腫瘤相關巨噬細胞與第二型環氧酵素在人類基底細胞癌致癌機轉之研究

Roles of Tumor-Associated Macrophages and Cyclooxygenase-2 in the patholgenesis of Human Basal Cell Carcinoma

邱政偉(Jeng-Wei Tjiu)

指導教授 ： 紀秀華 ； 郭明良

國立臺灣大學/醫學院/臨床醫學研究所/博士(2008年)

https://doi.org/10.6342/NTU.2008.00612

摘要

論文中文摘要
人類基底細胞癌(human basal cell carcinoma; BCC)是最常見的人類癌症。全世界每年有幾百萬例新發生的基底細胞癌。雖然說基底細胞癌幾乎不會轉移，但是局部侵犯與組織破壞相當的常見。第二型環氧酵素 (cyclooxyngease-2; COX-2)與腫瘤相關巨噬細胞(tumor-associated macrophages; TAM)已經知道會促進許多人類癌症的腫瘤新生(tumorigenesis)、血管新生(angiogenesis)、以及轉移(metastasis)。但是，COX-2與腫瘤相關巨噬細胞在人類基底細胞癌中扮演的角色仍然不清楚。我們利用免疫組織染色的方法發現人類基底細胞癌細胞本身表現COX-2的強度與其中浸潤的腫瘤相關巨噬細胞的數目，與基底細胞癌侵襲的深度與微細血管的密度(microvessel density; MVD)有關。再利用多變項線性迴歸分析控制其他可能影響到基底細胞癌侵襲與血管新生的因素，結果發現基底細胞癌本身COX-2表現強度與腫瘤相關巨噬細胞的數量是影響基底細胞癌侵襲深度與血管新生的獨立預測因子。這代表由臨床研究發現COX-2與腫瘤相關巨噬細胞與基底細胞癌的進展有重要的關聯性。
論文的第一部分，我們著重在研究基底細胞癌細胞本身過度表現COX-2對於基底細胞癌的影響。我們建立穩定轉殖並過度表現COX-2的人類基底細胞癌細胞株。COX-2過度表現的基底細胞癌細胞株中對抗細胞凋亡的Mcl-1與Bcl-2蛋白質的表現量顯著的上升，同時產生的對於紫外線B光照射誘發細胞凋亡的抗性。COX-2過度表現也會增加基底細胞癌細胞分泌血管內皮生長因子(vascular endothelial growth factor-A; VEGF-A)與鹼性纖維母細胞生長因子(basic fibroblast growth factor; bFGF)，因而促進基底細胞癌在活體內與活體外的血管新生。如果使用COX-2專一性的siRNA減少COX-2的表現量或是用COX-2的抑制劑NS-398抑制COX-2的功能，將會減少VEGF-A與bFGF的分泌量。當COX-2過度表現的基底細胞癌細胞打入SCID老鼠體內的時候，會產生比控制組更大的異種腫瘤(xenograft)。在COX-2過度表現的異種腫瘤中發現其新生血管也比控制組的異種腫瘤中的新生血管來的多。因此，我們證實了COX-2在人類基底細胞癌細胞過度表現的確會促進基底細胞癌細胞產生對細胞凋亡的抗性、增加其血管新生與腫瘤新生的能力，因而促進基底細胞癌細胞的進展。
運用臨床上手術切除下來基底細胞癌的檢體做免疫組織染色的結果發現，在COX-2過度表現的基底細胞癌細胞團塊附近有相當多的腫瘤相關巨噬細胞聚集。免疫組織染色結果的統計上也發現，基底細胞癌當中腫瘤相關巨噬細胞的數量與基底細胞癌細胞本身COX-2的表現量成顯著的正相關。加上我們已經發現COX-2在基底細胞癌細胞內過度表現可以促進基底細胞癌的腫瘤新生與血管新生。因此，我們第二部分研究的假設是腫瘤相關巨噬細胞經過活化基底細胞癌細胞的COX-2來達到促進基底細胞癌細胞侵襲與血管新生的作用。
腫瘤相關巨噬細胞是一種M2巨噬細胞。M2巨噬細胞是由Th2細胞激素活化的巨噬細胞會促進腫瘤的生長。人類THP-1細胞株在經由phorbol myristate acetat (PMA)處理過後會變成巨噬細胞，在以往的研究中常常用來作為腫瘤相關巨噬細胞的模型。我們證明了PMA-treated THP-1巨噬細胞是一種M2巨噬細胞，它們會表現M2的表現標記(CD204與CD206)，同時表現M2的cytokine profile。基底細胞癌細胞在與PMA-treated THP-1巨噬細胞共同培養之後，會增加基底細胞癌細胞COX-2的表現、與其侵襲性及活體外血管新生的能力。但是如果將基底細胞癌細胞內的COX-2表現用COX-2專一性的siRNA或是COX-2專一性抑制劑celecoxib阻斷的話，與PMA-treated THP-1巨噬細胞共同培養誘發基底細胞癌細胞的侵襲與血管新生就會顯著的減少。與巨噬細胞共同培養會經由活化基底細胞癌中p38 MAPK/ NF-κB訊息傳遞路徑活化基底細胞癌的COX-2表現。與巨噬細胞共同培養誘發基底細胞癌侵襲性的增加，是經由活化基底細胞癌中p38 MAPK/ NF-κB/ COX-2訊息傳遞路徑活化MMP-9的分泌與活性造成基底細胞癌侵襲增加。與巨噬細胞共同培養誘發基底細胞癌血管新生的增加，也是經由活化基底細胞癌中p38 MAPK/ NF-κB/ COX-2訊息傳遞路徑活化VEGF-A與bFGF的分泌而造成基底細胞癌血管新生的增加。為了讓我們的研究結果更加一般化，我們用人類週邊血液單核球(monocytes)細胞，將之分化為巨噬細胞後加入IL-4/IL-13，使其變為M2巨噬細胞。將單核球演變而來的M2巨噬細胞與基底細胞癌細胞共同培養，結果也會促進基底細胞癌細胞產生COX-2依賴的侵襲與血管新生，同時也會增加基底細胞癌細胞分泌MMP-9、VEGF-A、與bFGF。總結來說，腫瘤相關巨噬細胞會經由COX-2依賴的路徑來促進基底細胞癌細胞的侵襲性與血管新生。
本論文研究的結果指出COX-2與腫瘤相關巨噬細胞在人類基底細胞癌細胞的致癌機轉與其進展中的重要角色。COX-2與腫瘤相關巨噬細胞可以做為未來開發新的非手術性治療人類基底細胞癌新療法的標的。

關鍵字

人類基底細胞癌 ； 腫瘤相關巨噬細胞 ； 第二型環氧酵素 ； 侵襲 ； 血管新生

並列摘要

Human basal cell carcinoma (BCC) is the most common human cancer. BCC affects millions of people worldwide annually. Although BCC never metastasis, but local invasion and destruction are very common. Cyclooxygenase-2 (COX-2) and tumor-associated macrophages (TAM) are known to increase tumorigenesis, angiogenesis, and metastasis in many human cancers. However, the role of COX-2 and TAM remain elusive in human basal cell carcinoma. We found that both COX-2 expression in BCC epithelial cells and number of TAM associate with increased invasion depth and microvessel density (MVD) by immunohistochemistry (IHC). Multivariate linear regression analysis showed that both grades of COX-2 expression in BCC epithelial cells as well as number of TAM are independent predictors for depth of invasion and MVD in human BCC.
In the first part of the study, we focused first on the effects of COX-2 expression in human BCC cells. Overexpression of COX-2 in human BCC cell line conferred cells resistance to ultraviolet B (UVB) induced apoptosis by up-regulation of anti-apoptotic proteins Mcl-1 and Bcl-2. COX-2 overexpression also increased secretion of vascular endothelial growth factor-A (VEGF-A) and basic fibroblast growth factor (bFGF) and increased in vitro and in vivo angiogenesis. Blocking COX-2 fuction by COX-2 specific RNA interference (siRNA) or specific inhibitor NS-398 abrogated increased VEGF-A and bFGF secretion by BCC cells. When COX-2 overexpressing BCC cells were inoculated into SCID subcutaneously, they developed xenograft larger than vector control counterparts. Significant increased neovascularization was observed in COX-2 overexpressing xenograft. We confirmed COX-2 promote BCC progression by enhancing anti-apoptotic ability, angiogenesis and tumorigenesis.
Prominent TAM aggregated adjacent to COX-2 overexpressing tumor nests in BCC. In addition, increased number of TAM significantly correlated grading of COX-2 expression in BCC cancer cells. Taken the special adjacency and statistics together, we hypothesized that TAM might induce COX-2 expression in BCC cells and subsequently increase invasion and angiogenesis in BCC.
TAM is a kind of M2 macrophages, which is activated by Th2 cytokines. Human THP-1 cell line differentiated to macrophages after treatment with phorbol myristate acetate (PMA). We demonstrated PMA-treated THP-1 macrophages were a kind of M2 macrophages because they express M2 surface markers (i.e. CD204 & CD206) and a M2 cytokine profile. Non-contact coculture with PMA-treated THP-1 macrophages induced COX-2 expression, incrased invasiveness, and in vitro angiogenesis in human BCC cells. Once COX-2 activity was abrogated by COX-2 specific siRNA or specific inhibitor celecoxib, the increased invasiveness and angiogenesis induced by coculture were attenuated. Coculture with macrophages induced COX-2 expression in BCC by activating the p38 MAPK/NF-κB signaling pathway. Coculture with macrophages induced MMP-9 activation by p38 MAPK/ NF-κB /COX-2 cascades and increased invasion of BCC cells. Increased angiogenesis induced by coculture resulted from increased secretion of VEGF-A and bFGF by p38 MAPK/ NF-κB /COX-2 cascades. To verify our findings, we generate another kind of M2 macrophages from human peripheral monocytes. Coculture with monocyte-derived M2 macrophages also induced COX-2 dependent invasion and angiogenesis, as well as increased expression of MMP-9, VEGF-A and bFGF. We conclude that TAM might induce BCC invasion and angiogenesis via a COX-2 dependent manner in BCC cells.
Our study highlights the importance of COX-2 and TAM in the pathogenesis and progression of human BCC. COX-2 and TAM might be used as therapeutic targets for the non-surgical treatments of human basal cell carcinoma in the near future.

並列關鍵字

human basal cell carcinoma ； tumor-associated macrophages ； cyclooxygenase-2 ； invasion ； angiogenesis

參考文獻

Akita Y, Kozaki K, Nakagawa A, Saito T, Ito S, Tamada Y et al. Cyclooxygenase -2 is a possible target of treatment approach in conjunction with photodynamic therapy for various disorders in skin and oral cavity. Br J Dermatol. 2004 Aug; 151(2): 472-80.

Ando T, Shibata H, Suzuki T, Kurihara I, Hayashi K, Hayashi M et al. The possible role of apoptosis-suppressing genes, bcl-2 and mcl-1/EAT in human adrenal tumors. Endocr Res. 1998 Aug-Nov; 24(3-4): 955-60.

Arber N, Eagle CJ, Spicak J, Rácz I, Dite P, Hajer J et al. Celecoxib for the prevention of colorectal adenomatous polyps. N Engl J Med. 2006 Aug 31; 355(9): 885-95.

Arico S, Pattingre S, Bauvy C, Gane P, Barbat A, Codogno P et al. Celecoxib induces apoptosis by inhibiting 3-phosphoinositide-dependent protein kinase-1 activity in the human colon cancer HT-29 cell line. J Biol Chem. 2002 Aug 2; 277(31): 27613-21.

Azizi E, Bucana C, Goldberg L, Kripke ML. Perturbation of epidermal Langerhans cells in basal cell carcinoma. Am J Dermatopathol. 1987 Dec; 9(6): 465-73.