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.

Bufalin induces apoptosis via mitochondrial ROS-mediated caspase-3 activation in HCT-116 and SW620 human colon cancer cells.

OBJECTIVE: Bufalin has been reported to kill various types of cancer including human colorectal cancer. Our previous study demonstrated that bufalin induced cell death via autophagy in HT-29 and Caco-2 colon cancer cells, but the action of bufalin remains unclear. This study was conducted to investigate the role of bufalin in other colon cancer HCT-116 and SW620 cells as well as its potential mechanism. METHODS: The effect of bufalin in HCT-116 and SW620 colon cancer cells was detected by assessing cell viability and cell death. Apoptotic cells were analyzed by Western blot and trypan blue dye exclusion assay. Mitochondrial ROS production was analyzed by flow cytometry after DCFDA and DHR-123 staining. The potential mechanism was investigated via pharmacological inhibitors. RESULTS: Bufalin had high potency against HCT-116 and SW620 cells with IC50 values of 12.823 ± 1.792 nM and 26.303 ± 2.498 nM in HCT-116 and SW620 cells, respectively. Bufalin decreased cell viability, increased cell death as well as caspase-3 downstream target (cleaved PARP) accumulation, and these actions were significantly blocked by pan-caspase inhibitor zVAD-FMK. Mechanistically, ROS production, but neither the NAD(P)H oxidase, AMPK, ERK nor p38, is responsible for bufalin-induced apoptotic cell death. Moreover, bufalin-induced ROS generation is derived from mitochondria. CONCLUSION: Bufalin significantly induces apoptosis in HCT-116 and SW620 colon cancer cells via mitochondrial ROS-mediated caspase-3 activation. We believe that our novel findings will greatly alter our current understanding on the anti-cancer mechanism of bufalin in colon cancer cells and will pave the way for further exploiting the clinical application.

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.

An optimized protocol to detect protein ubiquitination and activation by ubiquitination assay in vivo and CCK-8 assay.

E3 ubiquitin ligases play a role in protein degradation, cellular localization, and activation, and their dysregulation is associated with human diseases. Here, we present a protocol to detect IGF2BP1 ubiquitination and activation by an E3 ubiquitin ligase FBXO45. We describe steps for preparing cells and transfecting plasmids. We detail the use of western blot to detect IGF2BP1 ubiquitination and a Cell Counting Kit-8 (CCK-8) assay to detect IGF2BP1 activation. This protocol is applicable to other proteins of interest. For complete details on the use and execution of this protocol, please refer to Lin et al. (2021).1.

In vivo analysis of FBXO45-mediated fibrosis and liver tumorigenesis in a chemically induced mouse model of hepatocellular carcinoma.

FBXO45, an E3 ubiquitin ligase highly expressed in liver tumors, is positively correlated with poor survival of hepatocellular carcinogenesis (HCC) patients, but whether FBXO45 drives HCC tumorigenesis remains largely unclear. Here, we describe a protocol that shortens the observation period for HCC tumorigenesis to assess the effects of FBXO45 in a DEN/CCl4-induced HCC mouse model. We describe steps for chemical induction of HCC in FBXO45-overexpressing mice, followed by tissue collection and pathology assessment via quantitative real-time PCR, histology, and immunohistochemistry. For complete details on the use and execution of this protocol, please refer to Lin et al. (2021).1.

Novel hydrocortisone magnetic molecularly imprinted polymers: Preparation, characterization, and application.

In this work, magnetic molecularly imprinted polymers (MMIPs) were prepared by a surface imprinting method using Fe3O4 nanoparticles as a support. The products were characterized by FT-IR spectroscopy, VSM, TGA, SEM, and TEM. Combined with HPLC, hydrocortisone in milk powder and milk were separated and purified, and their contents were monitored. The results showed that MMIPs with a particle size of approximately 1000 nm were successfully prepared. The adsorption mechanism of MMIPs was confirmed by kinetic adsorption and thermodynamic adsorption experiments; the maximum adsorption capacity was found to be 17.2 mg g-1, and adsorption equilibrium could be reached within 40 min. In the actual sample application, the recovery rates of milk powder and milk were 93.88-99.15% and 95.80-98.10%, respectively. These results showed that MMIPs had good performance in selectively identifying hydrocortisone and were suitable for determining hydrocortisone in milk products.

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 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.

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.

FBXW10-S6K1 promotes ANXA2 polyubiquitination and KRAS activation to drive hepatocellular carcinoma development in males.

The incidence rate of human hepatocellular carcinoma (HCC) is approximately three times higher in males than in females. A better understanding of the mechanisms underlying HCC development in males could lead to more effective therapies for HCC. Our previous study found that FBXW10 played a critical role in promoting HCC development in male mice and patients, but the mechanism remains unknown. Here, we found that FBXW10 promoted K63-linked ANXA2 polyubiquitination and activation in HCC tissues from males, and this process was required for S6K1-mediated phosphorylation. Activated ANXA2 further translocated from the cytoplasm to the cell membrane to bind KRAS and then activated the MEK/ERK pathway, leading to HCC proliferation and lung metastasis. Interfering with ANXA2 significantly blocked FBXW10-driven HCC growth and lung metastasis in vitro and in vivo. Notably, membrane ANXA2 was upregulated and positively correlated with FBXW10 expression in male HCC patients. These findings offer new insights into the regulation and function of FBXW10 signaling in HCC tumorigenesis and metastasis and suggest that the FBXW10-S6K1-ANXA2-KRAS-ERK axis may serve as a potential biomarker and therapeutic target in male HCC patients with high FBXW10 expression.

Therapeutic Effect of Catgut Implantation at Acupoint in a Mouse Model of Hepatocellular Carcinoma by Suppressing Immune Escape.

BACKGROUND: The occurrence and development of hepatocellular carcinoma (HCC) are closely related to immune function, as is the capacity of hepatoma cells to escape. Immunosurveillance is a key mechanism. Catgut implantation at acupoint (CIAA) is a promising acupuncture improvement method that can regulate immunity and has been widely used in the clinical treatment of a variety of diseases. The aim of this study is to observe the therapeutic effect of CIAA on HCC and to investigate the potential mechanism of immune escape. MATERIALS AND METHODS: A total of 40 mice were randomly divided into three groups: the HCC model group (n = 15), the CIAA treatment group (n = 15), and the control group (n = 10). HCC was chemically induced in 30 mice by the combination of DEN, carbon tetrachloride, and ethanol for 150 days. Among them, 15 were selected for CIAA treatment to ascertain the therapeutic effect. The mRNA expression levels of AFP, IL-10, PD-1, and CTLA-4 in three groups were examined by using RT-PCR. AFP and AKT expressions were measured by using western blotting. PD1, CTLA-4, IL-10, CD4+, and CD8+ protein expression levels were evaluated by using IHC. The mortality rate, body weight, and psychological conditions of three groups were also compared. RESULTS: The mRNA and protein expression levels of AFP, PD-1, CTLA-4, and IL-10 were significantly downregulated in the CIAA-treated mice in comparison with HCC mice. IHC assay shows that CD4+ and CD8+ expression levels were notably upregulated after CIAA treatment. Western blotting assay shows that AKT pathway was deactivated in CIAA-treated mice. CIAA notably reduced the mortality rate and inhibited weight loss caused by HCC and improved the overall psychological condition of the mice. CONCLUSIONS: Taken together, our data corroborate the effective potency of CIAA in the treatment of HCC by and inhibiting immune escape and deactivating the AKT pathway.

Elevated Sodium Pump α3 Subunit Expression Promotes Colorectal Liver Metastasis via the p53-PTEN/IGFBP3-AKT-mTOR Axis.

The sodium pump α3 subunit is associated with colorectal liver metastasis. However, the underlying mechanism involved in this effect is not yet known. In this study, we found that the expression levels of the sodium pump α3 subunit were positively associated with metastasis in colorectal cancer (CRC). Knockdown of the α3 subunit or inhibition of the sodium pump could significantly inhibit the migration of colorectal cancer cells, whereas overexpression of the α3 subunit promoted colorectal cancer cell migration. Mechanistically, the α3 subunit decreased p53 expression, which subsequently downregulated PTEN/IGFBP3 and activated mTOR, leading to the promotion of colorectal cancer cell metastasis. Reciprocally, knockdown of the α3 subunit or inhibition of the sodium pump dramatically blocked this effect in vitro and in vivo via the downregulation of mTOR activity. Furthermore, a positive correlation between α3 subunit expression and mTOR activity was observed in an aggressive CRC subtype. Conclusions: Elevated expression of the sodium pump α3 subunit promotes CRC liver metastasis via the PTEN/IGFBP3-mediated mTOR pathway, suggesting that sodium pump α3 could represent a critical prognostic marker and/or therapeutic target for this disease.

Elevated FBXO45 promotes liver tumorigenesis through enhancing IGF2BP1 ubiquitination and subsequent PLK1 upregulation.

Dysregulation of tumor-relevant proteins may contribute to human hepatocellular carcinoma (HCC) tumorigenesis. FBXO45 is an E3 ubiquitin ligase that is frequently elevated expression in human HCC. However, it remains unknown whether FBXO45 is associated with hepatocarcinogenesis and how to treat HCC patients with high FBXO45 expression. Here, IHC and qPCR analysis revealed that FBXO45 protein and mRNA were highly expressed in 54.3% (57 of 105) and 52.2% (132 of 253) of the HCC tissue samples, respectively. Highly expressed FBXO45 promoted liver tumorigenesis in transgenic mice. Mechanistically, FBXO45 promoted IGF2BP1 ubiquitination at the Lys190 and Lys450 sites and subsequent activation, leading to the upregulation of PLK1 expression and the induction of cell proliferation and liver tumorigenesis in vitro and in vivo. PLK1 inhibition or IGF2BP1 knockdown significantly blocked FBXO45-driven liver tumorigenesis in FBXO45 transgenic mice, primary cells, and HCCs. Furthermore, IHC analysis on HCC tissue samples revealed a positive association between the hyperexpression of FBXO45 and PLK1/IGF2BP1, and both had positive relationship with poor survival in HCC patients. Thus, FBXO45 plays an important role in promoting liver tumorigenesis through IGF2BP1 ubiquitination and activation, and subsequent PLK1 upregulation, suggesting a new strategy for treating HCC by targeting FBXO45/IGF2BP1/PLK1 axis.

Stereoselective Access to Spirooxindoles and Bisoxindoles Through Organocatalyzed Asymmetric Divergent Transformations of Isatin-derived MBH Carbonates.

An interesting ß-isoquinidine catalyzed divergent reaction was developed to produce either spirocyclopentene oxindoles, spirocyclopentadiene oxindoles or bisoxindoles in a high enantioselective fashion. The utility of this protocol was demonstrated by the versatile transformations of the products. This work not only represents the first highly stereoselective intermolecular catalytic asymmetric allylic alkylation reaction between two isatin-derived MBH carbonate molecules but also constitutes a rare example of isatin-derived MBH carbonate-based enantioselective and α-regioselective [3+2] cycloaddition reactions.

Dimeric aryl-substituted imidazoles may inhibit ALT cancer by targeting the multimeric G-quadruplex in telomere.

In 10-15% of cancers, telomere maintenance is provided by a telomerase-independent mechanism known as alternative lengthening of telomere (ALT), making telomerase inhibitors ineffective on these cancers. Ligands that stabilize telomeric G-quadruplex (G4) are considered to be able to inhibit either the ALT process or disrupt the T-loop structure, which would be promising therapeutic agents for ALT cancers. Notably, the 3'-terminal overhang of telomeric DNA might fold into multimeric G4 containing consecutive G4 subunits, which offers an attractive target for selective ligands considering large numbers of G4s widespread in the genome. In this study, a dimeric aryl-substituted imidazole (DIZ-3) was developed as a selective multimeric G4 ligand based on a G4-ligand-dimerizing strategy. Biophysical experiments revealed that DIZ-3 intercalated into the G4-G4 interface, stabilizing the higher-order structure. Furthermore, this ligand was demonstrated to induce cell cycle arrest and apoptosis, and thus inhibited cell proliferation in an ALT cancer cell line. Cancer cells were more sensitive to DIZ-3, relative to normal cells. Notably, DIZ-3 had little effect on the transcription of several G4-dependent oncogenes. This study provides a nice example for discovering dimeric agents to potentially treat ALT cancers via targeting telomeric multimeric G4.

Reduced IκBα promotes hepatocellular carcinoma cell proliferation and migration via regulation of NF-κB/Erbin axis.

Aberrantly low expression of NF-κB inhibitor α (IκBα) is observed in hepatocellular carcinoma (HCC), yet the underlying mechanism via which IκBα regulates HCC remains largely unknown. Therefore, to determine the potential function of IκBα in hepatocarcinogenesis, the present study used immunohistochemistry (IHC) staining to analyze the associations between IκBα protein expression and clinicopathologic characteristics of 107 patients with HCC. It was found that expression of IκBα was significantly associated with tumor recurrence. Moreover, IκBα protein expression was decreased in 107 HCC tissue samples and was positively associated with overall survival. Mechanistically, it was demonstrated that silencing of IκBα activated NF-κB in both Huh7 and HCCLM3 cells, followed by upregulation of Erbb2 interacting protein (Erbin) at both the mRNA and protein levels, confirmed by reverse transcription-quantitative PCR and western blotting, to promote cell proliferation and migration. Furthermore, knockdown of Erbin significantly attenuated NF-κB-mediated cell proliferation and migration. It was also identified that overexpression of Erbin in HCC tissues promoted both cell proliferation and migration, and was negatively associated with IκBα expression in 107 HCC tissue samples. Thus, these results indicated that downregulation of IκBα promoted HCC tumorigenesis via upregulation of NF-κB-mediated Erbin expression.

The FBXW7-SHOC2-Raptor Axis Controls the Cross-Talks between the RAS-ERK and mTORC1 Signaling Pathways.

FBXW7 is a tumor suppressive E3 ligase, whereas RAS-ERK and mechanistic target of rapamycin kinase (mTORC1) are two major oncogenic pathways. Whether and how FBXW7 regulates these two oncogenic pathways are unknown. Here, we showed that SHOC2, a RAS activator, is a FBXW7 substrate. Growth stimuli trigger SHOC2 phosphorylation on Thr507 by the mitogen-activated protein kinase (MAPK) signal, which facilitates FBXW7 binding for ubiquitylation and degradation. FBXW7-mediated SHOC2 degradation terminates the RAS-MAPK signals and inhibits proliferation. Furthermore, SHOC2 selectively binds to Raptor to competitively inhibit the Raptor-mTOR binding to inactivate mTORC1 and induce autophagy, whereas Raptor binding of SHOC2 inhibits the SHOC2-RAS binding to block the MAPK pathway and proliferation. Finally, SHOC2 is overexpressed in pancreatic cancer, which correlated with poor patient survival. SHOC2 mutations were found in lung cancer tissues with gain-of-function activity. Collectively, the SHOC2-Raptor interaction triggers negative cross-talk between RAS-ERK and mTORC1 pathways, whereas FBXW7 regulates both pathways by targeting SHOC2 for ubiquitylation and degradation.

Cardiac glycoside bufalin blocks cancer cell growth by inhibition of Aurora A and Aurora B activation via PI3K-Akt pathway.

In our previous study, cardiac glycosides including bufalin, a group of sodium pump (Na+/K+-ATPase) inhibitors widely used to treat heart failure for many years, have been demonstrated to induce a delay of mitotic entry and mitotic arrest in many cancer cells. However, the underlying mechanism remains poorly understood. Here, we reported for the first time that cardiac glycoside bufalin induced mitotic entry delay and prometaphase arrest by inhibition of activation of Aurora A/B. Furthermore, cardiac glycoside bufalin prevented Aurora A recruitment to mitotic centrosomes and Aurora B recruitment to unattached kinetochores. Mechanistically, bufalin and knockdown of sodium pump inhibited PI3K-Akt pathway, which in turn inhibit the activation of Aurora A/B, followed by a delay in mitotic entry and mitotic arrest. These actions were reversed by overexpression of Akt. In addition, ERK, mTOR, and ROS are not involved in bufalin-mediated downregulation of active form of Aurora A/B. Taken together, cardiac glycoside bufalin induces mitotic entry delay and mitotic arrest in cancer cells through inhibition of Aurora A/B activation via PI3K-Akt pathway. Based on this novel finding we could suggest that targeting PI3K-Akt pathway may have therapeutic value for the treatment of cancers associated with sodium pump overexpression.