FBXL16 Inhibits The Hippo Signaling Pathway By Promoting The Ubiquitination Of LATS1/2 To Promote The Biological Behavior Of Breast Cancer Cells

Objective:Breast cancer is the most frequently diagnosed malignant tumor in women worldwide,and the incidence rate is increasing.Currently,breast cancer has surpassed lung cancer to become the world’s largest cancer.Although substantial progress has been made in the early diagnosis and treatment of breast cancer,those with distant metastases are often incurable.And many breast cancer survivors experience lasting physical effects from surgery and radiation therapy,and may be at risk for cancer recurrence or development of a second primary malignancy,making the treatment of these patients an area of continued research.Advances in tumor genetics have transformed traditional breast cancer treatments,and identifying targeted mutations may help select which drugs are more likely to be effective against specific tumor types.Advances in some signaling pathways have led to the development and approval of many new drugs,changing the treatment prospects for patients with metastatic breast cancer.Although advances in tumor biology research have allowed us to make significant progress in breast cancer treatment,due to the complexity of tumors,there are still great challenges in real clinical research and practice,so it is necessary to enhance the understanding of tumor biology,understanding of oncogenic mechanisms and driving alterations to increase the ability to identify reliable prognostic and predictive biomarkers.In mammals,the key link in the activation of Hippo signaling pathway is to promote the phosphorylation of the transcription activator yes-associated protein（YAP）and its paralog WW domain–containing transcription regulator protein 1（TAZ）through the kinase cascade,and inhibiting their nuclear entry,thereby limiting tissue growth and cell proliferation.The key regulators are large tumor suppressor 1（LATS1）and large tumor suppressor 2（LATS2）kinases.LATS1/2 directly phosphorylate YAP and TAZ at multiple sites.The phosphorylated YAP/TAZ is localized in the cytoplasm,inhibiting its entry into the nucleus,and then degraded by the 14-3-3 protein through the ubiquitinated proteasome pathway.In contrast,unphosphorylated YAP/TAZ can enter the nucleus and interact with DNA-binding proteins,including those in the TEAD family,to trigger important transcriptional programs of genes that promote cell proliferation and migration.F-box and leucine-rich repeat protein 16（FBXL16）is a member of the F-box protein family with an N-terminal proline-rich domain,an F-box motif and a C-terminal LRR domain.At present,the role of FBXL16 in malignant tumors is still seldom studied.The Gene Expression Profiling Data Dynamic Analysis（GEPIA）database showed that compared with normal breast tissue,FBXL16 is highly expressed in breast cancer tissues,and compared with those with high FBXL16 expression,the survival rate of breast cancer patients with low expression of FBXL16 was increased.Genemania,Hitpredict and other websites have predicted that there may be a protein interaction between FBXL16 and LATS2 with high confidence.The LATS2 kinase and its homolog LATS1 are key regulators of the Hippo pathway,so we hypothesized that FBXL16 may interact with LATS2 and LATS1 to regulate their expression in breast cancer cells,regulate the activity of the Hippo signaling pathway,and then affect the biological behavior of breast cancer cells and provide new targets for the treatment of breast cancer.Methods:1.First,we searched multiple databases such as GEPIA,Bio GRID,Kaplan-Meier,Genemania,Hitpredict and STRING to evaluate the difference in the expression of FBXL16 in normal breast tissues and breast cancer tissues,and its relationship with the survival rate of breast cancer patients,and some proteins that may interact with FBXL16,providing a theoretical basis for this study.2.40 female breast invasive ductal carcinoma specimens and 32 breast cancer paracancerous specimens from the Department of Pathology,The First Affiliated Hospital of China Medical University,from January 2014 to December 2016,were randomly selected for immunohistochemical staining to investigate the role of FBXL16 in the expression in breast cancer.The patients were aged from 28 to 81 years old,and the existing follow-up data such as age,tumor size,histological grading,hormone receptor expression and lymph node metastasis were analyzed for correlation.Immunohistochemical staining was performed on all sections using the SP method,and semiquantitative scoring was performed based on staining intensity and extent of stained cells.Based on preliminary experiments,we knew that FBXL16 was localized in the cytoplasm in breast tissue,so we performed subsequent scoring of the cytoplasmic stained sections.The staining intensity of FBXL16 was as follows:0 point（no expression）,1 point（weak expression,light yellow particles）,2 points（moderate expression,yellow particles）,3 points（strong expression,brown-yellow particles）.The staining range is:0（no staining）,1（1-25%range staining）,2（26-50%range staining）,3（51-75%range staining）,4（76-100%range staining）.The scores of each section were multiplied to obtain a final score of0 to 12.Sections with a score greater than 3 were considered positive for FBXL16expression,and sections with a score less than 3 were considered negative.3.The cell lines used in this experiment mainly include normal mammary duct epithelial cells MCF-10A,breast cancer cell lines MCF-7（Luminal type）,SK-BR-3（Her-2overexpression type）and MDA-MB-231（Triple-negative type）were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.All cell cultures were performed under sterile conditions.MCF-10A cells were cultured in DMEM/F12（1:1）medium containing 5%horse serum,20 ng/ml EGF and 10μg/ml insulin.MCF-7 and SK-BR-3were cultured in DMEM high glucose medium containing 10%fetal bovine serum.MDA-MB-231 was cultured in L15 medium containing 10%fetal bovine serum.All cells were incubated in a 37℃,5%CO₂ incubator with fresh medium every 1-2 days.4.In order to detect the expression of FBXL16 in different cell lines and the nuclear and cytoplasmic localization of YAP,we carried out immunofluorescence experiments.Different cells were seeded in 24-well plates incubating at 37℃,5%CO₂ incubator for 18-24 hours.The 24-well plate was then taken out,fixed with 4%paraformaldehyde for 20minutes,punched with 0.2%Triton X-100 for 15 minutes,blocked with 3%BSA or goat normal serum for 1 hour at room temperature,and added with a specific primary antibody（FBXL16 1:100,YAP 1:100）,incubated overnight at 4℃.After 18 hours,the primary antibody was removed,fluorescent secondary antibody（1:100）was added in the dark,and incubated for 2 hours at room temperature in the dark.Finally,the nuclei were stained with DAPI for 10 minutes.The expression and localization of FBXL16 and YAP were observed under a fluorescence microscope,and images were collected.5.The transfection reagent used in this experiment is Attractene transfection reagent.Transfection was performed when the cell density reached about 70%.For a 2ml system culture dish or six-well plate,1.2μg of plasmids（PCMV6,Myc-DDK-FBXL16,LATS1,LATS2,HA-Ub）and 4.5μl of Attractene transfection reagent were added to 100μl of serum-free medium or MEM medium,standed at room temperature for 15 minutes,and then added it to the adherent cells,incubating in a 37℃,5%CO₂ incubator.The medium was replaced with fresh medium after 24 hours,and the cells were harvested after 48 hours.Lipofectamine 3000 was used for interference in this experiment.The interference was carried out when the cell density reached about 50%.For a 2ml system,7.5μl of lipo was placed in 250μl of serum-free medium or MEM medium,and standed for 5 minutes to obtain solution A.Put 7.5μl of NC-siRNA or FBXL16/LATS1/LATS2-siRNA interference sequence in 250μl of serum-free medium or MEM medium,which is B solution.Mix A and B solution,let it stand at room temperature for 15 minutes,add it to the cells incubating in a 37℃,5%CO₂ incubator,and replace the fresh culture medium after 24 hours.Cells were harvested after 48 hours.6.For cell protein extraction experiment,the cells after transfection or interference for 48h were collected.Total protein was extracted by adding NP40 cell lysate（the protease inhibitor PMSF and phosphatase inhibitor were added in advance in the lysate,the ratio was 100:1:1）,lysed on ice for 30 minutes,and centrifuged at 12,000rpm for 20minutes using a low-temperature（4℃）high-speed centrifuge.The obtained supernatant is the total protein.Adding 200μl of cytoplasmic protein extraction reagent A supplemented with PMSF to each 20μl of cell pellet,vortexing for 5 seconds to disperse the cell pellet,lysing on ice for 10 minutes,and then adding 10μl of cytoplasmic protein extraction reagent B,and vortexing for 5 seconds,lysing on ice for 1 minute,vortexing for 5 seconds,centrifuging at 12000rpm for 5 minutes in a low temperature（4℃）,and the obtained supernatant is the cytoplasmic protein.Use filter paper for the precipitation to completely absorb the residual supernatant,adding 50μl of nuclear protein extraction reagent,vortexing until the cell precipitate is completely dispersed,lysing on ice,and vortexing for15-30 seconds every 1-2 minutes for a total of 30 minutes,centrifuging at 12,000 rpm for10 minutes in a low-temperature（4℃）,and the obtained supernatant is nuclear protein.7.In order to clarify the interaction between FBXL16 and LATS1/LATS2 and the effect of FBXL16 on the ubiquitination levels of LATS1 and LATS2,we carried out co-immunoprecipitation experiments.Collecting total protein,we added 20μl of mixed A/G magnetic beads to each group of protein lysates,incubating at 4℃ on a rotary shaker for 2hours to block,and aspirating the supernatant.Add 1μg of specific primary antibody or Ig G antibody of the same property to each 1 mg of protein,and incubate overnight at 4℃ on a rotating shaker.Add Protein A+G magnetic beads after 18-24 hours,incubate for 4-6hours on a rotary shaker at 4℃,centrifuge at 1000rpm for 10 minutes in a low temperature（4℃）centrifuge,discard the supernatant,and wash the pellet with NP40 lysis buffer for three times.The same volume of 2×SDS loading buffer was added to the obtained immune complexes to make the final concentration 1×,boiled in boiling water for 10 minutes to fully denature the protein,and then detected by Western blot experiment.8.Western blot was used to detect protein expression.We measured the protein concentration with UV spectrophotometer or Bradford method.The same amount of protein was mixed with 6×SDS loading buffer to make the final concentration 1×,boiled in boiling water for 5 minutes to fully denature the protein.The protein samples with the same amount were added to 8%or 10%gel in a volume of 15μl,and proteins of different molecular weights were separated by SDS-PAGE electrophoresis,and then the proteins were transferred to PVDF membrane by wet transfer method.After washing with the membrane washing solution,the PVDF membrane was added to 5%skim milk in a shaker at room temperature to block for 2 hours,and then the PVDF membrane of the corresponding molecular weight was placed in the specific primary antibody and incubated overnight at 4℃ in a refrigerator.After 18-24 hours,the PVDF membranes were taken out,and then incubated with the corresponding secondary antibody labeled with HRP at room temperature for 2 hours.Then,the ECL method was used for luminescence,and images were collected,and the gray value of the protein bands was detected by Image J software.9.We filtered MCF-7 cells stably transfected with FBXL16 with G418.The cells were seeded in a 24-well plate and cultured for 16-18 hours.Different concentration gradients of G418（1-1100μg/ml）were added,and the medium was changed every two days.After culturing for 10 days,the concentration at which the cells just completely died,the screening concentration（400μg/ml）and the maintenance concentration（200μg/ml）were determined.The cells were seeded into 24-well plates and transfected.After 48 hours,the selected concentration of G418 was added.The culture medium containing the selected concentration of G418 was replaced every 2-3 days.Colonies were transferred to sterile petri dishes for continued cultivation.After about 28 days of screening,the transfection efficiency was measured by Western blot experiment,and the MCF-7 cell line stably expressing FBXL16 was obtained.10.In order to verify the in vivo biological function of FBXL16,we designed an in vivo tumorigenesis experiment in nude mice.The MCF-7 cells stably transfected with FBXL16were cultured,and the cells were harvested when they were in the logarithmic growth phase to prepare a cell suspension with a cell number of 5×10⁷/ml.200μl of untreated MCF-7 cells and MCF-7 cells stably transfected with FBXL16 were subcutaneously injected into the armpits of 4-5 weeks old BALB/c Nude mice that were randomly divided into two groups,and were reared aseptically for 25 days.Tumor volumes were measured and recorded at 0,5,10,15,20,and 25 days,respectively.The nude mice were sacrificed by cervical dislocation,taking pictures,taking out the tumor,and measuring the size and weight of the tumor.11.MTT assay was used to detect cell proliferation activity.100μl of cell suspension containing 3,000 cells were added to 96-well plates and cultured for 5 days.The original culture medium was replaced with serum-free medium containing 10μl of MTT at a fixed time every day,protecting from light,and incubating in a 37℃,5%CO₂ incubator for 4hours.Subsequently,the culture medium was discarded,150μl DMSO was added to dissolve MTT,and the cells were incubated on a shaker at room temperature for 10 minutes.The absorbance value at 490 nm wavelength was detected by a microplate reader,and a line graph was drawn.Colony formation assay was used to detect the ability of cell clone formation.Count 1,000 cells in each treatment group and add them to a six-well plate or petri dish containing 2 ml of culture medium.Fresh culture medium was replaced every 3-4 days.Incubating at 37℃,5%CO₂ cultured for about 10 days.Discard the medium,pre-cool methanol for 15 minutes,stain with hematoxylin for 10 minutes,and turn back to blue in tap water for 20 minutes.Images were acquired and colonies were counted.12.Transwell cell invasion assay was used to detect the invasive ability of cells.The correspondingly treated cells were prepared into a cell suspension with a culture medium containing 2%serum,the cells were counted,and 200ul of cell suspension was prepared and added to the upper chamber.Add fresh culture medium containing 20%serum to the lower chamber and incubate for 16-18 hours in a 37℃,5%CO₂ incubator.Pre-cooled methanol for 15 minutes,hematoxylin staining for 15 minutes,differentiated twice with 1%hydrochloric acid alcohol,and returned to blue in tap water for 10 minutes.Cells were counted with a microscope and images were collected.13.We analyzed the clinicopathological correlation of FBXL16 expression by Pearson’s chi-square test.The differences between the experimental group and the control group or between the experimental groups were analyzed by paired t test.Differences were considered statistically significant when P<0.05,and all experiments were repeated at least three times.Results:1.The results of the GEPIA database showed that compared with normal breast tissue,FBXL16 was highly expressed in breast cancer tissue,and compared with those with high FBXL16 expression,the survival rate of breast cancer patients in the low FBXL16 expression group was increased.The results of immunohistochemical staining showed that the expression of FBXL16 was low or not in normal breast ductal epithelium,and the expression of FBXL16 was significantly increased in breast cancer tissue,and it was mainly located in the cytoplasm.The results of clinicopathological analysis showed that the expression of FBXL16 in the 40 breast cancer tissues was positively correlated with the histological grading of the patients,and the results were statistically significant（P<0.05）.The results of cell line immunofluorescence showed that FBXL16 was mainly localized in the cytoplasm in both normal breast epithelium（MCF-10A）and different breast cancer cell lines MCF-7,SK-BR-3 and MDA-MB-231.The results of cell line Western blot showed that the expression of FBXL16 in three breast cancer cell lines was higher than that in normal mammary duct epithelial cells.2.In order to verify the biological function of FBXL16 in breast cancer cells,MCF-7 cells and SK-BR-3 cells were transfected and interfered with FBXL16 to regulate the protein expression of FBXL16 bidirectionally.The results of MTT and colony formation experiments showed that the cell proliferation ability was enhanced after transfection with FBXL16,and the cell proliferation ability was weakened after interference with FBXL16.Transwell assay was used to detect the invasive ability of the cells,and the results showed that the invasive ability of the cells was enhanced after transfection with FBXL16,and the invasive ability of the cells was weakened after interfering with FBXL16.3.In order to verify the in vivo biological function of FBXL16,tumorigenic experiments in nude mice were carried out.Untreated MCF-7 cells and MCF-7 cells stably transfected with FBXL16 were injected into nude mice.The results showed that overexpression of FBXL16 could significantly promote the tumor formation in nude mice.4.Genemania,Hitpredict and other websites have predicted that there may be a protein interaction between FBXL16 and LATS2 with high confidence,and LATS2 kinase and its homolog LATS1 are key regulators of the Hippo pathway.Therefore,after bidirectional regulation of FBXL16,we detected the protein expression levels of LATS2,LATS1 and key proteins of Hippo signaling pathway and downstream target genes,and the nucleoplasmic localization of YAP.The results showed that FBXL16 can inhibit the activation of Hippo signaling pathway.5.In order to clarify the interaction between FBXL16 and LATS2 or LATS1,co-immunoprecipitation experiments were carried out in MCF-7 and SK-BR-3 cell lines.The results showed that FBXL16 could bind to LATS2 and LATS1.Previous experiments have confirmed that FBXL16 can down-regulate the protein expression of LATS2 and LATS1.Therefore,after transfection of FBXL16 in MCF-7 cells,the ubiquitination level and protein stability of LATS2 and LATS1 were detected.The results showed that FBXL16can downregulate their expression by promoting ubiquitinated proteasome pathway.6.In order to clarify whether the regulation of FBXL16 on Hippo signaling pathway and the biological behavior of breast cancer cells depends on LATS1 and LATS2,a reverse experiment was performed.The expression of LATS1 and LATS2 was down-regulated while interfering with FBXL16 in MCF-7 cells.The key proteins of Hippo signaling pathway were detected by Western blot,and functional experiments were carried out.The results showed that down-regulation of LATS1/2 could completely reverse the activation of Hippo signaling pathway by down-regulation of FBXL16,indicating that the regulation of FBXL16 on Hippo signaling pathway is dependent on the activity of LATS1 and LATS2kinases.However,the results of functional experiments showed that down-regulation of LATS1/2 could only partially reverse the effects of down-regulation of FBXL16 on the biological function of breast cancer cells,suggesting that there are still other mechanisms for the regulation of biological behavior of breast cancer cells by FBXL16.Conclusion:1.The expression of FBXL16 is increased in breast cancer tissues and cell lines,and has clinicopathological correlation.2.FBXL16 can promote the biological behavior of breast cancer cells both in vivo and in vitro.3.FBXL16 can inhibit the activation of Hippo signaling pathway.4.FBXL16 can bind to LATS1/2 and downregulate their protein expression by promoting the ubiquitinated proteasomal degradation pathway.5.FBXL16 regulates the Hippo signaling pathway through LATS1/2,thereby regulating the biological behavior of breast cancer cells.