NLRP1 in Cutaneous SCCs: An Example of the Complex Roles of Inflammasomes in Cancer Development.

Protein complexes termed inflammasomes ensure tissue protection from pathogenic and sterile stressors by induction of inflammation. This is mediated by different caspase-1-induced downstream pathways, including activation of the pro-inflammatory cytokines proIL-1ß and -18, induction of a lytic type of cell death, and regulation of the release of other pro-inflammatory molecules. Aberrant inflammasome activation underlies the pathology of numerous (auto)inflammatory diseases. Furthermore, inflammasomes support or suppress tumor development in a complex cell-type- and stage-dependent manner. In human keratinocytes and skin, NLRP1 is the central inflammasome sensor activated by cellular perturbation induced, for example, by UVB radiation. UVB represents the main inducer of skin cancer, which is the most common type of malignancy in humans. Recent evidence demonstrates that activation of NLRP1 in human skin supports the development of cutaneous squamous cell carcinomas (cSCCs) by inducing skin inflammation. In contrast, the NLRP1 inflammasome pathway is restrained in established cSCCs, suggesting that, at this stage, the protein complex has a tumor suppressor role. A better understanding of the complex functions of NLRP1 in the development of cSCCs and in general of inflammasomes in cancer might pave the way for novel strategies for cancer prevention and therapy. These strategies might include stage-specific modulation of inflammasome activation or its downstream pathways by mono- or combination therapy.

Failure to reabsorb the primary cilium induces cellular senescence.

Aurora kinase A (AURKA) is necessary for proper primary cilium disassembly before mitosis. We found that depletion of caveolin-1 expression promotes primary cilia formation through the proteasomal-dependent degradation of aurora kinase A and induces premature senescence in human fibroblasts. Down-regulation of intraflagellar transport-88, a protein essential for ciliogenesis, inhibits premature senescence induced by the depletion of caveolin-1. In support of these findings, we showed that alisertib, a pharmacological inhibitor of AURKA, causes primary cilia formation and cellular senescence by irreversibly arresting cell growth. Suppression of primary cilia formation limits cellular senescence induced by alisertib. The primary cilium must be disassembled to free its centriole to form the centrosome, a necessary structure for mitotic spindle assembly and cell division. We showed that the use of the centriole to form primary cilia blocks centrosome formation and mitotic spindle assembly and prevents the completion of mitosis in cells in which cellular senescence is caused by the inhibition of AURKA. We also found that AURKA is down-regulated and primary cilia formation is enhanced when cellular senescence is promoted by other senescence-inducing stimuli, such as oxidative stress and UV light. Thus, we propose that impaired AURKA function induces premature senescence by preventing reabsorption of the primary cilium, which inhibits centrosome and mitotic spindle formation and consequently prevents the completion of mitosis. Our study causally links the inability of the cell to disassemble the primary cilium, a microtubule-based cellular organelle, to the development of premature senescence, a functionally and pathologically relevant cellular state.-Jeffries, E. P., Di Filippo, M., Galbiati, F. Failure to reabsorb the primary cilium induces cellular senescence.

The NLRP1 Inflammasome in Human Skin and Beyond.

Inflammasomes represent a group of protein complexes that contribute to host defense against pathogens and repair processes upon the induction of inflammation. However, aberrant and chronic inflammasome activation underlies the pathology of numerous common inflammatory diseases. Inflammasome assembly causes activation of the protease caspase-1 which in turn activates proinflammatory cytokines and induces a lytic type of cell death termed pyroptosis. Although NLRP1 (NACHT, leucine-rich repeat and pyrin domain containing 1) was the first inflammasome sensor, described almost 20 years ago, the molecular mechanisms underlying its activation and the resulting downstream events are incompletely understood. This is partially a consequence of the poor conservation of the NLRP1 pathway between human and mice. Moreover, recent evidence demonstrates a complex and multi-stage mechanism of NLRP1 inflammasome activation. In contrast to other inflammasome sensors, NLRP1 possesses protease activity required for proteolytic self-cleavage and activation mediated by the function-to-find domain (FIIND). CARD8 is a second FIIND protein and is expressed in humans but not in mice. In immune cells and AML (acute myeloid leukemia) cells, the anti-cancer drug talabostat induces CARD8 activation and causes caspase-1-dependent pyroptosis. In contrast, in human keratinocytes talabostat induces NLRP1 activation and massive proinflammatory cytokine activation. NLRP1 is regarded as the principal inflammasome sensor in human keratinocytes and UVB radiation induces its activation, which is believed to underlie the induction of sunburn. Moreover, gain-of-function mutations of NLRP1 cause inflammatory skin syndromes and a predisposition for the development of skin cancer. SNPs (single nucleotide polymorphisms) of NLRP1 are associated with several (auto)inflammatory diseases with a major skin phenotype, such as psoriasis or vitiligo. Here, we summarize knowledge about NLRP1 with emphasis on its role in human keratinocytes and skin. Due to its accessibility, pharmacological targeting of NLRP1 activation in epidermal keratinocytes represents a promising strategy for the treatment of the numerous patients suffering from NLRP1-dependent inflammatory skin conditions and cancer.

The Crosstalk between Nrf2 and Inflammasomes.

The Nrf2 (nuclear factor E2-related factor or nuclear factor (erythroid-derived 2)-like 2) transcription factor is a key player in cytoprotection and activated in stress conditions caused by reactive oxygen species (ROS) or electrophiles. Inflammasomes represent central regulators of inflammation. Upon detection of various stress factors, assembly of the inflamasome protein complex results in activation and secretion of proinflammatory cytokines. In addition, inflammasome activation causes pyroptosis, a lytic form of cell death, which supports inflammation. There is growing evidence of a crosstalk between the Nrf2 and inflammasome pathways at different levels. For example, Nrf2 activating compounds inhibit inflammasomes and consequently inflammation. This review summarizes what is known about the complex and predominantly antagonistic relationship of both stress-activated pathways.

Efficient Generation of CRISPR/Cas9-Mediated Knockout Human Primary Keratinocytes by Electroporation.

Due to their full differentiation capacity in vitro, the culture of human primary keratinocytes (HPKs) represents a physiological model for answering basic biological and dermatological research questions, including those related to skin diseases and the investigation of treatment options. When modified with the CRISPR/Cas9 gene editing approach and cultivated in organotypic 3D epidermal equivalents (EEs), these human cells have the potential to replace established mouse models. However, even when cultivated on feeder cells, HPKs have only a low proliferation capacity in 2D culture, limiting their application potential. This is particularly true for CRISPR/Cas9-modified HPKs, whose generation commonly requires selection of targeted cells, negatively affecting their lifespan. Here, we describe a robust protocol for the rapid, simple, and efficient generation of single- and multi-gene CRISPR/Cas9 knockout HPKs by electroporation of ribonucleoprotein (RNP) complexes, which comprise one or multiple guide RNAs (gRNAs) and Cas9 protein. Unlike DNA transfection or virus-based targeting strategies, electroporation of RNPs represents a targeting approach that minimizes immunological and toxic side effects. Using efficient gRNAs results in the generation of HPKs with a high yield of knockout cells, allowing for their immediate use in experiments without requiring the laborious process of selecting targeted cells or maintaining a feeder cell culture. Furthermore, the use of RNPs and their delivery via electroporation minimizes off-target and other unspecific effects, preventing unintended genomic alterations. Most importantly, CRISPR/Cas9 knockout HPKs generated with this protocol have the ability to form a fully differentiated epidermis in 3D, thus facilitating the understanding of specific protein functions in a highly physiological human skin model. Alternatively, this approach proves valuable for generating models of mono- or polygenic skin diseases via knockouts, providing insights into the underlying molecular mechanisms and facilitating the development of novel therapeutic approaches.

CARD8: A Novel Inflammasome Sensor with Well-Known Anti-Inflammatory and Anti-Apoptotic Activity.

Inflammasomes comprise a group of protein complexes with fundamental roles in the induction of inflammation. Upon sensing stress factors, their assembly induces the activation and release of the pro-inflammatory cytokines interleukin (IL)-1ß and -18 and a lytic type of cell death, termed pyroptosis. Recently, CARD8 has joined the group of inflammasome sensors. The carboxy-terminal part of CARD8, consisting of a function-to-find-domain (FIIND) and a caspase activation and recruitment domain (CARD), resembles that of NLR family pyrin domain containing 1 (NLRP1), which is recognized as the main inflammasome sensor in human keratinocytes. The interaction with dipeptidyl peptidases 8 and 9 (DPP8/9) represents an activation checkpoint for both sensors. CARD8 and NLRP1 are activated by viral protease activity targeting their amino-terminal region. However, CARD8 also has some unique features compared to the established inflammasome sensors. Activation of CARD8 occurs independently of the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), leading mainly to pyroptosis rather than the activation and secretion of pro-inflammatory cytokines. CARD8 was also shown to have anti-inflammatory and anti-apoptotic activity. It interacts with, and inhibits, several proteins involved in inflammation and cell death, such as the inflammasome sensor NLRP3, CARD-containing proteins caspase-1 and -9, nucleotide-binding oligomerization domain containing 2 (NOD2), or nuclear factor kappa B (NF-κB). Single nucleotide polymorphisms (SNPs) of CARD8, some of them occurring at high frequencies, are associated with various inflammatory diseases. The molecular mechanisms underlying the different pro- and anti-inflammatory activities of CARD8 are incompletely understood. Alternative splicing leads to the generation of multiple CARD8 protein isoforms. Although the functional properties of these isoforms are poorly characterized, there is evidence that suggests isoform-specific roles. The characterization of the functions of these isoforms, together with their cell- and disease-specific expression, might be the key to a better understanding of CARD8's different roles in inflammation and inflammatory diseases.

The mitochondrial DNA common deletion as a potential biomarker of cancer-associated fibroblasts from skin basal and squamous cell carcinomas.

Cancer-associated fibroblasts (CAFs) are components of the tumor microenvironment and represent appealing therapeutic targets for translational studies. Conventional protein-based biomarkers for CAFs have been reported to be limited in their specificity, rendering difficult the identification of CAFs from normal fibroblasts (NFs) in clinical samples and dampening the development of CAF-targeted therapies to treat cancer. In this study, we propose the mitochondrial RNA and the mitochondrial DNA (mtDNA) common deletion (CD) as novel indicators of CAF identity. We found that cancer-activation correlated with decreased levels of the mtDNA CD, a condition not due to altered mitochondria count or cellular redox state, but potentially linked to the generalized overexpression of mtDNA maintenance genes in CAFs. Decreased mtDNA CD content in CAFs was associated with moderate to strong overexpression of mtDNA-encoded genes and to slightly improved mitochondrial function. We identified similar patterns of upregulation of mtDNA-encoded genes in independent single-cell RNA seq data obtained from squamous cell carcinoma (SCC) patients. By using the identified nucleic acids-based indicators, identification of CAFs from NFs could be improved, leading to potential therapeutic benefits in advancing translational and clinical studies.

The gasdermins: a pore-forming protein family expressed in the epidermis.

Gasdermins comprise a family of pore-forming proteins, which play critical roles in (auto)inflammatory diseases and cancer. They are expressed as self-inhibited precursor proteins consisting of an aminoterminal cytotoxic effector domain (NT-GSDM) and a carboxyterminal inhibitor domain (GSDM-CT) separated by an unstructured linker region. Proteolytic processing in the linker region liberates NT-GSDM, which translocates to membranes, forms oligomers, and induces membrane permeabilization, which can disturb the cellular equilibrium that can lead to cell death. Gasdermin activation and pore formation are associated with inflammation, particularly when induced by the inflammatory protease caspase-1 upon inflammasome activation. These gasdermin pores allow the release of the pro-inflammatory cytokines interleukin(IL)-1ß and IL-18 and induce a lytic type of cell death, termed pyroptosis that supports inflammation, immunity, and tissue repair. However, even at the cellular level, the consequences of gasdermin activation are diverse and range from induction of programmed cell death - pyroptosis or apoptosis - to poorly characterized protective mechanisms. The specific effects of gasdermin activation can vary between species, cell types, the membrane that is being permeabilized (plasma membrane, mitochondrial membrane, etc.), and the overall biological state of the local tissue/cells. In epithelia, gasdermins seem to play crucial roles. Keratinocytes represent the main cell type of the epidermis, which is the outermost skin layer with an essential barrier function. Compared to other tissues, keratinocytes express all members of the gasdermin family, in part in a differentiation-specific manner. That raises questions regarding the specific roles of individual GSDM family members in the skin, the mechanisms and consequences of their activation, and the potential crosstalk between them. In this review, we summarize the current knowledge about gasdermins with a focus on keratinocytes and the skin and discuss the possible roles of the different family members in immunity and disease.

Abdominal rhabdoid tumor presenting with symptomatic spinal epidural compression in a newborn. A case report.

The occurrence of an abdominal tumor invading the spinal canal and causing symptoms of epidural compression is rare in an infant, and exceptional at birth. Peripheral neuroblastic tumors are by far the most common cause. Emergency chemotherapy is commonly curative, though permanent sequelae are possible. Although other malignancies may be involved, no case of rhabdoid tumors at birth has been reported. We describe the case of a neonate who presented symptoms of spinal epidural compression at birth secondary to a rhabdoid tumor. As expected with this highly malignant tumor, the patient experienced a rapidly progressive clinical course and died within three months of diagnosis.

High p62 expression suppresses the NLRP1 inflammasome and increases stress resistance in cutaneous SCC cells.

NLRP1 is the primary inflammasome sensor in human keratinocytes. Sensing of UVB radiation by NLRP1 is believed to underlie the induction of sunburn. Although constitutive NLRP1 activation causes skin inflammation and predisposes patients to the development of cutaneous SCCs, the NLRP1 pathway is suppressed in established SCCs. Here, we identified high levels of the autophagy receptor p62 in SCC cells lines and SCC tumors. Increased NF-κB activity in SCC cells causes p62 up-regulation. Suppression of p62 expression rescues UVB-induced NLRP1 inflammasome activation in early-stage SCC cells. p62 expression protects SCC cells from cytotoxic drugs, whereas NLRP1 sensitizes them. In summary, we identify p62 as a novel negative regulator of the NLRP1 inflammasome in human cutaneous SCC cells, in which suppression of NLRP1 by increased levels of p62 supports stress resistance of skin cancer cells.

NLRP1 Inflammasome Activation in Keratinocytes: Increasing Evidence of Important Roles in Inflammatory Skin Diseases and Immunity.

In 2007, it was shown that DNA sequence variants of the human NLRP1 gene are associated with autoimmune and autoinflammatory diseases affecting mainly the skin. However, at that time, the underlying cellular and molecular mechanisms were poorly characterized. Meanwhile, increasing evidence suggests that the NLRP1 inflammasome expressed by keratinocytes not only plays a part in the pathology of common inflammatory skin diseases and cancer development but also contributes to skin immunity. Understanding the mechanisms regulating NLRP1 activation in keratinocytes and the downstream events in human skin might pave the way for developing novel strategies for treating patients suffering from NLRP1-mediated skin diseases.

A Protumorigenic mDia2-MIRO1 Axis Controls Mitochondrial Positioning and Function in Cancer-Associated Fibroblasts.

Cancer-associated fibroblasts (CAF) are key regulators of tumorigenesis. Further insights into the tumor-promoting mechanisms of action of CAFs could help improve cancer diagnosis and treatment. Here we show that the formin mDia2 regulates the positioning and function of mitochondria in dermal fibroblasts, thereby promoting a protumorigenic CAF phenotype. Mechanistically, mDia2 stabilized the mitochondrial trafficking protein MIRO1. Loss of mDia2 or MIRO1 in fibroblasts or CAFs reduced the presence of mitochondria and ATP levels near the plasma membrane and at CAF-tumor cell contact sites, caused metabolic alterations characteristic of mitochondrial dysfunction, and suppressed the secretion of protumorigenic proteins. In mouse models of squamous carcinogenesis, genetic or pharmacologic inhibition of mDia2, MIRO1, or their common upstream regulator activin A inhibited tumor formation. Consistently, co-upregulation of mDia2 and MIRO1 in the stroma of various human cancers negatively correlated with survival. This work unveils a key role of mitochondria in the protumorigenic CAF phenotype and identifies an activin A-mDia2-MIRO1 signaling axis in CAFs with diagnostic and therapeutic potential. SIGNIFICANCE: Inhibition of mDia2/MIRO1-mediated mitochondrial positioning in CAFs induces mitochondrial dysfunction and suppresses tumor growth, revealing a promising therapeutic strategy to target tumor-stroma cross-talk.

The Pathways Underlying the Multiple Roles of p62 in Inflammation and Cancer.

p62 is a highly conserved, multi-domain, and multi-functional adaptor protein critically involved in several important cellular processes. Via its pronounced domain architecture, p62 binds to numerous interaction partners, thereby influencing key pathways that regulate tissue homeostasis, inflammation, and several common diseases including cancer. Via binding of ubiquitin chains, p62 acts in an anti-inflammatory manner as an adaptor for the auto-, xeno-, and mitophagy-dependent degradation of proteins, pathogens, and mitochondria. Furthermore, p62 is a negative regulator of inflammasome complexes. The transcription factor Nrf2 regulates expression of a bundle of ROS detoxifying genes. p62 activates Nrf2 by interaction with and autophagosomal degradation of the Nrf2 inhibitor Keap1. Moreover, p62 activates mTOR, the central kinase of the mTORC1 sensor complex that controls cell proliferation and differentiation. Through different mechanisms, p62 acts as a positive regulator of the transcription factor NF-κB, a central player in inflammation and cancer development. Therefore, p62 represents not only a cargo receptor for autophagy, but also a central signaling hub, linking several important pro- and anti-inflammatory pathways. This review aims to summarize knowledge about the molecular mechanisms underlying the roles of p62 in health and disease. In particular, different types of tumors are characterized by deregulated levels of p62. The elucidation of how p62 contributes to inflammation and cancer progression at the molecular level might promote the development of novel therapeutic strategies.

Electrophiles Against (Skin) Diseases: More Than Nrf2.

The skin represents an indispensable barrier between the organism and the environment and is the first line of defense against exogenous insults. The transcription factor NRF2 is a central regulator of cytoprotection and stress resistance. NRF2 is activated in response to oxidative stress by reactive oxygen species (ROS) and electrophiles. These electrophiles oxidize specific cysteine residues of the NRF2 inhibitor KEAP1, leading to KEAP1 inactivation and, subsequently, NRF2 activation. As oxidative stress is associated with inflammation, the NRF2 pathway plays important roles in the pathogenesis of common inflammatory diseases and cancer in many tissues and organs, including the skin. The electrophile and NRF2 activator dimethyl fumarate (DMF) is an established and efficient drug for patients suffering from the common inflammatory skin disease psoriasis and the neuro-inflammatory disease multiple sclerosis (MS). In this review, we discuss possible molecular mechanisms underlying the therapeutic activity of DMF and other NRF2 activators. Recent evidence suggests that electrophiles not only activate NRF2, but also target other inflammation-associated pathways including the transcription factor NF-κB and the multi-protein complexes termed inflammasomes. Inflammasomes are central regulators of inflammation and are involved in many inflammatory conditions. Most importantly, the NRF2 and inflammasome pathways are connected at different levels, mainly antagonistically.

Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9.

The culture of epidermal human primary keratinocytes (HPKs) represents a well-established model in biological and dermatological research. In addition, HPKs are used in three-dimensional organotypic cultures (OTCs), and gene therapeutic approaches have been reported for the treatment of patients suffering from epidermolysis bullosa, a severe blistering disease that can result in postnatal lethality. Therefore, there is a strong need for the development of techniques for the stable and specific genetic manipulation of HPKs, for example, by genome editing via the CRISPR/Cas9 approach. However, the main disadvantage of working with HPKs is the fact that these cells are prone to terminal differentiation and proliferate only for few passages in monoculture. As it is well known that the co-culture of HPKs with fibroblasts strongly increases the lifetime of the epidermal cells, we developed a protocol for the stable modification of HPKs by CRISPR/Cas9 via lentiviral transduction in the presence of 3T3-J2 fibroblasts as feeder cells. Selection of transduced HPKs is achieved with antibiotics in co-culture with antibiotic-resistant feeder cells. Modified HPKs generated by our protocol have the potential to generate epidermis-like structures in OTCs.

Inactivation of the Cytoprotective Major Vault Protein by Caspase-1 and -9 in Epithelial Cells during Apoptosis.

Inflammasome activation induces caspase-1-dependent secretion of the proinflammatory cytokine IL-1ß. In addition, caspase-1 activates the protein GSDMD in immune cells, causing pyroptosis, a lytic type of cell death. In contrast, UVB irradiation of human primary keratinocytes induces NLRP1 inflammasome activation, cytokine secretion, and caspase-1-dependent apoptosis, rather than pyroptosis. Here, we addressed the molecular mechanisms underlying the role of caspase-1 in UVB-induced cell death of human primary keratinocytes. We show that GSDMD is a poor substrate of caspase-1 in human primary keratinocytes and that its activation upon UVB irradiation supports secretion of IL-1ß. We screened for novel substrates of caspase-1 by a mass spectrometry-based approach and identified the specific cleavage of the major vault protein (MVP) at D441 by caspase-1 and -9. MVP is the main component of vaults, highly conserved ribonucleoprotein particles, whose functions are poorly understood. Cleavage of MVP is a common event occurring in human primary keratinocytes and fibroblasts undergoing apoptosis induced by different stimuli. In contrast, MVP cleavage could not be detected in pyroptotic cells. Cleavage of MVP by caspase-1 and -9 inactivates this cytoprotective protein. These results demonstrate a proapoptotic activity of caspase-1 and a crosstalk with caspase-9 upon inactivation of the cytoprotective MVP in apoptotic epithelial cells.

The NLRP1 Inflammasome Pathway Is Silenced in Cutaneous Squamous Cell Carcinoma.

The inflammasome protein NLRP1 is an important innate immune sensor in human keratinocytes, and, together with ASC and caspase-1, it mediates the activation and secretion of the proinflammatory cytokines IL-1ß and IL-18. These cytokines and inflammasomes can have partly opposing roles during tumorigenesis in mice. In contrast, ASC expression is impaired in different types of cancer in humans. In this study, we analyzed inflammasome activation and expression of inflammasome proteins, including their downstream cytokines, in squamous cell carcinomas, a type of nonmelanoma skin cancer derived from keratinocytes. We assessed mRNA and protein levels in human primary keratinocytes and skin carcinoma-derived SCC cell lines and detected a strong down-regulation of expression of NLRP1 inflammasome components, as well as reduced expression of the proinflammatory cytokines proIL-1ß and proIL-1α. Protein levels of NLRP1, ASC, caspase-1, and proIL-1ß were reduced in patient-derived SCC biopsy samples compared with healthy skin. Furthermore, the results suggest that expression of PYCARD (ASC), CASP1, IL1B, and NLRP1 is silenced by methylation in SCC cell lines. In conclusion, the down-regulation of the inflammasome pathway in SCCs might favor late tumor development in human skin.