The Effect And Mechanism Of ?Np63? On Bortezomib Resistance In Head And Neck Squamous Cell Carcinoma

Background Head and neck cancer(HNC)refers to a group of malignant tumors that originate from part of the upper respiratory and digestive system,which include oral cavity,oropharynx,hypopharynx,and larynx.According to the statistics of 2018 GLOBECAN,in 2018 alone,there were 705,781 new cases of head and neck cancer worldwide,and 358,144 patients died of HNC,accounting for 3.9% incidence and 3.7% mortality of systemic malignancies.Because HNC often has the same pathological origin,we often study tumors in different parts of head and neck as one type of disease.Head and neck squamous cell carcinoma(HNSCC)is the most common type of HNC.At present,more than 50% of patients with HNSCC have undergone local metastasis or are in advanced clinical stage at the time of consultation in our country.For these patients,traditional treatment has not increased the long-term survival rate.Small molecule targeted drugs are increasingly becoming an important adjuvant therapy.It can specifically recognize and kill tumor cells,and has potential application prospects.In the past ten years,the application of proteasome inhibitors in the treatment of hematological malignancies has greatly expanded.In 2003,bortezomib was approved by the FDA for the treatment of recurrent multiple myeloma and mantle cell lymphoma.Significant progress has been made in the treatment of MM and pediatric / adult acute leukemia cases,but slow progress has been made in the treatment of solid tumors.Maybe there are many reasons for this,but the inherent resistance mechanism in solid tumor cells is probably the most important reason.In general,mutations,overexpression,or persistent activation of certain proteins in a cell may contribute to drug resistance.These proteins can reduce tumor toxicity by activating or inhibiting certain cellular pathways so that tumor cells do not undergo apoptosis caused by drug.Tumor sequencing studies show that about 20%-25% of HNSCC have TP63 amplification.?Np63 subtype overexpression is more common,and is upregulated in more than 80% of HNSCC cases.The ?N subtype is a dominant inactive form of p63.By inhibiting the transcription of cell cycle regulatory protein P21,tumor cell proliferation can be promoted.In HNSCC,highly expressed ?Np63? can promote tumor cell survival and is an important prognostic marker for HNSCC.Our previous research on the drug sensitivity screening platform found that different HNSCC cell lines have significantly different sensitivities to bortezomib,and also found that individual relatively sensitive HNSCC cell lines have less ?Np63? expression.Based on this finding,we speculate that ?Np63? may be a key factor in the resistance of HNSCC cells to bortezomib.The correlation between ?Np63? and bortezomib resistance and the role of ?Np63? in drug resistance need further study.Under normal physiological conditions,the ?5 subunit of the proteasome exerts chymotrypsin-like activity to degrade ubiquitinated proteins.Bortezomib covalently binds to the ?5 subunit of the proteasome,inhibiting this part of the proteasome activity and causing a large number of unfolded proteins and misfolded proteins to aggregate instantaneously,which may induce endoplasmic reticulum stress.Endoplasmic reticulum stress disrupts cell homeostasis and can produce unfolded protein responses.Unfolded protein responses can induce excessive ROS production.In turn,ROS can also promote endoplasmic reticulum stress to eventually activate apoptosis and cause cell death.The ?5 subunit is the key point of the effect of bortezomib.We will investigate whether there is overexpression or mutation of the ?5 subunit in the HNSCC resistant strains for resistance bortezomib.Cytoglobin(CYGB)is a member of the globin family.It has a compact helix conformation and can reversibly bind to diatomic gas molecules such as O2,NO,CO.Other studies have confirmed that CYGB in normal squamous epithelial cells can clear excess ROS and protect cells.Excessive ROS production during bortezomib-induced apoptosis,whether CYGB mediates the process of clearing ROS,there is no research in this area,we speculate that CYGB of HNSCC strains may play a role in clearing excess ROS to inhibit apoptosis and promote cell survival.We will further study the role of CYGB in this process and whether there is an interaction between ?Np63? and CYGB.Research methods and resultsPart I: Validation of the relationship between ?Np63? expression level and drug sensitivity of bortezomib in HNSCCThe CCK8 method was used to detect the toxicity of bortezomib to four HNSCC cell lines(UM-SCC-17 A,UM-SCC-17 B,UM-SCC-11 and HN31),and IC50 values were measured.The expression of ?Np63? in four HNSCC lines was detected by Western-Blot.It was found that UM-SCC-11 and HN31 cells had high protein expression and relatively high IC50 values(9.48 n M and 10.78 n M,respectively);UM-SCC-17 A and UM-SCC-17B cells had low protein expression and relatively low IC50 values(3.97 n M and 3.66 n M,respectively).By gradually inducing the sensitive cell line UM-SCC-17 B in vitro,a drug-resistant cell line 17B-R-10 n M(IC50: 412.9n M)was obtained,and ?Np63? expression was significantly increased.Part II Assessing the role of ?Np63? in bortezomib-resistant HNSCC cell linesHN31 and 17B-R-10 n M were knocked down by lentiviral transfection to turn to stable transfected strains HN31sh?Np63? and 17B-R-10 n M-sh?Np63?.Verifing the transfection efficiency by Western-Blot,the toxicity of bortezomib to the transfected strain was detected by the CCK8 method.The results showed that: compared to HN31 sh Non,the IC50 of HN31sh?Np63? decreased from 10.48 n M to 5.21 n M;compared to 17B-LV?Np63?,the IC50 increased from 3.86 n M to 13.31 n M;compared to the control group,the 17B-R-10 n M-sh?Np63? was reduced from 407.2 n M to 70.69 n M.Nude axillary tumors were formed by inoculating the above-mentioned stable-transforming cell lines and drug-resistant cell lines.Bortezomib was injected into the tail vein for six weeks.The changes in tumor size and body weight of nude mice were observed,and the time of nude mouse death was recorded.Tumor size changes in nude mice showed that compared with HN31 sh Non + bortezomib group and HN31 sh Non + saline group,the tumor growth was fastest in the HN31 sh Non + saline group.In the 17B-LVNon + bortezomib group,the tumor began to grow slowly after 30 days.Compared with the 17B-LVNon + bortezomib group and the 17B-LV?Np63? + bortezomib group,the 17B-LV?Np63? + saline group grew faster(P <0.001).Compared with the parent strain + bortezomib group,the tumor volume of the 17B-R + bortezomib group grew faster(P <0.001).Nude mouse body weight showed that in each group,the HN31 sh Non + saline group,17B-LV?Np63? + saline group,and 17B-R + bortezomib group had the fastest weight loss in each group(P <0.001).Survival analysis results showed that the median survival times of the HN31 sh Non + saline group,HN31 sh Non + bortezomib group,and HN31sh?Np63? + bortezomib group were 49 days,60 days,and 76 days,respectively.The median survival time of the 17B-LV?Np63? + saline group,the 17B-LV?Np63? + bortezomib group,and the 17B-LVNon + bortezomib group were 49 days,57 days,and 73 days.The median survival times of the rice group and the 17B-P + bortezomib group were 47 days and 67 days.via PSMB5 in HNSCC Part III Mechanis m of ?Np63? induced resistance to bortezo m ibThe proteasome activity of HNSCC parent strains,induced drug-resistant strains,and each stable transfected strain was tested.The results showed that compared with the parent strain,the induced protease activity of the drug-resistant strains was enhanced,but There is no significant difference between different drug-resistant strains;compared with HN31-sh Non,HN31-sh?Np63? is significantly reduced;compared with 17B-LVNon,17B-LV?Np63? is significantly increased.Through SNP detection,genotyping results showed that no genetic mutations of PSMB5 were found in resistant strains.Western-Blot detection of PSMB5 expression showed that compared to the parental strains 17B-P,drug-resistant strains 17B-R-5n M and 17B-R-10 n M,PSMB5 expression was increased,but the difference between the two strains was not significant;Compared with 17B-LVNon,the PSMB5 of 17B-LV?Np63? increased significantly;compared with HN31-sh Non,the PSMB5 of HN31-sh?Np63? decreased.Immunohistochemical examination of nude mice showed that ?Np63? protein was mainly expressed in the nucleus,and PSMB5 was expressed in both the cytoplasm and the nucleus,and there was a positive correlation between the two,with a correlation index of 0.343(P = 0.002).Part IV Study Mechanism of ?Np63?-CYGB-ROS regulating HNSCC resistance to bortezomibFlow cytometry was used to measure the ROS level of HNSCC treated with bortezomib.The results showed that intracellular ROS was produced in large quantities after drug action,first increased and then decreased,and the peaks appeared in 5 hours;ROS production was increased in CYGB knocked down cell lines;the ROS produced by ?Np63? overexpressed cells were low,and the ROS produced by ?Np63? underexpressed cells were increased.CYGB methylation test results show that the methylation rate of the promoter of the HNSCC line CYGB used in this study is very low,and the total methylation rate of each strain is no more than 1%.By CHIP-seq detection,the results showed that CYGB was the transcriptional regulatory site of ?Np63?.The detection of the double luciferase reporter gene report showed that ?Np63? significantly enhanced the CYGB promoter activity.The expression of CYGB was detected by Western-Blot.The results showed that the expression of CYGB in drug-resistant strains was increased;compared with the control,CYGB in the HN31sh?Np63? cell line was significantly reduced;Immunohistochemical detection of nude mice tumors showed that: CYGB was mainly expressed in the cytoplasm and was positively correlated with ?Np63? with a correlation index of 0.464(P = 0.019).Conclusion1.?Np63? is a key factor in the resistance of HNSCC.High expression of ?Np63? can increase the resistance of HNSCC to bortezomib.Decreasing the expression of ?Np63? can reduce the resistance of head and neck squamous cell carcinoma to bortezomib.2.Relative to the parent strain,the proteasome activity and PSMB5 protein expression of the resistant strain were increased at relatively low drug resistance concentrations.However,with the increase of the drug resistance index,proteasome activity and PSMB5 protein expression no longer increase.3.?Np63? expression is related to PSMB5 expression in HNSCC.4.?Np63? regulates the expression of CYGB in HNSCC.CYGB is the target of transcription regulation of ?Np63?.It reduces apoptosis by reducing excess ROS produced by bortezomib,and thus exerts drug resistance.