A Novel Hydrogel-Based 3D In Vitro Tumor Panel of 30 PDX Models Incorporates Tumor, Stromal and Immune Cell Compartments of the TME for the Screening of Oncology and Immuno-Therapies.

Human-relevant systems that mimic the 3D tumor microenvironment (TME), particularly the complex mechanisms of immuno-modulation in the tumor stroma, in a reproducible and scalable format are of high interest for the drug discovery industry. Here, we describe a novel 3D in vitro tumor panel comprising 30 distinct PDX models covering a range of histotypes and molecular subtypes and cocultured with fibroblasts and PBMCs in planar (flat) extracellular matrix hydrogels to reflect the three compartments of the TME-tumor, stroma, and immune cells. The panel was constructed in a 96-well plate format and assayed tumor size, tumor killing, and T-cell infiltration using high-content image analysis after 4 days of treatment. We screened the panel first against the chemotherapy drug Cisplatin to demonstrate feasibility and robustness, and subsequently assayed immuno-oncology agents Solitomab (CD3/EpCAM bispecific T-cell engager) and the immune checkpoint inhibitors (ICIs) Atezolizumab (anti-PDL1), Nivolumab (anti-PD1) and Ipilimumab (anti-CTLA4). Solitomab displayed a strong response across many PDX models in terms of tumor reduction and killing, allowing for its subsequent use as a positive control for ICIs. Interestingly, Atezolizumab and Nivolumab demonstrated a mild response compared to Ipilimumab in a subset of models from the panel. We later determined that PBMC spatial proximity in the assay setup was important for the PD1 inhibitor, hypothesizing that both duration and concentration of antigen exposure may be critical. The described 30-model panel represents a significant advancement toward screening in vitro models of the tumor microenvironment that include tumor, fibroblast, and immune cell populations in an extracellular matrix hydrogel, with robust and standardized high content image analysis in a planar hydrogel. The platform is aimed at rapidly screening various combinations and novel agents and forming a critical conduit to the clinic, thus accelerating drug discovery for the next generation of therapeutics.

Gαi2-induced conductin/axin2 condensates inhibit Wnt/ß-catenin signaling and suppress cancer growth.

Conductin/axin2 is a scaffold protein negatively regulating the pro-proliferative Wnt/ß-catenin signaling pathway. Accumulation of scaffold proteins in condensates frequently increases their activity, but whether condensation contributes to Wnt pathway inhibition by conductin remains unclear. Here, we show that the Gαi2 subunit of trimeric G-proteins induces conductin condensation by targeting a polymerization-inhibiting aggregon in its RGS domain, thereby promoting conductin-mediated ß-catenin degradation. Consistently, transient Gαi2 expression inhibited, whereas knockdown activated Wnt signaling via conductin. Colorectal cancers appear to evade Gαi2-induced Wnt pathway suppression by decreased Gαi2 expression and inactivating mutations, associated with shorter patient survival. Notably, the Gαi2-activating drug guanabenz inhibited Wnt signaling via conductin, consequently reducing colorectal cancer growth in vitro and in mouse models. In summary, we demonstrate Wnt pathway inhibition via Gαi2-triggered conductin condensation, suggesting a tumor suppressor function for Gαi2 in colorectal cancer, and pointing to the FDA-approved drug guanabenz for targeted cancer therapy.

Receptor-interacting protein kinase 2 (RIPK2) stabilizes c-Myc and is a therapeutic target in prostate cancer metastasis.

Despite progress in prostate cancer (PC) therapeutics, distant metastasis remains a major cause of morbidity and mortality from PC. Thus, there is growing recognition that preventing or delaying PC metastasis holds great potential for substantially improving patient outcomes. Here we show receptor-interacting protein kinase 2 (RIPK2) is a clinically actionable target for inhibiting PC metastasis. RIPK2 is amplified/gained in ~65% of lethal metastatic castration-resistant PC. Its overexpression is associated with disease progression and poor prognosis, and its genetic knockout substantially reduces PC metastasis. Multi-level proteomics analyses reveal that RIPK2 strongly regulates the stability and activity of c-Myc (a driver of metastasis), largely via binding to and activating mitogen-activated protein kinase kinase 7 (MKK7), which we identify as a direct c-Myc-S62 kinase. RIPK2 inhibition by preclinical and clinical drugs inactivates the noncanonical RIPK2/MKK7/c-Myc pathway and effectively impairs PC metastatic outgrowth. These results support targeting RIPK2 signaling to extend metastasis-free and overall survival.

ROCK1 mechano-signaling dependency of human malignancies driven by TEAD/YAP activation.

Rho family mechano-signaling through the actin cytoskeleton positively regulates physiological TEAD/YAP transcription, while the evolutionarily conserved Hippo tumor suppressor pathway antagonizes this transcription through YAP cytoplasmic localization/degradation. The mechanisms responsible for oncogenic dysregulation of these pathways, their prevalence in tumors, as well as how such dysregulation can be therapeutically targeted are not resolved. We demonstrate that p53 DNA contact mutants in human tumors, indirectly hyperactivate RhoA/ROCK1/actomyosin signaling, which is both necessary and sufficient to drive oncogenic TEAD/YAP transcription. Moreover, we demonstrate that recurrent lesions in the Hippo pathway depend on physiological levels of ROCK1/actomyosin signaling for oncogenic TEAD/YAP transcription. Finally, we show that ROCK inhibitors selectively antagonize proliferation and motility of human tumors with either mechanism. Thus, we identify a cancer driver paradigm and a precision medicine approach for selective targeting of human malignancies driven by TEAD/YAP transcription through mechanisms that either upregulate or depend on homeostatic RhoA mechano-signaling.

Ruxolitinib reduces severe CRS response by suspending CAR-T cell function instead of damaging CAR-T cells.

The therapeutic effect of CAR-T is often accompanied by sCRS, which is the main obstacle to the promotion of CAR-T therapy. The JAK1/2 inhibitor ruxolitinib has recently been confirmed as clinically effective in maintaining control over sCRS, however, its mechanism remains unclear. In this study, we firstly revealed that ruxolitinib significantly inhibited the proliferation of CAR-T cells without damaging viability, and induced an efficacy-favored differentiation phenotype. Second, ruxolitinib reduced the level of cytokine release not only from CAR-T cells, but also from other cells in the immune system. Third, the cytolytic activity of CAR-T cells was restored once the ruxolitinib was removed; however, the cytokines released from the CAR-T cells maintained an inhibited state to some degree. Finally, ruxolitinib significantly reduced the proliferation rate of CAR-T cells in vivo without affecting the therapeutic efficacy after withdrawal at the appropriate dose. We demonstrated pre-clinically that ruxolitinib interferes with both CAR-T cells and the other immune cells that play an important role in triggering sCRS reactions. This work provides useful and important scientific data for clinicians on the question of whether ruxolitinib has an effect on CAR-T cell function loss causing CAR-T treatment failure when applied in the treatment of sCRS, the answer to which is of great clinical significance.

Antibody-Drug Conjugate Targeting c-Kit for the Treatment of Small Cell Lung Cancer.

Lung cancer is the leading cause of cancer-related deaths. Small cell lung cancer (SCLC) accounts for 15-25% of all lung cancers. It exhibits a rapid doubling time and a high degree of invasiveness. Additionally, overexpression of c-Kit occurs in 70% of SCLC patients. In this study, we evaluated an antibody-drug conjugate (ADC) that targets c-Kit, which is a potential therapeutic agent for SCLC. First, we generated and characterized 4C9, a fully human antibody that targets c-Kit and specifically binds to SCLC cells expressing c-Kit with a binding affinity of KD = 5.5 × 10-9 M. Then, we developed an ADC using DM1, a microtubule inhibitor, as a payload. 4C9-DM1 efficiently induced apoptosis in SCLC with an IC50 ranging from 158 pM to 4 nM. An in vivo assay using a xenograft mouse model revealed a tumor growth inhibition (TGI) rate of 45% (3 mg/kg) and 59% (5 mg/kg) for 4C9-DM1 alone. Combination treatment with 4C9-DM1 plus carboplatin/etoposide or lurbinectedin resulted in a TGI rate greater than 90% compared with the vehicle control. Taken together, these results indicate that 4C9-DM1 is a potential therapeutic agent for SCLC treatment.

6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 acts as a protein kinase to regulate glioblastoma progression by activating the AKT/forkhead box O1 pathway.

Abnormal expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) is closely related to the occurrence and development of tumors, and PFKFB4 has been shown to function as a protein kinase. However, the molecular mechanisms through which PFKFB4 functions in glioblastoma (GBM) remain poorly understood. Accordingly, in this study, we assessed the roles of PFKFB4 in GBM. Compared to in adjacent tissues, PFKFB4 was highly expressed in GBM, and its expression level was negatively correlated with the overall survival time. In addition, knockdown of PFKFB4 inhibited the proliferation and invasion of GBM cells and promoted apoptosis. In a xenograft tumor model, tumor growth was inhibited by knockdown of PFKFB4 using short hairpin RNA. Further studies demonstrated that PFKFB4 is involved in regulating the AKT signaling pathway. Thus, PFKFB4 acts as a protein kinase to regulate GBM progression by activating the AKT/forkhead box O1 pathway, which may be a potential therapeutic target in GBM.

Omega-3 fatty acids decrease CRYAB, production of oncogenic prostaglandin E2 and suppress tumor growth in medulloblastoma.

AIMS: Medulloblastoma (MB) is one of the most common malignant central nervous system tumors of childhood. Despite intensive treatments that often leads to severe neurological sequelae, the risk for resistant relapses remains significant. In this study we have evaluated the effects of the ω3-long chain polyunsaturated fatty acids (ω3-LCPUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on MB cell lines and in a MB xenograft model. MAIN METHODS: Effects of ω3-LCPUFA treatment of MB cells were assessed using the following: WST-1 assay, cell death probes, clonogenic assay, ELISA and western blot. MB cells were implanted into nude mice and the mice were randomized to DHA, or a combination of DHA and EPA treatment, or to control group. Treatment effects in tumor tissues were evaluated with: LC-MS/MS, RNA-sequencing and immunohistochemistry, and tumors, erythrocytes and brain tissues were analyzed with gas chromatography. KEY FINDINGS: ω3-LCPUFA decreased prostaglandin E2 (PGE2) secretion from MB cells, and impaired MB cell viability and colony forming ability and increased apoptosis in a dose-dependent manner. DHA reduced tumor growth in vivo, and both PGE2 and prostacyclin were significantly decreased in tumor tissue from treated mice compared to control animals. All ω3-LCPUFA and dihomo-γ-linolenic acid increased in tumors from treated mice. RNA-sequencing revealed 10 downregulated genes in common among ω3-LCPUFA treated tumors. CRYAB was the most significantly altered gene and the downregulation was confirmed by immunohistochemistry. SIGNIFICANCE: Our findings suggest that addition of DHA and EPA to the standard MB treatment regimen might be a novel approach to target inflammation in the tumor microenvironment.

Protocol for chronic hepatitis B virus infection mouse model development by patient-derived orthotopic xenografts.

BACKGROUND: According to the World Health Organization, more than 250 million people worldwide are chronically infected with the hepatitis B virus, and almost 800.000 patients die annually of mediated liver disorders. Therefore, adequate biological test systems are needed that could fully simulate the course of chronic hepatitis B virus infection, including in patients with hepatocellular carcinoma. METHODS: In this study, we will assess the effectiveness of existing protocols for isolation and cultivation of primary cells derived from patients with hepatocellular carcinoma in terms of the yield of viable cells and their ability to replicate the hepatitis B virus using isolation and cultivation methods for adhesive primary cells, flow cytometry and quantitative polymerase chain reaction. Another part of our study will be devoted to evaluating the effectiveness of hepatocellular carcinoma grafting methods to obtain patient-derived heterotopic and orthotopic xenograft mouse avatars using animal X-ray irradiation and surgery procedures and in vivo fluorescent signals visualization and measurements. Our study will be completed by histological methods. DISCUSSION: This will be the first extensive comparative study of the main modern methods and protocols for isolation and cultivation primary hepatocellular carcinoma cells and tumor engraftment to the mice. All protocols will be optimized and characterized using the: (1) efficiency of the method for isolation cells from removed hepatocellular carcinoma in terms of their quantity and viability; (2) efficiency of the primary cell cultivation protocol in terms of the rate of monolayer formation and hepatitis B virus replication; (3) efficiency of the grafting method in terms of the growth rate and the possibility of hepatitis B virus persistence and replication in mice. The most effective methods will be recommended for use in translational biomedical research.

Caffeic acid phenethyl ester targets ubiquitin-specific protease 8 and synergizes with cisplatin in endometrioid ovarian carcinoma cells.

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.

Chelerythrine inhibits the progression of glioblastoma by suppressing the TGFB1-ERK1/2/Smad2/3-Snail/ZEB1 signaling pathway.

AIMS: Glioblastoma (GBM) is the most common and aggressive intracranial tumor with poor prognosis. A large majority of clinical chemotherapeutic agents cannot achieve the desired therapeutic effect. Chelerythrine (CHE), a natural component with multitudinous pharmacological functions, has been proven to have outstanding antitumor effects in addition to antibacterial, anti-inflammatory, and hypotensive effects. However, the anti-GBM effect of CHE has not been reported to date. The purpose of this paper is to observe the anti-GBM effect of CHE and further explore the related mechanism. MATERIALS AND METHODS: GBM cell lines (U251 and T98G) and BALB/c nude mice were used in the experiments. Methyl thiazolyl tetrazolium (MTT) and clone formation assays were applied to detect the viability, proliferation and stemness of GBM cells. Flow cytometry was utilized to identify the effect of CHE on GBM apoptosis. Scratch and Transwell experiments reflected the migration and invasion of cells. In vivo, xenograft tumors were implanted subcutaneously in nude mice. The progression of tumors was assessed by ultrasound and magnetic resonance imaging. Finally, western blot, bioinformatics, and immunohistochemistry experiments were used to explore the molecular mechanisms in depth. KEY FINDINGS: In vitro tests showed that CHE inhibited the proliferation, stemness, migration, and invasion of GBM cells and induced apoptosis. In vitro, CHE was observed to restrain the progression of xenograft tumors. We eventually proved that the cytotoxicity of CHE was relevant to the TGFB1-ERK1/2/Smad2/3-Snail/ZEB1 signaling pathway. SIGNIFICANCE: CHE inhibited GBM progression by inhibiting the TGFB1-ERK1/2/Smad2/3-Snail/ZEB1 signaling pathway and is a potential chemotherapeutic drug for GBM.

KIR2DL4 promotes the proliferation of RCC cell associated with PI3K/Akt signaling activation.

BACKGROUND: Killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4) is a transmembrane glycoprotein that is expressed by natural killer (NK) cells and certain subsets of T cells. However, its expression profiles and functions in solid tumor progression remain poorly defined. METHODS: In the present study, using bioinformatics analysis, immunohistochemistry, immunoblotting, MTT cell viability assay, soft agar colony formation assay and a human renal cell carcinoma (RCC) cell xenograft model in nude mice, we examined whether KIR2DL4 is expressed by RCC and its possible roles in RCC progression. RESULTS: We confirmed that KIR2DL4 is overexpressed by RCC cells. MTT and soft agar cloning assays showed that KIR2DL4 knockdown delayed cell proliferation and viability in RCC cell lines, Caki-1 and 769-P, in vitro. By contrast, KIR2DL4 overexpression promoted Caki-1 cell proliferation both in vitro and in vivo, which was observed in a BALB/c-nu/nu xenograft mouse model. Moreover, RNA sequencing data demonstrated that the differentially expressed genes found between parallel-controlled and Caki-1 cells overexpressing KIR2DL4 were highly associated with cancer development, of which those related to the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway were particularly enriched, immunoblotting data showed that the level of AKT phosphorylation was higher or lower in KIR2DL4 overexpressing or KIR2DL4 knocking-down Caki-1 cells compared with that in the parallel-controlled cells. In addition, PI3K inhibitor wortmannin treatment and KIR2DL4-shRNA transfection further deregulated the levels of phosphorylated AKT and Caki-1 cell proliferation. CONCLUSIONS: Our results indicate that KIR2DL4 is also expressed by RCC cells, which promotes RCC progression associated with PI3K/AKT activation.

BRAF Inhibitors Reprogram Cancer-Associated Fibroblasts to Drive Matrix Remodeling and Therapeutic Escape in Melanoma.

The tumor stroma and its cellular components are known to play an important role in tumor response to treatment. Here, we report a novel resistance mechanism in melanoma that is elicited by BRAF inhibitor (BRAFi)-induced noncanonical activation of nuclear ß-catenin signaling in cancer-associated fibroblasts (CAF). Treatment with BRAFi leads to an expanded CAF population with increased ß-catenin nuclear accumulation and enhanced biological properties. This CAF subpopulation is essential for melanoma cells to proliferate and acquire resistance to BRAFi/MEK inhibitors (MEKi). Mechanistically, BRAFi induces BRAF-CRAF heterodimerization and subsequent activation of ERK signaling in CAFs, leading to inactivation of the ß-catenin destruction complex. RNA-seq identified periostin (POSTN) as a major downstream effector of ß-catenin in CAFs. POSTN compensates for the loss of ß-catenin in CAFs and mediates melanoma cell BRAFi/MEKi resistance. In melanoma cells, POSTN activates phosphoinositide 3-kinase (PI3K)/AKT signaling and subsequently reactivates the ERK pathway that was inhibited by BRAFi/MEKi. Collectively, these data underscore the role of BRAFi-induced CAF reprogramming in matrix remodeling and therapeutic escape of melanoma cells. SIGNIFICANCE: ß-Catenin activation in cancer-associated fibroblasts in response to BRAF inhibitors stimulates POSTN secretion to promote resistance in cancer cells, revealing POSTN as a potential matrix target in cancer therapy.

Pan-cancer analysis, cell and animal experiments revealing TEAD4 as a tumor promoter in ccRCC.

AIMS: Transcriptional enhanced associate domain (TEAD) transcription factor family, a very important family in the hippo signaling pathway, has been found to play oncogenic functions in the occurrence of various malignant tumors. However, the expression of TEADs in pan-cancer and the important role of TEAD4 in clear cell renal cell carcinoma (ccRCC) have not been analyzed. Herein, we aim to evaluate the expression of TEADs in pan-cancer, and focus on analyzing the role of TEAD4 in the progression of ccRCC. MAIN METHODS: Data from the Cancer Genome Atlas (TCGA) was used to analyze the expression of TEADs in pan-cancer and its clinical correlation. TEAD4 expression in ccRCC tissues, biological functions in vitro and in vivo were analyzed by immunohistochemistry (IHC), western blotting, RNAi and Xenograft assay. Mircode, BioGRID and g: Profiler website were used to build a ceRNA network and downstream pathway prediction. KEY FINDINGS: TEAD1, TEAD2, TEAD3 and TEAD4 were highly expressed in 3, 6, 5, and 12 types of cancer tissues, respectively, indicating that TEAD4 is most closely related to tumor progression. Among the cancers with high TEAD4 expression, the expression of TEAD4 has the greatest correlation with the poor prognosis of ccRCC. We also found the malignant phenotypes of ccRCC cells in vitro and vivo have been significantly suppressed by silencing TEAD4. SIGNIFICANCE: TEADs, especially TEAD4, were overexpressed in many human tumors. This study is the first to show that TEAD4 acts as an oncogene in ccRCC and may be an important factor in progress of ccRCC.

8-Geranyloxycarbostyril as a potent 15-LOX-1 inhibitor showed great anti-tumor effects against prostate cancer.

Carbostyrils are quinolone derivatives, with possible growth inhibition properties on cancer cells. Unlike many tumors, 15-Lipoxygenase-1 (15-LOX-1) is highly expressed in prostate cancer (PCa) cells and has oncogenic properties. Here, with the hypothesis that 6-, 7- and 8-geranyloxycarbostyril (GQ) have inhibitory properties on 15-LOX-1, their effects were assessed on PCa cells. Their cytotoxic effects were evaluated by MTT assay and mechanism of cell death was investigated using annexin V/PI staining. Finally, the anti-tumor properties of 8-GQ were assessed in immunocompromised C57BL/6 mice bearing human PCa cells. Accordingly, these compounds could effectively inhibit 15-LOX activity in PCa cells. MTT and flow cytometry tests confirmed their toxic effects on PCa cells, with no significant toxicity on normal cells, and apoptosis was the main mechanism of cell death. In vivo results indicated that use of 8-GQ at 50 mg/kg had stronger anti-tumor effects than 5 mg/kg cisplatin, with fewer side effects on normal tissues. Therefore, 8-GQ can be introduced as a potential drug candidate with 15-LOX-1 inhibitory potency, which can be effective in treatment of prostate cancer, and should be considered for further drug screening investigations.

KHDRBS3 promotes paclitaxel resistance and induces glycolysis through modulated MIR17HG/CLDN6 signaling in epithelial ovarian cancer.

Paclitaxel (PTX) resistance contributes to mortality in epithelial ovarian cancer (EOC). Aerobic glycolysis is elevated in the tumor environment and may influence resistance to PTX in EOC. KH domain-containing, RNA-binding signal transduction-associated protein 3 (KHDRBS3) is an RNA binding protein that is up-regulated in EOC, but its underlying mechanism in EOC is unclear. Here, we investigate the role of KHDRBS3 in glycolysis and increased resistance to PTX. Expression of KHDRBS3 and Claudin (CLDN6) were measured in EOC tissue and cells by quantitative real-time PCR, western blotting and immunohistochemistry. The biological functions of KHDRBS3, MIR17HG and CLDN6 were examined using MTT, colony formation, apoptosis and seahorse assays in vitro. For in vivo experiments, a xenograft model was used to investigate the effects of KHDRBS3 and MIR17HG in EOC. Here, we investigate the role of KHDRBS3 in glycolysis and increased resistance to PTX. The expression of KHDRBS3 was up-regulated in PTX-resistant cells. KHDRBS3 knockdown restrained the IC50 of PTX, cell proliferation, colony formation and glycolysis in SKOV3-R and A2780-R cells in vitro and enhanced PTX sensitivity in a xenograft mouse model in vivo. KHDRBS3 interacts with lncRNA MIR17HG, which is down-regulated in EOC tissue and cells. The effect of KHDRBS3 overexpression on PTX resistance and glycolysis was rescued by MIR17HG overexpression. Additionally, MIR17HG interacts with the 3'UTR of CLDN6 and negatively regulates CLDN6 expression. MIR17HG overexpression suppressed the IC50 of PTX and glycolysis by targeting CLDN6. Our results reveal a KHDRBS3-MIR17HG-CLDN6 regulatory axis that contributes to enhanced glycolysis in EOC and represents a potential target for therapy.

LncRNA Uc003xsl.1-Mediated Activation of the NFκB/IL8 Axis Promotes Progression of Triple-Negative Breast Cancer.

Aberrant activation of NFκB orchestrates a critical role in tumor carcinogenesis; however, the regulatory mechanisms underlying this activation are not fully understood. Here we report that a novel long noncoding RNA (lncRNA) Uc003xsl.1 is highly expressed in triple-negative breast cancer (TNBC) and correlates with poor outcomes in patients with TNBC. Uc003xsl.1 directly bound nuclear transcriptional factor NFκB-repressing factor (NKRF), subsequently preventing NKRF from binding to a specific negative regulatory element in the promoter of the NFκB-responsive gene IL8 and abolishing the negative regulation of NKRF on NFκB-mediated transcription of IL8. Activation of the NFκB/IL8 axis promoted the progression of TNBC. Trop2-based antibody-drug conjugates have been applied in clinical trials in TNBC. In this study, a Trop2-targeting, redox-responsive nanoparticle was developed to systematically deliver Uc003xsl.1 siRNA to TNBC cells in vivo, which reduced Uc003xsl.1 expression and suppressed TNBC tumor growth and metastasis. Therefore, targeting Uc003xsl.1 to suppress the NFκB/IL8 axis represents a promising therapeutic strategy for TNBC treatment. SIGNIFICANCE: These findings identify an epigenetic-driven NFκB/IL8 cascade initiated by a lncRNA, whose aberrant activation contributes to tumor metastasis and poor survival in patients with triple-negative breast cancer.

Mithramycin suppresses tumor growth by regulating CD47 and PD-L1 expression.

Mithramycin A (MIT) has reacquired extensive research attention due to its anti-solid tumor activity and improved pharmacological production. Mechanismly, MIT was broadly used as a c-Myc inhibitor, and c-Myc regulated CD47 and PD-L1 expression which has been demonstrated. However, how MIT affects immune check-point molecules remains unknown. In this study, we found CD47 expression was higher in melanoma of pan-tissue array. MIT inhibited CD47 expression both in mRNA and protein level in melanoma cells (SK-MEL-28 and B16). MIT inhibited c-Myc, Sp-1 and CD47 expression in a concentration-dependent way. MIT inhibited the surface CD47 expression and promoted the phagocytosis of SK-MEL-28 cells by THP-1 cells. We found MIT inhibited tumor growth in melanoma allograft mice and CD47 expression in tumor mass. We also found MIT upregulated PD-L1 expression in cancer cells possibly via inhibiting PD-L1 ubiquitination, increasing ROS and IFN-γ. Combination of MIT and anti-PD-1 antibody showed enhanced antitumor activity compared to MIT and anti-PD-1 antibody alone in MC38 allograft mice. Using immune checkpoint array we found MIT inhibited expression of FasL and Galectin3. These results suggest that MIT inhibits CD47 expression, while improves PD-L1 expression. Furthermore, the combination of MIT and anti-PD-1 antibody exerts potent antitumor effect.

Per- and polyfluoroalkyl substances target and alter human prostate stem-progenitor cells.

Per- and polyfluorinated alkyl substances (PFAS) are a large family of widely used synthetic chemicals that are environmentally and biologically persistent and present in most individuals. Chronic PFAS exposure have been linked to increased prostate cancer risk in occupational settings, however, underlying mechanisms have not been interrogated. Herein we examined exposure of normal human prostate stem-progenitor cells (SPCs) to 10 nM PFOA or PFOS using serial passage of prostasphere cultures. Exposure to either PFAS for 3-4 weeks increased spheroid numbers and size indicative of elevated stem cell self-renewal and progenitor cell proliferation. Transcriptome analysis using single-cell RNA sequencing (scRNA-seq) showed 1) SPC expression of PPARs and RXRs able to mediate PFAS effects, 2) the emergence of a new cell cluster of aberrantly differentiated luminal progenitor cells upon PFOS/PFOA exposure, and 3) enrichment of cancer-associated signaling pathways. Metabolomic analysis of PFAS-exposed prostaspheres revealed increased glycolytic pathways including the Warburg effect as well as strong enrichment of serine and glycine metabolism which may promote a pre-malignant SPC fate. Finally, growth of in vivo xenografts of tumorigenic RWPE-2 human prostate cells, shown to contain cancer stem-like cells, was markedly enhanced by daily PFOS feeding to nude mice hosts. Together, these findings are the first to identify human prostate SPCs as direct PFAS targets with resultant reprogrammed transcriptomes and metabolomes that augment a preneoplastic state and may contribute to an elevated prostate cancer risk with chronic exposures.

GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity.

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6 × 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors.