Opposing regulation of the STING pathway in hepatic stellate cells by NBR1 and p62 determines the progression of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) emerges from chronic inflammation, to which activation of hepatic stellate cells (HSCs) contributes by shaping a pro-tumorigenic microenvironment. Key to this process is p62, whose inactivation leads to enhanced hepatocarcinogenesis. Here, we show that p62 activates the interferon (IFN) cascade by promoting STING ubiquitination by tripartite motif protein 32 (TRIM32) in HSCs. p62, binding neighbor of BRCA1 gene 1 (NBR1) and STING, triggers the IFN cascade by displacing NBR1, which normally prevents the interaction of TRIM32 with STING and its subsequent activation. Furthermore, NBR1 also antagonizes STING by promoting its trafficking to the endosome-lysosomal compartment for degradation independent of autophagy. Of functional relevance, NBR1 deletion completely reverts the tumor-promoting function of p62-deficient HSCs by rescuing the inhibited STING-IFN pathway, thus enhancing anti-tumor responses mediated by CD8+ T cells. Therefore, NBR1 emerges as a synthetic vulnerability of p62 deficiency in HSCs by promoting the STING/IFN pathway, which boosts anti-tumor CD8+ T cell responses to restrain HCC progression.

PKCλ/ι inhibition activates an ULK2-mediated interferon response to repress tumorigenesis.

The interferon (IFN) pathway is critical for cytotoxic T cell activation, which is central to tumor immunosurveillance and successful immunotherapy. We demonstrate here that PKCλ/ι inactivation results in the hyper-stimulation of the IFN cascade and the enhanced recruitment of CD8+ T cells that impaired the growth of intestinal tumors. PKCλ/ι directly phosphorylates and represses the activity of ULK2, promoting its degradation through an endosomal microautophagy-driven ubiquitin-dependent mechanism. Loss of PKCλ/ι results in increased levels of enzymatically active ULK2, which, by direct phosphorylation, activates TBK1 to foster the activation of the STING-mediated IFN response. PKCλ/ι inactivation also triggers autophagy, which prevents STING degradation by chaperone-mediated autophagy. Thus, PKCλ/ι is a hub regulating the IFN pathway and three autophagic mechanisms that serve to maintain its homeostatic control. Importantly, single-cell multiplex imaging and bioinformatics analysis demonstrated that low PKCλ/ι levels correlate with enhanced IFN signaling and good prognosis in colorectal cancer patients.

Control of nutrient stress-induced metabolic reprogramming by PKCζ in tumorigenesis.

Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.

Simultaneous Loss of Both Atypical Protein Kinase C Genes in the Intestinal Epithelium Drives Serrated Intestinal Cancer by Impairing Immunosurveillance.

Serrated adenocarcinoma, an alternative pathway for colorectal cancer (CRC) development, accounts for 15%-30% of all CRCs and is aggressive and treatment resistant. We show that the expression of atypical protein kinase C ζ (PKCζ) and PKCλ/ι was reduced in human serrated tumors. Simultaneous inactivation of the encoding genes in the mouse intestinal epithelium resulted in spontaneous serrated tumorigenesis that progressed to advanced cancer with a strongly reactive and immunosuppressive stroma. Whereas epithelial PKCλ/ι deficiency led to immunogenic cell death and the infiltration of CD8+ T cells, which repressed tumor initiation, PKCζ loss impaired interferon and CD8+ T cell responses, which resulted in tumorigenesis. Combined treatment with a TGF-ß receptor inhibitor plus anti-PD-L1 checkpoint blockade showed synergistic curative activity. Analysis of human samples supported the relevance of these kinases in the immunosurveillance defects of human serrated CRC. These findings provide insight into avenues for the detection and treatment of this poor-prognosis subtype of CRC.

K63 polyubiquitination and activation of mTOR by the p62-TRAF6 complex in nutrient-activated cells.

The ability of cells to respond to changes in nutrient availability is critical for an adequate control of metabolic homeostasis. Mammalian target of rapamycin complex 1 (mTORC1) is a central complex kinase in these processes. The signaling adaptor p62 binds raptor, and integral component of the mTORC1 pathway. p62 interacts with TNF receptor associated factor 6 (TRAF6) and is required for mTORC1 translocation to the lysosome and its subsequent activation. Here we show that TRAF6 is recruited to and activates mTORC1 through p62 in amino acid-stimulated cells. We also show that TRAF6 is necessary for the translocation of mTORC1 to the lysosomes and that the TRAF6-catalyzed K63 ubiquitination of mTOR regulates mTORC1 activation by amino acids. TRAF6, through its interaction with p62 and activation of mTORC1, modulates autophagy and is an important mediator in cancer cell proliferation. Interfering with the p62-TRAF6 interaction serves to modulate autophagy and nutrient sensing.

NRF2 activates growth factor genes and downstream AKT signaling to induce mouse and human hepatomegaly.

BACKGROUND & AIMS: Hepatomegaly can be triggered by insulin and insulin-unrelated etiologies. Insulin acts via AKT, but how other challenges cause hepatomegaly is unknown. METHODS: Since many hepatomegaly-inducing toxicants and stressors activate NRF2, we examined the effect of NRF2 activation on liver size and metabolism using a conditional allele encoding a constitutively active NRF2 variant to generate Nrf2Act-hep mice in which NRF2 is selectively activated in hepatocytes. We also used adenoviruses encoding variants of the autophagy adaptor p62/SQSTM1, which activates liver NRF2, as well as liver-specific ATG7-deficient mice (Atg7Δhep) and liver specimens from patients with hepatic sinusoidal obstruction syndrome (HSOS) and autoimmune hepatitis (AIH). RNA sequencing and cell signaling analyses were used to determine cellular consequences of NRF2 activation and diverse histological analyses were used to study effects of the different manipulations on liver and systemic pathophysiology. RESULTS: Hepatocyte-specific NRF2 activation, due to p62 accumulation or inhibition of KEAP1 binding, led to hepatomegaly associated with enhanced glycogenosis, steatosis and G2/M cell cycle arrest, fostering hyperplasia without cell division. Surprisingly, all manipulations that led to NRF2 activation also activated AKT, whose inhibition blocked NRF2-induced hepatomegaly and glycogenosis, but not NRF2-dependent antioxidant gene induction. AKT activation was linked to NRF2-mediated transcriptional induction of PDGF and EGF receptor ligands that signaled through their cognate receptors in an autocrine manner. Insulin and insulin-like growth factors were not involved. The NRF2-AKT signaling axis was also activated in human HSOS- and AIH-related hepatomegaly. CONCLUSIONS: NRF2, a transcription factor readily activated by xenobiotics, oxidative stress and autophagy disruptors, may be a common mediator of hepatomegaly; its effects on hepatic metabolism can be reversed by AKT/tyrosine kinase inhibitors. LAY SUMMARY: Hepatomegaly can be triggered by numerous etiological factors, including infections, liver cancer, metabolic disturbances, toxicant exposure, as well as alcohol abuse or drug-induced hepatitis. This study identified the oxidative stress response transcription factor NRF2 as a common mediator of hepatomegaly. NRF2 activation results in elevated expression of several growth factors. These growth factors are made by hepatocytes and activate their receptors in an autocrine fashion to stimulate the accumulation of glycogen and lipids that lead to hepatocyte and liver enlargement. The protein kinase AKT plays a key role in this process and its inhibition leads to reversal of hepatomegaly.

p62 is a key regulator of nutrient sensing in the mTORC1 pathway.

The signaling adaptor p62 is a critical mediator of important cellular functions, owing to its ability to establish interactions with various signaling intermediaries. Here, we identify raptor as an interacting partner of p62. Thus, p62 is an integral part of the mTORC1 complex and is necessary to mediate amino acid signaling for the activation of S6K1 and 4EBP1. p62 interacts in an amino acid-dependent manner with mTOR and raptor. In addition, p62 binds the Rags proteins and favors formation of the active Rag heterodimer that is further stabilized by raptor. Interestingly, p62 colocalizes with Rags at the lysosomal compartment and is required for the interaction of mTOR with Rag GTPases in vivo and for translocation of the mTORC1 complex to the lysosome, a crucial step for mTOR activation.

Assessing eco-efficiency and the determinants of horticultural family-farming in southeast Spain.

Eco-efficiency is currently receiving ever increasing interest as an indicator of sustainability, as it links environmental and economic performances in productive activities. In agriculture these indicators and their determinants prove relevant due to the close ties in this activity between the use of often limited natural resources and the provision of basic goods for society. The present paper analyzes eco-efficiency at micro-level, focusing on small-scale family farms as the principal decision-making units (DMUs) of horticulture in southeast Spain, which represents over 30% of fresh vegetables produced in the country. To this end, Data Envelopment Analysis (DEA) framework is applied, computing several combinations of environmental pressures (water usage, phytosanitary contamination, waste management, etc.) and economic value added. In a second stage we analyze the influence of family farms' socio-economic and environmental features on eco-efficiency indicators, as endogenous variables, by using truncated regression and bootstrapping techniques. The results show major inefficiency in aspects such as waste management, among others, while there is relatively minor inefficiency in water usage and nitrogen balance. On the other hand, features such as product specialization, adoption of quality certifications, and belonging to a cooperative all have a positive influence on eco-efficiency. These results are deemed to be of interest to agri-food systems structured on small-scale producers, and they may prove useful to policy-makers as regards managing public environmental programs in agriculture.

Atypical protein kinase C (aPKCzeta and aPKClambda) is dispensable for mammalian hematopoietic stem cell activity and blood formation.

The stem-cell pool is considered to be maintained by a balance between symmetric and asymmetric division of stem cells. The cell polarity model proposes that the facultative use of symmetric and asymmetric cell division is orchestrated by a polarity complex consisting of partitioning-defective proteins Par3 and Par6, and atypical protein kinase C (aPKCζ and aPKCλ), which regulates planar symmetry of dividing stem cells with respect to the signaling microenvironment. However, the role of the polarity complex is unexplored in mammalian adult stem-cell functions. Here we report that, in contrast to accepted paradigms, polarization and activity of adult hematopoietic stem cell (HSC) do not depend on either aPKCζ or aPKCλ or both in vivo. Mice, having constitutive and hematopoietic-specific (Vav1-Cre) deletion of aPKCζ and aPKCλ, respectively, have normal hematopoiesis, including normal HSC self-renewal, engraftment, differentiation, and interaction with the bone marrow microenvironment. Furthermore, inducible complete deletion of aPKCλ (Mx1-Cre) in aPKCζ(-/-) HSC does not affect HSC polarization, self-renewal, engraftment, or lineage repopulation. In addition, aPKCζ- and aPKCλ-deficient HSCs elicited a normal pattern of hematopoietic recovery secondary to myeloablative stress. Taken together, the expression of aPKCζ, aPKCλ, or both are dispensable for primitive and adult HSC fate determination in steady-state and stress hematopoiesis, contrary to the hypothesis of a unique, evolutionary conserved aPKCζ/λ-directed cell polarity signaling mechanism in mammalian HSC fate determination.

Protocol to characterize mouse intestinal epithelial cell lineage using Opal multiplex immunofluorescence.

Applying Opal multiplex immunofluorescence (OMI) to characterize intestinal tissues of genetically engineered mouse models provides an excellent tool for studying complex processes. However, detecting appropriate signals from multiple target molecules is challenging. Here, we present a protocol to characterize mouse intestinal epithelial cell lineage using OMI. We describe steps for processing small intestine and colonic mouse tissues and designing and optimizing panels for OMI in mouse intestinal tissues. We then detail procedures for performing a quantitative evaluation of acquired images. For complete details on the use and execution of this protocol, please refer to Kinoshita et al.1.

Epithelial aPKC deficiency leads to stem cell loss preceding metaplasia in colorectal cancer initiation.

The early mechanisms of spontaneous tumor initiation that precede malignancy are largely unknown. We show that reduced aPKC levels correlate with stem cell loss and the induction of revival and metaplastic programs in serrated- and conventional-initiated premalignant lesions, which is perpetuated in colorectal cancers (CRCs). Acute inactivation of PKCλ/ι in vivo and in mouse organoids is sufficient to stimulate JNK in non-transformed intestinal epithelial cells (IECs), which promotes cell death and the rapid loss of the intestinal stem cells (ISCs), including those that are LGR5+. This is followed by the accumulation of revival stem cells (RSCs) at the bottom of the crypt and fetal-metaplastic cells (FMCs) at the top, creating two spatiotemporally distinct cell populations that depend on JNK-induced AP-1 and YAP. These cell lineage changes are maintained during cancer initiation and progression and determine the aggressive phenotype of human CRC, irrespective of their serrated or conventional origin.

Whole-mount staining of mouse colorectal cancer organoids and fibroblast-organoid co-cultures.

Imaging organoid culture provides an excellent tool for studying complex diseases such as cancer. However, retaining the morphology of intact organoids for immunolabeling has been challenging. Here, we describe a protocol for immunofluorescence staining in intact colorectal cancer organoids derived from mice. We also describe additional steps for co-culture with mouse fibroblasts to enable the study of interactions with other cellular components of the tissue microenvironment. For complete details on the use and execution of this protocol, please refer to Martinez-Ordoñez et al. (2023).1.

Hyaluronan driven by epithelial aPKC deficiency remodels the microenvironment and creates a vulnerability in mesenchymal colorectal cancer.

Mesenchymal colorectal cancer (mCRC) is microsatellite stable (MSS), highly desmoplastic, with CD8+ T cells excluded to the stromal periphery, resistant to immunotherapy, and driven by low levels of the atypical protein kinase Cs (aPKCs) in the intestinal epithelium. We show here that a salient feature of these tumors is the accumulation of hyaluronan (HA) which, along with reduced aPKC levels, predicts poor survival. HA promotes epithelial heterogeneity and the emergence of a tumor fetal metaplastic cell (TFMC) population endowed with invasive cancer features through a network of interactions with activated fibroblasts. TFMCs are sensitive to HA deposition, and their metaplastic markers have prognostic value. We demonstrate that in vivo HA degradation with a clinical dose of hyaluronidase impairs mCRC tumorigenesis and liver metastasis and enables immune checkpoint blockade therapy by promoting the recruitment of B and CD8+ T cells, including a proportion with resident memory features, and by blocking immunosuppression.

Enhanced SREBP2-driven cholesterol biosynthesis by PKCλ/ι deficiency in intestinal epithelial cells promotes aggressive serrated tumorigenesis.

The metabolic and signaling pathways regulating aggressive mesenchymal colorectal cancer (CRC) initiation and progression through the serrated route are largely unknown. Although relatively well characterized as BRAF mutant cancers, their poor response to current targeted therapy, difficult preneoplastic detection, and challenging endoscopic resection make the identification of their metabolic requirements a priority. Here, we demonstrate that the phosphorylation of SCAP by the atypical PKC (aPKC), PKCλ/ι promotes its degradation and inhibits the processing and activation of SREBP2, the master regulator of cholesterol biosynthesis. We show that the upregulation of SREBP2 and cholesterol by reduced aPKC levels is essential for controlling metaplasia and generating the most aggressive cell subpopulation in serrated tumors in mice and humans. Since these alterations are also detected prior to neoplastic transformation, together with the sensitivity of these tumors to cholesterol metabolism inhibitors, our data indicate that targeting cholesterol biosynthesis is a potential mechanism for serrated chemoprevention.

THBS1-producing tumor-infiltrating monocyte-like cells contribute to immunosuppression and metastasis in colorectal cancer.

Mesenchymal activation, characterized by dense stromal infiltration of immune and mesenchymal cells, fuels the aggressiveness of colorectal cancers (CRC), driving progression and metastasis. Targetable molecules in the tumor microenvironment (TME) need to be identified to improve the outcome in CRC patients with this aggressive phenotype. This study reports a positive link between high thrombospondin-1 (THBS1) expression and mesenchymal characteristics, immunosuppression, and unfavorable CRC prognosis. Bone marrow-derived monocyte-like cells recruited by CXCL12 are the primary source of THBS1, which contributes to the development of metastasis by inducing cytotoxic T-cell exhaustion and impairing vascularization. Furthermore, in orthotopically generated CRC models in male mice, THBS1 loss in the TME renders tumors partially sensitive to immune checkpoint inhibitors and anti-cancer drugs. Our study establishes THBS1 as a potential biomarker for identifying mesenchymal CRC and as a critical suppressor of antitumor immunity that contributes to the progression of this malignancy with a poor prognosis.

Par-4 inhibits Akt and suppresses Ras-induced lung tumorigenesis.

The atypical PKC-interacting protein, Par-4, inhibits cell survival and tumorigenesis in vitro, and its genetic inactivation in mice leads to reduced lifespan, enhanced benign tumour development and low-frequency carcinogenesis. Here, we demonstrate that Par-4 is highly expressed in normal lung but reduced in human lung cancer samples. We show, in a mouse model of lung tumours, that the lack of Par-4 dramatically enhances Ras-induced lung carcinoma formation in vivo, acting as a negative regulator of Akt activation. We also demonstrate in cell culture, in vivo, and in biochemical experiments that Akt regulation by Par-4 is mediated by PKCzeta, establishing a new paradigm for Akt regulation and, likely, for Ras-induced lung carcinogenesis, wherein Par-4 is a novel tumour suppressor.

A functional role for the p62-ERK1 axis in the control of energy homeostasis and adipogenesis.

In vivo genetic inactivation of the signalling adapter p62 leads to mature-onset obesity and insulin resistance, which correlate with reduced energy expenditure (EE) and increased adipogenesis, without alterations in feeding or locomotor functions. Enhanced extracellular signal-regulated kinase (ERK) activity in adipose tissue from p62-knockout (p62(-/-)) mice, and differentiating fibroblasts, suggested an important role for this kinase in the metabolic alterations of p62(-/-) mice. Here, we show that genetic inactivation of ERK1 in p62(-/-) mice reverses their increased adiposity and adipogenesis, lower EE and insulin resistance. These results establish genetically that p62 is a crucial regulator of ERK1 in metabolism.

Loss of PKC lambda/iota impairs Th2 establishment and allergic airway inflammation in vivo.

The differentiation of T cells along different lineages is central to the control of immunity. Here we have used a conditional gene knockout system to delete PKC lambda/iota selectively in activated T cells. With this system we have demonstrated that PKC lambda/iota is necessary for T-helper cell (Th2) cytokine production and optimal T-cell proliferation and allergic airway inflammation in vivo. Our data demonstrate that the activation of the transcription factors nuclear factor of activated T cells and NF-kappaB is impaired in PKC lambda/iota-deficient activated T cells. In addition, we present genetic knockout evidence in ex vivo experiments with primary T cells that PKC lambda/iota is critical for the control of cell polarity during T-cell activation. Therefore PKC lambda/iota emerges as a critical regulator of Th 2 activation.

Protein kinase Cλ/ι in cancer: a contextual balance of time and signals.

Nononcogenic cancer drivers often impinge on complex signals that create new addictions and vulnerabilities. Protein kinase Cλ/ι (PKCλ/ι) suppresses tumorigenesis by blocking metabolic pathways that regulate fuel oxidation and create building blocks for the epigenetic control of cell differentiation. Reduced levels of PKCλ/ι unleash these pathways to promote tumorigenesis, but the simultaneous activation of the STING-driven interferon cascade prevents tumor initiation by triggering immunosurveillance mechanisms. However, depending on the context of other signaling pathways, such as WNT/ß-catenin or PKCζ, and timing, PKCλ/ι deletion can promote or inhibit tumorigenesis. In this review, we discuss in detail the molecular and cellular underpinnings of PKCλ/ι functions in cancer with the perspective of the crosstalk between metabolism and inflammation in the tumor microenvironment.

Air-liquid organotypic assays to investigate cellular crosstalk in the tumor microenvironment of cancer cells.

Air-liquid organotypic culture models enable the study of the cellular crosstalk in the tumor microenvironment. This 3D assay recapitulates the tumor niche more faithfully than 2D culture systems and represents a versatile platform that can be easily adapted to different types of cancer cells, stromal components, or ECM composition. Here, we detail the steps to build an organotypic culture including the preparation of the organotypic structure, organotypic gels, cell seeding, gel casting, membrane processing, and image and data analysis. For complete details on the use and execution of this protocol, please refer to Linares et al. (2022).