The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9.

Siglec-9 is a sialic-acid-binding lectin expressed predominantly on myeloid cells. Aberrant glycosylation occurs in essentially all types of cancers and results in increased sialylation. Thus, when the mucin MUC1 is expressed on cancer cells, it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). Here we found that this cancer-specific MUC1 glycoform, through engagement of Siglec-9, 'educated' myeloid cells to release factors associated with determination of the tumor microenvironment and disease progression. Moreover, MUC1-ST induced macrophages to display a tumor-associated macrophage (TAM)-like phenotype, with increased expression of the checkpoint ligand PD-L1. Binding of MUC1-ST to Siglec-9 did not activate the phosphatases SHP-1 or SHP-2 but, unexpectedly, induced calcium flux that led to activation of the kinases MEK-ERK. This work defines a critical role for aberrantly glycosylated MUC1 and identifies an activating pathway that follows engagement of Siglec-9.

Targeting of Tumor-Associated Glycoforms of MUC1 with CAR T Cells.

We read with interest the manuscript by June and colleagues published recently in Immunity in which they describe targeting of aberrantly glycosylated tumor-associated cell membrane mucin MUC1 using chimeric antigen receptor-engineered human T cells (Posey et al., 2016). In that study, the authors used a second generation 4-1BB costimulatory-molecule-based chimeric antigen receptor (CAR) (Imai et al., 2004) in which targeting was achieved using a single-chain variable fragment (scFv) derived from the 5E5 antibody. This CAR selectively binds MUC1 that carries the Tn or sialyl (S)Tn glycan. Both of these truncated glycans are aberrantly expressed on the MUC1 glycoprotein in a spectrum of malignancies and consequently represent attractive targets for immunotherapeutic exploitation.

O-linked mucin-type glycosylation regulates the transcriptional programme downstream of EGFR.

Aberrant mucin-type O-linked glycosylation is a common occurrence in cancer where the upregulation of sialyltransferases is often seen leading to the early termination of O-glycan chains. Mucin-type O-linked glycosylation is not limited to mucins and occurs on many cell surface glycoproteins including EGFR, where the number of sites can be limited. Upon EGF ligation, EGFR induces a signaling cascade and may also translocate to the nucleus where it directly regulates gene transcription, a process modulated by Galectin-3 and MUC1 in some cancers. Here, we show that upon EGF binding, breast cancer cells carrying different O-glycans respond by transcribing different gene expression signatures. MMP10, the principal gene upregulated when cells carrying sialylated core 1 glycans were stimulated with EGF, is also upregulated in ER-positive breast carcinoma reported to express high levels of ST3Gal1 and hence mainly core 1 sialylated O-glycans. In contrast, isogenic cells engineered to carry core 2 glycans upregulate CX3CL1 and FGFBP1 and these genes are upregulated in ER-negative breast carcinomas, also known to express longer core 2 O-glycans. Changes in O-glycosylation did not significantly alter signal transduction downstream of EGFR in core 1 or core 2 O-glycan expressing cells. However, striking changes were observed in the formation of an EGFR/galectin-3/MUC1/ß-catenin complex at the cell surface that is present in cells carrying short core 1-based O-glycans but absent in core 2 carrying cells.

Tn and STn are members of a family of carbohydrate tumor antigens that possess carbohydrate-carbohydrate interactions.

The mucin-type O-glycome in cancer aberrantly expresses the truncated glycans Tn (GalNAcα1-Ser/Thr) and STn (Neu5Acα2,6GalNAcα1-Ser/Thr). However, the role of Tn and STn in cancer and other diseases is not well understood. Our recent discovery of the self-binding properties (carbohydrate-carbohydrate interactions, CCIs) of Tn (Tn-Tn) and STn (STn-STn) provides a model for their possible roles in cellular transformation. We also review evidence that Tn and STn are members of a larger family of glycan tumor antigens that possess CCIs, which may participate in oncogenesis.

Latest developments in MUC1 immunotherapy.

Currently, there is renewed interest in attempting to recruit the host immune system to eliminate cancers, and within this renewed activity, MUC1 continues to arouse interest. MUC1 has been considered a possible therapeutic target for the past 30 years as it is up-regulated, aberrantly glycosylated and its polarization is lost in many adenocarcinomas. Moreover, MUC1 is expressed by some haematopoietic cancers, including acute myeloid leukaemia and myeloma. Although multiple clinical trials have been initiated and immune responses have been documented, effective clinical benefit worthy of approval for general application has not as yet been achieved. However, this does not appear to have quelled the interest in MUC1 as a therapeutic target, as shown by the increase in the number of MUC1-based clinical trials initiated in 2017 ( Figure 1). As with all translational studies, incorporating new relevant research findings into therapeutic strategy is difficult. Decisions are made to commit to a specific strategy based on the information and data available when the trial is initiated. However, the time required for preclinical studies and early trials can render the founding concept not always appropriate for proceeding to a larger definitive trial. Here, we summarize the attempts made, to date, to bring MUC1 into the world of cancer immunotherapy and discuss how research findings regarding MUC1 structure and function together with expanded knowledge of its interactions with the tumour environment and immune effector cells could lead to improved therapeutic approaches. ppbiost;46/3/659/BST20170400CF1F1BST-2017-0400CF1Figure 1.Number of MUC1-targeted trials initiated each year.

O-linked mucin-type glycosylation in breast cancer.

Changes in mucin-type O-linked glycosylation are seen in over 90% of breast cancers where increased sialylation is often observed and a change from branched glycans to linear glycans is often seen. There are many mechanisms involved including increased/altered expression of glycosyltransferases and relocalisation to the endoplasmic reticulum of the enzymes responsible for the addition of the first sugar, N-acetyl-d-galactosamine. It is now becoming clear that these changes can contribute to tumour growth and progression by modulating the micro-environment through glycan-sensing lectins expressed on immune cells, by modulating interactions with tumour surface receptors and by binding to selectins. The understanding of how changes in mucin-type O-linked glycosylation influence tumour growth and progression reveals new potential targets for therapeutic intervention in the treatment of breast cancer.

Interactions of mucins with the Tn or Sialyl Tn cancer antigens including MUC1 are due to GalNAc-GalNAc interactions.

The molecular mechanism(s) underlying the enhanced self-interactions of mucins possessing the Tn (GalNAcα1-Ser/Thr) or STn (NeuNAcα2-6GalNAcα1-Ser/Thr) cancer markers were investigated using optical tweezers (OT). The mucins examined included modified porcine submaxillary mucin containing the Tn epitope (Tn-PSM), ovine submaxillary mucin with the STn epitope (STn-OSM), and recombinant MUC1 analogs with either the Tn and STn epitope. OT experiments in which the mucins were immobilized onto polystyrene beads revealed identical self-interaction characteristics for all mucins. Identical binding strength and energy landscape characteristics were also observed for synthetic polymers displaying multiple GalNAc decorations. Polystyrene beads without immobilized mucins showed no self-interactions and also no interactions with mucin-decorated polystyrene beads. Taken together, the experimental data suggest that in these molecules, the GalNAc residue mediates interactions independent of the anchoring polymer backbone. Furthermore, GalNAc-GalNAc interactions appear to be responsible for self-interactions of mucins decorated with the STn epitope. Hence, Tn-MUC1 and STn-MUC1 undergo self-interactions mediated by the GalNAc residue in both epitopes, suggesting a possible molecular role in cancer. MUC1 possessing the T (Galß1-3GalNAcα1-Ser/Thr) or ST antigen (NeuNAcα2-3Galß1-3GalNAcα1-Ser/Thr) failed to show self-interactions. However, in the case of ST-MUC1, self-interactions were observed after subsequent treatment with neuraminidase and ß-galactosidase. This enzymatic treatment is expected to introduce Tn-epitopes and these observations thus further strengthen the conclusion that the observed interactions are mediated by the GalNAc groups.

Targeting DNGR-1 (CLEC9A) with antibody/MUC1 peptide conjugates as a vaccine for carcinomas.

DCs are the most potent APCs and are the focus of many immunotherapeutic approaches for the treatment of cancer, although most of these approaches require the ex vivo generation and pulsing of DCs. We have targeted a subset of DCs in vivo using an Ab to DNGR-1, a C-type lectin dedicated to the cross-presentation of Ag expressed by subsets of DCs. HLA-A2 epitopes from the tumour-associated Ag, MUC1, were coupled to the anti-DNGR-1 Ab, and their efficacy in generating a Th1-cell response and inhibiting tumour growth was evaluated in a clinically relevant double transgenic mouse model expressing human MUC1 and A2K/b. Using this strategy, we demonstrate that an effective immune response to MUC1 can be generated, which results in a significant delay in the growth of MUC1-expressing tumours in both prophylactic and therapeutic settings. In addition, we also show, using PBMCs isolated from healthy volunteer blood, that target an MUC1 HLA-A2 epitope to human DNGR-1 in vitro can induce an MUC1-specific CD8(+) -T-cell response, which confirms the relevance of our in vivo murine results in the human setting.

Cancer-associated autoantibodies to MUC1 and MUC4--a blinded case­control study of colorectal cancer in UK collaborative trial of ovarian cancer screening.

Recent reports suggest that autoantibodies directed to aberrantly glycosylated mucins, in particular MUC1 and MUC4, are found in patients with colorectal cancer. There is, however, limited information on the autoantibody levels before clinical diagnosis, and their utility in cancer screening in the general population. In our study, we have generated O-glycosylated synthetic MUC1 and MUC4 peptides in vitro, to mimic cancer-associated glycoforms, and displayed these on microarrays. The assay's performance was tested through an initial screening of serum samples taken from patients at the time of colorectal cancer diagnosis and healthy controls. Subsequently, the selected biomarkers were evaluated in a blinded nested case­control study using stored serum samples from among the 50,640 women randomized to the multimodal arm of the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS), where women gave annual blood samples for several years. Cases were 97 postmenopausal women who developed colorectal cancer after recruitment and were age-matched to 97 women without any history of cancer. MUC1-STn and MUC1-Core3 IgG autoantibodies identified cases with 8.2 and 13.4% sensitivity, respectively, at 95% specificity. IgA to MUC4 glycoforms were unable to discriminate between cases and controls in the UKCTOCS sera. Additional analysis was undertaken by combining the data of MUC1-STn and MUC1-Core3 with previously generated data on autoantibodies to p53 peptides, which increased the sensitivity to 32.0% at 95% specificity. These findings suggest that a combination of antibody signatures may have a role as part of a biomarker panel for the early detection of colorectal cancer.

Cyclooxygenase-2 enzyme induces the expression of the α-2,3-sialyltransferase-3 (ST3Gal-I) in breast cancer.

Aberrant glycosylation is a common feature of malignant change. Changes in mucin-type O-linked glycosylation in breast cancer can result in the expression of truncated core 1-based sialylated glycans rather than the core 2-based glycans observed in normal mammary epithelium cells. This has been shown, in part, to be due to changes in the expression of glycosyltransferases, including the up-regulation of some sialyltransferases. Using the breast cancer cell line T47D, we have shown that PGE2, one of the final products of the cyclooxygenase-2 (COX-2) pathway, can induce the mRNA expression of the sialyltransferase α-2,3-sialyltransferase-3 (ST3Gal-I), resulting in increased sialyltransferase activity, demonstrated by a reduction in PNA lectin staining. Induction of COX-2 in the MDA-MB-231 breast cancer cell line also results in the increased expression of ST3Gal-I, leading to increased sialylation of the substrate of ST3Gal-I, core 1 Galß1,3GalNAc. This effect on sialylation could be reversed by the selective COX-2 inhibitor celecoxib. The use of siRNA to knock down COX-2 and overexpression of COX-2 in MDA-MD-231 cells confirmed the involvement of COX-2 in the up-regulation of ST3Gal-I. Moreover, analysis of the expression of ST3Gal-I and COX-2 by 74 primary breast cancers showed a significant correlation between the two enzymes. COX-2 expression has been associated with a number of tumors, including breast cancer, where its expression is associated with poor prognoses. Thus, these results suggest the intriguing possibility that some of the malignant characteristics associated with COX-2 expression may be via the influence that COX-2 exerts on the glycosylation of tumor cells.

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer.

INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

LYVE-1+ macrophages form a collaborative CCR5-dependent perivascular niche that influences chemotherapy responses in murine breast cancer.

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells that facilitate cancer progression. However, our knowledge of the niches of individual TAM subsets and their development and function remain incomplete. Here, we describe a population of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)-expressing TAMs, which form coordinated multi-cellular "nest" structures that are heterogeneously distributed proximal to vasculature in tumors of a spontaneous murine model of breast cancer. We demonstrate that LYVE-1+ TAMs develop in response to IL-6, which induces their expression of the immune-suppressive enzyme heme oxygenase-1 and promotes a CCR5-dependent signaling axis, which guides their nest formation. Blocking the development of LYVE-1+ TAMs or their nest structures, using gene-targeted mice, results in an increase in CD8+ T cell recruitment to the tumor and enhanced response to chemotherapy. This study highlights an unappreciated collaboration of a TAM subset to form a coordinated niche linked to immune exclusion and resistance to anti-cancer therapy.

Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation.

Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le(a) (SLe(a)) and Sialyl-Le(x) (SLe(x)) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung, breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLe(a)/SLe(x)-MUC1 and STn/SLe(a)/SLe(x)-MUC2 glycoforms in ≥50% of the cases, with a variable distribution among organs. Some new glycoforms-T/SLe(a)-MUC2, STn/T/SLe(a) SLe(x)-MUC5AC and STn/T/SLe(a)/SLe(x)-MUC6-were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O-glycan haptens alone.

Histone Methylases and Demethylases Regulating Antagonistic Methyl Marks: Changes Occurring in Cancer.

Epigenetic regulation of gene expression is crucial to the determination of cell fate in development and differentiation, and the Polycomb (PcG) and Trithorax (TrxG) groups of proteins, acting antagonistically as complexes, play a major role in this regulation. Although originally identified in Drosophila, these complexes are conserved in evolution and the components are well defined in mammals. Each complex contains a protein with methylase activity (KMT), which can add methyl groups to a specific lysine in histone tails, histone 3 lysine 27 (H3K27), by PcG complexes, and H3K4 and H3K36 by TrxG complexes, creating transcriptionally repressive or active marks, respectively. Histone demethylases (KDMs), identified later, added a new dimension to histone methylation, and mutations or changes in levels of expression are seen in both methylases and demethylases and in components of the PcG and TrX complexes across a range of cancers. In this review, we focus on both methylases and demethylases governing the methylation state of the suppressive and active marks and consider their action and interaction in normal tissues and in cancer. A picture is emerging which indicates that the changes which occur in cancer during methylation of histone lysines can lead to repression of genes, including tumour suppressor genes, or to the activation of oncogenes. Methylases or demethylases, which are themselves tumour suppressors, are highly mutated. Novel targets for cancer therapy have been identified and a methylase (KMT6A/EZH2), which produces the repressive H3K27me3 mark, and a demethylase (KDM1A/LSD1), which demethylates the active H3K4me2 mark, are now under clinical evaluation.

Autoantibodies to aberrantly glycosylated MUC1 in early stage breast cancer are associated with a better prognosis.

INTRODUCTION: Detection of serum biomarkers for early diagnosis of breast cancer remains an important goal. Changes in the structure of O-linked glycans occur in all breast cancers resulting in the expression of glycoproteins that are antigenically distinct. Indeed, the serum assay widely used for monitoring disease progression in breast cancer (CA15.3), detects a glycoprotein (MUC1), but elevated levels of the antigen cannot be detected in early stage patients. However, since the immune system acts to amplify the antigenic signal, antibodies can be detected in sera long before the antigen. We have exploited the change in O-glycosylation to measure autoantibody responses to cancer-associated glycoforms of MUC1 in sera from early stage breast cancer patients. METHODS: We used a microarray platform of 60mer MUC1 glycopeptides, to confirm the presence of autoantibodies to cancer associated glycoforms of MUC1 in a proportion of early breast cancer patients (54/198). Five positive sera were selected for detailed definition of the reactive epitopes using on chip glycosylation technology and a panel of glycopeptides based on a single MUC1 tandem repeat carrying specific glycans at specific sites. Based on these results, larger amounts of an extended repertoire of defined MUC1 glycopeptides were synthesised, printed on microarrays, and screened with sera from a large cohort of breast cancer patients (n = 395), patients with benign breast disease (n = 108) and healthy controls (n = 99). All sera were collected in the 1970s and 1980s and complete clinical follow-up of breast cancer patients is available. RESULTS: The presence and level of autoantibodies was significantly higher in the sera from cancer patients compared with the controls, and a highly significant correlation with age was observed. High levels of a subset of autoantibodies to the core3MUC1 (GlcNAcß1-3GalNAc-MUC1) and STnMUC1 (NeuAcα2,6GalNAc-MUC1) glycoforms were significantly associated with reduced incidence and increased time to metastasis. CONCLUSIONS: Autoantibodies to specific cancer associated glycoforms of MUC1 are found more frequently and at higher levels in early stage breast cancer patients than in women with benign breast disease or healthy women. Association of strong antibody response with reduced rate and delay in metastases suggests that autoantibodies can affect disease progression.

T cells reactive with HLA-A*0201 peptides from the histone demethylase JARID1B are found in the circulation of breast cancer patients.

The nuclear protein PLU-1/JARID1B/KDM5 is widely expressed in breast cancers while showing highly restricted expression in normal adult tissues. To investigate whether JARID1B is a potential target antigen for immunotherapy of breast cancer, we have analyzed the responses of CD8(+) T cells to JARID1B HLA-A*0201 peptides in vitro and used peptide multimers to detect the presence of JARID1B reactive T cells in the circulation of breast cancer patients. Peptides were selected using two web-based algorithms: criteria for inclusion being a high score in both prediction algorithms, and nonhomology with retinoblastoma binding protein-2 (RBP2/JARID1A/KDM5A). A 65-peptide panel was selected and assayed for binding strength by competition assay to obtain the IC(50). The immunogenicity in vitro of these peptides was assessed by T cell stimulation experiments, using autologous dendritic cells as APCs in the first rounds followed by autologous lymphoblasts. Fourteen of the peptides assayed produced cultures having >2% of the CD8(+) cells being IFN-γ(+) after 3-6 rounds of stimulation. An HLA-A*0201 cell line could activate the specific T cells if pulsed with peptide, but endogenous peptide levels were insufficient for activation. Nevertheless, multimer staining of circulating T cells from breast cancer patients showed a significantly higher percentage of multimer positive CD8(+) T cells, as compared to healthy adults for two of three JARID1B epitopes tested. One of these, peptide 73 (QLYALPCVL), was analyzed for memory phenotype, and found to have a significantly higher proportion of central memory T cells than the control group, demonstrating a previous exposure to the peptide.

Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm.

BACKGROUND: Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. RESULTS: Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser)3 linker precipitated at physiological pH 7.4. CONCLUSIONS: This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell trafficking studies. Assembling the single chain antibody with monomeric fluorescent protein linker facilitates optimal variable domain pairing and alters the isoelectric point of the recombinant 4D5-8 protein conferring solubility at physiological pH 7.4. The efficient intracellular expression of these functional molecules opens up the possibility of developing an alternative approach for tagging intracellular targets with fluorescent proteins for a range of molecular cell biology imaging studies.

Transforming growth factor-ß1 is constitutively secreted by Chinese hamster ovary cells and is functional in human cells.

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant proteins for clinical use as well as academic research. They are particularly important for the production of glycoproteins where bacteria cannot be used. TGFß1 is a potent cytokine highly conserved across species with multiple immunological and non-immunological effects. We have discovered that CHOK1, the CHO clone most commonly used by the pharmaceutical industry, constitutively secretes latent TGFß1 and that this hamster TGFß1 is active on human cells inducing profound immunological effects. As far as we are aware, the production of TGFß1 by CHOK1 cells has not been reported before in the literature. As TGFß1 exerts powerful and pleiotropic effects on diverse cell types, and as CHO cells are used to produce a large number of clinical and non-clinical products, our findings are highly relevant to studies that rely on recombinant proteins.

Apoptosis in the Pancreatic Cancer Tumor Microenvironment-The Double-Edged Sword of Cancer-Associated Fibroblasts.

Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis. This is attributed to the disease already being advanced at presentation and having a particularly aggressive tumor biology. The PDAC tumor microenvironment (TME) is characterized by a dense desmoplastic stroma, dominated by cancer-associated fibroblasts (CAF), extracellular matrix (ECM) and immune cells displaying immunosuppressive phenotypes. Due to the advanced stage at diagnosis, the depletion of immune effector cells and lack of actionable genomic targets, the standard treatment is still apoptosis-inducing regimens such as chemotherapy. Paradoxically, it has emerged that the direct induction of apoptosis of cancer cells may fuel oncogenic processes in the TME, including education of CAF and immune cells towards pro-tumorigenic phenotypes. The direct effect of cytotoxic therapies on CAF may also enhance tumorigenesis. With the awareness that CAF are the predominant cell type in PDAC driving tumorigenesis with various tumor supportive functions, efforts have been made to try to target them. However, efforts to target CAF have, to date, shown disappointing results in clinical trials. With the help of sophisticated single cell analyses it is now appreciated that CAF in PDAC are a heterogenous population with both tumor supportive and tumor suppressive functions. Hence, there remains a debate whether targeting CAF in PDAC is a valid therapeutic strategy. In this review we discuss how cytotoxic therapies and the induction of apoptosis in PDAC fuels oncogenesis by the education of surrounding stromal cells, with a particular focus on the potential pro-tumorigenic outcomes arising from targeting CAF. In addition, we explore therapeutic avenues to potentially avoid the oncogenic effects of apoptosis in PDAC CAF.

KDM5B protein expressed in viable and fertile ΔARID mice exhibit no demethylase activity.

Post­translational modification of histones serve a crucial role in the control of gene transcription. Trimethylation of lysine 4 on histone 3 is associated with transcription activation. There are currently six known methylases and six known demethylases that can control the methylation status of this site. Lysine demethylase 5B (KDM5B) is one such demethylase, which can repress gene expression. In particular KDM5B has been found to be overexpressed in a number of cancer types, and small­molecular weight inhibitors of its demethylase activity have been identified. Previous characterisation of Kdm5b knock­out mice has revealed that this genotype leads to either embryonic or neonatal lethality. However, the ΔA­T rich interaction domain (ΔARID)­KDM5B strain of mice, which have the ARID domain and five amino acids within the Jumonji (Jmj)N domain spliced out from KDM5B, remain viable and fertile. In the present study, ΔARID­KDM5B was found to have no demethylase activity as determined by in vitro demethylase assays and by immunofluorescence in transfected Cos­1 cells. Furthermore, molecular dynamic simulations revealed conformational changes within the ΔARID­KDM5B structure compared with that in WT­KDM5B, particularly in the JmjC domain, which is responsible for the catalytic activity of WT­KDM5B. This supports the experimental data that shows the loss of demethylase activity. Since Kdm5b knock­out mice show varying degrees of lethality, these data suggest that KDM5B serves a crucial function in development in a manner that is independent of its demethylase activity.