Generation of an Fsp1 (fibroblast-specific protein 1)-Flpo transgenic mouse strain.

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1-Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast-specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1-Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.

ZEB1 controls a lineage-specific transcriptional program essential for melanoma cell state transitions.

Cell plasticity sustains intra-tumor heterogeneity and treatment resistance in melanoma. Deciphering the transcriptional mechanisms governing reversible phenotypic transitions between proliferative/differentiated and invasive/stem-like states is required. Expression of the ZEB1 transcription factor is frequently activated in melanoma, where it fosters adaptive resistance to targeted therapies. Here, we performed a genome-wide characterization of ZEB1 transcriptional targets, by combining ChIP-sequencing and RNA-sequencing, upon phenotype switching in melanoma models. We identified and validated ZEB1 binding peaks in the promoter of key lineage-specific genes crucial for melanoma cell identity. Mechanistically, ZEB1 negatively regulates SOX10-MITF dependent proliferative/melanocytic programs and positively regulates AP-1 driven invasive and stem-like programs. Comparative analyses with breast carcinoma cells revealed lineage-specific ZEB1 binding, leading to the design of a more reliable melanoma-specific ZEB1 regulon. We then developed single-cell spatial multiplexed analyses to characterize melanoma cell states intra-tumoral heterogeneity in human melanoma samples. Combined with scRNA-Seq analyses, our findings confirmed increased ZEB1 expression in Neural-Crest-like cells and mesenchymal cells, underscoring its significance in vivo in both populations. Overall, our results define ZEB1 as a major transcriptional regulator of cell states transitions and provide a better understanding of lineage-specific transcriptional programs sustaining intra-tumor heterogeneity in melanoma.

Cooperative pro-tumorigenic adaptation to oncogenic RAS through epithelial-to-mesenchymal plasticity.

In breast cancers, aberrant activation of the RAS/MAPK pathway is strongly associated with mesenchymal features and stemness traits, suggesting an interplay between this mitogenic signaling pathway and epithelial-to-mesenchymal plasticity (EMP). By using inducible models of human mammary epithelial cells, we demonstrate herein that the oncogenic activation of RAS promotes ZEB1-dependent EMP, which is necessary for malignant transformation. Notably, EMP is triggered by the secretion of pro-inflammatory cytokines from neighboring RAS-activated senescent cells, with a prominent role for IL-6 and IL-1α. Our data contrast with the common view of cellular senescence as a tumor-suppressive mechanism and EMP as a process promoting late stages of tumor progression in response to signals from the tumor microenvironment. We highlighted here a pro-tumorigenic cooperation of RAS-activated mammary epithelial cells, which leverages on oncogene-induced senescence and EMP to trigger cellular reprogramming and malignant transformation.

Generation of a conditional mouse model to target Acvr1b disruption in adult tissues.

Alk4 is a type I receptor that belongs to the transforming growth factor-beta (TGF-ß) family. It takes part in the signaling of TGF-ß ligands such as Activins, Gdfs, and Nodal that had been demonstrated to participate in numerous mechanisms ranging from early embryonic development to adult-tissue homeostasis. Evidences indicate that Alk4 is a key regulator of many embryonic processes, but little is known about its signaling in adult tissues and in pathological conditions where Alk4 mutations had been reported. Conventional deletion of Alk4 gene (Acvr1b) results in early embryonic lethality prior gastrulation, which has precluded study of Alk4 functions in postnatal and adult mice. To circumvent this problem, we have generated a conditional Acvr1b floxed-allele by flanking the fifth and sixth exons of the Acvr1b gene with loxP sites. Cre-mediated deletion of the floxed allele generates a deleted allele, which behaves as an Acvr1b null allele leading to embryonic lethality in homozygous mutant animals. A tamoxifen-inducible approach to target disruption of Acvr1b specifically in adult tissues was used and proved to be efficient for studying Alk4 functions in various organs. We report, therefore, a novel conditional model allowing investigation of biological role played by Alk4 in a variety of tissue-specific contexts.

Tif1γ suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway.

Transcriptional intermediary factor 1γ (TIF1γ; alias, TRIM33/RFG7/PTC7/ectodermin) belongs to an evolutionarily conserved family of nuclear factors that have been implicated in stem cell pluripotency, embryonic development, and tumor suppression. TIF1γ expression is markedly down-regulated in human pancreatic tumors, and Pdx1-driven Tif1γ inactivation cooperates with the Kras(G12D) oncogene in the mouse pancreas to induce intraductal papillary mucinous neoplasms. In this study, we report that aged Pdx1-Cre; LSL-Kras(G12D); Tif1γ(lox/lox) mice develop pancreatic ductal adenocarcinomas (PDACs), an aggressive and always fatal neoplasm, demonstrating a Tif1γ tumor-suppressive function in the development of pancreatic carcinogenesis. Deletion of SMAD4/DPC4 (deleted in pancreatic carcinoma locus 4) occurs in approximately 50% of human cases of PDAC. We, therefore, assessed the genetic relationship between Tif1γ and Smad4 signaling in pancreatic tumors and found that Pdx1-Cre; LSL-Kras(G12D); Smad4(lox/lox); Tif1γ(lox/lox) (alias, KSSTT) mutant mice exhibit accelerated tumor progression. Consequently, Tif1γ tumor-suppressor effects during progression from a premalignant to a malignant state in our mouse model of pancreatic cancer are independent of Smad4. These findings establish, for the first time to our knowledge, that Tif1γ and Smad4 both regulate an intraductal papillary mucinous neoplasm-to-PDAC sequence through distinct tumor-suppressor programs.

The conditional expression of KRAS G12D in mouse pancreas induces disorganization of endocrine islets prior the onset of ductal pre-cancerous lesions.

BACKGROUND/OBJECTIVES: Pdx1-Cre; LSL-KRAS(G12D) mice develop premalignant pancreatic ductal lesions that can possibly progress spontaneously to pancreatic ductal adenocarcinoma (PDAC). Although Pdx1-Cre is expressed in the embryonic endoderm, which gives rise to all pancreatic lineages, the possible consequences of KRAS(G12D) expression in the endocrine compartment have never been finely explored. METHODS: We examined by histology whether Pdx1-driven expression of KRAS(G12D) could induce islets of Langerhans defects. RESULTS: We observed in Pdx1-Cre; LSL-KRAS(G12D) early disorganization of the endocrine compartment including i) hyperplasia affecting all the endocrine lineages, ii) ectopic onset of Ck19-positive (ductal-like) structures within the endocrine islets, and iii) the presence of islet cells co-expressing glucagon and insulin, all occurring before the onset of ducts lesions. CONCLUSIONS: This work indicates that expression of KRAS(G12D) in Pdx1-expressing cells during embryogenesis affects the endocrine pancreas, and highlights the need to deepen possible consequences on both glucose metabolism and PDAC initiation.

The human NUPR1/P8 gene is transcriptionally activated by transforming growth factor ß via the SMAD signalling pathway.

NUPR1 (nuclear protein 1), also called P8 (molecular mass 8 kDa) or COM1 (candidate of metastasis 1), is involved in the stress response and in cancer progression. In the present study, we investigated whether human NUPR1 expression was regulated by TGFß (transforming growth factor ß), a secreted polypeptide largely involved in tumorigenesis. We demonstrate that the expression of NUPR1 was activated by TGFß at the transcriptional level. We show that this activation is mediated by the SMAD proteins, which are transcription factors specifically involved in the signalling of TGFß superfamily members. NUPR1 promoter analysis reveals the presence of a functional TGFß-response element binding the SMAD proteins located in the genomic DNA region corresponding to the 5'-UTR (5'-untranslated region). Altogether, the molecular results of the present study, which demonstrate the existence of a TGFß/SMAD/NUPR1 activation cascade, open the way to consider and investigate further a new mechanism enabling TGFß to promote tumorigenesis by inducing stress resistance.

Dissecting the Origin of Heterogeneity in Uterine and Ovarian Carcinosarcomas.

Gynecologic carcinosarcomas (CS) are biphasic neoplasms composed of carcinomatous (C) and sarcomatous (S) malignant components. Because of their rarity and histologic complexity, genetic and functional studies on CS are scarce and the mechanisms of initiation and development remain largely unknown. Whole-genome analysis of the C and S components reveals shared genomic alterations, thus emphasizing the clonal evolution of CS. Reconstructions of the evolutionary history of each tumor further reveal that C and S samples are composed of both ancestral cell populations and component-specific subclones, supporting a common origin followed by distinct evolutionary trajectories. However, while we do not find any recurrent genomic features associated with phenotypic divergence, transcriptomic and methylome analyses identify a common mechanism across the cohort, the epithelial-to-mesenchymal transition (EMT), suggesting a role for nongenetic factors in inflicting changes to cellular fate. Altogether, these data accredit the hypothesis that CS tumors are driven by both clonal evolution and transcriptomic reprogramming, essential for susceptibility to transdifferentiation upon encountering environmental cues, thus linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences. Significance: We have provided a detailed characterization of the genomic landscape of CS and identified EMT as a common mechanism associated with phenotypic divergence, linking CS heterogeneity to genetic, transcriptomic, and epigenetic influences.

Epithelial-to-mesenchymal transition promotes immune escape by inducing CD70 in non-small cell lung cancer.

INTRODUCTION: Epithelial-to-mesenchymal transition (EMT) is associated with tumor aggressiveness, drug resistance, and poor survival in non-small cell lung cancer (NSCLC) and other cancers. The identification of immune-checkpoint ligands (ICPLs) associated with NSCLCs that display a mesenchymal phenotype (mNSCLC) could help to define subgroups of patients who may benefit from treatment strategies using immunotherapy. METHODS: We evaluated ICPL expression in silico in 130 NSCLC cell lines. In vitro, CRISPR/Cas9-mediated knockdown and lentiviral expression were used to assess the impact of ZEB1 expression on CD70. Gene expression profiles of lung cancer samples from the TCGA (n = 1018) and a dataset from MD Anderson Cancer Center (n = 275) were analyzed. Independent validation was performed by immunohistochemistry and targeted-RNA sequencing in 154 NSCLC whole sections, including a large cohort of pulmonary sarcomatoid carcinomas (SC, n = 55). RESULTS: We uncover that the expression of CD70, a regulatory ligand from the tumor necrosis factor ligand family, is enriched in mNSCLC in vitro models. Mechanistically, the EMT-inducer ZEB1 impacted CD70 expression and fostered increased activity of the CD70 promoter. CD70 overexpression was also evidenced in mNSCLC patient tumor samples and was particularly enriched in SC, a lung cancer subtype associated with poor prognosis. In these tumors, CD70 expression was associated with decreased CD3+ and CD8+ T-cell infiltration and increased T-cell exhaustion markers. CONCLUSION: Our results provide evidence on the pivotal roles of CD70 and ZEB1 in immune escape in mNSCLC, suggesting that EMT might promote cancer progression and metastasis by not only increasing cancer cell plasticity but also reprogramming the immune response in the local tumor microenvironment.

Opposite Roles for ZEB1 and TMEJ in the Regulation of Breast Cancer Genome Stability.

Breast cancer cells frequently acquire mutations in faithful DNA repair genes, as exemplified by BRCA-deficiency. Moreover, overexpression of an inaccurate DNA repair pathway may also be at the origin of the genetic instability arising during the course of cancer progression. The specific gain in expression of POLQ, encoding the error-prone DNA polymerase Theta (POLθ) involved in theta-mediated end joining (TMEJ), is associated with a characteristic mutational signature. To gain insight into the mechanistic regulation of POLQ expression, this review briefly presents recent findings on the regulation of POLQ in the claudin-low breast tumor subtype, specifically expressing transcription factors involved in epithelial-to-mesenchymal transition (EMT) such as ZEB1 and displaying a paucity in genomic abnormality.

EMT Transcription Factor ZEB1 Represses the Mutagenic POLθ-Mediated End-Joining Pathway in Breast Cancers.

A characteristic of cancer development is the acquisition of genomic instability, which results from the inaccurate repair of DNA damage. Among double-strand break repair mechanisms induced by oncogenic stress, the highly mutagenic theta-mediated end-joining (TMEJ) pathway, which requires DNA polymerase theta (POLθ) encoded by the POLQ gene, has been shown to be overexpressed in several human cancers. However, little is known regarding the regulatory mechanisms of TMEJ and the consequence of its dysregulation. In this study, we combined a bioinformatics approach exploring both Molecular Taxonomy of Breast Cancer International Consortium and The Cancer Genome Atlas databases with CRISPR/Cas9-mediated depletion of the zinc finger E-box binding homeobox 1 (ZEB1) in claudin-low tumor cells or forced expression of ZEB1 in basal-like tumor cells, two triple-negative breast cancer (TNBC) subtypes, to demonstrate that ZEB1 represses POLQ expression. ZEB1, a master epithelial-to-mesenchymal transition-inducing transcription factor, interacted directly with the POLQ promoter. Moreover, downregulation of POLQ by ZEB1 fostered micronuclei formation in TNBC tumor cell lines. Consequently, ZEB1 expression prevented TMEJ activity, with a major impact on genome integrity. In conclusion, we showed that ZEB1 directly inhibits the expression of POLQ and, therefore, TMEJ activity, controlling both stability and integrity of breast cancer cell genomes. SIGNIFICANCE: These findings uncover an original mechanism of TMEJ regulation, highlighting ZEB1 as a key player in genome stability during cancer progression via its repression of POLQ.See related commentary by Carvajal-Maldonado and Wood, p. 1441.

Chronic T cell receptor stimulation unmasks NK receptor signaling in peripheral T cell lymphomas via epigenetic reprogramming.

Peripheral T cell lymphomas (PTCLs) represent a significant unmet medical need with dismal clinical outcomes. The T cell receptor (TCR) is emerging as a key driver of T lymphocyte transformation. However, the role of chronic TCR activation in lymphomagenesis and in lymphoma cell survival is still poorly understood. Using a mouse model, we report that chronic TCR stimulation drove T cell lymphomagenesis, whereas TCR signaling did not contribute to PTCL survival. The combination of kinome, transcriptome, and epigenome analyses of mouse PTCLs revealed a NK cell-like reprogramming of PTCL cells with expression of NK receptors (NKRs) and downstream signaling molecules such as Tyrobp and SYK. Activating NKRs were functional in PTCLs and dependent on SYK activity. In vivo blockade of NKR signaling prolonged mouse survival, demonstrating the addiction of PTCLs to NKRs and downstream SYK/mTOR activity for their survival. We studied a large collection of human primary samples and identified several PTCLs recapitulating the phenotype described in this model by their expression of SYK and the NKR, suggesting a similar mechanism of lymphomagenesis and establishing a rationale for clinical studies targeting such molecules.

Comprehensive characterization of claudin-low breast tumors reflects the impact of the cell-of-origin on cancer evolution.

Claudin-low breast cancers are aggressive tumors defined by the low expression of key components of cellular junctions, associated with mesenchymal and stemness features. Although they are generally considered as the most primitive breast malignancies, their histogenesis remains elusive. Here we show that this molecular subtype of breast cancers exhibits a significant diversity, comprising three main subgroups that emerge from unique evolutionary processes. Genetic, gene methylation and gene expression analyses reveal that two of the subgroups relate, respectively, to luminal breast cancers and basal-like breast cancers through the activation of an EMT process over the course of tumor progression. The third subgroup is closely related to normal human mammary stem cells. This unique subgroup of breast cancers shows a paucity of genomic aberrations and a low frequency of TP53 mutations, supporting the emerging notion that the intrinsic properties of the cell-of-origin constitute a major determinant of the genetic history of tumorigenesis.

Generation of a conditional Flpo/FRT mouse model expressing constitutively active TGFß in fibroblasts.

Transforming growth factor (TGFß) is a secreted factor, which accumulates in tissues during many physio- and pathological processes such as embryonic development, wound healing, fibrosis and cancer. In order to analyze the effects of increased microenvironmental TGFß concentration in vivo, we developed a conditional transgenic mouse model (Flpo/Frt system) expressing bioactive TGFß in fibroblasts, a cell population present in the microenvironment of almost all tissues. To achieve this, we created the genetically-engineered [Fsp1-Flpo; FSFTGFßCA] mouse model. The Fsp1-Flpo allele consists in the Flpo recombinase under the control of the Fsp1 (fibroblast-specific promoter 1) promoter. The FSFTGFßCA allele consists in a transgene encoding a constitutively active mutant form of TGFß (TGFßCA) under the control of a Frt-STOP-Frt (FSF) cassette. The FSFTGFßCA allele was created to generate this model, and functionally validated by in vitro, ex vivo and in vivo techniques. [Fsp1-Flpo; FSFTGFßCA] animals do not present any obvious phenotype despite the correct expression of TGFßCA transgene in fibroblasts. This [Fsp1-Flpo; FSFTGFßCA] model is highly pertinent for future studies on the effect of increased microenvironmental bioactive TGFß concentrations in mice bearing Cre-dependent genetic alterations in other compartments (epithelial or immune compartments for instance). These dual recombinase system (DRS) approaches will enable scientists to study uncoupled spatiotemporal regulation of different genetic alterations within the same mouse, thus better replicating the complexity of human diseases.

Correction: Schwann cells support oncogenic potential of pancreatic cancer cells through TGFß signaling.

The original version of this article contained an error in the name of one of the co-authors (Kayvan Mohkam). This has been corrected in the PDF and HTML versions.

Tspan8-ß-catenin positive feedback loop promotes melanoma invasion.

Due to its high proclivity to metastasize, and despite the recent development of targeted and immune therapy strategies, melanoma is still the deadliest form of skin cancer. Therefore, understanding the molecular mechanisms underlying melanoma invasion remains crucial. We previously characterized Tspan8 for its ability to prompt melanoma cell detachment from their microenvironment and trigger melanoma cell invasiveness, but the signaling events by which Tspan8 regulates the invasion process still remain unknown. Here, we demonstrated that ß-catenin stabilization is a molecular signal subsequent to the onset of Tspan8 expression, and that, in turn, ß-catenin triggers the direct transcriptional activation of Tspan8 expression, leading to melanoma invasion. Moreover, we showed that ß-catenin activation systematically correlates with a high expression of Tspan8 protein in melanoma lesions from transgenic Nras; bcat* mice, as well as in deep penetrating naevi, a type of human pre-melanoma neoplasm characterized by a combined activation of ß-catenin and MAP kinase signaling. Overall, our data suggest that ß-catenin and Tspan8 are part of a positive feedback loop, which sustains a high Tspan8 expression level, conferring to melanoma cells the invasive properties required for tumor progression and dissemination.

Schwann cells support oncogenic potential of pancreatic cancer cells through TGFß signaling.

Pancreatic ductal adenocarcinoma (PDAC) is one of the solid tumors with the poorest prognosis. The stroma of this tumor is abundant and composed of extracellular matrix and stromal cells (including cancer-associated fibroblasts and immune cells). Nerve fibers invading this stroma represent a hallmark of PDAC, involved in neural remodeling, which participates in neuropathic pain, cancer cell dissemination and tumor relapse after surgery. Pancreatic cancer-associated neural remodeling is regulated through functional interplays mediated by physical and molecular interactions between cancer cells, nerve cells and surrounding Schwann cells, and other stromal cells. In the present study, we show that Schwann cells (glial cells supporting peripheral neurons) can enhance aggressiveness (migration, invasion, tumorigenicity) of pancreatic cancer cells in a transforming growth factor beta (TGFß)-dependent manner. Indeed, we reveal that conditioned medium from Schwann cells contains high amounts of TGFß able to activate the TGFß-SMAD signaling pathway in cancer cells. We also observed in human PDAC samples that high levels of TGFß signaling activation were positively correlated with perineural invasion. Secretome analyses by mass spectrometry of Schwann cells and pancreatic cancer cells cultured alone or in combination highlighted the central role of TGFß in neuro-epithelial interactions, as illustrated by proteomic signatures related to cell adhesion and motility. Altogether, these results demonstrate that Schwann cells are a meaningful source of TGFß in PDAC, which plays a crucial role in the acquisition of aggressive properties by pancreatic cancer cells.

Cellular Pliancy and the Multistep Process of Tumorigenesis.

Completion of early stages of tumorigenesis relies on the dynamic interplay between the initiating oncogenic event and the cellular context. Here, we review recent findings indicating that each differentiation stage within a defined cellular lineage is associated with a unique susceptibility to malignant transformation when subjected to a specific oncogenic insult. This emerging notion, named cellular pliancy, provides a rationale for the short delay in the development of pediatric cancers of prenatal origin. It also highlights the critical role of cellular reprogramming in early steps of malignant transformation of adult differentiated cells and its impact on the natural history of tumorigenesis.

The cell-of-origin dictates the genomic landscape of breast cancers.

Aberrant cell proliferation induced by activated oncogenes triggers oxidative stress and uncontrolled DNA replication, promoting genomic instability. We recently reported that human mammary stem cells exhibit the unique capacity to withstand an oncogenic activation by dint of an anti-oxidant program driven by the ZEB1 transcription factor. This pre-emptive program prevents the onset of chromosomal instability, leading to the development of tumors with unique pathological features.