Expression And Effects Of ROR2 In High-grade Serous Ovarian Carcinoma And Underlying Mechanism

BackgroundEpithelial ovarian cancer(EOC)is the most lethal cancer in female genital tumors due to absence of screening methods and limited treatments to recurrence.Among the main subtypes of this disease,high-grade serous ovarian carcinoma(HGSOC)accounts for majority part.Prognosis of patients with advanced stages(International Federation of Gynecology and Obstetrics,FIGO stage ???)has met marginal improvement even with unceasing development of technology and medicine.Hence,it's urgent to identify new disease markers and discover novel therapeutic strategies considering the current treatment status.ROR2,one of the receptor tyrosine kinase-like orphan receptors(RORs),belongs to the tyrosine kinase receptor family(RTKs).When the extracellular domain combines with related ligands,the intracellular domain activates signalling pathways,participating in cell proliferation,differentiation and migration.Character of ROR2 in tumors has faced a bit of controversy recently.More and more researches show ROR2 plays dual roles,either promoting or suppressing cancer,in tumors depending on tumor type and tumor context.ROR2 plays as tumor promoter in renal cancer,chronic lymphocytic leukemia,malignant melanoma,whereas its high expression was shown to be related with better prognosis in patients with colorectal cancer,liver cancer,medulloblastoma and endometrial cancer.Different roles are even played within the same kind of tumor.For ROR2 and its downstream cascades there is a need for more research.Endoplasmic reticulum stress(ERS)is a cellular dysfuction causing by accumulation of unfolded proteins in ER induced by factors like calcium imbalance,inflammation,oxidative stress and so on.ERS is an important mechanism for cells to survive during stress while excessive or severe one could cause cell apoptosis.Unfolded protein response(UPR)is originally a mechanism to relieve ERS via modulating three endoplasmic reticulum transmembrane receptor proteins:protein kinase-like endoplasmic reticulum kinase(PERK),inositol-requiring enzyme 1(IRE1),activating transcription factor 6(ATF6).If UPR is failed to relieve ERS,apoptosis is on the way.Three apoptotic pathways induced by ERS include CCAAT/enhancer-binding protein homologous protein(CHOP/GADD153)pathway,c-Jun N-terminal kinase(JNK)depending pathway and cysteinyl aspartate specific proteinase(caspase)family,among which CHOP and JNK act as a bridge in ERS and apoptosis.In this study,we detected the expression in HGSOC and analyzed its relationship with the clinicopathologic features to clear its clinical significance.We also detected the effects of ROR2 on the biological function of HGSOC in vitro and in vivo to reveal the role of ROR2 in HGSOC and the underlying mechanism.Part ? Expression and clinical significance of ROR2 inhigh-grade serous ovarian cancer tissuesObjective:1.To detect the expression of ROR2 in HGSOC,normal ovarian surface epithelium(OSE)and normal fallopian tube epithelium(FTE)tissues.2.To analyze the relationship betweent ROR2 and the clinicopathologic features to detect its effects on patients' prognosis.Methods:1.To detect the protein expression of ROR2 in tissues.Western Blot assay was used to detect the expression of ROR2 in 23 HGSOC tissues and 23 normal FTE tissues.2.To detect the mRNA expression of ROR2 in tissues.Gene expression profiles of GSE69428,GSE40595 and GSE18520 were downloaded from Gene Expression Omnibus(GEO).Dataset GSE69428 included HGSOC and paired normal FTE samples from 10 independent patients.Dataset GSE40595 included epithelial tumor samples from 32 HGSOC patients and 6 normal OSE samples.Dataset GSE18520 included 53 HGSOC tissue samples and 10 normal OSE samples.All the samples were isolated laser based microdissection using the Affymetrix human genome U133 Plus 2.0 microarray.The "limma" package was used to analyze mRNA expression of ROR2 between HGSOC and normal FTE or OSE samples.Adjusted P-value(adj.P)was applied to correct false-positives.3.To detect the location,expression and clinical significance of ROR2 in HGSOC tissues.Immunohistochemical staining(IHC)assay was used to detect the expression of ROR2 in 81 HGSOC tissues to analyze its relationship with clinicopathologic features.Results:1.The protein expression of ROR2 in HGSOC and normal fallopian tube tissues.Western Blot assay was used to detect the expression of ROR2 in HGSOC and normal fallopian tube tissues.The results showed the expression of ROR2 was significantly downregulated in HGSOC tissues compared to fallopian tube tissues(FigurelA,B),indicating that downregulation of ROR2 may contribute to the development of HGSOC.2.The mRNA expression of ROR2 in HGSOC and normal FTE or OSE tissues.Gene expression profiles of GSE69428,GSE40595 and GSE18520 were downloaded from Gene Expression Omnibus(GEO).The "limma" package was used to analyze differentially expressed genes(DEGs)between HGSOC and normal FTE or OSE samples.The result of GSE69428 showed mRNA of ROR2 in HGSOC tissues was significantly lower than that in the paired normal FTE tissues(adj.P=0.013).The results of GSE40595 and GSE18520 showed mRNA of ROR2 in HGSCO tissues was significantly lower than that in the normal ovarian surface epithelium(adj.P=8.5×10-3 and 7×10-5,respectively)(Figure2).3.Expression of ROR2 in HGSCO tissues and its clinical significance.IHC assay was used to detect the location and protein expression of ROR2 in 81 HGSCO tissues.The result showed ROR2 localized on the cellular membrane in HGSOC tissues(Figure3A).IHC staining score showed patients with advanced stages(FIGO stage???)or positive lymph nodes were prone to express lower ROR2(Figure3B,C),indicating that downregulation of ROR2 may correlate with poorer prognosis in HGSOC.Conclusions:1.ROR2 was downregulated in HGSOC tissues,indicating that downregulation of ROR2 may contribute to the development of HGSOC.2.Downregulation of ROR2 may correlate with poorer prognosis in HGSOC.Part ? Biological function of ROR2 in high-grade serousovarian cancer cellsObjective:1.To detect the expression of ROR2 in HGSOC cells.2.To detect the effects of ROR2 on the proliferation,apoptosis and cell cycle in HGSOC cells.3.To detect the effect of ROR2 on the growth of tumor in vivo.Methods:1.To detect the expression of ROR2 in HGSOC cells.Western Blot assay was used to detect the protein expression of ROR2 in 4 HGSOC cell lines.2.To detect the effects of ROR2 on biological beheviors in HGSOC cells.ROR2 adenovirus was transiently transfected into HEY,O V-90 and HO-8910 cells to construct ROR2 overexpression ovarian cancer cell model while cells transfected with negative control(NC)was used as control.RNA interference(RNAi)plasmid targeting ROR2 was transiently transfected into A2780 cell to constructed ROR2 knockdown cell model while cells transfected with empty vector was used as control.Western Blot assay were used to verify the overexpression.MTT assay,colony formation assay were used to detect the effects of ROR2 on cell growth.Flow cytometry assay and EdU assay were used to test cell apoptosis and proliferation.Western Blot assay was used to detect the expression of proteins related to apoptosis and cell cycle.3.To detect the effect of ROR2 on the growth of ovarian cancer in vivo.ROR2 stable overexpression cells were constructed with Lentivirus PCMV-ROR2.Cells transfected with PCMV-NC were used as control.Subcutaneous injection of stable overexpression cells into either side of the armpit of the same 4-week-old nude female mice to construct xenograft model.Tumor sizes were measured every other day 10 days after injection.Sizes of tumors were measured(Volume=[length × width2]/2)with vernier caliper every other day for drawing growth curves.Mice were sacrificed 33 days after injection and tumors were removed and weighted.Western Blot assay and IHC assay were used to detect the expression of ROR2 in xenograft tumor.Results:1.Expression of ROR2 in HGSOC cells.Western Blot assay was used to detect expression of ROR2 in 4 HGSOC cells.The result showed ROR2 barely express in HEY,OV-90 and HO-8910 cells,except in A2780 cell(Figure 1A,B).2.Effects of ROR2 on biological behaviors of HGSOC cells.After successful construction of ROR2 overexpression and knockdown cell model(Figure2A,B),MTT assay,colony formation assay,EdU assay and flow cytometry assay were used to detect the effects of ROR2 on biological behaviors in HGSOC cells.Results are as follows.2.1 Effect of ROR2 on growth of HGSOC cells.MTT assay was used to detect the effect of ROR2 on cell growth.Results showed,compared to the control group,growth speed was significantly downregulated after ROR2 overexpression while significantly upregulated after ROR2 knockdown(Figure3).2.2 Effect of ROR2 on colony formation of HGSOC cells.Colony formation assay was used to detect the effect of ROR2 on colony formation ability of cells.Results showed,compared to the control group,colony number of ovarian cancer cells was significantly decreased in ROR2 overexpression group while significantly increased in ROR2 knock down group(Figure4).2.3 Effect of ROR2 overexpression on apoptosis of HGSOC cells.Flow cytometry assay was used to detect the apoptosis rate of ovarian cancer cells.The result showed the apoptosis rate of ROR2 overexpression group was significantly increased compared to the control group(Figure5A,B).Western Blot assay showed ROR2 overexpression could upregulate Bax and promote cleavage of casapase3,caspase7 and PARP while downregulate Bcl-2(Figure5C,D),verifying the flow cytometry results.2.4 Effects of ROR2 overexpression on proliferation and cycle distribution of HGSOC cells.EdU assay was used to evaluate cell proliferation.The result showed proportion of proliferating cells in ROR2 overexpression group was significantly lower than the control group(Figure6A,B).Flow cytometry assay was used to explore cell cycle distribution.The result showed ROR2 overexpression could arrest ovarian cancer cells in G0/G1 phase(Figure7A,B),Western Blot assay showed ROR2 overexpression could suppress the expression of CDK4,CDK6,cyclin D1 and cyclin E(Figure7C,D).3.Effect of ROR2 on tumor growth in xenograft model.Stable overexpression cells were used to construct xenograft model for drawing tumor growth curve.The results showed volume and weight of tumors in the PCMV-ROR2 overexpression group were significantly lower than those in the PCMV-NC group(Figure8D,E,F).Western Blot assay and IHC assay further confirmed the successful overexpression of ROR2 in the ROR2 transfected group(Figure8G,H).Conclusions:1.ROR2 could significantly repress growth of HGSOC cells and suppress the tumor growth in xenograft model.2.ROR2 could induce cell apoptosis and cell cycle arrest in HGSOC cells.Part ? Mechanism of apoptosis induced by ROR2 inhigh-grade serous ovarian cancer cellsObjective:To explore the mechanism of apoptosis induced by ROR2 in HGSOC cells and authenticate it reversely.Methods:1.Whole transcriptome sequencing.Whole transcriptome sequencing was used to screen the differentially expressed genes(DEGs)between cells in ROR2 overexpression group and NC group.GO enrichment analysis were used to identify the signaling pathway.2.To detect the effects of ROR2 on ERS related protein.GO analysis showed mechanism by ROR2 was closely related to UPR.UPR is originally a mechanism to relieve ERS.After transfected with ROR2 overexpression adenovirus,Western Blot assay was used to detect the expression of ERS related proteins such as BIP,phosphorylated IRE1?(p-IRE1?),phosphorylated JNK(p-JNK),phosphorylated c-Jun(p-c-Jun)and CHOP.3."Recovery experiment" with IRE1? knockdown.Small interfering RNA(siRNA)targeted IRE la was used to knockdown the expression of IRE1?.Cells transfected with unintentional sequence(si-NC)were used as control.Western Blot assay was used to screen the most effective sequence for the following experiment.Flow cytometry assay and Western Blot assay were used to detect cell apoptosis and related protein expression in cells co-transfected with ROR2 and si-IRE1?.4."Recovery experiment" with IRE1?'s RNase repression.As IRE1? triggers cell apoptosis via its RNase.Kira6,an IRE1?'s RNase inhibitor,was used to inhibit its phosphorylation.MTT assay and Western Blot assay were used to screen appropriate concentration and application time.After pre-transfected with ROR2 overexpression or negative control adenovirus for 6h,2?M Kira6 was added into the culture medium.The cells were subjected to further incubation for 72h for flow cytometry assay and Western Blot assay.5.To verify the mechanism with xenografts in nude mice.IHC assay was used to detect the protein expression of BIP and CHOP of xenograft tumors to verify the mechanism in vivo.Results:1.Results of whole transcriptome sequencing.Compared to the NC group,there were 719 upregulated genes and 1260 downregulated genes in the ROR2 overexpression group(FigurelA).Gene ontology enrichment analysis of these differential genes showed that the enriched biological processes included response to unfolded protein,response to topologically incorrect protein,chaperone-mediated protein folding,cellular response to unfolded protein,protein refolding.Affected cell components included extracellular matrix,endoplasmic reticulum lumen,cell projection membrane.Impacted molecular function included protein binding involved in protein folding,unfolded protein binding,misfolded protein binding and chaperone binding(FigurelC).All the results indicated that changes induced by ROR2 overexpression involved UPR.2.ROR2 overexpression induced endoplasmic reticulum stress and modulated IRE1?/JNK/CHOP signalling.The expression of ERS relevant proteins in HGSOC cells were detected by Western Blot assay.ERS related proteins like BIP and phosphorylated IRE1? were upregulated by ROR2 overexpression.Furthermore,the pro-death factors like CHOP,phosphorylated JNK and phosphorylated c-Jun in the ROR2-overexpression group were significantly higher than the NC group(Figure2A,B).All the results indicated that ROR2 overexpression could induce ERS and modulate IRE1?/JNK/CHOP signalling,further causing apoptosis.3.IRE1? knockdown reversed the apoptosis and activation of IRE1?/JNK/CHOP pathway induced by ROR2 overexpression.Cells pre-transfected with si-IRE1? or si-NC for 24h were transfected with ROR2 overexpression or NC adenovirus for following 48h.Then cells were collected for flow cytometry assay and Western Blot assay.We found IRE1? knockdown could alleviate the apoptosis of cells induced by ROR2 overexpression(Figure3B).IRE1? knockdown could significantly down-regulate the level of p-IREla induced by ROR2 overexpression,therewith strongly inhibited JNK and c-Jun phosphorylation from IRE1? hyperactivation.Moreover,proteins related with apoptosis were detected by Western Blot.The change of Bcl-2 and Bax induced by ROR2 were reversed by IRE1? knockdown.IRE1? knockdown could also significantly inhibited caspase3,caspase7 and PARP cleavage upon ROR2 overexpression(Figure3C,D).4.Kira6 reversed the apoptosis and activation of IRE1?/JNK pathway induced by ROR2 overexpression.After pre-transfected with ROR2 overexpression or negative control adenovirus for 6h,2?M Kira6 was added into the culture medium.The cells were subjected to further incubation for 72h for flow cytometry assay and Western Blot assay.Kira6 could alleviate ROR2 induced apoptotic response in HEY and HO-8910 cells as shown by flow cytometry assay(Figure4D).The Western Blot assay showed Kira6 not only reversed the activation of IRE1?/JNK pathway,but also significantly reversed the change of apoptosis related proteins induced by ROR2.However,the upregulation of CHOP couldn't be significantly reversed by Kira6 treatment.Besides,the inhibition of PARP cleavage by Kira6 was not that significantly(Figure4E,F).5.Mechanism was verified with xenografts in nude mice:IHC assay was used to detect the protein expression of BIP and CHOP of xenograft tumors.The results showed BIP and CHOP were significantly higher in ROR2 overexpression group than the NC group(Figure5A,B),indicating growth suppression by ROR2 in xenograft tumors was related to ERS.Conclusions:1.Apoptosis induced by ROR2 was related to ERS.2.ROR2 induced cell apoptosis by activating IRE1?/JNK/CHOP pathway in HGSOC.