【摘要】：乳腺癌是女性最常見的惡性腫瘤之一,其高度異質(zhì)性是導(dǎo)致乳腺癌患者在臨床表現(xiàn)、治療反應(yīng)性和預(yù)后等方面存在顯著差異的主要原因。近年來,乳腺癌分子亞型概念的提出,對于指導(dǎo)臨床個體化治療和判斷預(yù)后起到重要作用。因此,明確乳腺癌患者分子亞型不僅為乳腺癌個體化治療方案提供支持,也為深入研究乳腺癌分子機制提供依據(jù)。腫瘤侵襲和轉(zhuǎn)移特征是導(dǎo)致乳腺癌患者復(fù)發(fā)和不良預(yù)后的主要原因,也是腫瘤治療難以克服的瓶頸問題。新近研究發(fā)現(xiàn)提示,腫瘤干細胞(CSCs)可能是導(dǎo)致侵襲轉(zhuǎn)移的主要根源,此群細胞在腫瘤細胞全體中含量甚少,但獨具自我更新、無限增殖、多向分化潛能及腫瘤重建的能力。本課題組前期研究發(fā)現(xiàn),ER-α36陽性乳腺癌細胞ALDH1表達陽性并具有干細胞樣表型,提示ER-α36在乳腺癌中具有重要意義。與腫瘤干細胞產(chǎn)生密切相關(guān)的重要機制上皮間質(zhì)轉(zhuǎn)化(EMT)也被認為是在腫瘤轉(zhuǎn)移過程中重要的細胞生物學(xué)事件。除此之外,腫瘤細胞轉(zhuǎn)移需要降解間質(zhì)的細胞外基質(zhì),而基質(zhì)金屬蛋白酶(MMPs)是降解細胞外基質(zhì)的關(guān)鍵酶。已有研究提示,不同分子亞型的乳腺癌侵襲轉(zhuǎn)移能力具有差異,然而調(diào)控不同亞型乳腺癌患者侵襲轉(zhuǎn)移的分子機制尚不清楚。蛋白酪氨酸磷酸酶1B(PTP1B)是一種非跨膜蛋白酪氨酸激酶,已被發(fā)現(xiàn)在胃癌、結(jié)直腸癌及前列腺癌等多種腫瘤中高表達,并參與了腫瘤的發(fā)生發(fā)展。而在乳腺癌中,PTP1B雖在腫瘤中高表達,但其在各種亞型乳腺癌中表達如何,是否及如何影響乳腺癌細胞的侵襲轉(zhuǎn)移能力尚不清楚�；诖�,我們首先對328例乳腺癌進行分子分型,檢測PTP1B在乳腺癌中的表達規(guī)律,通過敲低或過表達PTP1B的表達,觀察PTP1B是否可以影響乳腺癌細胞侵襲遷移能力。然后,探討了PTP1B影響乳腺癌細胞侵襲遷移的作用機制。最后,觀察PTP1B與乳腺癌分子亞型的關(guān)系,分析其與乳腺癌患者臨床病理學(xué)參數(shù)的關(guān)系及能否依據(jù)不同乳腺癌亞型進而指導(dǎo)乳腺癌患者個體化治療。主要結(jié)果和結(jié)論如下:1、乳腺癌分子亞型鑒定結(jié)果。(1)通過細胞免疫組化(ICC)聯(lián)合RT-PCR檢測ER、PR和HER2蛋白及m RNA表達,將12種正常及乳腺癌細胞系分為四種亞型。MCF7和T47D為Luminal A型(ER+和/或pr+,her2-);bt-474和uacc-732為luminalb型(er+和/或pr+,her2+);mcf10a、mda-mb-231、mda-mb-453、bt549、sum159和bcap37為basal-like型(er-,pr-,her2-);skbr3和jimt1為her2過表達型(er-,pr-,her2+)。(2)應(yīng)用ihc的方法檢測328例浸潤性乳腺癌組織中er、pr、her2和ki67蛋白表達水平情況,并對其進行分子分型。其中四種亞型分布狀態(tài)為:151例為luminala型,54例為luminalb型,94例為basal-like型,29例為her2過表達型。不同亞型乳腺癌患者總生存(overallsurvival,os)及無病生存(disease-freesurvival,dfs)均具有顯著性差異(p0.001,p0.001)。其中l(wèi)uminala型乳腺癌患者預(yù)后顯著好于其它任何亞型乳腺癌患者(p0.001)。2、ptp1b在乳腺癌中高表達并可以顯著促進乳腺癌細胞的侵襲和遷移能力。(1)ptp1b蛋白在乳腺癌組織中的表達顯著高于癌旁正常組織(p0.001)。蛋白表達主要分布在腫瘤組織或癌旁正常組織胞漿中。(2)ptp1b與乳腺癌淋巴結(jié)轉(zhuǎn)移顯著正相關(guān),其高表達患者淋巴結(jié)更易發(fā)生轉(zhuǎn)移(p0.01)。(3)在細胞系mcf7中,敲低ptp1b的表達后可以顯著抑制乳腺癌細胞系的侵襲和遷移能力(p0.01)。(4)在細胞系mda-mb-231中,過表達ptp1b的表達后可以顯著促進乳腺癌細胞系的侵襲和遷移能力(p0.01)。3、ptp1b通過調(diào)控pten和mmps調(diào)控乳腺癌細胞侵襲轉(zhuǎn)移能力。(1)敲低mcf7細胞ptp1b表達可以有效抑制akt磷酸化,這一改變主要是通過上調(diào)pi3k/akt上游通路中pten蛋白來實現(xiàn)的。相反,過表達ptp1b表達可以抑制pten的變化進而促進akt磷酸化。(2)敲低mcf7細胞ptp1b表達可以有效抑制基質(zhì)蛋白酶(mmp2和mmp7)mrna和蛋白水平的改變。然而,過表達ptp1b表達可以有效促進mmp2和mmp7mrna和蛋白水平的表達。(3)敲低或過表達ptp1b對細胞系mcf7和mda-mb-231中相關(guān)干性因子表達無顯著影響(oct4、nanog、sox2和aldh1,p0.05)。此外,通過成球培養(yǎng)法富集mcf7和mda-mb-231細胞中的乳腺癌干細胞,發(fā)現(xiàn)ptp1b的表達與乳腺癌干細胞無顯著相關(guān)性。(4)敲低或過表達ptp1b后不影響emt相關(guān)基因(e-cadherin、n-cadherin和vimentin,p0.05)mrna和蛋白表達。4、PTP1B在Luminal型乳腺癌中高表達且與不良預(yù)后正相關(guān)。(1)PTP1B蛋白在不同亞型乳腺癌組織中差異表達,在Luminal型和HER2陽性型中高表達,在Basal-like型中低表達(P0.05)。PTP1B蛋白在不同亞型乳腺及乳腺癌細胞系中亦呈現(xiàn)差異表達,在Luminal型和HER2過表達型細胞系中高表達,在Basal-like型細胞系中低表達。(2)PTP1B高表達與患者ER狀態(tài)顯著正相關(guān)(P0.05)。(3)PTP1B高表達與Luminal型乳腺癌患者不良預(yù)后相關(guān)(P0.05,DFS),而與其它亞型乳腺癌患者預(yù)后無顯著相關(guān)性(P0.05)。5、ER-α36在乳腺癌組織中的臨床意義。(1)ER-α36與乳腺癌患者淋巴結(jié)轉(zhuǎn)移正相關(guān),ER-α36高表達提示患者不良預(yù)后(P0.05)。(2)ER-α36蛋白在不同亞型乳腺癌組織中差異表達,在Luminal型和HER2陽性型中高表達,在Basal-like型中低表達(P0.05)。綜上所述,乳腺癌細胞系的亞型鑒定為乳腺癌科研工作者提供了良好的研究基礎(chǔ),對于乳腺癌患者個體化治療和機制深入研究提供依據(jù)。同時,乳腺癌分子亞型與患者預(yù)后顯著相關(guān),我們應(yīng)該推進和完善乳腺癌亞型的鑒定,以期更好的指導(dǎo)患者診治及判斷臨床預(yù)后。PTP1B在乳腺癌中高表達并與LNM相關(guān),其促進乳腺癌細胞的侵襲轉(zhuǎn)移的潛在機制主要是與PI3K/AKT通路和MMPs有關(guān),而非CSCs和EMT依賴的調(diào)控通路。ER-α36與乳腺癌患者淋巴結(jié)轉(zhuǎn)移正相關(guān),其高表達顯示患者不良預(yù)后。以上研究為認識不同分子亞型乳腺癌生物學(xué)特點、闡明其轉(zhuǎn)移機制提供了理論與實驗依據(jù)。
[Abstract]:Breast cancer is one of the most common malignant tumors in women. Its high heterogeneity is the main reason that causes significant differences in clinical manifestations, reactivity and prognosis in breast cancer patients. In recent years, the concept of the molecular subtypes of breast cancer has played an important role in guiding clinical individualized treatment and judging prognosis. The molecular subtypes of breast cancer patients not only provide support for the individualized treatment of breast cancer, but also provide a basis for the in-depth study of the molecular mechanism of breast cancer. The characteristics of tumor invasion and metastasis are the main causes leading to the recurrence and poor prognosis of breast cancer patients. Stem cells (CSCs) may be the main source of invasion and metastasis, and this group of cells has little content in all tumor cells, but it has a unique self renewal, unlimited proliferation, multidirectional differentiation potential and the ability to reconstruct the tumor. Previous studies in our group have found that ER- alpha 36 positive breast cancer cells are positive for ALDH1 expression and have stem cell like phenotypes, suggesting ER- Alpha 36 is of great significance in breast cancer. The important mechanism of epithelial mesenchymal transition (EMT), which is closely related to tumor stem cells, is also considered to be an important cell biological event in the process of tumor metastasis. In addition, tumor cell metastasis requires the degradation of extracellular matrix, and matrix metalloproteinase (MMPs) is the degradation of extracellular matrix. The key enzymes in the matrix have been suggested that the invasion and metastasis of different subtypes of breast cancer are different. However, the molecular mechanism of the invasion and metastasis of different subtypes of breast cancer is unclear. Protein tyrosine phosphatase 1B (PTP1B) is a non transmembrane protein tyrosine irritable enzyme, which has been found in gastric cancer, colorectal cancer and prostate cancer. The high expression of cancer and other cancers is involved in the development of the tumor. In breast cancer, although PTP1B is highly expressed in the tumor, it is not clear whether it is expressed in various subtypes of breast cancer and how and how it affects the invasion and metastasis of breast cancer cells. Based on this, we first made molecular typing for 328 cases of breast cancer and detected PTP1 The expression of B in breast cancer, by knocking down or overexpressing PTP1B expression, observed whether PTP1B could affect the invasion and migration of breast cancer cells. Then, the mechanism of the effect of PTP1B on the invasion and migration of breast cancer cells was explored. Finally, the relationship between PTP1B and the molecular subtypes of breast cancer was observed and the clinical pathology of breast cancer patients was analyzed. The relationship between the parameters and the individual breast cancer subtypes can be used to guide the individual treatment of breast cancer. The main results and conclusions are as follows: 1, the identification results of the molecular subtypes of breast cancer. (1) the expression of ER, PR and HER2 protein and m RNA by cellular immunization (ICC) combined with the expression of PR and HER2 protein and m RNA, and to divide 12 normal and breast cancer cell lines into four subtypes.MCF7 And T47D is Luminal A type (ER+ and / or pr+, her2-); BT-474 and uacc-732 are luminal B type (er+ and pr+, her2+). And the expression level of Ki67 protein and its molecular typing. The four subtypes were luminala, 54 luminal B, 94 basal-like and 29 HER2 overexpressed. The total survival (overallsurvival, OS) and disease-free survival (disease-freesurvival, DFS) in different subtypes of breast cancer were significant. The difference (p0.001, p0.001). The prognosis of luminala type breast cancer patients is significantly better than any other subtype of breast cancer (p0.001).2, PTP1B is highly expressed in breast cancer and can significantly promote the invasion and migration of breast cancer cells. (1) the expression of PTP1B protein in breast cancer tissues is significantly higher than that of normal paracancerous tissue (p0.001). (2) PTP1B was significantly correlated with lymph node metastasis in breast cancer, and the lymph node metastasis of the patients with high expression was more likely to metastasize (P0.01). (3) in cell line MCF7, the invasion and migration ability of breast cancer cell lines could be significantly inhibited after the expression of low PTP1B (P0.01). (4) in cell line mda-m In b-231, overexpression of PTP1B can significantly promote the invasion and migration capacity of breast cancer cell lines (P0.01).3. PTP1B regulates the invasion and metastasis of breast cancer cells by regulating PTEN and MMPs. (1) PTP1B expression in low MCF7 cells can effectively inhibit Akt phosphorylation. This change is mainly by up regulation of PTEN eggs in the pi3k/akt upstream pathway. On the contrary, overexpression of PTP1B expression can inhibit changes in PTEN and thus promote Akt phosphorylation. (2) the expression of PTP1B in low MCF7 cells can effectively inhibit the change of mRNA and protein levels of matrix protease (MMP2 and MMP7). However, overexpression of PTP1B expression can effectively promote the expression of MMP2 and mmp7mrna and protein levels. (3) knock low or over. The expression of PTP1B had no significant effect on the expression of related dry factors in cell lines MCF7 and MDA-MB-231 (Oct4, Nanog, Sox2 and ALDH1, P0.05). In addition, the enrichment of breast cancer stem cells in MCF7 and MDA-MB-231 cells was enriched by spheroid culture, and there was no significant correlation between PTP1B expression and breast cancer stem cells. (4) low or overexpressed PTP1B was not affected. The related genes (E-cadherin, N-cadherin and vimentin, P0.05) mRNA and protein expressed.4. PTP1B was highly expressed in Luminal type breast cancer and was positively related to poor prognosis. (1) the expression of PTP1B protein in different subtypes of breast cancer tissues was expressed in the Luminal type and HER2 positive type, and the low expression of the protein in the Basal-like type was different. Subtype breast and breast cancer cell lines also showed differential expression, high expression in type Luminal and HER2 overexpressed cell lines, low expression in Basal-like type cell lines. (2) high expression of PTP1B was significantly positively correlated with patients' ER state (P0.05). (3) high expression of PTP1B was associated with poor prognosis of Luminal type breast cancer patients (P0.05, DFS), and other subtypes. There was no significant correlation between the prognosis of patients with breast cancer (P0.05).5, ER- alpha 36 in breast cancer tissue. (1) ER- alpha 36 was positively correlated with lymph node metastasis in breast cancer patients, and high expression of ER- alpha 36 indicated poor prognosis (P0.05). (2) ER- alpha 36 protein was expressed differently in different subtype breast cancer tissues, and high table in Luminal and HER2 positive types The subtype identification of the breast cancer cell lines provides a good basis for breast cancer research workers and provides a basis for the individual treatment and mechanism of breast cancer patients. At the same time, the subtypes of breast cancer molecules are significantly related to the prognosis of the patients. We should promote and improve the prognosis of the breast cancer molecules. We should promote and improve the Basal-like. Identification of the subtypes of breast cancer in order to better guide the diagnosis and treatment of patients and determine the clinical prognosis of.PTP1B in breast cancer high expression and related to LNM. The potential mechanism for promoting invasion and metastasis of breast cancer cells is mainly related to the PI3K/AKT pathway and MMPs, not CSCs and EMT dependent modulation pathway.ER- alpha 36 and lymph node metastasis of breast cancer patients The above study shows the biological characteristics of different molecular subtypes of breast cancer and the theoretical and experimental basis for elucidating the mechanism of metastasis of different molecular subtypes of breast cancer.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R737.9

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10 劉霞;一種天然分子能阻止乳腺癌惡化[N];科技日報;2011年

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5 葛廣哲;樹，

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