Tissue-resident macrophages provide a pro-tumorigenic niche to early NSCLC cells.

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis3-5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions1. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial-mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8+ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions.

Phosphorylation of Pkp1 by RIPK4 regulates epidermal differentiation and skin tumorigenesis.

Tissue homeostasis of skin is sustained by epidermal progenitor cells localized within the basal layer of the skin epithelium. Post-translational modification of the proteome, such as protein phosphorylation, plays a fundamental role in the regulation of stemness and differentiation of somatic stem cells. However, it remains unclear how phosphoproteomic changes occur and contribute to epidermal differentiation. In this study, we survey the epidermal cell differentiation in a systematic manner by combining quantitative phosphoproteomics with mammalian kinome cDNA library screen. This approach identified a key signaling event, phosphorylation of a desmosome component, PKP1 (plakophilin-1) by RIPK4 (receptor-interacting serine-threonine kinase 4) during epidermal differentiation. With genome-editing and mouse genetics approach, we show that loss of function of either Pkp1 or Ripk4 impairs skin differentiation and enhances epidermal carcinogenesis in vivo Phosphorylation of PKP1's N-terminal domain by RIPK4 is essential for their role in epidermal differentiation. Taken together, our study presents a global view of phosphoproteomic changes that occur during epidermal differentiation, and identifies RIPK-PKP1 signaling as novel axis involved in skin stratification and tumorigenesis.

Loss of TGFbeta signaling destabilizes homeostasis and promotes squamous cell carcinomas in stratified epithelia.

Although TGFbeta is a potent inhibitor of proliferation, epithelia lacking the essential receptor (TbetaRII) for TGFbeta signaling display normal tissue homeostasis. By studying asymptomatic TbetaRII-deficient stratified epithelia, we show that tissue homeostasis is maintained by balancing hyperproliferation with elevated apoptosis. Moreover, rectal and genital epithelia, which are naturally proliferative, develop spontaneous squamous cell carcinomas with age when TbetaRII is absent. This progression is associated with a reduction in apoptosis and can be accelerated in phenotypically normal epidermis by oncogenic mutations in Ras. We show that TbetaRII deficiency leads to enhanced keratinocyte motility and integrin-FAK-Src signaling. Together, these mechanisms provide a molecular framework to account for many of the characteristics of TbetaRII-deficient invasive SQCCs.

Microbial alkyl- and aryl-sulfatases: mechanism, occurrence, screening and stereoselectivities.

This review gives an overview on the occurrence of sulfatases in Prokaryota, Eukaryota and Archaea. The mechanism of enzymes acting with retention or inversion of configuration during sulfate ester hydrolysis is discussed taking two complementary examples. Methods for the discovery of novel alkyl sulfatases are described by way of sequence-based search and enzyme induction. A comprehensive list of organisms with their respective substrate scope regarding prim- and sec-alkyl sulfate esters allows to assess the capabilities and limitations of various biocatalysts employed as whole cell systems or as purified enzymes with respect to their activities and enantioselectivities. Methods for immobilization and selectivity enhancement by addition of metal ions or organic (co)solvents are summarised.

Stereochemistry and Mechanism of Enzymatic and Non-Enzymatic Hydrolysis of Benzylic sec-Sulfate Esters.

The substrate scope of inverting alkylsulfatase Pisa1 was extended towards benzylic sec-sulfate esters by suppression of competing non-enzymatic autohydrolysis by addition of dimethyl sulfoxide as co-solvent. Detailed investigation of the mechanism of autohydrolysis in 18O-labeled buffer by using an enantiopure sec-benzylic sulfate ester as substrate revealed that from the three possible pathways (i) inverting SN2-type nucleophilic attack of [OH-] at the benzylic carbon represents the major pathway, whereas (ii) SN1-type formation of a planar benzylic carbenium ion leading to racemization was a minor event, and (iii) Retaining SN2-type nucleophilic attack at sulfur took place at the limits of detection. The data obtained are interpreted by analysis of Hammett constants of meta substituents.

Tumor-initiating stem cells of squamous cell carcinomas and their control by TGF-ß and integrin/focal adhesion kinase (FAK) signaling.

Cancer stem cells (CSCs) sustain tumor growth through their ability to self-renew and to generate differentiated progeny. These functions endow CSCs with the potential to initiate secondary tumors bearing characteristics similar to those of the parent. Recently the hair follicle stem cell marker CD34 was used to purify a CSC-like cell population from early skin tumors arising from treatment with 7,12-dimethylbenz[α]anthracene/12-o-tetradecanoylphorbol-13-acetate, which typically generates benign papillomas that occasionally progress to squamous cell carcinomas (SCCs). In the present study, we identify and characterize CSCs purified from malignant SCCs. We show that SCCs contain two highly tumorigenic CSC populations that differ in CD34 levels but are enriched for integrins and coexist at the SCC-stroma interface. Intriguingly, whether CD34(lo) or CD34(hi), α6(hi)ß1(hi) populations can initiate secondary tumors by serial limit-dilution transplantation assays, but α6(lo)ß1(lo) populations cannot. Moreover, secondary tumors generated from a single CSC of either subtype contain both CD34(lo) and CD34(hi) α6(hi)ß1(hi)CSCs, indicating their nonhierarchical organization. Genomic profiling and hierarchical cluster analysis show that these two CSC subtypes share a molecular signature distinct from either the CD34(-) epidermal or the CD34(hi) hair follicle stem cell signature. Although closely related, α6(hi)ß1(hi)CD34(lo) and α6(hi)ß1(hi)CD34(hi) CSCs differ in cell-cycle gene expression and proliferation characteristics. Indeed, proliferation and expansion of α6(hi)ß1(hi)CD34(hi) CSCs is sensitive to whether they can initiate a TGF-ß receptor II-mediated response to counterbalance elevated focal adhesion kinase-mediated integrin signaling within the tumor. Overall, the coexistence and interconvertibility of CSCs with differing sensitivities to their microenvironment pose challenges and opportunities for SCC cancer therapies.

Hedgehog signaling regulates the generation of ameloblast progenitors in the continuously growing mouse incisor.

In many organ systems such as the skin, gastrointestinal tract and hematopoietic system, homeostasis is dependent on the continuous generation of differentiated progeny from stem cells. The rodent incisor, unlike human teeth, grows throughout the life of the animal and provides a prime example of an organ that rapidly deteriorates if newly differentiated cells cease to form from adult stem cells. Hedgehog (Hh) signaling has been proposed to regulate self-renewal, survival, proliferation and/or differentiation of stem cells in several systems, but to date there is little evidence supporting a role for Hh signaling in adult stem cells. We used in vivo genetic lineage tracing to identify Hh-responsive stem cells in the mouse incisor and we show that sonic hedgehog (SHH), which is produced by the differentiating progeny of the stem cells, signals to several regions of the incisor. Using a hedgehog pathway inhibitor (HPI), we demonstrate that Hh signaling is not required for stem cell survival but is essential for the generation of ameloblasts, one of the major differentiated cell types in the tooth, from the stem cells. These results therefore reveal the existence of a positive-feedback loop in which differentiating progeny produce the signal that in turn allows them to be generated from stem cells.

Asymmetric Synthesis of ß-Substituted α-Methylenebutyro- lactones via TRIP-Catalyzed Allylation: Mechanistic Studies and Application to the Synthesis of (S)-(-)-Hydroxymatairesinol.

Asymmetric allylation of (hetero)aromatic aldehydes by a zinc(II)-allylbutyrolactone species catalyzed by a chiral BINOL-type phosphoric acid gave ß-substituted α-methylenebutyrolactones in 68 to >99% ee and 52-91% isolated yield. DFT studies on the intermediate Zn2+-complex - crucial for chiral induction - suggest a six-membered ring intermediate, which allows the phosphoric acid moiety to activate the aldehyde. The methodology was applied to the synthesis of the antitumour natural product (S)-(-)-hydroxymatairesinol.

An epigenetic switch controls an alternative NR2F2 isoform that unleashes a metastatic program in melanoma.

Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-to-mesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 - isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-binding domain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanoma progression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.

Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics.

In response to alphabeta1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require alphabeta1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of alphabeta1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL-PIX-PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK.

Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates.

The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>107 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2. A flexibly placed hydrophobic sequence motif emerges which is defined by higher order epistatic interactions between six residues, suggesting synergy that enables high connectivity in the sequence landscape. Through positive epistasis, MKK1 maintains function during mutagenesis, establishing the importance of co-dependent residues in mammalian protein kinase-substrate interactions, and creating a scenario for the evolution of diverse human signalling networks.

Integrated activity of PDZ protein complexes regulates epithelial polarity.

Polarized cells contain numerous membrane domains, but it is unclear how the formation of these domains is coordinated to create a single integrated cell architecture. Genetic screens of Drosophila melanogaster embryos have identified three complexes, each containing one of the PDZ domain proteins--Stardust (Sdt), Bazooka (Baz) and Scribble (Scrib)--that control epithelial polarity and formation of zonula adherens. We find that these complexes can be ordered into a single regulatory hierarchy that is initiated by cell adhesion-dependent recruitment of the Baz complex to the zonula adherens. The Scrib complex represses apical identity along basolateral surfaces by antagonizing Baz-initiated apical polarity. The Sdt-containing Crb complex is recruited apically by the Baz complex to counter antagonistic Scrib activity. Thus, a finely tuned balance between Scrib and Crb complex activity sets the limits of the apical and basolateral membrane domains and positions cell junctions. Our data suggest a model in which the maturation of epithelial cell polarity is driven by integration of the sequential activities of PDZ-based protein complexes.

Quinone reductase acts as a redox switch of the 20S yeast proteasome.

The proteasome has an essential function in the intracellular degradation of protein in eukaryotic cells. We found that the dimeric quinone reductase Lot6 uses the flavin mononucleotide (FMN)-binding site to bind to the 20S proteasome with a 1:2 stoichiometry-that is, one 20S proteasome molecule can associate with two quinone reductases. Furthermore, reduction of the FMN cofactor by either NADH or light irradiation results in the binding of the b-Zip transcription factor Yap4 to the Lot6-proteasome complex, indicating that recruitment of the transcription factor depends on the redox state of the quinone reductase. Here, we show that binding of Yap4 to the complex not only protects it from ubiquitin-independent proteasomal degradation, but also regulates its cellular localization. In non-stressed wild-type cells, we did not detect any Yap4 in the nucleus, whereas Yap4 was present in the nuclei from quinone-stressed yeast cultures. Thus, the Lot6-proteasome complex can be regarded as a redox switch in which the quinone reductase acts as a sensor for oxidative stress.

De Novo PITX1 Expression Controls Bi-Stable Transcriptional Circuits to Govern Self-Renewal and Differentiation in Squamous Cell Carcinoma.

Basal tumor propagating cells (TPCs) control squamous cell carcinoma (SCC) growth by self-renewing and differentiating into supra-basal SCC cells, which lack proliferative potential. While transcription factors such as SOX2 and KLF4 can drive these behaviors, their molecular roles and regulatory interactions with each other have remained elusive. Here, we show that PITX1 is specifically expressed in TPCs, where it co-localizes with SOX2 and TRP63 and determines cell fate in mouse and human SCC. Combining gene targeting with chromatin immunoprecipitation sequencing (ChIP-seq) and transcriptomic analyses reveals that PITX1 cooperates with SOX2 and TRP63 to sustain an SCC-specific transcriptional feed-forward circuit that maintains TPC-renewal, while inhibiting KLF4 expression and preventing KLF4-dependent differentiation. Conversely, KLF4 represses PITX1, SOX2, and TRP63 expression to prevent TPC expansion. This bi-stable, multi-input network reveals a molecular framework that explains self-renewal, aberrant differentiation, and SCC growth in mice and humans, providing clues for developing differentiation-inducing therapeutic strategies.

A novel mouse model demonstrates that oncogenic melanocyte stem cells engender melanoma resembling human disease.

Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.

KLF4 as a rheostat of osteolysis and osteogenesis in prostate tumors in the bone.

We previously showed that KLF4, a gene highly expressed in murine prostate stem cells, blocks the progression of indolent intraepithelial prostatic lesions into aggressive and rapidly growing tumors. Here, we show that the anti-tumorigenic effect of KLF4 extends to PC3 human prostate cancer cells growing in the bone. We compared KLF4 null cells with cells transduced with a DOX-inducible KLF4 expression system, and find KLF4 function inhibits PC3 growth in monolayer and soft agar cultures. Furthermore, KLF4 null cells proliferate rapidly, forming large, invasive, and osteolytic tumors when injected into mouse femurs, whereas KLF4 re-expression immediately after their intra-femoral inoculation blocks tumor development and preserves a normal bone architecture. KLF4 re-expression in established KLF4 null bone tumors inhibits their osteolytic effects, preventing bone fractures and inducing an osteogenic response with new bone formation. In addition to these profound biological changes, KLF4 also induces a transcriptional shift from an osteolytic program in KLF4 null cells to an osteogenic program. Importantly, bioinformatic analysis shows that genes regulated by KLF4 overlap significantly with those expressed in metastatic prostate cancer patients and in three individual cohorts with bone metastases, strengthening the clinical relevance of the findings in our xenograft model.

Cellular quiescence: How TGFß protects cancer cells from chemotherapy.

Using a functional proliferation reporter we identified quiescent tumor propagating cancer cells (TPCs) in intact squamous cell carcinomas, and found that TGFß signaling controls their reversible entry into a growth arrested state, which protects TPCs from chemotherapy. TPCs with compromised TGFß/Smad signaling can't enter quiescence and subsequently die from chemotherapy.

Structural and Mechanistic Analysis of the Choline Sulfatase from Sinorhizobium melliloti: A Class I Sulfatase Specific for an Alkyl Sulfate Ester.

Hydrolysis of organic sulfate esters proceeds by two distinct mechanisms, water attacking at either sulfur (S-O bond cleavage) or carbon (C-O bond cleavage). In primary and secondary alkyl sulfates, attack at carbon is favored, whereas in aromatic sulfates and sulfated sugars, attack at sulfur is preferred. This mechanistic distinction is mirrored in the classification of enzymes that catalyze sulfate ester hydrolysis: arylsulfatases (ASs) catalyze S-O cleavage in sulfate sugars and arylsulfates, and alkyl sulfatases break the C-O bond of alkyl sulfates. Sinorhizobium meliloti choline sulfatase (SmCS) efficiently catalyzes the hydrolysis of alkyl sulfate choline-O-sulfate (kcat/KM=4.8×103s-1M-1) as well as arylsulfate 4-nitrophenyl sulfate (kcat/KM=12s-1M-1). Its 2.8-Å resolution X-ray structure shows a buried, largely hydrophobic active site in which a conserved glutamate (Glu386) plays a role in recognition of the quaternary ammonium group of the choline substrate. SmCS structurally resembles members of the alkaline phosphatase superfamily, being most closely related to dimeric ASs and tetrameric phosphonate monoester hydrolases. Although >70% of the amino acids between protomers align structurally (RMSDs 1.79-1.99Å), the oligomeric structures show distinctly different packing and protomer-protomer interfaces. The latter also play an important role in active site formation. Mutagenesis of the conserved active site residues typical for ASs, H218O-labeling studies and the observation of catalytically promiscuous behavior toward phosphoesters confirm the close relation to alkaline phosphatase superfamily members and suggest that SmCS is an AS that catalyzes S-O cleavage in alkyl sulfate esters with extreme catalytic proficiency.