HAI-2 regulates prostate cancer cell migration and invasion via inhibiting matriptase activity

癌細胞轉移為由原先的腫瘤部位擴散到身體其他器官，是一個多重步驟的過程。目前，對於攝護腺癌細胞轉移的確切機制依然不清楚。間質蛋白酶(Matriptase)為上皮細胞衍生的第二型穿膜絲胺酸蛋白酶，它的活性是受到同源抑制蛋白質-第一型肝細胞生長因子活化抑制者(HAI-1)的調控。當間質蛋白酶和第一型肝細胞生長因子活化抑制者之間的比例，處於不均衡狀態會造成間質蛋白酶失調而活化，進一步地促進癌細胞的演進及轉移。一型和第二型肝細胞生長因子活化抑制者(HAI-1,HAI-2)為高度同源具Kunitz domain型的絲氨酸蛋白酶抑制劑。近年來，研究指出第一型和第二型肝細胞生長因子活化抑制劑能夠抑制促轉移的絲胺酸蛋白酶，並且減弱癌細胞的侵襲及移動力。儘管第一型和第二型肝細胞生長因子活化抑制者在可能具有抑制間質蛋白酶的功能部位上擁有高度保守性，第二型肝細胞生長因子活化抑制者對於調控間質蛋白酶活性及其生理功能依然不清楚。我們利用一組具轉移能力越來越強的人類攝護線惡化轉移細胞模式，包括103E, N1,N2細胞，來探討第二型肝細胞生長因子活化抑制者在攝護腺癌細胞中扮演的角色，及其如何調控間質蛋白酶的活性，進一步地調節癌細胞的移動及侵襲。們的結果發現第二型肝細胞生長因子活化抑制者的蛋白表現量與間質蛋白酶的活性，及攝護腺癌細胞的侵襲能力，呈現負相關。大量表現第二型細胞生長因子活化抑制者會減低癌細胞的生長，侵襲及移動力。此外，我們證實了在攝護腺癌細胞中，間質蛋白酶為第二型肝細胞生長因子活化抑制者的目標蛋白酶。第二型會與第一型肝細胞生長因子活化抑制者競爭，以直接地結合上間質蛋白酶來抑制其活性，進一步地降低癌細胞的移動與侵襲。利用點突變或是片段缺失突變所產生的第二型肝細胞生長因子活化抑制者突變體，進行研究，結果顯示當HAI-2(ΔKD1)或是 HAI-2(R48L)突變體大量表現時，都會降低其原有對間質蛋白酶的抑制功能；然而當大量表現 HAI-2(ΔKD2)，HAI-2(ΔTM)或HAI-2(R143L)突變體時，可以保留其抑制間質蛋白酶的角色。再者，我們的結果發現，第二型肝細胞生長因子活化抑制者可透過防止在間質蛋白酶上精胺酸614的位置上進行酶原切割，以抑制間質蛋白酶活性，因為這個位置的切割對於蛋白酶的活化是不可或缺的一步。這個發現顯示出第二型肝細胞生長因子活化抑制者透過抑制間質蛋白酶的酶原激活的過程，來達到降低攝護線癌細胞的侵襲及移動力。據在降低第二型肝細胞生長因子活化抑制者表現量的細胞中，發現間質蛋白酶的表現量也隨之降低的現像，顯示出第二型肝細胞生長因子活化抑制者同時也參與了間質蛋白酶的蛋白穩定度。在HEK293T細胞中，間質蛋白酶的蛋白表現量，在具有第二型肝細胞生長因子活化抑制者共同表現的狀況下，表現量會高出許多。此外，利用cycloheximide的處理下，在降低第二型肝細胞生長因子活化抑制者表現量的細胞中，間質蛋白酶的蛋白降解速率會比控制組細胞快上許多。合以上結果顯示出，第二型肝細胞生長因子活化抑制者藉由它的Kunitz domain1，抑制間質蛋白酶的活性，以執行其抗癌效力。第二型肝細胞生長因子同時也調控間質蛋白酶的蛋白表現量、酶原激活的切割、以及細胞外的釋放。因此，這些結果表明在攝護腺癌細胞中，因為第二型肝細胞生長因子活化抑制者的表現量降低，造成了持續活化的間質蛋白酶以及增強癌細胞的轉移能力。第二型肝細胞生長因子活化抑制者和間質蛋白酶的不平衡交互作用為攝護腺癌細胞得以移動與侵襲的重要因素。Metastasis is a multistep process with the spread of cancer cells from a primary tumor site to distant organs. Dysregulation of proteolysis on cell surface has been strongly proposed to play am important role in cancer cell invasion and metastasis. In this study, we delineated the roles of hepatocyte growth factor activator inhibitors (HAI-1and HAI-2) in prostate cancer cell migration and invasion, as well as identified their target protease(s) in human prostate cancer. With a human prostate progression model (103E, N1, and N2 cells), which has an increased metastasis potential.ur results showed that the protein level of HAI-2, but not HAI-1, was dramatically decreased in highly invasive N2 cells, compared to 103E cells, which was inversely related with matriptase activity and cancer cell invasion. Ectopic expression of HAI-2 resulted in reduced cell proliferation, migration and invasion. Moreover, we identified a type Ⅱtransmembrane serine protease matriptase was a HAI-2 regulated serine protease in human prostate cancer cells. HAI-2 was able to directly interact with matriptase and inhibited the protease activity, leading to reduced cell migration and invasion. Using several HAI-2 mutants with point and deletion mutations on its functional Kunitz domains (KDs) and transmembrane domain, our data showed that overexpression of HAI-2(ΔKD1) or HAI-2(R48L)mutant lost its inhibitory effects on matriptase activity and cancer cell migration and invasion, whereas overexpression of HAI-2(ΔKD2), HAI-2(ΔTM) or HAI-2(R143L) mutant retained its role against matriptase activity and these two biological events. Thus, the KD1 of HAI-2 played an essential role in modulating matriptase activity and prostate cancer cell migration and invasion. Furthermore, our data observed that HAI-2 affecting matriptase activation was through preventing a proteolytic cleavage at the residue Arg614 of matriptase, which is a critical step for protease activation. This finding indicated that HAI-2 reduced prostate cancer cell invasion and migration by inhibiting a proteolytic process for matriptase zymogen activation.AI-2 also participated in the protein stability of matriptase. This was supported by the results that in HAI-2-knockdown cells, the protein level of matriptase was reduced. Moreover, matriptase proteins were expressed at much higher levels in the presence of HAI-2 than matriptase alone in HEK293T cells. Under cycloheximide treatment, the degradation rate of matriptase is faster in HAI-2–knockdown cells than that in control cells.n summary, HAI-2 exerted its anti-tumor invasiveness through inhibiting matripase activity via its Kunitz domain 1. The roles of HAI-2 may play in regulating matriptase were to modulate matriptase’s protein expression, autoproteolytic cleavage for activation, and subsequent shedding. These results indicate that duringthe progression of human prostate cancer cells, HAI-2 expression was reduced, leading to constitutive matriptase activation and increase cell migration and invasion. Imbalance of HAI-2-matriptase system is strongly suggested to be involved in the prostate cancer cell invasion and tumor metastasis.致謝 i文摘要 iibstract iiihapter 1. Introduction 1.1 Prostate cancer 2.2 Proteolytic enzymes 3.3 Matriptase 4.4 HAI-1 (Hepatocyte Growth Factor Activator Inhibitor-1) 9.5 HAI-2 (Hepatocyte Growth Factor Activator Inhibitor-2) 13.6 Research motivation 16hapter 2. Materials and Methods 18.1 Materials 19.2 Methods 22.3 Table 31.4 Buffers 32hapter 3. Results 34.1 Human prostate cancer cell progression model 35.2 Protein levels of HAI-2 in human prostate cancer cell progression model. 36.3 Restoration of HAI-2 gene expression upon 5-Aza-dC treatment 37.4 Inhibitory roles of HAI-2 in prostate cancer cell invasion and migration 38.5 Characterization of functional HAI-2 domain on cell migration and invasion. 39.5 Inhibitory roles of HAI-2 in prostate cancer cell proliferation 40.6 Inhibitory roles of HAI-2 in prostate cancer cell invasion and migration were via inhibiting serine protease(s). 41.7 Endogenous expression levels of matriptase and HAI-1 in the prostate cancer cell progression model. 42.8 Matriptase and HAI-1 in human prostate cancer cell progression model. 43.9 Inhibitory roles of HAI-2 in matriptase activation 44.10 Requirement of Kunitz domain 1 of HAI-2 in matriptase inhibition. 46.11 Reciprocal effects of HAI-2 and matriptase on prostate cancer. 47.12 Decreased shedding of activated matriptase by HAI-2 48.13 HAI-2 prevents a proteolytic cleavage of matriptase 49.14 HAI-2 affects matriptase protein stability 51.15 The inhibitory role of HAI-2 on matriptase activation was not mediated by PI3K/AKT pathway 54.16 HAI-2 directly interacts with matriptase 55.17 Reciprocal effects of HAI-1 and HAI-2 on matriptase 57hapter 4. Discussion 58hapter 5. Figure 69hapter 6. Reference 9