PFKFB3 deprivation attenuates the cisplatin resistance via blocking its autophagic elimination in colorectal cancer cells.

Introduction: 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is highly expressed in several cancers and plays important roles during the whole pathological process of cancer. It is also involved in chemoresistance, while the intrinsic mechanism needs to be further revealed. Methods: The different responses to cisplatin (DDP) between wild type (WT) and DDP-resistant (DDR) colorectal cancer (CRC) cells were analyzed by several assays. Coumarin conjugated DDP (CP-DDP) was utilized to trace the distribution of DDP. Pharmacological and genetic methods were used to deprive autophagy and PFKFB3, and the effects were investigated. The mouse xenograft model was performed to confirm the effect of the PFKFB3 inhibitor on reversing DDP resistance. Results: DDR cells showed a lower capacity for apoptosis upon DDP treatment, but exhibited higher levels of autophagy and PFKFB3. CP-DDP partly co-localized with LC3, and its content lessened faster in DDR cells. Deprivation of both autophagy and PFKFB3 attenuated CP-DDP elimination, and reversed the DDP resistance. Moreover, PFKFB3 inhibition reduced DDP-induced autophagy. PFKFB3 inhibitor in combination with DDP led to a remarkable reduction in tumor growth in vivo. Discussions: Inhibition of PFKFB3 reduced the autophagy induced by DDP, and therefore extended the retention time of CP-DDP. Meanwhile, PFKFB3 deprivation reversed the DDP resistance and made it a potent therapeutic target for CRC.

MYBL2 is a Novel Independent Prognostic Biomarker and Correlated with TMB in pancreatic cancer.

Background: Pancreatic cancer continues to pose a significant threat due to its high mortality rate. While MYB family genes have been identified as oncogenes in certain cancer types, their role in pancreatic cancer remains largely unexplored. Methods: The mRNA and protein expression of MYB family genes in pancreatic cancer samples was analyzed using TNMplot, HPA, and TISBID online bioinformatics tools, sourced from the TCGA and GETx databases. The relationship between MYB family gene expression and survival time was assessed through Kaplan-Meier analysis, while the prognostic impact of MYB family gene expression was evaluated using the Cox proportional hazards model. Additionally, Spearman's correlation analysis was employed to investigate the correlation between MYB family genes and TMB/MSI. Results: The integration of data from various databases demonstrated that all MYB family genes exhibited dysregulated expression in pancreatic cancer. However, only the expression of the MYBL2 gene displayed a notable association with the grade and stage of pancreatic cancer. Furthermore, the MYBL2 gene exhibited significant variations in both univariate and multivariate factor analyses.Subsequent functional analyses revealed a significant correlation between MYBL2 expression in pancreatic cancers and various biological processes, such as DNA replication, tumor proliferation, G2M checkpoint regulation, pyrimidine metabolism, and the P53 pathway. Additionally, a notable positive association was observed between MYBL2 expression and tumor mutational burden (TMB), a predictive indicator for response to PD1 antibody treatment. Conclusion: MYBL2 may be a double marker for independent diagnosis and PD1 antibody response prediction of pancreatic cancer patients.

Targeting Super-Enhancer-Driven Transcriptional Dependencies Suppresses Aberrant Hedgehog Pathway Activation and Overcomes Smoothened Inhibitor Resistance.

Aberrant activation of the Hedgehog (Hh) signaling pathway plays important roles in oncogenesis and therapeutic resistance in several types of cancer. The clinical application of FDA-approved Hh-targeted smoothened inhibitors (SMOi) is hindered by the emergence of primary or acquired drug resistance. Epigenetic and transcriptional-targeted therapies represent a promising direction for developing improved anti-Hh therapies. In this study, we integrated epigenetic/transcriptional-targeted small-molecule library screening with CRISPR/Cas9 knockout library screening and identified CDK9 and CDK12, two transcription elongation regulators, as therapeutic targets for antagonizing aberrant Hh activation and overcoming SMOi resistance. Inhibition of CDK9 or CDK12 potently suppressed Hh signaling and tumor growth in various SMOi responsive or resistant Hh-driven tumor models. Systemic epigenomic profiling elucidated the Hh-driven super-enhancer (SE) landscape and identified IRS1, encoding a critical component and cytoplasmic adaptor protein of the insulin-like growth factor (IGF) pathway, as an oncogenic Hh-driven SE target gene and effective therapeutic target in Hh-driven tumor models. Collectively, this study identifies SE-driven transcriptional dependencies that represent promising therapeutic vulnerabilities for suppressing the Hh pathway and overcoming SMOi resistance. As CDK9 and IRS inhibitors have already entered human clinical trials for cancer treatment, these findings provide comprehensive preclinical support for developing trials for Hh-driven cancers. Significance: Dissecting transcriptional dependencies driven by super-enhancers uncovers therapeutic targets in Hedgehog-driven cancers and identifies strategies for overcoming resistance to smoothened inhibitors.

Acylcarnitines promote gallbladder cancer metastasis through lncBCL2L11-THOC5-JNK axis.

BACKGROUND: The progression of gallbladder cancer (GBC) is accompanied by abnormal fatty acid ß-oxidation (FAO) metabolism. Different types of lipids perform various biological functions. This study aimed to determine the role of acyl carnitines in the molecular mechanisms of GBC progression. METHODS: Distribution of lipids in GBC was described by LC-MS-based lipidomics. Cellular localization, expression level and full-length of lncBCL2L11 were detected using fluorescence in situ hybridization (FISH) assays, subcellular fractionation assay and 5' and 3' rapid amplification of the cDNA ends (RACE), respectively. In vitro and in vivo experiments were used to verify the biological function of lncBCL2L11 in GBC cells. Methylated RNA Immunoprecipitation (MeRIP) was performed to detect the methylation levels of lncBCL2L11. RNA pull-down assay and RNA immunoprecipitation (RIP) assay were used to identify lncBCL2L11 interacting proteins. Co-Immunoprecipitation (Co-IP) and Western blot assay were performed to validate the regulatory mechanism of lncBCL2L11 and THO complex. RESULTS: Acylcarnitines were significantly up-regulated in GBC tissues. High serum triglycerides correlated to decreased survival in GBC patients and promoted tumor migration. LncBCL2L11 was identified in the joint analysis of highly metastatic cells and RNA sequencing data. LncBCl2L11 prevented the binding of THOC6 and THOC5 and causes the degradation of THOC5, thus promoting the accumulation of acylcarnitines in GBC cells, leading to the malignant progression of cancer cells. In addition, highly expressed acylcarnitines stabilized the expression of lncBCL2L11 through N6-methyladenosine methylation (m6A), forming a positive feedback regulation in tumor dissemination. CONCLUSIONS: LncBCL2L11 is involved in gallbladder cancer metastasis through FAO metabolism. High lipid intake is associated with poor prognosis of GBC. Therefore, targeting lncBCL2L11 and its pathway-related proteins or reducing lipid intake may be significant for the treatment of GBC patients.

Community ecological succession of endophytic fungi associates with medicinal compound accumulation in Sophora alopecuroides.

Endophytic fungi of medicinal plants are symbiotic with the host and play an important role in determining metabolites. To understand the relationship between the accumulation of Sophora alopecuroides' medicinal bioactive compounds and the ecological succession of endophytic fungi, here we collected samples from S. alopecuroides at four developmental stages (adult, flowering, podding, and mature) and different organs (roots, stems, leaves, and seeds) at the mature stage. We then used high-performance liquid chromatography-mass spectrometry and high-throughput sequencing on the internal transcribed spacer region to identify the medicinal compounds and endophytic fungal communities in each sample. The endophytic fungal community characteristics and accumulation of medicinally bioactive compounds of S. alopecuroides varied with the host's developmental stages and organs, with the highest total alkaloids content of 111.9 mg/g at the mature stage. Membership analysis and network connection analysis showed a total of 15 core endophytic fungi in different developmental stages and 16 core endophytic fungi in different organs at the mature stage. The unclassified Ascomycota, Aspergillus, and Alternaria were significantly and positively correlated with the medicinal compounds of S. alopecuroides at the mature stage (r > 0.6 or r < -0.6; P < 0.05). In this study, we identified key endophytic fungal resources that affect the content of medicinally bioactive compounds in S. alopecuroides. This discovery could lay the foundation for enhancing the yield of medicinally bioactive compounds in S. alopecuroides and the development and application of functional endophytic fungi.IMPORTANCESophora alopecuroides is a traditional Chinese herbal medicine. The major medicinal chemicals are considered to be quinolizidine alkaloids. Quinolizidine alkaloids have been widely used for the treatment of tumors, dysentery, and enteritis. Previous studies have found that endophytic fungi in S. alopecuroides can promote the accumulation of host quinolizidine alkaloids. However, the relationship between the accumulation of S. alopecuroides' medicinal bioactive compounds and the ecological succession of endophytic fungi remains unclear. In this study, we screened the key endophytic fungal resources affecting the content of medicinally bioactive compounds and laid the foundation for subsequent research on the mechanism by which endophytic fungi promote the accumulation of medicinally bioactive compounds in S. alopecuroides.

GNL3L promotes autophagy via regulating AMPK signaling in esophageal cancer cells.

Guanine nucleotide-binding protein-like 3-like (GNL3L), a conserved GTP-binding nucleolar protein, participates in regulating cell proliferation, and associates with tumorigenesis and poor prognosis in several kind of cancers. However, the role of GNL3L in modulating autophagy remains unclear. Here, we verified that GNL3L was higher expressed in esophageal cancer (ESCA) biopsies than that in the corresponding normal biopsies by a human ESCA tissue array. Utilizing immunoblotting and real-time PCR assays, we analyzed the expression of GNL3L in several ESCA cell lines, and it was highly expressed in KYSE410 cells and rarely expressed in KYSE150 cells, respectively. GNL3L overexpression promoted cell viability and cell proliferation in KYSE150 cells. On the contrary, silencing of GNL3L resulted in opposite phenotypes in KYSE410 cells. Furthermore, GNL3L level correlated with autophagic flux and influenced the levels of autophagy core proteins. Meanwhile, GNL3L also affected the AMPK signaling pathway, which is a pivotal signaling pathway for autophagy regulation. In the GNL3L-silenced cells, the AMPK agonist AICAR partly rescued the autophagic flux. Inversely, both pharmacologically and genetically deprivation of AMPK attenuated the autophagic flux induced by GNL3L overexpression. Moreover, AMPK activity alteration influenced the effect of GNL3L in regulating cell proliferation. Collectively, these findings suggest that GNL3L positively regulates cell proliferation and autophagy in ESCA cells via regulating the AMPK signaling, making itself a promising therapeutic target for ESCA.

A Novel LSTM-Based Machine Learning Model for Predicting the Activity of Food Protein-Derived Antihypertensive Peptides.

Food protein-derived antihypertensive peptides are a representative type of bioactive peptides. Several models based on partial least squares regression have been constructed to delineate the relationship between the structure and activity of the peptides. Machine-learning-based models have been applied in broad areas, which also indicates their potential to be incorporated into the field of bioactive peptides. In this study, a long short-term memory (LSTM) algorithm-based deep learning model was constructed, which could predict the IC50 value of the peptide in inhibiting ACE activity. In addition to the test dataset, the model was also validated using randomly synthesized peptides. The LSTM-based model constructed in this study provides an efficient and simplified method for screening antihypertensive peptides from food proteins.

COPS3 inhibition promotes cell proliferation blockage and anoikis via regulating PFKFB3 in osteosarcoma cancer cells.

As a key component of the COP9 signalosome complex, which participates in a variety of physiological processes, COPS3 is intimately related to multiple cancers. It promotes cell proliferation, progression and metastasis in several cancer cells. However, whether COPS3 participates in regulating anoikis, a specific kind of apoptosis and functions as an essential modulator of cell metastasis, has not yet been studied. Here, we found COPS3 is highly expressed in several cancers especially in osteosarcoma (OS). Overexpression of COPS3 promoted cell proliferation, cell viability and migration/invasion in both control cells and oxaliplatin (Oxa) treated cells. On the contrary, knockdown of COPS3 further enhanced the cytotoxicity of Oxa. Utilizing bioinformatics analysis, we found that COPS3 was higher expressed in the metastatic group, and associated with the extra-cellular matrix (ECM) receptor interaction pathway, which involve in regulating anoikis. In an anoikis model, COPS3 expression varied and genetic modification of COPS3 influenced the cell death enhanced by Oxa. PFKFB3, an essential modulator of glycolysis, was found to interact with COPS3. Inhibition of PFKFB3 promoted apoptosis and anoikis enhanced by Oxa, and COPS3 overexpression failed to rescue this cell death. On the contrary, in the COPS3 knockdown cells, overexpression of PFKFB3 recovered the anoikis resistance, indicating COPS3 function upstream of PFKFB3. In summary, our results elucidated that COPS3 modulated anoikis via affecting PFKFB3 in OS cancer cells.

Idarubicin combats abiraterone and enzalutamide resistance in prostate cells via targeting XPA protein.

Although second-generation therapies like abiraterone (ABI) and enzalutamide (ENZ) benefit patients with castration-resistant prostate cancer (CRPC), drug resistance frequently occurs, eventually resulting in therapy failure. In this study, we used two libraries, FDA-approved drug library and CRISP/Cas9 knockout (GeCKO) library to screen for drugs that overcome treatment resistance and to identify the potential drug-resistant genes involved in treatment resistance. Our screening results showed that the DNA-damaging agent idarubicin (IDA) overcame abiraterone and enzalutamide resistance in prostate cancer cells. IDA treatment inhibited the DNA repair protein XPA expression in a transcription-independent manner. Consistently, XPA knockout sensitized prostate cancer cells to abiraterone and enzalutamide treatment. In conclusion, IDA combats abiraterone and enzalutamide resistance by reducing XPA protein level in prostate cancer.

Long-term exposure to genistein inhibits the proliferation of gallbladder cancer by downregulating the MCM complex.

Soy isoflavones are natural tyrosine kinase inhibitors closely associated with decreased morbidity and mortality of various tumors. The activation of tyrosine kinases such as ERBB2 is the mechanism by which cholecystitis transforms into gallbladder cancer (GBC), therefore, it is important to investigate the relationship between long-term exposure to soy isoflavones and the occurrence and progression of GBC. This case-control study (n = 85 pairs) found that the high level of plasma soy isoflavone-genistein (GEN) was associated with a lower risk of gallbladder cancer (≥326.00 ng/mL compared to ≤19.30 ng/mL, crude odds ratio 0.15, 95% CI 0.04-0.59; P for trend = 0.016), and that the level of GEN exposure negatively correlated with Ki67 expression in GBC tissue (n = 85). Consistent with these results, the proliferation of GBC cells was inhibited in the long-term exposure models of GEN in vitro and in vivo. The long-term exposure to GEN reduced the tyrosine kinase activity of ERBB2 and impaired the function of the PTK6-AKT-GSK3ß axis, leading to downregulation of the MCM complex in GBC cells. In summary, long-term exposure to GEN associated with soy products intake might play a certain role in preventing GBC and even inhibiting the proliferation of GBC cells.

Correlation in endophytic fungi community diversity and bioactive compounds of Sophora alopecuroides.

Sophora alopecuroides L. is a traditional Chinese medicine used for the treatment of several different disease states including bacillary dysentery and enteritis. But importantly, it also plays a role as an anti-tumor agent. That said, little is known about the role endophytes play regarding the clinically bioactive metabolites in S. alopecuroides. In order to explore the effects of endophytic fungi on the accumulation, quality, and correlation in the content of the medicinal compounds, the structural diversity of endophytic fungi in S. alopecuroides was analyzed. The relationship between endophytes and quinolizidine alkaloids (QAs), housed within the seeds of S. alopecuroides, which were interpreted based on established methods of high-throughput sequencing and high-performance liquid chromatography. A total of 1,034,418 effective sequence reads and 257 operational taxonomic units (OTUs) were obtained from 33 samples which were sourced from 11 different sampling sites and further classified into 9 phyla, 20 classes, 45 orders, 85 families, and 118 genera. Ascomycota was found to be the dominant phylum of endophytic fungi in S. alopecuroides, with a relative abundance ranging from 60.85 to 98.30%. Alternaria, Cladosporium, Filobasidium, and an unidentified Ascomycota were the core-shared endophytes, accounting for 49.96, 27.12, 14.83, and 7.88%, respectively. Correlation analysis showed that the Simpson's diversity index of endophytic fungal community in S. alopecuroides was significantly positively correlated with the Oxymatrine (OMA) content in different areas, while the Chao and Shannoneven indexes were significantly negatively correlated with OMA. The endophytic fungi of Alternaria were positively correlated with the content of OMA, Oxysophocarpine (OSC), and total QAs. This study has mastered the endophytic fungi resources of S. alopecuroides, explored potential functional endophytic fungi, and provided a scientific basis for using biological fertilization strategies to improve the quality of S. alopecuroides.

The role of PFKFB3 in maintaining colorectal cancer cell proliferation and stemness.

Since generally confronting with the hypoxic and stressful microenvironment, cancer cells alter their glucose metabolism pattern to glycolysis to sustain the continuous proliferation and vigorous biological activities. Bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) isoform 3 (PFKFB3) functions as an effectively modulator of glycolysis and also participates in regulating angiogenesis, cell death and cell stemness. Meanwhile, PFKFB3 is highly expressed in a variety of cancer cells, and can be activated by several regulatory factors, such as hypoxia, inflammation and cellular signals. In colorectal cancer (CRC) cells, PFKFB3 not only has the property of high expression, but also probably relate to inflammation-cancer transformation. Recent studies indicate that PFKFB3 is involved in chemoradiotherapy resistance as well, such as breast cancer, endometrial cancer and CRC. Cancer stem cells (CSCs) are self-renewable cell types that contribute to oncogenesis, metastasis and relapse. Several studies indicate that CSCs utilize glycolysis to fulfill their energetic and biosynthetic demands in order to maintain rapid proliferation and adapt to the tumor microenvironment changes. In addition, elevated PFKFB3 has been reported to correlate with self-renewal and metastatic outgrowth in numerous kinds of CSCs. This review summarizes our current understanding of PFKFB3 roles in modulating cancer metabolism to maintain cell proliferation and stemness, and discusses its feasibility as a potential target for the discovery of antineoplastic agents, especially in CRC.

AICAR enhances the cytotoxicity of PFKFB3 inhibitor in an AMPK signaling-independent manner in colorectal cancer cells.

Numerous studies have shown that 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), a pivotal enzyme in modulating glycolysis, plays vital roles in various physiological processes. PFKFB3 activity could be regulated by several factors, such as hypoxia and AMPK signaling; however, it could also function as upstream of AMPK signaling. Here, we showed that PFKFB3 inhibitor PFK-15 induced cell viability loss and apoptosis. Deprivation of PFKFB3 inhibited autophagy, while enhanced the ubiquitin-proteasome degradation pathway. Furthermore, PFK-15 reduced both the AMPK and AKT-mTORC1 signaling pathways, as the attenuated phosphorylation level of kinases themselves and their substrates. The addition of AICAR rescued the AMPK activity and autophagy, but enhanced PFK-15-induced cell viability loss. In fact, AICAR promoted the cytotoxicity of PFK-15 even in the AMPKα1/2-silenced cells, indicating AICAR might function in an AMPK-independent manner. Nevertheless, AICAR further reduced the AKT-mTORC1 activity down-regulated by PFK-15. Moreover, it failed to enhance PFK-15's cytotoxicity in the AKT1/2-silenced cells, indicating AKT-mTORC1 participated during these processes. Collectively, the presented data demonstrated that PFK-15 inhibited cell viability, AMPK, and AKT-mTORC1 signaling, and AICAR probably enhanced the cell viability loss aroused by PFK-15 in an AKT-dependent and AMPK-independent manner, thereby revealing a more intimate relationship among PFKFB3, AMPK, and AKT-mTORC1 signaling pathways.

Bortezomib potentiates antitumor activity of mitoxantrone through dampening Wnt/ß-catenin signal pathway in prostate cancer cells.

BACKGROUND: Bortezomib (BZM), alone or in combination with other chemotherapies, has displayed strong anticancer effects in several cancers. The efficacy of the combination of BZM and mitoxantrone (MTX) in treating prostate cancer remains unknown. METHODS: Anticancer effects of combination of BZM and MTX were determined by apoptosis and proliferation assay in vivo and in vitro. Expression of ß-Catenin and its target genes were characterized by western blot and Real-time PCR. RESULTS: BZM significantly enhanced MTX-induced antiproliferation in vivo and in vitro. Mice administered a combination of BZM and MTX displayed attenuated tumor growth and prolonged survival. BZM significantly attenuated MTX-induced apoptosis. Moreover, the combination of BZM and MTX contributed to inhibition of the Wnt/ß-Catenin signaling pathway compared to monotherapy. CONCLUSIONS: This study demonstrates that BZM enhances MTX-induced anti-tumor effects by inhibiting the Wnt/ß-Catenin signaling pathway in prostate cancer cells.

Alternol triggers immunogenic cell death via reactive oxygen species generation.

Alternol is a naturally occurring compound that exerts antitumor activity in several cancers. However, whether Alternol induces antitumor immune response remains unknown. In this study, we investigated whether Alternol induced immunogenic cell death (ICD) in prostate cancer cells. Alternol triggered ICD in prostate cancer cells, as evidenced by the release of damage-associated molecular patterns (DAMPs) (i.e., calreticulin, CALR; high mobility group protein B1, HMGB1; and adenosine triphosphate, ATP) and pro-inflammatory cytokine (i.e., interleukin [IL]-1α, IL-1ß, IL-6, and IL-8) expression. Alternol facilitated tumor-associated antigen uptake and cross-presentation, CD8 + T-cell priming, and T-cell infiltration in tumor-draining lymph nodes (LNs) and tumors. The presence of Alternol fostered antitumor immune response in vivo, resulting in delayed tumor growth and prolonged survival. Moreover, inhibition of reactive oxygen species (ROS) generation blocked Alternol-induced upregulation of pre-inflammation cytokines, endoplasmic reticulum (ER) stress, and consequent antitumor immune response. Overall, our data indicate that Alternol triggers ICD in prostate cancer cells, which is mediated by ROS generation.

Erratum: CCL16 maintains stem cell-like properties in breast cancer by activating CCR2/GSK3ß/ß-catenin/OCT4 axis: Erratum.

[This corrects the article DOI: 10.7150/thno.51000.].

Necroptosis pathway blockage attenuates PFKFB3 inhibitor-induced cell viability loss and genome instability in colorectal cancer cells.

Cancer cells prone to utilize aerobic glycolysis other than oxidative phosphorylation to sustain its continuous cell activity in the stress microenvironment. Meanwhile, cancer cells generally suffer from genome instability, and both radiotherapy and chemotherapy may arouse DNA strand break, a common phenotype of genome instability. Glycolytic enzyme PFKFB3 (6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3), plays essential roles in variety physiology and pathology processes, and generally maintain high level in cancer cells. Although this protein has been reported to involve in genome instability, its role remains unclear and controversial. Here, we showed that PFK-15, a PFKFB3 inhibitor, obviously induced apoptosis, cell viability loss, and inhibited cell proliferation/migration. Besides, PFK-15 was also found to induce necroptosis, as it not only up-regulated the phosphorylated RIP1, RIP3 and MLKL, but also enhanced the interaction between RIP3 and RIP1/MLKL, all of which are characterization of necroptosis induction. Both genetically and pharmacologically deprivation of necroptosis attenuated the cytotoxic effect of PFK-15. Besides, PFK-15 increased the γ-H2AX level and micronuclei formation, markers for genome instability, and inhibition of necroptosis attenuated these phenotypes. Collectively, the presented data demonstrated that PFK-15 induced genome instability and necroptosis, and deprivation of necroptosis attenuated cytotoxicity and genotoxicity of PFK-15 in colorectal cancer cells, thereby revealing a more intimate relationship among PFKFB3, necroptosis and genome instability.

Single-cell RNA-sequencing atlas reveals an MDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladder cancer.

BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.

CCL16 maintains stem cell-like properties in breast cancer by activating CCR2/GSK3ß/ß-catenin/OCT4 axis.

Rationale: Considerable evidence suggests that breast cancer metastasis and recurrence occur due to emergence of cancer stem cells (CSCs). In our previous study, we designed a high-throughput siRNA screening platform that identifies inflammation genes involved in the regulation of cancer cell stemness. We reported that CCL16 protein decreases OCT4 expression and reduces the ALDH+ subpopulation. However, the mechanism by which CCL16 maintains stem cell-like properties remains unclear. Methods: Tissue microarrays were used to evaluate CCL16 expression. Cancer stemness assays were performed in CCL16 knockdown and overexpressing cells in vitro and in a xenograft model in vivo. Human phosphokinase array, immunofluorescence and chromatin immunoprecipitation assays were performed to explore the underlying mechanism. Results: We report that CCL16 was overexpressed in breast tumors and significantly correlated with clinical progression. We found that silencing CCL16 in MDA-MB-231 and BT549 cells diminished CSC properties including ALDH+ subpopulation, side population, chemo-resistance, and sphere formation. Furthermore, mice bearing CCL16-silenced MDA-MB-231 xenografts had lower tumorigenic frequency and developed smaller tumors. Exploration of the underlying mechanism found that CCL16 selects CCR2 to activate p-AKT/GSK3ß signaling and facilitate ß-catenin nuclear translocation. Further, CCL16 binds to the OCT4 promoter and promotes OCT4 expression. In addition, shRNAs targeting CCR2 and XAV939 targeting ß-catenin abolished CCL16-mediated cancer stemness. Upstream, IL10 mediates STAT3 activation, which binds to the CCL16 promoter and enhances its expression. The STAT3-targeted inhibitor Stattic suppressed CCL16 expression in vitro and restrained tumor progression in vivo. Conclusions: We identified a potential CSC regulator and suggest a novel mechanism for how CCL16 governs cancer cell stemness. We propose that CCL16 could be an effective target for breast cancer therapy.

Adenosine kinase is critical for neointima formation after vascular injury by inducing aberrant DNA hypermethylation.

AIMS: Adenosine receptors and extracellular adenosine have been demonstrated to modulate vascular smooth muscle cell (VSMC) proliferation and neointima formation. Adenosine kinase (ADK) is a major enzyme regulating intracellular adenosine levels but is function in VSMC remains unclear. Here, we investigated the role of ADK in vascular injury-induced smooth muscle proliferation and delineated the mechanisms underlying its action. METHODS AND RESULTS: We found that ADK expression was higher in the neointima of injured vessels and in platelet-derived growth factor-treated VSMCs. Genetic and pharmacological inhibition of ADK was enough to attenuate arterial injury-induced neointima formation due to inhibition of VSMC proliferation. Mechanistically, using infinium methylation assays and bisulfite sequencing, we showed that ADK metabolized the intracellular adenosine and potentiated the transmethylation pathway, then induced the aberrant DNA hypermethylation. Pharmacological inhibition of aberrant DNA hypermethylation increased KLF4 expression and suppressed VSMC proliferation as well as the neointima formation. Importantly, in human femoral arteries, we observed increased ADK expression and DNA hypermethylation as well as decreased KLF4 expression in neointimal VSMCs of stenotic vessels suggesting that our findings in mice are relevant for human disease and may hold translational significance. CONCLUSION: Our study unravels a novel mechanism by which ADK promotes VSMC proliferation via inducing aberrant DNA hypermethylation, thereby down-regulating KLF4 expression and promoting neointima formation. These findings advance the possibility of targeting ADK as an epigenetic modulator to combat vascular injury.