Recombinant Human KDEL motif-containing protein 2 (KDELC2)

What is the molecular structure and function of KDELC2?

KDELC2 is a protein containing the KDEL (Lys-Asp-Glu-Leu) motif that appears to be associated with endoplasmic reticulum functions. Structurally, it contains regions that interact with components of cellular stress response pathways. Functionally, research indicates that KDELC2 plays roles in regulating ER stress, mitochondrial reactive oxygen species (ROS) production, and subsequent cellular responses that contribute to tumor progression. The protein has been shown to stimulate vasculogenic factor expression including ZEB2, VEGF-A, VEGF-R1, and PDGFA, which are crucial for angiogenesis . Methodologically, researchers typically assess KDELC2 function through knockdown or overexpression studies combined with molecular assays for downstream targets.

How does KDELC2 expression correlate with glioma classification?

KDELC2 expression demonstrates significant correlation with glioma tumor grade and molecular classification. Analysis of data from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) has revealed that KDELC2 mRNA expression is significantly higher in glioblastoma (GBM) compared to non-neoplastic brain tissue. Expression levels positively correlate with WHO tumor grades, with WHO grade IV tumors showing the highest expression (average score 76.18) compared to grade III (33.44), grade II (29.17), and grade I (0) . Additionally, KDELC2 expression is higher in the mesenchymal subtype than in classical and proneural subtypes of GBMs. Importantly, IDH wild-type gliomas across different grades consistently show higher KDELC2 expression than IDH-mutant tumors, and non-GCIMP (Glioma-CpG Island Methylator Phenotype) GBMs express higher levels than GCIMP GBMs .

What are the recommended techniques for manipulating KDELC2 expression in glioma models?

For effective KDELC2 manipulation in glioma research, RNA interference techniques using short hairpin RNA (shRNA) have been successfully employed. Specifically, shKDELC2#180 and #220 transfection has demonstrated efficient KDELC2 knockdown in both GBM8401 and U87MG cell lines, as confirmed by qRT-PCR quantification showing significant decrease in KDELC2 mRNA expression . For compensatory overexpression studies, recombinant OE-KDELC2 constructs have been utilized to reverse the effects of KDELC2 knockdown in experimental models. To verify successful manipulation, researchers should confirm changes in KDELC2 expression at both mRNA and protein levels through qRT-PCR and Western blotting respectively. For in vivo studies, orthotropic human glioblastoma xenograft mouse models using cells with manipulated KDELC2 expression have proven effective for studying its role in tumor angiogenesis and development .

How can researchers effectively evaluate KDELC2's impact on tumor angiogenesis?

To evaluate KDELC2's impact on angiogenesis, a multi-faceted approach combining molecular, cellular, and functional assays is recommended. At the molecular level, researchers should quantify angiogenic factors such as ZEB2, VEGF-A, VEGF-R1, and PDGFA through qRT-PCR after KDELC2 manipulation . At the cellular level, HUVEC tube formation assays provide valuable information on the functional consequences of KDELC2 expression; parameters to measure include branching and node growth when HUVECs are exposed to conditioned medium from glioblastoma cells with modified KDELC2 expression . Additionally, co-culture systems between glioblastoma cells and HUVECs can help assess direct cellular interactions. For in vivo evaluation, researchers can examine microvessel density in tumor xenografts through CD31 staining. The combined use of ER stress inhibitors (salubrinal and GSK2606414) and ROS scavengers (NAC and Mito-TEMPO) in these assays can further elucidate the mechanisms through which KDELC2 promotes angiogenesis .

What methodologies are most appropriate for investigating KDELC2's effects on tumor-associated macrophage polarization?

For investigating KDELC2's effects on tumor-associated macrophage (TAM) polarization, co-culture systems using THP-1 monocytic cells and glioblastoma cells with modified KDELC2 expression are recommended. To evaluate macrophage polarization, researchers should assess the expression of M1 markers (TNF-α, IL-12, CD38, IL-1β, IL-6) and M2 markers (IL-10) using qRT-PCR and ELISA . Flow cytometry for surface markers CD86 (M1) and CD163 (M2) provides additional characterization of macrophage phenotypes. Migration assays can help determine the chemoattractive potential of glioblastoma cells with different KDELC2 expression levels on macrophages. To understand the functional consequences of macrophage polarization, researchers should evaluate the conditioned medium from co-cultures on HUVEC proliferation and tube formation. For mechanistic insights, inhibitors targeting mitochondrial ROS (Mito-TEMPO) and the NLRP3 inflammasome (MCC950) can be employed to determine how these pathways mediate KDELC2-induced effects on macrophage polarization .

How does KDELC2 regulate cellular ROS and what downstream effects does this have on glioblastoma progression?

KDELC2 plays a critical role in regulating both cellular and mitochondrial reactive oxygen species (ROS) levels in glioblastoma cells. Research has demonstrated that KDELC2 knockdown significantly reduces intracellular and mitochondrial ROS levels in GBM8401 and U87MG cell lines . Mechanistically, this regulation appears to be linked to KDELC2's ability to modulate ER stress, as suppression of KDELC2 inhibits expression of ER stress factors including CHOP, PERK, sXBP1, ATF4, BIP, and EDEM1 . The downstream consequences of KDELC2-mediated ROS production are multifaceted. Elevated ROS levels lead to increased HIF-1α expression, which in turn upregulates angiogenic factors such as VEGF. When investigating this pathway, researchers should employ both cellular (DCF-DA) and mitochondrial (MitoSOX) ROS detection assays, and validate findings using ROS scavengers (NAC for cellular ROS; Mito-TEMPO for mitochondrial ROS). Immunofluorescence analysis for HIF-1α expression and Western blotting for ER stress markers provide additional mechanistic insights into how KDELC2-induced ROS promotes glioblastoma progression through angiogenesis and adaptation to nutrient-deprived conditions .

What is the relationship between KDELC2 expression and glioma stem cell properties?

KDELC2 expression significantly influences glioma stem cell (GSC) properties, which are associated with therapy resistance and high recurrence rates. Experimental evidence shows that KDELC2 knockdown in GBM8401 and U87MG cells results in smaller tumor spheroid formation compared to control cells after extended culture periods (11 and 17 days respectively) . At the molecular level, KDELC2 suppression downregulates multiple stemness factors. Immunofluorescence analysis and Western blotting reveal decreased expression of CD44 and OCT3/4 proteins in KDELC2-knockdown GBM cells . Additionally, qRT-PCR demonstrates significant downregulation of stemness-associated genes including CD133, SOX-2, Nanog, and pou5f1 following KDELC2 knockdown . These findings suggest that KDELC2 maintains or enhances stemness properties in glioblastoma cells, potentially contributing to therapeutic resistance and tumor recurrence. To investigate this relationship, researchers should employ 3D tumor spheroid formation assays, limiting dilution assays to assess self-renewal capacity, and comprehensive analysis of stemness markers at both protein and mRNA levels after KDELC2 manipulation.

How does KDELC2 influence cell cycle progression and apoptosis in glioblastoma cells?

KDELC2 plays a significant role in regulating cell cycle progression and apoptosis in glioblastoma cells. Flow cytometry analysis has shown that KDELC2 knockdown results in a significantly higher percentage of cells in the sub-G1 phase in both GBM8401 and U87 cell lines, indicating cell cycle delay . At the molecular level, Western blot analysis reveals that KDELC2 suppression enhances the expression of apoptotic markers caspase-3 and caspase-9, suggesting increased apoptotic activity . Additionally, KDELC2 knockdown downregulates multiple cell cycle checkpoints including cyclin-A2, cyclin-D1, cyclin-E2, and phosphorylated histone-3 . Conversely, KDELC2 overexpression promotes tumor proliferation by upregulating these cell cycle checkpoints. When investigating the role of KDELC2 in cell cycle regulation and apoptosis, researchers should employ flow cytometry with propidium iodide staining for cell cycle analysis, Annexin V/PI staining for apoptosis quantification, and Western blotting for key cell cycle and apoptotic proteins. These methodologies provide comprehensive insights into how KDELC2 modulates cellular proliferation and survival, contributing to glioblastoma aggressiveness.

What explains the varying strengths of correlation between KDELC2 expression and different molecular subtypes of glioma?

The varying strengths of correlation between KDELC2 expression and different molecular subtypes of glioma require careful interpretation. Data from TCGA and CGGA databases show that KDELC2 expression is significantly higher in IDH wild-type gliomas compared to IDH-mutant counterparts across multiple grades, with particularly strong differences in glioblastomas (average score 85.14 vs. 31.67) . Similarly, mesenchymal GBMs show higher KDELC2 expression than classical and proneural subtypes, and non-GCIMP GBMs express more KDELC2 than GCIMP GBMs . These variations may be explained by the underlying molecular pathways characteristic of each subtype. Mesenchymal GBMs typically display enhanced activation of inflammatory pathways and increased angiogenesis, aligning with KDELC2's role in promoting these processes. IDH mutations lead to production of 2-hydroxyglutarate, which affects epigenetic regulation and may suppress KDELC2 expression. The weaker correlation with MGMT methylation status suggests that KDELC2 functions independently of DNA repair mechanisms. When analyzing correlations between KDELC2 and molecular subtypes, researchers should employ multivariate statistical approaches to account for potential confounding factors and examine larger cohorts with comprehensive molecular characterization. Integration of multi-omics data (transcriptomics, methylomics, proteomics) can provide deeper insights into the regulatory networks controlling KDELC2 expression in different glioma subtypes.

What are the most promising therapeutic approaches targeting KDELC2 pathways in glioblastoma?

Based on current understanding of KDELC2's mechanisms, several therapeutic approaches warrant investigation. RNA interference strategies targeting KDELC2 have shown promise in preclinical models, with shKDELC2 significantly reducing angiogenesis, invasion, and stemness properties in glioblastoma cells . For clinical translation, development of siRNA delivery systems capable of crossing the blood-brain barrier represents a key research priority. Given KDELC2's role in promoting ER stress, combining KDELC2 inhibition with ER stress modulators like salubrinal and GSK2606414 may yield synergistic effects . Similarly, targeting downstream ROS production using mitochondria-specific antioxidants such as Mito-TEMPO could complement KDELC2 inhibition . The finding that KDELC2 influences tumor-associated macrophage polarization suggests that combinatorial approaches with immunomodulatory agents may be effective . When designing such studies, researchers should employ multiple glioblastoma models including patient-derived xenografts and syngeneic mouse models with intact immune systems to evaluate both direct anti-tumor effects and immunomodulatory outcomes. Methodologically, comprehensive assessment should include tumor growth kinetics, survival analysis, angiogenesis quantification, and characterization of the tumor immune microenvironment before and after treatment.

How might KDELC2 expression and function differ in other cancer types beyond glioblastoma?

While current research has focused primarily on KDELC2's role in glioblastoma, investigating its expression and function in other cancer types represents an important future direction. The mechanisms identified in glioblastoma—including promotion of angiogenesis, ROS production, ER stress modulation, and influence on tumor-associated macrophages—may be relevant across multiple malignancies, particularly those characterized by aggressive vascularization and inflammatory microenvironments . To explore this, researchers should first conduct comprehensive bioinformatic analyses of KDELC2 expression across cancer datasets such as TCGA and correlate expression with clinical outcomes. Cancer types showing significant KDELC2 overexpression should then be prioritized for functional studies. Methodologically, comparative analyses using cell lines from different cancer types with KDELC2 knockdown or overexpression would help identify common and tissue-specific effects. Particular attention should be paid to cancers with similar molecular characteristics to glioblastoma, such as IDH status and inflammatory signatures. Additionally, examination of KDELC2's role in cancer stem cell properties across different tumor types may reveal common stemness maintenance mechanisms that could be therapeutically targeted.

What methodological advances are needed to better understand KDELC2's protein interactions and post-translational modifications?

Current research has primarily focused on KDELC2's expression patterns and downstream effects rather than its direct protein interactions and regulatory mechanisms. To advance understanding, several methodological approaches should be pursued. Immunoprecipitation followed by mass spectrometry (IP-MS) would identify KDELC2's protein binding partners in glioblastoma cells, potentially revealing novel regulatory mechanisms and therapeutic targets. For structural studies, X-ray crystallography or cryo-electron microscopy of purified recombinant KDELC2 would provide insights into functional domains and potential drug binding sites. Investigation of post-translational modifications using phosphoproteomics, glycoproteomics, and ubiquitination profiling would elucidate how KDELC2 activity is regulated in different cellular contexts. Development of specific antibodies capable of distinguishing between modified forms of KDELC2 would facilitate these studies. To understand dynamic regulation, CRISPR-based transcriptional reporters and fluorescently tagged KDELC2 constructs would enable real-time monitoring of expression and localization in living cells. These methodological advances would provide a more comprehensive understanding of KDELC2 biology, potentially revealing new vulnerabilities for therapeutic exploitation in glioblastoma and other cancers.