DNA Methyltransferase 3B-Mediated Intratumoral Heterogeneity and Therapeutic Targeting in Breast Cancer Recurrence and Metastasis.

The mechanisms of how cancer cells are selected and evolve to establish distant metastatic colonies remain unclear. Tumor heterogeneity and lack of biomarkers are some of the most difficult challenges in cancer biology and treatment. Here using mouse models for triple-negative breast cancer (TNBC) metastasis, we report heterogeneous expression of DNA methyltransferase 3B (DNMT3B) in both mouse and human primary tumors. High levels of DNMT3B were correlated with poor clinical outcomes in multiple human breast cancer datasets. Mechanistically, clonal cells with high DNMT3B (DNMT3BH) showed higher vimentin (VIM) expression and displayed enhanced epithelial-to-mesenchymal transition capacity. Deletion of VIM diminished the metastatic phenotype of DNMT3BH cells. Importantly, in preclinical mouse models in which the primary tumors were surgically removed, perioperative targeting of DNMT3B in combination with chemotherapy markedly suppressed tumor recurrence and metastasis. Our studies identify DNMT3B-mediated transcription regulation as an important mediator of tumor heterogeneity and show that DNMT3B is critical for tumor invasion and metastasis, reinforcing its potential as a target for treating metastatic disease. IMPLICATIONS: Our findings of transcriptome changes mediated by DNMT3B provide new mechanistic insight for intratumor heterogeneity and chemoresistance, and therapeutic targeting of DNMT3B in combination with chemotherapy offer additional treatment options for metastatic disease especially for patients with TNBC.

The EGF Domains of MUC4 Oncomucin Mediate HER2 Binding Affinity and Promote Pancreatic Cancer Cell Tumorigenesis.

The HER2 receptor and its MUC4 mucin partner form an oncogenic complex via an extracellular region of MUC4 encompassing three EGF domains that promotes tumor progression of pancreatic cancer (PC) cells. However, the molecular mechanism of interaction remains poorly understood. Herein, we decipher at the molecular level the role and impact of the MUC4EGF domains in the mediation of the binding affinities with HER2 and the PC cell tumorigenicity. We used an integrative approach combining in vitro bioinformatic, biophysical, biochemical, and biological approaches, as well as an in vivo study on a xenograft model of PC. In this study, we specified the binding mode of MUC4EGF domains with HER2 and demonstrate their "growth factor-like" biological activities in PC cells leading to stimulation of several signaling proteins (mTOR pathway, Akt, and ß-catenin) contributing to PC progression. Molecular dynamics simulations of the MUC4EGF/HER2 complexes led to 3D homology models and identification of binding hotspots mediating binding affinity with HER2 and PC cell proliferation. These results will pave the way to the design of potential MUC4/HER2 inhibitors targeting the EGF domains of MUC4. This strategy will represent a new efficient alternative to treat cancers associated with MUC4/HER2 overexpression and HER2-targeted therapy failure as a new adapted treatment to patients.

The EMT Transcription Factor ZEB2 Promotes Proliferation of Primary and Metastatic Melanoma While Suppressing an Invasive, Mesenchymal-Like Phenotype.

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of Zeb2 hampered outgrowth of primary melanomas in vivo, whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of Zeb2 expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination. SIGNIFICANCE: ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/2983/F1.large.jpg.

Induction of DNMT3B by PGE2 and IL6 at Distant Metastatic Sites Promotes Epigenetic Modification and Breast Cancer Colonization.

Current cancer treatments are largely based on the genetic characterization of primary tumors and are ineffective for metastatic disease. Here we report that DNA methyltransferase 3B (DNMT3B) is induced at distant metastatic sites and mediates epigenetic reprogramming of metastatic tumor cells. Multiomics analysis and spontaneous metastatic mouse models revealed that DNMT3B alters multiple pathways including STAT3, NFκB, PI3K/Akt, ß-catenin, and Notch signaling, which are critical for cancer cell survival, apoptosis, proliferation, invasion, and colonization. PGE2 and IL6 were identified as critical inflammatory mediators in DNMT3B induction. DNMT3B expression levels positively correlated with human metastatic progression. Targeting IL6 or COX-2 reduced DNMT3B induction and improved chemo or PD1 therapy. We propose a novel mechanism linking the metastatic microenvironment with epigenetic alterations that occur at distant sites. These results caution against the "Achilles heel" in cancer therapies based on primary tumor characterization and suggests targeting DNMT3B induction as new option for treating metastatic disease. SIGNIFICANCE: These findings reveal that DNMT3B epigenetically regulates multiple pro-oncogenic signaling pathways via the inflammatory microenvironment at distant sites, cautioning the clinical approach basing current therapies on genetic characterization of primary tumors.

ZEB2 stably represses RAB25 expression through epigenetic regulation by SIRT1 and DNMTs during epithelial-to-mesenchymal transition.

BACKGROUND: Epithelial mesenchymal transition (EMT) is tightly regulated by a network of transcription factors (EMT-TFs). Among them is the nuclear factor ZEB2, a member of the zinc-finger E-box binding homeobox family. ZEB2 nuclear localization has been identified in several cancer types, and its overexpression is correlated with the malignant progression. ZEB2 transcriptionally represses epithelial genes, such as E-cadherin (CDH1), by directly binding to the promoter of the genes it regulates and activating mesenchymal genes by a mechanism in which there is no full agreement. Recent studies showed that EMT-TFs interact with epigenetic regulatory enzymes that alter the epigenome, thereby providing another level of control. The role of epigenetic regulation on ZEB2 function is not well understood. In this study, we aimed to characterize the epigenetic effect of ZEB2 repressive function on the regulation of a small Rab GTPase RAB25. RESULTS: Using cellular models with conditional ZEB2 expression, we show a clear transcriptional repression of RAB25 and CDH1. RAB25 contributes to the partial suppression of ZEB2-mediated cell migration. Furthermore, a highly significant reverse correlation between RAB25 and ZEB2 expression in several human cancer types could be identified. Mechanistically, ZEB2 binds specifically to E-box sequences on the RAB25 promoter. ZEB2 binding is associated with the local increase in DNA methylation requiring DNA methyltransferases as well as histone deacetylation (H3K9Ac) depending on the activity of SIRT1. Surprisingly, SIRT1 and DNMTs did not interact directly with ZEB2, and while SIRT1 inhibition decreased the stability of long-term repression, it did not prevent down-regulation of RAB25 and CDH1 by ZEB2. CONCLUSIONS: ZEB2 expression is resulting in drastic changes at the chromatin level with both clear DNA hypermethylation and histone modifications. Here, we revealed that SIRT1-mediated H3K9 deacetylation helps to maintain gene repression but is not required for the direct ZEB2 repressive function. Targeting epigenetic enzymes to prevent EMT is an appealing approach to limit cancer dissemination, but inhibiting SIRT1 activity alone might have limited effect and will require drug combination to efficiently prevent EMT.

TGF-ßRII Knock-down in Pancreatic Cancer Cells Promotes Tumor Growth and Gemcitabine Resistance. Importance of STAT3 Phosphorylation on S727.

Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers in the Western world because of a lack of early diagnostic markers and efficient therapeutics. At the time of diagnosis, more than 80% of patients have metastasis or locally advanced cancer and are therefore not eligible for surgical resection. Pancreatic cancer cells also harbour a high resistance to chemotherapeutic drugs such as gemcitabine that is one of the main palliative treatments for PDAC. Proteins involved in TGF-ß signaling pathway (SMAD4 or TGF-ßRII) are frequently mutated in PDAC (50⁻80%). TGF-ß signalling pathway plays antagonistic roles during carcinogenesis by initially inhibiting epithelial growth and later promoting the progression of advanced tumors and thus emerged as both tumor suppressor and oncogenic pathways. In order to decipher the role of TGF-ß in pancreatic carcinogenesis and chemoresistance, we generated CAPAN-1 and CAPAN-2 cell lines knocked down for TGF-ßRII (first actor of TGF-ß signaling). The impact on biological properties of these TGF-ßRII-KD cells was studied both in vitro and in vivo. We show that TGF-ßRII silencing alters tumor growth and migration as well as resistance to gemcitabine. TGF-ßRII silencing also leads to S727 STAT3 and S63 c-Jun phosphorylation, decrease of MRP3 and increase of MRP4 ABC transporter expression and induction of a partial EMT phenotype. These markers associated with TGF-ß signaling pathways may thus appear as potent therapeutic tools to better treat/manage pancreatic cancer.

Epithelial-to-Mesenchymal Transition: Epigenetic Reprogramming Driving Cellular Plasticity.

Epithelial-to-mesenchymal transition (EMT) is a process in which epithelial cells lose their junctions and polarity to gain a motile mesenchymal phenotype. EMT is essential during embryogenesis and adult physiological processes like wound healing, but is aberrantly activated in pathological conditions like fibrosis and cancer. A series of transcription factors (EMT-inducing transcription factor; EMT-TF) regulate the induction of EMT by repressing the transcription of epithelial genes while activating mesenchymal genes through mechanisms still debated. The nuclear interaction of EMT-TFs with larger protein complexes involved in epigenetic genome modulation has attracted recent attention to explain functions of EMT-TFs during reprogramming and cellular differentiation. In this review, we discuss recent advances in understanding the interplay between epigenetic regulators and EMT transcription factors and how these findings could be used to establish new therapeutic approaches to tackle EMT-related diseases.

The transcription factor Zeb2 regulates development of conventional and plasmacytoid DCs by repressing Id2.

Plasmacytoid dendritic cells (DCs [pDCs]) develop from pre-pDCs, whereas two lineages of conventional DCs (cDCs; cDC1s and cDC2s) develop from lineage-committed pre-cDCs. Several transcription factors (TFs) have been implicated in regulating the development of pDCs (E2-2 and Id2) and cDC1s (Irf8, Id2, and Batf3); however, those required for the early commitment of pre-cDCs toward the cDC2 lineage are unknown. Here, we identify the TF zinc finger E box-binding homeobox 2 (Zeb2) to play a crucial role in regulating DC development. Zeb2 was expressed from the pre-pDC and pre-cDC stage onward and highly expressed in mature pDCs and cDC2s. Mice conditionally lacking Zeb2 in CD11c(+) cells had a cell-intrinsic reduction in pDCs and cDC2s, coupled with an increase in cDC1s. Conversely, mice in which CD11c(+) cells overexpressed Zeb2 displayed a reduction in cDC1s. This was accompanied by altered expression of Id2, which was up-regulated in cDC2s and pDCs from conditional knockout mice. Zeb2 chromatin immunoprecipitation analysis revealed Id2 to be a direct target of Zeb2. Thus, we conclude that Zeb2 regulates commitment to both the cDC2 and pDC lineages through repression of Id2.

Tissue Factor Induced by Epithelial-Mesenchymal Transition Triggers a Procoagulant State That Drives Metastasis of Circulating Tumor Cells.

Epithelial-mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT-TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 4270-82. ©2016 AACR.

The mucin MUC4 is a transcriptional and post-transcriptional target of K-ras oncogene in pancreatic cancer. Implication of MAPK/AP-1, NF-κB and RalB signaling pathways.

The membrane-bound mucinMUC4 is a high molecularweight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-κB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-κB transcription factors via MAPK, JNK and NF-κB pathways and at the posttranscriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.

The oncogenic receptor ErbB2 modulates gemcitabine and irinotecan/SN-38 chemoresistance of human pancreatic cancer cells via hCNT1 transporter and multidrug-resistance associated protein MRP-2.

Pancreatic adenocarcinoma (PDAC) is one of the most deadly cancers because of a lack of early diagnostic markers and efficient therapeutics. The fluorinated analog of deoxycytidine, gemcitabine and emerging FOLFIRINOX protocol (5-fluorouracil (5-FU), irinotecan/SN-38, oxaliplatin and leucovorin) are the main chemotherapies to treat PDAC. The ErbB2/HER2 oncogenic receptor is commonly overexpressed in PDAC. In this context, we aimed to decipher the ErbB2-mediated mechanisms of chemoresistance to the two main chemotherapy protocols used to treat PDAC.ErbB2 knocking down (KD) in CAPAN-1 and CAPAN-2 cells led to an increased sensitivity to gemcitabine and an increased resistance to irinotecan/SN-38 both in vitro and in vivo (subcutaneous xenografts) This was correlated to an increase of hCNT1 and hCNT3 transporters and ABCG2, MRP1 and MRP2 ATP-binding cassette transporters expression and resistance to cell death. We also show that MRP2 is repressed following activation of JNK, Erk1/2 and NF-κB pathways by ErbB2. Finally, in datasets of human PDAC samples, ErbB2 and MRP2 expression was conversely correlated. Altogether, we propose that ErbB2 mediates several intracellular mechanisms linked to PDAC cell chemoresistance that may represent potential targets in order to ameliorate chemotherapy response and allow stratification of patients eligible for either gemcitabine or FOLFIRINOX treatment.

Overexpression of chemokine receptor CXCR2 and ligand CXCL7 in liver metastases from colon cancer is correlated to shorter disease-free and overall survival.

Our aim was to analyze the potential role of chemokine receptors CXCR2 and CXCR4 signalling pathways in liver metastatic colorectal cancer (CRC) relapse. CXCR2, CXCR4, and their chemokine ligands were evaluated in liver metastases of colorectal cancer in order to study their correlation with overall and disease-free survival of patients having received, or not received, a neoadjuvant chemotherapy regimen. Quantitative RT-PCR and CXCR2 immunohistochemical staining were carried out using CRC liver metastasis samples. Expression levels of CXCR2, CXCR4, and their ligands were statistically analyzed according to treatment with neoadjuvant chemotherapy and patients' outcome. CXCR2 and CXCL7 overexpression are correlated to shorter overall and disease-free survival. By multivariate analysis, CXCR2 and CXCL7 expressions are independent factors of overall and disease-free survival. Neoadjuvant chemotherapy increases significantly the expression of CXCR2: treated group 1.89 (0.02-50.92) vs 0.55 (0.07-3.22), P = 0.016. CXCL7 was overexpressed close to significance, 0.40 (0.00-7.85) vs 0.15 (0.01-7.88), P = 0.12. We show the involvement of CXCL7/CXCR2 signalling pathways as a predictive factor of poor outcome in metastatic CRC. 5-Fluorouracil-based chemotherapy regimens increase the expression of these genes in liver metastasis, providing one explanation for aggressiveness of relapsed drug-resistant tumors. Selective blockage of CXCR2/CXCL7 signalling pathways could provide new potential therapeutic opportunities.

Mucins and tumor resistance to chemotherapeutic drugs.

Epithelial cancer patients not considered eligible for surgical resection frequently benefit from chemotherapy. Chemotherapy is the treatment of cancer with one or combination of cytotoxic or cytostatic drugs. Recent advances in chemotherapy allowed a great number of cancer patients to receive treatment with significant results. Unfortunately, resistance to chemotherapeutic drug treatment is a major challenge for clinicians in the majority of epithelial cancers because it is responsible for the inefficiency of therapies. Mucins belong to a heterogeneous group of large O-glycoproteins that can be either secreted or membrane-bound. Implications of mucins have been described in relation to cancer cell behavior and cell signaling pathways associated with epithelial tumorigenesis. Because of the frequent alteration of the pattern of mucin expression in cancers as well as their structural and functional characteristics, mucins are thought to also be involved in response to therapies. In this report, we review the roles of mucins in chemoresistance and the associated underlying molecular mechanisms (physical barrier, resistance to apoptosis, drug metabolism, cell stemness, epithelial-mesenchymal transition) and discuss the therapeutic tools/strategies and/or prognosis biomarkers for personalized chemotherapy that could be proposed from these studies.

Colon cancer cells escape 5FU chemotherapy-induced cell death by entering stemness and quiescence associated with the c-Yes/YAP axis.

PURPOSE: Metastasis and drug resistance are the major limitations in the survival and management of patients with cancer. This study aimed to identify the mechanisms underlying HT29 colon cancer cell chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of patients with colon cancer with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery. RESULTS: We show that a clonal 5F31 cell population, resistant to 1 µmol/L 5FU, express a typical cancer stem cell-like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5FU concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, YES1 silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-free medium. Importantly, YES1 and YAP transcript levels were higher in liver metastases of patients with colon cancer after 5FU-based neoadjuvant chemotherapy. Moreover, the YES1 and YAP transcript levels positively correlated with colon cancer relapse and shorter patient survival (P < 0.05 and P < 0.025, respectively). CONCLUSIONS: We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival markers for colon cancer.

Membrane-bound mucin modular domains: from structure to function.

Mucins belong to a heterogeneous family of large O-glycoproteins composed of a long peptidic chain called apomucin on which are linked hundreds of oligosaccharidic chains. Among mucins, membrane-bound mucins are modular proteins and have a structural organization usually containing Pro/Thr/Ser-rich O-glycosylated domains (PTS), EGF-like and SEA domains. Via these modular domains, the membrane-bound mucins participate in cell signalling and cell interaction with their environment in normal and pathological conditions. Moreover, the recent knowledge of these domains and their biological activities led to the development of new therapeutic approaches involving mucins. In this review, we show 3D structures of EGF and SEA domains. We also describe the functional features of the evolutionary conserved domains of membrane-bound mucins and discuss consequences of splice events.

The mucin MUC4 and its membrane partner ErbB2 regulate biological properties of human CAPAN-2 pancreatic cancer cells via different signalling pathways.

The mucin MUC4 and its membrane partner the ErbB2 oncogenic receptor are potential interacting partners in human pancreatic tumour development. However, the way they function is still largely unknown. In this work, we aimed to identify the cellular mechanisms and the intracellular signalling pathways under the control of both ErbB2 and MUC4 in a human pancreatic adenocarcinomatous cell line. Using co-immunoprecipitation and GST pull-down, we show that MUC4 and ErbB2 interact in the human pancreatic adenocarcinomatous cell line CAPAN-2 via the EGF domains of MUC4. Stable cell clones were generated in which either MUC4 or ErbB2 were knocked down (KD) by a shRNA approach. Biological properties of these cells were then studied in vitro and in vivo. Our results show that ErbB2-KD cells are more apoptotic and less proliferative (decreased cyclin D1 and increased p27kip1 expression) while migration and invasive properties were not altered. MUC4-KD clones were less proliferative with decreased cyclin D1 expression, G1 cell cycle arrest and altered ErbB2/ErbB3 expression. Their migration properties were reduced whereas invasive properties were increased. Importantly, inhibition of ErbB2 and MUC4 expression did not impair the same signalling pathways (inhibition of MUC4 expression affected the JNK pathway whereas that of ErbB2 altered the MAPK pathway). Finally, ErbB2-KD and MUC4-KD cells showed impaired tumour growth in vivo. Our results show that ErbB2 and MUC4, which interact physically, activate different intracellular signalling pathways to regulate biological properties of CAPAN-2 pancreatic cancer cells.

Mucins and pancreatic cancer.

Pancreatic cancer is characterized by an often dramatic outcome (five year survival < 5%) related to a late diagnosis and a lack of efficient therapy. Therefore, clinicians desperately need new biomarkers and new therapeutic tools to develop new efficient therapies. Mucins belong to an ever increasing family of O-glycoproteins. Secreted mucins are the main component of mucus protecting the epithelia whereas membrane-bound mucins are thought to play important biological roles in cell-cell and cell-matrix interactions, in cell signaling and in modulating biological properties of cancer cells. In this review, we will focus on the altered expression pattern of mucins in pancreatic cancer, from the early neoplastic lesion Pancreatic Intraepithelial Neoplasia (PanIN) to invasive pancreatic carcinomas, and the molecular mechanisms (including genetic and epigenetic regulation) and signaling pathways known to control their expression. Moreover, we will discuss the recent advances about the biology of both secreted and membrane-bound mucins and their key roles in pancreatic carcinogenesis and resistance to therapy. Finally, we will discuss exciting opportunities that mucins offer as potential therapeutic targets in pancreatic cancer.