RCN1 Human

What is RCN1 and what are its primary functions?

RCN1 (reticulocalbin-1) is an endoplasmic reticulum (ER)-resident calcium-binding protein containing six conserved regions that enable calcium-binding activity. Its primary functions include maintaining intracellular calcium homeostasis, regulating ER stress responses, and participating in protein folding and quality control within the ER lumen. RCN1 plays important roles in cellular processes including proliferation, apoptosis, and migration across various cell types. The protein contains a signal peptide that directs it to the ER, where it predominantly resides, though studies have demonstrated it can be secreted into extracellular environments under specific conditions . Its calcium-binding properties are essential for maintaining proper ER function and calcium-dependent signaling pathways throughout the cell.

What experimental models are available for studying RCN1 function?

Several experimental models have been established to investigate RCN1 function across different biological contexts. Cell culture models include human cancer cell lines such as MOLM-13, NB4, OCI/AML3, and THP-1 for AML research; DU145 and LNCaP for prostate cancer studies; and Cal-27 and SCC-25 for oral cancer research . Genetic manipulation of RCN1 has been achieved through CRISPR/Cas9 technology to establish knockout cell clones (e.g., MOLM-13-23, MOLM-13-240, and MOLM-13-242) and through lentiviral shRNA vectors for gene knockdown . Animal models include xenograft models using immunodeficient mice (NCG and NSG strains) for studying tumor growth after RCN1 manipulation . Additionally, mouse models with Rcn1 gene deletion have been developed to examine RCN1's role in normal hematopoiesis versus leukemic cell survival. For phagocytosis studies, specialized phage display systems expressing RCN1 have been constructed to evaluate RCN1's role in microglial function .

How does RCN1 knockdown affect cell death mechanisms in different cancer types?

RCN1 knockdown induces distinct cell death mechanisms depending on the cancer type and cellular context. In acute myeloid leukemia (AML), RCN1 downregulation activates pyroptosis, a highly inflammatory form of programmed cell death. This process involves upregulation of type I interferon (IFN-1) through STING pathway activation, which subsequently increases expression of cleaved-caspase-1 and interleukin-1β (IL-1β). These changes trigger pyroptotic cell death via gasdermin D (GSDMD) signaling . Notably, this effect is specific to malignant cells, as RCN1 knockdown does not affect the viability of G-CSF-mobilized peripheral blood stem cells from healthy donors.

In prostate cancer, the cell death mechanisms differ based on the cell line. In DU145 cells, RCN1 depletion triggers caspase-dependent apoptosis through elevation of phosphatase and tensin homolog (PTEN) and subsequent inactivation of AKT . In contrast, LNCaP cells primarily undergo necroptosis following RCN1 knockdown, with predominant activation of CaMKII playing an important role in this process . These diverse outcomes highlight the context-dependent roles of RCN1 in regulating cell survival and death pathways, which must be considered when developing targeted therapeutic approaches.

What is the relationship between RCN1 and the endoplasmic reticulum stress response in cancer?

The relationship between RCN1 and endoplasmic reticulum (ER) stress is complex and appears to be cancer type-specific. In prostate cancer cells, RCN1 depletion induces ER stress, evidenced by the upregulation of GRP78 (a key ER chaperone), activation of PERK (protein kinase R-like ER kinase), and phosphorylation of eIF2α (eukaryotic initiation factor 2α) . These changes represent activation of the unfolded protein response (UPR), a cellular mechanism designed to alleviate ER stress by reducing protein synthesis and increasing protein folding capacity.

Interestingly, in AML cells, the situation differs. Studies indicate that reducing RCN1 does not lead to significant increases in GRP78, phosphorylated PERK, phosphorylated eIF2α, or CHOP (C/EBP homologous protein) in NB4 or OCI/AML3 cells . This suggests that ER stress and the UPR are not activated following RCN1 downregulation in these particular AML cells. Instead, the cell death mechanism primarily involves IFN-1 upregulation and pyroptosis. This differential response to RCN1 depletion across cancer types highlights the need for cell type-specific investigations when considering RCN1 as a therapeutic target and suggests that RCN1 has multiple, context-dependent functions beyond its role in ER calcium homeostasis.

How could RCN1 be targeted therapeutically in cancer treatment?

Therapeutic targeting of RCN1 in cancer shows promise based on preclinical research demonstrating differential effects on malignant versus healthy cells. Several approaches warrant investigation:

• RNA interference therapy: Lentiviral shRNA vectors targeting RCN1 have demonstrated efficacy in reducing RCN1 expression and inhibiting cancer cell viability in vitro . For in vivo applications, adenovirus vectors targeting RCN1 (Ad-sh-RCN1) have shown efficacy in murine xenograft models, restraining the growth of human AML .

• CRISPR/Cas9-based strategies: CRISPR/Cas9 technology has successfully created RCN1 knockout cancer cell lines with significantly reduced viability . This approach could be adapted for therapeutic applications using appropriate delivery systems.

• Small molecule inhibitors: Developing small molecules that disrupt RCN1's calcium-binding function or protein-protein interactions could provide another therapeutic avenue, though no specific inhibitors have yet been reported in the literature.

• Combination therapies: Given RCN1's role in multiple cellular processes, combining RCN1 inhibition with other cancer therapies might enhance efficacy. For instance, since RCN1 knockdown induces type I interferon responses in AML, combining RCN1 inhibition with immunotherapies that leverage interferon-mediated anti-tumor immunity could be synergistic .

The therapeutic potential of targeting RCN1 is supported by xenograft studies showing that NCG mice inoculated with RCN1-deficient THP-1 cells developed smaller tumors and had longer survival compared to those with control cells . Additionally, the specificity of RCN1 targeting is promising, as downregulation doesn't affect several non-tumor cell lines, including human embryonic kidney cells (293T/17), normal lung epithelial cells (BEAS-2B), and human foreskin fibroblasts (HFF-1) .

What techniques are most effective for studying RCN1 gene knockdown or knockout?

Multiple effective techniques for RCN1 manipulation have been validated in research settings:

CRISPR/Cas9 Gene Editing:

• Demonstrated success in creating complete RCN1 knockout cell clones (e.g., MOLM-13-23, MOLM-13-240, MOLM-13-242)

• Enables permanent genetic modification for long-term studies

• Recommended protocols include single guide RNA design targeting conserved RCN1 exons, followed by selection of edited clones via limiting dilution and Western blot verification

RNA Interference:

• Lentiviral shRNA vectors provide efficient RCN1 knockdown in multiple cell lines

• Allows titration of knockdown levels and inducible systems for temporal control

• Typically achieves 70-90% reduction in RCN1 protein levels within 48-72 hours post-transduction

Adenoviral Vectors:

• Particularly useful for in vivo applications as demonstrated in murine xenograft models

• Provides transient gene knockdown with high transduction efficiency

• Effective for targeted tissue delivery in animal models

Mouse Models:

• Deletion of mouse Rcn1 gene has been successfully implemented to study effects on normal hematopoiesis versus leukemic cell survival

• Can be combined with xenograft approaches using immunodeficient mouse strains (NCG/NSG)

For verification of knockdown/knockout efficiency, Western blotting remains the gold standard, with quantitative RT-PCR providing complementary mRNA expression data. Cell viability assays (CCK-8, MTT) are commonly employed to assess functional consequences, while flow cytometry enables detailed analysis of cell death mechanisms (apoptosis, pyroptosis, necroptosis) .

How can researchers effectively measure RCN1-induced cell death mechanisms?

Different cell death mechanisms require specific methodological approaches for accurate characterization:

For Pyroptosis Assessment (AML cells):

• Measure cleaved gasdermin D (GSDMD) by Western blotting as the definitive marker of pyroptosis

• Quantify release of inflammatory cytokines (IL-1β, IL-18) using ELISA

• Detect active caspase-1 using fluorescent-labeled inhibitor of caspases (FLICA) assay

• Evaluate type I interferon signaling through phosphorylated STAT1/STAT2 detection

• Assess cell membrane integrity using propidium iodide without permeabilization

For Apoptosis Measurement (e.g., DU145 prostate cancer cells):

• Flow cytometry with Annexin V/PI double staining to differentiate early/late apoptosis

• Detection of cleaved caspase-3/7/9 by Western blotting or fluorogenic substrate assays

• Analysis of mitochondrial membrane potential using JC-1 dye

• PTEN/AKT pathway activation status by Western blotting for phosphorylated forms

For Necroptosis Evaluation (e.g., LNCaP prostate cancer cells):

• Measure phosphorylated RIPK1/RIPK3/MLKL by Western blotting

• Use necroptosis inhibitors (necrostatin-1) to confirm mechanism specificity

• Analyze CaMKII activation status given its importance in RCN1-depleted LNCaP cells

• Assess cellular ATP levels as necroptosis correlates with rapid ATP depletion

Control experiments should include rescue experiments (e.g., PTEN silencing partially restores viability upon RCN1 loss in DU145 cells) and comparative analysis across multiple cell lines to highlight cell type-specific responses to RCN1 manipulation.

What are the best methods for analyzing RCN1's role in microglial phagocytosis?

To analyze RCN1's role in microglial phagocytosis, researchers should employ a combination of specialized techniques:

Phage Display Technology:

• Construction of Rcn1-Phage with verified expression through sequencing

• Use of control phages (GFP-Phage, Control-Phage) as experimental comparisons

• Quantification of phagocytosed phage through plaque assay measured as plaque forming units (pfu)/well

Recombinant Protein Production:

• Expression of GST-Rcn1 fusion proteins for binding studies

• Production of Rcn1-FLAG tagged proteins with and without signal peptide to study secretion and localization

• Purification of recombinant proteins for direct application in functional assays

Phagocytosis Assays:

• Co-culture of microglia with apoptotic neurons labeled with pH-sensitive dyes

• Time-lapse microscopy to track phagocytic events in real-time

• Immunofluorescence co-localization with phagosome markers

• Flow cytometry-based quantification of phagocytosed material

Binding Studies:

• Analysis of Rcn1 binding preference between apoptotic versus healthy neurons

• Measurement of Rcn1 secretion into culture medium

• Competition assays to identify binding partners and receptors

Genetic Manipulation:

• Knockdown/knockout of Rcn1 in microglial cells to assess functional consequences

• Rescue experiments with exogenous Rcn1 administration

These methodologies should be combined with appropriate controls and statistical analyses to comprehensively evaluate RCN1's function as a microglial phagocytosis ligand. The approaches described have successfully demonstrated that Rcn1 extrinsically promotes microglial phagocytosis of apoptotic but not healthy neurons, with phagocytosed neurons being targeted to phagosomes and co-localized with phagosome markers .

How does RCN1 influence the tumor microenvironment?

Recent research indicates that RCN1 plays significant roles in shaping the tumor microenvironment, particularly through its effects on immune cells. In oral squamous cell carcinoma (OSCC), knockdown of RCN1 has been shown to inhibit M2 macrophage polarization . This is particularly relevant as tumor-associated macrophages (TAMs) with an M2 phenotype generally promote tumor progression through immunosuppression, angiogenesis, and matrix remodeling. The ability of tumor cell-derived RCN1 to influence macrophage polarization was demonstrated in co-culture models of THP-1 macrophages and OSCC cells .

Additionally, in AML, RCN1 knockdown upregulates type I interferon responses , which are known to enhance anti-tumor immunity through multiple mechanisms including activation of natural killer cells, dendritic cells, and cytotoxic T lymphocytes. This suggests that RCN1 may normally suppress interferon-mediated anti-tumor immune responses. Future research should investigate whether RCN1 affects other components of the tumor microenvironment, such as cancer-associated fibroblasts, endothelial cells, or extracellular matrix composition. Understanding these broader effects will be crucial for developing RCN1-targeted therapies that can modulate the tumor microenvironment in addition to directly affecting cancer cell viability.

What is the relationship between RCN1 and type I interferon signaling in cancer?

The relationship between RCN1 and type I interferon (IFN-1) signaling represents a critical aspect of RCN1 biology in cancer. Research has demonstrated that RCN1 knockdown specifically upregulates IFN-1 expression by activating the STING pathway in AML cells . This increased IFN-1 signaling subsequently triggers pyroptosis through caspase-1 and gasdermin D (GSDMD) activation . The mechanism involves a cascade where reduced RCN1 leads to STING pathway activation, increased IFN-1 production, elevated cleaved-caspase-1 and IL-1β expression, and ultimately pyroptotic cell death.

This finding is particularly significant because type I interferons are known to have pleiotropic effects on cancer, including direct anti-proliferative activities, enhancement of immune recognition, and modulation of tumor cell immunogenicity. The discovery that RCN1 appears to suppress this pathway suggests it may contribute to tumor immune evasion. Future research should investigate whether this relationship between RCN1 and IFN-1 extends to other cancer types beyond AML and explore the molecular mechanisms by which RCN1 regulates the STING pathway. Understanding these interactions could lead to novel combination therapies that pair RCN1 inhibition with immunotherapies to enhance anti-tumor immune responses through interferon-mediated mechanisms.

How can single-cell analysis enhance our understanding of RCN1 function?

Single-cell analysis technologies offer powerful approaches to advance our understanding of RCN1 function across heterogeneous cell populations. These methodologies can address several limitations of bulk analyses:

• Cellular Heterogeneity Resolution: Single-cell RNA sequencing (scRNA-seq) would allow researchers to identify distinct cell subpopulations with differential RCN1 expression within tumors. This could reveal whether RCN1 expression correlates with specific cancer stem cell markers, differentiation states, or resistance phenotypes.

• Dynamic Response Tracking: Single-cell proteomics and phosphoproteomics could track the temporal dynamics of signaling pathway activation following RCN1 knockdown at the individual cell level, revealing cellular decision points leading to pyroptosis, apoptosis, or necroptosis.

• Microenvironment Interactions: Single-cell spatial transcriptomics technologies would enable mapping of RCN1 expression in relation to specific microenvironmental niches and neighboring stromal or immune cells. This could clarify how RCN1-expressing cancer cells interact with macrophages to promote M2 polarization .

• Therapeutic Response Prediction: Single-cell analyses before and after RCN1-targeting therapies could identify resistant cell populations and adaptive responses, informing more effective combination treatment strategies.

• Lineage Tracing: Combining RCN1 manipulation with cellular barcoding and single-cell sequencing would allow tracking of clonal dynamics and competitive fitness of RCN1-deficient versus RCN1-proficient cancer cells over time.

These approaches would address current knowledge gaps regarding cell-specific roles of RCN1 in heterogeneous tumors and provide higher-resolution insights into the molecular mechanisms underlying RCN1's diverse functions across different cellular contexts.

How does RCN1 protein structure relate to its calcium-binding function?

RCN1 belongs to the CREC (Cab45/reticulocalbin/ERC-45/calumenin) family of multiple EF-hand calcium-binding proteins localized primarily to the secretory pathway. The protein structure of RCN1 contains six EF-hand domains, which are helix-loop-helix motifs that coordinate calcium ions. These domains are highly conserved across species, highlighting their fundamental importance to RCN1 function. The calcium-binding capacity of RCN1 is central to its role in maintaining calcium homeostasis within the endoplasmic reticulum (ER).

The N-terminal region of RCN1 contains a signal peptide that directs the protein to the ER lumen. Following this region, the six EF-hand domains are arranged sequentially, each capable of binding a calcium ion with varying affinities. The C-terminal region typically contains an ER retention signal (HDEL) that maintains RCN1 within the ER lumen, though evidence suggests that under certain conditions, RCN1 can be secreted into the extracellular environment . The calcium-binding properties of RCN1 enable it to function as a calcium buffer within the ER, helping maintain appropriate calcium concentrations necessary for proper protein folding and ER function. Additionally, calcium binding may induce conformational changes in RCN1 that facilitate its interactions with other proteins, potentially explaining its diverse roles in cellular processes ranging from protein folding to signaling pathway regulation.

What protein-protein interactions are essential for RCN1's biological functions?

The protein-protein interactions of RCN1 play critical roles in its diverse biological functions, though many remain to be fully characterized. Several key interactions have been identified:

• ER-resident chaperones: RCN1 interacts with other calcium-binding chaperones and folding enzymes in the ER, contributing to protein quality control. While specific binding partners in cancer cells require further investigation, these interactions likely influence how RCN1 depletion affects ER stress responses differently across cancer types .

• STING pathway components: Given that RCN1 knockdown activates the STING pathway leading to type I interferon production in AML cells , RCN1 likely interacts with components of this pathway directly or indirectly to suppress its activation under normal conditions.

• Caspase-1 and GSDMD signaling: The effect of RCN1 on pyroptosis through caspase-1 and GSDMD signaling suggests potential interactions with inflammasome components or regulators, though these may be indirect through interferon-mediated signaling.

• PTEN/AKT pathway: In prostate cancer cells, RCN1 depletion leads to PTEN elevation and AKT inactivation , indicating RCN1 may interact with regulators of this pathway.

• CaMKII signaling: RCN1's influence on CaMKII activation in LNCaP cells suggests a potential interaction with calcium-dependent signaling molecules.

• Cell surface receptors on microglia: RCN1 can be secreted and preferentially binds to apoptotic neurons , implying interactions with specific receptors on microglia that recognize RCN1 as a phagocytosis ligand.

Further proteomic and interaction mapping studies are needed to comprehensively identify RCN1's interactome across different cellular contexts, which would provide deeper insights into its multifaceted functions in normal and disease states.

What are the most significant unanswered questions about RCN1 in human disease?

Despite recent advances in understanding RCN1's roles in cancer and cellular processes, several significant questions remain unanswered:

• Transcriptional regulation: What mechanisms drive RCN1 overexpression in multiple cancer types, and are there common transcriptional regulators across different cancers? Understanding these regulatory mechanisms could provide additional therapeutic targets.

• Cancer-specific functions: Why does RCN1 knockdown induce different cell death mechanisms (pyroptosis, apoptosis, necroptosis) in different cancer types ? Determining the molecular determinants of these differential responses would enhance therapeutic precision.

• Normal physiological roles: What are the essential functions of RCN1 in normal tissues, particularly in the central nervous system where it acts as a microglial phagocytosis ligand ? This knowledge would help predict potential side effects of therapeutic RCN1 targeting.

• Secretion mechanisms: Under what conditions is RCN1 secreted from cells despite having an ER retention signal, and how does this secretion contribute to intercellular communication and microenvironmental modulation?

• Biomarker potential: Can circulating RCN1 levels serve as a biomarker for cancer detection, prognosis, or treatment response monitoring? Preliminary evidence suggests a correlation between RCN1 expression and poor prognosis in OSCC , but broader clinical validation is needed.

• Resistance mechanisms: Do cancer cells develop resistance to RCN1 targeting, and what compensatory mechanisms might emerge following prolonged RCN1 inhibition?

Addressing these questions will be essential for developing RCN1-targeted therapies and understanding their potential impact across various disease contexts.

How can current RCN1 research findings be translated into clinical applications?

Translating current RCN1 research findings into clinical applications requires a strategic approach focused on several key areas:

• Diagnostic applications: Development of RCN1-based diagnostic assays could help identify patients with RCN1-overexpressing tumors. Immunohistochemical analysis of tumor biopsies for RCN1 expression might predict prognosis and inform treatment decisions, as suggested by OSCC studies showing correlation between RCN1 expression and clinical outcomes .

• Therapeutic development:

  • Optimization of RCN1 siRNA or shRNA delivery systems for clinical application, building on successful preclinical studies

  • Development of small molecule inhibitors targeting RCN1 function or critical protein-protein interactions

  • Creation of combination regimens pairing RCN1 inhibition with immunotherapies, particularly given RCN1's effects on type I interferon signaling and macrophage polarization

• Patient stratification: Identification of biomarkers predicting response to RCN1-targeted therapies. The differential cell death mechanisms induced by RCN1 knockdown across cancer types suggest that molecular profiling might help identify patients most likely to benefit from specific RCN1-targeting approaches.

• Safety assessments: Comprehensive evaluation of potential toxicities associated with RCN1 targeting, particularly focusing on tissues where RCN1 may play important physiological roles. Current evidence suggesting minimal effects on normal hematopoiesis and several non-tumor cell lines is promising but requires broader validation.

• Clinical trial design: Initial trial designs might focus on hematological malignancies such as AML where preclinical evidence is strongest , followed by expansion to solid tumors with validated RCN1 dependencies such as prostate cancer and OSCC .