T-Cell redirecting bispecific antibodies: a review of a novel class of immuno-oncology for advanced prostate cancer.

Novel T-cell immunotherapies such as bispecific T-cell engagers (BiTEs) are emerging as promising therapeutic strategies for prostate cancer. BiTEs are engineered bispecific antibodies containing two distinct binding domains that allow for concurrent binding to tumor-associated antigens (TAAs) as well as immune effector cells, thus promoting an immune response against cancer cells. Prostate cancer is rich in tumor associated antigens such as, but not limited to, PSMA, PSCA, hK2, and STEAP1 and there is strong biologic rationale for employment of T-cell redirecting BiTEs within the prostate cancer disease space. Early generation BiTE constructs employed in clinical study have demonstrated meaningful antitumor activity, but challenges related to drug delivery, immunogenicity, and treatment-associated adverse effects limited their success. The ongoing development of novel BiTE constructs continues to address these barriers and to yield promising results in terms of efficacy and safety. This review will highlight some of most recent developments of BiTE therapies for patients with advanced prostate cancer and the evolving data surrounding BiTE constructs undergoing clinical evaluation.

Comparative Evaluation of STEAP1 Targeting Chimeric Antigen Receptors with Different Costimulatory Domains and Spacers.

We have developed a chimeric antigen receptor (CAR) against the six-transmembrane epithelial antigen of prostate-1 (STEAP1), which is expressed in prostate cancer, Ewing sarcoma, and other malignancies. In the present study, we investigated the effect of substituting costimulatory domains and spacers in this STEAP1 CAR. We cloned four CAR constructs with either CD28 or 4-1BB costimulatory domains, combined with a CD8a-spacer (sp) or a mutated IgG-spacer. The CAR T-cells were evaluated in short- and long-term in vitro T-cell assays, measuring cytokine production, tumor cell killing, and CAR T-cell expansion and phenotype. A xenograft mouse model of prostate cancer was used for in vivo comparison. All four CAR constructs conferred CD4+ and CD8+ T cells with STEAP1-specific functionality. A CD8sp_41BBz construct and an IgGsp_CD28z construct were selected for a more extensive comparison. The IgGsp_CD28z CAR gave stronger cytokine responses and killing in overnight caspase assays. However, the 41BB-containing CAR mediated more killing (IncuCyte) over one week. Upon six repeated stimulations, the CD8sp_41BBz CAR T cells showed superior expansion and lower expression of exhaustion markers (PD1, LAG3, TIGIT, TIM3, and CD25). In vivo, both the CAR T variants had comparable anti-tumor activity, but persisting CAR T-cells in tumors were only detected for the 41BBz variant. In conclusion, the CD8sp_41BBz STEAP1 CAR T cells had superior expansion and survival in vitro and in vivo, compared to the IgGsp_CD28z counterpart, and a less exhausted phenotype upon repeated antigen exposure. Such persistence may be important for clinical efficacy.

Six Transmembrane Epithelial Antigen 1 as a Prognostic Biomarker in Carcinomas: A Meta-Analysis.

BACKGROUND: Six transmembrane epithelial antigen 1 (STEAP1) is aberrantly expressed in cancers and could therefore be a potential biomarker. This study examined the connection between STEAP1 expression and clinical features/prognosis in cancer patients. METHODS: Several databases were comprehensively searched for related published studies. The combination of hazard ratios (HRs), odd ratios (ORs), and 95% confidence intervals (95% CIs) was used to assess the role of STEAP1. The Cancer Genome Atlas (TCGA) dataset was used to estimate the prognostic value of STEAP1 in multiple cancer types, and several biological behaviors related to STEAP1 were evaluated by CancerSEA. RESULTS: Searches of electronic databases revealed 7 relevant trials with 765 patients. A significant connection was found between high STEAP1 expression and worse overall survival amongst cancer patients (HR = 1.87, 95% CI: 1.49-2.34, p < 0.001). In addition, a strong correlation was found between high STEAP1 expression and the occurrence of lymph node metastases (OR = 3.19, 95% CI: 1.26-8.09, p < 0.001). Analysis of TCGA datasets verified that a higher level of STEAP1 expression is linked with reduced survival in many kinds of cancer. At the single cell level, STEAP1 expression was correlated with some tumor biological behaviors, such as angiogenesis, quiescence, and stemness. CONCLUSIONS: STEAP1 could regulate various biological functions in tumors and predict prognosis as a novel biomarker in a number of cancer types.

Targeting STEAP1 as an anticancer strategy.

Although the six-transmembrane epithelial antigen of prostate 1 (STEAP1) was first identified in advanced prostate cancer, its overexpression is recognized in multiple types of cancer and associated with a poor prognosis. STEAP1 is now drawing attention as a promising therapeutic target because of its tumor specificity and membrane-bound localization. The clinical efficacy of an antibody-drug conjugate targeting STEAP1 in metastatic, castration-resistant, prostate cancer was demonstrated in a phase 1 trial. Furthermore, growing evidence suggests that STEAP1 is an attractive target for immunotherapies such as chimeric antigen receptor-T cell therapy. In this review, we summarize the oncogenic functions of STEAP1 by cancer type. This review also provides new insights into the development of new anticancer strategies targeting STEAP1.

Proteomic analysis of STEAP1 knockdown in human LNCaP prostate cancer cells.

Prostate cancer (PCa) continues to be one of the most common cancers in men worldwide. The six transmembrane epithelial antigen of the prostate 1 (STEAP1) protein is overexpressed in several types of human tumors, particularly in PCa. Our research group has demonstrated that STEAP1 overexpression is associated with PCa progression and aggressiveness. Therefore, understanding the cellular and molecular mechanisms triggered by STEAP1 overexpression will provide important insights to delineate new strategies for PCa treatment. In the present work, a proteomic strategy was used to characterize the intracellular signaling pathways and the molecular targets downstream of STEAP1 in PCa cells. A label-free approach was applied using an Orbitrap LC-MS/MS system to characterize the proteome of STEAP1-knockdown PCa cells. More than 6700 proteins were identified, of which a total of 526 proteins were found differentially expressed in scramble siRNA versus STEAP1 siRNA (234 proteins up-regulated and 292 proteins down-regulated). Bioinformatics analysis allowed us to explore the mechanism through which STEAP1 exerts influence on PCa, revealing that endocytosis, RNA transport, apoptosis, aminoacyl-tRNA biosynthesis, and metabolic pathways are the main biological processes where STEAP1 is involved. By immunoblotting, it was confirmed that STEAP1 silencing induced the up-regulation of cathepsin B, intersectin-1, and syntaxin 4, and the down-regulation of HRas, PIK3C2A, and DIS3. These findings suggested that blocking STEAP1 might be a suitable strategy to activate apoptosis and endocytosis, and diminish cellular metabolism and intercellular communication, leading to inhibition of PCa progression.

Development of a novel electrochemical biosensor based on plastic antibodies for detection of STEAP1 biomarker in cancer.

STEAP1 is a cell surface protein of the STEAP family whose main function focuses on intercellular communication and cell growth. STEAP1 is considered a promising putative biomarker and a candidate target for prostate cancer treatment. For specific and selective detection of STEAP1, a molecularly imprinted polymers (MIP) was developed on a screen-printed electrode (C-SPE) whose surface was modified with a nanocomposite based on carbon nanotubes decorated with dendritic platinum nanoparticles (CNTs- PAH /Pt). Then, the MIPs were produced on the modified C-SPE by electropolymerization of a mixture of STEAP1 and a monomer (pyrrole-2-carboxylic acid). Then, the protein was removed from the polymeric network by enzymatic treatment with trypsin, which created the specific template cavities for further STEAP1 detection. Electrochemical techniques such as EIS and CV were used to follow the chemical modification steps of C-SPE. The analytical performance of the biosensor was evaluated by SWV in PBS buffer and in lysates of neoplastic prostate cancer cells (LNCaP) extracts. The MIP material showing a linear range from 130 pg/ml to 13 µg/ml. Overall, the biosensor exhibits essential properties such as selectivity, sensitivity and reproducibility for its application in medical and clinical research diagnosis and/or prognosis of prostate cancer.

Inhibition of STEAP1 ameliorates inflammation and ferroptosis of acute lung injury caused by sepsis in LPS-induced human pulmonary microvascular endothelial cells.

BACKGROUND: Ferroptosis plays an important part in Acute lung injury (ALI) caused by sepsis. The six-transmembrane epithelial antigen of the prostate 1 (STEAP1) has potential effects on iron metabolism and inflammation but reports on its function in ferroptosis and sepsis-caused ALI are lacking. Here we explored the role of STEAP1 in sepsis-caused ALI and the possible mechanisms. METHODS AND RESULTS: Lipopolysaccharide (LPS) was added to human pulmonary microvascular endothelial cells (HPMECs) to form the sepsis-caused ALI model in vitro. The Cecal ligation and puncture (CLP) experiment was performed on C57/B6J mice to form the sepsis-caused ALI model in vivo. The effect of STEAP1 on inflammation was investigated by PCR, ELISA, and Western blot for the inflammatory factors and adhesion molecular. The reactive oxygen species (ROS) levels were detected by immunofluorescence. The effect of STEAP1 on ferroptosis was investigated by detecting malondialdehyde (MDA) levels, glutathione (GSH) levels, Fe2+ levels, cell viability, and mitochondrial morphology. Our findings suggested that STEAP1 expression was increased in the sepsis-induced ALI models. Inhibition of STEAP1 decreased the inflammatory response and ROS production as well as MDA levels but increased the levels of Nrf2 and GSH. Meanwhile, inhibition of STEAP1 improved cell viability and restored mitochondrial morphology. Western Blot results showed that inhibition of STEAP1 could affect the SLC7A11/GPX4 axis. CONCLUSION: Inhibition of STEAP1 may be valuable for pulmonary endothelial protection in lung injury caused by sepsis.

STEAP1 Knockdown Decreases the Sensitivity of Prostate Cancer Cells to Paclitaxel, Docetaxel and Cabazitaxel.

The Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) protein has been indicated as an overexpressed oncoprotein in prostate cancer (PCa), associated with tumor progression and aggressiveness. Taxane-based antineoplastic drugs such as paclitaxel, docetaxel, or cabazitaxel, have been investigated in PCa treatment, namely for the development of combined therapies with the improvement of therapeutic effectiveness. This study aimed to evaluate the expression of STEAP1 in response to taxane-based drugs and assess whether the sensitivity of PCa cells to treatment with paclitaxel, docetaxel, or cabazitaxel may change when the STEAP1 gene is silenced. Thus, wild-type and STEAP1 knockdown LNCaP and C4-2B cells were exposed to paclitaxel, docetaxel or cabazitaxel, and STEAP1 expression, cell viability, and survival pathways were evaluated. The results obtained showed that STEAP1 knockdown or taxane-based drugs treatment significantly reduced the viability and survival of PCa cells. Relatively to the expression of proliferation markers and apoptosis regulators, LNCaP cells showed a reduced proliferation, whereas apoptosis was increased. However, the effect of paclitaxel, docetaxel, or cabazitaxel treatment was reversed when combined with STEAP1 knockdown. Besides, these chemotherapeutic drugs may stimulate the cell growth of PCa cells knocked down for STEAP1. In conclusion, this study demonstrated that STEAP1 expression levels might influence the response of PCa cells to chemotherapeutics drugs, indicating that the use of paclitaxel, docetaxel, or cabazitaxel may lead to harmful effects in PCa cells with decreased expression of STEAP1.

Antibody-Drug Conjugates in Prostate Cancer: A Systematic Review.

The prognosis in the setting of metastatic castration-resistant prostate cancer patients (mCRPC) remains limited. Therefore, novel treatment strategies remain an unmet need. Antibody-drug conjugates (ADC) emerged as a new drug concept with the potential to deliver a cytotoxic payload with limited off-target toxicity and potentially bystander effect. Following the success of ADCs in breast cancer and urothelial tumours, their activity in prostate cancer is now under investigation. Thus, the aim of this systematic review was to identify published and ongoing prospective clinical trials regarding ADC treatment in prostate cancer. A systematic search of PubMed, MEDLINE, and Web of Science was conducted as per PRISMA guidelines to identify prospective clinical trials of ADCin prostate cancer. Trials are currently ongoing on ClinicalTrials.gov and in the EU. The Clinical Trials Register was also identified. Abstracts, publications in languages other than English, review articles, retrospective analyses, and phase I trials were excluded. A total of six phase I/II prospective clinical trials already published were included. Seven ongoing trials were also identified. All studies were in the refractory/advanced tumour setting, and two included only mCRPC patients. The ADC targets were prostate-specific membrane antigen (PSMA), trophoblast cell surface antigen-2 (TROP-2), six-transmembrane epithelial antigen of prostate-1 (STEAP-1), tissue factor (TF), delta-like protein 3 (DLL-3), B7-H3 family of proteins (B7-H3), and human epidermal growth factor receptor 2 (HER2). Regarding the efficacy of PSMA ADC treatment in the second-line or beyond mCRPC setting, a PSA ≥ 50% decline rate in 14% of all treated patients was reported. One patient achieved a complete response with TROP-2 ADC. Overall, a wide range of safety issues were raised, particularly in connection with neuropathy and hematologic toxicity. Novel therapies have been changing the scope of treatment in mCRPC. ADCs seem to provide efficacy benefits, even with potential toxicity. The results of most prospective ongoing studies are still awaited, and a longer follow-up time is warranted to evaluate the real impact of ADCs in PCa.

Prognostic Significance of Iron Metabolism Related Genes in Human Lung Adenocarcinoma.

Background: Iron metabolism related genes participate in cell proliferation, cell growth, and redox cycling in multiple cancers. Limited studies have revealed the roles and clinical significance of iron metabolism in the pathogenesis and prognosis of lung cancer. Methods: A total of 119 iron metabolism related genes were extracted from MSigDB database and their prognostic values were determined in The Cancer Genome Atlas lung adenocarcinoma (TCGA-LUAD) dataset and the Gene Expression Profiling Interactive Analysis 2 (GEPIA 2) database. Immunohistochemistry technique and correlations with immune cell infiltration, gene mutation and drug resistance were used to identify the potential and underlying mechanisms of STEAP1 and STEAP2 as prognostic biomarkers of LUAD. Results: The expression of STEAP1 and STEAP2 are negatively associated with the prognosis of LUAD patients both at the mRNA and protein level. The expression of STEAP1 and STEAP2 was not only negatively correlated with the trafficking degree of CD4+ T immune cells and positively related to most immune cells' trafficking degree, but also significantly associated with gene mutation status, particularly with mutations on TP53 and STK11. Four types of drug resistance showed significant correlation with the expression level of STEAP1 while 13 types of drug resistance were associated with the expression level of STEAP2. Conclusion: Multiple iron metabolism related genes including STEAP1 and STEAP2 are significantly associated with the prognosis of LUAD patients. STEAP1 and STEAP2 might affect the prognosis of LUAD patients partially through immune cell infiltration, gene mutation and drug resistance, which indicated they were independent prognostic factors for LUAD patients.

Specific Six-Transmembrane Epithelial Antigen of the Prostate 1 Capture with Gellan Gum Microspheres: Design, Optimization and Integration.

This work demonstrates the potential of calcium- and nickel-crosslinked Gellan Gum (GG) microspheres to capture the Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) directly from complex Komagataella pastoris mini-bioreactor lysates in a batch method. Calcium-crosslinked microspheres were applied in an ionic exchange strategy, by manipulation of pH and ionic strength, whereas nickel-crosslinked microspheres were applied in an affinity strategy, mirroring a standard immobilized metal affinity chromatography. Both formulations presented small diameters, with appreciable crosslinker content, but calcium-crosslinked microspheres were far smoother. The most promising results were obtained for the ionic strategy, wherein calcium-crosslinked GG microspheres were able to completely bind 0.1% (v/v) DM solubilized STEAP1 in lysate samples (~7 mg/mL). The target protein was eluted in a complexed state at pH 11 with 500 mM NaCl in 10 mM Tris buffer, in a single step with minimal losses. Coupling the batch clarified sample with a co-immunoprecipitation polishing step yields a sample of monomeric STEAP1 with a high degree of purity. For the first time, we demonstrate the potential of a gellan batch method to function as a clarification and primary capture method towards STEAP1, a membrane protein, simplifying and reducing the costs of standard purification workflows.

EFEMP1 binds to STEAP1 to promote osteosarcoma proliferation and invasion via the Wnt/ß-catenin and TGF-ß/Smad2/3 signal pathways.

Purpose: To investigate the prognostic value and function of six-transmembrane epithelial antigen of prostate 1 (STEAP1) in osteosarcoma and determine whether EFEMP1 mediates its effects. Methods: IHC (immunohistochemistry)/ICC (immunocytochemistry) in conjunction with RT-qPCR (quantitative real-time polymerase chain reaction) were employed to assess the expression of STEAP1 in paratumoral tissues, osteosarcoma, benign fibrous dysplasia, osteosarcoma cells, normal osteoblastic hFOB cells, as well as various invasive subclones. The association of STEAP1 with outcome was examined with Kaplan-Meier graph among the osteosarcoma population. The effects of the down-regulation and up-regulation of STEAP1 on the biological behavior of osteosarcoma cells were studied through in-vitro and in-vivo functional tests. Results: Up-regulation of STEAP1 in the osteosarcoma tissues, whose correlations with the malignant osteosarcoma phenotype and the poor patient outcome were positive. In addition, STEAP1 induced the epithelial-mesenchymal transition (EMT) via the Wnt/ß-catenin and TGF-ß/Smad2/3 pathways and facilitated the osteosarcoma cell infiltration and migration. An increase or decrease in EFEMP1 expression directly promoted or inhibited the expression of STEAP1. In osteosarcoma cells overexpressing EFEMP1, STEAP1 knockdown significantly inhibited cell invasion, EMT process, and increased activity of Wnt/ß-catenin and TGF-ß/Smad2/3 signaling pathways. Although exogenous EFEMP1 could stimulate the Wnt/ß-catenin and TGF-ß/Smad2/3 pathways to promote the EMT, it had not effect on osteosarcoma cells with STEAP1 knockdown. Collectively, similar to EFEMP1, STEAP1 acted like an oncogene in the osteosarcoma progression. Conclusion: EFEMP1 enabled the Wnt/ß-catenin and TGF-ß/Smad2/3 axises initiation and EMT elicitation by targeting STEAP1, thereby facilitating the osteosarcoma cell infiltration and migration. These results are expected to contribute to the search for new targeted drugs able to effectively inhibit invasion and metastasis and improve prognosis in osteosarcoma.

A chromatographic network for the purification of detergent-solubilized six-transmembrane epithelial antigen of the prostate 1 from Komagataella pastoris mini-bioreactor lysates.

The Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) is an integral membrane protein involved in cellular communications, in the stimulation of cell proliferation by increasing Reactive Oxygen Species levels, and in the transmembrane-electron transport and reduction of extracellular metal-ion complexes. The STEAP1 is particularly over-expressed in prostate cancer, in contrast with non-tumoral tissues and vital organs, contributing to tumor progression and aggressiveness. However, the current understanding of STEAP1 lacks experimental data on the respective molecular mechanisms, structural determinants, and chemical modifications. This scenario highlights the relevance of exploring the biosynthesis of STEAP1 and its purification for further bio-interaction and structural characterization studies. In this work, recombinant hexahistidine-tagged human STEAP1 (rhSTEAP1-His6) was expressed in Komagataella pastoris (K. pastoris) mini-bioreactor methanol-induced cultures and successfully solubilized with Nonidet P-40 (NP-40) and n-Decyl-ß-D-Maltopyranoside (DM) detergents. The fraction capacity of Phenyl-, Butyl-, and Octyl-Sepharose hydrophobic matrices were evaluated by manipulating the ionic strength of binding and elution steps. Alternatively, immobilized metal affinity chromatography packed with nickel or cobalt were also studied in the isolation of rhSTEAP1-His6 from lysate extracts. Overall, the Phenyl-Sepharose and Nickel-based resins provided the desired selectivity for rhSTEAP1-His6 capture from NP-40 and DM detergent-solubilized K. pastoris extracts, respectively. After a polishing step using the anion-exchanger Q-Sepharose, a highly pure, fully solubilized, and immunoreactive 35 kDa rhSTEAP1-His6 fraction was obtained. Altogether, the established reproducible strategy for the purification of rhSTEAP1-His6 paves the way to gather additional insights on structural, thermal, and environmental stability characterization significantly contributing for the elucidation of the functional role and oncogenic behavior of the STEAP1 in prostate cancer microenvironment.

Development of STEAP1 targeting chimeric antigen receptor for adoptive cell therapy against cancer.

Chimeric antigen receptors (CARs) that retarget T cells against CD19 show clinical efficacy against B cell malignancies. Here, we describe the development of a CAR against the six-transmembrane epithelial antigen of prostate-1 (STEAP1), which is expressed in ∼90% of prostate cancers, and subgroups of other malignancies. STEAP1 is an attractive target, as it is associated with tumor invasiveness and progression and only expressed at low levels in normal tissues, apart from the non-vital prostate gland. We identified the antibody coding sequences from a hybridoma and designed a CAR that is efficiently expressed in primary T cells. The T cells acquired the desired anti-STEAP1 specificity, with a polyfunctional response including production of multiple cytokines, proliferation, and the killing of cancer cells. The response was observed for both CD4+ and CD8+ T cells, and against all STEAP1+ target cell lines tested. We evaluated the in vivo CAR T activity in both subcutaneous and metastatic xenograft mouse models of prostate cancer. Here, the CAR T cells infiltrated tumors and significantly inhibited tumor growth and extended survival in a STEAP1-dependent manner. We conclude that the STEAP1 CAR exhibits potent in vitro and in vivo functionality and can be further developed toward potential clinical use.

Promoter Demethylation Upregulates STEAP1 Gene Expression in Human Prostate Cancer: In Vitro and In Silico Analysis.

The Six Transmembrane Epithelial Antigen of the Prostate (STEAP1) is an oncogene overexpressed in several human tumors, particularly in prostate cancer (PCa). However, the mechanisms involved in its overexpression remain unknown. It is well known that epigenetic modifications may result in abnormal gene expression patterns, contributing to tumor initiation and progression. Therefore, this study aimed to analyze the methylation pattern of the STEAP1 gene in PCa versus non-neoplastic cells. Bisulfite amplicon sequencing of the CpG island at the STEAP1 gene promoter showed a higher methylation level in non-neoplastic PNT1A prostate cells than in human PCa samples. Bioinformatic analysis of the GEO datasets also showed the STEAP1 gene promoter as being demethylated in human PCa, and a negative association with STEAP1 mRNA expression was observed. These results are supported by the treatment of non-neoplastic PNT1A cells with DNMT and HDAC inhibitors, which induced a significant increase in STEAP1 mRNA expression. In addition, the involvement of HDAC in the regulation of STEAP1 mRNA expression was corroborated by a negative association between STEAP1 mRNA expression and HDAC4,5,7 and 9 in human PCa. In conclusion, our work indicates that STEAP1 overexpression in PCa can be driven by the hypomethylation of STEAP1 gene promoter.

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure.

BACKGROUND: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. METHODS: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. RESULTS: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein's structural stability. CONCLUSIONS: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.

Fusion Protein Vaccine Based on Ag85B and STEAP1 Induces a Protective Immune Response against Prostate Cancer.

(1) Background: There are currently limited treatments for castration-resistant prostate cancer. Immunotherapy involving Sipuleucel-T has increasingly drawn attention for prostate cancer management. BCG plays a vital role in treating bladder cancer, mainly by inducing immune activation, but is rarely used for prostate cancer. (2) Methods: The TCGA database, PCR, and Western blotting were used to analyze the expression of STEAP1 in mouse and human tissues. Then, we constructed a fusion protein vaccine with Mycobacterium tuberculosis Ag85B and three repeated octapeptide epitopes of a six-transmembrane epithelial antigen of the prostate 1 (STEAP1186-193), Ag85B-3×STEAP1186-193. The uptake of the fusion protein vaccine by DCs was evaluated by confocal microscopy, and DC markers were detected using flow cytometry after incubation with the fusion protein. The immune response against prostate cancer was evaluated by the LDH assay and xenografts in vitro and in vivo. Then, the tumor microenvironment was determined using IHC and ELISA. In addition, the epitope was mutated using CRISPR-Cas9 to illustrate that the fusion protein elicited immunization against STEAP1. (3) Results: The TCGA database analysis, PCR, and Western blotting showed that STEAP1 was highly expressed in human and murine prostate cancer. After the uptake of the purified fusion protein vaccine by DCs, CD11c, CD80, CD86, and MHC II were upregulated and triggered a cytotoxic T lymphocyte (CTL) response against TRAMP-C1 and RM1 cells in vitro. Furthermore, the fusion protein vaccine inhibited tumor growth and improved the tumor microenvironment in vivo, with more CD3+ cells and fewer FOXP3+ cells in the tumor. Serum IFN-γ and IL-2 were significantly higher than in the control group, while IL-4 expression was lower, indicating that the fusion protein vaccine activated Th1 immunity. The immune response against prostate cancer was greatly suppressed when the antigen targets were knocked out using CRISPR-Cas9. (4) Conclusion: In summary, our results provide the first evidence that a vaccine based on a fusion protein consisting of Ag85B and a prostate cancer octapeptide epitope with complete Freund's adjuvant (CFA), triggers a robust immune response and inhibits tumor growth in murine prostate cancer.

Identification of a New Transcriptional Co-Regulator of STEAP1 in Ewing's Sarcoma.

Ewing's sarcoma (ES) is caused by a chromosomal translocation leading to the formation of the fused EWSFLI1 gene, which codes for an aberrant transcription factor EWSFLI1. The transcriptional targets of EWSFLI1 have been viewed as promising and novel drug targets in the treatment of ES. One such target is six transmembrane epithelial antigen of the prostate 1 (STEAP1), a transmembrane protein that is upregulated by EWSFLI1 in ES. STEAP1 is a hallmark of tumor invasiveness and an indicator of tumor responsiveness to therapy. EWSFLI1 binds to the STEAP1 promoter region, but the mechanism of action by which it upregulates STEAP1 expression in ES is not entirely understood. Upon analysis of the STEAP1 promoter, we predicted two binding sites for NKX2.2, another crucial transcription factor involved in ES pathogenesis. We confirmed the interaction of NKX2.2 with the STEAP1 promoter using chromatin immunoprecipitation (ChIP) analysis. We used single-molecule RNA imaging, biochemical, and genetic studies to identify the novel role of NKX2.2 in regulating STEAP1 expression in ES. Our results show that NKX2.2 is a co-regulator of STEAP1 expression and functions by interacting with the STEAP1 promoter at sites proximal to the reported EWSFLI1 sites. The co-operative interaction of NKX2.2 with EWSFLI1 in regulating STEAP1 holds potential as a new target for therapeutic interventions for ES.

Impact of glycerol feeding profiles on STEAP1 biosynthesis by Komagataella pastoris using a methanol-inducible promoter.

Currently, the lack of reliable strategies for the diagnosis and treatment of cancer makes the identification and characterization of new therapeutic targets a pressing matter. Several studies have proposed the Six Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) as a promising therapeutic target for prostate cancer. Although structural and functional studies may provide deeper insights on the role of STEAP1 in cancer, such techniques require high amounts of purified protein through biotechnological processes. Based on the results presented, this work proposes the application, for the first time, of a fed-batch profile to improve STEAP1 biosynthesis in mini-bioreactor Komagataella pastoris X-33 Mut+ methanol-induced cultures, by evaluating three glycerol feeding profiles-constant, exponential, and gradient-during the pre-induction phase. Interestingly, different glycerol feeding profiles produced differently processed STEAP1. This platform was optimized using a combination of chemical chaperones for ensuring the structural stabilization and appropriate processing of the target protein. The supplementation of culture medium with 6 % (v/v) DMSO and 1 M proline onto a gradient glycerol/constant methanol feeding promoted increased biosynthesis levels of STEAP1 and minimized aggregation events. Deglycosylation assays with peptide N-glycosidase F showed that glycerol constant feed is associated with an N-glycosylated pattern of STEAP1. The biological activity of recombinant STEAP1 was also validated, once the protein enhanced the proliferation of LNCaP and PC3 cancer cells, in comparison with non-tumoral cell cultures. This methodology could be a crucial starting point for large-scale production of active and stable conformation of recombinant human STEAP1. Thus, it could open up new strategies to unveil the structural rearrangement of STEAP1 and to better understand the biological role of the protein in cancer onset and progression.