Inhibition of PbeXTH1 and PbeSEOB1 is required for the Valsa canker resistance contributed by Wall-associated kinase gene MbWAK1.

Wall-associated kinases (WAKs) have been determined to recognize pathogenic signals and initiate plant immune responses. However, the roles of the family members in host resistance against Valsa canker, a serious fungal disease of apples and pears, are largely unknown. Here, we identified MbWAK1 in Malus baccata, a resistant germplasm differentially expressed during infection by Valsa mali (Vm). Over-expression of MbWAK1 enhanced the Valsa canker resistance of apple and pear fruits and 'Duli-G03' (Pyrus betulifolia) suspension cells. A large number of phloem, cell wall, and lipid metabolic process-related genes were differentially expressed in overexpressed suspension cell lines in response to Valsa pyri (Vp) signals. Among these, the expression of xyloglucan endotransglucosylase/hydrolase (XTH) gene PbeXTH1 and sieve element occlusion B-like (SEOB) gene PbeSEOB1 were significantly inhibited. Transient expression of PbeXTH1 or PbeSEOB1 compromised the expressional induction of MbWAK1 and the resistance contributed by MbWAK1. In addition, PbeXTH1 and PbeSEOB1 suppressed the immune response induced by MbWAK1. Our results enriched the molecular mechanisms for MbWAK1 against Valsa canker and resistant breeding.

Evolutionary and functional analysis reveals the crucial roles of receptor-like proteins in resistance to Valsa canker in Rosaceae.

Rosaceae is an economically important plant family that can be affected by a multitude of pathogenic microbes, some of which can cause dramatic losses in production. As a type of pattern-recognition receptor, receptor-like proteins (RLPs) are considered vital regulators of plant immunity. Based on genome-wide identification, bioinformatic analysis, and functional determination, we investigated the evolutionary characteristics of RLPs, and specifically those that regulate Valsa canker, a devastating fungal disease affecting apple and pear production. A total of 3028 RLPs from the genomes of 19 species, including nine Rosaceae, were divided into 24 subfamilies. Five subfamilies and seven co-expression modules were found to be involved in the responses to Valsa canker signals of the resistant pear rootstock Pyrus betulifolia 'Duli-G03'. Fourteen RLPs were subsequently screened as candidate genes for regulation of resistance. Among these, PbeRP23 (Chr13.g24394) and PbeRP27 (Chr16.g31400) were identified as key resistance genes that rapidly enhance the resistance of 'Duli-G03' and strongly initiate immune responses, and hence they have potential for further functional exploration and breeding applications for resistance to Valsa canker. In addition, as a consequence of this work we have established optimal methods for the classification and screening of disease-resistant RLPs.

Enhanced Valsa canker resistance conferred by expression of MdLecRK-S.4.3 in Pyrus betulifolia is largely suppressed by PbePUB36.

L-type lectin receptor-like kinases (L-LecRKs) act as sensors of extracellular signals and as initiators for plant immune responses; however, the function of LecRK-S.4 in plant immunity has not yet been extensively investigated. In the present study we found that MdLecRK-S.4.3 in apple (Malus domestica), a homologous gene of LecRK-S.4, was differentially expressed during infection by Valsa mali and Valsa pyri. Overexpression of MdLecRK-S.4.3 facilitated the induction of immune responses and enhanced the resistance to Valsa canker of fruits of apple and pear (Pyrus betulifolia), and of suspension cells of pear 'Duli-G03'. The expression of PbePUB36, a RLCK XI sub-family member, was significantly repressed in the MdLecRK-S.4.3-overexpressing cell lines. Overexpression of PbePUB36 interfered with the resistance to Valsa canker and the immune response caused by up-regulation of MdLecRK-S.4.3. In addition, we found that MdLecRK-S.4.3 interacted with BAK1 and/or PbePUB36 in vivo. Thus, whilst MdLecRK-S.4.3 activated various immune responses and positively regulated Valsa canker resistance, this could be largely compromised by PbePUB36. MdLecRK-S.4.3 interacted with PbePUB36 and/or MdBAK1 to mediate the immune responses. Our finding provides a basis for further examination of the molecular mechanisms underlying resistance to Valsa canker, and can contribute to resistance breeding.

Dynamic range expansion of spatial light modulators based on a module-nπ method.

It is known that the cumbersome 2π correction is needed in the traditional module-2π method (i.e., the phase wrapping method) due to the 2π deviation of the phase modulation depth of spatial light modulators (SLMs). To avoid the cumbersome 2π correction in the module-2π method, this paper proposes a module-n π method, and it can directly utilize any full-field phase modulation depth. First, for a Gaussian phase with a phase depth of 30 rad, wrapped by the module-3.6π, it is reconstructed with the root-mean-square (RMS) values of its phase response are 0.1006λ (for the Twyman-Green interferometer) and 0.1101λ (for the Shack-Hartmann wavefront sensor method), respectively, which proves that the monitoring accuracy is relatively consistent. Subsequently, some comparative experiments based on the traditional module-2π are performed. The experimental results show that the RMS values of its phase response are 0.8886λ (for a modulation depth of 11.3 rad) and 0.2261λ (for a modulation depth of 6.28 rad), respectively. All the results have proved that the SLM with a phase modulation depth exceeding 2π (e.g., 11.3 rad) has more prominent advantages. More specifically, increasing the SLM's phase modulation depth can effectively reduce the fringe orders of the wrapped patterns generated by the module-n π method. With the further reduction of the fringe orders, the influence of the fly-back zone error on the wavefront phase modulation is reduced, that is, the modulation accuracy is improved (the RMS values are reduced from 0.2261λ to 0.1006λ). Different from the traditional module-2π method, there is no need to consider the problems of the SLMs' over modulation or the insufficient modulation in the module-n π method. Furthermore, it avoids the cumbersome 2π correction process, which will make the use of the SLM more convenient.

Phase response measurement of spatial light modulators based on a Shack-Hartmann wavefront sensor.

It is known that the phase response of spatial light modulators (SLMs) measured by double-beam interferometers is sensitive to mechanical and environmental disturbances. This paper proposes a Shack-Hartmann wavefront sensor (SHWS) method to measure the phase response characteristics of the SLM. The results show that the phase modulation depth measured by the proposed method is 1.7581λ, and 1.7993λ by the Twyman-Green interferometer method. The difference in the phase modulation depth between the two methods is only 0.0412λ, and its relative error rate is 2.29%. It proves that the phase modulation accuracy obtained by the SHWS with lenslets of 73*73 used in this paper is equivalent to that of the Twyman-Green interferometer. Compared with the interferometer method, the SHWS method is simple, compact, and robust, has good real-time performance, and is relatively vibration insensitive. In the future, the SHWS method will play a more important role in the detection of the SLM's phase response.

p53 haploinsufficiency and increased mTOR signalling define a subset of aggressive hepatocellular carcinoma.

BACKGROUND & AIMS: p53 mutations occur frequently in human HCC. Activation of the mammalian target of rapamycin (mTOR) pathway is also associated with HCC. However, it is still unknown whether these changes together initiate HCC and can be targeted as a potential therapeutic strategy. METHODS: We generated mouse models in which mTOR was hyperactivated by loss of tuberous sclerosis complex 1 (Tsc1) with or without p53 haplodeficiency. Primary cells were isolated from mouse livers. Oncogenic signalling was assessed in vitro and in vivo, with or without targeted inhibition of a single molecule or multiple molecules. Transcriptional profiling was used to identify biomarkers predictive of HCC. Human HCC materials were used to corroborate the findings from mouse models. RESULTS: p53 haploinsufficiency facilitates mTOR signalling via the PTEN/PI3K/Akt axis, promoting HCC tumorigenesis and lung metastasis. Inhibition of PI3K/Akt reduced mTOR activity, which effectively enhanced the anticancer effort of an mTOR inhibitor. ATP-binding cassette subfamily C member 4 (Abcc4) was found to be responsible for p53 haploinsufficiency- and Tsc1 loss-driven HCC tumorigenesis. Moreover, in clinical HCC samples, Abcc4 was specifically identified an aggressive subtype. The mTOR inhibitor rapamycin significantly reduced hepatocarcinogenesis triggered by Tsc1 loss and p53 haploinsufficiency in vivo, as well as the biomarker Abcc4. CONCLUSIONS: Our data advance the current understanding of the activation of the PTEN/PI3K/Akt/mTOR axis and its downstream target Abcc4 in hepatocarcinogenesis driven by p53 reduction and Tsc1 loss. Targeting mTOR, an unexpected vulnerability in p53 (haplo)deficiency HCC, can be exploited therapeutically to treat Abcc4-positive patients with HCC. LAY SUMMARY: Tsc1 loss facilitates the p53 (haplo)insufficiency-mediated activation of the PTEN/Akt/mTOR axis, leading to the elevated expression of Abcc4 to drive HCC tumorigenesis and metastasis in mice. Inhibition of mTOR protects against p53 haploinsufficiency and Tsc1 loss-triggered tumour-promoting activity, providing a new approach for treating an aggressive subtype of HCC exhibiting high Abcc4 expression.

Rodent retinal microcirculation and visual electrophysiology following simulated microgravity.

How the absence of gravity affects the physiology of human beings is generating global research interest as space exploration, including missions aboard the International Space Station, continues to push boundaries. Here, we examined changes in retinal microcirculation and visual electrophysiology in mice suspended by their tails to simulate the cephalad movement of blood that occurs under microgravity conditions. Tail suspension was performed with a head-down tilt with a recommended angle of 30°. Mice in the control groups were similarly attached to a tether but could maintain a normal position. Morphologically, the 15-day tail-suspended mice showed retinal microvascular dilation, tortuosity, and a relatively long fluorescence retention; however, the average diameter of the major retinal vessels was not notably changed. In addition, optical coherence tomography showed their optic nerve head had an increased diameter. However, the mice could adapt to the change, with microcirculation and the optic nerve head recovering following 30-day tail suspension. Expression of rhodopsin and cone-opsins was not notably changed, and no retinal apoptotic-positive cells were detected between 15- and 30-day tail suspensions. Moreover, the three experimental groups of suspended mice showed normal retinal layers and thickness. Functionally, following 15-day tail suspension, scotopic electroretinograms showed a decline in the oscillatory potentials (OPs), but not in the b wave; simultaneously, the peak time of flash visual evoked potential component N1 was delayed compared to its baseline and the time-matched control. Following 30-day tail suspension, the OPs (O2) amplitude recovered to approximately 97% of its baseline or 86% of the time-matched control level. By simulating cephalad shifting of blood, short-term tail suspension can affect rodent retinal microcirculation, the optic nerve head, and disturb visual electrophysiology. However, the change is reversible with no permanent injury observed in the retina. The mice could adapt to the short-term change of retinal microcirculation, indicating new conditions that could be combined with, or could enhance, simulated microgravity for further studying the impact of short- or long-term outer space conditions on the retina.

Hybrid method for speckle noise reduction in digital holography.

In digital holography, the inherited speckle noise degrades imaging quality due to the coherent laser source. To overcome this problem, a hybrid method for speckle noise reduction is presented by combining a novel angular diversity approach with the block-matching and 3D filtering (BM3D) algorithm. A serial of holograms is first captured by the proposed recording approach, and then the image with high signal-to-noise ratio is obtained by averaging multiple reconstructed intensity images. Finally, the residual noise in the averaged image is further eliminated by the BM3D filtering algorithm. The speckle noise is significantly suppressed, and a nearly speckle-free image can be obtained. Experimental results demonstrate the effectiveness of the proposed method.

Phase curvature compensation in digital holographic microscopy based on phase gradient fitting and optimization.

We propose a numerical method for phase curvature compensation in digital holographic microscopy, in which the phase curvature is compensated for by subtracting a numerical phase mask from the distorted phase. The parameters of the phase mask are obtained based on phase gradient fitting and optimization, in which the initial mask parameters are obtained by fitting the phase gradient, and then more accurate mask parameters are determined using a spectrum energy search. The compensation can be executed in a hologram without extra devices or any prior knowledge of the setup and specimen. A computer simulation and experimental results demonstrated the feasibility of the proposed method.

Porphyromonas gingivalis, a periodontitis causing bacterium, induces memory impairment and age-dependent neuroinflammation in mice.

BACKGROUND: A possible relationship between periodontitis and Alzheimer's disease (AD) has been reported. However, there is limited information on the association between the Porphyromonas gingivalis (P. gingivalis) periodontal infection and the pathological features of AD. The hypothesis that P. gingivalis periodontal infection may cause cognitive impairment via age-dependent neuroinflammation was tested. RESULTS: Thirty 4-week-old (young) female C57BL/6 J mice were randomly divided into two groups, the control group and the experimental group. Thirty 12-month-old (middle-aged) were grouped as above. The mouth of the mice in the experimental group was infected with P. gingivalis. Morris water maze(MWM) was performed to assess the learning and memory ability of mice after 6 weeks. Moreover, the expression levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß in the mice brain tissues were determined by Quantitative real-time polymerase chain reaction (qRT-PCR), Enzyme Linked Immunosorbent Assay(ELISA) and immunohistochemistry. Our results showed that the learning and memory abilities of the middle-aged P. gingivalis infected mice were impaired. Moreover, the expression levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß in the brain tissues of the middle-aged P. gingivalis infected mice were increased. CONCLUSIONS: These results suggest that P. gingivalis periodontal infection may cause cognitive impairment via the release of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß in the brain tissues of middle-aged mice.

Diurnal biological effects of correlated colour temperature and its exposure timing on alertness, cognition, and mood in an enclosed environment.

Artificial lighting, which profits from the non-visual effects of light, is a potentially promising solution to support residents' psychophysiological health and performance at specific times of the day in enclosed environments. However, few studies have investigated the non-visual effects of daytime correlated colour temperature (CCT) and its exposure timing on human alertness, cognition, and mood. However, the neural mechanisms underlying these effects are largely unknown. The current study evaluated the effects of daytime CCT and its exposure timing on markers of subjective experience, cognitive performance, and cerebral activity in a simulated enclosed environment. Forty-two participants participated a single-blind laboratory study with a 4 within (CCT: 4000 K vs. 6500 K vs. 8500 K vs. 12,000 K) × 2 between (exposure timing: morning vs. afternoon) mixed design. The results showed time of the day dependent benefits of the daytime CCT on subjective experience, vigilant attention, response inhibition, working memory, emotional perception, and risk decisions. The results of the electroencephalogram (EEG) revealed that lower-frequency EEG bands, including theta, alpha, and alpha-theta, were quite sensitive to daytime CCT intervention, which provides a valuable reference for trying to establish the underlying mechanisms that support the performance-enhancement effects of exposure to CCT in the daytime. However, the results revealed no consistent intervention pattern across these measurements. Therefore, future studies should consider personalised optimisation of daytime CCT for different cognitive demands.

Putative pathogenic factors underlying Streptococcus oralis opportunistic infections.

Streptococcus oralis, belonging to the viridans group streptococci (VGS), has been considered a component of the normal flora predominantly inhabiting the oral cavity. In recent years, a growing body of literature has revealed that dental procedures or daily tooth brushing activities can cause the spread of S. oralis from the oral cavity into various body sites leading to life-threatening opportunistic infections such as infective endocarditis (IE) and meningitis. However, very little is currently known about the pathogenicity of S. oralis. Thus, the aim of this review is to update the current understanding of the pathogenic potential of S. oralis to pave the way for the prevention and treatment of S. oralis opportunistic infections.

Injectable PEG Hydrogels with Tissue-Like Viscoelasticity Formed through Reversible Alendronate-Calcium Phosphate Crosslinking for Cell-Material Interactions.

Synthetic hydrogels provide controllable 3D environments, which can be used to study fundamental biological phenomena. The growing body of evidence that cell behavior depends upon hydrogel stress relaxation creates a high demand for hydrogels with tissue-like viscoelastic properties. Here, a unique platform of synthetic polyethylene glycol (PEG) hydrogels in which star-shaped PEG molecules are conjugated with alendronate and/or RGD peptides, attaining modifiable degradability as well as flexible cell adhesion, is created. Novel reversible ionic interactions between alendronate and calcium phosphate nanoparticles, leading to versatile viscoelastic properties with varying initial elastic modulus and stress relaxation time, are identified. This new crosslinking mechanism provides shear-thinning properties resulting in differential cellular responses between cancer cells and stem cells. The novel hydrogel system is an improved design to the other ionic crosslink platforms and opens new avenues for the development of pathologically relevant cancer models, as well as minimally invasive approaches for cell delivery for potential regenerative therapies.

The RNA binding proteins LARP4A and LARP4B promote sarcoma and carcinoma growth and metastasis.

RNA-binding proteins (RBPs) are emerging as important regulators of cancer pathogenesis. We reveal that the RBPs LARP4A and LARP4B are differentially overexpressed in osteosarcoma and osteosarcoma lung metastases, as well as in prostate cancer. Depletion of LARP4A and LARP4B reduced tumor growth and metastatic spread in xenografts, as well as inhibiting cell proliferation, motility, and migration. Transcriptomic profiling and high-content multiparametric analyses unveiled a central role for LARP4B, but not LARP4A, in regulating cell cycle progression in osteosarcoma and prostate cancer cells, potentially through modulating key cell cycle proteins such as Cyclins B1 and E2, Aurora B, and E2F1. This first systematic comparison between LARP4A and LARP4B assigns new pro-tumorigenic functions to LARP4A and LARP4B in bone and prostate cancer, highlighting their similarities while also indicating distinct functional differences. Uncovering clear biological roles for these paralogous proteins provides new avenues for identifying tissue-specific targets and potential druggable intervention.

Elevated expression of WSB2 degrades p53 and activates the IGFBP3-AKT-mTOR-dependent pathway to drive hepatocellular carcinoma.

Dysregulation of wild-type p53 turnover is a key cause of hepatocellular carcinoma (HCC), yet its mechanism remains poorly understood. Here, we report that WD repeat and SOCS box containing protein 2 (WSB2), an E3 ubiquitin ligase, is an independent adverse prognostic factor in HCC patients. WSB2 drives HCC tumorigenesis and lung metastasis in vitro and in vivo. Mechanistically, WSB2 is a new p53 destabilizer that promotes K48-linked p53 polyubiquitination at the Lys291 and Lys292 sites in HCC cells, leading to p53 proteasomal degradation. Degradation of p53 causes IGFBP3-dependent AKT/mTOR signaling activation. Furthermore, WSB2 was found to bind to the p53 tetramerization domain via its SOCS box domain. Targeting mTOR with everolimus, an oral drug, significantly blocked WSB2-triggered HCC tumorigenesis and metastasis in vivo. In clinical samples, high expression of WSB2 was associated with low wild-type p53 expression and high p-mTOR expression. These findings demonstrate that WSB2 is overexpressed and degrades wild-type p53 and then activates the IGFBP3-AKT/mTOR axis, leading to HCC tumorigenesis and lung metastasis, which indicates that targeting mTOR could be a new therapeutic strategy for HCC patients with high WSB2 expression and wild-type p53.

Elevated FBXL18 promotes RPS15A ubiquitination and SMAD3 activation to drive HCC.

BACKGROUND: F-box and leucine-rich repeat protein 18 (FBXL18) is an E3 ubiquitin ligase that is reported to be involved in the tumorigenesis of various types of cancer. However, it remains unknown whether FBXL18 is correlated with hepatocarcinogenesis. METHODS AND RESULTS: In the current study, we found that FBXL18 was highly expressed in HCC tissues and positively associated with poor overall survival of HCC patients. FBXL18 was an independent risk factor for HCC patients. We observed that FBXL18 drove HCC in FBXL18 transgenic mice. Mechanistically, FBXL18 promoted the K63-linked ubiquitination of small-subunit ribosomal protein S15A (RPS15A) and enhanced its stability, increasing SMAD family member 3 (SMAD3) levels and translocation to the nucleus and promoting HCC cell proliferation. Moreover, the knockdown of RPS15A or SMAD3 significantly suppressed FBXL18-mediated HCC proliferation. In clinical samples, elevated FBXL18 expression was positively associated with RPS15A expression. CONCLUSION: FBXL18 promotes RPS15A ubiquitination and upregulates SMAD3 expression, leading to hepatocellular carcinogenesis, and this study provides a novel therapeutic strategy for HCC treatment by targeting the FBXL18/RPS15A/SMAD3 pathway.

Elevated FBXL6 expression in hepatocytes activates VRK2-transketolase-ROS-mTOR-mediated immune evasion and liver cancer metastasis in mice.

Metastatic hepatocellular carcinoma (HCC) is the most lethal malignancy and lacks effective treatment. FBXL6 is overexpressed in human hepatocellular carcinoma (HCC), but whether this change drives liver tumorigenesis and lung metastasis in vivo remains unknown. In this study, we aimed to identify FBXL6 (F-Box and Leucine Rich Repeat Protein 6) as a key driver of HCC metastasis and to provide a new paradigm for HCC therapy. We found that elevated FBXL6 expression in hepatocytes drove HCC lung metastasis and was a much stronger driver than Kras mutation (KrasG12D/+;Alb-Cre), p53 haploinsufficiency (p53+/-) or Tsc1 loss (Tsc1fl/fl;Alb-Cre). Mechanistically, VRK2 promoted Thr287 phosphorylation of TKT and then recruited FBXL6 to promote TKT ubiquitination and activation. Activated TKT further increased PD-L1 and VRK2 expression via the ROS-mTOR axis, leading to immune evasion and HCC metastasis. Targeting or knockdown of TKT significantly blocked FBXL6-driven immune evasion and HCC metastasis in vitro and in vivo. Notably, the level of active TKT (p-Thr287 TKT) was increased and was positively correlated with the FBXL6 and VRK2 expression levels in HCC patients. Our work provides novel mechanistic insights into FBXL6-driven HCC metastasis and suggests that targeting the TKT-ROS-mTOR-PD-L1/VRK2 axis is a new paradigm for treating patients with metastatic HCC with high FBXL6 expression.

Elevated FBXW10 drives hepatocellular carcinoma tumorigenesis via AR-VRK2 phosphorylation-dependent GAPDH ubiquitination in male transgenic mice.

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men, but the underlying mechanism remains to be further explored. Here, we report that ubiquitinated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is responsible for HCC tumorigenesis in males. Mechanistically, FBXW10 promotes GAPDH polyubiquitination and activation; VRK2-dependent phosphorylation of GAPDH Ser151 residue is critical for GAPDH ubiquitination and activation. Activated GAPDH interacts with TRAF2, leading to upregulation of the canonical and noncanonical NF-κB pathways, and increases PD-L1 and AR-VRK2 expression, followed by induction of immune evasion, HCC tumorigenesis, and metastasis. Notably, the GAPDH inhibitor koningic acid (KA) activates immune response and protects against FBXW10-driven HCC in vivo. In HCC clinical samples, the expression of active GAPDH is positively correlated with that of FBXW10 and VRK2. We propose that the FBXW10/AR/VRK2/GAPDH/NF-κB axis is critical for HCC tumorigenesis in males. Targeting this axis with KA is a potential therapeutic strategy for male HCC patients.

Degradation of helicase-like transcription factor (HLTF) by ß-TrCP promotes hepatocarcinogenesis via activation of the p62/mTOR axis.

Helicase-like transcription factor (HLTF) has been found to be involved in the maintenance of genome stability and tumour suppression, but whether its downregulation in cancers is associated with posttranslational regulation remains unclear. Here, we observed that HLTF was significantly downregulated in hepatocellular carcinoma (HCC) tissues and positively associated with the survival of HCC patients. Mechanistically, the decreased expression of HLTF in HCC was attributed to elevated ß-TrCP-mediated ubiquitination and degradation. Knockdown of HLTF enhanced p62 transcriptional activity and mammalian target of rapamycin (mTOR) activation, leading to HCC tumourigenesis. Inhibition of mTOR effectively blocked ß-TrCP overexpression- or HLTF knockdown-mediated HCC tumourigenesis and metastasis. Furthermore, in clinical tissues, decreased HLTF expression was positively correlated with elevated expression of ß-TrCP, p62, or p-mTOR in HCC patients. Overall, our data not only uncover new roles of HLTF in HCC cell proliferation and metastasis, but also reveal a novel posttranslational modification of HLTF by ß-TrCP, indicating that the ß-TrCP/HLTF/p62/mTOR axis may be a new oncogenic driver involved in HCC development. This finding provides a potential therapeutic strategy for HCC patients by targeting the ß-TrCP/HLTF/p62/mTOR axis.

Elevated FBXL6 activates both wild-type KRAS and mutant KRASG12D and drives HCC tumorigenesis via the ERK/mTOR/PRELID2/ROS axis in mice.

BACKGROUND: Kirsten rat sarcoma (KRAS) and mutant KRASG12D have been implicated in human cancers, but it remains unclear whether their activation requires ubiquitination. This study aimed to investigate whether and how F-box and leucine-rich repeat 6 (FBXL6) regulates KRAS and KRASG12D activity in hepatocellular carcinoma (HCC). METHODS: We constructed transgenic mouse strains LC (LSL-Fbxl6KI/+;Alb-Cre, n = 13), KC (LSL-KrasG12D/+;Alb-Cre, n = 10) and KLC (LSL-KrasG12D/+;LSL-Fbxl6KI/+;Alb-Cre, n = 12) mice, and then monitored HCC for 320 d. Multiomics approaches and pharmacological inhibitors were used to determine oncogenic signaling in the context of elevated FBXL6 and KRAS activation. Co­immunoprecipitation (Co-IP), Western blotting, ubiquitination assay and RAS activity detection assay were employed to investigate the underlying molecular mechanism by which FBXL6 activates KRAS. The pathological relevance of the FBXL6/KRAS/extracellular signal-regulated kinase (ERK)/mammalian target of rapamycin (mTOR)/proteins of relevant evolutionary and lymphoid interest domain 2 (PRELID2) axis was evaluated in 129 paired samples from HCC patients. RESULTS: FBXL6 is highly expressed in HCC as well as other human cancers (P < 0.001). Interestingly, FBXL6 drives HCC in transgenic mice. Mechanistically, elevated FBXL6 promotes the polyubiquitination of both wild-type KRAS and KRASG12D at lysine 128, leading to the activation of both KRAS and KRASG12D and promoting their binding to the serine/threonine-protein kinase RAF, which is followed by the activation of mitogen-activated protein kinase kinase (MEK)/ERK/mTOR signaling. The oncogenic activity of the MEK/ERK/mTOR axis relies on PRELID2, which induces reactive oxygen species (ROS) generation. Furthermore, hepatic FBXL6 upregulation facilitates KRASG12D to induce more severe hepatocarcinogenesis and lung metastasis via the MEK/ERK/mTOR/PRELID2/ROS axis. Dual inhibition of MEK and mTOR effectively suppresses tumor growth and metastasis in this subtype of cancer in vivo. In clinical samples, FBXL6 expression positively correlates with p-ERK (χ2 = 85.067, P < 0.001), p-mTOR (χ2 = 66.919, P < 0.001) and PRELID2 (χ2 = 20.891, P < 0.001). The Kaplan-Meier survival analyses suggested that HCC patients with high FBXL6/p-ERK levels predicted worse overall survival (log­rank P < 0.001). CONCLUSIONS: FBXL6 activates KRAS or KRASG12D via ubiquitination at the site K128, leading to activation of the ERK/mTOR/PRELID2/ROS axis and tumorigenesis. Dual inhibition of MEK and mTOR effectively protects against FBXL6- and KRASG12D-induced tumorigenesis, providing a potential therapeutic strategy to treat this aggressive subtype of liver cancer.