microRNA‐99a‐5p induces cellular senescence in gemcitabine‐resistant bladder cancer by targeting SMARCD1

Patients with advanced bladder cancer are generally treated with a combination of chemotherapeutics, including gemcitabine, but the effect is limited due to acquisition of drug resistance. Thus, in this study, we investigated the mechanism of gemcitabine resistance. First, gemcitabine‐resistant cells were established and resistance confirmed in vitro and in vivo. Small RNA sequencing analyses were performed to search for miRNAs involved in gemcitabine resistance. miR‐99a‐5p, selected as a candidate miRNA, was downregulated compared to its parental cells. In gain‐of‐function studies, miR‐99a‐5p inhibited cell viabilities and restored sensitivity to gemcitabine. RNA sequencing analysis was performed to find the target gene of miR‐99a‐5p. SMARCD1 was selected as a candidate gene. Dual‐luciferase reporter assays showed that miR‐99a‐5p directly regulated SMARCD1. Loss‐of‐function studies conducted with si‐RNAs revealed suppression of cell functions and restoration of gemcitabine sensitivity. miR‐99a‐5p overexpression and SMARCD1 knockdown also suppressed gemcitabine‐resistant cells in vivo. Furthermore, β‐galactosidase staining showed that miR‐99a‐5p induction and SMARCD1 suppression contributed to cellular senescence. In summary, tumor‐suppressive miR‐99a‐5p induced cellular senescence in gemcitabine‐resistant bladder cancer cells by targeting SMARCD1.     

A small molecule targeting CHI3L1 inhibits lung metastasis by blocking IL‐13Rα2‐mediated JNK‐AP‐1 signals

Our previous big data analyses showed a high level of association between chitinase 3 like1 (CHI3L1) expression and lung tumor development. In the present study, we investigated whether a CHI3L1‐inhibiting chemical, 2‐({3‐[2‐(1‐cyclohexen‐1‐yl)ethyl]‐6,7‐dimethoxy‐4‐oxo‐3,4‐dihydro‐2‐quinazolinyl}sulfanyl)‐N‐(4‐ethylphenyl)butanamide (K284), could inhibit lung metastasis and studied its mechanism of action. We investigated the antitumor effect of K284 both in vitro and in vivo. K284 (0.5 mg·kg⁻¹ body weight) significantly inhibited lung metastasis in in vivo models after injection of murine melanoma cells (B16F10) or adenocarcinomic human alveolar basal epithelial cells (A549). K284 significantly and concentration‐dependently also inhibited cancer cell proliferation and migration in the A549 and H460 lung cancer cell lines. We found that the binding of K284 to the chitin‐binding domain (CBD) of CHI3L1 prevented the binding of CHI3L1 to its receptor, interleukin‐13 receptor subunit alpha‐2 (IL‐13Rα2), thereby suppressing the CHI3L1 signal. This blocking of the CHI3L1‐IL‐13Rα2 signal caused the inhibition of c‐Jun N‐terminal kinase (JNK)‐activator protein 1 (AP‐1) signals, resulting in the prevention of lung metastasis and cancer cell growth. Our data demonstrate that K284 may serve as a potential candidate anticancer compound targeting CHI3L1.     

Role of L1CAM in retinoblastoma tumorigenesis: identification of novel therapeutic targets

The study presented focuses on the role of the neuronal cell adhesion molecule L1 cell adhesion molecule (L1CAM) in retinoblastoma (RB), the most common malignant intraocular childhood tumor. L1CAM is differentially expressed in a variety of human cancers and has been suggested as a promising therapeutic target. We likewise observed differential expression patterns for L1CAM in RB cell lines and patient samples. The two proteases involved in ectodomain shedding of L1CAM (L1CAM sheddases: ADAM10 and ADAM17) were likewise differentially expressed in the RB cell lines investigated, and an involvement in L1CAM processing in RB cells could be verified. We also identified ezrin, galectin‐3, and fibroblast growth factor basic as L1CAM signaling target genes in RB cells. Lentiviral L1CAM knockdown induced apoptosis and reduced cell viability, proliferation, growth, and colony formation capacity of RB cells, whereas L1CAM‐overexpressing RB cells displayed the opposite effects. Chicken chorioallantoic membrane assays revealed that L1CAM depletion decreases the tumorigenic and migration potential of RB cells in vivo. Moreover, L1CAM depletion decreased viability and tumor growth of etoposide‐resistant RB cell lines upon etoposide treatment in vitro and in vivo. Thus, L1CAM and its processing sheddases are potential novel targets for future therapeutic RB approaches.     

Calsequestrin 2 overexpression in breast cancer increases tumorigenesis and metastasis by modulating the tumor microenvironment

The spatial tumor shape is determined by the complex interactions between tumor cells and their microenvironment. Here, we investigated the role of a newly identified breast cancer‐related gene, calsequestrin 2 (CASQ2), in tumor–microenvironment interactions during tumor growth and metastasis. We analyzed gene expression and three‐dimensional tumor shape data from the breast cancer dataset of The Cancer Genome Atlas (TCGA) and identified CASQ2 as a potential regulator of tumor–microenvironment interaction. In TCGA breast cancer cases containing information of three‐dimensional tumor shapes, CASQ2 mRNA showed the highest correlation with the spatial tumor shapes. Furthermore, we investigated the expression pattern of CASQ2 in human breast cancer tissues. CASQ2 was not detected in breast cancer cell lines in vitro but was induced in the xenograft tumors and human breast cancer tissues. To evaluate the role of CASQ2, we established CASQ2‐overexpressing breast cancer cell lines for in vitro and in vivo experiments. CASQ2 overexpression in breast cancer cells resulted in a more aggressive phenotype and altered epithelial–mesenchymal transition (EMT) markers in vitro. CASQ2 overexpression induced cancer‐associated fibroblast characteristics along with increased hypoxia‐inducible factor 1α (HIF1α) expression in stromal fibroblasts. CASQ2 overexpression accelerated tumorigenesis, induced collagen structure remodeling, and increased distant metastasis in vivo. CASQ2 conferred more metaplastic features to triple‐negative breast cancer cells. Our data suggest that CASQ2 is a key regulator of breast cancer tumorigenesis and metastasis by modulating diverse aspects of tumor–microenvironment interactions.     

SENP3 loss promotes M2 macrophage polarization and breast cancer progression

Tumor‐associated macrophages (TAM) play a crucial role in promoting cancer progression. Upon cytokine stimulation, TAM preferentially polarize to the anti‐inflammatory and pro‐tumor M2 subtype. The mechanism underlying such preferential polarization remains elusive. Here, we report that macrophage‐specific deletion of the SUMO‐specific protease Sentrin/SUMO‐specific protease 3 promotes macrophage polarization towards M2 in bone marrow‐derived macrophage (BMDM) induced by interleukin 4 (IL‐4)/IL‐13 and in an ex vivo model (murine Py8119 cell line), as well as in a mouse orthotopic tumor model. Notably, Sentrin/SUMO‐specific protease 3 (SENP3) loss in macrophages accelerated breast cancer malignancy in ex vivo and in vivo models. Mechanistically, we identified Akt Serine/threonine kinase 1 (Akt1) as the substrate of SENP3 and found that the enhanced Akt1 SUMOylation upon SENP3 loss resulted in Akt1 hyper‐phosphorylation and activation, which facilitates M2 polarization. Analysis of clinical data showed that a lower level of SENP3 in TAM has a strong negative correlation with the level of the M2 marker CD206, as well as with a worse clinical outcome. Thus, increased Akt1 SUMOylation as a result of SENP3 deficiency modulates polarization of macrophages to the M2 subtype within a breast cancer microenvironment, which in turn promotes tumor progression.     

Cadherin‐3 is a novel oncogenic biomarker with prognostic value in glioblastoma

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. The prognosis of patients is very poor, with a median overall survival of ~ 15 months after diagnosis. Cadherin‐3 (also known as P‐cadherin), a cell–cell adhesion molecule encoded by the CDH3 gene, is deregulated in several cancer types, but its relevance in GBM is unknown. In this study, we investigated the functional roles, the associated molecular signatures, and the prognostic value of CDH3/P‐cadherin in this highly malignant brain tumor. CDH3/P‐cadherin mRNA and protein levels were evaluated in human glioma samples. Knockdown and overexpression models of P‐cadherin in GBM were used to evaluate its functional role in vitro and in vivo. CDH3‐associated gene signatures were identified by enrichment analyses and correlations. The impact of CDH3 in the survival of GBM patients was assessed in independent cohorts using both univariable and multivariable models. We found that P‐cadherin protein is expressed in a subset of gliomas, with an increased percentage of positive samples in grade IV tumors. Concordantly, CDH3 mRNA levels in glioma samples from The Cancer Genome Atlas (TCGA) database are increased in high‐grade gliomas. P‐cadherin displays oncogenic functions in multiple knockdown and overexpression GBM cell models by affecting cell viability, cell cycle, cell invasion, migration, and neurosphere formation capacity. Genes that were positively correlated with CDH3 are enriched for oncogenic pathways commonly activated in GBM. In vivo, GBM cells expressing high levels of P‐cadherin generate larger subcutaneous tumors and cause shorter survival of mice in an orthotopic intracranial model. Concomitantly, high CDH3 expression is predictive of shorter overall survival of GBM patients in independent cohorts. Together, our results show that CDH3/P‐cadherin expression is associated with aggressiveness features of GBM and poor patient prognosis, suggesting that it may be a novel therapeutic target for this deadly brain tumor.     

Recombinant nanobody against MUC1 tandem repeats inhibits growth,invasion, metastasis,and vascularization of spontaneous mouse mammary tumors

Alteration in glycosylation pattern of MUC1 mucin tandem repeats during carcinomas has been shown to negatively affect adhesive properties of malignant cells and enhance tumor invasiveness and metastasis. In addition, MUC1 overexpression is closely interrelated with angiogenesis, making it a great target for immunotherapy. Alongside, easier interaction of nanobodies (single‐domain antibodies) with their antigens, compared to conventional antibodies, is usually associated with superior desirable results. Herein, we evaluated the preclinical efficacy of a recombinant nanobody against MUC1 tandem repeats in suppressing tumor growth, angiogenesis, invasion, and metastasis. Expressed nanobody demonstrated specificity only toward MUC1‐overexpressing cancer cells and could internalize in cancer cell lines. The IC50 values (the concentration at which the nanobody exerted half of its maximal inhibitory effect) of the anti‐MUC1 nanobody against MUC1‐positive human cancer cell lines ranged from 1.2 to 14.3 nm. Similar concentrations could also effectively induce apoptosis in MUC1‐positive cancer cells but not in normal cells or MUC1‐negative human cancer cells. Immunohistochemical staining of spontaneously developed mouse breast tumors prior to in vivo studies confirmed cross‐reactivity of nanobody with mouse MUC1 despite large structural dissimilarities between mouse and human MUC1 tandem repeats. In vivo, a dose of 3 µg nanobody per gram of body weight in tumor‐bearing mice could attenuate tumor progression and suppress excessive circulating levels of IL‐1a, IL‐2, IL‐10, IL‐12, and IL‐17A pro‐inflammatory cytokines. Also, a significant decline in expression of Ki‐67, MMP9, and VEGFR2 biomarkers, as well as vasculogenesis, was evident in immunohistochemically stained tumor sections of anti‐MUC1 nanobody‐treated mice. In conclusion, the anti‐MUC1 tandem repeat nanobody of the present study could effectively overcome tumor growth, invasion, and metastasis.     

A novel AMPK activator shows therapeutic potential in hepatocellular carcinoma by suppressing HIF1α‐mediated aerobic glycolysis

Hepatocellular carcinoma (HCC) is characterized by rapid growth, early vascular invasion, and high metastasis. Currently available US Food and Drug Administration (FDA)‐approved drugs show low therapeutic efficacy, limiting HCC treatment to chemotherapy. We designed and synthesized a novel small molecule, SCT‐1015, that allosterically activated adenosine monophosphate‐activated protein kinase (AMPK) to suppress the aerobic glycolysis in HCC. SCT‐1015 was shown to bind the AMPK α and β‐subunit interface, thereby exposing the kinase α domain to the upstream kinases, resulting in the increased AMPK activity. SCT‐1015 dramatically reduced HCC cell growth in vitro and tumor growth in vivo. We further found that AMPK formed protein complexes with hypoxia‐inducible factor 1‐alpha (HIF1α) and that SCT‐1015‐activated AMPK promoted hydroxylation of HIF1α (402P and 564P), resulting in HIF1α degradation by the ubiquitin‐proteasome system. With declined HIF1α abundance, many glycolysis‐related enzymes were downregulated, suppressing aerobic glycolysis, and promoting oxidative phosphorylation. These results indicated that SCT‐1015 channeled HCC cells into an unfavorable metabolic status. Overall, we reported SCT‐1015 as a direct activator of AMPK signaling that held therapeutic potential in HCC.     

Deubiquitinase PSMD14 promotes ovarian cancer progression by decreasing enzymatic activity of PKM2

Dysregulation of deubiquitination has been reported to contribute to carcinogenesis. However, the function and mechanism of deubiquitinating enzyme 26S proteasome non‐ATPase regulatory subunit 14 (PSMD14) in the progression of ovarian cancer (OV), the deadliest gynecological cancer, still remains to be characterized. The present study demonstrated that PSMD14 was overexpressed in OV tissues and its higher levels correlated with a higher International Federation of Gynecology and Obstetrics (FIGO) stage in OV patients. A high level of PSMD14 expression was related to poor survival in OV patients. Knockdown and overexpression experiments elucidated that PSMD14 stimulated OV cell proliferation, invasion, and migration in vitro. Repression of PSMD14 suppressed OV tumor growth in vivo. PSMD14 inhibitor O‐phenanthroline (OPA) effectively attenuated malignant behaviors of OV cells in vitro and OV tumor growth in vivo. Mechanistically, we uncovered that PSMD14 was involved in post‐translational regulation of pyruvate kinase M2 isoform (PKM2). PSMD14 decreased K63‐linked ubiquitination on PKM2, downregulated the ratio of PKM2 tetramers to dimers and monomers, and subsequently diminished pyruvate kinase activity and induced nuclear translocation of PKM2, contributing to aerobic glycolysis in OV cells. Collectively, our findings highlight the potential roles of PSMD14 as a biomarker and therapeutic candidate for OV.     

Nuclear FABP7 regulates cell proliferation of wild‐type IDH1 glioma through caveolae formation

Isocitrate dehydrogenase 1 (IDH1) is a key enzyme in cellular metabolism. IDH1 mutation (IDH1mut) is the most important genetic alteration in lower grade glioma, whereas glioblastoma (GB), the most common malignant brain tumor, often has wild‐type IDH1 (IDH1wt). Although there is no effective treatment yet for neither IDH1wt nor IDHmut GB, it is important to note that the survival span of IDH1wt GB patients is significantly shorter than those with IDH1mut GB. Thus, understanding IDH1wt GB biology and developing effective molecular‐targeted therapies is of paramount importance. Fatty acid‐binding protein 7 (FABP7) is highly expressed in GB, and its expression level is negatively correlated with survival in malignant glioma patients; however, the underlying mechanisms of FABP7 involvement in tumor proliferation are still unknown. In this study, we demonstrate that FABP7 is highly expressed and localized in nuclei in IDH1wt glioma. Wild‐type FABP7 (FABP7wt) overexpression in IDH1wt U87 cells increased cell proliferation rate, caveolin‐1 expression, and caveolae/caveosome formation. In addition, FABP7wt overexpression increased the levels of H3K27ac on the caveolin‐1 promoter through controlling the nuclear acetyl‐CoA level via the interaction with ACLY. Consistent results were obtained using a xenograft model transplanted with U87 cells overexpressing FABP7. Interestingly, in U87 cells with mutant FABP7 overexpression, both in vitro and in vivo phenotypes shown by FABP7wt overexpression were disrupted. Furthermore, IDH1wt patient GB showed upregulated caveolin‐1 expression, increased levels of histone acetylation, and increased levels of acetyl‐CoA compared with IDH1mut patient GB. Taken together, these data suggest that nuclear FABP7 is involved in cell proliferation of GB through caveolae function/formation regulated via epigenetic regulation of caveolin‐1, and this mechanism is critically important for IDH1wt tumor biology.     

Fbxo45‐mediated NP‐STEP46 degradation via K6‐linked ubiquitination sustains ERK activity in lung cancer

Lung cancer is one of the most threatening malignant tumors to human health. Epidermal growth factor receptor (EGFR)‐targeted therapy is a common and essential means for the clinical treatment of lung cancer. However, drug resistance has always affected the therapeutic effect and survival rate in non‐small cell lung cancer (NSCLC). Tumor heterogeneity is a significant reason, yielding various drug resistance mechanisms, such as EGFR‐dependent or ‐independent extracellular signal‐regulated kinase 1 and/or 2 (ERK1/2) activation in NSCLC. To examine whether this aberrant activation of ERK1/2 is related to the loss of function of its specific phosphatase, a series of in vitro and in vivo assays were performed. We found that F‐box/SPRY domain‐containing protein 1 (Fbxo45) induces ubiquitination of NP‐STEP₄₆ , an active form of striatal‐enriched protein tyrosine phosphatase, with a K6‐linked poly‐ubiquitin chain. This ubiquitination led to proteasome degradation in the nucleus, which then sustains the aberrant level of phosphorylated‐ERK (pERK) and promotes tumor growth of NSCLC. Fbxo45 silencing can significantly inhibit cell proliferation and tumor growth. Moreover, NSCLC cells with silenced Fbxo45 showed great sensitivity to the EGFR tyrosine kinase inhibitor (TKI) afatinib. Here, we first report this critical pERK maintenance mechanism, which might be independent of the upstream kinase activity in NSCLC. We propose that inhibiting Fbxo45 may combat the issue of drug resistance in NSCLC patients, especially combining with EGFR‐TKI therapy.