal cell carcinoma.Cancer Discov. 2015; 5: 652-667https://doi.org/10.1158/2159-8290.CD-14-1507Crossref PubMed Scopus (258) Google Scholar,7Du W. Zhang L. Brett-Morris A. et al.HIF drives lipid deposition and cancer in ccRCC via repression of fatty acid metabolism.Nat Commun. 2017; 8: 1769https://doi.org/10.1038/s41467-017-01965-8Crossref PubMed Scopus (272) Google Scholar Despite these findings, the pathological relevance and molecular mechanisms underpinning lipid accumulation in ccRCC, specifically with respect to CE, remains elusive. In the latest issue of eBioMedicine,8Zhang S. Fang T. He Y. et al.VHL mutation drives human clear cell renal cell carcinoma progression through PI3K/AKT-dependent cholesteryl ester accumulation.eBioMedicine. 2024; https://doi.org/10.1016/j.ebiom.2024.105070Summary Full Text Full Text PDF Google Scholar Zhang et al. leveraged label-free Raman spectromicroscopy to elucidate the mechanisms underpinning lipid accumulation in ccRCC. The utilisation of Raman spectromicroscopy enables concurrent quantitative analysis of LD distribution and composition, making it a robust approach for studying lipid metabolism. By employing this method in tandem with validation via liquid chromatography-mass spectrometry, the authors observed aberrant LD accumulation, predominantly in the form of CE, in ccRCC tissue samples as compared to their normal adjacent counterparts. This observation resonates with previous reports on the high accumulation of CE in ccRCC. Intriguingly, CE accumulation was absent in a panel of commonly used ccRCC cell lines, prompting scrutiny regarding their suitability as models for studying CE accumulation in ccRCC. Instead, primary ccRCC cells were identified as an appropriate model due to their high abundance of CE, which was similar to levels detected in the source tissues. Nonetheless, careful experimental design is essential when employing these primary ccRCC cells due to the observed reduction in LDs and CE levels following cryopreservation and prolonged culture time. Further investigation revealed that the VHL-mutated primary ccRCC cells had significantly higher CE levels than those without the VHL mutation. The reintroduction of wild-type VHL into the VHL-mutated primary ccRCC cells, which induced HIFα degradation, led to a significant reduction in both the LD and CE levels. The shRNA-mediated knockdown of either HIF1α or HIF2α reinforced the notion that the accumulation of CE in ccRCC was primarily driven by HIF2α. These findings underscore the role of the VHL-HIFα pathway in regulating CE metabolism and accumulation in ccRCC. Mechanistically, the stabilization of HIF2α in VHL-mutated ccRCC cells resulted in the upregulation of PI3K/AKT/mTOR/SREBP pathway that increased cholesterol uptake and subsequent cholesterol esterification to CE for storage into LDs. These findings echo previous study that linked HIF2α with the activation of the PI3K/AKT/mTOR/SREBP pathway. This connection was demonstrated to be mediated by the KLF6-PDGFβ axis, which is crucial for cholesterol homeostasis and supporting ccRCC growth and progression.9Syafruddin S.E. Rodrigues P. Vojtasova E. et al.A KLF6-driven transcriptional network links lipid homeostasis and tumour growth in renal carcinoma.Nat Commun. 2019; 10: 1152https://doi.org/10.1038/s41467-019-09116-xCrossref PubMed Scopus (57) Google Scholar Moreover, chemical inhibition of cholesterol esterification using Avasimibe significantly depleted LD and CE levels and importantly, impaired ccRCC growth in vivo with no detectable signs of toxicity. These observations corroborate the functional significance of CE in supporting ccRCC pathogenesis. Furthermore, whole transcriptomic analysis of the Avasimibe-treated primary ccRCC cells identified several genes whose expression were dependent on membrane cholesterol levels, namely, ITGA6, ITGB1 and CAV1. It was proposed that the downregulation of these genes, upon Avasimibe-mediated CE depletion, contributed to the impaired ccRCC growth. Collectively, Zhang et al., provided important insights into the role of the ccRCC initiating-VHL-HIFα pathway in driving the enigmatic CE accumulation in this cancer type. Remarkably, the identification of an appropriate ccRCC in-vitro model for studying CE accumulation could advance this area of research and pave the way for more significant discoveries in the future. Given the dependency of ccRCC growth on cholesterol levels and functions, targeting cholesterol metabolism is considered to be a promising therapeutic option for ccRCC. Moving forward, it is imperative for future studies to comprehensively map the molecular networks governing cholesterol metabolism in ccRCC. A deeper understanding of these underlying mechanisms would help to identify other potential ccRCC "Achilles' heels". Such insights are beneficial for the development of robust treatment strategies for ccRCC. Literature search: M.A.M., S.N.H.M.Y., and S.E.S.; Data collection: M.A.M., S.N.H.M.Y., and S.E.S.; Data interpretation: M.A.M., and S.E.S.; Writing: M.A.M., and S.E.S. All authors read and approve the final manuscript. The authors declare no conflict of interest. This study was funded by Geran Universiti Penyelidikan (GUP), Universiti Kebangsaan Malaysia, GUP-2023-005. VHL mutation drives human clear cell renal cell carcinoma progression through PI3K/AKT-dependent cholesteryl ester accumulationCollectively, our study improves current understanding of the role of CE accumulation in ccRCC and opens up new opportunities for treatment. Full-Text PDF Open Access