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The risk of developing urothelial carcinoma of the bladder (UCB) in patients treated by radical nephroureterectomy (RNU) for an upper urinary tract urothelial carcinoma (UTUC) is 22% to 47% in the 2 years after surgery. Subject of debate remains whether UTUC and the subsequent UCB are clonally related or represent separate origins. To investigate the clonal relationship between both entities, we performed targeted DNA sequencing of a panel of 41 genes on matched normal and tumor tissue of 15 primary UTUC patients treated by RNU who later developed 19 UCBs. Based on the detected tumor‐specific DNA aberrations, the paired UTUC and UCB(s) of 11 patients (73.3%) showed a clonal relation, whereas in four patients the molecular results did not indicate a clear clonal relationship. Our results support the hypothesis that UCBs following a primary surgically resected UTUC are predominantly clonally derived recurrences and not separate entities., What's new? Patients treated by radical nephroureterectomy for upper urinary tract cancer have an increased risk of developing bladder carcinoma following surgery. It remains unclear, however, whether the upper urinary tract cancer and subsequent bladder carcinoma are clonally related or have separate origins. This targeted DNA sequencing study shows that almost 75% of patients have tumors that are clonally related, suggesting that seeding of tumor cells is the main mechanism of bladder carcinoma development following radical nephroureterectomy. This result underscores the need to minimalize the risk of seeding during surgery and/or diagnostic ureterorenoscopy plus biopsy, and to apply peri‐operative intravesical instillations with chemotherapy.

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3′‐triiodo‐l‐thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ‐Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRβ) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage‐specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3‐ and androgen‐mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth., What's new? Thyroid hormones may play a role in the progression of prostate cancer (PCa). In this study, the authors found that PCa cells had decreased expression of the thyroid‐hormone‐binding protein μ‐Crystallin (CRYM). Lower CRYM was also associated with poor prognosis in men with PCa. In addition, CRYM inhibited thyroid‐hormone and androgen signaling, as well as 18F‐fluoromethylcholine uptake by PCa cells. These results suggest that CRYM may act as a novel antagonist against factors that fuel PCa progression, and therefore thyroid signalling may offer a new therapeutic target in slowing aggressive prostate cancer.

The prognosis of patients with high‐grade or advanced‐stage endometrial cancer remains poor. As cancer stem‐like cells (CSCs) are thought to be associated with endometrial cancers, it is essential to investigate the molecular mechanisms that regulate endometrial CSCs. Dual‐specificity phosphatase 6 (DUSP6) functions as a negative‐feedback regulator of MAPK–ERK1/2 signaling, but its role in endometrial cancer remains unknown. We investigated whether DUSP6 is involved in cancer cell stemness using endometrial cancer cell lines and specimens from endometrial cancer patients. DUSP6 induced the expression of CSC‐related genes including ALDH1, Nanog, SOX2 and Oct4A, increased the population of cells in the G0/G1 phase, and promoted sphere formation ability. DUSP6 knockdown resulted in reduced cell invasion and metastasis, whereas DUSP6 overexpression inhibited apoptosis under serum‐free conditions. Moreover, DUSP6 decreased phosphorylated ERK1/2 and increased phosphorylated Akt levels, which potentially induces CSC features. In patients with endometrial cancers, DUSP6 expression was determined using immunohistochemistry, and based on the results, the patients were dichotomized into high‐ and low‐DUSP6‐expression groups. Progression‐free survival and overall survival were significantly shorter in the high‐DUSP6‐expression group. These results suggest that DUSP6 has potential value as a biomarker of CSCs and as a target of therapies designed to eliminate CSCs in endometrial cancer., What's new? Although cancer stem‐like cells (CSCs) are involved in human endometrial cancers, the underlying molecular mechanisms and biomarkers for CSCs in endometrial cancers remain elusive. Here, the authors found that DUSP6 plays an important role in regulating endometrial CSC phenotypes by increasing self‐renewal ability and starvation resistance. DUSP6 expression was required for inducing invasion and metastasis and resulted in ERK1/2 dephosphorylation and Akt phosphorylation, which potentially contribute to the promotion of CSC phenotypes. As DUSP6 expression was also positively associated with worse progression‐free and overall survival, DUSP6 represents a potential biomarker for endometrial CSCs and a therapeutic target in endometrial cancers.

Removal of colorectal adenomas is an effective strategy to reduce colorectal cancer (CRC) mortality rates. However, as only a minority of adenomas progress to cancer, such strategies may lead to overtreatment. The present study aimed to characterize adenomas by in‐depth molecular profiling, to obtain insights into altered biology associated with the colorectal adenoma‐to‐carcinoma progression. We obtained low‐coverage whole genome sequencing, RNA sequencing and tandem mass spectrometry data for 30 CRCs, 30 adenomas and 18 normal adjacent colon samples. These data were used for DNA copy number aberrations profiling, differential expression, gene set enrichment and gene‐dosage effect analysis. Protein expression was independently validated by immunohistochemistry on tissue microarrays and in patient‐derived colorectal adenoma organoids. Stroma percentage was determined by digital image analysis of tissue sections. Twenty‐four out of 30 adenomas could be unambiguously classified as high risk (n = 9) or low risk (n = 15) of progressing to cancer, based on DNA copy number profiles. Biological processes more prevalent in high‐risk than low‐risk adenomas were related to proliferation, tumor microenvironment and Notch, Wnt, PI3K/AKT/mTOR and Hedgehog signaling, while metabolic processes and protein secretion were enriched in low‐risk adenomas. DNA copy number driven gene‐dosage effect in high‐risk adenomas and cancers was observed for POFUT1, RPRD1B and EIF6. Increased POFUT1 expression in high‐risk adenomas was validated in tissue samples and organoids. High POFUT1 expression was also associated with Notch signaling enrichment and with decreased goblet cells differentiation. In‐depth molecular characterization of colorectal adenomas revealed POFUT1 and Notch signaling as potential drivers of tumor progression., What's new? Removal of colorectal adenomas is an effective strategy to reduce colorectal cancer (CRC) mortality rates. However, as only a minority of adenomas progress to cancer, such strategies may lead to overtreatment. While high‐risk adenomas, defined by specific DNA copy number aberrations, have an increased risk of progression, the mechanisms underlying colorectal adenoma‐to‐carcinoma progression remain unclear. This molecular characterization of colorectal adenomas, CRCs, and normal adjacent colon samples demonstrates that biological processes inherent to CRC are already more active in high‐risk adenomas compared to low‐risk adenomas. Moreover, the findings highlight POFUT1 and Notch signaling as potential drivers of colorectal tumor development.

G protein‐coupled estrogen receptor‐1 (GPER), a member of the G protein‐coupled receptor (GPCR) superfamily, mediates estrogen‐induced proliferation of normal and malignant breast epithelial cells. However, its role in breast cancer stem cells (BCSCs) remains unclear. Here we showed greater expression of GPER in BCSCs than non‐BCSCs of three patient‐derived xenografts of ER−/PR+ breast cancers. GPER silencing reduced stemness features of BCSCs as reflected by reduced mammosphere forming capacity in vitro, and tumor growth in vivo with decreased BCSC populations. Comparative phosphoproteomics revealed greater GPER‐mediated PKA/BAD signaling in BCSCs. Activation of GPER by its ligands, including tamoxifen (TMX), induced phosphorylation of PKA and BAD‐Ser118 to sustain BCSC characteristics. Transfection with a dominant‐negative mutant BAD (Ser118Ala) led to reduced cell survival. Taken together, GPER and its downstream signaling play a key role in maintaining the stemness of BCSCs, suggesting that GPER is a potential therapeutic target for eradicating BCSCs., What's new? G protein‐coupled estrogen receptor‐1 (GPER) mediates estrogen‐induced proliferation of normal and malignant breast epithelial cells. However, the role of GPER in breast cancer stem cells (BCSC) biology remains unclear. Here, using patient‐derived xenografts of ER–/PR+ breast cancer, the authors found higher expression of GPER in BCSCs than non‐BCSCs. Moreover, the results indicated that stemness features were sustained via GPER‐mediated PKA/BAD phosphorylation. Stimulation by the GPER ligand tamoxifen enhanced BCSC cell viability and population and BAD phosphorylation. The findings revealed a vital role of GPER‐mediated signaling pathways in BCSC survival, suggesting GPER as a potential therapeutic target for eradicating BCSCs.

E3 ubiquitin ligases primarily determine the substrate specificity of the ubiquitin‐proteasome system and play an essential role in the resistance to bortezomib in multiple myeloma (MM). Neural precursor cell‐expressed developmentally downregulated gene 4‐1 (NEDD4‐1, also known as NEDD4) is a founding member of the NEDD4 family of E3 ligases and is involved in the proliferation, migration, invasion and drug sensitivity of cancer cells. In the present study, we investigated the role of NEDD4‐1 in MM cells and explored its underlying mechanism. Clinically, low NEDD4‐1 expression has been linked to poor prognosis in patients with MM. Functionally, NEDD4‐1 knockdown (KD) resulted in bortezomib resistance in MM cells in vitro and in vivo. The overexpression (OE) of NEDD4‐1, but not an enzyme‐dead NEDD4‐1‐C867S mutant, had the opposite effect. Furthermore, the overexpression of NEDD4‐1 in NEDD4‐1 KD cells resensitized the cells to bortezomib in an add‐back rescue experiment. Mechanistically, pAkt‐Ser473 levels and Akt signaling were elevated and decreased by NEDD4‐1 KD and OE, respectively. NEDD4‐1 ubiquitinated Akt and targeted pAkt‐Ser473 for proteasomal degradation. More importantly, the NEDD4‐1 KD‐induced upregulation of Akt expression sensitized MM cells to growth inhibition after treatment with an Akt inhibitor. Collectively, our results suggest that high NEDD4‐1 levels may be a potential new therapeutic target in MM., What's new? While multiple myeloma (MM) remains an incurable disease, therapeutic advances have greatly improved its clinical management. Among the agents most effective against MM is the proteasome inhibitor bortezomib, though many patients eventually develop drug resistance. Here, decreased expression of the tumor suppressor NEDD4‐1 was found to serve a key role in modulating MM sensitivity to bortezomib. Reduced NEDD4‐1 expression was associated with increased PTEN/PI3K/Akt signaling and tumor growth. Experiments showed that NEDD4‐1 normally binds directly to Akt, enhancing its ubiquitination and thereby exerting anti‐MM effects. The data warrant further investigation of NEDD4‐1 reactivation as a novel therapeutic strategy for MM.

The olfactomedin 4 (OLFM4) gene has been analyzed as a tumor‐suppressor gene and a putative biomarker in many cancers. In our study, we analyzed the relationship of OLFM4 expression with clinicopathological features and with CpG site methylation in the OLFM4 gene promoter region in human primary prostate adenocarcinoma. OLFM4 protein expression was significantly reduced in prostate cancer tissue compared to adjacent normal tissue and was further significantly reduced in more advanced cancers. Bioinformatic studies with clinical datasets revealed that primary prostate adenocarcinoma patients with reduced OLFM4 mRNA expression exhibited higher Gleason scores and higher preoperative serum prostate‐specific antigen levels, as well as lower recurrence‐free survival. Three of the eight CpG sites in the OLFM4 gene promoter region were hypermethylated in cancerous prostate cells compared to adjacent normal cells, and reduced methylation of eight CpG sites was associated with increased OLFM4 mRNA expression in RWPE1 and PC‐3 cells. Furthermore, knockdown of OLFM4 gene expression was associated with enhanced epithelial–mesenchymal transition (EMT)‐marker expression in RWPE immortalized normal prostate cells. In contrast, restoration of OLFM4 expression in PC‐3 and DU145 prostate cancer cells lacking OLFM4 significantly inhibited both EMT‐marker expression and tumor cell growth in in vitro and in vivo models, indicating that OLFM4 may play a tumor‐suppressor role in inhibiting the EMT program, as well as tumor initiation and growth, in prostate cells. Taken together, these findings suggest that OLFM4 plays an important tumor‐suppressor role in prostate cancer progression and might be useful as a novel candidate biomarker for prostate cancer., What's new? Altered expression of the OLFM4 gene appears to be involved in many cancers. In this study of prostate cancers, the authors found that OLFM4 can suppress tumor initiation, growth and progression. Downregulation of OLFM4 was associated with higher serum PSA levels, higher Gleason scores, and lower recurrence‐free survival in prostate cancer patients. These results indicate that OLFM4 may play an important tumor‐suppressor role in the progression of prostate cancer, and may provide a novel prognostic biomarker for prostate cancer treatment.

Cancers occurring at the gastroesophageal junction (GEJ) are classified as predominantly esophageal or gastric, which is often difficult to decipher. We hypothesized that the transcriptomic profile might reveal molecular subgroups which could help to define the tumor origin and behavior beyond anatomical location. The gene expression profiles of 107 treatment‐naïve, intestinal type, gastroesophageal adenocarcinomas were assessed by the Illumina‐HTv4.0 beadchip. Differential gene expression (limma), unsupervised subgroup assignment (mclust) and pathway analysis (gage) were undertaken in R statistical computing and results were related to demographic and clinical parameters. Unsupervised assignment of the gene expression profiles revealed three distinct molecular subgroups, which were not associated with anatomical location, tumor stage or grade (p > 0.05). Group 1 was enriched for pathways involved in cell turnover, Group 2 was enriched for metabolic processes and Group 3 for immune‐response pathways. Patients in group 1 showed the worst overall survival (p = 0.019). Key genes for the three subtypes were confirmed by immunohistochemistry. The newly defined intrinsic subtypes were analyzed in four independent datasets of gastric and esophageal adenocarcinomas with transcriptomic data available (RNAseq data: OCCAMS cohort, n = 158; gene expression arrays: Belfast, n = 63; Singapore, n = 191; Asian Cancer Research Group, n = 300). The subgroups were represented in the independent cohorts and pooled analysis confirmed the prognostic effect of the new subtypes. In conclusion, adenocarcinomas at the GEJ comprise three distinct molecular phenotypes which do not reflect anatomical location but rather inform our understanding of the key pathways expressed., What's new? Adenocarcinomas that arise at the junction between the esophagus and the stomach are currently classified based on location. Here, the authors looked at patterns of gene expression of these cancers. They found that gastro‐esophageal junction adenocarcinomas can be sorted into three biological subtypes, independent of location, based on gene expression. Group 1 cancers have boosted stomach‐specific genes that combat the effects of acid reflux. Group 2 tumors express genes characteristic to the intestinal tract, and the genes active in Group 3 relate to inflammation. The differences in biological pathway expression means that these differences could be used to improve treatment.

In neuroblastoma (NB), genetic alterations in chromatin remodeling (CRGs) and epigenetic modifier genes (EMGs) have been described. We sought to determine their frequency and clinical impact. Whole exome (WES)/whole genome sequencing (WGS) data and targeted sequencing (TSCA®) of exonic regions of 33 CRGs/EMGs were analyzed in tumor samples from 283 NB patients, with constitutional material available for 55 patients. The frequency of CRG/EMG variations in NB cases was then compared to the Genome Aggregation Database (gnomAD). The sequencing revealed SNVs/small InDels or focal CNAs of CRGs/EMGs in 20% (56/283) of all cases, occurring at a somatic level in 4 (7.2%), at a germline level in 12 (22%) cases, whereas for the remaining cases, only tumor material could be analyzed. The most frequently altered genes were ATRX (5%), SMARCA4 (2.5%), MLL3 (2.5%) and ARID1B (2.5%). Double events (SNVs/small InDels/CNAs associated with LOH) were observed in SMARCA4 (n = 3), ATRX (n = 1) and PBRM1 (n = 1). Among the 60 variations, 24 (8.4%) targeted domains of functional importance for chromatin remodeling or highly conserved domains but of unknown function. Variations in SMARCA4 and ATRX occurred more frequently in the NB as compared to the gnomAD control cohort (OR = 4.49, 95%CI: 1.63–9.97, p = 0.038; OR 3.44, 95%CI: 1.46–6.91, p = 0.043, respectively). Cases with CRG/EMG variations showed a poorer overall survival compared to cases without variations. Genetic variations of CRGs/EMGs with likely functional impact were observed in 8.4% (24/283) of NB. Our case–control approach suggests a role of SMARCA4 as a player of NB oncogenesis., What's new? Mutations that affect chromatin remodeling can lead to cancer. In this paper, the authors investigated the impact of variations in chromatin remodeling genes and epigenetic modifier genes on neuroblastoma patients. They compared the frequency of these variations in NB cases with data from the Genome Aggregation Database (gnomAD). Neuroblastoma cases had a higher frequency of SMARCA4 and ATRX gene variations than the general population. Furthermore, NB patients with CRG/EMG mutations had poorer overall survival than NB cases without such mutations. These findings highlight the importance of chromatin remodeling in neuroblastoma as an avenue for new therapeutics.

Epithelial cell polarity regulator Crumbs3 (Crb3), a mammalian homolog within the Drosophila Crb gene family, was initially identified as an essential embryonic development factor. It is recently implicated in tumor suppression, though its specific functions are controversial. We here demonstrate that Crb3 strongly promotes tumor invasion and metastasis of human colon adenocarcinoma cells. Crb3 centrality to tumor migration was supported by strong expression at invasive front and metastatic foci of colonic adenocarcinoma of the patient tissues. Accordingly, two different Crb3‐knockout (KO) lines, Crb3‐KO (Crb3 −/−) DLD‐1 and Crb3‐KO WiDr from human colonic adenocarcinomas, were generated by the CRISPR‐Cas9 system. Crb3‐KO DLD‐1 cells exhibited loss of cellular mobility in vitro and dramatic suppression of liver metastases in vivo in contrast to the wild type of DLD‐1. Unlike DLD‐1, Crb3‐KO WiDr mobility and metastasis were unaffected, which were similar to wild‐type WiDr. Proteome analysis of Crb3‐coimmunopreciptated proteins identified different respective fibroblast growth factor receptor (FGFR) isotypes specifically bound to Crb3 isoform a through their intracellular domain. In DLD‐1, Crb3 showed membranous localization of FGFR1 leading to its functional activation, whereas Crb3 bound to cytoplasmic FGFR4 in WiDr without FGFR1 expression, leading to cellular growth. Correlative expression between Crb3 and FGFR1 was consistently detected in primary and metastatic colorectal cancer patient tissues. Taking these together, Crb3 critically accelerates cell migration, namely invasion and metastasis of human colon cancers, through specific interaction to FGFR1 on colon cancer cells., What's new? Epithelial cell polarity regulator Crumbs3 (Crb3) was initially identified as an essential embryonic development factor. More recently, it has been implicated in tumor suppression, though its specific functions remain controversial. Here, the authors demonstrate that Crb3 strongly promotes tumor invasion and metastasis of human colon adenocarcinoma cells. They identify among the binding partners of Crb3 the FGF receptors family, which is pivotal to tumor cell dynamics including proliferation, migration, and differentiation. Crb3 colocalizes with FGFR1 to activate downstream signaling and critically accelerate tumor migration and metastasis of human colon cancers.

Darolutamide is a novel androgen receptor (AR) antagonist with a distinct chemical structure compared to other AR antagonists and currently in clinical Phase 3 trials for prostate cancer. Using cell‐based transactivation assays, we demonstrate that darolutamide, its diastereomers and its main metabolite keto‐darolutamide are strong, competitive antagonists for AR wild type, and also for several mutants identified in prostate cancer patients for which other AR antagonists show reduced antagonism or even agonism. Darolutamide, its two diastereomers and main metabolite are also strong antagonists in assays measuring AR N/C interaction and homodimerization. Molecular modeling suggests that the flexibility of darolutamide allows accommodation in the W742C/L mutated AR ligand‐binding pocket while for enzalutamide the loss of the important hydrophobic interaction with W742 leads to reduced AR interaction. This correlates with an antagonistic pattern profile of coregulator recruitment for darolutamide. In vitro efficacy studies performed with androgen‐dependent prostate cancer cell lines show that darolutamide strongly reduces cell viability and potently inhibits spheroid formation. Also, a marked down‐regulation of androgen target genes paralleled by decreased AR binding to gene regulatory regions is seen. In vivo studies reveal that oral dosing of darolutamide markedly reduces growth of the LAPC‐4 cell line‐derived xenograft and of the KuCaP‐1 patient‐derived xenograft. Altogether, these results substantiate a unique antagonistic profile of darolutamide and support further development as a prostate cancer drug., What's new? Comparison of genomic landscapes from primary prostate cancer and metastatic tumor shows that resistance mechanisms are centered on androgen signaling and increased synthesis. Here, the novel androgen receptor (AR) antagonist darolutamide shows strong in vitro and in vivo efficacy in different prostate cancer models. Darolutamide retains its antagonistic properties at elevated androgen levels and for several AR mutants identified in therapy‐resistant patients. A unique binding profile inside the AR ligand‐binding domain linked to the flexibility of darolutamide is proposed. Altogether, these results substantiate a unique antagonistic profile of darolutamide and support further development as a prostate cancer drug.

The P2X7 receptor, an ATP‐gated ion channel, is critical for cancer cell growth, invasiveness, and angiogenesis. Previous studies indicate that P2X7 regulates osteoblast proliferation and osteodeposition and that high P2X7 expression has a pro‐growth effect in osteosarcoma. However, how it functions in osteosarcoma cell growth and metastasis is not clear. Thus, we elucidated molecular mechanisms of P2X7‐dependent positive regulation of osteosarcoma cell proliferation, invasion, migration, epithelial to mesenchymal transition (EMT), and angiogenesis using in vitro and in vivo models. We confirm that P2X7 is highly‐expressed in human osteosarcoma tumor tissues and HOS/MNNG, MG63, U2OS, SW1353 and SAOS‐2 cell lines. P2X7 receptor stimulation enhanced HOS/MNNG and SAOS‐2 cell proliferation, migration and invasion; but knockdown of P2X7 expression or receptor inhibition had opposite effects. P2X7 positively regulated glycogen content, epithelial to mesenchymal transition and stemness of HOS/MNNG cells. P2X7 activation promoted PI3K/Akt/GSK3β/β‐catenin and mTOR/HIF1α/VEGF signaling, thereby mediating pro‐tumor effects of osteosarcoma cells. Consistent with data from in vitro experiments, systemic administration of P2X7 agonist induced tumor growth, metastasis and tumor‐associated bone destruction in osteosarcoma‐bearing nude mice, whereas a P2X7 antagonist reversed these effects. Thus, the P2X7 receptor participates in regulation of osteosarcoma growth and metastasis and we offer evidence that P2X7 may be a promising therapeutic target for treating osteosarcoma., What's new? The ATP‐gated ion channel receptor P2X7 is increasingly recognized as a tumor‐promoting factor. In this study, P2X7 was found to be overexpressed in human osteosarcoma tissues and cells, with its activation enhancing osteosarcoma cell proliferation, migration, and invasion. P2X7 activation further induced epithelial‐mesenchymal transition (EMT), affected the stemness of osteosarcoma cells, and augmented angiogenesis. Experiments in mice showed that P2X7 also induces osteosarcoma‐associated bone destruction. Opposing effects were observed upon P2X7 inhibition. P2X7 activity was influenced in part by PI3K/Akt/GSK3β/β‐catenin and mTOR/HIF1α/VEGF signaling pathways. The data identify P2X7 as a target for the development of novel therapeutics against osteosarcoma.

Breast cancer remains the second cause of tumor‐related mortality in women worldwide mainly due to chemoresistance and metastasis. The chemoresistance and metastasis are attributed to a rare subpopulation with enriched stem‐like characteristics, thus called Cancer Stem Cells (CSCs). We have previously reported aberrant expression of the actin‐bundling protein (fascin) in breast cancer cells, which enhances their chemoresistance, metastasis and enriches CSC population. The intracellular mechanisms that link fascin with its downstream effectors are not fully elucidated. Here, loss and gain of function approaches in two different breast cancer models were used to understand how fascin promotes disease progression. Importantly, findings were aligned with expression data from actual breast cancer patients. Expression profiling of a large breast cancer dataset (TCGA, 530 patients) showed statistically significant correlation between fascin expression and a key adherence molecule, β1 integrin (ITGB1). In vitro manipulation of fascin expression in breast cancer cells exhibited its direct effect on ITGB1 expression. Fascin‐mediated regulation of ITGB1 was critical for several breast cancer cell functions including adhesion to different extracellular matrix, self‐renewability and chemoresistance. Importantly, there was a significant relationship between fascin and ITGB1 co‐expression and short disease‐free as well as overall survival in chemo‐treated breast cancer patients. This novel role of fascin effect on ITGB1 expression and its outcome on cell self‐renewability and chemoresistance strongly encourages for dual targeting of fascin‐ITGB1 axis as a therapeutic approach to halt breast cancer progression and eradicate it from the root., What's new? Residual cancer stem cells (CSCs) have the ability to regrow tumors and to metastasize to distant organs, resulting in disease relapse and increased cancer mortality. In breast cancer, CSC populations are enriched by aberrant expression of the actin‐bundling protein fascin, induction of which is also associated with chemoresistance and metastasis. In this study, fascin was found to upregulate β1 integrin (ITGB1) expression, an effect that proved critical to breast cancer cell adhesion and self‐renewal. Coexpression of fascin and ITGB1 was associated with decreased survival in chemotherapy‐treated breast cancer patients. The findings identify the fascin‐ITGB1 axis as a potential therapeutic target.

Toll‐like receptors (TLRs) play critical roles in host defense after recognition of conserved microbial‐ and host‐derived components, and their dysregulation is a common feature of various inflammation‐associated cancers, including gastric cancer (GC). Despite the recent recognition that metabolic reprogramming is a hallmark of cancer, the molecular effectors of altered metabolism during tumorigenesis remain unclear. Here, using bioenergetics function assays on human GC cells, we reveal that ligand‐induced activation of TLR2, predominantly through TLR1/2 heterodimer, augments both oxidative phosphorylation (OXPHOS) and glycolysis, with a bias toward glycolytic activity. Notably, DNA microarray‐based expression profiling of human cancer cells stimulated with TLR2 ligands demonstrated significant enrichment of gene‐sets for oncogenic pathways previously implicated in metabolic regulation, including reactive oxygen species (ROS), p53 and Myc. Moreover, the redox gene encoding the manganese‐dependent mitochondrial enzyme, superoxide dismutase (SOD)2, was strongly induced at the mRNA and protein levels by multiple signaling pathways downstream of TLR2, namely JAK‐STAT3, JNK MAPK and NF‐κB. Furthermore, siRNA‐mediated suppression of SOD2 ameliorated the TLR2‐induced metabolic shift in human GC cancer cells. Importantly, patient‐derived tissue microarrays and bioinformatics interrogation of clinical datasets indicated that upregulated expression of TLR2 and SOD2 were significantly correlated in human GC, and the TLR2‐SOD2 axis was associated with multiple clinical parameters of advanced stage disease, including distant metastasis, microvascular invasion and stage, as well as poor survival. Collectively, our findings reveal a novel TLR2‐SOD2 axis as a potential biomarker for therapy and prognosis in cancer., What's new? Many types of tumor cells have altered metabolism, which supports their rapid growth. Pattern‐recognition receptors such as Toll‐like receptors (TLRs) can have a broad range of effects on inflammation‐associated cancers such as gastric cancer (GC), and can influence proliferation, apoptosis, and migration. In this study, the authors identified a novel TLR2‐ SOD2 axis that promotes metabolic reprogramming of GC cells, mediated by multiple oncogenic signaling pathways. Increased expression of these proteins in gastric tumors may serve as a valuable biomarker for therapy and prognosis in GC.

Myxoid liposarcoma (MLS) shows extensive intratumoural heterogeneity with distinct subpopulations of tumour cells. Despite improved survival of MLS patients, existing therapies have shortcomings as they fail to target all tumour cells. The nature of chemotherapy‐resistant cells in MLS remains unknown. Here, we show that MLS cell lines contained subpopulations of cells that can form spheres, efflux Hoechst dye and resist doxorubicin, all properties attributed to cancer stem cells (CSCs). By single‐cell gene expression, western blot, phospho‐kinase array, immunoprecipitation, immunohistochemistry, flow cytometry and microarray analysis we showed that a subset of MLS cells expressed JAK–STAT genes with active signalling. JAK1/2 inhibition via ruxolitinib decreased, while stimulation with LIF increased, phosphorylation of STAT3 and the number of cells with CSC properties indicating that JAK–STAT signalling controlled the number of cells with CSC features. We also show that phosphorylated STAT3 interacted with the SWI/SNF complex. We conclude that MLS contains JAK–STAT‐regulated subpopulations of cells with CSC features. Combined doxorubicin and ruxolitinib treatment targeted both proliferating cells as well as cells with CSC features, providing new means to circumvent chemotherapy resistance in treatment of MLS patients., What's new? Despite improved survival of patients, existing therapies for Myxoid liposarcoma (MLS) present shortcomings as they fail to target all tumour cells. The nature of chemotherapy‐resistant cells in MLS remains unknown, however. Here, the authors show that myxoid liposarcomas are heterogeneous and contain subpopulations of cells with stem cell properties, including chemotherapy resistance. Moreover, JAK‐STAT signalling is active in MLS and regulates the size of the cancer stem cells‐like subpopulation via the SWI/SNF complex. The results shed light on the mechanisms of therapy resistance in MLS and point to JAK‐STAT inhibitors as a new avenue for targeted MLS therapies.

Bevacizumab, a VEGF‐targeting monoclonal antibody, may trigger an infiltrative growth pattern in glioblastoma. We investigated this pattern using both a human specimen and rat models. In the human specimen, a substantial fraction of infiltrating tumor cells were located along perivascular spaces in close relationship with endothelial cells. Brain xenografts of U87MG cells treated with bevacizumab were smaller than controls (p = 0.0055; Student t‐test), however, bands of tumor cells spread through the brain farther than controls (p < 0.001; Student t‐test). Infiltrating tumor Cells exhibited tropism for vascular structures and propensity to form tubules and niches with endothelial cells. Molecularly, bevacizumab triggered an epithelial to mesenchymal transition with over‐expression of the receptor Plexin Domain Containing 1 (PLXDC1). These results were validated using brain xenografts of patient‐derived glioma stem‐like cells. Enforced expression of PLXDC1 in U87MG cells promoted brain infiltration along perivascular spaces. Importantly, PLXDC1 inhibition prevented perivascular infiltration and significantly increased the survival of bevacizumab‐treated rats. Our study indicates that bevacizumab‐induced brain infiltration is driven by vascular endothelium and depends on PLXDC1 activation of tumor cells., What's new? Bevacizumab, a VEGF‐targeting monoclonal antibody, has been observed to trigger an infiltrative growth pattern in glioblastoma as an escape mechanism. The mechanisms underlying this gliomatosis‐like growth pattern, however, remain unclear. Here, the authors found that the infiltrative growth pattern occurs mostly along perivascular spaces and relies on the over‐expression of PLXDC1 by tumor cells and on the restoration of the endothelial component of blood brain barrier. Altogether, the data show that the brain infiltration induced by bevacizumab is mainly driven by the vascular endothelium. Importantly, inhibition of PLXDC1 prevents bevacizumab‐induced infiltrative growth, resulting in significant increase of survival.

Emerging data indicate that interferon‐induced transmembrane protein 1 (IFITM1) plays an important role in many cancers. However, it remains unclear whether IFITM1 is functionally indispensable in nonsmall cell lung cancer (NSCLC). Here, using NSCLC cell lines and patient‐derived samples, we show that IFITM1 is essentially required for the progression of NSCLC in vitro and in vivo. Specifically, IFITM1 depletion resulted in a significant reduction in sphere formation, migration, and invasion of NSCLC cells in vitro; these events were inversely correlated with the ectopic expression of IFITM1. In addition, tumor development was significantly impaired in the absence of IFITM1 in vivo. Mechanistically, epidermal growth factor receptor/sex‐determining region Y‐box 2 (EGFR/SOX2) signaling axis was compromised in the absence of IFITM1, and the ectopic expression of SOX2 partially rescued the defects caused by IFITM1 depletion. More importantly, using 226 patient‐derived samples, we demonstrate that a high level of IFITM1 expression is associated with a poor overall survival (OS) rate in adenocarcinoma but not in squamous cell carcinoma. Collectively, these data suggest that IFITM1 is a poor prognostic marker of adenocarcinoma and an attractive target to develop novel therapeutics for NSCLC., What's new? Interferon response genes play key roles in pathogen defense but emerging evidence also link them with cancer. The authors report that interferon‐induced transmembrane protein 1 (IFITM1) critically regulates epidermal growth factor receptor‐mediated signaling in nonsmall lung cancer models and is associated with a poor prognosis of patients with adenocarcinoma. This expands the function of this innate defense factor and might lead to improved clinical management of individuals afflicted with lung cancer.

Colorectal and glioblastoma cancer stem‐like cells (CSCs) are essential for translational research. Cell line authentication by short tandem repeat (STR) profiling ensures reproducibility of results in oncology research. This technique enables to identify mislabeling or cross‐contamination of cell lines. In our study, we provide a reference dataset for a panel of colorectal and glioblastoma CSCs that allows authentication. Each cell line was entered into the cell Line Integrated Molecular Authentication database 2.1 to be compared to the STR profiles of 4485 tumor cell lines. This article also provides clinical data of patients from whom CSCs arose and data on the parent tumor stage and mutations. STR profiles and information of our CSCs are also available in the Cellosaurus database (ExPASy) as identified by unique research resource identifier codes., What's new? Human cell lines obtained from cancer stem‐like cells represent an invaluable model for studying tumor properties. Cell line authentication by short tandem repeat (STR) profiling is an important tool to identify the potential mislabeling or cross‐contamination of cell lines. Here, the authors characterized 18 colorectal cancer stem‐like cell lines from 17 patients and 103 glioblastoma cancer stem‐like cell lines from 95 patients by STR profiling to create a reference dataset that allows the authentication of these cell lines and their identification through a unique research resource identifier. The results will help further ensure the reliability and reproducibility of research experiments.

In non‐small cell lung cancer (NSCLC), activating mutations in the epidermal growth factor receptor (EGFR) induce sensitivity to EGFR tyrosine kinase inhibitors. Despite impressive clinical responses, patients ultimately relapse as a reservoir of drug‐tolerant cells persist, which ultimately leads to acquired resistance mechanisms. We performed an unbiased high‐throughput siRNA screen to identify proteins that abrogate the response of EGFR‐mutant NSCLC to EGFR‐targeted therapy. The deubiquitinase USP13 was a top hit resulting from this screen. Targeting USP13 increases the sensitivity to EGFR inhibition with small molecules in vitro and in vivo. USP13 selectively stabilizes mutant EGFR in a peptidase‐independent manner by counteracting the action of members of the Cbl family of E3 ubiquitin ligases. We conclude that USP13 is a strong mutant EGFR‐specific cotarget that could improve the treatment efficacy of EGFR‐targeted therapies., What's new? Mutations in epidermal growth factor receptor (EGFR) are present in as many as 30 percent of patients with non‐small‐cell lung cancer (NSCLC). EGFR mutations render NSCLC sensitive to EGFR tyrosine kinase inhibitors, though over time many lung tumors become resistant to these therapies. This investigation shows that receptor stabilization and drug resistance in EGFR‐mutant NSCLC is mediated at least in part by the deubiquitinase USP13. Co‐targeting EGFR and USP13 increased NSCLC sensitivity to EGFR inhibitors in vitro and in vivo. The study identifies USP13 targeted therapy as a promising means for overcoming EGFR inhibitor resistance in EGFR‐mutant NSCLC.

Synchronous primary endometrial and ovarian cancers (SEOs) represent 10% of all endometrial and ovarian cancers and are assumed to develop as independent entities. We investigated the clonal relationship between endometrial and ovarian carcinomas in a large cohort classified as SEOs or metastatic disease (MD). The molecular profiles were compared to The Cancer Genome Atlas (TCGA) data to explore primary origin. Subsequently, the molecular profiles were correlated with clinical outcome. To this extent, a retrospective multicenter study was performed comparing patients with SEOs (n = 50), endometrial cancer with synchronous ovarian metastasis (n = 19) and ovarian cancer with synchronous endometrial metastasis (n = 20). Targeted next‐generation sequencing was used, and a clonality index was calculated. Subsequently, cases were classified as POLE mutated, mismatch repair deficient (MMR‐D), TP53‐wild‐type or TP53‐mutated. In 92% of SEOs (46/50), the endometrial and concurrent ovarian carcinoma shared at least one somatic mutation, with a clonality index above 0.95, supporting a clonal origin. The SEO molecular profiles showed striking similarities with the TCGA endometrial carcinoma set. SEOs behaved distinctly different from metastatic disease, with a superior outcome compared to endometrial MD cases (p, What's new? When primary endometrial and ovarian tumors are found simultaneously in the same patient, it has been assumed that they are separate cancers that developed independently. However, in this study, the authors found that these tumors share a clonal origin 92% of the time. They also found that these “synchronous” cancers tend to have a favorable prognosis, with far better outcomes than metastatic disease. Some subgroups, including TP53 mutations and extra‐utero‐ovarian disease, were independent predictors for poor clinical outcome, which may impact adjuvant treatment planning.

Currently, no effective targeted therapeutics exists for treatment of metastatic prostate cancer (PCa). Given that matrix metalloproteinases 9 (MMP9) and its associated vascular endothelial growth factor (VEGF) are critical for tumor vascularization and invasion under castration‐resistant condition, it is therefore of great importance to define the functional association and interplay between androgen receptor (AR) and MMP9 and their associated key survival and invasion pathways in PCa cells. Here, we found that there was a significant correlation between MMP9 and AR protein expression in primary and metastatic PCa tissues, and a trend that high level of MMP9 expression was associated with poor prognosis. We demonstrated that constitutive activation of AR increased expression of MMP9 and VEGF/VEGF receptors. We further showed that AR exerts its effect on MMP9/VEGF signaling axis through PIP5K1α/AKT. We showed that MMP9 physically interacted with PIP5K1α via formation of protein–protein complexes. Furthermore, elevated expression of MMP9 enhanced ability of AR to activate its target gene cyclin A1. The elevated sequential activation of AR/PIP5K1α/AKT/MMP9/VEGF signaling axis contributed to increased invasiveness and growth of metastatic tumors. Conversely, treatment with PIP5K1α inhibitor significantly suppressed invasiveness of PCa cells expressing constitutively activated AR, this was coincident with its inhibitory effect of this inhibitor on AR/MMP9/VEGF pathways. Our results suggest that AR and MMP9‐associated network proteins may be effectively targeted by blocking PIP5K1α/AKT pathways using PIP5K1α inhibitor in metastatic PCa., What's new? The androgen receptor (AR) is a key factor in prostate cancer (PCa) progression and metastasis. Given that matrix metalloproteinase 9 (MMP9) and the vascular endothelial growth factor (VEGF) pathway are critical for tumor vascularization and invasion under castration‐resistant condition, it may be of importance to define the functional interplay between AR and MMP9 and their associated key survival and invasion pathways in PCa cells. This study identifies novel cooperative mechanisms involving AR, the MMP9/ VEGF signaling axis and PIP5K1α/AKT pathways driving tumor invasion. The findings provide new information to guide the development of targeted therapy for invasive castration‐resistant prostate cancer.

LIM kinases modulate multiple aspects of cancer development, including cell proliferation and survival. As the mechanisms of LIMK‐associated tumorigenesis are still unclear, we analyzed the tumorigenic functions of LIM kinase 2 (LIMK2) in human bladder cancer (BC) and explored whether the newly identified LIMK2 3´‐UTR SNP rs2073859 (G‐to‐A allele) is correlated with clinical features. Expression levels of LIMK2 in 38 human BC tissues and eight cell lines were examined using quantitative real‐time PCR and immunohistochemistry. LIMK2 was overexpressed in most BC tissues (27/38, 71%) and BC‐derived cell lines (6/8), and was more frequently overexpessed in high‐grade than low‐grade BC (80% vs. 47%). The effects of LIMK2 on BC cell proliferation, survival and migration, were studied by overexpression and RNA interference approaches in vitro and in vivo. LIMK2 overexpression promoted proliferation, migration and invasion of BC cells, while LIMK2 depletion inhibited cell invasion and viability and induced growth arrest in vitro and in vivo. PCR‐Restriction Fragment Length Polymorphism (RFLP) was used to genotype LIMK2 SNP rs2073859 and multivariate logistic regression applied to assess the relationship between allele frequency and clinical features in 139 BC patients. Functional analyses localized SNP rs2073859 within the microRNA‐135a seed‐binding region and revealed significantly lower LIMK2 G allele expression. The frequency of A genotypes (AG + AA) was higher in the BC group than normal controls and correlated with risks of high‐grade and high‐stage BC. In conclusion, LIMK2 may function as an oncogene in human BC, while allele‐specific regulation by microRNA‐135a may influence disease risk., What's new? Genetic variations in microRNA (miRNA)‐binding regions are suspected to influence tumorigenesis. In this study, in tissues from bladder cancer (BC) patients, the single nucleotide polymorphism rs2073859 (G‐to‐A allele) in the 3’‐UTR miRNA‐135a‐binding site of the oncogene LIM kinase 2 (LIMK2) was associated with gene overexpression and higher clinical grade. A high frequency of rs2073859 A genotypes was detected in BC tissues. In BC cells and mice, LIMK2 overexpression promoted cell proliferation, migration, and invasion and accelerated BC tumor growth. The findings suggest that LIMK2 normally is downregulated by miRNA‐135a and becomes tumorigenic following miRNA‐135a disruption by rs2073859 variant A.

VEGF stimulates endothelial cells as a key molecule in angiogenesis. VEGF also works as a multifunction molecule, which targets a variety of cell members in the tumor microenvironment. We aimed to reveal VEGF‐related molecular mechanisms on breast cancer cells. VEGF‐knocked‐out MDA‐MB‐231 cells (231VEGFKOex3) showed rounded morphology and shorter perimeter (1.6‐fold, p < 0.0001). The 231VEGFKOex3 cells also showed impaired cell migration (2.6‐fold, p = 0.002). Bevacizumab treatment did not induce any change in morphology and mobility. Soluble neuropilin‐1 overexpressing MDA‐MB‐231 cells (231sNRP1) exhibited rounded morphology and shorter perimeter (1.3‐fold, p < 0.0001). The 231sNRP1 cells also showed impaired cell migration (1.7‐fold, p = 0.003). These changes were similar to that of 231VEGFKOex3 cells. As MDA‐MB‐231 cells express almost no VEGFR, these results indicate that the interaction between NRP1 and long isoform of VEGF containing a NRP‐binding domain regulates the morphology and migration ability of MDA‐MB‐231 cells. Genome‐wide gene expression profiling identified ARHGAP17 as one of the target genes in the downstream of the VEGF/NRP1 signal. We also show that VEGF/NRP1 signal controls filopodia formation of the cells by modulating Cdc42 activity via ARHGAP17. Among 1,980 breast cancer cases from a public database, the ratio of VEGF and SEMA3A in primary tumors (n = 450) of hormone‐receptor‐negative breast cancer is associated with ARHGAP17 expression inversely, and with disease free survival. Altogether, the bevacizumab‐independent VEGF/NRP1/ARHGAP17/Cdc42 regulatory network plays important roles in malignant behavior of breast cancer., What's new? Vascular endothelial growth factor (VEGF) acts on endothelial cells to promote blood vessel formation and tumor growth. In breast cancer, VEGF expression is associated with poor prognosis, though its effects on breast cancer cells are not well understood. Here, the long isoform of VEGF (VEGF165) was found to generate a signal through the transmembrane protein neuropilin‐1 (NRP1), without acting on VEGF receptors. ARHGAP17, a RhoGAP family protein that regulates Cdc42 activity and controls filopodia formation and cell migration ability, was identified as a VEGF165 signaling target. The VEGF/NRP1/ARHGAP17 network likely contributes to malignant cell behavior in hormone receptor‐negative breast cancer.

More than 90% of oral cancers are histopathologically squamous cell carcinomas (SCCs). According to clinical behavior and histopathological features, we hypothesize that oral SCC can originate from either oral squamous epithelium or minor salivary glands. Here, we examined whether some oral SCCs originate from minor salivary glands, and investigated whether these tumors show particularly aggressive biological behavior. The mRNA expression profiles of samples obtained from six patients with oral floor SCC (five men, one woman; mean age, 62.7 years) were analyzed using a microarray containing 32,878 probes. The six samples were divided into two groups by clustering of expression levels of 845 probes differentially expressed in normal oral squamous epithelium and normal salivary glands. The expression profile in four cases was similar to that of normal oral squamous epithelium, and in two cases was similar to that of normal salivary glands. Furthermore, we identified nine genes that reveal the origin of the oral SCC. Subsequently, we examined the expression levels of these nine marker genes by reverse transcriptase‐polymerase chain reaction to determine the origin of 66 oral SCCs. Twelve of the 66 oral SCCs were considered to originate from minor salivary glands, and these tumors showed high metastatic potential (p = 0.044, Chi‐square test). Furthermore, SCC derived from minor salivary glands showed a poor event‐free survival rate (p = 0.017, Kaplan–Meier analysis). In conclusion, determination of the origin of oral SCC is helpful in planning treatment for patients with oral SCC., What's new? In spite of several past attempts, no distinct factors controlling the biological aggressiveness of oral squamous cell carcinoma (SCC) have been identified. Determining the origin of the cancer cells could help predict biological aggressiveness, however. Here, the authors provide evidence that oral SCC has two different origins, as mucosal or salivary SCC. Furthermore, they identify nine genes able to reveal the origin of oral SCC. Based on their expression levels, the authors determined that twelve of the 66 oral SCCs in their sample originated from minor salivary glands. Salivary SCC tumors showed high metastatic potential and poor event‐free survival rate.

Pre‐clinical non‐small cell lung cancer (NSCLC) models are poorly representative of the considerable inter‐ and intra‐tumor heterogeneity of the disease in patients. Primary cell‐based in vitro models of NSCLC are therefore desirable for novel therapy development and personalized cancer medicine. Methods have been described to generate rapidly proliferating epithelial cell cultures from multiple human epithelia using 3T3‐J2 feeder cell culture in the presence of Y‐27632, a RHO‐associated protein kinase (ROCK) inhibitor, in what are known as “conditional reprograming conditions” (CRC) or 3T3 + Y. In some cancer studies, variations of this methodology have allowed primary tumor cell expansion across a number of cancer types but other studies have demonstrated the preferential expansion of normal epithelial cells from tumors in such conditions. Here, we report our experience regarding the derivation of primary NSCLC cell cultures from 12 lung adenocarcinoma patients enrolled in the Tracking Cancer Evolution through Therapy (TRACERx) clinical study and discuss these in the context of improving the success rate for in vitro cultivation of cells from NSCLC tumors., What's new? Traditionally, the primary culture of human cancer cells has been challenging. A new protocol known as 3T3 + Y has allowed cancer cell cultures to be established across multiple cancer types, but reports are inconsistent for non‐small cell lung cancer (NSCLC). Here, the authors sought to expand tumor cells from surgically resected primary human lung adenocarcinoma tumors within the TRACERx clinical study using 3T3 + Y. Contaminating normal epithelial cells were the predominant cell type in early passage cultures, although KRAS‐mutant tumor cells were cultured in one donor culture. The researchers urge caution in the use of NSCLC primary cell cultures and call for further investigation in 3T3 + Y applications.

Gain‐of‐function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have been shown to be dysregulated in GISTs and impact KIT expression. Little is known though on KIT‐independent targets of KIT‐regulating mRNAs. We sought to investigate how miR‐494 inhibits GIST proliferation and to identify novel target gene. We used microarray‐based gene expression analyses to identify pathways and target genes affected by miR‐494. The expressional relationship between survivin and miR‐494 was determined in 35 GIST tissues. Cell proliferation assay, FACS analysis, colony formation assay, promoter assays and chromatin immunoprecipitation (ChiP) were performed to clarify the roles of survivin in GIST progression. Gene expression microarray analysis revealed that miR‐494 inhibited GISTs by affecting multiple genes in the cell cycle pathway. Survivin (BIRC5) was a key target of miR‐494, and its expression showed an inverse correlation with miR‐494 expression in 35 GIST tissues (Pearson's correlation coefficient, r = −0.418, p = 0.012). Downregulation of survivin inhibited proliferation and colony formation, and resulted in cell cycle alteration. Induced survivin overexpression relieved miR‐494‐mediated inhibition of GIST progression. Targeting PI3K effectively suppressed proliferation of GISTs with downregulation of survivin. Survivin also regulated KIT expression at the transcription level. Immunohistochemical analysis using 113 GISTs revealed that survivin expression was significantly correlated with overall survival of GIST patients (p = 0.004). Our findings indicated that miR‐494 synergistically suppressed GISTs by concomitantly targeting survivin and KIT., What's new? Gain‐of‐function mutations of KIT are pathognomonic in sporadic gastrointestinal stromal tumors (GISTs). Several microRNAs have also been shown to be dysregulated in GISTs and impact KIT expression. However, still little is known on KIT‐independent targets of KIT‐regulating mRNAs. Here, the authors report that miR‐494 induces alteration of the cell cycle pathways in GISTs by downregulating survivin, which functions as a transcription factor of KIT, accompanying effective blockade of the PI3K pathway that both KIT and survivin belong to. Based on these findings, miR‐494 may play important roles during GIST tumorigenesis and be used as a potent agent to treat GISTs.

The expression of PD‐L1 in breast cancer is associated with estrogen receptor negativity, chemoresistance and epithelial‐to‐mesenchymal transition (EMT), all of which are common features of a highly tumorigenic subpopulation of cancer cells termed cancer stem cells (CSCs). Hitherto, the expression and intrinsic role of PD‐L1 in the dynamics of breast CSCs has not been investigated. To address this issue, we used transcriptomic datasets, proteomics and several in vitro and in vivo assays. Expression profiling of a large breast cancer dataset (530 patients) showed statistically significant correlation (p, What's new? Cancer cells that express the T‐cell inhibitory molecule programmed death‐ligand 1 (PD‐L1) readily escape immune attack. In addition, PD‐L1 expression contributes to chemoresistance and is associated with epithelial‐to‐mesenchymal transition, a process that generates cancer stem cells (CSCs). This study shows that in breast cancer, PD‐L1 expression further plays a direct part in maintaining CSC stemness. In breast cancer cells, PD‐L1 expression sustained stemness factors OCT‐4A and Nanog, via a PI3K/AKT‐dependent pathway, and promoted expression of the stemness controlling factor BMI1, independent of PI3K/AKT. Targeting PD‐L1 could help advance breast cancer therapy, owing to impacts on the pool of breast CSCs.

We have previously reported that the negative signaling regulator Similar Expression to FGF (hSef) is downregulated in prostate cancer and its loss is associated with clinical metastasis. Here, we explored the mechanistic basis of this finding. We first confirmed our clinical observation by testing hSef manipulation in an in vivo metastasis model. hSef stable expressing cells (PC3M‐hSef) or empty vector controls (PC3M‐EV) were injected subcutaneously into the lateral thoracic walls of NOD‐SCID gamma mice and lungs were harvested at autopsy. In this model, 6/7 PC3M‐EV xenografts had definitive lung micro‐metastasis whilst only 1/6 PC3M‐hSef xenografts exhibited metastasis recapitulating the clinical scenario (p = 0.03). Gene expression studies revealed key perturbations in genes involved in cell motility and epithelial to mesenchymal transition (EMT) along with alterations in cognate signaling pathways. These results were validated in an EMT specific PCR array whereby hSef over‐expression and silencing reciprocally altered E‐Cadherin expression (p =, What's new? The loss of negative signaling regulators may be implicated in prostate cancer metastasis, but the underlying mechanisms remain unclear. Here, by using a combination of xenograft, gene expression microarray, phosphokinase array and quantitative PCR techniques, the authors provide first evidence that the negative regulator hSef plays a key role in regulating epithelial to mesenchymal transition (EMT) in prostate cancer, which in turn results in changes in the metastatic ability of tumor cells. The results support the notion that the expression levels of hSef and other negative signaling regulators may be key biomarkers for the identification of triggers for metastasis.

The primary challenge facing treatment of epithelial ovarian cancer (EOC) is the high frequency of chemoresistance, which severely impairs the quality of life and survival of patients with EOC. Our study aims to investigate the mechanisms by which upregulation of NR2F6 induces chemoresistance in EOC. The biological roles of NR2F6 in EOC chemoresistance were explored in vitro by Sphere, MTT and AnnexinV/PI assay, and in vivo using an ovarian cancer orthotopic transplantation model. Bioinformatics analysis, luciferase assay, CHIP and IP assays were performed to identify the mechanisms by which NR2F6 promotes chemoresistance in EOC. The expression of NR2F6 was significantly upregulated in chemoresistant EOC tissue, and NR2F6 expression was correlated with poorer overall survival. Moreover, overexpression of NR2F6 promotes the EOC cancer stem cell phenotype; conversely, knockdown of NR2F6 represses the EOC cancer stem cell phenotype and sensitizes EOC to cisplatin in vitro and in vivo. Our results further demonstrate that NR2F6 sustains activated Notch3 signaling, resulting in chemoresistance in EOC cells. Notably, NR2F6 acts as an informative biomarker to identify the population of EOC patients who are likely to experience a favorable objective response to gamma‐secretase inhibitors (GSI), which inhibit Notch signaling. Therefore, concurrent inhibition of NR2F6 and treatment with GSI and cisplatin‐based chemotherapy may be a novel therapeutic approach for NR2F6‐overexpressing EOC. In summary, we have, for the first time, identified an important role for NR2F6 in EOC cisplatin resistance. Our study suggests that GSI may serve as a potential targeted treatment for patients with NR2F6‐overexpressing EOC., What's new? Chemoresistance is a major challenge in women afflicted with epithelial ovarian cancer (EOC), but molecular mechanisms of EOC chemoresistance remain unclear. Here the authors connect nuclear receptor subfamily 2 group F member 6 (NR2F6) with this process. They find NR2F6 upregulated in tissues from chemoresistant EOC patients. High NR2F6 expression promoted a cancer stem cell phenotype and suppressed cisplatin‐induced apoptosis by transcriptionally upregulating Notch3 signaling, thereby promoting EOC chemoresistance.

The Focal adhesion kinase (FAK) is a ubiquitous cytoplasmic tyrosine‐kinase promoting tumor progression and metastasis processes by acting in cancer cells and their tumor microenvironment partners. FAK overexpression in primary colon tumors and their metastasis is associated to poor colorectal cancer (CRC) patients’ outcome. Eight FAK mRNA alternative splice variants have been described and contribute to additional level of FAK activity regulation, some of them corresponding to overactivated FAK isoforms. To date, FAK mRNA alternative splice variants expression and implication in CRC processes remain unknown. Here, using different human CRC cells lines displaying differential invasive capacities in an in vivo murine model recapitulating the different steps of CRC development from primary tumors to liver and lung metastasis, we identified three out of the eight mRNA variants (namely FAK0, FAK28 and FAK6) differentially expressed along the CRC process and the tumor sites. Our results highlight an association between FAK0 and FAK6 expressions and the metastatic potential of the most aggressive cell lines HT29 and HCT116, suggesting that FAK0 and FAK6 could represent aggressiveness markers in CRC. Our findings also suggest a more specific role for FAK28 in the interactions between the tumors cells and their microenvironment. In conclusion, targeting FAK0, the common form of FAK, might not be a good strategy based on the numerous roles of this kinase in physiological processes. In contrast, FAK6 or FAK28 splice variants, or their corresponding protein isoforms, may putatively represent future therapeutic target candidates in the development of CRC primary tumors and metastasis., What's new? Overexpression of the focal adhesion kinase (FAK) is associated with poor outcome in patients with colorectal cancer but the role of the eight splice variants of FAK remains unknown. Here the authors correlated FAK splice variant expression in colorectal tumor cell lines with invasiveness in mouse models. FAK0 and FAK6 splice variant expression was associated with higher aggressiveness and metastatic potential, underscoring that distinct FAK splice variants may represent new targets in the development of drugs against colorectal cancer and associated metastasis.

miRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. Little is known on the origin of circulating miRNAs and their relationship with the tumor microenvironment in lung cancer. Here, we focused on the cellular source and relative contribution of different cell types to circulating miRNAs composing our risk classifier of lung cancer using in vitro/in vivo models and clinical samples. A cell‐type specific expression pattern and topography of several miRNAs such as mir‐145 in fibroblasts, mir‐126 in endothelial cells, mir‐133a in skeletal muscle cells was observed in normal and lung cancer tissues. Granulocytes and platelets are the major contributors of miRNAs release in blood. miRNAs modulation observed in plasma of lung cancer subjects was consistent with de‐regulation of the same miRNAs observed during immunosuppressive conversion of immune cells. In particular, activated neutrophils showed a miRNA profile mirroring that observed in plasma of lung cancer subjects. Interestingly mir‐320a secreted by neutrophils of high‐risk heavy‐smokers promoted an M2‐like protumorigenic phenotype through downregulation of STAT4 when shuttled into macrophages. These findings suggest a multifactorial and nonepithelial cell‐autonomous origin of circulating miRNAs associated with risk of lung cancer and that circulating miRNAs may act in paracrine signaling with causative role in lung carcinogenesis and immunosuppression., What's new? microRNAs play a central role in the complex signaling network of cancer cells with the tumor microenvironment. However, little is known on the origin of circulating miRNAs and their mechanisms of action. This study found a multifactorial and non‐epithelial cell‐autonomous origin of circulating miRNAs associated with lung cancer risk. The findings also suggest a link between an immunosuppressive and pro‐tumorigenic microenvironment and modulation of circulating miRNAs associated with lung cancer risk. The authors propose a novel mechanism whereby miRNA released by neutrophils induce macrophage polarization to support lung cancer growth, highlighting the potential for reprogramming macrophages toward an anti‐tumor polarization.

Viruses can inhibit host autophagy through multiple mechanisms, and evasion of autophagy plays an important role in immune suppression and viral oncogenesis. Merkel cell polyomavirus (MCPyV) T‐antigens are expressed and involved in the pathogenesis of a large proportion of Merkel cell carcinoma (MCC). Yet, how MCPyV induces tumorigenesis is not fully understood. Herein, we show that MCPyV T‐antigens induce miR‐375, miR‐30a‐3p and miR‐30a‐5p expressions, which target multiple key genes involved in autophagy, including ATG7, SQSTM1 (p62) and BECN1. In MCC tumors, low expression of ATG7 and p62 are associated with MCPyV‐positive tumors. Ectopic expression of MCPyV small T‐antigen and truncated large T‐antigen (LT), but not the wild‐type LT, resulted in autophagy suppression, suggesting the importance of autophagy evasion in MCPyV‐mediated tumorigenesis. Torin‐1 treatment induced cell death, which was attenuated by autophagy inhibitor, but not pan‐caspase inhibitor, suggesting a potential role of autophagy in promoting cell death in MCC. Conceptually, our study shows that MCPyV oncoproteins suppress autophagy to protect cancer cells from cell death, which contribute to a better understanding of MCPyV‐mediated tumorigenesis and potential MCC treatment., What's new? About four‐fifths of Merkel cell carcinomas harbor the Merkel cell polyomavirus (MCPyV) genome, though mutations in viral T antigens generally render MCPyV replication‐deficient. Here, the authors describe a network by which MCPyV oncoproteins and viral‐regulated miRNAs hijack autophagy machinery in MCC. In cells, expression of MCPyV T‐antigens induced miR‐375, miR‐30a‐3p, and miR‐30a‐5p expression. Subsequent experiments validated these miRNAs as autophagy regulators that specifically target the genes ATG7, SQSTM1, and BECN1. Autophagy suppression protected MCC cell survival. This novel insight into the role of MCPyV and autophagy regulation in MCC may be relevant in the generation of future therapeutic strategies.

Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti‐acid drugs, food additives and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. For example, no experimental evidence that aluminium promotes tumorigenesis in cultured mammary epithelial cells exists. We report here that long‐term exposure to concentrations of aluminium—in the form of aluminium chloride (AlCl3)—in the range of those measured in the human breast, transform normal murine mammary gland (NMuMG) epithelial cells in vitro as revealed by the soft agar assay. Subcutaneous injections into three different mouse strains with decreasing immunodeficiency, namely, NOD SCID gamma (NSG), NOD SCID or nude mice, revealed that untreated NMuMG cells form tumors and metastasize, to a limited extent, in the highly immunodeficient and natural killer (NK) cell deficient NSG strain, but not in the less permissive and NK cell competent NOD SCID or nude strains. In contrast, NMuMG cells transformed in vitro by AlCl3 form large tumors and metastasize in all three mouse models. These effects correlate with a mutagenic activity of AlCl3. Our findings demonstrate for the first time that concentrations of aluminium in the range of those measured in the human breast fully transform cultured mammary epithelial cells, thus enabling them to form tumors and metastasize in well‐established mouse cancer models. Our observations provide experimental evidence that aluminium salts could be environmental breast carcinogens., What's new? Aluminium salts, present in many industrial products of frequent use like antiperspirants, anti‐acid drugs, food additives, and vaccines, have been incriminated in contributing to the rise in breast cancer incidence in Western societies. However, current experimental evidence supporting this hypothesis is limited. Here, the authors report that long‐term exposure to concentrations of aluminium in the range of those measured in the human breast enables normal murine mammary gland (NMuMG) epithelial cells to form tumors and metastasis in well‐established mouse cancer models. The observations indicate that aluminium salts could be environmental breast carcinogens.

The relevance of the clinicopathological and molecular features of early gastric cancers (EGCs) having the microsatellite instability (MSI)‐high phenotype has not been clearly defined in sporadic gastric carcinogenesis. Here, we examined the clinicopathological and molecular characteristics of EGC according to MSI status in 330 patients with EGC (intestinal‐type adenocarcinoma). Tumors were classified as MSI‐high (45 cases), MSI‐low (9 cases), or microsatellite stable (MSS; 276 cases). The specimens were examined using a combination of polymerase chain reaction (PCR)‐microsatellite assays and PCR‐pyrosequencing to detect chromosomal allelic imbalances in multiple cancer‐related chromosomal loci, MSI, gene mutations (KRAS and BRAF) and methylation status [high methylation epigenome (HME), intermediate methylation epigenome and low methylation epigenome]. In addition, the expression levels of various target proteins were examined using immunohistochemistry. Interestingly, EGC with the MSI phenotype showed distinct papillary features. The expression of gastric mucin was more frequent in EGC with the MSI phenotype, while p53 overexpression was common in EGCs, irrespective of MSI status. The frequency of HME was significantly higher in EGCs with the MSI phenotype than in EGCs with the MSS phenotype. Although there was a low frequency of allelic imbalance in EGCs with the MSI phenotype, some markers of allelic imbalance were more frequently detected in EGCs with the MSI‐high phenotype than in EGCs with the MSS phenotype. KRAS and BRAF mutations were rare in EGCs. Thus, the MSI phenotype in EGC is a major precursor lesion in gastric cancer and is characterized by distinct clinicopathological and molecular features., What's new? The relevance of the clinicopathological and molecular features of early gastric cancers with the microsatellite instability (MSI)‐high phenotype remains to be clarified in sporadic gastric carcinogenesis. This study shows that early gastric cancers with the MSI‐high phenotype exhibit distinct histological features and accumulation of both genomic damage and MSI within the same tumors. In regions with genomic damage, the frequencies of 3p and 22q AI were significantly higher in the MSI‐high phenotype than in the microsatellite stable phenotype. The treatment strategies for patients with gastric cancers having the MSI‐high phenotype may thus need to differ from patients with colorectal cancer.

Mutations of KRAS, NRAS, BRAF and DNA mismatch repair (MMR) status have become an important part of the assessment of patients with colorectal cancer (CRC), while respective clinicopathologic features and prognostic significance in specific stages and related detection strategies remain unclear. We retrospectively analyzed clinicopathologic features and prognosis of 1,834 patients with Stage I–IV colorectal adenocarcinoma. Mutations in KRAS, NRAS and BRAF and DNA MMR status were determined. The mutation rates of KRAS, NRAS and BRAF were 46.4, 3.2 and 3.5%, respectively, and the mismatch repair gene deletion (dMMR) rate was 5.6%. In a multivariate analysis, female, advanced age, tumor type histology, mucinous carcinoma and positive tumor deposits were associated with a high KRAS mutation rate. A high BRAF mutation rate was associated with female, poor differentiation, lymphovascular invasion and positive tumor deposits. Factors associated with high dMMR rates included low age, large tumor size, poor differentiation, Stages I–III. Tumor site was independently associated with KRAS mutation, BRAF mutation and dMMR. KRAS and BRAF mutations were independent risk factors for shorter overall survival (OS) in Stage IV tumors but not in Stage I–III tumors. NRAS mutation was an independent risk factor for shorter OS in Stage I–II tumors. dMMR was independently associated with longer OS in Stage III tumors., What's new? Mutations in KRAS, NRAS, BRAF and DNA mismatch repair (MMR) status are important biomarkers in the assessment of patients with colorectal cancer (CRC). However, the clinicopathologic features associated with these mutations—and their impact on prognosis—are unclear, especially at earlier stages of CRC. In this large Chinese study, the authors analyzed variables such as gender, age, tumor histology, lymphovascular invasion, etc., that were associated with particular oncogene mutations and overall survival. These results should provide guidance for improved clinical strategies and enhance the usefulness of these biomarkers.

Standard therapy for advanced Prostate Cancer (PCa) consists of antiandrogens, which provide respite from disease progression, but ultimately fail resulting in the incurable phase of the disease: mCRPC. Targeting PCa cells before their progression to mCRPC would greatly improve the outcome. Combination therapy targeting the DNA Damage Response (DDR) has been limited by general toxicity, and a goal of clinical trials is how to target the DDR more specifically. We now show that androgen deprivation therapy (ADT) of LNCaP cells results in increased expression of TLK1B, a key kinase upstream of NEK1 and ATR and mediating the DDR that typically results in a temporary cell cycle arrest of androgen responsive PCa cells. Following DNA damage, addition of the TLK specific inhibitor, thioridazine (THD), impairs ATR and Chk1 activation, establishing the existence of a ADT > TLK1 > NEK1 > ATR > Chk1, DDR pathway, while its abrogation leads to apoptosis. Treatment with THD suppressed the outgrowth of androgen‐independent (AI) colonies of LNCaP and TRAMP‐C2 cells cultured with bicalutamide. Moreover, THD significantly inhibited the growth of several PCa cells in vitro (including AI lines). Administration of THD or bicalutamide was not effective at inhibiting long‐term tumor growth of LNCaP xenografts. In contrast, combination therapy remarkably inhibited tumor growth via bypass of the DDR. Moreover, xenografts of LNCaP cells overexpressing a NEK1‐T141A mutant were durably suppressed with bicalutamide. Collectively, these results suggest that targeting the TLK1/NEK1 axis might be a novel therapy for PCa in combination with standard of care (ADT)., What's new? Standard therapy for advanced Prostate Cancer (PCa) consists of anti‐androgens, which only provide temporary respite from disease progression to metastatic castrate‐resistant prostate cancer (mCRPC). Here, the authors show in the LNCaP cell model that the increased expression with ADT of TLK1B, a prosurvival checkpoint pathway that is enacted before conversion to androgen‐independent growth, offers a unique target for attacking more specifically PCa cells before their conversion to CRPC. Moreover, they suggest to re‐purpose thioridazine or other phenothiazine antipsychotic drugs as inhibitors of the TLK1 > Nek1 > ATR > Chk1 DNA Damage Response (DDR) axis for the early treatment of advanced PCa still responsive to ADT.

Ras proteins, most notably KRas, are prevalent oncogenes in human cancer. Plasma membrane localization and thereby signaling of KRas is regulated by the prenyl‐binding protein PDEδ. Recently, we have reported the specific anti‐proliferative effects of PDEδ inhibition in KRas‐dependent human pancreatic ductal adenocarcinoma cell lines. Here, we investigated the proliferative dependence on the solubilizing activity of PDEδ of human colorectal cancer (CRC) cell lines with or without oncogenic KRas mutations. Our results show that genetic and pharmacologic interference with PDEδ specifically inhibits proliferation and survival of CRC cell lines harboring oncogenic KRas mutations whereas isogenic cell lines in which the KRas oncogene has been removed, or cell lines with oncogenic BRaf mutations or EGFR overexpression are not dependent on PDEδ. Pharmacological PDEδ inhibition is therefore a possible new avenue to target oncogenic KRas bearing CRC., What's new? Oncogenic KRas mutations are present in about 45% of colorectal cancers (CRCs), where they are associated with poor prognosis. While KRas is an appealing therapeutic target, it has repeatedly eluded small‐molecule inhibitors. Here, the authors chose instead to target PDEδ, a prenyl‐binding protein that regulates the plasma membrane localization of KRas. In experiments in human colorectal cancer cells, PDEδ inhibition limited proliferation and survival in cells harboring KRas mutations, with no effect on wild‐type KRas cells, providing a new therapeutiv opportunity for CRC harbouring oncogenic KRas. In addition, PDEδ protein expression was correlated with oncogenic KRas activity within the CRC cell panel, suggesting that PDEδ protein‐level determination may be of prognostic relevance for CRC patients.

Langerhans cell histiocytosis (LCH) is the most common histiocytosis with constitutive activation of the RAS–RAF–MEK–ERK (MAPKinase) cell signaling pathway. We analyzed 89 cases of BRAF and MAP2K1 mutations by Sanger sequencing, of which 18 cases showed that these two gene mutations are negative. Whole genome sequencing of suitable specimens in these negative cases revealed a translocation from the 3 intron of PLEKHA6 to the 13 intron of NTRK3 in one case. We identified that this translocation could cause a novel fusion mutation, PLEKHA6‐NTRK3. Overexpression of the PLEKHA6‐NTRK3 mutant in NIH 3T3 cells enhanced MAPKinase pathway activation, promote cell growth. Our result suggested that a new mutation need be included in LCH molecular screening panel to better define its prevalence in LCH., What's new? Langerhans cell histiocytosis (LCH) is a rare immune and neoplastic disorder. While it is known as the most common histiocytosis with constitutive activation of the RAS‐RAF‐MEK‐ERK (MAPKinase) cell signaling pathway, its pathogenesis remains obscure. Here, whole‐genome sequencing of BRAF V600E‐negative and MAP2K1‐negative LCH cases revealed a translocation from the intron 3 of PLEKHA6 to the intron 13 of NTRK3 in one patient, identifying a novel fusion mutation. Overexpression of PLEKHA6‐NTRK3 in vitro enhanced MAPKinase pathway activation, promoting cell growth. The results support the inclusion of the fusion mutation in LCH molecular screening panel to better define its prevalence in patients.

ST3Gal1 is a key sialyltransferase which adds α2,3‐linked sialic acid to substrates and generates core 1 O‐glycan structure. Upregulation of ST3Gal1 has been associated with worse prognosis of breast cancer patients. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. In our study, we demonstrated that ST3GAL1‐silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. We identified vasorin (VASN) which was shown to bind TGF‐β1, as a potential candidate that links ST3Gal1 to angiogenesis. LC‐MS/MS analysis of VASN secreted from MCF7, revealed that more than 80% of its O‐glycans are sialyl‐3T and disialyl‐T. ST3GAL1‐silencing or desialylation of VASN by neuraminidase enhanced its binding to TGF‐β1 by 2‐ to 3‐fold and thereby dampening TGF‐β1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Examination of 114 fresh primary breast cancer and their adjacent normal tissues showed that the expression levels of ST3Gal1 and TGFB1 were high in tumor part and the expression of two genes was positively correlated. Kaplan Meier survival analysis showed a significantly shorter relapse‐free survival for those with lower expression VASN, notably, the combination of low VASN with high ST3GAL1 yielded even higher risk of recurrence (p = 0.025, HR = 2.967, 95% CI = 1.14–7.67). Since TGF‐β1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF‐β1 upregulates ST3Gal1 to circumvent the negative impact of VASN., What's new? The addition of sialic acid to glycoproteins is dysregulated in many cancers, and enhanced expression of one key enzyme, the sialyltransferase ST3Gal1, is associated with poor prognosis. Here, the authors identified the membrane protein vasorin as a new ST3Gal1 substrate and connect it with TGF‐β1‐induced signaling and angiogenesis in breast cancer. As silencing of ST3Gal1 dampened TGF‐β1 signaling and suppressed angiogenesis, development of ST3Gal1 inhibitors might be clinically useful to improve the prognosis of breast cancer patients.

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death among women worldwide. Breast cancer metastasis results in poor prognosis and increased mortality, but the mechanisms of breast cancer metastasis are yet to be fully resolved. Identifying distinctive proteins that regulate metastasis might be targeted to improve therapy in breast cancer. We previously described MOSPD2 as a surface membrane protein that regulates monocyte migration in vitro. In this study, we demonstrate for the first time that MOSPD2 has a major role in breast cancer cell migration and metastasis. MOSPD2 expression was highly elevated in invasive and metastatic breast cancer while it was absent or residual in normal tissue and in primary in situ tumors. In vitro experiments showed that silencing MOSPD2 in different breast cancer cell lines significantly inhibited cancer cell chemotaxis migration. Mechanistically, we found that silencing MOSPD2 profoundly abated phosphorylation events that are involved in breast tumor cell chemotaxis. In vivo, MOSPD2‐silenced breast cancer cells exhibited marked impaired metastasis to the lungs. These results indicate that MOSPD2 plays a key role in the migration and metastasis of breast cancer cells and may be used to prevent the spreading of breast cancer cells and to mediate their death., What's new? Motile sperm domain‐containing protein 2 (MOSPD2) is a key regulator of monocyte migration and is suspected of promoting cancer cell metastasis. In cancer patients, its expression is inversely correlated with survival. Here, MOSPD2 expression was found to be significantly elevated in metastatic breast cancer cells, with no or only residual expression in normal tissues and primary in situ tumors. in vitro MOSPD2 silencing inhibited breast cancer cell migration and impeded migration‐associated phosphorylation events. Its silencing in vivo impaired metastasis to the lungs, suggesting that MOSPD2 inhibition could be a useful therapeutic strategy for metastatic breast cancer in human patients.

Colorectal cancer results from the malignant transformation of colonic epithelial cells. Stromal fibroblasts are the main component of the tumour microenvironment, and play an important role in the progression of this and other neoplasias. Wnt/β-catenin signalling is essential for colon homeostasis, but aberrant, constitutive activation of this pathway is a hallmark of colorectal cancer. Here we present the first transcriptomic study on the effect of a Wnt factor on human colonic myofibroblasts. Wnt3A regulates the expression of 1,136 genes, of which 662 are upregulated and 474 are downregulated in CCD-18Co cells. A set of genes encoding inhibitors of the Wnt/β-catenin pathway stand out among those induced by Wnt3A, which suggests that there is a feedback inhibitory mechanism. We also show that the PKP2 gene encoding the desmosomal protein Plakophilin-2 is a novel direct transcriptional target of Wnt/β-catenin in normal and colon cancer-associated fibroblasts. PKP2 is induced by β-catenin/TCF through three binding sites in the gene promoter and one additional binding site located in an enhancer 20 kb upstream from the transcription start site. Moreover, Plakophilin-2 antagonizes Wnt/β-catenin transcriptional activity in HEK-293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/β-catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signalling and that Plakophilin-2 plays a role in the feedback control of this effect suggesting that the response to Wnt factors in the stroma may modulate Wnt activity in the tumour cells., This work was supported by the Spanish Ministerio de Economía y Competitividad and the European Regional Development Fund (Fondos FEDER) (SAF2013–43468-R, and SAF2014–53819-R), the Agencia Estatal de Investigacion-Fondos FEDER (SAF2016–76377-R, AEI/FEDER, UE), the Comunidad de Madrid (S2010/BMD-2344 Colomics2), the Instituto de Salud Carlos III–Fondos FEDER (RD12/0036/0021, and CIBERONC-CB16/12/00273) and the Nuclear Receptors Network (Nurcamein, SAF2015–71878-REDT).

The expression of the tumor suppressor p53 can influence the bioactivation of, and DNA damage induced by, the environmental carcinogen benzo[a]pyrene, indicating a role for p53 in its cytochrome P450 (CYP)‐mediated biotransformation. The carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), which is formed during the cooking of food, is also metabolically activated by CYP enzymes, particularly CYP1A2. We investigated the potential role of p53 in PhIP metabolism in vivo by treating Trp53(+/+), Trp53(+/−) and Trp53(−/−) mice with a single oral dose of 50 mg/kg body weight PhIP. N‐(Deoxyguanosin‐8‐yl)‐2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP‐C8‐dG) levels in DNA, measured by liquid chromatography‐tandem mass spectrometry, were significantly lower in liver, colon, forestomach and glandular stomach of Trp53(−/−) mice compared to Trp53(+/+) mice. Lower PhIP‐DNA adduct levels in the livers of Trp53(−/−) mice correlated with lower Cyp1a2 enzyme activity (measured by methoxyresorufin‐O‐demethylase activity) in these animals. Interestingly, PhIP‐DNA adduct levels were significantly higher in kidney and bladder of Trp53(−/−) mice compared to Trp53(+/+) mice, which was accompanied by higher sulfotransferase (Sult) 1a1 protein levels and increased Sult1a1 enzyme activity (measured by 2‐naphthylsulfate formation from 2‐naphthol) in kidneys of these animals. Our study demonstrates a role for p53 in the metabolism of PhIP in vivo, extending previous results on a novel role for p53 in xenobiotic metabolism. Our results also indicate that the impact of p53 on PhIP biotransformation is tissue‐dependent and that in addition to Cyp1a enzymes, Sult1a1 can contribute to PhIP‐DNA adduct formation., What's new? Many environmental carcinogens must be metabolized to form reactive electrophilic species capable of damaging DNA. The tumour suppressor p53 was shown to influence the cytochrome P450 (CYP)‐mediated biotransformation of the environmental carcinogen benzo[a]pyrene. The suspected carcinogen 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), which is formed during the cooking of food, is also metabolically activated by CYP enzymes. This study shows that expression of p53 influences the biotransformation of PhIP. Using a gene knockout mouse model, the authors show that the impact of p53 on PhIP‐DNA adduct formation is tissue‐dependent and that, in addition to cytochrome P450 enzymes, sulfotransferases contribute to the bioactivation of PhIP.

Our previous studies showed that the 1,25‐dihydroxyvitamin D (1,25‐D3) catabolizing enzyme, 1,25‐dihydoxyvitamin D 24 hydroxylase (CYP24A1) was overexpressed in colorectal tumours and its level correlated with increased proliferation. We hypothesised that cells overexpressing CYP24A1 have growth advantage and a diet rich in vitamin D and soy would restore sensitivity to the anti‐tumourigenic effects of vitamin D. Soy contains genistein, a natural CYP24A1 inhibitor. To determine causality between CYP24A1 and tumour growth, we established xenografts in male SCID mice with HT29 cells stably overexpressing either GFP‐tagged CYP24A1 or GFP. Mice were fed with either high (2500 IU D3/kg) or low vitamin D (100 IU D3/kg) diet in the presence or absence of soy (20% diet). In vitro, cells overexpressing CYP24A1 grew faster than controls. 1,25‐D3, the active vitamin D metabolite, reduced cell number only in the presence of the CYP24A1 inhibitor VID400. Regardless of the amount of vitamin D in the diet, xenografts overexpressing CYP24A1 grew faster, were heavier and more aggressive. Soy reduced tumour volume only in the control xenografts, while the tumours overexpressing CYP24A1 were larger in the presence of dietary soy. In conclusion, we demonstrate that CYP24A1 overexpression results in increased aggressiveness and proliferative potential of colorectal tumours. Irrespective of the dietary vitamin D3, dietary soy is able to increase tumour volume when tumours overexpress CYP24A1, suggesting that combination of vitamin D3 and soy could have an anti‐tumourigenic effect only if CYP24A1 levels are normal., What's new? Vitamin D3 supplementation is linked to reduced tumour growth in the colon, but colorectal tumours may also overexpress the vitamin‐D‐degrading enzyme CYP24A1, potentially undermining the benefits of vitamin D intake. We show that in a mouse xenograft model, CYP24A1 overexpression confers growth advantage to colorectal tumours, regardless of vitamin D intake. Combination of dietary vitamin D and soy increases volume and weight of tumours overexpressing CYP24A1. The findings warrant exploration of the effects of specific CYP24A1 inhibitors in CYP24A1‐overexpressing colorectal tumours.

Persistent activation of hedgehog (HH)/GLI signaling accounts for the development of basal cell carcinoma (BCC), a very frequent nonmelanoma skin cancer with rising incidence. Targeting HH/GLI signaling by approved pathway inhibitors can provide significant therapeutic benefit to BCC patients. However, limited response rates, development of drug resistance, and severe side effects of HH pathway inhibitors call for improved treatment strategies such as rational combination therapies simultaneously inhibiting HH/GLI and cooperative signals promoting the oncogenic activity of HH/GLI. In this study, we identified the interleukin‐6 (IL6) pathway as a novel synergistic signal promoting oncogenic HH/GLI via STAT3 activation. Mechanistically, we provide evidence that signal integration of IL6 and HH/GLI occurs at the level of cis‐regulatory sequences by co‐binding of GLI and STAT3 to common HH‐IL6 target gene promoters. Genetic inactivation of Il6 signaling in a mouse model of BCC significantly reduced in vivo tumor growth by interfering with HH/GLI‐driven BCC proliferation. Our genetic and pharmacologic data suggest that combinatorial HH‐IL6 pathway blockade is a promising approach to efficiently arrest cancer growth in BCC patients., What's new? Persistent activation of hedgehog (HH)/GLI signaling represents the main driver signal for the development of basal cell carcinoma (BCC), a common non‐melanoma skin cancer with rising incidence. Small molecule hedgehog pathway inhibitors are successfully used for the treatment of hedgehog‐driven BCC, but frequent drug resistance calls for improved strategies. Here, the authors identified the interleukin‐6 pathway as a novel synergistic signal promoting oncogenic HH/GLI via STAT3 activation. The synergistic interaction was required for the in vivo growth of hedgehog‐driven BCC. The study thus provides a rationale for effective combination treatments simultaneously targeting oncogenic hedgehog and interleukin‐6 signaling in BCC patients.

A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research., What's new? A variety of analytical approaches have indicated that melanoma cell line M14 and breast carcinoma cell line MDA‐MB‐435 originate from a common donor, but there is ongoing debate regarding which is the misidentified cell line. Here, authentication testing of M14 from 1975 (prior to the establishment of MDA‐MB‐435), with comparison to donor serum and lymphoblastoid cell line ML14, shows that M14 is the authentic cell line and MDA‐MB‐435 is a misidentified derivative. With clear provenance information and authentication testing of early samples, debates regarding the origins of problematic cell lines that are widely used in cancer research can be resolved.

TP53 mutations occur in half of all human tumours. Mutagen‐induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock‐in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen‐treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2–5 months) and much effort is expended maintaining TP53‐WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin‐3a, an MDM2 inhibitor that leads to stabilisation and activation of wild‐type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin‐3a to examine the effect on cell growth and p53 activation. Nutlin‐3a induced the p53 pathway in TP53‐WT HUFs and inhibited cell growth, whereas most TP53‐mutated HUFs were resistant to Nutlin‐3a. We then assessed whether Nutlin‐3a treatment could discriminate between TP53‐WT and TP53‐mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin‐3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin‐3a‐resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin‐3a‐sensitive clones were TP53‐WT. These data suggest that including a Nutlin‐3a counter‐screen significantly improves the specificity and efficiency of the HIMA, whereby TP53‐mutated clones are selected prior to sequencing and TP53‐WT clones can be discarded., What's new? Because half of human tumors carry mutations in the TP53 tumor suppressor gene, a mammalian cell culture system reproducing the diverse mutations seen in patients is necessary to properly study functional impact. Here the authors report a significant improvement to an existing model system using fibroblasts from human TP53 knock‐in mice (HIMA). They show that treating cells with Nutlin‐3a, an inhibitor of the destabilizing cofactor MDM2, efficiently selects for the growth of TP53‐mutated immortalized cells, while selecting against TP53‐wild type cells, thus increasing specificity and efficiency of TP53 mutagenesis.

Aberrant activation of Hedgehog (HH)/GLI signaling is causally involved in numerous human malignancies, including basal cell carcinoma (BCC) and medulloblastoma. HH pathway antagonists targeting smoothened (SMO), an essential effector of canonical HH/GLI signaling, show significant clinical success in BCC patients and have recently been approved for the treatment of advanced and metastatic BCC. However, rapid and frequent development of drug resistance to SMO inhibitors (SMOi) together with severe side effects caused by prolonged SMOi treatment call for alternative treatment strategies targeting HH/GLI signaling downstream of SMO. In this study, we report that 4SC‐202, a novel clinically validated inhibitor of class I histone deacetylases (HDACs), efficiently blocks HH/GLI signaling. Notably, 4SC‐202 treatment abrogates GLI activation and HH target gene expression in both SMOi‐sensitive and ‐resistant cells. Mechanistically, we propose that the inhibition of HDACs 1/2/3 is crucial for targeting oncogenic HH/GLI signaling, and that class I HDAC inhibitors either in combination with SMOi or as second‐line therapy may improve the treatment options for HH‐associated malignancies with SMOi resistance., What's new? Inhibition of signaling by Hedgehog polypeptide and the effector protein GLI is an effective therapeutic strategy for basal cell carcinoma and medulloblastoma. The frequent development of resistance to Hedgehog pathway inhibitors (HPI), however, calls for the identification of alternative drugs. This study identifies the class I histone deacetylase inhibitor 4SC‐202 as a novel Hedgehog/GLI antagonist. A clinically suitable small molecule, 4SC‐202, showed inhibitory efficacy, blocking GLI activation and Hedgehog target gene expression, even in cells with acquired HPI resistance. The findings suggest that 4SC‐202 could be effective against Hedgehog‐driven cancers that are refractory to standard targeted therapy.

We determined prognostic impact of KRAS, BRAF, PIK3CA and TP53 mutation status and mutation heterogeneity among 164 colorectal cancer (CRC) patients undergoing liver resections for metastatic disease. Mutation status was determined by Sanger sequencing of a total of 422 metastatic deposits. In univariate analysis, KRAS (33.5%), BRAF (6.1%) and PIK3CA (13.4%) mutations each predicted reduced median time to relapse (TTR) (7 vs. 22, 3 vs. 16 and 4 vs. 17 months; p, What's new? Preliminary evidence suggests that poor outcome after liver resection in metastatic colorectal cancer (CRC) is predicted by mutations in KRAS and BRAF and by intra‐individual heterogeneity involving copy number alterations that vary from one metastatic lesion to the next. Little is known, however, about the clinical implications of intra‐individual mutation heterogeneity in CRC. Here, in a comparison of KRAS and BRAF wild‐type status, mutational homogeneity, and mutational heterogeneity, mutation heterogeneity was found to be the strongest predict or of reduced disease‐specific survival following liver resection in metastatic CRC. Knowledge of intra‐individual mutation heterogeneity in KRAS and BRAF in CRC could facilitate therapeutic decisions.