乳癌幹細胞新血管生成潛力之機轉探討

在近代的社會中，癌症是個無形的殺手，尤其是乳癌對於女性的影響，更是不可忽略，由於乳癌難以治癒，所以每年在全世界都有數百萬名的女性為乳癌所苦。在先前的研究中學者們認為，癌症組織發展可以由任意一個癌細胞進行分化及增生達到產生一個新腫瘤的目的，這也就是所謂的“聚落式”模式。但是在最近的研究中指出，只有少部分的細胞有能力形成新的腫瘤，而這少部分的細胞又帶有類似正常幹細胞的性質並且與腫瘤的生成、自我更新、轉移以及抗藥性有非常大的相關性，這種細胞就被稱之為“癌症幹細胞”，由於這發現，也指出癌症的生長模式較相似於階級制而非先前研究所說的聚落式。另外在腫瘤生長的過程中，養分的供應也扮演著一個相當重要的角色，當腫瘤直徑大於2 mm的時候，單靠擴散作用已經沒有辦法供應腫瘤養分及氧氣，此時癌細胞就會釋出部分因子誘使周邊的內皮細胞以出芽的方式生成新的微血管，或是從骨髓中徵召血管內皮前趨細胞到腫瘤位進行排列形成新血管，而這些新生成的血管不只提供了腫瘤養分供應，它們也提供了腫瘤一個轉移的途徑造成病人預後變差。在神經膠質瘤以及卵巢癌中也發現到，癌症幹細胞會分化成內皮細胞族群，並且經由這些細胞排列形成微血管，由此我們便想討論，乳癌幹細胞在新血管生程中扮演的角色是什麼。我們首先自乳癌細胞株AS-B244中分離乳癌幹細胞 (ALDH +)和非乳癌幹細胞株(ALDH-)進行體外管狀結構生成測試，發現到只有乳癌幹細胞具有新血管生成的能力，接著我們使用反轉錄及聚合酶連鎖反應來對乳癌幹細胞mRNA表現進行觀察，發現到乳癌幹細胞株表現許多在人類臍帶內皮細胞株也會表現的基因包括有VEGF-A、VEGFR2、SDF-1α以及CXCR4。為了進一步研究乳癌幹細胞新血管生成的分子機制，我們首先觀察VEGFR2在其中的角色，使用PTK787對VEGFR2抑制之後可以看到PTK787對於AS-B244細胞的新血管生成有濃度依賴型的抑制，也說明了乳癌幹細胞新血管生成為VEGFR2依賴型的現象。我們也對AS-B244細胞處以槲黃素，槲黃素是一種植物中所含有的天然抗氧化物，對於小分子熱休克蛋白有抑制作用，並且有報導指出槲黃素也能抑制癌症新血管生成，在我們的實驗中可以得知，槲黃素也對AS-B244細胞的新血管生成有濃度依賴性的抑制作用。從西方墨點法的結果可以看到在Hsp27和VEGFR2有被抑制的情況下都能夠有效的抑制乳癌幹細胞新血管生成，並且可以發現到在槲黃素處理下，VEGFR2的mRMA表現量有減少，指出槲黃素有可能對於VEGFR2有轉錄上的抑制作用。我們進一步使用siRNA處理降低Hsp27蛋白表現也可以發現到對於AS-B244細胞有明顯的新血管生成抑制作用。由我們的結果可以得到以下結論，乳癌幹細胞新血管生成，可能是透過VEGFR2訊號傳遞路徑以及Hsp27來進行調控。

關鍵字

乳癌；癌症幹細胞；新血管生成

並列摘要

Cancer is an invisible killer in our modern worlds. Millions of women were suffered from breast cancer and it was hard to be cured. In former research, the heterogeneity of cancers has been suggested as a clonal evolution model which indicated that each single cancer cell could form a new tumor and differentiate into any cell type in order to support tumor growth. But recent studies have demonstrated that only a small population of cells within a tumor tissue was responsible for tumorigenesis and with malignant and stem cell-like characteristics such as differentiation, self-renewal, chemoresistance and metastasis. These cells were called as cancer stem cells (CSCs) and it shifts the hypothesis of tumor heterogeneity toward a hierarchy model. Nutrient supply plays an important role during tumor progression.Simple diffusion can’t support enough nutrients when tumor diameter was larger than 2mm. Cancer cells would try to release some factors to attract endothelial cells to bud from peripheral blood vessels and form new micro vascular structures to support tumor growth. Cancer cells also secret factors to attract endothelial progenitor cells from bone marrow to the tumor site and alignment to form new blood vessels. Micro blood vessels not only support tumor growth but also create a path for tumor metastasis. Recent studies indicated that glioma or ovarian CSCs could differentiate into endothelial-related cell types and form microvascular structures within tumor mass. Whether breast CSCs contribute to tumor vascularization remains unclear. We first compared the in vitro tube formation capacity between breast CSCs and non-CSCs from human breast cancer cell line, AS-B244, in matrigel-coated wells and the results demonstrated that only breast CSCs were able to form vessel-like structures. We next analyzed the expression of endothelial cells or angiogenesis related genes between breast CSCs and non-CSCs by RT-PCR and we found that breast CSCs expressed many angiogenesis-related genes, such as VEGF-A, VEGFR2, SDF-1 and CXCR4, with a similar level when compared to HUVEC. In order to understand the molecular mechanisms of the neovascularization potential of breast CSCs, we first tried to investigate the role of VEGFR2 by a small molecule inhibitor, PTK787. Pre-treatment of PTK787 caused inhibition of tube formation capacity of AS-B244 cells in a dose dependent manner. It suggests that the neovascularization potential of breast CSCs may be a VEGFR2 dependent phenomenon. Quercetin, a flavanoid derived from fruits and grains, shows highly inhibition on small heat shock proteins and it was reported to be an inhibitor of neovascularization of cancer cells. Pre-treatment of quercetin also suppress the tube formation capacity of AS-B244 cells in a dose dependent manner. By western blot analysis, the expression of Hsp27 and VEGFR2 was decreased. The expression of VEGFR2 mRNA was also inhibited by quercetin indicating that quercetin may transcriptionally inhibit VEGFR2 expression. We further investigated the role of Hsp27 in the neovascularization potential of breast CSCs. By siRNA-mediated gene silencing, knockdown of Hsp27 caused completely inhibition of tube formation capacity of AS-B244 cells. In conclusion, breast CSCs have neovascularization potential and it may be regulated by VEGFR2 and Hsp27.

並列關鍵字

Breast cancer ； Cancer stem cells ； Neovasculogenesis

參考文獻

Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, and Clarke MF.,

Prospective identification of tumorigenic breast cancer cells., Proc Natl Acad Sci

Al-Hajj M, and Clarke MF., Self-renewal and solid tumor stem cells., Oncogene.

2004; 23(43):7274–7282

DA, and Mor G., Stem-like Ovarian Cancer Cells can Serve as Tumor Vascular

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乳癌幹細胞新血管生成潛力之機轉探討

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