Aqueous-soluble components of sporoderm-removed Ganoderma lucidum spore powder promote ferroptosis in oral squamous cell carcinoma

Objective: Ferroptosis is a novel cell death process which displays a promising role in cancer treatment.However, clinically available drugs targeting ferroptosis are rarely used, and yet there are no studies reporting on inducing ferroptosis via Chinese herbal extracts. Here we explored the tumor inhibition effects of Ganoderma lucidum(G. lucidum) on oral squamous cell carcinoma(OSCC). Specifically, we aimed to clarify the biological mechanism of components in the dietary, aqueous-soluble sporoderm-removed G. lucidum spore powder(A-GSP).Methods: Preliminary transcriptome analysis revealed the significant enrichment of the ferroptosis pathway.Cellular Fe2+, glutathione(GSH), malondialdehyde(MDA), reactive oxygen species(ROS) and lipid peroxide levels were measured to identify ferroptosis occurrence. Western blotting was used to measure ferroptosis-related proteins. Changes in mitochondria morphology and function were observed with transmission electron microscopy(TEM) and ATP detection assays. Ferroptosis inhibitor ferrostatin-1 was then used to verify the anti-tumor effects of A-GSP. Finally, nude mice xenograft models of oral cancer confirmed that A-GSP inhibited tumor growth.Results: A-GSP promoted ferroptosis in oral cancer cells by inducing Fe2+influx, GSH depletion, as well as lipid peroxide and ROS accumulation. Ferroptosis-related proteins exhibited corresponding changes, particularly Acylco A synthetase long chain family member 4(ACSL4) increase and glutathione peroxidase 4(GPX4) decrease. A-GSP considerably lowered mitochondrial volume and ridge number, while significantly decreasing ATP production. Ferrostatin-1 reversed all of these A-GSP-induced changes. In vivo, A-GSP exerted a ferroptosismediated tumor-suppressing effect without observable adverse reactions.Conclusions: Our findings demonstrate the therapeutic potential of A-GSP for treating patients with OSCC by targeting ferroptosis.

AKT2^(S128)/CCTα^(S315/319/323)-positive cancer-associated fibroblasts (CAFs) mediate focal adhesion kinase (FAK) inhibitors resistance via secreting phosphatidylcholines (PCs)

Abnormal metabolism is regarded as an oncogenic hallmark related to tumor progression and therapeutic resistance.Present study employed multi-omics,including phosphoproteomics,untargeted metabolomics and lipidomics,to demonstrate that the pAKT2 Ser128 and pCCTαSer315/319/323-positive cancer-associated fibroblasts(CAFs)substantially release phosphatidylcholines(PCs),contributing to the resistance of focal adhesion kinase(FAK)inhibitors in esophageal squamous cell carcinoma(ESCC)treatment.Additionally,we observed extremely low levels of FAK Tyr397 expression in CAFs,potentially offering no available target for FAK inhibitors playing their anti-growth role in CAFs.Consequently,FAK inhibitor increased the intracellular concentration of Ca2+in CAFs,promoting the formation of AKT2/CCTαcomplex,leading to phosphorylation of CCTαSer315/319/323 sites and eventually enhancing stromal PC production.This activation could stimulate the intratumoral Janus kinase 2(JAK2)/Signal transducer and activator of transcription 3(STAT3)pathway,triggering resistance to FAK inhibition.Analysis of clinical samples demonstrated that stromal pAKT2 Ser128 and pCCTαSer315/319/323 are related to the tumor malignancy and reduced patient survival.Pseudo-targeted lipidomics and further validation cohort quantitatively showed that plasma PCs enable to distinguish the malignant extent of ESCC patients.In conclusion,inhibition of stroma-derived PCs and related pathway could be possible therapeutic strategies for tumor therapy.

CDK7-YAP-LDHD axis promotes D-lactate elimination and ferroptosis defense to support cancer stem cell-like properties

Reprogrammed cellular metabolism is essential for maintaining cancer stem cells(CSCs)state.Here,we report that mitochondrial D-lactate catabolism is a necessary initiating oncogenic event during tumorigenesis of esophageal squamous cell carcinoma(ESCC).We discover that cyclin-dependent kinase 7(CDK7)phosphorylates nuclear Yes-associated protein 1(YAP)at S127 and S397 sites and enhances its transcription function,which promotes D-lactate dehydrogenase(LDHD)protein expression.Moreover,LDHD is enriched significantly in ESCC-CSCs rather than differentiated tumor cells and high LDHD status is connected with poor prognosis in ESCC patients.Mechanistically,the CDK7-YAP-LDHD axis helps ESCC-CSCs escape from ferroptosis induced by D-lactate and generates pyruvate to satisfy energetic demands for their elevated self-renewal potential.Hence,we conclude that esophageal CSCs adopt a D-lactate elimination and pyruvate accumulation mode dependent on CDK7-YAP-LDHD axis,which drives stemness-associated hallmarks of ESCC-CSCs.Reasonably,targeting metabolic checkpoints may serve as an effective strategy for ESCC therapy.

PAFR/Stat3 axis maintains the symbiotic ecosystem between tumor and stroma to facilitate tumor malignancy

Stroma surrounding the tumor cells plays crucial roles for tumor progression.However,little is known about the factors that maintain the symbiosis between stroma and tumor cells.In this study,we found that the transcriptional regulator-signal transducer and activator of transcription 3(Stat3)was frequently activated in cancer-associated fibroblasts(CAFs),which was a potent facilitator of tumor malignancy,and formed forward feedback loop with platelet-activating factor receptor(PAFR)both in CAFs and tumor cells.Importantly,PAFR/Stat3 axis connected intercellular signaling crosstalk between CAFs and cancer cells and drove mutual transcriptional programming of these two types of cells.Two central Stat3-related cytokine signaling molecules-interleukin 6(IL-6)and IL-11 played the critical role in the process of PAFR/Stat3 axis-mediated communication between tumor and CAFs.Pharmacological inhibition of PAFR and Stat3 activities effectively reduced tumor progression using CAFs/tumor co-culture xenograft model.Our study reveals that PAFR/Stat3 axis enhances the interaction between tumor and its associated stroma and suggests that targeting this axis can be an effective therapeutic strategy against tumor malignancy.

Src heterodimerically activates Lyn or Fyn to serve as targets for the diagnosis and treatment of esophageal squamous cell carcinoma

Although Src is one of the oldest and most investigated oncoproteins,its function in tumor malignancy remains to be defined further.In this study,we demonstrated that the inhibition of Src activity by ponatinib effectively suppressed several malignant phenotypes of esophageal squamous cell carcinoma(ESCC)both in vitro and in vivo,whereas it did not produce growthinhibitory effects on normal esophageal epithelial cells(NEECs).Importantly,we combined phosphoproteomics and several cellular and molecular biologic strategies to identify that Src interacted with the members of Src-family kinases(SFKs),such as Fyn or Lyn,to form heterodimers.Src interactions with Fyn and Lyn phosphorylated the tyrosine sites in SH2(Fyn Tyr^(185)or Lyn Tyr^(183))and kinase domains(Fyn Tyr^(420) or Lyn Tyr^(397)),which critically contributed to ESCC development.By contrast,Src could not form heterodimers with Fyn or Lyn in NEECs.We used RNA sequencing to comprehensively demonstrate that the inhibition of Src activity effectively blocked several critical tumor-promoting pathways,such as JAK/STAT,mTOR,stemness-related,and metabolism-related pathways.Results of the real-time polymerase chain reaction(RT-PCR)assay confirmed that Lyn and Fyn were critical effectors for the Src-mediated expression of tumor growth or metastasis-related molecules.Furthermore,results of the clinical ESCC samples showed that the hyperactivation of pSrc Tyr^(419),Fyn Tyr^(185) or Tyr^(420),and Lyn Tyr^(183)or Tyr^(397)could be biomarkers of ESCC prognosis.This study illustrates that Src/Fyn and Src/Lyn heterodimers serve as targets for the treatment of ESCC.

Migfilin promotes migration and invasion in glioma by driving EGFR and MMP-2 signalings:A positive feedback loop regulation

Glioma is the most common type of primary brain tumors in the central nervous system（CNS）. Migfilin occurs in human glioma and enhances cellular motility via the epidermal growth factor receptor（EGFR）pathway. However, the underlying molecular mechanism is not fully understood. In this study, we found that Migfilin promoted matrix metalloproteinase-2（MMP-2） activity, and restrained the expression of tissue inhibitor of metalloproteinase 2（TIMP2）, which is an MMP-2 inhibitor. Functional and structural studies showed that the LIM1 domain of Migfilin was required for Migfilin-mediated TIMP2 expression inhibition and MMP-2 activity, and was also necessary in promoting cell motility. Furthermore, Migfilininduced EGFR phosphorylation was greatly reduced by MMP-2 inhibitor（GM6001） or si RNA, while Migfilin-induced MMP-2 activation was also blocked by the EGFR inhibitor（AG1478） or si RNA. MMP-2 and EGFR inhibitors and their si RNAs can block Migfilin-induced migration and invasion, respectively.These results demonstrated that EGFR and MMP-2 signalings may form a positive feedback loop to enhance Migfilin-induced migration and invasion. Finally, we detected that the expression of Migfilin,EGFR phosphorylation（Tyr1173） and MMP-2 activity had a positive correlation in the clinical glioma sample. Taken together, these results suggest that Migfilin is a critical regulator in cellular motility by driving the EGFR-MMP-2 feedback loop, and may be considered as a potential therapeutic target in glioma.

BAALC-AS1/G3BP2/c-Myc feedback loop promotes cell proliferation in esophageal squamous cell carcinoma

Background:Long non-codingRNAs(lncRNAs)have been found to be involved in the development of many cancers.In this study,we aimed to identify the molecular mechanisms of lncRNA BAALC antisense RNA 1(BAALC-AS1)in regulating the malignancy of esophageal squamous cell carcinoma(ESCC).Methods:The expression of BAALC-AS1 in cancer patients was analyzed using a tissue microarray.The protein and RNA levels of BAALC-AS1 were determined by Western blotting analysis and quantitative reverse transcription-PCR(RT-qPCR),respectively.The cell proliferation was determined by cell viability assays,bromodeoxyuridine incorporation,and flow cytometry.The relationships among BAALC-AS1,RasGAPSH3 domain-binding protein 2(G3BP2),and c-Myc were determined using RNA immunoprecipitation,RNA pull-down assays,and luciferase assays.Results:The expression of BAALC-AS1 was highly up-regulated and associated with malignant phenotypes in ESCC tissues and cell lines.In vivo and in vitro assays showed that BAALC-AS1 promoted ESCC cell proliferation,migration,and invasion.BAALC-AS1 directly interacted with G3BP2,and thereby inhibited the degradation of c-Myc RNA 3’-UTR by G3BP2,thus leading to the accumulation of c-Myc expression.Additionally,c-Myc acted as a transcription factor that can induce the expression of BAALC-AS1 by directly binding to its promoter region.Conclusions:BAALC-AS1/G3BP2/c-Myc feedback loop plays a critical role in the development of ESCC,which might provide a novel therapeutic target and facilitate the development of new therapeutic strategies for the treatment of ESCC.

Tumor-associated macrophage(TAM)-derived CCL22 induces FAK addiction in esophageal squamous cell carcinoma(ESCC)

Tumor cell dependence on activated oncogenes is considered a therapeutic target,but protumorigenic microenvironment-mediated cellular addiction to specific oncogenic signaling molecules remains to be further defined.Here,we showed that tumor-associated macrophages(TAMs)produced an abundance of C-C motif chemokine 22(CCL22),whose expression in the tumor stroma was positively associated with the level of intratumoral phospho-focal adhesion kinase(pFAK Tyr^(397)),tumor metastasis and reduced patient survival.Functionally,CCL22-stimulated hyperactivation of FAK was correlated with increased malignant progression of cancer cells.CCL22-induced addiction to FAK was demonstrated by the persistent suppression of tumor progression upon FAK-specific inhibition.Mechanistically,we identified that diacylglycerol kinaseα(DGKα)acted as a signaling adaptor to link the CCL22 receptor C-C motif chemokine receptor 4(CCR4)and FAK and promoted CCL22-induced activation of the FAK/AKT pathway.CCL22/CCR4 signaling activated the intracellular Ca^(2+)/phospholipase C-γ1(PLC-γ1)axis to stimulate the phosphorylation of DGKαat a tyrosine residue(Tyr^(335))and promoted the translocation of DGKαto the plasma membrane to assemble the DGKα/FAK signalosome,which critically contributed to regulating sensitivity to FAK inhibitors in cancer cells.The identification of TAM-driven intratumoral FAK addiction provides opportunities for utilizing the tumor-promoting microenvironment to achieve striking anticancer effects.

MAGE-C3 promotes cancer metastasis by inducing epithelial-mesenchymal transition and immunosuppression in esophageal squamous cell carcinoma

Background:Evading immune surveillance is necessary for tumor metastasis.Thus,there is an urgent need to better understand the interaction between metastasis and mechanisms of tumor immune evasion.In this study,we aimed to clarify a novel mechanism that link tumor metastasis and immunosuppression in the development of esophageal squamous cell carcinoma(ESCC).Methods:The expression of melanoma-associated antigen C3(MAGE-C3)was detected using immunohistochemistry.Transwell assays were used to evaluate the migration and invasion ability of esophageal squamous cell carcinoma(ESCC)cells.Metastasis assays in mice were used to evaluate metastatic ability in vivo.Lymphocyte-mediated cytotoxicity assays were performed to visualize the immune suppression function on tumor cells.RNA sequencing was performed to identify differentially expressed genes between MAGE-C3 overexpressing ESCC cells and control cells.Gene ontology(GO)enrichment analyses was performed to identify the most altered pathways influenced by MAGE-C3.The activation of the interferon-γ(IFN-γ)pathway was analyzed using Western blotting,GAS luciferase reporter assays,immunofluorescence,and flow cytometry.The role of MAGE-C3 in the IFN-γpathway was determined by Western blotting and immunoprecipitation.Furthermore,immunohistochemistry and flow cytometry analysis monitored the changes of infiltrated T cell populations in murine lung metastases.Results:MAGE-C3 was overexpressed in ESCC tissues.High expression of MAGE-C3 had a significant association with the risk of lymphatic metastasis and poor survival in patients with ESCC.Functional experiments revealed that MAGE-C3 promoted tumor metastasis by activating the epithelial-mesenchymal transition(EMT).MAGE-C3 repressed antitumor immunity and regulated cytokine secretion of T cells,implying an immunosuppressive function.Mechanistically,MAGE-C3 facilitated IFN-γsignaling and upregulated programmed cell death ligand 1(PDŋL1)by binding with IFN-γreceptor 1(IFNGR1)and strengthening the interaction between IFNGR1 and signal transducer and activator of transcription 1(STAT1).Interestingly,MAGE-C3 displayed higher tumorigenesis in immune-competent mice than in immune-deficient nude mice,confirming the immunosuppressive role of MAGE-C3.Furthermore,mice bearing MAGE-C3-overexpressing tumors showed worse survival and more lung metastases with decreased CD8+infiltrated T cells and increased programmed cell death 1(PD-1)^(+)CD8^(+) infiltrated T cells.Conclusion:MAGE-C3 enhances tumor metastasis through promoting EMT and protecting tumors from immune surveillance,and could be a potential prognostic marker and therapeutic target.