TGF-ß promotes heterogeneity and drug resistance in squamous cell carcinoma.

Subsets of long-lived, tumor-initiating stem cells often escape cancer therapies. However, sources and mechanisms that generate tumor heterogeneity and drug-resistant cell population are still unfolding. Here, we devise a functional reporter system to lineage trace and/or genetic ablate signaling in TGF-ß-activated squamous cell carcinoma stem cells (SCC-SCs). Dissecting TGF-ß's impact on malignant progression, we demonstrate that TGF-ß concentrating near tumor-vasculature generates heterogeneity in TGF-ß signaling at tumor-stroma interface and bestows slower-cycling properties to neighboring SCC-SCs. While non-responding progenies proliferate faster and accelerate tumor growth, TGF-ß-responding progenies invade, aberrantly differentiate, and affect gene expression. Intriguingly, TGF-ß-responding SCC-SCs show increased protection against anti-cancer drugs, but slower-cycling alone does not confer survival. Rather, TGF-ß transcriptionally activates p21, which stabilizes NRF2, thereby markedly enhancing glutathione metabolism and diminishing effectiveness of anti-cancer therapeutics. Together, these findings establish a surprising non-genetic paradigm for TGF-ß signaling in fueling heterogeneity in SCC-SCs, tumor characteristics, and drug resistance.

Skin stem cells orchestrate directional migration by regulating microtubule-ACF7 connections through GSK3ß.

Homeostasis and wound healing rely on stem cells (SCs) whose activity and directed migration are often governed by Wnt signaling. In dissecting how this pathway integrates with the necessary downstream cytoskeletal dynamics, we discovered that GSK3ß, a kinase inhibited by Wnt signaling, directly phosphorylates ACF7, a > 500 kDa microtubule-actin crosslinking protein abundant in hair follicle stem cells (HF-SCs). We map ACF7's GSK3ß sites to the microtubule-binding domain and show that phosphorylation uncouples ACF7 from microtubules. Phosphorylation-refractile ACF7 rescues overall microtubule architecture, but phosphorylation-constitutive mutants do not. Neither mutant rescues polarized movement, revealing that phospho-regulation must be dynamic. This circuitry is physiologically relevant and depends upon polarized GSK3ß inhibition at the migrating front of SCs/progeny streaming from HFs during wound repair. Moreover, only ACF7 and not GSKß-refractile-ACF7 restore polarized microtubule-growth and SC-migration to ACF7 null skin. Our findings provide insights into how this conserved spectraplakin integrates signaling, cytoskeletal dynamics, and polarized locomotion of somatic SCs.

Pioneer factors govern super-enhancer dynamics in stem cell plasticity and lineage choice.

Adult stem cells occur in niches that balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, stem cells outside their niche often display fate flexibility. Here we show that super-enhancers underlie the identity, lineage commitment and plasticity of adult stem cells in vivo. Using hair follicle as a model, we map the global chromatin domains of hair follicle stem cells and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters ('epicentres') of transcription factor binding sites undergo remodelling upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicentres, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, hair follicle stem cells dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicentres, enabling their genes to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of hair follicle stem cell super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense transcription-factor-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status but also stemness, plasticity in transitional states and differentiation.

Deletion of Adam6 in Mus musculus leads to male subfertility and deficits in sperm ascent into the oviduct.

The Adisintegrin and metalloprotease domain-containing (ADAM) family of proteins is involved in cell adhesion, migration, proteolysis, and signaling. Many ADAMs are required for reproduction; however, the role of Adam6 has remained largely unknown. In the course of humanizing the mouse immunoglobulin heavy chain (IgH) locus, we generated Adam6-deficient mice that demonstrate severe subfertility. We decided to elucidate the role of ADAM6 in fertility and explore the underlying mechanisms. Despite normal sperm development and motility, Adam6-deficient mice display diminished male fertility, have abnormal sperm adhesion, and most importantly cannot transition from uterus to oviduct. To test whether ADAM6 is required for sperm's binding to extracellular matrix (ECM) components, we used a panel of ECM components and showed that unlike normal sperm, Adam6-deficient sperm cannot bind fibronectin, laminin, and tenascin. Reintroduction of Adam6 into these deficient mice repaired sperm interaction with ECM, restored male fertility, and corrected the sperm transport deficit. Together, our data suggest that ADAM6, either alone or in complex with other proteins, aids sperm transport through the female reproductive tract by providing a temporary site of attachment of sperm to ECM components prior to ascent into the oviduct.

Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor of squamous cell carcinomas.

Mining modern genomics for cancer therapies is predicated on weeding out "bystander" alterations (nonconsequential mutations) and identifying "driver" mutations responsible for tumorigenesis and/or metastasis. We used a direct in vivo RNA interference (RNAi) strategy to screen for genes that upon repression predispose mice to squamous cell carcinomas (SCCs). Seven of our top hits-including Myh9, which encodes nonmuscle myosin IIa-have not been linked to tumor development, yet tissue-specific Myh9 RNAi and Myh9 knockout trigger invasive SCC formation on tumor-susceptible backgrounds. In human and mouse keratinocytes, myosin IIa's function is manifested not only in conventional actin-related processes but also in regulating posttranscriptional p53 stabilization. Myosin IIa is diminished in human SCCs with poor survival, which suggests that in vivo RNAi technology might be useful for identifying potent but low-penetrance tumor suppressors.

Mitotic internalization of planar cell polarity proteins preserves tissue polarity.

Planar cell polarity (PCP) is the collective polarization of cells along the epithelial plane, a process best understood in the terminally differentiated Drosophila wing. Proliferative tissues such as mammalian skin also show PCP, but the mechanisms that preserve tissue polarity during proliferation are not understood. During mitosis, asymmetrically distributed PCP components risk mislocalization or unequal inheritance, which could have profound consequences for the long-range propagation of polarity. Here, we show that when mouse epidermal basal progenitors divide PCP components are selectively internalized into endosomes, which are inherited equally by daughter cells. Following mitosis, PCP proteins are recycled to the cell surface, where asymmetry is re-established by a process reliant on neighbouring PCP. A cytoplasmic dileucine motif governs mitotic internalization of atypical cadherin Celsr1, which recruits Vang2 and Fzd6 to endosomes. Moreover, embryos transgenic for a Celsr1 that cannot mitotically internalize exhibit perturbed hair-follicle angling, a hallmark of defective PCP. This underscores the physiological relevance and importance of this mechanism for regulating polarity during cell division.

Ribosomal protein L29/HIP deficiency delays osteogenesis and increases fragility of adult bone in mice.

Mice lacking HIP/RPL29, a ribosomal modulator of protein synthesis rate, display a short stature phenotype. To understand the contribution of HIP/RPL29 to bone formation and adult whole bone mechanical properties, we examined both developing and adult bone in our knockout mice. Results indicated that bone shortening in HIP/RPL29-null mice is due to delayed entry of chondro-osteoprogenitors into the cell cycle. Structural properties of adult null bones were analyzed by micro-computed tomography. Interestingly, partial preservation of cortical thickness was observed in null males indicating a gender-specific effect of the genotype on cortical bone parameters. Null males, and to a lower extent null females, displayed increased bone material toughness to counteract decreased bone size. This elevation in a bone material property was associated with increased bone mineral density only in null males. Neither male nor female null animals could withstand the same maximum load as gender-matched controls in three-point bending tests, and smaller post-yield displacements (and thus increased bone brittleness) were found for null animals. These results suggest that HIP/RPL29-deficient mice exhibit increased bone fragility due to altered matrix protein synthesis rates as a consequence of ribosomal insufficiency. Thus, sub-efficient protein translation increased fracture risk in HIP/RPL29-null animals. Taken together, these studies provide strong genetic evidence that the ability to regulate and amplify protein synthesis rates, including those proteins that regulate the cell cycle entry during skeletal development, are important determinants for establishment of normal bone mass and quality.

Global growth deficiencies in mice lacking the ribosomal protein HIP/RPL29.

Because of their deleterious effects on developing organisms, ribosomal protein (RP) mutations have been poorly described in mammals, and only a few heterozygous mutations have been shown to be viable. This observation is believed to be due to the fact that each RP is an essential component in the assembly of a functional stable ribosome. Here, we created gene targeted mutant mice lacking HIP/RPL29, an RP associated with translationally active ribosomes in eukaryotes. In contrast to other RP mutants, HIP/RPL29 null mice are viable but are up to 50% smaller than their control littermates at weaning age. In null embryos, delayed global growth is first observed around mid-gestation, and postnatal lethality due to low birth weight results in distortion of the Mendelian ratio. Prenatal growth defects are not fully compensated for during adulthood, and null animals display proportionately smaller organs and stature, and reach sexual maturity considerably later when compared with their control siblings. Additionally, HIP/RPL29 null embryonic fibroblasts have decreased rates of proliferation and protein synthesis and exhibit reduced steady state levels of core RPs. Altogether, our findings provide conclusive genetic evidence that HIP/RPL29 functions as an important regulator of global growth by modulating the rate of protein synthesis.