Incorporating IL7 receptor alpha signaling in the endodomain of B7H3-targeting chimeric antigen receptor T cells mediates antitumor activity in glioblastoma.

CAR-T-cell therapy has shown promise in treating hematological malignancies but faces challenges in treating solid tumors due to impaired T-cell function in the tumor microenvironment. To provide optimal T-cell activation, we developed a B7 homolog 3 protein (B7H3)-targeting CAR construct consisting of three activation signals: CD3ζ (signal 1), 41BB (signal 2), and the interleukin 7 receptor alpha (IL7Rα) cytoplasmic domain (signal 3). We generated B7H3 CAR-T cells with different lengths of the IL7Rα cytoplasmic domain, including the full length (IL7R-L), intermediate length (IL7R-M), and short length (IL7R-S) domains, and evaluated their functionality in vitro and in vivo. All the B7H3-IL7Rα CAR-T cells exhibited a less differentiated phenotype and effectively eliminated B7H3-positive glioblastoma in vitro. Superiority was found in B7H3 CAR-T cells contained the short length of the IL7Rα cytoplasmic domain. Integration of the IL7R-S cytoplasmic domain maintained pSTAT5 activation and increased T-cell proliferation while reducing activation-induced cell death. Moreover, RNA-sequencing analysis of B7H3-IL7R-S CAR-T cells after coculture with a glioblastoma cell line revealed downregulation of proapoptotic genes and upregulation of genes associated with T-cell proliferation compared with those in 2nd generation B7H3 CAR-T cells. In animal models, compared with conventional CAR-T cells, B7H3-IL7R-S CAR-T cells suppressed tumor growth and prolonged overall survival. Our study demonstrated the therapeutic potential of IL7Rα-incorporating CAR-T cells for glioblastoma treatment, suggesting a promising strategy for augmenting the effectiveness of CAR-T cell therapy.

CAMSAP2 enhances lung cancer cell metastasis by mediating RASAL2 degradation.

AIMS: Cancer metastasis significantly contributes to mortality in lung cancer patients. Calmodulin-regulated spectrin-associated protein family member 2 (CAMSAP2) plays a significant role in cancer cell migration; however, its role in lung cancer metastasis and the underlying mechanism remain largely unknown. The present study aimed to investigate the impact of CAMSAP2 on lung cancer. MAIN METHODS: The clinical relevance of CAMSAP2 in lung cancer patients was assessed using public database. RNA interference experiments were conducted to investigate role of CAMSAP2 in cell migration through transwell and wound healing assays. Molecular mechanisms were explored by identifying the possible interacting partners and pathways using the BioGRID and KEGG pathway analyses. The impact of CAMSAP2 on Ras protein activator-like 2 (RASAL2)-mediated lung cancer metastasis was investigated through biochemical assays. Additionally, in vivo experimentation using a murine tail vein metastasis model was performed to comprehend CAMSAP2's influence on metastasis. KEY FINDINGS: A high expression level of CAMSAP2 was associated with poor overall survival in lung cancer patients and it positively correlated with cell migration in non-small cell lung cancer (NSCLC) cell lines. Knockdown of CAMSAP2 inhibited lung cancer cell motility in vitro and metastasis in vivo. Proteomic and biochemical analyses revealed the interaction between CAMSAP2 and RASAL2, which facilitates the degradation of RASAL2 through the ubiquitin-proteasome system. These degradation processes resulted in the activation of the extracellular signal-regulated kinase (ERK) signaling pathway, thereby promoting lung cancer metastasis. Collectively, the results of this study suggest that CAMSAP2 is a crucial regulator of cancer cell migration and metastasis and a promising therapeutic target for lung cancer.

Genetic polymorphisms influencing deferasirox pharmacokinetics, efficacy, and adverse drug reactions: a systematic review and meta-analysis.

Objective: Deferasirox is an iron-chelating agent prescribed to patients with iron overload. Due to the interindividual variability of deferasirox responses reported in various populations, this study aims to determine the genetic polymorphisms that influence drug responses. Methods: A systematic search was performed from inception to March 2022 on electronic databases. All studies investigating genetic associations of deferasirox in humans were included, and the outcomes of interest included pharmacokinetics, efficacy, and adverse drug reactions. Fixed- and random-effects model meta-analyses using the ratio of means (ROM) were performed. Results: Seven studies involving 367 participants were included in a meta-analysis. The results showed that subjects carrying the A allele (AG/AA) of ABCC2 rs2273697 had a 1.23-fold increase in deferasirox Cmax (ROM = 1.23; 95% confidence interval [CI]:1.06-1.43; p = 0.007) and a lower Vd (ROM = 0.48; 95% CI: 0.36-0.63; p < 0.00001), compared to those with GG. A significant attenuated area under the curve of deferasirox was observed in the subjects with UGT1A3 rs3806596 AG/GG by 1.28-fold (ROM = 0.78; 95% CI: 0.60-0.99; p = 0.04). In addition, two SNPs of CYP24A1 were also associated with the decreased Ctrough: rs2248359 CC (ROM = 0.50; 95% CI: 0.29-0.87; p = 0.01) and rs2585428 GG (ROM = 0.47; 95% CI: 0.35-0.63; p < 0.00001). Only rs2248359 CC was associated with decreased Cmin (ROM = 0.26; 95% CI: 0.08-0.93; p = 0.04), while rs2585428 GG was associated with a shorter half-life (ROM = 0.44; 95% CI: 0.23-0.83; p = 0.01). Conclusion: This research summarizes the current evidence supporting the influence of variations in genes involved with drug transporters, drug-metabolizing enzymes, and vitamin D metabolism on deferasirox responses.

Evaluating the Expression and Prognostic Value of Genes Encoding Microtubule-Associated Proteins in Lung Cancer.

Microtubule-associated proteins (MAPs) play essential roles in cancer development. This study aimed to identify transcriptomic biomarkers among MAP genes for the diagnosis and prognosis of lung cancer by analyzing differential gene expressions and correlations with tumor progression. Gene expression data of patients with lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) from the Cancer Genome Atlas (TCGA) database were used to identify differentially expressed MAP genes (DEMGs). Their prognostic value was evaluated by Kaplan-Meier and Cox regression analysis. Moreover, the relationships between alterations in lung cancer hallmark genes and the expression levels of DEMGs were investigated. The candidate biomarker genes were validated using three independent datasets from the Gene Expression Omnibus (GEO) database and by quantitative reverse transcription polymerase chain reaction (qRT-PCR) on clinical samples. A total of 88 DEMGs were identified from TCGA data. The 20 that showed the highest differential expression were subjected to association analysis with hallmark genes. Genetic alterations in TP53, EGFR, PTEN, NTRK1, and PIK3CA correlated with the expression of most of these DEMGs. Of these, six candidates-NUF2, KIF4A, KIF18B, DLGAP5, NEK2, and LRRK2-were significantly differentially expressed and correlated with the overall survival (OS) of the patients. The mRNA expression profiles of these candidates were consistently verified using three GEO datasets and qRT-PCR on patient lung tissues. The expression levels of NUF2, KIF4A, KIF18B, DLGAP5, NEK2, and LRRK2 can serve as diagnostic biomarkers for LUAD and LUSC. Moreover, the first five can serve as prognostic biomarkers for LUAD, while LRRK2 can be a prognostic biomarker for LUSC. Our research describes the novel role and potential application of MAP-encoding genes in clinical practice.

Accuracy of CCL20 expression level as a liquid biopsy-based diagnostic biomarker for ovarian carcinoma.

Objective: The study aimed to investigate the potentiality of chemokines, including MCP-1, CCL15, CCL20, and CXCL14, as biomarkers for differential diagnosis between benign tumors and ovarian cancer (OC). Methods: A cross-sectional study was conducted in women aged >18 years who had adnexal masses treated with elective surgery at the HRH Maha Chakri Sirindhorn Medical Center, Srinakharinwirot University, between 2020 and 2021. The preoperative MCP-1, CCL15, CCL20, and CXCL14 serum levels were measured using a sandwich enzyme-linked immunosorbent assay. Preoperative diagnosis was defined according to the risk of malignancy index. The histological diagnosis and cancer subtype were confirmed using pathological specimens. Results: Ninety-eight participants were preoperatively diagnosed with malignant tumors. The pathological diagnosis confirmed OC in 33 patients and disclosed 27 misdiagnosed cases, of which endometriotic cyst was the most common (44.44%). CCL20 and CA125 serum levels were significantly higher in the patients with cancer than in those with benign. In addition, CCL20 level could differentiate between benign and early-stage malignancy. Furthermore, only CCL20 levels could distinguish endometriotic cysts from OC, whereas CA125 levels could not. Concordant with the serum protein level, the increased mRNA level of CCL20 was observed in ovarian cancers comparing with that in benign tissues. We found that CCL20 levels could differentiate between benign tumors and OC with 60.61% sensitivity and 75.44% specificity at the optimal cutoff value of 38.79 pg/ml. Finally, the logistic regression model integrating CCL20, CA125, and menopause status promoted diagnostic accuracy by increasing the specificity to 91.23%. Conclusions: Our study revealed the potential usefulness of CCL20 level as a biomarker for diagnosing early-stage OC with endometriosis differentiation. We recommend further studies to confirm the accuracy of CCL20 levels with the current diagnosis in a large patient sample.

Triple-negative breast cancer influences a mixed M1/M2 macrophage phenotype associated with tumor aggressiveness.

Triple-negative breast cancer (TNBC) is characterized by excessive accumulation of tumor-infiltrating immune cells, including tumor-associated macrophages (TAMs). TAMs consist of a heterogeneous population with high plasticity and are associated with tumor aggressiveness and poor prognosis. Moreover, breast cancer cells can secrete factors that influence TAM polarization. Therefore, this study aimed to evaluate the crosstalk between cancer cells and macrophages in the context of TNBC. Cytokine-polarized M2 macrophage were used as control. Distinct from the classical M2 macrophage, TAMs generated from TNBC-conditioned media upregulated both M1- and M2-associated genes, and secreted both the anti-inflammatory cytokine interleukin IL-10 and the proinflammatory cytokine IL-6 and tumor necrosis factor- α. Theses TNBC-induced TAMs exert aggressive behavior of TNBC cells. Consistently, TCGA and MTABRIC analyses of human breast cancer revealed upregulation of M1- associated genes in TNBC comparing with non-TNBC. Among these M1-associated genes, CXCL10 and IL1B were revealed to be independent prognostic factors for disease progression. In conclusion, TNBC cells induce macrophage polarization with a mixture of M1 and M2 phenotypes. These cancer-induced TAMs further enhance tumor cell growth and aggressiveness.

Collagen XVII inhibits breast cancer cell proliferation and growth through deactivation of the AKT/mTOR signaling pathway.

Collagen XVII (COL17), a cell-matrix adhesion protein, has been found to be suppressed in breast cancer. Our previous data demonstrated a preventive role of COL17 in breast cancer invasiveness. The present study used the stable COL17-overexpressing MCF7 and MDA-MB-231 cells to reveal an anti-proliferative effect of COL17 on breast cancer cell through mTOR deactivation. Cell proliferation was negatively correlated with the expression level of COL17 in a concentration-dependent manner in both conventional and three-dimensional (3D) culture systems. The correlation was confirmed by decreased expression of the proliferative marker Ki67 in COL17-expressing cells. In addition, overexpression of COL17 reduced the clonogenicity and growth of the cells. We demonstrated that COL17 affects the AKT/mTOR signaling pathway by deactivation of AKT, mTOR and downstream effectors, particularly 4EBP1. Moreover, mice xenografted with high COL17-expressing cells exhibited delayed tumor progression and prolonged survival time. The high expression of COL17A1 gene encoding COL17 is associated with low-proliferation tumors, extended tumor-free period, and overall survival of breast cancer patients. In conclusion, our results revealed the novel function of COL17 using in vitro and in vivo models and elucidated the related pathway in breast cancer cell growth and proliferation.

Genome-wide association study (GWAS) of ovarian cancer in Japanese predicted regulatory variants in 22q13.1.

Genome-wide association studies (GWAS) have identified greater than 30 variants associated with ovarian cancer, but most of these variants were investigated in European populations. Here, we integrated GWAS and subsequent functional analyses to identify the genetic variants with potential regulatory effects. We conducted GWAS for ovarian cancer using 681 Japanese cases and 17,492 controls and found that rs137672 on 22q13.1 exhibited a strong association with a P-value of 1.05 × 10(-7) and an odds ratio of 0.573 with a 95% confidence interval of 0.466-0.703. In addition, three previously reported SNPs, i.e., rs10088218, rs9870207 and rs1400482, were validated in the Japanese population (P < 0.05) with the same risk allele as noted in previous studies. Functional studies including regulatory feature analysis and electrophoretic mobility shift assay (EMSA) revealed two regulatory SNPs in 22q13.1, rs2072872 and rs6509, that affect the binding affinity to some nuclear proteins in ovarian cancer cells. The plausible regulatory proteins whose motifs could be affected by the allele changes of these two SNPs were also proposed. Moreover, the protective G allele of rs6509 was associated with a decreased SYNGR1 expression level in normal ovarian tissues. Our findings elucidated the regulatory variants in 22q13.1 that are associated with ovarian cancer risk.

Identification of a novel p53 target, COL17A1, that inhibits breast cancer cell migration and invasion.

p53 mutation is a marker of poor prognosis in breast cancers. To identify downstream targets of p53, we screened two transcriptome datasets, including cDNA microarrays of MCF10A breast epithelial cells with wild-type p53 or p53-null background, and RNA sequence analysis of breast invasive carcinoma. Here, we unveil ten novel p53 target candidates that are up-regulated after the induction of p53 in wild-type cells. Their expressions are also high in breast invasive carcinoma tissues with wild-type p53. The GO analysis identified epidermis development and ectoderm development, which COL17A1 participates, as significantly up-regulated by wild-type p53. The COL17A1 expressions increased in a p53-dependent manner in human breast cells and mouse mammary tissues. Reporter assay and ChIP assay identified intronic p53-binding sequences in the COL17A1 gene. The MDA-MB-231 cells that genetically over-express COL17A1 gene product exhibited reduced migration and invasion in vitro. Similarly, COL17A1 expression was decreased in metastatic tumors compared to primary tumors and normal tissues, even from the same patients. Moreover, high COL17A1 expression was associated with longer survival of patients with invasive breast carcinoma. In conclusion, we revealed that COL17A1 is a novel p53 transcriptional target in breast tissues that inhibits cell migration and invasion and is associated with better prognosis.

Identification of a p53-repressed gene module in breast cancer cells.

The p53 protein is a sophisticated transcription factor that regulates dozens of target genes simultaneously in accordance with the cellular circumstances. Although considerable efforts have been made to elucidate the functions of p53-induced genes, a holistic understanding of the orchestrated signaling network repressed by p53 remains elusive. Here, we performed a systematic analysis to identify simultaneously regulated p53-repressed genes in breast cancer cells. Consequently, 28 genes were designated as the p53-repressed gene module, whose gene components were simultaneously suppressed in breast cancer cells treated with Adriamycin. A ChIP-seq database showed that p53 does not preferably bind to the region around the transcription start site of the p53-repressed gene module elements compared with that of p53-induced genes. Furthermore, we demonstrated that p21/CDKN1A plays a pivotal role in the suppression of the p53-repressed gene module in breast cancer cells. Finally, we showed that appropriate suppression of some genes belonging to the p53-repressed gene module contributed to a better prognosis of breast cancer patients. Taken together, these findings disentangle the gene regulatory network underlying the built-in p53-mediated tumor suppression system.

Identification of a p53 target, CD137L, that mediates growth suppression and immune response of osteosarcoma cells.

p53 encodes a transcription factor that transactivates downstream target genes involved in tumour suppression. Although osteosarcoma frequently has p53 mutations, the role of p53 in osteosarcomagenesis is not fully understood. To explore p53-target genes comprehensively in calvarial bone and find out novel druggable p53 target genes for osteosarcoma, we performed RNA sequencing using the calvarial bone and 23 other tissues from p53 +/+ and p53 -/- mice after radiation exposure. Of 23,813 genes, 69 genes were induced more than two-fold in irradiated p53 +/+ calvarial bone, and 127 genes were repressed. Pathway analysis of the p53-induced genes showed that genes associated with cytokine-cytokine receptor interactions were enriched. Three genes, CD137L, CDC42 binding protein kinase gamma and Follistatin, were identified as novel direct p53 target genes that exhibited growth-suppressive effects on osteosarcoma cell lines. Of the three genes, costimulatory molecule Cd137l was induced only in calvarial bone among the 24 tissues tested. CD137L-expressing cells exhibited growth-suppressive effects in vivo. In addition, recombinant Fc-fusion Cd137l protein activated the immune response in vitro and suppressed osteosarcoma cell growth in vivo. We clarified the role of CD137L in osteosarcomagenesis and its potential therapeutic application. Our transcriptome analysis also indicated the regulation of the immune response through p53.