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Myofibroblastic cancer-associated fibroblast (myoCAF)–rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species–producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti–PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. Significance: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.

Epithelial-mesenchymal transition (EMT) is an embryonic program that is reactivated in cancer and regulates the invasion and metastasis of tumor cells. Zinc finger E-box binding homeobox 2 (ZEB2) induces EMT by upregulating matrix metalloproteinases (MMP), yet MMP genes lack ZEB2 binding motif in their promoters. Recently, expression of MMPs was associated to the activation of ETS1 transcription factor; however, a link between ZEB2 and ETS proto-oncogene 1, transcription factor (ETS1) remains to be elucidated. Hence, we investigated the transcriptional regulation of ETS1 by ZEB2 after our initial observation that ZEB2 and ETS1 are coexpressed in hepatocellular carcinoma cells (HCCs). Chromatin immunoprecipitation and luciferase reporter assays clearly showed that ZEB2 binds to E-box sequences on the promoter of ETS1. Elevated expression of ETS1 was found in DLD-ZEB2 and A431-ZEB2 inducible systems, and knockdown of ZEB2 caused an explicit downregulation of ETS1 in shZEB2-SNU398 and shZEB2-SK-HEP-1 cells. Repression of ETS1 expression in ZEB2-induced conditions substantially impaired the migration and invasive capacities of DLD1 cells. Mechanistically, knockdown of ETS1 in ZEB2-expressing cells resulted in the downregulation of established ZEB2 targets TWIST and MMP9. Correlation analyses in HCC lines, cancer complementary DNA arrays, and The Cancer Genome Atlas RNA-sequencing data set revealed that ZEB2 and ETS1 are coexpressed, and their expressions in human tumors show a highly significant positive correlation. Our results demonstrated that ZEB2 acts as an upstream regulator of ETS1 and, in turn, ETS1 maintains ZEB2-induced EMT. These findings add another level of complexity to the understanding of ZEB2 in the invasion and metastasis of cancer cells, and put ZEB2/ETS1 axis as a novel therapeutic target in human malignancies.

Resistance to adjuvant chemotherapy is a major clinical problem in the treatment of colorectal cancer (CRC). The aim of this study was to elucidate the role of an epithelial to mesenchymal transition (EMT)‐inducing protein, ZEB2, in chemoresistance of CRC, and to uncover the underlying mechanism. We performed IHC for ZEB2 and association analyses with clinical outcomes on primary CRC and matched CRC liver metastases in compliance with observational biomarker study guidelines. ZEB2 expression in primary tumours was an independent prognostic marker of reduced overall survival and disease‐free survival in patients who received adjuvant FOLFOX chemotherapy. ZEB2 expression was retained in 96% of liver metastases. The ZEB2‐dependent EMT transcriptional programme activated nucleotide excision repair (NER) pathway largely via upregulation of the ERCC1 gene and other components in NER pathway, leading to enhanced viability of CRC cells upon oxaliplatin treatment. ERCC1‐overexpressing CRC cells did not respond to oxaliplatin in vivo, as assessed using a murine orthotopic model in a randomised and blinded preclinical study. Our findings show that ZEB2 is a biomarker of tumour response to chemotherapy and risk of recurrence in CRC patients. We propose that the ZEB2‐ERCC1 axis is a key determinant of chemoresistance in CRC., Here, we show that the expression of ZEB2 marks reduced overall and disease‐free survival in primary and secondary colorectal cancers (CRCs). ZEB2 overexpression promoted chemoresistance to oxaliplatin in vitro and in vivo by transcriptionally activating nucleotide excision repair genes ERCC1 and ERCC4. Overall ZEB2 is a promising biomarker predicting both therapy response and metastatic ability of CRCs.

Background Hepatocellular carcinoma (HCC) is common, but remains difficult to treat. Natural killer (NK) cells are cells of the innate immune system that have potent anti-cancer activity. Recent work has shown that stimulation with IL-12/15/18 leads to the generation of NK cells with enhanced functional and putative “memory” properties. We have investigated the activity of these NK cells against HCC cell lines in vitro and in a mouse model. Methods NK cells from healthy donors or individuals with HCC were activated with IL-12/15/18 in vitro and tested for cytotoxic activity against a panel of human HCC cell lines. IL-12/15/18 primed murine NK cells were then infused into a murine model of spontaneously arising HCC to test for anti-tumor activity. Results NK cells from patients and healthy controls had similar expression levels of activating and inhibitory NK cell receptors. However, proliferation of NK cells from HCC patients was weaker than healthy controls in response to IL-12/15/18 and IL-2 (p

Simple Summary AXL is a member of the TAM (TYRO3, AXL, MER) family of receptor tyrosine kinases. In normal physiological conditions, AXL is involved in removing dead cells and their remains, and limiting the duration of immune responses. Both functions are utilized by cancers in the course of tumour progression. Cancer cells use the AXL pathway to detect toxic environments and to activate molecular mechanisms, thereby ensuring their survival or escape from the toxic zone. AXL is instrumental in controlling genetic programs of epithelial-mesenchymal and mesenchymal-epithelial transitions, enabling cancer cells to metastasize. Additionally, AXL signaling suppresses immune responses in tumour microenvironment and thereby helps cancer cells to evade immune surveillance. The broad role of AXL in tumour biology is the reason why its inhibition sensitizes tumours to a broad spectrum of anti-cancer drugs. In this review, we outline molecular mechanisms underlying AXL function in normal tissues, and discuss how these mechanisms are adopted by cancers to become metastatic and drug-resistant. Abstract The TAM proteins TYRO3, AXL, and MER are receptor tyrosine kinases implicated in the clearance of apoptotic debris and negative regulation of innate immune responses. AXL contributes to immunosuppression by terminating the Toll-like receptor signaling in dendritic cells, and suppressing natural killer cell activity. In recent years, AXL has been intensively studied in the context of cancer. Both molecules, the receptor, and its ligand GAS6, are commonly expressed in cancer cells, as well as stromal and infiltrating immune cells. In cancer cells, the activation of AXL signaling stimulates cell survival and increases migratory and invasive potential. In cells of the tumour microenvironment, AXL pathway potentiates immune evasion. AXL has been broadly implicated in the epithelial-mesenchymal plasticity of cancer cells, a key factor in drug resistance and metastasis. Several antibody-based and small molecule AXL inhibitors have been developed and used in preclinical studies. AXL inhibition in various mouse cancer models reduced metastatic spread and improved the survival of the animals. AXL inhibitors are currently being tested in several clinical trials as monotherapy or in combination with other drugs. Here, we give a brief overview of AXL structure and regulation and discuss the normal physiological functions of TAM receptors, focusing on AXL. We present a theory of how epithelial cancers exploit AXL signaling to resist cytotoxic insults, in order to disseminate and relapse.

Background: Hepatocellular carcinoma (HCC) is a common and deadly cancer, however, very little improvement has been made towards its diagnosis and prognosis. The expression and functional contribution of the receptor tyrosine kinase ROR1 have not been investigated in HCC before. Hence, we investigated the expression of ROR1 in HCC cells and assessed its involvement in hepatocarcinogenesis. Methods: Recombinant bacterial ROR1 protein was used as an immunogen to generate ROR1 monoclonal antibodies. ROR1 transcript levels were detected by RT-qPCR and the protein expression of ROR1 in HCC was assessed by Western blotting by using homemade anti-ROR1 monoclonal antibodies. Apoptosis, cell cycle, trans-well migration, and drug efflux assays were performed in shRNA-ROR1 HCC cell clones to uncover the functional contribution of ROR1 to hepatocarcinogenesis. Results: New ROR1 antibodies specifically detected endogenous ROR1 protein in human and mouse HCC cell lines. ROR1-knockdown resulted in decreased proliferation and migration but enhanced resistance to apoptosis and anoikis. The observed chemotherapy-resistant phenotype of ROR1-knockdown cells was due to enhanced drug efflux and increased expression of multi-drug resistance genes. Conclusions: ROR1 is expressed in HCC and contributes to disease development by interfering with multiple pathways. Acquired ROR1 expression may have diagnostic and prognostic value in HCC.

Background S100 proteins have been implicated in various aspects of cancer, including epithelial-mesenchymal transitions (EMT), invasion and metastasis, and also in inflammatory disorders. Here we examined the impact of individual members of this family on the invasion of pancreatic ductal adenocarcinoma (PDAC) cells, and their regulation by EMT and inflammation. Methods Invasion of PDAC cells was analysed in zebrafish embryo xenografts and in transwell invasion assays. Expression and regulation of S100 proteins was studied in vitro by immunoblotting, quantitative PCR and immunofluorescence, and in pancreatic lesions by immunohistochemistry. Results Whereas the expression of most S100 proteins is characteristic for epithelial PDAC cell lines, S100A4 and S100A6 are strongly expressed in mesenchymal cells and upregulated by ZEB1. S100A4/A6 and epithelial protein S100A14 respectively promote and represses cell invasion. IL-6/11-STAT3 pathway stimulates expression of most S100 proteins. ZEB1 synergises with IL-6/11-STAT3 to upregulate S100A4/A6, but nullifies the effect of inflammation on S100A14 expression. Conclusion EMT/ZEB1 and IL-6/11-STAT3 signalling act independently and congregate to establish the expression pattern of S100 proteins, which drives invasion. Although ZEB1 regulates expression of S100 family members, these effects are masked by IL-6/11-STAT3 signalling, and S100 proteins cannot be considered as bona fide EMT markers in PDAC.

Colorectal cancer (CRC) is a key public health concern and the second highest cause of cancer related death in Western society. A dynamic interaction exists between CRC cells and the surrounding tumor microenvironment, which can stimulate not only the development of CRC, but its progression and metastasis, as well as the development of resistance to therapy. In this chapter, we focus on the role of fibroblasts within the CRC tumor microenvironment and describe some of the key methods for their study, as well as the evaluation of dynamic interactions within this biological ecosystem.

Background and Aims. Hepatocellular carcinoma is an aggressive malignancy of the liver and is ranked as the sixth most common cancer worldwide. There is still room for novel markers to improve the diagnosis and monitoring of HCC. Our observations in cancer databases that PLXNC1 is upregulated in HCC led us to investigate the expression profile of Plexin C1 mRNA and protein in HCC cell lines and tissues. Methods. A recombinant protein encompassing part of the extracellular domain of Plexin C1 was used as an antigen for monoclonal antibody development. Transcript and protein levels of Plexin C1 in HCC cell lines were determined by RT-qPCR and Western blotting, respectively. In vivo evaluation of Plexin C1 expression in HCC tissues was accomplished by immunohistochemistry studies in tissue microarrays. Results. A monoclonal antibody, clone PE4, specific to Plexin C1, was generated. In silico and in vitro analyses revealed a Plexin C1-based clustering of well-differentiated HCC cell lines. Staining of HCC and nontumoral liver tissues with PE4 showed a membrane-localized overexpression of Plexin C1 in tumors (p=0.0118). In addition, this expression was correlated with the histological grades of HCC cases. Conclusions. Plexin C1 distinguishes HCC cells of epithelial characteristics from those with the mesenchymal phenotype. Compared to the nontumoral liver, HCC tissues significantly overexpress Plexin C1. The newly generated PE4 antibody can be evaluated in larger HCC cohorts and might be exploited for the examination of Plexin C1 expression pattern in other epithelial malignancies.

ZEB2 is a transcriptional repressor that regulates epithelial-to-mesenchymal transition (EMT) through binding to bipartite E-box motifs in gene regulatory regions. Despite the abundant presence of E-boxes within the human genome and the multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets has been identified so far. Hence, we explored genome-wide ZEB2 binding by chromatin immunoprecipitation-sequencing (ChIP-seq) under endogenous ZEB2 expression conditions. For ChIP-Seq we used an anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells exhibiting a high endogenous ZEB2 expression. The ChIP-Seq targets were validated using ChIP-qPCR, whereas ZEB2-dependent expression of target genes was assessed by RT-qPCR and Western blotting in shRNA-mediated ZEB2 silenced SNU398 cells and doxycycline-induced ZEB2 overexpressing colorectal carcinoma DLD1 cells. Changes in target gene expression were also assessed using primary human tumor cDNA arrays in conjunction with RT-qPCR. Additional differential expression and correlation analyses were performed using expO and Human Protein Atlas datasets. Over 500 ChIP-Seq positive genes were annotated, and intervals related to these genes were found to include the ZEB2 binding motif CACCTG according to TOMTOM motif analysis in the MEME Suite database. Assessment of ZEB2-dependent expression of target genes in ZEB2-silenced SNU398 cells and ZEB2-induced DLD1 cells revealed that the GALNT3 gene serves as a ZEB2 target with the highest, but inversely correlated, expression level. Remarkably, GALNT3 also exhibited the highest enrichment in the ChIP-qPCR validation assays. Through the analyses of primary tumor cDNA arrays and expO datasets a significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 expression were detected in most of the tumors. We also explored ZEB2 and GALNT3 protein expression using the Human Protein Atlas dataset and, again, observed an inverse correlation in all analyzed tumor types, except malignant melanoma. In contrast to a generally negative or weak ZEB2 expression, we found that most tumor tissues exhibited a strong or moderate GALNT3 expression. Our observation that ZEB2 negatively regulates a GalNAc-transferase (GALNT3) that is involved in O-glycosylation adds another layer of complexity to the role of ZEB2 in cancer progression and metastasis. Proteins glycosylated by GALNT3 may be exploited as novel diagnostics and/or therapeutic targets.

Long non-coding RNAs (lncRNAs) are a diverse class of RNA transcripts which have limited protein coding potential. They perform a variety of cellular functions in health, but have also been implicated during malignant transformation. A further theme in recent years is the critical role of the tumour microenvironment and the dynamic interactions between cancer and stromal cells in promoting invasion and disease progression. Whereas the contribution of deregulated lncRNAs within cancer cells has received considerable attention, their significance within the tumour microenvironment is less well understood. The tumour microenvironment consists of cancer-associated stromal cells and structural extracellular components which interact with one another and with the transformed epithelium via complex extracellular signalling pathways. LncRNAs are directly and indirectly involved in tumour/stroma cross-talk and help stimulate a permissive tumour microenvironment which is more conducive for invasive tumour growth. Furthermore, lncRNAs play key roles in determining the phenotype of cancer associated stromal cells and contribute to angiogenesis and immune evasion pathways, extracellular-matrix (ECM) turnover and the response to hypoxic stress. Here we explore the multifaceted roles of lncRNAs within the tumour microenvironment and their putative pathophysiological effects.

Aberrant Hedgehog (Hh) signalling has been reported in a number of malignancies, particularly basal cell carcinoma (BCC) of the skin. Clinical trials of Hh inhibitors are underway in many cancers, and these have produced significant clinical benefit in BCC patients, although regrowth of new, or clinically aggressive, variants, as well as development of secondary malignancies, has been reported. ?v?6 integrin is expressed in many cancers, where it has been shown to correlate with an aggressive tumour phenotype and poor prognosis. We have previously reported ?v?6 up-regulation in aggressive, morphoeic BCC variants, where it modulates the stromal response and induces invasion. To examine a possible link between Hh and ?v?6 function, we generated BCC models, overexpressing Gli1 in immortalized keratinocytes (NTert1, HaCaT). Unexpectedly, we found that suppressing Gli1 significantly increased ?v?6 expression. This promoted tumour cell motility and also stromal myofibroblast differentiation through integrin-dependent TGF-?1 activation. Gli1 inhibited ?v?6 expression by suppressing TGF-?1-induced Smad2/3 activation, blocking a positive feedback loop maintaining high ?v?6 levels. A similar mechanism was observed in AsPC1 pancreatic cancer cells expressing endogenous Gli1, suggesting a common mechanism across tumour types. In vitro findings were supported using human clinical samples, where we showed an inverse correlation between ?v?6 and Gli1 expression in different BCC subtypes and pancreatic cancers. In summary, we show that expression of Gli1 and ?v?6 inversely correlates in tumours in vivo, and Hh targeting up-regulates TGF-?1/Smad2/3-dependent ?v?6 expression, promoting pro-tumourigenic cell functions in vitro. These results have potential clinical significance, given the reported recurrence of BCC variants and secondary malignancies in patients treated by Hh targeting.

The tumor microenvironment is a heterogeneous and dynamic network that exists between cancer and stroma, playing a critical role in cancer progression. Certain tumorigenic signals such as microRNAs are derived from the stroma and conveyed to cancer cells (and vice versa) in nanoparticles called exosomes. Their identification and characterization is an important step in better understanding cellular cross talk and its consequences. To this end we describe how to culture primary ex vivo derived fibroblasts from colorectal tissue, isolate their exosomes, extract exosomal RNA and perform microRNA profiling.

Fos-related antigen-1 (Fra-1) is a member of the Activator Protein-1 (AP-1) transcription factor superfamily that is overexpressed in a variety of cancers, including colon, breast, lung, bladder and brain. High Fra-1 levels are associated with enhanced cell proliferation, survival, migration and invasion. Despite its frequent overexpression, the molecular mechanisms that regulate the accumulation of Fra-1 proteins in tumour cells are not well understood. Here, we show that turnover of Fra-1, which does not require ubiquitylation, is cooperatively regulated by two distinct mechanisms—association with the 19S proteasomal subunit, TBP-1, and by a C-terminal degron, which acts independently of TBP-1, but is regulated by RAS–ERK (extracellular signal-regulated kinase) signalling. TBP-1 depletion stabilized Fra-1 and further increased its levels in tumour cells expressing RAS–ERK pathway oncogenes. These effects correlated with increased AP-1 transcriptional activity. We suggest that during Fra-1 degradation, association with TBP-1 provides a mechanism for ubiquitin-independent proteasomal recognition, while the C terminus of the protein regulates its subsequent proteolytic processing. This work was supported by grants from the National Health and Medical Research Council of Australia & SFI Deposited by bulk import

p53, p63 and p73 make a family of transcription factors that play a vital role in development and cancer. All p53 family members have more than one promoter producing Transactivating (TA) and Dominant Negative (DeltaN) isoforms and their mRNAs are subjected to extensive splicing at 3' end to produce multiple protein products. p53 is usually inactivated by point mutations during tumorigenesis, whereas the expression levels and p63 and p73 are modulated to give tumor cells a selective advantage. In this study, aiming to find novel targets of the p53 family members, we identified FGFR3 as a gene transcriptionally controlled by p63 and p73. FGFR3 has been implicated in development and tumor biology as activating mutations of this gene was described in skeletal disorders, non-invasive skin conditions and superficial bladder cancers. We found that TAp73, TAp63 and DeltaNp63 was capable of inducing FGFR3. siRNA mediated downregulation of DeltaNp63 decreased endogenous FGFR3 protein levels. Our findings of this new link between p53 family proteins and FGFR3 may help understanding the transition of superficial bladder cancers to an invasive phenotype.

TP73, as a TP53 homologue, drew the attention of tumor biologists because it is rarely mutated in human cancers and can induce cell cycle arrest and apoptosis by activating genes also regulated by p53. However, TP73 harbors an additional promoter that produces a dominant negative p73 protein (deltaNp73) having the opposite effect of the TAp73 protein. Thus, the regulation of p53 responsive genes in the absence of p53 relies on a critical balance between different p73 gene-derived proteins. Recent reports have described additional complexity in the mechanism of action of transcriptionally active p73 (TAp73) in the induction of cell death. The molecular mechanism through which p73 induces apoptosis involves (i) expression and changes in subcellular localization of scotin, producing an endoplasmic reticulum (ER) stress; and (ii) transactivation of PUMA and Bax, thus determining cell fate. On the contrary, deltaNp73 inhibits apoptosis, thus contributing to the oncogenic potential of neuroblastoma cells.

Cancer-associated fibroblasts are critical to tumor progression. There exists a dynamic crosstalk between cancer and stromal compartments, which maintains a permissive tumor microenvironment. Extracellular vesicles (EVs) play a significant role in this intercellular communication. Colorectal cancer (CRC) cells can be categorized according to epithelial-mesenchymal transition (EMT) status, and therefore metastatic capacity. We aimed to investigate the effect of EMT on EV-mediated cancer-fibroblast signaling. CRC cell lines (DLD-1, HCT116, SW620 and SW480) were characterized by western blotting to determine EMT status. EVs were isolated from conditioned media by serial centrifugation and validated by transmission electron microscopy, western blotting and nanoparticle tracking analysis. Fluorescently labeled EVs and cells were detected and evaluated by flow cytometry and fluorescence microscopy. Increasing concentrations of EVs from CRC cells were co-cultured with fibroblasts for 24h. Activation/inhibition of signaling pathways was examined by western blotting. EV microRNA (miRNA) profiles were obtained, validated by qPCR and submitted for target and pathway analysis. DLD-1, HCT116 and SW620 cells express E-cadherin and are considered epithelial, whereas SW480 lacks E-cadherin, expresses ZEB-1, and is considered mesenchymal. EVs were spherical, enriched in ALIX, TSG101, CD63 and had a mean diameter of 90nm. EVs from CRC cells were shown to transfer directly to primary ex vivo patient-derived fibroblasts and fibroblast cell lines. Transfer of EVs from epithelial CRC cells abrogated ERK activity in fibroblasts, even at the lowest concentration, and was associated with reduced fibroblast proliferation, whereas EVs from mesenchymal cells had no effect. MiRNA profiling of EVs from epithelial and mesenchymal CRC cells showed a 10-fold upregulation of miR-143-3p in epithelial compared to mesenchymal EVs. MiRNA target analysis and experimental validation show that miR-143-3p directly targets KRAS and HRAS, providing a potential miRNA-orchestrated mechanism of action for the downregulation of fibroblast ERK activity in the tumor microenvironment. Importantly, CRC cellular ERK activity is not reflected in fibroblasts treated with CRC EVs, suggesting that EVs do not directly transmit ERK protein or mRNA. However, miRNAs are the most stable EV cargo, and we show that epithelial but not mesenchymal CRC EVs contain upregulated miRNAs, which target critical components of the ERK pathway. Downregulation of ERK activity has been shown to induce fibroblast senescence, a phenotype linked to cancer progression. We hypothesize that differential regulation occurs because epithelial CRC cells are juxtaposed with fibroblasts in the tumor core, where senescent cancer associated fibroblasts are frequently observed, whereas mesenchymal CRC cells are at the invasive front or in the circulation. Citation Format: Rahul Bhome, Louise M. House, Tilman Sanchez-Elsner, Stephen M. Thirdborough, Emre Sayan, Alex H. Mirnezami. Metastatic and non-metastatic colorectal cancer cells differentially regulate fibroblast cell cycle via extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2982. doi:10.1158/1538-7445.AM2017-2982

Epithelial–mesenchymal transition (EMT) is an embryonic trans-differentiation programme that is implicated in organ formation by facilitating the formation of highly motile cells with stem cell capabilities.1 Most tissues of cells derived from ectoderm or mesoderm retain certain elements of EMT and mesenchymal properties are their differentiation markers. On the other hand, cells of organs derived from endoderm only experience EMT and its reversal (mesenchymal-epithelial transition) at certain phases of development and become differentiated secretory epithelial cells. As de-differentiation is a common feature of cancer progression, acquisition of mesenchymal markers accompanied with the loss of epithelial features can be observed in most carcinomas and considered as the reason of cancer spread (metastasis). For the reasons that (1) more than 85% of all cancers are of epithelial origin and (2) epithelial (differentiated) and mesenchymal (undifferentiated) carcinoma cells are morphologically very different, EMT has been mostly studied in carcinoma setting. However, there is growing evidence suggesting that cancers of organs derived from mesoderm (e.g., hematopoietic malignancies and sarcomas) or ectoderm (e.g., glioblastomas and melanomas) also activate EMT programs to acquire an undifferentiated state and become resistant to conventional therapies.2 A good example of this is a recent article by Sanchez-Tillo et al. that investigated the role of EMT-inducing transcription factor ZEB1 in mantle cell lymphoma (MCL) progression.3 MCL is a B-cell malignancy and considered as the rarest form of non-Hodgkin lymphoma.4 Despite this statistical data, it is responsible for a significant portion of B-cell malignancy related mortality because it is an aggressive cancer with a continuous relapse pattern.5 In addition, most MCL patients show poor response to chemotherapy. The translocation of cyclin D1 to immunoglobulin heavy chain enhancer locus t(11;14)(q13;q32) is the genetic hallmark of MCLs; however Wnt, p53, Notch pathway aberrations and pro-survival gene overexpression (Mcl1 and Bcl2 overexpression) are also commonly observed. Sanchez-Tillo et al. investigated the expression of EMT-inducing transcription factor ZEB1 in a cohort of MCL patient samples.3 They found ZEB1 overexpression in half of the samples with a strong correlation with nuclear β-catenin presence (Wnt signalling active). In addition, the clinico-pathological analysis of patient data revealed ZEB1 positivity as a marker for shorter overall survival. This led them to investigate the mechanisms of ZEB1 upregulation and functional contribution of ZEB1 expression to the pathology of MCL. ZEB1 promoter has been previously reported (by the same group) to contain two TCF–LEF binding sites allowing β-catenin–TCF4 binding in colorectal cancer setting.6 They validated the presence of endogenous β-catenin on the respective DNA motifs of ZEB1 promoter by chromatin immunoprecipitation and also showed that ZEB1 mRNA levels are changing upon stimulation with Wnt-3a or knockdown of endogenous β-catenin using two different MCL cell lines. Following this observation, they also investigated if modulation of β-catenin signalling by chemical inhibitors such as salinomycin can alter ZEB1 expression. Salinomycin treatment, indeed, was able to decrease endogenous ZEB1 expression. These results mark the potential to control acquired ZEB1 expression by targeting Wnt pathway and inhibit its functional consequences in MCL. The cellular consequences of ZEB1 overexpression in MCL have not been investigated, however, carcinomas expressing ZEB1 were shown to have canonical hallmarks of EMT such as increased invasiveness and metastasis, resistance to chemotherapy and decreased proliferation.7 Sanchez-Tillo et al. systematically tested all these aspects in MCL by knocking down ZEB1 or β-catenin using MCL cell lines. They found that ZEB1 downregulation decreases baseline cell viability and proliferation. The gene expression of ZEB1-expressing cells indicated activation of survival and repression of cell death pathways as validated by increased levels of myc, cyclin D1, CENPF, Ki67, Bcl2, MCL1 and decreased p53, bax and PMAIP1 mRNAs. In addition, they also investigated whether ZEB1 expression confers resistance to chemotherapy. In line with their previous data regarding changes in baseline cell viability, ZEB1 downregulation increased sensitivity to commonly used chemotherapeutic drugs used in MCL treatment, such as Doxorubicin, Cytarabine and Gemcitabine but not Vincristine. The effect of ZEB1 knockdown on chemosensitization was also confirmed by pre-treatment of MCL cell lines with Salinomycin. EMT-induced chemoresistance has been described in different carcinoma models; however, the cellular mechanism of this phenomenon still remains to be clarified.8 The reasons for chemoresistance can be at least 3-fold. Cancer cells can alter drug influx/efflux to favour less chemotherapeutic agents in the cell (e.g., activate multidrug resistance proteins), they can activate efficient detoxification (e.g., by glutathione S-transferase) or enable efficient DNA repair to escape DNA damage-induced apoptosis. In this article, Sanchez-Tillo et al. investigated the regulation of drug influx/efflux pathways by ZEB1. Using two different MCL cell lines and validating by IHC in primary MCL samples, they showed that critical drug transporters are regulated by ZEB1 to favour survival of Wnt signalling active cancer cells. The authors also extended their in vitro studies to animal models. Subcutaneous injection of Granta-519 cells expressing or silenced ZEB1 showed significantly different responses; ZEB1 silenced cells showing delays in tumour growth and enhanced sensitivity to Doxorubicin treatment. Finally, they explored whether salinomycin can synergize with Doxorubicin for an enhanced response to chemotherapy. Using established MCL cell lines and primary cells from MCL patients, they showed that sublethal doses of Salinomycin and Doxorubicin can synergize and effectively kill MCL cells. These results support their in vivo data and provide evidence that β-catenin-activated ZEB1 expression contributes to tumour progression and chemoresistance (Figure 1). Figure 1 Role of EMT-inducing transcription factor ZEB1 in MCL. In carcinoma cells, activation of distinct signalling pathways such as TGFβ, Wnt and receptor tyrosine kinase (RTK) converges to ZEB1 for a fully de-differentiated and mesenchymal (EMT) phenotype. ... What is next? A couple of things remained to be done and questions answered to make use of the full potential of these exciting findings. First of all, Sanchez-Tillo et al. did not show the clinico-pathological variables of their patient cohort. In recent years, anti-B cell-based immunotherapeutic regimens, such as the anti-CD20 antibody rituximab, gained attention as an alternative in the treatment of B-cell malignancies. The provision of detailed patient data may allow stratification of MCL cases and potentially mark subgroups that can or cannot be treated with chemo and/or immunotherapies. In addition, similar correlative studies have to be performed using independent patient cohorts to externally validate the clinical importance of ZEB1 in MCL and allow clinical translation. Another point to be clarified is the functional link between ZEB1-induced alterations in drug influx/efflux and the actual presence of drugs in MCL cells. Genotoxic agents such as Doxorubicin can be imported through cell membrane by passive diffusion and effectively retained in the cell as DNA-bound form so they cannot be exported by drug transporters. This question can be easily answered due to the fluorescence nature of Doxorubicin using normal and transformed B cells, in the presence and absence of ZEB1. Finally, activation of EMT pathways in carcinoma results in generation of malignant cells with stem cell properties. A critical question that remains to be answered is whether ZEB1-expressing MCL cells are de-differentiated enough to be considered as B-cell precursors. As a follow-up of this work, it will be fascinating to explore if the tumour-promoting features of ZEB1 in MCL cells are also conserved in other malignancies, particularly in other mesoderm-derived tissues such as sarcomas or cancers such as hepatoblastomas that β-catenin activation is an early event in tumourigenesis.

Despite recent advances, cancer remains a leading cause of death worldwide. In developed countries, the incidence of colorectal and breast cancer has been stable, but no improvement in prognosis has been observed if the patient presents with metastases at diagnosis. This fact highlights the importance of therapeutic approaches targeting cellular invasion and metastasis programmes as the next step in cancer treatment. During carcinoma progression a process called Epithelial-Mesenchymal Transition (EMT) results in enhanced invasion and motility which is directly linked with loss of epithelial polarity and epithelial junctions, migration permissive cytoskeleton alterations, and the acquisition of mesenchymal properties. The recent discovery of microRNAs (miRNAs) controlling key cellular pathways has opened a new era in understanding how EMT pathways are modulated. In this review, we classify EMT regulating proteins according to their cellular localization (membrane, cytoplasmic and nuclear), and summarize the current knowledge on how they are controlled by miRNAs and propose potential miRNAs for the transcripts that may control their expression.

G-protein coupled receptors (GPCRs) have been extremely successful drug targets for a multitude of diseases from heart failure to depression. This super-family of cell surface receptors have not, however, been widely considered as a viable target in cancer treatment. In the current study we demonstrate that a classical Gq/11-coupled GPCR, the M3-muscarinic receptor, was able to regulate apoptosis via receptors that are endogenously expressed in the human neuroblastoma cell line SH-SY5Y and when ectopically expressed in Chinese hamster ovary (CHO) cells. Stimulation of the M3-muscarinic receptor was shown to inhibit the ability of the DNA-damaging chemotherapeutic agent, etoposide, from mediating apoptosis. This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53. In contrast, stimulation of endogenous muscarinic receptors in SH-SY5Y cells did not regulate p53 expression but rather was able to inhibit p53 translocation to the mitochondria and p53 phosphorylation at serine 15 and 37. This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, sub-cellular trafficking and modification of p53 in a manner that is in part dependent on the cell type.

Survival rate of patients diagnosed with the invasive form of bladder cancer is low suggesting an urgent need to implement novel treatments. GTC (gemcitabine, paclitaxel and cisplatin) is a new chemotherapeutic regimen, which has shown promise in clinical trials. Given that receptor tyrosine kinases of the ErbB family are overexpressed in a high proportion of metastatic bladder tumours, approaches involving small-molecule inhibitors of ErbB receptors in combination with conventional cytostatic drugs are of potential interest. Here, we show that the dual inhibitor of ErbB receptors, lapatinib, enhances cytostatic and induces cytotoxic effects of GTC in two bladder cancer cell lines which differ with regard to expression levels of proteins taking part in the ErbB pathway. Lapatinib inhibited phosphorylation of ErbB receptors and also reduced the level of phosphorylated AKT. Flow cytometry analysis demonstrated that GTC treatment affects cell cycle distribution differently in the presence or absence of lapatinib. In RT112 cells, which express high levels of ErbB receptors and harbour wild-type p53, combined GTC/lapatinib treatment resulted in the phosphorylation of p53 at Ser46 and accumulation of sub-G1 cell populations. Our data indicate that a combinatorial approach involving GTC and lapatinib may have therapeutic potential in a subset of bladder tumours depending on the genetic context.

The epithelial-mesenchymal transition (EMT) contributes to cancer metastasis. Two ZEB family members, ZEB1 and ZEB2(SIP1), inhibit transcription of the E-cadherin gene and induce EMT in vitro. However, their relevance to human cancer is insufficiently studied. Here, we performed a comparative study of SIP1 and ZEB1 proteins in cancer cell lines and in one form of human malignancy, carcinoma of the bladder. Whereas ZEB1 protein was expressed in all E-cadherin-negative carcinoma cell lines, being in part responsible for the high motility of bladder cancer cells, SIP1 was hardly ever detectable in carcinoma cells in culture. However, SIP1 represented an independent factor of poor prognosis ( P = 0.005) in a series of bladder cancer specimens obtained from patients treated with radiotherapy. In contrast, ZEB1 was rarely expressed in tumor tissues; and E-cadherin status did not correlate with the patients' survival. SIP1 protected cells from UV- and cisplatin-induced apoptosis in vitro but had no effect on the level of DNA damage. The anti-apoptotic effect of SIP1 was independent of either cell cycle arrest or loss of cell-cell adhesion and was associated with reduced phosphorylation of ATM/ATR targets in UV-treated cells. The prognostic value of SIP1 and its role in DNA damage response establish a link between genetic instability and metastasis and suggest a potential importance for this protein as a therapeutic target. In addition, we conclude that the nature of an EMT pathway rather than the deregulation of E-cadherin per se is critical for the progression of the disease and patients' survival.

Although signal transducer and activator of transcription 1 (STAT1) mediated regulation of p53 transcription and apoptosis has been previously reported, modulation of other members of the p53 family of transcription factors remains poorly understood. In this study, we found that STAT1 and TA-p73 can interact directly and that p73-mediated Bax promoter activity was observed to be reduced by STAT1 expression in a p53-independent manner for which STAT1 Tyrosine-701 and Serine-727 are key residues. This study presents the first report physically linking STAT1 and TA-p73 signalling and highlights the modulation of the Bax promoter in the context of IFN-γ stimulation.

Background MicroRNAs (miRNAs) are increasingly implicated in the acquisition of metastatic capabilities. The aim of the present study was to determine the identity and function of deregulated miRNAs in colorectal cancer (CRC) associated stroma, and to study epigenetic mechanisms promoting CRC progression in the tumour microenvironment. Methods RT-PCR-based profiling of 20 laser capture microdissected colonic tissue specimens was used to identify stromal miRNAs differentially expressed in CRC compared with paired normal tissue. Candidate miRNAs were evaluated by in-situ hybridisation to determine cells of origin, and assessed for biological significance with in-vitro and three-dimensional organotypic functional assays. Findings 18 miRNAs were differentially expressed between CRC stroma and paired normal colonic stroma including miR-21, a known oncomiR in CRC. In-situ hybridisation showed that miR-21 was expressed exclusively in cancer-associated fibroblasts. Ectopic miR-21 expression in fibroblasts were associated with upregulated α-smooth mucle actin expression, implying that miR-21 supports myofibroblast transdifferentiation, a key prometastatic stromal event. Compared with controls, conditioned medium from miR-21 overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. Furthermore, three-dimensional organotypic co-cultures, developed to model in-vivo circumstances, showed that ectopic stromal miR-21 expression in fibroblasts was associated with a 2·5-fold increased invasion of CRC epithelium into stroma compared with controls (p Interpretation Deregulation of miR-21 is a stromal phenomenon and supports CRC progression through myofibroblast transdifferentiation, resistance to oxaliplatin cytotoxicity, and promotion of tumour cell invasion. Stromal miR-21 induced invasion is mediated by RECK downregulation and a reciprocal rise in MMP2 activity. This mechanism highlights the importance of miRNAs in the tumour microenvironment during CRC progression, and presents a novel perspective for the development of targeted therapeutic agents. To our knowledge, this study is the first functional demonstration of the pathological effect of deregulated stromal miRNAs in cancer. Further experiments are now required to validate these findings in vivo. Funding UK Medical Research Council, Cancer Research UK.