【摘要】：EGFR信號(hào)通路的異常激活與前列腺癌的惡性進(jìn)展、高Gleason分級(jí)及雄激素非依賴進(jìn)程密切相關(guān)。最新的研究發(fā)現(xiàn)，在前列腺腫瘤標(biāo)本中，僅有8%的EGFR發(fā)生突變，但卻有高達(dá)31%的標(biāo)本中EGFR發(fā)生高表達(dá)，提示EGFR的高表達(dá)是前列腺癌中EGFR信號(hào)通路異常激活的主要原因。EGFR在腫瘤組織中高表達(dá)主要有兩個(gè)原因，一是基因擴(kuò)增，另一個(gè)是蛋白降解受阻。該項(xiàng)研究并沒有發(fā)現(xiàn)組織水平EGFR高表達(dá)與EGFR基因擴(kuò)增具有顯著的相關(guān)性，提示EGFR蛋白分子的降解受阻可能是前列腺癌中EGFR異常表達(dá)的主要原因。但目前對(duì)于前列腺癌中EGFR蛋白穩(wěn)定性調(diào)控的分子機(jī)制還不明確，因此發(fā)現(xiàn)新的EGFR蛋白穩(wěn)定性的調(diào)節(jié)分子對(duì)于闡明前列腺癌惡性進(jìn)展的分子機(jī)制具有重要意義。SGEF是我們前期發(fā)現(xiàn)的前列腺癌中的一種潛在的癌基因，臨床研究表明SGEF隨著前列腺癌的惡性進(jìn)展表達(dá)水平逐漸升高，且我們發(fā)現(xiàn)SGEF能夠促進(jìn)前列腺癌細(xì)胞的增殖和存活能力，但我們對(duì)于SGEF在前列腺癌中發(fā)揮癌基因作用的具體分子機(jī)制還不清楚。我們的前期研究發(fā)現(xiàn)，敲低SGEF能夠抑制AKT分子的磷酸化，而AKT是EGFR下游的重要分子，因此我們推測(cè)SGEF有可能通過對(duì)EGFR蛋白穩(wěn)定性的調(diào)節(jié)進(jìn)而調(diào)控EGFR/AKT信號(hào)通路。 我們首先檢測(cè)了SGEF是否影響前列腺癌細(xì)胞中EGFR蛋白表達(dá)水平，我們發(fā)現(xiàn)敲低SGEF降低了前列腺癌細(xì)胞C4-2B和DU145中EGFR的蛋白表達(dá)水平，提示我們SGEF確實(shí)能夠調(diào)控前列腺癌細(xì)胞中EGFR蛋白表達(dá)水平。為了進(jìn)一步確定SGEF調(diào)控EGFR蛋白表達(dá)水平的機(jī)制，我們檢測(cè)了過表達(dá)SGEF對(duì)EGFR蛋白降解的影響，發(fā)現(xiàn)在293T細(xì)胞和COS7細(xì)胞中過表達(dá)SGEF能夠明顯EGFR蛋白的降解。隨后我們?cè)诙喾N前列腺癌細(xì)胞中檢測(cè)了敲低SGEF對(duì)EGFR蛋白降解的影響，我們發(fā)現(xiàn)在多種前列腺癌細(xì)胞中敲低SGEF均能明顯促進(jìn)EGFR蛋白的降解。這些結(jié)果說明SGEF能夠通過增強(qiáng)EGFR蛋白穩(wěn)定性來增強(qiáng)前列腺癌細(xì)胞中EGFR的蛋白表達(dá)水平。由于SGEF是RhoG特異性的鳥苷酸交換酶，，我們隨后檢測(cè)了SGEF是否以鳥苷酸交換酶活性依賴的方式增強(qiáng)EGFR蛋白的穩(wěn)定性。我們?nèi)笔B苷酸交換酶活性的SGEF依然能夠抑制EGFR蛋白的降解，同時(shí)發(fā)現(xiàn)持續(xù)激活型的RhoG不能夠回轉(zhuǎn)敲低SGEF對(duì)EGFR蛋白降解的促進(jìn)作用，這些結(jié)果說明SGEF是以鳥苷酸交換酶活性非依賴的方式增強(qiáng)EGFR蛋白的穩(wěn)定性。 EGFR在細(xì)胞中的降解受多重調(diào)控，EGFR與配體結(jié)合后，發(fā)生二聚化和自磷酸化，磷酸化的EGFR招募E3泛素酶Cbl促使EGFR發(fā)生泛素化。泛素化的EGFR在Eps15和epsin-1的介導(dǎo)下發(fā)生內(nèi)吞進(jìn)入細(xì)胞漿中。內(nèi)吞的EGFR先進(jìn)入早期內(nèi)體，然后在ESCRT復(fù)合物介導(dǎo)下進(jìn)入晚期內(nèi)體，最后在溶酶體中發(fā)生降解。我們隨后檢測(cè)了SGEF是通過哪一環(huán)節(jié)參與EGFR蛋白的降解調(diào)節(jié)。我們發(fā)現(xiàn)過表達(dá)SGEF并不影響EGFR的泛素化、內(nèi)吞和進(jìn)入早期內(nèi)體，而是抑制EGFR從早期內(nèi)體進(jìn)入晚期內(nèi)體。隨后我們?cè)谇傲邢侔┘?xì)胞中發(fā)現(xiàn)敲低SGEF不影響EGFR進(jìn)入早期內(nèi)體，而是促進(jìn)了EGFR從早期內(nèi)體進(jìn)入晚期內(nèi)體。這些結(jié)果說明了SGEF是通過抑制EGFR從早期內(nèi)體進(jìn)入晚期內(nèi)體進(jìn)而抑制EGFR蛋白降解的。 EGFR的高表達(dá)通常與EGFR信號(hào)通路的異常激活密切相關(guān)，我們隨后檢測(cè)了SGEF是否能夠調(diào)控EGFR信號(hào)通路。我們發(fā)現(xiàn)敲低SGEF明顯抑制了EGF誘導(dǎo)的EGFR和下游蛋白AKT的磷酸化。由于EGFR/AKT信號(hào)通路在前列腺癌細(xì)胞遷移中發(fā)揮著重要作用，我們利用劃痕實(shí)驗(yàn)檢測(cè)了SGEF對(duì)前列腺癌細(xì)胞遷移的影響，結(jié)果發(fā)現(xiàn)敲低SGEF明顯抑制了EGF誘導(dǎo)的細(xì)胞遷移。 EGFR/ERK1/2信號(hào)通路在細(xì)胞多種生命活動(dòng)中均發(fā)揮著重要作用。既然我們發(fā)現(xiàn)了SGEF能夠增強(qiáng)EGFR蛋白的穩(wěn)定性，我們隨后檢測(cè)了SGEF對(duì)EGFR/ERK1/2信號(hào)通路的影響。我們發(fā)現(xiàn)過表達(dá)SGEF能夠顯著增強(qiáng)EGF誘導(dǎo)的ERK1/2磷酸化，而敲低SGEF能夠明顯抑制EGF誘導(dǎo)的ERK1/2磷酸化，說明SGEF能夠增強(qiáng)EGFR/ERK1/2信號(hào)通路。隨后我們檢測(cè)了SGEF是否以RhoG依賴的方式增強(qiáng)EGFR/ERK1/2信號(hào)通路，我們發(fā)現(xiàn)敲低RhoG的表達(dá)并不能抑制SGEF對(duì)EGFR/ERK1/2信號(hào)通路的增強(qiáng)作用，說明SGEF能夠以RhoG非依賴的方式增強(qiáng)EGFR/ERK1/2信號(hào)通路。 Grb2在EGF誘導(dǎo)的ERK1/2激活的過程中發(fā)揮著重要的作用。由于Grb2的兩端含有SH3結(jié)構(gòu)域，而SGEF的N端含有Pro結(jié)構(gòu)域，而SH3結(jié)構(gòu)域與Pro結(jié)構(gòu)域之間存在著經(jīng)典的相互作用，因此我們推測(cè)SGEF有可能通過與Grb2相互作用進(jìn)而增強(qiáng)EGFR/ERK1/2信號(hào)通路。我們通過GST-pull down實(shí)驗(yàn)和免疫共沉淀實(shí)驗(yàn)驗(yàn)證了兩者之間的相互作用，隨后確認(rèn)了Grb2的SH3結(jié)構(gòu)域與SGEF的Pro結(jié)構(gòu)域介導(dǎo)了兩者之間的相互作用。但當(dāng)我們檢測(cè)缺失與Grb2相互作用的SGEF突變體對(duì)EGFR/ERK1/2信號(hào)通路的影響時(shí)，我們出乎意料的發(fā)現(xiàn)與野生型SGEF相比，SGEF突變體不但沒有減弱對(duì)EGFR/ERK1/2信號(hào)通路的增強(qiáng)作用，反而進(jìn)一步增強(qiáng)了EGF誘導(dǎo)的ERK1/2磷酸化，這就提示我們Grb2可能通過與SGEF相互作用拮抗SGEF對(duì)EGFR/ERK1/2信號(hào)通路的增強(qiáng)作用。隨后我們發(fā)現(xiàn)過表達(dá)Grb2確實(shí)能夠抑制SGEF對(duì)EGFR/ERK1/2信號(hào)通路的增強(qiáng)作用，而敲低Grb2促進(jìn)了SGEF對(duì)EGFR/ERK1/2信號(hào)通路的增強(qiáng)作用，并且發(fā)現(xiàn)Grb2拮抗SGEF對(duì)EGFR/ERK1/2信號(hào)通路的增強(qiáng)作用依賴與Grb2與SGEF之間的相互作用。 總之，本研究發(fā)現(xiàn)了SGEF能夠通過抑制EGFR從早期內(nèi)體進(jìn)入晚期內(nèi)體從而增強(qiáng)EGFR蛋白穩(wěn)定性。由于EGFR的高表達(dá)是前列腺癌細(xì)胞中EGFR信號(hào)通路異常激活的主要原因，SGEF對(duì)于EGFR蛋白穩(wěn)定性的調(diào)控可能是SGEF促進(jìn)前列腺癌惡性進(jìn)展的原因之一。同時(shí)由于多種生長(zhǎng)因子受體的降解方式與EGFR一致，SGEF有可能通過類似的方式抑制多種生長(zhǎng)因子受體的降解，發(fā)揮其癌基因的功能。同時(shí)我們發(fā)現(xiàn)SGEF能夠以RhoG非依賴的方式增強(qiáng)EGF誘導(dǎo)的ERK1/2激活，且Grb2能夠通過與SGEF相互作用拮抗SGEF對(duì)EGFR/ERK1/2信號(hào)通路的增強(qiáng)作用。由于Grb2一直被認(rèn)為在EGFR/ERK1/2信號(hào)通路中發(fā)揮正調(diào)控作用，本研究中發(fā)現(xiàn)了在SGEF過表達(dá)的情況下Grb2能夠負(fù)調(diào)控EGFR/ERK1/2信號(hào)通路，揭示出Grb2在EGFR/ERK1/2信號(hào)通路調(diào)控中功能的復(fù)雜性。
[Abstract]:The abnormal activation of the EGFR signaling pathway is closely related to the malignant progression of prostate cancer, the high Gleason classification and the androgen-independent process. The most recent study found that only 8% of the EGFR mutations in the prostate tumor specimens, but up to 31% of the samples, showed high EGFR expression, suggesting that the high expression of EGFR was the main cause of the abnormal activation of the EGFR signaling pathway in prostate cancer. The high expression of EGFR in tumor tissue is mainly two reasons, one is gene amplification, and the other is protein degradation. The study did not find a significant correlation between the high expression of EGFR and the amplification of EGFR gene, suggesting that the inhibition of EGFR protein molecule could be the main cause of the abnormal expression of EGFR in prostate cancer. However, the molecular mechanism of the stability and control of EGFR protein in prostate cancer is not clear, and therefore, it is of great significance to find the molecular mechanism of the stability of the new EGFR protein. SGEF is a potential oncogene in the early stage of prostate cancer, and the clinical study shows that the level of SGEF is gradually increasing with the malignant progression of prostate cancer, and we find that the SGEF can promote the proliferation and survival ability of prostate cancer cells. But the specific molecular mechanism of the role of SGEF in prostate cancer is unclear. Our previous studies have found that the knockdown of SGEF can inhibit the phosphorylation of AKT molecules, and AKT is an important molecule downstream of the EGFR, so we assume that the SGEF is likely to control the EGFR/ AKT signal pathway by modulating the stability of the EGFR protein. We first examined whether the SGEF has an effect on the level of EGFR protein expression in prostate cancer cells, and we have found that the knockdown of SGEF reduces the level of EGFR protein expression in prostate cancer cells C4-2B and DU145, suggesting that the SGEF is indeed capable of regulating the expression of EGFR protein in prostate cancer cells In order to further determine the mechanism of SGEF to regulate the level of EGFR protein expression, we have examined the effect of overexpression of SGEF on the degradation of EGFR protein, and it was found that overexpression of SGEF in 293T cells and COS7 cells could significantly reduce the decrease of EGFR protein. Solutions. Then we tested the effect of knock-on low SGEF on the degradation of EGFR protein in a variety of prostate cancer cells, and we found that the knockdown of the low SGEF in a variety of prostate cancer cells could significantly contribute to the reduction of the EGFR protein. These results indicate that the SGEF can enhance the protein expression of EGFR in prostate cancer cells by enhancing the stability of the EGFR protein Ping. Since the SGEF is a RhoG-specific bird-derived acid-exchange enzyme, we then examined whether the SGEF enhances the stability of the EGFR protein in a manner that depends on the activity of the avionic acid-exchange enzyme. Sex. The SGEF, which is missing the activity of the avionic acid-exchange enzyme, is still able to inhibit the degradation of the EGFR protein, while finding that the sustained-activated RhoG is not capable of turning on the promotion of the degradation of the EGFR protein by the low SGEF These results indicate that the SGEF enhances the stability of the EGFR protein in a non-dependent manner with the activity of the avionic acid-exchange enzyme. Sex. The degradation of EGFR in the cells is subject to multiple regulation, and after the combination of EGFR with the ligand, dimerization and autophosphorylation, and the EGFR recruitment E3 ubiquitin enzyme Cbl, which phosphorylate, cause the EGFR to occur. Ubiquitin. The ubiquitinated EGFR occurs endocytosis under the mediation of Epsilon-15 and epsin-1. The endocytosis of the EGFR enters the early inner body and then enters the late inner body under the mediation of the ESCRT complex, and finally, in the lysosome, Biodegradation. We then tested which link the SGEF was involved in the reduction of the EGFR protein We found that the overexpression of SGEF did not affect the ubiquitination of EGFR, endocytosis, and the entry of early endosomes, but rather the inhibition of EGFR from early endosomes to late During the period, we found that the knockdown of SGEF in prostate cancer cells did not affect EGFR's entry into the early inner body, but also promoted the entry of EGFR from the early inner body to the night During the period, these results indicate that the SGEF is the inhibition of the EGFR protein by inhibiting the entry of EGFR from the early internal body into the late inner body The high expression of EGFR is usually closely related to the abnormal activation of the EGFR signaling pathway, and we then detected whether the SGEF is capable of regulating the EGF R signal pathway. We found that the knockdown of SGEF significantly inhibited the EGF-induced EGFR and downstream protein AK T phosphorylation. Because the EGFR/ AKT signal pathway plays an important role in the migration of prostate cancer cells, the effect of SGEF on the cell migration of prostate cancer is detected by a scratch test, and the results show that the knockdown of SGEF significantly inhibited the induction of EGF. Cell migration. The EGFR/ ERK1/2 signaling pathway is in a variety of cell life activities It plays an important role. Since we have found that the SGEF is capable of enhancing the stability of the EGFR protein, we then tested the SGEF for EGFR/ ERK1/2 We have found that the expression of SGEF can significantly enhance the phosphorylation of ERK1/2 induced by EGF, while the knockdown of SGEF can significantly inhibit the phosphorylation of ERK1/2 induced by EGF, indicating that the SGEF can enhance the EGFR/ ERK. 1/2 signaling pathway. We then examined whether the SGEF enhanced the EGFR/ ERK1/2 signaling pathway in a RhoG-dependent manner, and we found that the expression of the knockdown RhoG does not inhibit the enhancement of the SGEF to the EGFR/ ERK1/2 signaling pathway, suggesting that the SGEF can enhance the EGFR/ ERK in a RhoG non-dependent manner. 1/2 signaling pathway. Grb2 is in the process of EGF-induced ERK1/2 activation Because the two ends of Grb2 contain the SH3 domain, and the N-terminal of the SGEF contains the Pro domain, and the SH3 domain and the Pro domain have a classical interaction, it is suggested that the SGEF can enhance the EGFR/ E by interacting with the Grb2. The RK1/2 signaling pathway was verified by the GST-pull down experiment and the immunoprecipitation experiment, and then confirmed that the SH3 domain of Grb2 was mediated by the Pro domain of the SGEF. Interaction between the two. But when we detected the effect of the deletion of the SGEF mutant interacting with the Grb2 on the EGFR/ ERK1/2 signaling pathway, we unexpectedly found that the SGEF mutant not only reduced the enhancement of the EGFR/ ERK1/2 signaling pathway compared to the wild-type SGEF, but further enhanced the EGF-induced ERK1/2 phosphorylation, suggesting that Grb2 may antagonize the EGFR/ ERK1/2 by interacting with the SGEF We found that the expression of Grb2 could inhibit the enhancement of the signaling pathway of the EGFR/ ERK1/2 signaling pathway by the expression of Grb2, while the low Gb2 promoted the enhancement of the EGFR/ ERK1/2 signaling pathway and found that the GGrb2 antagonized the enhancement of the EGFR/ ERK1/2 signaling pathway with Grb2 and SG In conclusion, the study found that the SGEF can be used to inhibit the entry of EGFR from the early inner body into the late inner body. The stability of EGFR protein is enhanced. Because of the high expression of EGFR is the main cause of abnormal activation of EGFR signaling pathway in prostate cancer cells, the control of the stability of the EGFR protein may be the leading role of the SGEF in promoting the stability of EGFR protein. One of the causes of the malignant progression of the adenocarcinoma. At the same time, the SGEF has the potential to inhibit the decline of a variety of growth factor receptors in a similar manner due to the fact that the degradation of the various growth factor receptors is consistent with the EGFR. We found that the SGEF can enhance the activation of the EGF-induced ERK1/2 in the non-dependent manner of RhoG, and the Grb2 can antagonize the EGFR/ ERK1 by interacting with the SGEF. The enhancement of the/2 signaling pathway. As Grb2 has been considered to play a positive regulatory role in the EGFR/ ERK1/2 signaling pathway, GGrb2 can negatively regulate the EGFR/ ERK1/2 signaling pathway in the case of overexpression of the SGEF, revealing that Grb2 is in the EGFR/ ERK1/2 signal
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.25

【參考文獻(xiàn)】

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1 Mikio Hoshino;Dennis W.Wolff;Margaret A.Scofield;Frank J.Dowd;;Upregulation of PIP3-Dependent Rac Exchanger 1(P-Rex1) Promotes Prostate Cancer Metastasis[J];生物物理學(xué)報(bào);2009年S1期

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