Multifaceted control of E-cadherin dynamics by the Adaptor Protein Complex 1 during epithelial morphogenesis

Intracellular trafficking regulates the distribution of transmembrane proteins including the key determinants of epithelial polarity and adhesion. The Adaptor Protein 1 (AP-1) complex is the key regulator of vesicle sorting, which binds many specific cargos. We examined roles of the AP-1 complex in epithelial morphogenesis, using the Drosophila wing as a paradigm. We found that AP-1 knockdown leads to ectopic tissue folding, which is consistent with the observed defects in integrin targeting to the basal cell-extracellular matrix adhesion sites. This occurs concurrently with an integrin-independent induction of cell death, which counteracts elevated proliferation and prevents hyperplasia. We discovered a distinct pool of AP-1, which localizes at the subapical Adherens Junctions. Upon AP-1 knockdown, E-cadherin is hyperinternalized from these junctions and becomes enriched at the Golgi and recycling endosomes. We then provide evidence that E-cadherin hyperinternalization acts upstream of cell death in a potential tumour-suppressive mechanism. Simultaneously, cells compensate for elevated internalization of E-cadherin by increasing its expression to maintain cell-cell adhesion.

Transfection of Vitamin D3-Induced Tolerogenic Dendritic Cells for the Silencing of Potential Tolerogenic Genes. Identification of CSF1R-CSF1 Signaling as a Glycolytic Regulator

The use of autologous tolerogenic dendritic cells (tolDC) has become a promising strategy to re-establish immune tolerance in autoimmune diseases. Among the different strategies available, the use of vitamin D3 for the generation of tolDC (VitD3-tolDC) has been widely tested because of their immune regulatory properties. To identify molecules and pathways involved in the generation of VitD3-tolDC, we established an easy and fast gene silencing method based on the use of Viromer blue to introduce siRNA into monocytes on day 1 of culture differentiation. The analysis of the effect of CD209 (DC-SIGN) and CD115 (CSF1R) down-modulation on the phenotype and functionality of transfected VitD3-tolDC revealed a partial role of CD115 in their tolerogenicity. Further investigations showed that CSF1R-CSF1 signaling is involved in the induction of cell metabolic reprogramming, triggering glycolysis to produce high amounts of lactate, a novel suppressive mechanism of T cell proliferation, recently found in autologous tolerogenic dendritic cells (ATDCs).

Loss of MGA repression mediated by an atypical polycomb complex promotes tumor progression and invasiveness

MGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated Mga in two mouse models of non-small cell lung cancer using a CRISPR-based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of non-canonical Polycomb ncPRC1.6 targets, including genes involved in metastasis and meiosis. Moreover, MGA deletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA-MAX, E2F6, and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes these ncPRC1.6 subunits. Lastly, we report that MGA loss also induces a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F target genes mediated by MGA-MAX associated with a non-canonical Polycomb complex.

Defective apoptotic cell contractility provokes sterile inflammation leading to liver damage and tumour suppression

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating Aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour suppressive mechanism.

Thalamocortical mechanisms regulating the relationship between transient beta events and human tactile perception

Transient neocortical events with high spectral power in the 15-29Hz beta band are among the most reliable predictors of sensory perception. Prestimulus beta event rates in primary somatosensory cortex correlate with sensory suppression, most effectively 100-300ms before stimulus onset. However, the neural mechanisms underlying this perceptual association are unknown. We combined human magnetoencephalography (MEG) measurements with biophysical neural modeling to test potential cellular and circuit mechanisms that underlie observed correlations between prestimulus beta events and tactile detection. Extending prior studies, we found that simulated bursts from higher-order, non-lemniscal thalamus were sufficient to drive beta event generation and to recruit slow supragranular inhibition acting on a 300ms time scale to suppress sensory information. Further analysis showed that the same beta generating mechanism can lead to facilitated perception for a brief period when beta events occur simultaneously with tactile stimulation before inhibition is recruited. These findings were supported by close agreement between model-derived predictions and empirical MEG data. The post-event suppressive mechanism explains an array of studies that associate beta with decreased processing, while the during-event faciliatory mechanism may demand a reinterpretation of the role of beta events in the context of coincident timing.

Characterisation of root-knot nematode resistance in black oat, Avena strigosa, ‘Terara’

Summary The mechanism of root-knot nematode (RKN) resistance in black oat ‘Terara’ and its stability under high temperatures were examined. To investigate the suppressive mechanism, we observed root invasion and development of RKN in ‘Terara’. Juveniles of Meloidogyne incognita, M. arenaria and M. javanica successfully invaded roots of ‘Terara’ 3 days after inoculation (DAI), whereas juveniles of M. hapla were hardly observed in black oat roots. The total number of M. incognita and the ratio of successfully developing juveniles were significantly lower in roots of ‘Terara’ than a susceptible cultivar from 7 DAI. A number of M. incognita developed to sausage-shaped juveniles in ‘Terara’; however, juveniles beyond that stage were seldom observed. Histological observation of feeding sites suggested the poor development of giant cells was involved in this arrested development. Juveniles of M. arenaria and M. javanica also only developed to the sausage-shaped stage at 21 DAI. These observations indicated that ‘Terara’ and resistant common oat cultivars suppressed RKN in a similar way. The effect of high temperature on resistance was evaluated using ‘Terara’ cultivated at 30°C throughout the experiment and at 32°C for the initial 5 DAI. The number of egg masses of M. incognita under the 32°C treatment was slightly higher than the 25°C control. However, it was still suppressed to 4.9% of the susceptible cultivar.

Loss of MGA mediated Polycomb repression promotes tumor progression and invasiveness

ABSTRACTMGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated Mga in two mouse models of non-small cell lung cancer using a CRISPR based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of atypical Polycomb PRC1.6, E2F and MYC-MAX targets. Similarly, MGA depletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA, E2F6 and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes PRC1.6 subunits. Lastly, we report that MGA loss has also a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F targets mediated by an atypical Polycomb complex containing MGA-MAX dimers.

Pharmacological CDK4/6 inhibition unravels a p53-induced secretory phenotype in senescent cells

Cellular senescence is a state of stable growth arrest that acts as a tumor suppressive mechanism. Several anti-cancer interventions function partly by inducing malignant cells into senescence. However, because of systemic administration and lack of specificity, anti-cancer treatments are associated with premature senescence of various non-malignant cells. Therapy-induced non-malignant senescent cells can have profound detrimental pro-tumorigenic and pro-disease functions via activation of a pro-inflammatory and NF-κB-mediated secretory phenotype (SASP). Inhibitors of the cyclin-dependent kinases 4/6 (CDK4/6i) has recently shown to have potent cytostatic effects with reduced toxicities. Here, we show that CDK4/6i lead non-malignant cells to a senescent state that lacks the pro-inflammatory and NF-κB-associated SASP. Interestingly, CDK4/6i-induced senescence overexpressed a number of genes encoding for secreted proteins, which we show being dependent on p53 transcriptional activity. CDK4/6i-induced p16+ senescent cells with a p53-associated (PASP), but not NF-κB-associated (NASP), secretory phenotype do not exert detrimental and pro-tumorigenic functions, but still retain the capacity to induce paracrine senescence and undergo clearance in vivo. Our data suggest that that senescent cells with a PASP but without a NASP may be well-tolerated and may represent a less toxic outcome for cancer interventions.

Entosis and apical cell extrusion constitute a tumor-suppressive mechanism downstream of Matriptase

The type II transmembrane serine protease Matriptase 1 (ST14) is commonly known as an oncogene, yet it also plays an understudied role in suppressing carcinogenesis. This double face is evident in the embryonic epidermis of zebrafish loss-of-function mutants in the cognate Matriptase inhibitor Hai1a (Spint1a). Mutant embryos display epidermal hyperplasia, but also apical cell extrusions, during which extruding outer keratinocytes carry out an entosis-like engulfment and entrainment of underlying basal cells, constituting a tumor-suppressive effect. These counteracting Matriptase effects depend on EGFR and the newly identified mediator phospholipase D (PLD), which promotes both mTORC1-dependent cell proliferation and sphingosine-1-phosphate (S1P)–dependent entosis and apical cell extrusion. Accordingly, hypomorphic hai1a mutants heal spontaneously, while otherwise lethal hai1a amorphs are efficiently rescued upon cotreatment with PLD inhibitors and S1P. Together, our data elucidate the mechanisms underlying the double face of Matriptase function in vivo and reveal the potential use of combinatorial carcinoma treatments when such double-face mechanisms are involved.