Multiplex Assay of Cytokines with Tunable Detection Ranges for the Precise Diagnosis of Breast Cancer.

Precise profiling of the cytokine panel consisting of different levels of cytokines can provide personalized information about several diseases at certain stages. In this study, we have designed and fabricated an "all-in-one" diagnostic tool kit to bioassay multiple inflammatory cytokines ranging from picograms per milliliter to µg/mL in a small cytokine panel. Taking advantage of the kit fabricated by the DNA-encoded assembly of nanocatalysts in dynamic regulation and signal amplification, we have demonstrated the multiplex, visual, and quantitative detection of C-reactive protein (CRP), procalcitonin (PCT), and interleukin-6 (IL-6) with limits of detection of 1.6 ng/mL (61.54 pM), 20 pg/mL (1.57 pM), and 4 pg/mL (0.19 pM), respectively. This diagnostic tool kit can work well with commercial kits for detecting serum cytokines from breast cancer patients treated with immunotherapies. Furthermore, a small cytokine panel composed of CRP, PCT, and IL-6 is revealed to be significantly heterogeneous in each patient and highly dynamic for different treatment courses, showing promise as a panel of quantitative biomarker candidates for individual treatments. So, our work may provide a versatile diagnostic tool kit for the visual detection of clinical biomarkers with an adjustable broad detection range.

Visualization and characterization of RNA-protein interactions in living cells.

RNA-protein interactions are the structural and functional basis of significant numbers of RNA molecules. RNA-protein interaction assays though, still mainly depend on biochemical tests in vitro. Here, we establish a convenient and reliable RNA fluorescent three-hybrid (rF3H) method to detect/interrogate the interactions between RNAs and proteins in cells. A GFP tagged highly specific RNA trap is constructed to anchor the RNA of interest to an artificial or natural subcellular structure, and RNA-protein interactions can be detected and visualized by the enrichment of RNA binding proteins (RBPs) at these structures. Different RNA trapping systems are developed and detection of RNA-protein complexes at multiple subcellular structures are assayed. With this new toolset, interactions between proteins and mRNA or noncoding RNAs are characterized, including the interaction between a long noncoding RNA and an epigenetic modulator. Our approach provides a flexible and reliable method for the characterization of RNA-protein interactions in living cells.

Expression and purification of RHC-EGFP fusion protein and its application in hyaluronic acid assay.

Hyaluronan is a widely distributed glycosaminoglycan which has multiple functions. Hyaluronic acid (HA) accumulation has been reported in many human diseases. Understanding the role of hyaluronan and its binding proteins in the pathobiology of disease will facilitate the development of novel therapeutics for many critical diseases. Current techniques described for the analysis of HA are mainly for HA quantification in solutions, not for the direct detection of HA in tissues or on cell surfaces. In our study, a fusion protein, named C-terminal domain of RHAMM-enhanced green fluorescence protein (RHC-EGFP), combined the HA-binding domain, C-terminal of receptor for hyaluronan-mediated motility, with EGFP, a widely used enhanced green fluorescence protein, was expressed and purified from Escherichia coli with high purity. Based on the sensitivity and convenience of fluorescence detection, methods for direct assay of HA in solutions, on cell surface or in tissues were established using RHC-EGFP. The binding specificity was also confirmed by competitive binding experiment and hyaluronidase degradation experiment. Our results provide an alternative choice for the specific and convenient assay of HA in various samples, and maybe helpful for further understanding of the fundamental and comprehensive functions of HA.

An Imbalance in Histone Modifiers Induces tRNA-Cys-GCA Overexpression and tRF-27 Accumulation by Attenuating Promoter H3K27me3 in Primary Trastuzumab-Resistant Breast Cancer.

tRNA-derived fragments (tRFs) play crucial roles in cancer progression. Among them, tRF-27 has been identified as a key factor in promoting naïve trastuzumab resistance in HER2-positive breast cancer. However, the origin of tRF-27 remains uncertain. In this study, we propose that the upregulated expression of specific cysteine tRNAs may lead to the increased accumulation of tRF-27 in trastuzumab-resistant JIMT1 cells. Mechanistically, the reduced inhibitory H3K27me3 modification at the promoter regions of tRF-27-related tRNA genes in JIMT1 cells, potentially resulting from decreased EZH2 and increased KDM6A activity, may be a critical factor stimulating the transcriptional activity of these tRNA genes. Our research offers fresh insights into the mechanisms underlying elevated tRF-27 levels in trastuzumab-resistant breast cancer cells and suggests potential strategies to mitigate trastuzumab resistance in clinical treatments.

Unveiling Alterations of Epigenetic Modifications and Chromatin Architecture Leading to Lipid Metabolic Reprogramming during the Evolutionary Trastuzumab Adaptation of HER2-Positive Breast Cancer.

Secondary trastuzumab resistance represents an evolutionary adaptation of HER2-positive breast cancer during anti-HER2 treatment. Most current studies have tended to prioritize HER2 and its associated signaling pathways, often overlooking broader but seemingly less relevant cellular processes, along with their associated genetic and epigenetic mechanisms. Here, transcriptome data is not only characterized but also examined epigenomic and 3D genome architecture information in both trastuzumab-sensitive and secondary-resistant breast cancer cells. The findings reveal that the global metabolic reprogramming associated with trastuzumab resistance may stem from genome-wide alterations in both histone modifications and chromatin structure. Specifically, the transcriptional activities of key genes involved in lipid metabolism appear to be regulated by variant promoter H3K27me3 and H3K4me3 modifications, as well as promoter-enhancer interactions. These discoveries offer valuable insights into how cancer cells adapt to anti-tumor drugs and have the potential to impact future diagnostic and treatment strategies.

tRF-27 competitively Binds to G3BPs and Activates MTORC1 to Enhance HER2 Positive Breast Cancer Trastuzumab Tolerance.

About 20% of breast cancer patients are positive for HER2. The efficacy of current treatments is limited by primary and secondary resistance to trastuzumab. tRNA-derived fragments (tRFs) have shown crucial regulatory roles in various cancers. This study aimed to evaluate the role of tRF-27 in regulating the resistance of HER2-positive breast cancer against trastuzumab. tRF-27 was highly expressed in trastuzumab-resistant cells, and its expression level could predict the resistance to trastuzumab. High expression of tRF-27 promoted the growth and proliferation of trastuzumab-exposed cells. RNA-pulldown assay and mass spectrometry were performed to identify Ras GTPase-activating protein-binding proteins 1 and 2 (G3BPs) (two proteins targeted by tRF-27); RNA-immunoprecipitation (RIP) to confirm their bindings; co-immunoprecipitation (co-IP) and RNA-pulldown assay to determine the binding domains between G3BPs and tRF-27.tRF-27 bound to the nuclear transport factor 2 like domain(NTF2 domain) of G3BPs through a specific sequence. tRF-27 relied on G3BPs and NTF2 domain to increase trastuzumab tolerance. tRF-27 competed with lysosomal associated membrane protein 1(LAMP1) for NTF2 domain, thereby inhibiting lysosomal localization of G3BPs and tuberous sclerosis complex (TSC). Overexpression of tRF-27 inhibited phosphorylation of TSCs and promoted the activation of mechanistic target of rapamycin complex 1(MTORC1) to enhance cell proliferation and entice the resistance of HER2-positive breast cancer against trastuzumab.

Exosome-Transmitted tRF-16-K8J7K1B Promotes Tamoxifen Resistance by Reducing Drug-Induced Cell Apoptosis in Breast Cancer.

Tamoxifen resistance remains a challenge in hormone receptor-positive (HR+) breast cancer. Recent evidence suggests that transfer ribonucleic acid (tRNA)-derived fragments play pivotal roles in the occurrence and development of various tumors. However, the relationship between tRNA-derived fragments and tamoxifen resistance remains unclear. In this study, we found that the expression of tRF-16-K8J7K1B was upregulated in tamoxifen-resistant cells in comparison with tamoxifen-sensitive cells. Higher levels of tRF-16-K8J7K1B were associated with shorter disease-free survival in HR+ breast cancer. Overexpression of tRF-16-K8J7K1B promotes tamoxifen resistance. Moreover, extracellular tRF-16-K8J7K1B could be packaged into exosomes and could disseminate tamoxifen resistance to recipient cells. Mechanistically, exosomal tRF-16-K8J7K1B downregulates the expression of apoptosis-related proteins, such as caspase 3 and poly (ADP-ribose) polymerase, by targeting tumor necrosis factor-related apoptosis-inducing ligand in receptor cells, thereby reducing drug-induced cell apoptosis. Therapeutically, the inhibition of exosomal tRF-16-K8J7K1B increases the sensitivity of breast cancer cells to tamoxifen in vivo. These data demonstrate that exosomal tRF-16-K8J7K1B may be a novel therapeutic target to overcome tamoxifen resistance in HR+ breast cancer.

Regulation of thiamine synthesis in Saccharomyces cerevisiae for improved pyruvate production.

Metabolic engineering of Saccharomyces cerevisiae for high-yield production of carboxylic acid requires a cytosolic pyruvate pool as precursor. In this study, a novel strategy to improve pyruvate production and reduce metabolic by-products via regulating thiamine synthesis was explored. Two of the thiamine biosynthesis regulatory genes, THI2 and THI3, were disrupted in the S. cerevisiae parent strain FMME-002. The mutants FMME-002ΔTHI2 and FMME-002ΔTHI3 both exhibited an enhanced pyruvate yield. Moreover, FMME-002ΔTHI2 achieved a relatively higher pyruvate production, and the highest concentration of pyruvate was achieved when 0.04 µ m thiamine was added. Enzyme assays and fermentation profiles of the THI2-complemented strain indicated that the observed metabolic changes represented intrinsic effects of THI2 deletion on the physiology of S. cerevisiae. Under optimal C:N ratio conditions, FMME-002ΔTHI2 produced pyruvate up to 8.21 ± 0.30 g/l, whereas the ethanol titre decreased to 2.21 ± 0.24 g/l after 96 h of cultivation. These results demonstrate the possibility of improving pyruvate production by regulating thiamine synthesis in S. cerevisiae.

A positive feedback loop: RAD18-YAP-TGF-ß between triple-negative breast cancer and macrophages regulates cancer stemness and progression.

As a key regulator of the DNA translesion synthesis (TLS) pathway, RAD18 is error-prone and contributes to the accumulation of DNA mutations. Our previous study showed that it plays an essential role in the progression of multiple tumors. However, the mechanism through which RAD18 influences triple-negative breast cancer (TNBC), especially the interaction between tumor cells and the tumor microenvironment, remains elusive. In this study, we showed that RAD18 expression is markedly higher in patients with high T stage TNBC and inversely correlated with prognosis. High expression of RAD18 facilitated a highly stem-cell phenotype through the Hippo/YAP pathway, which supports the proliferation of TNBC. In addition, the cytokine byproduct TGF-ß activates macrophages to have an M2-like tumor-associated macrophage (TAM) phenotype. Reciprocally, TGF-ß from TAMs activated RAD18 in TNBC to enhance tumor stemness, forming a positive feedback loop. Inhibition of YAP or TGF-ß breaks this loop and suppresses cancer stemness and proliferation In nude mice, RAD18 promoted subcutaneous transplanted tumor growth and M2-type TAM recruitment. Collectively, the RAD18-YAP-TGF-ß loop is essential for the promotion of the stemness phenotype by TNBC and could be a potential therapeutic target for TNBC.

Improved antitumor activity of TRAIL fusion protein via formation of self-assembling nanoparticle.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been known as a promising agent for cancer therapy due to its specific apoptosis-inducing effect on tumor cells rather than most normal cells. However, systemically delivered TRAIL suffers from a rapid clearance from the body with an extremely short half-life. Thermally responsive elastin-like polypeptides (ELPs) are a promising class of temperature sensitive biopolymers based on the structural motif found in mammalian tropoelastin and retain the advantages of polymeric drug delivery systems. We therefore expressed RGD-TRAIL fused with ELP (RGD-TRAIL-ELP) in E. coli. Purification of RGD-TRAIL-ELP was achieved by the conveniently inverse transition cycling (ITC). The purified RGD-TRAIL-ELP without any chemical conjugation was able to self-assemble into nanoparticle under physiological condition. Non-reducing SDS-PAGE results showed that trimer content of RGD-TRAIL-ELP increased 3.4-fold than RGD-TRAIL. Flow cytometry confirmed that RGD-TRAIL-ELP 3-fold enhanced apoptosis-inducing capacity than RGD-TRAIL. Single intraperitoneal injection of the RGD-TRAIL-ELP nanoparticle induced nearly complete tumor regression in the COLO-205 tumor xenograft model. Histological observation confirmed that RGD-TRAIL-ELP induced significant tumor cell apoptosis without apparent liver toxicity. These findings suggested that a great potential application of the RGD-TRAIL-ELP nanoparticle system as a safe and efficient delivery strategy for cancer therapy.

Fused hydrophobic elastin-like-peptides (ELP) enhance biological activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent with tumor-selective apoptotic activity. Formation of aggregates as trimer is the prerequisite for TRAIL's function as an apoptosis inducer. However the polymerization property of TRAIL has also brought difficulties for its production. RGD-TRAIL is an integrin-targeting TRAIL mutant with enhanced apoptosisinducing activity towards tumor cells both in vitro and in vivo. When expressed in E. coli, TRAIL or its mutant RGDTRAIL usually formed inclusion bodies. Their extreme aggregation propensity for aggregation destabilizes the protein, leading to poor recovery and therefore low yield from the purification process. The low purification efficiency of TRAIL retards its industrial application and large-scale production. To avoid the above problems during RGD-TRAIL production, we employed elastin-like polypeptides (ELPs) for the fusion-expression of recombinant RGD-TRAIL. Recombinant RGD-TRAIL-ELP was expressed in a soluble form and efficiently purified from the clarified cell extracts by three rounds of inverse transition cycling (ITC). SDS-PAGE and Western blotting analyses of purified RGD-TRAIL-ELP showed that RGD-TRAIL-ELP was successfully purified and the yield was up to 10 mg/L of bacterial culture. Apoptosis assay was performed in human colorectal carcinoma cells (COLO-205) and human breast cancer cell line (MDA-MB-231) to assess the potency of the fusion protein. Fusion with hydrophobic ELP effectively enhanced RGD-TRAIL's biological activity. The higher activity and appropriate particle size of RGD-TRAIL-ELP could be used for RGD-TRAIL delivery in tumor therapy.