Laminar Shear Stress-mediated ATOH8-VEGF Positive Loop Promote Colorectal Circulating Tumor Cells Survival

BackgroundColorectal cancer(CRC)is the third leading cause of cancer-related death in humans,due to the fact that about 20%of patients have distant metastasis at the time of diagnosis.Moreover,up to 60%of patients eventually develop distant metastasis during disease progression.Recent works suggested that hematogenous dissemination is the main way of metastasis in CRC.Tumor cells that are invasive and motile can enter the circulatory or lymphatic system and become circulating tumor cells(CTCs),which is a key step in tumor metastasis.Although most of these cells will perish,but a small proportion manages to resist blood flow shear stress(LSS)as well as the pressure of anoikis,and eventually infiltrates target organs,surviving as disseminated seeds for eventual metastasis.Therefore,research on the molecular mechanisms that support CTCs to resist adverse factors and maintain the intravascular survival yields clues for better understanding the metastatic process,with the aim of preventing tumor progression.Laminar shear stress(LSS)is defined as the internal frictional f'orce between the blood flow and the blood vessel wall or cell surface,which is an important parameter of hemodynamics.However,current research on laminar mechanics in the field of tumors is still relatively scattered and deficient.Our previous review has discussed the dynamic response of tumor cells to LSS related to cell survival.Previously,it is universally believed that LSS can promote tumor cell death.To be specific,LSS activates p38 MAPK/Smad1,5 pathway,causing autophagy and apoptosis of liver cancer and tongue cancer cells.However,researchers have confirmed that prostate cancer has a unique LSS adaptability compared to normal epithelial prostate cancer.Transient stimulation of high LSS to prostate cancer CTCs can help them resist mechanical deformation and improve survival rate after repeated exposure to LSS.In addition,studies have shown that PANX1 channels on breast cancer cells,which are mechanically sensitive,are capable of releasing ATP in response to LSS stimulation,therefore mediating intravascular survival by activating autonomic purine signaling pathways.Since the viability of CTCs is beneficial to metastatic seeding,further investigation into the regulation of CTCs resistance to LSS and its molecular mechanism is of vital significance for predicting tumor progression and targeting distant metastasis.It is noteworthy that the basic helic-loop-helix(bHLH)transcription factor expressed in eukaryotic cells has great contribution in cell differentiation and ontogenesis.Being a member of bHLH family,ATOH8 plays a "permitted" role in the development and differentiation of organs such as kidney,retina,muscle,and pancreas.The deficiency of ATOH8 expression may cause developmental disorders of corresponding organs.At present,there are few reports on the regulation of ATOH8 in tumor cells,especially in CTCs.Another important effect of ATOH8 is embodied as a shear stress-sensing molecule:10dyn/cm2 LSS in umbilical vein endothelial cells(HUEVC)and embryonic stem cells(hESC)can significantly up-regulate ATOH8 mRNA expression.Therefore,we speculate that ATOH8 may mediate the regulation of LSS on hematogenous metastasis of colorectal cancer,serving as a bridge molecule for oncology and laminar mechanics studies.The role of reactive oxygen species(ROS)and redox signals in mechanical transduction is well recognized.The degree of oxidative stress depends on the balance of ROS production and antioxidant defense.ROS production in the physiological range can protect cells from mechanical damage,but when the balance is altered to favour the latter,ROS will mediate apoptosis through mitochondria,death receptors and endoplasmic reticulum stresses.There had shown that LSS can reduce mitochondrial respiration rate,increase mitochondrial membrane potential to enhance mitochondrial ROS production.Furthermore,LSS is able to activate Akt in endothelial cells and raise the ROS level,leading to eventual apoptosis.Otherwise,glycolysis can produce more reducing metabolites to inhibit intracellular ROS levels,thus promoting resistance to oxidative stress damage.In addition,some researchers found that tumor cells in the circulatory system can undergo metabolic transition from mitochondrial respiration to glycolysis,which confers growth advantage and chemical resistance to circulating tumor cells,further suggesting that glycolysis is critical for maintaining survival in CTCs.Like Myc,HIF-la and other key transcription factors that activate the glycolytic process,ATOH8 also belongs to the bHLH transcription factor family.In summary,ATOH8 may mediate the reprogramming of glucose metabolism,which in turn modulate the survival of CTCs.Research contents1.1 Effect of LSS-sensing molecule ATOH8 on intravascular survival of CTCs.Colon cancer cell lines LoVo and SW480 were cultured in a shear stress loading device of 0,5,10,20 dyn/cm2,then we used western blotting,qPCR and immunofluorescence experiments to detect the expression levels of ATOH8.The ATOH8 stably transfected cell line was successfully established using lentivirus,after which the cells under quiescent state and shear stress stimulation were stained with live/dead cells in an attempt to obtain cell survival ratio.In addition,by injecting CT26-GFP and SW480-Luc cells into the tail vein of BALB/c and nude mice,we were able to observe the survival time and ratio of tumor cells in the circulatory system dynamically.Meanwhile,peripheral blood was collected from those mice and analyzed subsequently by flow cytometry for apoptosis rate of tumor cells.2.1 ATOH8 can transcriptionally activate HK2 and promote intravascular survival of CTCs.Immediately after analyzing the differential pathway between CRC metastases and primary lesions as well as subsequent metabolic pathway through GSEA,we performed western blotting and qPCR to identify the glycolytic genes with differential expression after overexpressing or silencing ATOH8.Eventually,HK2 was confirmed as a key metabolic enzyme for subsequent research.The activity of HK2 enzyme was then evaluated using the HK2 enzyme activity assay kit.In order to explore whether ATOH8 has a direct interaction with HK2,the dual luciferase reporter gene assay was performed to assess the binding of the ATOH8 and HK2 promoter regions.Next,we examined the expression changes of apoptosis indicators after overexpressing ATOH8 by western blotting.The effect of the addition of HK2 inhibitors 2-DG and 3-BrPA on the survival of circulating tumor cells was also investigated.3.1 The intermediate link of the upregulation of ATOH8 by LSS.In order to determine the intermediate link of the upregulation of ATOH8 by LSS,we applied multiple chips to analyze the cytokine types of the endothelial cells under the stimulation of LSS.We then co-cultured tumor cells with VEGF cytokines and measured the fluorescent expression of ATOH8 by immunofluorescence assay,intending to further explore the effect of VEGF on ATOH8.Additively,the enzyme activity of HK2 in the cells co-cultured with VEGF was monitored as well as the apoptosis rate of CTCs.While silencing ATOH8,VEGF was added into the culture media to verify the restorative effect of VEGF on ATOH8 expression,HK enzyme activity and apoptosis.4.1 Mechanism of ATOH8-VEGF promoting intravascular survival of CTCs.The content of cytokine VEGF in the supernatant of tumor cells was determined by enzyme-linked immunosorbent assay with overexpression or silence of ATOH8.Meanwhile western blotting and qPCR were conducted to monitor the changed expression of both VEGF and VEGFR-2.Apart from that,we investigated the changes in the downstream PI3K/Akt and ERK pathways when ATOH8 up-regulated the expression of VEGF and VEGFR-2.Then we evaluated the expression of ATOH8,HK2 and apoptosis of cells in reaction to VEGFR-2 inhibitor and Akt inhibitor AZD5363.Results1.LSS-sensing molecule ATOH8 promotes intravascular survival of CTCs.While cultured in a shear stress loading device of 0,5,10,20 dyn/cm2,the expression of ATOH8 in LoVo and SW480 cell lines increased with the rise of the shear stress,which is in line with the results obtained by immunofluorescence.Live/dead cells staining further revealed that overexpression of ATOH8 significantly enhanced tumor cell survival,both at rest and under shear stress.In animal experiments,ATOH8-overexpressing CT26-GFP cells survived longer in lung with more cells detected in the sinusoids of liver compared to the control group.Consistently,data from flow cytometry implied that a smaller proportion of ATOH8-overexpressing tumor cells undergo apoptosis.2.ATOH8 can transcriptionally activate HK2 and promote intravascular survival of CTCs.The GSEA analysis found that the glycolytic pathway of metastatic tumor cells was remarkably activated in comparison to primary colorectal cancer.And overexpression of ATOH8 dramatically increased the enzymatic activity of HK2.When ATOH8 was silenced by shRNA,the activity of HK2 also declined.According to the database,the 563-558 and the 639-644 base of the HK2 promoter region were E-box structures,which is highly probable to be the binding region of ATOH8 regulating HK2 transcription.It can be observed in the dual luciferase reporter gene asssay that the expression of HK2 luciferase was significantly increased with the overexpression of ATOH8,and this effect vanished with the deletion of the 563-558 base.In addition,when overexpressing ATOH8,the expression of Bax increased and Bcl-2 descended,while silencing ATOH8 obtained the opposite effect.Moreover,HK2 inhibitors 2-DG and 3-BrPA apparently reduced the survival rate of circulating tumor cells,while overexpression of ATOH8 partially restored the above results.3.Secretion of VEGF under LSS can up-regulate ATOH8 and its downstream glycolysis pathway.Multiple chip analysis revealed that laminar flow could stimulate the secretion of VEGF in endothelial cells.When tumor cells were co-cultured with VEGF cytokines,the fluorescence intensity of ATOH8 was markedly boosted.Furthermore,stimulation of VEGF can also strength the enzyme activity of HK2 and diminish apoptosis rate in circulating tumor cells.In the rescue experiment,VEGF was able to up-regulate ATOH8 expression,HK enzyme activity and reduce tumor cell apoptosis when ATOH8 was silenced.4.ATOH8-VEGFpositive feedback loop enhances intravascular survival of CTCs.As the enzyme-linked immunosorbent assay showed us,cell lines overexpressing ATOH8 possessed higher content of VEGF in the culture supernatant and increased expression of VEGF as well as VEGFR-2,with the phosphorylated Akt pathway activated-and vice visa.Otherwise,VEGFR-2 and Akt inhibitor were able to inhibit the expression of ATOH8 and HK2,thus inducing apoptosis.Consistently,overexpression of ATOH8 can partially restore the above results.ConclusionLSS enhances ATOH8 expression in colorectal cancer cells by activating"VEGF/VEGFR2/PI3K-Akt" axis pathway;the upregulation of ATOH8 subsequently activates HK2 signaling pathway,promotes VEGF expression,and eventually amplifies the LSS signal to promote colorectal cancer resistance.