血行性癌細胞(circulating tumor cells)表面纖連蛋白(fibronectin, FN ) 的組裝在與肺臟血管內皮細胞結合機制中扮演重要的角色。不具穿膜區域的胞外基質(extracellular matrix)中的一員FN需透過與細胞表面穿膜蛋白受器結合來促進FN的組裝，但血行性癌細胞膜上的FN受器尚未被研究。目前我們實驗室已發現鈉氫離子交換蛋白(Sodium/hydrogen exchanger；NHE)可扮演此受器的角色而促進FN的組裝與癌細胞肺轉移。而有文獻也指出，另一細胞表面穿膜蛋白CD44也會與FN結合。CD44 的數個exons中只variant exon 3 (V3)接有heparan sulfate glycosaminoglycans，而FN又含有數個heparin binding sites，此事實合理化了這兩分子的結合，但CD44與癌細胞轉移能力的關係卻極具爭議性。我們實驗室過去發現低轉移能力之人類乳癌及食道癌細胞中CD44扮演抑制轉移的角色。且在臨床上，含variant exon 3的isoforms (V3-CD44)在低轉移程度的癌症病人組織中表現較多。文獻又指出CD44可透過lipid raft被細胞內吞。因此，我們假設CD44可能利用v3與FN結合並藉由lipid rafts進行內吞來抑制NHE上FN的組裝並減少肺轉移。食道癌在台灣的發病率連年攀升，因此我們以食道癌為研究模式。首先，我們發現食道癌細胞表面組裝的程度主要受到FN內吞的影響而非自環境中攝取FN的能力。接著我們發現v3-CD44在食道癌細胞中的確可藉著與FN結合而促進整個V3-CD44/FN複合體在lipid raft區域內內吞入細胞中。另外，由外圍移位至lipid raft區域的NHE會促進含NHE、v3-CD44、高分子量FN之巨大複合體被食道癌細胞經由依賴V3-CD44途徑所內吞，並減少表面FN組裝。本實驗室的研究已知Rho A可以負調控FN組裝，我們接著發現因v3-CD44/FN的共同內吞而受抑制的細胞表面FN組裝可被Rho A下游之Rho kinase (ROK)蛋白所促進。最後，我們更發現v3-CD44可在Lipid raft以外區域扮演FN組裝之細胞表面受器。這些結果提供有力的證據來支持v3-CD44可利用與FN的結合與有lipid raft參與的內吞，而抑制FN組裝的假說。本論文部分揭開了懸浮(血行性)食道癌癌細胞調控細胞表面組裝FN的訊息傳遞分子機制，提供了未來遏止癌症轉移的可能治療標靶分子，且可能為食道癌癌症病人因延長壽命而帶來一線治癒的曙光。

The pericellular fibronctin (FN) assembled on circulating tumor cells (CTCs) plays an important role in mediating binding of CTCs to the lung endothelia. In order to be assembled on suspended tumor cell surfaces, FN, one of non-transmembrane extracellular matrix components, must first bind to surface transmembrane receptor(s). However, not any evidence has been provided in the studies of the surface transmembrane receptor(s) on suspended tumor cells for FN. We have preliminarily found that Sodium/hydrogen exchanger (NHE) may serve as such a FN receptor in promoting cell surface FN assembly, leading to cancer metastasis in the lungs. Interestingly, CD44, another cell surface transmembrane protein, is also capable of binding to FN. Among various exons of CD44, only variant exon 3 (V3) is glycosylated with heparan sulfate glycosaminoglycans, a potential FN-binding site within CD44 to bind to heparin binding sites within FN. Nevertheless, the relationship between CD44 expression and cancer metastasis is relatively controversial. Our preliminary results indicate a role of CD44 expressions of human breast cancer and esophageal squamous carcinoma cells (ESCCs) with low metastatic abilities in suppressing metastases. Clinically, it has also been found that isoforms harboring V3 (V3-CD44s) are highly expressed in cancer tissues of those cancer patients who have less metastatic complications and better prognosis. Moreover, it has been known that CD44 within lipid rafts may be endocytosed, leading us to the hypothesis that V3-CD44s suppress cancer metastasis via promoting lipid raft-engaged endocytosis of V3-CD44s/FN complexes and thus inhibiting NHE-mediated pericellular FN assembly on cancer cell surfaces. Since the morbidity and mortality of ESCC in Taiwan recently have been climbing high, we thus employ ESCC as our main study model. We first demonstrated that it is the ability of FN endocytosis, but not that of the FN-uptaking, that regulates pericellular FN assembly on ESCCs. Next, we showed that the V3-CD44 was endocytosed together with surface FN being formed as a large complex within lipid rafts. Moreover, we found that the translocation of NHE into lipid rafts may promote a V3-CD44-mediated endocytosis of large complexes including NHE, V3-CD44, and polymeric FN, leading to the reduction of the pericellular FN assembly of ESCCs. Preliminarily, we found that the activity of RhoA negatively regulates pericellular FN assembly of breast cancer cells. Here, we showed that a reduction of pericellular FN assembly via endocytosis of V3-CD44/FN complexes was provoked by Rho kinases (ROKs), the direct downstream effectors of RhoA. Finally, we found that, once located outside lipid rafts, V3-CD44 may serve as another cell surface receptor in promoting pericellular FN assembly. These results strongly support our hypothesis that V3-CD44 suppresses pericelllular FN assembly of ESCCs via complexing with FN within lipid rafts and promoting endocytosis of the V3-CD44/FN complexes. This thesis partially unveils the signaling regulatory mechanism for pericellular FN assembly of suspended ESCCs, provides candidate target molecules for therapeutics to impede cancer metastasis, and sheds lights on curing ESCC patients even in late stages by prolonging patients’ survivals.

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